WO2010131058A1 - Product for the protection against honeybee mites, and method for the preparation of the active substance - Google Patents

Product for the protection against honeybee mites, and method for the preparation of the active substance Download PDF

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Publication number
WO2010131058A1
WO2010131058A1 PCT/HU2010/000053 HU2010000053W WO2010131058A1 WO 2010131058 A1 WO2010131058 A1 WO 2010131058A1 HU 2010000053 W HU2010000053 W HU 2010000053W WO 2010131058 A1 WO2010131058 A1 WO 2010131058A1
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Prior art keywords
bar pressure
active substance
preparation
plant
active ingredient
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PCT/HU2010/000053
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French (fr)
Inventor
Judit VON BARTHELDNÉ ÁBRI
András Horváth
Ferenc Kolonics
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Von Bartheldne Abri Judit
Horvath Andras
Ferenc Kolonics
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Publication of WO2010131058A1 publication Critical patent/WO2010131058A1/en

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    • 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]
    • A01N65/10—Apiaceae or Umbelliferae [Carrot family], e.g. parsley, caraway, dill, lovage, fennel or snakebed
    • 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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings

Definitions

  • the present invention relates to protection against honeybee mites, and method for the preparation of the active substance.
  • the invention relates also to the application of the active ingredient of the product, in a preparation against honeybee mite pests.
  • the invention relates particularly to the preparation for protection against the disease caused by the big Asian mite Varroa (Varroa Destructor - earlier Varroa Jacobsoni), and the honeybee tracheal disease mite (Acarapsis woodi), method of preparation and application of the active ingredient.
  • Varroa mites eat the body liquid of both the developing and developed honeybees, and may cause dwarfism of the honeybees, infestation and death of the honeybee families. As a matter of fact, Varroa-infestation is the most threatening danger of apiculture all around the world.
  • the patent application No. HU 224 912 (and its foreign equivalents, i.e. the EP 0869714) describe a procedure and product to stem the infestation of honeybee families.
  • volatile oil or organic acid is used in slow release preparation at the habitat of the honeybee families.
  • the slow release gel preparation described contains at least 10 w% volatile oil or organic acid, which provides efficient amount of oil or acid in the course of dosing through at least one reproduction cycle of the Varroa mites.
  • the drawback of the solution described above is that the gel preparation is temperature dependent, and a common drawback of the two referred patent description is that the acids and volatile oils are irritating for the honeybees.
  • the body of the honeybees is irritated by the acids, and the volatile oils are offensive for them, because they are scent-sensitive, and many volatile oils induce bee- allergy.
  • the honeybees try to get rid of the substance offensive for them, with which they are in touch, they try to remove it mechanically from their body (with scratching, ,,barberdance", with the help of cleaning by their fellows), and therefore the mechanism of act of the preparations containing acids or volatile oils is specifically disadvantageous.
  • the volatile oils infiltrate into the beeswax too, i.e. after the application of thymol preferred according to the patent application No. HU 224 912 the wax can only be used after running.
  • the patent application No. HU 223 065 for freeing beehives and honeybees from mites describes a mechanic (beehive rotation) procedure, but this is not a sufficiently effective solution for the problem.
  • the registered design No. U0200333 describes an equipment for the treatment of honeybees against Varroa mite with means for heating, blowing and air moving of the active substance; but this needs the suitable active substance, which according to the current knowledge is not available from the technical standing.
  • the solution of the invention takes into consideration the whole requirement system which must be met to kill the dangerous pests according to current specifications, expectations, effectively, without exposing the honeybees to the effects unpleasant or harmful for them, and at the same time residual amount of the active ingredient doesn't reach the lower limit, doesn't have harmful effect to the consistency and consumer value of the honey.
  • the application of the active ingredient doesn't cause environmental injury.
  • alkaloids are saturated (coniine, N-methylconiine, conhydrine and pseudoconhydrine), and one is unsaturated, g-coniceine.
  • These alkaloids, particularly the coniine, are very toxic substances, acting mainly on the nervous system.
  • the invention is based mainly on the discovery, that the alkaloids with piperidine structure obtained from poison hemlock - prepared and applied in a correct way - can be used successfully in preparations for protection against mite pests of honeybees, as active substance of the preparations. Therefore in this aspect the invention relates to a preparation against mite pests of honeybees, characterized that it contains plant oil including alkaloids with piperidine structure obtained from poison hemlock as active ingredient.
  • the active substance obtained by the extraction procedure is applied to the surface of an absorbing carrier in at most 0,03 mg/solvent kg/carrier m 2 amount, suitably from
  • the absorbing carrier can be a fibrous paper of loose structure, like for example filter paper, blotting paper, or woven or not woven mass of natural basic material, preferably cotton textile or disordered threads.
  • the carrier is preferably cut to stripes, preparation of 5*20 cm stripes is advantageous form the aspects of application. If necessary, the preparation may contain one or more additives known per se, selected from diluents, emulsifiers or surfactants.
