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    公开号:ES2562637T9
    申请号:ES10755518.7T
    申请日:2010-03-18
    公开日:2017-05-30
    发明作者:Marcelo Santiago LAGOS GONZÁLEZ
    申请人:Proteus SA；
    IPC主号:
    专利说明:

    Method for industrial purification of biologically active ficotoxins
    Field of the Invention
    This invention relates to the industrial production, in large quantities, under controlled conditions and in continuous form of the paralytic phycoxins neosaxitoxin, saxitoxin and gonyaulatoxins (gonyaulatoxin 2 and gonyaulatoxin 3) from cyanobacteria producing paralytic ficotoxins, and in particular with the purification of said ficotoxins. With the central objective of obtaining a substantially pure compound that maintains its potent biological activity until the final product, where it is used as a raw material for the development of new medicines.
    These ficotoxins have application in cosmetic or pharmaceutical products, for example in cosmetic products to fight wrinkles and expression lines, or in pharmaceutical products of clinical application such as local anesthetics, medications for the control of pathologies associated with muscular hyperactivity, for the control of pain at the local and peripheral level, that is, products to improve the quality of life.
    Description of the prior art
    The phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins are active compounds produced by harmful algal blooms, of the genera Alexandrium sp., Piridinium sp., And Gimnodinium sp., (Lagos, N. (1998) Microalgal blooms: A global issue with negative impact in Chile, Biol. Res. 31: 375-386). In the last 15 years, it has been shown that in addition to being produced by marine dinoflagellates, these phycotoxins can also be produced by freshwater cyanobacteria, such as photosynthetic green-bluish algae.
    So far in the literature only 4 genera of cyanobacteria producing paralytic ficotoxins have been identified, and each produces a different mixture of ficotoxins, both in quantity and in types of ficotoxins produced, that is, they produce different profiles of paralytic ficotoxins (Lagos, N., Onodera, H., Zagatto, PA, Andrinolo, D., Azevedo, SMFQ, and Oshima, Y., 1999, The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. TOXICON, 37 : 1359-1373 Pereira, P., Onodera, H., Andrinolo, D., Franca, S., Araujo, F., Lagos, N., and Oshima, Y., 2000, Paralytic shellfish toxins in the freshwater cyanobacteria Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal TOXICON, 38: 1689-1702.
    The active substance in these paralytic ficotoxins acts as a specific blocker of the voltage-dependent sodium channels found in excitable cells (Kao, CY, 1966, Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomenon. Pharm. Rev 18: 997-1049). Due to the inhibition of sodium channels, nerve impulse transmission is blocked and thus the release of neurotransmitters at the level of the neuromotor plaque is prevented, which then prevents muscle contraction. Due to these physiological effects, these compounds are potentially useful in pharmacology, when used as inhibitors of muscle activity in pathologies associated with muscular hyperactivity, such as muscle spasms and focal dystonia, when applied in injectable form and locally. Additionally, because a blockade is generated at the level of nerve impulse transmission, when these compounds are applied as a local infiltration, they not only block the efferent neurotransmission pathways, but also block the afferent pathways and thus also produce inhibition of the sensory pathways, generating an anesthetic effect, both effects being inseparable when injected locally. This is a surprising effect, since both effects occur simultaneously (US Patent 4,001,413).
    The ficotoxins neosaxitoxina, saxitoxina and gonyaulatoxinas, at the moment are not commercially available like a massive product, in spite of his big potential applications with therapeutic and cosmetic aims. It is evident that an industrial production process of these compounds is required, to meet their growing demand, related to their large number of recently developed clinical and cosmetic uses and applications.
    Lubna Zaman describes the isolation of several phycotoxins including saxitoxin and gonyautoxin, conserving carbon compounds with the following elution and HPLC realization (Lubna Zaman, Osamu Arakawa, Ako Shimosu and Yoshio Onoue, 1997, Occurrence of paralytic shellfish poison in Bangladeshi freshwater puffers TOXICON, 35, 3, 423-431). However, the process includes the extraction in ethanol and HCl medium at pH 2. The phycotoxins lose their activity at low pH, the HCl medium being additionally difficult to remove from the purified product.
    US 2008/006579 A1 relates to a general method for concentrating biotransformed products catalyzed by cells in diatomaceous earth, in which the desired products are in the medium outside of


    said cells. Again, at the pH values <4 described, the phycotoxins are not active, while it is reported that higher pH values give an insufficient yield in the method of publication. The present invention, on the other hand, smooths the cell in order to obtain intracellular ficotoxins, and thus maintaining the biological activity of said ficotoxins.
    Ghazzarossian describes a method for the isolation of saxitoxin using Celite but also acidifying with HCl up to pH 2-3 (Ghazzarossian VE, Schantz EJ, Schnoes HK and Strong FM, 1974, Identification of a poison in toxic scallops from a Gonyaulax Tamarensis red tide. Biochm. And Biophys. Research Comunic. Academic Press Inc., 59, 4, 1219-1225).
    It is noteworthy that both Lubna Zaman and Ghazzarossian purify ficotoxins from infected fish tissue as a source, which determines the extraction procedure.
    The invention presented here corresponds to the extraction, fractionation and purification of the paralytic ficotoxins neosaxitoxin, saxitoxin and gonyaulatoxins, starting from an innovative procedure of continuous cell cultures, under controlled conditions and in large quantities from cyanobacterial strains (blue-green algae ). The central objective is the maintenance of the potent biological activity of this active principle throughout the industrial purification process.
    The cyanobacteria producing paralytic phycotoxins belong to the genera: Cylindrospermopsis sp, Mycrocistis sp, Anabaena sp, Gomphosphaeria sp, Oscillatoria sp, Aphanizomenon sp (Aphanizomenon issatchenkoi, Aphanizomenon flos-aquae, Aphanizomenon Lyibile and others.) None of these cyanobacteria has been used, so far, in the production of phycotoxins at industrial levels.
