Therapeutic uses of alpha-terpineol for the treatment of cardiovascular diseases.

Abstract

"Therapeutic uses of alpha-terpineol for the treatment of cardiovascular diseases" Cardiovascular diseases (CDs) are the leading cause of death worldwide due to anoxic injury. The complexity of CD pharmacotherapy, coupled with the lack of cardioprotective drugs, encourages the development of cardioprotective strategies. Thus, α-terpineol (TPN) is a monoterpene that exhibits vasorelaxant but not cardioprotective actions. This study aimed to evaluate the cardioprotective effect of TPN on cardiac lesions. For this purpose, rats were allocated into 6 groups and treated orally for 15 days, then induced to have a heart attack: (g1 = 0.9% saline); (g2 = infarcted 0.9% saline); (g3 = TPN 25 mg / kg); (g4 = TPN 50 mg / kg); (g5=tnp 75 mg / kg) and (g6=tpn 50 mg / kg without iso). On the 16th day, the animals were anesthetized and biochemical, morphometric, hemodynamic, baroreflex reactivity, and electrocardiographic assays were performed.The results showed that TPN increased the survival of animals to induced infarction, reduced the area of ​​necrosis in the myocardium, prevented electrocardiographic changes, and inhibited the release of cardiac markers. Furthermore, TPN inhibited the increase in heart rate induced by isoproterenol. Additionally, TPN produced coronary dilation and endothelial neovascularization in rat aortas. TPN induced intravenous bradycardia, and its mechanisms involve cardiac M2 receptors and blockade of central nervous system receptors (CVRs). It is concluded that TPN provides cardioprotection.

Description

1 / 71 Descriptive report of the invention patent for "Therapeutic uses of alpha-terpineol for the treatment of cardiovascular diseases"

[001] This patent application aims to make alpha-terpineol (TPN) a drug candidate for use in the prevention of cardiovascular diseases, acting both in the prevention of cardiac ischemia-reperfusion injuries and heart failure, and in the treatment of hypertension and cardiac arrhythmias. In summary, alpha-terpineol (TPN) is a cyclic monoterpene with a molar mass of 154.25 g / mol and a molecular formula of C10H18O of natural origin that can be isolated from a variety of aromatic plant species; it is a colorless, volatile, and odorous oil. TPN is considered one of the major compounds of the resin obtained from the stems of Protium hepthaphylum (MARQUES et al, 2010; MOBIN et al, 2016; MOBIN et al, 2017).Protium hepthaphylum is a plant native to Brazil, popularly known as "almécega" or "breu-branco," distributed throughout the Amazon region and the Brazilian Northeast. Its resin is used by Brazilian indigenous tribes in healing rituals for the popular treatment of various diseases through the inhalation of the burning resin obtained from this plant species (BREITBACH et al., 2013; MOBIN et al., 2017). The TPN used in this study was provided by the Federal Universities of Piauí and Sergipe (partner universities in this patent application), and its extraction and isolation methods for resins obtained from the stem of Protium hepthaphylum are patented at the National Institute of Industrial Property (INPI-BR) under protocol number INPI 00012. Previous studies have demonstrated that the essential oil obtained from P. hepthaphylum resins produces vasorelaxant effects (MOBIN et al., 2017).Additionally, TPN demonstrated vasorelaxant activities in arteries of spontaneously hypertensive rats and L-NAME, so it is believed that TPN is responsible for the vasorelaxant activities of the essential oil obtained from P. hepthaphylum. TPN also has acute antihypertensive and hypotensive actions in different hypertension models (RIBEIRO et al, 2010; SABINO et al, 2013). However, the... Petition 870190021822, dated 07 / 03 / 2019, page 10 / 118 2 / 71 The cardioprotective effects of alpha-terpineol remain unknown. Thus, this study aimed to evaluate the cardioprotective activity of alpha-terpineol (TPN) isolated from Protium hepthaphyllum resin against isoproterenol-induced cardiac lesions in normotensive (WT) and spontaneously hypertensive (SHR) rats. PROBLEM THAT THE INVENTION AIMS TO SOLVE

[002] Cardiovascular diseases (CDs) are pathologies that affect the cardiovascular system and include vascular, cardiac, cerebrovascular, cardiorenal diseases, and cardiovascular conditions originating from rheumatoid disorders (WHO, 2018). Recent studies show that cardiovascular diseases are among the 10 most common diseases affecting individuals worldwide, ranking first in the number of global deaths (WHO, 2017; WHO, 2018). It is estimated that cardiovascular diseases are responsible for approximately 17.9 million deaths annually, reflecting 31% of mortality when considering the cause-and-death relationship (WHO, 2017; WHO, 2018; BENJAMIN et al, 2018; LÜSCHER, 2018).The most common clinical outcomes leading to death in individuals affected by cardiovascular diseases were acute myocardial infarction, followed by stroke, which together accounted for 85% of mortality from all pre-existing cardiovascular diseases (WHO, 2017; WHO, 2018).

[003] In addition, the costs of hospitalizations and healthcare for patients suffering from heart attacks overburden public coffers. The European Union annually allocates 53% of the monetary fund destined for health solely for cardiovascular diseases, equivalent to 11 billion euros, with €18,875,775 allocated to heart attack treatments (EHNHEART, 2017).

[004] In the United States, the cost of hospitalizations for heart attack patients reflects 20% of their tax revenue, representing approximately $15,889.00 per hospitalized patient (AFANA et al, 2015). These data are consistent with our country; in Brazil, it is estimated that the treatment Petition 870190021822, dated 07 / 03 / 2019, page 11 / 118 The cost of standard 3 / 71 coronary stent implantation surgery is around R$ 8,211,654.50 annually for the Brazilian public health system, reflecting an expenditure of approximately R$ 23,461.87 per patient with surgical intervention or R$ 12,873.69 if surgical intervention is not necessary (MARQUES et al, 2012). In this context, recent studies have demonstrated the need for the implementation of new therapeutic strategies for cardioprevention (SPATH et al, 2016; SLUIJTER et al, 2014; HAUSENLOY et al, 2017). These strategies may involve: heart transplant techniques, pluripotent stem cell therapies in invasive procedures, or the use of pharmacotherapy (JACOBSON et al, 2000; SLUIJTER et al, 2014; HAUSENLOY et al, 2017). FIELD OF ACTIVITY

[005] The use of isolated alpha-terpineol for therapeutic purposes in cardiovascular diseases will encompass its application as a cardioprotective agent for the development of arterial hypertension and cardiac arrhythmias, as well as in the prevention of cardiovascular events such as cardiac injuries due to myocardial ischemia-reperfusion, heart failure, and sudden death. STATE OF THE ART

[006] Despite improvements in clinical and pharmacological management, AMI remains the leading cause of death worldwide, and the lack of a cardioprotective drug class in the literature is noteworthy (ZHOU, 2012). In this context, recent studies have demonstrated the need for the implementation of new therapeutic strategies for cardioprevention (SPATH et al, 2016; SLUIJTER et al, 2014; HAUSENLOY et al, 2017). HAUSENLOY et al. (2010) have pointed out useful recommendations for transposing pharmacological studies of cardioprotection in experimental models and their correlation with infarction developed in humans for the insertion of a future cardioprotective pharmacological class. These recommendations include: the selection of the animal used, the influence of comorbidities, the experimental infarction model developed (ex-vivo and in vivo), and its duration, among others. In addition to the variables... Petition 870190021822, dated 07 / 03 / 2019, page 12 / 118 4 / 71 analyzed, including: mortality, infarct area, biochemical marker levels (CK-MB and troponins), and cardiac function measurement. Thus, we emphasize the lack of cardioprotective drugs on the market.

[007] Recent efforts by researchers around the world to introduce compounds to the pharmaceutical market that can treat / prevent primary cardiovascular diseases and protect individuals from the aggravation of these diseases can benefit society as a whole by reducing the number of deaths and sequelae resulting from cardiovascular problems. In this sense, exenatide, a synthetic compound analogous to GLP-1, and exenatide 4, isolated from lizard venom, have shown cardioprotective actions in several experimental studies with small and large animals (SONNE et al, 2008; TIMMERS et al, 2009; HAUSENLOY et al, 2012).

[008] Administration of the selective ε1-adrenergic receptor antagonist, metoprolol, prevented reperfusion infarction by reducing the infarct area in large animals (IBANEZ et al, 2007). Combined therapy with a beta-blocker and an angiotensin-converting enzyme inhibitor produced a cardioprotective effect in an experimental study, involving the activation of the JAK2 / STAT-3 pathway as a mediator of cardioprevention (HAYBAR et al, 2018). Cyclosporine A, an immunosuppressant drug, has demonstrated cardioprotective action in ex-vivo and in vivo experimental studies involving the participation of mitochondrial permeability pore inhibition (PIOT et al, 2008; MEWTON et al, 2010).

[009] Some compounds of natural origin have also shown a significant effect on myocardial infarction, producing a relevant cardioprotective effect. Among them, myricetin, a flavonoid found in grapes of the species Vitis vinifera, exhibited a cardioprotective effect in vivo in the isoproterenol infarction model and involves the participation of the second messenger STAT-1 (TIWARI et al, 2009; SCARABELLI et al, 2009; GOUTAM et al, 2017). Resveratrol, a polyphenol obtained from grapes, is also indicated as a future candidate for a cardioprotective drug. Its cardiovascular effects have been studied over the last two decades, using various approaches. Petition 870190021822, dated 07 / 03 / 2019, page 13 / 118 5 / 71 experimental: ex vivo, in vitro, in vivo (WU et al, 2001; DERNEK et al, 2004; GURUSAMY et al, 2009; WU et al, 2011; DAS & MAULIK, 2011; MAGYAR et al; 2012; MOKNI et al, 2013).

[010] Thus, we believe that alpha-terpineol may contribute to the existence of a new class of drugs, and its patent protection may be a future drug candidate, enabling the prevention of cardiovascular diseases.

[011] The study did not evaluate the participation of antioxidant enzymes in the cardiovascular effect in the in vivo model;

[012] The coronary dilating effect was based only on postmortem studies, so it was not possible to evaluate the vasorelaxant effects in isolated and perfused coronary arteries;

[013] The toxicological assessment of the compound was based solely on the in vitro assay. DESCRIPTION OF THE APPROACH TO THE TECHNICAL PROBLEM Table 1: Comparison of the problem found in the prior art and the solution of the intended patent. PROBLEM SOLUTION Myocardial infarction as an aggravation of arterial hypertension Alpha-terpineol provides cardioprotection against acute myocardial infarction Arterial hypertension as a cardiovascular risk factor Alpha-terpineol has antihypertensive action in both acute and chronic modes Number of deaths from cardiovascular diseases due to infarction Alpha-terpineol increased the survival of animals undergoing infarction, preventing mortality Sudden death due to conduction arrhythmias Alpha-terpineol prevented the onset of isoproterenol-induced conduction arrhythmia Vascular dysfunction of arteries Alpha-terpineol improved Petition 870190021822, dated 07 / 03 / 2019, page 14 / 118 6 / 71 Post-infarction resistance: reactivity of post-infarction resistance arteries, re-establishing their vascular functions. Post-infarction hypertrophic heart failure: Alpha-terpineol inhibits the formation of post-infarction cardiac hypertrophy. DESCRIPTION OF THE FIGURES LIST OF FIGURES Table 2: Description of the figures

[014] Figure 1 Percentage of cell viability in J774 cell line macrophages in the MTT assay

[015] Figure 2 Percentage of survival after isoproterenol-induced acute myocardial infarction in (A) Wistar (B) SHR rats

[016] Figure 3 Microslices of isolated hearts from Wistar (WT) rats stained with 1% triphenyltetrazolium chloride (TTC 1%) after acute treatment

[017] Figure 4 Microslices of isolated hearts from spontaneously hypertensive (SHR) rats stained with 1% triphenyltetrazolium chloride (TTC 1%) after acute treatment

[018] Figure 5 Percentage of myocardial necrosis area after isoproterenol-induced acute myocardial infarction (85 mg / kg 2x sc) in (A) Wistar (B) SHR rats

[019] Figure 6 Micrographs of myocardium from WT rats after pretreatment with monoterpene (TPN) H&E staining

[020] Figure 7 Micrographs of myocardium from SHR rats after pretreatment with monoterpene (TPN) H&E staining Petition 870190021822, dated 07 / 03 / 2019, p. 15 / 118 7 / 71

[021] Figure 8 Serum creatine kinase MB (CK-MB) levels after isoproterenol-induced acute myocardial infarction (85 mg / kg 2x sc) in (A) Wistar (B) SHR rats

[022] Figure 9 Serum total creatine kinase (CK-NAC) levels after isoproterenol-induced acute myocardial infarction (85 mg / kg 2x sc) in (A) Wistar (B) SHR rats

[023] Figure 10 Electrocardiographic recordings after isoproterenol-induced acute myocardial infarction in WT rats in the 6 frontal plane leads in the groups

[024] Figure 11 Electrocardiographic recordings after isoproterenol-induced acute myocardial infarction in SHR rats in the 6 frontal plane leads in the groups

[025] Figure 12 C-reactive protein levels in the left ventricles after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc)) in rats (A) wistar (B) SHR Microslices of myocardium from WT rats, after prior treatment with the monoterpene (TPN)

[026] Figure 13 Serum levels of total cholesterol after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in rats (A) wistar (B) SHR Microslices of myocardium from SHR rats after prior treatment with the monoterpene (TPN)

[027] Figure 14 Serum triglyceride levels after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in rats (A) wistar (B) SHR Morphological aspects of isolated hearts from wistar (WT) rats after subacute treatment with TPN

[028] Figure 15 Hypertrophic evaluation post-induced myocardial infarction Morphological aspects of isolated hearts from spontaneously hypertensive rats using isoproterenol in WT rats. Petition 870190021822, dated 07 / 03 / 2019, p. 16 / 118 8 / 71 (SHR) after subacute treatment with TPN

[029] Figure 16 Hypertrophic assessment after isoproterenol-induced myocardial infarction in SHR rats

[030] Figure 17 Micrographs of myocardium from WT rats after prior treatment with the monoterpene (TPN) TG staining.

