CONJUGATES BETWEEN A BIOPOLYMER AND A PHOTOACTIVATED ANTIMICROBIAL AGENT FOR COMBATING FUNGAL DISEASES OF AGRICULTURAL INTEREST AND METHODS FOR PRODUCING SAID CONJUGATES

Abstract

This invention relates to the field of control of different fungal diseases in the agricultural industry. Specifically, the invention reports conjugates between biopolymers and a photoactivable agent, which when activated by light generates singlet oxygen, a species known for its antimicrobial capacity. The invention also relates to methods of production of these conjugates and their uses.

Claims

1-11. (canceled)

12. A fungicidal composition for inhibiting growth or combatting fungal infections of fungi of the genera Botritys, Penicillium, and Rhizopus, the fungicidal composition comprising a biopolymer selected from the group consisting of chitosan, modified chitosan, poly-lysine, alginate, modified alginate, cellulose, modified cellulose, and a derivative thereof, conjugated to a photoactivable agent selected from the group consisting of porphyrins, phthalocyanins, naphthalocyanins, chlorines, phenothiazines, acridines, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), and its derivatives, wherein the photoactivable agent generates oxygen radicals when activated by natural light.

13. The fungicidal composition of claim 12, wherein the biopolymer is at a concentration between 0.1 and 5%.

14. The fungicidal composition of claim 12, wherein the biopolymer is at a concentration between 0.1 and 2.5%.

15. The fungicidal composition of claim 12, wherein the photoactivable agent is at a concentration between 0.1% and 15%.

16. The fungicidal composition of claim 15, wherein the photoactivable agent is at a concentration between 0.1% and 1%.

17. The fungicidal composition of claim 16, wherein the biopolymer is at a concentration of 1% and the photoactivable agent is at a concentration of 1%.

18. The fungicidal composition of claim 12, wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.

19. A method for preparing the fungicidal composition of claim 12 comprising: a) combining the photoactivable agent (FA) with 4-maleimidophenyl isocyanate (PMPI) to obtain an FA-PMPI derivative; b) treating the biopolymer (BP) with thioglycolic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide to obtain a biopolymer product functionalized with reactive thiol groups (BP-SH); c) reacting the FA-PMPI derivative and the BP-SH in an aqueous solution to obtain a conjugate product; and d) optionally, dialyzing and lyophilizing the conjugate product for storage.

20. The method of claim 19, wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.

21. A method of treating or preventing gray rot, green rot, or soft rot fungal infections of a plurality of fruits or vegetables comprising contacting the fruits or vegetables with the fungicidal composition of claim 12.

22. A method of treating or preventing fungal infections caused by Botritys cinerea, Penicillium digitatum, and/or Rhizopus stoloniser of a plurality of fruits or vegetables comprising contacting the fruits or vegetables with the fungicidal composition of claim.

23. The fungicidal composition of claim 17, wherein the biopolymer is chitosan and the photoactivable agent is riboflavin or protoporphyrin IX.

24. The method of claim 19, wherein, in step a), the FA is combined with the PMPI at 45° C. in nitrogen atmosphere for 24 h to obtain the FA-PMPI derivative.

25. The method of claim 19, wherein, in step c), the FA-PMPI derivative and the BP-SH are reacted in the aqueous solution at pH 6 for 24 hours to obtain the conjugate product.

Description

BRIEF DESCRIPTION OF FIGURES

[0041] FIG. 1. Determination of the degree of deacetylation (GD) of hydrolyzed chitosan. Analysis performed by Nuclear Magnetic Resonance. GD determined by the ratio between the integration of signals between 4.5 to 3 ppm and the signal at 2 ppm.

[0042] FIG. 2. Chemical formulae and synthesis conditions of the precursors of the biofungicide. The first equation shows the chitosan on the left, the reagents and conditions necessary for the reaction to the medium and the thiolated chitosan product on the right. The second equation is riboflavin derivatization with PMPI. To the left is the PMPI and riboflavin respectively, to the middle the catalyst and the conditions used and to the right the product RF-PMPI.

[0043] FIG. 3. Separation of RF-PMPI reaction mixture by preparative chromatography on board (eluent Acetate: Methanol 9:1). The lower fractions correspond to unreacted riboflavin, the central fraction corresponds to the RF-PMPI product and the upper fraction corresponds to unreacted PMPI and the DBTDL catalyst.

