Introduction
============

Cardiac hypertrophy is the abnormal growth, or thickening, of the heart muscle, often due to chronic hypertension that leads to HF. While in left ventricular hypertrophy, the myocardial oxygen consumption is increased which results in decreased coronary blood flow reserve which results in angina pectoris, myocardial infarction, and sudden death ([@B24]). It is estimated that nearly 23 million people are affected by HF worldwide ([@B28]). In India, the prevalence of HF is around 4.6 million with an annual occurrence of nearly 1.8 million ([@B17]). Cardiac hypertrophy is a physiological adaptation which causes increased workload on the myocardium. Hypertrophic growth is due to many types of heart disease, including ischaemic heart disease, hypertension, heart failure, cardiac hypertrophy, and valvular disease ([@B10]).

Many bioactive compounds have been discovered from plants which have flavonoids, natural phenolic compounds, and phytoestrogens are strong antioxidant ([@B16]; [@B50]). Fruits and vegetables which are enriched with higher content of flavonoid prevents the development of cardiovascular disease ([@B16]). Cardiac hypertrophy is inhibited by different antioxidants caused by ROS ([@B48]; [@B42]). Quercetin (a polyphenolic flavonoid); and its analogs and its major metabolite modulate NO/cGMP-dependent vasorelaxation and regulate BP ([@B43]). Flavonoids such as Quercetin ([@B18]; [@B12]; [@B50]); Pentamethylquercetin ([@B15]); Hesperetin ([@B7]); Curcumin ([@B25]); Epigallocatechin-3 gallate (EGCG) ([@B14]); Myricetin ([@B47]) possess cardioprotective effect.

*Ulmus wallichiana* Planchon (Family: Ulmaceae), a traditional Indian medicinal plant used as astringent, demulcent, emollient, expectorant and diuretic ([@B19]). It contains quercetin analog flavonoids (2S,3S)-(+)-3′,4′,5,7-tetrahydroxydihydroflavonol-6-C-β-[D]{.smallcaps}-glucopyranoside **(K058)**; 6-Glucopyranosyl-3,3′,4′,5,7-pentahydroxyflavone **(K012)**; 6-Glucopyranosyl-4′,5,7-trihydroxyflavanone **(K068)**, and (2S,3S)-(+)-4′,5,7-trihydroxydihydroflavonol-6-C-β-[D]{.smallcaps}-glucopyranoside **(K100)** of known chemical structures (Supplementary Figure [1](#SM1){ref-type="supplementary-material"}) and composition (Supplementary Table [1](#SM1){ref-type="supplementary-material"}) ([@B37]; [@B41]). The characterization of compounds present in EE and BF of *U. wallichiana* namely K012, K058, K068, and K100 was reported by [@B27]. These flavonoids are potent for anabolic effect on osteoporotic bone ([@B40]), increases bone mass density, bone strength, and bone formation rate which enhances osteoblast differentiation and achieves peak bone in growing rats and has osteoprotective effect on ovariectomized rats ([@B41]). It also lowers blood glucose level in diabetic rats ([@B36]). The EE and BF of *U. wallichiana* contains quercetin analog flavonoids ([@B41]).

*Ulmus wallichiana* is used traditionally as diuretic ([@B19]). A related species *U. rhynchophylla*, native to China, known as Gou Teng in Chinese medicine, is used for eclampsia, headache, dizziness, convulsions, high fever, and hypertension (WHO). *U. macrocarpa* has antihypertensive and vasorelaxant activity ([@B32]). Therefore due to diuretic property of *U. wallichiana* it is used in hypertension as it contains quercetin (a polyphenolic flavonoid) and its major metabolites modulate NO/cGMP dependent vasorelaxation and regulates BP ([@B43]). Due to prolong increase in BP (hypertension) there is direct haemodynamic stress, leading to hypertrophy ([@B29]). Therefore, we have explored the effect of EE and BF of *U. wallichiana* in cardiac hypertrophy.

The effective dose of EE ranges from 500 to 750 mg/kg and that of BF ranges from 25 to100 mg/kg as reported in patent by [@B27] (co-author of this article). We have selected the above dose of EE (500 mg/kg) as it showed optimal decrease in BP. As this dose is high enough to be translated from rat to human, BF was isolated and used with effective dose of 50 mg/kg. BF enriched with high content of quercetin analog flavonoids compared to EE ([@B41]). Therefore the effective dose of EE is 500 mg/kg and that of BF is 50 mg/kg ([@B27]).

