ES2301293B1 - USE OF MICOSPORINE TYPE AMINO ACID (PORPHYRA 334) AS AN ANTIOXIDANT. - Google Patents

Abstract

Use of mycosporine-type amino acid (porfira 334) in products for the prevention of carcinogenic processes. The present invention is part of the biotechnology sector and describes the potential use of a mycosporin-like amino acid (MAA) specifically porfira 334 isolated from the red seaweed Porphyra leucostica , in addition to its possible application in pharmaceutical, nutraceutical, or functional food preparations, among others , for prevention of carcinogenic processes.

Description

Use of mycosporin-like amino acid (porfira 334) in products for the prevention of carcinogenic processes.

Technical sector

The present invention is part of the biotechnology sector and describes the potential use of a mycosporin-like amino acid (MAA), specifically porphyry 334 isolated from the red algae Porphyra leucostica , in addition to its possible application in pharmaceutical preparations, nutraceuticals, or functional foods, among others, for the prevention of carcinogenic processes.

State of the art

Ultraviolet radiation is one of factors biological factors that limit survival, physiology and growth of many organisms. Some of the multiple harmful effects of   UV radiation includes the alteration of DNA molecules and proteins, enzyme inactivation and radical formation free, which attack cell membranes and other molecules target altering its functionality. All aerobic organisms they have a wide variety of antioxidant defense systems both enzymatic and non-enzymatic that coordinate cooperatively and protect the body from the risks involved oxidative stress Among them, the activities stand out Enzymatic superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT); in addition to ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione (GSH), β-carotene, vitamin A, flavonoids and acids Phenolic among others.

Free radical is understood as any chemical species that contains one or more missing electrons in its external orbitals so that a compound can become in free radical capturing or losing an electron. Although they exist free radicals of very different nature, are the species that derive from the oxygen molecule (ROS) the most abundant in aerobic organisms highlighting breakdown products or the excitation of O2 as singlet oxygen1O2 and oxygen species that are partially reduced as the hydroxyl radical (OH •), superoxide anions (O 2 -) and hydrogen peroxide (H2O2). These molecules unstable travel the body taking electrons with what they recover their electrochemical stability, this makes them very dangerous because to achieve this attack stable molecules. A once the free radical has managed to take the electron that you need to pair your free electron, the other molecule will turn in turn into a free radical, thus initiating a cycle destructive to our cells.

Free radicals give rise to alterations important in molecules such as DNA, lipids and proteins, altering seriously the cycle and cellular functionality. DNA can suffer loss of bases as well as the breakage of one or both strands of the genetic material, alterations that can result in mutations irreversible Many proteins are able to absorb a large amount of oxidation without seemingly affected its function. However, there is no doubt that the consequences of alterations in some functions, for example, reception and signal transmission, ion transport, duplication and DNA repair, responses to stress conditions and the energy metabolism, transcription and translation can be Critics for the cell. Damage caused by OH \ and the 1 O 2 are irreversible and generally mark Proteins for degradation. Cell membranes too may be seriously damaged in oxidative stress since phospholipids that have fatty acids with several doubles bonds are very susceptible to oxidation by loss of a hydrogen (allyl) Once the carbon radical is generated in a fatty acid, it reacts with molecular oxygen forming a peroxyl radical. The peroxyl radical can take a hydrogen allyl to another methylene whereby the reaction is propagated. The hydroperoxides, which are stable compounds, if they come into contact with transition metal ions will produce more free radicals that will initiate and spread other chain reactions. So, the membranes are seriously damaged and therefore their functionality It is altered.

Free radicals are associated with a broad range of pathologies and diseases such as Alzheimer's or Parkinson's and conditions related to sun exposure such as appearance of cataracts, photoaging, inflammatory episodes and neoplasms. They are also responsible for the oxidation of food fats, which is the most deteriorating form important after the alterations produced by microorganisms With oxidation, odors and flavors appear stale, the color and texture are altered, and the value drops nutritious when missing some vitamins and fatty acids polyunsaturated In addition, products formed in oxidation They can become harmful to health.

