Cardioprotective effects induced by hydroalcoholic extract of leaves of Alpinia zerumbet on myocardial infarction in rats

Journal of Ethnopharmacology 242 (2019) 112037

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Cardioprotective effects induced by hydroalcoholic extract of leaves of Alpinia zerumbet on myocardial infarction in rats

T

Emanuel Tenório Paulinoa, Amanda Karine Barros Ferreiraa, Jessyka Carolina Galvão da Silvaa, Cintia Danielli Ferreira Costaa, Salete Smaniottoc, João Xavier de Araújo-Júniora,b, Edeíldo Ferreira Silva Júniorb, Janaína Herbele Bortoluzzid, Êurica Adélia Nogueira Ribeiroa,∗ a

Federal University of Alagoas, Institute of Pharmaceutical Sciences, Maceió, AL, Brazil Federal University of Alagoas, Chemical and Biotechnology Institute, Maceió, AL, Brazil c Federal University of Alagoas, Institute of Biology and Health Science, Maceió, AL, Brazil d Federal University of Alagoas, Chemical and Biotecnology Institute, Maceió, AL, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Alpinia zerumbet Isoproterenol Cardioprotection Myocardial infarction Rats

Ethnopharmacology relevance: The leaves of Alpinia zerumbet is used in folk medicine in Brazil to treat hypertension. However, the cardioprotective effect of this plant has not been studied yet. Aim of this study: To evaluate the cardioprotective effects of the hydroalcoholic extract of the leaves of Alpinia zerumbet (AZE) against isoproterenol (ISO)-induced myocardial infarction in rats. Material and methods: Rats were pretreated orally with AZE (300 mg/kg) for 30 days prior to ISO-induced myocardial infarction. The rats were sacrificed and hearts were collected and homogenized for biochemical analysis. At the end of the experiment, cardiac marker enzyme levels, histological and morphometric parameters, and hemodynamic measurements were assessed. Phytochemical compounds were verified by gas chromatography-mass spectrometry (GC-MS). Results: Rats administered with ISO showed a significant increase in cardiac marker enzymes, i.e., in creatine kinase-NAC (CK-NAC) and CK-MB. Triphenyltetrazolium chloride (TTC) staining exhibited an increase in infarct areas. In the animals treated with ISO induced a significant increase in heart rate. Pretreatment with AZE significantly inhibited these effects of ISO. Moreover, biochemical findings were supported by histopathological observations. The GC-MS analyses of AZE identified volatile oils, kavalactones, and phytosterols. Conclusions: Haemodynamic, biochemical alteration and histopathological results suggest a cardioprotective protective effect of oral administration of AZE in isoproterenol induced cardiotoxicity.

1. Introduction The myocardial infarction (MI) is a relevant public health problem all over the world. Studies show that in the next years, MI will be one of the leading causes of death and comorbidities for both men and women worldwide. According to the World Health Organization, on the estimated 32 million heart attacks and strokes that occur each year globally, roughly 12.5 million are fatal (Mendis et al., 2010). The development of MI is closely related to oxygen imbalance and ischemic phenomena (Thygesen et al., 2012). The ischemia occurs when blood supply is inadequate to the myocardium, producing damage, or the necrosis of myocardial muscle (Whellan, 2005). The medications used to treat myocardial infarction are β-blockers, anticoagulants,

antithrombotics, angiotensin-converting enzyme inhibitors, acetylsalicylic acid, nitrates, angiotensin II receptor blocker, calcium channel blockers, and magnesium supplementation. Some of the drugs used for the treatment of this disease have side effects, cost, nonavailability, and resistance development (Raja et al., 2016). Cardioprotection is defined as the preservation of the heart. It is essential to understand that this definition has significant theoretical implications because all adaptive and compensatory mechanisms that directly or indirectly contribute to myocardial maintenance must be classified as cardioprotective (Kuibler and Haass, 1996). The rat model of isoproterenol (ISO)-induced myocardial infarction is similar to an infarction in humans (Brooks and Conrad, 2009). This model offers a reliable non-invasive technique for studying the effects of potentials

∗ Corresponding author. Instituto de Ciências Farmacêuticas, Universidade Federal de Alagoas (UFAL), Campus A. C. Simões. Av. Lourival Melo Mota, s/n, Cidade Universitária, 57072-900, Maceió, AL, Brazil. E-mail addresses: [email protected], [email protected] (Ê.A. Nogueira Ribeiro).

https://doi.org/10.1016/j.jep.2019.112037 Received 1 December 2018; Received in revised form 27 May 2019; Accepted 21 June 2019 Available online 24 June 2019 0378-8741/ © 2019 Published by Elsevier B.V.