    The industrial production of phycotoxins from cyanobacteria must solve two technical problems, which although interrelated are different from each other. First, a large amount of phycotoxin-producing biomass must be generated. This technical problem is solved in the invention protected in patent application CL 722-2009, also filed by the authors of the present invention. Secondly, once the biomass and phycotoxins have been produced, these compounds must be purified under certain conditions suitable for maintaining the biological activity of the active compounds, preserving their potent biological activity and producing massive yields. This second technical problem is the one that solves the present invention.
    The proposed method is the purification of ficotoxins from a clonal culture of cyanobacteria, which produces a simple profile of paralyzing ficotoxins (comprising one or two ficotoxins or having a profile with a ficotoxin that represents more than 75% of the total composition of the profile), for example, a strain that only produces neosaxitoxin and saxitoxin or only 2/3 gonyaulatoxins, as the main components. Having yields greater than 75% of an active pharmacological principle is also a surprising effect, which is achieved in the present industrial procedure.
    The chemical structure of these ficotoxins has a general structure (I), and their particular structure is defined by the substituents R1 to R5, as indicated in the table:
    Compound R1R2R3R4R5
    Saxitoxin HHHCOONH2OH
    Neosaxitoxin OHHHCOONH2OH
    Gonyaulatoxin 1 OHHOSO3COONH2OH
    Gonyaulatoxin 2 HHOSO3COONH2OH
    Gonyaulatoxin 3 OHOSO3HCOONH2OH

    Compound R1R2R3R4R5
    Gonyaulatoxin 4 HOSO3HCOONH2OH
    Gonyaulatoxin 5 HHHCOONHSO3OH
    The revelation that these alkaloids are produced by cyanobacteria is very recent. Only in the last 15 years have they been shown to correspond to secondary metabolites found within the cells, which under certain conditions can be released into the culture medium. Therefore, in a continuous culture of cyanobacteria, two sources of these compounds are generated, the first in the cell pellet and the second in the culture medium where the cyanobacteria are grown.
    For the industrial production of these ficotoxins and to be able to purify them, it is essential to have a massive culture of these cyanobacteria, as indicated in patent application CL 722-2009. Prior to the procedure described in patent application CL 722-2009, the development of high volume industrial processes for obtaining phycotoxins had not been achieved. However, the ficotoxins were purified in basic research laboratories from shellfish contaminated with red tide, with the aim of obtaining some micrograms of these toxins as analytical standards (standards to be used as reference compounds in chemical analysis).
    To date, there are no publications describing purification procedures for these phycotoxins from contaminated shellfish, nor from dinoflagellates at industrial levels; In this case, the industrial levels are considered to be of an order of magnitude of grams for the production of the metabolite or phycotoxin. Nothing has been published about the isolation of ficotoxins from cyanobacteria, which is not surprising when it is considered that the cyanobacteria producing these ficotoxins have only recently been described in the literature (Lagos, 2003).
    In this patent application an industrial purification and production process of neosaxitoxin, saxitoxin and gonyaulatoxins 2/3 is presented, using an innovative biotechnological procedure from isolated, cloned cyanobacteria that optimizes the continuous industrial production of these ficotoxins under controlled conditions, to while maintaining their powerful biological activity throughout the industrial process to produce an industrial active ingredient useful for the development of new drugs.
    The integration of the production proposal with the technologies used in this invention considers the adjustment to the productive scale of cyanobacteria described above to produce the quantity necessary for massive pharmacological uses and the development of new drugs or active ingredients for cosmetic applications.
    The advantages of the present invention to massively produce these phycotoxins under controlled conditions from cyanobacteria are:
    • The availability of clones of selected cyanobacterial strains and producers of ficotoxins with a unique composition and simple toxin profiles.
    • Easy purification procedure of high performance of the phycotoxins.
    • Cost - production - very favorable performance ratio not achieved so far.
    Field of application for mass production of phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins
    The scope of these alkaloids includes their clinical applications as therapeutic agents in pathologies associated with muscular hyperactivity, such as muscle spasms and focal dystonia. Additionally, they can be used as local anesthetics in different broad-spectrum pharmaceutical preparations, which allows the classification of these ficotoxins as very useful products in the treatment of various pathologies, many of them of great commercial demand. On the other hand, they can also be applied in cosmetic products to combat wrinkles and expression lines, applications of obvious commercial demand. However, these phycotoxins are not commercially available in industrial quantities at the levels currently required. Therefore the present method of purification, fractionation and production of neosaxitoxin, saxitoxin and gonyaulatoxins, solves a biotechnological problem that remained without solution until now. The innovation proposed in this document, allows to meet the great demand in the national and international markets of the active principle of these pure phycotoxins, on a large scale for therapeutic and cosmetic uses and other products that improve the quality of life of people.
    These ficotoxins have very favorable physicochemical properties for applications in the field of medicine and the cosmetic industry. Their physical and chemical properties are as follows: they are soluble in water, stable at room temperature, highly resistant to acidic media and to extreme temperatures (above


    100 ° C), very low molecular weight (298-445 g / mol), which allows easier, less dangerous and painless applications, without allergic or immune responses, unlike other compounds, such as high molecular weight protein Botulinum toxin (Botox®).
    Due to these physical and chemical properties of phycotoxins, such as their low molecular weight and high chemical stability, it is possible to devise biotechnological applications and developments for many pharmaceutical products and preparations, such as the use in creams, gels, transdermal applications with apparatus for ultrasound, infrared and others, controlled release patches (patches attached to the skin for slow and continuous application). All these applications are possible due to the high stability of these compounds and the additional possibility of allowing chemical reactions with covalent bonds to other chemical compounds.
    These advantages position the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins as superior alternatives to botulinum toxin, a compound widely used in dermocosmetic applications, which is a protease that has the disadvantages of being unstable, high molecular weight, generates allergic immune response, produces structural damage (it digests nerve endings and surrounding endings, producing unwanted adverse effects and currently highly questioned worldwide) and digests itself in short periods of time.
