Use of mango leaf extract mangifera indica, for the treatment of neurodegenerative diseases (Machine

专利摘要:
Use of mango leaf extract mango indica, for the treatment of neurodegenerative diseases. The present invention relates to the use of a composition comprising a dried extract of mangitera indica linn, wherein said extract is characterized in that it comprises the phenolic components that are detailed below: {image-01} For the preparation of a medicament for the treatment of neurodegenerative diseases associated with vascular pathology of the central nervous system or with cerebral vascular dementia. (Machine-translation by Google Translate, not legally binding)
公开号:ES2630739A1
申请号:ES201600138
申请日:2016-02-16
公开日:2017-08-23
发明作者:Mónica GARCÍA ALLOZA；María Del Carmen INFANTE GARCÍA；María Teresa FERNÁNDEZ PONCE；Carmen Castro González；Juan José RAMOS；Casimiro MANTELL SERRANO；Lourdes CASAS CARDOSO；Enrique José MARTÍNEZ DE LA OSSA FERNÁNDEZ
申请人:Universidad de Cadiz；
IPC主号:

专利说明:

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USE OF INDICA HOSE HANDLE SHEET EXTRACT, FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES.
TECHNICAL SECTOR
This invention is related to the use of the bioactive phenolic extract from the mango leaf (Mangifera indica Linn, MGF from here) as a treatment and / or prevention of, complications of the central nervous system (CNS), related to the neurodegenerative process itself of central pathologies associated with aging such as dementia processes Additionally, it is also related to the use of the same extract for the elaboration of a pharmaceutical or nutraceutical composition useful in the treatment of CNS diseases that occur with inflammation, vascular damage and / or neurodegeneration, as occurs in Alzheimer's disease (AD) and vascular dementia (DVa). These are the most common forms of dementia and account for approximately 80% of cases. It is mbs, the border between the two is blurred and in many patients the EA type markers coexist with vascular alterations characteristic of DVa.
BACKGROUND OF THE INVENTION
The progressive increase in life expectancy is contributing secondarily to the increase in pathologies associated with age, among which AD and DVA play an important role, due to their prevalence and the associated economic, social and human costs. The neuropathological characteristics of AD include: neurofibrillary tangles formed by abnormally phosphorylated tau protein, accumulation of p-amyloid (PA) forming senile plaques (PS) and amyloid angiopathy (CAA) around the vessels, and neuronal and synaptic loss (Serrano-Well, A. et al Neuropathological alterations in Alzheimer disease Journal / Cold Sphng Harb Perspect Med 1 a006189. 2011) This is added to an inflammatory process, related to the presence of PS and ovillos Vascular dementia is the second most common cause of dementia. it is a heterogeneous pathology that encompasses multiple micro-infarcts, ischemic disease of small vessels, microvascular damage (Craft. S The role of metabolic disorders in Alzheimer disease and vascular dementia two roads converged Joumal / Arch Neurol 66 300-305. 2009) or deposit of PA in the form of amyloid angiopathy around the cerebral vessels (Greenberg. SM. Et al. Detection of isolated cerebrovascular beta-amyloid with Pittsburgh compound B Joumal / Ann Neurol 64: 587-591. 2008) The line between EA and DVa is diffuse blurred and in many
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Patients with vascular damage markers coexist with the characteristic histopathologic marks of AD.
At present, dementias, of different etiology, and specifically AD, do not have successful treatment: the use of anticholinesterase drugs and glutamatorgic antagonists that are symptomatic treatments and that although delay cognitive deterioration do not prevent the progression of the pathology in the CNS, nor the associated neurodegeneration The treatments based on amyloid hypnosis, such as inhibitors of pyy secretases, or anti-pA vaccines, have been studied extensively, however, today they still have serious clinical application problems. Hence, new therapeutic alternatives are urgently required for the population suffering from dementia processes (for review see (Sabbagh, M. and Cummings, J. Progressive cholinergic decline in Alzheimer's Disease consideration for treatment with donepezil 23 mg in patients with moderate to severe symptomatology Joumal / BMC Neurol. 11:21, 2011)).
BRIEF DESCRIPTION OF THE INVENTION
The authors of the present invention have faced the problem of providing a successful therapy against dementias such as AD or DVa. For this and in view of previous studies, rather than using a "magic bullet" that completely reverses its cause, the authors consider the use of agents that control various aspects related to inflammatory processes or vascular damage to be more useful for the treatment of this type of disease. Thus, previous studies have focused on the study of natural phenolic compounds. These are the secondary metabolite groups numerous mbs in plants, with mbs of 8,000 known structures (Soobrattee. MA, et al. Phenolics as potential antioxidant therapeutic agents: mechanism and actions Journal / Mutat Res 579 200-213, 2005) that exhibit high antioxidant and anti-inflammatory activity, among others (Nijveldt. R J .. et al Flavonoids: a review of probable mechanisms of action and potential applications. Journal / Am J Clin Nutr 74 418 -425 2001). Due to these properties, it is currently considered that the phenolic compounds can act as therapeutic agents in a large number of pathologies related to free radical damage and the inflammatory process, which is usually associated and that includes, among others, microglial activation, increase of the production of cytokines or endothelial damage. Phenolic compounds can be found in almost any part of plants, therefore agro-industrial by-products, such as leaves or bark, are a rich source of this type of compound.
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In the specific case of the agricultural by-products of the Mangifera indica Linn plant, its levels are attributed to its high concentration of high-antioxidant phenolic compounds (Ling, L. T, et al Standardized Mangifera indica extract is an ideal antioxidant. Joumal / Food Chemistry 113 1154-1159, 2008), considered as the compounds responsible for the multiple pharmacological properties of this plant. Among the most important polyphenols of the mango plant, in terms of antioxidant capacity and quantity, there are mangiferin and quercetin, although this plant contains other polyphenols in smaller proportions such as catechins, kaempferol, anthocyanins, public acid and benzoic acid La mangiferina, of the family of xanthones, it presents interesting nutraceitic and pharmacological properties, such as anti-inflammatory, antioxidant, antibacterial or promising activity in the treatment of diseases such as diabetes (Benard, O and Chi. And Medicinal properties of mangiferin, structural features, derivative synthesis, pharmacokinetics and biological activities Journal / Mini Rev Med Chem. 15: 582-594. 2015; Pal, PB. et al Mangiferin attenuates diabetic nephropathy by inhibiting oxidative stress mediated signaling cascade. TNFalpha related and mitochondnal dependent apoptotic pathways in streptozotocin- induced diabetic rats Journal / PLoS One 9: e107220, 2014 Wang. H L. et al Mangiferin facilites islet regeneration and beta-cell proliferation through upregulation of cell cycle and beta-cell regeneration regulators. Journal / lnt J Mol Sci 15: 9016-9035, 2014). Quercetin is a flavonoid that has antioxidant and anticancer properties as well as effects on the regulation of gibnical expression (Masibo, M. and He, Q Major Mango Polyphenols and Their Potential Significance to Human Health Journal / Comprenhensive Reviews in Food Science and Food Safety 7: 309-319, 2008) The literature describes antispasmodic, antipyretic, anti-inflammatory, antimicrobial, antifungal properties. antidiabetic, immunomodulatory, analgesic, antioxidant and antidiarrheal (Aderibigbe. A O., et al Evaluation of the antidiabetic action of Mangifera indica in mice Joumal / Phytother Res. 15: 456-458, 2001 Amazzal, L, et al. Mangiferin protects against 1-methyl-4-phenylpyridinium toxicity mediated by oxidative stress in N2A cells. Joumal / Neurosci Lett. 418: 159-164. 2007; Garrido, G .. et al. In vivo and in vitro anti-inflammatory activity of Mangifera indica L extract (VIMANG) Journal / Pharmacol Res 50: 143-149, 2004).
