green synthesis and cardioprotective activity of

www.wjpps.com │ Vol 9, Issue 11, 2020. │ ISO 9001:2015 Certified Journal │

1659

Haripriya et al. World Journal of Pharmacy and Pharmaceutical Sciences

GREEN SYNTHESIS AND CARDIOPROTECTIVE ACTIVITY OF

NYCTANTHUS ARBOR-TRISTIS ZNO NANOPARTICLE AGAINST

ISOPROTERENOL INDUCED MYOCARDIAL INFARCTION IN RATS

*Haripriya S. and Sudhakar P.

Department of pharmacology Swamy Vivekananda College of Pharmacy, Elayampalayam,

Tiruchengode - 637205, Namakkal (Dt), Tamilnadu, India.

ABSTRACT

Myocardial infarction is a life-threatening condition that occurs when

blood flow to the heart is abruptly cut off, thereby causing tissue

damage. The leaves of the Nyctanthus arbor-tristis mainly used in

ayurvedic, siddha, unani. Because of the presence of more potent

oleanolic acid in the leaves, the hydrochloric extract was therefore

investigated for its cardioprotective activity in Nano-form. The present

study was therefore aimed for Green synthesis and cardioprotective

activity of Nyctanthus arbor-tristisZnO Nanoparticle against

Isoproterenol induced myocardial infarction in rats. Thirty Albino

Wister male rats weighing 200-300gm were randomly assigned to

groups, each group containing six animals. I-Control, II- Negative

control, III-Standard, IV-HAE-NAT, V-ZnO NP-NAT. Myocardial

infarction was induced by intra peritoneal injection of Isoproterenol

85mg/kg in two consecutive dose on 14th

and 15th

day. In this study, ZnO NP against

isoproterenol induced animals exhibited significant induced in serum total cholesterol TG,

LDL level and decrease in HDL. And also serum cardiac activity markers CK-MB,

LDH,ALT,AST and ALP were elevated. It might be concluded Hydroalcoholic extract of

nyctanthus arbor tristis in cardioprotective activity which might be aid to reduce the

myocardial infarction, cardioprotective effect of ZnO NP was proved by reduction in cardiac

markers, altered lipid profile and histopathological studies.

KEYWORDS: Nanoparticle, Isoproterenol, cardioprotective, Green synthesis ZnO NP.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 11, 1659-1682 Research Article ISSN 2278 – 4357

*Corresponding Author

Haripriya S.

Department of

pharmacology Swamy

Vivekananda College of

Pharmacy, Elayampalayam,

Tiruchengode - 637205,

Namakkal (Dt), Tamilnadu,

India.

Article Received on

06 Sept. 2020,

Revised on 27 Sept. 2020,

Accepted on 18 October 2020

DOI: 10.20959/wjpps202011-17661


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INTRODUCTION

Myocardial infarction (MI) or heart attack is one of the leading causes of death all over the

world. It is caused due to an interruption in blood supply via the coronary circulation to any

part of myocardium, resulting in myocardial necrosis.[1]

Consequences of MI include

hyperlipidemia, peroxidation of membrane lipids and loss of plasma membrane integrity. The

pathogenesis of cardiac damage involves cell apoptosis, which is mainly influenced by

oxidative-ROS production. Reactive oxygen species (ROS) play a critical role in the

pathogenesis of cardiovascular injury associated with circulatory disturbance.[2]

Despite major therapeutic advances, MI remains the major cause of death in india and

increased mortality due to CVD is expected to be double by 2020.[3]

Hence Myocardial cell

protection and prevention of cell ischemia/necrosis have been therapeutic targets for a long

time. The allopathic medicines currently used to treat myocardial infarction have many side

effects. Hence new therapies are needed to treat myocardial damage limiting its adverse

effects and economical costs.

The model of Isoproterenol-induced myocardial necrosis has the mechanism of generating

ROS causing lipid peroxidation damage to the proteins due to production of carbonyl

derivatives.[4]

A disparity between the oxygen requirement of the myocardium and the ability

of the coronary artery to meet it results in the ischemic necrosis of heart muscle. The

pathophysiological changes following ISO administration are comparable to those taking

place in human MI.[5]

Hence this model is most widely used in order to study the beneficial

effects of various herbal drugs on cardiac function.[6]

Triterpenoids exists widely in nature and are the one of the major components of many

traditional medicinal herbs. Oleanolic acid (OA) is a triterpenoid compound that exists

widely in food and herbs.[7]

It has variety of biological effects, such anti-oxidants[8]

,

antifungal, anti-inflammatory, anti-hyperlipdemia, hepatoprotective, tumor prevention,

immunomodulatory[9]

, anti-HIV, anti-arrhythmic and cardiotonic.[10]

Due to its anti-oxidant,

anti-hyperlipedemic, antiarrhythmic, and cardiotonic effects, it will provide an accessible and

cheap traditional medicine source for treatment of myocardial ischemia in developing

countries. Hence current attention has been focused on phytoconstituents (OA) derived from

plant species as potential therapeutic agents in the prevention and management of

cardiovascular disease.


