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Abstract

Aims:

The aim of this study was to investigate the antihypertensive effect of leaves Mangifera indica L. using in vitro and in vivo assays.

Methodology:

The ethanol extract of leaves of M. indica was fractionated to dichloromethanic, n-butyl alcohol and aqueous fractions. The chemical composition of ethanolic extract and dichloromethanic fraction were evaluated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Antioxidant activity was evaluated in the DPPH scavenging activity assay. Angiotensin-converting enzyme (ACE) inhibitory activity was investigated using in vitro and in vivo assays. The chronic antihypertensive assay was performed in spontaneously hypertensive rats (SHRs) and Wistar rats treated with enalapril (10 mg/kg), dichloromethanic fraction (100 mg/kg; twice a day) or vehicle control for 30 days. The baroreflex sensitivity was evaluated through the use of sodium nitroprusside and phenylephrine. Cardiac hypertrophy was evaluated by morphometric analysis.

Results:

The dichloromethanic fraction exhibited the highest flavonoid, total phenolic content and high antioxidant activity. Dichloromethanic fraction elicited ACE inhibitory activity in vitro (99 ± 8%) similar to captopril. LC-MS/MS analysis revealed the presence of ferulic acid (48.3 ± 0.04 µg/g) caffeic acid (159.8 ± 0.02 µg/g), gallic acid (142.5 ± 0.03 µg/g), apigenin (11.0 ± 0.01 µg/g) and quercetin (203.3 ± 0.05 µg/g). The chronic antihypertensive effects elicited by dichloromethanic fraction were similar to those of enalapril, and the baroreflex sensitivity was normalized in SHR. Plasma ACE activity and cardiac hypertrophy were comparable with animals treated with enalapril.

Conclusions:

Dichloromethanic fraction of M. indica presented an antihypertensive effect, most likely by ACE inhibition, with benefits in baroreflex sensitivity and cardiac hypertrophy. Altogether, the results of the present study suggest that the dichloromethanic fraction of M. indica leaves may have potential as a promoting antihypertensive agent.

Keywords

antihypertensive activity angiotensin-converting enzyme baroreflex

Introduction

Mangifera indica L., Anacardiaceae, has been reported to be the second largest tropical fruit crop in the world [Barreto et al. 2008]. Different extracts of several parts of M. indica as fruit, bark, leaf, seed and peel have been reported to possess many different pharmacological activities and are worldwide used in folk medicine, mainly against diarrhea, kidney and urinary disorders, as complementary therapy to manage type 2 diabetes, against genitourinary inflammations, bronchitis and asthma, and locally applied in baths to treat scabies and syphilis [Masibo and He, 2008; Shah et al. 2010].

The biological effects of M. indica are attributed to the C-glucosyl-xanthone (mangiferin) [Shah et al. 2010; Matkowski et al. 2013]. Based on ethnopharmacological approach, a standardized aqueous extract of M. indica stem bark with antioxidant, anti-inflammatory and immunomodulatory properties has been developed in Cuba. It contains a defined and standardized mixture of components such as polyphenols, terpenoids, steroids, fatty acids and microelements [Núñez-Selles et al. 2002]. Other studies have reported the use of polyphenols in the treatment of hypertension [Patten et al. 2012; Persson, 2012], the inhibitory activity in the angiotensin-converting enzyme (ACE) and in the baroreflex of spontaneously hypertensive rats [Guerrero et al. 2012; Tangney and Rasmussen, 2013].

Cardiovascular disease, such as hypertension, is one of the most relevant causes of death in the world [Drazner, 2011; Cipriano et al. 2014; Feeman, 2014]. The renin–angiotensin–aldosterone system is a key factor of arterial blood pressure [Hargovan and Ferro, 2014]. Although many natural and synthetic ACE inhibitors (e.g. captopril, enalapril, and lisinopril) effectively regulate the blood pressure, diminishing the risk of cardiovascular diseases and stroke, they also have some disadvantages, such as easy digestion by protease in the body, and side effects, such as coughing, allergies, taste disturbances and skin rashes [Hansen et al. 1995; Braga et al. 2007; Guerrero et al. 2012; Lee et al. 2012; Persson, 2012; Kang et al. 2013]. Therefore, the development of new ACE inhibitors with strong antihypertensive activity is of substantial importance in alternative therapeutics.

In light of the exposed and the increasing need of new alternatives therapies, we speculated that chronic treatment with M. indica leaves may reduce the blood pressure of hypertensive animals via inhibition of ACE activity, resulting in the improvement of arterial baroreflex sensitivity. Here we report a comprehensive investigation undertaken with M. indica on the blood pressure and baroreflex of spontaneously hypertensive rats (SHRs). Our results demonstrated that M. indica reduced the blood pressure and these effects may be due, at least in part, to the inhibition of ACE.

Materials and methods

Plant material

Leaves of M. indica were collected in Viana-ES, Southeastern Brazil on April 2011 and identified by the botanist Ms Solange Schneider. A voucher specimen (UVVES2178) was deposited in the herbarium of the University Vila Velha-ES, Brazil. The plant material was dried (40°C) and grounded.