The high pressure extraction process of the polyphenols present in the Mangifera indica Linn leaf and its exact composition has been previously described in the patent ES 2464192 A1 Phenolic extracts of Mangifera indica Linn, method of obtaining and uses, Mantell Serrano, C. , Fernandez-Ponce, T, Casas Cardoso, L., Martinez de la Ossa, E describes a procedure that allows obtaining Mangifera indica Linn extracts enriched in phenolic compounds, such as mangiferin and quercetin through the use of green solvents. such as carbon dioxide, water. and ethanol, under sub- conditions
or supercritical at high pressure and temperature, avoiding the use of quantities of organic solvents considered harmful for health and the environment. The extract is characterized by presenting a phenolic mimic of mangiferin and quercetin of 1.93 and 0.88% respectively, as well as the presence of minority compounds.
The authors of the present invention have realized that the polyphenols present in the Mangifera indica Linn leaf are extraordinarily useful in the treatment and prophylaxis of neurodegenerative diseases associated with vascular pathology of the central nervous system such as Alzheimer's disease or with dementia cerebral vascular
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1: HPLC analysis. HPLC analysis by Sinergy Hidro RP reverse phase column for the phenolic extract obtained in example 1, with identification of the chromatographic peaks corresponding to the mangiferin and 3- (3-D-glucosyl-15 quercetin) phenolic compounds according to their time of retention (tr = 20min for mangiferin and tr = 34.4 min for 3-PD-glucosyl-quercetin).
Figure 2: Mass spectra. Mass spectra of phenolic compounds present in Mangifera indica Linn extracts made in quadrupole-ES negative LC-ES-MS SIM mode
20 Figure 3: LC-ES-MS and LC-UV analysis LC-ES-MS and LC-UV analysis for the patterns of possible phenolic compounds present in the sub- or supercritical extracts of Mangifera indica Linn leaves
Figure 4. Chronic treatment with FGM for 20 weeks improves cognitive disorders in db / db and APP / PS1 mice. A) The episode memory, assessed by means of the NOD test, showed an improvement of the db / db mice treated with MFG for all the parameters studied by ANOVA of a criterion followed by the Tuckey bo Tamhane test, as required: "qub" [F (3104) = 41 043, ** p <0.01 vs. other groups, ttp <0 01 vs Control-Sham and Control-MFG], "dbnde" [F (3107) = 20.752, ttp <0 01 vs. Control-Sham and Control-MGF] and "cubndo" [F (310o) = 17 451, ** p <0 01 vs other groups, ttP <0.01 vs 30 Control-Sham and Control-MGF], B) An effect similar we observed in APP / PS1 mice, model of EA, treated with FGM, which showed a significant improvement in the epic memory, assessed by NOD Differences detected by ANOVA of a criterion followed by Tuckey bo Tamhane test, as required: "qub" [F (3135) = 18.62, ** p <0.01 vs. other groups, ttp <0 01 vs. Control-Sham and Control-MGF]., "Dbnde" [F (3.28) = 22.87,
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** p <0.01 vs. other groups, ttP <0.01 vs. Control-Sham and Control-MGF] and "cudndo" [Fp.i23r2.61, p = 0.054] C) Space learning. valued in the acquisition phase of the Morris water maze, it also revealed an improvement in db / db animals treated with FGM. The daily analysis by ANOVA of a criterion followed by the Tuckey bo Tamhane test, as required, revealed a significant improvement in the time required for db / db animals treated with FGM to find the hidden platform (dial: [F (7156) = 2,319, p = 0 078], day 2 [F (3153) = 13 155, ttp <0 01 vs Control-Sham and Control-MGF, $ tp <0 01 vs. Control-Sham], day 3 [F (3145 ) = 20 5262, ** p <0 01 vs other groups, ttp <0.01 vs. Control-Sham and Control-MGF], day 4 [F (37153) = 24.062, ** p <0.01 vs. group challenge , ffp <0 01 vs Control-Sham and Control-MGF] D) A similar improvement profile can be observed in APP / PS1 mice treated with FGM in a chronic way Daily analysis by ANOVA of a criterion followed by Tuckey bo Tamhane test, according to it was required, reveld a significant improvement in the time that the APP / PS1 animals treated with FGM used to find the hidden platform (day 1 [F (3184) = 2 007, p = 0 115], day 2: [F (3,182 ) = 8.16, ttP <0 01 vs Control and Control-MGF], day 3: [F (3159) = 3.13, tp <0. 05 vs. Control and Control-MGF], day 4 [Fp.ie4r9.88, ** p <0.05 vs other groups, TTp <0 01 vs. Control]. E) When assessing the spatial memory in the retention phase of Morris's attic labyrinth, both 24 hours and 72 hours after the end of the acquisition, we observed a recovery in the time that the animals spent in the quadrant in which the platform was originally (quadrant 2): ANOVA analysis of a criterion followed by the Tuckey bo Tamhane test, as required (24h retention: [F (737) = 3 1 3, p = 0.037, fp <0 05 vs. Contro-sham and Control -MGF], retention 72 hours: [F (7156) = 2.319, p = 0 078], day 2 [F (334) = 3.29, * p = 0 032]). F) In the retention phase for APP / PS1 mice, we also observed a significant improvement after chronic treatment with FGM, using ANOVA of a criterion followed by Tuckey bo Tamhane test, as required (24h retention [F (737) = 3.13 , p = 0 037, tp <0.05 vs Control-Sham and Control-MGF, 72 h: [F (3 «0) = 4 40, ** p = 0.009 vs. other groups],
Figure 5. Chronic treatment with FGM improves brain atrophy typical of db / db mice. A) We observed a slight improvement in brain weight of db / db animals treated with FGM. although the differences were not significant by ANOVA of a criterion followed by Tuckey test b [F (333) = 161 65, ttp <0 01 vs. Control-Sham Control-MGF]. B) Representative image of violet crescent tincidn in the different groups under study, in which the reduction of cortical thickness can be seen in a db / db mouse, and the improvement after treatment with FGM Scale = 250 pm C) The analysis of the bark by means of violet cresyl tincidn revealed that the treatment with FGM is capable of reversing the cortical atrophy observed in db / db mice and we also observed improvement in db / db-FGM mice when we compare the hypodempic thickness Differences detected by ANOVA of a criterion followed test Tuckey b Cortex [F (3612) = 9 63, ** p <0 01 vs other groups], hippocampus
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[F, 3,3,7, = 4.29. $ tp = 0 06 vs Control-Sham] D) The increase in caspase activity 3/7 in the cortex in db / db mice is significantly reduced after treatment with FGM [F <3.22) = 4.24, * p = 0.016 vs rest of the groups]. Similarly in the hippocampus, caspase3 / 7 activity is reduced, although the differences are not statistically significant [F (322) = 1 52. p = 0 23], the data is the average of 6-8 mice and the differences statistics were detected by one-way ANOVA followed by a Tuckey b test
Figure 6. Chronic treatment with FGM decreases cortical hyperphosphorylation of tau in DM2 model mice (db / db). A) The increase in cortical tau phosphorylation observed in db / db mice is partially diminished after chronic treatment with FGM [F (320) = 4 37, ffpOO 016 vs Control-Sham and Control-FGM] B) Representative image of the levels of phospho-tau, total tau and actin in cortex from the 4 groups of animals under study: Control-Sham, Control-MGF, db / db-Sham and db / db-MGF C) Increased phosphorylation of cortical tau observed in APP / PS1 mice is partially diminished after crdmco treatment with FGM. although the differences were not statistically significant] [F (317) = 1.33, p = 0.295]