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Nyctanthes, also known as Harsingar, is an important member of Ayurveda, the traditional

Indian medicine science. It is blessed with a diverse spectrum of medicinal properties, such as

anti-helminthic, antimicrobial, antiviral, antileishmania, anti-allergic, anti-diabetic and anti-

cancerous. Juice of the leaves is used as digestives, antidote to reptile venoms, mild bitter

tonic, laxative, diaphoretic and diuretic.[11]

The phytochemical characterization of Nyctanthus

arbour-tristis reveals the presence of robust bioactive Triterpenoid compound, Oleanolic

acid.[12]

Hence this boosts up in investigating the effect of leaves on Isoproterenol-induced

cardiac injury. Previously reported articles on Oleonolic acid and myocardial damage

prompted our further research to deal with Cardioprotective activity of the plant.

Nanoparticles hold extraordinary and attractive properties due to their small sizes, large

surface area, free hanging bonds and superior reactivity. Nowadays, nanotechnology has a

vast range of application in diagnosis, drug delivery, food industry, paints, electronics, sports,

environmental cleanup, cosmetics, and sunscreens. Green synthesis approaches of herbal

extracts are gaining interest towards treatment of various diseases. Recently, plants and their

extracts based nanoparticles synthesis were considered to be the best techniques because of

easy availability, mass production and eco-friendly process. Zinc (Zn) an essential

micronutrient that exhibits antioxidant properties and protects cardiac cells against different

oxidative stressors.[13]

According to some previous studies, Zinc oxide nanoparticles (ZnO

NPs) are found to be non-toxic, biosafe, biocompatible making them an ideal candidate for

biological applications.

The plant Nyctanthes extracts have been reported to yield gold, silver and titanium dioxide

Nanoparticles. Two majorly researched substrates for biosynthesis of ZnONPs are zinc

acetate and zinc nitrate. This is, to the best of our knowledge, the first study reporting

synthesis of zinc oxide Nanoparticles using leaf extract of Nyctanthes arbor-tristis and zinc

acetate. The present study was therefore aimed to determine the Cardioprotective effect of

NA-ZnO NP against Isoproterenol induced Myocardial Infarction in Rats.

MATERIALS AND METHODS

Drugs and Chemicals

Isoprenaline hydrochloride (isoproterenol) was purchased from Sigma Chemical Co. (St.

Louis, MO, USA). All the chemicals and reagents used in this study were of analytical grade.

Propranolol was purchased from the manufacturer Cipla Pharmaceuticals, Mumbai.


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Collection and Authentication of Plant Material

Leaves of Nyctanthes arbor-tristis were collected from in and around the region of

Namakkal, Tamilnadu, in the month of November. The plant was authenticated by Dr. G.V.S.

Murthy, Joint Director, Botanical Survey of India, Coimbatore, Tamilnadu, India. A voucher

specimen is preserved in our laboratory for future reference.

Preparation of Plant Extract

The plants material was shade dried at room temperature. The dried plant materials were

subjected to size reduction to a coarse powder by using dry grinder and passed through sieve

no. 40 was used for extraction. Powdered plant material (500 gm) was extracted with 80%

methanol at room temperature for 72 hrs. The extract was filtered and concentrated to dryness

under reduced pressure and controlled temperature (400

C to 500

C) in a rotary evaporator

until all solvent was removed to give a dark colored molten extract. The extract was stored in

airtight containers in refrigerator maintained below 100

C until further use.


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Synthesis

Of Zno Nps[14]

1 mm Zinc acetate was dissolved in 50 ml Milli-Q water and Stirrer for 1 h respectively.

20 mL of NaOH solution was slowly added into the Zinc acetate solution

25 mL of plant extract was added

The colour of the reaction mixture was changed after 1 h of incubation time.

Solution was left in stirrer for 3 h Yellow colour appeared after the incubation time

confirmed the synthesis of ZnO NPs

Precipitate was separated and centrifugation at 8000 rpm at 60 °C for 15 min

Pellet was collected and dried hot air oven at 80 °C for 2 h and preserved.