[0044] FIG. 4. UV-visible absorption spectra from Riboflavin-based systems. Diluted RF and RF-PMPI from DMSO solution to aqueous solution and CH-RF in aqueous solution. Spectra measured at room temperature. Solid line: RF, Dashed Line: CH-RF. Scored Line: RF-PMPI. λ.sub.max absorption at 450 nm in all cases.

[0045] FIG. 5. Emission spectra from Riboflavin-based systems. Diluted RF and RF-PMPI from DMSO solution to aqueous solution and CH-RF in aqueous solution. Spectra obtained with an excitation λ of 450 nm..sub.λ max fluorescence at 524 nm in all cases. Spectra measured at room temperature. Solid line: RF, Dashed Line: CH-RF. Scored Line: RF-PMPI.

[0046] FIG. 6. Singlet oxygen generation profiles of riboflavin and chitosan conjugate. Profiles obtained by monitoring the fluorescence decrease of a oxygen-sensitive singlet acid 9.10-anthracenodiylbis(methylene)dimalonic (ABMA) probe. Samples adjusted to 0.1 u.a of absorption. Excitation wavelength at 450 nm and fluorescence tracking at 412 nm.

[0047] FIG. 7. RF-PMPI absorption curve A 450 nm for determination of RF content in CH-RF. Diluted RF-PMPI from DMSO concentrated solution. Conjugate (sample) measured at 0.1% m/v concentration.

[0048] FIG. 8. Fluorescence emission curve at 524 nm RF-PMPI for determination of RF content in CH-RF. Excitation wavelength at 450 nm. Diluted RF-PMPI from DMSO concentrated solution. Conjugate (sample) measured at 0.1% m/v concentration.

[0049] FIG. 9. Evaluation of fungicidal activity against Penicillium digitatum (1×10.sup.4 spores/mL) of RF, CH, CH/RF (physical mixture) and CH-RF (chemical conjugate) systems. All samples were pre-incubated for 1 hours. APDT samples were irradiated with white LED light for 1 hours. The growth of the fungus was carried to 30° C. for 7 days, on the seventh day the growth diameters were measured and the growth percentages were determined.

[0050] FIG. 10. Fluorescence microscopy photos of Penicillium digitatum spores in the presence of RF or CH-RF using 450 nm excitation light and a 510 nm fluorescence filter in 50. The same conditions of spore concentration and systems were used. 3 washes were performed with tween 20 0.1% solution prior to measurement.

[0051] FIG. 11. Growth percentages and representative photos of CH-RF fungicide activity against Botrytis cinerea under new growth conditions: 1×10.sup.6 spores/mL, growth with light regime of 12 hours and at 20° C. (PS stands for photosensitizer, in this case for riboflavin)

[0052] FIG. 12. Results of fungicidal activity of CH-RF against Penicillium digitatum under new growing conditions: 1×10.sup.4 spores/mL, growth with light regime of 12 hours and at 20° C. (PS stands for photosensitizer, in this case for riboflavin)

EXAMPLES

[0053] The following are examples of realization for this invention as described above:

Example 1

Preparation of Hydrolyzed Commercial Chitosan

[0054] A process of hydrolyzing the commercial chitosan used for the fungicide was carried out in order to improve its solubility and reactivity to subsequent processes. It has been observed that the degree of deacetylation of this is important for its biological properties, and therefore this parameter was evaluated. As shown in Table 1 and FIG. 1, it is observed that this polymer has a molecular weight of 2.4 kDa and a degree of deacetylation of 86%. Chitosan with this degree of deacetylation is the one that was used to produce the antimicrobial compounds described in the following examples.

Example 2

Synthesis and Characterization of Compounds with Antimicrobial Activity

[0055] The synthesis conditions of the 2 precursors of the biofungicide molecule are presented in FIG. 2. These correspond to a riboflavin derivative (RF-PMPI), to which the coupling agent PMPI and thiolate chitosane (CH-SH) are incorporated. This prior preparation of the precursors is intended to give the conditions of reactivity between these 2 (RF-PMPI and CH-SH) to form the chemical conjugate between chitosane and riboflavin (CH-RF).

