Preclinical Evaluation of Hydro Alcoholic Extract of fruits of Cascabela thevetia for its Cardio Toxic Activity

 

Somnath Saha, Sourav Roy, Souvik Pal, Mrityunjoy Majumdar

Netaji Subhash Chandra Bose Institute of Pharmacy, (NSCBIP) Tatla, Roypara, Chakdaha, Nadia, 741222,

West Bengal.

*Corresponding Author E-mail: somu9188@gmail.com, sourav.roy.g36@gmail.com, souvikpal5@gmail.com, majumdar777@gmail.com

 

ABSTRACT:

Cascabela thevetia is a medicinally active plant used to treat different diseases. The aim of the present study was to pharmacological investigation of the hydro alcoholic extract of the fruit kernel of Cascabela thevetia for its cardiotoxic activity. In this study the fruit kernel was extracted with 70% ethanol by using soxhlet apparatus and 12% yield was obtained. The phytochemical screening of the extract revealed the presence of alkaloids, steroids, saponins, flavonoids and tannins. In this study male Albino Wister rat weighing about 150-250 gm  were used. Different pre-estimated doses of Acetylcholine (Ach), Adrenaline (Adr) and hydroalcoholic extract of Cascabela thevetia fruit kernel (CT) were used to find out the effect on heart from the dose response curve. Acetylcholine when applied to heart it produced parasympathomimetic action and decreased the rate of diastolic depolarization (negative tropic action). Adrenaline (sympathomimetic) produced both positive inotropic and chronotropic activity. But CT showed unique cardiac activity in the isolated rat heart, when applied low to high dose in geometrical propagation manner, first it produced negative chronotropic action followed by negative inotropic action. But that same negative tropic activity has been blocked by using adrenaline along with CT. Atropine along with CT became unable to antagonize the activity of CT unlike acetylcholine. Therefore it can conclude that Cascabela thevetia is a potent cardiotoxic drug in moderate to higher doses, help to understand the purpose behind selecting this fruit as abusive and suicidal cases.

 

KEYWORDS: Cascabela thevetia, Soxhlet apparatus, Acetylecholine, Adrenaline, Cardiotoxic.

 

 


INTRODUCTION:

Since primitive times we have dependent on plants as medicines. WHO also told medicinal plants as the best sources to obtain a variety of new herbal drugs. Now a day's a number of researcher has evaluated pharmacological activities in various segments like anti-inflammatory1, wound healing2, antioxidant, antihypertensive3, diuretic4 and also in cardiovascular field.

 

Traditional medicine, which has compounds derived from medicinal plants are used by about 80% of individuals from developing countries. Human learned to pursue drugs in barks, seeds, fruit bodies, and other parts of the plants as a result of the many years of struggles against illnesses and consequently increase in the awareness of medicinal plant usage. Cascabela thevetia is an important medicinal material in Chinese folk medicine5. Previously it is assumed that all parts of the oleander plant (Cascabela thevetia) produces poisonous effects in human, animal and certain insects. Self-poisoning is a major clinical phenomena in rural Asia6, killing at least 300000 people every year, the majority of the following due to ingestion of pesticides or plants. The main active constituents are polysaccharides, cardenolides, glycosides, and triterpenoids. Seeds are used as antifungal7, anti-Inflammatory8, anti-termite9, antimicrobial agent10, piscicidal11, antidiarrheal activity12. All parts of this plant are poisonous but mainly kernel of this fruit is abused for suicidal attempt in various countries13,14. This paper explains the evidence-based information regarding the phytochemistry and pharmacological activity of CT in heart.

 

MATERIALS AND METHOD:

Preparation of extract:

The pieces of the kernels of Cascabela thevetia fruits were subjected to extraction process by using cold percolator. The extraction was done by using 70% ethanol for consecutive 72 hours. The CT has been collected and kept for evaporation under vacuum desiccators. 12% yield was obtained.

 

Preliminary Phytochemical investigation of the extract:

CT were subjected to qualitative analysis for the various phytochemical constituents like Alkaloids, Carbohydrates, Cardiac Glycoside, Tannins, Saponins, Flavonoids and proteins.15

 

Dose preparation:

The CT, acetylcholine, adrenaline and atropine were weighted 10 mg. and dissolved in 100 ml of distilled water. That is why the concentration of all the stock solutions will be 100µg/ml.

 

Krebs-henseleit’s solution preparation:

The experimental subject was albino Wister rat heart. The proper solution to make heart alive is Kerbs-henseleit’s solution. The composition of the solution is as follows:

 

Table 1: composition of Krebs-henseleit’s solution

SL. No.

Name of Compound

Amount (in 1000 ml)

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.

 

Heart Model:

Albino Wister rat was sacrificed by cervical dislocation method. The thorax was opened immediately, exposed and the heart along with arorta were isolated as soon as possible and the tissue was plunged in kerb’s hanslet solution. The heart was cannulated through aorta using artery cannula and mounted, tropic activity was recorded in sherrington rotating drum by using Starling’s heart liver. The heart was perfused at a constant pressure of  90 cm of water with kerb’s hanslet solution at 37°c and pH 7.4. The perfusion rate was maintained at 5 ml/min. The perfusion solution was saturated with O2 bubbles at 30 drops/ min.  The drugs like Adrenaline, extract, acetylcholine, atropine were administered in the injection port as per the geometrical propagation, agonism antagonism method to obtain their activity on heart. Kymograph was maintained at 0.25 rpm16.