  • the vegetable oil is packaged in a form suitable for application as a dry aerosol preparation, in a closed container, in a form of a water solution in at most 0,03 mg active ingredient/solvent concentration, diluted to 70 w% solution at the application.
  • a dry aerosol means application is carried out by filling it into the evaporation container, heating, and introducing into the hive.
  • this embodiment means closed, ready to use vials.
  • the alkaloids of the plant are synthesized by the conversion (cyclisation) of the eight carbon atoms' chain, deriving from four acetate units.
  • the nitrogen atom is connected in a certain stage of the biosynthesis, creating the piperidine structure.
  • the g-coniceine alkaloid is generated, and the other alkaloids, i.e. even the coniine, with the strongest biological activity, are formed from this alkaloid.
  • the poison hemlock the localisation of the biosynthesis was recently clarified, and the changes occurring in the course of the development of the plant.
  • the amount and ratio of the different poison hemlock alkaloids are influenced even by genetic factors, the age of the plant and ecological factors (temperature, humidity, the season, etc.). According to a previous investigation for example in the rainy season the g-coniceine, while in a drier season the coniine was the determinant alkaloid in the poison hemlock. According to the newest data the alkaloid-content of the roots is 0-0,5 percent, that of the leaves is 0,3-1 ,5 percent, that of the flowers is around 1 percent, the highest is in the unripe fruit (1 ,6-3,0 percent), while in the ripe fruits it is around 0,2-1 ,0 percent.
  • the active substance is extracted from the plant in such a way, that the plant starting material (suitably chopped) is mildly extracted a) in two steps, first at 85-90 bar pressure, and after the extraction of the active substance at 280-310 bar pressure, or b) by reaching gradually the 85-90 bar pressure, and keeping the pressure for a necessary period of time, and then at 28-35 bar.
  • the temperature is kept at 25-35 0 C in both steps.
  • the temperature-range mentioned above is kept.
  • the substance obtained is separated in two steps, first at 75-80 bar, at 28-35 0 C, and at 20 bar, at 20-27 0 C, this way the active substance is obtained as a 76-78 w% solution, the concentration of which is set to 45-50 % active substance with solvent, preferably water dilution.
  • solvent preferably water dilution.
  • the amount of the solution obtained is completed to 100 w% with water.
  • the portion of the poison hemlock above ground is harvested by mowing, and dried in a natural was as hay, stored until processing.
  • 1 kg poison hemlock hay is obtained from 5-6 kg of raw material.
  • the hay is ground, then extracted.
  • the extraction is carried out in a known way, in solvent, but preferably in carbon dioxide. This is favorable, because the carbon dioxide forms a reagent with the water present in the plant, which is especially significant in this case.
  • the product obtained is separated for the sake of stability, uniformity and even higher purity. According to the laboratory tests, the substance does not exhibit any organoleptically detectable, or any other change, even without deodorization.
  • the pressure chosen in the extraction step highly influences the quality and quantity of the product obtained, since by increasing the pressure, in addition to the volatile components, the less volatile components are also obtained in the first step.
  • the 25-35 0 C temperature range is kept, to ensure stability.
  • the procedure carried out in more mild conditions, at lower pressure may also be used, this has no effect to the scope of the invention.
  • the substance obtained by extraction is separated in two processes, the pressure values are changed in the separator. Namely, in the majority of experiments in the second separator 2 bar pressure is set.
  • the pressure of the first separator is selected to be 75-80 bar, to get the components of higher molecular weight, less volatile, and in the second separator the fraction richer in volatile oil. By setting the suitable pressure and temperature value, it can be reached that in the second separator a product with the physical characteristics of the volatile oil be obtained.
  • the pressure is set to 75-80 bar
  • the temperature is set to 28-35 0 C
  • the second separator 20-27 0 C temperature is kept at 20-35 bar, the samples are obtained in regular time- intervals, in order to be able to follow the yield of the components in the course of the extraction.
  • the components are separated by centrifugation.
  • the active substance content of the poison hemlock is 76-78 %. This is followed by dilution, in the course of which the active substance content is decreased to 45-50 %, and if necessary, the filtration through activated carbon, in order to remove the odor substances.
  • the solution transferred to the vaporizer stripe must be water soluble, or heat vaporizable, therefore emulsifying or surfactant additives, known per se can be added, i.e. alkyl-sulphonate or sodium-alkyl-ethoxy-sulphonate, in the amount of 5-15%.
  • emulsifying or surfactant additives known per se can be added, i.e. alkyl-sulphonate or sodium-alkyl-ethoxy-sulphonate, in the amount of 5-15%.
  • the solution obtained this was way is supplemented to 100% by the addition of water.
  • the active substance obtained this way can already be vaporized for about 24 hours, according to the effects of the temperature.
  • the vaporization stripe serves mainly prophylaxis.
