The blending of aliphatic polyolefins and aromatic poly (PET) based on different intrinsic viscosities (IV) was conducted in a torque rheometer. The comparison of blend components in terms of low density polythene (LDPE) and polypropylene (PP) in blending with PET was investigated, and the effects of the IV and proportion of PET on polymer blends are discussed in detail. Polymer blends with or without compatibilizer were examined by using a differential scanning calorimeter, thermogravimetric analyzer, rotary rheometer, field-emission scanning electron microscopy and a universal testing machine. It was found that the blending led to an increase in processability and a decrease in thermal stability for blends. The morphological analysis revealed that the incompatibility of blends was aggravated by a higher IV of PET, while this situation could be improved by the addition of compatibilizer. Results showed that there was an opposite effect for the tensile strength and the elongation at break of the polymer blend in the presence of a compatibilizer, wherein the influence of IV of PET was complicated. Polythene (PE) and polypropylene (PP), as representatives of aliphatic polyolefins, and poly (PET), as a representative of aromatic polymers, are the three most common commercialized synthetic polymers, owing to their excellent overall performance and affordable price. The yield of the three polymers in China reached more than 14, 20, 40 million tons in 2016, respectively. PET can be categorized into three types with different intrinsic viscosities (IV) or molecular weights according to the application field: fiber grade, bottle grade and industrial yarn, whose IV are about 0.65 dL/g, 0.85 dL/g and 1.0 dL/g and whose corresponding aims are to be applied in the field of textiles, containers bottles and belts, respectively [The blending of polyolefins and PET can be an alternative to improve cost-effectiveness for industrial applications . HoweverGenerally, the compatibilization of two immiscible polymers can be achieved by implementing a suitable grafting modification of polyolefin chains with reactive groups. The modification is achieved by chemical reactions between polar functional groups and polymer chains in melting blending . By enhag-GMA and PET. In yet another approach, a compatibilizer composed of styrene–ethylene–butylene–styrene (SEBS) grafted with MAH was used as the compatibilizer in the blending of PP and PET spun fibers [g-AA as a compatibilizer, a good interaction between the PP and PET surfaces was achieved as PET islands was deformed in micro-fibers after hot stretching [In the previous literature, GMA, MAH, acrylic acid (AA), ethylene-vinyl acetate copolymer (EVA), and maleimide (MI) are the common functional groups that can be either grafted onto polyolefins or copolymerized into compatibilizer. Recently, Jazani et al.  improvedn fibers . Resultsretching . g-MAH, SEBS-g-MAH and E–GMA, respectively. It was found that the compatibilizing effectiveness of E–GMA was the best. Raffa et al. [In most published works, PP is the dominant continuous phase in the blend system of polyolefins/PET. There is little research focusing on PE/PET blends, let alone on the compatibilizer, by grafting the functional groups onto PE. Lei et al.  studied a et al.  indicateg-MAH and PE-g-MAH, which offer the best convenience for designing polymer blend systems.In most cases, a variety of nanomaterials as a third component are added into two immiscible polymers to improve the compatibilizing effect . As a mag-MAH as a compatibilizer. A systematic performance investigation of the polymer blends with or without compatibilizer was performed to elucidate the processable correlation between several major polymer commodities. Based on the successful surface functionalization of polypropylene/polystyrene and their application in polymer blends, as described in our previous work ,22,23,24g-MAH and PP-g-MAH were purchased from Fine-blend Compatibilizer Jiangsu Co., Ltd. . Three poly (PET) chips with different intrinsic viscosities (IV) of 0.7 dL/g, 0.85 dL/g and 1.0 dL/g, were purchased from Zhejiang Guxiandao Polyester Dope Dyed Yarn Co., Ltd. . The moisture and carboxyl end group content of the PET chips are also listed as In order to decrease the hydrolytic decomposition, the PET chips were dried at 130 °C for 9 h in a vacuum oven before being blended into either LDPE or PP. The polymer blends were processed in a RM-200C torque rheometer . The processing temperatures were set to 260 °C for all blends in the rheometer, and the processing time was set to 5~10 min and the rotor speed was set to 50 r/min. The total weight of polymer blend was no more than 50 g. The weight ratio of the components in the blending are listed in cT) and before being kept for 3 min. Finally, the samples were heated to 300 °C at a rate of 10 °C/min to determine the melt point (mT). About 5 mg of the samples was heated from 25 to 600 °C at a rate of 10 °C/min with a nitrogen flow rate of 45 mL/min to determine the decomposition temperature .The thermal behavior of the blends was investigated using differential scanning calorimeter  and thermogravimetric analyzer  under nitrogen atmosphere. The drying samples were initially heated from 25 to 300 °C at a rate of 50 °C/min. After that, the samples were kept at the final temperature for 3 min to eliminate the thermal history, and then cooled to 50 °C at a rate of 20 °C/min to determine the crystallization temperature  using a stress-controlled rheometer  at 260 °C. The measurements of the granules and sheet samples were performed in air atmospheres. The loaded frequency sweep was undirectional from the angular frequency of ω = 500 1/s to 0.1 1/s.The banded samples of polymers were prepared from a blending system so that the morphological characteristics of the polymer blends could be examined using an ULTRATM 55 field-emission scanning electron microscopy  at an acceleration voltage of 1 kV. The samples were cooled in liquid nitrogen for 15 min and then taken out quickly to be brittle fractured. The fracture surface of PET was etched by using the mixed solution of phenol and tetrachloroethane (1/1 (wt %)), then the fracture surface was gilded because of the poor electrical conductivity.To test the mechanical property of the polymer blends, the round rod samples with diameter of 1.5 mm were stretched on a universal testing machine  at speed of 10 mm/min. Generally, the effective stretch length was about 2 mm, and then the tensile strength and elongation at break could be achieved.cT1) of the polymer blends, observed in cT2) was almost invisible in most cases. In contrast, there were two melting peaks in the heating DSC scans, which indicated the melting point of the polyolefin component (mT1) and PET component (mT2). Noticeably, the cT1, mT1 and mT2 for all polyolefin/PET blends were listed in cT1) from LDPE to LDPE/PET1, to LDPE/LDPET-g-MAH/PET, however, the cT1 of compatibilized PP/PET1 blends was lower than that of incompatible PP/PET1 blends. This might be ascribed to the plasticizing effect of compatibilizer on the PP/PET blend and the facilitation of crystallization [lization . On the cT1 of incompatible LDPE/PET  and derivative thermogravimetric (DTG) scans of polymer with polymer blends is reported in T5%) and the corresponding temperature of the maximum decomposition rate (cmaxT) of all polymer blends are also listed in T5% and cmaxT. These results are easy to understand if it is remembered that PET with a higher IV has a longer polymer chain. mT of PET was approximately equal to 260 °C, all the rheological measurements of polymers and polymer blends were conducted in air without inert gas protection, which was consistent with the blending process. Three kinds of PET displayed a pseudo-Newtonian behavior for the whole frequency range. The complex viscosity significantly increased with the IV of PET due to the longer polymer chain [er chain . Both thCompared with LDPE, the incompatible LDPE/PET blend showed a higher viscosity at low frequency and a lower viscosity at high frequency. However, the complex viscosity of compatibilized LDPE/PET blend was always lower than that of LDPE except at an extremely low frequency, and also lower than that of incompatible LDPE/PET blend within the whole frequency range. However, once the compatibilizer was formally incorporated into the system, the viscosity of the system decreased, which was due to the role of the compatibilizer in reducing the dispersity and inducing interaction at the interface between blend components, eventually increasing the mobility of the compatibilized system ,28. PP sg-MAH tended to be dispersed uniformly with diminishing size. All these confirmed that the compatibilizer had indeed improved the compatibility between polyolefins and PET. As mentioned above in the analysis of rheological behavior, the compatibilizer enhanced the interaction between the blend components which decreased the interfacial tension and suppressed the coalescence [lescence ,30,31,32The effect of the addition of different IV of PET on the blending system with or without compatibilizer was visualized by morphological characterization, as shown in Without compatibilizer, the elongation at break of both LDPE/PET blends and PP/PET blends declined with the increase of the IV of PET. Yet it increased remarkably after the compatibilizer was added to polyolefin/PET blends, especially for the highest IV of PET3 in blends. With longer polymer chains and higher steric effects, the PET with a higher enlarged interphase between the polyolefins and the PET, showed a decrease of elongation at break. The compatibilized polymer blends were expected to have a higher elongation at break because of the increased interphase adhesiveness and enhanced interaction. In this case, PET3 with better mechanical properties had a positive effect on the elongation at break of blends. This is different from the PE/PET blend with organoclay as a compatibilizer, as there may be a possible degradation of the clay surfactant during melt compounding . It was g-MAH was more likely to diffuse successfully into two blend components. It was also noticed that the tensile strength of all polymer blends generally declined, and the difference of tensile strength caused by either changing the IV of PET or changing the PET content also decreased. Therefore, it can be stated that the compatibilizer played a role in increasing interphase adhesiveness to prevent brittle fracture, thus increasing the mechanical properties of polymer blends.The variation of the tensile strength of polymer blends with PET content was largely different from the elongation at break, in that it was somewhat confused. In such a situation, the polymer blends were brittle fractured to subdue the tenacity of the materials. With the increase of the IV of PET, the tensile strength of the LDPE/PET blends was decreased, while it was gradually increased for PP/PET blends. According to the SEM micrographs, the interphase adhesiveness of incompatible LDPE/PET blends was relatively weaker than that of PP/PET blends. However, with the addition of compatibilizer, LDPE/PET blends showed better tensile properties since the shorter chain segment of LDPE-The blending of LDPE and PP with different intrinsic viscosities (IV) of PET was performed in the absence or presence of compatibilizer. The impact of the blending components on the thermal, rheological, morphological and mechanical properties was discussed in detail, and the following conclusions were drawn from the discussion: (1) The blending leads to a higher crystallization temperature of the polyolefin component, yet the melting peak both of the polyolefin component and of the PET component in the blends are little different from their pure polymer. While the thermal stability of polymer blends is generally better than PET but worse than LDPE, and blending of PP with PET had no significant effect on the thermal stability of PP. The thermal performance of polyolefin/PET was slightly decreased by either the increase of the IV of PET or the decrease of PET content. It was found that the compatibilizer can improve the thermal stability of the blending system by enhancing the interface adhesion. (2) The complex viscosity of incompatible polymer blends is slightly higher than that of compatibilized blends. The results show that it is increased with the increase of the IV of PET, yet decreased with the increase of PET content increasing in blends. The complex viscosity of the blend with compatibilizer can be similarly increased by either increasing the IV of PET or increasing the PET content in blend. g-MAH as a compatibilizer. As the IV of the PET increases in the polymer blends, PET can be easily gathered to enlarge the scale of the dispersed phase, resulting in worse nonuniform dispersity, while it can be improved by adding compatibilizer.(3) PET disperses nonuniformly in LDPE in the form of granules; the cross-section of the incompatible blend shows brittle fracture. The number of holes in the blend increases with the increase of PET content. In contrast, the cross-section tends to be indistinct in the presence of LDPE-(4) It was demonstrated that with the increase of the IV of PET, the elongation at break of the polymer blend decreases slightly and the tensile strength of the LDPE/PET blend also decreased accordingly, while the tensile strength of the PP/PET blend gradually increased. It is worth noting that the higher content of PET in the blend results in a smaller elongation at break and lower tensile strength. When the compatibilizer was added to the blends, an increase of elongation at break and a decrease of tensile strength could be seen simultaneously, and the difference of tensile strength caused by either changing the IV of PET or changing the PET content declined.