    Recently published clinical applications for the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins, are opening markets for mass demand applications: in the cosmetic field, for backaches, migraines, anal fissures, pain control in laparoscopic surgery and neuropathic pain control in In general, with millions of potential patients, raising an incalculable future demand for these compounds.
    Table 1 shows the advantages of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins when compared, for example, with botulinum toxin (Botox®), which produces a similar physiological response and therefore has similar applications to those of ficotoxins in therapy clinical and cosmetic, although the molecular mechanisms of action of ficotoxins and botulinum toxin are very different.
    TABLE 1: Advantages of phycotoxins compared to botulinum toxin
    Properties Botox®Phycotoxins
    Active toxin Botulinum toxinneoSTX *; STX *; GTX *
    Molecular weight 900,000Oscillating between 298 and 450
    Chemical stability UnstableVery stable
    Storage FrozenRoom temperature
    Mechanism of action ACH release inhibition *Nerve impulse inhibitor, inhibitor of the release of any neurotransmitter
    Activation time 5-15 daysImmediate (minutes)
    Duration 2 to 4 monthsDose dependent
    Topical application and other modes of application It's not possibleIn cream, in gels, in patches, iontophoretically, ultrasound.
    neoSTX: STX neosaxitoxin: GTX saxitoxin: ACH gonyaulatoxin: acetylcholine
    The relative ease in switching users of Botox® to the use of potential products derived from ficotoxin (including neosaxitoxin, saxitoxin and gonyaulatoxins) due to painless, lower-cost and instant-effect applications, generates competitive advantages of Botox ficotoxins ®.
    Objects of the invention
    The general object of the invention is the extraction, purification and industrial production of biologically active ficotoxins, compounds and secondary metabolites produced by cyanobacteria that have high added and commercial value and are not commercially available at industrial production levels. The surprising thing about this invention is its ability to obtain these biologically active compounds throughout the industrial process, being a unique and surprising biotechnological process.
    A specific object of the invention is a process of purification, massive, industrial and high-yield production of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins in biologically active form, from practically unlimited levels of cyanobacteria produced in continuous cultures and under controlled conditions .


    Another specific object of the invention is to achieve a process of purification of phycotoxins from wet and / or frozen cyanobacterial sediments, eliminating the pigments and other major secondary metabolites that accompany the phycotoxins during the purification process.
    Description of the drawings
    Figure 1 shows a chromatogram corresponding to 10 microliters of Aphanizomenon gracile cyanobacteria extract, injected into an analytical high performance liquid chromatography (HPLC) equipment. The detection method used was online fluorescent detection. Peak 1, Rt = 2,640 minutes, cyanobacterial pigments. Peak 2, Rt = 7,980 minutes, neosaxitoxin. Peak 3, Rt = 11,970 minutes, saxitoxin.
    Figure 2 is a chromatogram corresponding to 10 microliters of purified Aphanizomenon gracile extract purified by preparative high performance liquid chromatography of molecular exclusion (molecular size separation). The detection method used was online fluorescent detection. Peak 1, Rt = 3,067 minutes, cyanobacterial pigments. Peak 2, Rt = 9.373 minutes, neosaxitoxin. Peak 3, Rt = 12,953 minutes, saxitoxin.
    Figure 3 shows a chromatogram of an analytical pattern that includes as main components neosaxitoxin and saxitoxin, with a small amount of a mixture of gonyaulatoxins (NL2 standard), determined and quantified by analytical high resolution liquid chromatography with fluorescent online detection. First peak Rt = 3,653 minutes, mixture of gonyaulatoxins; second peak 2, Rt = 5,040 minutes, neosaxitoxin; third peak, Rt = 7,440 minutes, saxitoxin.
    Figure 4 is a chromatogram of a sample of purified neosaxitoxin from extracts of Aphanizomenon gracile cyanobacteria (Rt = 4,933 minutes). This corresponds to the fraction eluted from preparative high resolution liquid chromatography using ion exchange columns with online fluorescent detection.
    Figure 5 shows a chromatogram of analytical patterns of gonyaulatoxins. From left to right: Peak 1: GTX4 (gonyaulatoxin 4); Peak 2 GTX1 (Gonyaulatoxin 1); Peak 3 GTX5 (Gonyaulatoxin 5); Peak 4 GTX3 (Gonyaulatoxin 3); Peak 5 GTX2 (Gonyaulatoxin 2).
    Figure 6 shows a chromatogram of a sample of cyanobacterial extract C. raciborskii. Peak 1, Rt = 8,873 minutes, Peak 2, Rt = 10,927 minutes, Peak 3, Rt = 13,260 minutes; Peak 4, Rt = 16,647.
    Figure 7 is a chromatogram of a fraction of C. raciborskii partially purified by preparative high performance liquid chromatography of molecular exclusion with dominant presence of the GTX3 and GTX2 epimers, respectively.
    Figure 8 shows a chromatogram of the pure final fraction of the GTX3 and GTX2 epimers obtained from cyanobacteria in continuous culture of C. raciborkii.
    Figure 9 shows an elution graph of a preparative molecular exclusion chromatography showing the fractions obtained and where the range of fractions where the different compounds present (organic compounds, phycotoxins and salts) are identified.
    Figure 10 shows an elution graph of an anion exchange preparative chromatography showing the fractions obtained and where the range of fractions where the different compounds present (phycotoxins and salts) are identified.
    Figure 11 shows an elution graph of a preparative cation exchange chromatography showing the fractions obtained and where the range of fractions where the different compounds present (phycotoxins and salts) are identified.
    Figure 12 shows an elution graph of a preparative molecular exclusion chromatography showing the fractions obtained and where the range of fractions where the different purified ficotoxins (saxitoxin and neosaxitoxin) appear.
    Brief Description of the Invention
    This document describes the industrial purification and purification of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins in a biologically active form, from cultures of cyanobacteria producing these ficotoxins.
    The cyanobacteria used in the present invention belong to the genera: Cylindrospermopsis sp, Mycrocistis


    sp, Anabaena sp, Gomphosphaeria sp, Oscillatoria sp, Aphanizomenon sp (Aphanizomenon issatchenkoi, Aphanizomenon flos-aquae, Aphanizomenon gracile).
