A mixture of high molecular weight compounds obtained from sugarcane wax (saccharum officinarum l.)

Abstract

To provide a method for producing a mixture of high-molecular weight compounds obtained from sugarcane wax and having neuroprotective action and antioxidant action.SOLUTION: Saponifying sugarcane wax with an alkaline or alkaline-earth hydroxide, subsequently subjecting the saponified wax obtained to extraction with CO2 in supercritical conditions, subjecting both the alcohol and aldehyde fraction extracted with CO2 and the mixture of residual salts to a purification step with hexane and acetone, analyzing the fractions obtained by gas chromatography and spectrophotometry in order to measure and determine the content of salts, alcohols and aldehydes, and then mixing in the proportions required to achieve the specific composition of the mixture.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the technical field of the food and pharmaceutical industries by providing a mixture of high molecular weight substances (HMWS) with antioxidant and neuroprotective properties that can be used as an active ingredient (AI) in nutritional supplements or pharmaceutical preparations, said AI being obtained from sugarcane wax (Saccharum officinarum L.) and containing purified salts of fatty acids, fatty alcohols and high molecular weight aldehydes in specific ratios that ensure a synergistic effect, where the pharmacological effect of the mixture described in the present invention is greater than the sum of the pharmacological effects of the group of compounds that compose it.

[0002] Because several diseases related to the nervous system affect the quality of life and can lead to the death of patients, the need for new, safe, and effective neuroprotective therapeutic approaches is a current issue. Among the many diseases affecting the cells of the nervous system, the present inventors focus on spinal muscular atrophy, primary lateral sclerosis, progressive spinobulbar muscular atrophy, and amyotrophic lateral sclerosis. The latter is the best-known and most dangerous neurodegenerative disease due to its fatal prognosis. This condition is widely known in the public sphere for having caused the deaths of famous personalities such as scientist Stephen Hawking, athletes Lou Gehrig, Gianluca Signorini, and Stefano Borgonov. It is characterized by the gradual loss of function and eventual death of neurons responsible for muscle movement, resulting in progressive muscle paralysis.

[0003] Taking into account the fact that neurological disorders affect the quality of life, some of which may cause the death of patients and constitute a major health problem on a global scale, and that existing conventional treatments are unable to cure them or prevent their progression to the desired extent, it is necessary to search for new effective neuroprotective agents for the prevention and treatment of these medical conditions.

[0004] Meanwhile, the search for antioxidants has also become a hot topic. The production of reactive oxygen species and free radicals typically occurs during cellular metabolism and, under normal physiological conditions, is repaired by antioxidant systems that maintain redox balance and cell survival. Nevertheless, several factors, such as exposure to pollutants and the environment, lifestyle, and various pathologies, can alter this balance, resulting in an excess and accumulation of free radicals, which can cause oxidative stress (OE) (Poljsak, 2011). OE has been shown to play an important role in the development and progression of various chronic degenerative pathologies, such as atherosclerosis, cancer, heart disease, and neurotrophic disorders (Maldonado, 2010; Farlane, 2004; Guichardant, 2009). Therefore, the consumption of antioxidants can prevent or reduce the deterioration of cells and functional groups in living organisms caused by OE, further benefiting the prevention of these conditions (Oxilia, 2010). [Background technology]

[0005] Although there are several patents and publications related to obtaining extracts from sugarcane wax (S. officinarum L.), the preparation of mixtures with neuroprotective and antioxidant effects has not been disclosed, nor have the compositions shown in this invention been shown to have such effects. Most of the published studies have presented the preparation of pure mixtures of alcohols for the treatment of conditions related to lipid profiles, platelet hyperaggregability, and coronary conditions. For these same purposes, mixtures of alcohols with fatty acids (predominantly fatty alcohols), pure mixtures of fatty acids, and combinations of these substances with different drugs have also been prepared with the aim of enhancing the above-mentioned pharmacological effects.

[0006] In the first published study on this topic, we found that saponification of sugarcane wax and extraction with organic solvents yields a mixture of fatty alcohols (≥90%) that is effective in treating atherosclerotic complications such as hypercholesterolemia, platelet hyperaggregability, ischemia, and thrombosis, while preventing gastric ulcers and improving sexual activity in men (Laguna, US5856316). The significant pharmacological effects exhibited by this mixture have motivated the obtaining from this wax of other mixtures with a high content of fatty alcohols for these same pharmaceutical purposes (Almagro, US20070295326A1, Ribeiro BRPI0702137A and Matkin, US20060013842 A1), as well as other techniques such as fluid extraction in supercritical conditions (ShintakuBRPI0701341A2), multiphase hydrolysis extraction (Somaiya 1058 / MUM / 2005) and reaction of wax with hydrogen in the presence of a catalyst (Somaiya WO2010103549).