Mycosporin-type amino acids consist of a cyclohexenone or cyclohexenimine ring, conjugated with a nitrogenous substituent of an amino acid or its amino alcohol that acts as a chromophore allowing the absorption of certain shortwave radiation. The metabolites that are isolated in fungi have an absorption range between 310 and 320 nm and have cyclohexenone rings exclusively, being known by the name of mycosporins in reference to their origin. On the contrary, the metabolites that are isolated from marine organisms and algae contain cyclohexenimine rings, with maximum absorptions between 310 and 360 nm and are known as mycosporine-type amino acids or MAAs . Still, mycosporine-glycine and mycosporine taurine are aminocyclohexenones isolated from marine organisms. There are currently 13 different mycosporins described in fungi and 23 MAAs in marine organisms. They are small molecules, with molecular weights around 330 Da and have high photostability. They behave like amphoteric molecules, similar to amino acids, so that they have positive and negative charges on the same molecule. They show physicochemical characteristics of ionic compounds, for example, high effusion point and high water solubility.

There are many functions that have been attributed to these molecules in the organism: from organic osmolyte in Chlorogloeopsis cyanobacterial communities, photosynthetic accessory pigments or precursors of these, to determining molecules in reproductive processes of some species of fish, however it is the photoprotective role against UV radiation the most accepted and documented since apparently they act partially protecting cellular components and physiological processes. A number of studies have evaluated these types of molecules for their activity as topical photoprotectors, vehiculating natural extracts with a high percentage of MAAs and seeing their SPF and their photoprotective potential in plant cells, human keratinocytes, etc. (US Patent 6787147; WO 02/39974; WO 03/020236). Works have also been published that refer to the antioxidant properties of extracts obtained from algae and corals.

Dunlap and Yamamoto in 1995 (Comp. Biochem. Physiol. 112: 105-114) pointed to a possible antioxidant activity of mycosporine-glycine by in vitro assays of lipid peroxidation (phosphatidylcholine method) from extracts of marine organisms containing MAAs, while other iminoMAAs such as porphyra 334, shinorine, palythine, asterine 330 and palythinol were oxidatively robust and did not participate in oxidation-reduction reactions. However, as indicated, these tests were carried out with algal extracts that, in addition to MAAs, contained a high percentage of other cellular components such as polysaccharides, enzymes, etc., so it is not possible to firmly affirm the antioxidant activity of mycosporine-glycine (or M-gly) based on that work.

Nakayama et al. In 1999 (J. Am. Oil Chem. Soc., 76: 649-653) isolated a new mycosporin-like amino acid from the red algae Porphyra yezoensis called usujilene, already identified in Palmaria palmata but not in any species of Porphyra . Their results indicated that such MAA showed antioxidant activity against linoleic acid autoxidation (Thiobarbituric acid and ferric thiocyanate methods), donating certain hydrogens to LOO lipid radicals and giving rise to resonance stabilized MAA molecules as well as
α-tocopherol.

Suh et al. In 2003 (Photochem. Photobiol 78: 109-113) also suggest the function M-gly antioxidant, but probably acting together with other active MAAs. The M-gly, among others, could play an important role participating in the O_ {2} ^ {}} system-generated removal endogenous photosynthesizers.

Yakovleva et al. In 2004 (Comp. Biochem. Physiol., 139: 721-739 examined the importance of M-gly as a functional antioxidant against thermal stress in two corals, Platygyra ryukyuensis and Stylophora pistillata , based on the correlation between degree of susceptibility and endogenous concentration of M-gly.

Also the patent US2004228875 refers to the antioxidant properties of extracts obtained from algae of the genus Porphyra , although without concluding on the possible role played by MAAs.

More recent publications analyze the antioxidant properties of extracts obtained from algae but without specifying the participation of MAAs (Yuan et al., 2005, Food Chem. Toxicol., 43: 1073-1081; Kuda and collaborators, 2005, J. Food Compos. Anal., 18: 625-633).

