DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2017.06

Research Article

Antihypertensive activity of Beta vulgaris on dexamethasone induced hypertension in rats

Dignesh Patel1, Rupali Patil1, Alkesh Patel2*

1Department of Pharmacology, M. G. V.'S Pharmacy College, Panchavati, Nashik, India

2Department of Pharmacology, Parul Institute of Pharmacy, Vadodara, India

*For correspondence

Mr. Alkesh Patel,

Department of Pharmacology, Parul Institute of Pharmacy, Vadodara, India. Email: alkesh.patel@ paruluniversity.ac.in

 

 

 

 

 

 

 

 

 

 

Received: 29 December 2016

Revised: 16 January 2017

Accepted: 27 January 2017

ABSTRACT

Objective: Investigation of methanolic extract for detecting the presence of Betanin by phytochemical analysis, to induce experimental hypertension and to carry out measurement of BP by invasive (direct) method (IBP) and vascular reactivity to various catecholamines after completion of treatment schedule.

Methods: The animals were divided into six main groups. Each group contains 5 animals. Group I received vehicle , group II received dexamethasone injection (20 µg/kg/day, s.c.) for 14 days , group III received extract of Beta vulgaris (100 mg/kg/day, p.o.) for 14 days , group IV received extract of Beta vulgaris (300 mg/kg/day, p.o.) for 14 days , group V received dexamethasone injection (20 ug/kg/day, s.c.) and extract of Beta vulgaris (100 mg/kg/day, p.o.) for 14 days and group VI received dexamethasone (DEXA) injection (20 µg/kg/day, s.c.) and extract of Beta vulgaris (300 mg/kg/day, p.o.) for 14 days. After 14 days of dosing period body weight, ECG and changes in vascular reactivity to various catecholamines were recorded using Powerlab 4SP (AD Instrument, Australia).

Results: Animals treated with dexamethasone along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in heart rate compared to Dexamethasone treated group. Dexamethasone administered rats showed a significant elevation (p<0.05) in systolic blood pressure (SBP), vascular reactivity changes to Catecholamine as compared to control group. Beta vulgaris extract (100, 300 mg/kg/day, p.o.) treatment for 14 days in dexamethasone administered rats significantly (p<0.05) reduced systolic blood pressure, vascular reactivity changes to catecholamines as compared to dexamethasone administered group.

Conclusions: The methanolic-HCl extract of Beta vulgaris has antihypertensive activity in dexamethasone model.

Keywords: Hypertension, Dexamethasone, Beta vulgaris

Introduction

Hypertension is defined conventionally as a sustained increase in blood pressure up to 140/90 mmHg. Hypertension is the prevalent cause of cardiovascular disease that leads to heart failure, stroke, renal failure and ultimately death. The syndrome of hypertension is more than just an elevation of arterial pressure, although it is this aspect that the general public is most familiar. The occurrence of hypertension increases with old age. About 50% of people between the ages of 60 and 69 years old found with hypertension, and the prevalence is further increased beyond age 70.1 The striking impact of aging was seen among participants in the Framingham Heart Study in that normotensive person at either age 55 or 65 after 20 years follow-up it was found that, hypertension developed in 90% of those who were now aged 75 or 85 (providing 2 cohorts).2 Hypertension caused 57% of all stroke deaths and 24% of all coronary heart disease deaths in India.3

High blood pressure (BP) is estimated to account for 6% of deaths worldwide and is the most common treatable risk factor for cardiovascular disease (CVD).4 During the last 3 decades, hypertension therapy has improved well, contributing to a decrease in the incidence of mortality due to stroke and coronary heart disease (CHD).

According to analysis of worldwide data for the global burden of Hypertension, 20.6% of Indian men and 20.9% of Indian women were suffering from HTN in 2005 that is expected reach up to 22.9%and 23.6% for Indian men and women, respectively by 2025.5

Materials and Methods

Animals

Adult male albino Wistar rats of either sex, weighting 150-200 g, obtained from Bharat Serum and Vaccines Ltd., Thane, India, were used for the study. They were housed in polypropylene cages lined with husk, renewed every 48h under 12:12 h light dark cycle at around 25 ± 5°C. They were fed with commercial pellet rat chow and given water ad libitum. The experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, and approved by the MGV's Institutional Animal Ethics Committee (Protocol number: MGV/PC/XXVI/01/2011-12).

Drugs and chemicals

Adrenaline (Adr), noradrenaline (NA), phenylephirne (PE), serotonin (5-HT) and Urethane were purchased from Sigma-Aldrich, st. Louis, MO, USA. All other chemicals used in the study were of analytical grade.

Plant

Roots of Beta vulgaris were obtained from local market, Nasik and were identified by Dr. J. Jayanti, Scientist, Botanical Survey of India, Pune where a voucher specimen (DRP-1) has been retained.