CHARACTERIZATION OF ZnO NANOPARTICLES

Uv –Visible Spectroscopy

For UV- Visible spectra of synthesized ZnO nanoparticles were re-suspended in equal

amount of sterilized de-ionised water and spectrum scans were performed using Shimadzu

UV-1800 Spectrophotometer. 2 ml solution of the nanoparticles was taken in quartz cuvette.

The scan range was set between 200-800nm and the background was minimised using de-

ionised water.

Transmission Electron Microscopy (Tem)

TEM technique was used to visualize the morphology of the nanoparticles and determination

of the size, shape and arrangement of particles. The ZnO nanoparticles was suspended in

sterile deionised water, sonicated for 15 min and diluted to yield slightly turbid suspension.

The suspension was then coated onto a copper grid and allowed to dry. TEM images were

taken on the Philips CM200 7500 model with resolution 2.4 A0 operating at voltage 20-

200kv.

Scanning Electron Microscopy (Sem)

The morphological features of synthesized ZnO nanoparticles from NAT plant extract were

studied by Scanning Electron Microscope (JSM-6480 LV). After 24Hrs. of the addition of

ZnO the SEM slides were prepared by making a smear of the solutions on slides. A thin layer


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of platinum was coated to make the samples conductive. Then the samples were characterized

in the SEM at an accelerating voltage of 20 KV.

X Ray Diffraction Analysis (Xrd)

The XRD pattern of the synthesized nanoparticles was then recorded using Analytical

PW3040/60 XpertPRO model X-ray diffractometer. A thin flim of the sample was made by

dipping a glass plate for XRD studies. The instrument was operated at a current of 30mA and

voltage of 40KV.The size was calculated using Scherer formula.

= 0.94 𝝀

β cosθ

Where,

D = crystal size,

λ = wavelength of X-ray,

Ѳ = Bragg’s angle

β= Full width at half maxima (FWHM) of spectral peak (in radians)

5.5.5. FOURIER TRANSFORM INFRARED (FT-IR) SPECTROSCOPY

The characterisation of phytoconstituents of Nyctanthes arbor-tristis hydro alcoholic extract

involved in the reduction and stabilization of zinc nanoparticles was investigated by FT-IR

analysis (Shimadzu- IR Affinity-1) and the spectra was scanned in the range of 4000-500

cmˉ1 range at a resolution of 4 cmˉ

1.The sample was prepared by grounding the ZnO

nanoparticles uniformly in a matrix of dry KBr, compressed to form an almost transparent

disc. KBr was used as a standard to analyse the sample.

EVALUATION OF CARDIO PROTECTIVE EFFECT

Experimental Animals

The colony inbred female albino wistar Rats , Weighing 150-300gm were obtained from

Central Animal house of Swamy Vivekananda college of pharmacy, Elayampalayam,

Namakkal -637 205. The animals were kept under standard environmental conditions of

12/12light/dark rhythm, maintained under controlled room temperature (23±2ºC) and a

relative humidity of 60%± 10%, in polypropylene cages. They were fed with standard pellet

diet and water ad libitum. The immature animals were acclimatized under laboratory

conditions three days prior to initiation of the experiment. The cages were cleaned daily by

changing the husk bedding.


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The experimental protocol was approved by the institutional Animal Ethical Committee

(IAEC) of Swamy Vivekananda college of pharmacy, Elayampalayam, Namakkal -637

205.Care and use of laboratory animals were confirmed to CPCSEA guidelines.

IAEC Approval No.: SVCP/IAEC/PG/2/04/2018

Induction of Myocardial Infarction Using Isoproterenol[15]

Myocardial Infarction was induced by dissolving Isoproterenol hydrochloride in normal

saline and was injected subcutaneously (S.C) 85mg/kg body weight for two consecutive days

into rats at an interval of 24 hrs to induce experimental MI (14th

& 15th

day).

Animal Grouping

The experimental rats were divided into four groups of six animals each and treated as

follows:

Group 1: Normal control animals receiving normal saline (2ml/kg/day) once daily for 15

days.

Group 2: Negative-control animals receiving normal saline (2ml/kg/day) once daily for 15

days and treated with isoproterenol (ISO) (85mg/kg, s.c.) on 14th

&15th

day.

Group 3: Animals receiving standard drug propranolol (10mg/kg/day) orally 15 days and

challenged with isoproterenol (ISO) (85mg/kg, s.c.) on 14th

&15th

day.