[031] Figure 18 Micrographs of SHR rat myocardium after pretreatment with the monoterpene (TPN) TG staining

[032] Figure 19 Antioxidant activity of TPN in the in vitro radical scavenging activity test against reactive nitric oxide species (RNS) as a function of time

[033] Figure 20 Hemodynamic changes after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in WT rats

[034] Figure 21 Hemodynamic changes after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in SHR rats

[035] Figure 22 Myocardial oxygen demand after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc)) in rats (A) wistar (B) SHR

[036] Figure 23 Microslices of aortas from wistar rats in transverse view (WT) after pretreatment with monoterpene (TPN) H&E staining

[037] Figure 24 Microslices of aortas from spontaneously hypertensive rats (SHR) in transverse view after pretreatment with monoterpene (TPN) H&E staining.

[038] Figure 25 Microslices of aortas from wistar rats in longitudinal view (WT) after pretreatment with monoterpene (TPN) TG staining. Petition 870190021822, dated 07 / 03 / 2019, page 17 / 118 9 / 71

[039] Figure 26 Microslices of aortas from spontaneously hypertensive rats (SHR) in longitudinal view after pretreatment with the monoterpene (TPN) staining TG.

[040] Figure 27 Morphological aspects of the coronary arteries of Wistar (WT) rats after treatment with the monoterpene

[041] Figure 28 Morphological aspects of the coronary arteries of Wistar (WT) rats after treatment with the monoterpene

[042] Figure 29 Vascular reactivity to contraction obtained by the concentration-response curve of phenylephrine after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in SHR rats

[043] Figure 30 Vascular reactivity to relaxation obtained by the concentration-response curve of sodium nitroprusside after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in SHR rats

[044] Figure 31 Total calcium levels after acute myocardial infarction induced by isoproterenol (85 mg / kg 2x sc) in WT rats (A) serum (B) ventricle (C) aorta and in SHR rats (D) serum (E) ventricle (F) aorta

[045] Figure 32 Cardiovascular effects induced by TPN in spontaneously hypertensive rats not anesthetized and not infarcted at doses of 1.5 and 10 mg / kg iv

[046] Figure 33 Cardiovascular effects induced by TPN in spontaneously hypertensive rats anesthetized and not infarcted at doses of 1.5 and 10 mg / kg iv

[047] Figure 34 Cardiovascular effects induced by TPN in spontaneously hypertensive rats not anesthetized and not infarcted after hexamethonium (20 mg / kg iv) or atropine (2 mg / kg iv) at doses of 1.5 and 10 mg / kg iv

[048] Figure 35 Molecular docking in the analysis of the monoterpene compound. Petition 870190021822, dated 07 / 03 / 2019, page 18 / 118 10 / 71 (TPN)

[049] Figure 36 Molecular docking of TPN at the dihydropyridine action site in L-type calcium channels

[050] Figure 37 Molecular docking of TPN at the action site in muscarinic M2 receptors

[051] Figure 38 Schematic representation of the cardioprotective effect induced by TPN and its mechanisms of action

[052] Figure 39 Chromatogram of Alpha-terpineol by GC / MS LIST OF TABLES Table 4: Description of the tables

[053] Table 1 Total Leukocyte Count (TLC) in myocardium of WT rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[054] Table 2 Total Leukocyte Count (TLC) in myocardium of SHR rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[055] Table 3 Total Pyknotic Nuclei Count (TPNC) in myocardium of WT rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[056] Table 4 Total Pyknotic Nuclei Count (TPNC) in Myocardium of SHR rats after prior treatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc))

[057] Table 5 Total count of nuclei in karyolysis (CTNc) in myocardium of WT rats after pretreatment with the monoterpene TPN and subsequent exposure to the agent. Petition 870190021822, dated 07 / 03 / 2019, page 19 / 118 11 / 71 Sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[058] Table 6 Total count of karyolysis nuclei (CTNc) in myocardium of SHR rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[059] Table 7 Total count of karyorrhexis nuclei (CTNcx) in myocardium of WT rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc)

[060] Table 8 Total count of karyorrhexis nuclei (CTNcx) in myocardium of SHR rats after pretreatment with the monoterpene TPN and subsequent exposure to the sympathomimetic inducer isoproterenol (85 mg / kg 2x sc

[061] Table 9 Antioxidant profile of monoterpene (TPN) in in vitro antioxidant radical scavenging activity test against DPPH, ABTS and iron in the Fe3+ oxidation state

[062] 10 Table 11 Comparison of Emax and pD2 values ​​of isolated mesenteric artery rings from SHR rats without functional endothelium in response to contractions with phenylephrine (10-10-10-5 M)

[064] 12 Table 12 Comparison of Emax and pD2 values ​​of isolated mesenteric artery rings from SHR rats without functional endothelium in response to relaxation with sodium nitroprusside (10-10-10-7 M). TECHNIQUE DESCRIPTION Petition 870190021822, dated 07 / 03 / 2019, page 20 / 118 12 / 71

[065] To evaluate the cardioprotective effects of alpha-terpineol and its mechanisms, in vivo, in vitro, ex vivo, and in silico approaches were used. The techniques used for pharmacological evaluation were performed as described below. Assessment of toxicity in macrophages

[066] For cytotoxicity assessment, we used the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) colorimetric cell viability assay as described by Mosman, 1983. Briefly, J774 cell line macrophages were placed in 96-well microplates at a concentration (5.0 x 10⁵ cells / well), supplemented with RPMI nutrient solution (solution created by the Roswell Park Memorial Institute) 10% fetal bovine serum and gentamicin 40 pg / kg, then incubated for 4 hours (37°C), adherent cells were suspended at least twice before the start of the cytotoxicity experiment.

[067] After the cell line viability maintenance period, the monoterpene was diluted in DMSO and then inserted into the microwells containing the cell cultures. The assay was performed in triplicate at concentrations [1, 10, and 100 pg / mL] respectively. Triton X-100 1% was used as a control for cell death, while RPMI nutrient medium was used as a control for cell viability. DMSO was also tested as a vehicle in the cell lines. Each microwell was treated with 200 pL of an MTT solution, then the cell cultures were incubated in ovens at 37°C for 48 hours. Finally, after 48 hours, the optical density was read in a spectrophotometer at 540 nm. Animals

[068] In all pharmacological protocols described in this work, 140 young adult rats (Rattus norvegicus, albino variety), male Wistar strain (WT) and 140 young adult rats (Rattus norvegicus, albino variety), male spontaneously hypertensive strain (SHR), both healthy, weighing between 150 and 250 grams, were used, which came from the Central Animal Facility of UFAL, Maceió, AL. Petition 870190021822, dated 07 / 03 / 2019, p. 21 / 118 13 / 71

[069] The animals were previously housed in collective plastic cages, containing five animals per cage, maintained under a 12-hour light and dark photoperiod, minimal noise, ambient temperature of approximately 25°C, and humidity maintained by air conditioning, as recommended by the Canadian Council on Animal Experimentation (Olfert, 1993). The rats were fed standard balanced rodent feed (Nuvilab, BR) and filtered water ad libitum. The bedding was changed three times a week. Before the start of the experiment, the animals underwent an adaptation period in the sectorial animal facility of the Cardiovascular Pharmacology Laboratory of the School of Nursing and Pharmacy (LFCESENFAR) for seven days. All experimental protocols were approved by the animal research ethics committee of UFAL, under protocol number 09 / 2015.Alpha-terpineol was kindly provided by the collaborating institutions in this study (UFPI and UFS), and a gas chromatographic analysis coupled with mass spectrometry was performed at our institution for structural confirmation of the compound (Figure 39). Preparation of alpha-terpineol solution for pharmacological studies.

[070] A stock solution containing alpha-terpineol was prepared from a calculated dose of 75 mg / kg. Alpha-terpineol was weighed and added to a 1% cremophor A25 solution (Sigma, Aldrisch, USA LOT BCBG1152V) previously prepared with distilled water. A 0.9% NaCl solution was placed in 10 mL round-bottom flasks, and 4 drops of the cremophor solution were added. The flask was then inserted into a magnetic plate (IKA C-MAG H57), and a micromagnet was placed in contact with the solution. Alpha-terpineol was then slowly added while the solution was vigorously shaken (500 rpm) for 5 minutes without heating. After 5 minutes, an emulsion was formed, and then the solution was administered to rats by dilution in descending order of dose. The volume of the solution was prepared according to the number of animals to be treated, following the dose criteria described below. Petition 870190021822, dated 07 / 03 / 2019, p. 22 / 118 14 / 71 Pharmacological approaches

[071] To evaluate the cardioprotective potential of the monoterpene (TPN), we used the isoproterenol-induced acute myocardial infarction method, as described by Rajadurai & Prince, 2006. The animals were randomly allocated into 6 experimental groups, containing 5 rats per group:

[072] I-Saline group (group that received 0.9% saline solution orally for 15 days)

[073] II-Group infarcted by isoproterenol (group that received 0.9% saline solution PO for 15 days + isoproterenol 85 mg / kg subcutaneously on days 14 and 15)

[074] III-TPN 25 mg / kg + ISO (group receiving TPN at a dose of 25 mg / kg PO for 15 days + isoproterenol 85 mg / kg subcutaneously on days 14 and 15)

[075] IV-TPN 50 mg / kg + ISO (group receiving TPN at a dose of 50 mg / kg PO for 15 days + isoproterenol 85 mg / kg subcutaneously on days 14 and 15)

[076] V-TPN 75 mg / kg + ISO (group receiving TPN at a dose of 75 mg / kg PO for 15 days + isoproterenol 85 mg / kg subcutaneously on days 14 and 15)

[077] VI-TPN 50 mg / kg without ISO (group receiving TPN at a dose of 50 mg / kg PO for 15 days) Evaluation of survival in isoproterenol-induced myocardial infarctions.

[078] The survival rate of animals following lethal infarctions induced by isoproterenol was estimated using the following mathematical expression: Equation 01 % Survival = (number of non-lethal heart attacks - number of fatal heart attacks) x 100

[079] Those obtained due to long chronic treatment prior to exposure to isoproterenol and / or due to trauma from oral administration by gavage were excluded. Determination of the area of ​​myocardial necrosis

[080] After euthanasia by bloodletting under anesthesia (Sodium thiopental 45 mg / kg ip), the animals underwent a thoracotomy and the hearts were excised from the thoracic space. The hearts were cleaned of adherent tissues, and the cardiac chambers were carefully washed with solution Petition 870190021822, dated 07 / 03 / 2019, page 23 / 118 15 / 71 0.9% saline solution, and immediately frozen (-18°C) for 15 minutes, then the myocardium was dissected transversely to the septal axis. The cardiac microslices were 3-4 mm thick.

[081] Each microslice was incubated in a 1% triphenyltetrazolium chloride solution containing 1M phosphate buffer with pH= 7.4. Hermetically sealed glass containers were immersed in a water bath at 37°C for 20 minutes. The microslices were placed on glass slides and photographed using a stereoscope under light (Leica, KL 300-LED). The images were imported into ImageJ Pro Plus 7.0 software (Media Cybernetics Inc., Silver Spring, MD, USA), where the percentage of necrotic and viable areas was determined. Resting electrocardiogram assessment

[082] After chronic treatment (16th day), the animals were anesthetized with a mixture of anesthetic agents (Ketamine 80 mg / kg + Xylazine 4 mg / kg ip). Then, subcutaneous incisions were made for electrode insertion and electrocardiographic recordings were made. The electrodes were connected to an amplifier (Bioamp, powerlab, ADinstruments, Dunedin, New Zealand) that sent the recordings to a microcomputer that allowed real-time monitoring of the electrocardiographic recording using Labchart 8.0 software. Cardiac electrical activity was evaluated in the 6 peripheral leads in the frontal plane (DI, DII, DIII, aVR, aVL and aVF).

