prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9362/1/Nadia Afsheen... · Web viewIn the name of ALLAH, the Gracious, The Merciful, All prays belong to ALLAH, Lord of All

In the name of ALLAH, the Gracious, The Merciful, All prays belong to ALLAH, Lord of All words,

The Gracious, The Merciful. Master of the Day of Judgment. Thee alone do we worship and

Thee alone do we Implore for help. Guide us in the right path….

The path of those on whom Thou hast Bestowed Thy blessings, Those who have not incurred. Thy

Displeasure, and Those Who have not gone astray.

1

Biochemical Profiling and Cardioprotective Potential of Various Combinations of Medicinal Plants

By NADIA AFSHEEN

(M. Phil. UAF)

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHYIN

BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF SCIENCES,UNIVERSITY OF AGRICULTURE,

FAISALABAD2016

2

DECLARATION

I hereby declare that the content of the thesis “Biochemical profiling and cardioprotective

potential of various combinations of medicinal plants” are product of my own research and no

part has been copied from any published source (except the references, standard mathematical

models/equation/protocols). I further declare that this work has not been submitted for the award

of any other diploma/degree. The university may take action if the information provided is found

inaccurate at any stage.

SIGNATURE OF THE STUDENT

3

The Controller of Examinations,

University of Agriculture,

Faisalabad.

“We, the supervisory Committee, Certify that the contents and form of thesis submitted by Miss

Nadia Afsheen, Reg. no. 2007-ag-526, have been found satisfactory and recommend that it

should be processed for evaluation, by the External Examiner(s) for the award of degree”.

Supervisory committee:

1. Chairman ------------------------------------ (Prof. Dr. Khalil-ur-Rehman)

2. Member ----------------------------------- (Prof. Dr. Khalid Mahmood Khan)

3. Member ------------------------------------ (Dr. Muhammad Anjum Zia)

4. Member ------------------------------------ (Dr. Nazish Jahan)

4

DEDICATED To

MY ADORABLE AND AFFECTIONATE

“PARENTS”“Mr. & Mrs. Rana Irshad

Ahmed”WHO

Taught me the First Word to SpeakThe First Alphabet to Write

The First Step to TakeAnd

Who those burnt themselves to make me a candle&To

My BelovedHusband

“Rana Shafaqat Ali”

5

6

AcknowledgementsFirst and the foremost, I would like to give my humble thanks and praise to the Almighty Allah for His

grace and blessings throughout my entire life and in the completion of this dissertation particularly.

Without the Blessings of Almighty Allah and His teachings taught by Prophet Muhammad (PBUH), my

life is nothing.

During the completion of this thesis, there were many kinds of supports I have got. I would like to express

my deepest thanks and gratitude to my supervisor Prof. Dr. Khalil-ur-Rehman, (Department of

Biochemistry University of Agriculture, Faisalabad), for the valuable guidance, assistance and

constructive advice throughout the entire project. I would like to make my sincere appreciation and

pleasure and sincerest thanks to my committee members Dr. Khalid Mehmood Khan, Dr. Muhammad

Anjum Zia (Department of Biochemistry University of Agriculture Faisalabad) and Dr. Nazish Jahan

(Department of Chemistry, University of Agriculture, Faisalabad), for valuable assistance and guidance.

I also pay homage to all teachers goal of my academic carrier with light of knowledge and enable me to

touch a section in my life.

Very special thanks to Higher Education Commission (HEC) of Pakistan for its moral & financial

support, without its assistance this dissertation was merely a dream.

No acknowledgement would ever adequately express my delegation to my beloved parents Mr. & Mrs.

Rana Irshad Ahmed and my parents in law Mr. & Mrs. Rana Sadiq for their day and night prayers

which boost my moral to fly high to accomplish my goal. The names of my parents will always be in front

of my eyes, as I will look on the cover of my life. I also express my gratitude to my beloved Husband,

Rana Shafaqat Ali for his sincere help and inspiring assistance.

I feel my immense pleasure to express my deepest gratitude and sincere thanks to my brother Rana Asif

and brother-in-law Rana Rafaqat Ali, Dr. Shahid Hafeez and Rana Moeen and for their encouraging

and inspiring cooperation all the time. I have no words to express my sweet sensation to my beloved

Sisters, my cousins and my all family for their moral boost, encouragements and countless prayers for

me to achieve higher ideal of life.

I have no words to express my sweet sensations to my loving friends Saman Hina and Sofia Parveen,

Zynab Ahmed who are near and dear to me. Finally, May Allah’s blessings be upon all these people with

His countless favors and I would like to pray for their happy and peaceful lives (Ameen)!

NADIA AFSHEEN

7

CONTENTSChapter

No. Title Page No.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 6

3. MATERIALS AND METHODS 16

4. RESULTS AND DISCUSSIONS 35

5. SUMMARY 130

LITERATURE CITED 132

8

LIST OF TABLES

Table No. Title Page No.

3.1Selected parts of plants for evaluation of cardioprotective

potential 17

3.2 Protocol of the mutgenicity assay 22

3.3Experimental design suggested by Response Surface

Methodology to optimize the dose of Salbutamol23

3.4The Central Composite Design for the treatment of selected

medicinal plants25

3.5 Formation of different herbal combinations 26

3.6 Dehydration treatment procedure of histopathology 33

3.7 Clearing treatment procedure of histopathology 33

3.8 Infiltration treatment procedure of histopathology 33

3.9Detailed protocol of Hematoxylin and Eosin (H&E) staining for

histopathology34

4.1Angiotensin converting enzyme inhibitory activity (%) of studied

medicinal plants 37

4.2 DPPH radical scavenging activity of selected medicinal plants 53

4.3 Hemolytic activity (%) of extracts of selected medicinal plants 58

4.4The mutagenicity of standard, Background and extracts of

selected medicinal plants63

4.5Analysis of variance (ANOVA) for the fitted model of CK-MB,

LDH and SGOT activity as a function of independent variables68

4.6Effects of optimized dose of salbutamol on different cardiac

markers suggested by Response Surface Methodology69

4.7

Analysis of variance (ANOVA) for response surface

methodology of CK-MB (IU/L) as a function of independent

variables

73

9

4.8Optimized concentrations of medicinal plants for CK-MB (IU/L)

against salbutamol induced Myocardial infarction74

4.9


methodology of SGOT (IU/L) as a function of independent

variables

77

4.10Optimized concentrations of medicinal plants for SGOT (IU/L)


4.11


methodology of LDH (IU/L) as a function of independent

variables

81

4.12Optimized concentrations of medicinal plants for LDH (IU/L)


4.13

Analysis of variance (ANOVA) for Response Surface

Methodology of HDL (mg/dL) as a function of independent

variables

86

4.14Optimized concentrations of medicinal plants for HDL (mg/dL)

against salbutamol induced myocardial infarction87

4.15


methodology of LDL (mg/dL) as a function of independent

variables

91

4.16Optimized concentrations of medicinal plants for LDL (mg/dL)

against salbutamol induced myocardial infarction92

4.17


methodology of TGs (mg/dL) as a function of independent

variables

95

4.18Optimized concentrations of medicinal plants for triglycerides

(mg/dL) against salbutamol induced myocardial infarction96

4.19


methodology of TC (mg/dL) as a function of independent

variables

99

10

4.20

Optimized concentrations of medicinal plants for Total

Cholesterol (mg/dL) against salbutamol induced myocardial

infarction

100

4.21


methodology of SOD (IU/mg) as a function of independent

variables

104

4.22


methodology of GPX (IU/mg) as a function of independent

variables

106

4.23


methodology of Catalase (IU/mg) as a function of independent

variables

108

4.24Hematological analysis of different groups of rats treated with

various concentrations of selected medicinal plants109

4.25Formulation of different herbal combinations of selected

medicinal plants 112

4.26Hemodynamic analysis of herbal combination against surgically

induced myocardial infarction120

11

LIST OF FIGURES

Figure No. Title Page No.

2.1 Schematic representation of the progression of myocardial necrosis after coronary artery occlusion 7

2.2Role of reactive oxygen species and its prevention by Natural

antioxidants 9

4.1Graphical presentation of angiotensin converting enzyme

inhibition (%) of studied medicinal plants 37

4.2 Full mass spectrum of Terminalia arjuna 40

4.3MS-MS of 685.58 with CID showing Termiarjunoside I at

667.50 m/z 40

4.4 Mass spectrum of T. arjuna showing Quercetin at 301.08 m/z 41

4.5 Mass spectrum of T. arjuna showing Gallic acid at 169.08 m/z 41

4.6 MS-MS CID (30.00) of peak 169 m/z 41

4.7 Mass spectrum indicating the presence of Myricetin 42

4.8MS/MS of T. arjuna of peak 317 at CID (21.00) showing

Ferulic acid at 193 m/z and Catechin at 289 m/z 42

4.9Mass spectrum of C. oxyacantha showing proanthocynidine at

593.17 m/z 43

4.10MS2 CID (20.00) of peak 591.42 showing Ursolic acid at

457.25 m/z 44

4.11Mass spectrum of C. oxyacantha showing Crateagolic acid at

471.08 m/z 44

4.12 MS2 of 381 of C. oxyacantha with CID (20.00) showing Quercetin at 301.17 m/z 45

4.13Mass spectrum of R. serpentina showing Yohimbine at 355.33

and Ajmaline at 327.25 m/z 46

4.14 R. serpentina showing MS-MS at peak 327 with CID (25.00) 46

12

4.15MS2 of peak 327 with CID (25.00) showing Ajmailicine at

353.25 m/z 47

4.16Mass spectrum of R. serpentina showing serpentine at 349.25

m/z 47

4.17 Mass spectrum of A. sativum 48

4.18 MS2 CID 25.00 of 896 of A. sativum showing Myricetin at 319.25 m/z 48

4.19 Mass spectrum of A. sativum showing Apigenin at 327.25 m/z 49

4.20 Full Mass spectrum of C. sativum 49

4.21 Mass Spectrum of C. sativum showing Caffeic acid at 179.08 m/z and isorhamnetin-3-O-glucoside at 477.17 m/z 50

4.22 Mass spectrum of Coriandrum sativum showing apigenin-6-C-glucoside at 593.25 m/z 50

4.23 Mass Spectrum of E. cardamom showing Terpinylacetate at 195.17 m/z 51

4.24 Mass Spectrum of E. cardamom showing Sabinene at 137.08 m/z 51

4.25 Mass Spectrum of P.nigrum showing pipercide at 219.08 m/z 52

4.26Graphical presentation of DPPH radical scavenging activity of

selected medicinal plants 54

4.27 DNA plasmid pBR322 55

4.28

Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extracts [Lane 1: pBR322 DNA + 30mM H2O2+ P1 (100 µg/mL), Lane 2: pBR322 DNA + 30mM H2O2+ P1 (500 µg/mL), Lane 3: pBR322 DNA + 30mM H2O2+ P1 (1000 µg/mL), Lane 4: pBR322 DNA + 30mM H2O2+ P2 (100 µg/mL), Lane 5: pBR322 DNA + 30mM H2O2+ P2 (500 µg/mL), Lane 6: pBR322 DNA + 30mM H2O2+ P2 (1000 µg/mL), Lane 7: pBR322 DNA + 30mM H2O2+ P3 (100 µg/mL), Lane 8: pBR322 DNA + 30mM H2O2+ P3 (500 µg/mL), Lane 9: pBR322 DNA + 30mM H2O2+ P3 (1000 µg/mL), Lane 10: pBR322 DNA + 30mM H2O2+ P4 (100 µg/mL), Lane 11: pBR322 DNA + 30mM H2O2+ P4 (500µg/mL), Lane 12: pBR322 DNA + 30mM H2O2+ P4 (1000 µg/mL)

56

13

4.29

Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extracts: [Lane13: pBR322 DNA + 30mM H2O2+ P5 (100 µg/mL), Lane 14: pBR322 DNA + 30mM H2O2+ P5 (500 µg/mL), Lane 15: pBR322 DNA + 30mM H2O2+ P5 (1000 µg/mL), Lane 16: pBR322 DNA + 30mM H2O2+ P6 (100 µg/mL), Lane 17: pBR322 DNA + 30mM H2O2+ P6 (500 µg/mL), Lane 18: pBR322 DNA +30mM H2O2+ P6 (1000 µg/mL), Lane 19: pBR322 DNA + 30mM H2O2+ P7 (100 µg/mL), Lane 20: pBR322 DNA + 30mM H2O2+ P7 (500 µg/mL), Lane 21: pBR322 DNA + 30mM H2O2+ P7 (1000 µg/mL)]

57

4.30 Graphical presentation of % Hemolysis of extracts of selected medicinal plants at different concentrations 60

4.31 Standard S. typhimurium TA 98 62

4.32 Background plate 62

4.33 Mutagenicity of plants extracts 63

4.34 Response surface plots of CK-MB vs. time and concentration 65

4.35 Response surface plot of SGOT vs time and concentration 66

4.36 Response surface plot of LDH vs. time and concentration 67

4.37Graphical representation of optimized concentration of medicinal plants for CK-MB (IU/L) against salbutamol induced Myocardial infarction

72

4.38Graphical representation of optimized concentration of medicinal plants for SGOT (IU/L) against salbutamol induced Myocardial infarction

76

4.39Graphical presentation of optimized concentration of medicinal plants for LDH (IU/L) against salbutamol induced Myocardial infarction

81

4.40Graphical presentation of optimized concentration of medicinal plants for HDL (mg/dL) against salbutamol induced Myocardial infarction

85

4.41Graphical presentation of optimized concentration of medicinal plants for LDL (mg/dL) against salbutamol induced Myocardial infarction

90

4.42Graphical presentation of optimized concentration of medicinal plants for Triglycerides (mg/dL) against salbutamol induced Myocardial infarction

94

4.43Graphical presentation of optimized concentration of medicinal plants for T. cholesterol (mg/dL) against salbutamol induced Myocardial infarction

99

14

4.44 Graphical representation of doses optimization of medicinal plants for SOD 104

4.45 Graphical representation of doses optimization of medicinal plants for GPX 106

4.46 Graphical representation of doses optimization of medicinal plants for CAT 108

4.47

Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on CK-MB level (IU/L) in the serum of experimental groups through the preventive mode of treatment

115

4.48

Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on SGOT level (IU/L) in the serum of experimental groups through the preventive mode of treatment

117

4.49

Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on LDH level (IU/L) in the serum of experimental groups through the preventive mode of treatment

118

4.50 Graphical representation of hemodynamic parameters of various groups treated with different herbal combinations 122

4.51 The histopathological representation of cardiac tissue of normal control group 123

4.52 The histopathological representation of cardiac tissue surgically induced MI control group 124

4.53 The histopathological representation of cardiac tissue of HC1 treated group 125




15

ABSTRACTMyocardial infarction (MI) is the most dreaded menace and its incidences are increasing

gradually. Although many of the major and minor risk factors impart a crucial role in the onset of

MI, however the hypertension and hyperlipidemia are its major risk factors. In spite of

significant pharmacological advancements regarding drug development has been made, but most

of the available drugs have a long list of side effects which limit their use in clinical medicine.

Hence there is a dire need to integrate complementary and alternative medications into the

practice of conventional medicines, for the treatment of MI. The research was planned to be

carried out into two sections including in vitro and in vivo analysis. In vitro analysis involved the

screening of medicinal plants by Angiotensin Converting Enzyme inhibition assay. Among all

the selected medicinal plants, methanolic extracts of Terminalia arjuna, Piper nigrum,

Coriandrum sativum, Allium sativum, Rauvolfia serpentina, Eletaria cardamom and Crataegus

oxyacantha showed maximum ACE inhibition potential. These medicinal plants were further

subjected to LC-MS analysis which proved the existence of vital phytoconstituents and phenolic

acids in extracts. The antioxidant execution of selected medicinal plants has performed by DPPH

and DNA protection assay. The dose dependant response for antioxidative potential i.e, the

activity of all the medicinal plants in term of % age inhibition increased with increase in

concentration. The toxicity assay of selected medicinal plants exhibited no hemolytic effect and

considered to be safe herbal product for effective fighting against various diseases. Section- II

comprised of In vivo analysis was conducted in three phases. The phase-I included the

preliminary trial, in which the RSM optimized the dose of salbutamol (80 mg/kg b. wt.) to

induce myocardial infarction. In phase-II, the optimal concentrations of selected medicinal plants

were evaluated against salbutamol induced myocardial infarction by using Response Surface

Methodology. In case of Phase-III, the optimized doses of selected medicinal plants were used

to formulate four different herbal combinations with appropriate ratio. The herbal combination

(HC4) showed maximum restoration of cardiac markers (CK-MB, AST and LDH) and

haemodynamic parameters (MAP, HR, LVEDP). The histopathological examination also

confirmed the cardioprotective potential of HC4. Thus the HC4 being safe, inexpensive and

cardioprotective herbal combination, could be considered an alternate of synthetic drug.

Keywords: Myocardial Infarction, Angiotensin converting enzyme, LCMS, Herbal

combinations.

16

CHAPTER #1 INTRODUCTIONCardiovascular diseases (CVDs) which usually stem from vascular dysfunctions are the

major factors causing morbidity and mortality in developing countries (Lee and Kim, 2014).

Cardiovascular diseases cause 17.1 million deaths every year and this number will raise up to 20

million in 2020 (Velavan et al., 2008; Gunjal et al., 2010; Upaganlawar et al., 2011). Although it

is considered as a disease of developed countries, but its incidence is increasing in the

developing world as well (Torabian et al., 2009; El-Sayed et al., 2011). In Pakistan, the

condition has become very critical as CVDs are responsible for 25% of deaths (Maruthappan and

Shree, 2010; Radhika et al., 2011; Manimegalai and Venkatalakshmi, 2012).

A variety of synthetic drugs for the treatment and management of CVD are now

available. Significant improvements regarding synthetic drug development have been made.

Contrary to this advancement these available drugs are taking their toll in the form of side effects

(Thippeswamy et al., 2009; Gielen and Landmesser, 2014). Hence there is a need to integrate

conventional drugs into the practice of synthetic drugs, for the treatment of CVD (Ittagi et al.,

2014).

Among CVD, the Myocardial infarction (MI) is the most dreaded menace. MI is defined

as extended myocardial ischemia with necrosis of myocytes due to disruption of blood supply to

a part of heart resulting in death of cardiac tissue (Kumar and Gurusamy 2014; Subhashini et al.,

2011; Ittagi et al., 2014). Multiple biochemical alterations, lipid peroxidation, free radical

damage, hyperglycemia and hyperlipidemia occur during MI (Siddiq et al., 2012; Krushna et al.,

2009; Alamgeer et al., 2015). The patients of MI mostly presents as chest pain, palpitations,

sweating and anxiety. These symptoms may be gradual or instantaneous (Thygesen et al., 2007;

Radhika et al., 2011). MI causes loss of some of the functioning of myocardium, hence

increasing the work load of the healthy myocardial tissue causing its hypertrophy. The cardiac

hypertrophy leads to compensation of perfusion pressure of ventricles. If work load on the heart

increases for a long time, it will result in compromised cardiac functioning and heart failure

(Cohen and Shah 2004).

The prevalence of major risk factors of cardiovascular diseases including hypertension,

diabetes and impaired glucose tolerance is increasing in Pakistan (Jafary et al., 2007; Jaffery et

al., 2014). This increase in the prevalence of risk factors may be responsible for increase in the

morbidity and mortality rate in Pakistan (Pham et al., 2007; Jaffery et al., 2014). Although a

17

variety of major and minor risk factors are involved in the onset and progression of CVD

(Zahidullah et al., 2012) but the hypertension and hyperlipidemia are considerd as most

remarkable risk factors. The hypertension doubles the rate of coronary artery disease and triples

the rate of congestive heart failure (Mendis et al., 2011).

The blood pressure in body is monitored mainly by Renin Angiotensin Aldosterone

System (RAAS) and to some extent by Kallikrein Kinin system (Hammoud et al., 2007). The

Angiotensin converting enzyme (ACE) (EC 3.4.15.1) is one of the members of a series of

enzymatic reactions in RAAS which converts angiotensin I to angiotensin II. Angiotensin II acts

as a potent vasoconstrictor for vascular smooth muscle cells (Chen et al., 2009; Hernan-

dezLedesma et al., 2011). It also acts on proximal convulated tubules of kidney and causes Na+

and water retention, leading to increase in blood pressure. Thus inhibition of the ACE lowers the

blood pressure by opposing its effects (Ansor et al., 2013; Sharifi et al., 2013). The drugs used in

the management of hypertension include diuretics, β-blockers, calcium channel blockers,

angiotensin II receptor blockers and ACE inhibitors. Among these ACE inhibitors are commonly

used to manage hypertension (Sharifi et al., 2013). A number of synthetic ACE inhibitors

including Captopril, Lisinopril, Enalpril and Rampril have been used as first line of treatment for

hypertension. These ACE inhibitors have many common side effects like cough, dizziness,

headach, weakness, renal failure and angioneuretic edema. Hence there is a need to explore the

herbal alternatives having same mechanism of action as that of ACE inhibitor but with no side

effects (Balasuriya and Rupasinghe 2011). Mostly the natural ACE inhibitors are protein

hydrolysates and peptides attained from animal and plant sources (Belovic et al., 2011; Belovic

et al., 2013). A number of crude and purified herbal extracts have been used to assess ACE

inhibition potential (Chen et al., 2009; Hernan-dezLedesma et al., 2011). Active substances

present in medicinal plants may act as ACE inhibitors and reduce the blood pressure to normal

(Sharifi et al., 2013).

Major biochemical changes during MI due to hyperlipidemia and peroxidation lead to

loss of plasma membrane integrity. The hyperlipidemia is highly responsible for the

development and complcations of atherosclerosis and coronary heart diseases (Gosain et al.,

2010; Joshi and Jain 2014). Proatherogenic cholesterols are risk factors for thrombotic

cardiovascular diseases including MI (Rudenko et al., 2010; Mohanty et al., 2009; Radhika et

al., 2011) but the antiatherogenic cholesterol HDL possess protective effect (Hajizadeh et al.,

18

2011; Joshi and Jain 2014). There are several synthetic drugs such as HMG-Co-A reductase

inhibitors, Fibrates, Niacin, Bile acid binding resins and Probucol that may help to lower plasma

lipid. Owing to high costs and more side effects, associated with synthetic drugs, the scientists

and researchers are trying to substitute the natural components as an alternate of synthetic drugs

(Mahmood et al., 2010; Joshi and Jain 2014; Khursheed et al., 2010).