Group 4: Animals receiving hydroalcholic extract NAT–ZnO NP (500mg/kg/day) orally for

15 days and challenged with isoproterenol (ISO) (85mg/kg, s.c.) on 14th

&15th

day.

Group 5: Animals receiving higher dose NAT –ZnO NP (30 mg/kg/day) orally for 15 days

and challenged with isoproterenol (ISO) (85mg/kg, s.c.) on 14th

&15th

day.

Acute Toxicity Studies

Acute toxicity study was conducted in accordance with Organisation for Economic

Cooperation and Development (OECD) guidelines for testing of acute oral toxicity (AOT) by

up and down procedure (UDP) OECD- No: 423


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PHYSICAL EVALUATION

Measurement of Body Weight

Body weight of each rats in all groups were measured weekly till end of the treatment using a

weighing balance and the changes were recorded.

Biochemical Estimation

At the end of experimental period on 14th

day rats were fasted overnight (12 h) and blood

samples were collected via retro-orbital sinus puncture under mild anaesthesia. Serum was

obtained by centrifugation of samples at 3000 rpm for 10 min and used for further plasma

lipid profile and cardiac specific injury markers estimations.

Serum Lipid Profile

Serum total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and Low

density lipoproteins (LDL) were analysed by using commercially available laboratory kits

(ARKRAY Healthcare Pvt. Ltd., surat, India).


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Serum Cardiac Specific Injury Markers

Activity levels of creatine phospokinase-MB (CK-MB), lactate dehydrogenase (LDH),

Alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP)

in serum were estimated using commercially available kits (AGAPPE Diagnostics LTD,

Kerala, India).

Histopathological Evaluation

Histological evaluation was performed on lower portion of the heart tissue. Fresh heart

tissues were excised and then fixed in 10% formalin for 24 hr. The fixative was removed by

washing through running tap water for overnight. After dehydration through a graded serious

of alcohols, the tissues were cleaned in methyl benzoate, embedded in paraffin wax. Section

were cut into 5 μm thickness and stained with hematoxylin and eosin. After repeated

dehydration and cleaning, the sections were mounted and observed under light microscope

with 100x magnification for histological changes.

Statistical Analysis

The results of cardio protective activities are expressed as mean ± SEM from four animals

from each group. Results were analysed statistically by one way ANOVA followed by post

hoc Dunnet’s test by using SPSS V.17(student trail version).The difference was considered

significant when p<0.05.

RESULT

Characterization of Zno Nanoparticles

Uv –Visible Spectroscopy

The green synthesized zinc oxide nanoparticles showed absorbance from 250 to 265 nm and

the absorbance centred at 260nm in UV-visible spectroscopy.


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TRANSMISSION ELECTRON MICROSCOPY (TEM)

It is spherical shape. The average particle size measured from the TEM images is found to be

50nm


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SCANNING ELECTRON MICROSCOPY (SEM)

The images showed the presence of both individual as well as the aggregated ZnO NPs. The

ZnO NPs are mainly uniform spherical shaped with the average range of particle size

distribution from 40 nm to 80 nm.

POWER X RAY DIFFRACTION ANALYSIS (XRD)

Diffraction peaks were observed at the 2h values of 28.55°, 31.76°, 32.62°, 34.42°,36.2°,

47.53°, 50.66°, 56.58° and 62.84° corresponding to lattice planes

(70),(86),(90),(113),(114),(145),(200),(208) and (235) respectively.

Using Bragg’s Law equation

Sinθ=nλ where,

θ is Bragg’s angle of diffraction,

λ is X-ray wavelength, i.e. 1.5406 A ° and n= 1.

using Debye–Scherrer formula

D ¼ 0:89k=b cosθ where

0.89= Scherrer’s constant,

k= X-ray wavelength

= 0.89x1.5406

0.1061x0.8064

= 16.27

The value of particle size was found to be 16.2 nm


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2-theta (deg)

Inte

nsity (

cp

s)

20 40 60 80 100

0.0e+000

1.0e+004

2.0e+004

3.0e+004

4.0e+004

5.0e+004

6.0e+004

Fourier Transform Infrared Spectroscopy

In FTIR, various peaks at 3262, 2923.56, 1567.84, 1406.82 and 1035.59cm-1

. The peaks at

3262 correspond to H bonded OH stretch and N-H stretch. Peak at 2923.56 corresponds to C-

H stretch. The peak 1567.84 refers amine –NH vibration stretch in protein amide. The peak at

1406.82 refers C-C stretch. The 1035.59 peak results from C-O-C stretching in aromatic

amine.