Preparation of extract6

The extraction was carried out by maceration process. Roots of Beta vulgaris (1 kg) were purchased from local market, cleaned with water. The roots were uniformly chopped. The chopped pieces were subjected to maceration with 1% methanolic HCl for two days with frequent shaking the macerating flask. The macerated product was then air dried for removal of methanol (yield: 5.2% w/w).

Phytochemical investigation6

  1. The extracted samples were heated in 2 M HCl for 5 min at 100º C. The colour was vanished, this showed the presence of betacyanin.
  2. To the extracted sample solution 2M NaOH was added drop wise and the colour changed to yellow, which shows the presence of betacyanin.

Acute toxicity study

Acute toxicity study was performed according to OECD guideline 425 for oral dose in that Beta vulgaris extract dissolved in water and administered at 2000 mg/kg dose and observed. After administration, Irwin's test was conducted in which animals were observed for gross behavioral changes like responsiveness, awareness, irritability, passivity, grooming, and restlessness with neurological profile like spontaneous motor activity, reactivity and tremor, touch and pain stimuli response was also evaluated. Autonomic profile like writhing, defecation and urination was evaluated.

Experimental design

The animals were divided into six main groups. Each group contains 5 animals. Group I received vehicle, group II received dexamethasone injection (20 mcg/kg/day, s.c.) for 14 days, group III received extract of Beta vulgaris (100 mg/kg/day, p.o.) for 14 days, group IV received extract of Beta vulgaris (300 mg/kg/day, p.o.) for 14 days, group V received dexamethasone injection (20 mcg/kg/day, s.c.) and extract of Beta vulgaris (100 mg/kg/day, p.o.) for 14 days and group VI received dexamethasone injection (20 mcg/kg/day, s.c.) and extract of Beta vulgaris (300 mg/kg/day, p.o.) for 14 days. After 14 days of dosing period body weight, ECG and changes in vascular reactivity to various catecholamines were recorded using Powerlab 4SP (AD Instrument, Australia).

Estimation of parameters

a. Measurement of invasive blood pressure

After completion of treatment schedule, rats from each group were anesthetized with urethane (120 mg/100 g). Tracheotomy was performed; left common carotid artery was cannulated and means arterial blood pressure was recorded using pressure transducer direct method on chart data system (Power Lab/4sp AD Instrument, Australia). Heparinized saline (100 IU/ml) was filled in the transducer and in the fine polyethylene catheter cannulated to the carotid artery to prevent clotting. After 15 min of stabilization, mean arterial blood pressure and heart rate were recorded.7

b. Pressure-rate index (PRI)

The PRI, a parameter used as an index of myocardial oxygen demand, was calculated as the product of MABP x HR/10008.

c. Vascular reactivity to Catecholamines9

The animals were anesthetized with urethane (120 mg/100 gm). The body temperature was monitored and maintained at 37°C during the experimental protocol. The neck was opened with a ventral midline incision to perform tracheotomy and rat was ventilated with room air. The left femoral vein was used for infusion of saline and various catecholamines during the experiment. The right carotid artery was cannulated and the cannula was filled with heparinized saline (100 IU/ml) and using pressure transducer the systolic blood pressure was recorded. The animals were allowed to stabilize for 15 min before recording the ECG and heart rate and after that vascular reactivity to Adr (1 µg/kg) , NA (1 µg/kg), PE (1 µg/kg) and 5-HT (1 µg/kg) were recorded.

Results and Discussion

Acute toxicity study

At the dose of 2000 mg/kg, no any sign of toxicities were found in all animals.

Heart rate

The heart rate in control animals was recorded as 237.7 ± 13.74 beats per minute. The DEXA administered group exhibited significant increase in heart rate compared to control animals. Animals treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in heart rate compared to DEXA treated group.

Figure 1: Effect of Beta vulgaris on heart rate in dexamethasone induced hypertension in rats.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris.

Figure 2: Effect of Beta vulgaris on mean invasive blood pressure in dexamethasone induced hypertension in rats.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. MIBP: Mean invasive blood pressure.

Measurement of invasive blood pressure (mean arterial blood pressure)

The blood pressure in control animals was recorded as 73.6 ± 5.87 mmHg. The DEXA administered group showed significant increase in the blood pressure compared to control animals. No significant alterations in blood pressure were observed in the animals treated with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) compared to control animals. Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Pressure-rate index (PRI)

The PRI in control animals was recorded as 17.68 ± 2.124 mmHg/min. The DEXA administered group significantly increases the PRI as compared to control animals. Animals treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in PRI compared to DEXA treated group.

Figure 3: Effect of Beta vulgaris on pressure rate index in dexamethasone induced hypertension in rats.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. PRI: Pressure rate index.

Vascular Reactivity to Catecholamines

a. Effect of Beta vulgaris on adrenaline (1 mcg/kg i.v.) induced change in vascular reactivity.

