INTRODUCTION:

Hypertension is defined as a condition in which pressure in the blood vessels is higher than normal (1). Hypertension is the most common cardiovascular disease and is a major public health issue in developed as well as developing countries. In hypertension systolic blood pressure (SBP) rises above of 140 mmHg while diastolic blood pressure (DBP) is above of 90 mmHg. Hypertension is the largest attributable risk factor for mortality worldwide, and is responsible for more than half of all instances of stroke and coronary heart disease (CHD).

The problem is increasing, with predictions that one-third of adults worldwide will have hypertension by 2025. Despite public health programmes and effective pharmacotherapy for hypertension in developed economies, approximately 25% of adults have hypertension. It remains untreated in up to 50% of these individuals, and, disappointingly, BP is controlled to guideline driven targets in only 50% of those hypertensive patients advised to take treatment. Hypertension is generally symptom less, but increases the risk of various other cardiovascular diseases like stroke, heart attack and non-cardiovascular diseases like renal damage, end stage of renal failure, etc. BP rises with increasing age, and this is thought to reflect environmental and lifestyle factors, as well as changes in hemodynamic caused by arterial stiffness in the major elastic arterial vasculature, especially the aorta (Bhagani, et al., 2018). Hypertension is one of the leading cause of global burden of disease. It is one of the major cause of cardiovascular mortality, which is estimated to be 1.5 million deaths per year in India.

MATERIAL AND METHOD:

Animals:

Male wistar rats of weight range 180 to 200 grams were received from Krystal Biological Solutions pvt ltd. They were housed in a clean, well-managed animal house maintained at a temperature of 22±1˚C and 60-70% relative humidity under 12 hours light and 12 hours dark cycle. After acclimatization for 7 days, the mice were divided into 6 groups each group containing 6 rats kept in a polypropylene cage with husk as a bedding material. The animals were provided with food and water adlibitum. The use of animals for the experimental study was approved from the Institutional Animal Ethics Committee and all the experiments were carried out between 8:00 am to 5:00 pm. Minimization of animal suffering and providing the utmost suitable environment to the animals was taken care of.

Chemicals:

Extracts of Brassica oleracea and Apium graveolens were obtained from Green heaven India, Nagpur Maharashtra. Fructose were purchased from Research lab, Mumbai; Methanol purchased from Himeda, Mumbai; Heparin from Caprin, Mumbai; Diethyl ether purchased from Merck, Mumbai; N-Hippuryl-His-Leu hydrate purchased from Sigma Aldrich, Bangalore; Hydrochlorothiazide purchased from Mylan, Mumbai; Urea purchased Research lab fine chem industries, Mumbai; Captopril from Wockhaedt limited, Mumbai.. All other chemicals used were of analytical grade and highest purity.

Instrumentation:

Centrifuge, UV-spectrophotometer, Heating water bath, Sonication water bath, pH meter, Analytical weighing balance, Student biopac lab, Metabolic cage.

Selection of Solvent:

On the basis of solubility study methanol was selected as the solvent for dissolving BO and AG.

Acute Oral Toxicity Study:

After approval from Institutional Animal Ethics Committee (IAEC), three female rats were housed in polypropylene cage (28 × 21 × 14cm). The combination of Brassica oleracea and Apium graveolens was subjected to acute oral toxicity study as per the OECD guidelines 423. After acclimatization for 7 days, three female rats were dosed at a dose of 2000mg/kg (1000 mg/kg of Brassica oleracea and 1000mg/kg of Apium graveolens) of combination. Thereafter the dosed animals were observed for 14 days for any changes in skin, fur, eyes, mucus membrane, behavior and body weight was recorded weekly.

In-Vivo Study: Evalution of antihypertensive activity:

Induction of acute renal hypertension (2K1C):

The method first described by Gold blatt et al. (1934) was used with some modifications. 2K1C (2 Kidneys 1 Clip) was used for the 4-week treatment model due to better animal survivability. For the 2K1C surgery, the rats were anesthetized with urethane (1.5g/kg i.p.) and an incision in the left side of the abdomen was made for renal artery exposure. A silver clip of 0.2mm internal diameter was placed over the left renal artery near the aorta leading to a constriction greater than 50% and the incision was sutured. The rats belonging to group II to group V were treated with 2K1C for the induction of hypertension.

Table No. 1. Protocol for 2KIC

Sr. No	Groups	Treatment		No of animals
1	Group I	Normal Control (Saline)		06
2	Group II	2K1C + (Saline)		06
3	Group III	2K1C + Captopril		06
4	Group IV	2K1C + Combination of extract(100 mg/kg, p.o)		06
5	Group V	2K1C + Combination of extract (200 mg/kg, p.o)		06
6	Group VI	2K1C + Combination of extract (400mg/kg, p.o)		06
Total Animal			36

After the induction of hypertension different hemodynamic parameters were measured by using carotid artery cannulation invasive method with BIOPAC student’s lab. After measurement of animal the animal was killed by euthanasia.