The sirtuins form a family of evolutionarily conserved nicotinamide adenine dinucleotide (NAD)-dependent deacetylases. Seven sirtuins (SIRT1–SIRT7) have been described in mammals, with specific intracellular localization and biological functions associated with mitochondrial energy homeostasis, antioxidant activity, proliferation and DNA repair. Physical exercise affects the expression of sirtuin in skeletal muscle, regulating changes in mitochondrial biogenesis, oxidative metabolism and the cellular antioxidant system. In this context, sirtuin 1 and sirtuin 3 have been the most studied. This review focuses on the effects of different types of exercise on these sirtuins, the molecular pathways involved and the biological effect that is caused mainly in healthy subjects. The reported findings suggest that an acute load of exercise activates SIRT1, which in turn activates biogenesis and mitochondrial oxidative capacity. Additionally, several sessions of exercise (training) activates SIRT1 and also SIRT3 that, together with the biogenesis and mitochondrial oxidative function, jointly activate ATP production and the mitochondrial antioxidant function. The seven sirtuins present in mammals SIRT1–SIRT7) are a family of class III histone deacetylases –SIRT7 ar that regThe wide spectrum of cellular activities of sirtuins in the organism places them as therapeutic targets to combat metabolic, neurodegenerative and proliferative diseases. In this context, in underdeveloped and developed countries, diseases related to obesity, such as insulin resistance, type 2 diabetes and dyslipidemia have become a public health problem. Among the strategies to prevent or combat such diseases is exercise. Exercise positively affects the activity and/or expression of sirtuins, resulting in better oxidative metabolism efficiency, increased biogenesis and mitochondrial function, as well as maintenance of the antioxidant system [In the last two decades, evidence has accumulated about the effect of exercise on the expression and/or activity of sirtuins. Much of this knowledge has emerged from research in animal and cellular models, and to a lesser extent from studies in humans, in most cases, in healthy humans. In this review we summarize the evidence about the effects of different types of exercise on SIRT1 and SIRT3 in skeletal muscle, mostly in healthy individuals, and the role of such sirtuins as regulators of the benefits of exercise.Skeletal muscle not only functions to generate force and movement, it is now known that it is a tissue that acts as an endocrine organ secreting cytokines and transcription factors into the bloodstream, thus regulating the function of other organs. Furthermore, skeletal muscle is a metabolically active tissue with an important role in the maintenance of the metabolic homeostasis of the organism. Skeletal muscle comprises approximately 40% of the total body mass, and it is the major site for insulin-stimulated glucose up-take as well as the main energy consumer of lipid catabolism ,8. MetabPhysical exercise causes cellular metabolic stress, which impacts the sirtuins. In this context, the most studied sirtuins have been SIRT1 and SIRT3; SIRT1 is located intracellularly in the nucleus and SIRT3 is located in the mitochondria. The study models that have been used range from cellular cultures and rodents to humans. In 2009, Palacios et al. reported that mouse skeletal muscle SIRT3 responds dynamically to six weeks of voluntary exercise to coordinate the downstream molecular response. They showed that exercise increases the SIRT3 protein content, as well as the associated phosphorylation of cAMP response element-binding (CREB), the up-regulation of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and the citrate synthase (CS) activity. They also demonstrated that SIRT3 is more expressed in type I muscle fiber. The researchers found that in Sirt3 knockout mice, the phosphorylation of AMP-activated protein kinase (AMPK) and CREB and the mRNA of PGC-1α was down regulated. Hence, they suggested that these key cellular molecules could be important components of the biological signals regulated by SIRT3 . Based oKnowing that the metabolic stress induced by exercise causes changes in the NAD+ and subsequently in the sirtuins, and after an interesting review, White and Schenk (2012) suggested that the increased ATP demand during exercise leads to an increase in the NAD+ level and NAD+/NADH ratio, which provides increased substrate for SIRT1 and SIRT3. The increase in ATP production is facilitated by SIRT3, which deacetylates and activates enzymes of the tricarboxylic acid (TCA) cycle, β-oxidation, and electron transport chain. At the same time, SIRT3 acutely reduces mitochondrial protein synthesis, which maximizes the availability of reducing equivalents for ATP production. On the other hand, in response to exercise, SIRT1 contributes to mitochondrial biogenesis through PGC1α-dependent and -independent mechanisms .d-ribofuranosyl-imidazole-4-carboxamide (AICAR) treatment, suggesting that the SIRT3 content is regulated in an AMPK–independent manner. The researchers did not find a relationship between mitochondrial SIRT3 and fatty acid oxidation; however, they suggested the possibility that the post-translational activation of SIRT3, instead of the protein content, plays an acute regulatory role in oxidative metabolism in skeletal muscle in vivo [In 2012, Gurd et al. reported that SIRT3 is proportional to the PGC-1α protein and the oxidative capacity of the mammalian muscle, which means that there is more SIRT3 in the heart, followed by type I muscle fiber, and finally, in type II muscle fiber. They also confirmed that in the muscle of mammals, the subcellular localization of SIRT3 is the mitochondria  and that in vivo .The connection between the regulation of the production of ATP and SIRT3 was studied by Vassilopoulos et al. (2014); the data obtained by these researchers suggests that acetyloma signaling contributes to the homeostasis of mitochondrial energy through SIRT3-mediated deacetylation of ATP synthase protein. Their results demonstrated that the F1 portion of the ATP synthase complex contains multiple SIRT3-dependent reversible acetyl-lysines that are altered under various metabolic stress conditions, including exercise, in both mice and human samples .Previously it has been suggested that SIRT3 carries out the antioxidant function by deacetylation of manganese superoxide dismutase (MnSOD) . Shi et In the same way, understanding the role of sirtuins as regulators of the benefits of exercise in human health has been pivotal to many investigations and obtaining greater knowledge. The section below is divided by subheadings. It provides a concise and precise description of the experimental results and their interpretation as well as the experimental conclusions that can be drawn.Several investigations have focused on analyzing the effect of exercise on the increase of sirtuin expression, and measuring mRNA expression and/or the increase of protein or even the activity of sirtuin Although it is difficult to compare these studies, here are some of the important findings. Lanza et al. 2008) were pioneers in trying to explain the effect of exercise and influence of age on the expression of sirtuins. They undertook a cross-sectional investigation in which they analyzed healthy young and older subjects that were divided into sedentary and trained (subjects had performed at least 1 h of exercise 6 days per week over the past 4 years or longer). The research reported that regular endurance exercise partly normalizes age-related mitochondrial dysfunction, and the researchers observed that regular training, regardless of age, increased the activity of the CS and la expression of proteins involved in mitochondrial biogenesis including PGC-1α, nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM) as well as SIRT3 . Villano08 were pAnother cross-sectional study was carried out by Koltai et al. (2018) with master athletes and sedentary subjects, all of whom were over 60 years old. Greater expression of forkhead box O1 (FOXO1) and SIRT1 mRNA was observed, as well as higher content of MnSOD and SIRT3 proteins in the master athletes compared with sedentary participants . Only miThe above is explained because SIRT3 deacetylates the medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase, thus controlling the metabolism of fatty acids . As mentThe investigations into the effects of physical exercise can be grouped by type of exercise: acute exercise (a single exercise load), exercise with successive loads, and exercise training , aerobic or resistance exercise).In analyzing In addition to considering the influence of the methodology followed in each investigation on the results obtained, it is important to note that the results will vary depending on the type of participants. Radak et al. reported that the expression of SIRT1 mRNA increased in skeletal muscle samples from sedentary subjects taken immediately after a single exercise load compared to active subjects in whom there was no difference in mRNA .In most studies, besides studying the effect of exercise on sirtuins, the impact on molecules or metabolic pathways related to sirtuins has also been analyzed. Guerra et al.  showed tA constant observation is that acute exercise does not increase either the protein or the mRNA of SIRT3 ,27,28,29Only Dumke et al. have analyzed the effect of an exercise load for three consecutive days. They found that the intervention caused acute increases in mRNA of the upstream elements as Pgc-1α and Sirt1, and chronic increases in the downstream targets as cytochrome C and CS, thus increasing mitochondrial biogenesis and oxidative enzyme capacity .The observations derived from the effect of consecutive loads on the SIRT1 are close to the pattern observed in the training interventions with either HIIT or aerobic training. In general, research has found that several sessions of HIIT increase the protein or activity of SIRT1 and along with it the PGC-1α protein, either in the whole muscle or in the cell nucleus ,32,33. NThe PGC-1α is a transcriptional co-activator that plays a crucial role in coordinating mitochondrial gene transcription. In most interventions with HIIT it was found that the nuclear PGC-1α protein increased, which indicates that PGC-1α is active . GreaterHIIT also increases the activity of COX, CS ,30,31, βResearch on the effects of aerobic training has focused on analyzing the response of SIRT3. It is generally observed that SIRT3 protein increases in the muscle of sedentary subjects after several training sessions ,36. BranAs already described, SIRT3 deacetylates key molecules that include elements of the TCA cycle and proteins involved in oxidative phosphorylation ,48,49 asThe findings of Johnson et al. also contributed to the ROS management line. Their participants were young and elderly sedentary people: the endurance training had an age-dependent response. The results showed that the young participants increased SIRT3 and MnSOD proteins without changes in the catalase, in addition, in the young individuals also increased the NAMPT protein, an upstream regulator of SIRT3. On the other hand, older adults after endurance training also increased the SIRT3 protein, although in a smaller proportion than the young, it was also observed that the enzymatic activity of MnSOD and catalase increased; NAMPT protein did not increase with training, but when analyzing the chronic effect of exercise (> 4 years), a higher content