Figure 7. Chronic treatment with FGM decreases the presence of spontaneous hemorrhages in DM2 model mice (db / db). A) The increase in cortical hemorrhage load (% affected area), typical of db / db mice, decreases significantly after chronic treatment with FGM [F (3 m) = 12.70, ** p <0.01 vs. other groups] This fact is due to an increase in hemorrhages [F (3112) = 12.48, ** p <0 01 vs other groups], since the size is not affected [F, 3 3147) = 0 541 , p = 0.654] Differences detected by ANOVA of a criterion followed by the Tuckey test b B) Representative image of the cortex of a control mouse, a control mouse treated with FGM, a db / db mouse and a db / db MGF mouse, where a decrease in spontaneous hemorrhages (marked with green arrows) is observed in db / db-MGF mice Scale = 50 pm C) A similar profile is observed in the hippocampus, and although the decrease in hemorrhagic load did not reach significant differences [F <3 52) = 12.70, p = 0.087], the bleeding density was significantly lower after treatment with FGM of db / db mice [F (355) = 4 23. ** p = 0.009 vs. other groups], As in the cortex, the non-FGM treatment did not influence the size of the hemorrhages [F (3 4a9) = 0 541, p = 0262], Differences detected by ANOVA of a criterion followed by Tuckey b test
Figure 8. Chronic treatment with FGM decreases the inflammatory process in the CNS in db / db and APP-PS1 mice. A) The increase in cortical microglial load in db / db mice is controlled by treatment with FGM [F (31585) = 12 06, ** p <0 01 vs other groups, ttP <0 01 vs. Control-Sham and Control-MFG] and this effect is due to a reduction in
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microglial cell size [F (38i23 »= 12 06, ** p <0 01 vs. other groups, ttP ^ .OI vs. Control-Sham and Control-MFG], while the number of microglial cells / mm2 it was not altered [F (31561 | = 43.03, ttP <0 01 vs. Control-Sham and Control-MFG]. Differences detected by ANOVA of a criterion followed by Tuckey bo Tamhane test as required B) A similar profile was observed in the hippocampus, where treatment with FGM reduced the microglial load [F (3312) = 7.19, ** p <0.01 vs. other groups] and the size of the microglial cells [F (3-1767) = 5.87, * * p <0 01 vs other groups] while their density was not altered [F (31313) = 14 60, ** p <0 01 vs other groups, tTp <0.01 vs. Control-Sham] Differences detected by ANOVA of a criterion followed by Tuckey bo Tamhane test as required. C) Representative images of the immunostaining of cortical microglia (green) with anti-IBA1 antibody in which a decrease in microglial load is observed and the size of the microglial cells after treatment with FGM. D) The microglial load increased in the cortex of APP-PS1 animals. Manguiferin treatment significantly reduced the microglial load in the cortex. This effect was detected in areas free of senile plaques [F (3, 1693) = 157 30, ** p <0.01 vs. other groups of the groups, ttP <0.01 vs control-sham and control-MGF, fctp <0 01 vs control-sham] and although a similar reduction could be observed near the plates, it was not statistically significant (p = 0 367). The reduction in microglial load was due to a reduction in the number of microglial cells away from senile plaques [F (3.1748) = 912.83, ** p <0 01 vs. rest of groups, ttp <0.01 vs control-sham and control-MGF], (p = 0 480 near plates) while cell size was maintained between different groups (near senile plates p = 0 06, far of senile plaques [F (83.10954) = 2.46, p = 0.06] The data represent the average of 5-6 mice and the differences were detected by a T-student test for independent samples or one-way ANOVA followed by test b of Tuckey E) A similar profile was observed in the hippocampus where the microglial load was reduced in the mice treated with FGM These differences were statistically significant away from the senile plaques [F (3, 333) = 44 03, ** p < 0 01 vs. other groups, ftp <0.01 vs control-sham and control-MGF,] and although a similar profile could be observed in the vicinity of the plates, the differences did not become statistically significant (p = 0 085). The reduction in microglial load was due to the small number of microglial cells (far from senile plaques [F (3,332) = 145.13, ** p <0.01 vs. other groups, tTp <0.01 vs. sham-control and MGF-control ], (p = 0 441 near senile plaques.) The individual cell size was maintained between the different groups (near senile plaques p = 0 073, away from senile plaques [F (3,2166) = 2 25, p = 0.081], the data represent an average of 5-6 mice and the differences were detected by a Student's T-test for independent samples or or by one-way ANOVA followed by a Tuckey b test
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is an alternative for the treatment of CNS complications associated with vascular damage and / or neurodegenerative processes, by treatment with a phenolic extract obtained from leaves and / or branches of Mangifera indica Linn by means of high pressure tbcmcas ( characterized in the patent ES 2464192 A1).
This extract in the CNS reduces the inflammatory process, limits cortical and hypocombic atrophy, reduces tau hyperphosphorylation and controls the presence of spontaneous hemorrhages. All this translates into an improvement of the cognitive alterations characteristic of dementia processes, such as EA or DVa
Below we detail in detail the way of obtaining the extract of the invention (obtained from leaves and / or branches of Mangifera indicates Linn by means of high pressure techniques), the examples incorporate additional information regarding this extract
Obtaining procedure
i) Preparation of the leaves and branches of Mangifera indica Linn:
It leaves from leaves and / or branches of Mangifera indica Linn wet or dry. Drying can be done in the environment or in homo. The raw material is stored under refrigeration or freezing conditions and subsequently ground to reduce its size.
ii) Extraction of leaves and / or branches of Manqifera indica Linn with sub- or supercritical solvents:
The dried and ground branches and leaves undergo a continuous extraction process with high-pressure green solvents
As a solvent system, mixtures of supercritical carbon dioxide (C02-SC) with water and / or hydro-alcoholic and / or alcoholic solvents are used. As alcohols, methanol or ethanol are used, preferably using ethanol as a GRAS solvent (Generally Recognized as Safe) The percentage of CO2 is set between 0-100% w / w, that of water between 0-100% w / w and in the same way that of ethanol, giving rise to different solvent mixtures. The solvent system is subjected to a flow between 5 -40 g / min depending on the size of the extraction plant.
The use of C02 in the extraction process increases the efficiency of the process, reduces the extraction times, mimics the use of liquid solvents, reduces the concentration stages, and allows to preserve the properties of the bioactive compounds.
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The extraction temperature is set between a range of 35-140 ° C, considering a temperature higher than the critical point of C02 and not too high to avoid degradation of thermolabile compounds. A range between 80-100 ° C is preferably used.
The pressure is set between 10-40 MPa, considering a pressure higher than the critical point of C02, preferably in a range between 10-20 MPa.