Free radicals are required during many biochemical processes in living organisms

(Velavan et al., 2007) but the amplified production of toxic Reactive Oxygen Species exerts

severe oxidative stress on myocardium leading to ischemic heart disease, cardiomyopathy,

cardiac failure and arrhythmia (Ittagi et al., 2014). ROS also formed in ischemic tissues cause

lipid peroxidation of membrane and DNA damage. These alterations lead to anatomical and

physiological damages of cardiac cells as well. In this process, mitochondrial, endoplasmic

reticular and extraceluular Ca2+ is released in cytosole through damaged cell membrane hence

increasing cytosolic Ca2+. This cytosolic Ca2+ activates: 1) ATPase, exhausting ATP and

increasing cyclic AMP, 2) Phospholipase, decreasing phospholipids, 3) protease, disrupting

membrane and cytoskeleton proteins, 4) endonucleases, causing nuclear chromatin damage

(Prabha et al., 2014). ROS increases angiotensin II which causes hypertension and also activate

NADPH oxidase. This increase in NADPH oxidase further rise the ROS (Sharifi et al., 2013)

leading to progression of ischemic injury (Manjunatha et al., 2011).

The antioxidant potential of herbal medicines has shielding effect against cardiovascular

diseases (Ayesha et al., 2013). This will be natural protective strategy and would be freely

available with low cost as compared to synthetic drugs (Dianat et al., 2014). The use of

indigenous medicinal plants against variety of diseases is successfully in practice by

complementary and alternative medical practitioner but without knowing their pharmacokinetics

and therapeutic details. However it is also predicted that about one quarter of accepted medicines

have been orignated from medicinal plants (Adaramoye, 2009).

Pakistan is bestowed with a wide range of plants species with unique biodiversity in

different climatic zones. There are about 6000 wild plants available; out of these 600 are used as

medicinal plants (Khan et al., 2012). These medicinal plants have been used in scientific

research for the therapeutical intentions in human beings (Vasu et al., 2009; Shreya et al., 2013).

The bioactive substances like tannins, alkaloids, carbohydrates, terpenoids, steroids and

flavonoids found in medicinal plants impart a crucial role in physiological and biochemical

19

pathways during different ailments (Edoga et al., 2005, Slusarczyk et al., 2009; Soni and Sosa,

2013). Various phytoconstituents present in medicinal plants like aloin, mangiferin, bromelain,

curcuminoids, glycosides, ginkgolides, crocin, neriine, emblicanin, piperine etc are well

documented for their cardioprotective potential (Ramesh et al., 2008; Ragavendran et al., 2011;

Lakshmy et al., 2014). These phytoconstituents may lessen the rate of heart diseases and other

degenerative tissue injuries (Palasuwan and Soogarun 2014). Epidemiological studies also

claimed that there is a considerable synergism between intake of fruits, vegetables and herbs.

Thus the medicinal plants and other natural food constituents are now the focus of attention for

decreasing the risk factors of MI (Goyal et al., 2010; Ojha et al., 2010). The number of drugs and

chemotherapeutics extracted from plants are now used as medicine with reliability. That’s why

the use of traditional medicinal plants in most developing countries is increasing (Alsarhan et al.,

2014).

Currently available synthetic cardioprotective drugs exhibit a number of side effects and

are out of reach for poor community. Many researches have studied the cardioprotective

potential of some medicinal plants which are safe and inexpensive (John 2014; Beaulah et al.,

2014; Susila et al., 2013; Ramadoss et al., 2012; Ittagi et al., 2014). Therefore the natural

cardioprotective drugs divert the attention of entire world population towards green medicines.

Its therapeutic efficacy is because of the primary and secondary metabolites (Beaulah et al.,

2014). According to World Health Organization (WHO) herbal medicines are used as primary

health care in 80% of total world’s population and recommended for therapeutic uses as safe

alternative medicines all over the world (Menaka et al., 2011).

Keeping in view the above facts the study was planned to assess biochemical profiling

and cardioprotective potential of medicinal plants by inducing myocardial infarction, chemically

as well as surgically in experimental animals. The research work was divided into two sections

comprised of in vitro and in vivo studies. The in vitro studies included the screening of medicinal

plants for ACE inhibition potential. After screening, the said medicinal plants were subjected to

biochemical profiling including, LC-MS analysis, antioxidant and toxicological assays. The

animal models impart crucial role in drug discovery for management of various diseases. The

chemically provoked myocardial infarction in animals is a well known model to study the

important outcomes of various medicinal plants against cardiac disorder (Ahsan et al., 2014;

Sahreen et al., 2011). Hence after in vitro characterization of medicinal plants, the In vivo

20

analysis was planned comprising of three phases. In phase-I, the dose of salbutamol was

optimized to induce myocardial infarction. The Phase-II involved the dose optimization of

cardioprotective medicinal plants by using Response Surface Methodology (RSM). In

combination bioactive phytoconstituents of medicinal plants imparts synergic therapeutic

potential (Li et al., 2009). In case of Phase-III, herbal combinations were made by using the

optimized concentrations of selected medicinal plants to evaluate their cardioprotective potential

through surgically induced MI. The hemodynamic parameters, activities of cardiac enzymes in

serum and antioxidants enzymes in cardiac tissue of different groups of experimental animals

were analyzed to evaluate the best herbal combination which showed the maximum potential

against MI and also suppress the risk factors associated to MI.

Aims and Objectives:

The objective of this research was to get an alternative, innocuous and effective herbal

formulation that enable to ameliorate the MI and its risk factors.

21

CHAPTER # 2 REVIEW OF LITERATURE

Anatomically the heart comprises of arterial, ventricular and conductive muscle fibers

(Harika et al., 2014). The cardiac muscle fibers, which are anatomically as well as

physiologically similar to that of skeletal muscle, are formed by typical myofibrils containing

actin and myosin filaments. During cardiotoxicity any damage to the heart muscle can lead to

small changes in blood pressure, arrhythmias and cardiomyopathy (Koti et al., 2009).

Cardiomyopathy and the subsequent loss of heart function is continuation of a variety of

cardiovascular pathologies. Generally in cardiomyopathy the heart muscle becomes enlarged,

thick, or rigid and in unusual cases the muscle tissue itself is substituted with scar tissue thus

consequently heart would be unable to pump blood throughout the body.

2.1 Cardiovascular Diseases:

Cardiovascular diseases (CVD) are complex multifactorial disease (Rahman and Lowe

2006). Not only under developing countries like Pakistan, but the developed countries also could

not able to give the successful solution for the management and treatment of CVD (Aslam et al.,

2015). According to the World Health Organization, CVD are one of the major reasons of

fetality and account for 30% of all deaths in 2005 (Lee and Kim 2014). The Global Burden of

Disease estimated that 29.6% of all worldwide deaths were caused by CVD in 2010 (Nichols et

al., 2014). It is estimated that by year 2020, it will account for one third of the deaths all over the

world (Rajalakshmy et al., 2011). Many risk factors such as elevated low density lipoprotein,

low levels of high density lipoprotein, diabetes mellitus and hypertension increase the prevalence

of CVD (Toth, 2007). Among these the hypertension is a major risk factor contributes to the

development of CVD by 13%. WHO estimation showed that 45% of CVD deaths are associated

with hypertension as about 15-37% of the adult population suffered from high blood pressure,

while at 60 years of age this prevalence increased to 50% of population (Banjari et al., 2013).

The oxidized form of low density lipoprotein-cholesterol (LDL-c) and high concentration

of cholesterol is also one of the major risk factors of heart diseases that lead to progressive

atherosclerosis (Attar, 2006; Mansour et al., 2009; Maruthappan and Shree 2010). This is

responsible for blockage of coronary arteries and infarction of cardiac muscles (Maruthappan

and Shree 2010; Radhika et al., 2011; Manimegalai and Venkatalakshmi, 2012).

22

2.2: Myocardial infarction:

It is well established that among the CVD, the Myocardial infarction (MI) is necrosis of

myocardium and perpetually followed by several biochemical alterations like hyperglycemia,

hyperlipidaemia and lipid peroxidation (bhandari et al., 2008). Moreover, during pathological

progression of MI, there is a large scale alteration in normal biochemical processes because of

metabolic shift (Taegtmeyer et al., 2004). This “metabolic shift” leads to dysfunctioning of

energy metabolism, oxidative stress and inflammation in plasma membrane (Jiang et al., 2011).

The thrombotic occlusion in coronary arteries may also lead to myocardial cell death (Jiang et

al., 2014; Graham et al., 2007; Devlin and Henry 2008). As the plaque increases in size, the

coronary arteries get narrower and cause the hindrance in blood flow to cardiac musculature.

This reduced blood flow and oxygen supply effects heart structurally leading to MI, ischemia,

unstable angina and sudden death (Dhevi et al., 2014).

Fig. 2.1 Schematic presentation of the development of myocardial infarction after blockage of coronary artery

The process of necrosis starts from a small zone of myocardium beneath the endocardium

surface. This necrosed region is oxygenated by diffusion of blood, circulating into the ventricle.

This region of myocardium (shaded) depends on the occluded vessel for its perfusion (Fig. 2.1).

23

The necrotic region of cardiac musculature losses its integrity and viability, this myocardiam is

called infracted and the process is called myocardial infarction.

In developing countries, over 80% of deaths take place due to CVD in underdeveloped

countries (Susila et al., 2013). The classical symptoms of myocardial infarction included nausea,

vomiting, chest pain, palpitations, acute coronary syndrome, anxiety, sweating or feelings of

impending doom. The onset of these symptoms is usually gradual and rarely instantaneous

(Thygesen et al., 2007; Radhika et al., 2011).

2.3: Salbutamol induced myocardial infarction:

The cardioprotective potential of medicinal plants against chemically induced MI in

animal models has been executed in many studies (Siddiq et al., 2012; Beaulah et al., 2014).

This method is simple for executing the biochemical and histological alteration during acute

myocardial infarction (Gomathi et al., 2014; Ittagi et al., 2014; Prabha et al., 2014; Ramadoss et

al., 2012). Salbutamol is synthetic beta adrenergic receptor agonist that causes severe oxidative

stress in myocardium (Shiny et al., 2005; Hina et al., 2010; Jahan et al., 2012; Kumar and

Gurusamy 2014; Zafar et al., 2015) and alterations in membrane permeability which is

responsible for loss of anatomical and physiological integrity of myocardium (Ramadoss 2012;

Beaulah et al., 2014).

The mechanism of action of salbutamol, to induce MI, is to create hyperlipidemia by

enhancing adenylate cyclase activity. This results in increased cyclic AMP formation and

accumulation of lipid. The cytotoxic action of salbutamol causes lipolysis and peroxidation of

endogenous lipids. These biochemical and pathological changes including hyperlipidemia,

peroxidation and loss of plasma membrane integrity lead to MI (Khursheed et al., 2010).

Considerable clinical and experimental evidences also suggested that the generation of free

radicals and ROS are involved in the pathogenesis of salbutamol induced MI. Therefore,

salbutamol induced MI would be a well established model to study the protective potential of

medicinal plants against MI (Kumar and Gurusamy 2014). Several studies have demonstrated

that nutrients, antioxidant and/or complementary medicine strengthen the LDL oxidation

susceptibility and increase the antiatherosclerotic impact of high density lipoproteins which is

performing a key role in prevention of cardiovascular diseases (Rivas-Arreola, 2010).

24

2.3: Oxidative stress and Natural antioxidants:

Free radicals are essential for chemical signaling, detoxification, energy supply and

immune functions during normal physiological function. However excess amount of free radicals

initiate the oxidation of biomolecules that may lead to numerous diseases along with cell injury.

The production of reactive oxygen species (ROS) is balanced by the endogenous antioxidative

defense system. The deleterious oxidative stress is generated as a result of imbalance between

the production and elimination of ROS (Alam et al., 2013). This oxidative stress on myocardium

may lead to the development of MI (Yun et al., 2013). The free radicals and ROS damage the

cell membrane and consequently attributed to the structural and biochemical alterations which

ultimately lead to cell death (Tappia et al., 2001; Prabha et al., 2014).

Free radical scavenging activity

Fig. 2.2 Role of reactive oxygen species and its prevention by Natural antioxidants

The ROS can adversely damage tissues by reacting with polyunsaturated fatty acids,

nucleotides and critical sulfhydryl bonds (Kaja et al., 2014). Antioxidants delay the oxidation

process by inhibiting the initiation of series of oxidizing reactions (Ayesha et al., 2013). Owing

to the presence of antioxidants, medicinal plants have emerged as substantial therapeutic agents

to cure various diseases like, cardiovascular diseases, cancer and diabetes (Biapa et al., 2007).

Most phenolic compounds are usually present in medicinal plants have been reported as free

radical scavengers and good therapeutic agent (Zhang and Zuo 2006; Mradu et al., 2012). Thus

25

Hypercholestrolemia, Diabetes Mellitus, Hypertension

Free radicals

Altered vasomotionVascular smooth muscle growth

Adhesion molecule expression

Lipid oxidation Apoptosis

Cardiovascular diseases

Natural antioxidants (Curcuma longa, Crataegus oxyacantha, Terminalia arjuna, Coriandrum sativum, Trigonella foenum)

to understand the therapeutic potential of medicinal plants, it is necessary to study the existence

of phytoconstituents of herbal medicines (Wang and Zuo 2011).

The medicinal plants are nature’s gift to have disease free healthy life (Begum 2009;

Gomathi et al., 2014). Therefore, the scintists and researchers are taking interest in the

assessment of prophylactic and therapeutic effects of medicinal plants in order to decrease CVD

as they are economical, effective and harmless (Ramadoss et al., 2012; Berman, 2000). More

than 100,000 of the active compounds have been found in medicinal plants (Souravi and

Rajasekharan 2014). The Phytoconstituents being part of medicinal plants are the natural

bioactive compounds, and found to be the integral part of defense system against various

diseases and stress conditions (Mathangi and Prabhakaran 2013; Nonita and Mylene 2010).

Experimental and epidemiological studies also proved that there is an inverse relation between

intake of phytoconstituents and progression of diseases (Devasagayam et al., 2004). Human

health could be maintained by consumption of medicinal plants (Lichtenstein et al., 2006;

Halliwell, 2012; Othman et al., 2014) because a number of plant-derived phenolic compounds

having antioxidant potential are able to challenge oxidative stress in hman body (Halliwell, 2012;

Othman et al., 2014).

Nature designs the metabolism of our body to oppose the excess of free radicals by the

formation of endogenous antioxidants. Their effectiveness is increased by endogenous free

radical scavenging enzymes and vitamins. The redox stress triggers the activation of immune

cells which release proinflammatory cytokines, reactive oxygen and nitrogen species that lead to

damage of biological molecules and inducing imbalance in physiological and pathological

pathways (Lonkar and Dedon, 2011; Babu et al., 2013). Any alteration in the balance of

oxidative metabolites and antioxidant scavangers, may lead to many disorders (Rashed, 2014).

For these reasons, information on the antioxidant properties of natural product is becoming

relavant to the nutrition and nutraceutical fields. Owing to its complexity, the single method is

unable to provide comprehensive picture of the antioxidant profile therefore, Multimethod

approach is required to assess the antioxidant activity (Rivas-Arreola et al., 2010).

In 1985 Farnsworth et al., identified 119 secondary metabolites which were used as

therapeutic agents. The World Health Organization recommended that out of 225 basic and

essential drugs 11% are obtained from natural precursors. These Phytochemicals are known to

possess antibacterial (Nair et al., 2005), antifungal (Khan and Wassilew, 1987), antioxidant

26

(Wong et al., 2009), antidiabetic (Singh and Gupta, 2007; Kumar et al., 2008), anti-inflammatory

(Kumar et al., 2008) and radio-protective activity (Jagetia et al., 2005). Owing to these

properties the medicinal plants are commonly used for therapeutic purpose.

2.4: Medicinal plants:

According to the WHO the medicinal plants could be used for various therapeutic

purposes due to presence of active phytoconstituents (Brussels, 2001; Kumari et al., 2011).

Pakistan is rich in medicinal herbs because of its varied climate and wide distribution over a

large area. About 600 plant species are identified as medicinal plants having potent therapeutics

(Husain et al., 2008). In Pakistan medicinal plants are mostly used in crude form by hakims.

Thus it should be processed for the extraction of various active constituents by pharmaceutical

industries and researchers (Mahmood et al., 2003; Husain et al., 2008).

More than 2000 plants being a part of Traditional systems of medicine are used to treat

the people suffering from cardiovascular diseases (Arya and Gupta 2011; Rajalakshmy et al.,

2011). About 300 species of medicinal plants are used worldwide in the pharmaceutical

industries (Deshmukh et al., 2012; Harisaranraj et al., 2009) because of the presence of many

bioactive substances which are used for treatment and management of various diseases (Hu et

al., 2008; Harisaranraj et al., 2009; Mitta et al., 2012).

2.5: A promising approach for natural therapy:

A variety of compounds in traditionally used medicinal plants having therapeutic

properties are now the focous of attention for many researchers (Reddy et al., 2010). Many plant

products are rich in polyphenolics which are different in chemical structure, characteristics and

widely recognized as naturally occurring antioxidants (Rajadurai and Prince 2007). The active

compounds in their natural formulations are more potant, as they contain both dotes and

antidotes (Khopde et al., 2001; Krushna et al., 2012). Moreover the side effects of synthetic

medicines motivated the researchers to explore the therapeutic potentials of medicinal plants to

treat myocardial infarction. Being a natural source the plant extracts have been used for

medicinal purposes without any side effects. The prophylactic and therapeutic effects of many

plants in reducing isoprenaline induced cardiotoxicity have been discussed (Prashee et al., 2008;

Panda and Naik 2008). It also revealed that the antioxidants present in medicinal plants not only

suppress the formation of ROS but also have a modulatory effect (Merzenich et al., 2009). That’s

why herbal medicine is increasingly attaining appreciation from medical professionals due to

27

advancement in understanding of the mechanisms by which herbs positively influence health and

quality of life (Panda and Naik 2008).

2.6: Medicinal Plants with Cardioprotective Properties:

Many medicinal plants have been found to possess beneficial cardioprotective effects

(Siddiq et al., 2012; Beaulah et al., 2014). Ayurveda has identified many plants which possess

cardiotonic and cardioprotective effects. Some of them are Allium sativum, Allium cepa,

Asparagus racemosus, Caesalpinia bonducella, Cassia fistula, Curcuma longa, Emblica

officinalis, Garcinia indica, Hemidesmus indicus, Ocimum sanctum, Phyllanthus amarus,

Terminalia arjuna, Trigonella foenum-graecum, Vitis vinifera, Withania somnifera and Zingiber

officinalis (Tilak-Jain and Devasagayam 2006; Niero et al., 2010; Silva and Fernandes 2010;

Rajalakshmy et al., 2011; Hassan, 2012, Alaribe, 2008). Thus, it is usually referred that the

“extracts of plants” not the plants themselves or their parts are used for medicinal effects. These

substances have therapeutical potential; hence could be used for different ailments with respect

to human physiology (Nwachukwu et al., 2010).

In this study the sixteen medicinal plants were selected on the basis of folk literature data

showing the effectiveness and therapeutical potential against cardiovascular diseases and its

related risk factors. All these plants were screened and out of these seven medicinal plants were

chosen for further biochemical and cardioprotective potential against myocardial infarction.

Crataegus oxyacantha:

Crataegus oxyacantha, commonly known as Hawthorn, a member of Rosaceae family

(Amy 2006; Verma et al., 2007). Hawthorn berries supported for its beneficial cardiovascular

properties due to the high content of bioflavonoids (oligomeric procyanidins, vitexin, quercetin,

and hyperoside) (Mohanty et al., 2013). Hawthorn preparations have been used to treat the early

stages of congestive heart failure, hypertension and total plasma cholesterol. The in vitro analysis

demonstrated that Hawthorn is more active antioxidant than vitamins E and C (Saadatian et al.,

2014). Previous investigations have confirmed the efficacy, non toxicity and reliability of this

plant and its active ingredients (Verma et al., 2007; Ebrahimzadeh and Bahramian, 2009).

Eletaria cardamom:

Eletaria cardamom is commonly known as ‘chhoti elaichi’ and is also rightly known as

the ‘Queen of Spices’. Fruits and seeds of the Cardamom are economically important parts of the

plant and also act as antiseptic, carminative, anti-spasmodic and diuretic (Kumari and Nirmala,

28

2015). Cardamom is effective as an antioxidant and could increase the levels of glutathione, a

natural antioxidant in body (Amma et al., 2010). It also acts as remedy in case of digestive

problems, urinary complaints, asthma, bronchitis and several other human ailments (Kaushik et

al., 2010; Ali and Shahnaz 2014; Abbas and Maliki 2011). Its valuable part, the seeds are used to

cure tumors of the uterus (Krishnamurthy, 2010) dyspepsia, nausea and even during pregnancy

(Jazila et al., 2007; Savan and Zehra 2013).

Coriandrum sativum:

Coriandrum sativum is herbaceous plant well known for its diuretic, carminative,

digestive, anthelmintic, antioxidative, hepatoprotective and antibacterial activities (Pandey et al.,

2011). C. sativum has been used as an antifungal, antioxidant, hypolipidemic, antimicrobial,

hypocholesterolemic and anticonvulsant substance (Joshi et al., 2014). The major compounds

present in essential oil are 𝛼-pinene, 𝛾-terpinene, geranyl acetate and geraniol (Nadeem et al.,

2013). C. sativum showed protection against the deleterious effects in lipid metabolism (Chithra

and Leelamma, 1999). C. sativum have strong antioxidant potential which might be responsible

for its therapeutic potential (Kousar et al., 2011).

Terminalia arjuna:

Terminalia arjuna belongs to family Combretaceae, is a large and evergreen tree

commonly known as Arjuna (Rameshkumar et al., 2014). It is traditionally claimed that the bark

of T. arjuna is mostly used for medicinal purposes. The cardioprotective potential of T. arjuna

bark against isoproterenol induced myocardial injured supported this traditional claim (Jahan et

al., 2012; Sivakumar and Rajeshkumar, 2014). Phytochemicals including tannins, triterpenoids,

saponins, arjunic acid, arjunolic acid and arjungenin in the bark of T. arjuna, are responsible for

its medicinal properties (Manna et al., 2007). The bark also helps in maintaining the cholesterol

level near to normal due to its antioxidant potential. It also showed significant antidiabetic,

cardioprotective and antimicrobial activities (Jahan et al., 2011a; Ramya et al., 2008; Jahan et

al., 2011b; Jahan et al., 2012). Two new cardenolide cardiac glycosides were extracted from the

root and seed of T. arjuna. The mechanism of action of cardenolides is to increase the

intracellular sodium and calcium and increasing force of myocardial contractility (Amol et al.,

2014).