of this protein was observed in active than in sedentary older adults. The researchers concluded that endurance training increases muscle and mitochondrial antioxidant capacity in young and adult individuals . The effWe tested the effect of 12 weeks of resistance training in obese youths on the content of SIRT3 protein. The SIRT3 remained unchanged, suggesting that this protein might not be activated during resistance training because this type of training preferentially uses phosphocreatine and muscle glycogen for ATP synthesis in the sarcoplasm. This means that resistance training does not induce sufficient metabolic stress to activate oxidative phosphorylation, and consequently, the metabolic pathways that induce the activation of SIRT3 . Other aIn conclusion, the effects of exercise on SIRT1 and SIRT3 in human skeletal muscle will depend on the type of exercise . The effMeanwhile, the effect of interventions with aerobic training has been studied only on SIRT3. The results indicate that after several sessions of aerobic training there is an increase of SIRT3 in sedentary individuals and increases in PGC-1α. In addition, NAMPT also increases as an effect of exercise, allowing the synthesis of NAD+, the main substrate of sirtuins.Due to the methodological complexity, the investigations have focused on some part of the metabolic pathway. The information we currently have is complemented by the results of all these investigations, even the animal and cellular models. It is known that exercise activates each one of the sirtuin pathways in different ways, but the outcome is common: to improve the mitochondrial health, which places them as therapeutic targets for the treatment of metabolic diseases such as obesity, insulin resistance and type 2 diabetes. So, in order to identify exercise training that can efficiently achieve greater improvement in these metabolic states, it will be necessary to perform more research that evaluates the effect of different types of exercise on both sirtuins and the health benefits in metabolically compromised individuals of different ages and different fitness level.


Dioscorea deltoidea (laboratory code DM-05) on the physiological and biochemical parameters of vascular endothelial function in hypoestrogen-induced endothelial dysfunction after bilateral ovariectomy. It was shown that the use of DM-05 at a dose of 1 mg/kg makes it possible to prevent the development of arterial hypertension (the level of systolic blood pressure (SBP) decreases by 9.7% (p < 0.05) and diastolic blood pressure (DBP) by 8.2%), to achieve a decrease in the coefficient of endothelial dysfunction by 1.75 times against the background of a hypoestrogenic state. With DM-05, an increase in the concentration of stable nitric oxide metabolites (NOx) by 45.6% (p < 0.05) and an increase in mRNA endothelial nitric oxide synthase (eNOS) expression by 34.8% (p < 0.05) was established, which indicates a positive effect of furostanol glycosides on the metabolism of nitric oxide after ovariectomy. Positive dynamics in the histological structure of the heart and the abdominal aorta indicate the pronounced endothelio- and atheroprotective effects of DM-05.Currently, there is no doubt surrounding a theory that the cardiotropic effects of sex hormones can be due to their direct effect on the cardiovascular system. In recent years, interest in the study of steroid glycosides has increased. We studied the effects of furostanol glycosides (protodioscin and deltozid) from the cell culture of the  Atherosclerotic cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the world. According to the World Health Organization, there were 56.9 million deaths worldwide in 2016, and more than half of these (54%) were due to the top 10 causes of death. Ischemic heart disease and stroke are the world’s most significant killers, accounting for a combined 15.2 million deaths in 2016. These diseases have remained the leading causes of death globally in the last 15 years. Oxidative stress, endothelial activation, and endothelial dysfunction have been largely identified as the main alterations involved in the pathogenesis of macrovascular diseases .Currently, endothelial dysfunction is understood as an imbalance between mediators, which normally ensure the optimal course of all endothelium-dependent processes . When enThe concept that the influence of sex steroids to one degree or another extends to the functional state of all organs and systems, including the cardiovascular system, is actively developing, and researchers are finding numerous confirmations. Sex differences in the development of cardiovascular diseases have been described in humans and animals. These differences explain the low incidence of cardiovascular diseases  in women during the reproductive period . All sexIn addition, there is evidence that phytoestrogens, even in a small amount, characteristic of the “Western” diet, have a cardioprotective effect. It was found that the habitual use of two phytoestrogens, isoflavone from soybeans and lignans from flaxseed, is associated with less rigidity of the aortic wall in postmenopausal women . These cDioscorea deltoidea cell culture (laboratory code DM-05).Our attention, which is focused on pharmacological effects in a model of endothelial dysfunction caused by bilateral ovariectomy, was inspired by a composition containing furostanol glycosides (protodioscin and deltoside in a ratio of 3/2) obtained using a super-producer strain of Dioscorea deltoidea cell culture in a rat model of endothelial dysfunction after bilateral ovariectomy.The objective of this study is a pharmacological evaluation of the endotheliotropic effects of furostanol glycosides from Dioscorea deltoidea (DM-05) at a dose of 1 mg/kg were administered intraperitoneally once a day for 42 days. According to the protocol, the anesthetized animals were taken to the experiment on the 43rd day.According to the study design, hypoestrogen-induced endothelial dysfunction was simulated by bilateral ovariectomy in female Wistar rats. The studied furostanol glycosides from the cell culture of p < 0.05) and diastolic blood pressure (DBP) by 30.5% (p < 0.05). At the same time, intraperitoneal administration of DM-05 at a dose of 1 mg/kg prevents the development of arterial hypertension; the level of SBP decreases by 9.7% (p < 0.05), and DBP decreases by 8.2%  by 24.9% ( by 8.2% .p < 0.05), which reflects the development of endothelial dysfunction and change in the ratio of endothelium-dependent to endothelium-independent vasodilation. In the group with intraperitoneal administration of DM-05 within 42 days after bilateral ovariectomy, the concentration of stable metabolites of nitric oxide was significantly increased by 45.6% (p < 0.05), in comparison with the group with no treatment, and approached the NOx concentration in the control group , vascular changes in the form of a spastic state of the arterioles, and a thickening of their walls were noted in the heart . When stThe pronounced decrease in blood pressure and CED shown in this study was reflected in the morphological characteristics of the pathology—with the intraperitoneal administration of DM-05 (1 mg/kg), a statistically significant decrease in the cross-sectional diameter of cardiomyocytes was demonstrated .The morphological study of histological sections of the wall of the abdominal aorta of the animals in the control group showed the preservation of the vessel architectonics. The intima was lined with a single layer of squamous epithelium or endothelium. Endotheliocytes were located along the edge of the intima on the basement membrane, one after another, flattened in shape. The cytoplasm of the cells was oxyphilic, and the nuclei were rod-shaped, located in the center of the cell and oriented longitudinally relative to the long axis of the cell.In the media, brightly oxyphilic, sinusoidally shaped, fenestrated elastic membranes were well-visualized, and between these membranes, dark-basophilic cross-cut nuclei of circularly located smooth myocytes were defined. The cell density was low. In the outer shell (adventitia), loosely located collagen and elastic fibers were determined, and the fibroblastic cells were located between these fibers. There were no destructive changes, no squamous cell infiltration, no microthrombosis, and no signs of edema .In histological examination of the abdominal aorta, in the group with hypoestrogen-induced endothelial dysfunction, morphological changes were revealed, and these changes included signs of perivascular and pericellular edema, as well as inflammation of all the membranes of the vessel wall (aorta). When Van Gieson’s method was performed, signs of endothelial dysfunction were observed, and these were morphologically manifested in the fact that, in some parts of the intima, the intermittence of the endothelial layer was visualized, and lymphocytes were also visualized c,d. The The complex of changes in the heart and the abdominal aorta allows us to evaluate them as corresponding to a period of persistent increase in blood pressure with the development of vascular damage due to estrogen deficiency caused by bilateral ovariectomy in female Wistar rats.Amid administration of furostanol glycosides from DM-05 (1 mg/kg) for 42 days against the background of a hypoestrogenic state, pronounced positive dynamics of the histological structure of the abdominal aorta was found. In this group, the preservation of the architectonics of the layers of the vessel wall was found. The ratio of the thickness of the layers showed no visual differences in comparison with the data of the control group. Preservation of the endothelial lining, the location of the endotheliocytes in one layer on the basement membrane, a flat shape of the cells, and weakly oxyphilic cytoplasm were observed. The nuclei were rod-shaped, oriented along the blood vessel. No signs of pericellular and perivascular edema were detected. A slightly increased cell density per unit area of the section was observed, although without signs of destruction, which occurred in animals without pharmacological therapy after bilateral ovariectomy .According to some authors, surgical menopause is associated with a higher risk of vascular changes than natural menopause. In a study involving 144,260 postmenopausal women, premature menopause (compared to the absence of premature menopause) was associated with a significantly increased risk of developing and progressing coronary heart disease, heart failure, aortic stenosis, mitral regurgitation, atrial fibrillation, ischemic stroke, peripheral artery disease, and venous thromboembolism. For natural premature menopause, the risk ratio was 1.36; for surgical premature menopause, the risk ratio was 1.87 .Sexual differences in decreased endothelial function are explained by changes in sex hormones with aging. In women, there is a progressive violation of endothelial function at all stages of menopause, which is partially associated with a decrease in estradiol level. The increased risk of cardiovascular disease in this group of women is primarily due to the reaction of tissues to a severe hypoestrogenic state in the absence of a physiological adaptation of the female body to new conditions ,15.In hypertension, the walls of great arteries become thicker and tougher, and so-called vascular remodeling processes occur . In the The prospective randomized Women’s Health Initiative (WHI) and the Early Versus Late Intervention Trial (ELITE) showed that starting menopausal hormone treatment (MHT) within 