Animals and cell culture

Adult Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) (Rattus norvegicus; 250–300 g), aged 12–13 weeks were maintained under standard laboratory conditions with a 12-hour light–dark cycle and free access to food and water. All experiments were conducted in accordance with the Brazilian Committee for animal care and use (COBEA) guidelines and approved by the Ethics, Bioethics and Animal Welfare of the University Vila Velha (CEUA-UVV; Protocol 238/2012). The H9c2 cell line was obtained from the American Type Culture Collection (ATCC number CRL-1446^TM, Manassas, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin.

Extraction and fractionation procedure

The plant material (120 g) was defatted with pentane (1 l) followed by extraction with ethanol (400 ml) by ultrasound extraction. The liquid extract was then dried in a rotary evaporator under reduced pressure (Fisaton 801, São Paulo, Brazil) at 40°C until a residue was formed (79.3 g). An aliquot of this extract (74.3 g) was suspended in 500 ml of water and sequentially partitioned into dichloromethanic fraction (yield 9.0 g), n-butyl alcohol fraction (yield 18.6 g) and water-soluble fraction (yield 6.0 g) for subsequent analysis. Bioassay-guided fractionation revealed that the dichloromethanic partitioned fraction was the most active in the in vitro ACE inhibitory activity; hence, it was selected for further experiments.

Subfractionation of the dichloromethanic fraction

The dichloromethanic fraction (8.1 g) was subfractionated by open column chromatographic on Sephadex LH-20^® (1.8 cm × 24 cm, 6 g, Sigma Aldrich, St Louis) as stationary phase and methanol as eluent. The subfractions were screened by thin-layer chromatography (TLC) and those with similar composition were combined to give a total of 30 subfractions (SFD1–SFD30).

ESI(-)-FT-ICR MS analysis of SFD10

SFD10 yielded acicular crystals and was characterized using negative-ion electrospray ionization (ESI(-)) coupled with Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) [Freitas et al. 2013]. Details are given in the online Supplementary Material.

Analysis of ethanol crude extract and dichloromethanic fraction by LC MS/MS

The identification and quantification of the main secondary metabolites in the ethanol crude extract and dichloromethanic fraction was analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS; Applied Biosystems) as previously described by Zhu and colleagues [Zhu et al. 2012], with minor modifications. Details of the modifications are depicted in the online Supplementary Material.

Determination of total phenolic content and total flavonoids

Total phenolic contents were determined spectrophotometrically according to the Folin-Ciocalteu reagent method [Krepsky et al. 2012]. Quantification was based on the standard curve generated with pyrogallol (3.125–37.5 µg/ml) and the results were expressed in milligrams of pyrogallol equivalents (PE) per 100 grams dry weight (mg PE/100 g). Determination of flavonoids was performed according to a colorimetric assay with aluminium chloride [Krepsky et al. 2012]. The content of total flavonoids was calculated using a calibration curve of quercetin (1–12 µg/ml) and the results expressed in grams of quercetin equivalents per 100 g of dry weight. All determinations were carried out in triplicate.

DPPH radical scavenging assay

The antioxidant activity of the ethanol extract and the partition fractions were evaluated by monitoring its ability in quenching the stable free-radical DPPH according to Scherer and Godoy [Scherer and Godoy, 2009]. The antioxidant activity was expressed as the antioxidant activity index (AAI). The assays were carried out in triplicate and antioxidant activity was compared with the commonly used synthetic antioxidant butylated hydroxyanisole (BHA).

In vitro colorimetric ACE inhibition assay

The in vitro potential ACE inhibitory activity of the ethanol extract and the partition fractions was evaluated as previously described by Endringer and colleagues [Endringer et al. 2014]. The method is based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with trinitrobenzenesulfonic acid toform 2,4,6-trinitrophenyl-glycyl-glycine, whose absorbance is determined at 415 nm in a microtiter plate reader. For the calculation the equation % inhibition= (100 − 100A)/B) was used, where A is the absorbance measured at 415 nm in the presence of an inhibitor and B is the absorbance of the blank solution.

In vitro cytotoxicity assay and in vivo acute toxicity

The in vitro cytotoxic activity of dichloromethanic fraction was evaluated in H9c2 cell line using the classical MTT assay [Mosmann, 1983]. Details are given in the online Supplementary Material. The acute oral toxicity of dichloromethanic fraction was performed as described in the 2001 guide from the Organisation of Economic Cooperation and Development (OECD) 423. Blood samples were collected for biochemical and hematologic tests. After, the animals were euthanized with a high dose of anesthetic, and their organs were macroscopically evaluated by a pathologist who was not informed about the protocol. The hematological analyses of total erythrocytes, leukocytes, hemoglobin, protein, and hematocrit as well as the mean corpuscular volume (VCM), corpuscular hemoglobin (HCM) and corpuscular hemoglobin concentration (CHCM) were performed with a hemocytometer (CC550^®, Celm, São Paulo, Brazil). The biochemical analyses of alanine transaminase (ALT), aspartate transaminase (AST), urea, creatinine and troponin I levels were performed with commercial kits (Bioclin^®, Minas Gerais, Brazil; Biocon^®, Minas Gerais, Brazil).