The anti-hypertrophic activity of *U. wallichiana* was remained unexplored in cardiac hypertrophy model. In our lab, we have explored the antihypertensive activity of EE and BF of *U. wallichiana* ([@B45]). Therefore, in the present work, we have investigated the anti-hypertrophic activity of EE and BF of *U. wallichiana* in ISO induced cardiac hypertrophy.

Materials and Methods {#s1}
=====================

Chemicals and Reagents
----------------------

Isoprenaline hydrochloride, Olive oil, Gum Acacia, *N*-\[3-(2-Furyl) acryloyl\]-[L]{.smallcaps}-phenylalanyl-glycyl-glycine (FAPGG), 2′,7′-Dichlorofluorescein diacetate and TRIzol reagent were procured from Sigma--Aldrich Chemical Co., St. Louis, MO, USA. Sodium Chloride was procured from Merck Specialities Private Limited, Mumbai, India. Tris (Hydroxymethyl) Aminomethane was procured from SRL Pvt. Ltd., Mumbai, India. High capacity RNA to cDNA Reverse Transcriptase kit was procured from Applied Biosystems, USA. 2X SYBR green Premix Ex Taq (Takara Bio, Japan), List of rat primers for Real-Time (RT) qPCR (Eurofins Scientific, Luxembourg) as shown in **Table [1](#T1){ref-type="table"}**, Renin ELISA Kit (Shanghai, China), Human/Mouse/Rat Angiotensin II Enzyme Immunoassay Kit (RayBiotech, Inc.), Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), cGMP Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), Renin Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) were purchased.

###### 

Primers of cardiac hypertrophy related genes for RT qPCR.

  Gene     Forward primer           Reverse primer
  -------- ------------------------ -------------------------
  rANP     GAGGAGAAGATGCCGGTAG      CTAGAGAGGGAGCTAAGTG
  rBNP     TGATTCTGCTCCTGCTTTTC     GTGGATTGTTCTGGAGACTG
  rTNF-α   CCCAGACCCTCACACTCAGAT    TTGTCCCTTGAAGAGAACCTG
  rIL-6    CCCTTCAGGAACAGCTATGAA    ACAACATCAGTCCCAAGAAGG
  rNOS3    AAAGGAAGTGCAGCAAAAGG     CCCATGAGTGAGGCAGAGAT
  rTGFβ1   CCTGGAAAGGGCTCAACAC      CAGTTCTTCTCTGTGGAGCTGA
  rACE2    TCAAGGGAAAAGAACCAGACA    GGTTTCAAATCACTCACCCATAC
  rAdrb1   AGAGCAGAAGGCGCTCAAG      AGCCAGCAGAGCGTGAAC
  rMas1    ACGTCCCCAGACCAGTCAT      TGAGGAGTTCTTGTGCTGGA
  rMMP9    CCTCTGCATGAAGACGACATAA   GGTCAGGTTTAGAGCCACGA
  rGAPDH   TGGGAAGCTGGTCATCAAC      GCATCACCCCATTTGATGTT

Plant Material
--------------

Stem bark of *U. wallichiana* was collected and authenticated by [@B4] of CSIR--Central Drug Research Institute (CDRI), Lucknow, India and extraction procedure was reported ([@B41]; [@B4]; [@B27]). EE and BF from stem bark of *U. wallichiana* were obtained from Medicinal and Process Chemistry division of CSIR--CDRI, Lucknow, India. The fingerprinting of EE and BF of *U. wallichiana* was done by us using LC-MS/MS -- Mass spectrometer QTrap 5500-AB (Applied Biosystems/MDS Sciex, Canada), using an electrospray ionization source in negative ion mode (ESI) and published ([@B45]).

Animals
-------

The experiments were carried out with male normotensive wistar rats (180--220 g) procured from the Laboratory Animal Division of CSIR--CDRI, Lucknow, India. Experiments were performed according to protocols approved by Institutional Animal Ethics Committee \[(IAEC) no. IAEC/2012/92N\] of CSIR--CDRI and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. Rats were maintained under standard housing conditions (room temperature 25 ± 2°C and humidity 60 ± 5%) with a 12 h light and dark cycle. Food and water were available *ad libitum*.

