INTRODUCTION:

Cardiovascular Disease (CVD):

Cardiovascular diseases (CVDs) are the most predominant reason of death and debility worldwide. Statistics from WHO indicates that the load of chronic diseases including coronary heart disease, diabetes, cancer and obesity share 59% of the 56.5 million deaths reported. CVD is defined as any serious, abnormality of heart or blood vessels (arteries, veins). Cardiovascular diseases incorporate stroke, coronary heart disease, congenital heart disease, peripheral vascular disease, endocarditis, and many other conditions.^1,2

Some major cardiovascular diseases are described as follow:

· Angina Pectoris

· Atherosclerosis

· Heart Failure

Among ischemic heart disease, acute myocardial infarction is the most eminent one and it occurs due to imbalance between coronary blood supply and myocardial demand.³ MI is generally known as heart attack, it is a disease that occurs when blood is interrupted towards a part of heart which causes death of the heart tissue. Acute MI is considered by chest pain, sweating, weakness, vomiting, arrhythmia and loss of consciousness and even sudden death.⁴An increased risk of coronary heart disease is associated with high levels of total cholesterol and low-density lipoprotein-Cholesterol and decreased levels of high-density lipoprotein-Cholesterol in serum.⁵

MI is a multifactorial ailment linked with many factors such as heredity, hyperlipidemia, obesity, hypertension, environmental and life style variables such as stress, smoking and alcohol consumption. A healthy and balanced diet is important for both prevention and treatment of cardiovascular disease.⁶

Cardioprotective drugs:

Cardioprotective drugs are agents that inhibit or minimize harmful consequences of impaired cardiac functions such as sudden coronary death (SCD) due to early post-occlusion ventricular fibrillation (EPVF), development of incapacity myocardial necrosis. Traditional systems of medicine list more than 2,000 plants and some of these are providing remarkable relief to the people suffering from cardiovascular diseases. WHO reports evidenced that around 80% of Global population is still trust on herbal drugs and some herbal medicines have been clinically use in modern time.⁷In this study comparative effect of herbal drug (Areca catechu) with synthetic drug (Metoprolol) have been explored for the treatment of Myocardial Infarction.

Areca catechu (Arecaceae) is widely distributed in South and Southeast Asia. The fruits of Areca catechu are used as chewable items and in many traditional herbal medicine.^8,9 Many chemical active compounds have been extracted and isolated from A. catechu polyphenols (catechin, epicatechin) alkaloids (arecoline, arecolidine) flavonoids (isorhamnetinchrysoeriol, luteolin, quercetin) tannins (procyanidin A1,procyanidin B1, procyanidin B2) fat (lauric acid, palmitic acid, myristic acid,).^10,11 Areca catechu have various pharmacological actions like Antioxidant activity, Antiparasitic activity, Anti-inflammatory, Antihypertensive activity, Hypolipidemic activity, Antimicrobial activity, CNS Stimulant.¹²This plant is traditionally used in various Unani and Ayurvedic formulations.

MATERIAL AND METHODS:

Plant Material:

Areca Catechu seeds collected from local market of Lucknow, Uttar Pradesh. The drug was authenticated by Dr. Mohammad Arif, Faculty of Pharmacy, Integral University, Lucknow with a Voucher no. IU/PHAR/HRB/17/06.

Preparation of extract:

The plant material was made devoid of adulterants, seeds were compressed to open up the crest of kernel and soaked in 2 L of aqueous-ethanol solvent (50%) for cold maceration for an episode of 7 days at room temperature. Subsequent to which the filtrate was collected through a piece of porous cloth and filter paper. This extract was dried in an oven at 90°C and then stored at -4ºC until use. For experimental use the stock solution was prepared by suspending the extract in 2% w/v sodium carboxy methyl cellulose (CMC) on the day of the experiments.¹³

Preparation of solution:

a) Areca catechu extract solution:

Areca catechu extract were weighed accurately and dissolved in CMC (1%) and freshly prepare extract was used for dose treatment.

b) Metoprolol tartrate solution:

Metoprolol Tartrate (Pure standard drug) powder was solubilized in 0.9% normal saline and solution was freshly prepared as standard drug solution.

c) Isoprenaline hydrochloride solution:

Isoprenaline Hydrochloride drug was solubilized in 0.9% normal saline and freshly prepared solution was used for induces necrosis in rat heart.