[0007] Other extracts and mixtures have also been obtained from sugarcane wax with the aim of providing pharmaceutical products with hypocholesterolemic, cardioprotective and antiplatelet properties, such as those proposed by Gonzales and coworkers (US 6,486,205 B2) formed by fatty acids of 26 to 36 carbon atoms; Kutney and Wessman (US 20050234025), who propose the combination of at least one alcohol or fatty acid of this wax with a sterol and / or a derivative of ascorbic acid (without specifying the ratio between these substances); and those proposed by Matkin and coworkers (US 20060013842), who propose a mixture of a minimum of 60% fatty alcohol, a maximum of 40% fatty acid and possibly salicylic acid, which may also be obtained from other waxes, animals or plants. Similarly, extracts containing alcohols and other fatty substances are obtained from other natural waxes, such as beeswax, sorghum and millet, which reflect the same pharmacological effects on lipid profiles mentioned above (Perez EP1189605B1 and Hargrove et al. US20060127449).

[0008] Regarding the use of fatty acids in neuroprotective compositions, the inventors have found that Miller (US20110280852A1) proposed activating unsaturated fatty acids between 18 and 22 carbon atoms as nitroacids or ketoacids, and Paquin and Russell (US20020128316 and US20090280199) proposed the use of triglycerides (or their free acids) and omega-3 fatty acids mixed with other substances, respectively. In any case, neuroprotective and antioxidant compositions consisting of mixtures of fatty acids having more than 26 carbon atoms in specific proportions in the form of salts, which also contain specific proportions of high molecular weight fatty alcohols and aldehydes, have not previously been disclosed. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US5856316 [Patent Document 2] US20070295326A1 [Patent Document 3] BRPI0702137A [Patent Document 4] US20060013842 A1 [Patent Document 5] BRPI0701341A2 [Patent Document 6] WO2010 / 103549 [Patent Document 7] US6486205 B2 [Patent Document 8] US20050234025 [Patent Document 9] US20060013842 [Patent Document 10] EP1189605B1 [Patent Document 11] US20060127449 [Patent Document 12] US20110280852A1 [Patent Document 13] US20020128316 [Patent Document 14] US20090280199 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, neurological disorders are serious health problems that affect the quality of life and may lead to death, but existing conventional treatments cannot cure them or prevent their progression to the desired extent. Therefore, it is necessary to search for new and effective neuroprotective agents for the prevention and treatment of these medical conditions. Meanwhile, since there are several factors that cause excess and accumulation of free radicals, which affect the onset and progression of various chronic degenerative pathologies, the search for antioxidants is also necessary and is a current issue. [Means for solving the problem]

[0011] As a possible solution to this problem, the subject of the present invention is a mixture of high molecular weight substances (HMWS) with antioxidant and neuroprotective properties. This mixture can be used as an active ingredient (AI) in nutritional supplements or pharmaceutical preparations. The AI ​​contains purified salts of fatty acids, fatty alcohols, and high molecular weight aldehydes obtained from sugarcane (Saccharum officinarum L.) wax in specific ratios that ensure a synergistic effect, and the pharmacological effect of the mixture described in the present invention is greater than the sum of the individual pharmacological effects of the group of compounds that make it up.

[0012] (Summary of the Invention) The AIs presented as subject matter of the present invention are characterized by containing the following groups of compounds in specific proportions: fatty acids (60-80% by weight), fatty alcohols (10-30% by weight) and high molecular weight aldehydes (10-30% by weight). The main components, fatty acids, are present as alkali or alkaline earth salts in the following proportions: C26:0 = 0.3-5%, C27:0 = 0.3-5%, C28:0 = 20.0-40.0%, C29:0 = 1.0-3.0%, C30:0 = 12.0-25.0%, C31:0 = 0.5-2.0%, C32:0 = 5.0-15.0%, C33:0 = 0.5-3.0%, C34:0 = 5.0-18.0%, C35:0 = 0.3-1.5%, C36:0 = 1.0-8.0%. The primary aliphatic alcohols present in the mixture are homologous compounds having between 24 and 34 carbon atoms, of which 1-octacosanol (C28) is preferred, and the aldehydes are unsaturated αβ having more than 48 carbon atoms. This new AI can be formulated for oral administration in solid form (tablets, capsules) or liquid (suspension), for which it is mixed with excipients accepted by the pharmaceutical industry.

[0013] It should be noted that the proportions of high molecular weight substances found in the subject matter of the AI ​​of the present invention are not naturally achieved by a simple extraction and purification process, but are the result of mixing specific amounts of extracted and purified fractions of each substance group, depending on the purity obtained from each fraction. Thus, the final composition that is the subject matter of the present invention is the result of a standardization process in which the content of each group of compounds is brought to the specific concentrations and proportions described herein, in order to ensure a synergistic effect between the components and the desired pharmacological effect, as determined by pharmacological studies.

[0014] In this regard, the potent pharmacological effect of the composition is far superior to the effect of administering the three groups of substances of the present invention separately, which is confirmed in the examples and demonstrates the occurrence of a synergistic effect, which could not be predicted from the state of the art. Thus, the subject AI of the present invention significantly exceeds the pharmacological antioxidant effects of both the fatty acid extract and the extract of fatty alcohols and high molecular weight aldehydes, and exhibits a potent neuroprotective effect not observed for any of the three substances that make up this IA (AI?).

