Evidence of the Cardioprotective Effects of Aloysia polystachya in Isoproterenol-Induced Myocardial Infarction in Rats

Abstract

Aloysia polystachya is a plant species that is widely used in Brazilian folk medicine for the treatment of different disorders that affect the cardiovascular system. The aim of the study was to investigate the cardioprotective effects of an ethanol-soluble fraction of A. polystachya (ESAP) on isoproterenol-induced myocardial infarction in rats. Different groups of rats (n = 8) were orally treated with ESAP (30, 100, and 300 mg/kg), carvedilol (10 mg/kg), or vehicle (filtered water; 1 mL/100 g) for 7 days. Naive rats received no treatment. On the morning of day 6, acute myocardial infarction was induced by the acute oral administration of isoproterenol (100 mg/kg). On the morning of day 8, all rats underwent electrocardiography and transthoracic echocardiography. Blood samples were then collected, and serum levels of creatine kinase-MB fraction (CK-MB) and cardiac troponin T (cTNT) were quantified. ESAP significantly reduced electrocardiographic changes, improved the ventricular ejection fraction, and reduced serum levels of CK-MB and cTNT in infarcted rats. The cardioprotective effects of ESAP could be exploited as an effective tool against isoproterenol-induced myocardial infarction in rats.

INTRODUCTION

Aloysia polystachya (Griseb) Moldenke (Verbenaceae) is a plant species that is widely distributed in South America, especially in Paraguay and Brazil.¹ Popularly known as “Burrito,” this species is commonly used for its aroma and to give a minty flavor to traditional Brazilian beverages (e.g., “Tereré”). In folk medicine, healers in the region of Grande Dourados, Mato Grosso do Sul state, Brazil, recommend this species for the treatment of various disorders that affect the cardiovascular system.² A. polystachya is also used in traditional medicine to treat hypertension, anxiety, gastrointestinal, and respiratory disorders, as sedative and as digestive.^2
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A recent phytochemical study of an ethanol-soluble fraction of A. polystachya (ESAP) that was obtained from the leaves of A. polystachya that were collected in this region detected the abundant presence of organic acids, nucleosides, methoxylated flavones, and glycosylated compounds, including phenolic acids, phenylpropanoids, iridoids, and monoterpenes.⁵ Marchetti et al⁶ and Carmona et al⁷ also detected the presence of forsythoside A, plantainoside C, purpureaside D, martynoside, acteoside, carvone, and limonene in the leaves of this species.

Several biological activities have been ascribed to this species. Anxiolytic- and antidepressant-like,^4,7,8 anticancer,⁹ anesthetic,¹⁰ and repellent¹¹ effects have been reported with different preparations of A. polystachya. Marques et al⁵ found that prolonged oral ESAP administration in spontaneously hypertensive rats reversed renal, hemodynamic, electrocardiographic, and cardiac morphological changes that were induced by hypertension.

Despite evidence of the popular use and beneficial effects of A. polystachya for the treatment of heart disease, its effects on the prevention and evolution of myocardial infarction (MI) remain unknown. Thus, the present study investigated the cardioprotective effects of ESAP in a rat model of isoproterenol-induced MI.

MATERIALS AND METHODS

Drugs and salts

Ketamine and xylazine hydrochloride were purchased from Syntec (São Paulo, SP, Brazil). Heparin was purchased from Hipolabor (Belo Horizonte, MG, Brazil). Carvedilol (CVDL) and isoproterenol (ISO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were obtained in analytical grade.

Plant material and extract preparation

A. polystachya leaves were collected in the Dourados region, Mato Grosso do Sul state, Brazil (21°13’15”S, 54°48’21”N). A voucher specimen was authenticated by Dr. Zefa Valivina Pereira and deposited in the Federal University of Grande Dourados (UFGD) herbarium (no. 5536). The plant name was confirmed in “The Plant List” database (http://www.theplantlist.org/; accessed April 13, 2021). The infusion was obtained by Marques et al.⁵ In this work, the infusion was treated with ethanol (95%) at a proportion of 1:3 (v/v), resulting in a precipitate and ethanol-soluble fraction (ESAP; 13% yield). The ESAP was concentrated, freeze-dried, and stored at −18°C. The detailed phytochemical composition of the ESAP was recently reported.⁵

Cardioprotective studies

Animals

We used 48 male Wistar rats 20 weeks old. All animals were obtained from the vivarium at UFGD. All rats were kept in an animal room with controlled temperature (22°C ± 2°C) and humidity (54% ± 2%) under a 12-h light/12-h dark cycle. Food and water were provided ad libitum. All animal procedures were approved by the Ethics Committee on Animal Experimentation, UFGD (protocol No. 21/2019).

Induction of acute MI, treatments, and clinical evaluations

The rats were randomly divided into six different groups (n = 8/group). The naive and negative control (NC) groups were treated only with vehicle (1 mL/100 g body weight). The positive control (PC) group received CVDL orally (10 mg/kg). The ESAP groups received 30, 100, and 300 mg/kg ESAP by gavage. All treatments were performed once daily for 7 days.

