INTRODUCTION:

Cardiovascular diseases (CVDs) are the major health problems in the present situation. According to WHO and 2020 prediction, CVDs are the major reason for death globally. A major number of people passed away because of heart attack and stroke. Many peoples under the age of 50 years are died due to heart disease and day by day it increases. Early deaths tend to develop extra pressure on the middle-class family, society, and economy. There are many risk factors that lead the CVDs like an unhealthy diet, obesity, hypertension, diabetes, hyperlipidemia, etc. Nowadays medicines are so costly and have so many side effects. But herbal medicine can play a vital role in the remedy of CVDs.

Sometimes, we can see that herbal medicine with synthetic drugs is used for treating CVDs. Because many herbs are easily available and have no life-threatening side effects^1,2.

Plants have been used as remedies since the dawn of humanity. Medicinal plants, according to the WHO, are the best resource for obtaining a variety of new herbal medications. A growing number of researchers are evaluating pharmacological properties in a variety of areas, including anti-inflammatory³, antihypertensive, antioxidant⁴, wound healing⁵, diuretic⁶, and cardiovascular⁷. Treatment with herbal plants is allowed for very safe as there are no or least side effects. The tremendous advantage is that any age groups and the sexes are capable of the use of herbal treatments alone and these remedies come from nature directly. Globally, about 80% of people confide on herbal medicine for their primary treatments. Medicinal plants are deliberated as a high resource of components that can be applied in drug development. Since archaic times, people looked for drugs in nature for their treatments. CVDs⁸.

Hippocrates - Father of Medicine – said that abandon off our medications in the chemist’s container if we can recover the sufferer with diet. Intake of nutraceuticals and herbal derivatives, looked after by a healthy lifestyle and dietary routines, and ordinary corporal activity exhibit a beneficial impact in patients diagnosed with cardiac diseases¹. Among the plants, Moringa oleifera has been used as folk medicine in cardiac disease like hypertension⁹.

Moringa oleifera belongs to the family Moringaceae. Moringa oleifera Lam is one of the most broadly developed plants in the Moringaceae family which can be utilized as vegetable and restorative plants. Also, M. oleifera leaves could fill in as the nourishing wellspring of certain nutrients and minerals like calcium. This plant has been utilized as a conventional medication in numerous nations including Thailand to treat a few side effects, for example, skin diseases, asthma, bronchitis, hack and sore throat¹⁰. It is native to the Indian subcontinent and commonly found in tropical and subtropical countries. It is distributed in Southwest Asia, Africa, Northern India, Pakistan, Nepal, Nigeria, and many other countries¹¹. According to previous studies, Moringa oleifera exhibited hepatoprotective activity^12,13, antioxidant^14,15,16,wound healing activity^14,17, anti-inflammatory activity^14,17, cardiac ameliorative effect¹⁵, analgesic activity¹⁷, local anesthetic activity¹⁷, gastric ulcer protective activity¹⁷, antinociceptive activity¹⁷, antifungal activity¹⁸, antiviral activity¹⁸, antifertility activity¹⁸, anti-hypertension¹⁸, hypocholesterolemic activity¹⁸, anticancer activity¹⁸, antileishmanial activity¹⁸, anti-diabetic^18,19, anti-hyperlipidemicactivity^18,20, micromeriticactivity^20,21, and immunomodulatory activity²².

Thiscurrent article describes the evidence-based research regarding Moringa oleifera's phytochemistry and cardiotropic activity on albino Wistar rats.

MATERIAL AND METHOD:

Plant material and preparation of extract:

The leaves of Moringa oleifera were collected from the local area of Chakdaha, West Bengal, India. Fresh mature leaves were collected, washed with clean and fresh water for removing the dust particles. The dried leaves were processed under shade drying, the dried leaves were subjected for extraction.

The extraction process of Moringa oleifera leaves was done with 70% ethanol by using the cold percolation technique for consecutive 48 hours. The extract has been collected and kept for evaporation under a vacuum evaporator. The extract of Moringa oleifera leaves was collected and a stock solution of 100µg/ml was prepared.

