ES2926873B2 - BIOACTIVE COMPOUND EXTRACTED FROM PHLOMIS PUPUREA, OBTAINING METHOD, ANTIFUNGAL COMPOSITION CONTAINING IT AND ITS USE - Google Patents

Description

DESCRIPTION

BIOACTIVE COMPOUND EXTRACTED FROM PHLOMIS PURPUREA,

OBTAINING METHOD, ANTIFUNGAL COMPOSITION CONTAINING IT AND

ITS USE

Technical sector of the invention

The invention falls within the field of the agroforestry industry, more specifically in the phytosanitary industry and more particularly in the antimicrobial and antifungal industry. It specifically deals with new bioactive compounds and formulations based on said compounds, used mainly for the treatment of microbial and fungal infections, such as infection by Phytophthora cinnamomi, a phytopathogen. Specifically, said formulations are based on an extract from the Phlomis purpurea plant, which can be used as an agent that inhibits and kills Phytophthora cinnamomi (P. cinnamomi), among other species of this oomycete genus.

Background of the invention

There are currently 3.5 million hectares of pasture in Spain, with Extremadura being the autonomous community with the largest area (1.2 million). The meadow is an ecosystem characterized by the presence of trees of the genus Quercus (mostly holm oaks -Quercus ilex- and cork oaks -Quercus suber-), which constitutes a great source of biological resources, favoring the development of rural populations, as well as the wealth creation through food products with high added value, the main exponent of which is acorn-fed Iberian ham.

A multitude of factors, such as global warming, abiotic and biotic contamination, the overexploitation of water resources and poor environmental management have caused a significant increase in phytosanitary problems that affect holm oaks and cork oaks. This translates into the presence of more than 5,000 outbreaks of the so-called "La Seca" in Extremadura, which have caused the death of approximately 1,000,000 holm oaks and cork oaks with a total affected area of 100,000 Ha (IPROCOR, 2016. Instituto Cork, Wood and Coal, JUNTAEX).

Despite the fact that there are several works that describe this problem, none of them coincides in the factors responsible for "La Seca", except that in all of them describes the presence of a phytopathogen, called Phytophthora cinnamomi. This microorganism, belonging to the Oomycetes class and the Chromista kingdom (Hardham and Blackman, 2018. Pathogen Profile Update: Phytophthora cinnamomi. Molecular Plant Pathology, 19(2):260-285), carries out its entire life cycle in the soil (asexual and sexual phases), where humidity is a fundamental factor in its development and propagation.

P. cinnamomi can grow as a saprophyte on dead organic matter or as a parasite, affecting some 5,000 species of woody, shrubby, and herbaceous plants (Jung T, Chang TT, Bakonyi J, Seress D, Pérez-Sierra A, et al. (2017 ). Diversity of Phytophthora species in natural ecosystems of Taiwan and association with disease symptoms. Plant Pathology, 66:194-211), among which are holm and cork oaks. P. cinnamomi usually infects roots, but it can also invade woody stems, especially through wounds on the periderm. Its growth in the root system causes root rot and interferes with the absorption of crude sap, which results in the appearance of chlorosis on the leaves, among other effects. Following infection with P. cinnamomi, hosts may die rapidly or may be asymptomatic for many years.

In addition to its great capacity for expansion, P. cinnamomi presents a high degree of resistance against conventional eradication treatments. Most fungicides have no effect on the species of the genus Phytophthora, with the exception of metalaxyl (González M, Caetano P, Sánchez ME (2017). Testing sustemic fungicides for control of Phytophthora oak root disease. Forest Pathology, 47 ( 4) \e 12343) The application of these fungicides, rather than triggering the elimination of the pathogen, induces various natural resistance mechanisms in the plant (Romero MA, González M, Serrano MS, Sánchez ME (2019). Trunk injection of fosetyl-aluminium controls the root disease caused by Phytophthora cinnamomi on Quercus ilex woodlands. Annals of Applied Biology, 174 ( 3): 313-318). On the other hand, it is important to highlight the negative effects on the environment after the use of pesticides, such as soil and water contamination, so it is advisable to reduce the use of this type of compound.

