WO2015079080A1

WO2015079080A1 - Method for stabilising lupinus spp seed processing water, extraction of alkaloids and use thereof as an environmentally-friendly biocide

Info

Publication number: WO2015079080A1
Application number: PCT/ES2014/000197
Authority: WO
Inventors: José MarÍa FERNÁNDEZ BOLANOS; Inés MAYA CASTILLA; Sergio MARTOS DELGADO; Rosa PÉREZ OTERO
Original assignee: Universidad De Sevilla
Priority date: 2013-11-28
Filing date: 2014-11-27
Publication date: 2015-06-04
Also published as: ES2539644A1; ES2539644B2

Abstract

The invention relates to the agro-chemistry and agriculture sectors and to a method for stabilising and preserving Lupinus spp. seed processing water, using sulphites or bisulphites, and to the use thereof as biocide. The invention also relates to the separation of the alkaloid and non-alkaloid fractions from said water using macroporous diatomaceous earth and to the use thereof as an environmentally-friendly biocide.

Description

TITLE

Procedure of stabilization of the waters of processing of seed of Lupinus spp, extraction of alkaloids and its use as ecological biocide.

OBJECT OF THE INVENTION

The present invention, as expressed in the statement of this specification, is intended to provide solutions with respect to the treatment of the processing waters of the seeds that act as biocides, since due to the large amount of organic matter they contain they are susceptible to suffer gradual blackening, decomposition and rot. Thus, in this patent we describe the stabilization of these process waters and their use as an ecological biopesticide.

The technique object of the present patent falls within the area of agrochemistry and agriculture. The Activity Sector in which the invention would be applied corresponds to that of plant protection products isolated from plants, specifically from different species of Lupinus.

BACKGROUND IN THE STATE OF THE TECHNIQUE

According to the state of the art, there are many scientific publications that describe the presence of quinolizidine type alkaloids in both Lupinus spp. (Resta, D .; Boschin, G .; D'Agostina, A .; Arnoldi, A. Mol. Nutr. Food Res., 2008, 52, 490-495) as in the aerial parts of the plant (Net, AT ; Oliveira, CQ; Ilha, V .; Pedroso, M .; Burrow, RA; Dalcol, II; Morel, AFJ Mol. Struct., 2011, 1004, 174-177; Cook, D .; Lee, ST; Pfister, JA; Stonecipher, CA; Welch, KD; Green, BT; Panter, KE Phytochem. Anal., 2012, 23, 278-284).

The alkaloids, most of them quinolizidine, account for between 0.4% and 5% by weight of the seed (Ganzera, M .; Krüger, A .; Wink, MJ Pharm. Biomed. Anal., 2010, 53, 1231 -1235), and give it a bitter taste. These metabolites are synthesized by Lupinus plants as a defense mechanism against insects and herbivores (Kordan, B .; Dancewicz, K .; Wroblewska, A .; Gabrys, B. Phytochem. Lett, 2012, 5, 71-77), since they have neurotoxic properties. In humans, a high intake is necessary to have symptoms (Litkey, J .; Dailey, MW Am. J. Emerg. Med., 2007, 25, 215-217).

Lupanin is a quinolizidine alkaloid characteristic of the genus Lupinus since it is present in all species, often being the majority alkaloid. (Ganzera, M .; Krüger, A .; Wink, M. J. Pharm. Biomed. Anal., 2010, 53, 1231-1235) It has an anticholinergic effect on the central nervous system, reversibly blocking acetylcholine neurotransmitter receptors. Some of the most common families of insecticides, such as nicotinoids and carbamates, act similarly. An antimicrobial activity of alkaloid-rich extracts has also been described (Villa-Ruano, N .; Pacheco-Hernández, Y .; Rubio-Rosas, E .; Ruiz-González, N .; Cruz-Duran, R. Lozoya-Gloria , E .; Zurita-Vásquez, G .; Franco-Monsreal, J. Arch. Biol. Sci., 2012, 64, 1065-1071; Kügükboyaci, N .; Ózkan, S .; Tosun, F. Rec. Nat. Prod., 2012, 6: 1, 71-74) where, among others, lupanin and 13-hydroxychupanin are present, as well as an antifungal and allelopathic activity (Erdemoglu, N .; Semiha, O .; Fatma, T. Phytochem. Rev. 2007, 6, 197-201).