5 to 10 years of menopause is fundamental to the success of estrogen’s cardioprotection in postmenopausal women without adverse effects. Age stratification of the WHI data has shown that starting hormone treatment within the first decade after menopause is both safe and effective, and the long-term WHI follow-up studies are supportive of cardioprotection. This is especially true in estrogen-treated women who underwent surgical menopause. According to Naftolin et al., without continuing efforts at improving the drug regimens and removing harmful agents and practices from menopausal health care, it is inevitable that there will be increased aggregate misery, cost, and societal impact of age-related ovarian failure. The findings discussed above should be a call to action to further the search for effective lifestyle regimens, educational programs, diagnostic methods, and hormonal and non-hormonal agents with which the increased rate of CVD in postmenopausal years can be confronted .In recent years, interest in steroid glycosides has increased, and research has been pursued in several directions. These compounds are used for the synthesis of hormonal medications in the pharmaceutical industry, and the interest in them as substances with a wide range of biological effects on living organisms is growing.Dioscorea species with a high content of this steroid are used as raw materials for its production. In addition to Dioscorea rhizomes, agaves, various types of nightshade, fenugreek, yucca, and a number of other plants are used as industrial raw materials abroad.A component of steroid glycosides, sapogenins, serves as feedstock for the synthesis of steroid hormones and their analogues. The highest importance among these sapogenins, from which hormonal medications are obtained, is attributed to diosgenin, the leading position of which is due to the fact that the rhizomes of wild-growing and cultivated Dioscorea deltoidea cell culture (laboratory code DM-05). A unique feature of this strain is the complete absence of spirostanol glycoside production in suspension culture, and the protodioscin and deltoside furostanol glycoside production in a ratio of 3/2, which is due to the absence of oligofurostanoside-specific alpha glycosidase in DM-05.The object of this study was to obtain a composition containing furostanol glycosides using a super-producer strain of In the present study, we showed that bilateral ovariectomy in Wistar rats after 42 days leads to a significant increase in systolic and diastolic blood pressure, changes in the ratio of the response of endothelium-dependent to endothelium-independent vasodilation with a statistically significant increase in the coefficient of endothelial dysfunction by more than two times, compared to the control group. This, together with our data regarding the decrease in the concentration of final stable nitric oxide metabolites, and with the data regarding the change in the histological structure of the heart and the abdominal aorta, indicates a complex of functional, biochemical, and morphological changes associated with the progressive loss of endogenous estrogens 42 days after hypoestrogenic state simulation.The results obtained in this study confirm our hypothesis about the effectiveness of furostanol glycosides in endothelial dysfunction and arterial hypertension after ovariectomy in rats. We showed that the intraperitoneal administration of DM-05 at a dose of 1 mg/kg prevents the development of arterial hypertension and eliminates the imbalance of endothelium-dependent and endothelium-independent vasodilation with a decrease in the CED in the hypoestrogenic state. At the same time, positive changes in the histological structure of the heart and the abdominal aorta indicate a pronounced endothelio- and atheroprotective effect of DM-05.Understanding the biochemical pathways that regulate the metabolism and bioavailability of nitric oxide is necessary in assessing the effectiveness of the pharmacological correction of endothelial dysfunction . To charx concentration and the increase in the expression of eNOS mRNA in the use of furostanol glycosides from DM-05 indicate an effective correction of metabolic disorders of nitric oxide after ovariectomy.The increase in NOAn increase in eNOS expression helps to establish the equilibrium of transcriptional and post-transcriptional molecular mechanisms that belong to both proapoptotic and antiapoptotic pathways. In addition, the bioavailability of NO is determined not only by eNOS levels but also by the presence of substrate and eNOS cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROSs), which can inactivate eNOS . The incBased on the literature data and the results of this study, we think that the use of furostanol glycosides can slow down or prevent the formation of endothelial dysfunction and cardiovascular disease in postmenopausal women. To create a complete concept of the pharmacological activity of furostanol glycosides, the athero- and cardioprotective effects of DM-05 on models of cardiovascular diseases in estrogen deficiency require further detailed study. An important task of further research is the precise study of the pathways of DM-05′s influence on the content and metabolism of NO. DM-05 might affect the level of eNOS phosphorylation or the enzyme compartmentalization, increasing its activity.The experiments were approved by the Belgorod State National Research University, Local Ethics Committee, Belgorod (Protocol #03/19). Ethical principles of handling laboratory animals were observed in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, CETS No. 123. The animals were housed in an animal facility with a 12 h day/12 h night cycle and provided a standard laboratory diet and water. Experiments were carried out on 30 Wistar rats  weighing 250 ± 25 g. For the study, the rats were taken with no external signs of disease, having passed the quarantine regime.(1)control group—a group of falsely operated animals (control);(2)a group with simulated endothelial dysfunction after bilateral ovariectomy (OE);(3)Dioscorea deltoidea (OE + DM-05).a group with a correction of pathology with furostanol glycosides from a cell culture of The following groups were included in the experiment :® 50 mg/kg of rat body weight, i.p.): the anterior abdominal wall was opened, and the wound was then sutured in layers without removing the ovaries. Animals of the control group were intraperitoneally injected with NaCl 0.9% 0.1 mL/100 g once a day for 42 days.A false operation was performed on the animals of the control group under anesthesia : the anterior abdominal wall was opened, the tube and peritoneal parts of the ovaries were ligated, and they were then removed [Endothelial dysfunction was simulated by bilateral ovariectomy in female Wistar rats. Bilateral ovariectomy was performed under general anesthesia  from a ® 6 mg/100 g + Chloral hydrate 15 mg/100 g), the left carotid artery was catheterized for intravascular measurement SBP and DBP using the MP150 data acquisition and analysis system . In continuous blood-pressure measurement mode, vascular tests of endothelium-dependent (acetylcholine 40 μg/kg) and endothelium-independent  vasodilation were performed. Vasoactive agents were administered at 15 min intervals through a catheter inserted in the femoral vein. The level of endothelial dysfunction in the experimental animals and the level of its correction by the studied drugs were valued by CED expressed in relative units.On the 43rd day from the start of the experiment, in each animal under anesthesia  recovery curve at a functional test with nitroprusside administration; SBPAH is the area of the triangle above the BP recovery curve at a functional test with acetylcholine administration. Points of the smaller side of this triangle include the point of BP before the test and the point of the maximum reduction of BP, and the longer side is the time of BP restoration ,19,20.After functional vascular tests, the animals were euthanized, a blood sample was taken from the left ventricle of the heart to determine the final stable nitric oxide metabolites, and the heart and abdominal aortic section were taken for histological studies, as well as for evaluation of eNOS gene mRNA expression.® NP80 spectrophotometer . The output of the RNA was approximately 1000 ng/μL.Aortic tissue was harvested, homogenized, and incubated for 10 min at 37 °C in the “Extract RNA” solution. After sample lysing in the reagent, it was subjected to chloroform purification, and the resulting RNA precipitate was washed with isopropanol and 70% ethanol. The concentration of the resulting RNA was measured on an IMPLEN NanoPhotometerReverse transcription was performed using the MMLV RT SK021 kit in accordance with the manufacturer’s protocol . The mixture was gently mixed and heated at 70 °C for 2 min for the melting of RNA secondary structures and the subsequent annealing of the OligoDT primer . After that, the samples were transferred to ice. The entire reaction mixture was incubated within 60 min at 40 °C in a T100™ ThermalCycler . To stop the reaction, the mixture was heated at 70 °C for 10 min. The resulting cDNA was diluted to a concentration of 1 ng/μL. The level of gene expression was evaluated relative to the values of the Gapdh reference gene.N-naphthylethylenediamine in water) and Solution II (1% solution of sulfanilamide in 30% acetic acid). Both solutions were stored in the dark at 4 °C for several months. To prepare a solution of vanadium chloride, 400 mg of VCl3 was dissolved in 50 mL of 1 N HCl, followed by filtration through a paper filter. Freshly prepared solution was always used.We used a modification of the method for determining stable NO metabolites, which allows one-step quantitative determination of total nitrates and nitrites after deproteinization of blood serum . The prix) was determined by the colorimetric method by the development of color in the diazotization reaction of sulfonamide nitrite, which is part of the Griss reagent. To construct the calibration curve, we used a 1 M solution of NaNO2 in water, which was stored at a temperature of −20 °C; before use, it was diluted 1000 times and a series of dilutions was prepared to construct a curve [The level of NO metabolites . The blocks with the standard orientation of the pieces were poured at the station, so as to pour biological material into EC 350 paraffin . To ensure standardization, paraffin filling was carried out in the form of multiblocks of 5–6 pieces. Slices for histological examination with a thickness of 5 μm were made on a semi-automatic rotary microtome with a system of transportation and a spreading of slices “NM 340E” . Hematoxylin and eosin staining was carried out in a machine for staining histological sections and smears . Descriptive histological studies were performed under an Axio Scope A1 microscope .t criterion) or non-parametric (Mann–Whitney test) methods, depending on the type of distribution. Differences were determined at a 0.05 significance level. Statistical analyses were performed using Statistica 10.0 software [For all data, descriptive statistics were used, and the data were checked for normal distribution. Distribution type was determined using the criterion of Shapiro–Wilk. In case of a normal distribution, the average value (M) and standard error of the mean (m) were calculated. In cases of abnormal distribution, the median (Me) and the quartile range (QR) were calculated. Between-group differences were analyzed by parametric (Student’s software .