The increase in blood pressure by adrenaline (1 mcg/kg i.v.) in control animals was recorded at 78.2 ± 2.728 mmHg. The DEXA administered group exhibited significant increase in the blood pressure compared to control animals. No significant alterations were found in animals treated with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days). Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

b. Effect of Beta vulgaris on noradrenaline (1 mcg/kg i.v.) induced change in vascular reactivity.

The increase in blood pressure by NA (1 mcg/kg i.v.) in control animals was recorded as 92.4 ± 2.977 mm of Hg. The DEXA administered group exhibited significant increase in the blood pressure compared to control animals. Animal group treated with Beta vulgaris (300 mg/kg p.o. for 14 days) per se showed significant decrease in blood pressure compared to control group. Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Figure 4: Effect of Beta vulgaris on adrenaline (1 mcg/kg i.v.) induced change in vascular reactivity.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. MIBP: Mean invasive blood pressure.

Figure 5: Effect of Beta vulgaris on noradrenaline (1 mcg/kg i.v.) induced change in vascular reactivity.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. MIBP: Mean invasive blood pressure.

c. Effect of Beta vulgaris on phenylephrine (1mcg/kg i.v) induced change in vascular reactivity

The increase in blood pressure by PE (1 mcg/kg i.v.) in control animals was recorded as 91 ± 3.194 mmHg. The DEXA administered group exhibited significant increase in the blood pressure compared to control animals. Animal group treated with Beta vulgaris (300 mg/kg p.o. for 14 days) per se showed significant decrease in blood pressure compared to control group. Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg, p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Figure 6: Effect of Beta vulgaris on phenylephrine (1 mcg/kg i.v) induced change in vascular reactivity.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. MIBP: Mean invasive blood pressure.

d. Effect of Beta vulgaris on 5-HT (1 mcg/kg i.v.) induced change in vascular reactivity.

The increase in blood pressure by 5-HT (1 mcg/kg i.v.) in control animals was recorded as 59 ± 4.794 mm of Hg. The DEXA administered group exhibited significant increase in the blood pressure compared to control animals. Animal group treated with Beta vulgaris (300 mg/kg p.o. for 14 days) showed significant decrease in blood pressure compared to control group. Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Hypertension is the most common primary diagnosis and most common cause of death in the United States. The incidence and prevalence of hypertension is about 50% higher in African-American adults compared with their counterparts who are white or Mexican-American.10 It is estimated that 50 million Americans are affected by hypertension. Of these 50 million, only 70% (35 million) are aware of their condition. In addition, only 50% (17.5 million) of those aware of their condition are receiving treatment. Remarkably, only 25% of all hypertensive patients have their blood pressure under control.11 The life-long risk of developing hypertension in normotensives after the sixth decade of life is approximately 90%.

Figure 7: Effect of Beta vulgaris on 5-HT (1 mcg/kg i.v.) induced change in vascular reactivity.

All values are expressed as mean ± SEM, n=5. All data are subjected to One-Way ANOVA followed by Dunnett's test. * p<0.05 when compared to control and # p<0.05 when compared to DEXA group. Vertical lines represent SEM. CTR: Control. DEXA: Dexamethasone. BV: Beta vulgaris. MIBP: Mean invasive blood pressure.

According to one survey, only 25% of the people taking modern treatment of hypertension are able to keep their arterial BP within normal range. The reasons for this are many, including the adverse effects of drugs and relatively high cost of treatment. The Ayurvedic approach gives emphasis on the individualization of the treatment and it also makes sure that the treatment is affordable for the hypertension and many of the cardiovascular diseases.

Despite of the vast literature of drugs being available for the treatment of hypertension, the last two decades have observed the introduction of a large number of newer antihypertensive drugs. The worldwide increasing demand for medicine from natural sources has prompted to search for plants with potential antihypertensive effect. Beta vulgaris (Beet root) is claimed to possess potential antioxidants betanin and also the inorganic nitrite which are claimed to have a good antihypertensive activity.12

Several models have been proposed for the induction of hypertension in rats. Here in the present study dexamethasone was used for the induction of hypertension in rats. Dexamethasone has been proposed to induce hypertension by several pathways. Hypertension may be associated with an increase in oxidative stress as a possible mechanism for the increased vascular tone and organ injury. It was also reported that an increased production of reactive oxygen species and endothelial cell death in the microcirculation were the causes of hypertension in rats.

Tracings

Figure 8: Tracing of MIBP record of control group.