Fructose Induced Hypertension:

Increase in dietary carbohydrate intake can rise blood pressure in experimental animals. The increase intake of either sucrose or glucose was shown to enhance the development of either spontaneous hypertension or salt hypertension in rats^(4,10,12). First reported that hypertension could be induced in normal rats by feeding a high fructose diet. Fructose feeding was also found to cause insulin resistance, hyperinsulinemia, and hypertrigly eridemia in normal rats^(5,6), studied that the concentration and duration-dependence of fructose-induced hypertension in rats. Procedure followed for induction of hypertension in animals were housed two per cage on a 12 h light 12-h dark cycle and are allowed free access to standard laboratory diet and drinking fluid. Drinking fluid consists 10% fructose solution. Body weight, food intake of animals measured every week^(7).

a) Cardiac parameter:

i) Systolic Blood Pressure

ii) Diastolic Blood Pressure

iii) Mean Arterial Blood Pressure

b) Biochemical Parameters:

i) Triglyceride

ii) LDL

iii) HDL

Table No. 2. Protocol for Fructose Induced Hypertension

Sr. No	Groups	Treatments	No of Animals
1	Group I	Normal Saline	06
2	Group II	10% fructose p.o	06
3	Group III	10% fructose + captopril p.o	06
4	Group IV	10% fructose + Combination of extract (100 mg/kg)	06
5	Group V	10% fructose + Combination of extract ( 200 mg/kg )	06
6	Group VI	10% fructose + Combination of extract ( 400 mg/kg)	06
	Total		36

After the induction of hypertension blood pressure and different biochemical parameters were measured by using carotid artery cannulation invasive method with BIOPAC student’s lab. After measurement of the animal were sacrificed with the help of euthanasia.

Biochemical analysis:

Procedure for Triglycerides Estimation:

Take three cuvette of blank, standard and test respectively. Add 1ml of triglycerides reagent in all 3 cuvette. The triglycerides standard 10ul was added to cuvette having standard and test sample. And add 1ml of triglycerides reagent and standard to test sample cuvette. Prepared sample was mixed and incubated at 37 C⁰ for 5 minutes. Absorbance of test (T) and standard (S) was taken against reagent blank at 540-560 nm. And calculation has done by using following:

Triglyceride in mg/ dl = Abs.T / Abs.S * 200

Assay Procedure for LDL estimation:

Take three cuvette of blank, standard and test respectively. Add 600ul of LDL-cholesterol-1 reagent in all 3 cuvette. Then 600ul LDL-cholesterol-1 and 200ul LDL-cholesterol-2 and 6ul LDL-C calibrator were added to cuvette having standard sample. And add 600ul of LDL-cholesterol-1 reagent and 200ul LDL-cholesterol-2 to test sample cuvette. Prepared sample was mixed and incubated at 37 C⁰ for 5 minutes. Absorbance of test (T) and standard (S) was taken against reagent blank at 620 nm. And calculation has done by using following:

LDL (mg/dl) = Abs T / Abs C * Conc. of Calibrate

Assay Procedure for HDL estimation:

Take three cuvette of blank, standard and test respectively. Add 1000ul of HDL-C-cholesterol reagent in all 3 cuvette. Then 10ul HDL-C-calibrator were added to cuvette having standard sample. Prepared sample was mixed and incubated at 37 C⁰ for 5 minutes. Absorbance of test (T) and standard (S) was taken against reagent blank at 620 nm. And calculation has done by using following:

HDL-C (mg/dl) = Abs T / Abs C * Conc. of Calibrator

STATISTICAL ANALYSIS:

Data was expressed as the mean ± standard error of the mean (SEM). The statistical significance of difference between the mean values for the treatment groups was analyzed by One way and Two Way ANOVA (analysis of variance) followed by Bonferroni test using Graph pad prism-7 software.

Results- in-vitro study

ACE inhibition:

Both Brassica oleracea and Apium graveolens have beean reported for their ACE (Angiotensin converting enzyme) inhibitory activity. Hence the combination of both was tested to determine the inhibition of ACE in terms of Inhibitory concentration 50 (IC50). The IC 50 value of Brassica Oleracea was found to be 520.03 and Apium graveolens was found to be 400.22 and combination of both was found to be 350.32

In-Vivo Study -Acute Oral Toxicity:

Acute oral toxicity study of the combination of Brassica oleracea and Apium graveolens in (1: 1) was done according to OECD guidelines 423. The combination was found to be safe up to a dose of 2000 mg/kg as indicative from no significant changes in the bod weight, skin, fur, eyes, mucus membrane, salivation and other physiological and behavioral parameters of dosed rats, observed during a period of 14 days. Therefore the combination of Brassica oleracea and Apium graveolens was found to be safe up to a dose of 2000 mg/kg.