Study Protocols
---------------

Male wistar rats were injected subcutaneously (s.c.) with ISO 5 mg/kg/day for 10 days ([@B51]) with or without 500 mg/kg/day of EE and 50 mg/kg/day of BF of *U. wallichiana*. The rats were randomly divided into four groups and each group contains six animals each.

1.  Control group: olive oil (vehicle) was given s.c. for 10 days.

2.  ISO group: Isoprenaline, 5 mg/kg/day was administered subcutaneously for 10 days, to induce cardiac hypertrophy.

3.  ISO + EE group: EE was administered orally for 14 days at a dose of 500 mg/kg/day of *U. wallichiana* after development of cardiac hypertrophy.

4.  ISO + BF group: Oral administration of BF for 14 days at a dose of 50 mg/kg/day of *U. wallichiana* after development of cardiac hypertrophy.

Blood Pressure Measurement
--------------------------

### NIBP Measurement

To measure changes in arterial blood pressure in conscious rat, NIBP technique was used (Columbus instrument, Model NIBP-8). Conscious rats were acclimatized in the restrainer for 15 min three times a day for a period of 1-week before the starting of the testing schedule. The trained animals were placed in a restrainer and tails of rats were exposed to hot air blower. The rats were allowed to acclimatize inside the cage for 15 min before starting the actual blood pressure measurement. Occlusion and sensor cuffs were placed around the base of the tail and the cuffs were inflated and deflated several times to measure tail arterial blood pressure. A minimum of 8--10 cycles of inflation and deflation were made and the measurement of tail arterial blood pressure was recorded and the data was averaged. The data were recorded and analyzed with software.

### IBP Measurement

Arterial blood pressure was measured by invasive method in the urethane (1.2 g/kg) anesthetized rats. The tracheotomy was performed. The catheter was inserted into the carotid artery to monitor the alteration in the arterial blood pressure. The other end of the catheter was connected to the computerized Data Acquisition System (DAS), AD-Instruments with compatible software.

Determination of Heart Weight to Body Weight Ratio, Heart Weight to Tail Length Ratio, and Heart Weight to Tibia Length Ratio
-----------------------------------------------------------------------------------------------------------------------------

After the measurement of IBP the rats were sacrificed and heart weight, tail length, and tibia length were measured. The heart weight index was calculated by dividing the heart weight by the body weight ([@B51]), the heart tail index was calculated by dividing the heart weight by tail length ([@B6]), and the heart tibia index was calculated by dividing the heart weight by tibia length ([@B31]).

Renin Inhibition Assay
----------------------

The renin inhibition assay was performed by Renin Inhibitor Screening Assay Kit (catalog number: 10006270; Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. The renin inhibition was expressed as percentage inhibition or percent initial activity as a function of the inhibitor concentration to determine the IC~50~ value.

Estimation of Plasma Renin Level in Plasma
------------------------------------------

The plasma renin assay was measured by rat Renin (Renin) ELISA kit (catalog number: E02R0022; BlueGene Biotech, Shanghai, China) according to the manufacturer's instructions. To determine renin level, the blood was collected from the retro--orbital plexus of rat in EDTA containing tubes. Plasma was harvested by using centrifugation at 1000 × *g* for 15 min at 4°C. Plasma (100 μL) was used for estimation of renin concentration.

Angiotensin Converting Enzyme (ACE) Assay
-----------------------------------------

Angiotensin converting enzyme activity was determined by a spectrophotometric mono-reagent assay. The reaction mixture containing serum (10 μL) and the substrate solution (200 μL) were kept at 37°C in Elisa plate reader (BIOTEK, USA) and reaction was monitored at 340 nm for 30 min. Serum ACE activity was expressed as unit/L ([@B13]).

Determination of Angiotensin II (Ang II) Level in Plasma
--------------------------------------------------------

The plasma AG II level was estimated by Human/mouse/Rat Ang II Enzyme Immunoassay Kit (catalog\#: EIA-ANGII, EIAM-ANGII, EIAR-ANGII; RayBiotech, Inc.) according to the manufacturer's instructions.

Estimation of Nitrate/Nitrite (NO) Level in Plasma
--------------------------------------------------

The plasma NO was estimated by Nitrate/Nitrite Colorimetric Assay Kit (catalog number: 780001; Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. To determine nitrate/nitrite level in plasma, 40 μL of plasma was taken and diluted with 40 μL of Assay Buffer to adjust the volume to 80 μL of sample and used for NO determination.