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ANIMAL STUDIES

Changes In Body Weight

Body weight of the animals of all groups was measured at trial period, initial and a final body

weight change in treatment group was compared with control group. Initially there were no

significant changes in body weight between treatment groups when compared to the control

group. On 15th

day weight of ZnO NAT treated group showed significantly increase in body

weight compared to other groups.

Table NO: 1 Changes In Body Weight.

Treatment Initial body weight(g) Final body weight(g)

Normal control 164.5± 10.03 225.3±16.26

Diseases control 173.3±12.07 232.4±14.73a * * *

Standard (Propronolol) 163.6±11.16 215.0±10.57 b * * *

HE of NAT(500mg/kg) 167.3±8.73 235.21±6.54 b * * *

ZnO NAT (30mg/kg) 176.7±10.23 242.3±15.79 b* * *

Values are expressed as mean ± SEM, n=6. Comparisons were made between:

a- Group I vs II, III, IV and V; b- Group II vs I,III ,IV and V

Symbols represent statistical significance: #=P<0.001, $=P<0.01,* = P<0.05

Graph No. 1: Changes In Body Weight

Effect of Nat Zno-Nps on Isoproterenol-Induced Changes In Serum Cardiac Specific

Injury Markers

The activities of cardiac functional marker enzymes (CPK, LDH, AST, ALT and ALP) in the

serum of isoproterenol alone treated group shown significant increases as compare to the

normal control group rats. Though the pretreatment of ZnO NPs NAT (30mg/kg) related


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groups significantly prevented the depletion of myocardial enzymes as compared to the

negative control. ZnO NPs NAT (30mg/kg) treatment group did not show any significant

effect on the activity of ALP enzyme.

Table NO: 2 Effect of Hena on Marker Enzymes of Rats Myocardial Serum.

Group AST ALT ALP LDH CK-MB

Normal control 17.01±1.48

15.56±2.25

95.20±3.14

213.2±12.59

65.95±15.04

Diseases control 50.02±4.25a#

52.07±3.17 a#

228.7±9.05a#

328.9±16.6 a#

140.6±4.45 a#

Standard

(Propronolol) 50.80±1.05

a# 33.01±1.05

a# b# 130.9±7.09

a#b* 269.46±11.0

a# 76.08±1.02

HE of

NAT(500mg/kg) 39.06±2.34

a# 49.42±3.15

b# 145.04±3.15a

** 298.09±1.64

a#b* 123.35±9.08

a#

ZnO NAT

(30mg/kg) 16.9±1.08

b# 22.36±1.36

b# 118.78±2,08

b# 210.8±2.08

b* 112.06±1.67

a# b*


a- Group I vs II, III ,IV and V b- Group II vs I,III ,IV and V Symbols represent statistical

significance: #=P<0.001, $=P<0.01,* = P<0.05

Graph 2: Effect of Hena on Marker Enzymes of Rats Myocardial Serum.

Effect of Zno Np-Nat on Isoproterenol- Induced Changes In Serum Lipid Profile

The results of ZnO NPs NAT on isoproterenol induced changes in serum lipid profile are

cited in Table 2. The level of serum total cholesterol, triglycerides, LDL significantly

increases and decrease in the levels of HDL in isoproterenol treatment as compartment to

control group. The treatment ZnO NPs NAT of for 15 days however, significantly restored

the lipid profile to near normally as compared to the negative control.


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Table No: 3: Effect of Zno Np-Nat on Isoproterenol- Induced Changes In Serum Lipid

Profile.

Treatments TC (mg/dl) TG(mg/dl) HDL(mg/dl) LDL(mg/dl)

Normal control 108.17 ± 4.98 95.67 ± 4.09 49.33 ± 5.38 99.83 ± 4.71

Diseases control 177.67 ± 8.11a# 144.50 ± 5.46 a

# 33.17 ± 3.99 a* 128.83 ± 7.85 a*

Standard

(Propronolol) 169.5 ±5.32 134.42 ±7.01 33.17 ±5.87 119.6 ±4.23

HE of

NAT(500mg/kg) 127.31 ± 7.18a* b

# 115.83 ± 6.98 a* b

$ 38.00 ± 4.03 112.66 ± 7.01

ZnO NAT

(30mg/kg) 108.24 ± 6.18 b

# 96.00 ± 6.92 b

# 48.67 ± 5.00 b* 97.17 ± 5.71 b*


a- Group I vs II, III and IV. And V b- Group II vs III and IV, V

Symbols represent statistical significance: #=P<0.001, $=P<0.01,* = P<0.05

Graph No. 3: Effect of Zno Np-Nat on Isoproterenol-Induced Changes In Serum Lipid

Profile.