  • the stripe is fastened to the exit slot, since the honeybee leaving is rather making discovery on the dewy flowers. Passes through the ribbon, nearly taking a bath in the active substance, and when going back, it carries the dew into the bee-hive, which through the humid honeybee landed on the ribbon takes up the active substance, this way facilitating the introduction of the active substance into the bee-hive.
  • the time-interval of application can only be 2 hours.
  • the preparation suitable for dry aerosol treatment is recommended primarily in case of bigger infested families, for disaster prevention, because in this case speed is of vital importance in preserving the condition of the family.
  • 1-2 drops of the diluted active substance, that can be introduced into the bee-hive with a vaporizer instrument is used, normally without the use of excipients.
  • the relative humidity of the bee-hive is around 80%, ventilation is provided by the flutter of wings of the honeybees.
  • the amount, concentration of the active substance, the speed of evaporation, and the temperature of vaporization are interconnecting parameters, co-ordination of which can be selected on the basis of the necessary knowledge of an expert.
  • the poison hemlock is a characteristic plant of the side of ditch, neglected pastures, meadows.
  • the portion of the poison hemlock above ground is harvested by mowing, followed by drying in a natural way, at a ventilated place, protected from rain. This way 1 kg poison hemlock hay is obtained from 5-6 kg of raw material.
  • the 14,5% air-dry poison hemlock hay is chopped to 2,5-3 mm pieces, and this chop is introduced into the atmospheric, direct steam-heated water vapor extractor.
  • the extractor is charged with 5 kg chopped substance, and 0,5 liter water distilled is added.
  • the solution obtained this way contains only 20-22 % water and 30-36 w% other, liquid fat-like substances.
  • the substance from the first operation separator is further separated, but already at a reduced pressure (20-22 bar) and at low (20-26 0 C) temperature.
  • the active substance content of the product obtained with this procedure is identical with the active substance content of the poison hemlock (since it is temperature-dependent), it is 76-78% solution, containing the coniine in volatile form.
  • the effectivity of the preparation of the invention is tested with pharmacological examinations.
  • the examination is carried out the following way: clean white sheets are put below the poison hemlock plant, and after subsiding of the honeybees, by examining the sheets under microscope numerous Varroa mite-bodies were found. Following this, the sheets were changed for many days after each insect visit, and in each case Varroa bodies, individuals gradually becoming ataxic were prognosed.
  • young dead mites were found in the hatches, mainly in the older drone hatches.
  • the dead honeybees were investigated microscopically. In the families having higher degree deaths, mite- corpses were also present in almost each case. In the covered hatches the mite could be found, sucking the pupas or larvae.
  • Sampling sites Boros Albert apiculture Kisvarda
  • the aerosol or vaporizing stripe solution of the invention was tested in laboratory conditions, in 2 parallels, with 10*100 live honeybees taken from mite infested families. Honeybees were put in sieves covered with glass plate, and treatment was carried out from below. White indicator paper was put under the sieve to detect the remains falling down.
  • the dry aerosol treatment is recommended mainly for bigger infested families, since in this case the speed is of vital importance in preserving the condition of the family.
  • active substance of desired concentration introduced by a vaporizer instrument, with the amount determined by the given application conditions is used, normally without the use of excipients.
  • the active substance evaporates continuously for 24 hours from the stripe put into the hive, and this way it can better infiltrate even the uncovered cells.

Abstract

The present invention relates to protection against honeybee mites, and method for the preparation of the active substance and the application of the active ingredient of the product in a preparation for protection against honeybee mite pests. The main point of the preparation that it contains vegetable oil, containing alkaloids with piperidine structure obtained from poison hemlock plant as active ingredient. The procedure of the invention relates to extracting alkaloids with piperidine structure from poison hemlock plant, when the portion of the plant above ground is harvested, its humidity content is decreased and suitably chopped, and the solvent extraction is carried out in two steps a) first at 85-90 bar pressure, and after dissolving the active substance at 280-310 bar pressure, or b) first at 85-90 bar pressure, then at 28-35 bar pressure, in both cases the temperature is kept at 25-35 °C, and the substance obtained is separated in two steps, first at 75-80 bar pressure, at 28-35 °C, then at 20 bar pressure, at 20-27 °C, the concentration of the active substance obtained as 76-78 w% solution is preferably adjusted to 45-50% active substance content with water dilution.

Description

Product for the protection against honeybee mites, and method for the preparation of the active substance
The present invention relates to protection against honeybee mites, and method for the preparation of the active substance. The invention relates also to the application of the active ingredient of the product, in a preparation against honeybee mite pests. The invention relates particularly to the preparation for protection against the disease caused by the big Asian mite Varroa (Varroa Destructor - earlier Varroa Jacobsoni), and the honeybee tracheal disease mite (Acarapsis woodi), method of preparation and application of the active ingredient.