Preliminary phytochemical investigation of the plant extract:

The extract obtained from the Moringa oleifera leaves was subjected to preliminary phytochemical investigation like Alkaloids, Carbohydrates, Flavonoids, Glycosides, Saponins, Steroids, Phenolic acids, as well as Proteins²³.

Experimental animal:

Albino Wistar rat weighing around 120-150gm was used for the study. The animal was purchased from an authorised animal breeder. They had provided with commercial food pellets as well as tap water ad libitum. The experiment was conducted between 10 am to 6 pm. The rat was housed in CPCSEA approved Netaji Subhas Chandra Bose Institute of Pharmacy animal house (approval no:1502/PO/a/11/CPCSEA) under standard laboratory conditions with 12-hour light/12-hour dark cycles and a room temperature of 22±2°C.

Dose preparation:

The extract, acetylcholine, adrenaline, as well as atropine were weighted 10mg. In 100mL of distilled water, each product is dissolved. That's why the concentration of all the products will be 100µg/ml.

Krebs-henseleit’s solution preparation:

Krebs-henseleit's solution was used for the alive isolated heart of the Albino Wistar rat. The composition of the solution per 1000ml is showing in the table number 1.

Table 1: Ingredients of Kreb’s-henseleit’s Solution

Sl. No.	Name of the compound	Quantity
1	NaCl	6.9 gm
2	KCl	1.28 gm
3	NaHCO₃	2.1 gm
4	CaCl₂	0.28 gm
5	MgSO₄ . 7H₂O	0.35 gm
6	Glucose	2.0 gm
7	K₂H₃PO₄	0.16 gm

All the weighted materials were mixed with 1000ml of distilled water one by one except Cacl₂. Cacl₂ was mixed with distilled water in a separate beaker. Then the cacl₂ solution was poured into the remaining solution slowly and stirred well to prevent the formation of chelates. Mixed the solution properly. After that prepared solution was elevated to 37°C temperature for the experimental work.

Design of experiment (Isolated Heart Model):

Albino Wister rat was sacrificed by the cervical dislocation method. The thoracic cavity was opened so that the heart was exposed. The vena cava was cannulated followed by the heart was isolated and mounted along with the adequate supply of the kerbs-henseleit’s solution and aeration. The reservoir of the solution was maintained at 37°C by using the thermostat. The flow of physiological solution was controlled by a regulator and to maintain the uniform flow through the heart. The apex of the ventricle was attached with a sterling heart lever and the inotropic and chronotropic activity was recorded on Sherrington rotating drum. Parasympathetic drug-like acetylcholine, atropine, and sympathetic drugs like adrenaline, isoprenaline, and propranolol were subjected to record their influence on tropic activities on isolated heart preparation. Different dilution of extract of Moringa oleifera was applied on the same heart to compare its activity with the aforesaid endogenous agonists and the exogenous antagonists²⁴.

RESULT AND DISCUSSION:

Preliminary phytochemical investigation:

The phytochemical analysis result of 70% ethanolic extract of the Moringa oleifera leaf shows the presence ofAlkaloids, Carbohydrates, Flavonoids, Glycosides, Saponins, Steroids, Phenolic acids, and Proteins.

Effects of various agents along with extract on heart model:

In this project work, Moringa oleifera leaves extract was subjected for evaluation of cardio-activity. To simply its route of action on the heart was compared with parasympathetic and sympathetic pathways. Prototype of parasympathomimetic, parasympatholytic, sympathomimetic, sympatholytic agents was evaluated simultaneously on isolated rat heart.

Dose response curve of Acetylcholine:

Acetylcholine (ach) is a neurotransmitter at autonomic and somatic types. Acetylcholine produces its action by binding with muscarinic (M1, M2, M3), nicotinic (NN, NM) receptors. In the heart M2 receptor is present and controls the activity of acetylcholine on the heart. Acetylcholine is a para-sympathomimetic agent that produces hyperpolarization on the sinoatrial (SA) node and causes a decrease in the rate of phase IV depolarization followed by impulse production is decreased. The situation resembles bradycardia. The refractory period on the atrioventricular (AV) node and bundle of his is increased and causes the negative chronotropic effect. Decreased AV nodal conduction or blocked evidence from the electrocardiogram (ECG). PR interval increases in certain exchange followed by negative inotropic effect. The resting potential of myocardial energy is abbreviated due to vagal innervation – inhomogeneity conduction produces arterial fibrillation or flutter. Acetylcholine causes dilation of blood vessels. Muscarinic receptor (M3) is present on vascular endothelial cells causes endothelium-dependent relaxing factor (EDRF) mediated vasodilation^25,26.

