ES2417279B2 - Use of oxilipins and their derivatives as anti-inflammatory agents - Google Patents

Abstract

Use of oxylipins and their derivatives as anti-inflammatory agents. # The invention relates to the use of monohydroxylated fatty acids, in particular oxylipins derived from {al} -linolenic and linoleic acids, as well as the use of microalgae fractions, extracts or biomass which They contain these acids, for use as anti-inflammatory and chemopreventive in inflammatory bowel disease.

Description

USE OF OXYLIPINES AND THEIR DERIVATIVES AS AGENTS

ANTI-INFLAMMATORIES

The present invention relates to the use of monohydroxy fatty acids, as well as the use of fractions, extracts or biomass of microalgae containing said acids, for use as anti-inflammatory and chemopreventive in inflammatory bowel disease. Therefore, the present invention could be encompassed within the pharmaceutical sector.

STATE OF THE TECHNIQUE

Oxylipins are a set of structurally diverse molecules that derive from the oxidation of polyunsaturated fatty acids (PUFAs). These metabolites are found in animals, plants, algae, bacteria and fungi, and generally act in signaling pathways related to various responses to environmental and pathological factors. One type of oxylipins are fatty acids that incorporate one or more hydroxyl groups in their structure (hydroxylated fatty acids, hydroxy acids).

Oxylipins in general, and hydroxylated fatty acids in particular, have important biological activities in higher and human animals.

It has been shown that various mono-, di- or trihydroxylated acids, formed from acids, eicosapentaenoic acids (C20: 5 n-3; EPA) and docosahexaenoic acids (C22: 6 n-3; DHA), possess a potent anti-inflammatory activity and immunoregulatory. These highly bioactive derivatives, also known as resolvins and docosatriennes, are considered responsible for several of the beneficial effects of EPA and DHA acids in the prevention, treatment and treatment of inflammatory and cardiovascular diseases. In recent years various patents have been filed that describe a variety of hydroxylated derivatives of arachidonic acids (C20: 4 n-6; ARA), EPA and DHA, the routes to

through which they are formed, as well as methods for obtaining and using them

in the treatment of inflammatory processes (US2006 / 0293288).

13-Hydroxyoctadecatrienoic acid (13-HOTE) is derived from the oxidation of a-linolenic acid (C18: 3 n-3; ALA) and has been detected in human plasma, although its physiological function is not established. The potential of 13-HOTE as an anti-inflammatory agent in the

treatment of osteoarthritis and rheumatoid arthritis, given its ability to suppress in vitro the expression of matrix metalloproteinases induced by

interleukin-1¡3 (Schulze-Tanzil on: al. 2002, Histo /. Histophato /. 17: 477485).

In humans, the oxidation of linoleic acid (C18: 2 n-6; LA) is predominantly mediated by 15-lipoxygenase-1 (15-LOX-1) to cause the

13-hydroxyoctadecadienoic acid (13-HODE), an oxylipin that has

proven to possess numerous physiological effects. Acid (13S) -HODE is a natural ligand of PPARs (peroxisome proliferator-activated receptors), transcription factors that are involved in pathologies

like obesity, diabetes and cancer. Through interaction with

PPARs, 13-HODE induces apoptClsis in HCT-116 colon cancer cell lines (Zuo et al. 2006, Oncogene 25: 1225-1241). On the other hand,

It has been shown that 13-HODE acid has antitumorgenic effects in colorectal cancer and that increased expression

of 15-LOX-1 (enzyme that generates 13-HODE) decreases proliferation and emigration capacity in the cell lines of colorectal carcinoma HCT-116 and HT-29 (<; imen et al.; W09, Cancer Sci. 100: 2283-2291). Likewise, it has been described that 13-HODE acid modulates proliferation

cutaneous and that its topical application reverses epidermal hyperproliferation

induced (Ziboh et al. 2000, Am. J. Clin. Nutr. 71: 3615-3665). In the

Several patents have been published in recent years, including those

related to the use of 13-HOOE acid as a marker to assess oxidative stress in mammals (W02005 / 059566) or its topical use for the treatment of eye disorders (WO 2006 / 065916A 1).