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E.T. Paulino, et al.

follows: initially 40 °C for 3 min, after heating from 40 °C to 325 °C at a rate of 10 °C/min, and finally 325 °C for 5 min. The complete analysis run time was approximately 36.5 min. The carrier gas used was helium (Linde Gases, Jaboatão dos Guararapes, Brazil, 99.9% purity) at 1 mL/ min and the sample volume injected was 1.0 μL of the 25 mg/mL of the final extract in n-hexane. The spectrometer was operated in electron impact mode (EI) with 70 eV detection volts and scan range 40–1000 m/z. The control of the GC-MS system and the data processing was achieved using integrated GC solution software, version 2.31.00. The compounds were identified by comparing their mass spectral fragmentation with standard reference spectra from the NIST 05 database (NIST Mass Spectral Database, PC Version 5.0, 2008) or by GC-MS co-chromatography with previously isolated authentic standards. Moreover, data obtained from the literature were used for the identification of the compounds. The areas of the peaks were normalized by a minimum area of 50.000 TIC (total ion chromatogram). When the peaks (present in the chromatogram) related to the mass spectra were not found in the NIST library, they were identified through molecular reconstruction, based on the particular fragments, identified in the mass spectra (SUPPLEMENTARY MATERIAL S2).

cardioprotective agents (Upaganlawar et al., 2011) because ISO is a synthetic catecholamine and β-adrenergic agonist (Remião et al., 2001). ISO in high doses cause intense cardiac activity, imbalance of oxygen supply, increased production of radical species and myocardial infarction biomarkers, myocarditis, myocardial necrosis focus, and cardiac remodeling in response to adaptive mechanisms in the hearts (Bhagat et al., 1978). A significant number of individuals with cardiovascular diseases make use of alternative therapies, such as medicinal plants, herbal medicines, and supplements (Ara Tachjian et al., 2010). The literature reports that plant extracts, phytochemicals compounds, and phytotherapeutic agents exhibited cardioprotective activity (Ohja et al., 2012; Priscilla and Prince, 2009; Stanely et al., 2008). Evaluation of the cardiovascular effects of the extract and the description of the pathways of action remain a logical research strategy in the search for new drugs to treat myocardial infarction (Wong et al., 2017). The Alpinia zerumbet (Pers.) B.L.Burtt. & R.M. Smith = Alpinia speciosa is a plant belonging to the family Zingiberaceae. It is native to East Indies and widely distributed in South America, Oceania, and Asia. It is commonly named as “colônia" in Brazil. In the northeast region of Brazil, Infusates and teas made from leaves and flowers of A. zerumbet are commonly used in folk medicine because of its antihypertensive and diuretic properties (Cartaxo et al., 2010). Several cardiovascular actions of extracts from A. Zerumbet have been reported, such as hypotensive, vasorelaxant and antihypertensive effects (De Moura et al., 2005; Lahlou et al., 2003), antioxidant properties (Wong et al., 2008), antiplatelet activities (Teng et al., 1990), and cardio-depressive effect (Santos et al., 2011). All these cardiovascular actions may contribute to a possible cardioprotective effect induced by the extract. In the present study, we tested the cardioprotective effect of the hydroalcoholic extract of leaves of Alpinia zerumbet against ISOinduced MI using rat models.

2.4. Animals Male Wistar rats (250–350 g; 10 weeks old), obtained from central bioterium of the Federal University of Alagoas, were used in experiments. The rats were housed at 25 ± 5 °C in an animal house under a 12 h light-dark cycle, with access to food and water. Experimental protocols and procedures were approved by the ethics committee (01082/2009-01 SUPPLEMENTARY MATERIAL S3). 2.5. Induction of myocardial infarction

2. Material and methods

Myocardial infarction was induced in experimental rats by subcutaneous injection of 85 mg/kg of ISO daily for two consecutive days.

2.1. Drugs and chemicals

2.6. Experimental protocol

All chemical reagents were purchased from Sigma Aldrich Co., USA. Biochemical kits were acquired from Labtest® (Lagoa Santa, MG, Brazil).

The rats were randomly distributed to three groups of six rats each: Saline group (SC group) received saline solution 0.9% mg/kg/d orally; Isoproterenol group (ISO group) received saline solution 0.9% mg/kg/d orally and isoproterenol 85 mg/kg s.c. on days 25 and 26; and AZE + ISO group received extract 300 mg/kg/d orally and isoproterenol 85 mg/kg s.c. on days 25 and 26. All drugs and saline were given once a day at a fixed time, by oral gavage, for 26 days. At the end of this period, i.e., 24 h after the last injection of ISO, the rats were anesthetized (pentobarbital sodium, 45 mg/kg, i.p.) and killed by aortic exsanguination. The blood was collected and centrifuged (5000 rpm, 10 min). The serum separated was used for the determination of diagnostic marker enzymes. The hearts were excised via a single thoracotomy incision for macroscopic, morphometric, and histopathology assays. After this, we performed three sets of experiments. The first one focused on biochemical and blood pressure analyzes. At the end of the study, the hearts were dissected for the morphometric assays. In the second set of experiments, we performed the determination and quantification of myocardial infarct size by the TTC assay. Finally, in the last set, we completed the histopathological appraisal. A total of 45 rats were used in these experiments.