M. tuberculosis is recognized as the primary target of isoniazid (INH), a first-line antibiotic for tuberculosis treatment. To identify the specific interactions of INH-NAD adduct and its derivative adducts in InhA binding pocket, molecular docking calculations and quantum chemical calculations were performed on a set of INH derivative adducts. Reliable binding modes of INH derivative adducts in the InhA pocket were established using the Autodock 3.05 program, which shows a good ability to reproduce the X-ray bound conformation with rmsd of less than 1.0 Å. The interaction energies of the INH-NAD adduct and its derivative adducts with individual amino acids in the InhA binding pocket were computed based on quantum chemical calculations at the MP2/6-31G (d) level. The molecular docking and quantum chemical calculation results reveal that hydrogen bond interactions are the main interactions for adduct binding. To clearly delineate the linear relationship between structure and activity of these adducts, CoMFA and CoMSIA models were set up based on molecular docking alignment. The resulting CoMFA and CoMSIA models are in conformity with the best statistical qualities, in which r2cv is 0.67 and 0.74, respectively. Structural requirements of isoniazid derivatives that can be incorporated into the isoniazid framework to improve the activity have been identified through CoMFA and CoMSIA steric and electrostatic contour maps. The integrated results from structure-based, ligand-based design approaches and quantum chemical calculations provide useful structural information facilitating the design of new and more potentially effective antitubercular agents as follow: the R substituents of isoniazid derivatives should contain a large plane and both sides of the plane should contain an electropositive group. Moreover, the steric and electrostatic fields of the 4-pyridyl ring are optimal for greater potency.The enoyl-ACP reductase enzyme (InhA) from  Mycobacterium tuberculosis (M. tuberculosis) still remains a major cause of illness and death worldwide, especially in Asia and Africa. Globally, 9.2 million new cases and 1.7 million deaths from TB occurred in 2006. The number of new cases was still increasing between 2005 and 2006, from 9.1 to 9.2 million (an increase of 0.6%) [para-aminosalicylic acid, ethionamide, and cycloserine [Tuberculosis (TB) caused by loserine ,4,5. Howloserine ,9,10. Actrans-enoyl-acyl carrier protein reductase (InhA) displaying a long-chain fatty acid elongation activity. Inhibition of this activity by INH blocks the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope [+ to form covalent adducts like the parent INH. Therefore, insight into the nature of these bioactive forms of INH derivative prodrugs in the InhA binding pocket should provide more detailed information for designing agents that greatly inhibit InhA. In the present study, molecular docking and quantum chemical calculations were employed to elucidate the potential antitubercular binding modes and main interactions of INH derivative adducts. In order to understand the structural requirement of InhA inhibitors, the relationship between structure and activity of these compounds was elucidated by CoMFA and CoMSIA methods [Isoniazid (INH) has the greatest bactericidal activity and is used almost from the outset of tuberculosis chemotherapy ,12,13. Tenvelope ,15,16,17envelope ,20,21,22envelope ,24. INH-envelope . To reveenvelope ,27,28. Aenvelope ,30,31. Tenvelope . Ligand-envelope ,34,35,36 methods ,38. The Molecular docking calculations were employed to predict the potential binding mode of INH derivative adducts in the InhA binding pocket. To ensure that the binding modes of adduct inhibitors obtained from molecular docking calculations are reliable the docking parameters in Autodock 3.05 program were validated. The structure of the INH-NAD adduct in the X-ray crystal structure was extracted and docked back into the binding pocket. The superimposition between the docked conformation and the X-ray crystal structure of INH-NAD adduct is shown in M. tuberculosis of these compounds are shown in M. tuberculosis with a log (1/MIC) value of 7.70, where MIC is the in vitro minimum inhibitory concentration in µg/mL unit. The docked conformation of the INH-NAD adduct into the active site of InhA is indeed similar to that observed in the X-ray crystal structure. The 4-pyridyl ring of the INH-NAD adduct is buried in the cavity formed by the Ala191, Gly192, Trp222, Tyr158, Phe149 and Pro193 residues, as shown in 2 and a nitrogen atom of the adenine part interact with the carbonyl oxygen group of Asp64 and the NH backbone of Val65 to form two hydrogen bonds. According to the observed interactions of INH-NAD adduct, the main interactions found in the binding are hydrogen bond interactions, therefore they may play an important role in the binding of adduct inhibitors.INH and its derivatives were used for molecular docking calculations. The chemical structures and experimental biological activities against the BCG strain of 1 possessing a furan ring at the R substituent position, the inhibitory activity differs slightly from the activity of INH, with log (1/MIC) of 7.22. The NAD part of compound 1 is aligned well with the NAD part of INH, as shown in 1 are insignificantly changed (< 0.1 Å), compared with INH, as shown in 1 as compared with the 4-pyridyl ring of the INH adduct, the π-π interaction with the aromatic sidechain of Phe149 is lost. However, a H-π interaction with the aromatic sidechain of Phe149 is formed to compensate for the lost interaction. These results reinforce the slight difference on the inhibitory activity of compound 1 as compared with INH.With regard to compound 2-7, the NAD parts of these compounds lie in the same position as the NAD part of INH, as shown in For the other highly active compounds, compounds 8-23, mostly interact with Pro193 to form hydrophobic interactions and interact with Ala191, Gly192 and Tyr158 to form van der Waals interactions. Unfavorably, a steric effect between the R substituents and the aromatic sidechain of Trp222 could be observed. Two of six member ring substituents, phenyl and pyridyl rings, are favored to form a hydrophobic interaction with Pro193, such as compounds 12, 15, 17 and 20. However, the bulkier atoms or group of atoms in the six member ring substituents could collide with surrounding amino acid, in particular with Trp222. To reduce the steric effect, the position of the NAD part of these compounds may be shifted as compared with INH, leading to an increase in the hydrogen bond distances of the NAD part, especially for the two hydrogen bonds of the nicotinamide ring with Asp148, as shown in The different R substituents of the INH derivative adducts are located inside the hydrophobic cavity generated by the residues of Ala191, Gly192, Trp222, Tyr158, Phe149 and Pro193. The R substituents of moderately active compounds, compounds 13 and 19, no steric effect could be found with the surrounding amino acids in the R substituent cavity. The important hydrogen bonds of the NAD part also remain in the same quality as in INH, as shown in For the five member ring substituents contained in compounds 24-35 show lower activity against strains of M. tuberculosis. The van der Waals interactions are the major interaction of the R substituents of these compounds. Steric effects between the substituents and the sidechain of Pro193, Phe149, Tyr158 and Trp222 could be found. The bicyclic substituents in compounds 28, 31 seem to be the large substituents. These large substituents bump into amino acids in the R substituent cavity, leading to a shift in the position of the NAD part of these compounds, compared with INH. Moving the NAD part position leads to a loss of the hydrogen bond interactions of the nicotinamide ring with Asp148 and pyrophosphate part with Thr196, as shown in Compounds 33-35 display the lowest activity against strains of M. tuberculosis. The R positions of these compounds are substituted by a piperidine ring. The bulky piperidine ring could only form a van der Waals interaction with amino acids in the R substituent cavity. A steric effect of this substituent with the sidechains of Pro193, Phe149, Tyr158 and Trp222 is also observed. The bulky groups at the N position on piperidine ring of compounds 33-35 reinforce the steric effect of these compounds. Because of the steric effect, the hydrogen bonding interactions of nicotinamide part with Asp148 and pyrophosphate part with Thr196 of compounds 33, 34 are eliminated, as shown in Compounds 35 possesses the piperidine ring in the R substituent, the NAD part of this compound shows good alignment with the NAD part of the INH-NAD adduct, as shown in 35 may be related to the activation process. The activation process of these compounds proceeds via free radical formation. The ability of R substituents to stabilize the acyl radical should influence the efficiency for coupling of the acyl radical with NAD+ to form an adduct inhibitor for InhA. The rich aromatic group has a higher capacity to stabilize the acyl radical than a non-aromatic group. The electronic charge of the acyl radical can be delocalized through resonance of the aromatic ring. This makes it more stable, whereas the electronic charge delocalization of acyl radical in the non-aromatic system could not occur. Therefore, the aromatic pyridyl ring of INH shows higher potency to stabilize the acyl radical than the non-aromatic piperidine ring of compound 35, thereby explaining the lower inhibitory activity of this compound, compared with INH. Accordingly, the low potency for stabilizing the acyl radical of R substituents may be additional cause for the poor activity against M. tuberculosis of other compounds that contain the R substituent like compound 35.Interestingly, although compound 2 and NO2 completely reduces the inhibitory activity of compounds because of their steric effect in the R substituent cavity.The hydrogen bonds of the NAD part with Asp148, Thr196, Ile21, Ser20 and Asp48 are major interactions for adduct binding. For the R substituent, the π-π or H-π interaction with the aromatic sidechain of Phe149 and the H-π interaction with the aromatic sidechain of Tyr158 are also required to enhance the binding of these inhibitors. Moreover, the capacity of R substituents for stabilizing the free radical intermediate in the activation process is interesting. To facilitate the activation process, R substituents should reveal a high aromatic property. Importantly, R substituents must not present a steric effect in the R substituent cavity. The 4-pyridyl ring and furan ring seem to be optimal for binding of InhA inhibitors. The replacement of hydrogen atoms on these ring with bulkier atoms or groups of atoms such as Cl, I, Br, NH1 and the weakly active compound 33 were selected for quantum chemical calculations. Moreover, to explain the effect of the activation process on the inhibitory activity of these inhibitors, the interaction energy of compound 35 was also calculated. Based on quantum chemical calculations at MP2/6-31G(d) level of theory, the interaction energies of INH-NAD adduct fall in the range of -33.01 to 13.20 kcal/mol. The 4-pyridyl ring, the R substituent of INH-NAD adduct, shows an