PT105914B - USE OF AN ABSTRACT OF OLIVE OIL INHIBITOR OF ACETILCOLINESTERASE IN THE TREATMENT OF COLINERGIC DISTURBATIONS - Google Patents

Abstract

The present invention relates to the application of an oil-based extract of maize by hydrogen peroxide with an inhibitory activity of the enzyme acetylcholinesterase (ACHEI) to be used as an inhibitor of this enzyme in the prevention / treatment of neurological disorders / collinergic disorders. THIS COMPARISON IS A NON-TOXIC NATURAL EXTRACT OF THE EXTRACTED OLIVE OIL BAG, MAINLY CONSTITUTED BY THE PHENOLIC COMPOUND HYDROXYTOLYOL, COULD CONTAIN TIROSOL AND ITS DERIVATIVES AND DERIVATIVES FROM HYDROXYTUANOL, BETWEEN OTHER PHENOLIC COMPOUNDS, OBTAINED FROM OLIVE OIL, BY-PRODUCTS OF THE OLIVE OIL PRODUCTION INDUSTRY . The mentioned extract comprises an anti-acetylcholine synthase activity superior to the activity of hydroxyl isoleated and purified (standard) at the same concentration. THIS NON-TOXIC NATURAL EXTRACT MAJORLY CONSTITUTED BY HYDROXYTANOLSOL MAY BE USED IN INDUSTRIAL FORMULATIONS, ESPECIALLY FOR THE FOOD AND / OR PHARMACEUTICAL INDUSTRIES.

Description

The present invention relates to the application of a natural and non-toxic excretion of the king tom action, which is disrupted by hydroxide rosacea with inhibitory activity only in industrial formulations, namely for the food industries. and / or pharmaceutical, · with fine therapeutics.

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Several epidemiologic studies suggest that table milk and table amitone significantly affect the benefits of the Mediterranean diet in use with respect to the decreased incidence of degenerative pathologies, namely coronary artery disease.

However, several cisatifying studies have come to the point that the protective effect of these products is due not only to the presence of a high content of oleic acid, but also: the anti-wetting properties of the polyphenyl compounds they contain. Among these, hydronitrile 1 (3, A-diuidvoxifsn.iletaual - DOEET; desisicated because it is a maloritable component of the physical form of acceptance and also for its high autonomous capacity nv.ênge.ld et dl - 2001).

Studies published by various research groups have explored the biological activities of rosal hydrozit (Manos et al., 1999; Rletjena et al., 299 © Cloffr et al., 2010 © notably, the in-situ prevention of low-lipepretein oxidation. density (LDL) and platelet aggregation, acting on the cytotoxic effect of oxygen-receptive species. © © in various human cellular systems, and the α-proliferative effect on © - © cells induces apuptosis (delia. Regiene et al. 2000®

Yes, one. Sigrifiable part of td.droxytitosei and other phenolic compounds of. fine in the by-products (residues) resulting from the production of the accept, © cement, the most modern process for the production of olive oil, without adding water, generates a by-product which is a semi-solid residue called rich olive pomace. in these compounds. Based on this fact, several patents have been published for the recovery of drug compounds, particularly hydroxytyrosol and tyrosol, to obtain these compounds isolated and of a washed degree. This is what ® r gives documents Pl .® © .7-0 A2, EF 19 © 8 © Al and í® 1389901 B © which refer to methods for obtaining hydroxy-shoots ©, pari, obtained from olive products and by-products.

also, the patent ®C 02/1 © 19 and the document © 80 03/910 © 51 Al refer to one. The method for obtaining hydroxytyrosis was derived from water from the olive vegetation by acid incubation in order to convert the oleutopine to the hydras®, thiophaside of fractionation so as to separate the hydroxyphtosei from the remaining composition. This latter patent utlliragão suck the product obtained as food supplement for the protection of tired skin lesions flap radiation iraviaiata _f d based on the potent antioxidant capacity hicroxldruscl.

Another patent (US d003 / 0236202 ÃX / SF 1434638 Bld) discloses the alkylation to the natural hydroxytyrosal and iso-olated olive compounds and also derivatives derived from hydratititosi synthesis, namely esters, for the protection against neurodegenerative diseases, such as Alzbimer s Parkiasou, enters others.