29

Rauvolfia serpentina:

Rauvolfia serpentina belongs to the family Apocynaceae is an evergreen, woody and

perennial shrub having tuberous roots (Deshmukh et al., 2012; Singh et al., 2009). The common

name of plant is Sarpagandha (Mallick et al., 2012). The metabolites present mainly in the

leaves, roots and rhizomes of R. serpentina are taking attention of practitioners due to its

medicinal importance (Poonam et al., 2013; Mittal et al., 2012). R. serpentina is considered an

important medicinal herb in the pharmaceutical world because of numerous alkaloids in its roots.

The roots have been reported to treat cardiovascular diseases, hypertension, arrhythmia

(Kirillova et al., 2001; Kumari et al., 2013) and human leukemia (Dey et al., 2010). Reserpine is

the main alkaloid that shows complex pattern of activity in brain and act as anti-diuretic (Rani et

al., 2014; Mittal et al., 2012; Kumari et al., 2013). Reserpine, the active ingredient of R.

serpentina is responsible for antihypertensive potential due to its action on central nervous

system (Kumari et al., 2013). Researches also showed that the root of R. serpentina has anti

hypercholesterolemic effects with no side effects (Qureshi and Shamsa 2009).

Piper nigrum:

Piper nigrum (Black pepper) belongs to the family Piperaceae, known as ‘King of

Spices’ (Srinivasan, 2007). The Piperine, an alkaloid of P. nigrum improves the bioavailability

of a variety of structurally and functionally diverse drugs (Khajuria et al., 2002; Duangjai et al.,

2013). It also has small amounts of various bioactive compounds (Hussain et al., 2011) which

may be responsible for its high antioxidant and free radical scavenging property (Nahak and

Sahu 2011). The methanolic extract of P. nigrum showed a considerable protection against

chemically induced cardiotoxicity due to presence of various antioxidantspresent in it (Aruna et

al., 2014; Wakade et al., 2008). During an experimental high fat diet along with black pepper

were given to rats which showed the elevated level of HDL-c, reduced the LDL-c and VLDL-c

levels in the plasma as compared to rats treated merely with high fat feed (Vijayakumar et al.,

2002). Black pepper, being rich in vanadium, is responsible for its cardioprotective potential

during myocardial infarction and hypertrophy (Shenuarin and Fukunaga, 2009).

Allium sativum:

Allium sativum is present extensively in all over the world and used as a popular

medication for treatment of a various diseases like dermatitis, rheumatism, abdominal disorders

and diabetes mellitus. In many of the experimental studies Garlic has been most encouraging as a

30

complementary therapy for hypertension and cardiovascular diseases (Capraz et al., 2006). The

vasoactive ability of garlic was also confirmed by in vitro study, whereby red blood cells convert

garlic organic polysulfides into endogenous cardioprotective signaling molecule (Papu et al.,

2012). The two major risk factors including high blood pressure and high blood serum

cholesterol levels that may lead to heart disease and the therapeutic action of garlic directly

reduced the impact of these risks. In India, 432 patients suffering from coronary artery disease

were grouped and half of them were supplied with garlic juice in milk depicted curative

therapeutic potential as compared to other groups that were not supplied with garlic juice (Papu

et al., 2012). Hence the literature presented the garlic a bioactive agent for prevention and

treatment of cardiovascular and other metabolic diseases (Khan et al., 2008). Moreover, garlic

also responsible for antioxidant potential against MI in rats (Asdaq and Inamdar, 2010; Anoush

et al., 2009).

31

CHAPTER # 3 MATERIALS AND METHODSThe research was planned to explore the Biochemical profiling and cardioprotective

potential of medicinal plants in various combinations. The major part of the research was

conducted in the Clinico-Medical Biochemistry Lab (CMBL), Department of Biochemistry,

University of Agriculture, Faisalabad (U.A.F). The surgical procedure of the experimental

animal was conducted in the Department of Clinical Medicine and Surgery, U.A.F. A part of

research was also accomplished in National Institute of Biotechnology and Genetic Engineering

(NIBGE) Faisalabad. The sixteen medicinal plants were selected on the basis of information

available in the literature showing the cardioprotective potential and also able to minimize the

risk factors of myocardial infarction. This research was an effort to develop an herbal

combination possessing good cardioprotective potential but with less or no side effect. For this

purpose the research work was divided in two sections, in vitro and in vivo analysis. The in vitro

analysis involved the screening of medicinal plants for their Angiotensin Converting Enzyme

(ACE) inhibition potential. The plants which exhibited the good ACE inhibition potential were

selected for further biochemical profiling including, LC-MS analysis, antioxidant and

toxicological evaluation. Second part of the research was comprised of in vivo studies and

subdivided into three phases. In phase-I, the dose of salbutamol was optimized that can induce

myocardial infarction. Phase-II consisted of the series of experiments in which the dose of

cardioprotective medicinal plants were optimized. In phase-III, different herbal combinations of

studied medicinal plants were formulated against surgically induced MI to get the best

formulation having cardioprotective potential and also able to restrain the risk factors related to

MI.

3.1 Collection of Medicinal plants:

Different parts of medicinal plants were collected from Botanical Garden of University of

Agriculture, Faisalabad and from the local herbal market. All the plants’ parts were identified by

the Plant Taxonomist in the Department of Botany, University of Agriculture, Faisalabad,

Pakistan. The parts of the plants, selected for research work are listed in Table. 3.1. These parts

of the plants were washed and allowed to dry under shadow at room temperature and ground and

sieved to get fine powder.

32

Table. 3.1 Selected parts of plants for evaluation of cardioprotective potential

Parts used Plants

Seeds Trachyspermum ammi, Coriandrum sativum, Foeniculum vulgarea, Bunium

bulbocastanum, Eletaria cardamom and Ocimum sanctum

Roots Rauvolfia serpentina, Cichorium intybus and Curcuma longa

Leaves Piper nigrum, Aloe vera and Lepidium sativum

Fruits Allium sativum, Crataegus oxyacantha and Terminalia chebula

Bark Terminalia arjuna

3.1.1 Preparation of Herbal Extract:

The powdered plants (5 g of each) were macerated in methanol (50 mL). The macerate

was kept in orbital shaker for four days. The supernatant was poured and the residue was

remacerated with methanol. The pooled supernatants were combined and filtered with

Whatman’s filter paper No. 1. The filtrate was concentrated by rotary evaporator under reduced

pressure and dried by using lyophilizer (Jahan et al., 2012).

Section-I (In vitro analysis)3.2 Screening of medicinal plants:

The methanolic extracts of selected medicinal plants were screened by using Angiotensin

Converting Enzyme inhibition assay. Although various in vitro methods are available for

evaluation of ACE inhibition activity but the most elaborated method established by Cushman

and Cheung, 1971 (Gao et al., 2010; Rinayanti et al., 2013) was followed.

3.2.1 ACE inhibition assay:3.2.1.1 Principle:

The Hippuryl L-Histidyl Leucine (HHL) is hydrolysed by Angiotensin Converting

Enzyme. The Hippuric acid formed in the reaction is estimated by measuring the absorbance at

228 nm. The difference between absorbance in the absence and presence of inhibitor is

proportional to the inhibitory activity of tested sample.

3.2.1.2 Reagents: The main reagents used in the ACE inhibition assay included, 1) Angiotensin converting

enzyme (EC 3.4.15.1) that was extracted from rabbit’s lungs, 2) Hippuryl Histidyl Leucine (Hip

His Leu) and Captopril purchased from Sigma Aladrich (U.S.A).

3.2.1.3 Angiotensin converting enzyme extraction:

33

Angiotensin converting enzyme (ACE) was extracted from rabbit’s lungs powder. For the

formation of rabbit’s lungs acetone powder, the lungs were obtained from freshly slaughtered

rabbits and washed with 0.8% saline solution. The lungs were homogenized with phosphate

saline buffer by tissue homogenizer and filtered through cheese cloth. More volume of buffer

was added and centrifuged at 4000 rpm for 10 minutes. The residues were washed several times

with acetone to complete dehydration. The acetone dip residues were placed overnight for

evaporation. The dry rabbit’s lungs acetone powder was ground to get fine powder and preserved

in polythene bag at 4oC (Nemerson, 1969; Luna et al., 2009). The dry lung acetone powder was

used for crude extraction of ACE. One gram of this lung powder was mixed in 10 mL of 100

mM phosphate buffer (pH 8.3). It was stirred on magnetic stirrer overnight and centrifuged at

4000 rpm for 45 minutes. The supernatant was dialyzed against 100 mM phosphate buffer of pH

8.3 by using dialyzing membrane of 12 KD cutoffs and lyophilized to get ACE extract.

3.2.1.4 Preparation of Captopril solution:

Captopril (25 mg) was grounded and extracted with distilled water (25 mL) in ultrasonic bath

for 10 minutes. The obtained extract was filtered by using filter paper (0.45 μm pore size).

Captopril solution (1 mg/mL) was used as the positive control (Duncan et al., 1999; Donath-

Nagy et al., 2011).

3.2.1.5 Procedure:

ACE solution (50 μL) and 50 μL of borate buffer were incubated at 37°C for 10 min. The

reaction mixture was incubated for 80 min at 37°C after addition of 8.3 mM Hip His Leu (150

μL). After that 250 μL of 1 M HCl was added to terminate the reaction. The hippuric acid

formed in this reaction was extracted by 1500 μL of ethyl acetate and centrifuged at 800 rpm for

15 min. The upper layer (750 μL) was poured into test tube and dried under laminar air flow at

37°C. At the end 1 mL of distilled water was used to dissolve hippuric acid and the absorbance

was measured at 228 nm by UV/Vis spectrophotometer (Cintra 303, GBC Scientific Equipment,

Australia). The reaction blank was also prepared by following the same process except the HCl

was added before the enzyme (Belovic et al., 2013). Extracts of plants and captoprill standards

were prepared by replacing the same quantity of buffer with samples. The sample blank was

prepared as the reaction blank, by replacing buffer to the tested sample. The ACE inhibition was

calculated by given formula.

% IACE = 100[ (A-B) – (C-D)]

34

(A-B)Where “A” depicts the absorbance in the presence of ACE, “B” represents the absorbance

of the reaction blank, “C” is the absorbance in the presence of ACE and inhibitor and “D”

indicates the absorbance of the sample blank. (HernandezLedesma et al., 2011; Cushman and

Cheung, 1971).

3.3 Biochemical profiling:

Biochemical profiling through LC-MS analysis, antioxidant assay and toxicological

evaluation of screened medicinal plants, possessing high ACE % age inhibition, was performed.

3.3.1 Liquid chromatography mass spectrometry (LC-MS):

These medicinal plants were analyzed by using Liquid Chromatography combined with

Electron Spray Ionization Mass Spectrometry (LC-ESI-MS) from National Institute of

Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan. The plant extracts

prepared in section 3.1.1 were filtered by using a 0.45 µm syringe filter before analysis. Surveyor

plus HPLC System equipped with Surveyor auto (Thermo Scientific, San Jose, CA, USA) were

used for separation. A Luna Reverse Phase C-18 analytical column (4.6×150 mm, 3.0 µm

particle size) (Phenomenex, USA) was attached with pump. LC-MS grade methanol and

acidified water (0.5 % formic acid v/v) were used as the mobile phase A and B respectively.

Solvent elution consisted of gradient system run at a flow rate of 0.3 mL/min. The gradient

elution was programmed as follows. A 20 minutes re-equilibration time was used after each

analysis. The column was maintained at 25ºC and the injection volume was 5.0 µL. The effluent

from the HPLC column was directed to electron spray ionization mass spectrometer (LTQ XLTM

linear ion trap Thermo Scientific River Oaks Parkway, USA). Negative ion mode with spectra

posessing mass range from m/z 260 to 2000 was used for analysis of parameters (Adom and Liu,

2002). The accurate mass spectra data of the molecular ions was processed through X-caliber

software (Thermo Fisher Scientific Inc, Waltham, MA, USA) (Jiao and Zuo, 2009; Zuo et al.,

2002).

3.3.2 Antioxidant assay:

In order to evaluate the antioxidant potential of methanolic extracts of plants, the “1, 1-

Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging assay" and “DNA protection assay”

were used.

3.3.2.1 1, 1, Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging assay:

35

DPPH free radical scavenging assay was used to explore the antioxidant potential of

extracts of selected plants (Sahu et al., 2013). The extracts of secreened plants, prepared in

section 3.1.1 were further used to perform the DPPH free radical scavenging assay.

3.3.2.1.1 Procedure:

The antioxidant activity was resoluted by using DPPH as a free radical. Stock solutions

(10 mg/mL) of extracts of plants were prepared in methanol. Different concentrations (20, 40,

60, 80 and 100 µg/mL) of extracts of selected plants and methanolic solution of DPPH (0.1 mM)

were mixed in equal volume. The mixture was kept for 30 minutes in dark and the absorbance

was noted at 517 nm against a blank solution. Ascorbic acid acts as a standard. The DPPH

inhibition (% ) was measured by using given formula.

DPPH Inhibition (%) = [1 - A1/A0] x 100

Where “A1” is the absorbance of sample and “A0” is the absorbance of control (Yadav et al.,

2014; Ayesha et al., 2013).

3.3.2.2 DNA protection assay:

The antioxidant activity was also confirmed by DNA protection assay following the

method given by Riaz et al, (2012). pBR 322 DNA plasmid (0.5 µL) was diluted by using

sodium phosphate buffer (50 mM, pH 7.4). The diluted pBR 322 DNA (3 µL) was treated with 5

µL of different concentrations (100, 500 and 1000 µL) of extracts of all the plants. After that 4

µL of 30% H2O2 was added to make the volume up to 15 µL with sodium phosphate buffer (pH

7.4). The comparative variation in the migration between the native and oxidized DNA was

determined on 1% agarose by horizontal DNA gel electrophoresis using a wide mini system

(Techview, Singapore). 1% agarose was ready by mixing 1g agarose in 100 mL of 1X×TAE

buffer and placed it in microwave oven for two minutes. It was cooled and poured in casting

plate. After solidification, gel was kept in the sodium phosphate buffer and samples were loaded

in the wells one by one. The gel was stained with ethidium bromide and documented by Syngene

model Gene Genius unit (Syngene, Cambridge, UK).

3.3.3 Toxicity assay:

The toxicological evaluation of some of the screened medicinal plants was performed

through “Hemolytic activity” and “Mutagenicity assay”.

3.3.3.1 Hemolytic activity:

36

The haemolytic activity, a useful starting point for toxicological evaluation, provides the

primary information about the interaction between bioactive compounds of medicinal plants and

biological entities (Da Silva et al., 2004).

3.3.3.1.1 Preparation of erythrocytes suspension:Five milliliter of blood was withdrawn from individuals having good health and

centrifuged at 1500 rpm for three minutes. Plasma (supernatant) was discarded and the washing

of pellet was performed thrice with PBS (pH 7.2±0.2) by centrifugation at 1500 rpm for 5 min.

The cells were resuspended in normal saline and maintained the cell count at 108 cell/mL (Kumar

et al., 2011).

3.3.3.1.2 Procedure:Cell suspension (0.5 mL) was mixed with 0.5 mL various concentrations of extracts of

said plants (100, 500 and 1000 μg/mL in PBS). The incubation of mixture was done for 30 min

at 37°C in an incubator (Sanyo, MIR-254, Japan) and centrifuged at 1500 rpm for 10 min in a

laboratory centrifuge (22331 Hamburg). The free haemoglobin in the supernatant was measured

in UV-Vis spectrophotometer (Dynamica, Halo BD-20, UV-Vis spectrophotometer, Australia) at

540 nm. Phosphate buffer saline and Triton-X were used as negative and positive hemolytic

control respectively. The hemolysis in term of percentage by the plants extracts was quantified

by using the given formula,

% Hemolysis = At - An

Ac

Where “At” is the absorbance of test sample, “An” showed the absorbance of the saline

control and “Ac” is the absorbance of the water control (Kumar et al., 2011).

3.3.3.2 Mutagenicity assay:

The mutagenicity test was performed by using “Ames bacterial reverse mutation assay”.

3.3.3.2.1 Test bacterial strain:

The mutant strain S. typhimurium TA98 was maintained on nutrient agar and incubated at

37˚C for 18-24 hr prior to the test (Razak et al., 2007).

3.3.3.2.2 Preparation of reagent mixture:

Reagent mixture was prepared by mixing Devis Mingoili salt (21.62 mL), D-Glucose

(4.75 mL), Bromocresol purple (2.38 mL), D-Biotin (1.19 mL) and L-Histidine (0.06 mL)

37

aseptically in a sterile bottle. The mutagen sodium azide (0.5 µg/100mL) was used for S.

typhimurium TA 98.

3.3.3.2.3 Herbal extraction:

The extracts of all the selected plants prepared in section 3.1.1 were reconstituted in

Dimethyl Sulfoxide (DMSO) to form 10, 000 µg/mL stock solution.


Herbal extracts, reagent mixture, sterile distilled water and standard mutagen were mixed

in bottles with the quantity indicated in Table. 3.2 and inoculated with homogenous culture broth

of S. typhimurium. The contents of each bottle were dispensed into each well of a 96 well micro

titration plate and plates were incubated at 37˚C for 4 days.

3.3.3.2.5 Interpretation of result:

The yellow wells were considered as positive and the purple wells were considered as

negative. The herbal extract was said to be mutagenic if the positive well are prominently higher

in number as compared to the positive well in the background plate.

Table. 3.2 The protocol for mutagenicity assay

Treatment Volume (mL)Mutagen standard

Extract

Reagent mixture

Deionized water

S. typhimurium

Blank - - 2.5 17.5 -Background

- - 2.5 17.5 0.005

Standard mutagen

0.1 - 2.5 17.4 0.005

Test samples

- 0.005 2.5 17.5 0.005

3.4 Section-II (In vivo study)

3.4.1 Selection of animals:

The experimental animals including rats and dogs were housed in cages and acclimatized

for one week under laboratory conditions (27°C in 12 hr dark/light cycle). They were fed with

standard feed and water. The husk in the cages of rats was changed thrice a week to ensure

hygienist. All the animals were kept in Animal House, Department of Clinical Medicine and

Surgery, University of Agriculture, Faisalabad.

38

3.4.2 Phase-I: Dose optimization of salbutamol:The rats were orally administered with varying concentrations of salbutamol as suggested

by “Central Composite Design” of Response Surface Methodology (RSM-CCD) for two

consecutive days. Furthermore different time intervals from 0 to 116 hr, for blood sampling were

also proposed by RSM-CCD (Table. 3.3). All the blood samples were centrifuged to separate the

serum for the analysis of cardiac markers (CK-MB, SGOT and LDH). The procedures for the

estimation of cardiac markers have been given in section 3.4.6. The response of cardiac markers

was analyzed by the same statistical tool “RSM” to get the optimal dose of salbutamol at which

it could induce MI.

Table. 3.3 Experimental design suggested by Response Surface Methodology to optimize the dose of Salbutamol

Sr. No. Time of blood sampling (hr)X1

Conc. of salbutamol (mg/kg)

X2

1 0 502 96 503 0 1254 96 1255 0 886 116 887 48 348 48 1419 48 8810 48 8811 48 8812 48 8813 48 88

3.4.3 Phase-II: Dose response experiment: A pilot study:

In the dose response experiment the rats were randomly divided into following groups.

The rats in control group were fed with normal diet throughout the experimental period of 23

days. In positive control group the rats were treated with normal diet for 21 days and after that

the optimized dose of salbutamol in phase-I was given orally twice at an interval of 24 hr.

Moreover seven treatment groups were run and each treatment group was pretreated with

different concentrations of its respective plant as suggested by “Central Composite Design”

39

(Table. 3.4), once daily for three weeks. After that the optimized dose of salbutamol was

administered for two consecutive days. The blood sampling was performed after 24 hr of

administration of salbutamol. The cardiac biomarkers (CK-MB, SGOT and LDH) as well as lipid

profile (HDL-c, LDL-c, TC and TG) were analysed to assess the cardioprotective worth of

selected medicinal plants at various concentrations. At the end of experiment the rats were

sacrificed and their hearts were excised for the estimation of antioxidants (SOD, GPX and CAT).

The protocols of all these biomarkers have been given under the section 3.4.6.

40

Table. 3.4 The Central Composite Design for the treatment of selected medicinal plants

Grps. Plants Conc. (mg/kg)



G1 T. arjuna 80 P. nigrum 110 A. sativum 200 T. arjuna 80 P. nigrum 170 A. sativum 200 T. arjuna 110 P. nigrum 200 A. sativum 140 T. arjuna 170 P. nigrum 200 G6 R. serpentina 80 T. arjuna 200 P. nigrum 140 R. serpentina 80 T. arjuna 200 G4 C. sativum 80 R. serpentina 110 T. arjuna 140 C. sativum 80 R. serpentina 170

G2 C. oxyacantha 80 C. sativum 110 R. serpentina 200

C. oxyacantha 80 C. sativum 170 R. serpentina 200

C. oxyacantha 110 C. sativum 200 R. serpentina 140 C. oxyacantha 170

C. sativum 200 G7 E. cardamom 80 C. oxyacantha 200 C. sativum 140

E. cardamom 80 C. oxyacantha 200 G5 A. sativum 80 E. cardamom 110

C. oxyacantha 140 A. sativum 80 E. cardamom 170 G3 P. nigrum 80 A. sativum 110 E. cardamom 200

P. nigrum 80 A. sativum 170 E. cardamom 200 E. cardamom 140

3.4.4 Phase-III (Herbal combination therapy):In phase-III the synergestic cardioprotective potential of selected medicinal plants was

observed. For this purpose the dogs were selected as experimental animals and myocardial

infarction was induced surgically. The dogs were divided into three groups. The first group of

dogs was the control group, to which normal diet was fed for 23 days. The second group was

surgically induced MI control group, in which the dogs were treated with normal diet for 22 days

and after that the ligation of left anterior descending coronary artery was performed on 23rd day.