[0015] The procedure for obtaining the desired pharmaceutical composition of the present invention is characterized by an initial step of hydrolysis of raw or refined cane wax using alkali or alkaline earth hydroxides, followed by a selective extraction process of alcohols and aldehydes, and purification steps of the remaining salts, as well as the alcohol and aldehyde fractions, all of which are carried out using organic solvents or CO2 in supercritical state, with final standardization of the content of the three groups of substances whose specific proportions are described, ensuring the desired pharmacological effect. [Effects of the Invention]

[0016] The advantage of this composition over other existing compositions (also obtained from sugarcane wax) is that it has greater efficacy as an antioxidant and a neuroprotective effect not present in any of the previous compositions. The present invention has industrial applicability and is unique in that the composition is novel and cannot be deduced from the state of the art that a mixture of three groups of substances in the specified ratios and concentrations will exhibit the pharmacological benefits and synergistic effects. The present invention contributes to the food and pharmaceutical industries, as the resulting composition can be used as a dietary supplement due to its beneficial effects against oxidative stress, and as a medicine to prevent neurological diseases and those related to oxidative stress disorders. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0017] Preparation of a mixture of high molecular weight substances (HMWS) with neuroprotective and antioxidant properties: 1 kg of unrefined cane (sugarcane stalk) wax was subjected to a basic hydrolysis process using potassium hydroxide, and then the saponified wax was subjected to extraction with CO2 under supercritical conditions. Both the CO2-extracted alcohol and aldehyde fraction and the residual salt mixture were subjected to a purification process using hexane and acetone. The resulting fractions were analyzed by gas chromatography and spectrophotometry to determine the salt, alcohol and aldehyde content and then mixed in the proportions required to achieve the following: 76% fatty acids as potassium salts, 13% fatty alcohols and 11% high molecular weight aldehydes; where the fatty acid proportions were C26:0 1.5%, C27:0 1.4%, C28:0 30.6%, C29:0 1.2%, C30:0 13.5%, C31:0 1.0%, C32:0 8.3%, C33:0 1.7%, C34:0 11.6%, C35:0 1.4% and C36:0 3.8%. [Example]

[0018] Preparation of a mixture of high molecular weight substances (HMWS) with neuroprotective and antioxidant properties: 1 kg of refined cane wax was subjected to basic hydrolysis process with calcium hydroxide, then the saponified wax was subjected to extraction with hot hexane, the organic fraction was cooled and the recrystallized solid was subjected to process purification by successive washing with ethanol & acetone. Both the extracted and purified fractions and the remaining salt mixture were analyzed by gas chromatography and spectrophotometry to determine the salt, alcohol, and aldehyde content and then mixed in the proportions required to achieve the following: 70% fatty acids as calcium salts, 19% fatty alcohols, and 11% high molecular weight aldehydes, where the fatty acids were in the following proportions: C26:0 1.2%, C27:0 1.1%, C28:0 26.3%, C29:0 1.1%, C30:0 15.3%, C31:0 0.7%, C32:0 6.9%, C33:0 1.5%, C34:0 11.4%, C35:0 1.0%, and C36:0 3.7%. [Example]

[0019] Evaluation of neuroprotective and antioxidant effects in a cerebral ischemia-induced model: To evaluate the neuroprotective effect of the mixture of high molecular weight substances of the present invention, an experimental model of global cerebral ischemia caused by carotid artery occlusion and reperfusion was used. The composition of high molecular weight substances (HMWS) obtained in Example 1 was subjected to a preclinical study using animals, and its pharmacological effect was compared with that of pure extracts of fatty acids (AcGS), fatty alcohols (AlcG), and high molecular weight aldehydes (AldG) in salt form, each administered at a dose equivalent to the proportion present in 200 mg / kg of HMWS active ingredient. Furthermore, the effect of HMWS was compared with that of pure extract of free fatty acids (AcGL) administered at the same dose.

[0020] Male gerbils (60-80 g) were used and allowed to adapt to laboratory conditions (temperature 20-25°C, relative humidity 60±5%, 12-h light / dark cycle) for 7 days with free access to water and food. The various substances to be evaluated were prepared as suspensions in an acacia / water vehicle (1%).

[0021] After isolation, the gerbils were divided into eight groups: a negative control group that received only vehicle and no cerebral ischemia (CI) was induced, and seven groups with induced CI. One of the induced CI groups was a positive control treated with vehicle alone, while the other six groups were treated with HMWS (200 mg / kg), AcGS (132 mg / kg), AlcG (40 mg / kg), AldG (28 mg / kg), AcGL (200 mg / kg), and aspirin (60 mg / kg) as a reference substance. All treatments were administered orally via intragastric intubation (0.5 mL / 70 g body weight) 1 h before induction of ischemia. Global cerebral ischemia was induced by bilateral ischemia and reperfusion. To do this, the gerbils were anesthetized in halothane atmosphere, and an incision was made in the ventral midline of the neck to expose the two common carotid arteries. The arteries were isolated and separated by placing black silk sutures around them, and then allowed to recover from anesthesia for 30 minutes. At the end of this period, the silk sutures were removed, and pressure clips were placed on each carotid artery to completely block blood flow for 5 minutes. The clips were then removed, and blood was allowed to recirculate for 24 hours.