The present invention differs from the state of the art in that it does not comprise the separation or purification of the chloroxltirosol by any eromatographic process or other type of clean technologies such as nanofiltration, ultrafiltration or superoretic extraction. The present invention is directed to the use of a natural water-soluble axtract supplied to a company that owns a olive oil processing plant. The present invention comprises the use of said extraoto, constituted by aldrxltiroscl and other fendileone corundum, the hydrochloride is the major fetal compound, figs 1 to 4), as an inhibitor of the enzyme acetylollnetarass (ACheld for the prevention and / or treatment of neurodegenar diseases). , which causes Alzheimer's disease ..

The present invention differs from U.S. Patent 2003/6233202 A1 / 6143433: 313 in that it utilizes a complex natural extract of olive pomace rather than pure compounds isolated or synthesized. In addition, the present invention describes, for the first time in the state of adolescence, the hydroxyl compounds, as well as said non-toxic natural complex exhaustion of the Î ± hydroxytyrool rich olive pomace, having inesteronic anti-acetyl activity. This activity being greater in the complex extract of the bagasse of a s items, whereas in the hydroxyl t.trol alone to purified it can be activated as an inhibitor of the ima a or ti.I. fines are focused on the prevention and / or treatment of eolimergine disorders / nano-reproductive disorders, such as bicheimer disease.

h hoença ds hl mm ima r is a progressive, nsurodugenerar pathology that removes princeIt is important for the elderly population, responsible for some of the cases of dementia in people older than eighteen years old. Ps main symptoms associated with this disease involve cognitive dysfunction, mainly breast misses. One of the most promising ways to treat this disease is to increase the level of acetylin {ÃCh} in the brain using inhibitors of the above acetylcholinesterase (AChé;>

A number of studies have shown that the acost.il col inosters.se CAtldb is responsible for decreasing cerebrali neurotransmission in the synapse of the nerve, causing cholinergic neurons, which are responsible for cognitive fungi. Increasing the level of acetylcholine may be helpful in improving the symptomatology of hypertension disease, such as memory loss or the difficulty of learning. It is inhibition of the above auet11calinasterase promotes increased concentration of acetylcholine in the synopses, slowing the progression of symptoms associated with the disease of ha ha hama.

LITERATURE _: SCIENTIFIC AND ORGANIZING THE SEARCH FOR NEW SOILYLCHOLINOSTERASE INHIBITORS IN NATURAL EXTRACTS OF OH PLANTS IN ETH., 2001? Ferre ira et al., 2006, Fale et al., 2009? Porfirlo et al., 2010;., Which have good toxicity and have no undesirable side effects when administered. The present invention differs from the state of the art in that it refers to an extract of a self-rising residue, known as the extract of the olive extract, which is identified as being an inhibitor of the enzyme ACnE, which residue is mainly comprised of tidaoxitzrosoi, a natural and non-toxic compound. The present invention differs from the state of the art in that it relates to the use of said extract in the protection against neurodegenerative diseases, namely Alateimar disease, on the grounds that hydroxyt 1 rosei is the xisioritary compound of this extract which confers its Inhibited activity of the enzyme acetylolinasterase (AChEj, the enzyme responsible for the decrease in acetii colma, cerebral ineurotransmitter) in the nerve synapse, and this decrease in acetylcholin correlates with the severity of Aizhemem disease.

It is known from the state of the art that treatment of Alxuaimet's disease with inhibitors of this enzyme improves symptoms by enhancing cholinergic functions and increasing the amount of amino acid present in oolinerggiox siuapsas. The present invention further differs from the state of the art. by identifying what compound is the natural extract responsible for. For its anti-acetyl calinosterase activity, it is rather a phenolic compound, while normally the state of the art only refers to extracts of plants with inhibitory nativity of AdhB, but for the most part not identical. which compounds are responsible for AChE inhibitory activity, as they are reported, these are usually alkaloids, compounds which are toxic only have the oxidant antifreeze

For example, Ferreiro et al., 2000 reported that natural extracts of eranolieus and cleavensis and different plantar species have AChe inhibitory capacity and anti-incident activity. However, it does identify which compounds have these autities, and the anti-ACh activity may be. due to the presence of the alkaloids, the egg is badly eaten, while the antioxidant activity is due to the fendiic compounds, as is also common.

It is well-known in the art that, despite the numerous pasquina tests of Füolic compounds with ACúll activity, they are rare in that they exhibit such activity and do not harm the hydroxylate (T. nasi.ru et al., 1011} and of these, It is not known whether they affect the brain at all times. It is also in accordance with the state of the art that, when taken at this time, the rosal membrane is rapidly absorbed and passes through the nematode-spherical barrier at relatively high levels.