In vivo ACE inhibitory activity assay

Adult WKY rats were randomly divided into two groups (n = 10), weight and deeply anesthetized by intraperitoneal (i.p.) administration of an association between ketamine (80 mg/kg) and xylazine (15 mg/kg) and catheterized (femoral artery and vein) according to Andrade and colleagues [Andrade et al. 2012]. The ACE inhibitory activity assay was performed as described by Mangiapane and colleagues [Mangiapane et al. 1994, with minor modifications. The following groups were used: captopril (C; n = 5) received an intravenous (IV) dose of 30 mg/kg; and the dichloromethanic fraction (n = 5) received an IV dose of 100 mg/kg. Before and after of the administration of captopril or the dichloromethanic fraction, the animals received increasing doses of Ang I (0.03, 3 and 300 µg/kg, IV). The mean arterial pressure (MAP) was measured before and after each Ang I administration to obtain the difference in pressure (ΔMAP). The subsequent dose of Ang I was administered after MAP returned to baseline. The inhibition of ACE was evaluated as the difference between ΔMAP for each dose of Ang I before and after the administration of captopril or dichloromethanic fraction.

In vivo acute hypotensive effect of dichloromethanic fraction

The WKY rats (n = 5) were weighed, anesthetized and catheterized (femoral artery and vein) according to Andrade and colleagues [Andrade et al. 2008], and hypotensive effect was determined by a dose–effect curve. Before and after the administration of the dichloromethanic fraction (1–300 mg/kg), the animals received acetylcholine (Ach 5.0 µg/kg, IV). The subsequent dose of Ach was administered after MAP returned to baseline. The percentage of maximum decrease in MAP was determined after each single dose [Soncini et al. 2011].

In vivo chronic antihypertensive effects

WKY rats and SHRs were divided into six groups (n = 5, each): negative normotensive (WKYC) and hypertensive (SHRC) controls that received saline (NaCl 0.9%, i.p.); positive normotensive (WKYE) and hypertensive (SHRE) controls that received enalapril (10 mg/kg, i.p.); and normotensive (WKYM) and hypertensive (SHRM) groups that received dichloromethanic fraction (200 mg/kg, i.p.). The animals were treated twice a day, at the same time, for 30 days. The animals were weighed at day zero (initial weight [IW]) and on the last day of the experiment (final weight [FW]). To measure the initial systolic blood pressure (SBP) of conscious rats, a tail-cuff manometer was used (IITC Life Science Inc., Woodland Hills, CA, USA) [Andrade et al. 2008].

Hemodynamic and arterial baroreflex assessment

Adult WKY rats were weighed, anesthetized by intraperitoneal (i.p.) administration of an association between ketamine (80 mg/kg) and xylazine (15 mg/kg) and catheterized (femoral artery and vein) according to Andrade and colleagues [Andrade et al. 2008]. After at least 24 hours, the basal MAP and heart rate (HR) of the animals were determined and arterial baroreflex was evaluated according to Beutel and colleagues [Beutel et al. 2005]. In brief, MAP and HR were controlled beat to beat in awake animals, and the variation of these parameters was evaluated by the application of phenylephrine (100 μg/ml) and sodium nitroprusside (180 μg/ml) with an infusion pump (EFF311B, Insight, Ribeirão Preto, São Paulo, Brazil) at a rate of 0.1 ml for 30 seconds. The relation between the changes in MAP and the respective alteration of the HR was evaluated by regression. The regression coefficient (slope of the curve) expressed as beats per minute (bpm)/mmHg was used as baroreflex sensitivity index of phenylephrine (BRS_PE) or sodium nitroprusside (BRS_NP).

Evaluation of ACE activity on serum and histological analyses

After the hemodynamic recording, the animals were euthanized by decapitation with a guillotine. Blood was collected, and the serum was separated by centrifugation. The serum was kept at −80°C until analysis. The evaluation of ACE activity in serum was performed with a colorimetric assay as described previously [Franquni et al. 2013], with the following modifications; instead of cardiac homogenate, 10 µl of serum was used. For the evaluation of cardiac hypertrophy, the hearts were removed, washed with saline, dried with filter paper and weighed. The histological analyses for cardiac hypertrophy determination were evaluated by the number of myocyte nuclei per high power field, area of the cardiac myocyte nuclei (µm²), and heart weight to body weight ratio (HW/BW) [Franquni et al. 2013].

Statistical analysis

The results were expressed as the mean ± standard error of the mean (SEM). Data were analyzed with a one-way analysis of variance (ANOVA) followed by Turkey’s post hoc test. Tests were adjusted for multiple comparisons, and significance was defined as p < 0.05. The software GB-Stat and SlideWrite was used for statistical analyses and figures, respectively.