Chemicals and Enzymatic kits:

Normal saline (0.9%) (Albert David Ltd, Ghaziabad), Metoprolol Tartrate (Pure drug) (Sigma Aldrich, USA), Isoprenaline Hydrochloride, Petroleum Ether (40-60) (S.D. Fine Chemicals, Mumbai), Ethanol (Jiangsu Huaxi Ltd, China), Formaldehyde (Fisher scientific Ltd, Navi Mumbai), Serum AST diagnostic kit, Serum ALT diagnostic kit, Serum Creatinine diagnostic kit (Span Diagnostics Ltd, Surat), Serum LDH diagnostic kit (Accurex Biomedical Pvt Ltd Thane, Mumbai), all the chemicals used were of analytical grade.

Experimental Animals:

Rats (150-200g) were procured from central animal house of CSIR-CDRI Lucknow. The animals were housed in standard climatic condition and provided with food and water ad libitum. All experiments were accomplished in harmony with the guidelines of Committee for the Purpose of Control and Supervision on Experimental Animal (CPCSEA). Ethical clearance for this research work was obtained from Institutional Animal Ethical Committee (IAEC), Faculty of Pharmacy, Integral University, Lucknow. (Reg. No. IU/IAEC/18/03).

Isoprenaline induced model for myocardial infraction:

The model of isoprenaline-induced myocardial infarction is a widely used experimental model for cardio-toxicity to study the advantageous effects of various drugs on cardiac function. Rats receiving subcutaneous injections (s.c.) of isoproterenol on two successive days that will develop myocardial infarction depending on the dose and the weight of the animals. The inspection is to develop a pharmacological technique for producing myocardial necrosis of standard harshness in animals. Isoprenaline (ISO) is a synthetic β-adrenergic agent, causes ischemic necrosis in rats, which closely resembles human myocardial infarction.¹⁴

Treatment protocol:

Wistar rats of (150-200g) were distributed into five groups with six (n=6) rats in each group. The group I and group II were served as normal control and toxic control group, group I was received normal saline throughout the experiment and group II was received normal saline with isoprenaline (85mg/kg, s.c.) on 14^thand 15^th day. Group III and IV was served as low and high dose extract treated groups, it was received 100 and 200mg/kg, p.o Areca catechu extract and isoprenaline on 14^thand 15^th day respectively. Group V was served as standard group, treated with metoprolol (10mg/kg, p.o.) for 13 days and isoprenaline on 14^thand 15^th day as shown in table 1.

Table 1: Protocol for treatment and isoprenaline induction

Groups	Number of animals	Treatment to be given
I. Normal control (NC)	6	Rats were treated with normal saline (0.5ml/day.p.o) throughout the study period.
II. Isoprenaline Control	6	Rats were fed with NPD (Normal Pellet Diet) and water ad libitum all through the study Period (13 days) and isoprenaline (85mg/kg s.c.) was administered at the interval of 24 hours on the14^thand 15^th day of study.
III. Areca catechu (100mg/kg)	6	Rats were fed with NPD and water ad libitum along with Areca catechu extract (0.5 ml/day, p.o.) in low dose all through the study period then isoprenaline (85mg/kg s.c.) was administered at the interval of 24 hours on the 14^thand 15^th day of study.
IV. Areca catechu (200mg/kg)	6	Rats were fed with NPD all through the study period (13 days) along with Areca Catechu extract (0.5 ml/day, p.o.) in high dose and isoprenaline (85mg/kg s.c.) administered at the interval of 24 hours on the 14^th and 15^th day of study.
V. Metoprolol (10mg/kg)	6	Rats were administered with metoprolol (0.5 ml/day, p.o.) and fed with NPD and Pretreated all through the study period (13 days) then isoprenaline (85mg/kg s.c.) was administered at the interval of 24 hours on the 14^thand 15^th day of study.