In relation to other C18 hydroxylated acids, a

patent application on obtaining and using as agents

anti-inflammatory and antineurode ~ lenetives of mono derivatives or

dihydroxylates of stearidonic acid (C18: 4 n-3; SOA) and ylinollanic acid (C18: 3 n-6; GLA), such as 10-hydroxy SOA or 7-hydroxy GLA acids (US2007 / 0248586Al); (b) a patent application on the use of C18 and C20 hydroxylated fatty acids, such as (9S) -HOOE, (8S) -HETrE or (8S} -HEPE, for the preparation of medicinal products applicable to

therapeutic neutralization of an organoboronate type drug

(W02006 / 059083); (c) a patent application for a biologically active composition containing, among other principles, 9HOTE acid (W02006 / 010560A2).

Currently some fine chemical companies market a range of hydroxylated derivatives of fatty acids of

18 to 22 carbons, including 13-HOTE and 13-HOOE acids. for use in biochemical studies (eg Cayman Chemical Company, USA).

Regarding the methods of obtaining these acids, it has been

described a stereoselective synthesis of acid (1 3S} -HOTE, and various methods of synthesis of acid (13S) -HOOE, as well as the preparation of this compound through enzymatic oxidation of LA. More recently a request for patent on obtaining 13-HOOE by means of a synthesis that involves the oxidation of LA with corn lipoxygenase (WO <! 008 / 133398A 1).

Microalgae are a group of unicellular organisms, both prokaryotic and eukaryotic, found in terrestrial and marine aquatic environments, and which are mostly photosynthetic. Its enormous biodiversity, chemical composition, short generation times and the

possibility of industrial cultivation make microalgae

potentially a very interesting source of products with applications in the food, cosmetic and pharmaceutical industry.

There are few data on microalgae oxilipins. The

Isolation of hydroxy acids in lalS microalgae of the Cyanophyceae class

Lygnbya majuscula (malynolic acid), Anabaena flos-aquae f. flos-aquae (9R-HOTE and 9R-HODE), Oscil / aloria redeki HUB 051 (9-HODE and 13HODE) and Nostoc sp. PCC7120 ([lR-HOTE and 9R-HODE) and in the microalgae of the Chlorophyceae class Dunaliella acidophila (12R-HOTE and 9S-HOTE).

On the other hand, in diatoms (Bélcilliarophyceae) a

variety of oxylipins, mainly polyunsaturated aldehydes, involved in the chemical defense of these microalgae.

The only reference on the use of natural sources to obtain

Isolated hydroxylated fatty acids, or oils containing said acids, refers to derivatives of stearidonic acid (SOA) or acid and

linolenic (GLA) (US 2007 / 02485B6Al). This patent application includes

the use of animals, plants and microorganisms to obtain oils containing oxilipins. In regards to microorganisms.

said patent includes the use of, finished species of microalgae (eg species of the genus Scenedesmlls) as well as their genetic manipulation

(eg species of the genus Crypthecodinium).

No data have been found on the use of microalgae as a source of oxylipins derived from acids, a-linolenic and / or linoleic acids in general and from 13-HOTE and 13-HODE hydroxy acids in particular.

DESCRIPTION OF THE INVENTION

The authors of this application have found:

(to)

that various species of cultivated microalgae of the Chlorophyceae and Trebouxiophyceae class contain oxylipins (13S, 9Z, 11 E, 15Z) -13-hydroxyoctadBca-9,11, 15-trienoic [(13S) -HOTE] and / or (13S, 9Z, 11 E) -13-hydroxyoctadeca-! L, 11-dienoic [(13S) -HODE],

(b)

that these compounds present in vitro activity as anti-inflammatory agents, and

(and)

that the administration to animal models of a preparation based on microalgae containing the aforementioned oxylipins attenuates the damage of inflammatory bowel disease.

Accordingly, the present invention relates to the use of 13-HOTE and / or 13-HODE acids and compounds derived therefrom, as well as fractions, extracts or biomass of the microalgae containing them, in the preparation of dietary or pharmaceutical compositions for use in the treatment and / or prevention of inflammatory processes in general, and of inflammatory bowel disease in particular.

Therefore, a first aspect of the present invention relates to the use of a compound of general formula (1) (hereafter compound of the invention):

_ ~ C02R2

R, O

(one)

where: == represents a double or single link; R, is selected from the list comprising hydrogen, a group

alkyl (C, -C1O), alkenyl (CrC1O), alkynyl (C, -C, o) or acyl; Y

R2 is selected from hydrogen or an alkyl group (C, -C, or).

or any of its pharmaceutically acceptable salts or stereoisomers. for the preparation dH a pharmaceutical composition for the treatment and / or prevention of inflammatory diseases of the tract

digestive.