The third group was treatment group which was further divided into four subgroups. Each

subgroup was pretreated with its respective herbal combination (Table. 3.5) for 22 days. On day

23 all the dogs of treatment group underwent left anterior descending coronary artery ligation.

After completion of surgical procedure the blood samples were taken at various time intervals (0

41

to 48 hr) to analyze the biomarkers (CK-MB, SGOT and LDH). At the end of the experiment the

dogs were sacrificed by an overdose of anesthesia and hearts were excised for histopathological

studies. The procedures and principles of the said biomarkers are given in section 3.4.6.

Table. 3.5 Formation of different herbal combinations

Groups R.serpentin

a

E.cardamo

m

P.nigru

m

A.sativu

m

T.arjun

a

C.oxyacanth

a

C.

sativum

Herbal ratio

HC1 1 0.5 1 0.5 - - 0.5

HC2 1 0.5 1 0.25 - 1 0.5

HC3 1 1 0.5 - 1 - 0.5

HC4 0.5 - - 0.5 1 0.5 1

3.4.5 Surgical induction of myocardial infarction:The dogs were sedated with pentabarbitone sodium (60 mg/kg) and additional 4mg/kg

were given when required. To keep the heart rate elevated during surgical procedure and to

reduce the bronchotracheal secretions atropine was given subcutaneously at a dose of 0.1 mg/kg

once before surgery. The animals were ventilated with room air from a positive pressure by using

compressed air at the rate of 90 stroke/min and tidal volume of 10 mL/kg. Neck was opened and

left tracheotomy was performed to open the thoracic cavity. Anatomy of Left Anterior

Descending (LAD) coronary artery was ligated 4-5 mm and end of this ligature was passed

through polyethylene tube to form snare. At the end of surgery the heart was returned to its

normal position in thoracic cavity (Ojha et al., 2010; Ojha et al., 2012).

3.4.5.1 Estimation of hemodynamic variables:

The mean arterial pressure (MAP) and heart rate of dogs in all the groups was calculated.

The left thoracic cavity was opened by an incision at the fifth intercostal space and the heart was

exposed. A metal cannula was introduced in cavity of left ventrical from the posterior epical

region of heart for measuring left ventricular dynamics throughout the surgical procedure

(Nandave et al., 2013; Ojha et al., 2010; Ojha et al., 2012).

3.4.6 Biochemical analysis:

The principles and protocols of all the studied biochemical parameters have given under

the following headings.

3.4.6.1 Creatine Kinase MB (CK-MB):

42

3. 4.6.1.1 Reagent preparation:

The reaction mixture was organized by adding four parts (400 µL) of reagent 1 (R1) and

one part (100 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added

and the absorbance was measured at 340 nm after 5 minutes.

3.4.6.2 Serum Glutamate Oxaloacetate Transaminase (SGOT):

Serum Glutamate Oxaloacetate Transaminase (SGOT) was determined through kinetic

method.

3.4.6.2.1 Principle:

The kinetic method was optimized for determination of aspartate amino transferase

without perodoxal phosphate in accordance with International Federation of Clinical Chemistry

(I.F.C.C) recommendation. Aspartate aminotransferase (AST/GOT) catalyzed α- Ketoglutarate

and L-Aspartat by the coupled reaction of malate dehydrogenase and the relative coenzyme

(NADH), oxaloacetate is reduced to malate with the co-enzyme oxidation.

3.4.6.2.2 Reagent preparation:

Reaction mixture was prepared by mixing 9 parts (450 µL) of reagent 1 (R1) and 1 part

(50 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added and the

absorbance was measured at 340 nm after 120 seconds.

3.4.6.3 Lactate Dehydrogenase (LDH):

3. 4.6.3.1 Principle:

Lactate Dehydrogenase was measured in accordance with Deutsche Gesell schaft for

Klinische Chemie (DGKC) recommendation. The oxidation rate of NADH is proportional to the

LDH catalytic activity. The LDH activity of the sample was calculated by taking the absorbance

at 340 nm.


Reaction mixture was prepared by mixing 4 parts (400 µL) of reagent 1 (R1) and 1 part

(100 µL) of reagent 2 (R2). In the reaction mixture 50 µL serum samples were added and the

absorbance was taken at 340 nm after 30 seconds.

3. 4.6.4 Cholesterol:3. 4.6.4.1 Principle:

43

Cholesterol esterase oxidized the cholesterol ester releasing hydrogen peroxide.

Hydrogen peroxide reacted with a phenol substitute and 4-aminopyrine. The intensity of red

compound formed is directly proportional to the total cholesterol in the sample.


The reaction mixture contained reagent 2 (1000 µL), standard solution (1000 µL) and

sample (1000 µL). In the reaction mixture 50 µL serum samples were added and the absorbance

was measured.

3. 4.6.4.3 Calculation:

Cholesterol (mg/dL) = (A) Sample×200 (A) Standard

3.4.6.5 Triglycerides:


The quantitative determination of Triglycerides was done by measuring the intensity of

red color produced. It is directly proportional to triglycerides in the sample.

3.4.6.5.2 Reagent preparation: The reagent R1 and R2 were mixed with 10:1 ratio respectively and 10 µL samples was mixed and incubated for 5 min at 37˚C and the absorbance was taken.

3.4.6.5.3 Calculation:

Triglycerides (mg/dL) = (A) Sample×200 Standard

3.4.6.6 High density lipoprotein (HDL):


HDL-Cholesterol is obtained during the preparation of LDL and VLDL, where the HDL

remained in solution. At the end the color formation was observed and absorbance was measured

at 546 nm that was proportional to HDL cholesterol concentration in sample.


Reaction mixture was made by mixing 1.0 mL BioMed Cholesterol reagent and sample.

The mixture was then incubated for 5 min at 37˚C and absorbance was measured.

3.4.6.7 Low density lipoprotein (LDL):


44

High density lipoprotein and very low density lipoprotein remained in the supernatant,

the LDL is precipitated by addition of Heparine and after centrifugation it is measured

enzymatically. The concentration of LDL cholesterol is calculated by the difference of total

cholesterol and cholesterol in the supernatant.


Sample (50 µL) and precipitating reagent (500 µL) were mixed at room temperature and

centrifuged for 20 minutes.

3.4.6.7.3 Calculation:Cholesterol in supernatant (mg/dL) = Sample×conc. Std. (mg/dL)

StandardThe standard concentration is the concentration of the total cholesterol in the cholesterol

standard solution.

LDL cholesterol (mg/dL) = Total cholesterol (mg/dL) - cholesterol in the supernatant

3.4.6.8 Antioxidant assay:

Antioxidant enzymes were evaluated from cardiac tissue by using following protocol.

3.4.6.8.1 Enzyme assay of superoxide dismutase (SOD):

The potential of superoxide dismutase was estimated by its ability to inhibit the photo

reduction of Nitro Blue Tetrazol (NBT) following the method of Gianopolitis and Ries (1977).

3.4.6.8.2 Required reagents for enzyme assay:

1 M EDTA solution containing 0.3 mM NaCN.

0.067 mM potassium phosphate buffer, pH 7.8.

1.5 mM NBT

0.12 mM Riboflavin.

3.4.6.8.3 Preparation of 0.067 mM Phosphate buffer, pH 7.8:

0.14 g of KH2PO4 was taken in a flask and then 0.74 g of K2HPO4 was added in it. The

volume was made up to 80 mL in flask by adding distilled water.

3.4.6.8.4 Preparation of EDTA and NaCN solution:

0.16 g of EDTA was taken in flask and then 0.08 mg NaCN was added in it. The distilled

water was added to make the volume 5.4 mL.

3.4.6.8.5 Preparation of riboflavin solution:

45

0.06 mg riboflavin was taken in flask and distilled water was added to make its volume

up to 1.3 mL and stored in cold dark bottle.

3.4.6.8.6 Preparation of Nitro Blue Tetrazol (NBT) solution:

3.23 mg NBT was taken in flask and its volume was made up to 2.64 mL with the

addition of distilled water and stored in cold dark bottle.


The buffer (1 mL) was taken in cuvette as blank and absorbance was measured at 560 nm

in spectrophotometer. After that 5-6 cuvettes were set in a light box having light bulb of 30 watt.

1 mL of buffer, 0.05 mL of enzyme extract and 0.016 mL of ribolflavin was added to each

cuvette and incubated in light box for 12 minutes. The cuvettes were moved to

spectrophotometer where EDTA/NaCN solution (0.067 mL) and NBT (0.033 mL) were added to

illuminate reaction mixture. The absorbance was measured after 20 seconds of the reaction. The

specific activity was measured in IU/mL/min/mg of protein by multiplying enzyme activity with

the protein contents (mg) of the extract.


% age inhibition = Blank-sample (Abs)×100 Blank (Abs)

Specific Activity= Enzyme Activity ×mg of protein

3.4.6.9 Glutathion peroxidase:

3.4.6.9.1 Preparation of enzyme extract:

To remove the RBCs, the disected organs were washed with 0.2 M phosphate buffer of

pH 6.5 and homogenized in blender. After that the samples were centrifuged for 15 minutes at

10,000 rpm and 4ºC. The supernatants were stored at 4ºC for enzyme assay (Civello 1995).

3.4.6.9.2 Reagents for enzyme assay:

0.2M phosphate buffer, pH 6.5

Hydrogen peroxide

Guaiacol

3.4.6.9.3 Preparation of 0.2 M phosphate buffer (pH 6.5)

4g NaH2PO4 and 1g Na2HPO4 was dissolve in distilled water. Then volume was maid up

to 200 mL.

3.4.6.9.4 Preparation of buffer substrate solution:

46

Guaiacol (750 µL) and phosphate buffer (47 mL) were mixed well. After agitation, H2O2

(0.3 mL) was added to buffer solution.


A cuvette having 3 mL of blank solution was put into spectrophotometer at wavelength of

470 nm. The 0.06 mL of enzyme extract was added to it and the absorbance was noted after 3

min. Enzyme activity was calculated and this activity was multiplied with the protein contents

(mg) of the extract to measure the specific activity (U/mL/min/mg of Protein).


Activity U/mL = ΔA/3____26.6×60/300

Specific activity= enzyme activity× mg of protein

3.4.6.10 Catalase:3.4.6.10.1 Isolation of catalase enzyme:3.4.6.10.1 Procedure:

The organs were weighed and the phosphate buffer was addedwith ratio of 1:4.

Homogenized it by using pestle and mortar.

To remove the biomass homogenized tissue was passed through muslin cloth.

The obtained liquid was filtered with Whatman’s filter paper No. 1 and centrifuged at

10,000 rpm for 15 minutes.

Both supernatants and sediments were separated and stored at 4˚C.

3.4.6.10.3 Enzyme activity:

The activity of catalase was measured by its ability of to decompose H2O2 at 240 nm

(Chance and Mehlay, 1977).

3.4.6.10.4 Reagents:

60 mM phosphate buffer (pH 7.0)

10 mM hydrogen peroxide

3.4.6.10.5 Preparation of 60 mM phosphate buffer (pH 7.0):

0.224 g of NaH2PO4 and 0.1632 g of Na2HPO4 were added in distilled water to make the

total volume up to 50 mL.

3.4.6.10.6 Preparation of buffer substrate solution:

47

Buffer substrate solution of 10mM of H2O2 was prepared in 60 mM phosphate buffer by

dissolving 0.442 H2O2 in 60 mM phosphate buffer.

3.4.6.10.7 Procedure: A cuvette having 2 mL of blank solution was kept into spectrophotometer at the

wavelength of 240 nm.

Another cuvette having buffered substrate solution was put in the spectrophotometer.

0.05 mL enzyme extract was added into buffered substrate solution.

The second cuvette was incubated for three minutes to complete the reaction.

The absorbance was measured at 240 nm.

3.4.6.10.8 Calculation:Enzyme activity was calculated then this activity was multiplied with the protein content

(mg) of the extract and specific activity was measured in U/mL/min/mg of protein.

Activity (U/mL) = ΔA/min×dilution×2mL 0.04mM-1cm-1×0.05mL

Where, ΔA Absorbance at 240 nmMin: Reaction time buffer0.04 mm-1cm-1 Standard factor0.05 mL Enzyme concentration used2 mL Amount of enzyme and substrateSpecific activity: Enzyme activity× mg of protein

3.4.6.11 Histopathology:

Reserved organs were processed for histopathaological examination (Bancfort and

Gamble) by following steps as described below.

3.4.6.11.1 Washing and dehydration:

After fixation in 10% buffered formaline 5 mm chunky pieces of tissue were washed by

tap water overnight to remove all debrisis. Slices of tissues were then dehydrated as follows.

Table. 3.6 Dehydration treatment procedure of histopathology

48

Dehydrating agents Time (hour)

Alcohol 70% 8

Absolute alcohol-1 2

Absolute alcohol-2 2

Alcohol 85% 4

Alcohol 95% 4

3.4.6.11.2 Cleaning:

The elimination of dehydrating agent and substitution with some fluid was done during

cleaning process.

Table. 3.7 Clearing treatment procedure of histopathology

Clearing agent Time (minutes)

Xylene+Alcohol (50+50) 30

Xylene-1 15

Xylene-2 15

3.4.6.11.3 Infiltration:

Infiltration was carried out in liquid paraffin at 59-60 ºC as shown below:

Table. 3.8 Infiltration treatment procedure of histopathologyInfiltrating agent Time (hrs)

Paraffin-1 2

Paraffin-2 2

Paraffin-3 2

3.4.6.11.4 Embedding:

Tissues in Steel mold were poured with wax and let it solidify at -1 to 5 ˚C in the plastic

moults. On solidification of paraffin, the steel moult was removed from the block.

3.4.6.11.5 Sectioning and Mounting:

Microtome was used for tissue sectioning.

The thickness of section was about 4-5 µm.

Tissue blocks were faced by wheel of microtome to form smooth ribbon of sections.

These sections were kept in the warm water (~50˚C).

49

Thin smeared of egg albumin was made onto glass slides and the slides were swelled by

dipping the slides under the section floating in water bath.

Slides were dried in incubator at 45-55˚C for 30 minutes to remove fragments of the

paraffin.

3.4.6.11.6 Staining:

Staining was performed by using Hematoxylin and Eosin stain (H&E stain) by adopting

protocol as given in Table. 3.9.

Table. 3.9 Detailed protocol of Hematoxylin and Eosin (H&E) staining for histopathology

Staining agents Time (minutes)Xylene-1 3Xylene-2 3Absolute Alcohol-1 3Absolute Alcohol-2 3Alcohol 70% 3Water 5Hematoxylin 5-8Acid Alcohol 1-2 dropsWater 3Ammonia Alcohol 3Water 3Alcohol 70% 3Eosin y 1-2Alcohol 70% 3Absolute Alcohol-1 3Absolute Alcohol-2 3Xylene-1 3Xylene-1 3

Afterward a drop of mixture of distyrene, a plasticizer and xylene was added on the cover slip

and placed on the stained section.

3.4.7 Statistical analysis:

The statistical analysis was done by ANOVA and Response Surface Methodlogy (Myers

et al., 2002).

CHAPTER # 4 RESULTS AND DISCUSSION

50

The research work was conducted in the Clinico-Medical Biochemistry Laboratory,

Department of Biochemistry, University of Agriculture, Faisalabad. The research work was

divided into two sections. The first included in vitro and the second was comprised of in vivo

studies. In the in vitro section, screening of some medicinal plants through ACE inhibition assay

was made. After that, the medicinal plants those showed high % age of ACE inhibition were

further subjected to biochemical profiling including LC-MS analysis, antioxidant and

toxicological evaluation. The in vivo study (section-II) was comprised of three phases. In phase-

I, the concentration of salbutamol required to induce cardiotoxicity in terms of MI, was

confirmed. The phase-II involved the evaluation of different concentrations of selected medicinal

plants for their therapeutic potential against MI. The phase-III included the assessment of

synergistic cardioprotective potential of different herbal combinations in surgically induced MI.

The findings of all the said experiments have been described under the following headings.

Section-I (In vitro study):

4.1 Screening of medicinal plants:

Untreated hypertension may leads to various complications including coronary and

peripheral heart diseases and even stroke (Chen et al., 2009; HernandezLedesma et al., 2011).

The Angiotensin Converting Enzyme (ACE) inhibitors are one of the most valuable medications

for the management of hypertension (Sharifi et al., 2013). The medicinal plants possessing ACE

inhibition potential would be the better alternative of synthetic drugs for the treatment of

hypertension, which is one of the major risk factors of MI.

4.1.1 Angiotensin converting enzyme inhibition assay:

The ACE inhibition of both methanolic and ethanolic extracts of various parts of some

selected medicinal plants has been demonstrated in Table. 4.1. The comparative ACE inhibition

(%) of methanolic and ethanolic extract of each selected medicinal plant has been presented

graphically in Fig. 4.1. The methanolic extracts of the selected medicinal plants showed

relatively higher ACE inhibition potential relative to the corresponding ethanolic extracts (Fig.

4.1). The difference in ACE inhibition potential of both types of extracts might be due to the

reason, that the ethanol masked the inhibitory activity of the examined samples (Belovic et al.,

2013). The methanolic extracts of Trachyspermum ammi (50.76±1.342), Terminalia arjuna

(56.23±3.427), Piper nigrum (63.03±0.153), Coriandrum sativum (63.033±0.153), Foeniculum

vulgarea (50.26±0.75), Allium sativum (52.9±2.621), Rauvolfia serpentina (63.467±3.198),

51

Eletaria cardamom (53.467±0.55) and Crataegus oxyacantha (64.267±0.2) showed more than

50% ACE inhibition potential (Table. 4.1). All these medicinal plants with good ACE inhibition

activity have already been reported to possess cardiotonic, diuretics and antilipidemic potential

(Tassell et al., 2010; Kousar et al., 2012; Jahan et al., 2012). Although the captoprill showed

maximum inhibition of ACE (Fig. 4.1), but the rising cost and side effects of this and other

contemporaneous antihypertensive drugs stimulate the assessment of medicinal plants as cheaper

and safer natural alternatives (Filho, 2006).

The high ACE inhibition of medicinal plants might be due to the presence of secondary

metabolites like terpenoids and polyphenolic compounds (Balasuriya and Rupasinghe 2011).

Most of the flavonoids were reported to be competitive inhibitors that can compete with the

substrate in binding to the active site of the enzyme (Balasuriya et al., 2011). The presence of

polyphenolics like procyanidin, catchein and epicatechin in T. arjuna (Goretta et al., 2006) is

responsible for its antihypertensive potential (Dwivedi and Chopra 2014). The dimers and

hexamers of the epicatechins present in T. arjuna were found to be competitive inhibitors of

angiotensin converting enzyme (Goretta et al., 2003).

Procyanidin, the active bioflavonoid of Crataegus oxyacantha was claimed to have the

vasorelaxant effect (Verma et al., 2007) due to its ACE inhibition potential (Sharifi et al., 2013).

The dimers and tetramers of procyanidins are responsible for its competitive ACE inhibition

(Ottaviani et al., 2006). The extract of C. oxyacantha can normalize the blood pressure, both

systolic (from 160 to 150 mm Hg) and diastolic (from 89 to 84 mm Hg) (Murray, 1995; Mills

and Bone 2000) in hypertensive patients in a similar fashion to synthetic ACE inhibitors

(Meschino 2014).

The apigenin, a flavon, present in A. sativum is responsible for its antihypertensive

potential (Loizzo et al., 2009; Balasuriya and Rupasinghe 2011). Daily use of Allium sativum

may keep the blood pressure normal due to presence of the Phe-Tyr (dipeptide) at its N-terminal

sequence. The peptides are responsible for ACE inhibition by chelating zinc, which being a part

of ACE is mandatory for ACE activity (Kumar et al., 2011).

Table. 4.1 Angiotensin Converting Enzyme inhibitory activity (%) of studied medicinal

plants

52

Botanical names Family name Parts used

% age ACE inhibition of

Methanolic extract

% age ACE inhibition of

Ethanolic extractCaptoprill 85.87±1.503 81.08±2.98

Trachyspermum ammi

Apiaceae Seed 50.76±1.342 44.03±1.95

Terminalia arjuna Combretaceae Bark 56.23±3.427 23.9±1.082

Piper nigrum Piperaceae Leaves 63.03±0.153 31.67±0.907

Curcuma longa Zingiberaceae Powder 31.1667±3.427 22.7±0.754

Coriandrum sativum Umbelliferae Seed 63.033±0.153 49.67±0.305

Foeniculum vulgarea Umbelliferae Seed 50.267±0.75 30±0.36

Bunium bulbocastanum

Apiaceae Seed 44.2±1.136 29.4±0.721

Allium sativum Amaryllidaceae Fruit 52.9±2.621 38.633±0.551

Rauvolfia serpentine Apocynaceae Root 63.467±3.198 56.033±0.636Eletaria cardamom Zingiberaceae Seed 53.467±0.55 39.66±0.666

Lepidium sativum Curciferae Leaves 36.767±2.914 18.3±0.794

Ocimum sanctum Lamiaceae Seed 49.167±0.838 12.933±1.001

Cichorium intybus Compositae Root 18.367±2.35 16.76±0.152

Crataegus oxyacantha Rosaceae Fruit 64.267±0.2 21.9±0.567

Terminalia chebula Combretaceae Fruit 16.466±1.32 16.43±0.61

Aloe vera Liliaceae Leaves and gel

38.1±0.954 26±2

53

Captoprill

T. ammi

T. arju

n

P. nigrum

C. longa

C. sativu

m

F. vulgarea

B. bulbocasta

num

A. sativu

m

R. serpen

tine

E. cardamom

L. sativu

m

O. sanctu

m

C. intyb

us

C. oxyc

antha.

T. cheb

ula

A. vera

0

10

20

30

40

50

60

70

80

90

100

Methanolic extractEthanolic extract

% a

ge A

CE in

hibi

tion

Fig. 4.1 Graphical presentation of angiotensin converting enzyme inhibition (%) of studied

medicinal plants

The antihypertensive property of Rauvolfia roots are attributed to reserpine due to its

inhibitory action on central and peripheral nervous system (Kumari et al., 2013). The binding of

reserpine stops the normal accumulation of catecholamine and serotonin (Nammi et al., 2005)

which is involved in maintaining the blood pressure, heart rate, cardiac contraction and

peripheral resistance (Kumari et al., 2013).

Generally ACE inhibition of extracts of medicinal plants is due to chelation of metal ion

co-factor with secondary metabolites. Other possible mechanism is the formation of hydrogen

bridges between the inhibitor and amino acids near the active site of ACE (Balasuriya et al.,

2011).

