DESCRIPTION

The invention relates to an allicin-based formulation having high stability, non-phytotoxic at the doses of use, easily injectable into vascular plants, phytopharmaceutical compositions or kits comprising said formulation and uses thereof.

STATE OF THE PRIOR ART

In agriculture, in the last years, the demand for eco-sustainable solutions of low environmental impact is ever-increasing. Said context opened a market niche for natural products as a resource for new strategies in phytosanitary defense (Slusarenko A. et al, 2008. Eur J Plant Pathol 121 , 313-322).

The therapeutic use of garlic extract is known since ancient times, and medical studies demonstrated its considerable antimicrobial activity against gram-negative and gram positive bacteria, fungi, intestinal human parasites and viruses; moreover, its anticarcinogenic, antidiabetic properties, and its properties against cardiac complications are known. Even in the agronomic field, garlic extract proved effective in vitro and in vivo against oomycetes (e.g., Phytophthora spp. and Pythium ultimum). In particular, Ports et al (2008. Eur J Plant Pathol 122, p. 196-206) describe the therapeutic effects of garlic juice against Phytophthora infestans when sprayed onto tomato plant leaves, with better results compared to on-soil treatment. Garlic extract also proved effective against ascomycete (e.g., Alternaria spp., Botrytis cinerea, Colletotrichum lindemuthianum, Fusarium solani, Magnaporthe spp., Plectosphaerella cucumerina) and basidiomycete fungi (e.g., Rhizoctonia solani), and bacteria ( Agrobacterium tumefaciens, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris ; Curtis H. et al 2004. Physiol Mol Plant P 65, 79-89; Slusarenko et al., 2008, above). Finally, a possible insecticidal/insect repellent (e.g., against maize weevil, psylloidea and diptera; Prowse G. M. et al, 2006. Agric For Entomol 8, 1-6; Nwachukwu I.D., Asawalam E. F., 2014. Afr J Biotechnol 13, 1123-1130; US 20060110472 A1), fertilizing and biostimulant (Hayat S. et al, 2016 Frontiers in Plant Science 7, 1235. doi: 10.3389/fpls.2016.01235) activity thereof has been reported. Depending on the garlic variety, extraction method, solvent type and polarity and storage modes used, the chemical species and the amounts of organosulfur compounds in the extract are variable. These differences condition its biological activity (Fujisawa H. et al 2008. J Agr Food Chem 56, 4229-4235). Among the active principles (ingredients) present in the extracts, allicin represents one of the molecules with the greatest antimicrobial activity. Said effectiveness is due to the rapid chemical reaction of the S-S bond with -SH and S-S groups contained in various proteins of the pathogens, leading also to the inhibition of certain enzymes (Ankri and Mirelman, 1999. Microbes Infect, 125- 129). Other mechanisms involved in its biological activity are the rapid permeability through membranes and the ability to react with free radicals. Moreover, in an aqueous, preferably acidic, means, allicin is degraded into various products, among which ajoenes that, along with allicin, demonstrated considerable antifungal, antibacterial, antiviral and antiprotozoal activity (Naganawa et al., 1996. Appl Environ Microb 62, 4238-4242). The antimicrobial activity of allicin was also described by Ankri S. and Mirelman D. (1999, Microbes and infection 2, 125-129): antimicrobial activity toward a wide range of Gram negative and Gram-positive bacteria, as well as an antifungal effect against fungi typically causing pathologies in humans (e.g., Candida albicans) was associated to allicin. It is known, as published in Reiter et al (2017. Molecules 22, 1711 pp 1-14), that allicin is effective as antibacterial toward human lung pathogenic bacteria; the study was performed also on mammalian cells to study the possible use of the substance in humans.

As to allicin extraction methods from garlic, the paper published by Arsanlou and Bohlooli in Food Chemistry 120 (2010, p. 179-183) reveals allicin presence in whole green garlic plant (and not only in bulbs), teaching that the entire plant may be considered as a good source of allicin. Miron et al describe, in Analytical Biochemistry 331 (2004, p. 364-369), methods for the preparation and purification of allicin and report the results of pharmacokinetic studies on the molecule. Document CN 106957883 instead relates to garlic processing and to a method for synthesizing ajoene from allicin. The document specifically describes that allicin, though endowed with potent antimicrobial activities, is an extremely unstable compound that decomposes at standard temperatures and conditions.

Allicin in fact has some weak points, such as the low miscibility in aqueous solutions and the very short half-life (Ankri and Mirelman, 1999, above). Given its wide efficacy spectrum, various formulations containing allicin, pure or in combination with other active principles, were designed; allicin was carried in polymer matrices, nanoparticles or liposomes, and in various pharmaceutical presentations (e.g., tablets, oral granules, gels or sprays for topical application, balms or vapors for inhalators), with medical, veterinary or agroalimentary purposes. In the agronomic field, encapsulation proved a successful method for stabilizing the molecule, making it useful as biopesticide in-soil or by sprinkling (Slusarenko et al., 2008, above; W02002056683 A2; CN104351181 A). Compared to traditional pathogens-fighting methods, endotherapy, i.e. , injections into the vascular system of plants (Montecchio, 2013 Jove-J Vis Exp 80, 51199), has many positive aspects that comply with the contents of Directive 2009/128/EC, legislative decree No. 150/2012, and the current National Action Plan for the sustainable use of phytosanitary products. Garlic extract, applied by endotherapy, was tested against fungi (e.g., Hymenoscyphus fraxineus ; Dal Maso et al., 2014. Urban For Urban Green 13 (4), 697-703) and bacteria (e.g., Pseudomonas syringae pv. aesculr, Mabbett, 2015. EssentialARB 2015, 16-18), but its injection rate can be very slow (Dal Maso et al., 2014, above); moreover, the S-S bond of allicin preferably reacts with other organosulfur compounds present in the garlic extract matrix, forming various derivatives and making allicin concentration very variable (Shen et al., 2002. Eur J Plant Pathol 121 , 313-322). Hence, the state of the art leaves the following problems unsolved:

limited stability of allicin molecule under conditions of use, with particular reference to endotherapy

low solubility of allicin molecule in aqueous solvents

- difficult injectability of allicin-containing commercial products.

Given the relevance of the perspectives of allicin use in the therapeutic treatment of plants, and given the above-reported limitations, suggestions enabling to solve one or more of these problems currently unsolved by the state of the art are extremely useful.

SUMMARY OF THE INVENTION

The Authors of the present invention have made an innovative allicin-based formulation, also suitable for endotherapic use, exhibiting high stability and antimicrobial effectiveness, absence of phytotoxicity at the doses of use and high absorption rate in the injection into vascular plants.

In particular, the formulation object of the invention exhibits the following advantageous features compared to the state of the art:

-increase of molecule stability, thanks to the addition of coformulants;

-increase of injection rate with the addition of coformulants;

-standardized dosage of the active principle;

-injectability at high dosages, such as to enable injection of reduced volumes thereof; -synergistic effect of coformulants towards pathogens;

-compatibility with the chemico-physical characteristics of sap;

-non-phytotoxic at the doses of use.

In the present description a formulation is disclosed comprising, as active principle having a phytopharmaceutical effect, allicin alone or in combination with other active principles or natural extracts, and coformulants with properties of antioxidants, surfactants and solubility enhancers, able to maintain the stability of the active principle and speed up its endotherapic absorption.

The formulation appears, in a liquid state, as a soluble concentrate (SL), in accordance with FAO (Food and Agricultural Organization of the United Nations) international denomination.

Object of the present invention are: -a liquid formulation comprising:

-allicin, having a final concentration from 0.3 to 0.5 % w/v;

-surfactants, having a final concentration from 0.01 % w/v to the solubility limit at room temperature;

-saline buffer;

-solubility enhancers, having a final concentration from 0 to 5% w/v;

-antioxidants, from 0.005% w/v to the solubility limit at room temperature, wherein the final pH of said formulation is in the range 2-5 and wherein the final concentration of iron salts in said formulation is not greater than 5 mM;

-a phytopharmaceutical composition comprising the formulation;

-the use of said formulation for the preparation of phytopharmaceutical compositions;

-the use of said formulation or of said composition in the endotherapic treatment of woody plants or palm trees;

-a kit for the treatment of plants by injection into the vascular system, comprising one or more vials containing said formulation and optionally one or more phytopharmaceutically acceptable solvents or solvents containing said phytopharmaceutical composition;

-a method for the endotherapic treatment of woody plants and palm trees, comprising at least one step wherein said mixture or said phytopharmaceutical composition is injected into the vascular system of said plants.

-a process for preparing said formulation comprises the following steps:

a) keeping allicin, obtained by extraction from natural sources or via chemical synthesis, at a temperature below -18°C until the moment of use; b) checking the purity of allicin through chromatographic analysis, such as for example high-pressure liquid chromatography with spectrophotometric detection with diode array and/or mass spectrometry (HPLC-DAD-MS);

c) preparing a mixture of said antioxidants and surfactants by means of solubilization in a suitable saline buffer;

d) solubilizing said allicin as per points a) and b) into a suitable surfactant agent and, optionally, adding one or more suitable solubility enhancers by mechanical stirring at a temperature lower than 10°C, thereby obtaining an emulsion or a solution;

e) adding said emulsion or said solution obtained in d) to said solution obtained in c);

f) filtering the mixture obtained in step e) maintaining the temperature lower than 4°C; -a process for preparing said pharmaceutical composition comprising the steps a) to f) above and a further step g) wherein said mixture is coformulated with at least one phytopharmaceutically acceptable excipient or carrier. 1 - Mobility and efficacy

The endotherapic use of the present formulation is preferably suited to the fight against pathogens affecting leaves, flowers or fruits, or mainly developing into the vascular system. 2 - Antioxidants function and synergies

Organosulfur compounds exhibit reduced stability in the presence of oxygen; in particular, oxygen induces breakage of the thiosulfinate double bond 0=S-S present in the allicin molecule, a bond weaker than a S-S bond (Putnoky S., Caunii A., Butnariu M., 2013. Chemistry Central Journal 7, 1-6). In the present formulation, the addition of antioxidants slows down allicin degradation and amplifies its effectiveness against plant pathogens.

3 - Absorption rate

The combination of mono-, di- and tri-carboxyl acids and salts, preferably of monovalent metals, enables to speed up the formulation absorption (Solorzano

E.R., Dal Maso E., Pastore P., Montecchio L, Frasconi M., Bogialli S., 2017. Nuovo formulato linfa-compatibile. Italian Patent Application N° 102017000109583), thereby reducing the time of contact of allicin in the formulation with environmental oxygen.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1. In the graph, the half-life time of the various solutions containing allicin 0.3-0.5 % w/v, tested in an oven at a temperature of 37°C in order to accelerate the degradative effect, is indicated. Said solutions are: garlic extract (Conquer),“pure allicin”,“allicin in water”,“allicin in acid aqueous means” also comprising citric acid 1 % w/v,“allicin-based formulation”. The composition of the allicin-based formulation, object of the invention, is reported in Table 2. Half-life time measurement was carried out by means of HPLC-DAD- MS measuring.

Figure 2. In the graph, histograms indicate the average absorption rate in A. chinensis for each solution, with the related standard error. Demineralized water and garlic extract were introduced in the assay as controls. The diluted garlic extract is at pH 3.5-3.8. The composition of the allicin-based formulation object of the invention is reported in Table 2.

Figure 3. In the graph, histograms indicate the average absorption rate in A. hippocastanum for each solution, with the related standard error. Demineralized water and garlic extract were introduced in the assay as controls. The diluted garlic extract is at pH 3.5-3.8. The composition of the allicin-based formulation object of the invention, is reported in Table 2.

Figure 4. In the graph, histograms indicate the average absorption rate in O. europaea for each solution, with the related standard error. Demineralized water and garlic extract were introduced in the assay as controls. The diluted garlic extract is at pH 3.5-3.8. The composition of the allicin-based formulation, object of the invention, is reported in Table 2.

GLOSSARY

Antimicrobial agents: substances synthetically produced or naturally used to kill or inhibit the growth of microorganisms, including bacteria, viruses or fungi, or parasites, in particular protozoa (Regulation EC No 1831/2003 on additives for use in animal nutrition).

Dosage: amount of active principle required to achieve, by its administration, a certain effect.

Half-life: the time required for the concentration of a substance to decrease by half of its initial value.

Endotherapy: plant endotherapy is a technique used to prevent or treat pathologies (such as, e.g., parasitic and fungal ones) or nutritional deficiencies on arboreal/woody plants. This methodology consists in the administration (through "injections into the trunk") of sap-compatible fluids directly into the xylem system of a vascular plant. Said fluids contain the active principles required for therapy, such as specific plant protection products specially studied and prepared. Therefore, with endotherapy a first stage of absorption of active substance in the sap flow, and subsequently a stage of substance translocation to the remainder of the plant, are had. Depending on the method used, two types of endotherapy can be distinguished: infusion, i.e., a procedure only based on transpiration flow for liquid absorption, and injection, i.e. an endotherapic method entailing the application of an external force (e.g., pump, or syringe plunger). By “endotherapic” for the purposes of the present invention it is meant a product suited for administration by endotherapy.

Natural (with reference to a molecule). By the term“natural” for the purposes of the present invention it is meant a compound extracted from natural sources; conversely, a “synthesis” or“synthetic” product is a molecule artificially obtained by chemical synthesis in a laboratory.

Polyoxyethylene derivatives. A derivative is a compound obtained from another compound, of which the former maintains its general structure (A Dictionary of Chemistry, 2016. Oxford University Press, Oxford UK, p. 160). By the term “polyoxyethylene derivatives” there are meant compounds derived from polyoxyethylene surfactants; polyoxyethylene compounds (synonyms: polyethylene oxide, polyethylene glycol) are a class of compounds with formula H0(CH ₂ CH ₂ 0) _n H (Gangolli S. (Ed.), 1999. The dictionary of substances and their effects. Vol. 6. The Royal Society of Chemistry, Cambridge UK, p. 428; Nico M. van OS. (Ed.), 1998. Nonionic Surfactants: Organic Chemistry. Marcel Dekker Inc., New York, p. VIII), ethylene glycol polymers. In general, the term“polyoxyethylene derivatives” is the term commonly used in the literature to define nonionic surfactants containing a polyoxyethylene hydrophilic component (such as, e.g., PEG ethers).

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention relates to a liquid formulation comprising: -allicin, having a final concentration from 0.3 to 0.5 % w/v;

-surfactants having a final concentration from 0.01 % w/v to the solubility limit at room temperature;

-saline buffer,

-solubility enhancers, having a final concentration from 0 to 5% w/v.

-antioxidants, from 0.005% w/v to the solubility limit at room temperature, wherein the final pH of said formulation is in the range 2 - 5 and wherein the final concentration of iron salts in said formulation is not greater than 5 mM.

The formulation of the invention appears, in the liquid state, as a soluble concentrate (SL), in accordance with FAO (Food and Agricultural Organization of the United Nations) international denomination.

According to one embodiment of the invention, the antioxidants present in the formulation, are at least one of: metal scavenging antioxidants, natural phenolic non- flavonoid antioxidants, synthetic phenolic non-flavonoid antioxidants; natural phenolic flavonoid antioxidants; synthetic phenolic flavonoid antioxidants; carotenoid antioxidants; antioxidant vitamins; natural antioxidant plant extracts or a mixture of at least two of said antioxidants.

In one particular embodiment, said metal scavenging antioxidants are selected from: citric acid, EDTA (ethylenediaminetetraacetic acid), flavonoids, carnosine or a mixture thereof.

According to one embodiment, the natural phenolic non-flavonoid antioxidants can be selected from: acids (gallic, caffeic, cinnamic, nordihydroguaiaretic, etc.), resveratrol, tannins, lignans, tocopherols, tocotrienols or a mixture thereof; whereas said synthetic phenolic non-flavonoid antioxidants are selected from: propyl gallate, butyl hydroxyanisole, butylhydroxytoluene or a mixture thereof.

In particular, said phenolic flavonoid antioxidants can be selected from one or more of: quercetin, pinocembrin, calcone or a mixture thereof.

According to one embodiment of the invention, said carotenoid antioxidants are selected from: lycopene, beta-carotene, zeaxanthin or a mixture thereof.

In the formulation of the invention, as mentioned above, antioxidant vitamins are present. The technician in the field may select any suitable antioxidant vitamin known in the state of the art, a non-binding example of said antioxidant vitamins is given by vitamin C, E or a mixture thereof.

As indicated above, in the formulation of the invention, it is possible to use, among antioxidants, natural antioxidant plant extracts.