At the end of 13 days of pre-treatment with test drug (metoprolol) rats were weighed and isoprenaline hydrochloride injection was administered at the dose of (85 mg/ kg/day, s.c.) twofold at a break of 24 hours (i.e., on 14^th and 15^th day of extract treatment). The rats were sacrificed on 16^thday, blood was collected by retro-orbital plexus using capillary tubes under mild ether anaesthesia and allowed to clot at room temperature for the evaluation of various biochemical parameters. The collected blood was centrifuged for 15 minutes at 3000 rpm at 30°C to collect serum which was used for the estimation of marker enzymes. The hearts were immediately dissected out, chilled, and perfused with Ice-cold saline.¹⁵

Estimation parameters:

a) Gross examination of rat heart:

The dissected hearts were washed down with Ice-cold saline. The hearts were visually examined for the inflammation, Scar formation, colour, redness, capillary dilatation in whole heart and grading was done.¹⁶

b) Heart weight: body weight ratio:

The rats were euthanized, weighed and recorded. Rats were pinned on its back onto the slat with stretched extremities (inner side of hands and foot). The rats were wiped with 70% ethanol to control hair. Exclusion of heart was done through immediate dissection of aortic root over aortic valves and superior vena cava on the top of the atria. Adjoining mediastinal fat layers were removed from the heart cautiously with forceps. Heart blood was squeezed from heart by patting the heart on spongy pad or surgical compress and done until the heart become totally dry. The dry heart was carefully weighed and recorded. Then the heart was positioned in fixative. ¹⁷

c) Biochemical estimations:

Blood samples were taken at the end of experimental period and serum was separated for further estimation of different enzymes associated to myocardial infarction such as aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), creatine kinase-MB fraction (CK-MB). All the study was performed with the help of commercially accessible kits acquired from SPAN Diagnostics Ltd, Surat, Accurex Biomedical Pvt Ltd Thane, Mumbai and measured by spectrophotometer, Shimadzu. TROPONIN-I release was estimated by Troponin–I Rapid test kit purchased from Reckon diagnostic Pvt Ltd.¹⁸

Statistical analysis:

One-way ANOVA was adopted for Statistical analysis followed by Dunnett “t” test (GraphPad Instat, USA).

Histopathological study:

The heart was isolated at the end of the study and washed with ice cold saline. The tissue was stored in 10% buffered neutral formalin solution. After fixation tissues were embedded in paraffin-wax, trimmed at five micrometre thick sections and stained with haematoxylin and eosin. The slides were viewed under light microscope and photomicrographs were captured.

RESULTS:

Gross examination of heart:

The visually examined heart was observed for redness, inflammation, capillary dilatation, Scar formation, colour and grading of the heart has been done as shown in table 2.

Morphological grading for heart tissue damage:

Grade 0 = No Lesion

Grade 1= Inflammation, redness, capillary dilations.

Grade 2 = Edema, yellowish ventricle portion

Grade 3 = Atrium and ventricle turns yellow, scar formation

Grade 4 = Diffuse heart, absolute scar formation, increased necrosis portion.

Table 2: Observation table for morphological damage of heart tissues

Groups	Grading of cardiac damage
Normal control	Grade 0
Isoprenaline Control	Grade 4
Areca catechu extract (100mg/kg)	Grade 1
Areca catechu extract (200mg/kg)	Grade 3
Standard Metoprolol (10mg/kg)	Grade 2

The isoprenaline (ISO) group demonstrated marked inflammation, scar formation, diffused heart as compared to Normal control (NC) group. The standard Metoprolol treated (10mg/kg) group illustrated marked reduction in edema, capillary dilation, and scar formation, with mild redness as compared to Isoprenaline (ISO) group. The Areca catechu extract (200mg/kg) showed notable decline in inflammation, redness, capillary dilatation and scar formation as compared to test Areca catechu extract (100mg/kg) when both extracts were compared to isoprenaline (ISO) group.

The heart weight and heart weight/body weight ratio (table 3) were examined in various treatment group. The isoprenaline (ISO) group confirmed noticeable raise in heart weight which is due to hypertrophy, as compared to Normal control (NC) group.