Results

Total phenolic, flavonoids and tannin content

Polyphenolics are known to have many important biological activities, including antioxidant activity, vasodilatory properties, antibacterial, immune-stimulating and anti-inflammatory among many others [Zhang et al. 2011; Tangney and Rasmussen, 2013]. The analyses of total phenolic, flavonoids and tannins reveal that the dichloromethanic fraction exhibited the highest content of total phenolic 96.42 ± 0.43 g/100 g, total flavonoids 6420.4 ± 131.6 g/100 g, nonadsorbed phenolic 38.71 ± 2.14 g/100 g and total tannins 57.7 ± 2.29 g/100 g when compared with the ethanol extract and the dry leaves (see Table 1S of the online Supplementary Material).

DPPH radical scavenging assay

The antioxidant activity of polyphenolic compounds are mainly due to redox properties, which enable them to act as reducing agents hydrogen donors, singlet oxygen quenchers, heavy metal chelators and hydroxyl radical quenchers [Scherer and Godoy, 2009]. The radical scavenging activity expressed AAI of ethanol extract (4.64 ± 0.33), n-butyl alcohol (6.69 ± 0.24) and aqueous fraction (3.24 ± 0.86) were similar; however, the dichloromethanic fraction exhibited a high antioxidant activity (1.80 ± 0.21) compared with the commonly used synthetic antioxidant BHA (4.47 ± 0.22) (see Table 2S of the online Supplementary Material).

Determination of in vitro ACE inhibitory activity of M. indica ethanol extract and partionated fractions

The highest in vitro ACE inhibitory activity was observed for dichloromethanic fraction (99.5 ± 7.2%), followed by aqueous fraction (93.6 ± 6.0%), ethanol extract (72.9 ± 3.7%) and n-butyl alcohol fraction (24.0 ± 2.1%) tested at 100 µg ml⁻¹ concentration. The ACE inhibitory activity was found to be similar to the standard ACE drug, captopril (83 ± 20 %). Considering the total polyphenols, flavonoids and tannin content, the antioxidant and mainly the in vitro ACE inhibitory activity, the dichloromethanic fraction was selected for further extensive biological studies.

Identification of the main secondary metabolites by LC-ESI(-)MS/MS

Ethanol extract of M. indica was standardized in ferulic acid (20.1 ± 0.03 µg/g), gallic acid (143.5 ± 0.02 µg/g) and quercetin (176.1 ± 0.04 µg/g); while the dichloromethanic fraction was standardized in ferulic acid (48.3 ± 0.02 µg/g), caffeic acid (159.8 ± 0.04 µg/g), gallic acid (142.5 ± 0.05 µg/g), apigenin (11.0 ± 0.02 µg/g) and quercetin (203.3 ± 0.03 µg/g) using LC-ESI(-)MS/MS experiments.

Chemical identification of SFD10 by ESI(-)-FT-ICR MS and ESI(-)-FT-ICR MS/MS

Figure 1S of the online Supplementary Material shows ESI(-)-FT-ICR MS for SFD10, where a mixture of glycosylated polyphenol compounds was identified: ions of m/z 421.0780 and 421.1142 detected in deprotonated form, [M–H]^-, with M = C₁₉H₁₈O₁₁ and C₂₀H₂₂O₁₀ and DBE = 11 and 10, respectively. All m/z values present an accuracy mass < 1 ppm. All results are summarized in Table 1S of the online Supplementary Material and two possible structures, mangiferin [Schieber et al. 2003] and foliamangiferosideo [Wang et al. 2012], were proposed as reported previously. The DBE = 11 for the ion [C₁₉H₁₈O₁₁ - H]^- corresponds to sum of one glucoside ring (DBE = 1) and one xanthone group (DBE = 10). The latter is composed of one ketone group, two phenolic rings and one etheric ring whereas the DBE = 10 for ion [C₂₀H₂₂O₁₀ – H]^- corresponds to one glucoside ring (DBE = 1), one ketone group (DBE = 1), and two phenolic rings (DBE = 8). The structures and the connectivity of the mangiferin and the foliamangiferosideo polyphenol compounds were confirmed from the ESI(-)-MS/MS experiments, where the profile of their fragments are shown in Table 3S of the online Supplementary Material.

In vitro cytotoxicity and in vivo acute toxicity

The in vitro cytotoxic activity of dichloromethanic fraction from M. indica was investigated in H9c2 cell line. Tested concentration in the ranged from 2.5 to 160 µg/ml exhibited only marginal cytotoxicity, displaying less than 22.31 ± 3.25% of cell death at 160 µg/ml. This indicates a negligible in vitro cytotoxic activity. No deaths were observed in the in vivo acute toxicity experiments after administration of an oral dose of dichloromethanic fraction of 2000 mg/kg. All animals were subjected to gross necropsy. Pathology was evaluated in a blinded pattern and there was no evidence of changes. The hematological and biochemical parameters are depicted in Table 1. No significant differences were observed in the treated animals compared with the vehicle control group. Altogether these results show evidence of an absence of cytotoxic activity in vitro and no toxicological effects even at high doses.

Table 1.

Results of hematological and biochemical analyses after 15 days of treatment with acute doses of 2000 mg/kg of dichloromethane fraction from Mangifera indica L.