It is known, that humans use and enjoy the products produced by honeybees. Pollination activity of the honeybees is an essential element both for the sustainable environment and the agricultural production, and what is more, for the human life. Just that is why protection of the health of honeybees is also an essential element of deliberate environmental protection.
In the last decades alarming, major honeybee deaths were reported all over the world, about since 1975, and many people are worried about the honeybee
(Apis mellifera). Large number of honeybee families died in all the countries of the globe, i.e. the number of honeybee families died in the US is estimated to be around 700.000, representing almost 30% of the population there. Natural death rate, which represents almost the same percentage, is not included in this death rate.
Each activity of the honeybee individual is directed to assure survival, reproduction of the family. Removal of the sick hatching, and death of the sick individual out of the beehive - doesn't return to the beehive - is also part of the defense mechanism. The situation becomes tragic, if the high loss of individuals occurs in a relatively short period of time, the family cannot regenerate itself.
The Varroa mites eat the body liquid of both the developing and developed honeybees, and may cause dwarfism of the honeybees, infestation and death of the honeybee families. As a matter of fact, Varroa-infestation is the most threatening danger of apiculture all around the world.
Damage of the Varroa mites was observed in Hungary in 1975, in Bekes County, and by 1985 the infestation covered practically the whole territory of the country.
As a consequence of globalization newer and newer diseases, pathogens must be taken into consideration, which arrive to us from other countries, continents.
Fight against the Varroa mite can only be successful with a systematic defense strategy. Numerous methods were developed or applied in the past years, but none of the methods applied resulted in a complete success.
Fight against the Varroa mite can only be successful with a systematic defence strategy. Numerous methods were developed or applied in the past years, but none of the methods applied resulted in a complete success. At the beginning amitrase smoking stripes were used, which helped a lot, but over a period of years their quality decreased, therefore lost the confidence of bee-keepers. Use of home-made stripes, lamp wicks, straps, containing mite- killing substances and amitrase smoking started at that time. The uncontrolled active ingredient content and the frequent, improperly timed treatments lead to mixed results. In this period, the presence of honeybee viruses was observed in the Hungarian apiculture too.
To stop spreading the disease several products were tested, containing flumethrin, tau-fluvalinate, acrinathrin pyrethroids active ingredient, or contained cumaphos, amitrase, malathion, chlorphenvinos, bromo-propylate, fenpyroximate, etc., but after a certain period of time mite resistance developed against most of these substances. Most of the active ingredients are dangerous pesticides, and application of many of them was even banned for the goal mentioned above.
The patent application No. HU 209 044 (and its foreign equivalents) describes phosphoric acid ester treatment for the protection of honeybees against Varoe Jacobsoni.
The patent application No. HU 224 912 (and its foreign equivalents, i.e. the EP 0869714) describe a procedure and product to stem the infestation of honeybee families. In the procedure volatile oil or organic acid is used in slow release preparation at the habitat of the honeybee families. The slow release gel preparation described contains at least 10 w% volatile oil or organic acid, which provides efficient amount of oil or acid in the course of dosing through at least one reproduction cycle of the Varroa mites. The drawback of the solution described above is that the gel preparation is temperature dependent, and a common drawback of the two referred patent description is that the acids and volatile oils are irritating for the honeybees. The body of the honeybees is irritated by the acids, and the volatile oils are offensive for them, because they are scent-sensitive, and many volatile oils induce bee- allergy. The honeybees try to get rid of the substance offensive for them, with which they are in touch, they try to remove it mechanically from their body (with scratching, ,,barberdance", with the help of cleaning by their fellows), and therefore the mechanism of act of the preparations containing acids or volatile oils is specifically disadvantageous. It must be noted also, that the volatile oils infiltrate into the beeswax too, i.e. after the application of thymol preferred according to the patent application No. HU 224 912 the wax can only be used after running.
Nowadays chemical treatments - because of the drug residues - are regulated by decrees. Higher than permitted drug residue results in destruction of the honey, not to mention the ethical damage caused by the irresponsible bee- keeper.
The patent application No. HU 223 065 for freeing beehives and honeybees from mites describes a mechanic (beehive rotation) procedure, but this is not a sufficiently effective solution for the problem. The registered design No. U0200333 describes an equipment for the treatment of honeybees against Varroa mite with means for heating, blowing and air moving of the active substance; but this needs the suitable active substance, which according to the current knowledge is not available from the technical standing.
The solution of the invention takes into consideration the whole requirement system which must be met to kill the dangerous pests according to current specifications, expectations, effectively, without exposing the honeybees to the effects unpleasant or harmful for them, and at the same time residual amount of the active ingredient doesn't reach the lower limit, doesn't have harmful effect to the consistency and consumer value of the honey. The application of the active ingredient doesn't cause environmental injury.