It can be concluded, therefore, that the role antioxidant of iminoMAAs as such, that is, purified or isolated in a high degree of purity, it is not well known, nor is it its behavior at the level of free radical sequestration water soluble.

An antioxidant is defined as a substance that in low concentrations compared to an oxidizable substrate, delays or prevents its oxidation. The present invention describes the potentiality of the porphyra 334 MAA isolated from Porphyra leucosticta as a radical scavenger and lipid peroxidation inhibitor. In the tests performed, the new antioxidant is compared with another known antioxidant, α-tocopherol. The described compound could be used in therapeutic applications, and in non-medical applications for the stabilization of compounds susceptible to oxidative deterioration, in the preservation of food or related products, and in nutritional, nutraceutical, functional foods or parapharmacy supplements for their antioxidant properties for prevent oxidative stress.

MAAs are found naturally in isolated organisms in the order of mg / g PS, highlighting porphyra 334 found in the Porphyra leucosticta algae in the order of 3-6 mg / gPS. As we know, seaweed has been used as human food since ancient times, especially in eastern countries and whose culture is expanding worldwide. Porphyra (whose vulgar name in Japan is nori) is one of the most important seaweed used as human food. In recent years Porphyra has been listed in Japanese fisheries statistics as the third catch in order of importance and has a high content of valuable edible proteins so that this study could give added value to certain types of food
natural

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Detailed description of the invention

The present invention presents an isolated compound of Porphyra leucosticta with the following structure and useful as an antioxidant and free radical sequestrant.

one

Compounds of the mycosporin type amino acid type have been purified in the aqueous phase starting from Porphyra leucosticta . The compounds have been detected and characterized by HPLC. A UV-visible detector (photodiode detector 996) was used, which measured the absorbance for each sample between 290 and 400 nm. Once the chromatogram was extracted at 330 nm, the peaks were identified by co-chromatography according to their spectra and retention times, compared to MAAs standards.

Preparatory scale extraction was performed dissolving 60-80 g (PF) of biological material in 1 liter of 20% v / v methanol and incubating in a thermostatic bath to 45 ° C for 2 hours. Subsequently, the extract is centrifuged at 14000 rpm for 15 min and rotary evaporation at 45 ° C to eliminate part of the methanol in the sample.

The purification was done in three steps Consecutive in which chromatographic techniques of absorption by the application of active carbon, precipitation of  polysaccharides by adding 100% methanol to the sample and separation final by ion exchange chromatography. Finally I know they obtained aqueous solutions of MAA in high degree of purity in concentrations of the order of mM.

Antioxidant capacity of mycosporin-like amino acids

To measure activity as a hijacker of water-soluble radicals the ABTS method has been used peroxidase, which allows to determine the antioxidant activity total (TAA) of a sample understood as a parameter that allows quantify the capacity of a sample, natural or processed, of sequester free radicals present in an aqueous solution.

Porphyra 334 isolated from Porphyra leucosticta has no significant antioxidant activity at any pH tested as an inhibitor of water-soluble free radical production (ABTS +).

Capacity as a lipid peroxidation inhibitor

Porphyra 334 isolated from Porphyra leucosticta was studied as an inhibitor of lipid peroxidation in vitro using the β-carotene bleaching technique. The method of decolorization of β-carotene is widely used to determine the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption in order to determine their degree of self-preservation in a natural state. In this test, α-tocopherol (α-TOC) was used as a positive control.

Porphyra 334 isolated from Porphyra leucosticta shows moderate antioxidant activity at the level of lipid peroxidation inhibition. It is thus constituted as an antioxidant of moderate activity in vitro .

Abduction of superoxide radicals

The protocol that was carried out was based on Marklund & Marklund (1974, Eur. J. Biochem., 47: 469-474) with some modifications. Relations of dose-response for the MAAs under study is determined at different concentrations.