Parameters	Dichloromethane fraction	Vehicle control
Erythrocytes (million/µl)	7.8 ± 0.30	7.8 ± 0.80
Hemoglobin (g/dl)	13.17 ± 0.33	12.5 ± 1.66
Hematocrit (%)	41.7 ± 0.88	41.6 ± 2.4
VCM	47.64 ± 7.03	49.5 ± 3.04
HCM	17.02 ± 1.0	14.2 ± 2.34
CHCM	31.65 ± 1.33	28.53 ± 3.57
Total protein (%)	6.33 ± 0.24	6.5 ± 0.28
Leukocytes (% × 1000/mm³)	5.86 ± 0.20	8.5 ± 1.23
ALT (UI/l)	51.16 ± 1.56	53.2 ± 2.88
AST (UI/l)	59.9 ± 2.9	54.4 ± 4.1
Urea (mg/dl)	33.6 ± 2.75	39.34 ± 2.75
Creatinine (mg/dl)	0.80 ± 0.075	0.68 ± 0.13
Troponin I	Negative	Negative

Values are expressed as the mean ± SD.

VCM, mean corpuscular volume; HCM, corpuscular hemoglobin; CHCM, corpuscular hemoglobin concentration; ALT, alanine transaminase; AST, aspartate transaminase.

Acute hypotensive activity evaluation

Dose-dependent hypotensive effect of the dichloromethanic fraction in normotensive animals (WKY) is shown in Figure 1(B). The observed decrease in the MAP for acetylcholine was −60.7 ± 3 mmHg, and the doses of 1, 50, 100, 200 and 300 mg/kg of dichloromethanic fraction decreased in a dose-dependent manner by −13.7 ± 3 mmHg, −27 ± 3 mmHg, −38 ± 4 mmHg, −54 ± 3 mmHg, −58 ± 4 mmHg, respectively (Figure 1B). There were no changes in the reduction in MAP after an administration of 200 and 300 mg/kg of dichloromethanic fraction, therefore the dose 200 mg/kg was chosen for further studies. Thereafter acetylcholine was administered again resulting in a decrease of MAP of −66 ± 2 mg/kg indicating that application of increasing doses of dichloromethanic fraction not alter the response induced by acetylcholine.

Figure 1.

Acute hypotensive effect and ACE inhibitory activity of dichloromethanic fraction in vivo. Panel A: Increase in MAP (ΔMAP) in WKY rats following an in bolus injection of Ang I (0.03, 3 and 300 μg/kg) before and after the administration of captopril (30 mg/kg) or dichloromethanic fraction (M 200 mg/kg). Values are expressed as the mean ± S.E.M. ++p<0.01 in comparison with the increase caused by Ang I before the administration of captopril; **p<0.01 in comparison with the increase caused by Ang I before the administration of MIDI; ##p<0.01 in comparison with the increase caused by Ang I after the administration of captopril. Panel B: Dose-dependent decrease in the MAP after exposure of 1, 50, 100, 200 and 300 mg/kg of dichloromethanic fraction in normotensive animals. **p<0.01 in comparison with the previous dose.

In vivo ACE inhibitory activity

As shown in Figure 1(A), the bolus application of Ang I in WKY animals induced an incremental increase, with similar magnitude in MAP before the administration of captopril or dichloromethanic fraction of M. indica (41 ± 5 mmHg, 89 ± 4 mmHg, 147 ± 3 mmHg and 49 ± 5 mmHg, 92 ± 9 mmHg, 158 ± 12 mmHg, respectively). A significant reduction of the increased MAP induced by Ang I was observed after administration of captopril (6 ± 2 mmHg, 21 ± 5 mmHg and 30 ± 4 mmHg) and dichloromethanic fraction (5 ± 2 mmHg, 22 ± 4 mmHg and 54 ± 3 mmHg) (p < 0.01). After the administration of dichloromethanic fraction, increase in the MAP induced by Ang I was higher than that obtained after captopril treatment for at least two tested doses (0.03 and 3 µg/kg).

In vivo chronic antihypertensive effect

Chronic treatment with dichloromethanic fraction of M. indica (100 mg/kg, twice a day) reduced the MAP in SHR animals (Figure 2A) in similar manner to that observed with enalapril treatment (SHRC: 209 ± 3 mmHg versus SHRE: 150 ± 3 mmHg, SHRM: 145 ± 9 mmHg; p < 0.01 compared with SHRC animals). Similar to enalapril, the dichloromethanic fraction treatment did not decreased the blood pressure to basal levels in SHR animals (p < 0.01) compared with the WKY animals (Figure 2A). No differences were observed in the MAP between the groups when using WKY animals (WKYC: 107 ± 2 mmHg, WKYE: 106 ± 4 mmHg, WKYM: 105 ± 3 mmHg). Furthermore, there was no difference in HR between the groups (SHRC: 303 ± 4 bpm, SHRE: 310 ± 17 bpm, SHRM: 284 ± 10 bpm, WKYC: 283 ± 19 bpm, WKYE: 285 ± 22 bpm, WKYM: 307 ± 19 bpm).

Figure 2.