Estimation of cGMP Level in Plasma
----------------------------------

The cGMP concentrations were estimated using an enzyme immunoassay (EIA) kit (catalog number: 581021; Cayman Chemical, Ann Arbor, MI, USA), as per the manufacturer's instructions. To determine the cGMP levels in plasma, 2 mL of ice-cold ethanol was added to 500 μL of plasma, and the solution was centrifuged at 1500 × *g* for 10 min. The supernatant was evaporated by vacuum centrifugation (Turbo Vap LV, Caliper, USA) and used for the cGMP estimation.

Determination of Reactive Oxygen Species (ROS)
----------------------------------------------

Reactive oxygen species was estimated in heart tissue. Heart tissue was homogenized in ice-cold 40 mMtris buffer (pH 7.4) and samples were centrifuged at 100,000 × *g* at 4°C in an Eppendorf centrifuge (5819 R Germany) for 15 min. The resulting heart homogenate was incubated with DCFDA (5 μM) for 30 min at 37°C. Formation of fluorescent product DCF was monitored by fluorometer at an excitation wavelength of 488 nm and emission wavelength of 530 nm.

Histopathological Examination
-----------------------------

After measurement of hemodynamic parameters of rats, their hearts were quickly and filled with 4% paraformaldehyde solution, and kept in it for 24 h. The heart samples were embedded in paraffin blocks. Samples were sliced of 5 μm thickness and stained with hematoxylin and eosin (H&E) for assessing myocardial fibrosis. Myocardial fibrosis and relative cell surface area were evaluated in each of the heart tissue sections using a morphometric procedure using ImageJ software.

RNA Extraction and Relative Gene Expression Analysis by Real-Time PCR
---------------------------------------------------------------------

### Total RNA Extraction

Total RNA was extracted from 0.1 to 0.2 g of rat heart tissues by using TRIzol reagent as per manufacturer's protocol. The quality and quantity of the isolated RNA were assessed using the 260/280 nm absorbance ratio (1.8--2.0 indicates a highly pure sample) using NanoDrop 2000c spectrophotometer (USA). Total RNA was stored at -80°C until used.

### Synthesis of cDNA and RT qPCR Quantification

RNA was transcribed into cDNA in a 20 μL reaction using a High Capacity RNA to cDNA Reverse Transcriptase kit, analyzed, and amplified. 2x master mixes were prepared using 2 μl 10x Reverse Transcription buffers, 0.8 μL 25x dinucleotide triphosphate (dNTP) mix (100 mM), 2 μL 10x Reverse Transcription random primers, and 1 μL MultiScribe Reverse Transcriptase. For a 20 μL reaction mixture under the following conditions: 5.8 μL master mix; 2 μL of 2 μg RNA sample; 12.2 μL nuclease free water. Following was the program for reverse transcriptase PCR using SureCycler 8800 (Agilent Technologies, USA): 25°C for 10 min; 37°C for 120 min; 85°C for 5 min; hold at 4°C. The product obtained was diluted 4× for real-time PCR. Samples were seeded in triplicate in 96-well reaction plates (LightCycler 480). RT qPCR was performed on Light Cycler 480 II (Roche Diagnostics) with SYBR Green fluorescent label. Samples (10 μL final volume) contained the following: 2x SYBR Premix Ex Taq 1 μL of each primer; 3 μL of cDNA sample, and 5 μL of nuclease free water. Cycle threshold (Ct) values were used to calculate the amount of amplified PCR product relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control (reference gene).

Statistical Analysis
--------------------

Statistical analysis was performed with Prism software version 5.01 (Graph Pad Software, San Diego, CA, USA) and Curve Expert Professional version 2.0.3. Results are expressed as mean ± SEM. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Newman--Keuls Multiple comparison test and *t*-test. A value of *p* \< 0.05 was considered to be statistically significant.

Results
=======

*U. wallichiana* Attenuated Heart Weight to Body Weight Ratio, Heart Weight to Tail Length Ratio, and Heart Weight to Tibia Length Ratio
----------------------------------------------------------------------------------------------------------------------------------------

Heart to body weight, heart weight to tail length, and heart weight to tibia length ratio were significantly increased in the ISO groupas compared to control group. The treatment of extract and fraction of *U. wallichiana* significantly decreased heart to body weight, heart weight to tail length, and heart weight to tibia length ratio in ISO+EE and ISO+BF group compared to disease control (**Figures [1A--C](#F1){ref-type="fig"}**), respectively. The images of whole heart have been provided in the Supplementary Figure [3](#SM1){ref-type="supplementary-material"}.