The extraction time is in the range of 1 to 3 hours, preferably between 1.5-2 hours, for a solvent flow in a range of 5-40 g / min depending on the size of the extraction plant
The extractive process with C02-SC is carried out protected from light and in the absence of oxygen, this prevents the onset of oxidation reactions and prevents possible degradation of the phenolic compounds. Additional advantage offered by the use of C02 in the solvent system
The overall extraction yield obtained with the extractive process ranges from 1-50 g / 100g of dry matter The use of C02-SC as a single solvent allows very low extraction yields The addition of polar modifiers such as water and / or ethanol allows obtaining higher extraction yields, reaching yields of 42% when a mixture of C021 0.5: 0.5 is used as a solvent system
Extracts obtained at this stage can already be used in food and cosmetic applications because they have a minimum phenolic content between 1.93% and 0.88% of mangiferin and quercetin respectively, as well as powerful antioxidant properties with a minimum IAA of 4.0 , superior to the tocopherol IAA
iii) Obtaining dried extracts of leaves and / or branches of Mangifera Indica Linn
The sub-or supercritical extracts of Mangifera indica Linn obtained can be dried in various ways, taking into account that it is always carried out preferably at temperatures below 40 ° C. Evaporation evaporation, hydrogen drying, lyophilization, lyophilization are contemplated as forms of drying. or precipitation drying using supercritical fluids Nitrogen drying is preferably used at room temperature that prevents oxidation of antioxidant compounds
The extracts are stored refrigerated or frozen, preferably at -4 ° C, and protected from light to avoid possible changes in the phenolic content, as well as in the antioxidant / anti-radical properties of the extracts.
Table 1 shows that the predominant phenolic compounds present in the Mangifera indica Linn extract obtained by high pressure techniques were first 3-C-0-D-glucosyl iriflofenone, followed by the public acid and thirdly the mangiferin. This extract favors metabolic control, limiting body weight and maintaining activity 0-
Pancreatic, characterized by levels of peptide C This translates into sustained levels of insulin. In addition, in the CNS it reduces the inflammatory process, limits cortical and hypocritical atrophy. Tau hyperphosphorylation decreases and controls the presence of spontaneous hemorrhages. All this translates into an improvement of the cognitive alterations characteristic of dementia processes, such as those associated with metabolic disorders, AD or DVa.
Tablal. Characteristic composition of Mangifera indica Linn extract obtained by high-pressure techniques
 Compound  Phenolic content (g / 100 g extract)
 gaicic acid  6.29 ± 0.13
 3-C-P-D-glucosyl iriflophenone  14.28 ± 0.10
 3-C- (2-0-p-hydroxybenzoyl) -p-D-glucosyl iriflophenone  1.88 ± 0.07
 mangiferina  5.52 ± 0.16
 3-C- (2-6-di-0-galoyl) -P-D-glucosyl iriflophenone  2.06 ± 0.12
 tetra-O-galloyl glucose  0.10 ± 0.02
 3-D-galactosyl-quercetin  0.61 ± 0.08
 3-p-D-glucosyl-quercetin  0.97 ± 0.10
 3-O-xylosil quercetin  0.32 ± 0.02
 3-O-L-arabopyranosyl-quercetin  0.23 ± 0.01
 1,2,3,4,6-penta-O-galoyl-p-D-glucose  0.08 ± 0.01
At this point it is important to understand that dementia processes are the most serious consequence of neuronal and symptomatic loss of the elderly brain. Clinical heterogeneity and neuropathological complexity make the classification of dementia processes controversial, although the most recent studies refer to 5 large groups: AD, a-synucleopathies (dementia with Lewy bodies and Parkinsonian dementia), frontotemporal dementia, vascular dementia and mixed dementias (Raz, L, et al. The neuropathology and cerebrovascular mechanisms of dementia Journal / J Cereb Blood Flow Metab. 2015). Although each of these conditions has specific neuropathological characteristics, neuronal and syndromic loss, and cerebrovascular alterations are a common characteristic to all of them at some point in the disease (Raz. L, et al. The neuropathology and cerebrovascular mechanisms of dementia Journal / J Cereb Blood Flow Metab. 2015). It's mds. Previous studies have shown that cerebrovascular disease is common in elderly patients suffering from dementia (Gorelick. P. B., et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart
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association / american stroke association Journal / Stroke 42: 2672-2713, 2011. ladecola. C. The pathobiology of vascular dementia. Joumal / Neuron. 80: 844-866. 2013; Toledo, J. B .. et al. Contribution of cerebrovascular disease in autopsy confirmed neurodegenerative disease cases in the National Alzheimer's Coordinating Center Joumal / Brain. 136: 2697-2706. 2013) and that there is a synergy between neuron, glia and vascular cells (ladecola, C. The overlap between neurodegenerative and vascular factors in the pathogenesis of dementia. Journal / Acta Neuropathol. 120: 287-296. 2010: Zlokovic. BV Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders Joumal / Nat Rev Neurosci 12: 723-738. 2011) underlining the role of vascular pathology in neuronal damage and derived cognitive impairment. Given that dementias in general do not have successful treatment, the control of cerebrovascular alterations can be a benefit for patients suffering from AD or other dementias, as previously suggested (Toledo. JB, et al Contribution of cerebrovascular disease in autopsy confirmed neurodegenerative disease cases in the National Alzheimer's Coordinating Center. Joumal / Brain. 136: 2697-2706. 2013). In this sense and as illustrated in the examples of the invention, the extract of the invention has been successfully tested in the control of said cerebrovascular alterations that we will call in general terms as neurodegenerative diseases associated with vascular pathology of the central nervous system or who have cerebral vascular dementia.
In the context of the present invention, vascular alterations or vascular pathology mean cerebrovascular pathology of small vessel, as a consequence of the inflammatory process, presence of free radicals, endothelial rupture and / or vascular musculature that lead to spontaneous bleeding.
In the context of the present invention, dementia processes are understood as the deterioration of the learning and memory processes presented by demented patients and reproducing the experimental models presented, including cognitive alterations at the level of epic and spatial memory.
In the context of the present invention, neuroinflammatory processes are understood as the alteration of microglial activation, as a marker of central inflammation, observed in patients with AD and DVa, and which we also observe in our experimental models (APP / PS1 and db / db ).
Therefore, based on all these considerations. A first aspect of the invention relates to the use of a composition comprising a dry extract of Mangitera indica Linn, where said extract is characterized in that it comprises the phenolic components detailed in Table 1, for the preparation of a medicament for the treatment. therapeutic or prophylactic of neurodegenerative diseases associated with pathology
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Vascular system of the central nervous system or those with DVa Preferably, the present invention focuses on the prophylaxis of such diseases. Preferably, said extract is further characterized in that by means of the DPPH test (2,2-Diphenyl-1-picrilhydroxy) it has an antioxidant activity IC50 (amount of antioxidant necessary to reduce up to 50% the initial concentration of DPPH) minimum of 5, 3 ug / mL. More preferably, said extract is further characterized by being obtained or obtainable by any of the methods of obtaining detailed in the present invention, but preferably a method characterized in that it comprises the following steps
a Mangifera indica linn leaves and / or bark of the Kent variety are obtained and they are dried, frozen and ground;
b The product of step a) is subjected to a continuous extraction process in a high-pressure equipment using as a solvent system a mixture of C02 and aqueous, alcoholic or hydroalcoholic solvents at a flow of C02 in the range between 5-40 glmin g / min, where the extraction conditions are set at a temperature between 35-140 ° C and a pressure between 10-40 MPa and where the extraction is performed for a time of 1-3 hours, preferably between 1.5- 2 hours, in an inert atmosphere and protected from light, and
C. The extract obtained in step b) is evaporated at room temperature to dryness to obtain the dry extract of Mangifera indica Linn
In a preferred embodiment of the first aspect of the invention, the diseases
neurodegeneratives associated with CNS vascular pathology or with DVa
selected from the list consisting of a-synucleopathy, frontotemporal dementia or Pick's disease, DVa and mixed dementias. Preferably a-synucleopathies are selected from the list consisting of dementia with Lewy bodies and parkinsomana dementia
In another preferred embodiment of the first aspect of the invention, due to diseases
neurodegeneratives associated with CNS vascular pathology or with DVa
refer to diseases that occur with the overproduction of abnormally phosphorylated tau protein. Preferably, the EA.