2.2. Plant material and preparation of the extract The leaves of Alpinia zerumbet were collected in Maceió (Alagoas, Brazil) and were identified in the herbarium of the Environmental Institute of Alagoas State (IMA), Brazil (SUPPLEMENTARY MATERIAL S1). A voucher specimen was deposited in this herbarium and given the number 25070. The leaves were dried under shade (28 °C for six days) and after they were grounded into a coarse powder. The obtained powder (4 kg) was subjected to extraction by maceration method at room temperature with a hydroalcoholic mixture (10% water and 90% ethanol). Then, it was filtered and concentrated at 40 °C using vacuum evaporator, affording 3.5 kg of the hydroalcoholic extract of the leaves of Alpinia zerumbet (AZE) with a yield of 85.65%. 2.3. GC-MS analysis The gas chromatography-mass spectrometry (GC-MS) analyses were recorded on a Shimadzu GC-2010 instrument (Kyoto, Japan) coupled to a mass spectrometry detector QP-2010, using a split/splitless capillary injection system at 325 °C, with the split ratio of 20:1. A low-polarity phase, diphenyl dimethylpolysiloxane, Rtx-5MS (Restek Corporation, U.S.) capillary column (30 m × 0.25 mm × 0.25 μm) was used for all GC separations. The temperature program used for the determination of the hydroalcoholic extract from leaves of Alpinia zerumbet was as

2.7. Biochemical analysis Plasma levels of total creatine kinase (CK-NAC and CK-MB) were ascertained by using commercially available kits (Labtest®, Lagoa Santa, MG, Brazil). A stable reagent mixture was prepared by adding 80 μL of Reagent 1 (imidazole buffer, N-acetyl cysteine, ADP, AMP, pentaphosphate diadenosine, magnesium acetate, NADP, hexokinase, and 2

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sodium azide) and 20 μL of Reagent 2 (Glucose, phosphate creatine, sodium azide, anti-CK-M to inhibit CK-MM on CK-MB kit or CK-NAC kit without antibody to subtypes of CK). Then, 50 μL of serum was added, so that the absorbance was measured at 340 nm via spectrophotometer (UV-VIS mini-1240 Shimadzu). The enzyme activity was calculated by the equation below and expressed as IU/L.

CK Activity =

Absob 2 − Absorb 1(standard) X [Caliper ] Absorb 2 − Absorb 1 (Samples ) Fig. 1. Levels of biochemical activities in the groups: SC (0.5 mL, P.O.); ISO (85 mg/kg 2x s.c.) and AZE (300 mg/kg 26 d P.O. + ISO 85 mg/kg 2x s.c.). The results were express means ± SEM (n = 5 rats/group). Statistical analysis ANOVA one-way followed Newman-keuls post-test considered significant when ∗∗ P < 0.01 e ***P < 0.001.

2.8. Determination and quantification of myocardial infarct size The hearts were sliced approximately into a thickness of 3 mm. The slices were incubated for 20 min in a 1% solution of buffered triphenyltetrazolium chloride (TTC), preheated to 37 °C, and immersed in 10% buffered formalin phosphate. They were photographed with magnification to identify the necrotic zones (yellow coloration) and the non-infarcted region (uncolored). The areas of necrosis in each slice were determined by Image J Pro Plus 7.0 software.

statistical analyses were performed using one-way ANOVA followed by Newman-Keuls post-test to groups or student's t-test to unpaired samples. The graphs were plotted using Graph Pad Prism Version 5.0 software (Graph Pad software San Diego, USA). The results with p < 0.05 were considered significant.

2.9. Morphometric analyzes After the chronic treatment, the rats were anesthetized with sodium thiopental (45 mg/kg i.p.) and killed by stunning and bleeding. The hearts were removed and cleaned from connective tissue and fat. The arterial and venous branches were removed, then the cardiac chambers were rinsed in 0.9% saline solution. In the following, the hearts were weighed on a precision scale (Shimadzu AYY220), and the heart weight to body mass ratio was calculated. Afterwards, they were cut along the longitudinal axis. The ventricular wall thickness was measured using a calibrated digital vernier caliper. The following morphometric parameters were determined: the wet heart weight (AWH mg), the ratio of heart weight to body weight (Wh/Wb mg/g), the relative increase in heart weight (RWH%), and the difference in left ventricular growth (ΔTLV mm2).