![**Effect of *Ulmus wallichiana* on (A)** Heart weight to body weight ratio, **(B)** Heart weight to tail length ratio, and **(C)** Heart to tibia length ratio in ISO induced cardiac hypertrophy in wistar rats. Data were analyzed by using one-way ANOVA followed by Newman--Keuls Multiple comparison test. Data are shown as Mean ± SEM; α, control vs. ISO; β, ISO vs. EE; γ, ISO vs. BF; ^∗^*P* \< 0.05, ^∗∗^*P* \< 0.01, ^∗∗∗^*P* \< 0.001.](fphar-07-00510-g001){#F1}

Hemodynamics
------------

Blood pressure and HR were significantly increased in ISO induced cardiac hypertrophy as compared to normotensive Wistar group (**Figures [2A--C](#F2){ref-type="fig"}**). The treatment of extract and fraction of *U. wallichiana* significantly decreased the SBP in ISO+EE and ISO+BF compared to diseased control (**Figure [2A](#F2){ref-type="fig"}**) and it also decreased the HR in ISO+EE and ISO+BF compared to diseased control (**Figure [2D](#F2){ref-type="fig"}**), respectively. (In support IBP data is given in Supplementary Figures [2A](#SM1){ref-type="supplementary-material"}--[D](#SM1){ref-type="supplementary-material"}.)

![**Effect of *U. wallichiana* on BP (A)** SBP, **(B)** DBP, **(C)** MAP, and **(D)** HR by NIBP measurement technique in ISO induced cardiac hypertrophy in wistar rats. Data were analyzed by using one-way ANOVA followed by Newman--Keuls Multiple comparison test. Data are shown as Mean ± SEM; α, control vs. ISO; β, ISO vs. EE; γ, ISO vs. BF; ^∗∗^*P* \< 0.01, ^∗∗∗^*P* \< 0.001.](fphar-07-00510-g002){#F2}

Renin Inhibition
----------------

Renin inhibition assay *in vitro* was conducted to assess the renin inhibitory properties of EE and BF of *U. wallichiana*. The IC~50~ value for EE was found to be 12.5 μg/mL and that of BF was 5.5 μg/mL (**Figures [3E,F](#F3){ref-type="fig"}**)

![**Effect of *U. wallichiana* on (A)** renin concentration, **(B)** angiotensin converting enzyme (ACE) activity, **(C)** NO concentration, **(D)** Ang II concentration, **(E)** inhibition of human recombinant renin by EE and **(F)** inhibition of human recombinant renin by BF in ISO induced cardiac hypertrophy in wistar rats. Data were analyzed by using one-way ANOVA followed by Newman--Keuls Multiple comparison test. Data are shown as Mean ± SEM; α, control vs. ISO; β, ISO vs. EE; γ, ISO vs. BF; ^∗^*P* \< 0.05, ^∗∗^*P* \< 0.01, ^∗∗∗^*P* \< 0.001.](fphar-07-00510-g003){#F3}

*U. wallichiana* Reduced Plasma Renin Concentration
---------------------------------------------------

The plasma renin concentration was increased significantly in ISO group. Administration of EE and BF of *U. wallichiana* significantly decreased plasma renin concentration in ISO+EE and ISO+BF group compared to diseased control (**Figure [3A](#F3){ref-type="fig"}**) group, respectively.

*U. wallichiana* Decreased ACE Activity
---------------------------------------

The plasma ACE activity was increased significantly in ISO group. Further administration of EE and BF of *U. wallichiana* significantly decreased ACE activity in ISO+EE and ISO+BF group compared to diseased control (**Figure [3B](#F3){ref-type="fig"}**) group, respectively.

*U. wallichiana* Attenuated AG II Concentration in Plasma
---------------------------------------------------------

The plasma Ang II level was increased significantly in ISO group. Further administration of EE and BF of *U. wallichiana* significantly decreased ACE activity in ISO+EE and ISO+BF compared to diseased control (**Figure [3D](#F3){ref-type="fig"}**) group, respectively.