 

RESULT AND DISCUSSION:

Preliminary phytochemical investigation: The CT were subjected for preliminary phyto-chemical investigation and the following observations were found-

 

Table 2: Test name and result of phytochemical investigation

 

Test Name

Result

Test for  terpinoid

Libermann- Burchard test

Positive

Legal’s test

Positive

Baljet test

Positive

Keller killani’s test

Positive

Test for proteins and amino acid

Millons’s test

Positive

Biuret test

Positive

Test for alkaloids

Mayer’s test

Positive

Dragendroff’s test

Positive

Wagner’s test

Positive

Test for saponins

Water shaking method

Positive

Test for tannins

Ferric chloride test

Positive

Lead acetate test

Positive

Test for flavonoids

Ferric Chloride test

Positive

Lead acetate test

Positive

Magnesium ribbon test

Positive

Zinc hydrochloric acid test

Positive

 

Dose Response curve of adrenaline:

 

Fig. 1: Dose response curve of adrenaline

 

Here in this experimental model 100µg/ml of adrenaline stock solution was used and Adrenaline shows positive inotropic and positive chronotropic effect in isolated rat heart. The response was gradually increased in dose dependant manner.  As the dose increases the response length (force of contraction) and number of peak /minute (rate of contraction) both increases. In the dose of 0.2 ml the adrenaline shows its highest effect (maximum response). As the number of receptor is limited in isolated tissue, in higher dose(0.4ml), it has showed its saturation peak (supramaximal response).

 

 

 

 

Dose Response curve of CT:

 

Fig.2: Dose response curve of CT

 

In this experimental model CT 100µg/ml was used as a stock solution and shows an overall negative chronotropic as well as inotropic effect on the same heart. As the dose increases the response length (force of contraction) and number of peak /minute (rate of contraction) both decreases. At first 0.0125 ml of drug has shown negative chronotropic effect but force of contraction remain unchanged. 0.025 ml of dose has shown negative chronotropic effect as well as decrease in force of contraction. There was an overall negative tropic activity was observed in dose dependant manner. At the dose 0.4 ml shows almost cardiac arrest in the isolated heart.

 

Effect of adrenaline over the CT:

 

Fig.3: Effect of adrenaline over CT

 

The CT has shown negative tropic effect on the heart in dose dependant manner and at 0.4ml it has shown almost cardiac arrest (vide fig 2). Therefore 0.4 ml dose of CT has been selected to evaluate the effect of adrenaline over the extract. When 0.1 ml of adrenaline added with 0.4 ml of CT, there was a prominent alteration, the failing heart has started responding due to improvement in the tropic activity.  At the dose 0.2 ml of adrenaline along with 0.4 ml of CT, it produced positive inotropic effect along with tachycardia.

 

Effect of acetylcholine over CT:

 

Fig.4: Effect of acetylcholine over CT

0.0125 ml of CT produced negative chronotropic effect but inotropic effect was unchanged. At the same  dose of Ach, bradycardia was visible, but total cardiac arrest was not found. When 0.0125ml of CT with 0.0125ml Ach was injected to the isolated heart, total cardiac arrest occurred. The cardiac contractility was completely abolished may be due to synergistic effect of CT with Ach, which was reverted by applying 0.05 ml of Adr in the isolated heart preparation. Here synergism between CT and Ach was obvious because none of them has produced complete cardiac arrest at 0.025ml of dose separately (vide fig 2and fig5).

 

Combined effect of Acetylcholine, Adrenaline, Atropine and CT on rat heart:

 

Fig.5: Combined effect of acetylcholine, adrenaline and CT

 

Atropine shows antagonistic action of acetylcholine as it is an anticholinergic drug. 0.025 ml of Atropine has blocked the Ach mediated bradycardia. Ach produces negative tropic action, CT also do the same but while atropine has been injected separately with Ach and CT, atropine antagonizes the activity of Ach but not CT. this demarcation indicate the MOA of CT is not cholinergic receptor mediated. When CT, adrenaline and atropine at same dose (0.025 ml) applied together, only the activity of adrenaline become gradually prominent with time. Here the activity of atropine was omitted, as there was not present any cholinergic drug. So it can be assumed that the negative tropic activity of CT was nullified by adrenaline, as the time increases the positive tropic activity of adrenaline becomes more prominent.

 

From all the above evidence it can be established that CT has shown negative tropic activity which was synergistic with Ach, but not antagonized by atropine. At the same time the activity of CT was nullified in presence of adrenaline. From all of these observation, it can be assumed that the activity of CT on heart is mediated by some chemical constituents which may have sympatholytic activity. CT never showed arrhythmia(α adrenergic mediated) on heart but negative tropic(β anti-adrenergic mediated) activity was prominent17,18,19. Therefore it can be assumed this activity is very resemble with β anti-adrenergic compounds but not with α adrenergics.

 

 

CONCLUSION:

CT was evaluated on isolated rat heart preparation for its cardio toxic activity. Ach, adrenaline, atropine was injected in perfused heart along with CT in different fashion and from all the findings it was observed that the cardio toxic activity of CT was not antagonized by atropine even cardiac arrest was accelerated in addition with Ach with the former. The cardio toxicity produced by CT was reverted with adrenaline.

 

ACKNOWLEDGEMENT:

It gives me great pleasure to acknowledge, with deep appreciation, to all those who have extended their cooperation throughout the period of my project work. I take this opportunity to express my deep gratitude to the principal, management and dept. of pharmacology, Netaji Subhas Chandra Bose Institute of Pharmacy (NSCBIP) for guiding and helping me throughout this research.

 

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Received on 15.03.2019           Modified on 16.04.2019

Accepted on 18.05.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(8):3755-3758.

DOI: 10.5958/0974-360X.2019.00643.7