[0056] To obtain the pure precursor for chemical characterization and then its binding with chitosane, a separation was performed by preparative chromatography on board, the band in the middle of the plate corresponds to the RF-PMPI derivative, obtaining a

TABLE-US-00001 TABLE 1 Characterization of commercial chitosan and hydrolyzed chitosan t Mv c System (s) nr nsp [n] g/mol g/mL α K CH 132 5.07 4.07 3.69E+04 39585 6.0E−05 0.96 1.424 CH.sub.H 51 1.96 0.96 2.53E+03 2426 3.0E−04 — — Solvent 26 — — — — — — —
successful separation (FIG. 3).

[0057] Additionally, a characterization of the absorption and emission spectra of riboflavin, the RF-PMPI derivative and the chemical conjugate was performed. It is important to know the absorption spectrum because in this way it is known what type of light the molecule absorbs to photoactivate. It absorbs in the region of blue light and UV (white and solar light have these light components present). The fluorescence spectrum is used to know in which region of the light spectrum this molecule emits light, useful for later experiments. As shown in FIGS. 4 and 5, the conjugate fluoresces less than riboflavin, this phenomenon does not affect its performance as a fungicide.

[0058] Subsequently, an additional characterization was performed in which the life-times and fluorescence anisotropy of riboflavin and riboflavin derivatives were determined. The data obtained are shown in Table 2, and these results show that highlighting riboflavin is chemically bound to the polymeric matrix of chitosan.

TABLE-US-00002 TABLE 2 Life-time and fluorescence anisotropy of RF-based systems System T (ns) α.sub.i r.sub.0 φ (ns) r.sub.∞ RF 4.8   0.278 0.276 0 RF-PMPI 1.6 0.033 0.276 0.374 0 3.9 0.146 CH.sub.H-RF 1.58 0.046 0.237 0.485 0.011 3.75 0.132

Example 3

Determination of Singlet Oxygen Generation by Compounds

[0059] In this example, the capacity of singlet oxygen generation mediated by riboflavin and chitosan-riboflavin conjugate was evaluated. As shown in FIG. 6, the results obtained show that the CH-RF conjugate has a higher capacity for singlet oxygen generation than riboflavin. This result is a good indicator that the CH-RF conjugate will have a good fungicide effect.

Example 4

Quantization of Riboflavin in Conjugates

[0060] The results from Table 3 and FIGS. 7 and 8 were obtained with the aim of quantifying how much riboflavin binds to chitosan in the final conjugate. A determination was made by absorption curve, by molar extinction coefficient using the Lambert-Beer Law formula and finally by fluorescence curve.

[0061] Concentrations calculated in 3 different ways are within the same order of magnitude, which is good indication that the amount of riboflavin that was achieved by chitosan is within the micromolar magnitude. Therefore, the percentage composition of the final conjugate is: [0062] GLCN: 2.11 m ×10.sup.−4 moles, 70% [0063] GlcNAc: 0.10-×10.sup.−4 moles, 14% [0064] Thiol groups: 61.10-×10.sup.−6 moles, 15% [0065] Riboflavin: 4.3-×10.sup.−6 moles, 1%

[0066] The conjugate possesses 1% riboflavin as a photoactive agent.

Example 5

In vitro Fungicidal Activity of the Chitosan-Photoactive Agent Formulations

[0067] It can be seen in FIG. 9 that the conjugate is the system that has the most antifungal activity against the fruit pathogen fungus Penicillium digitatum. The antifungal activity of the chemical conjugate between riboflavin and chitosan is greater than that of the separate components, and it was observed that the effect is effectively enhanced when subjected to a light regime. It should be noted that this synergistic surprising activity has not been reported previously.

[0068] Riboflavin has green fluorescence, therefore in FIG. 10 specifically it can be seen that riboflavin alone has no interaction with the spores of the fungus, whereas the conjugate does have interaction with the surface structures of the fungus, with the characteristic fluorescence of riboflavin, it can then be concluded that the chemical conjugation of riboflavin with chitosan causes this transport to the photoactive agent to the vicinity of the spore, so the photoactive effect is generated “in situ”.

[0069] Subsequently, tests were carried out on Penicillium digitatum and Botrytis cinerea to see the photodynamic fungicidal activity of the conjugate, under conditions closer to the industry and optimal for the growth of both pathogens (20° C., light regime 12/12, Potato Agar culture medium). The results obtained for Botrytis cinerea and Penicillium digitatum can be seen in FIGS. 11 and 12, respectively.

[0070] These new results indicate that the conjugate possesses antifungal activity against these 2 pathogens and that in both cases this antifungal effect is enhanced by light (irradiation for one hour with white LED light). In the specific case of Botrytis cinerea, the biofungicide eliminates 100% growth at a concentration of 1% (m/v) under ambient light, whereas, under an irradiation of 1 hours, only 0.5% in concentration of the biofungicide is needed to control 100% of the pathogen. In the case of Penicillium digitatum, total control is obtained with a concentration of 1% under 1 hours of irradiation.

Example 6

Control of Gray Mold (Botrytis cinerea) in Table Grapes

[0071] Additionally, the efficacy of the formulation developed in the present invention, which comprises the light-activable biopolymer, under more challenging conditions, was evaluated. Three treatments were used to demonstrate the technical advantages of this polymer: (1) witness with wound and inoculated with sterile water, (2) standard wound treatment and inoculated with the pathogen, and (3) treatment with the composition of the molecule described in this request to 0.7%, with wound and inoculated with the pathogen.

[0072] The berries used were obtained from a commercial orchard, which had a maturity of more than 16% of soluble solids, and these berries were collected from clusters that had never been treated with fungicides.

[0073] Berries were taken randomly from bunches with adhered pedicel, and were superficially disinfected with sodium hypochlorite at 0.5% for 1 min, followed by 95% ethanol for 30 seconds and rinsed twice with sterile distiled water, ensuring that no residue remains on the berries. They were then allowed to dry at room temperature under laminar flow chamber. The berries collected were distributed in a number of 10 berries with pedicel on metal grids in appropriately sized polyethylene boxes (e.g.: 20×15×10 cm), placing absorbent paper towel moistened with 40 ml sterile water under the grids to assemble wet chambers. The water level was adjusted depending on the size of the container.

[0074] A fixed point was punctured in each berry with a hypodermic syringe and then a 10 μl drop of 10.sup.6 conidia/ml B. cinerea suspension was mounted on the wound using a micropipette.

[0075] Subsequently, wet chambers were closed and maintained at 20° C. and relative humidity (RH)≥90% avoiding the displacement of the drop on the surface of the berry for 24 hours.

[0076] Finally, the treatments were applied (except T1), reselling chambers and arranging containers randomly in a storage chamber at 20° C. and relative humidity (RH)≥90% for 7 days. The incidence of the disease (gray mold) will be assessed 7 days after each treatment is applied.

[0077] Results: [0078] Disease control efficiency T3>Disease control efficiency T2

Example 7

Control of Green Mold (Penicillium digitatum) in Citrus Fruits

[0079] An experimental strategy very similar to the previous example was used. Briefly, fruits were obtained from a commercial orchard at its point of harvest, of uniform size and color, without defects in the shell or deformations, and the fruits were not treated with fungicide prior to collection. The fruits were then disinfected with sodium hypochlorite at 0.5% for 5 minutes, followed by 95% ethanol for 30 seconds and rinsed twice with sterile distiled water, ensuring that no residue remains on the fruits. They were allowed to dry at room temperature. 12 fruits were then taken and distributed in wet polyethylene chambers of an appropriate size according to the size of the fruit. The water level under the wet chamber louvers was adjusted according to the size of the container.

[0080] A puncture was made with the tip of a scalpel not more than 3 mm deep by 3 mm wide in the equatorial area of the fruit. After the above action, the wound was inoculated with a 10 μl drop of conidial suspension of P. digitatum of 10.sup.4 conidia/ml using a micropipette.

[0081] Subsequently, wet chambers were closed and maintained at 20° C. and relative humidity (RH)≥90% avoiding the displacement of the drop on the surface of the berry for 24 hours.

[0082] Finally, the treatments were applied (except T1), reselling chambers and arranging containers randomly in a storage chamber at 20° C. and relative humidity (RH)≥90% for 7 days. The incidence of the disease (green mold) will be assessed 7 days after each treatment is applied.

[0083] It is expected that the development of patients after treatment 3 (T3) is significantly lower than the other treatments. In particular, T3 is expected to be more efficient in inhibiting disease development compared to T2.

[0084] Results: Disease control efficiency T3>Disease control efficiency T2.