RHIPICEPHALUS SANGUINEUS

[001] This invention relates to a shampoo comprising a Piper tuberculatum extract, as well as the use of shampoo in controlling Rhipicephalus sanguineus, and methods for preparing the shampoo.

[002] Background

[003] Dangerous pests such as ticks (such as Rhipicephalus sanguineus) and fleas are commonly found on dogs and are difficult to control, often resulting in the transmission of infectious agents to humans. Currently, no effective vaccines are available and dog owners rely on ingestible medicine, repellent collars or spot solutions. In addition, ticks have developed resistance to a high percentage of chemicals used in existing solutions. There is also a changing consumer preference for organic, environmentally friendly solutions as pet owners become more aware of the toxic chemicals used in existing products.

[004] The In-vitro acaricidal activity of a 70% ethyl alcohol extract of Piper tuberculatum was tested against Rhipicephalus (Boophilus) microplus, also known as the "common cattle tick", in Hailyng Barrios, Byron Flores, Christiane Duttmann, Brenda Mora-Sanchez, Jessica Sheleby-Elias, William Jiron & Jose L. Balcazar (2022): In vitro acaricidal activity of Piper tuberculatum against Rhipicephalus (Boophilus) microplus, International Journal of Acarology, DOI: 10.1080/01647954.2022.2050808. However, testing was only against this specific tick. Use on dogs, for example in controlling Rhipicephalus sanguineus, was not mentioned.

[005] In connection with the specific problem of controlling Rhipicephalus sanguineus, it should be noted that the Rhipicephalus genus comprises eighty-five species. The genus population structure is extremely complex, and even within the species there are high degrees of variability. This variability is reflected in the biology, ecology, vector competence and capacity of Rhipicephalus sanguineus to different pathogens. Thus, the efficacy of a particular composition against one species of the Rhipicephalus genus provides no indication of whether that composition could also be utilised against other species within that genus.

[006] An improved composition for use in controlling Rhipicephalus sanguineus has been sought.

[007] Statement of invention

[008] This invention relates to a shampoo comprising a Piper tuberculatum extract. In particular, the shampoo may be a canine shampoo.

[009] In the context of the invention, the phrase "Piper tuberculatum extract" is used to refer to the solid obtained when water, or a mixture of water and at least one alcohol, is contacted with Piper tuberculatum plant material for a defined period of time in order to extract components from the Piper tuberculatum plant material, and the water, or mixture of water and at least one alcohol, is removed (for example by evaporation) to produce the solid. In particular, the at least one alcohol may comprise ethanol and/or isopropyl alcohol, more particularly the at least one alcohol may comprise isopropyl alcohol. In particular, the mixture may comprise at least 50 %v/v of the at least one alcohol, more particularly at least 60 %v/v, even more particularly at least 65 %v/v

[0010] In particular, the shampoo as defined above may additionally comprise a shampoo base comprising one or more of:

(a) an anionic surfactant,

(b) a non-ionic surfactant,

(c) sodium chloride,

(d) honey, and

(e) water.

More particularly, the shampoo base may comprise components (a)-(c) and (e). Even more particularly, the shampoo base may comprise all of components (a)-(e).

[0011] In particular, the anionic surfactant may be a sodium lauryl ether sulfate such as Texapon®. More particularly, the non-ionic surfactant may be cocamide DEA such as Comperlan. [0012] In particular, the shampoo base may comprise 8-14 wt% of the anionic surfactant, more particularly 9-13 wt%, even more particularly 10-12 wt%. In particular, the shampoo base may comprise 1-7 wt% of the non-ionic surfactant, more particularly 2-6 wt%, even more particularly 3-5 wt%.%. In particular, the shampoo base may comprise 2-5 wt% sodium chloride, more particularly 3-4 wt%. %. In particular, the shampoo base may comprise 1-4 wt% honey, more particularly 2-3 wt% honey. In particular, the shampoo base may comprise 40-90 wt% water, more particularly 70-90 wt%, even more particularly 75-85 wt%. More particularly, the balance of the shampoo base may comprise water.

[0013] In particular, the shampoo may comprise (i) an aqueous solution of the Piper tuberculatum extract, and (ii) the shampoo base. More particularly, the aqueous solution of the Piper tuberculatum extract may comprise 50-500 mg of the Piper tuberculatum extract per ml of the solution, more particularly 100-300 mg/ml, even more particularly 150-250 mg/ml.