In this experiment ach in different dosages i.e. 0.0125, 0.0250, 0.0500, 0.1 were used on the isolated rat heart. In 0.0125ml of acetylcholine, the heart produces 20 beats and 32 drops per minute. In the next dose i.e., 0.0250ml, 9 beats, and 15 drops per minute were observed. In 0.05ml 2 beats and 5 drops per minute were produced. In the next higher dose, the heart has shown complete cardiac failure. Acetylcholine produces negative inotropic (droplet counts) and negative chronotropic (beats per minute) in a dose-dependent manner and in higher doses i.e., 0.1ml due to SA nodal hyperpolarization the heart stops beating and produce cardiac failure.

Table 2: Observations of Acetylcholine

Acetylcholine (ml)	Drops	Beats
0.0125	32	20
0.0250	15	12
0.0500	5	3
0.1000	-	Cardiac failure

Fig. 1: DRC of acetylcholine

Dose response curve of atropine over acetylcholine:

Atropine is an anticholinergic drug and it blocks the m2 receptor (competitive inhibitor). On the SA node, by which vagal tone decreases followed by tachycardia. As atropine is an antagonist of the parasympathetic system, it does not produce any action in absence of ach^25,26. So, here in the experimental, the model was designed to evaluate the effect of atropine over acetylcholine.

Atropine 0.0250ml dose was added with acetylcholine 0.0250ml and found 44 beats and 60 drops per minute. From here, it is evidenced that atropine produces tachycardia over the acetylcholine-induced heart.

Fig. 2: DRC of atropine over acetylcholine

Dose response curve of adrenaline:

Adrenergic transmission is prominent to the sympathetic system of the autonomic nervous system. Three closely related catecholamines namely adrenaline, noradrenaline and dopamine act on the sympathetic system.

Noradrenaline is stored in synaptic vesicles in the adrenergic nerve terminal. The stimulus initiates to release of catecholamines by exocytosis followed by noradrenaline or adrenaline are get released and bind with adrenergic receptors abundantly present in our body. Adrenergic receptors are classified as α (α1, α2) and β (β1, β2, β3). All the receptors are G-protein coupled receptors causes increasing or decreasing of the intracellular production of second messenger i.e. cyclic cAMP or IP3/ DAG or in some cases altering the Ion channel regulation. β1 is present in the heart (K+ or Ca++) at the same time β2 is present in the blood vessel. α1 is present in postjunctional or effector organs including the heart and blood vessel.

Adrenaline (ADR) increases heart rate because of the increasing slope of the phase Ⅳ depolarisation of nodal cells in the SA node. As latent pacemaker activity in AV node and Purkinje fiber increased; a result, arrhythmia is produced and blood pressure (BP) rises. Sudden rise in blood pressure BPreflex unmasks the latent pacemaker activity. Positive inotropes are established systole is shortened more than diastole. Cardiac output is increased. SA node generates more impulse, conduction through AV node, Purkinje fiber is increased followed by positive chronotropic effect. β1 is mainly responsible for producing a positive inotropic and positive chronotropic effect on the heart.

Catecholamines produce vasoconstriction (α1) vasodilation (ß2) action on blood vessel depending on the drug (ARD→α1+ ß2, NA→α1, ISO→ ß2), its dose and effector organ. Adrenaline causes a rise in systolic pressure but the diastolic pressure falls down. Mean blood pressure increases. Noradrenaline causes increasing diastolic, systolic, and mean blood pressure (no ß2 action). Isoprenaline (ISO) shows rising systolic pressure but falling diastolic pressure (ß1 cardiac stimulation, ß2 vasodilation) the mean blood pressure falls down^25,26.