US 2003/0230202 Al / SF 1414651 si, suggests the use of pure olive isolated compounds, namely hydroxytyrosol, or derivatives thereof obtained by synthesis, in protection against neurodeuenerative diseases, in particular Alxhemerer's disease, based on the fact that (hydroxytylresal being a strong antipaxis and inhibitor of monoamline oxlds.se (Mau-B)). Patent no. substantially purified based on the fact that tldruxltirusoi is an antioxidaute paper. Fletjens et al. (2000) and Cbafcroan et al. (2010) also refer to the neuroprotective effect of nitroxyrosol due to its antioxidant effect, having an action to delay oxidative action. daacanha et aU (2 (111j suuexe due to the reduced risk of Alzheímer's disease or other related neurodégens.) are due to the rapidity and that the major fendly constituents of olive and olive oil, namely ο bidmuxitirosui, have to inhibit the aggregation of premi at Tau.

The present invention differs from the state of the art, as described above for the first time suggesting the use of an extract from the axuim bagasse, a residue of the production of the bulk of the bulk of the oxide. go. The sun in the prevention of the treatment of neurological disease / neurological disorders, such as the case of hlzheimer disease, among others, by the inhibition of the disease. above a 1 l.uoiiriesturass.

Inhibitors of the acase cola west rase enzyme are used clinically for the prevention / treatment of various pathologies including severe myasthenia, glaucoma, onset, and Alsheimer's disease. Several compounds are approved by Fel to alleviate these symptoms, however. however, synthetic acetylcholinesterase inhibitors, such as taurine and donepezil, are serious in other indications such as aepa. toroxiu age and gastrointestinal complaints. Furthermore, in order for these compounds to become effective inbounds, they must be bonded to the anti-center of the joint, since novel binding bonds may lead to. very dull consequences, incorring mo rt e.

Several plant motifs were investigated by the father. its potential to treat mutually active problems. Gaiantemine was the first single compound with a ml exhibiting ACnÉ inhibitory activity and is clinically useful, although it has only minimal effects on the alkaloid. For example, · Oinkco biioba EOb 7ul> (Tenubind) natural tract and a natural potemu plant axtract. A free mmmm weed manta, marketed for the treatment of Alsheimer type et al., 2011 ; Sumia, 2002; Manrer et al., 19m. Thus, there is a great interest in finding new non-allaloid ACaE inhibitors, with fendile compounds being potential candidates, and the present invention demonstrating AChE inhibitory activity of a natural aqueous extract of the residue resulting from the production of a myth (bagasse de ase it anaà esmoei a Leme consisting of fmolluos compounds, but hydrochloride derivatives or hydrolyzose derivatives), consisting of a fendi leu nautotoxic compound, as demonstrated by Soni at al. (20déj, and also described by It is first seen in the present invention to have ACnE inhibitory activity, being the major response to the inhibitory activity of aztastate.

Figure 1 shows the profile of the horns (moles obtained by capillary aleotrophoresis of the lyophilized extract). The compounds identified by the migration of the migration times and UV spectra with the migration times to 11V spectra of previously anaerated standards. by capillary eleutruforssn, are indicated with 1 mm The golmyous structures of the identified compounds are shown together with the respective mist

spectra UV .. as well as < the peroantagám of eucerdânola between the the back identifie cated and shallow · default active commercial THE Figure. 2 illustrates the a .1 ec t r o f a r r the respective spectrum UV and structure chemistry for the standard of

nitroxltlrosol. provided by Fxttaaynthèss.

Figure 3 Illustrates the electrofrogram corresponding to the co-injection of the extract with the hydroxythool standard. The chemical structures of the ideal compounds are replicated alongside sporting OV spectra.

The figure. 4 illustrates the spectra obtained by FTIR retrospectively for the hydroxytyrosol standard, the aseitone pomace extract or the standard shoots, presented individually (figure ia}] and overlapping (figure 4ó (; ..

UB

The present invention relates to splits as such. Extract obtained from the assiduous that is obtained after the production of atits, called bagasse exhalation of items, even the rumen of enxi.ma at quotio 3.1 in this phase, to be used in the prevention / treatment of neurodegenerative diseases. cholinaroin disorders. Said excerpt is provided by an accepted production waste processing company (dC.A3dti, in the form of an aqueous solution, which is filtered and lyophilized to give a pasty brown product which is smelled of aromatherapy). The product _and product thus obtained is composed mainly of hydroxytyrol and, among other things, aspherical compounds, numbered as the derivatives (oirusol and hydroxytyrosol derivatives). Figure 1 shows the profile of fused compounds obtained by axillary capillary ulcer. As a result of the lyophilate, a very clean profile can be found, characterized mostly by a hydroxytyrosis [> 70i of the utraste ;, as identified by comparing the W spectrum of the peak obtained at 1>. UV to a standard commercial hydroelectric standard supplied by Extrasyntbése, whose electrogram and UV spectrum are shown in Figure 11 below. 99.952 A. The presence of b1 dioxitri cl in this extract as deaf the compound malar, was further confirmed by the collation in the extrudate capillary eluctrofurusa equipment with the hydroxytrusol standard, observing if a increased peak area obtained at It-nti and an agreement of 99.91 [figure 5].