Porphyra 334 isolated from Porphyra leucosticta at concentrations of 1 mM 50% inhibits the oxidation kinetics of pyrogallol.

By acting as an antioxidant and sequestrant of free radicals, this compound, in extracts or preparations that contain, could be used in preparations or formulations Pharmaceuticals for the prevention and therapeutic treatment of diseases or conditions related to free radicals, in parapharmacy products, in functional foods, supplements Nutritional and nutraceutical preparations, and in the industry food as antioxidant potential (additive).

Description of the drawings

Figure 1. Area (%) of eluted peaks and concentrations expressed in mg g-1 PS of different MAAs present in methanolic extracts of the algae Porphyra leucosticta , Gymnogongrus devoniensis, Gelidium sesquipedale and lichen Lichina pygmaea . The presence of a type of majority MAA in each organism (> 66%) is observed together with other minor MAAs and traces of unidentified substances.

Figure 2. Chromatograms of aqueous extracts of MAAs eluted from the column loaded with DOWEX resin.

Figure 3. Table. Dose (µM) - antioxidant activity response (%) of porphyra 334 isolated from Porphyra leucosticta with respect to 10 µM of α-tocopherol by the method of decolorization of β-carotene. The values represent the mean values and standard deviation of 3 experiments. Porphyra 334 isolated from Porphyra leucosticta is a good antioxidant at a concentration of 100-200 µM.

Figure 4. Sequestration capacity of superoxide radicals generated by the porphyra 334 pyrogallol method isolated from Porphyra leucosticta . The mean and standard deviation of three experiments are represented.

Embodiments of the invention Preparative scale purification of porphyra 334 from the red seaweed Porphyra leucosticta

Compounds of the mycosporin type amino acid type have been purified in the aqueous phase starting from Porphyra leucosticta . The compounds have been detected and characterized by HPLC (Waters 600). The column used for the separation of the MAAs in the HPLC was a C 8 (Sphereclone?, Phenomenex, Aschaffenburg, Germany), packed with porous silica microparticles of 5 mm in diameter with surface derivatized with an aliphatic chain of 8 carbon atoms (octadecyl silane). Its size was 250 x 4.6 mm. A pre-column (Phenomenex, Aschaffenburg, Germany) related to the column used was used. The mobile phase used was 2.5% methanol (v / v, HPLC quality) plus 0.1% acetic acid (v / v) isocratically pumped at a flow rate of 0.5 ml min -1. A UV-visible detector (photodiode detector 996) was used, which measured the absorbance for each sample between 290 and 400 nm. Figure 1 shows the percentages in area of the chromatographed peaks of different algal extracts, some identified as MAAs and others unknown. The objective of the purification was to isolate the major MAA in Porphyra leucosticta in the aqueous phase, in addition to removing traces and other types of unidentified compounds.

Once the chromatogram was extracted at 330 nm, the peaks were identified by co-chromatograph according to their spectra and retention times, compared with MAA standards, provided by Professor Dr. Ulf Karsten (University of Rostock, Germany) extracted from different organisms Marine: Mastocarpus stellatus (shinorine), Porphyra yezoensis (porphyra-334), Bostrychia scorpioides (palythine), the eyes of the coral trout Plectropomus leopardus (asterine 330) and the lichen Lichina pygmaea collected in France (M-glycine). The chromatograms of the extracts collected after passing through the DOWEX50 ion exchange column are shown in Figure 2. As can be seen, in the case of the extract from the P. leucosticta algae, initially with the presence of 4 different MAAs, subsequently only two of them appear, porphyra 334 and shinorine, so similar in structure, functional groups and size that their separation was really difficult. The ratio between shinorine and porphyra 334 remained constant throughout the purification process (1: 8.5 approx.).

Preparatory scale extraction was performed dissolving 60-80 g (PF) of biological material in 1 liter of 20% v / v methanol and incubating in a thermostatic bath to 45 ° C for 2 hours. Subsequently, the extract is centrifuged at 14000 rpm for 15 min and rotary evaporation at 45 ° C to eliminate part of the methanol in the sample.