54

4.2 Biochemical profiling:

Biochemical profiling of the methanolic extracts of seven plants including T. arjuna, C.

oxyacantha, R. serpentina, C. sativum, A. sativum, E. cardamom and P. nigrum which showed

higher ACE inhibition potential, was performed through LC-MS, antioxidants and toxicological

evaluation.

4.2.1 Liquid chromatography mass spectrometry (LC-MS):

The HPLC analysis generated extremely narrow peaks thus the high speed data handling

performance was required in the MS segment of the analysis. This LC/MS greatly contributes for

the identification of some of the phenolics and flavonoids compounds (Zhang and Zuo 2004;

Ogura and Sakamoto 2012). Moreover, the purpose was to have detailed analysis of some of the

peak fragmentation to discriminate between different compounds by Collision Induced

Dissociation (CID) using MS technique. Thus the structural elucidation of specific bioactive

compounds responsible for different therapeutic potential was made through LC-MS by the

courtesy of National Institute of Biotechnology and Genetic Engineering (NIBGE), Pakistan.

4.2.1.1 Biochemical profiling of Terminalia arjuna:

LC-MS analysis of methanolic extract of Terminalia arjuna was performed to evaluate

the phytoconstituents including phenolics, flavonoids and alkaloids. The full mass spectrum

obtained by LC-MS analysis was presented in Fig. 4.2. The mass spectrum depicted the high

peaks at 413.42, 511.50, 321.33, 589.33 and 685.58. The CIDMS-MS-ESI fragment ion of

685.58 peak resulted in three abundant peaks at 667.50, 523.33 and 457.25. The peak of 667.50

indicated the presence of Termiarjunoside 1,3,9,22-tetraol-12-en-28-oicacid-3-D-

glucopyranoside (Fig. 4.3). The presence of Termiarjunoside I from the bark of T. arjuna was

also reported in study by Ali et al. (2006). The fast atom bombardment mass spectroscopy

(FABMS) of T. arjuna also displayed a molecular ion peak at m/z = 666 [M]+ indicating the

presence of Termiarjunoside I, with a molecular formula of C36H58O11, which was also supported

by 13C and Distortionless enhancement by polarization transfer (DEPT) NMR spectra.

55

Fig. 4.2 Full mass spectrum of Terminalia arjuna

Fig. 4.3 MS-MS of 685.58 with CID showing Termiarjunoside I at 667.50 m/z

The mass spectrum obtained from LC-MS analysis in Fig. 4.4 revealed the highest peak

at 301.08 which may indicate the presence of quercetin in T. arjuna. The quercetin prominantly

decreased the impairment of cardiac functions following ischemia by improving mitochondrial

function (Kumar and Agnihotri 2008).

The Fig. 4.5 depicted the high peaks at 301.08, 523.42 and 169.08 (m/z). The peak at

169.08 indicated the presence of gallic acid which was further confirmed by MS-MS using CID

(30.00). The peak at 125.08 is the concequence of the removal of COO - from gallic acid (Fig.

4.6). Gallic acid (GA) is also found to be strong antioxidant which impart a vital role in number

of biological and pharmacological activities (Kim, 2007; Nikolic et al., 2011).

56

Fig. 4.4 Mass spectrum of T. arjuna showing Quercetin at 301.08 m/z

Fig. 4.5 Mass spectrum of T. arjuna showing Gallic acid at 169.08 m/z

Fig. 4.6 MS-MS CID (30.00) of peak 169 m/z

57

Fig. 4.7 Mass spectrum indicating the presence of Myricetin

The mass spectrum of T. arjuna depicted the presence of myricetin at peak 317.25 (Fig.

4.7). The MS-MS of the peak 317.25 by CID (21.00) showed the highest peaks at 302.08,

241.08 and 179.06. However the peaks at 193 and 289 may indicate the presence of ferulic acid

and catechin respectively. The mass spectrum and structures of these compounds were presented

in Fig. 4.8. Ferulic acid is responsible for wide range of therapeutic effects against cancer,

diabetes, lungs and cardiovascular diseases (Paiva et al., 2013).

Fig. 4.8 MS/MS of T. arjuna peak 317 at CID (21.00) showing Ferulic acid at 193 m/z and

Catechin at 289 m/z

The ferulic acid present in T. arjuna is not only a good antioxidant against protein

oxidation but also have some potential against lipid peroxidation in various biological systems

(Kanski et al., 2002). The HPLC analysis of T. arjuna bark by Jahan et al. (2012) exhibited

excellent concentration of polyphenols and phenolic acids including ferulic acid followed by

gallic acid, caffeic acid and catechin (Jahan et al., 2012).

58

The cardioprotective potential of flavonoids is because of its various actions as the intake

of flavonoids stop the endothelial dysfunction hence responsible to reduce arterial pressure

(Narayana et al., 2001; Kurosawa et al., 2005). Flavonoids may also help in uptake of oxdatively

modify LDL-c due to its free radicals scavenging property. Various studies showed that the

quercetin suppress the LDL oxidation and exert considerable vasorelaxation (Burns et al., 2000).

The presence of such potent bioactive compounds in T. arjuna is responsible for its

cardioprotective and antioxidant activity (Jahan et al., 2012).

4.2.1.2 Crataegus oxyacantha:The LC-MS analysis of C. oxyacantha was executed to assess the phytoconstituents

which help to cure the myocardial infarction. The mass spectrum of C. oxyacantha showed the

peaks at 591.33, 553.42 and 593.17. The peak at 593.17 indicated the presence of bioactive

compounds proanthocynidine with positive mode of ESI (Fig. 4.9). The MS-MS of peak 593 in

Fig. 4.10 gave the highest peaks 429.25, 457.17, 411.25 and 401.17, where the peak at 457.17

might indicate the presence of ursolic acid.

Fig. 4.9 Mass spectrum of C. oxyacantha showing proanthocynidine at 593.17 m/z

59

Fig. 4.10 MS2 CID (20.00) of peak 591.42 showing Ursolic acid at 457.25 m/z

The ursolic acid has also been reported to act as competitive inhibitor of Na+/K+ ATPase

at the digitaloid binding site (Verma et al., 2007). The presence of ursolic acid in C. oxyacantha

is responsible for its ACE inhibiting and cardioprotective potential (Lacaille et al., 2001). The

LC-MS-ESI also revealed the spectrum and structure of cratagolic acid at peak of 417 (m/z) as

given in Fig. 4.11. The ESI is commonly used for ionization of molecules where the sample was

ionized by application of high electric charge. Moreover ESI covers a broad range of

metabolites, since it operates ionization in negative and positive modes (Mallick et al., 2012).

Fig. 4.11 Mass spectrum of C. oxyacantha showing Crateagolic acid at 471.08 m/z

The CID MS-MS of the peak 381 of C. oxyacantha showed the highest peak of 191,

207.08 and 249.17. The peak at 301.17 may give the idea of the presence of quercetine (Fig.

4.12).

60

Fig. 4.12 MS2 of 381 of C. oxyacantha with CID (20.00) showing Quercetin at 301.17 m/zThe most important active constituents identified in C. oxyacantha are the phenols and

the flavonoids (Verma et al., 2007; Meschino et al., 2014). These bioactive phytoconstituents

showed cardioprotective potential by inhibiting phosphodiesterase which increases the

intracellular cyclic nucleotides (Verma et al., 2007).

4.2.1.3 Rauwolfia serpentina:

R. serpentina is a versatile medicinal plant and several researchers have investigated it

due presence of various phytoconstituents (Mittal et al., 2012; Singh et al., 2009; Mallick et al.,

2012; poonam et al., 2013). Various secondary metabolites such as alkaloids, phenols, tannins

and flavonoids present in the roots of R. serpentina are responsible for its cardioprotective

potential (Morales, 2000b). The LC-MS analysis of roots extract of R. serpentina was performed.

The full mass spectrums along with the highest peaks at 327.25 and 355.33 indicated the

presence of ajmaline and yohimbine respectively (Fig. 4.13).

61

Fig. 4.13 Mass spectrum of R. serpentina showing yohimbine at 355.33 and ajmaline at

327.25 m/z

Fig. 4.14 R. serpentina showing MS-MS of peak 327 with CID (25.00)

Ajmaline being a sodium channel blocker when given intravenously illustrated the

therapeutic potential (Brugada et al., 2000; Kostin et al., 1986). It has also been claimed to

stimulate respiration and intestinal movements. The action of ajmaline on systemic and

pulmonary blood pressure is similar as that of serpentine (Gawade et al., 2012). The MS-MS

with CID of 25.00 at peak 327 produced different fragments ion peaks (Fig. 4.15). Among these

peaks the peak at 353.25 m/z may indicate the presence of ajmailacine. The ajmalicine is derived

from tryptophan. It prevents strokes and also helps in maintaining blood pressure (Srivastava et

al., 2006).

62

Fig. 4.15 MS2 of peak 327 with CID (25.00) showing Ajmailicine at 353.25 m/z

The Fig. 4.16 showed the mass spectrum and structure of serpentine at the peak 349.52.

Serpentine is relatively weak tertiary base and is useful to cure hypertension, cardiovascular and

neurological diseases (Gawade et al., 2012; Weiss et al., 2000). R. serpentina is a hopeful

medication in the world of pharmacy due to the existance of considerable bioactive constituents

in roots (Rolf et al., 2003).

Fig. 4.16 Mass spectrum of R. serpentina showing Serpentine at 349.25 m/z

4.2.1.4 Allium sativum:

A. sativum was subjected to LC-MS analysis to evaluate the presence of

phytoconstituents that might be responsible for cardiovascular diseases, dyslipidemia and

hypertension. The LC-MS analysis of A. sativum depicted the highest peaks at 896.92, 917.75

and 782.58 (Fig. 4.17). The MS-MS of 896.92 with CID (25.00) gave the peak at 319.25,

indicated the presence of myricetin. The mass spectrum along with structure of myricetin is

presented in Fig. 4.18. The myricetin due to its specific chemical structure counteracts oxidative

63

stress generated as a result of Reactive Oxygen Species (ROS) (Mariee et al., 2012; Larson et al.,

2012).

Fig. 4.17 Mass spectrum of A. sativum

Fig. 4.18 MS2 CID 25.00 of 896 of A. sativum showing Myricetin at 319.25 m/zThe mass spectrum in Fig. 4.19 showed the presence of apigenin at peak 269.08. The

apigenin showed its peak at negative mode of ESI. Naturally occurring apigenin is found mostly

in hydroxylated form and has been demonstrated to inhibit tumor cell proliferation, angiogenesis

and induce apoptosis (Fang et al., 2007).

64

Fig. 4.19 Mass spectrum of A. sativum showing Apigenin at 327.25 m/z4.2.1.5 Coriandrum sativum:

LC-MS analysis of seeds extract of C. sativum was performed to evaluate the active

phytoconstituents including phenolics, flavonoids and alkaloids. The full mass spectrum is given

in Fig. 4.20. The Fig. 4.21 was the presentation of mass spectrum and structure of Caffeic acid at

peak 179.08 and Isorhamnetin-3-O-glucoside at 478.17 m/z. The Caffeic acid is the main

representative of the phenolic acids showing peak at negative mode of electron spray ionization.

The mass spectrum of C. sativum also showed apigenin-6-C-glucoside at peak 593.25 m/z with

negative mode of electron spray ionization (Fig. 4.22).

Fig. 4.20 Full Mass spectrum of C. sativum

65

Fig. 4.21 Mass Spectrum of C. sativum showing Caffeic acid at 179.08 m/z and Isorhamnetin-3-O-glucoside at 477.17 m/z

Fig. 4.22 Mass spectrum of Coriandrum sativum showing Apigenin-6-C-glucoside at 593.25 m/z

Caffeic acid is a potent antioxidant and has several therapeutic properties including

antiviral, antioxidants, anti-inflammatory and anticarcinogenic. It has been reported that caffeic

acid inhibits both lipoxygenase activity and suppresses lipid peroxidation thus completely blocks

the production of Reactive Oxygen Species (Jayanthi and Subash 2010).

4.2.1.6 Eletaria cardamom:

Cardamom fruit is used against cardiac disorders, renal and vesicular calculi, dyspepsia,

debility, halitosis and gastrointestinal disorders (Husain and Ali 2014). Phytochemical

66

investigation of cardamom has revealed highly bioactive components. Therefore, phytochemical,

biological and chemical compositional studies of this genus should be intensified (Savan and

Kucukbay 2013). In this research the mass spectrum obtained by LC-MS analysis of E.

cardamom represented the high peaks at 195.17 133.06 and 333.33. The peak at 195.17 indicated

the presence of terpinyl acetate (Fig. 4.23).

Fig. 4.23 Mass Spectrum of E. cardamom showing Terpinylacetate at 195.17 m/z The mass spectrum also depicted the presence of sebinen at 137.08 peak (Fig. 4.24). The

literature also reported the presence of terpinyl acetate in E. cardamom extract (Menon et al.,

1999 and Singh et al., 2008).

Fig. 4.24 Mass Spectrum of E. cardamom showing Sabinene at 137.08 m/z

67

4.2.1.7 Piper nigrum:The methanolic extract of P. nigrum is subjected to LC-MS analysis to determine its

bioactive compounds that impart crucial role in cardioprotection. The pippercide, an active

ingredient of P. nigrum showed its peak at 219.08. The mass spectrum and structural

representation of pipercide is shown in Fig. 4.25. Pipercide, important secondary metabolite,

play a vital role in disease resistance (Sruthi et al., 2013).

Fig. 4.25 Mass Spectrum of P.nigrum showing Pipercide at 219.08 m/z

4.2.2 Antioxidant assay:

Antioxidant potential of extracts of selected medicinal plants (Rauvolfia serpentina,

Terminalia arjuna, Coriandrum sativum, Piper nigrum, Eletaria cardamom, Allium sativum and

Crataegus oxyacantha) was determined through “DPPH free radical scavenging activity” and

“DNA protection assay”.

4.2.2.1 DPPH free radical scavenging activity:

The DPPH free radical scavenging activity (in term of % age inhibition) of R. serpentina,

T. arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha at various

concentrations (20, 40, 60, 80 and 100 µg/mL) was recorded (Table. 4.2). The T. arjuna

(62.5±1.624) and A. sativum (60.7±2.83) showed high antioxidant potential at the least

concentration of 20 µg/mL as compared to the same concentrations of other selected plants

(Table. 4.2). The C. sativum and P. nigrum showed higher than 50% inhibition at the

concentration of 40 µg/mL. However the R. serpentina depicted maximum antioxidant potential

(56.2±0.871) at the concentration of 100 µg/mL as compared to other concentrations of this

plant. While the E. cardamom presented relatively low antioxidant potential (47.7±2.52) even at

68

its higher concentration of 100 µg/mL. In case of C. oxyacantha, the increase in antioxidant

potential was very low with increase in concentration from 20 to 40 µg/mL, but it rapidly

increased with further increase in concentration from 60 to 100 µg/mL. The antioxidant potential

of C. oxyacantha is mainly due to the presence of procynidine and quercetine in it (Sokol-

Letowska et al., 2007; Kashyap et al., 2012; Guo et al., 2003; Mary et al., 2010).

Table. 4.2 DPPH free radical scavenging activity of selected medicinal plants

Plants extract Concentration (µg/mL)

20 µg/mL 40 µg/mL 60 µg/mL 80 µg/mL 100 µg/mL

R. serpentina 29.307±1.279

36.1±1.605 42.5±1.473 48.4±1.64 56.24±0.871

T. arjuna 62.5±1.624 65.78±2.678 71.57±3.25 89.607±1.14 91.093±0.349

C. sativum 31.1±2.505 58.067±0.95 64.267±1.419 75.53±1.45 77.83±0.21

P nigrum 37.97±1.285 53.73±2.618 58.03±1.88 67.13±1.25 76.77±4.12

E. cardamom 13.20±1.923 16.54±1.47 24.48±2.54 39.87±2.44 47.7±2.52

A. sativum 60.7±2.835 56.13±3.362 70.4±4.53 75.03±2.16 80.33±2.80

C. oxyacantha 33.8±2.212 44.73±1.124 65.033±1.32 77.23±1.42 92.31±1.06

The high antioxidant potential of T. arjuna is because of the presence of its flavonoids

(Nema et al., 2012). The LC-MS analysis also revealed the abundance of Termiarjunoside I,

catchein, gallic acids, quercetin and myricetin in the methanolic extract of T. arjuna bark

(4.2.1.1) that strongly endorsed its antioxidant potential (Mandal et al., 2013). The high contents

of antioxidant in the bark of T. arjuna contribute significantly to its therapeutic profile and also

used in various degenerative diseases (Nema et al., 2012).

The graphical presentation depicted the dose dependant response for free radical

scavenging potential i.e, the activity of all the extracts of plants in term of % age inhibition

increased with increase in concentration (Fig. 4.26). The dose dependant response of extracts of

various plants for their antioxidant potential has also been supported by other studies (Shukla et

al., 2013; Soni and Sosa et al., 2013; Yadav et al., 2014; Ayesha et al., 2013). This dose

dependant manner might be due to gradual increase in phenolic components, phenolic acids and

phenolic diterpenes which have very strong antioxidant power (Soni and Sosa, 2013).

69

R. serpentine T. arjun C. sativum P. nigrum E. cardamom A.sativum C. oxycantha0

10

20

30

40

50

60

70

80

90

100

20 µg/ml40 µg/ml60 µg/ml80 µg/ml100 µg/ml

% a

ge in

hibi

tion

Fig. 4.26 Graphical presentation of DPPH radical scavenging activity of selected medicinal

plants

Free radical production in the biological system is a routine and continuous phenomenon

and the healthy individuals balance it by the antioxidative defense system (Gulcin et al., 2005;

Soni and Sosa 2013). The oxidative stress is produced when there is high free radical generation

as a result of reduction in antioxidant levels (Kratchanova et al., 2010). The antioxidant level

could be maintained by phytoconstituents present in medicinal plants, which claimed to act as

powerful chain breaking antioxidants (Padmanabhan and Jangle 2012) hence have a positive

correlation with antioxidant activity (Sahu et al., 2013).

Different kind of mechanisms including inhibition of enzymes, chelation of metal ions

and scavenging of free radicals are involved in antioxidative properties of flavonoids (Mandal et

al., 2013; Soumia et al., 2014). The selected medicinal plants could be beneficial to mankind by

virtue of their effective antioxidant activity which may impart cardioprotective potential against

various cardiovascular diseases.

70

4.2.2.2 DNA protection assay:

The DNA protection assay of extracts of various selected plants (Terminalia arjuna,

Crataegus oxyacantha, Piper nigrum, Rauvolfia serpentina, Allium sativum, Coriandrum

sativum and Eletaria cardamom) was performed by using pBR322 plasmid DNA (Fig. 4.27).

The pBR322 plasmid has been used in number of researches to investigate the DNA protection

of various plants against H2O2 damage (Bhawya and Anilakumar 2011; Guha et al., 2011; Kalita

et al., 2012; Kutlu et al., 2014).

Fig. 4.27 DNA plasmid pBR322

The protective effect of different concentrations (100, 500 and 1000 µg/mL) of extracts

of said plants was evaluated against pBR322 plasmid DNA damaged by oxidative stress. The

response of seven selected methanolic extracts of medicinal plants with varying concentrations

on DNA damage along with positive control (H2O2 and FeSO4) has been presented in Fig. 4.28

and 4.29. The free radicals produced in response of H2O2 and FeSO4 caused the strand cleavage

of pBR322 plasmid DNA and resulted in DNA band streaking (Fig. 4.28). All the plants

exhibited protection of plasmid DNA against H2O2 damage as compared to the plasmid DNA

merely treated with H2O2. High phenolic compounds, in extracts of all plants could be considered

as the key reason behind the antioxidant potential of the plants (Kalita et al., 2012; Golla et al.,

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http://www.hindawi.com/61020285/

2014). The strength of DNA protection increased in concentration dependant manner of extracts

of all the plants, which showed the protective effect of extracts against H2O2 induced damage.

The least concentration (100 µg/mL) of P. nigrum showed the band streaking in lane: 7 of Fig.

4.28, while the lane: 8 and 9 exhibited the good protection of P. nigrum against pBR322 plasmid

DNA damage at the corresponding concentration of 500 and 1000 µg/mL. In case of E.

cardamom, the concentrations of 100 µg/mL and 500 µg/mL in lane: 19 and 20 of Fig. 4.29 did

not show the protection, while the concentration of 1000 µg/mL showed the marked DNA

protection against H2O2 damage. Shyur et al. (2005) has also investigated the dose dependent

DNA protection of some selected medicinal plants and the results showed significant inhibition

of free radical and superoxide anion with increase in concentration of medicinal plants.

Fig. 4.28 Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30 mM H2O2 in the presence and absence of different plants extract. [Lane 1: pBR322 DNA + 30mM H2O2+ P1 (100 µg/mL), Lane 2: pBR322 DNA + 30mM H2O2+ P1 (500 µg/mL), Lane 3: pBR322 DNA + 30mM H2O2+ P1 (1000 µg/mL), Lane 4: pBR322 DNA + 30mM H2O2+ P2 (100 µg/mL), Lane 5: pBR322 DNA + 30mM H2O2+ P2 (500 µg/mL), Lane 6: pBR322 DNA + 30mM H2O2+ P2 (1000 µg/mL), Lane 7: pBR322 DNA + 30mM H2O2+ P3 (100 µg/mL), Lane 8: pBR322 DNA + 30mM H2O2+ P3 (500 µg/mL), Lane 9: pBR322 DNA + 30mM H2O2+ P3 (1000 µg/mL), Lane 10: pBR322 DNA + 30mM H2O2+ P4 (100 µg/mL), Lane 11: pBR322 DNA + 30mM H2O2+ P4 (500µg/mL), Lane 12: pBR322 DNA + 30mM H2O2+ P4 (1000 µg/mL)

72

Fig. 4.29 Lane13: pBR322 DNA + 30mM H2O2+ P5 (100 µg/mL), Lane 14: pBR322 DNA + 30mM H2O2+ P5 (500 µg/mL), Lane 15: pBR322 DNA + 30mM H2O2+ P5 (1000 µg/mL), Lane 16: pBR322 DNA + 30mM H2O2+ P6 (100 µg/mL), Lane 17: pBR322 DNA + 30mM H2O2+ P6 (500 µg/mL), Lane 18: pBR322 DNA +30mM H2O2+ P6 (1000 µg/mL), Lane 19: pBR322 DNA + 30mM H2O2+ P7 (100 µg/mL), Lane 20: pBR322 DNA + 30mM H2O2+ P7 (500 µg/mL), Lane 21: pBR322 DNA + 30mM H2O2+ P7 (1000 µg/mL)

P1= T. arjuna, P2= C. oxyacantha, P3= P. nigrum, P4= R. serpentina, P5= A. sativum, P6 =C. sativum and P7= E. cardamom.