[0014] In particular, the shampoo may comprise 0.5-2 parts by volume of the aqueous solution comprising the Piper tuberculatum extract and 2-4 parts by volume of the shampoo base. More particularly, the shampoo may comprise about 1 part by volume of the aqueous solution comprising the Piper tuberculatum and about 3 parts by volume of the shampoo base. In particular, the shampoo may comprise 10-100 mg/ml of the Piper tuberculatum extract, more particularly 25-75 mg/ml, even more particularly 40-60 mg/ml.

[0015] This invention also relates to a method of controlling Rhipicephalus sanguineus comprising applying to a canine animal a shampoo as defined above. In addition, this invention relates to a method of treating Rhipicephalus sanguineus infestation in a canine animal comprising applying to the canine animal a shampoo as defined above.

[0016] This invention also relates to the use of a shampoo as defined above in controlling Rhipicephalus sanguineus. In addition, this invention relates to a shampoo as defined above for use in controlling Rhipicephalus sanguineus. This invention also relates to the use of a shampoo as defined above in treating Rhipicephalus sanguineus infestation. In addition, this invention relates to a shampoo as defined above for use in treating Rhipicephalus sanguineus infestation. In particular, the treatment may be of a canine animal.

[0017] This invention also relates to the use of a Piper tuberculatum extract, for example as defined above, in controlling Rhipicephalus sanguineus. In addition, this invention relates to a Piper tuberculatum extract, for example as defined above, for use in controlling Rhipicephalus sanguineus. This invention also relates to the use of a Piper tuberculatum extract, for example as defined above, in treating Rhipicephalus sanguineus infestation. In addition, this invention relates to a Piper tuberculatum extract, for example as defined above, for use in treating Rhipicephalus sanguineus infestation. In particular, the treatment may be of a canine animal.

[0018] This invention also relates to a method for preparing a shampoo as defined above, the method comprising the steps of:

(a) providing an aqueous solution comprising a Piper tuberculatum extract, and

(b) mixing the aqueous solution with a shampoo base comprising one or more of an anionic surfactant, a non-ionic surfactant, sodium chloride, honey, and water.

[0019] In particular, step (b) may comprise mixing the aqueous solution with a shampoo base comprising an anionic surfactant, a non-ionic surfactant, sodium chloride and water. More particularly, step (b) may comprise mixing the aqueous solution with a shampoo base comprising an anionic surfactant, a non-ionic surfactant, sodium chloride, honey, and water. Even more particularly, the shampoo base may be as defined above.

[0020] In particular, step (a) may comprise preparing an aqueous solution comprising a Piper tuberculatum extract. More particularly, preparing the aqueous solution may comprise the steps of:

(i) adding water, or a mixture of water and at least one alcohol, to Piper tuberculatum plant material,

(ii) allowing the water, or the mixture of water and the at least one alcohol, to contact the Piper tuberculatum plant material for a defined period of time, (iii) removing the water, or the mixture of water and the at least one alcohol, to provide a Piper tuberculatum extract, and

(iv) adding water to the Piper tuberculatum extract to produce an aqueous solution comprising a Piper tuberculatum extract.

More particularly, step (i) may comprise adding a mixture of water and at least one alcohol to Piper tuberculatum plant material. In particular, the at least one alcohol may comprise ethanol and/or isopropyl alcohol, more particularly the at least one alcohol may comprise isopropyl alcohol. In particular, the mixture of water and at least one alcohol may comprise at least 50 %v/v of the at least one alcohol, more particularly at least 60 %v/v, even more particularly at least 65 %v/v.

[0021] In particular, step (i) may comprise drying Piper tuberculatum plant material, milling the Piper tuberculatum plant material to form a Piper tuberculatum powder, and then adding water, or a mixture of water and at least one alcohol, to the Piper tuberculatum powder. More particularly, the amount of Piper tuberculatum powder to which the water, or the mixture of water and at least one alcohol, was added may be 50-500 mg of plant material per ml of the mixture, more particularly 100-300 mg/ml, even more particularly 150-250 mg/ml. In particular, between steps (ii) and (iii) the method may comprise the step of filtering the water, or the mixture of water and the at least one alcohol, and the Piper tuberculatum plant material.