HISTOPATHOLOGICAL OBSERVATIONS

Histopathological examination of myocardial tissue from normal control animals exhibited

clear integrity of myocardial membrane. Histopathological findings confirmed the induction

of myocardial infarction by isoproterenol. Heart tissues from isoproterenol treated rats

showed widespread myocardial structure disorder and sub endocardial necrosis with capillary

dilatation and leukocyte infiltration as compared to normal control rats. Treatment of

hydroalcoholic extract of NAT 500mg/kg showed mild muscle separation and few

inflammatory cells, lower dose of 30mg/kg treatment showed no change in histo-architecture

of heart tissue as compared to normal control rats.


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(A) Normal Control (B) Disease control (IP)

(C) Standard (Propranolol ) (D) HE-NAT

E) ZnO-NAT

Effect of Zno –Nat On The Histolocical Morphology Of Rat Heart Shown By

Hematoxin And Eosin (X100)

In histopathological studies the control rats showed the regular arrangement with clear

striations of myocardial fibers without any histological alterations because of degeneration or

necrosis. Group II-Negative control induced rats showed pathological changes in heart

including several congestions, myocardial necrosis and inflamed cells. Group-III Heart was

Inflamed

cells

Mild

necrosis

Necrosis

Mild

muscle

separation

Mild inflamed

cells

Muscle

separation


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having near normal appearance with mild changes in congestions and necrosis in rats treated

with standard drug propranolol. Same pattern was obtained in rats treated with

hydroalcoholic extract of HE-NAT 500 mg/kg and ZnO-NAT 30 mg/kg its induced showed

marked reduction in edema,. So this research proved that the hydroalcoholic extract of the

Nyctanthus arbor-tristis plant can protect the heart from the myocardial infarction condition.

DISCUSSION

Green synthesis of nanoparticles is an eco-friendly approach which might pave the way for

researchers across the globe to explore the potential of different herbs in order to synthesize

nanoparticles. Some special features such as high surface area, having 1-100 nanometres in

size, and easy penetration into the cells and proteins, sensing, and detection of biological

environments, make inorganic nanoparticles as potential candidate for applications in

biomedical fields. Among this Zinc oxide nanoparticles (ZnO- NP) as a material of low

toxicity, because zinc is an essential trace element in the human body and is commonly

present in foods or added as a nutritional supplement. The fact that zinc appears to have

protective effects in coronary artery diseases and cardiomyopathy is attributed to its critical

role in redox signalling pathway, whereby certain triggers such as ischemia and infarction

leads to release of zinc from proteins and cause myocardial damage.[16]

Oleanolic acid is a lipophilic β-blocker in nature. β -adrenergic blockers have long been

useful adjuvants in the management of myocardial ischemic syndromes.[17]

The use of

oleanolic acid significantly prevented an increase in the systolic blood pressure and cardiac

lipid peroxidation level. Oleanolic acid is known to possess beneficial effects on the

regulation of the cardiovascular homeostasis.[18,19]

However, the exact of Nyctanthus arbor-

tristis is the source of oleanolic acid which was used and synthesised the ZnO green nano

particle for evaluating cardio protection action.

In UV-Visible spectra the green synthesized zinc oxide nanoparticles showed absorbance

spectra at 260nm. It enlightens that the nanoparticles were found to be symmetrical with

spherical polydispersed in nature.[20]

TEM analysis was performed in order to investigate the morphological and distribution of our

green synthesized zinc oxide nanoparticles. TEM analysis revealed that all the nanoparticles

were found in general as spherical shape. The average particle size measured from the TEM

images is found to be 50nm. The analysis of the scanning electron microscopy (SEM) images


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predicts the formation and the morphology of stable zinc oxide nanoparticles obtained from

the current green approach. The images showed the presence of both individual as well as the

aggregated ZnONPs.[21]

The ZnONPs are mainly uniform spherical shaped with the average

range of particle size distribution from 40 nm to 80 nm.

Fourier transform infrared (FT-IR) spectroscopy helps establish the identity of various

phytochemical constituents involved in the reduction and stabilization of the nanoparticles.