The purification is done in three steps Consecutive in which chromatographic techniques of absorption by the application of active carbon, precipitation of  polysaccharides by adding 100% methanol to the sample and separation final by ion exchange chromatography (Dowex 50 resin W x 8-100). For elution of the amino acid type Mycosporin porphyra 334 was used as eluent double-distilled water, with a slightly alkaline pH (7.2). They were finally obtained aqueous solutions of MAA in high degree of purity in concentrations of the order of mM.

Antioxidant capacity at the level of radical sequestration ABTS water soluble

To measure activity as kidnappers of water-soluble radicals the ABTS method has been used peroxidase, which allows to determine the antioxidant activity total (TAA) of a sample understood as a parameter that allows quantify the capacity of a sample, natural or processed, of sequester free radicals present in an aqueous solution. This parameter is oriented to give activity information antioxidant that can present a specific sample with independence of the partial activities that each  one of its components or the effects of synergism that could settle.

He 2,2'-Azino-bis- (3-ethyl-benzothiazoline-6-acid sulfonic) or ABTS is a compound that has great stability chemical, high water solubility and maximum absorption in the UVA band at 342 nm. This compound in the presence of H 2 O 2 and peroxidases enzymes derive to a radical metastable (ABTS +) with a characteristic absorption spectrum and different from ABTS, presenting maximum absorption in the region UV spectral and visible at 413, 645, 727 and 811 nm.

ABTS is a product that presents great stability over a wide pH range, showing the same spectrum of absorbance at pH 4 and pH 8.5. Likewise, the formation of the radical ABTS + is also carried out in that pH range but the enzymatic activity of peroxidase itself that is pH dependent of the reaction medium so that upon alkalization it the activity decreases, thus increasing the delay period or "lag time ". The activity of our enzyme could be adjusted to a exponential curve so that it is maximum at pH 4.5 and ceases to be active at pH higher than 10. Our tests will run at pH 6-8.5 so that we ensure the activity of the enzyme.

The quantification of kidnapping capacity Free radicals of a sample are carried out by bleaching assays in which the formation of ABTS + gives place to a characteristic coloration that will decrease so proportional to the amount of substances capable of trapping these radicals that are added to the reaction volume. This Color loss can be measured by kinetic tracking of loss of absorbance at 413 nm (wavelength that does not interfere with other molecules) over a minute using as peroxidase HRP and as a negative control ascorbic acid (THE C). The reaction medium is composed of buffer 50 mM phosphate pH 6, 7.5, 8, 2 mM H2O2, 2 mM ABTS, enzyme 0.25 µM HRP and sample at increasing concentrations.

The calculation of TAA is established according to the relationship between the slopes (Abs / min) of enzymatic assays in which The course of the reaction is estimated in the absence of antioxidants (positive control), and in the presence of different concentrations of substances with possible antioxidant activity. In this way, the pending kinetic control would correspond to a TAA of zero percent, calculating based on this the percentages of inhibition of the other curves.

Capacity as a lipid peroxidation inhibitor

Lipid peroxidation is a well-established mechanism of cell damage in plants and animals, as well as food spoilage (thickening). This process leads to the production of lipid peroxides and degradation aldehydes that leads to loss of cell membrane function and integrity. Porphyra 334 isolated from Porphyra leucosticta was studied as an inhibitor of lipid peroxidation in vitro using the β-carotene bleaching technique.

The method of fading β-carotene is widely used for Determination of the antioxidant capacity of various substances in lipophilic medium, most of them extracted from fruits, vegetables and other products intended for food consumption for be able to determine its greater or lesser degree of self-preservation in natural state. It is a spectrophotometric method that measures the inhibition that an antioxidant causes on the discoloration of the β-carotene in an emulsified aqueous system with Tween 20 and linoleic acid.