Numerous plant extracts having antioxidant activities are known in the state of the art. A non-binding example of extracts suitable for the carrying out of the invention is given by one or more from extract of: rosemary, green tea, grape seeds, species belonging to the genus Larrea, or a mixture thereof.

As indicated above, the formulation of the invention also comprises surfactants. In one preferred embodiment, said surfactants are preferably nonionic surfactants.

A non-limiting example of said surfactants is given by polyoxyethylene derivatives of fatty acids such as sorbitan esters and/or ethers or polysorbates, which, depending on the hydrophilic properties allowed by the number of oxyethylene groups bonded to the sorbitan molecule, are classified as polysorbate 60 or polysorbate 80, known on the market as Tween 60 and Tween 80.

The formulation of the invention can further comprise one or more monovalent salts and/or one or more other preservative compounds.

One example of said salts is given by: potassium polysorbate, sodium/potassium benzoate.

According to the invention, other preservative compounds may be selected by the expert in the field from any preservative compound suitable for on-plant therapy, such as, e.g., sorbic acid, benzoic acid, lactic acid.

In one particular embodiment, the formulation of the invention, already comprising allicin, which has a phytopharmacological effect, can further comprise at least one additional phytopharmacological active principle.

Any phytopharmacological active principle administrable by endotherapy could be used. Said active principle could be, e.g., in the form of aqueous solution of active principles useful in endotherapy, such as, for instance, an aqueous solution of potassium phosphite, or an aqueous solution of salicylic acid. Additional non-limiting examples of aqueous solutions of formulations of water-miscible active principles suitable for the carrying out of the formulation according to the invention can be solutions of commercial formulations of Vertimec EC (abamectin), Imidachem and Confidor (imidacloprid), Tecto 20S (tiabendazole) type.

The invention also provides a phytopharmaceutical composition comprising the formulation as described above and at least one excipient or phytopharmacologically acceptable carrier. A non-limiting example of phytopharmacologically acceptable carrier is the sap-compatible formulation as described and claimed in the Italian Patent Application N° 102017000109583.

The phytopharmaceutical composition according to the invention also can comprise, as described above for the formulation, one or more phytopharmacologically acceptable active principles.

Each of the embodiments indicated for the above formulation, alone or in any combination therebetween, apply to the phytopharmaceutical composition of the invention.

In case of a phytopharmaceutical composition, the additional one or more active principles could be formulated in a mixture with the formulation of the invention or could be provided so as to be suspended or dissolved into the formulation upon administration. Furthermore, object of the invention is the use of the formulation in any one of the embodiments provided above, for preparing phytopharmaceutical compositions.

Further object of the invention is also the use of the formulation in any one of the embodiments provided above, or of the formulation composition in any one of the embodiments provided above for the endotherapic treatment of woody plants or palm trees. According to the invention, in such a use said mixture or said composition is administered by injection into the vascular system of said plants or of said palm trees.

In particular, by a BITE (Blade for Infusion in TrEes) device as described, for instance, in Patent Applications PD2011A000245, EP2012/063680, WIPO WO/2013/010909, or in Montecchio 2013“A Venturi Effect Can Help Cure Our Trees” J Vis Exp. 2013; (80): 51199.

Said device, as known in the literature and as described, e.g., in the above-mentioned documents, can be advantageously used for administering the mixture, the formulation or the phytopharmaceutical composition according to the invention.

A further object of the invention is a kit for the treatment of plants by injection into the vascular system, comprising one or more vials containing: the formulation or the phytopharmaceutical composition as defined in any one of the embodiments given above or any one combination of one or more of said embodiments.

According to the invention, in one particular embodiment, said formulation or said composition provided in the kit further comprise one or more phytopharmacologically acceptable active principles as described above.

Moreover, object of the invention is a method for the endotherapic treatment of woody plants and of palm trees, comprising at least one step wherein a formulation or a pharmaceutical composition as defined in any one of the embodiments provided above, or any one combination of one or more of said embodiments, is injected into the vascular system of said plants.

In one preferred embodiment, said injection is carried out using BITE technology (Blade for Infusion in TrEes, Pat. EP 2731764).