When == represents a double bond the general formula would be the

following:

~~ CD, R,

R, D

(the)

When == represents a simple link the general formula would be the

next (lb):

~~ CD, R2

DR,

(lb)

The wavy link in the formulas indicates that the configuration of that

Carbon can be R or S, or a racemic mixture.

The compounds of the present invention, represented by the formula

(1), described above may include any of its salts, ester,

sorvate or its stereoisomers, in particular any of its enantiomers (R or S) or a racemic mixture.

The term "pharmaceutically acceptable salts" refers to any pharmaceutical salt or any other compound that, being administered to a receptor, is capable of providing (directly or indirectly) a compound described herein. However it will be observed

that pharmaceutically unacceptable salts are also within the scope of the invention since the latter may be useful in the preparation of pharmaceutically acceptable salts. Salt preparation,

Stereoisomers and derivatives can be carried out by means of methods known in the art.

The term "alkyl" refers, in the present invention, to chains

saturated, linear, branched or branched hydrocarbons, having 1 to 10

carbon atoms, for example, methyl, ethyl, n-propyl, ¡-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Preferably the alkyl group has between 1 and 6 carbon atoms. The alkyl groups can

be optionally substituted by one or more substituents such as

alkynyl, alkenyl, halo, hydroxy, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "alkenyl" refers, in the present invention, to unsaturated, linear or branched hydrocarbon chains, having from 2 to 10

carbon atoms, preferably from 2 to 6, and which contain one or more double carbon-carbon bonds and which may optionally contain some triple bond, for example, vinyl, 1-propenyl, allyl, isoprenyl, 2

butenyl, 1,3-butadienyl, etc. alkenyl radicals may be optionally substituted by one or more substituents such as alkyl, alkynyl, halo, hydroxy, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "alkynyl" refers to hydrocarbon chains, linear or

branched, from 2 to 10 carbon atoms, preferably from 2 to 6, and that

they contain at least one or more, triple carbon-carbon bonds and which may optionally contain some double bond, for example, ethylino, propynyl, butynyl, etc. Alkynyl radicals may optionally be

substituted by one or more substituents such as alkyl, alkenyl, halo,

hydroxyl, alkoxy, carboxyl, cyano, or carbonyl, acyl, alkoxycarbonyl, amino, nitro or mercapto.

The term "acyl group" refers to a hydrocarbon chain, linear or branched, saturated or unsaturated, in which the first carbon in the chain is a carbonyl group, that is, the acyl group has the formula

5 -CO-R ', where R' is an alkyl, alkenyl or alkynyl group as has been

previously described.

In a preferred embodiment of the present invention, R, is hydrogen. In another preferred embodiment of the present invention, R2 is hydrogen.

In a more preferred embodiment R :, and R2 are hydrogen.

In an even more preferred embodiment the compounds of the invention are

the following: (13S, 9Z, 11 E, 15Z) -13-hydroxyoctadeca-9,11, 15-trienoic 15 [(13S) -HOTE]

~~ C02H

OH and (13S, 9Z, 11 E) -13-hydroxyoctadeca-9, 11- <lienoic [(13S) -HODE]

~~ CO, H

OH

These anti-inflammatory compounds of the invention can be obtained from certain species of lTIicroalgae by extracting the biomass with organic solvents. The extract can be separated by chromatographic techniques and fractions containing the

anti-inflammatory compounds Depending on the purification steps, it is possible to obtain the pure compounds. For example, him

compound (13S} -HOTE has been obtained from the dry biomass of the microalgae Chlamydomonas debaryana. The isolated compound has

high activity as an inhibitor of the production of TNF-a, an important proinflammatory cytokine.

The anti-inflammatory agent can be used in pure form or as a constituent of an extract or a composition based on the microalgae biomass, typically an extract or biomass composition where the concentration of the anti-inflammatory agent is known so that it

I can control the dose. In addition, other components present in the extract or biomass can reinforce the effects of the compound

anti-inflammatory

Therefore, another embodiment of the present invention relates to the use of the

compound of formula (1), as described above, in a pure form or in an extract or biomass of microalgae.