Table 3: Heart weight/ body weight ratio

Groups	Body weight (gm)	Heart weight (gm)	Heart/body weight ratio (x 10³)
Normal control	200	0.68	3.4
Isoprenaline Control	190	0.90	4.73
Areca catechu extract (100mg/kg)	2100	0.86	4.10
Areca catechu extract (200mg/kg)	225	0.83	3.69
Standard Metoprolol (10mg/kg)	215	0.79	3.67

The standard Metoprolol treated (10mg/kg) group demonstrated reduction in heart weight and heart/body weight ratio as compared to Isoprenaline (ISO) group. Areca catechu extract (200mg/kg) demonstrated significant decrease in heart weight and decrease in heart/body weight ratio as compared to Isoprenaline group. When both Areca catechu extracts (100mg/kg, 200mg/kg) were compared with Isoprenaline (ISO) group, the higher dose Areca catechu extract (200mg/kg) showed significant reduction in heart weight and heart/body weight ratio as compared to lower dose Areca catechu extract (100mg/kg).

The effects of aqueous ethanolic Areca catechu extract oral treatments on serum marker enzymes AST, ALT, LDH for 15 days are outlined in Table 4. Rats treated with ISO confirms an extremely significant increase (p<0.001) in serum marker enzyme activities as compared to the normal rat (NC) group. Rats pre-treated with metoprolol (STD group) showed a highly significant (p<0.001) diminution in cardiac marker enzyme as compared to Isoprenaline (ISO) group.

Pre-treatment with high dose Areca catechu extract (200mg/kg) to rats for 15 days, followed by ISO subcutaneous injection on the 14th and 15th days, elicited a highly significant (p<0.001) reduction in the ISO-induced augmented activities of AST, ALT, LDH, and CPK. The high dose of Areca catechu extract (200mg/kg) as compared to Isoprenaline (ISO) group showed significance (p<0.01) in reducing ISO elevated serum enzyme activities.

Table 4: Biochemical estimations

Treatment	AST (IU/L)	ALT (IU/L)	LDH (IU/L)	CK (mg/dl)
Normal Control	48.475±0.3965	29.27±0.7314	368.59±1.086	0.963±0.0063
Isoprenaline control (85mg/kg)	72.450±0.43^a	73.7825±1.204^a	596.58±0.874^a	1.943±0.0167^a
Areca catechu extract (100mg/kg/day, p.o.) + Isoprenaline (85mg/kg)	50.22±2.67^c	19.452±29.585^c	492.58±0.868^c	1.343±0.0163^c
Areca catechu extract (200mg/kg/day, p.o.)+ Isoprenaline (85mg/kg)	46.80±0.52^d	23.535±0.5000^d	429.145±4.398^d	1.285±0.0105^d
Standard metoprolol (pure) (10 mg/kg/day, p.o.)	38.24±0.4128^b	19.4525±0.268^b	388.50±3.174^b	1.13±0.0179^b

Values are mean ± SEM for five animals in each group ^aP< 0.001 When ISO group is compare with NC group; ^bP< 0.001 When standard group is compare with ISO group; ^cP< 0.01 When experimental group is compare with ISO group; ^dP< 0.001 When experimental group is compare with ISO group; AST- Aspartate amino transferases; ALT- Alanine transaminase; LDH - Lactate dehydrogenase CK-MB- Creatine kinase; NC-Normal control; ISO-Isoprenaline; STD- Standard; Areca catechu extract (100mg/kg); Areca catechu extract (200mg/kg)

Table 5: Release of TROPONIN –I in various treatment groups: +ve - Presence of troponin in serum, -ve - Absence of troponin in serum

Group	Normal Control	Isoprenaline control	*Areca catechu extract* (100mg/kg)	*Areca catechu extract* (200mg/kg)	Standard Metoprolol (10mg/kg)
I	-ve	+ve	+ve	-ve	-ve
II	-ve	+ve	+ve	-ve	-ve
III	-ve	+ve	-ve	+ve	+ve
IV	-ve	+ve	+ve	-ve	-ve
V	-ve	+ve	-ve	+ve	+ve