H2O2 generates ●OH radicals, which cause massive oxidative damage. The ●OH radicals

bound to DNA strand may leads to base modification, deoxy sugar fragmentation and strand

breakage. Moreover, ●OH and other ROS causes oxidation of lipids which is responsible to

generate end products, like malondialdehyde and unsaturated aldehydes, that can attach to DNA

and produce mutagenic adducts (Guha et al., 2011). These oxidative modifications of DNA have

been suggested to contribute in the development of aging and various diseases (Bhawya and

Anilakumar, 2011).

The treatment of C. oxyacantha against hydroxyl radical induced DNA damage suggested

the DNA protection against oxidative damage. As the C. oxyacantha fruit is a traditional

medicine, hence used to cure the cardiovascular diseases because of its antioxidant potential

(Park et al., 2010). The mechanism by which the plants protect the DNA against H2O2 damage is

ambiguous because there are several potential inhibition pathways. However the antioxidants

may control the oxidative stress by reacting directly with H2O2 or reacting with intermediates

formed from enzymes and H2O2 (Kanwal et al., 2011).

73

A large number of plants have been examined to protect DNA against oxidative stress

due to UV induced photolysis of H2O2 (Rajkumar et al., 2010; Guha et al., 2011). The aqueous

extract of Curcuma amada showed the concentration dependant increase in protective potential

against oxidative stress induced in pBR322 plasmid DNA (Vishnupriya et al., 2012). However,

the established view is that the antioxidant potential of medicinal plants are related to DNA

protection activity by various mechanisms including chelation of transition metal (Asker et al.,

1996) inhibition of enzymes required for the initiation reaction of DNA cleveage (Bibi et al.,

2011).

4.2.3 Toxicity assay:

Toxicological testing is an essential prerequisite for the evaluation of medicinal plants as

therapeutics agent (Kumar et al., 2011). The cytotoxicity extracts of selected medicinal plants

(R. serpentina, T. arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha)

was evaluated by conducting “Hemolytic assay” and “Mutagenic assay”.

4.2.3.1 Hemolytic assay:

Hemolytic assay of the selected methanolic extracts was performed against human

erythrocytes using triton X-100 as positive control. The hemolytic activity of extracts of various

plants is shown in term of percentage hemolysis and given as mean ± SD of three replicates. The

percentage hemolysis of individual plants at different concentrations (100, 500 and 1000 μg/mL)

was expressed in Table. 4.3. None of the plant extract showed any hemolytic effect against

erythrocytes thus all the results showed no hemolysis. So pharmacologically these plants are safe

to use for human beings as a source of therapeutic drug. The T. arjuna and C. oxyacantha

showed very low % age hemolysis as compared to other selected medicinal plants. The P.

nigrum showed 8.10±0.232 and 9.21 ±0.106 % age hemolysis at the corresponding concentration

of 500 and 1000 μg/mL. However, extracts of all the plants showed dose dependent increase in

hemolytic activity, which showed that further increase in concentration, may cause hemolysis.

Thus further investigations are needed to evaluate hemolysis at higher concentrations.

74

Table. 4.3 Hemolytic activity (%) of extracts of selected medicinal plants

SamplesHemolysis (%)

100µg/mL 500 µg/mL 1000 µg/mL

C. sativum 5.47±0.281 6.03±0.131 7.30±0.012 -

C. oxyacantha 2.86±0.237 4.45±0.281 5.71±0.0321 -

T. arjuna 2.49±0.164 3.18±0.071 5.15±0.046 -

R. serpentina 4.20±0.082 5.26±0.051 5.39±0.102 -

A. sativum 3.46±0.067 5.18±0.201 7.26±0.341 -

E. cardamom 4.78±0.201 5.92±0.291 7.92±0.210 -

P. nigrum 6.30±0.014 8.10±0.232 9.21±0.106 -

Positive control Triton X 100

97.38±0.11

Negative control PBS

1.02±0.82

Hemolysis is due to obliteration of red blood cells which resulted from lysis of membrane

lipid bilayer. The hemolytic activity is related to chemical composition, concentration and

potency of each extract (Zohra and Fawzia, 2014). Several reports indicated that the membranes

of human erythrocytes have varying stability as determined from the mean corpuscular fragility.

The extracts of plants may affect the membrane of red blood cells in a positive way (Freitas et

al., 2008) but many plants may also have serious adverse effects, which include initiation of

hemolytic anemia (Zohra and Fawzia, 2014). Hemolytic activity of the aqueous extracts of

different Acacia species were accessed against human erythrocytes and showed low to mild

hemolytic effect. The hemolytic percentage of extracts was found to be increased with increase

in concentration of extracts of plants (Sulaiman and Gopalakrishnan, 2013).

75

C. sativu

m

C. oxya

cantha

T. arju

na

R. serpen

tina

A. sativu

m

E. card

amom

P. nigr

um

Positive

control Tr

iton X 100

Negative

control P

BS0

20

40

60

80

100

120

100µg/mL500 µg/mL1000 µg/mL

Samples

Hem

olys

is (%

)

Fig. 4.30 Graphical presentation of % Hemolysis of extracts of selected medicinal plants at

different concentrations

The aqueous extracts of Aerva lanata, Calotropis gigantea and Elaeocarpus ganitrus

with various concentrations of 125, 250, 500, 1000 μg/mL were analyzed for the hemolytic

activity against human erythrocytes. All the samples exhibited very low hemolytic effect as

compared to positive control and also showed the dose dependent increase in hemolytic activity

(Kumar et al., 2011).

Solvent extracts of Syzigum cuminii, Cratevan urvula and Achyranthes aspera also have

been studied for their hemolytic activity and reported to possess no haemolytic effect (Mathur et

al., 2011; Priya et al., 2010). The phytochemicals constituents present in extracts of various

medicinal plants might be responsible for their antihemolytic activity (Kumar et al., 2011;

76

Lakshmi et al., 2014). Actually, investigation of the toxicity of certain medicinal plants is

considered to be important because of their uses for medicinal purpose (Das et al., 2010).

4.2.3.2 Mutagenicity assay (Ames test):

The Ames test was applied to confirm whether the selected medicinal plants were

mutagenic or not (Zeiger et al., 2001; Kim et al., 2010; Abudayyak et al., 2015), so that these

may be used as safe therapeutics for various CVD.

The mutagenic probability of selected medicinal plants including R. serpentina, T.

arjuna, C. sativum, P. nigrum, E. cardamom, A. sativum and C. oxyacantha was evaluated by

using test strain S. typhimurium TA 98. The standard S. typhimurium TA 98 showed considerable

mutagenicity with high number (94/96) of positive wells (yellow) (Fig. 4.31). The mutagenic

property of all the selected medicinal plants was counted and compared with background plate

(11/96 positive wells) (Fig. 4.32). The medicinal plants are assumed to be mutagenic if the

number of positive wells is two folds higher as compared to the background plate (Razak and

Aidoo, 2011). The C. sativum, T. arjuna and R. serpentina showed 8/96 while the E. cardamom,

A. sativum and P. nigrum presented 16/96 yellow wells in microplate (Table. 4.4). Among these

the C. oxyacantha showed good results with no positive well in microplate. Therefore none of

the selected medicinal plants exhibited the mutagenic property. The variation in response of

these examined medicinal plants might be due to the differences in their active constituents (Aqil

et al., 2008).

77

Fig. 4.31 Standard S. typhimurium TA 98

Fig. 4.32 Mutagenicity in Background plate

Fig. 4.33 Mutagenicity of extracts of selected medicinal plants. A= C. sativum, B= C.

oxyacantha, C= E. cardamom, D= P. nigrum, E= A. sativum, F= T. arjuna, G= R. serpentina

Table. 4.4 The mutagenicity of standard, Background and extracts of selected medicinal

plants

Sr. No. Plant extracts +ve/total Results Interpretation

78

1 Standards 94/96 + Mutagenic2 Background 11/963 C. sativum 08/96 - Non mutagenic

4 C. oxyacantha 0/96 - Non mutagenic5 E. cardamom 16/96 - Non mutagenic6 P. nigrum 16/96 - Non mutagenic

7 A. sativum 16/96 - Non mutagenic

8 T. arjuna 8/96 - Non mutagenic9 R. serpentina 8/96 - Non mutagenic

The Ames test is based on the number of Histidine revertants, produced by crude plant

extracts in S. typhimurium strain (Taylor et al., 2003; Luseba et al., 2007). Hence the absence of

mutagenic response of these selected medicinal plants against S. typhimurium bacterial strains is

an astounding approach in determining the safe use of these medicinal plants (Ghazali et al.,

2011).

Luseba et al. (2007) also observed the non mutagenicity of some medicinal plants

through Ames in S. typhimurium (TA98) test strain. This non-mutagenic property of medicinal

plants might be owing to the existance of active phytoconstituents. Lack of mutagenicity

suggested that the medicinal plants are safe tool for effective fighting against various diseases

(Luseba et al., 2007).

4.5 Section- II (In vivo analysis):

Section-II is related to in vivo analysis and it has been completed in three Phases. In

phase-I, the preliminary trial was run to optimize the dose of salbutamol at which it induced

myocardial infarction. The phase-II involved the optimization of various concentrations of

selected medicinal plants (R. serpentina, T. arjuna, C. sativum, P. nigrum, E. cardamom, A.

sativum and C. oxyacantha) to get more suitable cardiopotant dose against salbutamol induced

myocardial infarction. In Phase-III of in vivo analysis, the cardioprotective potential of herbal

combinations was evaluated against surgically induced myocardial infarction to get the more

cardiopotent herbal product.

4.5.1 Phase-I:

4.5.1.1 Preliminary trial (Dose optimization of Salbutamol):

79

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In preliminary trial the rats were administered with different concentrations of salbutamol

(mg/kg) for two consecutive days and the blood samples were taken at different time intervals

(hr). The varying concentrations and time intervals, both were suggested by “Central Composite

Design” of RSM. The cardiac markers including CK-MB, SGOT and LDH, being diagnostic

features were analysed to estimate the severity of myocardial infarction (Dianita et al., 2015).

The relationship between dependant (cardiac markers) and independent variables (concentrations

of salbutamol and time intervals) was graphically presented by 3D Response surface plots in

Figures. 4.34-36.

The interaction of different concentrations of salbutamol and time intervals of blood

sampling on the level of CK-MB has been given in Fig. 4.34. The increased in concentration of

salbutamol from 34 to 88 mg/kg.b.wt. after 48 hr of salbutamol administration resulted in

elevation in CK-MB level from 152 to 297 IU/L. Further increase in concentration from 88 to

141 mg/kg of salbutamol showed comparatively low elevation in the CK-MB level. However

after 116 hr of salbutamol administration (88 mg/kg.b.wt), there was considerable decline in

enzyme level from 293 to 184 IU/L. The possible explaination could be that the CK-MB level

fall to normal with passage of time intervals after 48 to 72 hr of MI development (Dianita et al.,

2015). In case of rats, administered the salbutamol at concentration of 50 and 125 mg/kg did not

show the considerable elevation (148 and 198 IU/L) in the level of CK-MB after intervals of 96

hr. The variation in quantity of enzymes released depends upon the degree of cellular damage

(Azmat et al., 2012; Khan et al., 2014).

80

Design-Expert® Sof tware

CK-MB299

144

X1 = A: TimeX2 = B: Conc

0.00

24.00

48.00

72.00

96.00

50.00

68.75

87.50

106.25

125.00

140

180

220

260

300

CK

-MB

A: Time B: Conc

Fig. 4.34 Response surface plot of CK-MB vs. time and concentration

The Fig. 4.35 described the relation between SGOT (dependent variable) conc. and time

intervals (independent variables). The salbutamol at the concentrations of 34, 88 and 141 mg/kg

b.wt depicted the inclined in the level of SGOT with corresponding values of 51, 128 and 132

IU/L after 48 hr of salbutamol administration. This might be due to the reason that salbutamol

causes leakage of SGOT from cardiomyocytes into the blood. It occurs due to collapse of cellular

and subcellular compartments that reflect pathological alterations in myocardium (Jaffe et al.,

2006). After 96 hr of salbutamol administration at the concentration of 50 and 125 mg/kg, the

SGOT level was comparatively low. This decline in SGOT level might be due to the increase in

time intervals as the SGOT level shifted towards normal with passage of time (Dianita et al.,

2015).

81


SGOT264

36

X1 = A: TimeX2 = B: Con.

0.00

41.00

82.00

123.00

164.00

50.00

68.75

87.50

106.25

125.00

50

110

170

230

290

SG

OT

A: Time B: Con.

Fig. 4.35 Response surface plot of SGOT vs time and concentration

In case of LDH, the increase in concentrations of salbutamol from 34 to 88 mg/kg b.wt.

resulted in increase in level of LDH from 271 to 512 IU/L respectively after 48 hr interval of

salbutamol administration. The LDH is detectable from 8 to12 hours of post MI and reached at

its peak at 24-72 hours (Rosenblat et al., 2012). Further increase in time interval of 116 hr after

salbutamol administration the rats treated with salbutamol at the concentration of 88 mg/kg

depicted the considerable fall in the level of LDH from 512 to 298 IU/L (Fig.4.36). This decline

in LDH level supported the fact that serum levels of LDH approaches to normal value after 96

hours after MI (Wang et al., 2006). The level of LDH was 278 and 357 IU/L in response to the

corresponding concentrations of 50 and 125 mg/kg b.wt. after 96 hr intervals of salbutamol

administration. This increase in the level of cardiac markers in serum indicated the altered

membrane permeability and leakage of these enzymes into blood stream (Abirami and

Kanagavalli, 2013).

The increase in enzyme level is due to the oxidative stress and myocardial cell necrosis

caused by salbutamol (Dianita et al., 2015; Zafar et al., 2015). The extent of leakage of cardiac

markers (CK-MB, LDH and SGOT) indicated the onset of myocardial infarction and act as

sensitive markers of myocyte injury (Nandave et al., 2007).

82


LDH518

265

X1 = A: TimeX2 = B: Conc

0.00

24.00

48.00

72.00

96.00

50.00

68.75

87.50

106.25

125.00

260

325

390

455

520

LD

H

A: Time B: Conc

Figure: 4.36 Response surface plot of LDH vs. time and concentration

The cardiotoxicity due to administration of salbutamol is reflected by the increased

cardiac markers, lipid profile and alleviated antioxidant enzymes as compared to normal control

group (Zafar et al., 2015). The histopathological examination of heart after salbutamol

administration also endorsed the development of myocardial infarction in another animal trial

(Kousar et al., 2012; Aslam et al., 2015).

The analysis of variance (ANOVA) presented the statistical significance of the fitted

quadratic polynomial model (Table. 4.5). The F values of model were, 48.18, 27.27 and 122.30

for CK-MB, SGOT and LDH respectively which suggested the significance of quadratic model

for cardiac markers. The model is adequate to present the relationship between the response of

dependent (cardiac markers) and independent variables (concentrations of salbutamol and time

intervals). The value of determination coefficient (R2) for CK-MB, SGOT and LDH was 0.9718,

0.9512 and 0.9887 respectively with non significant lack of fit at P>0.05, which means that the

statistical model could explain more than 97.18 of the result. Meanwhile, a relatively lower value

83

of coefficient of variation, 6.86, 12.9 and 4.20 for CK-MB, SGOT and LDH respectively showed

a better precision and reliability of the experiment.

Table. 4.5 Analysis of variance (ANOVA) for the fitted model of CK-MB, LDH and SGOT activity as a function of independent variables

Parameter Source SS Df MS F Value P>F

CK-MB R2=0.9718 CV=6.86

Model 54546.23 5 10909.25 48.18 <0.0001A-Time 1188.22 1 1188.22 5.25 0.0557B-Conc 4585.08 1 4585.08 20.25 0.0028

AB 400.00 1 400.00 1.77 <0.0001

A2 34111.31 1 34111.31 150.65 <0.0001

B2 20304.00 1 20304.00 89.67 <0.0001Residual 1585.00 7 226.43

Lack of Fit 1564.20 3 521.40 100.27 <0.0521Pure Error 20.80 4 5.20

L

SGOT R2=0.9512CV=12.96

Model 17167.89 5 3433.58 27.27 0.0002A-Time 969.95 1 969.95 7.7 0.0275B-Con 3459.58 1 3459.58 27.48 0.0012

AB 387.04 1 387.04 3.07 0.123A2 11359.67 1 11359.67 90.22 < 0.0001B2 2036.11 1 2036.11 16.17 0.0051

Lack of Fit 731.36 3 243.79 6.5 0.0541Residual 881.36 7 125.91

Pure Error 150 4 37.5

LDHR2=0.9887

CV=4.20

Model 1.571E+005 5 31423.93 122.30 < 0.0001A-Time 2680.09 1 2680.09 10.43 <0.0145B-Conc 7444.98 1 7444.98 28.97 <0.0010

AB 1521.00 1 1521.00 5.92A2 99216.21 1 99216.21 386.13

B2 64680.21 1 64680.21 251.72Residual 1798.64 7 256.95

Lack of Fit 1777.44 3 592.48 111.79 0.0013Pure Error 21.20 4 5.30

The purpose of dose optimization of salbutamol was to find out the optimal concentration

which indicated the onset of myocardial infarction in experimental animals. The optimal time of

blood sampling (hr) and concentration of salbutamol (mg/kg) was suggested by RSM (Table.

4.6). Hence the RSM predicted the 80 mg/kg as optimum dose of salbutamol at which it may

elevate the SGOT level up to 99 IU/L after 20 hr of salbutamol administration while in case of

84

our experiment the concentration of 141 mg/kg.b.wt. elevated the SGOT level at its maximum

extant (132 IU/L). In case of our experimental study the concentration of 88 mg/kg elevated the

level of CK-MB (293 IU/L) and LDH (515 IU/L). The RSM suggested the concentration of 80

mg/kg.b.wt which raised the level of CK-MB and LDH up to 265 and 467 IU/L after 23 hr of

two concecutive doses (Table. 4.6) which was near to our experimental approach. The

cardioprotective potential of Coriandrum sativum against salbutamol induced cardiac injury also

favored the increased in CK-MB level up to 203 IU/L after administration of two doses of

salbutamol (Kousar et al., 2012).

Table. 4.6 Effects of optimized dose of salbutamol on different cardiac markers suggested by Response Surface Methodology

Parameter Time (hr) Concentration(mg/Kg) Optimized dose (IU/L) Desirability

CK-MB 23 80 265 0.907

SGOT 20 80 99 0.786

LDH 23 80 467 0.912

Exhaustion in levels of cardiac LDH and CK-MB isoenzymes during myocardial

infarction indicated the alteration in membrane integrity and causes the leakage of these

biomarkers into blood stream (Nandave et al., 2013). The formation of free redicals due to

oxidative stress resulted in Increase in cardiac enzymes which consequently responsible for

variations in membrane permeability thus ultimately lead to the failure of myocardial membranes

functions (Barman et al., 2013). The optimum dose (80 mg/kg) of salbutamol suggested by RSM

was further used to establish the experimental model of chemically induced MI.

4.6. Phase-II

4.6.1 Dose response experiment (A pilot study):

The dose response evaluation of selected parts of the medicinal plants like roots of R.

serpentina, bark of T. arjuna, seeds of C. sativum and E. cardamom, leaves of P. nigrum and

fruit of A. sativum and C. oxyacantha, was carried out followed by in vitro characterization. In

this phase different groups of rats were pretreated with said medicinal plants with varying

concentrations as suggested by RSM. After administration of two optimized doses of salbutamol

(Phase-I) the blood samples of rats were drawn to evaluate different biomarkers including

85

cardiac markers (CK-MB, SGOT and LDH), lipid profile (LDL, HDL, T. Cholesterol and

Triglycerides) and antioxidant enzymes (SOD, CAT and GSH). The results of all these

biomarkers are described accordingly under the following headings.

4.6.1.1.1 Cardiac markers:

The elevated levels of CK-MB, SGOT and LDH during myocardial infarction act as

highly sensitive diagnostic markers (Sabeena et al., 2004; Gurgun et al., 2008; Mastan et al.,

2009; Siddiq et al., 2012). The effect of different concentrations of medicinal plants against MI

was evaluated by estimation of cardiac markers (Fig. 4.37-39). These cardiac markers were

analysed by RSM to have the optimum concentration of each medicinal plant (Table. 4.8, 4.10,

4.12).

4.6.1.1.1.1 CK-MB:

CK-MB is considered as standard for diagnosis of MI and hence preferred in selective

situations (Jagannadha et al., 2010). The CK-MB level reaches at its peak after 24 hr of MI

development and fall to normal with passage of time intervals after 48 to 72 hr (Dianita et al.,

2015).

The CK-MB level in normal control group was found 150 IU/L. The positive control

group, to which only salbutamol (80 mg/kg) was given, showed considerable elevation (296

IU/L) in the CK-MB level. This might be due to the reason that salbutamol causes leakage of

CK-MB from cardiomyocytes into the blood stream. It occurs due to collapse of cellular and

subcellular compartments that reflect pathological alterations in myocardium (Jaffe et al., 2006).

Moreover, the rats in different treatment groups were treated with various concentrations (80,

110, 140, 170 and 200 mg/kg) of its respective medicinal plants. The effect of these medicinal

plants on the level of CK-MB has been given graphically (Fig. 4.37).

The first group was pretreated with different concentrations of T. arjuna. The increased

in concentration from 80-140 mg/kg b.wt. of T. arjuna maintained the level of CK-MB from

188-151 IU/L which display a decline of 12.38%. While the increase in concentration of this

plant up to 170 mg/kg showed very nominal effect i.e 153 IU/L level of CK-MB. However, with

further increase in concentration from 170 to 200 mg/kg, the level of CK-MB was maintained up

to 162 IU/L. Therefore the findings of the study gave the impression that 140 mg/kg b.wt is a

good dose to maintain the level of CK-MB. In another study the T. arjuna bark at the

concentration of 200 mg/kg b.wt, could maintained the CK-MB level with value of 146 IU/L

86

near to normal (Jahan et al., 2012). This cardioprotective potential of T. arjuna is credited to the

potent antioxidants and their free radical scavenging activity (Aslam et al., 2015).