    The cyanobacteria are preferably obtained from a continuous, massive, semi-automatic culture under controlled conditions such as that described in the Chilean patent application CL 722-2009, filed simultaneously with the present application by the same authors.
    The industrial purification of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins from cyanobacteria in a continuous culture, is carried out using a method that includes extraction, fractionation, partition with solvents, participation in solid phases and partition in liquid-solid contact surfaces of these phycotoxins (neosaxitoxin, saxitoxin and gonyaulatoxins) from sediments of culture cells or culture supernatant. It is a continuous, sequential and semi-automatic procedure, which generates industrial quantities of an active ingredient that maintains its biological activity during the entire purification procedure, until it reaches its final product. The process involves chemical and biochemical procedures at various stages of fractionation using differential centrifugation, extraction with aqueous and organic solvents, phase partition of hydrophobic solvents, partition in solid phases, column purification and various types of preparative high resolution chromatographs (cation exchange , anionic exchange and molecular exclusion), until partially purified extracts are obtained, without pigments, corresponding to fractions with simple phycotoxin profiles and pure ficotoxins from these fractions (Figures 1 to 8). All these stages are part of a single biotechnological procedure and part of an industrial procedure only developed in Chile.
    Detailed description of the invention
    The invention relates to a purification method for the industrial production of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins, maintaining their biological activity throughout the industrial purification process, from cyanobacteria producing these ficotoxins.
    The cyanobacteria that produce these ficotoxins belong to the genera: Cylindrospermopsis sp., Mycrocistis sp., Anabaena sp., Gomphosphaeria sp., Oscillatoria sp., Aphanizomenon spp. (Aphanizomenon issatchenkoi, Aphanizomenon flos-aquae, Aphanizomenon gracile).
    The starting material is a unialgal culture of a species of cyanobacteria; What is a unialgal culture is essential for obtaining these purified ficotoxins with high yields. This is part of the process of growth and scale adjustment to an industrial level, to obtain a high amount of phycotoxins in the starting material.
    Cyanobacteria are obtained by the development of a continuous, massive, semi-automatic method and procedure of cultivation under controlled conditions that sustains a permanent logarithmic growth of cyanobacteria, described in the Chilean patent application CL 722-2009, filed simultaneously with the present application by The same authors.
    During the culture process of these cyanobacterial species, the ficotoxins accumulate inside the cells, but are also released into the culture medium, thus having two sources of ficotoxins.
    For example, in the culture of Aphanizomenon gracile, the main component released from cyanobacterial cells is neosaxitoxin (Figures 2 and 4).
    Therefore, there are two possible sources of obtaining phycotoxins from an industrial culture of cyanobacteria:
    - from the filtered supernatants (without cells or filaments) of the cyanobacterial growth medium;
    -from the wet sediments obtained by centrifugation of the developing crop, which is composedby cyanobacterial cells and filaments.
    The production of the target phycotoxins, which in each case depends on the producing cyanobacterium grown in each case, can be obtained for any of the aforementioned cyanobacteria, both from the supernatants and from the sediments obtained.
    The industrial production of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins, from cyanobacteria in continuous culture is achieved using a method that includes extraction, fractionation, partition with solvents, partition in solid phases and partition in liquid-solid phases of these ficotoxins (neosaxitoxin , saxitoxin and gonyaulatoxins) from culture cells (sediment). The procedure involves chemical and biochemical procedures at various stages of fractionation using differential centrifugation, solvent extraction


    aqueous and organic, phase partition of hydrophobic solvents, solid phase partition, column purification and various types of preparative high resolution chromatographs (cation exchange, exchange and molecular exclusion), until partially purified extracts are obtained, without pigments corresponding to fractions with Simple phycotoxin profiles and pure phycotoxins from these fractions (Figures 1 to 8). The fundamental approach is aimed at obtaining a stable active ingredient that maintains the full potential of its biological activity, so that it can be used as the basis for the development of new pharmaceutical products.
    In a simplified form, the method of purification of the neosaxitoxin, saxitoxin and gonyaulatoxin of the invention comprises the following steps:
    to. provide a source of phycotoxins, specifically a culture of cyanobacterial clones, which is separated in a culture medium and a wet sediment of cyanobacterial cells or a frozen sediment of cyanobacterial cells;
    b. lyse said sediment using homogenization, solvent extraction, ball mill, freeze / thaw cycles, ultrasonication or enzymatic lysis;
    C. subject the material obtained to cold extraction and organic / aqueous phase separation, specifically 50% by volume of a mixture of chloroform: methanol 1: 1 and 50% by volume of 1 mM acetic acid, at pH between 4 and 5, and subsequently separating the aqueous and organic phases using chloroform: methanol 1: 1 by volume, which is repeated between 1 and 5 times;
    d. obtaining a concentrate of the aqueous phase obtained in step (c);
    and. centrifuge the concentrated aqueous phase to obtain a supernatant;
    F. pass the supernatant through a solid matrix of diatomaceous earth, where the phycotoxins are retained, and wash with a wash solution; then obtain a ficotoxin eluate with an elution solution;
    g. pass the eluate containing the phycotoxins through an activated carbon matrix, where the ficotoxins are retained again, and then wash with distilled water to remove the retained pigments and impurities; the phycotoxins must be eluted therefrom with an elution solution;
    h. pass the eluate from the previous stage containing the phycotoxins again through a solid matrix of diatomaceous earth, wash and elute once more with an extraction solution;
    i. evaporate the organic components of the aqueous eluate from the previous step, to obtain a partially purified extract of phycotoxins;
    j. subject the partially purified phycotoxin extract obtained in the previous stage to a biochemical process of separation and fractionation by preparative HPLC (HPLC-Prep: preparative high resolution liquid chromatography) in several stages, according to the physical or chemical fractionation principle used, which It can include sequentially: molecular exclusion, anion exchange, cation exchange and again molecular exclusion.