attractive interaction with Phe149 and Tyr158 with an attraction energy of 1.21 and 2.09 kcal/mol. These attraction energies correlate well with the observed interactions from the X-ray crystal structure of INH-NAD bound in the InhA active site. The 4-pyridyl ring forms a π-π interaction with the aromatic sidechain of Phe149 and a H-π interaction with the aromatic sidechain of Tyr158. The nicotinamide part provides the main attraction energy of 4.31 kcal/mol with Asp148. This attraction energy agrees with the weak hydrogen bonds produced from two hydrogen atoms on the nicotinamide ring and the carbonyl backbone of Asp148. In this part, a high repulsion interaction with Ile194 could be observed with a repulsion energy of 13.20 kcal/mol. From the X-ray crystal structure, the carbonyl amide of the nicotinamide part lies near the carbonyl backbone of Ile194. The repulsion between two groups with the same charge may occur. Nicotinamide ribose displays a highly attractive interaction of 11.15 kcal/mol with Lys165, due to the electrostatic interaction of nicotinamide ribose hydroxy group with Lys165. The pyrophosphate part presents higher attraction energy than other parts of the INH-NAD adduct. The highest attraction energy of 33.01 kcal/mol is found between the pyrophosphate part and Ser20. Moreover, other highly attractive interactions are also produced between the pyrophosphate part and Ile21 and Thr196, with attraction energies of 23.92 and 24.48 kcal/mol, respectively. These high attraction energies of the pyrophosphate part relate to the strong hydrogen bond interactions of the phosphate oxygens with Ser20, Thr196 and Ile21. These results confirm that the hydrogen bond interactions are the key interactions in this region. Among all attraction energies of the adenine part, the interaction with Asp64 shows the highest attraction energy , consistent with the hydrogen bond interaction of NH2 on the adenine part with the carbonyl sidechain of Asp64. Indeed in the X-ray crystal structure, the NH backbone of Val65 can form a hydrogen bond with the nitrogen atom of the adenine ring. The interaction energy of this interaction is low . Based on the interaction energy calculation results, all high attraction energies correlate with the hydrogen bond interactions of the INH-NAD adduct in the InhA binding pocket observed in the X-ray crystal structure. Therefore, we can conclude that the hydrogen bond interactions are crucial for binding of the INH-NAD adduct, particularly the hydrogen bond interactions of the pyrophosphate part.To investigate the key interactions for binding of INH-NAD adduct and its derivatives, INH, the highly active compound M. tuberculosis, with a log (1/MIC) of 7.70. The inhibitory activity of compound 1 at a log (1/MIC) of 7.22 is comparable with INH-NAD adduct. These two compounds show structural difference in the R substituent. The furan and 4-pyridyl rings are maintained at the R substituent position of compound 1 and the INH-NAD adduct, respectively. The binding mode of compound 1 in the InhA active site was studied by means of molecular docking calculations. To quantitatively elucidate the R substituent influence on its activity, the interaction energy of compound 1 was calculated and compared with the calculated interaction energy of the INH-NAD adduct. The interaction energy of compound 1 is presented in 1 attractively contacts with Phe149 with an attraction energy of 0.93 kcal/mol. This attractive interaction correlates with the H-π interaction of the hydrogen atom on the furan ring with the aromatic sidechain of Phe149. Moreover, this attractive interaction is comparable with the π-π interaction of the 4-pyridyl ring of INH-NAD adduct with the aromatic sidechain of Phe149. Additionally, slightly attractive interactions with Pro193 and Trp222 could be found with attraction energies of 0.89 and 1.48 kcal/mol, respectively. However, the furan ring of compound 1 is missing the attractive interaction with Try158 of about 1 kcal/mol. This result correlates with the docking results in which the smaller furan ring loses the H-π interaction with Try158 compared with the 4-pyridyl ring of the INH-NAD adduct. For other parts of compound 1, nicotinamide, nicotinamide ribose, pyrophosphate, adenine ribose and adenine ring, the main interaction energies are all similar to those of the INH-NAD adduct. These results suggest that the slightly lower inhibitory activity of compound 1 as compared with INH-NAD adduct could be attributed to the loss of attractive interaction of the furan substituent with Try158 because of its smaller size.In the data set of our study, the INH-NAD adduct is the highest potency compound against 33, the least active compound, was taken to calculate the interaction energies and then these calculated interaction energies were compared with the interaction energy of the INH-NAD adduct, the most active compound. The interaction energy of this compound is given in 33 possesses a piperidyl ring in the R substituent position. The interactions of the piperidyl ring substituent with Phe149, Tyr158, Ala191, Pro193 and Trp222 show high repulsion energies . These high repulsion energies relate to the steric effect of the R substituent, as found from the docking results. As in the docking results, the steric effect changes the position of the NAD part of compound 33 leading to an alteration of the hydrogen bond patterns of this compound. Supporting the molecular docking results, the attraction energy of the important hydrogen bonds between pyrophosphate and Ser20 are decreased about 20 kcal/mol as compared with the INH-NAD adduct. The docked structure of compound 33. Moreover, the attraction energy of pyrophosphate with Ile16 is also reduced by about 5 kcal/mol. In addition of the reduction of the main attraction energy, a repulsion energy of 14.11 kcal/mol between the adenine ring and Val65 is additionally detected. Therefore, based on the calculated interaction energy, the high repulsion energy between the R substituent and surrounding amino acid could account for the lowest activity of compound 35 shows inhibitory activity like compound 33 with log (1/MIC) of 3.22. The binding mode of this compound is similar to that of the INH-NAD adduct, as described in the previous discussion of the docking results. To elucidate the important binding interactions of this compound, the interaction energy of compound 35 was calculated as given in 35 exhibits slightly attractive interactions with Tyr158 and Trp222. Repulsive interactions of the piperidyl ring with Phe149, Ala191, Gly192 and Pro193 could be observed with repulsion energy of less than 6.72 kcal/mol. Nevertheless, all interaction energies of other parts of compound 35 are quite comparable with those of INH-NAD. Moreover, the repulsion energy between the nicotinamide and Ile194 is decreased about 10 kcal/mol. Based on the calculated interaction energy, the binding affinities of compound 35 and INH in the InhA binding pocket should be similar. Therefore, the lower activity of compound 35 may depend on the activation process that requires the high capacity of substituents for stabilizing the free radical intermediate. In this case, the aromatic pyridyl rings of INH-NAD are more suitable for the activation process than the non-aromatic piperidyl ring of compound 35, supporting the lower inhibitory activity of this compound.Compound 2) and the cross-validation (r2cv) of 0.94 and 0.67 for CoMFA model and 0.96 and 0.74 for CoMSIA model. The selection of the best CoMSIA is based on the highest r2cv. Thus, the best CoMSIA model includes steric and electrostatic fields. Considering all statistical parameters, the best model CoMSIA including steric and electrostatic fields shows slightly more predictive ability than the best CoMFA model. The statistical parameters of the best CoMFA and CoMSIA models generated based on molecular docking alignment are shown in The correlations between experimental and predicted activities are shown in The CoMFA and CoMSIA models reveal the importance of steric and electrostatic fields through the contour maps shown in M. tuberculosis. Nevertheless, the R substituent size is limited by the neighboring steric region. As the X-ray structure of the INH-NAD adduct bound in InhA, three amino acid residues, Ala191, Gly192 and Trp222 are located near the steric region. Therefore, any larger R substituent may collide with these residues. From molecular docking calculations, the moderately active compounds and weakly active compounds display steric effects with Trp222.The CoMFA steric field reveals two yellow contours and one large green contour near the R substituent, as shown in 1, 3, 6, 13 and 16 that contain a five member ring substituent show lower activity than INH bearing the 4-pyridyl ring. The five member ring of compound 1 is completely buried in the large green contour but is not present any atom in the steric region represented by a yellow contour. As compared with the 4-pyridyl plane of INH, the plane of the five member ring is rather small. The results suggest that the plane size of the five member ring substituent does not generate any steric effect and it is not large enough for improving the inhibitory activity. Compounds 9, 18, 23, 25, 28, 31 and 32 possess the large plane of bicyclic rings at the R substituent position. Although the bicyclic rings of these compounds are favorable for a green contour, the activities of these compounds are not better as compared with INH. From molecular docking calculations, these substituents are too large, which leads to generation of a steric effect with Trp222. As a result of the steric hindrance, the hydrogen bond interactions of the pyrophosphate parts of these compounds with Thr196 are eliminated. Compounds bearing the piperidine ring, 33-35, show the lowest activity. The piperidine ring of these compounds is bulkier than the 4-pyridyl ring of INH. The piperidine ring of compound 34 lies in the yellow contour, resulting in unfavorable active compound against strains of M. tuberculosis. These results indicate that steric effect is an additional cause of the lower activity of these compounds. Compounds 4. The results show that the fluorine atom does not enhance the inhibitory activity of this compound because this atom is close to the unfavorable blue contour. In accordance with the CoMFA and CoMSIA models for improving the inhibitory activity, hydrogen atoms on both sides of the 4-pyridyl ring should be substituted by more electropositive groups. The phenyl ring of compound 5 is like the 4-pyridyl ring of INH except for the fact the nitrogen atom (N4) of the 4-pyridyl ring is replaced by the carbon atom of a phenyl ring (C4). The alignment of compound 5 and the INH-NAD adduct obtained from molecular docking calculations reveals that the N4 of the pyridyl ring lies in the same position as C4 of the phenyl ring. The hydrogen atom attached to the C4 of the phenyl ring of compound 5 is placed in the red contour. Regarding inhibitory activity, compound 5 shows lower inhibitory activity than INH. These results imply that the hydrogen atom attached to the C4 of the phenyl ring is not favourable. Compounds 11, 14, 20 and 22 contain R substituents like compound 5. The C4 of the phenyl ring of these