A second aspect of the present invention specifies that the composition referred to in the first aspect of the invention is a pharmaceutical composition that also comprises pharmaceutically acceptable excipients and optionally other active ingredients.
A third aspect of the present invention specifies that the composition referred to in the first aspect of the invention is a food, nutraceutical or food composition.
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medicated food composition Preferably said composition is used as a food supplement.
The following examples illustrate the present invention but in some cases limit it
EXAMPLES
Example 1. Obtaining the extract
Preparation of the extract of leaves of Manaifera indica Linn obtained with a mixture CO ^ water / ethanol in a ratio of 1.0: 0.5: 0.5 as the solvent system.
The dried, frozen and ground leaves of Mangifera indica Linn of the Kent variety undergo a continuous extraction process in a high pressure Thar Tehcnologies Model SF100 100 mL capacity. A mixture of C02 / water / ethanol in a 1.0: 0.5: 0.5 ratio is used as a solvent system at a C02 flow of 5 g / min. The extraction conditions are set at a temperature of 80 ° C and a pressure of 12 MPa. The extraction is performed for a period of 3 hours, in an inert atmosphere and protected from light
After the extraction stage, the extract obtained is evaporated with nitrogen at room temperature to dryness. It is stored in freezing at -4 ° C protected from light to prevent its degradation.
The yield of the extraction for this example 1 is 38.8 g of extract / 100g dried leaves
The content of mangiferin and 3-p-D-glucosyl-quercetin in the extract of this example 1 was determined by injecting 50 pL of extract at 1000 ppm in an Agilent HPLC 1100 series equipment, with a C18 column of Thermo Electron reverse phase. A mobile phase was used as the separation method Solvent A: 2% acetic acid in water and Solvent B methanol at a flow rate of 0.9 mL / min, and with a gradient of 0-2 min, 5% B; 2-7 min, 5-25% B, 711 min, 25% B. 11-19 min, 25-32% B; 19-27 min, 32% B: 27-28 min, 32-40% B; 28-38 min, 40% B; 38-50 mm, 40-100% B, 50-60 min, 100% B; 60-70 min, 100-5% B; 5 min, 5% B isocritical, at a wavelength of 340nm
Each compound was identified according to its retention time (tr = 20min for mangiferin and tf = 34.4 min for 3-PD-glucosyl-quercetin), and quantified according to the respective calibration curve, Abs = 54.252 ■ C —100.12 for mangiferina, and
A = 54.252 • C - 100.12 A = 87.077 • C - 130.11 Abs = 87.077 ■ C- I30.il for quercetin
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The extract obtained from this example 1 had a content of 3.78 ± 0.01 g of Mangiferin / 100g of extract and 1.23 ± 0.02 g of 3- (3-D-glucosyl-quercetin / 100g of extract ( Figure 1: HPLC analysis by reverse phase column Sinergy Hidro RP for the extract obtained in example 1, with identification of the peaks of mangiferin and 3-PD-glucosyl-quercetin).
Other phenolic compounds present in the extract were also qualitatively identified by the LC-MS method. 100 pL of a dilution of the extract was injected at a concentration of 300 ppm in a negative quadrupole-ES equipment, using a Phenomenex C18 column of reverse phase Synergi 4 pm Hydro -RP 150x3mm with pre-column C18, a mobile phase of A: Water at 0.1% formic acid and B 0.1% acetonitrile of folic acid, at 0.8 mL / min, using an elucibn gradient of: 0-0.2 min, 0% B; 0.2-0.3 min, 0-7% B, 0.3-14.7 min, 7-8.5% B; 14.7-40 min. 8.5-19% B; 40-45 min. 19-33% B; 45-48 min, 33-50% B; 48-50 min, 50-95% B; 50-57 min, 95-0% B; 57-63 min, 0% B; at a wavelength of 278 nm 0-18 min, 340 nm 18-43 min, 278 nm 43-45 min, 340 nm up to 50 min. The ionization was carried out in SCAN ES negative mode, 100V fragmentor and range m / z of 50-2000 urn a The identification of the phenolic compounds was made taking into account the retention time and the ions of each compound determined by their respective spectrum of masses
In the extract obtained in this example 1, the presence of mangiferin, 3-PD-glucosyl-quercetin, public acid, methyl gallate, 3-D-galactosyl-quercetin, 3-OaL-arabinopyranosyl-quercetin and 1,2 are identified. 3,4,6-penta-O-galoyl-pD-glucose, mbs no of quercetin (Figure 2: LC-ES-MS analysis in a negative quadrupole-ES SCAN mode, fragmentor 100V and range m / z- 50 - 2000 uma for the mango leaf extract obtained in example 1)
The antioxidant capacity of the Mangifera indica Linn extract was also determined with the DPPH * test, expressed in terms of IAA Antioxidant Activity Index. This extract from Example 1 presented a potent antioxidant activity with an IAA value = 4.84, higher than the Tocopherol IAA = 3.65.
Example 2. Definition of the extract.
Characterization of the sub- or supercritical extracts of Mangifera indica Linn
The extracts of Mangifera indica Linn are characterized by temendo considering their phenolic content and their antioxidant capacity, to determine their possible use in the cosmetic and food industry
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i) Determination of the fendolic content of the sub- or supercritical extracts of Mangifera indicates Linn:
The determination of the fendolic content of the extract is carried out by means of high performance liquid chromatography HPLC (High Performance Liquid Chromatography), in an Agilent HPLC 1100 series (Agilent Technologies, United States), with a reverse phase C18 column (Thermo Electron Corporation) and a UV / vis detector.
The separation method uses as a mobile phase a solution of acetic acid in 2% water (solvent A) and methanol (solvent B), at a flow rate of 0.9 mL / min, an injection volume of 50 pmL, and using the following elution conditions: 0-2 min, 5% B isocratic; 2-7 min, linear gradient 5-25% B; 7-11 min, 25% B isocratic; 11-19 min, linear gradient 25-32% B, 19-27 min, 32% B isocritical; 27-28 min, linear gradient 32-40% B; 28-38 min, 40% B isocratic and finally a washing and reconditioning stage of the column is included (38-50 min, linear gradient 40-100% B, 50-60 min, 100% B isocritical; 60-70 min , linear gradient 100-5% B, and 5 min, 5% B isocritical)
Fendilic compounds are detected at a wavelength of 340 nm according to their retention time, and are quantified using calibration curves for the corresponding patterns A standard chromatogram of the majority fendyl compounds present in mango, mangiferin and 3 extracts -PD-glucosyl-quercetin is shown in Figure 1 HPLC analysis by Sinergy Hidro RP reverse phase column for the fendolic extract obtained in example 1, with identification of the chromatographic peaks corresponding to the mangiferin and 3-PD-glucosyl compounds -quercetin according to its retention time (tf = 20min for mangiferin and tr = 34.4 min for 3-pD-glucosyl-quercetin).
The calibration curves of the major compounds present in the extracts, mangiferin and 3-p-D-glucosyl-quercetin, are expressed as Abs = 54,252 • C - 100.12, and
A = 54.252 • C - 100.12 A = 87.077 * C - 130.11 Abs = 87.077 • C- 130.11,
respectively Where C is the concentration expressed in pg / ml For each sample triplicate measurements are made
Finally, the extraction performance of the fendil compounds is expressed in terms of g / 100g dry extract. Mangifera indica Linn extracts obtained according to the extraction procedure, have a minimum content in mangiferin and quercetin of 1.93% and 0.88% respectively
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However, the antioxidant activity of the extracts does not depend solely on the high content of these two compounds, but on the synergy between the different fendolic compounds extracted during the process
ii) Qualitative determination of the fendolic compounds present in the sub- or supercritical extracts of Mangifera mdica Linn:
To confirm that the extracts contain mangiferin and 3-PD-glucosyl-quercetin, as well as the presence of other fendil compounds, a qualitative analysis is performed by liquid chromatography coupled to LC-MS mass spectrometry using a Quadrupole-ES mass detector (Quadrupolar -ionization time-of-flight mass spectrometry).