3. Results

2.10. Histopathology appraisal

Fig. 2 (A-C) shows the staining of heart tissue slices by 2,3,5-TTC. The TTC stained heart slices of the group control (SC group) revealed utterly viable tissue (with red color), determining an intact myocardial tissue (Fig. 2A). In the isoproterenol induced myocardial infarcted rats (Isoproterenol group), the infarcted regions of heart tissue are plainly visible as yellow spots on the myocardium (Fig. 2B). Pretreatment with extract (AZE group) induced a significant reduction in infarct size in isoproterenol-induced myocardial infarcted rats, exposed by the absence of yellow pigmentation in the heart slices of infarcted rats (Fig. 2C). Fig. 3 describes the quantification of areas of necrosis in heart tissue slices. The SC group did not show any infarct region (0%). The ISO group showed a significant (P < 0.001) increase in necrotic zones (59.0 ± 8%) and pretreatment with extract (AZE group) significantly (P < 0.001) reduced infarct regions (5.7 ± 1.1%) in isoproterenolinduced myocardial infarcted rats.

3.1. Effect of AZE on cardiac marker enzymes Fig. 1 shows the effect of AZE on activities of CK-NAC and CK-MB in the serum of experimental groups of rats. ISO administration showed a significant increase in the activities of CK-NAC and CK-MB in the heart when compared to the control group (ISO vs. SC). However, pretreatment with AZE (300 mg/kg/day for 26 days) in ISO-treated rats significantly prevented the altered levels of cardiac marker enzymes (ISO vs. AZE + ISO) (Fig. 1). 3.2. Effect of AZE on myocardial infarct size measurements

The hearts were removed, fixed in Bouin's solution for 12 h, and dehydrated in alcohol (starting from 70% to absolute ethanol). They were embedded in paraffin and serial sectioned at a thickness of 4 μm, and then stained with hematoxylin, eosin, and Manson Trichrome. The serial histological sections were obtained and examined under a light microscope. More specifically, the following parameters were evaluated for each histological sections: Leukocyte migration, total leukocyte count, and collagen deposition. 2.11. Hemodynamic measurements All groups analyzed were anesthetized with sodium thiopental (45 mg/kg, i.p.) and the catheter was embedded into the abdominal aorta via the left femoral artery (for the recording of arterial blood pressure). For the administration of substances, another catheter was inserted into the inferior vena cava via the left femoral vein. Then, to measure blood pressure, the arterial catheter was connected to a pressure transducer (BLPR, AECAD, SP, Brazil). This transducer was connected to an amplifier-recorder (Model 04P, AECAD, SP, Brazil) and a personal computer equipped with an analog to digital converter board. Using AQCAD software (AVS Projects, SP, Brazil), data were sampled every 500 Hz. For each cardiac cycle, the computer calculated systolic blood pressure (SAP), diastolic blood pressure (DAP), mean arterial pressure (MAP), and pulse interval (referred to as heart rate - HR).

Fig. 2. Samples represented photographs in TTC 1% test. Transversal hearts sections on groups (A) SC (0.5 mL, P.O.), (B) ISO (85 mg/kg 2x s.c.), (C) AZE (300 mg/kg 26 d P.O. + ISO 85 mg/kg 2x s.c.).

2.12. Statistical analyses All data were expressed with means ± standard errors (SEM) and 3

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and collagen deposition (Fig. 5B–E; Fig. 6B–E). The pretreatment with extract (AZE group) was able to prevent all the alterations cited above, therefore, inhibit cardiac remodeling (Fig. 5A–D; Fig. 6A–D). Furthermore, leukocyte infiltration increased significantly in the ISO group compared to the SC group. The pretreatment with extract reduced significantly this infiltration (Fig. 5C–F, Table 1). Thus, we have found that AZE treatment was able to produce cardioprotective effects. 3.4. Effect of AZE on hemodynamic parameters Fig. 3. Necrotic size areas (%) in the groups: SC (0.5 mL, P.O.); ISO (85 mg/kg 2x s.c.) and AZE (300 mg/kg 26 d P.O. + ISO 85 mg/kg 2x s.c.). The results were express means ± SEM (n = 5 rats/group). Statistical analysis ANOVA one-way followed Newman-keuls post-test considered significant when ∗∗ P < 0.01 e ***P < 0.001.

In the control animals treated with saline (SC group), the recorded blood pressure values were as follows: MAP 124.8 ± 9.2 mmHg, SAP 142.0 ± 7.6 mmHg, DAP 107.1 ± 12.5 mmHg, and HR 224.9 ± 6.5 bpm. In ISO group occurred a significant decrease in MAP, SAP, and DAP, while HR was increased as compared to SC group. In AZE + ISO group, the HR (253.3 ± 8.2 bpm) was decreased significantly as compared to ISO group and was similar to SC group (Fig. 7). The result indicated that AZE pretreatment for 26 days was effective in preventing the ISO-induced tachycardia.

In Fig. 4A, we noted a significant increase in wet heart weight of infarcted rats. This increase was significantly attenuated after treatment with the extract. The heart weight to body weight ratio was significantly greater in ISO group (7.07 ± 0.55 mg/g) compared to SC group (4.89 ± 0.25 mg/g; P < 0.05). The pretreatment with extract significantly (P < 0.001; Group AZE) reduced the heart weight to body weight ratio compared to ISO group (Fig. 4B). Similarly, the relative increase in cardiac weight was also significantly reduced (Fig. 4C). Fig. 4D showed the difference of left ventricular wall thickness between ISO and AZE groups. The pretreatment with AZE (300 mg/kg/day for 26 days) significantly decreased ventricular wall thickness in infarcted rats. These results suggest that the extract was able to prevent the cardiac hypertrophy induced by isoproterenol.

3.5. GC-MS analysis In the present work, ten compounds were detected in the extract prepared from Alpinia zerumbet (Fig. 8 and Table 2). Among them, the most abundant were two kavalactones (5–6 dehydrokawain and 7,8dihydro-5,6-dehydrokawain), one phytosterol (Tocopherol and β-sitosterol), and volatile oils (Terpineol and isomeric mixture). 4. Discussion Cardioprotection is a term used to describe the preservation of cardiac structures. This preservation constitutes or involves all mechanisms that contribute to reduce or prevent myocardial damage. In this context, the cardioprotection includes primary and secondary prevention of coronary heart disease, cardiosurgical procedures, thrombolysis, and acute myocardial infarction, as well as the cardioprotective effects of the compounds on cardiac lesions after experimental drug intervention (Kuibler and Haass, 1996).

3.3. Effect of AZE on histopathological changes of rat myocardium Figs. 5 and 6 showed that the extent of histopathological changes in myocardial tissues in normal, ISO-treated, and AZE-treated experimental animals. The control animals treated with saline (SC group) showed a normal histoarchitecture of the myocardium (Fig. 5A–D; Fig. 6A–D). On the other hand, ISO administration in control group (ISO group) showed degeneration on myofibrils, myonecrosis focus, edema,

Fig. 4. Morphometrical analyses on the groups: SC (0.5 mL, P.O.); ISO (85 mg/kg 2x s.c.) e AZE (300 mg/kg 26 d P.O. + ISO 85 mg/kg 2x s.c.). (A) = Average Weight of Hearts(mg). (B) = Average Weight hearts/Average Weight body(mg/g). (C) = Relative increases of weight hearts (%). (D) = Δ Thickness Ventricular Left (mm). The results were express means ± SEM (n = 5 rats/group). Statistical analysis ANOVA one-way followed Newman-keuls post-test considered significant when ∗ P < 0.05 ∗∗P < 0.01 and ***P < 0.001.

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Fig. 5. Histopathological of myocardial tissue of control and ISO treated experimental animals. (A and D) control-SC, (B and E) isoproterenol-ISO (85 mg/kg, s.c), (C and F) AZE (ISO 85 mg/kg 2x s.c.+ AZE 300 mg/kg 26 x V.O) Section of myocardium from Group A (A and D) showing normal architecture. Section of the myocardium from Group B (B and E) rats reveals focal myonecrosis and leukocyte infiltration (myocarditis). Section of myocardium from Group C (C and F) rats reveals less intensity and low distribution of focal myonecrosis and leukocyte infiltrations. *LI = Leukocyt

Several studies have shown that the ISO has been extensively used to produce acute myocardial infarction in different doses (Mali and Bodhankar, 2010; Kumar et al., 2011; Kurian et al., 2005). This catecholamine, when administered to animals in high doses, causes severe oxidative stress in the myocardium resulting in infarct-like necrosis of the heart muscles which are similar to those found in acute myocardial infarction and sudden death in humans (Baroldi, 1974). ISO causes subendocardial myocardial ischemia, hypoxia, metabolic and biochemical changes, inflammation, degeneration, necrosis, and cardiac remodeling (Rona, 1985). Thus, ISO-induced sympathetic hyperactivity mimics the

Table 1 Number of infiltrated leukocyte into myocardium. The results were expressed as means ± S.E.M on groups (SC, n = 3), (ISO, n = 3) and (AZE, n = 3). Statistical analyses ANOVA one-way following Newman-Keuls test. Significant when ∗∗∗P < 0.001 (ISO vs SC) and (AZE vs ISO). Parameter

SC

ISO

AZE

Leukocyte numbers/0.038 mm2

413 ± 0.7

3.517 ± 4.5∗∗∗

1.376 ± 2.6∗∗∗

Fig. 6. Histopathological examination of the myocardium of control and ISO treated experimental animals. (A and D) control-SC, (B and E) isoproterenol-ISO (85 mg/kg, s.c), (C and F) AZE (ISO 85 mg/kg 2x s.c.+ AZE 300 mg/kg 26 x V.O) Section of myocardium from Group A (A and D) showing normal architecture. Section of the myocardium from Group B (B and E) rats reveals collagen deposition stained in blue and edema. Section of myocardium from Group C (C and F) rats reveals less intensity and low distribution of collagen between cardiac fibers. A, B, C: 40x magnification and H &E staining. D, E, F: 20x magnification and Masson's trichrome staining. C = collagen áreas. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 7. Hemodynamic repercussions in the groups: SC (0.5 mL, P.O.); ISO (85 mg/kg 2x s.c.) and AZE (300 mg/kg 26 d P.O. + ISO 85 mg/kg 2x s.c.). The results were express means ± SEM (n = 5 rats/group). Statistical analysis ANOVA one-way followed Newman-keuls post-test considered significant when ∗P < 0.05 ∗∗P < 0.01 e ***P < 0.001.

Fig. 8. Chromatogram obtained by gas chromatography/mass spectrometry-coupled to identified natural compounds of the hydroalcoholic extract from leaves Alpinia zerumbet (AZE). Natural compounds identified were represented by numbers.* = dilution solvent.★ = natural compounds unkown. 6

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Table 2 Natural compound identified on hydroalcoholic extract from leaves of Alpinia zerumbet by gas chromatography coupled mass spectroscopy. Nº

Substance

1

Molecular structure

Molecular mass (g/mol)

Molecular formula

Phytochemical class

Retention time

Peak area %

Terpinen-4-ol

154.24 l

C10H18O

Cyclic monoterpene

12.183

9.2%

2

α-terpineol

154.24

C10H18O

Cyclic monoterpene

12.379

0.9%

3

Octanoic acid

144.21

C8H16O2

Fatty acid

16.417

2.4%

4

7,8-dehydro-5,6-dehydrokawain

230.26

C14H14O3

Kavalactone

23.377

8.3%

5

Epicatechin

226.

C15H14O2

Flavanol

24.923

1.2%

6

5-6-dehydrokawain

228.25

C14H14O3

Kavalactone

25.355

19.4%

7

Alnustone

262.00

C19H18O

Diarylheptanoid

26.594

1.7%

8

Cardamonin

270.0

C16H14O

Chalcone

28.201

2.3%

9

Vitamin E (Tocopherol)

430.70

C29H50O2

Tocopherols

30.877

9.1%

10

β-sitosterol

414.00

C29H50O

Phytosterols

30.267

13.4%

depletion of myocardial enzymes (Rohini et al., 2013; Goyal et al., 2015). The infarct size is an important parameter to evaluate the severity of MI (Takagawa et al., 2007). The coloration with 2,3,5-TTC is a wellaccepted and straightforward method for determining the size of myocardial infarction, thus providing a reliable index of necrosis. This substance forms a red formazan precipitate with lactate dehydrogenase of the viable myocardial tissue, but the infarcted myocardium fails to stain with 2,3,5-TTC. Therefore, there is a direct association between the extent of myocardial infarct size and mortality (Hemalatha and Prince, 2015). In our results, we observed that ISO administration increased infarct size (59%) with less dye absorbing capacity, indicating significant leakage of lactate dehydrogenase enzyme. AZE pretreatment reduced infarct size (5.7%), demonstrating a mild leakage of lactate dehydrogenase. These results suggest that Alpinia zerumbet might prevent myocardial infarction and mortality in ISO-induced myocardial infarcted rats. According to some previous studies, morphometric changes occur in the MI, among them, we can mention the concentric hypertrophy. This hypertrophy is characterized by an expansion in the water content, more fluid accumulation in intramuscular space, and necrosis of cardiac muscle fibers, resulting in an increase in the ventricular wall thickness (French and Kramer, 2007). These functional and structural changes