By "compound in a pure form" or "pure compound" is meant the compound obtained from a natural extract by successive steps of purification or chemically synthesized by methods known to one skilled in the art.

By "microalgae biomass" is meant a material obtained after separation of the mass of microalgae from the culture medium and subsequent drying.

By "microalgae extract" is meant the mixture of organic compounds, among which are the compounds of the invention, obtained by treating the dried biomass of microalgae with

organic solvents, preferably methanol, followed by evaporation of the solvent. For extract we also consider a

fraction thereof, that is, "Fraction" means a mixture of organic compounds obtained by separating an extract by chromatographic techniques.

In a preferred embodiment, the microalgae are of the class

Chlorophyceae or Trebouxiophyceale. More preferably they are of the genus Ch / amydomonas, Nannoch / oris or Chlorella and even more preferably of the species Ch / amydomonas debaryana or Ch / orella sorokiniana.

Another aspect of the present invention relates to the use of microalgae of the Chlorophyceae class, preferably of the genus Ch / amydomonas, more preferably of the speci, and Ch / amydomonas debaryana, for obtaining the compound of general formula (1) described above.

Another aspect of the present invention relates to the use of microalgae of the Trebouxiophyceaei class, preferably of the genera

Nannoch / oris or Ch / orella, more preferably of the species Ch / orella

sorokiniana for obtaining the compound of general formula (1)

previously described.

The compounds of the invention, as demonstrated in the examples, can be used as anti-inflammatory agents.

By "inflammatory diseases of the digestive tract" refers to

diseases that occur with acute and / or chronic inflammatory processes, of high or mild intensity, produced in the digestive tract, considering as such the system of organs capable of ingestion, digestion, absorption and absorption of food in an animal, Especially in a human. These diseases are particularly associated with immunological disorders.

In particular by "inflammatory bowel diseases" refers in the present invention to those that occur in the digestive tract, especially in the small and large intestine, and which can cause

large number of symptoms in the individual suffering from it, such as weight loss, blood in stool and / or diarrhea, among others, which lead to a considerable deterioration in their quality of life. These diseases can be selected from, but not limited to, ulcerative colitis or Crohn's disease.

In addition, based on recent evidence demonstrating the development of certain tumor processes Gomo as a result of poorly controlled previous inflammatory pathologies, including inflammatory bowel disease and colorectal cancer, the compounds of the invention can be used for the prevention of colon cancer.

Regardless of the form in which it is found, pure or in an extract or biomass of microalgae, but in particular when it is the pure compound, the compound of the invention is formulated to facilitate its application and bioavailability, preferably with acceptable carriers or excipients. pharmaceutically

The compound of the invention can be used with other active ingredients or drugs that potentiate the anti-inflammatory effects or offer other additional benefits. The other drugs may be part of the same composition or may be provided in a separate composition for administration at the same time or at different times.

In addition, the compound of the pure invention or an extract or a composition based on biomass can be formulated alone or with other ingredients, in the form of powders, granules, tablets, suspensions, solutions or emulsions that may contain other compounds commonly used in the scope of the preparation of this type of products.

The dosage of the compound of the invention will vary according to the

particular formulation, the specific inflammatory condition to be treated and the mode of application.

Throughout the present description, the term "treatment" refers to eliminating, reducing or decreasing the cause or effects of the disease. For the

Purposes of this invention, treatment includes, but is not limited to, alleviating, reducing or eliminating one or more symptoms of

the illness; reduce the degree of disease, stabilize (i.e., not worsen) the state of the disease, delay or slow the progression

of the disease, alleviate or improve the disease status and remit

(either total or partial).

Another aspect of the present invention relates to the use of a compound

of formula (1) described above and in any of its forms,

as an additive in functional foods or food supplements or

dietary

Thus, the compounds described in the present invention can be used, in addition to pharmaceutical compositions, in foods (including those labeled as functional or nutraceutical) or in food or dietary supplements not only to prevent the occurrence of inflammatory processes but also to improve the body's defenses as part of the immune system.

In the present invention it is understood as functional walimento "to a

food or food supplement including so-called "nutraceuticals",

It has a beneficial effect on health.

By "food or dietary supplements" they refer to non-food compositions, that is, they are not food, but they contribute to the

diet a beneficial effect on health, these supplements may contain other types of supplements such as vitamins, antioxidants, among

others. These compositions may not be administered in the same manner as described for pharmaceutical compositions.