For this analysis, commercial patterns of glycolic acid, methyl gallate, mangiferin, 3-D-galactosyl-quercetin, 3-PD-glucosyl-quercetin, 3-OaL-arabinopyranosyl-quercetin, 1,2,3,4,6 -penta-O-galloyl-PD-glucose, and quercetin In order to optimize the ionization conditions, the patterns were injected individually, one by one, directly into the negative ionization mass spectrometer ES (Electro Spray) with a m / z range of 50-2000 uma A mobile phase consisting of a 0.1% solution of water of the thermal acid and a 0.1% solution of acetonitrile of a thermal acid, in isocritical mode 50:50, was used at a flow rate of 1, 0 mL / min, and an injection volume of 10pL,
The following table shows the masses of the corresponding ions for each pattern of phenolic compound (m / z) obtained from the corresponding LC-ES-MS analysis for each pattern. (Figure 2: Mass spectra of fendolic compounds present in Mangifera indica Linn extracts made in quadrupole-ES negative LC-ES-MS SIM mode |
Sedates LC-ES-MS [M-H] ‘(m / z)
 Phenolic compound  m / z [M-H] tr (min)
 Gallic acid  169 4.06
 methyl gallate  183 10.13
 mangiferina  421 25.94
 3-D-galactosyl-quercetin  463 38.81
 3-p-D-glucosyl-quercetin  463 39.66
 3-O-L-arabopyranosyl-quercetin  433 42.39
 1,2,3,4,6-penta-O-galoyl-P-D-glucose  939 43.50
 quercetin  301 47.86
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Subsequently, anbysis was performed by liquid chromatography coupled to HPLC-MS mass spectrometry to separate and identify the different phenolic compounds. An LC-ES-MS was used with a Phenomenex C18 reverse phase Synergi 4 pm Hydro-RP 150x3mm column with pre-column. C18 As the mobile phase, a solution A of 0.1% water of phosphoric acid and a solution B of 0.1% acetonitrile of thermal acid was used, at a flow rate of 0.8 mL / min, and using a gradient elucibn of: 0-0.2 min, 0% B, 0.20.3 min, linear gradient 0-7% B; 0.3-14.7 min, linear gradient 7-8.5% B; 14.7-40 min, linear gradient 8.5-19% B, 40-45 min, linear gradient 19-33% B; 45-48 min, linear gradient 33-50% B, 48-50 min, linear gradient 50-95% B and finally a washing and reconditioning stage of the column (50-57 min, linear gradient 95-0% B; 57-63 mm, 0% B), at a fixed wavelength at t = 0 min 278 nm, t = 18 min at 340 nm, t = 43 min at 278 nm, t = 45 min 340 nm, an injection volume of 20pL. As a detector, a quadrupole is used in SIM ES Negative mode and fragment windows of the selected ions of the mass spectra of the standards: 0-7 min: 100V (169,125), 7-18 min: 120V (183,124), 18-33 min: 150V (421.301) 33-41.1 min: 160V (463.301.300), 41.1-43 min: 150V (433.301.300), 43-45 min: 240V (939.769.617), 45-63 min: 150V ( 301,151,179).
A type chromatogram of a mixture of the standards is shown in Figure 3: Analysis LC-ES-MS and LC-UV for the patterns of possible phenolic compounds present in the sub- or supercritical extracts of Mangifera indica Linn
Hi) Determination of antioxidant activity with the radical a-a-difeml- (3-picnhydroxy (DPPH) of the sub- or supercritical extracts of Mangifera indica Linn.
To measure antioxidant activity with the radical aa-diphenyl- (B-picrilhidrazil (DPPH *) (Brand Williams et al. (1995) Use of a free radical method to evaluate antioxidant activity Lebensmittel. Wissenschaft. Und Technologie 28. 25-30 ), the decrease in absorbance at 515 nm, every two minutes until steady state, of a solution of 3.9 mL of DPPH 6 10-5 M in methanol and 10 pL of the antioxidant extract is measured The extract is evaluated at different concentrations between 0 and 5000 ppm To obtain the reference of an antioxidant, the antioxidant activity of the commercial pattern of atocopherol is also evaluated.
The concentration of DPPH * (CDpph) in the reaction medium is calculated from the calibration curve determined by linear regression with the following equation:
.-tbs = 12.709 • CDPPH - + - 0.002
The percentage of remaining DPPH * is calculated according to the following equation:
% DPPH remaining = C0PPllT / CDppHti x 100
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The EC50 or efficient concentration that inhibits 50% of the DPPH radical is calculated graphically using a nonlinear regression of the concentration vs. curve. % DPPH remaining at steady state The antioxidant activity of the extracts is expressed as the Index of Antioxidant Activity (IAA) that is calculated considering the final concentration of DPPH * and EC50 as expressed by the following equation:
IAA = DPPH Final Concentration (^) / ^ so (^)
Plant extracts have low antioxidant activity when IAA <0.5, moderate when IAA is between 0.5 and 1.0, potent when IAA is between 1.0 and 2.0, and very strong when IAA> 2 , 0
The extracts object of this patent have a minimum IAA value of 4.0, and higher than the IAA value obtained for a-tocopherol (AAI = 3.65). This indicates the potent antioxidant activity of Mangifera indica Linn sub- or supercritical extracts, and their potential use as an antioxidant in food and cosmetics. In any case, these extracts may also present other interb activity / properties for the food, dietary industry. , cosmetics and pharmaceuticals that will also be included in this invention. Thus, for example, it is expected that extracts containing high content of mangiferin and quercetin will present activities described for isolated molecules such as anticancer, antibacterial, antifungal, or certain preventive capacity of neurological or cardiac diseases
Example 3. Effect of FGM treatment on episddic memory
As shown in Figure 4, the chronic treatment with the extract of MGF (50mg / kg, pelletized in food) obtained according to the procedure described (hereinafter MGF), meant an improvement in the cognitive processes of animals db / db treated Adembs, in a classic model of EA, such as the mouse APP / PS1, we also observed a significant improvement of cognitive processes after chronic treatment with MGF extract between 6 and 26 weeks of life These tests began at 24 weeks of age, they were performed consecutively, and concluded at 26 weeks of life, immediately before the animals were slaughtered. We assessed the episbical memory through the NOD test. The animals went through a habituation phase to the record box (length 22cm x 44cm wide x 40cm high) on day 1, followed by a phase of habituation to objects (which were not used during the test, on day 2). On day 3 the acquisition phase was carried out and for 5 minutes the animal was placed in the box with 4 blue balls in triangular arrangement. After 30 minutes of rest the animal returned to the box for 5 minutes, this time with 4 red cones arranged in a rectangle. The test took place 30 minutes later and the
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The animal was placed in the box with two copies of the object of the second test sample (recent objects), placed in the same position and two copies of the objects of test sample 1 (old objects) placed one of them in the same position (old object not displaced) and the other in a new position (old object displaced). Integrated episbdic memory was analyzed for “qub", '' dbnde "and" cubndo "(Dere. £., Et at. Episode-like memory in mice: simultaneous assessment of object, place and temporal order memory Joumal / Brain Res Brain Res Protoc 16: 10-19, 2005) '' Qub "is defined as the difference in the scan time of recent and known objects," dbnde "is defined as the difference in the scan time of displaced and non-displaced objects and "cubing" is defined as the difference in the time of exploration of the old objects not displaced and the recent objects not displaced. After the analysis of the results, we observed that the cognitive deterioration of the db / db animals improved after the treatment with FGM. , and these differences were significant in the case of the "qub" and "covering" paradigms (Figure 4A). Similarly, the APP / PS1 mice treated with FGM improved significantly in the NOD, after the chronic treatment with FGM (Figure 4B).