From Figure 1 it can be seen that the lyophilized extrudate with the same name to contain thyrose, albeit a much smaller amount, as provided by Comparison of the UV spectrum of the peak obtained is only 8,733 minutes with the uV assumption for the standard <ie. commercial tyrosol supplied by fdtraaynrhese, showing an agreement of 99> 71. The chemical structures of the idantified compounds are depicted innately of the respective DV spectra in Figures 1 to 3. Figure 4 illustrates: the respectrically obtained FTIR spectra for the bidruxitifusel standard, the aseitone bagasse extract and the uirosol standard, shown in FIG. individually [Fig. 4a}} and overlapping [Fig. b]}, The existing cation within the spectrum of the aridone bagasse extract of the hydroxytyroso reveals that this extract is only comprised of rhydroxytyrosol and hydroxy derivatives.

The objective of the present invention is to use the said hydroxititose-containing extract> 708 in its composition as an inhibitor of the acetylculiesthetase above, with inhibition of the enzyme greater than 507 in a concentration of 1 mg / ml. This extract may be used in industrial formulations. , in particular in the food and pharmaceutical industries, by incorporating food formulations (eg microencapsulation of the extract), in the form of a food supplement or as a naturally occurring drug,

Bféátóa iswéfidcg pasha a du cúmawiúmx of the wtmamdo riem in. hmdmumitiXd & nX

The use of rich mixtures of a hydroxytyrosol as inhibitors of the enzyme silylculinusterase for use in the prevention and treatment of neurodegenerative diseases or of collagenous disorders has added to the new potential beyond those already well known. The biological effects of hydroxytyrosoi, both free and sturified, have been documented, in particular its high antioxidant capacity., The absence of toxicity and rapid absorption by various organs into tissues, including the ability to be rapidly absorbed and pass through the barrier itself. Highly significant amounts of this compound make natural extracts rich in this compound try to be of interest for therapeutic purposes.

surrounded is an extract obtained from the sugarcane bagasse, a plant biomass consisting of a byproduct of. the production industry of the acceptor, presents aorous value and opens new market perspectives for the valorization of this residue. It thus contributes to the value .traction of a regional agricultural product by ensuring a more competitive and sustainable agricultural invoice, in addition to other applications likely to be of this biomass but of lower added value.

and «o» pwpamagão .1. Saatérla-prima is one of the residues obtained after the production of ascites, supplied to an olive oil production processing company (UCAfebi), which supplies the extra-aged olive pomace and supplies one. extract resulting from the ash extract extracted as a water solution. This solution is filtered and diluted to give a pasty product of greenish brown color and full aromatic treatment. The treatment of olive pomace in the processing company increases the initial hldruxltirusol content by converting it. of the European oil, the existing Feuolo oyster compound, in hydroxytyrosol, thus making this residue mainly a product of hydroxytyrosol having a potential added value,

Gbtwplm dto Mtasú. xieo w cohesive to s fem.àlócw,

u.umsuuiamsan h & dtoxitiro s o 1

The lyophilized extract P is characterized in that it has in its composition compound specific phytices according to the tiger erythrocyte and these compounds are idealized (larger 17% non-tyrosol minority lambs derivatives such as, ti tosei, ni.drouttirosol of hydacrityrosei, among other ideutifed <

Amtxvidádn mrkà <'aee7Xmel.ina®'7.râsâ.

The evaluation of the inhibitory activity of the ethiolinolinesteraae above is determined in vitro using an adaptation of the method described by ingkaniuau st al. ; Ingkaniaan, K. ,, Temkitthasua, 7, cpuonohun, 7, Yuyçem,

T., & Thougnoi, 7 7x7 o Fthnopharmacoiogy, 17 7 3 7777 ..