*U. wallichiana* Increased Plasma Nitrite/Nitrate (NO) Concentration
--------------------------------------------------------------------

The concentration of NO was decreased significantly in ISO group. Further administration of EE and BF of *U. wallichiana* significantly increased NO in ISO+EE and ISO+BF group compared to diseased control (**Figure [3C](#F3){ref-type="fig"}**) group, respectively.

*U. wallichiana* Increased cGMP Concentration
---------------------------------------------

The concentration of cGMP was decreased significantly in ISO group. Further administration of EE and BF of *U. wallichiana* significantly increased cGMP concentration in ISO+EE and ISO+BF group compared to diseased control (**Figure [4A](#F4){ref-type="fig"}**) group, respectively.

![**Effect of *U. wallichiana* on (A)** cGMP concentration, **(B)** fluorescence intensity, **(C)** myocardial fibrosis, **(D)** relative cell surface area, and **(E)** relative mRNA expression level of ANP, BNP, IL-6, NOS3, TNF-α, TGFβ1, MMP9, β1 adrenergic receptor (β1 AR), ACE2 and Mas in ISO induced cardiac hypertrophy in wistar rats. Data were analyzed by using one-way ANOVA followed by Newman--Keuls Multiple comparison test. Data are shown as Mean ± SEM, α, control vs. ISO; β, ISO vs. EE; γ, ISO vs. BF; ^∗^*P* \< 0.05, ^∗∗^*P* \< 0.01, ^∗∗∗^*P* \< 0.001.](fphar-07-00510-g004){#F4}

*U. wallichiana* Attenuated ROS Level
-------------------------------------

The ROS activity was increased significantly in ISO group. Further administration of EE and BF of *U. wallichiana* significantly increased ROS activity in ISO+EE and ISO+BF group compared to diseased control (**Figure [4B](#F4){ref-type="fig"}**) group, respectively.

*U. wallichiana* Reduced Expression of Cardiac Hypertrophic Markers
-------------------------------------------------------------------

To investigate whether EE and BF of *U. wallichiana* confers cardio-protection in ISO treated group. We have determined the cardiac gene expression of ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR, ACE2, Mas relative to ISO treated group. ISO caused significant up regulation of ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR and down regulation of NOS3, ACE2, Mas as compared to control group. On treatment with the EE and BF of *U. wallichiana* the ANP, BNP, TNF-α, IL-6, TGFβ1, MMP9, β1-AR was significantly reduced in ISO+EE and ISO+BF group and significantly increased in NOS3, ACE2, Mas expression as compared to diseased control group, respectively, (**Figure [4E](#F4){ref-type="fig"}**).

*U. wallichiana* Decreased Cardiac Fibrosis and Relative Cell Surface Area
--------------------------------------------------------------------------

The regularly arranged myocardial fibers with clear striations were observed in the control group (**Figure [5A](#F5){ref-type="fig"}**). The photomicrograph of heart from normal group shows no pathological alteration. The histological sections of the heart obtained from ISO group showed enlargement of cardiomyocyte showing increased cell radius with eccentric positioning of nucleus indicating hypertrophy. Fibrocytic proliferation was also invariably observed along with cellular degenerative changes characterized by scanty cytoplasm and vacuolation. Focal infiltration of inflammatory cells is observed in few animals. There was significant increase in myocardial fibrosis in ISO group (**Figure [5B](#F5){ref-type="fig"}**). There was also significant increase in relative cell surface area in ISO group. On administration of EE and BF of *U. wallichiana* significantly decreased myocardial fibrosis in ISO+EE and ISO+BF group compared to diseased control (**Figures [4C](#F4){ref-type="fig"}** and **[5C,D](#F5){ref-type="fig"}**); decreased relative cell surface area in ISO+EE and ISO+BF group as compared to diseased control (**Figures [4D](#F4){ref-type="fig"}** and **[5C,D](#F5){ref-type="fig"}**) group, respectively. A representative photo has also been given in the Supplementary Figure [4](#SM1){ref-type="supplementary-material"}.