[0022] Neurological function was assessed 4 hours after ischemia / reperfusion (according to McGraw, 1977) as follows: 0: absence of symptoms; 1: curve of trunk or setae of hair; 2: Ptosis; 3: Circular movements (rotational behavior); 4: Hind leg extension and 5: Convulsions. The locomotor activity of the gerbils was assessed in an open field 24 hours after ischemia (Katsumata et al., 2006). To do this, each gerbil was placed in the center of a box measuring 60.5 cm long x 30 cm high x 46 cm wide, with the bottom edge at 14.5 cm. 2 The area was divided into 12 quadrants (quarters). Over a 6-minute period, the number of times the gerbils crossed the different quadrants with their front legs and the number of times they stopped or leaned was quantified.

[0023] After behavioral observations were completed, the gerbils were anesthetized in a halothane atmosphere, and blood samples were taken from the vena cava, collected in plastic tubes, and mixed with EDTA (10%). The brains were immediately removed for histological analysis. Blood was centrifuged at 3000 rpm for 10 min to obtain plasma, where biochemical quantification of lipid peroxidation and protein oxidation was performed. To determine lipid peroxidation, the formation of substances reactive to thiobarbituric acid (SRATB) was quantified in plasma (according to Ohkawa et al., 1979) and expressed as nmol malondialdehyde (MDA) / mg protein.

[0024] To determine protein oxidation, sulfhydryl (SH) groups were determined (technique described by Miao-Lin Hu, 1994). Aliquots of 50 μL plasma and 950 μL 10 mM DTNB were taken and incubated at room temperature for 20 minutes. The absorbance of the supernatant was read at 412 nm. A blank was prepared with DTNB and the absorbance was measured at 13,600 cm. -1 M -1 The total number of SH groups was calculated using the absorption rate and expressed in mmol. Protein concentrations were determined by a modified Lowry method (Marxwell, 1987).

[0025] For histological analysis, brains were fixed in 10% buffered formaldehyde, dehydrated, and embedded in paraffin, and sections containing the hippocampus were stained with hematoxylin and eosin. Sections from each brain were scored by a light microscopy scoring system to determine damage to pyramidal cells in the CA1 region of the hippocampus, averaged bilaterally. This histopathological scoring system (based on the method used by Bartus et al., 1998) is as follows: 0 = normally pigmented, closely packed cells with a rounded soma and a well-pigmented central nucleus; 1 = some shrinkage and irregularity of cell shape with areas of pale chromatin lysis surrounded by a peripheral ring of cytoplasm; 2 = some obvious cell loss with areas of pyknotic cells; 3 = moderate cell loss and pyknotic nuclei; 4 = Loss of Nissl substance, showing significant depletion of neurons and only occasional neurons among numerous microglia.

[0026] result Even short-term global ischemia leads to selective neurodegeneration in vulnerable brain regions, such as the cortical cortex (AC) region of the hippocampus. CA1 pyramidal neurons, in particular, are the most vulnerable to ischemia / reperfusion injury (Sharma, 2005; Kirino, 2000). A model of global ischemia induced by bilateral ligation of the two common carotid arteries for 5 minutes and their reperfusion for 24 hours constitutes a useful model for evaluating substances that may have beneficial effects in ischemic stroke (Ravinder, 2009). Neurological assessment using the McGraw clinical symptom score and increased locomotor activity measured in the open field test constitute indicators of histological damage to brain tissue (McGraw, 1977; Katsumata, 2006).

[0027] The results showed the presence of clinical symptoms and increased locomotion in positive control animals, changes that were antagonized by aspirin, which is consistent with what has been reported for this model and supports its relevance in our experimental conditions. This study demonstrated that the HMWS composition (200 mg / kg) has the ability to significantly reduce clinical symptoms (91.6% inhibition) and hyperactivity (86.1% inhibition) induced by global cerebral ischemia in Mongolian gerbils. Treatment of HMWS with each of the pure extracts of its individual components (AcGS, AlcG, and AldG) resulted in a moderate but significant reduction in clinical symptom scores (36.6, 21.6, and 11.6%, respectively) and hyperactivity (33.4, 21.7, and 11.1%, respectively) compared with the positive control group. Comparison of HMWS (200 mg / kg) with each of the pure extracts of its individual components was significant, demonstrating greater efficacy and superior to the combined effect of the three extracts, demonstrating a synergistic or potentiating effect between the three components when treated together in the AI ​​HMWS (Table 1).

[0028] On the other hand, administration of HMWS (200 mg / kg) resulted in significantly higher protection in clinical symptoms than pure AcGL extract (200 mg / kg), indicating better neurological protection by the extract combining the presence of fatty acids, fatty alcohols and fatty aldehydes in salt form.

[0029] Table 1: Effects on clinical symptom scores and locomotor activity (open field) in Mongolian gerbils with global cerebral ischemia / reperfusion (I / R). [Table 1] Mean ± SEM (standard error of the mean), SC: clinical symptoms *p<0.05; **p<0.01; ***p<0.001; compared with the positive control group at p<0.05 compared to AcGS and AcGL, bp <0.001 compared to AlcG and AldG (Mann Whitney U) I / R: global cerebral ischemia / reperfusion HMWS: High molecular weight substances obtained in the examples AcGL: Pure free fatty acids AcGS: Pure extract of fatty acids in salt form AlcG: A pure extract of fatty alcohols AldG: a pure extract of high molecular weight aldehydes ASA: Aspirin

[0030] Table 2 shows the results of the histological study. Negative control (also referred to as "negative control") brains showed no changes, while all positive control (also referred to as "positive control") brains showed loss and damage of numerous pyramidal cells in the CA1 region. Treatment with the reference substance aspirin (60 mg / kg) significantly reduced the histological score of brain damage by a moderate amount (32.4%), which supports the validity of the results in our experimental conditions.