DISCUSSION:

Hypertension is one of the commonly occurring disease both developed and developing countries. Community surveys in industrialized countries have shown a prevalence of 15-33% in persons aged 30 years. The disease continues to be a leading cause of morbidity and mortality from coronary artery disease and stroke, In this respect, herbal drugs are helpful and render encouraging results in comparison to synthetic drugs due to their fewer or no side effects and easy availability. The screening of various plants according to their traditional uses and nutritional value based on their therapeutic value leads to discovery of newer and safer alternative drug treatment for management of hypertension.

In present study polyherbal mixture of two selected herbs was evaluated for antihypertensive activity. The two herbs selected were Brassica oleracea and Apium graveolens. The broccoli contains vitamin C, glucosinolate and flavonoids and it reported as an antioxidant, antihypertensive, ACE inhibitor. While Celery seeds contains anthocyanin, chlorophyll, carotenoids, limonene, sedanelodis, 3-butylphthalide and it reported as calcium channel blockers, vasodilator, and ACE inhibitor. As these two herbs are reported to act on different mechanisms that are responsible for hypertension, the effect of combination of these two herbs was done and evaluated for antihypertensive activity. The phytochemical studies was conducted by detecting presence of qurcetin and rutin as biomarkers in the polyherbal mixture showed the presence of most of the biologically active compounds in the plant and stable in polyherbal mixture. In-vitro assay of both the drugs was performed for their ACE inhibition activity, the IC50of combination was found to be lesser than individual drugs. So what additive effect or lesser concentration of both is useful.

Model used to evaluate antihypertensive activity is (2K1C) is a renovascular type animal model for hypertension. In 2K1C model, the renal artery is constricted on only one side while the other artery (or kidney) left untouched. This results in a sustained increase in BP due to increased plasma rennin activity, which in turn increases circulating angiotensin‑II, and it is potent vasoconstrictor⁽¹⁹⁾. However, there is no salt and water retention because other kidney being intact. Thus, the resultant hypertension at this stage is renin‑angiotensin dependent only. Moreover, it has been shown that other vasoactive agents may also be involved in producing hypertension in 2K1C, such as: Thromboxane and prostaglandin F2α. In renovascular hypertension, there is an alteration in sympathetic function with increased sympathetic drive and impaired catecholamine’s extraction. The sympathetic nervous activity may be augmented by the angiotensinergic mechanism in 2K1C hypertension⁽²⁰⁾

In present study the polyherbal formulation at dose 200 mg/kg and 400mg/kg significantly reduced the blood pressure and mean arterial blood pressure wherein dose 400mg/kg was found to be more effective than 200 mg/kg as compare to hypertensive rats. These findings suggest that Polyherbal Mixture may act via ACE inhibitor activity and thereby block or antagonises the action of angiotensin II. Another model reveals the evaluation of antihypertensive activity in fructose induced hypertension. Following ingestion, fructose is rapidly absorbed from the intestine via the fructose-specific glucose transporter, GLUT5 (L.T Tran et al., 2009). High fructose feeding play role on endothelial dysfunction, nitric oxide, renin system, sympathetic nervous system, oxidative stress, uric acid. High fructose diet up-regulate sodium and chloride transporters therefore resulting salt overload and increases blood pressure^(6).

The polyherbal mixture of extracts at dose level of 200 mg/kg and 400mg/kg exhibited significant diuretics activity. Also increased volume and concentration of Na⁺, K⁺ and Cl⁻ in urine when compared to the normal control group was observed. The lipid parameters like HDL, LDL, and Triglyceride, plays an important role in cardiovascular diseases. Increase LDL is associated with increase in risk of morbidity and mortality the herbs used in polyherbal are reported to decrease lipid parameter individually but the effect of combination of these herb is not reported. In the presence study serum lipid parameters were measured. No significant difference in the lipid parameter as compared group as well as standard was observed these suggest the polyherbal mixture do not produce significant effect on lipid metabolism.

CONCLUSION:

The present study was undertaken with the aim to evaluate the antihypertensive activity of combination of herbs extract Brassica oleracea and Apium graveolens in 2K1C model and fructose induced hypertension model in Wistar rats. The antihypertensive potential of Brassica oleracea and Apium graveolens were evaluated in terms of LDL, Triglycerides, Creatinine, HDL status in rats. The outcome of the present study reveals that both the drugs significantly inhibited ACE enzyme activity. Both the drugs was able to decrease LDL, Triglycerides, Creatinine and increase in HDL level in Wistar rats. Combination of two drugs showed antihypertensive activity in dose dependent manner. The dose 400mg/kg was found to be more effective than 100 mg/kg and 200mg/kg. Conclusively, based on findings of present study it can be stated that the Brassica oleracea and Apium graveolens could be a potential source for the treatment and management of antihypertension.

ACKNOWLEDGMENTS:

The authors are thankful to Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune and to Dr. S. R. Naik, Adjunct Professor, and DYPIPSR.

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Received on 20.07.2019 Modified on 05.09.2019

Research J. Pharm. and Tech 2020; 13(5):2069-2075.

DOI: 10.5958/0974-360X.2020.00372.8