    In this way pure phycotoxin preparations are obtained, suitable for pharmaceutical and cosmetic applications.
    The method of the present invention provides for the first time in the state of the art, a method for purifying ficotoxins, such as neosaxitoxin, saxitoxin and gonyaulatoxins, from cyanobacteria producing these ficotoxins.
    This method comprises providing an adequate amount of a source of phycotoxins, such as a culture of cyanobacteria, the culture medium where the cyanobacteria grow in said culture or a sediment of cyanobacteria.
    If a cyanobacterial culture is used, the cells are separated from the culture medium, for example using a centrifugation step. An aqueous liquid phase corresponding to the culture medium and a wet sediment corresponding to the cyanobacteria are thus obtained. The cyanobacterial sediments can be frozen or processed directly, since the freezing stage does not affect the subsequent purification of the phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins.


    The sediments obtained are lysed using appropriate methods known in the state of the art, such as homogenization, solvent extraction (organic phase / aqueous phase), ball mill, freeze / thaw cycles, ultrasonication, enzymatic lysis, and the like.
    The material from the cyanobacterial lysis is subjected to a cold extraction using a mixture of organic solvents in aqueous phase (chloroform: methanol 1: 1 with 10 mM acetic acid comprising 50% by volume of the above mixture), and subsequently, the organic and aqueous phases are separated using an organic phase at pH 5 comprising chloroform: methanol 1: 1 by volume, which is repeated between 1 and 5 times. All the aqueous phases obtained are collected to form a single aqueous phase with which the purification process is continued.
    The aqueous phase obtained in the previous stage or the culture medium supernatant is concentrated using, for example, a rotary evaporator at room temperature, until a concentration of between 5 and 20 times the original volume is reached. The concentrate is subjected to centrifugation at a relative centrifugal force ranging from 15,000 x g to 25,000 x g during periods ranging from 10 to 40 minutes. The centrifuge supernatant is passed through a column of diatomaceous earth, washing the column with 5 to 15 times its volume of an appropriate solution, such as 50 mM acetic acid. The phycotoxins are eluted with an appropriate solution, such as an alcohol extraction mixture, ethanol: water: 5 mM acetic acid in a ratio of 2: 1: 1 (vol / vol / vol). The eluate from the previous stage is passed through activated carbon columns, which are then washed with distilled water to remove retained pigments and impurities. In the column, the phycotoxins are retained and eluted with an appropriate solution, such as a mixture of alcohol elution, ethanol: water: 1 mM acetic acid in a ratio of 3: 2: 1 (vol / vol / vol) at pH 5. The eluate from the previous stage is again passed through a column of diatomaceous earth. The columns are washed with an appropriate solution of 50 mM acetic acid using a volume equivalent to 10 times the volume of the matrix and the phycotoxins are eluted with an appropriate solution, such as by solvent extraction with an appropriate alcoholic solution at pH 5 , for example a mixture of chloroform / methanol / water;
    1: 1: 1 (vol / vol / vol). The eluate from the previous stage is left in an aqueous phase, evaporating the organic solvents, using for example a "Speed Vac" equipment (Savant, NY, USA). A partially purified ficotoxin extract is thus obtained.
    The partially purified extract from the previous stage is subjected to preparative HPLC (preparative high resolution liquid chromatography) in several stages which can include sequentially: molecular exclusion, anion exchange, cation exchange and again molecular exclusion. The realization of several successive chromatographs guarantees an analytical purity suitable for the requirements of the pharmaceutical industry. However, in certain cases, depending on the degree of purity required, the phycotoxins can be purified using only one HPLC-Prep stage.
    It should be understood that the mentioned values of the proportions and ratios of the components of each solution, the concentrations of components or the pH values may vary in a range of ± 5% without altering the result of the invention.
    In more detailed and schematic form, the method of purification of phycotoxins of the invention can be defined in the following steps:
    to. Provide an adequate amount of a source of phycotoxins, such as the cultivation of a pure cyanobacterium, according to the disclosure of the Chilean application “Culture of cyanobacteria able to produce neosaxitoxin and saxitoxin at industrial levels” (CL 722-2009), presented simultaneously with This application by the same authors.
    • Separate the cells and the culture medium by centrifugation. Volumes of 1 L of culture obtained from each reactor are used and centrifuged at 10,000 x g for 25 minutes, to obtain a supernatant and a wet precipitate or sediment of cyanobacteria.
    • Optional stage of freezing the cell pellets. The cell pellets can be frozen to be purified later or the purification can begin immediately.
    b. Line the sediment using a preferred method of sediment lysis and then weigh and add the same volume of the organic solvent mixture in aqueous phase by weight of the sediment (chloroform: methanol; 1: 1
    + 10 mM acetic acid in a 50% mixture with the previous mixture), to destroy the cell wall and cytoplasmic membrane. This is done in a slightly acidic medium (pH 5.0). A slightly acidic medium is an important and unique requirement that allows the active substance to remain active.
    C. Subject the material from the cell lysis to a cold extraction and phase separation (organic / aqueous).


    • Repeat the extraction of the organic phase obtained previously.
    • Combine the two organic phases obtained.
    • Extract the aqueous phase twice with organic phase at pH 5.0. The same volume of chloroform solution is used: methanol; 1: 1 vol / vol.
    5 d. Concentrate the extracted aqueous phase 10 times its volume (one tenth of the initial volume is obtained), using for example a rotary evaporator at room temperature.
    and. Centrifuge the concentrate, preferably at 20,000 x g for 30 minutes.