[0031] HMWS treatment (200 mg / kg) significantly reduced (89.8% inhibition) the histological score of brain damage induced by bilateral ischemia-reperfusion in Mongolian gerbils (also known as gerbils). Individual pure extracts containing AcGS (132 mg / kg), AlcG (40 mg / kg), and AldG (28 mg / kg) significantly reduced the histological score compared to the positive control. Comparison between HMWS and each of its individual component extracts showed greater efficacy not only for each pure extract but also for the sum of the three. This result is consistent with that observed regarding clinical symptoms and reinforces the synergistic nature of the three components present in AI HMWS.

[0032] Administration of HMWS (200 mg / kg) was also more effective than pure AcGL extract (200 mg / kg) in protecting against histological damage, confirming what was observed regarding clinical symptoms and supporting better neurological protection with extracts combining the presence of fatty acids, fatty alcohols, and fatty aldehydes in salt form (Table 2).

[0033] Table 2: Effects of ischemia / reperfusion (I / R) on the histological scores of the gerbil brain. [Table 2] Mean data ± SEM (standard error of the mean) *p<0.05; ** p<0.01; *** p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U)

[0034] The results of the study on oxidative variables related to the global cerebral ischemic process induced by ischemia and reperfusion in Mongolian gerbils are shown in Table 3. The ischemia and reperfusion process increased the plasma concentrations of MDA (an indicator of lipid peroxidation) and sulfhydryl (SH) groups (an indicator of protein oxidation) in the positive control group compared with those in healthy animals in the negative control group.

[0035] Oral administration of HMWS (200 mg / kg) significantly prevented the increase in plasma concentrations of MDA and SH groups (97.7% and 80% inhibition, respectively) compared with the positive control group. Individual pure extracts containing AcGS (132 mg / kg) and AlcG (40 mg / kg) significantly reduced plasma concentrations of MDA (41.5% and 25.9% inhibition, respectively) and SH groups (80% and 36% inhibition, respectively) compared with the positive control. However, pure extracts containing AldG (28 mg / kg) produced slight reductions in MDA (11% inhibition) and SH groups (12% inhibition), without reaching statistical significance. Comparisons between HMWS and each of its individual component extracts demonstrated greater efficacy not only for each pure extract but also for the three combined. Thus, we demonstrate that the antioxidant effect of this novel HMWS substance protects both lipid and protein structure, demonstrating a synergistic effect among its three components.

[0036] Meanwhile, AcGL extract (200 mg / kg) also produced antioxidant effects by significantly reducing plasma levels of MDA (40% inhibition) and SH groups (36% inhibition) compared with the positive control. However, comparison of HMWS (200 mg / kg) with AcGL (200 mg / kg) showed superior antioxidant efficacy of HMWS, supporting its greater neuroprotective efficacy, as demonstrated by both clinical symptoms and histological brain damage scores.

[0037] Treatment with ASA (60 mg / kg) did not modify or affect any of these oxidative variables, corresponding to its action profile, since its neuroprotective efficacy is based on its antiplatelet activity and not on its antioxidant effect.

[0038] Table 3: Effects of cerebral ischemia / reperfusion (I / R) on plasma concentrations of MDA and sulfhydryl groups in Mongolian gerbils. [Table 3] Mean data ± SEM (standard error of the mean) *p<0.05; ***p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U) [Example]

[0039] Evaluation of neuroprotective effects in a spinal cord ischemia-induced model: The neuroprotective effect of the HMWS mixture, the object of the present invention, was also investigated using an experimental model of induced ischemia in the spinal cord. The composition obtained in Example 2 was subjected to a preclinical study, and its pharmacological effect was compared with that of pure extracts of fatty acids (AcGS), fatty alcohols (AlcG), and high molecular weight aldehydes (AldG) in the form of salts, administered at a dose of 200 mg / kg of the HMWS active ingredient, equivalent to that of each of these substances. Furthermore, the effect of HMWS was compared with that of a pure extract of free fatty acids (AcGL), administered at the same dose.

[0040] Male New Zealand rabbits (1.8–2.2 kg) were used and adapted to laboratory conditions (temperature 20–25°C, relative humidity 60±5%, 12-h light / dark cycle) for 15 days with free access to water and food.

[0041] The substances HMWS, AcGS, AlcG, AldG, AcGL, and aspirin (ASA) were prepared as suspensions in an acacia / water vehicle (1%). After isolation was completed, rabbits were divided into eight groups: a negative control group that received only the vehicle and no ischemia was induced; seven groups with spinal cord ischemia: a positive control group treated with vehicle only; and six groups treated with HMWS (200 mg / kg), AcGS (132 mg / kg), AlcG (40 mg / kg), AldG (28 mg / kg), AcGL (200 mg / kg), and aspirin (2 mg / kg) as a reference substance. All treatments were administered orally via an intragastric tube (1 mL / 2 kg body weight) for 10 days.