clinical condition of individuals at cardiac risk of primary coronary diseases such as acute myocardial infarction due to ischemia-reperfusion injury, as well as secondary myocardial ischemic disorders such as coronary reocclusion, post-infarct heart failure, and unstable angina (Upaganlawar et al., 2011; Liaudet et al., 2014). Hence, ISO-induced MI is widely used as a standard model to study the cardioprotective effects of drugs, antioxidants, and medicinal plants (Wong et al., 2017). The present investigation was aimed to evaluate whether AZE might affect ISO-induced MI in rats. The creatine kinase (CK) is an enzyme that catalyzes the phosphorylation of creatine. This enzyme consists of three isoenzymes with activity in the muscle (CK-MM), heart (CK-MB), and brain (CK-BB). The detection and quantitation of the total CK-NAC and CK-MB are useful in the diagnosis of MI. Thus, elevated serum CK-MB activity is used as a sensitive criterion for myocardial injury (Puleo et al., 1990). In our study, we observed a significant elevation in the levels of diagnostic marker enzymes (CK-NAC and CK-MB) in plasma of ISO group. These results are in accordance with earlier works (MorardiArzeloo et al., 2016; Prabhu et al., 2012). Pretreatment with AZE decreased the activities of serum CK-NAC and CK-MB in ISO-treated rats, indicating the cardioprotective property of extract. These results are in agreement with newer studies, which showed that extracts of two plants belonging to family Zingiberaceae were also able to prevent the 7

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Authors’ contribution

have been observed in ISO-induced myocardial infarcted rats (Wong et al., 2017). AZE pretreatment significantly reduced heart weight, heart weight/body weight ratio, and the thickness of the left ventricle in ISO-treated rats. These results indicate that chronic treatment with extract was able to prevent cardiac hypertrophy induced by ISO. A light microscopic investigation was also performed to determine the cardioprotective effect of AZE on ISO-induced myocardial necrosis. In the histopathological study, in the control group exhibited an intact and united cell membrane with no sign of edema, inflammation, and infiltration of inflammatory cells. Besides, microscopic evaluation of cardiac tissue in ISO group showed the presence of edema, coagulative myonecrosis, myofibrillar degeneration, leukocyte infiltration, necrosis zone, and collagen deposition, indicating infarct lesions as reported in other studies mentioned in the literature. The pretreated group with AZE showed a decrease of necrosis zone, edema, degeneration on myocytes, and a significant reduction leukocytic infiltration. These results are consistent with the earlier reports. Therefore, the histopathological findings suggested that the Alpinia zerumbet could adequately prevent isoproterenol-induced myocardial damage. ISO acts on both β1-and β2-adrenergic receptors to induce myocardial infarction by various mechanisms. This synthetic catecholamine increases heart rate due to dromotropic, lusitropic, chronotropic, and positive inotropic effects, generating insufficient blood flow to the heart and myocardial damage (Averin et al., 2010). The increase in heart rate may also be due to relative ischemia or hypoxia because of hyperactivity of the β-receptors in the myocardium and cytosolic Ca2+ overload (Engelhardt and Ahles, 2014). In the present study, the detected changes in hemodynamic parameters after administration of isoproterenol in rats was comparable to earlier reported ones. Isoproterenol injection significantly increased the HR compared to that of the saline group. Literature data show that the propranolol, a β-adrenergic receptor antagonist, exhibits cardioprotective effects in infarcted rats, preventing the increased heart rate and serum activities of CK-NAC and CK-MB. In this context, our results were similar to propranolol. Some studies have shown that calcium channel blockers such as verapamil have a cardioprotective effect in in vivo rat models of MI (Sandmann, 2001). Therefore, it is important to mention that the essential oil from Alpinia speciosa induced a cardio-depressive action which is related to the L-type Ca2+ current blockade. Thus, we also suggest that the decrease in heart rate is probably due to a reduction in Ca2+ entry through voltage-dependent L-type Ca2+ channels. However, more pharmacological studies will be needed to clarify the mechanism (s) responsible for the cardio-depressive action. Another important detail is that the isoproterenol induces myocardial ischemia owing to the excessive production of free radicals resulting from the oxidative metabolism of catecholamines (Remião et al., 2001). Previous works explained that the antioxidant activity of various extracts prevents MI in rats (Wong et al., 2017). In experimental studies, Alpinia zerumbet showed to exhibit an antioxidant property (Chan et al., 2017). We can assume that the cardioprotective effect induced by AZE is due to possible antioxidant action in the myocardium. Based on this premise, we can suggest that the possible mechanisms for the cardioprotective effects of AZE would be cardio-depressive effect elicited by to L-type Ca2+ current blockade, as well as, its antioxidant action, preventing the leakage of cardiac diagnostic marker enzymes, including CK-NAC and CK-MB, and membrane disruption. Nevertheless, further experiments are needed to clear up the mechanisms involved.