[083] Electrocardiographic signals were stabilized for a minimum period of 15 minutes and then the electrocardiographic recording in each lead was recorded for 10 minutes. To assess the presence of acute myocardial infarction lesions and / or cardiac changes secondary to infarction, at least 3 leads referring to the electrocardiographic waves, segments and intervals together were considered. Biochemical evaluation

[084] After euthanasia by blood transfusion under anesthesia (Ketamine 80 mg / kg + Xylazine 4 mg / kg ip). The animals underwent a thoracotomy and a cardiac puncture was performed. The collected blood was centrifuged at 5,000 rpm for Petition 870190021822, dated 07 / 03 / 2019, page 24 / 118 16 / 71 minutes and serum was obtained as a sample for biochemical assays (total creatine phosphokinase, creatine phosphokinase MB isoform, total cholesterol, triglycerides, and calcium).

[085] The hearts were also excised from the thorax, cleaned of adherent tissues, and the cardiac chambers washed with 0.9% saline solution. The hearts were dissected, and the right ventricles and aortas were homogenized with a 200 mM PBS solution. The ventricular and aortic homogenates were centrifuged under refrigeration at 15,000 rpm for 10 minutes, and the supernatant was used for calcium assays. The supernatant of the ventricular homogenate was used to measure C-reactive protein. Dosage of serum markers of acute myocardial infarction

[086] For the determination of serum markers of acute myocardial infarction, we used the dosages of total creatine phosphokinase and MB isoform, according to the technique described by Szasz et al., 1976. Briefly, a reagent mixture (working reagent) was prepared by adding 80 pL Reagent 1 (Imidazole buffer 125 nmol / L, N-acetylcysteine ​​25 nmol / L, ADP 2.5 mmol / L, AMP 6.25 mmol / L, diadenosine pentaphosphate 12.5 pmol / L, magnesium acetate 12.5 mmol / L, NADP 2.5 mmol / L, hexokinase 5,000 U / L and sodium azide 0.095%) + 20 pL Reagent 2 (Glucose 100 mmol / L, creatine phosphate 150 mmol / L, sodium azide 0.095%, anti-CK-M to inhibit CK-MM in the CK-MB kit. The CK-NAC kit did not have antibodies to inhibit isoforms. 50 pL of serum was incubated and immediately entered into a spectrophotometer for reading two absorbances in kinetic mode (0 and 5 minutes for CK-MB and 0 and 2 minutes for CK-NAC), the absorbances were read at 340 nm in the spectrophotometer (Shimadzu UV-VIS mini-1240).Next, creatine phosphokinase activities were estimated using the mathematical expression: Equation 02 CK Activity (U / L) = Absob2 - Absorb1 (vadrons) x [caHbrator] Absorb 2-Absorb 1 (samples) Dosage of lipid levels Petition 870190021822, dated 07 / 03 / 2019, page 25 / 118 17 / 71

[087] Lipid levels were estimated by measuring total cholesterol and triglyceride levels in blood serum. Cholesterol dosage

[088] 1000 pL of reagent 1 (Phosphate buffer 50 mmol, phenol 24 mmol / L, sodium cholate 500 pmol / L, sodium azide 15 mmol / L, 4-aminoantipyrine 500 pmol / L, cholesterol esterase 250 U / L, cholesterol oxidase 250 U / L and peroxidase 1000 U / L) was mixed with 10 pL of the sample, inserted into a spectrophotometer (Shimadzu mini-1240 UV-VIS), and absorbances were read at 505 nm in colorimetric mode. Then, total cholesterol was calculated: Equation 03 Cholesterol (mg / dL) = (Absorbance of the sample / Absorbance of the standard) x 200 Triglyceride dosage

[089] For the determination of serum triglyceride levels, the method described by Trinder et al., 1969, was used. Thus, 1000 pL of reagent 1 (Phosphate buffer 50 mmol / L, ionized magnesium 4 mmol / L, 4-chlorophenol 2.7 mmol / L, 4-aminoantipyrine pmol / L, ATP 1.8 mmol / L, lipoprotein lipase 1,400 U / L, glycerol kinase 1,000 U / L, glycerol phosphate oxidase 1,500 U / L, peroxidase 900 U / L and sodium azide 0.095%) was mixed with 10 pL of the sample, which was then entered into a spectrophotometer (Shimadzu mini-1240 UV-VIS) in colorimetric mode, and absorbances were read at 505 nm. Next, triglyceride levels were calculated: Equation 04 Triglycerides (mg / dL) = (Absorbance of the sample / Absorbance of the standard) x 200 Calcium level measurement

[090] For the determination of calcium levels, the colorimetric method based on the titrimetric reaction was used. Thus, 1000 pL of the working reagent formed by 900 pL of reagent 1 (phosphate buffer [920 mmol / L]) + 100 pL of reagent 2 (O-cresolphthalein 320 pmol / L, 8-hydroxyquinoline 13 mmol / L and hydrochloric acid 130 mmol / L) were added to 10 pL of sample (blood serum, supernatant of aortic homogenate). Petition 870190021822, dated 07 / 03 / 2019, page 26 / 118 18 / 71 or ventricular), then they were inserted into a spectrophotometer, where absorbances were read at 570 nm. Calcium levels were estimated using the formula expressed as follows: Equation 05 Calcium (mg / dL) = (Absorbance of the standard / Absorbance of the samples) x 10 C-reactive protein dosages

[091] C-reactive protein was estimated using the semi-quantitative immunolatex method. The supernatant of the ventricular homogenate was chosen as the sample for C-reactive protein assay. Thus, it was inserted into microplates containing 96 wells. 2.5 pL of a latex suspension coated with anti-CRP monoclonal antibody was incubated in each microwell, and additionally 2.5 pL of the pure sample, then 195 pL of distilled water was added to the microplates, and the absorbances were read in the 540 nm range. In order to avoid the prozone effect, the samples were diluted up to 64 times their initial concentration. Absorbances were read kinetically for 2 minutes. Samples with concentrations below 5 mg / L were considered negative, due to the total absence of agglutination, while those with concentrations above 5 mg / L were considered positive. The concentration of the samples was calculated using the formula below: Equation 06 CRP (mg / dL) = 5 mg / LX sample dilution value Histopathological evaluation

[092] For histopathological evaluation, the aortic branches and hearts were removed from the thorax. The aortas were cleaned and dissected of adherent connective tissues, while the hearts were washed in saline solution and dissected longitudinally parallel to the septal axis. The hearts were submerged in a 10% formalin solution where they remained in Petition 870190021822, dated 07 / 03 / 2019, page 27 / 118 19 / 71 overnight for 12 hours, then a new formalin solution was substituted and the histological specimens remained for 12 hours.

[093] After 24 hours of fixation, the specimens were washed in running water for 30 minutes and immediately dehydrated in increasing degrees of ethanol, sequentially cleared by xylene and immersed in heated paraffin baths for complete embedding. The paraffin blocks were cut into microslices (4-5 µm) in a semi-serial manner, and then each microslice was adhered to glass slides containing albumin; each slide formed was then stained with hematoxylin-eosin and / or Gomori's trichrome. In the aortic branches, alterations were observed on the tunics while the diameter of the aortic lumen was measured. Cardiac damage was assessed according to the degree and extent of lesions as described by Rona, 1963.The following parameters were evaluated: edema, separation and disorganization of myofibrils, leukocyte invasion, inflammation (myocarditis), hemorrhage, formation of necrotic zones, cytoplasmic vacuolization, alterations in the nucleus (pyknosis, karyolysis and karyorrhexis) and hypertrophic cardiac remodeling. Morphological and morphometric assessment

[094] After the hearts were removed, they were cleaned of adherent tissues and the heart chambers were washed with 0.9% saline solution. The hearts were weighed according to Scherle's method. After weighing, the hearts were photographed in their natural state and then carefully dissected, the atria were removed and the ventricles were separated, measured in thickness and weighed. The weight of the heart and ventricles were balanced with the body weight, as well as with the weight of the femur of the rats. The following were evaluated: anatomical location of the occurrence of induced infarcts, as well as changes in morphometric parameters: body weight, heart weight, heart weight / body weight, heart weight / femur weight, relative increase in heart weight, left ventricular weight / heart weight, right ventricular weight / heart weight, left ventricular thickness, right ventricular thickness and Δ of ventricular thicknesses. Petition 870190021822, dated 07 / 03 / 2019, page 28 / 118 20 / 71 Assessment of radical kidnapping activity

[095] The antioxidant activity of the monoterpene was performed using an in vitro approach based on its free radical scavenging capacity. Kidnapping activity of radical DPPH0

[096] The antioxidant action of the monoterpene against the DPPH radical was evaluated as described by Abe et al., 1998. A solution of the DPPH radical was prepared by adding 200 µl of DPPH0 [600 pmol / L] to a methanol solution containing 500 μL of sodium acetate buffer at a concentration of 0.1 M (pH = 5.5). A concentration-response curve was then obtained in the presence of the monoterpene or standards (gallic acid, caffeic acid, and quercetin). The mixture was incubated for 30 min and placed in a spectrophotometer; absorbances were measured at 517 nm. Trolox, a synthetic antioxidant, was used as a positive standard, while MilliQ water or methanol were used as blank controls for the IC50 calculation. ABTS's kidnapping activity

[097] Briefly, the ABTS' radical scavenging potential was performed as described by Hsu et al., 2011. A solution for generating the ABTS' radical was prepared by adding 200 μL of ABTS' [2.5 mg / mL] in aqueous solution to 800 μL of 0.1 M phosphate buffer (pH = 7.4). After preparing the solution containing the free radical, a concentration-response curve was obtained in the presence of the monoterpene or known natural antioxidants (gallic acid, caffeic acid, and quercetin). The mixture was then incubated for 30 min and entered into a spectrophotometer; absorbances were measured at 734 nm. Trolox was used as a positive control, while MilliQ water was used as a blank control to calculate the IC50. Reducing activity of Fe3+ ions

[098] The iron ion reducing power assay was performed in accordance with Huang et al., 2005. Initially, [Fe(TPZT)2]2+ was formed by mixing 625 Petition 870190021822, dated 07 / 03 / 2019, page 29 / 118 21 / 71 pL of 1% K3Fe(CN)6 and 375 pL of the monoterpene and standards were incubated at 50°C in phosphate buffer (pH = 3.6). Then, 625 pL of 10% C2HCl3O2 and 250 pL of 1% FeCl3 were added to the mixture. After 20 minutes, the ability of the monoterpene or the natural antioxidant standards (gallic acid, caffeic acid, and quercetin) to inhibit the formation of [Fe(TPZT)2]3+ was evaluated by measuring the absorbances at 700 nm using a spectrophotometer. Trolox was used as a positive antioxidant control. Complexation assay with Fe3+

[099] The Fe3+ complexation capacity was evaluated as described by Diniz et al., 1994. Initially, 200 pL of ferrozine (C2oHi2N4Na06S2) at a concentration of 5 mM and 200 pL FeSO4 [0.6 mM] were added to a solution. Subsequently, 800 pL of the monoterpene or natural antioxidants (gallic acid, caffeic acid, and quercetin) were incubated with the mixture, and the mixture was placed in a spectrophotometer. Complex formation was monitored in the absorbance range of 562 nm. EDTA was used as a positive complexing agent. Sequestration activity of reactive nitrogen species

[100] Nitrogen radical scavenging activity (NO7ONOO') was evaluated according to the method presented by Ebrahimzadeh et al., 2008 et al. Previously, Griess' reagent, which was capable of generating reactive nitrogen species, was prepared by adding 1% sulfonylamide previously diluted in 5% phosphoric acid to 1% N-(1-naphthylethylenediamine) dihydrochloride and sodium nitroprusside at a concentration of 14 mg / mL. Then a single concentration of the monoterpene [500 pg / mL] or natural standard antioxidants was incubated with 1 mL of Griess' reagent and 4 mL of 1 M Na2NO3. 2.5 mL of the mixture was aliquoted and inserted into the spectrophotometer for absorbance reading at 700 nm at the following times: 0, 20, 40, 60, 80, 100, 120, 140, and 160 minutes. Trolox was used as a control. Petition 870190021822, dated 07 / 03 / 2019, p. 30 / 118 22 / 71 antioxidant, while sodium nitroprusside was used as a negative control, generating reactive nitrogen species. Hemodynamic assessment

[101] To evaluate the hemodynamic changes induced by the monoterpene after chronic treatment in the presence or absence of isoproterenol infarction (animals mentioned in section 2.1), we used the technique described by Parasuraman & Raveendran, 2012. On day 16, the animals were anesthetized with a mixture of Ketamine 80 mg / kg + Xylazine 4 mg / kg ip and then underwent surgery for the insertion of intravascular polyethylene catheters. The polyethylene (PE) catheters had a PE-10 segment with an internal and external diameter of 0.28 and 0.61 mm, respectively, which were welded to a PE-50 segment with an internal and external diameter of 0.58 and 0.96 mm, respectively. The catheters were filled with heparinized saline solution (1:20 v / v) and then implanted into the abdominal aorta and inferior vena cava via the left femoral artery and vein.After insertion and fixation, the arterial catheter was connected to a pre-calibrated pressure transducer (Statham P23 ID; Gould, Cleveland, OH, USA), coupled to an amplifier (Model TBM-4M, WPI, Sarasota, FL, USA) and connected to a microcomputer equipped with an analog-to-digital converter board (CIODAS16 / JR, Computer Boards, Inc., Mansfield, MA, USA). The AQCAD program (São Paulo, Brazil) was used to acquire the recordings.