FT-IR spectrum for dried and powdered ZnO NPs was obtained using Perkin Elmer FT-IR

Spectrophotometer Frontier using the technique of Attenuated Total Reflectance (ATR) in the

range of 4000–400 cm. The sample for the infrared analysis was carefully prepared to

exclude any possibility of the presence of any unbound plant extract residue. The similarities

between the spectra with some marginal shifts in peak position clearly indicate the presence

of the residual plant extract in the sample as a capping agent to the ZnO NPs. FT-IR

spectroscopy was performed. The FTIR spectra resulted in various peaks at 3262, 2923.56,

1567.84, 1406.82 and 1035.59 cm-1

. The peaks at 3262 correspond to H bonded OH stretch

and N-H stretch. Peak at 2923.56 corresponds to C-H stretch. The 1567.84 peak refers amine

–NH vibration stretch in protein amide. The peak at 1406.82 refers C-C stretch. The 1035.59

peak results from C-O-C stretching in aromatic amine.

Crystal lattice indices and particle size calculations were performed using the X-ray

diffraction pattern of ZnO NPs. Diffraction peaks were observed at the 2h values of

28.55º,31.76º,32.62º,34.42º,36.25º,47.53º,50.66º,56.58º and 62.84º corresponding to lattice

planes (70),(86),(90),(113),(114),(145),(200),(208) and (235).The value of particle size was

found to be 16.2 nm which falls within the size range of 15-48 nm reported by TEM and

XRD.

Many of today’s diseases including cardiac diseases have been linked to oxidative stress

which is initiated by the reaction of free radicals with biological macromolecules such as

proteins, lipids and DNA.[22]

Generally antioxidants, preferably from natural sources, have

been considered as effective treatments.

Isoproterenol produces relative ischemia or hypoxia due to myocardial hyperactivity and

coronary hypotension and induce myocardial ischemia due to cytosolic Ca2+

overload. The

oxidative stress may be exerted through quinone metabolites of isoproterenol, which reacts

with oxygen to produce ROS and interfere with glutathione reductase, superoxide dismutase


1677


and ATP pumps. Radioactive oxygen species (ROS) are formed at an accelerated rate in ISO-

treated myocardium. Cardiac myocytes, endothelial cells and infiltrating neutrophils

contribute to this ROS production and can lead to cellular dysfunction and necrosis23

.

Myocardial necrosis induced by ISO is probably due to a primary action on the sarcolemmal

membrane, followed by stimulation of adenylate cyclase, activation of Ca+ and Na+

channels, exaggerated calcium inflow and excess of excitation-contraction coupling

mechanism leading to energy consumption cellular death. Free radicals generated by ISO

initiate lipid peroxidation of the membrane bound polyunsaturated fatty acids, leading to

impairment of membrane structural and functional integrity24

. Hence in study MI was

induced successfully by using Isoproterenol.

Isoproterenol induced myocardial necrosis has been reported to alter membrane permeability

and to cause leakage of marker enzymes of cardiac damage (LDH, CK-MB, AST, ALT and

ALP) into the blood stream.[25]

Increase in the activity of these enzymes is diagnostic

indicators of myocardial infarction and are indicative of cellular damage and loss of

functional integrity of cell membrane. Result of this study assures that significant elevated

level of marker enzymes LDH, CK-MB, AST, ALT and ALP in IP alone treated group as

compared to the normal control. On the contrary, all the treatment protected the structure and

functional integrity of myocardial membrane as evident from the significant reduction in the

elevated levels of these serum marker enzymes in the rats when compared to the

isoproterenol treated rats. In comparison between Nyctanthus arbor-tristis hydro alcoholic

extract (NAHE) and ZnO NAT treatment; ZnO NAT treatment shown more significant

protection action against isoproterenol induced elevated marker enzymes of cardiac damage.

It shows the better cardioprotective property of synthesised ZnO Nyctanthus arbor-tristis

nano particle.

Lipids play an important role in cardiovascular diseases. A significant elevation in the total

cholesterol and triglycerides was observed in serum of isoproterenol treated rats.

Isoproterenol treated rats also showed an increase in LDL fraction along with a decrease in

HDL-cholesterol. Increased total cholesterol, LDL cholesterol, TG and decrease HDL

cholesterol are associated with raised risk for myocardial infarction. All the treatment

successfully restored the elevated triglycerides, LDL-cholesterol; total cholesterol levels and

also increases the HDL cholesterol levels in serum. In comparison between the treatment


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groups Nyctanthus arbor-tristis ZnO nano particle treatment shows improved hypolipidemic

property.