Finally, object of the invention is a process for preparing the formulation as defined in any one of the embodiments provided above or any one combination of one or more of said embodiments comprising the following steps:

a) keeping allicin, obtained by extraction from natural sources or via chemical synthesis, at a temperature below -18°C until the moment of use; b) checking the purity of allicin through chromatographic analysis, such as for example high-pressure liquid chromatography with spectrophotometric detection with diode array and/or mass spectrometry (HPLC-DAD-MS);

c) preparing a mixture of said antioxidants and surfactants by means of solubilization in a suitable saline buffer;

d) solubilizing said allicin as per points a) and b) into a suitable surfactant agent and, optionally, one or more suitable solubility enhancers by mechanical stirring at a temperature lower than 10°C, thereby obtaining an emulsion or a solution;

e) adding said emulsion or said solution obtained in d) to said solution obtained in c);

f) filtering the mixture obtained in step e) maintaining the temperature lower than 4°C.

Said allicin at point a) could be natural or synthetic allicin obtained from standard procedures commonly used by the technician in the field, such as, e.g., but in a non limiting manner, by enzyme production in situ or by purification through conventional techniques, such as, e.g., chromatography techniques.

The technician in the field will anyhow know how to obtain allicin required for the carrying out of the invention, by simply making use of known techniques available to the public. In particular, in the above-described process, said preparing in c) can be facilitated by heating at a temperature from 30 to 50 °C for 10-60 minutes or by subjecting said mixture of antioxidants to ultrasound treatment for a period of from 3 to 30 minutes, or by mechanical stirring.

Finally, object of the invention is also a process for preparing a phytopharmaceutical composition wherein the formulation as defined in any one of the embodiments provided above, or any one combination of one or more of said embodiments, prepared by the above-described process, is mixed with one or more phytopharmaceutically acceptable carriers or excipients and/or with one or more additional phytopharmacological active principles as defined above.

EXAMPLES

In the following examples, garlic extract, widely reported in the literature as antimicrobial, was used as control to compare and optimize the new formulations object of the present patent application.

Example 1 - In-field efficacy of an allicin-containing garlic extract against Pseudomonas actinidiae and equal in vitro efficacy with synthetic allicin

Preliminarily to the test in vitro, an in-field test was carried out to ascertain the effectiveness against Pseudomonas actinidiae of the commercial product then used as control in subsequent tests, i.e. a garlic extract (Conquer, Neem Biotech, Cardiff UK). In May 2016, 30 Actinidia chinensis plants of Soreli cv with presence of P. actinidiae were subjected to endotherapic treatment with water (control), or garlic extract containing allicin at a concentration of 1650 ppm or of 3300 ppm. Each solution was injected considering a liquid volume equal to 1 ml_/cm of trunk circumference and 10 replicates per treatment. At injection (first detection) and 30 days later (second detection), 10 leaves per treatment were harvested and photographed. For each leaf, the necrotized area was quantitated (in percent relative to the total area) using Gimp v. 2.8. and ImageJ v. 1.46r (Wajne Rasband, National Institutes of Health, USA). Data were statistically processed in R cran by using the Kruskal-Wallis test.

To consider bacterial viability, leaf extracts of the second detection were then processed in a laboratory in order to isolate P. actinidiae, keeping apart the samples deriving from individual plants. In particular, the plant extract, once obtained, was seeded onto King’s B medium (KB) in accordance with EPPO (2014). Bacterial DNA was then extracted (Ferrante and Scortichini, 2010) and subjected to molecular analysis both with single PCR (primers PsaF1 and PsaR2; Rees-George et al., 2010) and with duplex-PCR (primers KN-F, KN-R; AvrDdpx-F, AvrDdpx-R; Gallelli et al., 2011), with a subsequent electrophoretic run.

In the subsequent assay in vitro, the Inventors wanted to compare the efficacy of a synthetic allicin solution with garlic extract (Conquer, Neem Biotech, Cardiff UK) concentrations being equal. Garlic extract was kept frozen at a temperature below -18°C until the moment of use. For allicin synthesis, 1.9 g of perbenzoic acid (AP) and 1.45 g of diallyl disulfide (DADS) are weighted with an analytical scale and respectively solubilized into 100 and 50 ml of HPLC grade chloroform. The AP-containing solution is slowly added dropwise, over a time not lower than 30 minutes, to the chloroformic DADS solution, kept under continuous stirring in an ice bath and protected from direct light. Subsequent washings with 5% sodium bicarbonate and demineralized water are carried out in order to eliminate starting reagent residues and neutralize the solution. Chloroform is evaporated in a rotary evaporator at a temperature below 35 °C. The experimental yield should not be lower than 78% compared to the theoretical yield. Immediately after synthesis, solution density is checked and allicin is identified by means of high-resolution mass spectrometry. Product purity is assessed by using HPLC-DAD-MS.

Minimum inhibitory concentrations (MICs) of the solutions tested against Pseudomonas actinidiae were determined with the broth microdilution method in microplate, following CLSI directives. Absorbance at time zero and after 24/48 hours was measured with a spectrophotometer (Micro-Plate Reader 318MC, Shanghai Sanco Instrument CO., Ltd.) at 630 nm. At the end of the test, the minimum concentration for which a minimum Absorbance increase of 0.05 did not occur was considered as a MIC value. All tests were replicated thrice.

As results of the field test, the degree of infection did not prove different among treatments at first detection (determination); on the contrary, in the second detection statistically significant differences (p<0.05) were singled out, based on Kruskal-Wallis test, rank sum test (chi-squared) and Pairwise comparisons between the two garlic extract-based treatments and the control. In particular, the analysis indicated that, in the time period under examination, development of necroses caused by P. actinidiae was significantly slowed down with injection into the trunk of garlic extract compared to the controls. Though considering the low isolation rate of P. actinidiae from leaf samples, associated with the natural infection rate, the bacterium was re-isolated only from leaves collected from controls (20% of positive samples); on the contrary, it was never re isolated from leaves collected from plants injected with the garlic extract containing allicin at the concentration of 1650 ppm or of 3300 ppm.

The results of the test in vitro reported in Table 1 show the identity of in vitro behavior of garlic extract and synthetic allicin.

Table 1. in vitro efficacy of synthesis allicin and of garlic extract. MICs (Minimum Inhibitory Concentrations) for P. actinidiae are indicated.

Example 2 - Improvement of allicin solution stability with coformulant addition

Allicin stability is strongly influenced by solution temperature and pH. The optimal pH for the purposes of stability is of 3 to 6. Thermal stability studies suggest that, to preserve allicin in aqueous extracts, a temperature as low as possible is preferable (Wang et al. , 2014).

As stability assay, the following allicin solutions were compared:

1 -“pure allicin”

2 -“allicin in water”, at the concentration of 0.3-0.5 % w/v

3 -“allicin in acidic aqueous means”, comprising allicin at a concentration of 0.3-0.5 % w/v and citric acid 1 % w/v

4 -“allicin-based formulation”, the composition of which is reported in Table 2

5 - garlic extract with allicin at the concentration of 0.3-0.5 % w/v (Conquer). Table 2. Composition of the allicin-based formulation. Concentrations are expressed as % w/v. Citrate buffer was used to bring to a final volume the described ingredients.

Results are reported in Figure 1 , showing allicin stability, in terms of half-life time, under different conditions, highlighting the increase in the case of the proposed formulation. Half-life times measurement was carried out by HPLC-DAD-MS measuring. Example 3 - Synergistic effect of coformulants contained in the allicin-based formulation against Pseudomonas actinidiae (in vitro)

MICs of the solutions against P. actinidiae were determined with the broth microdilution method in microplate, following CLSI directives. Individually tested (assayed) solutions were: synthetic allicin 3300 ppm, garlic extract with allicin 3300 ppm, citric acid 4096 ppm, ascorbic acid 4096 ppm, adipic acid 4096 ppm. Absorbance at time zero and after 24/48 hours was measured with a spectrophotometer (Micro-Plate Reader 318MC, Shanghai Sanco Instrument CO., Ltd.) at 630 nm. At the end of the test, the minimum concentration for which a minimum Absorbance increase of 0.05 did not occur was considered as a MIC value. All tests were replicated thrice.

Upon determining the MIC for each solution, the isobolographic analysis of interactions was carried out in accordance with the methods reported in preceding studies (Tallarida, 2002; Luszczki et al., 2005). For each combination of three components/active principles (e.g., A, B, C) the theoretical isobole for additivity was calculated:

cA/MICA + cB/MICB + cC/MICC= 1

where MICA, MICB and MICC represent the MICs of the three solutions with the components/active principles A, B, C, and c denotes the concentration. Other combinations of the three solutions A, B, C were then considered by varying the fixed- dose ratios, FR; from the intersection of the plane represented by these latter individual combinations and that of the theoretical isobole for additivity were obtained, to be compared with the experimental ones to determine a possible synergy or antagonism. All experiments in vitro on isobolographic analysis were conducted with the broth microdilution method in microplate, following CLSI directives. Absorbance at time zero and after 24/48 hours was measured with a spectrophotometer (Micro-Plate Reader 318MC, Shanghai Sanco Instrument CO., Ldt) at 630 nm. At the end of the test, the minimum concentration for which a minimum absorbance increase of 0.05 did not occur was considered as a MIC value. All tests were replicated thrice.

Test results are reported in Table 3. The MICs of the solutions with a single component, and subsequently of the mixtures are indicated. For three combinations of synthesis allicin with citric acid and ascorbic acid a synergistic effect against P. actinidiae was found. No synergy was found in cases in which synthesis allicin had been replaced by garlic extract.

Table 3. MICs resulting from isobolographic analysis. ALL-s = synthesis allicin; ALL-e = allicin in garlic extract; CIT = citric acid; ASA = ascorbic acid; ADI = adipic acid.

Example 4 -allicin-based formulation absorption rate in arboreal species

Based on the synergistic effects of coformulants against Pseudomonas actinidiae, a synthetic allicin-based formulation was optimized (Table 2). The absorption rate of said formulation and of the garlic extract (Conquer, Neem Biotech, Cardiff UK) was tested on Actinidia chinensis, Aesculus hippocastanum and O/ea europaea, with 3 replicates per solution; demineralized water was used as control. The formulation and the garlic extract were suitably diluted so as to contain both 0.3-0.5 % w/v of allicin and injected. In particular, the suitably diluted garlic extract was injected as is and at pH 3.5-3.8 with citric acid to make the results comparable with those obtained with the formulation at the same pH value. Injections were carried out by natural infusion with the BITE (Blade for Infusion in TrEes) tool, as described in Italian Patent Application PD2011A000245 and in PCT W02013010909. Injection rates obtained with the garlic extract were comparable thereamong. Injection rates obtained with the formulation are significantly higher for all three species assessed (see Figures 2, 3, 4).

Example 5 - In-field and in vitro phytotoxicity test of the allicin-based formulation For the in-field phytotoxicity test, the synthetic allicin-based formulation and the garlic extract (Conquer, Neem Biotech, Cardiff UK) were injected on Actinidia chinensis, with 10 replicates per solution; demineralized water was used as control. The composition of the carrier solution with allicin is reported in Table 2. The formulation and the garlic extract diluted so as to contain both 0.3-0.5 % w/v of allicin, were previously brought to the same pH (3.5-3.8) to make the results comparable. Injections were made by natural infusion with the Bite (Blade for Infusion in TrEes) tool, as described in Italian Patent Application PD2011A000245 and in PCT W02013010909. The effects commonly associated with trunk injection of phytotoxic solutions are:

At foliage level, presence of discolorations, burns, curlings and defoliation;

At injection points level, discharge of sap/exudate from the injection hole, cambium death, bark detachment off the cambium and presence of more or less wide longitudinal splits (Lodi et al., 1988; Acimovic et al., 2016).

At +30 days from injections (Prasad and Travnick, 1976), any presence of such symptoms on A. chinensis subject of the test was assessed.

For the phytotoxicity assay in vitro, the two allicin-containing solutions were assessed with young leaves of Salix alba, singled out as model species (Clausen and Trapp, 2017), with 10 replicates per solution. Demineralized water was used as control. Any presence of phytotoxicity symptoms was tested after 24 hours, by observing any presence of desiccation, chlorosis or necrosis on the leaf surface (Vikrant et al., 2006; Martos et al., 2008; EPPO, 2014).

The results are reported in Table 4; as to the controls (demineralized water), no phytotoxicity symptoms were found in-field, nor in vitro.

Table 4. Results of phytotoxicity tests with the two allicin-containing solutions.