Therefore, another aspect of the present invention relates to a food

functional or a dietary or dietary supplement comprising a

compound of formula (I) according to SB described in the present invention and in any of its forms.

Throughout the description and claims the word "comprises"

and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be apparent in

part of the description and part of the practice of the invention. The

The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

Next, the invention will be illustrated by a series of tests performed by the inventors.

EXAMPLE 1

This example illustrates how 13-HOTE and 13-HODE acids have been isolated from microalgae and the methods used for their identification.

Materials and methods

Column chromatography was malified with Merck silica gel (70-230 µm).

The HPLC separations were performed on a LaChrom-Hitachi device

with LiChrospher Si-60 (Merck) columns and an L-7400 UV detector. The solvents used were of high quality for use in HPLC. NMR spectra were recorded on a Varian INOVA 600 spectrometer using CD, OD as solvent. the chemical shifts were referenced with respect to the solvent signal (8 "3.30 and Be 49.0). COY, HSQC, HMBC, and NOESY spectra were performed using the

Varian standard pulse sequences.

Extraction and isolation of products The dry biomass of the microalgae Chlamydomonas debaryana was extracted

with a 1: 1 mixture of acetone-methanol. After evaporation of

solvent under reduced pressure, the extract obtained was subjected to separation by silica gel column chromatography eluted with hexane-diethyl ether mixtures of increasing polarity and subsequently with mixtures of methanol chloroform of increasing polarity. Fractions

eluted with diethyl ether and with chloroform-methanol (9: 1 and 8: 2) were suspended in methanol-water (9 ': 1) and passed through RP-18 cartridges. the solution obtained was evaporated under reduced pressure and the resulting mixture was separated by normal phase HPlC used as

eluent mixtures of chloroform-methanol or hexane-isopropanol-acetic acid until the pure compounds are obtained.

Compound Identification (13S) -HOTE acid was identified from the spectra of 'H-NMR and 13C-NMR (Table 1), COZY, HSQC, HMBC and NOESY. the configuration

absolute in C-13 was established from the value of the optical rotation

(Yadav al. 1992, Tefrahedron 58: 4465-4474).

The acid (13S) -HODE was identified from the spectra of 'H-NMR and 13C-NMR (Table 2), COZY, HSQC, HMBC and NOESY. the absolute configuration in C-13 was established from the value of the optical rotation (Yadav et al. 1992, Telrahedron 58: 4465-4474).

Table 1. Dalos of 'H and 13C-NMR of 13-HOTE acid (600 MHz, CD30D)

.and

5 '' <: m B 'H (m, J in Hz) faith 5 'lC m 6 'H (m, Jen Hz)

1 2 3 4 5 6 7 8 9

171.7. 35.01 26.11 JO.l'l JO.2 '1 JO.3'1 30.71 28.61 133.0d 227 (l. 7.4) 1.59 (m) 1.32 (m) 1.32 (m) 1.32 (m) 138 (m) 219 (dl 77.7.7) 5.40 (dllO.7.7.7) 10 11 12 13 14 15 16 17 18 129.3 d 126.6 d 136.7d 73.3d 36.31 125.5 d 1346d 21.71 14.5e 5.96 (dd, 10.9,10.9) 6.49 (dd, 15.3.11 .0) 5.63 (dd, 15.3.6.6) 410 (di, 6.4.6.4) 2.29 (m). 2.24 (m) 5.35 (dII, 10.7,7.3,1.5) 5.45 (dII, 10.7,7.3,1.6) 2.05 (de, 7.5,7.5) 0.95 (l. 7.5)

• Exchangeable assignments

Table 2. Dalos of 'H and' 3C-NMR d, 13-HODE acid (600 MHz, CD30D)

tea

fi 1lCm B'H (m, Jon Hz)  tea fi 13 (: m B 'H (m, Jen Hz)

1 2 3 4 5 6 7 8 9

171.7. 35.01 26.1 I JO.1 'I JO.2' 1 JO.3'1 30.71 28.61 132.9d 2.27 (l. 7.5) 1.59 (m) I.JO (m) I.JO (m) 1.JO (m) 1.39 (m) 2.18 (dl 7.5.7.5) 5.40 (dl 10.8.78) 10 11 12 13 14 15 16 17 18 129.4 d 126.5 d 137.3 d 73.4 d 38.41 2631 33.01 23.71 14.4c 5.97 (dd, 11 .1.11 .1) 6.48 (dd, 15.2.11 .0) 5.60 (dd, 15.1.6.8) 4.07 (dl6.4.6.4) 1.52 (m), 146 (m) 1.JO (m) I.JO (m) 1.JO (m) 090 (1.7.0)

-

Exchangeable Assignments

EXAMPLE 2 This example illustrates the activity d, and the pure compounds in tests of

in vitro anti-inflammatory activity.