[0042] To induce ischemia in the spinal cord, rabbits were anesthetized with thiopental (20 mg / kg iv). The abdomen was depilated and swabbed with antiseptic solution, and a ventral midline incision was made to penetrate the retroperitoneal cavity. The aorta and left renal artery were incised and exposed. The aorta was ligated by placing a clip just below the left renal artery for 20 minutes, after which it was removed and the reperfusion period began. The incision was sutured. At 4 and 24 hours after reperfusion, the animals' neurological deficits were assessed blindly by two independent observers (according to Zivin et al., 1982). The scale used was as follows: Grade 0: Complete paralysis Grade 1: Partial neuropathy Grade 3: Normal

[0043] The animals were sacrificed 24 hours after reperfusion. Bone marrow sections were extracted for histological analysis. Once the spinal cord (lumbar) segments were extracted, they were fixed in 10% buffered formaldehyde. The samples were dehydrated in increasing concentrations of alcohol, embedded in paraffin, and cut into 4-μm sections with a horizontal micrometer. The sections were stained with hematoxylin and eosin. They were then observed under an Olympus BH2 microscope. Spinal cord lesions were evaluated blindly using the criteria described by De Girolami, 1982, and Zivin, 1982. 3- No injury 2-Mild: When only 1-10 necrotic neurons are detected in the gray matter sample, involving less than 33% of the material. 1-Moderate: When only 10-20 necrotic neurons are detected in the gray matter sample in 33-66% of the relevant section area. 0-Severe: When the gray matter sample is only detected to contain more than 20 necrotic neurons in more than 66% of the involved section area.

[0044] result: Abdominal aorta occlusion and reperfusion resulted in neurological damage in rabbits, resulting in paralysis of the hind limbs and an inability to jump. Table 4 shows the scores for clinical symptoms of neurological deficit and mortality. After 20 minutes of abdominal aorta occlusion, the positive control animals showed these symptoms, with scores significantly lower than those of the negative control group (healthy animals), and also showed an 80% mortality rate. Administration of the reference substance, aspirin (2 mg / kg), significantly prevented both clinical symptoms and death, supporting the results obtained in the experimental conditions. Treatment with repeated doses of HMWS (200 mg / kg) significantly increased clinical symptom scores and reduced overall mortality at 4 and 24 hours after abdominal aortic reperfusion. Treatment with AcGS (132 mg / kg), AlcG (40 mg / kg), and AldG (28 mg / kg) significantly protected against clinical symptoms at 4 and 24 hours, but only AcGS reduced mortality by 50% compared with the positive control group (80%). Comparison of HMWS (200 mg / kg) with pure extracts of its individual components also showed statistical significance, indicating that HMWS was more effective than each of the individual components. Furthermore, extracts containing AcGL (200 mg / kg) also protected against clinical signs and mortality, but comparison with HMWS showed the greater efficacy of the latter.

[0045] Therefore, comparison of the treatment results showed greater efficacy of HMWS compared to the others in improving clinical symptoms 4 and 24 hours after reperfusion and in reducing mortality. The HMWS composition significantly protected against mortality, such that the frequency of death in the group treated with this substance (0.0%) was significantly lower than that in the positive control (80%), indicating a 100% protective effect.

[0046] Histological analysis (Table 5) showed severe lesions in the spinal cord of animals in the positive control group, characterized by neuronal necrosis and vacuolization of the neuropil. Aspirin provided modest but significant protection compared to the positive control.

[0047] Treatment with HMWS (200 mg / kg) significantly inhibited histological damage in the spinal cord caused by neurological deficits, achieving the highest efficacy of all applied treatments (93.3% inhibition). Treatment with AcGS (132 mg / kg), AlcG (40 mg / kg), and AldG (28 mg / kg) moderately protected against histological damage (40, 24, and 11.6% inhibition, respectively), with HMWS efficacy being higher than each of these, and even greater than the combined effect of the three. These results indicate the presence of a synergistic effect of the three substances present in the IA HMWS to protect against neurological deficits in the spinal cord of rabbits subjected to ischemia and reperfusion. Furthermore, the protection of HMWS was higher than that observed with pure AcGL extract, demonstrating the benefits of this new AI.

[0048] In conclusion, treatment with HMWS compositions demonstrated the advantage of enhancing neuroprotective benefits, effectively reducing clinical symptoms, mortality, and histological damage scores in the spinal cord.

[0049] Table 4: Effects on symptoms and mortality in rabbits with spinal cord injury induced by ischemia / reperfusion (I / R). [Table 4] Mean data ± SEM (standard error of the mean) *p<0.05; ** p<0.01; *** p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U) +p<0.05; ++p<0.01; compared to positive control cp<0.01 compared with AcGS and AcGL; dp<0.05 compared with AlcG and AldG (Fisher exact test)

[0050] Table 5: Effect of ischemia / reperfusion (I / R) on the histological scores of the spinal cord in rabbits. [Table 5] Mean data ± SEM (standard error of the mean) *p<0.05; ** p<0.01; *** p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U) [Example]

[0051] Evaluation of neuroprotective effects in toxicant-induced neuronal injury models: The neuroprotective effect of the HMWS mixture, the subject of the present invention, was investigated using an experimental model of kainic acid-induced nerve damage. The composition obtained in Example 2 was subjected to a preclinical study, and its pharmacological effect was compared with that of a pure extract of fatty acids (AcGS), fatty alcohols (AlcG), and high molecular weight aldehydes (AldG) in the salt form, prepared in doses equivalent to the doses of each substance in the HMWS active ingredients. Furthermore, it was compared with that of a pure extract of free fatty acids (AcGL).