Êurica Ribeiro and Emanuel Paulino conceived, designed the study and wrote the manuscript. Emanuel Paulino, Amanda Ferreira, Jessyka da Silva and Cintia Costa performed the in vitro and in vivo experiments. Salete Smaniotto performed histopathology appraisal, and João AraújoJúnior, Edeíldo Ferreira Silva Júnior, and Janaína Bortoluzzi performed the chemical experiments. All authors read and approved the final manuscript. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from FAPEAL, CAPES, and CNPq. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.112037. References Ara Tachjian, M.D., Viqar Maria, M.B.B.S., Arshad Jahangir, M.D., 2010. Use of herbal products and potential interactions in patients with cardiovascular diseases. J. Am. Coll. Cardiol. 55, 515–525. Averin, A.S., Zakharova, N.M., Ignat’ev, D.A., Tarlachokov, S.V., Nakipova, O.V., 2010. Isoproterenol effects on the contractility of papillary muscles in the heart of ground squirrel. Biophysics 5, 800–806. Baroldi, G., 1974. Letter: myocardial necrosis: the need for definition. J. Mol. Cell. Cardiol. 6, 401–402. Bhagat, B., Sullivan, J.M., Fischer, V.W., Nadel, E.M., Dhalla, N.S., 1978. cAMP activity and isoproterenol-induced myocardial injury in rats. Recent Adv. Stud. Card. Struct. Metabol. 12, 465–470. Brooks, W.W., Conrad, C.H., 2009. Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates. Comp. Med. 59, 339–343. Cartaxo, S.L., Souza, M.M.A., Albuquerque, U.P., 2010. Medicinal plants with bioprospecting potential used in semi-arid northeastern Brazil. J. Ethnopharmacol. 131, 326–342. Chan, E.W.C., Wong, S.K., Chan, H.T., 2017. Alpinia zerumbet, a ginger plant with a multitude of medicinal properties: an update on its research findings. J. Chin. Pharm. Sci. 26, 775–788. De Moura, R.S., Emiliano, A.F., de Carvalho, L.C., Souza, M.A., Guedes, D.C., Tano, T., 2005. Antihypertensive and endothelium-dependent vasodilator effects of Alpinia zerumbet, a medicinal plant. J. Cardiovasc. Pharmacol. 46, 288–294. Engelhardt, S., Ahles, A., 2014. Polymorphic variants of adrenoceptors: pharmacology, physiology and role in disease. Pharmacol. Rev. 66, 598–637. French, B.A., Kramer, C.M., 2007. Mechanisms of post-infarct left ventricular remodeling. Drug Discov. Today Dis. Mech. 4 (3), 185–196. Goyal, N.S., Sharma, C., Umesh, U.B., Patil, C.R., Agrawal, Y.O., Kumari, S., Arya, D.S., Ojha, S., 2015. Protective effects of cardamom in isoproterenol-induced myocardial infarction in rats. Int. J. Mol. Sci. 16, 27457–27469. Hemalatha, K.L., Prince, P.S.M., 2015. A biochemical and 2, 3, 5-triphenyl tetrazolium chloride staining study on the preventive effects of zingerone (vanillyl acetone) in experimentally induced myocardial infarcted rats. Eur. J. Pharmacol. 746, 198–205. Kumar, B., Kannan, M., Quine, S., 2011. Litsea deccanensis ameliorates myocardial infarction in wistar rats: evidence from biochemical and histological studies. J. Young Pharm. 3, 287–296. Kurian, G.A., Philip, S., Varguese, T., 2005. Effect of aqueous extract of the Desmodium gangeticum DC root in the severity of myocardial infarction. J. Ethnopharmacol. 3, 457–461. Kuibler, W., Haass, M., 1996. Cardioprotection: definition, classification, and fundamental principles. Heart 75, 330–333. Lahlou, S., Interaminense, L.F.L., Leal-Cardoso, J.H., Duarte, G.P., 2003. Antihypertensive effects of the essencial oil of Alpinia zerumbet and its main constituent, terpinen-4-ol, in doca-salt hypertensive conscious rats. Fundam. Clin. Pharmacol. 17, 323–330. Liaudet, L., Calderari, B., Pacher, P., 2014. Pathophysiological mechanisms of catecholamine and cocaine-mediated cardiotoxicity. Heart Fail. Rev. 19, 815–824. Mali, V.R., Bodhankar, S.L., 2010. Cardioprotective effect of Lagenaria siceraria (LS) fruit powder in isoprenaline-induced cardiotoxicity in rats. EUJIM 2, 143–149. Mendis, S., Thygesen, K., Kuulasmaa, K., Giampaoli, S., Blackett, K.N., Lisheng, L., 2010. World health organization definition of myocardial infarction: 2008–09 revision. Int. J. Epidemiol. 165, 1–8. Moradi-Arzeloo, M., Farshid, A.A., Tamanddonfard, E., Asri-Rezaei, S., 2016. Effects of histidine and vitamin C on isoproterenol-induced acute myocardial infarction in rats.

5. Conclusion The biochemical, morphometric, histological and hemodynamic evidence show that the pretreatment with the hydroalcoholic extract of the leaves of Alpinia zerumbet has protective nature against ISO-induced myocardial injury.

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