Linoleic acid self-oxidizes at high velocity in the presence of hydrogen atoms especially activated Β-carotene, precursor of the Vitamin A, is also known as lipophilic antioxidant that prevents lipid peroxidation in sequestering membranes singlet oxygen molecules and peroxyl lipid radicals. He β-carotene, when in presence of linoleic acid, yields electrons delaying the stage of initiation of the linoleic acid autooxidation process as well as limiting the propagation phase of damage by eliminating simultaneously formed peroxide radicals. If we add a new one substance with possible antioxidant capacity to the reaction medium which contains linoleic acid and β-carotene, this new substance will tend to oxidize it preferably at β-carotene, competing with this for the kidnapping of these radicals.

Β-carotene has a maximum absorption at 470 nm. This maximum varies when the molecule it oxidizes because it loses double bonds and the chromophore structure of the molecule is altered, thus losing its characteristic Orange color and can be detected spectrophotometrically. The absorbance of the reaction medium will remain unchanged throughout of time in the presence of antioxidant substances, warning a drop in the absorbance of the sample when measured in the absence of antioxidants Thus, the measure of antioxidant capacity of a substance will be inversely proportional to the fall of slope of the curve that describes the oxidation of β-carotene (measured at 470 wavelength nm).

In this trial, a positive control was used the α-tocopherol (α-TOC).

The activity of the solution was evaluated according to the degree of discoloration of β-carotene, applying the formula proposed by Hidalgo et al. (1994, Phytochemistry, 37: 1585-1587) with some modifications:

AA = [P \ Sample - P \ control / P \ Pattern - P \ control] \ cdot 100

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P refers to the slopes of the curves of discoloration obtained (Abs / time). For this we adjust by linear regression the part of the kinetic curve that describes linear behavior The correlation coefficients for each Replica of each sample were all greater than 0.98.

Abduction of superoxide radicals

The superoxide radicals (O2 -) are mediators of autooxidation reactions of some compounds. The most of the time these oxidized compounds are characterized by have a characteristic and quantifiable absorption spectrum by spectrophotometry

Pyrogallol (1,2,3-benzenotriol) is a substance that is autooxidizes rapidly in the presence of oxygen especially in alkaline solutions At pH 7.9 SOD inhibits 99% of the reaction indicating a practically total participation of the superoxide anion O 2 - in the reaction. Oxidized pyrogallol has a maximum absorption at 420 nm so that the capacity of the MAAs to sequester superoxide radicals was measured as loss of absorbance of monitored kinetic assays spectrophotometrically (Shimadzu UV 1603) for one minute of reaction. The protocol that was carried out was based on Marklund & Marklund (1974, Eur. J. Biochem., 47: 469-474) with some modifications. Mix reaction contained 0.4 mM pyrogallol and MAA at different 50 mM concentrations of phosphate buffer at pH 8.2, containing 1 mM of diethylenetriaminepentaacetic acid in a final volume of 1 ml incubation. The temperature was stable at 20 ± 1 ° C The positive control was the kinetic curve of generating oxidized pyrogallol radicals in the absence of antioxidants for compare them with different concentrations of SOD as an antioxidant known. Dose-response relationships for MAAs under study were determined at different concentrations. The ability to sequester superoxide radicals of the purified extracts was evaluated following the following formula:

AA = 100 - [P \ sample \ cdot 100 / P \ control]

P refers to the slopes of the curves oxidation kinetics of pyrogallol (Abs / time).

Claims (2)

1. Use of the amino acid extract type mycosporin porphyra 334 extracted from the red algae Porphyra leucosticta for the preparation of a product for the prevention of carcinogenic processes. 2. Use of the porphyra 334 mycosporin-type amino acid extract extracted from the red seaweed Porphyra leucosticta according to the preceding claim for the preparation of parapharmacy products, pharmaceutical products, cosmetic products, nutraceutical preparations or functional foods for the therapeutic treatment of carcinogenic processes.