Cardioprotective activity of Piper betle juice in isoproterenol induced hypertrophic rat models

Impaired electrolytes (preferably Na, K, Cl−, HCO3) is one of the key fundamental complications associated with cardiac hypertrophy (enlarged heart) mediated heart failure. Piper betle (P. betle family Piperaceae) due to its potent antioxidant activity is used in various pharmacological applications. The present study evaluates the cardioprotective (anti-hypertrophic) potential of aqueous (juice) extract of P. betle in restoring electrolyte homeostasis, thereby its ability to curb the fundamental progression of hypertrophy. Isoproterenol (ISO 10 mg/kg b.w., i.p., 7 days) induced cardiac hypertrophic rats were developed that were simultaneously treated with the standard drug losartan (50mg/kg b.w., orally., 7days) and aqueous juice extract of P. betle (30mg/kg b.w., orally., 7 days). The biochemical estimation of glucose, protein, cholesterol, cardiac marker enzymes like SGOT, SGPT, LDH, enzymatic antioxidants namely SOD, catalase, GPx and electrolytes (Na, K, Cl− and bicarbonate) in serum were statistically performed along with the histopathological analysis of left ventricles (H&E stain). The present study showed increased levels of glucose, protein, cholesterol, cardiac marker enzymes, reduced enzymatic antioxidants with reduced electrolytes during cardiac hypertrophy (ISO) that were re lected in a histopathological analysis by thickened abnormal myocardial architecture which was reversed similar to normal in plant juice administered rats. Hence, this study demonstrated the bene icial effect of P. betle (aqueous juice) on electrolytes (Na, K, Cl−, HCO3), thereby imposing an interventional effect against cardiac hypertrophy.


INTRODUCTION
Cardiac hypertrophy (CH) is the clinical condition in which the heart is enlarged due to a constitutive uncontrolled state of the adaptive response of cardiomyocytes towards vigorous functional stress. CH is characterized by the thickening of the ventricular wall, increased heartbeat and enlargement (mostly left ventricle) that leads to diastolic and systolic dysfunctions. It has become one of the major causes of morbidity and mortality in the world (Zhao et al., 2017). The two types of cardiac hypertrophy, namely physiological and pathological, are due to aggressive exercise (as seen in athletes mostly), pressure, volume, overload and endocrine disorders, respectively (Yan et al., 2015).
In India, the mortality rate in cardiovascular diseases (CVD) has been increased by 59% from 23 million (1990) to 37 million (2010) (Prabhakaran et al., 2016). The treatment for CH is usually based on the electrocardiographic (ECG) or echocardiographic (ECHO) or magnetic resonance imaging (cardiac MRI) that estimates left ventricular mass (Buss et al., 2012). The treatment options involve surgical procedures, lifestyle changes and medication, which include β-blockers, Calcium channel blockers and anti-hypertensive drugs (Zwieten, 2000). Among the various mechanism of pathophysiology underlying CH, electrolytes being the fundamental player of the cardiac electrophysiological events is impaired during cardiac hypertrophy, primarily potassium. Supplements to improvise the electrolyte imbalances are suggested for treating cardiac hypertrophy and CVDs (Wang et al., 2005).
Piper betle belongs to the Piperaceae family, widely distributed in the tropical and sub-tropical regions of the world. The leaves of P. betle are used as carminative and wound healing in the traditional system of medicine (Atiya et al., 2017). Phytochemical investigation of the plant revealed that the presence of protein, amino acids, saponins, tannins, lavonoids and glycosides (Rekha et al., 2014).

Chemicals
All the chemicals were purchased from Hi-media laboratories, India. Isoproterenol was purchased from Sigma-Aldrich. The standard drug losartan (50 mg tablets) was purchased commercially from a local pharmacy, India. The biochemical estimation kits were purchased from Arkay Healthcare Pvt. Ltd and from Agappe Pvt. Ltd., India.

Plant collection and extraction
P. betle was identi ied and authenticated by the Botanical Survey of India (BSI/SRC/5/23/2019/Tech./2694) and the leaves of P. betle were collected during the month of December 2019 from the local area of Krishnagiri district, Tamil Nadu, India. The collected leaves were shade dried and ground to a coarse powder, extracted using cold maceration with distilled water for about 72 hours and then iltered to prepare the aqueous (juice) (Alam et al., 2013), which was subjected to further studies.
In the end, the serum and heart tissue (left ventricle) were isolated for further biochemical and histopathological analysis.

Histopathological analysis
The excised heart tissues were cleaned and preserved in 10% formalin immediately until tissue processing (left ventricle) as transverse, 5µm thick paraf in sections. The sections were stained with hematoxylin and eosin and the cellular con iguration of the heart tissues were examined by scanning the stained slides (40 X) (Mahmoud et al., 2015).

Statistical analysis
Data obtained were expressed as mean ± standard deviation. Statistical analysis was performed by using the Student t-test in SPSS.25 statistical package and values of P<0.05 were considered as significance level (Cochran and Snedecor, 1946).

RESULTS AND DISCUSSION
P. betle an edible and potent antioxidant herb (Prabu et al., 2012) is studied enormously in treating various diseases, but its intervening effect towards CH is less explored. Besides the increased necrosis and cellular in lammation, electrolyte imbalance plays a fundamental key role in the progression of CH (Urso et al., 2015).

Effect of P. betle on hypertrophic index
CH characterized by thickened left ventricle due to increased protein synthesis could contribute to the increased heart weight that serves as the primary phenotypic index of CH as indicated by the increased HW/BW ratio in ISO administered rats when compared to normal and plant juice administered groups (Yang et al., 2016) as shown in Table 1.