    F. Treat the aqueous supernatant phase of the centrifugation stage with a solid matrix of diatomaceous earths at a stage that involves passing the supernatant through a column of diatomaceous earth. Be
    10 wash the column, with 50 mM acetic acid with an amount of preferably 10 times the volume of the column and the retained ficotoxin in the column is extracted with an alcohol extraction solution or elution buffer (ethanol: water: 5 mM acetic acid ; 2: 1: 1 vol / vol / vol).
    g. Now pass the column eluate from the previous stage through activated carbon columns, which
    They are then washed with distilled water to remove retained pigments and impurities. In the columns the ficotoxins that are eluted with a mixture of alcohol elution (ethanol: water: 1 mM acetic acid) are retained
    3: 2: 1 vol / vol / vol, pH 5.0 This fractionation eliminates a large fraction of the most abundant low molecular weight pigments and molecules in the lysate of cyanobacteria, such as amino acids, nucleotide bases, peptides, sugars (monosaccharides and disaccharides).
    h. Again, perform a differential elution from solid matrices of diatomaceous earth. Columns are loaded
    20 diatomaceous earth with eluted extract of activated carbon. Again, the phycotoxins are retained in the solid matrix by hydrophobic interaction. These columns are initially washed to elute a large fraction of the low molecular weight components, which are removed. The retained ficotoxins are subsequently eluted from the column by solvent extraction with a mixture of chloroform: methanol: water
    1: 1: 1 vol / vol / vol. The use of diatomaceous earth columns generates a total stratified fractionation. This
    The massive procedure for large quantities does not generate the fractionation achieved with these columns when used in a discontinuous technique. Another surprising effect is represented by the replacement of the discontinuous technique by a column separation, which unexpectedly produces a large initial preliminary purification of the massive industrial material, with the consequent cost savings and a great increase in efficiency.
    i. Leave the eluate of the second column of diatomaceous earth in an aqueous phase, evaporating the solvents
    30 organic, for example using a "Speed Vac" equipment (Savant, NY, USA). A partially purified ficotoxin extract is thus obtained.
    j. Subject this partially purified extract to a preparative HPLC (HPLC-Prep: preparative high resolution liquid chromatography).
    A) Separation by preparative HPLC by size: For this separation, for example, columns of
    35 high pressure stainless steel, filled with fine Bio-Gel P-2 (Bio-Rad) 45-90 micrometers (wet). These columns are 10 centimeters in diameter and 85 centimeters long, and are filled with pressure with dry Bio-Gel P-2 resin and have an exclusion size of 1800 Daltons. This resin separates, in the volume of vacuum (Vo), any molecule of greater size and having a molecular weight greater than 1800 Daltons. In this way they pass in the initial fractions (in front of the execution), all macromolecules and even
    40 small peptides of 10 amino acids, retaining the compounds of lower molecular weight such as phycotoxins (from 298 to 445 Daltons). Using this molecular exclusion resin, 90% of unwanted organic material is removed, including a large number of own pigments that are characteristic of these cyanobacteria. All these compounds and soluble macromolecules leave the column in the first fractions (first 5 liters eluted). The intermediate fractions very close to the final fractions are those that
    45 contain the phycotoxins. In the final fractions, much of the salt present in the extracts is removed. The use of this resin for molecular exclusion is a totally innovative concept, especially in the form used in this case. The fractionation columns are of particular design of this invention not only because of their size but also in the way in which the columns are filled. This purification stage is transcendental, efficient and provides practically a partially purified active ingredient. All the subdivisions of
    50 preparative liquid chromatography used hereinafter, uses this innovative biotechnological method of column filling with a solid matrix at high pressure, which generates virtually unlimited contact surfaces that are totally suitable for industrial active principle purification procedures. Also in these preparative HPLC procedures, the favorable speed of the procedure should be mentioned, a fundamental issue to achieve a pure biologically active compound. Figure 9 shows a graph of


    elution of a preparative chromatography, which exemplifies this stage of molecular exclusion. The fractions containing ficotoxins are concentrated and then subjected to a second separation corresponding to an anion exchange procedure. Molecular exclusion is also useful for eliminating the greatest amount of salts present in these extracts, which were not completely eliminated in the phase fractionations initially performed in this purification procedure. This is important, since these salts could interfere with ion exchange procedures. Therefore, in this separation, not only macromolecules and molecules with a molecular weight above 1800 Daltons are removed, but most of the low molecular weight salt components are also eliminated (sodium 23, chlorine 35, etc.). ).
    B) Anion exchange: Similar columns are mounted to the preparative molecular exclusion columns, but now using an anion exchange resin. In this case, Cellex-D (Bio-Rad) with an exchange capacity of 0.66 milliequivalents per gram is used. In this preparative chromatography, the phycotoxins with a net positive charge (+2 and +1) at neutral pH, elute in the first fractions (initial 2.5 liters), since they are not retained in the column because they have a positive charge. Therefore, these phycotoxins elute in the vacuum volume of the column. In these columns, only those compounds that are negatively charged are retained and eluted at the end of chromatography in fractions that do not contain ficotoxins and are therefore removed. The initial fraction containing the total amount of ficotoxins is concentrated again (industrial lyophilizer) and then applied in preparative cation exchange columns. This fraction contains substantially pure ficotoxins with a net charge +2 (neosaxitoxin and saxitoxin) and +1 (gonyaulatoxins).
    C) Cation exchange: In this preparative chromatography (using columns with the same dimensions as the previous ones, filled with Bio-Rex® 70 from Bio-Rad), the phycotoxins will be retained, which will be separated into two main groups: those that have net charge +2 and those with net charge +1. In the initial fractions, the remaining small fraction of negatively charged salts (measured by electrical conductivity) is eluted, then gonyaulatoxins are eluted, and finally saxitoxin and neosaxitoxin are eluted. These last two separate ficotoxins are separated and a fraction containing a mixture of both with a ratio of 2/3 of neosaxitoxin and 1/3 of saxitoxin. The last fraction that elutes contains substantially pure neosaxitoxin.
    D) Finally, several final concentrated fractions of the cation exchange separation are separated again in the Bio-Gel P-2 molecular exclusion columns (using a procedure identical to that described in A), to remove the remaining salts and obtain saxitoxin pure eluted in distilled water.
    It should be understood that the values mentioned for the proportions and ratios of the components of each solution, the concentrations of components or the pH values may vary in a range of ± 5% without altering the result of the invention.
    The phycotoxins suitable for purification with the method described above are neosaxitoxin, saxitoxin and gonyaulatoxins. Obtaining a particular toxin will depend on the cyanobacterial strain from which purification is performed, because certain strains preferably produce a particular type of ficotoxin. As a criterion of separation and fractionation, the retention times characteristic of the target compound (phycotoxins) are considered, as presented below in the following examples of the present application.
    The purity of each stock solution (batch) can be determined by visible and ultraviolet spectroscopy, and also using mass spectrometry. Final detection and quantification can be performed by spectrofluorometry. This last analytical method for quantitative detection is specific for all ficotoxins, such as neosaxitoxin, saxitoxin and gonyaulatoxins (Lagos, 1998. Microalgal blooms: a global issue with negative impact in Chile. Biol. Res. 31: 375-386).
    Examples
    Example 1: Phycotoxins obtained and purified from Aphanizomenon gracile maintained in continuous cultures under controlled conditions.
    As already indicated, in Aphanizomenon gracile cultures that use a single clone by selecting, neosaxitoxin is the main ficotoxin produced. In this particular case, this clone also produces a smaller amount of saxitoxin.
    The production of neosaxitoxin from the cyanobacteria Aphanizomenon gracile was made from an initial amount of 10 milligrams of wet cells of Aphanizomenon gracile. The sediment (obtained in step a) was subjected to the method described in the specification, that is:
    b. for 10 mg of cell pellet, 10 ml of the same volume by weight of the mixture of organic solvents in aqueous phase (chloroform: methanol; 1: 1 + 50 mM acetic acid in a 50% proportion with the previous mixture) was added , to destroy the cell wall and cytoplasmic membrane, in a slightly acidic medium (pH 5.0).

    C. The lysed cells of stage b are subjected to cold extraction and phase separation (organic / aqueous).
    d. The aqueous phase extracted according to step c, is concentrated 10 times in volume (one tenth of the initial volume is obtained), using for example a rotary evaporator at room temperature.
    and. The concentrate is centrifuged at 20,000 x g for 30 minutes.
    F. 2 ml of the centrifugal supernatant is passed through a diatomaceous earth column. This column is washed with 10 times its volume, (in this case 20 ml) with 50 mM acetic acid. After washing, the retained neosaxitoxin in the column is extracted with 5 ml of an alcohol extraction mixture (ethanol: water: 5 mM acetic acid; 2: 1: 1 vol / vol / vol).
    g. The column eluate obtained in step f is passed through activated carbon columns, which are then washed with distilled water to remove retained pigments and impurities. In the columns the phycotoxins are retained and eluted with 10 ml of a mixture of alcoholic elution (ethanol: water: 1 mM acetic acid;
    3: 2: 1 vol / vol / vol) pH 5.
    h. The eluate is passed through a column of diatomaceous earth and washed with 50 mM acetic acid. Subsequently, the toxin is eluted from the column with a mixture of 5 ml of chloroform: methanol: water; 1: 1: 1 vol / vol / vol.
    i. The eluate of the second column of diatomaceous earth is left in an aqueous phase, evaporating the organic solvents with a "Speed Vac" equipment (Savant, NY, USA). A partially purified neosaxitoxin extract is obtained.
    j. The partially purified extract is subjected to a preparative HPLC of molecular exclusion (preparative high resolution liquid chromatography).
    Each reactor after 2 days of culture produces an average of 652.4 micrograms of total toxin per liter collected. The average neosaxitoxin / saxitoxin ratio is 8.47. In percentages, an average of 11.8% of saxitoxin and 88.2% of neosaxitoxin is obtained. The main objective is the production of neosaxitoxin, which is sought, increased and protected because this compound is the active ingredient patented in clinical applications as a drug or cosmetic. Neosaxitoxin has a biological effect that is 25% more potent than that of saxitoxin and is also the most potent phycotoxin described so far.
    Figures 1,2 and 4 show chromatograms of HPLC series detected by in-line fluorescent detection, which describe a phycotoxin profile produced by the Aphanizomenon gracile species. Figures 1 and 2 describe the HPLC profiles of fractions presenting different degrees of purification from Aphanizomenon gracile extracts. Again, it is important to remember that these are high performance liquid chromatography analytical chromatograms that are performed to detect and quantify ficotoxins. They are not preparative chromatograms of the industrial process. These chromatograms are only useful to show the degree of purity and the content of each purified fraction.
    Figure 1 shows a chromatogram of an unpurified extract of Aphanizomenon gracile made to know and quantify the profile of ficotoxins present in this original extract. The chromatogram shows a simple profile with major proportions of neosaxitoxin and less saxitoxin (less than 13%).
    Figure 2 shows the purification of 10 microliters of an extract of Aphanizomenon gracile in continuous culture and under controlled conditions partially purified by preparative high performance liquid chromatography of molecular exclusion (molecular size separation). In this partially purified extract a peak is already present at Rt = 9.373 minutes that saturates the chromatogram and corresponds to neosaxitoxin, indicating the presence of a large amount of neosaxitoxin in this fraction.
    Figure 4 shows a sample of purified neosaxitoxin from extracts of Aphanizomenon gracile cyanobacteria (Rt = 4,933 minutes) obtained in the eluted fraction of HPLC with ion exchange columns, corresponding to the last stage of purification (final stage of method described above). A single peak is obtained that corresponds to a single component that in this case is pure neosaxitoxin.


    TABLE 2: Production of neosaxitoxin and saxitoxin depending on the number of filaments and the wet weight of the Aphanizomenon gracile sediment.
    neoSTX mM STX mMneoSTXg / mlSTXg / mlneoSTX pg / fil.STX pg / filFil. cyano./ml*
    8.98 3.502.831.052.420.901,170,000.00
    13.66 4.914.301.473.441.181,250,000.00
    3.42 1.031.080.311.710.49630,000.00
    17.82 4.785.611.439.422.41596,000.00
    17.81 4.025.611.2113.172.83426,000.00
    7.66 1.472.410.444.940.91488,000.00
    10.87 2.193.420.6610.251.97334,000.00
    10.52 2.073.310.624.380.82756,000.00
    10.77 1.963.390.597.471.30454,000.00
    17.24 2.995.430.9013.122.16414,000.00
    5.31 0.411.670.125.190.38322,000.00
    12.26 2.173.860.659.471.59408,000.00
    9.84 1.943.100.5817.423.27178,000.00
    STX: neoSTX saxitoxin: neosaxitoxin Fil. cyano .: cyanobacterial filaments * Cyanobacteria filaments correspond to associations of 20 to 100 cyanobacterial cells. These cyanobacteria form filaments in solution and these filaments are counted using the magnification of an inverted phase microscope.