On the morning of day 6, MI was induced in the ESAP, NC, and PC groups by the acute oral administration of ISO (100 mg/kg). On the morning of day 8, all rats were intramuscularly anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) and underwent electrocardiography (ECG) using a 12-lead ECG recorder (WinCardio, Micromed, Brasília, Brazil) according to Guarnier et al.¹² Transthoracic echocardiography was then performed using an Eco1 Hipermedic echocardiograph (Hipermedic, Porto Alegre, RS, Brazil). Immediately after echocardiography, blood samples were collected through the jugular vein. Serum was obtained, and levels of creatine kinase-MB fraction (CK-MB) and cardiac troponin T (cTNT) were quantified using an automated biochemical analyzer (Cobas Integra 400 plus, Roche).

Statistical analyses

The results are expressed as the mean ± standard error of the mean (SEM) of n = 8 rats/group. The statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Values of P < .05 were considered statistically significant.

RESULTS AND DISCUSSION

In the present study, acute treatment with ISO (100 mg/kg) significantly increased serum levels of CK-MB and cTNT in normotensive rats after 24 h of administration, indicating direct myocardium damage (Table 1). We detected a significant increase in ECG QT and QRS segments (Table 2) and decreases in the systole diameter and ventricular ejection fraction (Table 1). Treatment with CVDL and ESAP (at all doses tested) prevented all changes, maintaining values of the various biochemical, electrocardiographic, and echocardiographic parameters similar to those in naive animals.

Table 1.

Effects of the Ethanol-Soluble Fraction from Aloysia polystachya Leaves and Carvedilol on Biochemical and Echocardiographic Parameters

Parameter	Naive	NC	ESAP (30 mg/kg)	ESAP (100 mg/kg)	ESAP (300 mg/kg)	CVDL (10 mg/kg)
Biochemical
CK-MB (U/L)	66.33 ± 10.09	201.23 ± 22.11^a	140.2 ± 15.16^ab	133.22 ± 19.44^ab	141.32 ± 22.23^ab	87.2 ± 14.22^bc
cTNT (μg/L)	0.12 ± 0.02	0.82 ± 0.03^a	0.52 ± 0.02^ab	0.46 ± 0.03^ab	0.50 ± 0.02^ab	0.13 ± 0.03^bc
Echocardiographic
HR (bpm)	232.10 ± 21.60	153.50 ± 24.93^a	196.63 ± 37.22	198.88 ± 26.18	191.25 ± 29.36	224.25 ± 22.95^b
SD (mm)	0.13 ± 0.03	0.17 ± 0.02^a	0.13 ± 0.02^b	0.13 ± 0.02^b	0.12 ± 0.02^b	0.13 ± 0.02b
DD (mm)	0.41 ± 0.07	0.42 ± 0.06	0.42 ± 0.08	0.39 ± 0.03	0.41 ± 0.03	0.44 ± 0.07
FDV (mL)	0.18 ± 0.07	0.19 ± 0.08	0.19 ± 0.09	0.16 ± 0.03	0.17 ± 0.04	0.18 ± 0.05
FSV (mL)	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01
SV (mL)	0.18 ± 0.02	0.17 ± 0.02	0.18 ± 0.03	0.14 ± 0.03	0.16 ± 0.04	0.19 ± 0.04
EF (%)	96.26 ± 1.34	83.01 ± 2.60^a	96.71 ± 2.71^b	95.30 ± 2.68^b	96.19 ± 2.35^b	97.39 ± 2.60^b

Statistical analyses were performed using one-way ANOVA followed by Tukey's test. Values are expressed as mean ± SEM of eight animals per group.

P < .05 compared to the naive rats.

Denotes P < .05 compared to the NC.

Denotes P < .05 compared to the ESAP (30, 100, or 300 mg/kg).

ANOVA, analysis of variance; CK-MB, creatine kinase-MB fraction; cTNT, cardiac troponin T; CVDL, carvedilol; DD, diastole diameter; ESAP, ethanol-soluble fraction of A. polystachya; EF, ejection fraction; FDV, final diastolic volume; FSV, final systolic volume; HR, heart rate; NC, negative control; SD, systole diameter; SEM, standard error of the mean; SV, stroke volume.

Table 2.

Effects of the Ethanol-Soluble fraction from Aloysia polystachya Leaves and Carvedilol on Electrocardiographic Parameters