Observing the behavior, customs, dances of the bees, and analyzing the attendance of the different plant species it was found that contrary to the faithfulness to flowers and colors, there is a plant, which is not preferred, or even very toxic for humans and animals, but the bees, humble-bees and wasps surprisingly prefer to visit, and take a bath in its pollen. This plant is the Conium maculatum, belonging to the Umbelliferae family, the poison hemlock.
Arabian and Greek doctors were using the active substances of the poison hemlock both in human and veterinary therapy for the treatment of certain tumors, joint pains, although the therapeutic and toxic concentrations are very close to each other.
But it was observed, that although significant amount of pollen remained on the bees even after performing the cleaning procedures, the individuals didn't make the "barberdance", didn't request cleaning from another mate, but flew directly into the beehive, and when coming out of it presented the correct direction towards the poison hemlock for their mates with dancing [Orosi Pal Zoltan: Mehek kόzόtt, 49. oldal, Bόrze Kft. (1991)]. The plant, which is poisonous both for humans and animals is unexpectedly atoxic for the bees, and they even like it. As it is known, the substances found in poison hemlock with strong biological effect are alkaloids with piperidine structure. Four of the five most important alkaloids are saturated (coniine, N-methylconiine, conhydrine and pseudoconhydrine), and one is unsaturated, g-coniceine. These alkaloids, particularly the coniine, are very toxic substances, acting mainly on the nervous system.
In studying the observed bee families and bee-hives it was unexpectedly found, that the Varroa mite, which is extremely dangerous for the bees, dies by the effect of these active alkaloid substances, but at the same times the lethal effect of the plant doesn't manifest at the bees. Therefore the invention is based mainly on the discovery, that the alkaloids with piperidine structure obtained from poison hemlock - prepared and applied in a correct way - can be used successfully in preparations for protection against mite pests of honeybees, as active substance of the preparations. Therefore in this aspect the invention relates to a preparation against mite pests of honeybees, characterized that it contains plant oil including alkaloids with piperidine structure obtained from poison hemlock as active ingredient.
According to an embodiment of the preparation of the invention, the active substance obtained by the extraction procedure is applied to the surface of an absorbing carrier in at most 0,03 mg/solvent kg/carrier m2 amount, suitably from
40-50% water solution. The absorbing carrier can be a fibrous paper of loose structure, like for example filter paper, blotting paper, or woven or not woven mass of natural basic material, preferably cotton textile or disordered threads. The carrier is preferably cut to stripes, preparation of 5*20 cm stripes is advantageous form the aspects of application. If necessary, the preparation may contain one or more additives known per se, selected from diluents, emulsifiers or surfactants.
In commercializing it must be taken into consideration, that the product is toxic for humans, therefore it can be marketed packaged in closed plastic boxes or in aluminium boxes, indicating the instructions.
According to another embodiment of the preparation of the invention the vegetable oil is packaged in a form suitable for application as a dry aerosol preparation, in a closed container, in a form of a water solution in at most 0,03 mg active ingredient/solvent concentration, diluted to 70 w% solution at the application. Using it as a dry aerosol means application is carried out by filling it into the evaporation container, heating, and introducing into the hive. In practice, this embodiment means closed, ready to use vials.
Numerous procedures are known for the isolation of active substances from plant starting materials, which generally include harvesting of the plant, decreasing its moisture content (enrichment of the dry substance), chopping and solvent extraction.
It was found - and this discovery is the basis of the procedure of the invention - that from the aspect of the invention selection of the temperature range and pressure used in the course of the extraction procedure is very important, because if the parameters chosen are not suitable, then as a result of the extraction procedure the active substance is obtained in unsuitable yield and in insufficiently effective form. As it was mentioned, it contains substances with strong biological effect, with piperidine-structure (coniine, N-methylconiine, conhydrine pseudoconhydrine, g-coniceine). The piperidine alkaloids (hundreds of them are already known) are synthesized from essential amino acid, i.e. from lysine, acetate or mevalonate molecules. The five poison hemlock alkaloids can be derived from acetate-groups.
According to the investigations the alkaloids of the plant are synthesized by the conversion (cyclisation) of the eight carbon atoms' chain, deriving from four acetate units. The nitrogen atom is connected in a certain stage of the biosynthesis, creating the piperidine structure. In the course of the biosynthesis starting from acetate molecules the g-coniceine alkaloid is generated, and the other alkaloids, i.e. even the coniine, with the strongest biological activity, are formed from this alkaloid. In the poison hemlock the localisation of the biosynthesis was recently clarified, and the changes occurring in the course of the development of the plant. It turned out for example that in the seedling stage both in the root tips and in the other tissues of the plant, biosynthesis of the alkaloids and the vegetable oils could be demonstrated. Later on, the tissue parts demonstrating expansion type growth are superior in their significant biosynthesizing ability. Both in the plant body and in the flowers intensive biosynthesis is going on, and later on, ten days after fertilisation, the presence of the alkaloids can be demonstrated even in the different layers of the pericarp.
