WO2023233200A1 - Compositions comprenant un extrait de quassia amara pour lutter contre les acariens destructeurs varroa - Google Patents

Compositions comprenant un extrait de quassia amara pour lutter contre les acariens destructeurs varroa Download PDF

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Publication number
WO2023233200A1
WO2023233200A1 PCT/IB2023/000317 IB2023000317W WO2023233200A1 WO 2023233200 A1 WO2023233200 A1 WO 2023233200A1 IB 2023000317 W IB2023000317 W IB 2023000317W WO 2023233200 A1 WO2023233200 A1 WO 2023233200A1
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Prior art keywords
composition
solution
alcohol
quassia amara
mites
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PCT/IB2023/000317
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English (en)
Inventor
Christiane DÜTTMANN
Byron Flores SOMARRIBA
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Quassia Labs Ltd
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Publication of WO2023233200A1 publication Critical patent/WO2023233200A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/02—Acaricides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones

Definitions

  • compositions comprising a hydroalcoholic solution comprising a Quassia amara extract, the solution comprising at least 80 %v/v of at least one alcohol, as well as to the use of such compositions in controlling Varroa destructor mites, and methods for preparing the solution.
  • Varroa destructor mites are parasitic mites that attack and feed on honey bees.
  • the Varroa mite is considered the parasite with the most pronounced economic impact on the beekeeping industry.
  • Varroa mites are commonly found in bee colonies globally, resulting in major economic losses for both beekeepers and farmers of crops, which rely on pollination. Detection often results in intense activities to control the spread of the mite and prevent the infestation of colonies and collapse of the honey industry.
  • WO 2016/185379 A1 and WO 2016/185380 A1 relate the use of hydroxyapatite as a carrier of bioactive substances, including Quassia amara, for application to plants with vascular diseases.
  • WO 2010/089567 A2 relates to herbal compositions comprising various components, including Quassia, for the control of hematophagous parasites. This document does not mention Varroa mites, and the compositions are prepared by cell culturing.
  • This invention relates to a composition
  • a composition comprising:
  • the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, even more particularly at least 95 %v/v of the at least one alcohol.
  • the at least one alcohol may comprise ethanol and/or isopropyl alcohol, even more particularly ethanol.
  • hydroalcoholic solution comprising a Quassia amara extract is used to refer to the solution obtained when a solvent, for example a mixture of water and at least one alcohol, is contacted with Quassia amara plant material for a defined period of time in order to extract components from the Quassia amara plant material.
  • a solvent for example a mixture of water and at least one alcohol
  • the composition may comprise 0.1-5 parts by volume of the hydroalcoholic solution as defined above, more particularly 0.5-2 parts by volume, even more particularly about 1 part by volume.
  • the composition may comprise 15-25 parts by volume of the aqueous sucrose solution, more particularly 18-22 parts by volume, even more particularly about 19 parts by volume of the aqueous sucrose solution.
  • the aqueous sucrose solution may comprise 40-60 wt% sucrose, more particularly 45-55 wt% sucrose, even more particularly about 50 wt% sucrose.
  • this invention relates to a spray comprising a spray device and a composition as defined above.
  • the spray may be for use in controlling Varroa destructor mites.
  • the spray may comprise a chamber which contains the composition as defined above.
  • the spray device may be a hand pressure spray device.
  • This invention also relates to a method of controlling Varroa destructor mites comprising applying to a bee colony a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol.
  • this invention relates to a method of controlling Varroa destructor mites comprising applying to a bee colony a composition as defined above.
  • This invention also relates to a method of treating Varroa destructor mite infestation in a bee colony comprising applying to the bee colony a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol.
  • this invention relates to a method of treating Varroa destructor mite infestation in a bee colony comprising applying to the bee colony a composition as defined above. In particular, the applying may be by spraying.
  • This invention also relates to the use of a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, in controlling Varroa destructor mites.
  • this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, for use in controlling Varroa destructor mites.
  • This invention also relates to the use of a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, in treating Varroa destructor mite infestation in a bee colony.