It was hypothesize that xanthine oxidase (XO) may be a potential source of oxidants induced by glucocorticoid induced hypertension. Mammalian cells are capable of generating metabolites of oxygen, referred to as reactive oxygen species (ROS) via the action of several enzymes. In vascular cells, ROS are predominantly produced by the NADPH oxidases, uncoupled nitric oxide synthase, xanthine oxidase and by mitochondrial sources. In hypertension, ROS production by these sources is increased, and this not only contributes to hypertension, but also causes vascular disease and dysfunction. ROS production in other organs, particularly the kidney and the centers within the brain, likely participate in blood pressure regulation13. Perturbations in the various pathophysiological systems affecting blood pressure such as plasma volume, renin-angiotensinaldosterone system, sympathetic activity, vasopressor and vasodepressor systems have been proposed as contributing to dexamethasone-induced hypertension.

Figure 9: Tracing of MIBP record of DEXA group.

Although there are no published data on the role of NO based on acute NO synthase blockade or sequential blockade of vasoactive systems in DEX-HT, there is accumulating evidence suggesting a role for NO deficiency in the pathogenesis of DEX-HT. Plasma nitrate/nitrite, a marker of total body NO synthesis, are reduced in rats and mice made hypertensive by dexamethasone treatment. The reduced availability of NO might result from a range of influences on the NO biosynthetic pathways: i) alteration in the activity and expression of NO synthases ii) decreased availability of tetrahydrobiopterin (BH4), a NOS cofactor, iii) decreased NO precursor L-arginine and iv) increased NO removal via its interaction with superoxide to form peroxynitrite14. Ingestion of dietary nitrate (beetroot juice) results in increased plasma nitrite concentration via bioconversion in vivo. This bioactive nitrite substantially decreases BP, inhibits platelet aggregation and prevents endothelial dysfunction in healthy volunteers. These findings suggest that dietary nitrate likely plays a major role in mediating the beneficial effects of a vegetable-rich diet. Beetroot commonly have high inorganic nitrate (NO3) content. In humans, following absorption through the stomach wall, ~25% of consumed nitrate enters the enterosalivary circulation where it is reduced to nitrite (NO2) by bacterial nitrate reductases from facultative anaerobes on the dorsal surface of the tongue. This nitrite is swallowed and in the acidic environment of the stomach is reduced to nitric oxide (NO) or re-enters the circulation as nitrite. Indeed, it has been hypothesized that dietary nitrate represents an intravascular source of the pleiotropic, vasoprotective molecule nitric oxide (NO), that supplements conventional NO generation by NO synthases.15

The DEXA administered group showed significant increase in the blood pressure compared to control animals. Normal animals treated with Beta vulgaris (100 and 300 mg/kg) showed normal blood pressure. Rats treated with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) and DEXA a showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Figure 10: Tracing of MIBP record of BV 100 mg/kg group.

Figure 11: Tracing of MIBP record of BV 300 mg/kg group.

Figure 12: Tracing of MIBP record of DEXA + BV 100 mg/kg group.

Figure 13: Tracing of MIBP record of DEXA + BV 300 mg/kg group.

For pressure rate index the DEXA administered group significantly increase the PRI as compared to control animals. Animals treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in PRI compared to DEXA treated group. For heart rate the DEXA administered group significantly increase in heart rate as compared to control animals. Animals treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in heart rate compared to DEXA treated group.

For vascular reactivity to catecholamines, the DEXA administered group exhibited significant increase in the blood pressure compared to control animals. Normal animals treated with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed normal blood pressure. Rats treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in blood pressure compared to DEXA treated group.

Conclusions

The present investigation highlights the effect of methanolic extract of Beta vulgaris, prepared from root of Beta vulgaris on BP induced by dexamethasone. The effect of Beta vulgaris on pressure rate index, heart rate, blood pressure, vascular reactivity to various catecholamines were studied. For pressure rate index the DEXA administered group significantly increase the PRI as compared to control animals. Normal animals treated with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) + DEXA showed a significant (p<0.05) decrease in PRI compared to DEXA treated group. For heart rate the DEXA administered group significantly increase in heart rate as compared to control animals. Animals treated with DEXA along with Beta vulgaris (100 and 300 mg/kg p.o. for 14 days) showed a significant (p<0.05) decrease in heart rate compared to DEXA treated group.

Dexamethasone administered rats showed a significant elevation (p<0.05) in systolic blood pressure (SBP), vascular reactivity changes to Adr, NA, PE, and 5-HT as compared to control group. Beta vulgaris extract (100, 300 mg/kg/day, p.o.) treatment for 14 days in dexamethasone administered rats significantly (p<0.05) reduced systolic blood pressure, vascular reactivity changes to Adr, NA, PE, and 5-HT as compared to dexamethasone administered group. The basal arterial blood pressure, pressor responses to Adr, NA, PE and 5-HT were not significantly altered in Beta vulgaris extract (100, 300 mg/kg/day, p.o) treated rats as compared to control rats .

Thus in conclusion the methanolic-HCl extract of Beta vulgaris has antihypertensive activity in dexamethasone model.

Funding: No funding sources

Conflict of interest: None declared

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