The second group of rats was pretreated with different concentrations of C. oxyacantha.

The dose dependent response was observed when treated with concentration of 80 to 170 mg/kg

of C. oxyacantha with level of CK-MB from 223-162 mg/dL. The rats pretreated with

concentration of 200 mg/kg depicted slight elevation in CK-MB level (170 IU/L) as compared to

the concentration of 170 mg/kg. Thus the concentration of 170 mg/kg would be considered as the

dose of choice against MI. The C. oxyacantha berry is probably deemed as best known

cardiotonic. Hence it helps to improve the blood supply to heart by dilating peripheral and

coronary blood vessels and attenuating symptoms in early period of heart failure (Zafar et al.,

2015).

The third group of rats was pretreated with different concentrations of P. nigrum to get its

optimal concentration against myocardial infarction. With increase in concentrations the level of

CK-MB was also maintained gradually. The dose of 80 mg/kg b.wt. of P. nigrum could not

maintain the level of CK-MB (262 IU/L) with in limits in contrast to the same dose of other

selected medicinal plants. The treatment with concentrations of 170 and 200 mg/kg depicted

almost same effect on the levels of CK-MB with values of 180 and 188 IU/L respectively.

The fourth group was treated with different concentrations of C. sativum. The dose

dependent response was observed to maintaine the level of CK-MB appreciably. The

concentration of 200 mg/kg b.wt showed the substantial maintenance in the level of CK-MB

(171 IU/L) as compared to the positive control (296 IU/L) presented in Fig. 4.37. This reduction

in enzyme level could be due to its protective action on membrane integrity and restricting the

leakage of this enzyme (Aslam et al., 2015).

The fifth group pretreated with different concentrations of A. sativum also showed the

increase in the maintenance of CK-MB level with increase in its concentrations. There was not

much difference between the response of 140 and 170 mg/kg which showed almost equal

protection with CK-MB level of 162 and 168 IU/L. The concentration of 200 mg/kg appreciably

restored the level of CK-MB (157 IU/L) near to normal value. A. sativum has therapeutic

potential for hypertension and hypercholesterolemia. German Commission and the World Health

Organization have approved the A. sativum for its hyperlipidemia and atherosclerotic vascular

changes (Blumenthal et al., 2000).

87


CK-MB

B1 T. arjunB2 C. oxy canthaB3 P. nigrumB4 C. sativ umB5 A. sativ umB6 R. serpentineB7 E. cardamom

X1 = A: Conc.X2 = B: Plant

Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

CK

-MB

140

172.5

205

237.5

270

Fig. 4.37 Graphical presentation of optimized concentration of medicinal plants for CK-

MB against salbutamol induced Myocardial infarction

The pretreatment with R. serpentina showed the prominent effect in keeping the level of

CK-MB from 214 IU/L to 163 IU/L with increased in corresponding concentrations from 80 to

170 mg/kg. There was abrupt elevation in the level of CK-MB (175 IU/L) at the concentration of

200 mg/kg. This increase in CK-MB level might be due to the intake of high concentration of R.

serpentina which also cause a depletion of nor-epinephrine resulting in tranquilizing effect.

Moreover very high dose can also cause a loss of nerve coordination (Huang et al., 1999). The

seventh group was treated with E. cardamom to get the optimized dose at which it showed

preventive potential against salbutamol induced MI. The E. cardamom showed the dose

dependent response as the increase in concentrations considerably maintained the level of CK-

MB. In case of the dose of 80 mg/kg the CK-MB level was maintained at 261 IU/L while there

88

was better maintenance of CK-MB (219 IU/L) when the rats were treated with concentration of

110 mg/kg. However the concentration of 200 mg/kg b.wt depicted the maximum preventive

potential as it maintained the level of CK-MB up to 179 IU/L.

Overall the T. arjuna showed better maintenance even at lower concentration of 80

mg/kg as compared to the same dose of other selected medicinal plant. Cardioprotective potential

of T. arjuna extract can be correlated with presence of polyphenolic fraction and its antioxidant

activity (Jahan et al., 2012). The bark of T. arjuna reported to possess cardioprotective

(Gauthaman et al., 2001; Singh et al., 2008; Sivakumar and Rajeshkumar 2014; Aslam et al.,

2015; Zafar et al., 2015), hypocholesterolemic and antioxidant effect (Jahan et al., 2011a).

The experimental data was statistically analyzed using the Design-expert 7.0 for ANOVA

(Table. 4.7) which explained the significance of quadratic model. The F-value (437.43) depicted

the model is significant and also indicated the interaction strength of each parameter. The closer

the R2 (0.9945) value to unity, better the model fits to the actual data (Wani et al., 2013). The

value of Predicted R-Squared (0.9898) was close to the Adjusted R-Squared value (0.9922) as

expected. This may indicate a small block effect or a possible problem.

Table. 4.7 Analysis of variance (ANOVA) for response surface methodology of CK-MB

(IU/L) as a function of independent variables

Source SS Df MS F Value Prob > FModel 45186.46 14 3227.60 437.43 < 0.0001 Significant

A-Conc. 25348.74 1 25348.74 3435.46 < 0.0001 .B-Plant 11898.25 6 1983.04 268.76 < 0.0001

AB 2697.00 6 449.50 60.92A2 5242.48 1 5242.48 710.50

Residual 250.87 34 7.38Lack of Fit 250.37 20 12.52 350.52 < 0.0691 Non

SignificantPure Error 0.50 14 0.036Cor Total 45437.33 48R-Squared 0.9945 Standard Deviation 2.72

Adj R-Squared 0.9922 Mean 190.84Pred R-Squared 0.9898 Coefficient of Variation (CV) % 1.42Adeq Precision 72.594 Prediction Error Sum of Squares (PRESS) 463.20

The optimum concentration of selected medicinal plants predicted by Response Surface

Methodology is given in Table. 4.8. The RSM suggested 165, 172 and 164 mg/kg b. wt. as

89

optimum concentration for T. arjuna, C.oxyacantha and R. serpentina respectively that may

sustain CK-MB level near to normal which was related to experimental findings (140-170

mg/kg.b.wt). According to experimental approach the concentration of 170 mg/kg.b.wt. of P.

nigrum maintained the enzyme level against salbutamol induced MI while the response surface

methodology suggested the concentration of 186 mg/kg that may able to maintain the level of

CK-MB near to normal. The RSM predicted the optimum concentration of 170 mg/kg for A.

sativum while the experimental value depicted the concentration of 170-200 mg/kg to keep the

level of CK-MB within range. The RSM endorsed the concentration of 183 and 190 mg/kg for

C.sativum and E. cardamom which was near to our experimental approach (200 mg/kg). The

ability of medicinal plants to maintain the CK-MB level might be due to the presence of

flavanoids and antioxidants (Abirami and Kanagavalli 2013).

Table. 4.8 Optimized concentrations of medicinal plants for CK-MB (IU/L) against

salbutamol induced Myocardial infarction

Sr.

No.

Plants Optimum concentrations of selected medicinal plants (mg/kg)

CK-MB (IU/L)

Based upon biochemical evaluation

Suggested by RSM

1 T. arjuna 140-170 165 150 (165)*

2 C.oxyacantha 140-170 172 163

3 P.nigrum 170 186 182

4 C.sativum 200 183 170

5 A. sativum 170-200 170 155

6 R. serpentina 140-170 164 164

7 E. cardamom 200 190 178

Among all the studied medicinal plants, T. arjuna showed maximum restoration of CK-

MB at the optimum concentration. Cardioprotective potential of T. arjuna extracts can be

correlated with polyphenolic fraction and antioxidant activity (Jahan et al., 2012).

4.6.1.1.1.2 SGOT:

The SGOT is an index of cardiac damage and is also a clinical biomarker during

myocardial infarction (Rafatullah et al., 2008; Mahaswari et al., 2008). The normal control group

90

illustrated 36 IU/L of SGOT while the positive control group, merely treated with salbutamol,

showed 96 IU/L level of SGOT. The considerable elevation in the levels of SGOT in salbutamol

provoked group indicated the onset of myocardial injury. Amount of SGOT released depends

upon the degree of cellular damage (Alla et al., 2007; Azmat et al., 2012; Khan et al., 2014). A

number of medicinal plants have been reported to maintain the elevated level of SGOT during

cardiac damage (Orhue and Nawanze, 2004). The graphical presentation (Fig. 4.38) showed the

response of different treatments on SGOT against salbutamol induced MI. The first treatment

group of rats was administered with different concentrations of T. arjuna. A decreasing trend of

SGOT from 75 to 45 IU/L was found, with increase in concentration of T. arjuna from 80 to 170

mg/kg. The rats pretreated with the concentration of 140 and 170 mg/kg showed parallel effect

on the enzymatic level (45 IU/L). The effective maintenance of SGOT is due to the presence of

active phytoconstituents in T. arjuna bark (Gauthaman et al., 2001). The high concentration (200

mg/kg) of T. arjuna could not maintained the level of SGOT (55 IU/L) as compared to the

concentrations of 140 and 170 mg/kg b.wt. This depicted the slight deviation from dose

dependant response that might be due to the side effects of high dose of T. arjuna.

The second group of rats was pretreated with different concentrations of C. oxyacantha

(Fig. 4.38). All the concentrations of C. oxyacantha showed effective maintenance of SGOT in

rats against salbutamol even at its least concentration of 80 mg/kg.b.wt, as compared to the same

dose of other selected medicinal plants. High cardioprotective effect with 49 IU/L of SGOT was

observed with the concentration of 200 mg/kg. There was not any report related to adverse

effects of low doses of C. oxyacantha but its higher doses may increase the risk of hypotension

and sedation (Verma et al., 2007). The German Commission has also approved the use of C.

oxyacantha as a heart remedy and has become a part of many prescriptions for the common

cardiovascular disorders (Verma et al., 2007).

The third group of rats treated with P. nigrum maintained the SGOT level at 53 IU/L with

the corresponding concentration of 170 mg/kg.bwt. Further increase in concentration from 170 to

200 mg/kg did not show any considerable difference in order to maintain the level of SGOT (60

IU/L). None of the concentrations of P. nigrum from 80 to 140 mg/kg b.wt. could maintain the

level of SGOT as compared to normal control group. Therefore 170 mg/kg of P. nigrum might

be considered as the effective concentration that may cope with complications related to MI. The

fourth group of rats treated with C. sativum showed the dose dependent behaviour on the level of

91

SGOT as the increase in concentrations resulted in gradual decline in the enzyme level. The

outcomes of results gave an idea that the concentration of 200 mg/kg may exert a remarkable

protection from the toxic effect of salbutamol.

The A. sativum given to fifth group of rats, showed the better maintenance of SGOT level

with increase in its concentrations from 180 to 200 mg/kg b.wt. (Fig. 4.38). The concentration of

200 mg/kg maintained the level of SGOT at 46 IU/L that depicted very close effectiveness to the

response of concentration of 170 mg/kg.b.wt. The sixth group pretreated with R. serpentina

considerably sustained the level of SGOT almost within normal range (35 to 55 IU/L) at the

corresponding concentrations of 140-200 mg/kg b.wt. Hence this range could be considered the

effective range to maintain the enzyme level near to normal as compared to positive control

group (96 IU/L).


SGOT



Plant species

80 110 140 170 200

Conc.

SG

OT

40

53

65

78

91

Fig. 4.38 Graphical representation of optimized concentration of medicinal plants for

SGOT against salbutamol induced Myocardial infarction

The seventh group of rats pretreated with E. cardamom showed the decrease in SGOT

level from 88 IU/L to 54 IU/L with corresponding concentration of 80 to 170 mg/kg. Further

increase in concentration up to 200 mg/kg b.wt. could not sustain the level of SGOT (64 IU/L)

92

near to control group. Previously considerable maintenance of enzymes level has been observed

in the rats pretreated with 100 and 200 mg/kg.b.wt. of E. cardamom with concomitant

administration of isoproterenol. Moreover, the cardioprotective effect was supported by better

histopathological changes, which specify the salvage of cardiomyocytes from the harmful effects

of isoproterenol (Goyal et al., 2015).

The response of above said medicinal plants at varying concentration gave an idea that

the C. oxyacantha and T. arjuna are relatively more effective for maintaining the SGOT level.

This maintenance in enzyme level could be due to the presence of antioxidant and polyphenols in

these medicinal plants, thereby preventing the secretion of enzymes from myocardium (Jahan et

al., 2012). The bark of T. arjuna enhances the generation of endogenous antioxidant compounds

of rats’ hearts and prevent oxidative stress associated with ischemic reperfusion injury of the

heart (Singh et al., 2008).

The Analysis of Variance (ANOVA) indicated the model F-value of 114.98, which

implies the significance of quadratic model (Table 4.9). The value of R2 (0.9793) for SGOT was

closer to unity, indicating better the empirical model fits the actual data (Fan et al., 2008).

Moreover, the accuracy and reliability of the experiment was also confirmed by the coefficient of

variation (3.71) for SGOT. This suggested that the predicted quadratic model defined well the

behavior of the studied markers.

Table. 4.9 Analysis of variance (ANOVA) for response surface methodology of SGOT (IU/L) as a function of independent variables

Source SS Df MS F Value Prob > F Model 8591.59 14 613.69 114.98 < 0.0001 Significant

A-Conc. 4741.56 1 4741.56 888.41 < 0.0001 .B-Plant 1909.99 6 318.33 59.64 < 0.0001

AB 399.52 6 66.59 12.48A2 1540.52 1 1540.52 288.64

Residual 181.46 34 5.34Lack of Fit 174.95 20 8.75 18.80 < 0.0864 Non

Significant Pure Error 6.52 14 0.47Cor Total 8773.05 48 R-Squared 0.9793 Standard Deviation 2.31

Adj R-Squared 0.9708 Mean 62.34Pred R-Squared 0.9633 Coefficient of Variation (CV) % 3.71Adeq Precision 36.813 Prediction Error Sum of Squares (PRESS) 321.95

93

The RSM depicted optimized concentration of 165, 157, 173 and 170 mg/kg b.wt for

maximum protective potential of T. arjuna, C. oxyacantha, P.nigrum and E. cardamom

respectively and the biochemical analysis showed the effective concentration ranged from 140-

170 mg/kg. The RSM suggested optimal concentration of 178 mg/kg of C. sativum, which fall in

our experimental range of 170-200 mg/kg which noticeably maintained the level of SGOT. In

case of A. sativum and R. serpentina the range of 140-200 mg/kg b.wt. gave the best respons

while the RSM suggested 174 and 156 mg/kg b.wt.

Table. 4.10 Optimized concentrations of medicinal plants for SGOT (IU/L) against


Sr. No. Plants extract Optimum concentrations of selected medicinal

plants (mg/kg)

SGOT (IU/L)

Based upon

biochemical evaluation

Suggested by RSM

1 T. arjuna 140-170 165 502 C.oxyacantha 140-170 157 41

3 P.nigrum 140-170 173 56

4 C.sativum 170-200 178 52

5 A. sativum 140-200 174 46

6 R. serpentine 140-200 156 52

7 E. cardamom 140-170 170 59

4.6.1.1.1.3 LDH:

Lactate dehydrogenase (LDH) is a cardiospecific enzyme, existed in myocardium and

released into the blood stream following myocytes injury and disintegration of the subcellular

and cellular compartments (Mnafgui et al., 2015).

94

The normal and positive control groups presented 250 and 519 IU/L level of LDH

respectively. The high level of LDH in positive control group is due to the reason that salbutamol

causes leakage of LDH from cardiomyocytes into the blood stream. Different treatment groups

were administered with varying concentrations of selected medicinal plants and their effects on

the level of myocardium specific LDH (H4) have been presented graphically in Fig. 4.39. The

first treatment group was administered with different concentrations of T. arjuna prior to the

salbutamol intoxication. The increase in concentration from 80 to 170 mg/kg resulted in decrease

in the level of LDH (437 to 299 IU/L). The concentrations of 140 and 200 mg/kg b.wt. of T.

arjuna did not show much variation in maintaining the level of LDH against salbutamol induced

MI. However 170 mg/kg.b.wt of T. arjuna would be considered as the dose of choice that

depicted the effective response to uphold the LDH level (299 IU/L) near to normal. The

maintenance of LDH by T. arjuna has been widely explored in the scientific researches all over

the world owing to its powerful antioxidant activities (Aslam et al., 2015). Traditional remedial

system also documented the cardioprotective properties of T. arjuna (Nema et al., 2012).

The second group treated with different concentrations of C. oxyacantha illustrated the

dose dependent response to maintain the level of LDH against salbutamol intoxication. The

increase in concentration from 80 to 170 mg/kg showed the gradual decline in the level of LDH

from 399 to 283 IU/L. The concentration of 200 mg/kg of b.wt. depicted the closs effectiveness

to the concentration of 170 mg/kg.b.wt. in order to maintain the level of LDH. Thus the

concentration of 170 mg/kg b.wt. could be recommended as therapeutic dose.

In third treatment group, increased in concentrations of P. nigrum from 80 to 170 mg/kg

b.wt resulted in increase in protective potential against salbutamol induced MI with respect to

LDH level. The concentration of 200 mg/kg did not show any protective effect as it kept the

level of LDH at 316 IU/L. Therefore the concentration of 170 mg/kg b.wt. was the only dose of

choice that could keep the level of LDH near to normal.

The C. sativum given to forth group of rats showed the dose dependant response in order

to sustain the level of LDH near to normal control group. The increased in concentration from 80

to 200 mg/kg decreased the level of LDH from 421 to 305 IU/L. Several phytochemicals and

pharmacological studies of the various parts of C. sativum also supported its cardioprotective

potential against MI (Momin et al., 2012; Iqbal et al., 2012).

95

In case of treatment group five, different concentrations of A. sativum was given to rats

and found that the increase in concentration of A. sativum resulted in increase in protective

potential to maintain the level of LDH against salbutamol induced damage. The rats pretreated

with concentration of 200 mg/kg b.wt exhibited substantial maintenance of LDH at 287 IU/L,

which could be considered as therapeutic dose to maintain the level of LDH.

The sixth group pretreated with R. serpentina also showed the dose dependant response

with respect to decrease in the LDH level (442 to 306 IU/L) when treated with concentration of

80 to 170 mg/kg b.wt. The concentration of 200 mg/kg.b.wt showed an inconsequential

maintenance in the level of LDH (334 IU/L). The findings suggested that none of the

concentration of R. serpentina could maintain the level of LDH against salbutamol induced MI.

Thus comparatively the concentration of 170 mg/kg could be considered as better dose to

maintain the level of LDH.


LDH



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

LDH

270

335

400

465

530

96

Fig. 4.39 Graphical presentation of optimized concentration of medicinal plants for LDH

against salbutamol induced Myocardial infarction

The seventh treatment group of E. cardamom also presented the dose dependant decline

in enzyme level against MI. The least concentration of 80 mg/kg of E. cardamom did not show

any protective effect on the LDH level (519 IU/L) while further increase in concentrations from

110 to 200 mg/kg effectively decreased the level of LDH gradually from 420 to 292 IU/L. Goyal

et al. (2015) also demonstrated the cardioprotective potential of E. cardamom with concentration

of 200 mg/kg b.wt. against MI by bring back the level of endogenous antioxidants, maintaining

histopathology of myocardium, thus enhancing cardiac function.

The overall response of all the treatment groups gave a picture that C. oxyacantha and T.

ajuna restored the LDH level better even at their lower concentrations as compared to other

groups pretreated with various concentrations of selected medicinal plants. The reduction in

LDH leakage, by C. oxyacantha pretreatment endorsed the protection of the cell membrane from

myocardial injury (Kashyap et al., 2012; Panda and Naik 2009).

The ANOVA for the fitted quadratic model was presented in Table. 4.11. The value of

determination coefficient (R2) was 0.9832 which means that the calculated model was able to

explain 98.32% of the results. The significance of the model was also judged by F-test, which

showed that model had a high F-value (142.17).

Table. 4.11 Analysis of variance (ANOVA) for response surface methodology of LDH

(IU/L) as a function of independent variables

Source SS Df MS F Value Prob > FModel 2.076E+005 14 14828.25 142.17 < 0.0001 Significant A-Conc. 1.655E+005 1 1.655E+005 1587.14 < 0.0001 . B-Plant 15205.97 6 2534.33 24.30 < 0.0001 AB 11629.36 6 1938.23 18.58 A2 15226.65 1 15226.65 145.99Residual 3546.09 34 104.30Lack of Fit 3545.59 20 177.28 4963.82 < 0.0781 Non

significant Pure Error 0.50 14 0.036Cor Total 2.111E+005 48R-Squared 0.9832 Standard Deviation 10.21Adj R-Squared 0.9763 Mean 360.26Pred R- 41.125 Coefficient of Variation (CV) % 2.83

97

SquaredAdeq Precision 10.823 Prediction Error Sum of Squares (PRESS) 6086.98

The RSM suggested the optimized concentrations of selected medicinal plants which may

able to cure the MI (Table. 4.12). The RSM suggested that T. arjuna and C. oxyacantha may

restore the level of LDH near to normal at the corresponding optimum concentration of 186 and

179 mg/kg which was in accordance to our experimental range from 140 to 200 mg/kg. The

RSM recommended the concentration of 193 mg/kg b.wt. for P.nigrum which showed slight

deviation from our experimental findings (140-170 mg/kg b.wt). The RSM endorsed the

optimum concentration of 181 mg/kg as effective dose for C. sativum that was under our

experimental range of 170-200 mg/kg. The optimize dose suggested by RSM for A.sativum and

E. cardamom was 200 mg/kg which is in accordance to our experimental findings (200 mg/kg).

Table. 4.12 Optimized concentrations of medicinal plants for LDH (IU/L) against


The T. arjuna and C.oxyacantha presented the effective response to maintain the level of

cardiac enzymes (CK-MB, SGOT and LDH) during myocardial infarction. Jahan et al. (2012)

treated different groups of rabbits with T. arjuna extracts (200 mg/kg b.wt.) which significantly

stopped the isoproterenol induced leakage of all cardiac diagnostic markers (CK-MB, LDH and

AST), sensitive index to assess the degree of myocardial necrosis. The amount of these cardiac

98

Sr. No. Plant extract Optimum Concentration (mg/kg) of selected

medicinal plants

LDH

(IU/L)

Based upon

experimental evaluation

Suggested by RSM

1 T. arjuna 140-200 186 298

2 C.oxyacantha 140-200 179 284

3 P.nigrum 140-170 193 302

4 C.sativum 170-200 181 310

5 A. sativum 200 200 296

6 R. serpentina 200 180 317

7 E. cardamom 200 200 296

biomarkers in heart shows the changes in plasma membrane permeability. Due to therapeutic

potential of medicinal plants, these are considered valuable remedies. Plants having flavanoids

have been reported to possess strong antioxidant properties which is responsible for its

cardioprotective potential (Abirami and Kanagavalli 2013).