In view of the difficulties that arise from the application of conventional treatments, it is necessary to find solutions that increase the probabilities of success in eliminating P. cinnamomi.

It has been seen over time that in the pastures, in addition to being organisms sensitive to P. cinnamomi, there are plant species resistant to the microorganism, such as: ash ( Fraxinus angustifolia), white heather ( Erica arborea), oats ( Avena sativa), wheat ( Triticum aestivum), matagallo ( Phlomis purpurea) and rosemary ( Rosmarinus officinalis). Recent research indicates that this type of plants could synthesize compounds with antifungal activity that, in addition to protecting the plant itself, would help the regeneration of infected land and the defense of holm oaks and cork oaks in those areas (Mateus MC, Neves D, Dacunha B, Laczko E, Maia C, Teixeira R, Cravador A (2016). Structure, anti- Phytophthora and anti-tumor activities of a nortriterpenoid from the rhizome of Phlomis purpurea (Lamiaceae). Phytochemistry, 731:158-164).

The most interesting studies carried out on the beneficial activity of any of these plant species have focused on the matagallo plant ( Phlomis purpurea), present in the southwest of the Iberian Peninsula, and its ability to produce bioactive compounds with antifungal potential. It has been observed that this species has the capacity to inhibit the growth of phytopathogens, and more specifically, its roots secrete compounds capable of protecting in vitro from infection by P. cinnamomi . Currently, only one of these compounds has been determined: the terpenoid phlomispurpentaolone (Neves D, Caetano P, Oliveira, Maia C, Horta M, et al. (2014). Anti-Phytophthora cinnamomi activity of Phlomis purpurea plant and root extracts European Journal of Plant Pathology, 138(4):835-846; Mateus et al., 2016).

However, in an effort to find solutions to this problem of "La Seca" that greatly affects the pasture, the authors of the present invention have detected in recent works the presence of other compounds, of a chemical nature not yet known up to now, with greater antifungal capacity than the terpenoid phlomispurpentaolone.

In short, obtaining new bioactive compounds not characterized up to now, with inhibitory capacity against P. cinnamomi , involves the application of new natural bioactive molecules, extracted from the matagallo plant ( P. purpurea ). This proposal would apply to all plants and trees that are sensitive and susceptible to be attacked by P. cinnamomi, such as avocado ( Persea americana), oak ( Quercus spp.) and chestnut ( Castanea sativa), and not only the species of special interest: holm oaks ( Q. ilex) and cork oaks ( Q .up).

DESCRIPTION OF THE INVENTION

The present invention refers, first of all, to a bioactive compound, belonging to the family of triterpenoids, which is defined for the first time in this report. Said compound has been identified in an extract derived from the Phlomis purpurea plant, preferably from the meadows in the south of Badajoz, obtained from any part of the plant, aerial or radicular, but preferably from its roots, and preferably in larger specimens. one year of life, which is where it seems to be most concentrated. This compound has a molecular structure compatible with pentacyclic triterpenes derived from oleanoic acid, but with its own distinctive characteristics, both of this oleanolic acid and of the phlomispurpentaolone compound, with a molecular mass of 490.3294 Da.

The compound object of protection, which has not been previously described, is characterized by the following formula:

and is defined generically as: a pentacyclic triperpene compound that contains five non-aromatic rings, four of them formed by 6 carbon atoms and only one of them connected, at one of its ends (carbon 17), through a spiroatom (C) to a fifth ring formed by 5 carbon atoms; where:

- the first ring, called ring A, contains 2 methyl groups on carbon 4 (carbons 23 and 24), which are both hydroxylated;

- in ring A, in addition, carbons 2 and 3 are hydroxylated;

- carbons 8 (carbon 26), 10 (carbon 25) and 14 (carbon 27) of rings B and C are methylated;

- in ring C there is a double bond between carbons 12 and 13;

- in ring E, carbon 20 has two methyl groups (carbons 28 and 29); and - in ring D, carbon 18 has a keto group (C=0).

Said compound is defined by a molecular weight of 474.3345 Da and, unlike other chemically similar compounds, such as oleanoic acid, it does not have a carboxyl group attached to ring D, but a keto group attached to carbon 18 of said ring, and its E ring is five carbon atoms. Also, unlike the already known compound phlomispurpentaolone, this compound described here with a molecular weight of 474.3345 Da has the aforementioned keto group on carbon 18 of ring D and lacks the hydroxyl group that phlomispurpentaolone has on carbon 11 of ring C.

The new compound has been identified as one of the components of an extract from the Phlomis purpurea plant, which is where it was obtained by isolation. Therefore, the first known way to obtain this new compound is its isolation and purification from an extract from said plant, which includes, among other components, the one indicated here.

The extract to which the text refers here is an extract obtained by common and known methods from any part of the Phlomis purpurea plant, preferably its root, which is where a higher concentration of these metabolites or active compounds is located.

Specifically, the concentration of the compound defined herein in the dry matter of the Phlomis purpurea plant, preferably in its dry root, is between 50 and 150 pg/g of dry matter, more preferably between 110 and 140 pg/g of dry matter.

It should be noted that when a range of values is cited herein, its lower and upper limits are always included in the scope of the invention, as specific embodiments thereof.

The invention thus includes a method for obtaining the bioactive compound described above, which is an isolation method: that is, it is purified from an extract of the P. purpurea plant, in which said compound is contained. Specifically, the method object of the present invention comprises:

- prepare a suspension of the extract obtained from plant matter of the P. purpurea plant in a solvent selected from the group consisting of ethanol, methanol and ethyl acetate, in a weight ratio of solvent and extract of 1:10 (1 gram root for every 10 grams of ethanol);

- subjecting the above suspension to solid phase extraction (SPE), preferably by fractionation on solid phase columns with C18 stationary phase matrix, and subsequent elution with an increasing gradient of methanol in water from 20% to 100% methanol, until obtaining individual fractions in which the used solvent is eliminated;

- drying the fractions from the previous stage (preferably by rotary evaporation) and resuspending again in an organic solvent, preferably selected from the group consisting of methanol, ethanol, acetone and ethyl acetate; and

- subjecting the resuspensions to high performance liquid chromatography (HPLC) using C18 stationary phase columns, with a mobile phase gradient made up of one of the following solvent combinations: methanol with water or acetonitrile with water. The mobile phase consisting of acetonitrile and water always contains 0.1% formic acid, while the combination of methanol and water may or may not contain 0.1% formic acid. These HPLC gradients are used until the bioactive compound (molecular weight 474.3345 Da) is purified (see fractionation details in Table 1: methanol with water is used in HPLC purification programs 1, 2 and 3; while that acetonitrile with water and 0.1% formic acid is used in the HPLC purification program 4).

As has been said, the starting material in this process is an extract (preferably liquid, suspended in a solvent) obtained by common and known methods from aerial or radicular plant matter of the P. purpurea plant, that is, regardless of any part of the plant but preferably its root, since it is where a higher concentration of these metabolites or active compounds is located.

Preferably, the specimens from which the extract is obtained come from the meadows in the south of Badajoz, and even more preferably they are more than one year old. An example of the common methods for obtaining the extract of the P. purpurea plant would be:

- drying the vegetable matter of the plant and macerating with a solvent selected from the group consisting of ethanol, methanol and ethyl acetate, more preferably ethanol, in a proportion of solvent (by volume, ml) and vegetable matter (by weight, g ) of 10:1;

- Submit the mixture of vegetable matter and solvent to stirring, for its homogenization, and then subject to centrifugation, at a speed between 5,000 and 10,000 rpm, preferably 9000 rpm, for a time between 5 and 10 min, preferably 5 minutes .

Preferably, once the centrifugation is finished, the supernatant is separated, dried (preferably by rotary evaporation) until a solid product in the form of a pellet is obtained, which is resuspended in a solvent selected from the group consisting of ethanol, methanol and acetate. ethyl, thus obtaining the liquid extract of P. purpurea. When the extraction process is carried out more than once, these same actions are applied but pooling the supernatant of all the centrifugations carried out.

In general, the solvents for each stage of both the method for obtaining the extract and the method for obtaining the bioactive compound are selected without distinction from each other at each stage. Thus, the solvent in which the plant extract is suspended in the first stage of obtaining the bioactive compound can be the same or different from the solvent in which the extract is diluted before beginning the process of obtaining said bioactive compound. , and at the end of its preparation.

The bioactive compounds identified in this invention are characterized by having antifungal inhibitory activity. Said activity may be due to the inhibition of essential cellular processes in the pathogen P. cinnamomi. Preferably, they present activity against the phytopathogenic fungus P. cinnamomi, which is why they are configured as two relevant metabolites in the fight against "La Seca" and its main causal agent, in regard to the Quercus species, although they can be used to treat any disease caused by fungi in one of the species selected from the group consisting of: avocado ( Persea americana), oak ( Quercus spp.) and chestnut ( Castanea sativa), in addition to the species of special interest here treated: holm oaks ( Q. ilex) and cork oaks ( Q. suber).

Another of the relevant aspects of the invention presented here consists in the fact that said antifungal activity has not only been found for the previously defined bioactive compound, but has also been confirmed for other already known pentacyclic triperpene compounds of similar structure. In this way, the present invention covers the phytopathogenic use in plants of a group of compounds defined by the following Markush formula, acting as active agents that inhibit the activity of fungi:

where R1 is selected between OH and =O.

In one of the compounds or variants of the defined group, which can be called here Compound 1 (molecular weight 474.3345 Da), carbon 18 has a keto group attached, said compound then being the main object of this invention described above. In another variant, here called Compound 2, carbon 18 has a hydroxyl group attached (molecular weight 476.3502 Da); this second compound has already been defined by Liu P, Yao Z, Zhang W, Takaishi Y, Duan HQ (2008). Novel Nortriterpenes from Phlomis umbrosa. Chem Pharm Bull ( Tokyo), 2008 July; 56 ( 7): 951-5, although not its antifungal activity or its use against phytopathogens in plants.

compound 2

It has been verified that this group formed by two compounds can inhibit the activity of invasive species of the genus Phytophthora, which are oomycetes. Preferably, this group of compounds is used to inhibit the activity of the P. cinnamomi fungus, in any plant, and specifically in one of the species selected from the group consisting of: avocado ( Persea americana), oak ( Quercus spp.) and chestnut. ( Castanea sativa), in addition to the species of special interest: holm oaks ( Q. ilex) and cork oaks ( Q. suber), due to the fact that they are affected by “La Seca”.

Another object of protection covered by the present invention is a composition antifungal containing at least one of the compounds defined above as active agents or any mixture thereof (ie Compound 1, Compound 2 or a mixture of both), with fungicidal action. This antifungal composition can preferably be in liquid or solid form; regardless of its form of presentation and the excipients that accompany the bioactive agent, the composition contains said bioactive compound in a proportion between 5 and 200 mg/kg of antifungal composition, when it is in solid form, or between 2 and 200 mg/ liter of antifungal composition when it is in liquid form.

In a particular embodiment, in which the composition is liquid, it may preferably comprise the bioactive agent in an amount between 5 and 200 mg per liter of composition, more preferably in an amount between 25 and 75 mg/l composition, being in the most preferred case 50 mg/l composition. Said composition can be a solution of the active agent in a component selected from the group consisting of:

- one or more vegetable gels, selected from the group consisting of: safflower flower, sunflower and other plants from the group of composite and oilseeds; - a polyalcoholic solution of one or more compounds selected from the group consisting of polysaccharides, xylitol, sorbitol and mannitol; and

- water.

In another preferred embodiment, in which the composition is presented in solid form, said composition comprises the described bioactive agent in an amount between 5 and 200 mg/kg of composition, more preferably in an amount between 25 mg/kg of composition and 75 mg/kg of composition, being more preferably 50 mg/kg; in this case, the bioactive agent (Compound 1, Compound 2 or a combination of both) can be accompanied by one or more of the excipients selected from the group consisting of: dibasic calcium phosphate, talc, silica and one or more synthetic polymers.

The application or use of Compounds 1, 2 or their combination covers, in one of its embodiments, a method for treating a fungal infection in plants, preferably the infection caused by an oomycetes fungus of the genus Phytophthora, more preferably the fungus P. cinnamomi ; which comprises applying the composition antifungal containing the bioactive agent described by the above Markush formula, in a concentration less than 200 pg/ml of liquid composition, preferably less than 100 pg/ml of liquid composition and more preferably at a concentration of 50 pg/ml of liquid composition . Alternatively, the method comprises applying the antifungal composition containing one or more of the two bioactive agents described by the above Markush formula (Compound 1, Compound 2 or a combination of both), in a concentration of less than 200 pg/g of solid composition. , preferably less than 100 pg/g of solid composition and more preferably at a concentration of 50 pg/g of solid composition. The application of the composition can be foliar or radicular (in the soil), although it is preferably applied to the soil, to combat the disease directly on the roots.

This result is unexpected and advantageous, since in previous studies of the prior art (Mateus et al., 2016) the antifungal activity of extracts from the P. purpurea plant had been attributed to the existence of another molecule, phlomispurpentaolone. However, in the current work it has been verified (see examples) that this molecule, phlomispurpentaolone, only has activity above concentrations of 200 pM (98 mg/l), which implies having to purify and apply a high amount of the itself for antifungal potential. On the contrary, in the present invention, the identified bioactive compound, and the compound of similar structure defined here as compound 2, are capable of inhibiting P. cinnamomi (Minimal Inhibitory Concentration or MIC) at concentrations lower than 200 pg/ml of the fungicidal composition, preferably less than 150 pg/ml of the fungicidal composition that contains it as active agent, more preferably less than 100 pg/ml in that same composition that contains it and in the most preferred case at concentrations of 50 pg/ml in said composition. Similarly, when the antifungal composition is in solid form, the MIC is less than 200 pg/g of the fungicidal composition, preferably less than 150 pg/g of the fungicidal composition that contains it as an active agent, more preferably less than 100 pg. /g in that same composition that contains it and, in the most preferred case, at concentrations of 50 pg/g in said composition, in solid state.

Brief description of the figures

Figure 1: Filtered search of the Phlomis purpurea root chromatogram for the masses corresponding to compound 1. After isolating this molecule, the mass of the same in negative ionization. In descending order: 519.3328 Da; 473.3276 Days and 509.3040 Days.

Figure 2: Chromatogram of the analysis by fragmentation of the formate adduct of Compound 1 (519.3328 Da) object of the present invention.

Figure 3: Filtered search of the Phlomis purpurea root chromatogram for the masses corresponding to compound 3. After isolating this molecule, its mass is checked in negative ionization. In descending order: 521.3484 Da; 475.3422 Days and 511.3197 Days.

Figure 4: Chromatogram of the fragmentation analysis of the formate adduct of compound 3 (521.3484 Da).

Figure 5: Example of a positive bioassay. Control (left) and sample (right).

Examples of realization

Example 1: Selection and collection of plant matter from the Phlomis purpurea plant

In order to analyze the antifungal capacity of the P. purpurea plant, specifically against P. cinnamomi, and following the indications of Neves D, Caetano P, Oliveira, Maia C, Horta M, et al. (2014). Anti-Phytophthora cinnamomi activity of Phlomis purpurea plant and root extracts. European Journal of Plant Pathology, 138(4):835-846, samples were collected from the roots. In the first working hypotheses, it was expected to find a higher concentration of antifungal compounds in the root zone, since it is the one that is in contact with the aforementioned pathogenic fungus. Also hypothetically, at that time said antifungal activity was exclusively related to the compound phlomispurpentaolone, due to the background of the prior art, which was intended to be analyzed in depth.

For the collection of matagallo plants, the province of Badajoz was selected, more specifically the La Parrilla farm, located in the municipality of Jerez de los Caballeros, near the Valuengo reservoir.

Plant material collection activities began with the arrival of the first rains, in October. Initially, 553 grams of matagallo plant roots were collected on the La Parrilla farm, and a sample was also collected of 115 grams of leaves and stems of matagallo plants from this same farm.

Example 2: Obtaining an extract from the plant matter of P. purpurea collected in Example 1

The extraction process used is based on the one described by Neves et al. (2014), macerating all the tissues in ethanol for their extraction, as previously mentioned in the Description of the invention section. The complete process is as follows:

Once the plant material was collected, the samples were dried. To do this, the soil present in the roots was removed by immersing them in water, and the clean roots were then left on absorbent paper for a minimum period of one week. Once the roots were dehydrated, they were chopped. For this, pruning shears were used, and they were cut until fragments less than 0.5 cm in length were obtained. These fragments were crushed by means of a blender until obtaining root powder.

Subsequently, to achieve the extraction of the compounds from said dry and pulverized matter, 97% ethanol was preferably used.

Specifically, 60 g of Phlomis purpurea root were pulverized and extracted with 97% ethanol in a ratio of 10:1: 10 ml of ethanol for each gram of pulverized root. The mixture was stirred for 1h and centrifuged at 9000rpm at room temperature for 5min. The supernatant was separated and, on the same root sample, the same amount of 97% ethanol was added again for a second extraction equal to the previous one. The supernatant of both extractions was combined, dried by rotary evaporation, and the obtained pellet was resuspended in 10 ml of 20% ethanol in water, thus obtaining the liquid extract of P. purpurea that was used for subsequent analysis.

Example 3: Fractionation of the plant extract obtained from P. purpurea in Example 2 and purification and quantification of the components identified in said extract

From the liquid extract (in this case, 97% ethanol) of the roots of Phlomis purpurea obtained in example 2, the purification and quantification of the compounds was carried out. contained in the extract.

The possible bioactive compounds present in these ethanolic extracts were obtained by means of an extraction process in solid phase columns (SPE), specifically Phenomenex Strata C18-E, 55 pm, 70Á, 10 g, 60 mL, followed by an isolation process by HPLC-DAD, monitored by HPLC-MS, following the protocol developed in the Mass Spectrometry Unit of the Scientific-Technical Services of the University of Oviedo (see Table 1).

Thus, the sample was resuspended in a 20% methanol solvent in water and subsequently fractionated on an SPE column (Phenomenex Strata C18-E, 55 pm, 70A, 10 g, 60 mL), previously conditioned with 100 ml of methanol. 20% in water. Subsequently, 10 ml of the concentrated sample of the plant were added to the SPE column, and this was eluted from the column with 50 ml per fraction, in an ascending phase of methanol (20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90% and 100%); a final wash may also be performed, preferably with 100% acetone elution. At the end of the SPE column fractionation process, 10 liquid fractions were obtained from the initial sample, herein designated C1-C10 respectively.

Fractions C7 and C8, both 50 ml, were collected, rotary-evaporated to dryness and resuspended again in 850 µl of 100% methanol. The resuspended SPE fractions were then subjected to a series of HPLC-DAD fractionations in order to purify the compounds of interest (Table 1).

Table 1.- Parameters corresponding to the HPLC-DAD programs used to purify the compounds of interest*

* HPLC-DAD protocol from the BIONUC research group.

v The percentage of the organic phase is indicated. Water accounts for the remainder up to 100%.

Bioactive HPLC-DAD fractions were analyzed by HPLC-MS on a Zorbax Eclipse Plus C18 type column (50 mm x 2.1 mm, 1.8 pm particle size), using negative ion detection mode to determine their mass. in daltons (Da).

From the HPLC-MS analysis, a first compound was identified, here called Compound 1, defined by a mass of 474.3345 Da, and which was detected at minute 25.7 of the chromatogram of each root extract.

In order to corroborate that the peak detected in that minute corresponds to this compound 1, a filter was performed on the HPLC-MS chromatogram by the known mass of the compound. As in the HPLC-MS program, the negative mode was used to search for its mass with one less proton (473.3276 Da), or adducts with formate (519.3328 Da) or with chlorine (509.3040 Da): see Figures 1 and 2.

The second compound detected, which has been designated Compound 2 herein (see Markush formula), has a mass of 476.3502 Da and was also detected at minute 25.5 of the chromatogram of each root extract. To corroborate that the peak detected in that minute corresponds to this compound, a filter was performed by the known mass of the compound. As in the HPLC-MS program of the previous examples, the negative mode was used, having to look for its mass with one less proton (475.3422 Da), or adducts with formate (521.3484 Da) or with chlorine (511, 3197 Da): see Figures 3 and 4.

From 60 g of P. purpurea root used, 0.7 mg of compound 1 and 7.5 mg of compound 2 were obtained. Therefore, it can be inferred that in each gram of dehydrated root there are 11.7 pg of compound 1 and 125.3 pg of compound 2.

Example 4: Preparation of the formulation containing the antifungal active compound: as solid root fertilizer and/or liquid foliar fertilizer

To prepare the antifungal liquid formulation, the dissolution of the antifungal compound in a gel or liquid medium, preferably liquid due to its ease of preparation, was required. The medium, liquid excipient, consisted of a polyalcoholic solution of polysaccharides, preferably xylitol, sorbitol and mannitol. The formulation was carried out with the addition of 50 mg of the bioactive compound to 1 liter of this solution.

In the case of the solid formulation, the selected excipient was dibasic calcium phosphate, the amount of antifungal compound to be incorporated per kg of dibasic calcium phosphate being 50 mg/kg.

Example 5: Calculation of the Minimum Inhibitory Concentration Test (MIC) against the phytopathogenic fungus P. cinnamomi

For MIC assays, 192 pL of solution were used, at a concentration of 41.1 pg of compound/ml of 20% methanol in water, in 18 ml of medium. The final concentration was 0.43 pg of compound/mL of culture medium.

The application of bioactive compounds 1 and 2 was carried out in two ways: A) in liquid form, sprayed and absorbed by the foliar part of the tree, and B) solid form, which was applied to the roots under the projection of the tree Top.

A) For the liquid formulation, an application between 30 and 70 liters, more preferably 50 liters, per tree (oak or cork oak) was needed, since with this volume the entire foliage of an adult holm oak was perfectly covered.

The minimum inhibitory concentration of the bioactive compounds analyzed was found in a range of 0.5 to 200 pg/ml, more preferably in a range of 40-50 pg/ml, therefore making an estimate of the amount of micrograms of compound that was needed to treat 10,000 m2 of pasture with a density of 40 trees/ha, it was obtained that 50 liters x 40 = 2,000,000 mi and x 50 pg = 100,000,000 pg of biocomposite per 10,000 m2.

B) In the case of the formulation in solid form, it was obtained that with a dose between 30 and 90 kg per tree the projection of the crown was covered, and more preferably with a dose between 50-80 kg per tree, in this way the treatment could reach the root system of the tree and act against P. cinnamomi. As an example, if 80 kg were applied, it resulted in 80,000 g/tree x 40 trees/10,000 m2, requiring 3,200,000 g per 10,000 m2 and with a preferable minimum inhibitory concentration of 50 pg/ml, obtaining a total of 160,000,000 pg of the biocomposite that was used as a bioactive agent per 10,000 m2.

Thus, it was verified that the MIC of Compounds 1 and 2 against this pathogenic fungus varied in a range between 0.355 pg/ml of culture medium and 2.84 pg/mL.

Thus, 60.54% inhibition in the growth of P. cinnamomi was obtained. Complete inhibition would occur at 0.71 pg compound/ml medium.

As a result of these tests, the following was concluded: the main advantages of the composition were to eliminate the infection by the pathogenic fungus Phytopthora cinamomi and to save holm and cork oaks. In addition, as it is a 100% natural product, with extracts of native plants from the pasture where it was applied, there were no problems of any kind, being a 100% ecological solution.

Claims (6)

1. A bioactive compound, belonging to the triterpenoid family, which is a pentacyclic triterpene with a molecular weight of 474.3345 Da and formula:

that contains five non-aromatic rings, four of them formed by 6 carbon atoms and only one of them connected, at one of its ends (carbon 17), through a spiroatom (C) to a fifth ring formed by 5 carbon atoms. carbon; where: - the first ring, called ring A, contains 2 methyl groups on carbon 4 (carbons 23 and 24), which are both hydroxylated; - in ring A, in addition, carbons 2 and 3 are hydroxylated; - carbons 8 (carbon 26), 10 (carbon 25) and 14 (carbon 27) of rings B and C are methylated; - in ring C there is a double bond between carbons 12 and 13; - in ring E, carbon 20 has two methyl groups (carbons 28 and 29); and - in ring D, carbon 18 has a keto group (C=O).2 2. A method for obtaining the bioactive compound defined in claim 1 from an extract of the P. purpurea plant, characterized in that it comprises: - preparing a suspension of the extract obtained from vegetable matter of the P. purpurea plant in a solvent selected from the group consisting of ethanol, methanol and ethyl acetate, in a ratio by weight of solvent and extract of 1:10; - subjecting the previous suspension to solid phase extraction and subsequent elution with an increasing gradient of methanol in water from 20% to 100% methanol, until obtaining individual fractions in which the solvent is eliminated used; - drying the fractions of the previous stage and resuspending again in an organic solvent; and - subjecting the resuspensions to high performance liquid chromatography using C18 stationary phase columns, with a mobile phase gradient made up of one of the following solvent combinations: methanol with water or acetonitrile with water and 0.1% formic acid; until purifying the bioactive compound with a molecular weight of 474.3345 Da. 3. The method according to claim 2, wherein the solid phase extraction is carried out by fractionation on solid phase columns with C18 stationary phase matrix. 4. The method according to any one of claims 2 or 3, wherein the starting extract of the P. purpurea plant is a liquid extract obtained from aerial or root vegetable matter of the plant. 5. The method according to claim 4, wherein the extract comes from the roots of the plant. 6. The method according to any one of claims 2 to 5, wherein the starting extract is obtained by means of a process comprising: - dry the vegetable matter of the plant and macerate with a solvent selected from the group consisting of ethanol, methanol and ethyl acetate, in a proportion of 10 ml of solvent per 1 gram of vegetable matter; - subjecting the mixture of vegetable matter and solvent to stirring, for its homogenization, and then subjecting it to centrifugation, at a speed between 5,000 and 10,000 rpm for a time between 5 and 10 min; - after centrifugation, separate the supernatant and dry until a solid product is obtained; and - resuspend the solid product in a solvent selected from the group consisting of ethanol, methanol and ethyl acetate, obtaining the liquid extract of P. purpurea.