Example 4. Effect of FGM treatment on learning and spatial and working memory.
The animals were also evaluated in a clinical test to check the learning capacity and spatial and working memory. The Morris Aquatic Labyrinth Test This test began 24 hours after the conclusion of the NOD test. The labyrinth consists of a water tank of 0.95 m in diameter, divided into four quadrants marked with geometric figures on the walls. The escape platform is located 1-2 cm below the surface and is camouflaged with calcium carbonate dissolved in water, at a Ta of 21 +/- 1 ° C. The animal learns the location of the platform following the geographic tracks of the Pool exterior. The test is carried out in two phases The acquisition consists of 4 daily tests for 4 consecutive days with the submerged platform During this phase, the platform is located in quadrant 2 The animal swims freely for a maximum time of 60 seconds, with a 10min interval between trials If you cannot find the platform, place it on it for 10 seconds. The time taken and the distance traveled to reach the platform are recorded and analyzed with SMART software (Letica, Espafla). In the db / db animals treated with FGM, we observed a reduction in the time they needed to find the hidden platform during the 4 days of training, in order to improve the learning capacity of these animals (Figure 4C). Similarly, the spatial learning problems of mice
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APP / PS1 improved significantly over the 4 days of the experiment, after treatment with FGM (Figure 4D).
In the retention phase. Remove the platform, let the mouse swim for a minute and measure the time and distance traveled in each quadrant. The retention phase will take place at 24 hours. and once more at 72 hours, after the last acquisition trial. In this case we also observe that the memory problems presented by db / db mice, spending significantly less times in the quadrant in which the platform was previously located. they were recovered in db / db mice treated chronically with FGM (Figure 4E) Similarly, when we analyze APP / PS1 animals in the retention phase. We observed an improvement in those treated with FGM (Figure 1F)
Example 5. Effect of FGM treatment on cerebral atrophy
Chronic treatment with FGM limits the cerebral atrophy observed in db / db mice, when we compare the brain weights of the animals under study after slaughter (Figure 5A). In order to deepen the possible impact of different structures, we proceeded to perform a violet crescent tincibn in all the study groups. After fixation in parformaldehldo to 4% of the left hemisphere for 2 weeks, we performed coronal sections for histomorphological studies. Coronal codes were made from 30 pm to 1.5, 0 5, -0 5, -1 5, -2 5 and -.3 5 mm Bregma, with the help of a histological atlas. The sections were mounted in gelatinized slides, subsequently immersed in 70% ethanol for 15 minutes, to pass them to a 0.5% violet cresil solution for 10 minutes. After washing in distilled water, they were immersed in a solution of absolute ethanol and acetic acid (0 25%) for 7 minutes. Finally, successive washes were performed in ethanol and xylol, and the sections were mounted with DPX. These sections were photographed and analyzed in an Olympus Bx60 microscope (Japan) coupled to an Olympus DP71 camera. From the images the total pitch of each section was quantified. as well as the breasts of the cortex, hippocampus and ventricle. All measurements were performed using Adobe Photoshop Elements and Image J software. In the db / db animals treated with FGM we observed the reversal of cortical atrophy, typical of db / db mice (Figures 5B and 5C). To a lesser extent, we observed a similar profile in the hippocampus of animals treated with FGM chronically (Figure 2C). The apoptosis rate was quantified by analyzing caspase activity 3/7. Homogenates of bark or hippocampus were used in which caspase activity was measured by the caspase-Glo3 / 7 test (Promega, Madrid Espafia), according to the manufacturer's instructions Briefly, 5-7 mg of tissue were homogenized in PBS supplemented with a cocktail of proteases (sigma USA) and diluted to 2pg / pL. 50 pi of Promega caspase glo 3/7 solution were added and the
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Samples were incubated for 1 hour at room temperature and protected from light. The lumimscence was measured in a Biotek Synergy Mx microplate reader and the data were expressed as a percentage of control values. As shown in Figure 5D, activity increases in db / db mice and treatment with FGM reduces caspase activity in the cortex and in the hippocampus. The reduction observed in the cortex is statistically significant.
Example 6. Effect of FGM treatment on tau hyperphosphorylation
The tau protein is necessary for microtubule stabilization and therefore for neuronal integrity. When hyperphosphorylated, as in different dementia processes: AD, frontotemporal dementia, dementia with Lewy bodies, or in CNS alterations associated with diabetes, the neuronal integrity is compromised From which we proceed to determine the levels of abnormally phosphorylated tau protein in all the groups under study Tau phosphorylation, as a marker of neuronal damage, was evaluated by western blotting in the cortex. The tissue was homogenized in lysis buffer (Cell Signaling, USA) supplemented with a cocktail of inhibitors of phosphatases and proteases (Sigma, USA). The homogenate was sonicd and centrifuged at 4 ° C for 5 min to subsequently collect the supernatant. The amount of protein was determined by the Bradford method (Biorad, Germany). Proteins were separated on a 10% acrylamide-bisacrylamide gel followed by a PVDF membrane transfer (Schleicher & Schuell, Keene, NH). The membranes were blocked with blocking buffer (Invitrogen, USA) for 1 hour and incubated. overnight at 4 ° C with primary antibodies (AT8 ion for phospho-tau or tau-total) diluted 1.1000 in blocking solution. The membranes were washed and incubated with the secondary antibodies. Finally, the membranes were washed and the serial was detected using the chemilumimscent substrate Novex AP (Invitrogen, USA) and a Kodak Biomax Light Film development film (Sigma, USA) Immunoblots were quantified by calculating the optical density of each protein band of the films scanned using Image J software. Each band was normalized by actin protein The results were expressed as ratio hyperphosphorylated tau protein levels / total tau levels. When comparing the groups under study, we observed that the increase in tau hi Perfosphorylated in db / db animals was controlled in animals treated chronically with FGM (Figures 6A and 6B) Similarly, tau phosphorylation decreased in APP / PS1 mice, although this decrease was not statistically significant (Figure 3C ).