Histopathological examination of myocardial tissue in control illustrated clear integrity of the

myocardial cell membrane and no inflammatory cell infiltration was observed. Isoproterenol

injected rats showed coagulative necrosis, separation of cardiac muscle fibres and infiltration

of inflammatory cells.[26]

The control rats showed the regular arrangement with clear

striations of myocardial fibers without any histological alterations because of degeneration or

necrosis. Negative control induced rats showed pathological changes in heart including

several congestions, myocardial necrosis and inflamed cells. Heart was having near normal

appearance with mild changes in congestions and necrosis in rats treated with standard drug

propranolol. Same pattern was obtained in rats treated with hydro alcoholic extract of HE-

NAT 500 mg/kg and ZnO-NAT 30 mg/kg showed marked reduction in oedema. So this

research proved that the hydroalcoholic extract of the Nyctanthus arbor-tristis plant can

protect the heart from the myocardial infarction condition.

From the present study it is clear that the ZnO nanoparticles in Nyctanthus arbor tristis show

the significant cardioprotective activity against ISO induced MI it might be due to the

presence of the phyto constituent oleanolic acid. This could be due to the anti-oxidative

property of oleanolic acid against ROS induced by ISO. The results of this study imply that

Nyctanthus arbor tristis treatment proved to be effective in reducing the extent of myocardial

damage by decreasing lipid peroxidation, prevent the overloading of myocardium with lipids

and its β-blocking activity. In this presence of phytochemicals such as oleanolic acid,

alkaloids, Flavanoids, Saponin, Aminoacid, may plays a vital role aganist ISO induced

myocardial damage by its cardioprotective activity.

CONCLUSION

Myocardial infarction is the common presentation of the ischemic heart disease. Even though

clinical care is improved, public awareness is raised and health innovations are widely used,

myocardial infarction still remains the leading cause of death worldwide. In our study

isoproterenol was used to induce MI in rats, because of this pathological changes mimics the

human MI.


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Green synthesis of nanoparticles has gained significant importance in recent years and has

become one of the most preferred methods. Green synthesis of nanoparticles makes use of

environmental friendly, non-toxic and safe reagents.

In the present study Green synthesis of zinc oxide nanoparticles using hydroalcoholic flower

extract of Nyctanthes arbor-tristis is the source of oleanolic acid. Synthesis nanoparticles

were optimized and resultant nanopowder was characterized using UV- Visible spectroscopy,

SEM, TEM, XRD and FT-IR. Results confer the nano size of the synthesised ZnO NAT.

The treatment with ZnO NAT potently restored serum lipid profile i.e. the elevated

triglycerides, LDL-cholesterol, total cholesterol levels and also increases the HDL cholesterol

levels at a dose dependent manner against the isoproterenol induced MI as compare to the

plain extract of Nyctanthes arbor-tristis.

And also result of this study indicates that the treatment of ZnO NAT prevent the elevation of

cardiac activity marker enzymes LDH, CK-MB, AST, ALT and ALP against IP induced MI.

It might be indicates the cardioprotective effect of Nyctanthus arbor tristis. Further

histopathological analysis result conforms the significant cardioprotective effect of hydro

alcoholic leaf extract of Nyctanthus arbor tristis against IP induced MI.

Nyctanthes arbo-tristis plant contains potential antioxidant phytochemical such as

Triterpenoids, polyphenols, flavonoids and related compounds have received increasing

attention for their potential role in prevention of human diseases. Phytotherapeutics need a

scientific approach to deliver the components in a sustained manner to increase patient

compliance and avoid repeated administration is of the novel approach in nanotechnology.

Nano-sized drug delivery systems of herbal drugs have a potential future for enhancing the

activity and overcoming problems associated with plant medicines. ZnO NAT is a more

potent as compared to Hydroalcoholic extract of Nyctanthus arbo tristis.

Based on the present study it could be concluded that subcutaneous injections of

isoproterenol induced myocardial infarction in rats as identified by the release of myocyte

injury markers and altered lipid profile in serum. Most Cardioprotective effect of ZnO NAT

was proved by reduction in cardiac marker enzymes, altered lipid profile and

histopathological studies. In comparison to the hydro alcoholic extract ZnO nano particle of

NAT treatment poses more potent cardioprotective effect. Further investigation is needed to


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explore the active principles and exact mechanism of action ZnO nano particle of Nyctanthus

arbor tristis plant in prevention and treatment of cardio vascular disorders.

ACKNOWLEDGEMENT

I thank the management of Swamy Vivekanandha college of pharmacy for providing the

facilities to carry out this work.

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