[0022] In particular, in step (iii) the removing may be by evaporation. More particularly, in step (iv) the amount of water added to the Piper tuberculatum extract may be such that the aqueous solution comprises 50-500 mg of Piper tuberculatum extract per ml of the mixture, more particularly 100-300 mg/ml, even more particularly 150-250 mg/ml.

[0023] In particular, the defined period of time may be 1-14 days, more particularly 2-10 days, even more particularly 4-8 days. In particular, step (ii) may include heating the mixture of water and the at least one alcohol. More particularly, the heating may be to at least 30 °C, even more particularly to at least 40 °C

[0024] In particular, in step (b) of the method as defined above for preparing a shampoo as defined above, 0.5-2 parts by volume of the aqueous solution comprising the Piper tuberculatum extract are mixed with 2-4 parts by volume of the shampoo base. More particularly, about 1 part by volume of the aqueous solution comprising the Piper tuberculatum extract is mixed with about 3 parts by volume of the shampoo base. In particular, the amount of Piper tuberculatum extract in the shampoo may be 10-100 mg/ml, more particularly 25-75 mg/ml, even more particularly 40-60 mg/ml.

[0025] In addition, this invention relates to a shampoo obtainable by the method defined above.

[0026] Brief description of the drawings

[0027] This invention will be further described by reference to the following Figures which are not intended to limit the scope of the invention claimed, in which:

Figure 1 shows graphs comparing the presence of ticks in the ears of the dogs in the control and experimental ("Shampoo Piper") groups during the three weeks of testing,

Figure 2 shows graphs comparing the presence of ticks on the neck of the dogs in the control and experimental ("Shampoo Piper") groups during the three weeks of testing,

Figure 3 shows graphs comparing the presence of ticks on the paws of the dogs in the control and experimental ("Shampoo Piper") groups during the three weeks of testing,

Figure 4 shows graphs comparing the presence of ticks on other body parts of the dogs in the control and experimental ("Shampoo Piper") groups during the three weeks of testing, and

Figure 5 shows graphs comparing the presence of dry ticks on the dogs in the control and experimental ("Shampoo Piper") groups initially and after two weeks of testing.

[0028] Detailed description

[0029] Preparation for In-vivo testing [0030] 18 dogs were identified in Leon City, Nicaragua, and surrounding areas. The characteristics of the dogs were: male or female of all ages, easy to handle, with infestation of ticks at various parts of the body.

[0031 ] Preparation of shampoos with and without Piper tuberculatum

[0032] Leaves, flowers and seeds of the Piper tuberculatum plant were collected and then dried naturally for two weeks. A hydro-alcoholic solution of Piper tuberculatum at 20% (600 g Piper per 3 litres of isopropyl alcohol 70 %v/v) was prepared as follows. All parts of the plant were ground with a blender to produce a fine powder. 600 g of the powder was mixed with 3000 ml of 70 %v/v isopropyl alcohol (to give a concentration of 200 mg/ml). This hydro-alcoholic solution was left to rest overnight, and it was then filtered first with a cotton filter and then with Whatman™ grade 1 filter paper. The resulting filtrate was divided into six 500 ml portions, which were placed in the incubator at 50 °C for 6 days until all the liquid had evaporated. The residual sediment (i.e. the Piper tuberculatum extract) from each of the six portions weighed 35 g. 175 ml of water was then added to each of the six portions, giving an aqueous solution of Piper tuberculatum having a concentration of 200 mg/ml.

[0033] 3 litres of a shampoo base was then prepared as follows:

1. 320 g of Texapon was beaten until it turned white

2. 120 g of Comperland was added to the prepared Texapon with stirring

3. 104 g of sea salt was dissolved in 300 ml of water to produce a brine

4. 80 g of honey was dissolved in 176 ml of water

5. The brine and the honey water were added to the Texapon and Comperland mixture

6. 1900 ml of water was added with fast stirring

7. The resulting shampoo base was left to stand for 12-24 hours to allow the foam to set

[0034] An experimental shampoo according to the invention was then prepared by mixing the 3 litres of shampoo base with 1000 ml of the 200 mg/ml aqueous Piper solution (giving a total volume of 4 litres of the experimental shampoo). The concentration of the final product was a 5% (50 mg/ml) Piper shampoo. [0035] In-vivo testing

[0036] Two products were tested, the shampoo base as a control group and the experimental shampoo with Piper tuberculatum extracts at 5% (50 mg/ml), in order to determine effectiveness in controlling ticks. The dogs were processed in two groups as set out below.

[0037] Group of dogs washed with the experimental shampoo comprising the hydro- alcoholic tuberculatum extract 10 dogs were washed completely with the experimental shampoo. Before bathing, the body of each dog was checked to determine tick infestation, especially the neck, head, ears and paws, as well as the rest of the body. The developmental stage of the ticks was determined to confirm new infestations.

[0038] Control group washed with shampoo without the hydro-alcoholic Piper tuberculatum extract The 7 dogs in the control were washed with the shampoo base using the same procedure as the group washed with the experimental shampoo.

[0039] Repetitions of the tested group and concentration - The washing with the shampoo in the experimental and control groups was carried out once a week for three weeks. A week after the final wash, each dog was checked to determine tick infestation. If any dog still had ticks, they received a conventional chemical composition. The owners were then advised of further prevention measures. One dog had to be removed from the study due to illness.

[0040] Results

[0041] Before each wash, the body of each dog was checked to identify tick infestation of the species Rhipicephalus sanguineus. The results are reported in four groups based on the infestation site, i.e. ears, neck, paws and other parts. The presence of any dry ticks, dead and still stuck on the dogs body, was also recorded.

[0042] Results for body infestation: Ears - When checking for the presence of ticks in the ears of the canines, we found that 9 of 10 dogs to be washed with the experimental shampoo had ticks in their ears in week one. However, by the third week of washing only 3 of these 10 dogs had ticks in their ears. According to the statistical test of McNemar, this result reflects a significant decrease (p<0.05). On the other hand, in week one of the control group 4 of 7 dogs were observed to have ticks in their ears. The number of infested dogs in the control group increased to 7 of 7 by the third week. This suggests that in the absence of the hydro-alcoholic Piper tuberculatum extract the infestation increased, although not significantly (p>0.05). This data is shown in Figure 1.

[0043] Results for body infestation: Neck - When checking for the presence of ticks on the necks of the dogs, we found that 9 of 10 dogs to be washed with the experimental shampoo had ticks on their neck in week one. However, by the third week of washing only 1 of these 10 dogs had ticks on its neck (McNemar, p<0.05). On the other hand, in week one of the control group all 7 dogs were observed to have ticks on their neck. This level of incidence was maintained during the three weeks of testing (p>0.05). This data is shown in Figure 2.

[0044] Results for body infestation: Paws - When checking for the presence of ticks on and underneath the paws of the canines, we found that 9 of 10 dogs to be washed with the experimental shampoo had ticks in their paws in week one. However, by the third week of washing only 1 of these 10 dogs had ticks on their paws (McNemar, p<0.05). In week one for the control group, 4 of 7 dogs were observed to have ticks on their paws. This level of incidence was increase to 6 of the 7 dogs during the three weeks of testing (p>0.05). This data is shown in Figure

3.

[0045] Results for body infestation: Other body parts - When checking for the presence of ticks on the other parts of the dogs' bodies, we found that 7 of 10 dogs to be washed with the experimental shampoo had ticks in week one. However, by the third week of washing this frequency decreased to only 3 of the 10 dogs (McNemar, p>0.05). On the other hand, in week one of the control group 5 of the 7 dogs were observed to have ticks on their bodies. This level of incidence was maintained during the three weeks of testing (p>0.05). This data is shown in Figure

4. [0046] Dry ticks - From the second week of follow-up, the dogs were checked for the presence of dead (dry) ticks. No dry ticks were observed on any of the dogs in the control group. However, 9 of 10 dogs washed with the experimental shampoo showed presence of dried ticks, which is significantly higher according to the exact Fisher statistic test (p<0.05). This data is shown in Figure 5.

[0047] Conclusions

[0048] These results clearly show the effectiveness of using a shampoo comprising a Piper tuberculatum extract to control Rhipicephalus sanguineous. The shampoo was well accepted by the dogs and showed no adverse effects or allergies in those dogs. In addition, the shampoo did not cause any adverse effects to the skin of the person applying it to the dogs.