[0052] Male Wistar rats (250-300 g) were used and adapted to laboratory conditions (temperature 20-25°C, relative humidity 60±5%, 12-h light / dark cycle) for 7 days with free access to water and food.

[0053] The substances HMWS, AcGL, AcGS, AlcG, AldG, and aspirin were prepared as suspensions in an acacia / water vehicle (1%). After isolation, rats were divided into seven groups: a negative control group (no neuronal damage, as they received only the vehicle) and six groups (with neurotoxicity). Of the latter, one was a positive control (treated with vehicle only), and the other five groups were treated with HMWS (200 mg / kg), AcGS (132 mg / kg), AlcG (40 mg / kg), AldG (28 mg / kg), and AcGL (200 mg / kg). All treatments were administered orally via intragastric intubation (5 mL / kg body weight) 30 min before neurotoxicity induction.

[0054] Neurotoxicity was induced in rats by injecting kainic acid (6 mg / kg; i.p.). One hour later, the rats' exploratory activity was assessed in an open field (Fernandez, 1987). To do this, the rats were placed in the center of the box (60.5 cm long x 30 cm high x 46 cm wide, with a 14.5 cm base). 2 The animals were placed in a quadrant (divided into 12 quadrants) and the number of times they crossed different quadrants (C) as well as the number of times they stopped or leaned (P) within 6 minutes after being placed in the apparatus was quantified.

[0055] After the behavioral experiments were completed, rats were sacrificed in a halothane atmosphere, and the brains were rapidly extracted and fixed in 10% buffered formaldehyde. Subsequently, tangential brain sections were embedded in paraffin, cut, and stained with 1% acid fuchsin for 30 seconds to detect neuronal death (Lee, 2000). The presence of acidophilic neurons (kainic acid positive) is a variable indicating neuronal damage (Lee, 2002). To count kainic acid positive cells, pyramidal neurons in the CA1 and CA3 hippocampi of each rat were extracted from the center of these regions at 250 μm. 2 The area was examined by light microscopy (Savaskan, 2002).

[0056] result: Kainic acid is a glutamate analogue that, when administered systemically or intracerebrally, induces neurotoxicity through selective neurodegeneration, mediated by its receptors (Coyle, 1983; Borg, 1991). Depending on the dose administered, kainic acid can induce non-convulsive or convulsive seizures. Consequently, non-convulsive doses induce changes in spontaneous behavior and impair processes related to the maintenance of attention, allowing the analysis of these effects on animal behavior (Mikulecka, 1999).

[0057] Kainic acid administration induced a decrease in both components of exploratory activity (crossing and stopping) and overall exploratory activity compared with the negative control group, corresponding to behavioral changes reported in the literature and lending validity to the model in our experimental conditions. A single oral administration of HMWS (200 mg / kg) increased both components as well as total activity compared with the positive control group, achieving the highest efficacy of all treatments. Treatment with AcGS (132 mg / kg), AlcG (40 mg / kg), and AldG (28 mg / kg) increased crossing and stopping, as well as total activity, but only the latter increased crossing and stopping, and total activity, reaching statistical significance. HMWS (200 mg / kg) was more effective on all measured behavioral variables than each of its individual components (AcGS, AlcG, and AldG), even exceeding the combined effects of the three substances. Furthermore, the efficacy of HMWS in increasing hybridization, arrest, and their total was higher than that of AcGL extract (200 mg / kg), confirming the advantages of this new AI (Table 6).

[0058] Table 7 shows the results of histological studies. As expected, systemic administration of kainic acid resulted in neuronal loss associated with this excitotoxin-induced effect in the rat hippocampus by inducing the loss of pyramidal neurons in hippocampal areas CA1 and CA3 (Mikulecka, 1999; Perez, 1996). Administration of HMWS (200 mg / kg) significantly reduced the extent of neuronal death (85.3% inhibition). Pure extracts of AcGS (132 mg / kg), AlcG (40 mg / kg), and AldG (28 mg / kg) moderately reduced neuronal death (35, 22.05, and 11.7% inhibition, respectively). Comparison of the effects of HMWS and each of its individual components showed significant differences, indicating the greater efficacy of this extract when presented in combination with the three substances. At the same time, its efficacy was greater than the sum of the individual effects of each substance, indicating the presence of a synergistic effect in the neuroprotection of HMWS in this model.

[0059] On the other hand, AcGL extract (200 mg / kg) also exerted neuroprotective effects by moderately protecting against kainic acid-induced neuronal death (30.8% inhibition), in which case HMWS extract also showed superior efficacy.