![**Representative photomicrographs of heart dyed with haematoxylin and eosin (H&E) stain (10× and 40× magnification). (A)** Control, **(B)** ISO, **(C)** ISO+EE, **(D)** ISO+BF groups, respectively.](fphar-07-00510-g005){#F5}

Discussion
==========

In the present study, the *U. wallichiana* attenuated cardiac hypertrophic response induced by ISO, a non-selective β-adrenoceptor agonist. The EE and BF of *U. wallichiana* attenuated cardiac hypertrophy by decreasing SBP and HR and inhibiting plasma renin, ACE, Ang II and augmented plasma NO and cGMP concentration (Supplementary Table [2](#SM1){ref-type="supplementary-material"}). The EE and BF of *U. wallichiana* have antihypertensive activity ([@B45]). ISO in chronic dose not only produced cardiac hypertrophy but also altered the gross structure of the heart. There was an increase in weight of the heart, but no significant effect on weight of the body. The effect of EE and BF of *U. wallichiana* was studied as anti-hypertrophic. The dose of EE (500 mg/kg) and BF (50 mg/kg) of *U. wallichiana* contains quercetin analog flavonoids K058: 9.79 and 1.34 mg, K012: 4.33 and 0.24 mg, K068: 0.625 and 0.15 mg, and K100: 2.49 and 0.41 mg, respectively.

The EE and BF of *U. wallichiana* decreased the heart rate, renin release, and Ang II level there by decreasing the cardiac output. ISO, a directly acting catecholamine, binds with the β-adrenergic receptor in heart and causes significant increase in heart rate ([@B3]), increased plasma renin and Ang II level ([@B23]). ISO, a sympathetic β-adrenergic receptor agonist, stimulates renin release via adenylate cyclase-cyclic AMP system ([@B44]). The stimulatory effect of ISO on renin release was inhibited by the EE and BF of *U. wallichiana* due to the presence of quercetin analog flavonoids. Renin overproduction increases the level of Angiotensin I in the blood, leading to high BP. The IC~50~ value for EE was found to be 12.5 μg/mL and that of BF was 5.5 μg/mL, showing renin inhibition in the present study.

Cardiac mast cell secretes renin initiating the formation of angiotensinogen. Cardiac mast cells releases renin on degranulation by ROS. Renin initiates the activation of a local renin-angiotensin system which culminates in the formation of Ang II ([@B38]). The quercetin analog flavonoids acts as antioxidant and inhibits release of pro-inflammatory cytokines, IL-6 from mast cell. Therefore the EE and BF of *U. wallichiana* attenuates the ROS production, due to its antioxidant activity, inhibiting the TNF-α and IL-6 and it has renin inhibition property (**Figures [3E,F](#F3){ref-type="fig"}**). Thus EE and BF may be responsible to inhibit the release of renin from the cardiac mast cells.

Angiotensin converting enzyme, an enzyme that regulates RAAS pathway, contains zinc and a zinc ion ([@B33]). The flavonoids quercetin analog forms chelate complex with zinc and inhibit the ACE enzyme ([@B1]; [@B11]). The ACE2 and Mas receptors are involved in the cardiac hypertrophy as their reduced expression is found in hypertrophied heart ([@B52]). ACE2, by degradation of Ang II, forms Ang (1--7) which opposes actions of Ang II by binding to the Mas receptor leading to vasodilation, anti-hypertrophic and anti-fibrotic effect ([@B8]). The EE and BF of *U. wallichiana* increased the ACE2 and Mas receptor gene expression as compared to the ISO group.

In RAS pathway, increased renin and ACE enzyme causes increased level of Ang II. Angiotensin II activates NAD(P)H oxidase leading to oxidative stress ([@B49]) causing decrease in bioavailability of NO resulting in endothelial dysfunction and vasoconstriction of arterioles leading to hypertension. Emerging evidence suggests that the nitricoxide (NO)/guanosine 3′5′-cyclic monophosphate (cGMP) pathway plays an important role in the modulation of cardiac hypertrophy ([@B34]). Therefore the EE and BF of *U. wallichiana* increased the bioavailability of NO/cGMP leading to vasodilation and act as anti-hypertrophic ([@B46]). As NO has anti-hypertrophic effect which is mediated by cGMP which involves cGMP-dependent protein kinase I activation. Plasma NO level was decreased by ISO, an indirect indicator of endogenous NO production.

NO/sGC/cGMP/cGMP dependent protein kinase G acts as cardioprotective. *U. wallichiana* causes increase in the NO level, probably via signal transduction pathway which results in cGMP generation. Cardiovascular diseases are associated with the impairment of Nitric oxide/soluble guanylate cyclase/cGMP signaling ([@B20]). NO/cGMP/ANP act as anti-hypertrophic. ANP and BNP are present in cardiac tissues ([@B26]), and act as cardiac hypertrophic markers ([@B2]).

