KR100473499B1 - Sterilization Composition - Google Patents

Abstract

A fungicide with a water-soluble polymer and the active ingredient 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene is provided for use as a chemical fungicide in media for the control of bacterial or fungal contaminants.

Although the invention can be used for the sterilization of solid or liquid media, it is particularly useful for the production of in vitro culture plants. And surprisingly, the use of the present invention can perfectly replace the conventional sterilization procedures required by the medium. Furthermore, it is possible to replace the addition of hormones necessary for the fixation of in vitro plants of crops.

Description

Bactericidal composition

The present invention relates to the field of microbiology, in particular to bactericidal compositions containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene as the active principle. 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene is used as a chemical fungicide in culture media for the control of bacterial or fungal contaminants.

For example, in the production of plants cultured in vitro, contamination of the medium causes substantial loss of the entire production process because only the instantaneous contamination is removed by autoclaving. In some cases other antibiotics, such as cefotaxim, novobiocin and dihydrostrept omycin, are used to prevent contamination, but are not satisfactory.

In contrast, patent application WO89 / 11793 discloses the use of 2- (2-bromo-2-nitroethenyl) -furan as a broad range of antimicrobial agents.

The compound 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene has already been disclosed (ZN Nazarova, 1972. "Furylnitroolefins of Potentially Biological Activity" Synthesis "; Khim. Farm. Zh. 6 (10), pp. 5-8 and ZN Nazaroba et al., 1972." Physicochemical properties and reactivity of furylnitroolefins, Zh.org. 404-11), and the compound 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene is also known as a potent agent with antibacterial and antifungal activity (European patent application) No. EP-A 0 678 516).

The present invention provides bactericidal compositions that can be beneficially used to control bacterial or fungal contaminants in the medium.

Although the invention can be used for the sterilization of solid or liquid media, it is particularly useful for the production of in vitro culture plants. And surprisingly, the use of the present invention can perfectly replace the conventional sterilization processes required by the medium. Furthermore, it is possible to replace the addition of hormones necessary for the fixation of in vitro plants of crops.

The major limitations on the use of this active ingredient as a fungicide in solid medium are the solubility and reduced dissolution rate for near zero water in liquid medium. A possible solution to this problem may be solid dispersion. This solid dispersion can reduce particle size down to the molecular level.

The method presented in the present invention provides a bactericidal composition containing the compound 1- (5-brom ofur-2-yl) -2-bromo-2-nitroethene as an active ingredient as a bactericide. The method is also a dispersion by fusion or co-precipitation of this compound in a physiologically inert and easily soluble matrix. This substrate is formed by polyethylene glycol and other polymers of high molecular weight.

In order to effect dispersion by dissolution of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene, the choice of preparation methods and temperature as well as agitation times is a function of the active ingredients and the choice of polymers. It is based on point and thermal stability.

Compound 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene melts at 89-90 ° C without degradation and is thermally stable up to 110 ° C.

However, co-precipitation in solvents is recommended because the completion of solid dispersion systems of the active ingredient with polymers with melting points above 110 ° C. affects the thermal stability of the active ingredient.

In order to disperse by dissolution of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene, the physical mixture of PEG-6000 with an active ingredient in a ratio of 1:20 to 1: 1 is added. Heat at 80 ° C. until dissolution and stir constantly for 15 minutes until the melt is homogenized as a whole. Thereafter, the molten mixture is poured into a thin layer on a cold stainless steel sheet and spread out to ensure the speed of the condensation process. The solid obtained is ground in a mill and screened to a particle size smaller than 0.2 mm.

The range of melting point of the active ingredient and the solid dispersion of PEG-6000 varies with the composition of the mixture. That is, the melting point of the solid dispersion due to the dissolution dispersion of 30%, which is consistent with the active ingredient-PEG-6000 eutectic composition whose composition is binary, is 53-54 ° C.

In order to perform dispersion by coprecipitation of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene, the mixture of the active ingredient and the polymer in the ratio of 1:20 to 1: 1 is 10-60 minutes. Dissolved in a heated polar solvent over a temperature range between 40 and 60 ° C. Thereafter, vacuum-rotoevaporated with temperature control at 40-80 ° C. The solid obtained is ground in a mill and screened to a particle size smaller than 0.2 mm.

For the completion of a liquid medium containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene, the composites were prepared using Solutol IIS-15, Cremopher and the like. It was added to an aqueous formulation using the same extensible compound. These compositions were prepared to find the high stability and solubility of the composites and concentrations that did not adversely affect the organisms cultured in the medium.

Once a stable composition containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene is obtained using the method described above, other studies have been carried out, particularly in the propagation of plants cultured "in vitro". It is done to know the optimal dosage when used as a fungicide of the solid medium used for the purpose. Phytotoxicity was measured for necrosis, chlorosis, multiplication coefficient and junction-junction distance (to potato).

For the cultures studied (bananas, potatoes and sugar cane), the optimal dose of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene was in the range between 30 and 45 μg / ml. .

The following examples are intended to illustrate the invention and do not limit the scope of the invention.

Example 1: The effect of temperature and time on the purity of the solid dispersion obtained by the dissolution method.

The effect of temperature and time on the purity of the compositions containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene obtained by the dissolution method has been studied.

It was observed that the purity of the active ingredient in the obtained solid dispersion depends on the temperature used and the stirring time.

Table 1 shows the percent purity of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene obtained by solid dispersion of this compound (30:70, equivalent to 30) in PEG-6000. . At this time, the melting temperature was 80 ℃ to 120 ℃, the stirring time was 15 minutes to 60 minutes.

The study in all cases was done as described below.

For a temperature of 80 ° C. and a stirring time of 15 minutes, the physical mixture of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene and PEG-6000 (30:70) is heated until it melts And stirred constantly for 15 minutes until full melt homogenization was reached. The molten mixture was poured into flakes on a cold stainless steel sheet. The solid obtained was then ground in a mill and screened to a particle size smaller than 0.2 mm.

The purity of the active ingredient in the dispersion obtained under the above conditions was measured by gas chromatography, which was 99.89%.

TABLE 1

Effect of temperature and time on the purity of the solid dispersion obtained by the dissolution method

Example 2 Determination of Dissolution Characteristics of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene after Dispersion Processes by Dissolution or Coprecipitation

Dissolution properties are important for obtaining homogenized solutions / suspensions that are water soluble in the active ingredient.

Table 2 is a comparison of the dissolution rates of the active ingredient either with a humidified or micronized standard particle size or with a solid dispersion with 30% PEG-6000. It will be clear how the active ingredient properties can be significantly improved to ensure dispersion rate and homogenization in the medium.

TABLE 2

Determination of the Dissolution Rate of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene in Various Form Media

Example 3 Determination of Chemical Stability of Active Ingredients Dispersed / Diluted in PEG

The chemical stability of the dissolved-dispersed active ingredients in polyethylene glycol was studied using gas chromatography in a quantitative assay.

Chemical stability studies lasting 385 days demonstrate that no decomposition occurs.

TABLE 3

Determination of Shelf Chemical Stability of Solid Dispersion at 30% PEG-6000 by Gas Chromatography

Example 4 Activity of Formulations Containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene Against Selected Microorganisms

After completing the formulation study, in vitro assays were conducted to determine the product's activity against agricultural pathogens and contaminating microorganisms present in the production of in vitro culture plants. The study of bacteria was carried out in tubes modified by E. Siveira (Measurement of Minimal Concentration Inhibitors (MCI) of Actual Bioactivity, Boletin Cientifico ISCM IV (2), 1986). By serial dilution in (Bailey.WR and Scott.ES Signs Microbiology, 4th Edition, pp. 313-329, CV Cosby, St. Louis, 1974). And fungal activity was measured by diameter growth of colonies (Casadens II: Metodo de investigaciones. Instituto Iberoamericano de cooperacion para Agriculture. San Jose de Costa Rica, 1985).

The concentration used to measure the bioactivity of the product was 1-100 μg / ml. Medium, temperature and incubation time intervals were tailored to the conditions or characteristics of the microorganisms analyzed.

_{LD_50} was calculated in μg / ml by the Probit's method (Finney DJ. Probit Assay, 3rd edition, University of Cambridge Press, 1971).

The results obtained from this study are shown in Table 4.

TABLE 4

In vitro experiments with associated _{LD_50} values for agriculturally important microorganisms

Example 5 Determination of the Activity of 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene on Media Used for Production of In Vitro Cultured Plants

After demonstrating the in vitro activity of the active ingredient against fungal or bacterial contaminants of bananas, potatoes, and sugar canes in vitro, we proceeded as follows:

Proper washing of the flasks and lids to be used for the production of in vitro plants was done by washing them with sufficient water using a detergent and immersing the bottles in 0.5% sodium chloride for at least 3 minutes. The flasks were then turned upside down and allowed to dry completely before 24 hours.

Once the medium was prepared and reached approximately 60-80 ° C., the required amount of formulation was added to obtain a concentration of 35 μg / ml, and the required volume was poured into each flask undergoing the growth process after 24 hours.

The concentration was chosen based on the effectiveness in the studied media and the absence of phytotoxic effects.

The range studied was 1-50 μg / ml. The activity of in vitro cultures of bananas, potatoes and sugarcane was measured for the period necessary for cultivation after 72 hours to perform sequential culture in growth medium. The same study was carried out in fixation media.

Phytotoxicity was measured for necrosis, chlorosis, multiplication coefficient and junction-junction distance (for potatoes). The optimal dose for the three cultures studied was about 30-45 μg / ml.

Two scale-up studies were conducted on Musa sp. Cultures in proliferation. Percent mortality during the three studies was 0.81% for the size-increase done with the addition of the active ingredient and 1% for the standard method.

The results obtained in these studies are shown in Table 5.

TABLE 5

Activity of Solid Dispersions Containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene Against Contaminants in Medium Used for Production of In Vitro Plants

From these studies, it can be inferred that the contaminations behaved similarly to those of controls that had been physically sterilized by autoclave media, and that contamination began to increase from day 11. In addition, the growth coefficient was noticeably increased when working with chemical fungicides.

Fixing medium without the addition of autoclaving or root-inducing hormones had a stronger and more abundant outward appearance in a shorter period of time, showing better visual effects than fixation medium by standard methods. The percent polluted surveyed was meaningless compared to the control.

TABLE 6

Activity of Solid Dispersions Containing 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene on the Development of In Vitro Plants

Two stage size-steam studies were conducted on innocent species cultures in the proliferative state.

In addition, the study was conducted on mitigated species with Grain-Nane clones and FHIA-03 clones in fixation medium without the addition of root-derived hormones (A.I.A.). Both clones worked better in chemically sterilized strains, so large amounts of stronger roots were observed after 5 days. In contrast, the control by the standard method showed a small degree of development during the same period.

Claims (14)

1- (5-bromofur-2-yl) -2-bromo-2-nitroethine and 1- (5-bromofur-2-yl) -2-bromo-2-nitroethene) and polyethylene glycol 6000 (PEG- Sterile composition comprising 6000). Producing a bactericidal composition of claim 1 comprising applying a process of solid dispersion to a large amount of 1- (5-bromoper-2-yl) -2-bromo-2-nitroethine. Way. The method according to claim 2, Wherein said process of solid dispersion is dispersion by fusion. The method according to claim 3, Preparing a mixture of 1- (5-bromoper-2-yl) -2-bromo-2-nitroethine, active ingredient and PEG-6000; Heating the mixture to about 60 to 80 ° C .; Stirring the mixture until uniformity of the fused mixture is obtained; Spreading the molten mixture in a thin layer on the solidifying medium; And A method of producing a sterile composition comprising the step of obtaining a solid. The method according to claim 2, Wherein said process is co-precipitation using a solvent. The method according to claim 5, Dissolving a mixture of 1- (5-bromoper-2-yl) -2-bromo-2-nitroethine, active ingredient and PEG-6000; Subjecting the mixture to vacuum rotation evaporation; And A process for producing a bactericidal composition comprising obtaining a solid. The method according to claim 5 or 6, The solvent used is a method for producing a sterilizing composition, characterized in that the polar solvent. The method according to claim 7, Wherein the solvent used is selected from the group comprising ethanol, chloroform, methanol or isopropyl alcohol. A method of using a composition comprising 1- (5-bromoper-2-yl) -2-bromo-2-nitroetine and PEG-6000 as a sterilizing agent for control of contamination of culture media. The method according to claim 9, A method of using bactericidal compositions in connection with the production of non-vegetarian cultured plants. 1- (5-bromoper-2-yl) -2-bromo-2-nitroetine and PEG-6000 as a rootage in culture media for the production of plants cultured in vivo The method using the composition to make. Sterilization of the medium used in the production of in vitro cultured plants comprising the addition of a composition comprising 1- (5-bromoper-2-yl) -2-bromo-2-nitroetine and PEG-6000 How to. The method according to claim 12, The fungicide is also a method for sterilization, characterized in that it is a rotage agent in the medium used in the production of in vitro cultured plants. The method according to claim 12 or 13, The optimal dosage of 1- (5-bromoper-2-yl) -2-bromo-2-nitroethine used is in the range of 30 to 45 μg / ml.