[0060] Thus, the fact that the HMWS composition protects against neuronal cell death in this model confirms its neuroprotective effect observed in the model described in the previous example, and its superior efficacy relative to the rest of the treatment, as well as the synergistic effects between its components, and represents a potential benefit in the treatment of neurodegenerative diseases.

[0061] Table 6: Kainic acid (KA)-induced neurotoxicity. Effects on exploratory activity (open field) in rats. [Table 6] Mean data ± SEM (standard error of the mean) *p<0.05; ** p<0.01; *** p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U)

[0062] Table 7: Kainic acid (KA)-induced neurotoxicity. Effects on neuronal cell death in rats. [Table 7] Mean data ± SEM (standard error of the mean) (# of cells positive for acid fusin staining) *p<0.05; ** p<0.01; *** p<0.001 compared with the positive control group p<0.05 compared to AcGS and AcGL, bp<0.001 compared to AlcG and AldG (Mann Whitney U) [Example]

[0063] Tablet preparation: 5 kg of the composition obtained in Example 1 was mixed with 5 kg of sodium carboxymethylcellulose, 30 kg of lactose, and 10 kg of microcrystalline cellulose in a rotating stainless steel drum at 5 rpm for 10 minutes. Subsequently, 1 kg of magnesium stearate was added to the previous mixture, and the resulting granules were mixed in the rotating drum at 5 rpm for 5 minutes. The final mixture was compressed in a tablet press at a rate of 30,000 tablets per hour to obtain tablets with an average weight of 450 mg. According to USP methods, these tablets had a rounded convex shape with a diameter of 12 mm on both sides, their disintegration time in water was less than 20 minutes, their friability was less than 1%, and their hardness was 44-60 N. [Example]

[0064] Tablet Coating: The tablets obtained in the previous examples can be coated. To do this, they were mixed in a propeller mixer with the following ingredients to form a coating suspension: 25 kg of cellulose acetophthalate in a 28% aqueous solution, 0.6 kg of talc, and 0.9 kg of titanium dioxide. The final weight of the coated tablets was 470 mg. A colorant can be added to the coating solution, and the coated tablets can be polished with wax or paraffin. [Example]

[0065] To obtain the capsules: The granules obtained in Example 7 can alternatively be encapsulated. For this purpose, size 1 hard gelatin capsules were filled with 430 mg of the granules obtained in Example 7, thereby obtaining capsules with the following characteristics: average weight 455.0 + / - 30 mg, disintegration in water in less than 20 minutes. [Example]

[0066] Preparation of the suspension: 200 g of the composition shown in Example 2, 40 kg of sorbitol, and 5.00 g of preservative (a mixture of methylparaben and propylparaben dissolved in alcohol) were mixed with 158 liters of water in a stainless steel reactor equipped with a stirring propeller. 2 kg of apple extract was then added, and stirring was continued. The resulting suspension was packaged in amber glass bottles with plastic lids at 115 mL volumes, with one tablespoon (15 mL) recommended as the dosage unit.

Claims

[Claim 1] 1. A method for producing a mixture of high molecular weight compounds having neuroprotective and antioxidant properties obtained from sugarcane wax, comprising: Raw or refined sugarcane wax (Saccharum officinarum) is used as a raw material. The sugarcane wax is saponified with an alkaline or alkaline earth hydroxide, and the resulting saponified wax is then sintered in a supercritical fluid. 2 Extraction with CO 2 Both the alcohol and aldehyde fraction extracted in step (a) and the remaining salt mixture are subjected to a purification step with hexane and acetone, and the resulting fractions are analyzed by gas chromatography and spectrophotometry to measure the salt, alcohol and aldehyde contents, and the salt, alcohol and aldehyde contents are determined, and then mixed in the proportions required to achieve the following mixture proportions: A manufacturing method using raw or refined sugarcane wax (Saccharum officinarum) as a raw material, which comprises saponifying the sugarcane wax with an alkali or alkaline earth hydroxide, subsequently subjecting the resulting saponified wax to extraction with hot hexane, cooling the organic fraction, subjecting the recrystallized solid to process purification by successive washings with ethanol and acetone, analyzing both the extracted and purified fraction and the remaining salt mixture by gas chromatography and spectrophotometry to determine the salt, alcohol and aldehyde content, and subsequently mixing the mixture in the proportions required to achieve the following mixture proportions: Composition of the mixture: 60-80% by weight of fatty acids having 26-36 carbon atoms in the form of their alkali or alkaline earth salts, 10-30% by weight of primary aliphatic alcohols having 24-34 carbon atoms, and 10-30% by weight of αβ-unsaturated aldehydes having more than 48 carbon atoms; The fatty acids are C26:0 0.3-5%, C27:0 0.3-5%, C28:0 20.0-40.0%, C29:0 1.0-3.0%, C30:0 12.0-25.0%, C31:0 0.5-2.0%, C32:0 5.0-15.0%, C33:0 0.5-3.0%, C34:0 5.0-18.0%, C35:0 0.3-1.5%, and C36:0 1.0-8.0%; The primary aliphatic alcohols are homogeneous primary aliphatic alcohols of 24 to 34 carbon atoms, with 1-octacosanol (C28) predominating; Alpha-beta unsaturated aldehydes are predominantly alpha-beta unsaturated aldehydes having more than 48 carbon atoms.

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