Effect of P. betle on glucose, total protein and total cholesterol
ISO elevates glucose, protein and cholesterol levels by impairing insulin activity and AMPK activities. Decreased albumin (hypoalbuminemia), considered as risk factors for cardiac hypertrophy , is observed in ISO, which could be attributed to the inhibitory effects of ISO upon albumin (Doss and Kuberapandian, 2019). Similarly, in this study, raised glucose, protein and cholesterol levels and decreased albumin (hypoalbuminemia), as shown in Table 2, were observed in the hypertrophic groups were reduced after treatment with the plant extract and losartan.

Effect of P. betle on serum cardiac marker enzymes
Increased cellular stress with decreased antioxidants is indicated by the elevated levels of SGOT, SGPT and LDH during CH, thereby imposing risk upon cardiac function. Similar results were observed in most studies, but LDH in a recent study was reported with decreased levels in ISO treated groups due to metabolic alterations of AMPK. So, the relationship between AMPK-LDH warrants further investigation in terms of CH (Doss and Kuberapandian, 2019;Long, 2006;Doss et al., 2018). Cardiac markers enzymes, namely SGOT, SGPT, LDH, were increased in serum of isoproterenol induced rats and were reduced by the administration of aqueous (juice) extract of P. betle as shown in Table 3.

Effect of P. betle on enzymic antioxidants
Eugenol is a reported anti-hypertrophic phytocompound (Choudhary et al., 2006). Similar compounds such as isoeugenol, chavibetol, hydroxychavicol (Vikash et al., 2012) present in this plant are potent antioxidants and cardioprotective compounds, which could be attributed to the anti-hypertrophic potential of P.betle juice which requires further investigations. Free radicals trigger the cellular stress underlying the metabolic and cellular alterations involved in the pathophysiology of CH (Widowati et al., 2013;Maulik and Kumar, 2012). ISO being cardiotoxic induces oxidative stress marked by the decreased levels of enzymic antioxidants, which upon treatment using P.betle is involved in the restoration of SOD, CAT and GPx when compared to ISO administered rats (Doss and Kuberapandian, 2019;Wexler, 1979;Wu et al., 2009). Decreased enzymic antioxidants (SOD, catalase, GPx) and increased level of lipid peroxidation were seen in the isoproterenol induced rat models. Administration of aqueous (juice) extract of P. betle showed increased levels of SOD, catalase and GPx, as shown in Table 4.

Effect of P. betle on serum electrolytes
ISO administration caused decreased levels of serum electrolytes, which can be considered as a risk factor for CH that may progress to ventricular arrhythmias and even myocardial infarction (Brembilla-Perrot et al., 1993;Okin et al., 2012). To our knowledge, P.betle is reported to have bene icial effects upon Na + /K + , Ca 2+ and Mg 2+ pumps in erythrocytes (Chitra and Vidya, 2006) but its effect upon serum electrolytes in association with CH pathology is irst reported in this study. ISO administration resulted in reduced serum levels of sodium, potassium, chloride and bicarbonate as indicated in Table 5 and their ranges were brought to near normal values when treated with plant extract and losartan.

Histopathological analysis of left ventricle
Cardiotoxicity induced by ISO due to the increased free radicals and metabolic shifts causes cellular stress that results in increased myocardial degeneration with reduced cardiomyocytes (Doss and Kuberapandian, 2019;Padma et al., 2013). In CH, the enlargement of the heart is depicted by thickened ventricle (preferably left ventricle) with distorted myocardial architecture, as shown similarly in this study. The strong antioxidant, anti-       in lammatory potentials and metabolic restoration ability (herein via serum electrolytes) showed reduced in lammatory and degeneration of the heart tissue (left ventricle). In the isoproterenol administered group Figure 1(b), the enlargement of heart tissue was observed by the presence of thickened cellular architecture with distorted myo ibrils, as shown in Figure 1. In the aqueous (juice) extract administered rats administered group Figure 1(d), reduced cellular thickening and necrosis were observed similar to the standard drug losartan administered rats Figure 1(c) that showed better myo ibrillar rearrangement similar to the control group Figure 1(a).
Figure 1(a) represents an organized myocardial network of nucleated (N) cells (group 1 -normal). Figure 1(b) indicates the thickened myocardial architecture (TMA) with invisible nucleated cellular structure as a marked feature of cardiac hypertrophy. Figure 1(c) and Figure 1(d) indicate the presence of visibly nucleated cells with rejuvenating striated heart tissue organization and less thickened myocardial architecture (LTM) when compared to normal and hypertrophic tissues.

CONCLUSION
The present study primarily concludes that the Piper betle aqueous (juice) extract possesses the ability to reverse the cellular effects induced by isoproterenol induced cardiac hypertrophy animal model.
This has been con irmed by both biochemical and histopathological studies in this work. It is shown to restore electrolytes to near-normal levels. Further studies are required to check the electrophysiological bene its of P.betle and to isolate its bioactive compounds like chavibetol, hydroxychavicol, isoeugenol for checking its anti-hypertrophic effect. As P.betle possesses effect upon restoring electrolyte balance, this study also suggests that it can be extended to the treatment of blood pressure and other electrolyte mediated complications.

ACKNOWLEDGEMENT
We thank PSG Institute of Medical Sciences and Research and PSG Institutional Animal Ethics Committee, Coimbatore and the CPSCEA, India, for providing animal ethical clearance. Heartful thanks to the Botanical Survey of India (BSI) for the plant authentication. Thanks to Ponmani and Co Pvt Ltd., Coimbatore for providing Isoproterenol. Thanks to Arkray Healthcare Pvt Ltd., India, Agappe Diagnostics Ltd., India for providing us with the kits and Ratthan Lab Pvt Ltd., Coimbatore for the histopathological facility

Con lict of interests
The authors declare that they have no con lict of interest for this study.

Funding Support
The authors declare that they have no funding sup-