  • this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, for use in treating Varroa destructor mite infestation in a bee colony.
  • This invention also relates to the use of a composition as defined above in controlling Varroa destructor mites. In addition, this invention relates to a composition as defined above for use in controlling Varroa destructor mites. This invention also relates to the use of a composition as defined above in treating Varroa destructor mite infestation in a bee colony. In addition, this invention relates to a composition as defined above for use in treating Varroa destructor mite infestation in a bee colony.
  • This invention also relates to a method for preparing a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, the method comprising the steps of:
  • the at least one alcohol may comprise ethanol and/or isopropyl alcohol, more particularly ethanol.
  • the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, more particularly at least 95 %v/v of the at least one alcohol.
  • the amount of Quassia amara plant material to which the mixture of water and at least one alcohol was added may be 100-1000 mg of plant material per ml of the mixture, more particularly 250-750 mg/ml, even more particularly 400-600 mg/ml.
  • the amount of Quassia amara plant material to which the mixture of water and at least one alcohol was added may be 50-300 mg of plant material per ml of the mixture, in particular 75-275 mg/ml, more particularly 150-250 mg/ml, even more particularly 175-225 mg/ml.
  • the defined period of time may be 6-48 hours, more particularly 12-36 hours, even more particularly 18-30 hours.
  • this invention relates to a hydroalcoholic solution comprising a Quassia amara extract, wherein the solution comprises at least 80 %v/v of at least one alcohol, obtainable by the method defined above.
  • the invention also relates to the compositions, methods and uses defined above utilising such a hydroalcoholic solution.
  • This invention also relates to a method for preparing a composition as defined above, the method comprising the steps of:
  • step (b) may comprise mixing 0.5-2 parts by volume, more particularly about 1 part by volume, of the hydroalcoholic solution. More particularly, step (b) may comprise mixing 18-22 parts by volume, more particularly about 19 parts by volume, of the aqueous sucrose solution.
  • the composition of the aqueous sucrose solution may be as defined above.
  • step (a) may comprise preparing a hydroalcoholic solution by the method as defined above.
  • this invention relates to a composition obtainable by the method defined above.
  • the invention also relates to the methods and uses defined above utilising such a composition.
  • this invention relates to a composition
  • a composition comprising:
  • Vegetable oils are defined as oils extracted from seeds or from other parts of fruits. Examples of seed oils include soybean oil and grape seed oil. Examples of oils from other parts of fruits include olive oil and palm oil. In some embodiments, the vegetable oil may be olive oil, although other vegetable oils can be used.
  • the hydroalcoholic solution may comprise at least 90 %v/v of the at least one alcohol, even more particularly at least 95 %v/v of the at least one alcohol.
  • the at least one alcohol may comprise ethanol and/or isopropyl alcohol, even more particularly ethanol.
  • the composition may comprise 80-90 % by volume of the hydroalcoholic solution as defined above, more particularly 82-88 % by volume, even more particularly 84-85 % by volume.
  • the composition may comprise IQ- 20 % by volume of the vegetable oil, more particularly 12-18 % by volume, even more particularly 14-15 % by volume of the vegetable oil.
  • the composition may be in the form of an emulsion.
  • the composition may additionally comprise an emulsifier.
  • the emulsifier may comprise castor oil. Suitable emulsifiers include Rimulgan® and LV42®.
  • the composition may comprise 0.2-1.1 % by volume of the emulsifier, more particularly 0.4-0.9 % by volume, even more particularly 0.6-0.7 % by volume of the emulsifier.
  • this invention relates to a porous material, in particular a sponge, impregnated with a composition as defined above.
  • This invention also relates to a method of controlling Varroa destructor mites comprising contacting a bee colony with the composition of the further embodiment as defined above.
  • This invention also relates to a method of treating Varroa destructor mite infestation in a bee colony comprising contacting the bee colony with the composition of the further embodiment as defined above.
  • the contacting may be by evaporation.
  • This invention also relates to the use of the composition of the further embodiment as defined above in controlling Varroa destructor mites. In addition, this invention relates to the composition of the further embodiment as defined above for use in controlling Varroa destructor mites. This invention also relates to the use of the composition of the further embodiment as defined above in treating Varroa destructor mite infestation in a bee colony. In addition, this invention relates to the composition of the further embodiment as defined above for use in treating Varroa destructor mite infestation in a bee colony. [0034] This invention also relates to a method for preparing a composition (i.e. that of the further embodiment) as defined above, the method comprising the steps of:
  • step (b) may comprise mixing 82-88 % by volume, more particularly 84-85 % by volume, of the hydroalcoholic solution. More particularly, step (b) may comprise mixing 12-18 % by volume, more particularly 14-15 % by volume, of the vegetable oil.
  • the vegetable oil may be as defined above.
  • step (a) may comprise preparing a hydroalcoholic solution by the method as defined above.
  • this invention relates to a composition obtainable by the method defined above.
  • the invention also relates to the methods and uses defined above utilising such a composition.
  • Figure 1 shows a graph of Varroa mite Infestation Rate (IR %) in adult bees before and after the application of a Quassia amara aqueous solution
  • Figure 2 shows a graph of Varroa mite Infestation Rate (IR %) in adult bees before and after the application of a Quassia amara hydroalcoholic solution
  • Figure 3 shows a graph of Varroa mite Extension of Invasion (El %) in brood cells before and after the application of a Quassia amara aqueous solution
  • Figure 4 shows a graph of Varroa mite Extension of Invasion (El %) in brood cells before and after the application of a Quassia amara hydroalcoholic solution
  • Figure 5 shows a graph comparing Varroa mite Infestation Rate (IR %) in adult bees for the aqueous and hydroalcoholic solutions, both before and after the application of the Quassia amara solution
  • Figure 6 shows a graph depicting the correlation between beehive condition and reduction in Infestation Rate (IR)
  • Figure 7 shows a graph comparing the average number of dead Varroa mites between the different experimental groups during the 8 hours post-exposure to the evaporation formulation
  • Figure 8 shows a line graph comparing the average number of dead Varroa mites between the different experimental groups during the 8 hours postexposure to the evaporation formulation
  • Figure 9 shows a line graph comparing the percentage of dead Varroa mites between the different experimental groups during the 8 hours post-exposure to the evaporation formulation
  • Figure 10 shows comparing Varroa mite Infestation Rate (IR %) in adult bees for the evaporation formulation (referred to as "oasis”), both before and after application.
  • a hydroalcoholic solution comprising an extract of Quassia amara was prepared as follows. 2 kg of branches of the Quassia amara tree were weighed. The dry branches were cut into pieces approximately 10 cm long and 2 cm wide and placed in a container. 4 litres of 95 %v/v ethanol were added (i.e. 500 mg of plant material to 1 ml of liquid, 500 mg/ml). After 30 hours resting at room temperature, the resulting hydroalcoholic solution comprising a Quassia amara extract was filtered and stored in the dark. A hydroalcoholic solution using isopropyl alcohol instead of ethanol has also been produced and successfully tested.
  • the hydroalcoholic solution comprising a Quassia amara extract was then diluted for testing.
  • An aqueous sugar solution was prepared by mixing 1 kg of sugar per 1 litre of warm water, and then allowing the solution to cool. 50 ml of the hydroalcoholic solution comprising a Quassia amara extract was then mixed with 950 ml of the sugar solution to get a volume of 1000 ml (i.e. a dilution to 25 mg/ml).
  • the strength of a beehive can be estimated by a subjective method that relies on visual estimates of various parameters. This method is less accurate than a quantitative mode, but is far less disruptive to the bees. It allows the investigator to preserve the social cohesion and health of tested colonies, and even collect data on variables that determine the status quo of the colony.
  • the hive was opened and the hydroalcoholic dilution of Quassia amara mixed with the sugar solution was sprayed directly onto the bees moving in the spaces between the frames.
  • the strength of the hive was taken into account in applying the composition. If the hive was weak, the amount of composition used was reduced.