The amount and ratio of the different poison hemlock alkaloids are influenced even by genetic factors, the age of the plant and ecological factors (temperature, humidity, the season, etc.). According to a previous investigation for example in the rainy season the g-coniceine, while in a drier season the coniine was the determinant alkaloid in the poison hemlock. According to the newest data the alkaloid-content of the roots is 0-0,5 percent, that of the leaves is 0,3-1 ,5 percent, that of the flowers is around 1 percent, the highest is in the unripe fruit (1 ,6-3,0 percent), while in the ripe fruits it is around 0,2-1 ,0 percent.
In our investigations, it was found that all parts of the plant above ground can be used for obtaining the product, independently from the season.
According to the procedure of the invention the active substance is extracted from the plant in such a way, that the plant starting material (suitably chopped) is mildly extracted a) in two steps, first at 85-90 bar pressure, and after the extraction of the active substance at 280-310 bar pressure, or b) by reaching gradually the 85-90 bar pressure, and keeping the pressure for a necessary period of time, and then at 28-35 bar. In the two-step procedure, carried out at higher pressure, the temperature is kept at 25-35 0C in both steps. The same way, in case of the extraction carried out mildly, at lower temperature (requiring 10-15 hours) the temperature-range mentioned above is kept. After the extraction step the substance obtained is separated in two steps, first at 75-80 bar, at 28-35 0C, and at 20 bar, at 20-27 0C, this way the active substance is obtained as a 76-78 w% solution, the concentration of which is set to 45-50 % active substance with solvent, preferably water dilution. Preferably after filtration through activated carbon after the addition of 5-15% emulsifying or surfactant additive, if necessary, the amount of the solution obtained is completed to 100 w% with water.
Therefore in detail, according to the procedure, the portion of the poison hemlock above ground is harvested by mowing, and dried in a natural was as hay, stored until processing. This way 1 kg poison hemlock hay is obtained from 5-6 kg of raw material. The hay is ground, then extracted. The extraction is carried out in a known way, in solvent, but preferably in carbon dioxide. This is favorable, because the carbon dioxide forms a reagent with the water present in the plant, which is especially significant in this case. The product obtained is separated for the sake of stability, uniformity and even higher purity. According to the laboratory tests, the substance does not exhibit any organoleptically detectable, or any other change, even without deodorization.
In the two-step procedure first 85-90 bar is applied, until no more substance dissolves. Then the pressure is raised to 280-310 bar. The product obtained in the first step is an oil, and the same amount of water, forming two phases. The product obtained in the second step has an oily feel, creamy nature, the substance obtained in both phases has a very characteristic hemlock (mouse urine) scent. The pressure chosen in the extraction step highly influences the quality and quantity of the product obtained, since by increasing the pressure, in addition to the volatile components, the less volatile components are also obtained in the first step. By comparing the extracts obtained by the extraction of the ground and unground samples, it was experienced that extraction of the product from the ground plant, but in case of grinding higher amounts the material may warm up, and damage or loss may occur. In the course of the procedure the 25-35 0C temperature range is kept, to ensure stability. Optionally the procedure carried out in more mild conditions, at lower pressure may also be used, this has no effect to the scope of the invention. The substance obtained by extraction is separated in two processes, the pressure values are changed in the separator. Namely, in the majority of experiments in the second separator 2 bar pressure is set. The pressure of the first separator is selected to be 75-80 bar, to get the components of higher molecular weight, less volatile, and in the second separator the fraction richer in volatile oil. By setting the suitable pressure and temperature value, it can be reached that in the second separator a product with the physical characteristics of the volatile oil be obtained. In the first separator the pressure is set to 75-80 bar, the temperature is set to 28-35 0C, and in the second separator 20-27 0C temperature is kept at 20-35 bar, the samples are obtained in regular time- intervals, in order to be able to follow the yield of the components in the course of the extraction. The components are separated by centrifugation. The active substance content of the poison hemlock is 76-78 %. This is followed by dilution, in the course of which the active substance content is decreased to 45-50 %, and if necessary, the filtration through activated carbon, in order to remove the odor substances. The solution transferred to the vaporizer stripe must be water soluble, or heat vaporizable, therefore emulsifying or surfactant additives, known per se can be added, i.e. alkyl-sulphonate or sodium-alkyl-ethoxy-sulphonate, in the amount of 5-15%. The solution obtained this was way is supplemented to 100% by the addition of water. The active substance obtained this way can already be vaporized for about 24 hours, according to the effects of the temperature.
Application of the vaporization stripe serves mainly prophylaxis. In the course of the application in the morning the stripe is fastened to the exit slot, since the honeybee leaving is rather making discovery on the dewy flowers. Passes through the ribbon, nearly taking a bath in the active substance, and when going back, it carries the dew into the bee-hive, which through the humid honeybee landed on the ribbon takes up the active substance, this way facilitating the introduction of the active substance into the bee-hive. In this case, since both the air and the exit create more humid conditions, the time-interval of application can only be 2 hours.