Methodology

Immune system cells respond to various stimuli by secreting a large amount and diversity of cytokine dH. In the inflammatory process, TNF-a stands out among these cytokines, which plays a fundamental role

in the earliest stages of the process (Popa et al. 2007, J. Lipid Res.

48: 751-762).

The measurement of anti-inflammatory activity was carried out on the line of

THP-1 human monocytic leukemia, after inducing its differentiation to

macrophages with phorbol acetate myristate (PMA) 0.2 ~ M, and stimulating cytokine production, for 24 hours, with lipolysaccharide (LPS) at a concentration of 1 ~ g ml-1. To the differentiated cells the pure compounds 13-HOTE and 13-HODE obtained from microalgae were added

(example 1) at a concentration of 50 J.lM, concentration that is not

cytotoxic for THP-1. TNF-a production was quantified by enzyme immunoassay (ELlSA) using commercial kits (Calbiochem, EMD Chemicals Inc. Germany).

TNF-a cytokine inhibition Stimulation with LPS increased TNF-a production from 4.5 ±

3.0 to 111 .62 ± 3.1 ng mr ', while in the presence of compounds 13-HOTE and 13-HODE it was reduced to 27.05 ± 0.5 and 65.45 ±

0.5 ng mr '. respectively, which implies an inhibition of

TNF-a production of 79 and 44%, respectively. Therefore, these

Pure compounds have a high anti-inflammatory activity, especially the 13-HOTE compound.

The effect of the 13-HOTE and 13-HODE compounds on the production of TNF-a by TPH-1 monocytes not differentiated to macrophages was also studied. Stimulation with LPS increased TNF-a production from 9.9 ± 3.3 to 31.5 ± 9.9 nI) mr ', while in the presence of the compounds it was reduced to 9.8 ± 1.3 and 17.7 ± 0.79 ng mr', for the cases 13- HOTE and 13-HODE, respectively, which is a

TNF-a production inhibition of 100 and 64%, respectively. Therefore, the anti-inflammatory action of these compounds is exerted on both rhinocytes and macrophages, and is especially notable in

the 13-HOTE compound.

EXAMPLE 3

This example illustrates the in vivo anti-inflammatory activity of the biomass of

microalgae containing 13-HOTE and 13-HODE.

An acute trial (5 days) was performed in Wístar rats, assessing the activity

of a lyophilisate of microalgae against intestinal colitis induced by

Trinitro-benzenesulfonium acid hapten (TNBS; Morris et al. 1989, Gastroenterology 96: 795-803). Each gram of microalgae lyophilisate contained 0.54 mg of 13-HOTE and 0.44 mg of 13-HODE. TNBS was applied

rectally and lyophilisate under study (in saline suspension

with 5% Tween 80, and at concentrations of 100 and 400 mg kg "of animal)

It was administered orally, using an intragastric Latvian probe. The lyophilisate was administered from day 1 to 5, and colitis was induced on the third day of treatment. This oxperimental model results in localized inflammation and alterations in the colon with characteristics similar to those found in chronic inflammatory diseases in

humans. The results obtained from this study can guide

on the preventive and / or curative capacity of the compounds, in a range of active doses, influencing the anti-inflammatory capacity they had.