When adrenaline has given in the heart produces sympathomimetic action in a typical dose-dependent manner. Here in the experimental model, we have used adrenaline 100µg/ml as a stock solution. 0.05ml of adrenaline applied on isolated rat heart and found a minimum increasing force of contraction and rate of contraction i.e., 90 drops and 72 beats per minute. 0.1 ml of adrenaline has shown 106 drops and 82 beats per minute whereas 0.2 ml of adrenaline has shown a maximal response of 130 drops and 98 beats per minute. But as the number of receptors is limited in isolated tissue the next higher (0.4ml) dose has shown saturation peak (supramaximal response) 128 drops and 95 beats per minute.

Table 3. Observations of adrenaline

Adrenaline (ml)	Drops	Beats
0.05	90	72
0.1	106	82
0.2	130	98
0.4	128	95

Fig. 3. DRC of adrenaline

Dose response curve of Moringa oleifera:

Moringa oleifera leaves extract preparation (100µg/ml) applied in isolated rat heart and shows typical action on tropic activity. 0.1ml has produced 92 drops (inotropic effect) and 80 beats

(Chronotropic effect) per minute. In the next higher dose i.e., 0.2ml, 76 drops, and 68 beats per minute have been observed. In 0.4ml, there is still a reduction in force of contraction, 65 drops and 60 beats per minute respectively. Moringa oleifera has shown a significant negative inotropic effect and negative chronotropic effect in a dose-dependent manner. 0.025ml of atropine applied along with 0.025ml of acetylcholine, atropine blocks the acetylcholine mediated hyperpolarization in SA node followed by bradycardia and ultimately there is an overall increased rate of contraction i.e., 24 beats per minute, as atropine is an antagonist of acetylcholine. It only produces such action in presence of parasympathomimetic agents. 0.1ml of Moringa oleifera is added with 0.025ml of acetylcholine and found an increased rate of contraction. From the above findings which are clear Moringa oleifera may have some of the parasympathomimetic principles in it.

Table 4: Observations of Moringa oleifera

*Moringa oleifera* extract (ml)	Drops	Beats
0.1	92	80
0.2	76	68
0.4	65	60

Fig. 4.4. DRC of Moringa oleifera

In this research, the hydroalcoholic extract of Moringa oleifera leaves was evaluated to establish its antihypertensive potential through the negative tropic effect on isolated rat heart. In an isolated heart model, most of the neuronal, endogenous innervation is excluded. All the autonomic innervation was evaluated by applying such agents externally. The dose-response curve of Moringa oleifera was obtained and compared with standard parasympathomimetic, parasympatholytic, sympathomimetic agents. Moringa oleifera has shown effect resemble with parasympathomimetic agents to some extent. This effect only established the traditional use of Moringa oleifera in hypertension. Though this experiment is designed to authenticate the traditional use of Moringa oleifera on heart and blood vessels, needless to say, drumsticks and its leaves are popularly used as food all over India and other continents also may be beneficial in the view of keeping our heart and blood pressure controls naturally.

CONCLUSION:

The Moringa oleifera leaves were collected, washed, shade dried, and extracted with 70% ethanol. A stock solution (100µg/ml) was prepared by using distilled water. Acetylcholine, adrenaline, Moringa oleifera leaf extract were applied on isolated rat heart and dose-response curves of the substances were recorded. Moringa oleifera leaf extract has shown a substantial negativeinotropic and chronotropic effects, which may be beneficial in the treatment of hypertension. The above findings establish the traditional use of Moringa oleifera in hypertension and other cardiac disorders.

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors would like to express their most sincerely appreciation to Dr. Arnab Samanta, Principal, Netaji Subhas Chandra Bose Institute of Pharmacy. Authors also acknowledge the support and help of all non-teaching staffs of Netaji Subhas Chandra Bose Institute of Pharmacy.

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Received on 12.04.2022 Modified on 02.07.2022

Research J. Pharm. and Tech 2023; 16(7):3417-3421.

DOI: 10.52711/0974-360X.2023.00565