In vivo chronic antihypertensive activity and ex vivo serum ACE activity of dichloromethanic fraction of M. indica. Panel A: Mean arterial pressure (MAP) of animals subject to chronic treatment with dichloromethanic fraction of M. indica (WKYM and SHRM), enalapril (WKYE and SHRE) or vehicle control (WKYC and SHRC) for 30 days. **p<0.01 compared to WKY animals, ##p<0.01 compared to SHRC group. Panel B: Ex vivo serum ACE activity from animals treated with dichloromethanic fraction, enalapril or vehicle for 30 days. **p<0.01 in relation to WKYC animals, ##p<0.01 compared to SHRC group. Values are expressed as mean ± S.E.M.

The study of ex vivo serum ACE activity after chronic treatment of animals treated with dichloromethanic fraction of M. indica exhibited a reduced activity of this enzyme in SHR animals (SHRC: 122 ± 11%, SHRE: 69 ± 3%; SHRM: 68 ± 10%, p < 0.01; Figure 2B) and that there was no difference in ACE activity between the SHRM and SHRE groups. The ACE activity in the WKYM and WKYE groups were comparable, however it was lower than that observed in the WKYC group (WKYC: 80 ± 5%, WKYE: 60 ± 7%, WKYM: 62± 11%).

Arterial baroreflex sensitivity

A relationship between baroreceptor sensitivity and blood pressure behavior has often been suggested. Our study demonstrated that a treatment with dichloromethanic fraction of M. indica normalized the baroreflex sensitivity in SHRM animals after phenylephrine (BRS_PE: SHRC = −0.50 ± 0.07, SHRE = −0.79 ± 0.02, SHRM = −0.78 ± 0.06, p < 0.01 compared with SHRC group; Figure 3A) and after sodium nitroprusside (BRS_NP: SHRC = −1.20 ± 0.17, SHRE = −1.62 ± 0.07, SHRM = −1.61 ± 0.09; p < 0.01 compared with SHRC group, Figure 3B) applications. Similar effects were observed between SHRM and SHRE treated animals. There was no difference between the hypertensive animals treated with enalapril or dichloromethanic fraction and the normotensive animals. The sensitivity of the baroreflex was similar among the normotensive animals (BRS_PE: WKYC = −0.80 ± 0.08, WKYE = −0.77 ± 0.05, WKYM = −0.76 ± 0.05; BRS_NP: WKYC = −1.67 ± 0.05, WKYE = −1.71 ± 0.12, WKYM = −1.63 ± 0.09; Figure 3). Therefore, an increase in the baroreceptor sensitivity was observed in the SHR animal after dichloromethanic fraction and the results can be compared with those indices found in the enalapril treated group (p < 0.01).

Figure 3.

Changes in the arterial baroreflex sensitivity after chronic treatment with dichloromethanic fraction of M. indica (100 mg/kg), enalapril (10 mg/kg) or vehicle (NaCl 0.9 %) twice a day for 30 days after the administration of phenylephrine (BRSPE; 100 μg/mL; Panel A) and sodium nitroprusside (BRSNP: 180 μg/mL; Panel B). The values are expressed as the mean ± S.E.M. **p<0.01 SHRC group compared to the other groups.

Cardiac hypertrophy and body weight

A reduction in the degree of cardiac hypertrophy in SHRM treated group was observed which promoted an improvement in the HW/BW ratio (SHRC: 4.342 ± 0.10 mg/g, SHRE: 3.192 ± 0.17 mg/g, SHRM: 3.103 ± 0.21 mg/g; p < 0.01 compared with SHRC animals; Figure 4I). There was no difference between the values of treated SHR animals and normotensive animals (WKY: 2.911 ± 0.13 mg/g, WKYE: 2.962 ± 0.19 mg/g, WKYM: 2.812 ± 0.18 mg/g).

Figure 4.

Histological analysis from hearts in SHR and WKY animals after dichloromethanic fraction of M. indica (100 mg.kg-1), enalapril (10 mg.kg-1) or vehicle (NaCl 0.9 %) twice a day for 30 days. Microscopic slides were stained with hematoxylin/eosin (H&E), and the analysis was performed at a 400X magnification (A: WKYC; B: WKYE; C: WKYM; D: SHRC; E: SHRE; F: SHRM). Panel G: bar graph corresponding to the number of myocyte nuclei/high power field. Panel H: bar graph representing the area of the cardiac myocyte nuclei. Panel I: Heart weight to body weight (HW/BW) ratio. Values are expressed as the mean ± S.E.M. **p<0.01 compared to WKY animals, ##p<0.01 in relation to SHRC group.

Morphometric analysis showed that in the SHRM and SHRE groups the number of myocyte nuclei (Figure 4G) per high-power field (SHRM 5.13 ± 0.18 and SHRE 4.73 ± 0.19) and the area of cardiac nuclei (Figure 4H) (SHRM 660.9 ± 20 μm² and SHRE 702.4 ± 31 μm²) were different compared with the SHRC group (4.12 ± 0.08 and 932.3 ± 38 μm²; p < 0.01) however, those effects were not observed in the normotensive animals (WKYC: 4.7 ± 0.4 and 634.3 ± 16 μm², WKYE: 4.91 ± 0.24 and 609 ± 40 μm², WKYM: 4.8 ± 0.35 and 615 ± 20 μm²). No difference was observed between SHRM and SHRE groups.