The cardiac mast cell induces MMP activation due to oxidative stress as well as TNF-α which causes degradation of collagen causing cardiac fibrosis in cardiac hypertrophy ([@B22]; [@B35]). ROS also have potent effects on the extracellular matrix, stimulating cardiac fibroblast proliferation and activating MMPs. EE and BF of *U. wallichiana* attenuates the increased expression of TGFβ1, MMP9, and ROS activity. TNF-α, IL-6 are cytokines which mediates various physiological and pathological functions like inflammation, cellular survival, growth, differentiation, and apoptosis. TNF-α in the myocardium produce inflammation and apoptosis via TNFR1 receptor ([@B39]) where as IL-6 also produce cardiac hypertrophy, fibrosis, and inflammation ([@B9]; [@B30]). The flavonoids and quercetin analogs present in EE and BF of *U. wallichiana* act as antioxidant ([@B21]; [@B5]) thus decreasing ROS activity.

Conclusion
==========

We suggest that the quercetin analog flavonoids present in the EE and BF of *U. wallichiana* attenuated cardiac hypertrophy (**Figure [6](#F6){ref-type="fig"}**) showing cardioprotective effect. The BF showed pharmacological response and potent at a lower dose (50 mg/kg/day) as compared to EE (500 mg/kg/day) as BF contains higher concentration of flavonoids than EE ([@B41]).

![**Schematic diagram showing the mechanism of action of EE and BF of *U. wallichiana* in ISO induced cardiac hypertrophy.** ACE, angiotensin converting enzyme; cGMP, cyclic guanosine monophosphate; NOS, nitric oxide synthase.](fphar-07-00510-g006){#F6}

Thus, the EE and BF of *U. wallichiana* showed cardioprotective effect against ISO induced cardiac hypertrophy. Based on these findings the marker compounds present in *U. wallichiana* can be isolated to study the mechanism of action and it may be a novel therapeutic agent for treatment of cardiac hypertrophy.

Author Contributions
====================

AS, KH, and JG designed the research work; AS, SL, DM, GV, AG, and SK performed the research experiments; RM, HB, KH, and JG contributed new reagents/analytic tools; AS, SL, DM, KH, HB, RM, and JG analyzed data; AS, KH, and JG wrote the paper.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** JG is thankful to Department of Science and Technology, SERB (Govt. of India), Department of Biotechnology (Govt. of India), and CSIR (Govt. of India) for financial support \[GAP0114, BSC0103, BSC0201\].

The authors are thankful to the Director, CSIR-CDRI for his/her constant encouragement, support, and funding. RM is thankful to CSIR for Emeritus Scientist award. CDRI communication number: 9392.

Supplementary Material
======================

The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fphar.2016.00510/full#supplementary-material>

###### 

Click here for additional data file.

BF

:   butanolic fraction

BNP

:   brain natriuretic peptide

BP

:   blood pressure

cGMP

:   cyclic guanosine monophosphate

DBP

:   diastolic blood pressure

DCFDA

:   2′,7′-Dichlorodihydrofluorescein diacetate

DCF

:   2′,7′-Dichlorofluorescein

EE

:   ethanolic extract

FAP

:   N-\[3-(2-furyl) acryloyl\]-[L]{.smallcaps}-phenylalanine

GG

:   glycylglycine

HF

:   heart failure

HR

:   heart rate

IBP

:   invasive blood pressure

IL-6

:   Interleukin 6

ISO

:   isoprenaline

MAP

:   mean arterial pressure

NIBP

:   non-invasive blood pressure

NO

:   total nitrate/nitrite

qPCR

:   quantitative polymerase chain reaction

RAAS

:   Renin-angiotensin-aldosterone system

ROS

:   reactive oxygen species

SBP

:   systolic blood pressure

TNF-α

:   tumor necrosis factor alpha

[^1]: Edited by: *Kalin Yanbo Zhang, University of Hong Kong, Hong Kong*

[^2]: Reviewed by: *Xiao Yu Tian, The Chinese University of Hong Kong, Hong Kong; Feng Feng, China Pharmaceutical University, China*

[^3]: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology