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Example 7. Effect of FGM treatment on spontaneous hemorrhages in the CNS
To verify the effect that chronic treatment with FGM has on vascular pathology, we carry out a quantification of hemorrhages by means of blue tintibn. Prussia 6 sections of each animal attached to those used for cresyl violet staining were mounted on a gelatinized portal (coronal codes of 30 pm to 1.5, 0 5, -0.5, -15, -2.5 and -.3.5 mm of Bregma, with the help of a histological atlas). The sections were dehydrated and incubated in Prussian blue solution (20% HCI and 10% potassium ferrocyanate) for 30 minutes. They were rinsed with distilled water and rehydrated in phosphate buffer. Once dried, they were immersed in a neutral red solution (1% in 1% acetic acid) for 5 minutes. The tissue was rinsed and dehydrated with increasing concentrations of ethanol. Finally they were immersed in xylol and we covered them with DPX. The sections were photographed with an Olympus DP71 camera attached to an Olimpus Bx60 microscope (Japbn). The images were analyzed using Photoshop Elements and Image J software to quantify the number of hemorrhages / mm2, the size of each hemorrhage and the hemorrhagic load (% of pitch occupied by hemorrhages) in cortex and hippocampus. In the cortex we observed that the treatment with FGM decreases the hemorrhagic load (cortical pitch affected by hemorrhages) in db / db mice and this effect is due to the decrease in the number of new hemorrhages, while their size remains unchanged (Figures 4A and 4B). A similar profile is observed in the hippocampus of the db / db mice treated with FGM, in which we observed some reduction in the hemorrhagic load, as a consequence of the decrease in the number of hemorrhages, while the size remained unchanged (Figure 4C). In APP / PS1 mice no spontaneous hemorrhages were found
Example 8. Effect of FGM treatment on the inflammatory process in the CNS
Inflammation is closely related to DM2 and also in the brains of patients with brain lesions or dementias, such as Alzheimer's, the inflammatory process has increased. Other studies have also shown that inflammation in the CNS may be involved in dysfunction of the blood-brain barrier and contribute to vascular damage. Therefore, as inflammatory markers in the CNS, we carried out immunostains for microglia in the cortex and hippocampus. To do this, sections 30 pm thick were taken, contiguous to the previous sections. The sections were blocked with sine albumin. 3% bovine (Sigma Aldrich) 5% in PBS with 0 5% Tritbn X. Subsequently, they were incubated with the anti-microglia antibody IBA-1 (1: 1000, Sigma Aldrich) After washing in PBS, the sections were incubated with the secondary antibody conjugated with Alexa Fluor anti-Rabbit (Molecular Probes, USA) at a concentration
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1: 1000 Finally, the sections were mounted with GVA mounting solution (Invitrogen, USA) in gelatinized slides and photographed with an Olympus U-RFL-T Laser fluorescence microscope (Olympus, Japan). The images were acquired using the MMIcellTools software and analyzed with Image J to quantify the number of cells / mm2, the individual size of the hemorrhages and the microglial load. In the cortex of db / db mice we observed a significant increase in microglial load (% area covered by microglia), as previously described, and this effect was significantly diminished in db / db mice treated with FGM chronically (Figures 7A and 7B). Although we observed some reduction in the number of microglial cells / mm2 in the db / db-FGM animals, the effect was especially significant when we compared the size of the individual cells. In this case we observed that the size of the microglia in the db / db animals It was significantly increased, indicative of the process of microglial activation and associated inflammation. However, after treatment with FGM we observed that the size of the microglia returned to control values (Figures 8A and 8C). A similar profile is observed in the hippocampus, where treatment with FGM is able to control the increase of microglial load in the hippocampus of db / db mice (Figure 5B). Similarly, this process is mediated by a slight decrease in the number of microglial cells / mm2 and a significant decrease in their size (Figure 5B).
In APP / PS1 animals, we observed that the microglial load increased in the cortex, indicative of the process of microglial activation and associated inflammation. Treatment with manguiferin significantly reduced the microglial load in the cortex. This effect was detected in PS-free areas and despite the fact that a similar reduction could be observed near the plates, it was not statistically significant. The reduction of the microglial load was due to a reduction in the number of microglial cells away from the plates. The size of the cells was maintained between the different groups. it was observed in the hippocampus that the microglial load was reduced in the mice treated with FGM. These differences were statistically significant away from the senile plaques, and although a similar profile could be observed in the vicinity of the plaques, the differences did not reach be statistically significant As in the cortex, the reduction of The microglial load was due to the small number of microglial cells. These results indicate that in the two models of neurodegenerative pathologies used, treatment with FGM reduces the inflammation induced by the neurodegenerative process.

权利要求:
Claims (3)
[1]
1. Use of a composition comprising a dry extract of Mangitera mdica Linn, where said extract is characterized in that it comprises the phenolic components detailed below;
 Compound  Phenolic content (g / 100 g extract)
 Gallic acid  6.29 ± 0.13
 3-C-3-D-glucosyl iriflophenone  14.28 ± 0.10
 3-C- (2-0-p-hydroxybenzoyl) -p-D-glucosyl iriflophenone  1.88 ± 0.07
 mangiferina  5.52 ± 0.16
 3-C- (2-6-di-0-galoyl) -P-D-glucosyl iriflophenone  2.06 ± 0.12
 tetra-O-galloyl glucose  0.10 ± 0.02
 3-D-galactosyl-quercetin  0.61 ± 0.08
 3-3-D-glucosyl-quercetin  0.97 ± 0.10
 3-O-xylosil quercetin  0.32 ± 0.02
 3-O-L-arabopyranosyl-quercetin  0.23 ± 0.01
 1,2,3,4,6-penta-O-galoyl-p-D-glucose  0.08 ± 0.01
for the preparation of a medicament for the prophylactic or therapeutic treatment of neurodegenerative diseases associated with vascular pathology of the central nervous system or with cerebral vascular dementia.
[2]
2. The use of the extract of claim 1, wherein said extract is further characterized in that its antioxidant activity by means of the DPPH test (2,2-Diphenyl-1- picrilhydrole) has an IC50 (amount of antioxidant necessary to reduce to 50 % initial concentration of DPPH) minimum of 5.3 ug / mL
The use of the extract according to any one of claims 1 to 2, wherein said extract is further characterized by being obtected or obtained by a method characterized in that
a Mangifera indica Linn leaves and / or bark of the Kent variety are obtained and dried, frozen and ground;
b The product of step a) is subjected to a continuous extraction process in a high pressure equipment using a mixture of CQ2 as solvent system and
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aqueous, alcoholic or hydroalcoholic solvents at a flow of C02 in the range between 5-40 glmin g / min, where the extraction conditions are set at a temperature between 35-140 ° C and a pressure between 10-40 MPa and where the extraction is carried out for a time of 1-3 hours, preferably between 1, 5 -2 hours, in an inert atmosphere and protected from light, and
C. The extract obtained in step b) is evaporated at room temperature to dryness to obtain the dry extract of Mangifera indica Linn
4 The use according to any one of claims 1 to 3, where neurodegenerative diseases associated with CNS vascular pathology or with DVa are selected from the list consisting of a-synucleopathies, frontotemporal dementia or Pick's disease, vascular dementia and dementias mixed
[5]
5. The use according to claim 4, wherein the a-synucleopathies are selected from the list consisting of dementia with Lewy bodies and Parkinsonian dementia
6 The use according to any one of claims 1 to 3, where neurodegenerative diseases associated with CNS vascular pathology or with DVa refer to diseases that occur with the overproduction of abnormally phosphorylated tau protein
7 The use according to claim 6, where EA is selected for diseases that occur with the overproduction of abnormally phosphorylated tau protein.
The use according to any one of claims 1 to 7, wherein said composition is a pharmaceutical composition that also comprises pharmaceutically acceptable excipients.
The use according to any one of claims 1 to 7, wherein said composition is a food composition, nutraceutical or a medicated food composition.

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公开号 | 公开日

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ES2630739B1|2018-06-06|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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ES2464192B1|2012-11-30|2015-06-11|Universidad De Cádiz|Phenolic extracts of Mangifera indica Linn, procedure for obtaining and uses.|

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ES201600138A|ES2630739B1|2016-02-16|2016-02-16|Use of Mangifera indica mango leaf extract, for the treatment of neurodegenerative diseases|ES201600138A| ES2630739B1|2016-02-16|2016-02-16|Use of Mangifera indica mango leaf extract, for the treatment of neurodegenerative diseases|