Weight variations, intestinal adhesions were analyzed

(scales 0-3), diarrhea (scale 0-1) and the total weight ratio of the isolated colon (g cm- '). Damage assessments were also made

determining the macroscopic and microscopic damage in the cojan of each

animal. Intracolonic administration of 10 mg of TNBS produced in the

Animals an important picture of acute colitis. with a significant loss of body weight and increased diarrhea. Changes in the colonic fraction were associated with a significant increase (p <0.01)

in the relation pesollongitud of the colon (0_169 ± 0_01 9 cm-'in treated with TNBS, compared to 0.081 ± 0.004 9 cm-' in healthy control). The results obtained in animals treated with lyophilisate suggested, in conjunction,

Changes indicative of some improvement. The lyophilisate moderately reduced the loss of body weight and caused a marked decrease in

presence of diarrhea and adhesions at the highest dose (400 mg kg - ') _ With regard to the weight / length ratio of the colon, a decrease was observed with both doses, being significant (p <O. Ol) at

100 mg kg-1 dose. The data obtained after measuring the

Ulcerated surface reflect that both doses of lyophilisate (100 and 400 mg

kg-1) significantly attenuated the extent and severity of colonic damage. These doses were able to reduce the ulcerated surface in

more than 30% compared to the TNBS control.

For histological study, samples fixed and included in paraffin,

they were cut at 5-7 IJm thick and stained with hematoxylin / eosin. In the histological sections obtained after treatment with the lyophilisate, lower peeling of the epithelial cells was detected and a better conservation of the glandular structure was observed. In addition, a decrease in inflammatory infiltrate was detected with respect to the control

TNBS and a smaller size of the superficial necrotic layer.

The study was completed with two biochemical tests on colon samples; myeloperoxidase enzyme (MPO) activity was determined; This activity is related to the degree of inflammation, since it is a constitutive enzyme of the neutrophils, and allows to estimate the degree of neutrophilic infiltration. The MPO was quantified by a test

colorimetric, based on the oxidation dependent on H, O, of a donor

of artificial electrons (TMB). The results are expressed as units

of enzyme activity per tissue rng (U mg- '). The results

indicated that the highest concentration of lyophilisate decreased

10 significantly (p <O. Ol) the inflammatory infiltrate. A decrease in MPO was observed from 7.37 ± 0.42 U mg- '(TNBS) to 6.89 ± 0.54 U mg-' (TNBS and 100 mg kg- 'of lyophilisate) and up to 2.55 ± 0.28 U mg-' (TNBS and 400 mg kg- 'of lyophilisate), 2.15 ± 0.31 U mg-' being the value

corresponding to healthy controls.

All these results show the in vitro anti-inflammatory activity of

13-HOTE and 13-HODE acids and the in vivo capacity of a preparation

which contains them to prevent damage similar to those observed in inflammatory bowel diseases in humans.

Claims (16)

1.-Use of a compound of general formula (1): R, O (one) where: = represents a double or single link; R, is selected from the list comprising hydrogen. an alkyl group (C, -C'D). alkenyl (C, -C1O). alkynyl (C, -C'D) or an acyl group; and R, is selected from hydrogen or an alkyl group (C, -C1O). or any of its pharmaceutically acceptable salts or stereoisomers acceptable. except the compound where - it is a single bond and R1 and R2 are hydrogen. for the preparation of a pharmaceutical composition for the treatment and / or prevention of inflammatory diseases of the digestive tract. 2. Use according to claim 1. wherein R is hydrogen. 3. Use according to any of claims 1 or 2. wherein R is hydrogen. 4. Use according to any of claims 1 to 3. where it represents a double bond. 5. Use according to any of claims 1 to 3. wherein It represents a simple link. 6. Use according to claim 1, wherein the compound is of the formula: ~~ C02H OH 7. Use according to any of I: as claims 1 to 6, wherein the disease is intestinal. 8. Use according to any of the preceding claims, wherein the digestive tract disease is selected from ulcerative colitis and Crohn's disease. 9. Use according to any of the preceding claims, wherein the compound of formula (1) is in a pure form or in an extract or biomass of microalgae. 10. Use according to the preceding claim, wherein the microalgae is of the Chlorophyceae or Trebouxiophyceae class. 11. Use according to the preceding claim, wherein the microalgae is of the genus Chlamydomonas. 12. Use according to the preceding claim, wherein the microalgae is of the  Chlemydomonas debarys'na species. 13. Use according to claim 10, wherein the microalgae is of the genus Nannochloris or Chlore / la 14. Use according to the preceding claim, wherein the microalgae is of the species Chlore / the sorokiniana. 15. Use of a compound of formula (1) as described in any of claims 1 to 14, as an additive in functional foods or food or dietary supplements. 17. Functional food comprising a compound of formula (1) as described in any of claims 1 to 6. 18. Food or dietary supplement comprising a compound of formula (1) as described in any of claims 1 to 10 6.