BW data demonstrated no weight changes in the SHRC animals, while the other groups exhibited a BW gain (FW/IW: WKYC: 310 ± 9/247.3 ± 11 g; WKYE: 307 ± 11/268.2 ± 8 g; WKYM: 296 ± 9/263 ± 10 g; SHRC: 258 ± 11/247 ± 13 g; SHRE: 294 ± 7/255 ± 9 g; SHRM: 276 ± 6/227 ± 5 g).

Discussion

Several studies have shown that stem bark and leaf extracts from M. indica exhibit a wide range of pharmacological effects such as antioxidant, anticancer, antimicrobial, antiatherosclerotic, antiallergenic, anti-inflammatory, analgesic and immunomodulatory among many others; however, the underlying mechanisms involved in these effects are mostly not yet well described [Sanchez et al. 2000; Beltran et al. 2004; Engels et al. 2009; Abu Bakar et al. 2010; Ajila et al. 2010; Chieli et al. 2010; Kanwal et al. 2010; Agarwala et al. 2012]. In our investigations we studied the possible effects of M. indica as an antihypertensive drug. The results clearly indicated that the dichloromethanic fraction from the leaves of M. indica exhibited antihypertensive activity, despite the complexity of the renin–angiotensin system (RAS) with many components such as peptides and enzymes, the underlying mechanism of action may be due to ACE inhibitory activity. Moreover, dichloromethanic fraction of M. indica normalized baroreflex sensitivity, reduce the MAP and the degree of cardiac hypertrophy.

The contents of secondary metabolites present in M. indica have been studied extensively and a very high concentration of polyphenols compounds such as benzoic acid, gallic acid, propyl benzoate, methyl and propyl gallate, epicatechin, catechin and mangiferin, as the major compound, are described in the literature [Núñez-Selles et al. 2002; Barreto et al. 2008; Ajila et al. 2010; Luo et al. 2012; Liu et al. 2013]. In agreement, our results demonstrate the high content of polyphenols and flavonoids as the predominant phytoconstituents in the ethanol extract of M. indica. Quercetin, gallic acid and ferulic acid were found to be the major constituents. Historically, antioxidant activity has been associated with the presence of polyphenols [Tangney and Rasmussen, 2013] and based on AAI proposed by Scherer and Godoy [Scherer and Godoy, 2009] the dichloromethanic fraction presented a strong antioxidant activity. In addition, this antioxidant activity observed may contribute to the cardioprotective activity of M. indica extract.

Natural products are recognized as important sources of ACE inhibitors. Recently, polyphenols, mainly flavonoids, have gained a great amount of interest because of their potential for cardiovascular protection. Indeed, many epidemiological studies have correlated the consumption of foods and beverages rich in flavonoids with a reduced risk of cardiovascular disease [Guerrero et al. 2012; Umamaheswari et al. 2012]. Studies have demonstrated that certain flavonoids can have an inhibitory effect on ACE activity, which plays a key role in the regulation of arterial blood pressure [Tangney and Rasmussen, 2013]. In agreement, the dichloromethanic fraction of M. indica exhibited a high ACE in vitro inhibitory activity and those results may be associated, at least in part, with the highest presence of phenolic content, as the major constituents, in the studied fraction. The in vitro ACE inhibitory activity of dichloromethanic fraction could be evidenced in vivo by the reduced of the increase of MAP induced by the bolus application of Ang I. Interestingly, a significant decrease in the MAP was lower than that obtained after the standard captopril treatment for at least two tested doses. In a similar way green tea (Camellia sinensis L.) and Rooibos (Aspalathus linearis Dahlg.) inhibit ACE in vitro and in vivo and authors proposed a mixed inhibitor mechanism similarly to the ACE inhibitor enalaprilat [Persson, 2012].

Chronic treatment with dichloromethanic fraction of M. indica (100 mg/kg, twice a day) exhibited in vivo antihypertensive activity evidenced by reduction of the MAP in similar manner that observed after enalapril treatment. However, we cannot exclude that enalapril has been more potent than the extract, as the latter is composed of a mix of compounds. The effects of a standard aqueous extract (Vimang)® obtained from the stem bark of M. indica and the isolated mangiferin were previously investigated in vivo in WKY rats and SHRs. Vimang® extract and mangiferin exhibited anti-inflammatory activity, however only the Vimang® extract reduced the vasoconstrictor responses. The observed results in the extracts were attributed due the presence of different polyphenols in the Vimang® extract [Beltran et al. 2004]. Similar results were reported with the hydroalcoholic extract from the fruits of Nitraria sibirica Pall [Senejoux et al. 2012] and aqueous extract of Averrhoa carambola L. [Soncini et al. 2011], where the observed effects were, at least partially, correlated to the flavonoids contents.