In the second possibility of the application of the vaporizing stripe, it is put to the bottom of the bee-hive, and left there for 24 hours. The reason for this is that the honeybees try to keep a constant temperature (30-32 °C) inside the bee-hive, providing steady evaporation. To provide greater efficiency the stripe is put below the so called nest (hatching).
The preparation suitable for dry aerosol treatment is recommended primarily in case of bigger infested families, for disaster prevention, because in this case speed is of vital importance in preserving the condition of the family. In this case 1-2 drops of the diluted active substance, that can be introduced into the bee-hive with a vaporizer instrument is used, normally without the use of excipients. The relative humidity of the bee-hive is around 80%, ventilation is provided by the flutter of wings of the honeybees. The amount, concentration of the active substance, the speed of evaporation, and the temperature of vaporization are interconnecting parameters, co-ordination of which can be selected on the basis of the necessary knowledge of an expert.
After evaporation the exits are closed, 15 minutes later full ventilation is carried out, even by opening the roof of the bee-hive. The bee-hive stays like this for about half an hour. Then mainly the mites on the workers and drones dye, but because of the respiration function the number of the tracheal mites can also be significantly reduced.
For better understanding, the procedure of the invention is demonstrated hereinunder by an example, but without limiting the scope of the invention to the substance illustrated by the example.
Example
The poison hemlock is a characteristic plant of the side of ditch, neglected pastures, meadows. The portion of the poison hemlock above ground is harvested by mowing, followed by drying in a natural way, at a ventilated place, protected from rain. This way 1 kg poison hemlock hay is obtained from 5-6 kg of raw material. Following this the 14,5% air-dry poison hemlock hay is chopped to 2,5-3 mm pieces, and this chop is introduced into the atmospheric, direct steam-heated water vapor extractor. The extractor is charged with 5 kg chopped substance, and 0,5 liter water distilled is added. Following screwing down the lid of the container heating is started, taking care, that the temperature of the chop in the container shouldn't exceed the 25-35 °C temperature limit, because in opposite case we don't get two-phase water solution, but an oily mass, which, although contains the alkaloid, cannot be used in the next steps. To get a uniform solution the pressure is gradually raised to 85-90 bar, this is reached in about 5 hours, this pressure is kept for 3 hours at 25-35 °C, following this the pressure is gradually decreased, in about 5 hours, to 28-35 bar. The substance obtained this way is transferred to the separator from the florentine container, where, keeping the set 28-35 °C temperature range and the separation is carried out first at 75-80 bar pressure. The solution obtained this way contains only 20-22 % water and 30-36 w% other, liquid fat-like substances. For further separation, the substance from the first operation separator is further separated, but already at a reduced pressure (20-22 bar) and at low (20-26 0C) temperature. The active substance content of the product obtained with this procedure is identical with the active substance content of the poison hemlock (since it is temperature-dependent), it is 76-78% solution, containing the coniine in volatile form.
Pharmacological example
The effectivity of the preparation of the invention is tested with pharmacological examinations.
The examination is carried out the following way: clean white sheets are put below the poison hemlock plant, and after subsiding of the honeybees, by examining the sheets under microscope numerous Varroa mite-bodies were found. Following this, the sheets were changed for many days after each insect visit, and in each case Varroa bodies, individuals gradually becoming ataxic were prognosed. By examining the families taking pollen bath, young dead mites were found in the hatches, mainly in the older drone hatches. After winter, after the bee-hive opening, the dead honeybees were investigated microscopically. In the families having higher degree deaths, mite- corpses were also present in almost each case. In the covered hatches the mite could be found, sucking the pupas or larvae. Sampling sites: Boros Albert apiculture Kisvarda
Gombos Ferenc apiculture Tiszatelek
Nemeth Andras apiculture lbrany
Csepany Laszlό apiculture Nagyhalasz
The aerosol or vaporizing stripe solution of the invention was tested in laboratory conditions, in 2 parallels, with 10*100 live honeybees taken from mite infested families. Honeybees were put in sieves covered with glass plate, and treatment was carried out from below. White indicator paper was put under the sieve to detect the remains falling down.
After the aerosol treatment 72, 81 , 75, 74, 74, 80, 76, 81 , 73, 72, mite remains, 76 on the average were counted. In the course of the treatment, and within 24 hours 2, 5, 11 , 3, 5, 2, 14, 6, 3, 8 honeybees died. After the vaporizing stripe treatment, where the active ingredient is released as a function of temperature, 71 , 75, 68, 82, 55, 81 , 76, 43, 68, 71 mite remains, 69 on the average were counted. In the course of the treatment, and within 24 hours 3, 7, 5, 1 , 3, 5, 7, 5, 3, 3 honeybees died.
Our studies have proven, that contrary to the warm-blooded living creatures the coniine alkaloid of the poison hemlock doesn't have lethal effect to the insects, but the vaporizing stripe treatment has proven to be milder, and the vapor could enter even the open cells, and significantly thinned the mite larvae present.