  • the composition was applied to all frames with bees, using between 60 and 80 ml of the composition per hive. Given that any Varroa mites in the capped brood cells of the hive are not affected by the composition sprayed into the spaces between the frames, the procedure was repeated 4 times at intervals of 4 days in order to kill any hatched mites. Thus, the complete process per hive consisted of four applications.
  • the observed characteristics of strength estimation for the seven beehives tested are shown in Table 3 below.
  • the observed characteristics were codified using the Likert scale (assigned values in brackets in Table 2).
  • the codification permits a correlation analysis with the results of varroosis.
  • the strength values for the hives varied from 13 to 21 , 21 being the strongest hive.
  • Table 4 below shows the estimated quantity of bees on the frames according to parameters from Delaplane et al (2013).
  • Table 5 shows the Infestation Rate (IR %) of adult bees in the seven beehives before and after application of the composition (i.e. comprising the hydroalcoholic solution comprising a Quassia amara extract and the sugar solution).
  • Beehives 1 , 3 and 7 showed a significant decrease in the infestation of mites in adult bees.
  • Beehives 2, 5 and 6 maintained a low IR (we observed a difference of only one or two mites in the samples).
  • the only beehive that demonstrated a higher IR after applying the composition was beehive number 4.
  • Aethina tumida the Small Hive Beetle, another beekeeping pest.
  • the conditions of the wooden material were severe, so the increase in this beehive with Varroa mites is believed to have been influenced by these factors.
  • the aqueous solution comprising a Quassia amara extract was then diluted for testing.
  • An aqueous sugar solution was prepared by mixing 1 kg of sugar per 1 litre of warm water, and then allowing the solution to cool. 1 litre of the aqueous solution comprising an extract of Quassia amara was then mixed with 3 litres of the sugar solution to get a volume of 4 litres.
  • the effectiveness of the composition being tested is determined by the absolute decrease of Varroa mites in the brood cells of the worker bees. This is shown for the composition comprising the hydroalcoholic solution for the seven hives tested in Table 6 below. It is particularly notable that the extent of Varroa invasion in hive number 7 decreased to zero. This means that the reproductive phase of the Varroa mite was significantly interrupted.
  • Figure 6 shows the correlation between beehive strength and the reduction of IR of varroosis.
  • a means of assessing the strength of each beehive was utilised as described above. The lowest possible result using this method was 8 points and maximum was 22 points. In the In-vivo testing the range was from 13 to 21 points.
  • a higher reduction of IR was observed in the weaker hives (numbers 1 and 3) and in the hive with the higher IR at the beginning of the survey (beehive number 7). This is a positive feature of the product, because high infested and weak colonies need the most help to control varroosis.
  • the hives with a low level of infestation maintained their IR in a range of +/-0.5%.
  • a formulation which is applied by spraying also disclosed herein is a formulation which is applied to the hive by evaporation.
  • a sponge is soaked with the formulation, which is then placed in the hive and the formulation is allowed to evaporate and thereby disperse through the hive.
  • the evaporation formulation comprises more alcohol.
  • the evaporation formulation comprises a vegetable oil (e.g. olive oil), generally instead of the aqueous sucrose solution.
  • the acaricidal effectiveness of the evaporation method using Quassia amara extract was determined by In-vitro testing as follows. Small sponges with different concentrations of the composition were put on the gauzes of the prepared Petri dishes (each containing five mites). The prepared dishes were kept in the dark at an ambient temperature of about 32 °C without ventilation. The test emulsion ( ⁇ 50ml) was a 15% by volume emulsion of a hydroalcoholic extract of Quassia amara (prepared substantially as described below, but to provide two different concentrations, 100 mg/ml and 200 mg/ml) with olive oil: 42.5 ml extract - 7.5ml olive oil - 15 drops emulsifier.
  • Group 2 Hydroalcoholic extract (200 mg/ml)
  • the control group comprises five Petri dishes each containing five Varroa destructor mites without any added substance.
  • two Petri dishes were prepared with pure ethanol applied to a sponge placed upon the gauze that covered each of the Petri dishes (each dish containing five Varroa destructor mites). Thus, there was a total of 35 control mites.