compounds attaches to Cl, I, Br and NO2 that are more electronegative than the hydrogen atom in compound 5. Although these groups are favorable for a red contour, the activities of these compounds are not increased as compared with compound 5. From molecular docking calculations, these compounds lose the important hydrogen bonds of the pyrophosphate moiety because of the steric effect of Cl, I, Br and NO2 with Trp222. These results imply that the nitrogen atom on the 4-pyridyl ring is optimal for both the steric and electrostatic fields.The CoMFA contours of the electrostatic field are shown in To enhance the activity of INH derivative adducts, the R substituents should contain a large plane. Both sides of the plane should present electropositive groups. However, size of the R substituents is controlled by the steric region above the substituent plane. Accordingly, the steric and electrostatic fields of the 4-pyridyl ring are optimal for greater potency of compounds. The improvement of electrostatic properties on the 4-pyridyl ring by replacement of the hydrogen atom on both sides of the 4-pyridyl ring with more electropositive groups should be done carefully because a steric effect can also be generated.2 were chosen from the original reference [M. tuberculosis were reported in Isoniazid and its 35 derivatives, a total of 36 compounds, with the general formula RC(O)NHNHeference . The inhIn the present study, the chemical structures of INH derivatives are presented in the form of INH derivative adducts, the active forms of these inhibitors. The X-ray crystal structure of the INH-NAD adduct in the InhA enzyme (pdb code 2ZID) was used as the initial geometry to build of the active adducts of the INH derivatives. All chemical structures of adducts were drawn with the Gaussview 3.07 program . The NADThe X-ray crystal structure of the INH-NAD adduct complexed to InhA (pdb code 2ZID) was used for molecular docking calculations. Docking calculations were carried out by the Autodock 3.05 program using the Lamarckian Genetic Algorithm (LGA) . The INH1) and the lowest active adducts (compounds 33 and 35) were selected to calculate their interaction energies. The whole structure of the INH-NAD adduct and its derivatives was fragmented into small parts consisting of the R substituent, nicotinamide, nicotinamide ribose, pyrophosphate, adenine and adenine ribose. The ends of each part were terminated by hydrogen atoms. The amino acids located within a radius of 6 Å from each part of adducts were adopted. The peptide chain was divided at the C and N terminal into the individual amino acids. The ends of individual amino acids were capped with hydrogen atoms. The interaction energies of each part with individual amino acids in the InhA binding pocket were determined by using the MP2 method with the 6-31G (d) basis set. The interaction energy (IE) of adducts with each amino acid is defined as follows:adduct + each amino acid is the energy of the complex structure between adduct and each amino acid. Eadduct and Eeach amino acid are the energies of adduct and each amino acid, respectively.To elucidate the important interactions of INH derivative adducts in the InhA binding pocket, individual interaction energies between adducts and the surrounding amino acids were computed. The X-ray structure of the INH-NAD adduct, docked conformations of the highly active adduct  field and electrostatic  field. The probe atom was placed at each lattice point and their steric and electrostatic interactions with each atom in compounds were all calculated with CoMFA standard scaling and then compiled in a CoMFA table. The computed field energies were truncated at 30 kcal/mol for both electrostatic and steric fields. CoMSIA similarity index descriptors were derived with the same lattice box as used for the CoMFA calculations. Five CoMSIA descriptors, steric, electrostatic, hydrophobic, hydrogen bond donor and hydrogen bond acceptor, were evaluated. An attenuation factor of 0.3 was used in the CoMSIA study to generate a contour map with prominent molecular features. The partial least square (PLS) approach was employed to derive a linear relationship, in which CoMFA and CoMSIA descriptors were used as independent variables and log (1/MIC) values were used as dependent variables. To obtain the optimal number of components to be used in the final analysis, the cross-validation was performed using the leave-one-out method with a 2.0 kcal/mol column filter to minimize the influence of the noisy columns. Sequentially, a final non cross-validated analysis was performed using the optimal number of components previously identified and was then employed to analyze the results. The q2 or r2cv values were used to evaluate the predictive ability of the CoMFA and CoMSIA models. The structural alignment of compounds into their bioactive conformations is essential in the set up of CoMFA and CoMSIA models. In the present study, the molecular alignment was derived from the reasonable positions of compounds in the InhA binding pocket obtained from the molecular docking calculations. The CoMFA and CoMSIA descriptor fields around the aligned compounds were generated automatically by Sybyl/CoMFA and CoMSIA routine at each lattice intersection on grid spacing of 2 Å. A spMolecular docking calculations and interaction energy calculations were performed on a series of INH derivative adducts to achieve a better understanding of the crucial interactions for binding affinity of these adducts in the InhA binding pocket. The obtained results from the two approaches demonstrate that hydrogen bond interactions play an important role on the adduct binding in InhA, especially the hydrogen bonds of pyrophosphate part. To reinforce the adduct binding, the R substituents of adducts should be optimal for its cavity. Too bulky substituents generate steric effects with high repulsion energy. Conversely, too small substituents did not generate the steric effect but the preferable interactions, π-π and H-π interactions, are missing. To clearly elucidate the structural requirements of adducts that favor binding interactions in the InhA binding pocket, robust CoMFA and CoMSIA models were established. The best CoMFA and CoMSIA models include the steric and electrostatic fields suggesting that the antitubercular activity of these adducts depend on their steric and electrostatic characteristics. CoMFA and CoMSIA steric and electrostatic fields reveal that the R substituents should contain a large plane and both sides of the plane should contain an electropositive group. However, size of R substituents is controlled by the steric region above the substituent plane. Finally, the integrated results from these approaches provide valuable concepts that can be utilized for designing new and more potential effective antitubercular drugs.


To evaluate the potential of magnetic hyperthermia using aminosilane-coated superparamagnetic iron oxide nanoparticles in glioblastoma tumor model.in vitro assays, the C6 cells cultured and transduced with luciferase were analyzed by bioluminescence in the absence/presence of alternating magnetic field, and also with and without aminosilane-coated superparamagnetic iron oxide nanoparticles. In the in vivo study, the measurement of bioluminescence was performed 21 days after glioblastoma induction with C6 cells in rats. After 24 hours, the aminosilane-coated superparamagnetic iron oxide nanoparticles were implanted in animals, and magnetic hyperthermia was performed for 40 minutes, using the best conditions of frequency and intensity of alternating magnetic field tested in the in vitro study  and verified the difference of bioluminescence before and after magnetic hyperthermia.The aminosilane-coated superparamagnetic iron oxide nanoparticles were analyzed as to their stability in aqueous medium and their heating potential through specific absorption rate, when submitted to magnetic hyperthermia with different frequencies and intensities of alternating magnetic field. In magnetic hyperthermia in vitro and in vivo analysis, resulted in cell death of 52.0% and 32.8%, respectively, detected by bioluminescence.The aminosilane-coated superparamagnetic iron oxide nanoparticles were stable, and their heating capacity increased along with higher frequency and intensity of alternating magnetic field. The magnetic hyperthermia application with 874kHz and 200 Gauss of alternating magnetic field determined the best value of specific absorption rate (194.917W/g). When these magnetic hyperthermia parameters were used in The magnetic hyperthermia was promissing for the therapeutical process of glioblastoma tumors in animal model, using aminosilane-coated superparamagnetic iron oxide nanoparticles, which presented high specific absorption rate. Glioblastoma represents one of the most devastating diseases of the central nervous system due to its extremely aggressive behavior, and no satisfactory responses to the varied therapeutic modalities so far.Glioblastoma (GBM) accounts for 47.7% of all malignant brain tumors, and only 5.6% of patients affected survive up to 5 years after diagnosis, as per the statistical report of the Central Brain Tumor Registry of the United States (CBTRUS),1 with Among the preclinical models of tumor induction, those that use C6 cells show advantages due to histological aspects and their similarities with findings in humans. These include nuclear polymorphism, high rates of mitosis, tumor necrosis foci, intratumor hemorrhage, invasion of the parenchyma, and neoangiogenesis.Preclinical studies in GBM tumors allowed understanding and evaluation of the different strategies for the therapeutic process.,4 Am among which, magnetic hyperthermia (MHT). Its relevance results from significant antitumor effects in models of GBM, as well as in other types of tumors. In the MHT technique, when directed towards the tumor and exposed to an alternating magnetic field (AMF) with appropriate intensity frequencies, the magnetic nanoparticles are heated until reaching a therapeutic temperature of 42-45°C, capable of affecting the tumor mass without hindering the surrounding normal tissue.Since currently there is no effective treatment for GBM, alternative therapeutic mechanisms have been developed,7 amon Preclinical studies showed the efficacy of several biocompatible sizes and coatings of SPIONs, although in many of these studies the efficacy of MHT was evaluated in models of GBM tumors induced in the flanks of the animals instead of intracerebral induction. Applications, both of the inductive cells of the tumor as well as the SPIONs through stereotaxis, are the most adequate options for the representation of the GBM model submitted to an MHT treatment. They mimic what really happens, and due to the need to place a large quantity of SPIONs located in the tumor to increase the heating potential of thermotherapy.Superparamagnetic iron oxide nanoparticles (SPIONs) have a great potential for application in the MHT, based on their magnetic properties of energy transformation, generating heat.9 Prec One of the magnetic nanoparticles that presents with a great potential is SPIONs coated with aminosilane (SPIONsAmin), as they present with a greater value of saturation magnetization (790.93 A/m) and influences