The dry aerosol treatment is recommended mainly for bigger infested families, since in this case the speed is of vital importance in preserving the condition of the family. In this case, as it was mentioned, active substance of desired concentration, introduced by a vaporizer instrument, with the amount determined by the given application conditions is used, normally without the use of excipients.
Application technology:
Two different application ways and timings are possible for the alkaloids, forming the base of the preparations of the inventions:
1. Early spring, in July, October, before the morning flight, with the vaporizing stripe fixed to the exit slot, which must be removed after 2 hours, and must be destroyed. One vaporizing stripe contains the amount of the active substance enough for a family.
2. The active substance evaporates continuously for 24 hours from the stripe put into the hive, and this way it can better infiltrate even the uncovered cells.
3. In July, October, with the application of a vaporizing instrument (the principle of vaporization by heating), by creating dry aerosol, the amount of the substance calculated for families is vaporized, and the exit slots are closed for about 15 minutes. By taking into consideration that the treatment may include factors dangerous for humans, only trained personnel can carry out the treatments. In the course of treatment the required wearing is protective clothe, gas mask, protective gloves.
It is deemed to be advisable to carry out the aerosol treatments in the course of the otherwise obligatory yearly veterinary treatment, in the presence of a veterinarian.

Claims

1. Preparation for protection against mite pests of honeybees, characterized that it includes vegetable oil containing alkaloids with piperidine structure as active ingredient, obtained from poison hemlock.
2. Preparation according to Claim 1 , characterized that it contains the active ingredient obtained from poison hemlock by extraction procedure and transferred to the surface of an absorbent carrier, in at most 0,03 mg/solvent kg/m2 carrier.
3. Preparation according to Claims 1 and 2, characterized that it contains one or more additives selected from diluents, emulsifiers, surfactants.
4. Procedure according to any of the preceding claims, characterized that the carrier is a fibrous paper of loose structure, like for example filter paper, blotting paper, or woven or not woven mass of natural basic material, preferably cotton textile or disordered threads.
5. Preparation according to Claim 1 , characterized that it contains the at most 50% solution of the active ingredient in a closed container, for example in an ampoule.
6. Procedure for extracting alkaloids with piperidine structure from hemlock plant, when the portion of the plant above ground is harvested, its humidity content is decreased and suitably chopped in the order requested, characterized that the solvent extraction is carried out by extracting the suitably chopped plant starting material in two steps a) first at 85-90 bar pressure, and after dissolving the active substance at 280-310 bar pressure, or b) first at 85-90 bar pressure, then at 28-35 bar pressure, in both cases the temperature is kept at 25-35 0C, and the substance obtained is separated in two steps, first at 75-80 bar pressure, at 28-35 0C, then at 20 bar pressure, at 20-27 0C, the concentration of the active substance obtained as 76-78 w% solution is preferably adjusted to 45-50% active substance content with water dilution.
7. Preparation according to Claim 6, characterized that the diluted active substance is filtered through active carbon, optionally 5-15 w% emulsifier or surfactant additive is added, and the amount of the solution obtained is supplemented with water to 100 w%.
8. Application of vegetable oil containing alkaloids with piperidin structure, obtained from poison hemlock as active ingredient, in a preparation for protection against mite pests of honeybees.
PCT/HU2010/000053 2009-05-15 2010-05-13 Product for the protection against honeybee mites, and method for the preparation of the active substance WO2010131058A1 (en)

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CN102273491A (en) * 2011-06-22 2011-12-14 华南农业大学 Insecticidal activity of three cicuta L. plant extractives
CN102550612A (en) * 2011-12-16 2012-07-11 天长市中能国泰能源技术有限公司 Ecological environment-friendly pesticide
WO2012170420A3 (en) * 2011-06-06 2013-02-28 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation
US9545110B2 (en) 2013-01-07 2017-01-17 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation
US11229211B2 (en) 2018-05-14 2022-01-25 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation

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ARIANA ARDESHIR ET AL: "Laboratory evaluation of some plant essences to control Varroa destructor (Acari: Varroidae).", 2002, EXPERIMENTAL AND APPLIED ACAROLOGY, VOL. 27, NR. 4, PAGE(S) 319-327, ISSN: 0168-8162, XP002592119 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012170420A3 (en) * 2011-06-06 2013-02-28 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation
CN102273491A (en) * 2011-06-22 2011-12-14 华南农业大学 Insecticidal activity of three cicuta L. plant extractives
CN102550612A (en) * 2011-12-16 2012-07-11 天长市中能国泰能源技术有限公司 Ecological environment-friendly pesticide
US9545110B2 (en) 2013-01-07 2017-01-17 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation
US11229211B2 (en) 2018-05-14 2022-01-25 John I. Haas, Inc. Compositions and methods for controlling a honey bee parasitic mite infestation

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