Normal Control Isoprenaline control Areca catechu extract (100mg/kg)

Areca catechu extract (200mg/kg) Metoprolol (10mg/kg)

Figure 1: Visual photographs of rat heart: a- Normal control group; b- ISO control group; c- Areca catechu extract group (100mg/kg); d- Areca catechu extract group (200mg/kg); e- Metoprolol group

Photomicrograph of rat heart pre-treated with Areca catechu extract (100mg/kg) group showed modest degree of myonecrosis and less infiltration of inflammatory cells but myophagophytosis and subendocardium vacuolar changes are present and are clearly visible in (Fig 2. c) at 10x (prominently). Pre-treated rat heart with Areca catechu extract (200mg/kg) treated group showed lesser infiltration of inflammatory cells and little degree of myonecrosis as well as a decreased myophagophytosis and sub endocardium vacuolar changes are present and clearly visible in (Fig 2.d) at10x (prominently). Standard (STD) Metoprolol (10mg/kg) pre-treated heart showed minute myonecrosis, myophagocytosis and lymphocytic infiltration, oedema and diminutive infiltration of inflammatory cells are clearly visible in (Fig 2.e) at10x (prominently).

DISCUSSION:

Myocardial infarction is characterized by an unbalanced coronary blood supply and demand which results in myocardial ischemic injury and damages the cardiomyocytes.¹⁹ Cardiovascular disease is major global health issue reaching epidemic proportion in Indian subcontinent with low and middle-income countries contribute 78% of all death.²⁰ Myocardial cell fortification and anticipation of cell ischemia or necrosis have been therapeutics targets for a long time. New treatment is required to treat ischemia for the reason that modern therapeutic management has merely inadequate impact on endurance and yearly cost.²¹ Many of the prescribed medications for myocardial infarction have side effects but the herbal medication has fewer side effects or no side effect. A great many cardiovascular drugs have the potential to induce adverse reactions.²²

The present investigation was aimed to explore and assess the cardioprotective consequences of Areca catechu on isoprenaline induced myocardial infarction in rats. AST, ALT, LDH, CK-MB and TROPONIN concentration in myocardium serves as diagnostic marker enzyme of myocardial infarction, once myocardium is damaged, releases of its content into the extra-cellular fluid serves as the diagnostic enzyme marker of myocardial damage tissue.²³

This study observed a significant increase in the level on marker enzyme (AST, ALT, LDH, and TROPONIN) in serum of isoprenaline treated rats. It is obvious that isoprenaline induced rat showed elevated levels of ALT, AST, LDH and CK-MB as compared to control rats. These finding could be a consequence of a reduction in the quantity of viable myocytes due to enhanced cell death in heart as these animals showed the highest level of AST, ALT, LDH and CK MB.²⁴ Among both dose of Areca catechu, treatment with the highest dose Areca catechu extract (200mg/kg) decreased the level of these marker enzyme quite significantly when compared to low dose Areca catechu extract (100mg/kg) pointing clearly that Areca catechu could be a cardioprotective against the MI.

CONCLUSION:

From the study, it could be concluded that though significant improvement was observed in myocardial infarction rats during the extract treatment. The aqueous ethanolic extract of Areca catechu leaves have been shown to possess cardioprotective effect against isoproterenol-induced myocardial infarction in rats that was supported by biochemical and histopathological studies of the heart tissue sections of experimental rats.

This might be due to the free radical scavenging ability and higher number of antioxidants present in the Areca catechu seeds. Thus, the aqueous ethanolic extract of Areca catechu seeds could be recommended as a potential cardioprotective drug.

ACKNOWLEDGMENTS:

Author thanks Prof. Syed Waseem Akhtar, Hon. Chancellor and Prof. Aqil Ahmad, Hon. Vice Chancellor for providing excellent research facility in the university. The university has provided a manuscript communication number for further communication (IU/R&D/2020-MCN000927).

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Received on 03.05.2020 Modified on 01.07.2020

Research J. Pharm. and Tech. 2021; 14(6):3067-3073.

DOI: 10.52711/0974-360X.2021.00536