    The yields obtained correspond to a surprisingly unexpected production of pure phycotoxins (neosaxitoxin and saxitoxin) per milligram of wet cyanobacteria, when compared with the production of 5 filaments and sediments of cyanobacteria under the usual culture conditions described so far in culture bottles small without continuous microaeration and with light cycles: day and without light: night.
    In the example described in this case, cyanobacteria are always in a logarithmic growth phase, with permanent illumination 24 hours a day and with permanent collection of cyanobacteria, inducing permanent growth, by providing new nutrients in a volume that is equivalent to volume
    10 collected at each pickup.
    Example 2: Ficotoxins obtained and purified from Cylindrospermopsis raciborskii maintained in continuous cultures under controlled conditions.
    The Cylindrospermopsis raciborskii cyanobacterium was cultured according to the continuous, massive, semi-automatic culture method, under controlled conditions in permanent logarithmic growth, described in the application for
    15 Chilean patent filed simultaneously with the present application by the same authors.
    The unpurified extract of the strain Cylindrospermopsis raciborskii has a chromatographic profile according to the figure
    6. Cylindrospermopsis raciborskii has a toxin profile, where GTX3 and GTX2 predominate with the longest retention times in the spine (the last two peaks on the right, respectively).
    Cylindrospermopsis raciborskii extract obtained from continuous culture is subjected to the procedure of
    20 purification corresponding to the steps mentioned above, under the same conditions described for example 1 and already in the preparative HPLC stage of molecular exclusion, the GTX3 and GTX2 epimers are almost exclusively obtained, as shown in Figure 7.
    The final fraction of the method of purification of ficotoxins from Cylindrospermopsis raciborskii has only GTX3 and GTX2, as shown in figure 8 and the fractions containing each
    25 gonyaulatoxin to obtain large amounts of pure GTX3 and GTX2.
    权利要求:
    Claims (7)
    [1]
    1. Method for industrial purification of biologically active phytotoxins neosaxitoxin, saxitoxin and gonyaulatoxins from cyanobacteria, said method comprising the steps of:
    a) providing a source of phycotoxins, specifically a culture of cyanobacterial clones, which is separated in a culture medium and a wet sediment of cyanobacterial cells or a frozen sediment of cyanobacterial cells;
    b) lyse said sediment using homogenization, solvent extraction, ball mill, freeze / thaw cycles, ultrasonication or enzymatic lysis;
    c) subjecting the material from the lysis of cyanobacterial cells obtained in step b) to cold extraction and organic / aqueous phase separation, specifically 50% by volume of a mixture of chloroform: methanol
    1: 1 and 50% by volume of 1 mM acetic acid, at pH between 4 and 5, and then separate the organic and aqueous phases using chloroform: methanol 1: 1 by volume, which is repeated between 1 and 5 times ;
    d) obtaining a concentrate of the aqueous phase obtained in step (c);
    e) centrifuge the concentrate obtained in step d) to obtain a supernatant;
    f) run the supernatant through a column of diatomaceous earth, and wash the column with a wash solution; then obtain a ficotoxin eluate with an elution solution;
    g) passing the eluate from the previous stage through activated carbon columns, and then washing said activated carbon columns with distilled water to remove retained pigments and impurities; the phycotoxins are eluted with an elution solution;
    h) passing the eluate from the previous stage again through a column of diatomaceous earth; the columns are washed with a solution as in step f), and the phycotoxins are eluted with an extraction solution;
    i) leaving the elutate of the previous stage in an aqueous phase, evaporating the organic solvents, to obtain a partially purified phycotoxin extract;
    j) subject the partially purified extract of the previous stage to preparative high performance liquid chromatography in several stages to obtain pure biologically active ficotoxin.
    [2]
    2. Method according to claim 1, wherein the extraction of step (c) is repeated 3 times.
    [3]
    3. Method according to claim 1, wherein the washing solution of steps f) and h) is 50 mM acetic acid, in step (f) the columns are washed with 5 to 15 times their volume and the solution of Elution is a mixture of alcoholic extraction.
    [4]
    4. Method according to claim 1, wherein the elution solution of step g) is an alcohol elution mixture.
    [5]
    5. Method according to claim 1, wherein the solution of step h) is a mixture of chloroform: methanol: water
    1: 1: 1 vol / vol / vol.
    [6]
    6. The method according to claim 1, wherein the chromatographic steps of step j) are sequentially molecular exclusion, anion exchange, cation exchange and molecular exclusion.
    [7]
    7. Method according to any one of claims 1 to 6, wherein the phytotoxin-producing cyanobacteria belong to the genera Cylindrospermopsis sp, Mycrocistis sp, Anabaena sp, Gomphosphaeria sp, Oscillatoria sp, Aphanizomenon sp and Lyngbya wollei.








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    KR20110130420A|2011-12-05|
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    HRP20160079T1|2016-04-08|
    CA2754154C|2013-12-17|
    DK2412714T3|2016-02-01|
    BRPI1013548B1|2020-06-09|
    JP2012521408A|2012-09-13|
    EP2412714B9|2016-11-23|
    BRPI1013548B8|2021-05-25|
    DK2412714T5|2017-02-27|
    JP5728774B2|2015-06-03|
    ES2562637T3|2016-03-07|
    EP2412714A1|2012-02-01|
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    PCT/IB2010/051187|WO2010109386A1|2009-03-24|2010-03-18|Method for the industrial purification of biologically active phycotoxins|
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