The desmargamiento broths derived from the industrial preparation of lupine, are abundant in alkaloids and other water-soluble metabolites present in the seed of Lupinus spp., Hence the interest in the use of such broths as an insecticide (Ortiz Falantes, J. 2008, WO 2008003795) and as plant growth promoters (Przybylak, JK; Ciesiolka, D .; Wysocka, W .; García-López, PM; Ruíz-López, MA; Wysocki, W .; Gulewicz, K. Ind. Crops Prod. , 2005, 21, 1-7).

Aqueous seed extracts of Lupinus spp. they also have insecticidal activity (Bermúdez-Torres, K .; Martínez Herrera, J .; Figueroa Brito, R .; Wink, M .; Legal, L. BioControl, 2009, 54, 459-466), nematicide, (Yildiz, S Afr. J. Biotechnol., 201 1, 10, 13252-13255) antifungal and antibacterial (Erdemoglu, N .; Semiha, O .; Fatma, T. Phytochem. Rev. 2007, 6, 197-201). It has been determined (Stobiecki, M .; Blaszczyk, B .; Kowalczyk-Bronisz, SH; Gulewicz, KJ Appl. Toxicol., 1993, 13, 347-52) the toxicity in mice of both aqueous extracts of Lupinus spp. . containing 10% alkaloids (LD50> 4000 mg kg-1 of animal weight), as of different fractions.

Recently antimicrobial activity has been described (Carreira, A .; Monteiro, S .; Ferreira, RB Brit. UK Pat. Appl., 2012, GB 2484509 A 20120418.), antifungal and as a plant growth promoter of an extract of proteins obtained from seeds, cotyledons or seedlings (Ferreira, RB; Monteiro, S .; Teixeira, AR; Loureiro, V. PCT Int. Appl., 2007, WO 2007010459 A2 20070125). This activity has been assigned to the 20 kDa BLAD peptide (Lupinus Alba Doce Band) generated during germination from β-conglutin. (Monteiro, S .; Freitas, R .; Rajasekhar, BT; Teixeira, AR; Ferreira, RB PLoS One, 2010, 5, 1 1)

By way of conclusion, this invention provides with respect to the state of the art, a process of stabilization of the processing waters of the seed of Lupinus spp., Which avoids a progressive degradation with a gradual darkening, appearance of insoluble matter and loss of activity as an insecticide

Likewise, this invention also addresses the separation of the alkaloids present in these broths which implies:

Solid phase extraction of the alkaloids using columns or cartridges filled with macroporous diatomaceous earth, replacing the traditional liquid-liquid extraction procedures.

- A soluble fraction is obtained in aprotic organic solvents (for example dichloromethane) consisting of the alkaloids present in the waters and another non-alkaloid fraction (very poor or lacking alkaloids) that is extracted from the column by treatment with non-polar protic solvents. aqueous (for example ethanol or methanol).

Finally, in this invention the application as a pesticide of both the alkaloid fraction and the non-alkaloid fraction is described.

EXPLANATION OF THE INVENTION

By way of explanation of the invention "Procedure of stabilization of the waters of processing of seed of Lupinus spp, extraction of alkaloids and its use as ecological biocide" it tries to unfold sequentially the following objectives:

- Develop a procedure for the stabilization of dewatering waters derived from the industrial preparation of lupine (Lupinus spp.). - Establish a procedure for solid phase separation of the alkaloids present in the waters.

- Use alkaloid and non-alkaloid extracts as pesticides.

For this, the procedure proposed with the present invention is deployed based on the following steps;

A. The centrifugation of the seed release broths of Lupinus spp. to eliminate insoluble matter if any.

B. The addition to the soluble fraction obtained after centrifugation of any of the following elements:

Sodium benzoate and sodium pyrosulfite E223.

Potassium pyrosulfite E224,

Sodium hydrogen sulphide E222.

Sulfite sodium E221.

C. Conservation at 5 º C and in the dark in order to prolong the activity.

D. Alternative extraction of the alkaloid and non-alkaloid fraction.

Below is a complete tour of the procedure where each of the aforementioned stages are described.

PHASE 1 - Color stabilization against microorganisms

Natural plant extracts often suffer progressive degradation that leads to the modification of their properties and the proliferation of microorganisms. In the case of the broths obtained in the detachment of the lupine seed, there is a gradual darkening that evidences the degradation of the product, in addition to presenting the inconvenience of losing visual attractiveness when marketing the product.

The dewatering broths are subjected to centrifugation to eliminate possible solid matter in suspension. The solid fraction is discarded and the liquid fraction (supernatant) has been stabilized using various combinations of products as preservatives.

The study of the stabilization of the color and appearance of the supernatant has been carried out.

to. We have verified that there is a marked blackening of the sample containing 2 g / L of sodium benzoate after one month at room temperature with both the sample in a closed vial and exposed to air.

b. We have compared the activity of sodium sulphite (E221) and sodium pyrosulfite, also called sodium pyrosulfite (E223) on color stabilization, verifying that pyrosulfite is more effective than sulphite. Thus, for example, the color of the supernatant after two months under indirect light and containing an amount of 0.2 mg / mL of pyrosulfite (test 7, Table 1) is lighter than if we use 2 mg / mL of sulphite (test 5, Table one). Using 2 mg / mL of pyrosulfite, the sample is virtually colorless after 2 months.

The greater activity of the pyrosulfite is due to its ability to give bisulfitic addition products by reaction with the carbonyl group of the reducing sugars. In this way, the initial stages of the Maillard reaction (or non-enzymatic browning) are inhibited, resulting in a complex route of chemical transformations in which sugars and amino acids / proteins are involved and that originates colored products.

We have also studied the influence of other factors such as temperature and sunlight, checking that both negatively affect color conservation. Samples stored at 5 ° C were less colored than those stored at room temperature. Samples exposed to direct sunlight (tests 10-14, Table 1) have a darker color than those exposed to indirect light. Observing how the intensity of color decreases with increasing concentration of pyrosulfite.

Regarding the preservation of the broth against microorganisms, we have verified that in lupine release broths without additives, putrefaction occurs in less than a week with proliferation of microorganisms and the appearance of an unpleasant smell.

We have found that both benzoate and sodium pyrosulfite preserve the rot sample for at least one week at room temperature. The combination of both preservatives in concentrations of 1.0 mg / mL (Table 2) preserves the sample for at least one month.

Therefore, the combined addition of sodium pyrosulfite (to stabilize the color and prevent the growth of microorganisms) and sodium benzoate (against microorganisms) turns out to be very effective. For example, at a concentration of 1 g / L of sodium bisulfite and 1 g / L of sodium benzoate, no darkening of the broths or proliferation of microorganisms is observed after one month at room temperature. Other concentrations and even other combinations of stabilizers were also effective for that purpose.

PHASE 2-Solid phase alkaloid extraction

Dewatering broths subjected to centrifugation and discarded the solid fraction, the liquid fraction (supernatant) is basified to pH 12, and returned to centrifuge The supernatant obtained is introduced into a macroporous diatomaceous earth column and after a few minutes for the liquid to be distributed along the column, CH ₂ CI ₂ is passed through the column collecting the organic phase (called alkaloid fraction) , while the aqueous phase remains retained inside the column.

One or more extractions are made with dichloromethane until it is observed that no more matter is recovered. Next, 96% ethanol is passed through the column, recovering the matter retained in the column dissolved in water-alcohol (called a non-alkaloid or water-soluble fraction).

Both fractions are concentrated to dryness under reduced pressure. The alkaloid fraction is obtained in a pale yellow oily form and the non-alkaloid fraction has a brown amorphous solid appearance. Both fractions were subjected to analysis by GC-MS and evaluated as insecticides.

PHASE 3-Analysis of alkaloids

The analyzes of the alkaloid fraction were performed on an Agílent GC 6890N gas chromatograph, equipped with an automatic sampler (Autosampler 7683 Agilent series) and a high resolution capillary column Agilent J&W Scientifics DB-5ms (30m × 0.25mm × 0.25μm ). The gas chromatograph is coupled with a Micromass AutospecQ mass spectrometer, where the ionization source operates in electronic impact mode (El) at 70 eV. Data is acquired from 50 to 650 m / z.

PHASE 4-Activity as an insecticide

The efficacy as an insecticide of the alkaloid and non-alkaloid fractions obtained from the waters of lupinus seed detachment was evaluated. As a model test, these fractions were analyzed as an insecticide in the control of aphids Toxoptera citricidus Kirkaldi, the aphid that transmits the citrus sadness virus (CTV), and other insect species. As examples of the tests carried out, we show the results obtained using different extracts on four species of homoptera: the aphids Toxoptera citricidus and Toxoptera aurantii, the green aphid of the apple tree Aphis pomi and the psyllid of the eucalyptus Ctenarytaina spatulata. The experiments were carried out on a Potter tower with a nozzle with a nozzle of 0.6985 mm at a working pressure of 0.7 kg / cm2. 1 ml of the undiluted insecticides was sprayed on each 9 cm diameter Petri dish containing the insects adhered to their corresponding plant support. Colonies of aphids of the same species and characteristics were used as controls, but were treated with 1 ml of distilled water. The excess treatment was allowed to dry at room temperature, and the colonies were subsequently introduced into plastic containers with a perforated lid to allow aeration and a wet florist sponge at the bottom to prevent drying. The containers were then placed in a culture chamber with controlled conditions of 23 ± 2 ° C temperature, 70 ± 5% relative humidity and 16 hours of photoperiod light. The efficacy of the products was evaluated 24 hours after treatment (24 hdt), and then at 48 and 72 hours, recording the death of the individuals and considering as dead the aphids that showed no motor activity when touched with an entomological needle. .

In the case of Toxoptera citricidus a colony was placed with nymphs and adult apes attached to an Eureka lemon tree outbreak; Each colony consisted of 20 ± 5 individuals and the tests were performed in triplicate. In the case of Toxoptera aurantii in camellia bud, Aphis pomi in apple tree, and Ctenarytaina spatulata in terminal bud of Eucalyptos globulus, 50 nymphs of each species were placed.

The percentages of mortality obtained were corrected using Abbott's formula, which allows the natural mortality effect present in the control treatment to be separated from the observed mortality.

% Efficacy of Abbott = [(Vt -Ve) / Vt] x100 where Vt = live individuals in the control and Ve = live individuals in the products tested. Description of the drawings. -

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical implementation thereof, a set of drawings is attached as an integral part of said description. where, for illustrative and non-limiting purposes, the following has been represented:

Figure 1. Total ion chromatogram of the GC-MS analysis in full scan mode showing the alkaloids present: Spartein (1), Amodendrine (2), Albin (3), Angustifoline (4), a-isolupanin (5), Lupanin (6), N-methylalbine (7), Multiflorine (8), 17-Oxolupanin (9), 13a-Hydroxylpanpanin (10), 13a-Hydroximultiflorine (11), 13a-Tigloyloxylupanin (12).

Figure 2. Structure of the alkaloids detected

Figure 3. Insecticidal activity of the lupinus albus seed cooking broth and the alkaloid and non-alkaloid fractions against aphids Toxoptera citricidus

Figure 4. Insecticidal activity of the cooking broth of the seeds of Lupinus albus and of the alkaloid and non-alkaloid fractions against different species of insects

EXAMPLE OF PREFERRED EMBODIMENT

In a preferred embodiment of the invention "Process of stabilization of the seed processing waters of Lupinus spp, extraction of alkaloids and their use as an ecological biocide", the combined addition of sodium bisulfite (1 g / L) and sodium benzoate ( 1g / L) to the seed release broths proved to be very effective in preventing the darkening of the broth and the proliferation of microorganisms even after two months at room temperature.

To remove the alkaloids from the release waters, 20 mL of broth is centrifuged for 10 minutes at 3,500 rpm. The supernatant is separated and basified with 2M NaOH until pH 12, and centrifuged again for 10 minutes at 3,500 rpm. The supernatant obtained is introduced into an Extrelut® NT 20 column. After at least 15 minutes in the column, 2 x40 mL of CH ₂ CI ₂ is passed through the column collecting the organic phase (alkaloid phase). Once the entire organic phase has left, they are passed through the column 40 ml_ of 96% ethanol whereby the matter retained in the column is recovered as a water-alcohol solution (non-alkaloid or water-soluble fraction).

Both fractions are brought to dryness under reduced pressure. Once concentrated, the alkaloid fraction is obtained in a pale yellow oily form whose average weight is 300 mg. The non-alkaloid fraction has an aspect of brown amorphous solid whose average weight is 3 g. Both fractions were redissolved for analysis by GC-MS and its evaluation as an insecticide.

The analyzes of the alkaloid fraction were performed on an Agilent GC 6890N gas chromatograph, equipped with an automatic sampler (Autosampler 7683 series from Agilent) and a high resolution capillary column Agilent J&W Scientifics DB-5ms (30m × 0.25mm × 0.25μm ). The gas chromatograph is coupled with a Micromass AutospecQ mass spectrometer, where the ionization source operates in electronic impact mode (El) at 70 eV. Data is acquired from 50 to 650 m / z.

The analysis is carried out on 20 mL of the soluble fraction obtained after centrifuging the broth supplied from lot 2B of 24-4-09 containing 4.6 g of dry matter. From these 20 mL, 301 mg (6.5% of the dry matter) is extracted with an alkaloid mixture that was analyzed by gas chromatography-mass spectrometry (GC-MS), detecting and identifying the alkaloids detailed in the Table 3 and whose structures are represented in Figure 2. In addition, the retention time, molecular peak and other characteristic ions whose relative intensity appears in parentheses are described in the table.

Note the abundant presence of lupanin (67%) in addition to other quinolizidine alkaloids as shown in Table 3. The chromatogram of the alkaloid fraction is shown in Fig 1 and the structure of the alkaloids detected in the mixture is shown in the Fig. 2. The presence of lupanin as a major component of the alkaloid mixture is confirmed by the ¹³ C-NMR and ¹ H-NMR spectra.

The efficacy as an insecticide of the alkaloid fractions at pH 5 and pH 12 and the non-alkaloids obtained from the waters of unclogging the lupinus seed against aphids of Toxoptera citricidus Kirkaldi were evaluated.

The insecticidal activity is shown in Table 4 and in the corresponding bar chart (Figure 3), where the average Abbott efficacy values are collected for the tests carried out with the dewatering broth (entry 1), alkaloid fraction after neutralizing with acetic acid up to pH 5 (entry 2), the alkaloid fraction without neutralizing with acetic acid (pH 12, entry 3), the non-alkaloid fraction (entry 4), and the control (water-treated control, entry 5), at 24 and 48 hours after the intervention. The results were not collected at 72 hours because almost all aphids had died within the previous interval. Nor is any differentiation established between nymph and adult stages of the aphid because no variation was observed in the effect obtained by the preparations.

These results demonstrate the excellent behavior as bioinsecticide of both the alkaloid and non-alkaloid fractions (Figures 3 and 4). The maximum efficiency corresponds to the alkaloid fraction at pH 5, followed by the alkaloid fraction at pH 12 and unfractionated unwanted waters. The non-alkaloid fraction also shows remarkable efficacy, since at 72 h all insects have died, acting by a mechanism of action different from that caused by the alkaloids.

The results of the extracts on the aphid Toxoptera aurantii in camellia bud are shown in Table 5, on the aphid Aphis pomi in apple tree in Table 6, and on the psyllid Ctenarytaina spatulata in terminal bud of Eucalyptos globulus are shown in the Table 7.

Claims

1. Procedure of stabilization of the waters of seed processing of Lupinus spp and extraction of alkaloids characterized by being carried out based on a procedure that addresses the following stages:

Sodium benzoate and sodium pyrosulfite E223.

Potassium pyrosulfite E224,

Sodium hydrogen sulphide E222.

Sulfite sodium E221.

C. Conservation at 5 º C and in the dark in order to prolong the activity.

D. Alternative extraction of the alkaloid and non-alkaloid fraction.

2. Use as an ecological biocide, both insecticide and fungicide, of the aqueous extracts obtained by the method described in claim 1 without undertaking the extraction of the alkaloid and non-alkaloid fraction.

3. Procedure for stabilizing the water for processing seed of Lupinus spp and extracting alkaloids according to claim 1, characterized in that in step D, the procedure for extracting the alkaloids contained in the aqueous extracts obtained in the treatment (soaking, cooking or washing) of the seed of Lupinus sp, is carried out according to the following process:

A. The use of a matrix of macroporous diatomaceous earth as a support for the liquid-liquid extraction of alkaloids contained in aqueous extracts of Lupinus sp.

B. Elution of the alkaloids with an organic solvent immiscible with water.

C. The subsequent addition to the matrix of a polar solvent (ethanol, water) for the recovery of an alkaloid free fraction.

4. Use as an ecological biocide, both insecticide and fungicide, of the alkaloid fraction obtained by the procedure described in claims 1 and 3.

5. Use as an ecological biocide, both insecticide and fungicide, of the non-alkaloid fraction obtained by the procedure described in claims 1 and 3.