The lyophilized, rich, lyophilized, rich sample is dissolved in water with a determined concentration as such and added to a given syrup canopy. action covers a coaste of space purple fears three. To this point, a solution is added by counting the above to the ethylesterase and incubated for 15 minutes at room temperature. Next I postponed join one of tsrmi.ua of vol. a solution 1 se trate iodate of ac et 11 thiochol lua 77 and of the area dg EI ima ncide 1, 5 ^f - dithiobis - (2 - ui t robe siiòi ca j [DTPa) .. In another cuvette, the resulting blank (without AEChE) is faxed and then the absorbance at 75 nm for the first 4 to 5 minutes of the reaction is measured. A control reaction using water instead of the sample solution is considered to be 175 of the 777 climatic activity. This assay is carried out in various samples and in triplicate for each concentration.

The percentage inhibition of aostilrolinestarasa above is calculated by comparing the reaction rates (substrate hydrolysis) of the samples to the control;

where I (%) is the assay inhibition rate, is the initial reaction rate which contains the sample at the highest concentration and is the initial reaction rate of the control.

A. concentration of. aruus, whose inhibition of unrimental activity is 501 XX® is determined by regression obtained by plotting the percentage inhibition versus sample concentration for the various concentrations of the samples.

This method and substrate acetylthiodide iodide (Acchl) is hydrated by the method Achl) to give tincoline. g® reacts with Î ±, β-dithiols® [[2aitrobensium] Î »XXB acid or Elimac® prodnxincX Xu i t reagent. ® ® n oa t o - 5 -me rcap t c 11 ® o 11 na e ® 11 is - 2 ® i t r be n ® (XX S), which can be tested at 405 nm.

To a 1 mL capacity spectrophotometer cuvette, 425 µl of HEPEt- buffer and 100 µl of 0.4 mg / mL acaituna bagasse extract solution were added. To this mixture is added a solution containing 24X gives A® E and thioon® Incubate for 15 ml®Â® room temperature. Then 7S bp Achc and 47 5 μΐ DThB are added. The other blank was the corresponding blank [without AC®} and then the absorbance was measured at 405 µm during the first 4 a. After 5 minutes of the reaction, a control removal was performed using water instead of the master solution. The inhibition of the enzyme ethylcoesterase was 100%.

1 ml sspectxofotbamtrc cuvette cougar.

of capacity, 225 pt of HF9E3 buffer and 105 µl of an olive pomace extract solution were added.

entrapment of: ç, 9 mg / mt. The mixture was then added to a solution containing 25 µl of water and flooded for 15 minutes at room temperature. mm then Achi nt is added and 475 gL d of other upvetts made the white run s ρ nu dent s (without idbE) and em. Then the absctvence a.

the first 4 to o minutes of

A water was effected in the sample solution. The percentage inhibition of the core sample was 99.4 71: 0.949.

In a 1 ml capacity spectrophotometer uuvate, 325 µl of HSrSS buffer and 100 µl of a hi.druxitirosol (entrasynthesis) standard solution were added at 0.4 mg / mt. A solution was added while maintaining 35 µl of AebE and incubated for 15 minutes at room temperature. Then 75 ml was added. of Achi and 475 æl of DTPBt In another cave, the blunt white was made (without aasu, and then the absorbance at 40S nm was measured during the first 4 to 5 minutes of the reaction. A control reaction was performed using water instead of sample collection d The percentage of irrigation rate of the acetylene: ε.inesr.erase was 3 ', 29x5, Lei.

Claims (3)

1 x Non-Therapeutic Axtra-infected Anabolic Pellet Extract characterized in that it can be used for the pre-treatment / treatment of neurodegenerative diseases / colourous disorders, such as but not limited to slaucoma, severe myatenia and slcheimer disease. Aqueous extract of olive pomace extracted for therapeutic use according to claim 1, characterized in that the maicritary acid is hydroxytyrosol, 3, Aqueous bagasse extract extracted for therapeutic use and according to claims 1 and 2, as the major compound is in a concentration greater than 7%. Extracting the aqueous residue extracted axeltona for therapeutic use according to claims 1 to 3 caractcríráuc adlciunalmante contain other pair falls comp _o: f 'and smooth node .. Aqueous oil extract extract for therapeutic use according to any one of claims 1 to 2, wherein the other fungi compounds may be tyrosol and its derivatives and hydroxytyrosol derivative, among others. t, Extracted aqueous extract of aneitone pomace for therapeutic use according to hollow. claims 1 to 5 characterized in that it can be in any form and any solid matrix or. liquid gives any nature. ;. Aqueous extracellular azase bagasse for therapeutic use according to claims 1 to 4 may be used in formulations for the food, pharmaceutical and / or chemical industry.