Recent studies have suggested that baroreflex mechanisms contribute to long-term control of sympathetic activity and blood pressure [Jordan et al. 2012; Menne et al. 2013]. Head and colleagues [Head et al. 2002] showed the inverse relationship between the physiological function of the baroreflex and levels of angiotensin II (Ang II), and this fact is in agreement with our results where animals treated with dichloromethanic fraction of M. indica normalized their baroreflex sensitivity. This improvement may be related to observed inhibition of ACE and reduction of MAP. Other studies using ACE inhibitors reported the improving human baroreflex sensitivity in patients with hypertension [Mancia et al. 1982]. Using different models of hypertension, plant extracts have demonstrated to improve the baroreflex and this effect have been mainly attributed to phenolic contents [Parveen et al. 2012; Khaliq et al. 2013]. Our results corroborates with Monteiro and colleagues, who observed that quercetin, a well-known antioxidant, also demonstrated to reduce the hypertension in spontaneously hypertensive rats and improves baroreflex sensitivity via reduction of oxidative stress [Monteiro et al. 2012]. Therefore, it could be a mechanism for the cardio protection observed on SHR animals treated with M. indica.

In addition, the in vivo effects of M. indica were not limited to the inhibition of ACE, reduction of MAP and normalize the baroreflex sensitivity. Histological analysis from the hearts after chronic treatment reveal a reduction in the degree of cardiac hypertrophy in spontaneously hypertensive rats treated with dichloromethanic fraction promoted by an improvement in the HW/BW ratio. These effects are of great importance because cardiac hypertrophy can be related to both the reduction of cardiac output and to the development of heart failure [Dickhout et al. 2011].

Although we did not directly evaluate the vasodilator action of the dichloromethanic fraction of M. indica, we cannot exclude this action from the mechanism by which this extract induces its antihypertensive effect. The hypotensive assay, where the extract induces a fall in blood pressure after IV injection, similar to those induced by Ach, is an evidence of its vasodilator action. In addition, the direct relaxant effect of aqueous M. indica extract was observed by the reduction of the contractions induced by noradrenaline in ring segments of mesenteric resistance arteries in both WKY and SHR [Beltran et al. 2004]. However, further studies are necessary to properly evaluate the vasodilator action of M. indica and, as well, the mechanisms by which this action occurs [Silva et al. 2011].

Conclusion

A comprehensive study was undertaken to investigate the possible in vitro and in vivo benefits of Mangifera indica leaves extract in hypertension. Our results demonstrated that chronic treatment with M. indica of spontaneously hypertensive rats has antihypertensive effects, at least in part, via the inhibition of ACE activity, resulting in a reduction in MAP as well as a reversion of the changes in cardiac hypertrophy and baroreflex sensitivity. The presence of phenolic and flavonoids compounds at concentration in the dichloromethanic fraction may contribute to the observed biological effects. The obtained results are encouraging for further studies about the isolation and identification of the active principles, and to evaluate the possible synergism among extract components for the antihypertensive observed in this study.

Footnotes

Acknowledgements

FAPES/Brazil (Fundação de Amparo à pesquisa e inovação do Espírito Santo) is acknowledged for a research fellowship (TUA). We also thank Tommasi analítica for their assistance with the LC-MS/MS analysis. Professor Leandro Abreu for the biochemical analysis.

Funding

This work was supported by the Comissão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Conflict of interest statement

The authors have no conflicts of interest to declare.

Supplementary Material

Online Supplementary Material accompanies this paper.

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Phytochemical and in vitro and in vivo biological investigation on the antihypertensive activity of mango leaves ( Mangifera indica L.)

Abstract

Aims:

Methodology:

Results:

Conclusions:

Keywords

Introduction

Materials and methods

Plant material

Animals and cell culture

Extraction and fractionation procedure

Subfractionation of the dichloromethanic fraction

ESI(-)-FT-ICR MS analysis of SFD10

Analysis of ethanol crude extract and dichloromethanic fraction by LC MS/MS

Determination of total phenolic content and total flavonoids

DPPH radical scavenging assay

In vitro colorimetric ACE inhibition assay

In vitro cytotoxicity assay and in vivo acute toxicity

In vivo ACE inhibitory activity assay

In vivo acute hypotensive effect of dichloromethanic fraction

In vivo chronic antihypertensive effects

Hemodynamic and arterial baroreflex assessment

Evaluation of ACE activity on serum and histological analyses

Statistical analysis

Results

Total phenolic, flavonoids and tannin content

DPPH radical scavenging assay

Determination of in vitro ACE inhibitory activity of M. indica ethanol extract and partionated fractions

Identification of the main secondary metabolites by LC-ESI(-)MS/MS

Chemical identification of SFD10 by ESI(-)-FT-ICR MS and ESI(-)-FT-ICR MS/MS

In vitro cytotoxicity and in vivo acute toxicity

Acute hypotensive activity evaluation

In vivo ACE inhibitory activity

In vivo chronic antihypertensive effect

Arterial baroreflex sensitivity

Cardiac hypertrophy and body weight

Discussion

Conclusion

Footnotes

Acknowledgements

Funding

Conflict of interest statement

Supplementary Material

References