  • the aim of this testing is to determine the effectiveness of Quassia amara formulation as a bio-acaricide to control varroosis in which the formulation does not have direct contact with the bees (as would be the case if using a spraying or dribbling method).
  • the results set out the number of the dead mites, as well as the percentage of the mortality of the mites, during the evaluation of the different concentrations used in the emulsion of the hydroalcoholic extract of Quassia amara with olive oil.
  • the results are set out in graphical form.
  • the absolute number of dead Varroa mites is shown in Figures 7 and 8.
  • the relative mortality of Varroa destructor mites is shown in Figure 9.
  • Post-hoc testing by HSD Tukey test for pairwise comparison is shown in Table 7 below.
  • sponges were impregnated with the emulsion of the hydroalcoholic extract of Quassia amara.
  • the sponges (made of 70 wt% cellulose and 30 wt% cotton) were divided into two parts of the same size. These parts were then moistened with water and squeezed to remove excess water.
  • the preparation of the emulsion was based on the results of the In-vitro testing.
  • Rimulgan was used as the emulsifier, although an alternative emulsifier may be used.
  • the In-vivo test was carried out with 16 hives, using the composition on eight of them and leaving another eight hives as a control group (i.e. without use of the composition). For each hive, two sponges were used (one sponge divided in 2 parts); the prepared emulsion (400 ml) was poured onto the 16 cut sponges. The In- vivo testing in the beehives was then carried out using these sponges.
  • the In-vivo test started with the sampling of adult bees to determine the infestation rate (IR) of varroosis in the 16 selected beehives located in two apiaries. At the same time, two impregnated sponges were placed into the brood chamber of eight of the 16 beehives. This was repeated for two further weeks, with an interval of one week between each use of the composition. One week after the last use, another sampling was carried out to determine IR post-use. After a further week, the hives were checked and their behaviour analysed to determine the tolerability to the composition.
  • IR infestation rate
  • the aim of the testing is to determine the effectiveness of Quassia amara as a bio-acaricide to control varroosis utilising an application method that is minimally invasive for the bees and is less time-consuming for the beekeeper.
  • the procedure involves placing sponges impregnated with an emulsion of the hydroalcoholic extract of Quassia amara with olive oil in the hive. This was repeated once a week for three weeks. The infestation rate of varroosis before and after the use of the composition was determined in order to demonstrate the efficacy of this method.

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Abstract

La présente invention se rapporte à une composition comprenant : (a) une solution hydroalcoolique comprenant un extrait de Quassia amara, la solution comprenant au moins 80 % v/v d'au moins un alcool, et (b) une huile végétale. L'invention se rapporte également à une composition comprenant : (a) une solution hydroalcoolique comprenant un extrait de Quassia amara, la solution comprenant au moins 80 % v/v d'au moins un alcool, et (b) une solution aqueuse de saccharose. De plus, l'invention se rapporte à un procédé de préparation d'une solution hydroalcoolique comprenant un extrait de Quassia amara, la solution comprenant au moins 80 % v/v d'au moins un alcool.
PCT/IB2023/000317 2022-05-30 2023-05-26 Compositions comprenant un extrait de quassia amara pour lutter contre les acariens destructeurs varroa WO2023233200A1 (fr)

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WO2010089567A2 (fr) 2009-02-06 2010-08-12 Roy Walter Brown Composition à base de plantes destinée à la lutte contre les parasites hématophages
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ITMI20110799A1 (it) * 2011-05-10 2012-11-11 Antonella Roberti Formulazione spot-on a base di composti naturali ad attivita' adulticida
WO2016185379A1 (fr) 2015-05-18 2016-11-24 Nbvd S.A. Utilisation de l'hydroxyapatite comme support d'éléments nutritionnels et d'extraits végétaux pour traiter les plantes
WO2016185380A1 (fr) 2015-05-18 2016-11-24 Nbvd S.A. Utilisation d'hydroxyapatite comme support de substances bioactives pour le traitement des maladies vasculaires des plantes

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