[102] The frequency chosen for data sampling was 500 Hz. A venous catheter was used for bolus drug administration. The hemodynamic parameters evaluated were: mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR). Hemodynamic recordings were kept for 30 minutes after stabilization of cardiovascular parameters. Determination of myocardial oxygen demand Petition 870190021822, dated 07 / 03 / 2019, page 31 / 118 23 / 71

[103] The estimation of myocardial oxygen demand, as an index for ischemia-reperfusion injuries, was determined by the values ​​expressed in myocardial reperfusion pressure rates in resting rats (animals mentioned in section 2.1) as a function of heart rate as a reflection of the cardiac work performed. Myocardial oxygen demand was then obtained by the mathematical expression: Equation 07 BMD (mmHg / min) = (MAP) x HR / 1000 Assessment of vascular reactivity in mesenteric arteries

[104] To evaluate the effects of the monoterpene on vascular reactivity in resistance arteries, rats treated with the monoterpene and induced to infarction (animals mentioned in section 2.1) were euthanized by exsanguination under anesthesia (Ketamine 80 mg / kg + Xylazine 4 mg / kg ip) on day 16. Then, through an incision in the animal's abdomen, the superior mesenteric artery was removed. Rings of the first segment of the artery (1-2 mm) were obtained free of connective and adipose tissue. The rings were kept in vats containing 5 mL of Tyrode's solution at 37°C and carbonated with carbogen. The rings were suspended by cotton threads attached to a force transducer (Fort 10, WPI, Sarasota, USA), which was connected to an acquisition system (Miobath-4, WPI, Sarasota, USA) for recording isometric tensions. Each ring was subjected to a constant tension of 0.75 g for a period of 60 minutes.During this time, the nutrient medium was changed every 15 minutes to prevent interference from metabolites (Altura; Altura, 1970). The endothelium-free rings were obtained mechanically through friction between the inner walls of the vessel. The presence of endothelium was verified by the relaxation of the rings after the addition of 10 μM of acetylcholine. Rings with relaxation greater than 80% after pre-contraction with 10 μM of phenylephrine were considered to have endothelium. Rings with relaxation less than 10% were considered to have no endothelium (FURCHGOTT & ZAWADZKI, 1980). Petition 870190021822, dated 07 / 03 / 2019, page 32 / 118 24 / 71

[105] After a stabilization period of 60 minutes, a cumulative concentration-response curve of phenylephrine [10-10-10-5M] was obtained. Subsequently, a cumulative concentration-response curve to sodium nitroprusside [10-11-10-5M] was developed. The pharmacological parameters to assess changes in vascular reactivity were: PD2 and Emax. Direct measurement of blood pressure and heart rate in SHR rats

[106] Intra-aortic pressure parameters were measured in non-anesthetized hypertensive rats, according to the technique described by Kramer & Remie, 2005. Briefly, spontaneously hypertensive rats (200-250g), which had not undergone chronic treatment, were anesthetized (Sodium thiopental 45 mg / kg ip) and underwent a surgical procedure for the implantation of intravascular polyethylene catheters. Catheter implantation was performed as described in section 2.1. After insertion and fixation, the catheter was tunneled subcutaneously and exteriorized through an incision in the posterior cervical region of the animal (scapulae). 24 hours after the surgical procedure, the non-anesthetized animals were connected to a device for pressure recording.The arterial catheter was connected to a precalibrated pressure transducer (Statham P23 ID; Gould, Cleveland, OH, USA), coupled to an amplifier (Model TBM-4M, WPI, Sarasota, FL, USA) and connected to a microcomputer equipped with an analog-to-digital converter board (CIODAS16 / JR, Computer Boards, Inc., Mansfield, MA, USA). The AQCAD program (São Paulo, Brazil) was used to acquire the recordings. The chosen sampling frequency was 500 Hz. Meanwhile, the venous catheter was used for bolus drug administration. Evaluation of cardiovascular activities induced by monoterpenes

[107] The animals were kept in acclimatization for at least 30 minutes to stabilize hemodynamic parameters. Then NPS (10 μg / kg, iv) was administered to verify the effectiveness of catheter implantation. After 15 minutes, increasing doses of TPN (1, 5 and 10 mg / kg, iv) were administered randomly at sufficient time intervals to Petition 870190021822, dated 07 / 03 / 2019, page 33 / 118 25 / 71 that MAP and HR returned to their baseline values. MAP and HR values ​​were calculated before (baseline values) and immediately after TPN administration; their variations were expressed as a percentage of mean arterial pressure and heart rate, for each dose, calculated using equations 08 (1) and 09 (2), respectively: Equation 08 (1) PAM values ​​(%) = (PAM after - PAM before) x 100 PAM before Equation 09 (2) aaloeed de FC (%) = (FC after-FC before)x 100 FC before Evaluation of the involvement of the central nervous system in the hypotensive and bradycardic responses induced by the monoterpene.

[108] In order to evaluate the participation of the central nervous system in the cardiovascular effects induced by the monoterpene, a new dose-response curve for the monoterpene was obtained in spontaneously hypertensive rats under the effect of sodium thiopental 45 mg / kg, a centrally acting barbiturate (MURRAY et al, 2006). Evaluation of ganglionic involvement in hypotensive and bradycardic responses induced by monoterpenes.

[109] After a control recording obtained as described in item 4.1.14, the animals were treated with hexamethonium (20 mg / kg, iv), a non-depolarizing ganglionic blocker (OWYANG, 1997). After 30 minutes, a new dose-response curve for TPN was obtained. Changes in MAP and HR induced by TPN were compared before (baseline values) and after treatment with hexamethonium. Evaluation of muscarinic involvement in hypotensive and bradycardic responses induced by monoterpenes.

[110] After a control recording obtained as described in item 4.1.14, the animals were treated with atropine (2 mg / kg, iv), a non-selective antagonist of muscarinic receptors (MITCHELSON, 1984). After 15 minutes, Petition 870190021822, dated 07 / 03 / 2019, page 34 / 118 26 / 71 A new dose-response curve for TPN was obtained. Changes in MAP and HR induced by TPN were compared before (baseline values) and after treatment with atropine. In silico assay of molecular docking Preparation of Binders

[111] The 1D structure of the monoterpene was drawn using the free software ACD / ACD / ChemSketch®, available at: http: / / www.acdlabs.com / resources / freeware / . Then, converted to 3D, and energy minimization determined using the free software ArgusLab® version 4.0.1 developed by Thompson (1994-2004) available at: http: / / www.arguslab.com / arguslab.com / ArgusLab.html. Using the free software AUTODOCK TOOLS 1.5.6® developed by Sanner, 1999, available at: http: / / autodock.scripps.edu / . Partial atomic charges were assumed according to Gasteiger's mathematical model. Furthermore, σ bonds were considered flexible and π bonds rigid. Selection of Macromolecules

[112] Macromolecules frequently studied as therapeutic targets were selected for the design of new drug candidates acting on the cardiovascular system and enzymes from studies of antioxidant systems. Thus, the following PDB-ID targets were selected: 4MQS (M2 receptor linked to its agonist), 3UON (M2 receptor linked to its antagonist), 4DAJ (M3 receptor), 1J95 (Potassium KCa2+ channel), 3NAF (BkCa channel), 4GPO (β1 receptor), 3NOS (Nitric Oxide Synthase enzyme linked to L-arginine), 4BKE (Albumin), 2LQC (N-terminal L-type Ca+2 channel), 2LQP (C-terminal L-type Ca+2 channel), 4DEY (Voltage-sensitive L-type Ca+2 channel), 2BE6 (Dihydropyridine docking site in voltage-sensitive L-type Ca+2 channels), 2RH1 (Type 2 β-Adrenergic receptor). 1QQW (Catalase enzyme), 2SOD (Superoxide Dismutase enzyme), 1GP1 (Glutathione Peroxidase enzyme) and 1XOS (Phosphodiesterase 4 enzyme) obtained from the RSCBProtein Data Bank (PDB). Petition 870190021822, dated 07 / 03 / 2019, page 35 / 118 27 / 71 Preparation of Macromolecules

[113] In this step, the AUTODOCK TOOLS 1.5.6® software was used again, where all water molecules were eliminated and all hydrogens were added to the macromolecules. Partial atomic charges were added using Kollman's mathematical model and all atoms were assumed to be of type AD4. Finally, the macromolecules were converted to the extension. Gridbox parameters

[114] The search map was established with dimensions 126 A2 and spacing of 0.375 A, which was calculated using AUTOGRID4

[20] . The affinity and electrostatic potentials of the maps were calculated for each type of atom of the ligands, according to their natures. The coordinates were established by centering the “gridbox” inside each macromolecule. Docking parameters

[115] The Lamarckian Genetic algorithm was chosen for the docking procedures. The number of executions of these algorithms was set to 100, and for the population evaluation, a value of 150 was assumed. All other parameters were kept as “default” by AUTODOCK4. Search for Docking Vina®

[116] The free software AUTODOCK VINA® (developed by Trott & Olson, 2010), available at: http: / / vina.scripps.edu / , was used, where all the coordinates used were the same as those mentioned in section 3.5.15.4. The exhaustiveness of the method was maintained at 8 and the number of models generated was set at 100. Docking Validation Petition 870190021822, dated 07 / 03 / 2019, page 36 / 118 28 / 71

[117] The method validation was interpreted in terms of the root mean square standard deviation (RMSD) between the conformations adopted by the monoterpene, with the cut-off for this < 2 A. Determination of cLogP

[118] The free software OSIRIS PROPERTY® was used to calculate cLogP, available at: http: / / organic-chemistry.org / prog / peo / , developed by Sander at Actelion Pharmaceuticals Ltd. Statistical analysis

[119] Values ​​are expressed as mean ± standard error of the mean (sem). The unpaired Student's t-test was used for comparisons between two variables. Meanwhile, one-way ANOVA was used for comparisons of more than two variables, followed by the Newman-Keuls multiple comparison test. A p-value < 0.05 was adopted for the variable results. This represents a one in twenty chance of the event having occurred by chance as a result of sampling error. Statistical analysis was performed using GraphPad Prism statistical software version 5.0.

[120] For the optimization of analytical antioxidant parameters, analytical sensitivity was used as a criterion based on the slope of the respective analytical curve under a given study condition. The evaluated analytical curve was constructed with at least five points according to the relationship: %IF = aCAO + b, where %IF is the percentage of inhibition, CAO is the concentration of the antioxidant compound, a is the slope, and b is the linear coefficient. The linear correlation coefficient (r) was calculated to evaluate the arrangement of the points regarding the adequacy of linear behavior. A similar procedure was performed for the proposed method and the comparison methods aimed at quantifying the antioxidant capacity in different samples by interpolating the data.

[121] The calculations relating to the limits of detection (3σ) and quantification (10σ) were performed according to the following equations, LOD = 3sb / ac and Petition 870190021822, dated 07 / 03 / 2019, page 37 / 118 29 / 71 LOQ = 10sb / ac, where sb is the standard deviation of the analytical blank (n = 10), while ac corresponds to the slope of the analytical curve used. The relative standard deviation (RSD) was calculated according to the equation: RSD = (sp / xp) x 100, where sp is the standard deviation for a given analytical standard within the linear range and xp corresponds to the mean value found for this standard (n = 10). The evaluation of the results of the different methods used was performed using the paired Student's t-test, ANOVA (Analysis of Variance), and linear correlation procedures between the results obtained. For all procedures, a normal distribution of the data (random error) and a confidence interval of 95% were considered. RESULTS OBTAINED

[122] This study aimed to evaluate the potential cardioprotective activity of the monoterpene TPN and its intrinsic mechanisms of pharmacological activity, in a model of acute myocardial infarction induced by isoproterenol. For this purpose, experimental approaches were used: in vivo, in vitro, ex-vivo and in silico.

[123] RDC 251 / 1997 of the National Health Council mandates that in preclinical studies for the development of candidate molecules for new drugs and their prototypes, prior toxicological aspects must be studied as a requirement to guarantee the safety of medicines (CNS, RDC 251 / 1997). According to Hansen & Bross 2010, the use of cell viability assays in immune cell lines is used as a strategy adopted to evaluate the toxicity of new drugs (HANSEN & BROSS, 2010).

[124] In addition, Postnikova and colleagues described the close relationships between screening assays of new therapeutic agents based on in vitro cell viability models with primary immune cells and the toxic effects of drugs in clinical studies (POSTNIKOVA et al., 2018). Petition 870190021822, dated 07 / 03 / 2019, page 38 / 118 30 / 71

[125] Thus, the cytotoxic activity of the compound was tested in J774 macrophage cell cultures to investigate the compound's toxic actions, aiming at the insertion of the evaluated monoterpene as a possible candidate for a cardiovascular drug in subsequent pharmacological evaluation in an in vivo model.

[126] According to the cytotoxicity scale described in ISO 10993-5 of 1999, TPN varied its response in a concentration-dependent manner; at the lowest concentration tested it demonstrated a non-cytotoxic effect (cell viability > 90%), while at the highest concentration tested it proved to be moderately cytotoxic (cell viability = 50-79%) (ISO 10993-5, 1999). It is worth noting that it was not possible to determine the IC50% of the compound, since the cytotoxicity levels did not reach 100% cytotoxicity at the concentrations evaluated (Figure 1). These findings together corroborate that TPN, when used at the concentrations chosen in the present study, is not inducing significant toxic effects.

[127] Based on this result, the cardioprotective activity of TPN in in vivo tests in acute myocardial infarction in an isoproterenol-induced ischemia-reperfusion injury model was evaluated.

[128] Thus, the ability of TPN to induce survival in rats against the induction of infarcts produced with isoproterenol was evaluated. The results show that treatment with TPN increased the survival of normotensive (WT) and hypertensive (SHR) rats against infarct induced with isoproterenol.

[129] Repeated administration of high-dose ISO (85 mg / kg sc 2x) can induce lethal infarctions in vivo in rats, despite isoproterenol being considered an effective method in the search for new experimental therapies because it does not produce high lethality, presenting an LD50 = 680 mg / kg (RAHMATHULLA & DEVI, 2013; SADDIQUI et al, 2016). In in vivo tests, it was found that in the infarcted control groups, there was an increase in infarction-induced deaths with isoproterenol. However, prior treatment with TPN was able to significantly increase the percentage of survival to lethality. Petition 870190021822, dated 07 / 03 / 2019, p. 39 / 118 31 / 71 induced by isoproterenol at the doses tested in both experimental cell lines (Figure 2).

[130] The increase in survival in in vivo tests of lethal infarctions is indicative that TPN inhibited the worsening of cardiac lesions produced by catecholamines, producing cardioprotection. Treatment with TPN 50 mg / kg, in the absence of isoproterenol, did not produce significant changes in the percentage of survival compared to their respective saline and infarcted controls in both rat strains (Figure 2).

[131] To verify the cardioprotective effect induced by TPN on the myocardium, the degree and extent of myocardial lesions produced by isoproterenol were evaluated in the triphenyltetrazolium colorimetric test. It has already been described that ischemic injury in the heart produces cell death dependent on mitochondrial pathways (NING et al., 2000; MARIN-GARCÍA, et al., 2001). Thus, necropsy evaluations are useful to verify the magnitude of infarctions on the heart, being routine in forensic medicine institutes (KOTABAGI et al., 2000; KAKIMOTO et al., 2013).

[132] It has also been described that isoproterenol is capable of inducing AMI, causing a reduction in viable cardiac area through mechanisms of activation of cellular necrosis, causing alterations in mitochondrial functionality and thus playing a crucial role in the pathological development in ischemia-reperfusion lesions (SANCHÉZ et al., 1997).

[133] Nevertheless, the use of triphenyltetrazolium to assess cardiomyocyte cell viability has been widely employed in preclinical pharmacological evaluations of cardioprotection (MOHAN et al., 2010; AMAN et al., 2012; CHEN et al., 2015). Triphenyltetrazolium salts are colorless in aqueous solutions and are converted to formazan in a manner dependent on mitochondrial NADPH reductase systems, becoming a red solution. Viable cells acquire a red coloration that is deposited in the cytosol in the form of insoluble crystals and represents the remaining viable myocardium, while degraded cells have denatured oxidoreductase systems in the mitochondrial matrices, thus, the conversion of triphenyltetrazolium salts Petition 870190021822, dated 07 / 03 / 2019, page 40 / 118 32 / 71 do not occur, and the necrotic regions remain pale yellow or white in color (KLEIN et al., 1981; KHALIL et al., 2006). The results obtained demonstrated that TPN was able to inhibit the development of necrotic areas in a dose-dependent manner in both rat strains compared to the infarcted rat groups (Figure 3 and Figure 4).

[134] Upon macroscopic analysis, we observed that the saline groups in both cell lines exhibited red coloration throughout the transverse cardiac extent, while the infarcted control groups of both cell lines showed white or yellow areas corresponding to the necrotic areas formed during ischemia-reperfusion injuries, while other areas remained red, corresponding to the remaining myocardium. Pretreatment with TPN showed a higher proportion of red regions than yellow ones, demonstrating the property of inhibiting the formation of necrotic areas in all transverse cardiac extents analyzed in the apex-base direction (Figure 3, Figure 4 and Figure 5).

[135] Treatment with TPN 50 mg / kg in the absence of isoproterenol did not produce significant changes in myocardial viability when compared to their respective saline and infarcted controls in both experimental strains (Figure 3, Figure 4 and Figure 5). The results presented above indicate that subacute treatment with TPN at the tested doses, in the in vivo model in normotensive and hypertensive rat strains, and also that there is a dose-dependent relationship in the cardioprotection induced by TPN.

[136] It has already been pointed out that isoproterenol is capable of producing alterations in cardiac microstructures, which are demonstrated in histopathological analyses (RONA, 1985). It is described that frequent exposures to isoproterenol are capable of inducing the following alterations in cardiac histoarchitecture: increased leukocyte invasion in the myocardium, myofibrillar degeneration and separation, edema, myocardial hemorrhage, and degenerative nuclear alterations: pyknosis, karyorrhexis and karyolysis, in addition to hypertrophic cardiac remodeling without Petition 870190021822, dated 07 / 03 / 2019, page 41 / 118 33 / 71 Cell neogenesis, as an adaptive survival response to ischemic injury aggressions (WEXLER & JUDD, 1970; LOBO-FILHO, et al., 2011; RAMATHULLA, et al., 2013).

[137] The histopathological results presented in this study show that the infarct produced by isoproterenol was able to mimic all the pathological processes of ischemia-reperfusion injuries, inducing an increase in leukocyte invasion in the myocardium compared to saline controls in both experimental cell lines, producing intense myocarditis, in addition, it produced fibrillar degenerations and ruptures, edema and progressive and degenerative nuclear changes in hematoxylin and eosin stained heart slides (Figure 6 and Figure 7).

[138] When performing the total leukocyte count, it was found that there was a significant increase in the number of total polymorphs compared to the saline controls in both experimental lines (table 1 and table 2) the same occurred when quantifying the presence of nuclei in pyknosis (table 3 and table 4) karyorrhexis (table 5 and table 6) and karyolysis (table 7 and table 8).

[139] The results of prior treatment with TPN show a significant reduction in all histological parameters analyzed at the tested doses, when in the presence of isoproterenol, indicating that TPN was able to prevent changes in histoarchitecture (Figures 6 and 7, Tables 1-8).

[140] The histopathological results found in this study were similar to the cardioprotective studies of natural products described in the literature (SHUKLA, et al., 2010; BRAMACHARI. et al 2014; OJHA, et al.,2016). Thus, some molecules derived from natural products have attracted pharmaceutical attention due to the cardioprotective potential produced against previously induced infarction in preclinical evaluations, preventing histological changes. Petition 870190021822, dated 07 / 03 / 2019, page 42 / 118 34 / 71 TABLE 1 - Total Leukocyte Count (TLC) in myocardium of WT rats after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg + ISO TPN 50 mg / kg - ISO TPN 75 mg / kg - ISO TPN 50 mg / kg without ISO Number of leukocytes = ____________ 0.038 mm2 302 = 9.7 2,596 = 36.70 2,314 = 17.51 ​​856 = 6.31 334 = 4.4 490 = 3.7 Statistical analysis: One-way ANOVA followed by Newman-Keuls post-hoc test. Significance was considered when tp>0.0001 (infarcted vs. saline) and ***p>0.0001 (TPN vs. infarcted). N=5 TABLE 2 - Total Leukocyte Count (TLC) in myocardium of SHR patients after prior treatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg-ISO TPN 50 mg / kg-ISO TPN 75 mg / kg-ISO TPN 50 mg / kg without ISO N- of leukocytes 0.038 mm2 6084 5.4 4124 4 12.24 2462 4 10.15 857 4 6.32 510 4 3.4 490 4 3.4 Statistical analysis: one-way ANOVA followed by post-hoc Newman-Keuls test. Significance was considered when p > 0.0001 (infarcted vs. saline) and p > 0.0001 (TPN vs. infarcted). N=5 TABLE 3 - Total pyenosis nuclei count (TNCp) in myocardium of WT rats after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Parameter Saline Infarct TPN LTN TPN TPN (ISO) 25 mg / kg-ISO 50 mg / kg 4lS0 75 mg / kg-ISO 50 mg / kg without ISO 84 = 2.20 716 = 30.85 656 = 33.98 306 = 15.20 158 = 2.03 168 = 2.7 .V- of nucian DfCTNV = 0.838 mm2 Statistical analysis: ANOVA one-way VA followed by the Newman-Keuls post-test. Significance was considered when fp>0.0001 (Infarcted vs. saline). *p>0.05 and **p>0.0001 (TPN vs. infarction). N=5 TABLE 4 - Total pyenosis nuclei count (TNCp) in SHR rat myocardium after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg 41 SO₂ TPN 50 mg / kg ISO TPN 75 mg / kg ISO TPN 50 mg / kg without ISO Number of nuclei CTNP ~ 0.038 mm² 178 = 5.6 698 = 11.02 470 = 2.68 304 = 3.42 210 = 1.78 208 = 0.8 Statistical analysis: one-way ANOVA followed by post-hoc Newman-Keuls test. Significance was considered when fp>0.0001 (infarcted vs. saline) and ****p>0.0001 (TPN vs. infarcted). N=5 TABLE 5 - Total karyolysis nuclei count (CTNc) in myocardium of WT rats after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg 410mg / kg ISO TPN 50 mg / kg ISO TPN 75 mg / kg ISO TPN 50 mg / kg without ISO Number of nuclei CTNc ~ 0.038 mm2 624 3.21 1.0404 12.76 496 441.5 208 = 4.10 1004 1.30 110 4 0.8 Statistical analysis: One-way ANOVA followed by Newman-Keuls post-hoc test. Significance was considered when fp > 0.0001 (infarcted vs. saline) and ****p > 0.0001 (TPN vs. infarcted). N=5 Petition 870190021822, dated 07 / 03 / 2019, page 43 / 118 35 / 71 TABLE 6 - Total karyolysis nuclei count (CTNc) in SHR rat myocardium after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg - ISO TPN 50 mg / kg - ISO TPN 75 mg / kg + ISO TPN 50 mg / kg without ISO N- of nuclei CTNc = n „„„----5- 0.038 mm2 164 = 1.7 930 = 8.79 446 = 4.30**** 208 = 4.7**** 124 = 1.63***“ 130 = 0.31**** Statistical analysis: one-way ANOVA followed by post-hoc Newman-Keuls test. Significance was considered when p > 0.0001 (infarcted vs. saline) and p > 0.0001 (TPN vs. infarcted). N=5 TABLE 7 - Total karyorrhexis nuclei count (CTNcx) in myocardium of WT rats after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg 2x sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg - ISO TPN 50 mg / kg - ISO TPN 75 mg / kg + ISO TPN 50 mg / kg without ISO Number of nuclei CTNcx = ——--5- 0.038 mm1 3 Oi 1.4 198 = 4.97 176 = 1.32**** 78 = 0.86**** 46 = 0.40**** 68 = 0.48**** Statistical analysis: one-way ANOVA followed by post-hoc Newman-Keuls test. Significance was considered when fp>0.0001 (infarcted vs. saline) and ****p>0.0001 (TPN vs. infarcted). N=5 TABLE 8 - Total karyorrhexis nuclei count (CTNcx) in SHR rat myocardium after pretreatment with the monoterpene (TPN) and subsequent exposure to the sympathomimetic infarction-inducing agent isoproterenol (85 mg / kg² sc). Infarcted Saline Parameter (ISO) TPN 25 mg / kg-HSO TPN 50 mg / kg+ISO TPN 75 mg / kg+ISO TPN 50 mg / kg without ISO N- of nuclei CTNcx = „ ---5- 0.038 mm2 42± 0.96 236i4.43 110 = 1.09**** 78 T0.76**** 70 = 0.89**** 66 i 0.74**'* Statistical analysis: one-way ANOVA followed by post-hoc Newman-Keuls test. Significance was considered when fp>0.0001 (infarcted vs. saline) and ****p>0.0001 (EPN vs. infarcted). N=5

[141] However, in humans, heart attacks are clinically diagnosed based on a combination of analyses: laboratory (through the detection of biological markers), electrocardiographic (through changes in cardiac electrical waves and segments), hemodynamic and imaging (SBC, 2017; PANDEY & GUPTA, 2011).

[142] In this context, to evaluate the cardioprotective potential of TPN, changes in biochemical markers, as well as cardiac electrical activity and hemodynamic parameters were measured after infarction induced with isoproterenol in the experimental cell lines.

[143] Creatine phosphokinase is an enzyme formed by a set of three isoforms. CK-MB is most abundant in cardiac muscle and its release in large quantities into the blood is associated with cardiomyocyte death due to ischemia-reperfusion injuries in myocardial infarction, and for this reason, it is used together with total CK (CK-NAC) in the early diagnosis of myocardial infarction. Petition 870190021822, dated 07 / 03 / 2019, page 44 / 118 36 / 71 myocardium since the 1970s (WHITE et al., 1985). Although new methodologies have been proposed for the early diagnosis of AMI, the use of total creatine phosphokinase and MB remains among the most useful, sensitive, and effective methods in the routine clinical management of myocardial infarction in recent years (CHAN & LEONG, 2010; MYTHILI & MALATHI, 2015; HAJAR, 2016).

[144] Similarly, the use of isoproterenol as a method to mimic AMI in experimental models has proven effective in increasing total creatine phosphokinase and its specific isoform (CK-MB) in blood samples from small laboratory animals (BERNAUER, 1978; LOBOFILHO, et al., 2011; KOLMANOVÁ, et al., 2015). For this reason, total creatine phosphokinase and its isoforms have been extensively used as a pharmacological identification method in studies of the cardioprotective activity of natural products or synthetic compounds (WONG, et al., 2017).

[145] The results described in this work showed that induction with isoproterenol was able to significantly increase the levels of total creatine phosphokinase and MB isoform in rats of both experimental strains compared to their respective saline controls, similar to findings in the literature (Figure 8 and Figure 9). Pretreatment with TPN was able to significantly inhibit the release of these enzymatic markers in the blood serums of both experimental strains compared to their respective infarcted controls, demonstrating a cardioprotective effect (Figure 8 and Figure 9). Pretreatment with TPN in the absence of isoproterenol did not significantly alter the levels of total creatine phosphokinase and MB compared to saline control animals, revealing that treatment with TPN does not produce cytotoxic myocardial lesions.

[146] The electrocardiogram has been considered the most useful examination for the rapid and early diagnosis of myocardial injury lesions (GOLDBERGER et al., 2018). Electrocardiographic changes in infarction are usually characterized according to the evolutionary phase and location of myocardial lesions. Depending on the evolutionary phase, ischemic lesions Petition 870190021822, dated 07 / 03 / 2019, page 45 / 118 37 / 71 Reperfusion lasting less than 20 minutes of hypoxia / anoxia usually produces changes in the T wave of the electrocardiogram, reducing the amplitude or inverting it in the electrical plane (LUNA, et al. 2012). More persistent myocardial lesions initiate reentry mechanisms and, for this reason, cause changes in the ST segment of the electrocardiogram, characterized by ST segment depression in lead DI and reciprocal ST elevation in lead DII on the ECG, commonly called mirror images (ZIMETBAUM, et al., 2003; O'GARA, et al., 2013).

[147] Coronary branch occlusion with ischemia exceeding 6 hours usually produces Q wave deepening in the same leads where ST segment changes manifest, and is considered a sign of transmural necrosis. However, necrosis with anoxia lasting more than 12 hours initiates a process of R wave loss, altering the RR interval in the same leads. However, changes in the RR interval can also represent changes in heart rhythm and may be non-pathological. Therefore, it is necessary to evaluate the set of electrocardiographic changes in order to identify lesions from injury early, for better choice of clinical management of the infarction (ZIMETBAUM, et al., 2003; O'GARA, et al., 2013).

[148] Other changes secondary to infarction can be seen on the electrocardiogram, such as: changes in sinus rhythm, altering atrial depolarization which is marked by changes in amplitude and duration in the P wave (LUNA, 2012) and conduction blocks characterized by activation of reentry mechanisms diverting the conduction of the post-acute myocardial infarction action potential, with changes in the PR interval (SHAMROTH & BRADLOW, 1964; GINEFRA, et al., 2005; GOLDBERGER, 2006).

[149] Regarding their location, changes in precordial leads tend to be more precise in relation to the injured wall and depth of the lesions. However, in peripheral leads, ST segment changes usually occur in ischemic lesions of the Petition 870190021822, dated 07 / 03 / 2019, page 46 / 118 38 / 71 left anterior ventricular wall if the following occurs: ST segment elevation in leads I and aVL associated with ST segment elevation in V2, indicating block in the first diagonal branch, or ST segment depression in aVL, indicating occlusion of the distal branch in the left descending coronary artery, as well as ST segment elevation in aVR, indicating occlusion in the proximal septal branch of the left descending coronary artery (PANDEY & GUPTA, 2011).

[150] Ischemic lesions occur in the lower left ventricular wall only if: ST segment elevation in lead III is greater than in lead II, indicating occlusion of the right circumflex coronary artery branch, or if ST segment elevation is greater in lead II than in lead III, indicating occlusion of the left circumflex coronary artery branch (PANDEY & GUPTA, 2011).

[151] It has already been clearly described that isoproterenol is capable of inducing acute myocardial infarction by mimicking the electrocardiographic changes expected in ischemia-reperfusion injuries occurring in humans and rats (KELA, et al., 1980; YADAV, 2014).

[152] In this work, it was observed that isoproterenol induced significant changes on the electrocardiogram compared to its respective controls compatible with the lesions of AMI injury (Figures 10 and 11). Regarding the duration of the lesions, isoproterenol induced ischemic lesions, altering the T wave, in lead DI in the infarcted group of the Wistar strain, and in leads DII, aVL, aVR and aVF in the infarcted group of the spontaneously hypertensive strain; however, it also produced changes in the ST segment, appearing mirrored in leads DI and DII in both strains of infarcted rats, as well as producing the deepening of the pathological Q wave in DI in both strains of infarcted rats (Figure 10 and 11).

[153] Isoproterenol was also able to produce secondary changes in the electrocardiogram. In the group of infarcted Wistar rats, there was a change in sinus rhythm, causing a delay in atrial depolarization, Petition 870190021822, dated 07 / 03 / 2019, page 47 / 118 39 / 71 seen in lead DII, while in spontaneously hypertensive infarcted rats, sinus arrest was observed, as seen by the absence of the P wave in leads DI, DII, and aVF, reaching the isoelectric point. Regarding atrioventricular block, isoproterenol induced conduction delay in the atrioventricular system, producing first-degree infra-Hisian atrioventricular conduction block, due to the delay in the passage of the PR interval action potential in lead aVF of the electrocardiogram in both infarcted rat strains compared to their respective saline controls (Figure 10 and Figure 11).

[154] Regarding location, it was observed that isoproterenol induced a greater degree of lesion on the left inferior wall in infarcted rats from the spontaneously hypertensive rat lineage because the ST segment elevation was greater in derivatization DIII than in DII. While in infarcted rats from the Wistar lineage, it was not possible to predict the location of the infarcts, as no significant elevation was observed in derivatization DIII.

[155] Pretreatment with TPN was able to inhibit changes on the ST segment of the electrocardiogram when infarction was induced at all doses tested. TPN also significantly reduced the deepening of the pathological Q wave, demonstrating a cardioprotective effect in persistent injury lesions. (Figure 10 and Figure 11).

[156] Thus, it was found that TPN was able to prevent myocardial ischemia-reperfusion injuries greater than 20 minutes, but was not effective in preventing injuries with shorter times.

[157] Pretreatment with TPN was also able to prevent isoproterenol-induced first-degree atrioventricular conduction block. Therefore, TPN significantly prevented PR interval increase in aVF derivatization, protecting the heart from rhythm disturbances in response to ischemia-reperfusion injury. Regarding sinus rhythm, TPN showed a tendency to prevent sinus arrhythmia, as well as to prevent sinus arrest. Petition 870190021822, dated 07 / 03 / 2019, page 48 / 118 40 / 71

[158] Furthermore, pretreatment with TPN, in the absence of isoproterenol, did not produce significant changes in the electrocardiogram, but reduced isoproterenol-induced tachycardia in the RR interval, without altering the heart rhythm.

[159] In recent years, C-reactive protein, a mucoprotein produced by the liver, released into the bloodstream and its high titers in organs, has been considered an important biomarker for acute myocardial infarction in the inflammatory phase (DEDOBBELEER et al, 2004; MARTINS et al, 2011; CHACKO et al., 2018), while at the same time it has been considered an important risk factor for myocardial ischemia (COZLEA et al., 2013; CREA & MORROW, 2018).

[160] Similarly, isoproterenol has demonstrated the ability to elevate C-reactive protein levels in rats associated with myocarditis induced by AMI mimicked by this β-adrenergic agonist (ZAAFAN, M et al, 2012; SHUKLA, et al, 2015). For this reason, low levels of C-reactive protein have been considered an important biomarker in cardioprotective studies of natural products against isoproterenol-induced AMI (UPAGANLAWAR et al, 2011).

[161] The results of this work show that isoproterenol was able to induce a significant increase in ventricular levels of C-reactive protein, demonstrating that there was induction of myocardial inflammation associated with AMI produced by the subacute administration of repeated doses of isoproterenol compared to their respective saline controls (Figure 12). These results confirm the results previously shown in the migration and invasion of polymorphonuclear leukocytes in the total leukocyte count in myocardium, demonstrating the effectiveness of isoproterenol in inducing myocarditis in rats by the isoproterenol infarction method (Tables 1 and 2).

[162] Pretreatment with TPN significantly prevented the increase in C-reactive protein levels at the doses tested (Figure 12). Thus, the efficacy of TPN in producing cardioprotection in the inflammatory phase of isoproterenol-induced myocardial infarction is demonstrated. Similarly, treatment Petition 870190021822, dated 07 / 03 / 2019, page 49 / 118 Subacute treatment with nifedipine also significantly reduced C-reactive protein levels in the presence of isoproterenol (Figure 12). Pretreatment with TPN in the absence of isoproterenol did not produce significant changes in C-reactive protein levels when compared to their respective saline controls (Figure 12).

[163] Recent studies have pointed out that dyslipidemia is an important risk factor for ischemic heart disease, thus high levels of total cholesterol and triglycerides are unfavorable for cardiac prognosis in humans, increasing clinical mortality outcomes (ANAND et al, 2008; KIANI et al; 2016; RATHORE et al; 2018). The protective potential of TPN in these risk factors, monitoring total cholesterol and triglyceride levels in the serum of rats.

[164] Isoproterenol has been described as capable of causing an imbalance in carbohydrate and lipid metabolism in rats during the development of experimental acute myocardial infarction (LIU et al, 2013; WONG et al, 2017). It is already clearly described that beta-adrenergic agonists are capable of increasing fatty acid recirculation due to lipolytic stimulation in adipocytes, thanks to the action exerted on type III β-adrenergic receptors, activating phospholipase as a second messenger and consequent phosphorylation of protein kinase type A (PKA), and clear involvement of voltage-dependent calcium channels, increasing cytosolic calcium influx (PHILIPSON, 2002; SAMANI et al, 2016) and intracellular calcium stores, with involvement of adenylyl cyclase (AC) in the reprogramming of lipid metabolism (MAUS et al., 2017).

[165] Thus, lipid levels have been monitored in studies of the cardioprotective activity of natural products in the isoproterenol model (UPAGANLAWAR et al, 2011; WONG et al, 2017). In our study, isoproterenol was able to induce an increase in total cholesterol levels when compared to its respective saline controls (Figure 13). Similarly, the induction of AMI was able to increase serum levels of Petition 870190021822, dated 07 / 03 / 2019, page 50 / 118 42 / 71 triglycerides in rats when compared with their respective saline controls (Figure 14).

[166] Pretreatment with TPN significantly inhibited the increase in total cholesterol and triglyceride levels at all doses tested. Thus, TPN is able to act in the prevention of risk factors for ischemic heart disease, demonstrating its cardioprotective action, reducing the risk of mortality in the experimental study (Figure 13 and Figure 14).

[167] At the same time, subacute treatment with TPN in the absence of isoproterenol did not produce any changes on lipid levels, therefore, it did not significantly alter total cholesterol or triglyceride levels in any of the rat strains compared with their respective saline controls (Figure 13 and Figure 14).

[168] It is also clearly described that after AMI, the tissue repair stimulus is activated, inducing the formation of concentric cardiac hypertrophy, as an adaptation mechanism, after the stimulus of aggression and cellular defense (ERTL & FRANTZ, 2005). This mechanism occurs without the genesis of new cardiomyocytes and is due to the deposition of collagen in the extracellular matrix (JUGDUTT, 2003). This mechanism of cardiac hypertrophy by ventricular remodeling is considered a defense against hypoxia / anoxia and prevents fatality in myocardial infarction. However, in the long term, cardiac hypertrophy leads to reduced ventricular compliance and induces ventricular dysfunction and heart failure, producing morbidities and cardiovascular risk (FRENCH & KRAMER, 2007; ZORNOFF et al, 2009; GALLI & LOMBARDI, 2016; AZEVEDO et al., 2016; HASSEL et al, 2017).

[169] Isoproterenol-induced infarction is capable of mimicking post-acute myocardial infarction cardiac hypertrophy, without the genesis of new cardiomyocytes, with increased collagen deposition in the extracellular matrix (REMIÃO, 2002; LIAUDET et al, 2014). Ventricular dysfunction of cardiac remodeling contributes to the progression of cardiomyopathy and induces heart failure over time with the involvement of the renin-angiotensin system activation, increasing morbidity and mortality (SETHI & DHALLA, Petition 870190021822, dated 07 / 03 / 2019, page 51 / 118 43 / 71 1995; GRIMM et al, 1998; BROOKS & CONRAD, 2009; LOBO-FILHO et al, 2011).

[170] In our experiments, isoproterenol was also able to induce an increase in ventricular morphometry, producing left ventricular hypertrophy (Figure 15 and Figure 16). When analyzing the ventricles in histological experiments stained with Gomori's trichrome, a dye that highlights the presence of collagen in the extracellular matrix, it was observed that there was an increase in the deposition of interstitial collagen, replacing the cardiac cells in the areas of ventricular necrosis (Figures 17 and Figure 18).

[171] Pretreatment with TPN inhibited morphometric increase, indicating prevention of post-infarction hypertrophy compared to the respective infarcted groups (Figure 15 and Figure 16). The same was observed in histological sections stained with Gomori's trichrome; it was found that pretreatment with TPN inhibited the development of isoproterenol-induced fibrosis, reducing the interstitial collagen of the extracellular matrix (Figures 17 and Figure 18).

[172] Similarly, pretreatment with TPN in the absence of isoproterenol did not produce significant changes in ventricular morphometry. Thus, it is evident that there was no development of cardiac hypertrophy in animals treated with TPN in the absence of isoproterenol (Figure 15 and Figure 16). When histological sections of the ventricles of rats treated with TPN in the absence of isoproterenol were analyzed, no increase in collagen deposition in the extracellular matrix was observed in either experimental strain (Figure 17 and Figure 18).

[173] After verifying the cardioprotective effect of TPN, this work aimed to verify which possible mechanisms of action were related to the cardioprotective activity induced by TPN. Several mechanisms of action can be correlated with the cardioprotective effects, most of which are due to enzymatic or non-enzymatic antioxidant action (DHALLA et al, 2000; PUGSLEY et al 2003; FARÍAS et al, 2017), activation of mitochondrial pathways, increasing oxidative phosphorylation and increasing metabolism in the Krebs cycle (CARREIRA et al, 2011; Petition 870190021822, dated 07 / 03 / 2019, page 52 / 118 44 / 71 DONGWORTH et al, 2014; FORINI et al, 2015; PANEL et al, 2017), inhibition of apoptosis and cell death pathways (LI et al, 2006; KOSHINUMA et al, 2014) or actions dependent on the activation of cardiovascular targets (GARCÍADORADO et al, 2017; LEE et al, 2017; HAUSENLOY et al, 2017).

[174] In addition, several natural products are able to activate cardioprotective pathways through multiple mechanisms, proving effective in the cardioprotection produced against induced MI (UPAGANLAWAR et al, 2011; WONG et al, 2017). Thus, we evaluated the intrinsic mechanisms of the cardioprotective action of TPN described here.

[175] It is clearly described that AMI produces a significant increase in the generation of oxygen-derived free radicals, as well as nitrogen-derived species (SIMPSON & LUCCHESI 1985; ZWEIER & TALUKDER, 2006; VERNARDOS et al, 2007). Similarly, isoproterenol is able to induce an increase in free radicals during infarction development in isolated cardiomyocytes and in laboratory animals (ZHANG et al, 2005; REMIÃO et al, 2002; BOVO et al, 2012; BOVO et al, 2015). In contrast, antioxidant compounds have been shown to play an important role in cardioprotection against myocardial reperfusion injury through the scavenging of free radicals (JEROUDI et al, 1994).

[176] Thus, the radical scavenging potential induced by TPN was evaluated in in vitro tests, demonstrating its antioxidant action. However, the associations of the results of the in vitro tests reveal that TPN has reduced radical scavenging activity and therefore little antioxidant effect. Thus, we refute the hypothesis that TPN has significant antioxidant action as a proposed mechanism for the cardioprotective effects demonstrated here.

[177] In comparison, the various natural compounds tested in the in vitro model that had cardioprotective activity described in the literature (KAGLI et al, 2005; TAN et al, 2005; HO et al, 2014; LI et al, 2013; PRINCE et al, 2009; RYU et al, 2016) obtained superior antioxidant effects to those induced by TPN, Petition 870190021822, dated 07 / 03 / 2019, page 53 / 118 45 / 71 thus the evidence of the antioxidant action induced by TPN playing a fundamental role in the cardioprotective effect of TPN in the in vivo study described here becomes low or nil (Figure 19, Table 9 and Table 10). TABLE 9 - Antioxidant profile of the monoterpene (TPN) in tests of radical scavenging activity against DPPH radicals, ABTS and iron reducing power (Fe3+). ifetodoloeia employed ABTS DPFH - 30% methanol · water (mv) DFPH - ?0%metanulagna (m / r) Reduction of Fe(III) € ,με mL C , με mL % 1„_ Cmi', με ml1 € ,με mL Aabs 700 400 1.3 100 -1.9 300 -2.9 100 0.034 TABLE 10 - Antioxidant profile of the monoterpene (TPN) in complexing activity tests against iron in the Fe3' oxidation state Compounds __ Complexation capacity of FeCHTj Fail a Linear, FT of points Analytical curve «HjU + I r EC.,, TPN 2.5 - 10.0 4 ^ = 9.00(=0.4)^ / -2.51(=2.29) 0.9980 5:8 EDTA 1.0 - 5.0 4 %I = 17.12 (=0.74) Cp / - 4- 5.9 8 (=2.12) 0.9981 2,<5

[178] Given this premise, we decided to evaluate the repercussions Cardiovascular effects produced by TPN in the presence or absence of isoproterenol after subacute treatment. It is already reported in the literature that isoproterenol is a synthetic catecholamine that pharmacologically activates beta-adrenergic receptors (MARTINSSON et al, 1989) and thus reduces blood pressure due to its vasodilatory action, reducing total peripheral resistance, mediated by β2-adrenergic receptors (KIEFFER & ABEL, 2016), and increases cardiac output due to increased heart rate through the induction of positive chronotropic, inotropic, dromotropic, and lusitropic effects on the heart, mediated by β12-adrenergic receptors (AVERIN et al, 2010; ENGELHARDT & AHLES, 2014; KIEFFER & ABEL, 2016).

[179] The increase in heart rate induced by isoproterenol plays a key role in calcium overload and strenuous cardiac work and therefore plays a crucial role in isoproterenol-induced ischemia-reperfusion injury (BLOOM & DAVIS, 1972; LOHSE et al, 2003; BRUM et al, 2006; RAMATHULLA & DEVI, 2013). Petition 870190021822, dated 07 / 03 / 2019, p. 54 / 118 46 / 71

[180] In our experiments, subcutaneous administration of isoproterenol reduced mean arterial pressure, systolic and diastolic pressure and increased heart rate, compared to their respective saline control groups (Figure 20 and Figure 21), this is due to vascular β2adrenergic activity, reducing total peripheral resistance. Isoproterenol was also able to induce an increase in heart rate (Figure 20 and Figure 21) these effects are attributed to the positive chronotropic, inotropic, dromotropic and lusitropic activities induced by isoproterenol.

[181] Pretreatment with TPN did not produce significant changes in blood pressure values ​​in the two experimental cell lines in the presence of isoproterenol when compared to their respective infarcted controls (Figure 20 and Figure 21). However, they showed a reduction in blood pressure parameters compared to the saline control groups (Figure 20 and Figure 21). These effects are due to beta-adrenergic activation induced by isoproterenol. However, pretreatment with TPN significantly inhibited the increase in heart rate induced by isoproterenol (Figure 20 and Figure 21). These results show the ability of TPN to modulate cardiac actions, preventing the activation of chronotropism, inotropism, dromotropism, and positive lusitropism induced by isoproterenol.

[182] Subacute treatment with TPN in the absence of isoproterenol induced a reduction in blood pressure in normotensive rats, as well as producing an antihypertensive effect in rats of the spontaneously hypertensive strain compared with their respective saline controls (Figure 20 and Figure 21).

[183] ​​The present work also verified that the administration of isoproterenol was able to increase myocardial oxygen demand in infarcted rats when compared to their respective saline controls, inducing myocardial hypoxia (Figure 22). However, pretreatment with alpha-terpineol was able to inhibit the increase in myocardial oxygen demand in the presence of isoproterenol at the doses tested, blocking the hypoxia induced by isoproterenol (Figure 22). Petition 870190021822, dated 07 / 03 / 2019, page 55 / 118 47 / 71

[184] It is established that AMI induces endothelial dysfunction in rats (CHEN et al, 2011; KANDHAI-RAGUNATH et al 2016), altering the morphology in large vessels (KONG et al, 2013) and the functionality of small arteries, making mesenteric arteries hyporeactive and coronary arteries hyperreactive (STASSEN et al, 1997; COUTO et al, 2012)

[185] When analyzing the histological sections of thoracic aortas, it was observed that there was an apparent increase in the diameter of the aortic lumen after isoproterenol-induced infarction, however, it was not statistically different compared to the saline control groups (Figure 23 and Figure 24). Pre-treatment and subacute treatment with TPN produces an apparent increase in the diameter of the thoracic aortic lumen in the presence or absence of isoproterenol-induced infarction, but it was not statistically different between the groups in both experimental lineages (Figures 23 and Figure 24).

[186] When histological sections of thoracic aortas in the longitudinal axis were analyzed, it was observed that isoproterenol-induced infarction produced a reduction in the vascular intima layer (Figures 25 and 26). However, subacute treatment with TPN produced a significant increase in the vascular intima in the presence or absence of isoproterenol-induced infarction (Figures 25 and 26). These results lead us to believe that TPN is capable of inducing angiogenic responses, increasing the vascular inner layers.

[187] The tunica intima is often described as the vascular layer that corresponds to the vascular endothelium as a major component (GERRITY & CLIFF, 1972; KHAN et al; 2000; LIBBY et al, 2011) and for this reason the increase in this layer has been correlated with cardiovascular benefits (HEENEMAN et al, 2003). Thus, we believe that neovascularization and angiogenic mechanisms may correlate with the cardioprotective effects induced by TPN, increasing the availability of endothelium-derived relaxing factors such as nitric oxide, and thus promoting improvements in vascular functions, contributing to the cardioprotective action. Petition 870190021822, dated 07 / 03 / 2019, p. 56 / 118 48 / 71

[188] The changes produced by isoproterenol on the macroscopic morphology of arterial branches were investigated, and in the hearts of rats infarcted with isoproterenol, no significant changes were observed in the left descending coronary artery (Figure 27 and Figure 28). However, subacute treatment with TPN induced an apparent dilating effect on the left descending coronary artery in a dose-dependent manner, in the presence or absence of isoproterenol (Figure 27 and Figure 28). Therefore, it is assumed that TPN may influence changes in coronary vascular tone and may contribute to the reduction of myocardial ischemia-reperfusion lesions.

[189] When investigating the vascular reactivity of mesenteric arteries, it was found that isoproterenol-induced infarction did not significantly alter the potency and maximum effect of the vasocontractile stimulus in response to phenylephrine, an α1-adrenergic agonist, in isolated superior mesenteric arteries of spontaneously hypertensive rats compared to the respective saline control (Figure 29 and Table 11). Thus, infarction by the sympathomimetic method did not change the contractile vascular reactivity in hypertensive rat strains. However, when vascular reactivity induced by the vasorelaxant stimulus of sodium nitroprusside, a spontaneous nitric oxide donor, was verified in isolated and perfused mesenteric arteries of spontaneously hypertensive rats, it was found that there was a change in both the potency and the maximum relaxing effect induced by sodium nitroprusside (Figure 30 and Table 12).

[190] The results shown here reveal that isoproterenol-induced infarction was able to induce dysfunction in the vascular response to relaxing stimuli. It has already been described that isoproterenol can activate ε2-adrenergic receptors in resistance arteries, reducing vascular tone and producing a reduction in blood pressure (GARLAND et al, 2011; ENGELHARDT & AHLES, 2014). It has also been shown that sodium nitroprusside reduces total peripheral resistance due to its nitric oxide-donating action (LOVREN & TRIGGLE 2000; HOTTINGER et al, 2014). Petition 870190021822, dated 07 / 03 / 2019, p. 57 / 118 49 / 71

[191] Subacute treatment with TPN was able to significantly reduce the maximum effect (Emax) of phenylephrine in the presence of isoproterenol-induced infarction without altering the pharmacological potency (pD2) in a dose-independent manner; however, in the absence of infarction, TPN did not statistically reduce the vasocontractile response of phenylephrine compared to the saline control (Figure 29 and Table 11). Meanwhile, treatment with TPN was able to increase the vasorelaxant effect of sodium nitroprusside, both in maximum effect (Emax) and pharmacological potency (pD2), in a dose-independent manner in the presence of isoproterenol (Figure 30 and Table 12). But, in the absence of isoproterenol, subacute treatment with TPN was not significantly different in the pharmacological parameters evaluated compared to the saline control (Figure 29 and Figure 30, Table 11 and Table 12). TABLE 11 - Comparison of Emax pD2 values ​​of isolated rat superior mesenteric artery rings without functional endothelium, in response to contractions with phenylephrine (10'1°105M). pD2 Emax Experimental Groups (M) (g) Saline 6.22 ± 0.08 0.67 ±0.02 Infarcted (ISO) 6.25 ±0.09 0.68 ±0.04 TPN 25 mg / kg + ISO 6.04 ± 0.07 0.56 ± 0.02* TPN 50 mg / kg + ISO 6.35 ±0.09 0.63 ±0.01 TPN 75 mg / kg + ISO 6.24 ± 0.07 0.46 ± 0.03 TPN 50 mg / kg without ISO 6.20 ± 0.04 0.63 ±0.02 Values ​​are expressed as mean ± SEM. Statistical analysis: one-way ANOVA followed by Newman-Keuls post-hoc test, significant when *p> 0.05 (TPN vs infarcted) ****p>0.0001 (TPN vs infarcted) or (TPN vs saline). TABLE 12 - Comparison of Emax pD2 values ​​of isolated rat superior mesenteric artery rings without functional endothelium, in response to relaxation with NPS (3 x 10'11- 10'7M). Experimental pD2 Groups Emax (M) (g) Saline 7.84 ± 0.06 0.62 ± 0.02 Petition 870190021822, dated 07 / 03 / 2019, page 58 / 118 50 / 71 Infarcted (ISO) 8.22 ± 0.07** 0.74 ± 0.02* TPN 25 mg / kg + ISO _ _ ___ mg / kg + ISO 8.05 ± 0.05 0.55 ± 0.06*** TPN 50 mg / kg without ISO 8.08 ± 0.07 0.61 ± 0.02 Values ​​are expressed as mean ± SEM. Statistical analysis: one-way ANOVA followed by Newman-Keuls post-hoc test, significant when *p> 0.05 (Infarcted (ISO) vs saline / TPN vs infarcted (ISO) **p>0.01 (Infarcted (ISO) vs saline / TPN vs infarcted (ISO) ***p>0.001 (TPN vs infarcted)

[192] In this way, we verified that TPN was able to modulate vascular actions, improving the vasorelaxant responses of nitroprusside, preventing vascular dysfunction in hypertensive rats. The TPN results presented here corroborate the study by SABINO, 2013, which shows that TPN is able to produce changes in the tone of isolated mesenteric arteries from L-NAME hypertensive rats, involving endothelium-independent targets of action, such as voltage-dependent calcium channels (SABINO, 2013).

[193] Thus, we decided to evaluate the involvement of calcium signaling in cardioprotective responses by measuring intracellular calcium levels in isolated aortas and ventricles of rats. In addition, serum calcium levels were measured in the presence or absence of isoproterenol-induced infarction. It is widely described that acute myocardial infarction produces changes in calcium signaling in the heart, inducing intracellular calcium overload, triggering mitochondrial cell death and consequent apoptotic signaling (BLOOM & DAVIS, 1972; BRUM et al., 2006). Isoproterenol-induced infarction mimics this effect on myocardial calcium influx (REMIÃO, 2001).

[194] While substances that prevent this overload, either through stimulation of SERCA or by blocking voltage-dependent calcium channels, prevent acute myocardial infarction (SANDMANN et al, 2001; EL-ANI, 2007).

[195] Isoproterenol-induced acute myocardial infarction significantly increased total ventricular calcium levels compared with Petition 870190021822, dated 07 / 03 / 2019, page 59 / 118 51 / 71 ventricles of control rats were treated with saline (Figure 31B and Figure 31E). While subacute treatment with TPN significantly inhibited intracellular calcium overload in the ventricles (Figure 31B and Figure 31E). Subacute treatment with TPN in the absence of isoproterenol produced a significant reduction in intracellular calcium levels in the ventricles (Figure 31B and Figure 31E). These results indicate the involvement of calcium influx through voltage-dependent calcium channels in the cardioprotective effect induced by TPN.

[196] When analyzing intracellular calcium levels in aortas, it was observed that isoproterenol increased calcium concentrations only in aortas of hypertensive rats when compared with their respective saline control (Figure 31C and Figure 31F), indicating calcium overload. However, treatment with TPN reduced calcium levels (Figure 31C and Figure 31F). While in rat aortas, subacute treatment with TPN affected calcium concentrations only in Wistar rats.

[197] These results suggest the involvement of voltage-dependent calcium channels in the vascular modulation promoted by TPN. Although the vasorelaxant effects induced by TPN in the studies of RIBEIRO, 2012 are attributed to the presence of endothelium in arteries of hypertensive rats, involving the stimulation of the NOS-cGMP signaling pathway. In contrast, the studies of SABINO, 2013 demonstrate the involvement of calcium channels in the vasorelaxant responses of TPN in arteries of L-NAME-induced hypertensive rats. The results described here may contribute to the findings of both studies, revealing that TPN may involve the blockade of calcium channels in arteries of normotensive and spontaneously hypertensive rats.

[198] It is well reported that different types of calcium channels are involved in the maintenance of spontaneous hypertension, such as an increase in voltage-dependent calcium channels, an increase in intracellular calcium levels without an increase in calcium release from the sarcoplasmic reticulum (GRAY et al 1993; ARII et al, 1999; LIU et al, 2005) and a reduction in arterial relaxation due to activation of potassium channels. Petition 870190021822, dated 07 / 03 / 2019, pages 60 / 118 52 / 71 are sensitive to increased intracellular calcium (WESTON et al, 2010). However, a reduction in the sensitivity of the dihydropyridine activation site of voltage-dependent calcium channels has also been shown in spontaneously hypertensive rats compared to Wistar rats (EBATA et al, 1991; FUKUDA et al, 2014).

[199] Thus, the evidence of the involvement of calcium channels in the vasorelaxant effect of TPN pointed out by SABINO et al., 2013, although not found by the studies of RIBEIRO et al., 2012, is due to the nature of the type of animal chosen for the vasorelaxant studies. L-NAME hypertensive animals belong to the Wistar strain and become hypertensive due to a reduction in the availability of nitric oxide by supplementation with a nitric oxide synthase inhibitor, while spontaneously hypertensive rats have a modified genetic origin (FAZANS et al., 2001). These differences in the sensitivity of voltage-dependent calcium channels in the two experimental strains pointed out by EBATA, 1991, are also evidenced in the calcium concentrations in rat aortas in our studies and for this reason may diverge in the modulation induced by TPN between the experimental models, similar to the studies of RIBEIRO, 2012 and SABINO, 2013.

[200] Studies by REMIÃO, 2001 show that after 24 hours of isoproterenol-induced infarction there is a significant reduction in serum calcium levels and normalization of other electrolytes in Wistar rats (REMIÃO, 2001). Our studies corroborate the findings of REMIÃO, 2001. Through Figure 31A, it can be seen that serum calcium levels were significantly affected by isoproterenol-induced infarction compared to the respective saline control. However, the same was not observed in hypertensive and infarcted rats (Figure 31D). The divergence in the results obtained is due to differences in the nature of calcium channels and their functionality in spontaneously hypertensive rat strains when compared to Wistar rat strains (GRAY et al 1993; ARII et al, 1999; LIU et al, 2005 EBATA et al, 1991; FUKUDA et al, 2014). Petition 870190021822, dated 07 / 03 / 2019, pages 61 / 118 53 / 71

[201] Pretreatment with TPN was able to restore serum calcium levels, but did not reach baseline levels compared to the saline control group (Figure 31A). In the absence of isoproterenol, pretreatment with TPN reduced serum calcium levels compared to the saline control group in Wistar rats (Figure 31A). In hypertensive rats, TPN increased serum calcium levels only at the lowest dose tested in the presence of isoproterenol when compared to control groups, and nifedipine did not affect serum calcium concentration.

[202] Given the cardiovascular changes induced by TPN in the presence and absence of isoproterenol, it became necessary to investigate the cardiovascular effects and their mechanisms induced by intravenous administration of TPN in spontaneously hypertensive rats.

[203] Intravenous administration of TPN induced dose-dependent hypotension and bradycardia in spontaneously hypertensive, non-anesthetized, and non-infarcted rats (Figure 32). TPN-induced bradycardia increased over time as a function of dose (Figure 32). The use of sodium thiopental (45 mg / kg i.p.) was able to attenuate the bradycardic effect induced by TPN (Figure 33). Sodium thiopental, a commonly used anesthetic, is able to depress autonomic reflexes of the nervous system, thus reducing the activation of the cardiovascular system (PEISS & MANNING, 1964). In this context, TPN-induced bradycardia appears to exert some influence on the cardiac autonomic system.

[204] To assess whether TPN reduces cardiac tone indirectly, hexamethonium, a ganglionic blocker antagonist of nicotinic receptors, was used, which reduces the actions of the autonomic nervous system, decreasing the homeostatic control of the cardiovascular system through the vagus nerve (BELESLIN 1972; DING 2017). The presence of hexamethonium (20 mg / kg iv) altered hypotension to a lesser degree, but influenced TPN-induced bradycardia (Figure 34), thus, the bradycardic responses to TPN are influenced by indirect mechanisms to reduce cardiac tone, Petition 870190021822, dated 07 / 03 / 2019, pages 62 / 118 54 / 71 possibly through the ganglia of the autonomic nervous system via vagal innervation.

[205] C fibers are unmyelinated fibers that connect to the atrium from the nucleus of the solitary tract of the brainstem (CHENG et al., 1999; CHENG & POWLEY, 2000). Stimulation of these fibers generates bradycardia through cholinergic activation (NOSAKA et al., 1979; CLOZEL et al., 1985; JONES et al., 1995) increasing substance P concentrations in cardiac nerve endings, however this effect can be inhibited by the action of atropine (WOOLLEY et al., 1987; HAXHIU-POSKURICA, 1992), but the same does not occur with hexamethonium (WOOLLEY et al., 1987).

[206] Thus, we used atropine, a non-selective antagonist of muscarinic receptors, which exerts influence mainly on cardiac tone, increasing cardiac contractility via M2 receptors with inhibition of cAMP (PERERA et al, 2017). The presence of atropine (2 mg / kg iv) significantly attenuated TPN-induced bradycardia, without altering hypotension (Figure 34), thus the bradycardic responses induced by TPN are mediated by muscarinic receptors.

[207] Muscarinic receptors have been identified as playing a key role in cardioprotection in ischemia-reperfusion injuries, producing beneficial effects for the cardiovascular system due to their multiple functions in the body (SUN et al, 2011).The variety of muscarinic receptor subtypes can suppress micro-messenger RNA signaling, preventing the chain reaction of cardiac injury signals initiated and emitted via micro-RNA, thus exerting a cardioprotective effect (PAN et al, 2012). They can also preserve connexin phosphorylation and reduce the expression of cyclooxygenases as inflammatory mediators in myocardial infarction, producing a delay in cardiac injury lesions (ZHAO et al, 2010). Furthermore, they can have direct effects on cardiovascular function, producing coronary vasodilation and preventing ischemic injury via M3 receptors in coronary arteries due to increased nitric oxide availability (RADICO et al, 2014). Petition 870190021822, dated 07 / 03 / 2019, pages 63 / 118 55 / 71 cardiac effects, preventing uncontrolled cardiac output, cardiac oxygen supply, and ventricular pressures (HARDOUIN et al 2002). For these reasons, cholinergic pathways have been identified as new therapeutic targets for cardiac diseases such as acute myocardial infarction (ROY et al, 2014).

[208] In this context, we evaluated the interaction capacity of TPN with various molecular targets of cardiovascular interest through an in silico approach, obtaining a cLog P= 2.3 and a hydroxyl group in the alpha position linked to the aromatic ring as the most reactive group in the molecule (Figure 35). We verified that TPN has a higher electron affinity energy for the type 2 muscarinic receptor and its affinity energy behaved similarly to muscarinic receptor agonists in the agonist / antagonist activity comparison assay (Figure 37).

[209] TPN also demonstrated electron affinity energy comparable to nifedipine at the dihydropyridine action site in L-type calcium channels (Figure 36), and demonstrated electron affinity for albumin as a possible plasma carrier. Thus, the respective work demonstrates the cardioprotective action induced by TPN and its possible mechanisms in a summarized manner in Figure 19 (Figure 38). ADVANTAGES OF THE PATENT

[210] The consumption of cardioprotective drugs (alpha-terpineol is intended to become a member of this drug class through this application) represents the possibility of preventing the onset of cardiovascular diseases in individuals who have cardiovascular risk factors such as: hereditary factors or lifestyle with a high risk of heart disease. It also has the advantage of reducing public health budget costs related to cardiovascular diseases, reducing mortality and morbidity in cardiac patients, reducing the use of multiple drugs in cardiac individuals, improving the quality of life of these patients, and finally, valuing the native flora of Brazil. REFERENCES Petition 870190021822, dated 07 / 03 / 2019, pages 64 / 118 56 / 71

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Claims

CLAIMS 1. Use of α-terpineol characterized as being for the manufacture of a medicament intended for the treatment of acute myocardial infarction.

2. Use of α-terpineol, according to claim 1, characterized by being able to reduce the area of ​​myocardial necrosis associated with acute myocardial infarction.

3. Use of α-terpineol, according to claim 1, characterized by increasing the survival rate in an acute myocardial infarction model. Petition 870260056323, dated 10 / 06 / 2026, p. 9 / 9

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