	Naive	NC	ESAP (30 mg/kg)	ESAP (100 mg/kg)	ESAP (300 mg/kg)	CVDL (10 mg/kg)
Segments (ms)
PR	35.00 ± 4.96	36.43 ± 17.08	42.88 ± 12.17	50.88 ± 12.11	45.13 ± 10.82	40.88 ± 6.83
QRS	46.25 ± 3.41	145.71 ± 9.57^a	59.25 ± 15.76^b	65.75 ± 21.72^b	79.88 ± 34.79^b	49.75 ± 12.67^b
QT	104.5 ± 10.77	150.14 ± 8.95^a	128.13 ± 21.97	121.00 ± 20.00	131.00 ± 29.97	106.00 ± 10.72^b
QTC	196.25 ± 22.93	278.29 ± 25.72^a	229.75 ± 39.95	235.77 ± 26.42	233.25 ± 36.24	205.13 ± 18.53^b
Waves (mV)
P	0.06 ± 0.03	0.05 ± 0.03	0.04 ± 0.03	0.07 ± 0.03	0.06 ± 0.02	0.05 ± 0.02
Q	-0.04 ± 0.01	-0.05 ± 0.02	-0.03 ± 0.01	-0.03 ± 0.01	-0.04 ± 0.02	-0.03 ± 0.01
R	0.21 ± 0.08	0.19 ± 0.05	0.22 ± 0.04	0.24 ± 0.06	0.25 ± 0.05	0.26 ± 0.06
S	-0.02 ± 0.01	-0.02 ± 0.01	-0.02 ± 0.01	-0.01 ± 0.01	-0.03 ± 0.02	-0.03 ± 0.02

Statistical analyses were performed using one-way ANOVA followed by Tukey's test. Values are expressed as mean ± SEM (standard error of the mean) of 8 animals per group.

P < .05 compared to the naive rats.

Denotes P < .05 compared to the NC.

Cardiovascular diseases currently represent ∼30% of all deaths worldwide. Among the main causes of death and other important cardiovascular sequelae, MI is notable. Myocardial infarction can lead to necrosis and the persistent scarring of cardiomyocytes, causing additional morbid pathological sequelae.¹³ The diagnosis of MI is based on measurements of serum markers, including CK-MB and cTNT. The detection of intracoronary thrombi and electrocardiographic changes and abnormalities of cardiac motility and the ejection fraction may be important indications of MI.^13
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Cardiac troponin is detectable in three forms: cTNT, troponin I (cTNI), and troponin C (cTNC). cTNT is a component of the heterotrimeric complex to which tropomyosin is associated and directly responsible for the myocardial contractile process. This protein is exclusive to cardiomyocytes. Elevations of cTNT suggest cardiac muscle injury. cTNT levels generally begin to increase within 2–4 h after the onset of the myocardial necrosis process, reaching maximum values after 24–48 h. Troponin has the same diagnostic sensitivity as the CK-MB enzyme 12–48 h after the onset of infarction symptoms, but troponin is essential for patients with diseases that reduce the specificity of the CK-MB.¹⁴

Another useful tool for IM diagnosis is ECG. Although some infarcted patients have a normal ECG, some ECG changes are significantly related to MI, including new significant ST-segment–T wave or new left bundle branch block. Furthermore, the development of pathological Q waves may also be present.¹³ Although echocardiography is important for detecting several cardiac diseases, it is not always suitable for detecting acute MI. Echocardiography is often useful for monitoring complications that occur after MI, including ventricular wall rupture, mitral valve regurgitation, and other alterations in ventricular function.¹⁵

Isoproterenol is known to increase oxidative stress¹⁶ and lipid peroxidation in cardiac muscle cells.¹⁷ It also mediates calcium overload,¹⁸ renin-angiotensin release,¹⁹ and inflammation²⁰ in the myocardium, resulting in infarct-like necrosis. Beta-blocking drugs, such as CVDL, are routinely used to prevent MI. Its cardioprotective effect is related to the direct reduction of cardiac overload. In addition, beta blockers also reduce pre- and post-load by reducing renin release by renal juxtaglomerular cells, resulting in lower activation of the renin-angiotensin-aldosterone system.²¹

Although we did not investigate the molecular mechanisms of the cardioprotective effects of ESAP, some previous studies suggest some possibilities. In a recent study, Marques et al⁵ reported the presence of organic acids, methoxylated flavones, and glycosylated compounds, including phenolic acids, phenylpropanoids, iridoids, and monoterpenes, in the ESAP. These authors showed that prolonged treatment with the ESAP in spontaneously hypertensive rats modulated the tissue antioxidant system in heart, kidney, and aorta samples, resulting in significant antioxidant effects.

Several studies have shown that different natural products, including flavonoids and other phenolic compounds, effectively prevent ISO-induced MI through antioxidant activities and the prevention of lipid peroxidation.²² Thus, luteolin,²³ safranal,²⁴ curcumin, and quercetin²⁵ deserve to be highlighted. The present findings suggest that the ESAP may exert a cardioprotective response through its antioxidant effects and protection against lipid peroxidation in the myocardium. Future studies should further test possible nutraceutical products based on the ESAP for the prevention of ISO-induced toxicity.

CONCLUSION

The present study suggests that the ESAP has cardioprotective effects against ISO-induced MI in rats. The cardioprotective effects of the ESAP may be useful for preventing the occurrence of MI.

Footnotes

AUTHORs' CONTRIBUTIONS

The authors confirm that the article has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship. Further confirms that the order of authors listed in the article has been approved by all.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

This research was funded by Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Brazil; No. 59/300.046/2015 and 71/700.135/2018) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil; No. 407685/2018–9).

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