the heating capacity, in comparison with SPIONs coated with other materials, for instance, carboxymethyl-dextran (227.13 A/m). Using SPIONsAmin in assays with MHT, Jordan et al., showed that SPIONs enabled the formation of more stable deposits of nanoparticles around the entire tumor, relative to SPIONs coated with dextran. Additionally, these SPIONsAmin were evaluated and used for clinical applications in patients with tumors, and did not harm cortical neurons in animal model studies.There are some aspects that still need to be assessed relative to efficacy of MHT therapy, such as the physical and chemical properties of the nanoparticles, coatings, size, type of magnetic nanoparticle, AMF parameters (frequency and intensity of oscillating field), concentration of nanoparticles, time of application of MHT, as well as few studies using models of intracerebral glioblastoma.,13 Oin vitro and in vivo, the properties of SPIONs coated with aminosilane associated with different parameters of MHT in the therapeutic efficiency of GBM in preclinical models. Therefore, we evaluated the characteristics of the appropriate AMF for MHT applied in the treatment of GBM tumors, due to the efficiency in heating of the SPIONsAmin, resulting from the intensity of the magnetic field and the frequency of oscillation, as well as the assessment of the efficacy of MHT therapy in vitro and in vivo, by means of the bioluminescence (BLM) technique.In this sense, the present study seeks to elucidate, To evaluate the potential of the magnetic hyperthermia using nanoparticles of iron oxide coated with aminosilane in models of glioblastoma tumors.Amin   B. In theIONsAmin C. In thereotaxis D, followreotaxis E, which Amin with a magnetite nucleus (Fe3O4) , with a density of ∼1.25g/cm3, coupled with the functional amine (NH2) group, and HD 100nm and zeta potential +20mV.SPIONsAmin, the dynamic light scattering (DLS) technique was used, with the Zetasizer Nano S  system. The assessment was made at the concentration of 100mgFe/mL, and data were collected at 25°C, for a 60-second equilibrium period, with recordings every 30 minutes for 24 hours.To evaluate the polydispersion of the HD and stability of SPIONsamin with the MHT technique, an AMF system model DM100  was used, combining four frequencies , and five intensities of magnetic field  to obtain the heating curves.To determine the heating potential of SPIONsAmin at the concentration of 5mg/mL dispersed in an aqueous medium, allocated in a thermally isolated recipient. The temperature of the SPIONsAmin was monitored through an optical fiber system (Luxtron 3204), initiating all the analyses at 19°C.We used 100μL of SPIONsThe heating curves obtained were used to calculate the values of SAR in W/g for each frequency and magnetic field applied, according to water is the specific heat of the water, ΔT/δt is the initial inclination of the temperature in °C/s, and CFe is the concentration of iron in g/mL.where H® Invitrogen Corporation, CA, USA), supplemented with 10% bovine fetal serum (BFS) , and incubated at 37°C (5% of CO2) until reaching a confluence of 70%, when it then was trypsinized to be used in the proposed trials.The C6 cell line was cultivated in RPMI medium (GIBCO(8) resulting in the acquisition of cells transfected with luciferase for the evaluation of treatment with the BLM technique.The C6 cells were transduced with a luciferase lentiviral vector as per a previously established protocol,18 res6 C6 cells suspended in 200µL of RPMI 10% BFS were evaluated in triplicate under four conditions: C6 cells (Control Group), C6 cells marked with SPIONsAmin, C6 cells submitted to MHT, and C6 cells marked with SPIONsAmin submitted to MHT. Marking of the C6 with SPIONsAmin was performed with 600mg Fe/mL for 18 hours. In the samples submitted to MHT, we used the parameters of 200 Gauss and 874kHz for a period of 40 minutes. Efficiency of the MHT technique (cell viability) was evaluated with BLM, in which 100µL of luciferin were added to each sample, along with the posterior acquisition of images employing the IVIS Lumina III  equipment.Samples containing 10Instituto de Ensino e Pesquisa Albert Einstein (CEUA 3126-17). This vivarium is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animals were kept in individual boxes lined with autoclaved sawdust, with water and balanced feed ad libitum, following a light-dark cycle of 12 hours (7 am to 7 pm), with a temperature of 21°C (±2°C), as per international specifications.Ten male Wistar rats at 2 months of age (weighing 290 to 350g) were used. The animals were acclimated at the Surgical Experimentation and Training Center (CETEC) of the 6/10µL of culture medium) was done by stereotaxis according to the established protocol. The point of implantation of cells in the right frontal cortex was determined and marked on the bone surface following orientations of Swansen's stereotaxical atlas: anteroposterior = 2.0mm; latero-lateral = 2.0mm; depth = 2.5mm.The animals were anesthetized with ketamine (90mg/kg)and xylazine (12mg/kg). Implantation of C6 cells The he group.,21st day with the BLM technique, using IVIS equipment in which the animals were maintained with inhaled anesthesia of isofluorane (2% saturation) with a flow of 2.5L/min of oxygen. The BLM signal was acquired after 5 minutes of administration of 100μL luciferin solution (1mM in PBS).Before being submitted to the MHT process, the control tumor evaluation was performed on the 21nd day after tumor induction, 50µg of iron were applied contained in 10µL of the colloidal suspension of SPIONsAmin, divided into four equidistant points, 3mm from the central region of the tumor mass using stereotaxis equipment. After 20 minutes of the SPIONsAmin application, the animals were positioned inside the coil of the MHT equipment and submitted to the planned heating, composed of two parts. During the first part, the frequency of 874kHz and a field of 200 Gauss were used until reaching 42°C; during the second part, the frequency was maintained and the intensity of the field was modulated to maintain the temperature at 42°C, totaling up a period de 40 minutes of MHT. Mapping of the intratumor temperature was measured with an optical fiber (Luxtron 3204) with temperature probes (diameter of 0.55mm).On the 22After MHT application, the animals were reassessed by means of BLM, and thus was determined the difference of the signal between the images before and after therapy in photons/second.Amin dispersed in water are shown in Amin are polydispersed in the hydrodynamic size, in which the peak of maximal intensity corresponding to the mean HD was 110±5nm, with no significant difference between the measurements (p>0.05) acquired in 24 hours. Therefore, we can consider SPIONsAmin stable during the period analyzed, as shown in The measurements of stability performed with the DLS technique on the SPIONsAmin obtained when submitted to AMF are shown in The heating curves of the SPIONsAmin at 874kHz and 200 Gauss afforded a higher SAR (194.917W/g) among all the analyses  (Amin (2.32x108 photons/s)   II as to otons/s) III. Thesotons/s) -IV.st day after tumor induction, the baseline evaluation of the tumor by BLM without the application of luciferin showed that the BLM equipment alone is not capable of biasing the analysis result  Thus, the values of SAR found in our study were consistent with literature, in which the highest value of SAR was 194.917W/g for the colloidal suspension of the SPIONsAmin submitted to AMF of 874kHz and 200 Gauss, proving adequate for the in vivo application of the MHT therapy.After guaranteeing the stability of the SPIONs heating.27 Heain vivo, it was noted, through the BLM images in the in vitro sample, that AMF did not influence the viability of the C6 cells in the absence of SPIONsAmin, or the viability of the cells in the presence of SPIONsAMIN in AMF. The BLM technique, due to its sensitivity, is used in the evaluation of tumor growth and of possible metastases, and has a good correlation with other imaging techniques, such as magnetic resonance and histology in monitoring of tumor growth.Before the application of MHT tastases,31 andAmin administered locally in the tumor mass can be influenced by several factors, such as the dispersion of SPIONsAmin throughout the tissues surrounding the administration site, which affects the heating produced by the SPIONsAmin in the presence of AMF. Additionally, heating can be influenced by physiological aspects that are implicit in the calculation of heat transfer (Pennes equation), such as local metabolism, blood perfusion rates, coefficient of heat transfer in the tissues involved, specific mass of the blood, among others. In our study, we obtained 32.5% efficiency with a single application of MHT, using the lowest mass value of SPIONsAmin (50µg Fe) in comparison with other studies that applied in the range of 0.5 to 3mg Fe in glioma tumors induced in the flank or subcutaneous regions. Use of high concentrations of iron nanoparticles can cause toxicity to neighboring tissues, as well as affect other organs associated in the biodistribution and elimination of nanoparticles through the liver, kidneys, and spleen.The efficiency of MHT of the SPIONsg others.,33 IAs a limitation of the study, we can point out that the efficiency of MHT through the evaluation of cell viability was not tested with a shorter AMF application time, or in its effect over time. Nonetheless, there are challenges to be overcome in order to increase the efficiency of MHT for clinical application. The increase in efficiency of MHT therapy depends of the prior determination of AMF parameters, which produce the adequate value of nanomaterial SAR, as well as the appropriate evaluation of in vitro and in vivo with molecular imaging techniques, such as BLM. Finally, we can affirm that SPIONsAmin have the potential for MHT therapy in the treatment of GBM tumors.Preclinical studies of MHT showed promising results when combined with another treatment, such as radiation therapy or chemotherapy.35 Nonin vitro study and of 52% in the in vivo study. Therefore, application of magnetic hyperthermia proved to be promising for the therapeutic process of glioblastoma tumors using aminosilane-coated superparamagnetic iron oxide nanoparticles, which demonstrates favorable magnetic characteristics for the process of magnetic hyperthermia. Nevertheless, a careful assessment of other parameters involved, such as magnetic properties, polydispersion of superparamagnetic iron oxide nanoparticles, morphology, dose applied of superparamagnetic iron oxide nanoparticles, site of administration, periodic applications of the treatment, among others is necessary.The present study evaluated the potential of magnetic hyperthermia using the aminosilane-coated superparamagnetic iron oxide nanoparticles for the treatment of glioblastoma in the animal model. The value of the specific absorption rate characterized for aminosilane-coated superparamagnetic iron oxide nanoparticles submitted to the action of an alternating magnetic field, indicates a good capacity for heating in order to reach the temperature. The bioluminescence technique showed, after application of magnetic hyperthermia therapy, a reduction in cell viability by 32.8% in the