4.6.1.1.2 Antilipidemic profile:The lipid profile is a group of clinical tests comprising of total cholesterol, triglycerides,

HDL-c and LDL-c. The hypertriglyceridemia and low level of HDL-c are associated with the

development of cardiovascular diseases (Spalding et al., 2009). Hence the lipid profile along

with other risk factors is used to assess the risk level of development of cardiovascular disease

(Beaulah et al., 2014). The antilipidemic potential of varying concentrations (80, 110, 140, 170

and 200 mg/kg b.wt.) of the selected medicinal plants was evaluated and its findings were

presented graphically in Figures 4.40-43.

4.6.1.1.2.1 HDL-c:The HDL-c is inversely associated with risks of CHD and is a key component for

predicting the chances of development of cardiovascular diseases. The HDL-c shuttles the

cholesterol back to the liver for recycling hence preventing the cholesterol deposition in

bloodstream. Problem arises when there is no enough HDL to carry the cholesterol back to liver

hence creating an imbalance that may lead to heart diseases (Rader and Hovingh 2014).

The normal control group showed the HDL-c level (53 mg/dL) more than the positive

control group (23 mg/dL), indicating the reduction of good cholesterol due to intoxication of

salbutamol. The salbutamol might be responsible for accumulation of cholesterol in Cardiac

membranedue to rapid mobility of LDL-cholesterol from blood into the myocardial membranes

(Sangeetha and Quine 2006).

The response of varying concentrations of selected medicinal plants towards HDL-c

against salbutamol induced MI has been indicated graphically (Fig. 4.40). The HDL-c level was

28 and 24 mg/dL in rats pretreated with T. arjuna at the concentration of 80 and 110 mg/kg

respectively. This means that the said doses could not maintain the level of HDL-c relative to

normal control group. The HDL-c level was found well maintained at all the high doses of 140,

170 and 200 mg/kg.b.wt of T. arjuna with corresponding HDL values of 33, 48 and 37 mg/dL.

The best response regarding the maintenance of HDL-c was observed in rats pretreated with

concentration of 170 mg/kg. The excellent hypolipidemic potential of T. arjuna bark is due to its

99

ability to increase in hepatic clearance of cholesterol, down regulation of lipogenic enzymes and

inhibition of HMG- CoA reductase (Patil et al., 2011).

The second group was treated with different concentrations of C. oxyacantha through

preventive mode. The rats to which the dose of 80 mg/kg was given did not show any

considerable maintenance of HDL level (27 mg/dL). Increase in concentration of C. oxyacantha

from 110 to 200 mg/kg revealed the effective maintenance of HDL level from 37 to 53 mg/dL

respectively. The concentration of 200 mg/kg b.wt. of C. oxyacantha was considered as the dose

of choice to sustain the level of HDL near to normal control. The C. oxyacantha acts as lipid

regulating agent (Verma et al., 2007) and the cardioprotective potential of it is due to its

antioxidative strength and presence of oligomeric procyanidins (Chatterjee et al., 1996).


HDL



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

HD

L

21

30

39

48

57

Fig. 4.40 Graphical presentation of optimized concentration of medicinal plants for HDL

(mg/dL) against salbutamol induced Myocardial infarction

100

The rats treated with P. nigrum at the concentrations of 80, 170 and 200 mg/kg depicted

very close effectiveness in order to maintain the level of HDL with corresponding values of 34,

36 and 33 mg/dL. However comparatively, the concentrations of 110 and 140 mg/kg showed

better impact on the HDL level (40 and 46 mg/dL).

The fourth group of rats was pretreated with different concentrations of C. sativum. The

dose dependant response of C. sativum was observed to maintain the level of HDL-c near to

normal value (54 mg/dL). The concentration of 200 mg/kg of C. sativum showed maximum

potential to maintaine the level of HDL-c (54 mg/dL). The preventive treatment of C. sativum

against salbutamol induced MI is due to its natural antioxidant property, which reduces the

complications related to cardiac diseases (Kousar et al., 2011).

The response of A.sativum at varying concentrations towards HDL-c against salbutamol

induced toxicity has been presented graphically. The concentration of 110 and 140 mg/kg did not

show the effective response to control the level of HDL (24 and 25 mg/dL) against MI while

further increase in concentration (200 mg/kg) substantially sustained the HDL level up to 49

mg/dL. The A. sativum is reported to maintain the cholesterol level, blood pressure and also

delayed the progression of atherosclerosis thus preventing the heart diseases (Rottblatt et al.,

2002).

The sixth treatment group treated with R. serpentina sustained the HDL-c at the level of

27, 29, 36, 39 and 54 mg/dL at corresponding concentrations of 80, 110, 140, 170 and 200 mg/kg

b.wt. This showed the graduall maintenance in the level of HDL-c during MI. Therefor, the 200

mg/kg b.wt. of R. serpentina roots might be considered as therapeutic dose. R. serpentina has

been reported to enrich with almost 50 indole alkaloids (Deshmukh et al., 2012) that may be

responsible for maintenance of HDL-c level.

The E. cardamom given to seventh group of rats also showed the dose dependent

response. The increase in concentration resulted in considerable maintenance of HDL-c level.

However the concentrations of 110, 140 and 170 mg/kg depicted very close effectiveness to

maintain the level of HDL-c with values of 35, 38 and 40 mg/dL respectively. The E. cardamom

at its concentration of 200 mg/kg maintained the HDL level (46 mg/dL) near to normal control

group (53 mg/dL). Hence the concentration of 170 and 200 mg/kg b.wt. would be recommended

as the potant dose to maintain the level of HDL-c during MI. This suggested the cardamom as a

beneficial mediator in delaying the progression and development of MI.

101

It is assumed that HDL can eliminate the cholesterol within arteries and transfer it back to

the liver for reutilization, that’s why HDL-c is considered as “good cholesterol.” The effective

maintenance in the level of HDL-c during MI by selected medicinal plants might be due to the

reasons that these plants enhance the production of HDL-c or increase the activity of the protein

lipase (Prince et al., 2008; Hamid et al., 2013; Gomathi et al., 2014; Shatoor et al., 2014). Many

of the medicinal plants are reported to maintain the lipid profile during MI (Jahan et al., 2011;

Murugesan et al., 2012; Adi et al., 2013).

The statistical analysis was carried out through ANOVA and the findings have been

presented in Table. 4.13. The ANOVA showed the Pred R2 of 0.9235 is closed to the Adjusted R-

Squared 0.8639 as expected which indicated a small block effect (Loong et al., 2014). The

adequate precision measures the signal to noise ratio (Liu et al., 2010). Here the adequate

precision was 12.515 which indicated the importance of model.

Table. 4.13 Analysis of variance (ANOVA) for Response Surface Methodology of HDL

(mg/dL) as a function of independent variables

Source SS Df MS F Value Prob > F

Model 3756.24 21 178.87 15.51 < 0.0001 Significant A-Conc. 2237.79 1 2237.79 194.08 < 0.0001B-Plant 354.98 6 59.16 5.13 0.0012 AB 694.16 6 115.69 10.03 < 0.0001 A2 2.24 1 2.24 0.19 0.6631

Residual 311.31 27 11.53Lack of Fit 278.31 13 21.41 9.08 0.0501 Non

significantPure Error 33.00 14 2.36Core Total 4067.55 48

R-Squared 0.9235 Standard Deviation 3.40Adj R-Squared 0.8639 Mean 37.27Pred R-Squared 0.7738 Coefficient of Variation (CV) % 9.11Adeq Precision 12.515 Prediction Error Sum of Squares (PRESS) 920.01

The RSM was applied to get the optimal concentrations of selected medicinal plants

(Table. 4.14). The experimental values of T. arjuna ranged from the concentration of 170-200

102

mg/kg depicted the maximum cardioprotective potential and the RSM supported this range as it

suggested the concentration of 200 mg/kg as optimal dose. The concentration of 110 to 200

mg/kg of C.oxyacantha tried to maintain the HDL level while the RSM recommended the

optimal concentration of 200 mg/kg which was in accordance to given finding. The experimental

value of P. nigrum depicted that the concentration of 140 mg/kg prominently maintained the

HDL level near to normal but the RSM suggested the concentration of 110 mg/kg showing the

deviation from our experimental approach. The RSM presented the concentration of 200 mg/kg

for C.sativum and A. sativum to keep the level of HDL in normal range against salbutamol that

was also related to our findings. The recommended dose of R. serpentina by RSM was 180

mg/kg while the experimental finding suggested the maximum potential in rats pretreated with

concentration of 170-200 mg/kg. In case of E. cardamom the RSM proposed the concentration of

170 mg/kg while in our experimental analysis concentration of 140 to 200 mg/kg showed

effectiveness in order to maintain the level of HDL near to normal. Thus these suggested

concentrations could be used to maintain the HDL level during MI.

Table. 4.14 Optimized concentrations of medicinal plants for HDL (mg/dL) against salbutamol induced myocardial infarctionSr. No. Plants extract Optimum Conc. (mg/dL) of selected medicinal

plants

HDL (mg/dL)

Based upon biochemical

evaluation

Suggested by RSM

1 T. arjuna 170-200 200 39.11842 C.oxyacantha 110-200 200 53.80093 P.nigrum 140 110 38.2934 C.sativum 200 200 53.2935 A. sativum 200 200 42.78516 R. serpentine 170-200 180 50.92797 E. cardamom 140-200 170 45.1343

4.6.1.1.2.2 Low density lipoprotein cholesterol LDL:

103

A high level of LDL-c in serum is considered as a main risk factor for development of

coronary heart disease (Lamarche et al., 1997; Austin et al., 1988). It is also involved in

activating the inflammatory state which ultimately causes the heart diseases (Colpo 2005).

The positive control groups revealed high LDL-c level with the value of 154 mg/dL as

compared to normal control group (33 mg/dL). The elevated level of LDL-c in positive control

group indicated the salbutamol induced hyperlipidemia. Highly oxidative metabolites of

salbutamol accelerate the rate of peroxidation in membrane phospholipids. Salbutamol is

responsible for release of free fatty acids into plasma by the action of phospholipase A2 (Panda

and Naik 2009).

The effects of various concentrations of selected medicinal plants in different treatment

groups on LDL level against MI have been shown in Fig. 4.41. The first group of rats was treated

with various concentrations of T. arjuna. The rats treated with concentration of 80 and 110

mg/kg b.wt. showed LDL-c level 80 and 63 mg/dL respectively. Both of these concentrations

could not maintain the level of LDL considerably as compared to normal control group (33

mg/dL). However the rats treated with concentration of 140, 170 and 200 mg/kg b.wt. of T.

arjuna revealed considerable maintenance in the level of LDL-c (53, 51 and 55 mg/dL).

Although there is no remarkable difference in response of these concentrations therefore the

concentration of 140 mg/kg, being the lower concentration, may be considered as the dose of

choice to maintain the LDL level.

The second group was treated with various concentrations of C. oxyacantha showed the

dose dependant response as the increase in concentration from 80 to 170 mg/kg b.wt. tend to

decrease the LDL-c level from 62 to 46 mg/dL. The concentration of 170 mg/kg showed

maximum potential against salbutamol intoxication and could be considered as a potant dose to

maintain the level of LDL-c (46 mg/dL) near to normal control group. The antilipidemic

potential of C. oxyacantha may be due to stabilization of heart membrane and scavenging of

highly oxidized metabolites produced by salbutamol (Zafar et al., 2015).

The third group of rats was treated with various concentrations (80, 110, 170 and 200

mg/kg b.wt.) of P. nigrum. The increase in concentrations from 80 to 200 mg/kg depicted the

considerable decline in LDL level from 120 to 41 mg/dL. Therefore 200 mg/kg b.wt. would be

considered the effective concentration to maintain the LDL level near to normal control group.

104

The protective effect of P. nigrum is because of its antioxidant property that defence against

oxidation of LDL (Vijver 1997).

The treatment of rats with different concentrations of C. sativum showed that the

concentration of 80 mg/kg did not maintain the LDL level (105 mg/dL) against salbutamol

intoxication. However the rats pretreated with concentrations of 110, 140 and 170 mg/kg showed

the LDL-c level at 52, 56 and 48 mg/dL respectively. This showed that the concentrations of 170

mg/kg b.wt. is the dose of choice to maintain the level of LDL-c. The oral administration of

aqueous extracts of coriander seeds was reported to decrease the metabolic syndrome,

atherosclerotic indices and increased the cardioprotective potential (Aissaoui et al., 2011).


LDL



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

LDL

36

58

80

102

124

Fig. 4.41 Graphical presentation of optimized concentration of medicinal plants for LDL

(mg/dL) against salbutamol induced Myocardial infarction

The group of rats treated with A. sativum with various concentrations from 80 to 170

mg/kg depicted the decline in the level of LDL from 98 to 55 mg/dL. The rats pretreated with

concentration of 200 mg/kg b.wt. was less effective to maintain the LDL level (62 mg/dL) in

105

comparision to the 170 mg/kg b.wt. Thus the concentration of 170 mg/kg sustain the level of

LDL near to normal against salbutamol induced MI and could be referred as dose of choice. The

observed cardioprotective potential might be due to presence of flavanoid and its antioxidant

activity (Balasuriya and Rupasinghe 2011).

The group treated with various concentrations of 80, 110 170 and 200 mg/kg b.wt. of R.

serpentina showed the level of LDL-c 79, 66, 61 and 64 respectively. However the rats treated

with concentration of 140 mg/kg of R. serpentina effectively maintained the LDL-c at 58 mg/dL

near to normal.

The group of rats pretreated with different concentrations of E. cardamom showed the

decrease in the LDL-c level (116 to 61 mg/dL) with increase in concentrations from 80 to 140

mg/kg b.wt. However further increase in concentration up to 200 mg/kg b. wt. did not show any

considerable effect against salbutamol intoxication as it showed the LDL-c level 73 mg/dL. The

concentration of 140 mg/kg maintained the LDL level (61 mg/dL) up to some extant. Therefore

the concentration of 140 mg/kg of E. cardamom would be considered as therapeutic dose.

The results suggested that the C. sativum, T. arjuna and C. oxyacantha showed good

maintenance of LDL level as compared to other selected medicinal plants against salbutamol

induced MI. The lipid lowering effect of these medicinal plants may be due to stabilization of

heart membrane and scavenging of highly oxidized metabolites produced by salbutamol (Zafar

et al., 2015). The prior administration of extracts of various plants also showed significant

reduction in elevated serum lipid profile during myocardial infarction and responsible for normal

structural and architectural integrity of cardiac myocytes (Siddiq et al., 2012).

The experimental data were statistically analyzed using the Design-expert 7.0 (Table

4.15) The ANOVA depicted the significance of quadratic model with F value of 53.22. The

linear terms of concentrations (A) and plants (B) are also significant which showed that both

have important impact on the LDL profile. In addition, the coupling term AB is also significant

which indicated a positive interaction between the two variables (Conc. and Plants) to maintain

the tendency of LDL-c towards normalization (Noshadi et al., 2012). Moreover the values of the

determination coefficient R2 (0.9564) and the adjusted determination coefficient (0.9384)

showed the significance of model. Comparatively low value of the CV 7.52 % indicated a

improved accuracy and consistency of experimental study.

106

Table. 4.15 Analysis of variance (ANOVA) for response surface methodology of LDL


Source SS Df MS F Value Prob > F

Model 19560.68 14 1397.19 53.22 < 0.0001 Significant

A-Conc. 9533.46 1 9533.46 363.14 < 0.0001

B-Plant 3280.41 6 546.73 20.83 < 0.0001

AB 3717.43 6 619.57 23.60 < 0.0001

A2 3029.38 1 3029.38 115.39 < 0.0001

Residual 892.59 34 26.25

Lack of Fit 678.59 20 33.93 2.22 0.0654 Non significan

Pure Error 214.00 14 15.29

Cor Total 20453.27 48

R-Squared 0.9564 Standard Deviation 5.12

Adj R-Squared 0.9384 Mean 68.12

Pred R-Squared 0.9112 Coefficient of Variation (CV) % 7.52

Adeq Precision 27.358 Prediction Error Sum of Squares (PRESS) 1815.72

The optimal concentrations of selected medicinal palnts suggested by response surface

mehdology heva been presented in Table. 4.16. The RSM suggested 160, 144 and 170 mg/kg for

T. arjuna, C. oxyacantha and P. nigrum respectively that was related to our experimental

therapeutic range 140-170 mg/kg b.wt. The concentrations proposed by RSM for C. sativum, A.

sativum and E. cardamom was 173, 168 and 175 mg/kg respectively that were close to the

107

experimental finding (170 mg/kg b. wt). The RSM proposed concentration of 152 mg/kg for R.

serpentina to maintain the LDL level with in range that was near to experimental finding (140

mg/kg).

Table. 4.16 Optimized concentrations of medicinal plants for LDL (mg/dL) against

salbutamol induced myocardial infarction

Sr. No. Plants extract Optimum Conc.(mg/kg) of selected

medicinal plants

LDL(mg/dL)


evaluaton

Suggested by RSM

1 T. arjuna 140-170 160 482 C.oxyacantha 140-170 144 433 P.nigrum 140-170 200 414 C.sativum 170 173 465 A. sativum 170 168 566 R. serpentina 140 152 547 E. cardamom 170 175 64

4.6.1.1.2.3 Triglycerides:

Triglycerides are rich in apo C-III that delays the lipolysis of VLDL and inhibits its

uptake and clearance from plasma (Aminoff 2004). Elevated level of triglycerides increases 1.9

and 1.8 folds risk of coronary heart diseases in men and women respectively (Nadeem et al.,

2013).

The normal and positive control group showed the level of triglyceride with

corresponding values of 46 and 131 mg/dL. The effect of different concentrations of selected

medicinal plants on the level of triglycerides has been presented in Fig. 4.42. The group of rats

treated with bark extract of T. arjuna at varying concentrations against salbutamol induced MI

showed that the concentration of 170 mg/kg b.wt. considerably maintained the level of TGs (57

108

mg/dL) near to normal. The excellent hypolipidemic effect of T. arjuna bark seems to be

mediated through increased hepatic clearance of cholesterol and inhibition of HMG- CoA

reductase (Oben et al., 2006; Kanakavalli et al., 2014).

The C. oxyacantha showed the dose dependent response, as the increase in concentrations

from 80 to 170 mg/kg resulted in the decline in level of TGs from 89 to 57 mg/dL. However the

concentration of 200 mg/kg showed the slight deviation from the dose dependent response by

keeping the level of TGs at 66 mg/dL. Thus the concentration of 170 mg/kg of C. oxyacantha

would be recommended as therapeutic dose to maintain the level of TGs. This finding was also

supported by Weikl et al. (1996) who studied the effect of C. oxyacantha at the concentration of

160 mg/kg during human clinical trial. The findings depicted that there was no changes in

electrolytes, liver enzymes and ESR and therefore the concentration of 160 mg/kg was

considered as safe dose. The higher concentrations may increase the chances of hypertension but

there are no reports of side effects with low doses. (Verma et al., 2007).


TG



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

TG

46

62.75

79.5

96.25

113

Fig. 4.42 Graphical presentation of optimized concentration of medicinal plants for

Triglycerides (mg/dL) against salbutamol induced Myocardial infarction

109

The rats treated with concentration of 80 mg/kg of P. nigrum did not show the effective

response to maintain the TGs level (81 mg/dL) against salbutamol induced MI as compared to

normal control group (43 mg/dL). However the concentration of 200 mg/kg considerably

maintained the level of TGs (48 mg/dL) near to normal and could be recommended as

therapeutic dose to maintain the level of TGs.

The group treated with C. sativum also showed the dose dependant decline in the level of

TGs from 86 to 53 mg/dL with corresponding increase in concentration from 80 to 170 mg/kg.

The concentrations of 200 mg/kg did not show any considerable effect to maintain the TGs level

(66 mg/dL) in comparision to the concentration of 170 mg/kg b.wt. and could not be used as

therapeutic dose. The concentration of 170 mg/kg depicted the maximum therapeutic potential

and could be used to maintain the level of TGs near to normal against salbutamol intoxication.

The ability of C. sativum to maintain the level of TGs might be due to presence of

phytoconstituents in it (Momin et al., 2012). These phytoconstituents also play a crucial role in

ethnomedicine, pharmaceutical and food industries (Burdock and Carabin, 2009). The dried

seeds of C. sativum have been used as herb in ethnomedicine for the treatment of a variety of

diseases (Chithra and Leelamma, 1999; Momin et al., 2012).

The group of rats treated with various concentrations of A. sativum prior to salbutamol

intoxication showed that the concentration of 80 mg/kg was unable to sustain the level of TG (99

mg/dL) near to normal control group (43 mg/dL). All other concentrations of A. sativum from

110 to 200 mg/kg b.wt. decreased the triglycerides level from 77 to 55 mg/dL. Therefore, the

concentration of 200 mg/kg b.wt. would be recommended as the effectiv dose of A. sativum.

The rats treated with various concentrations (80-200 mg/kg b.wt.) of R. serpentina

resulted in decrease in TGs level (104 to 73 mg/dL). This showed that there was no considerable

maintenance in TGs level as compared to the concentrations of other selected medicinal plants.

Similarly the rat treated with various concentrations (80-200 mg/kg b.wt.) of E. cardamom

demonstrated that none of the concentration was able to maintain the level of TGs within normal

range. Even the rats pretreated with concentration of 200 mg/kg depicted poor effect in order to

maintain the level of TGs (67 mg/dL).

The good lipid lowering effect of T. arjuna, C. sativum and C. oxyacantha is because of

constraint of liver cholesterol synthesis, higher bile acid discharge and enhancement of receptor

110

mediated catabolism of LDL cholesterol (Hamid et al., 2013; Gomathi et al., 2014). Moreover,

plant extracts also reported to activate the generation of HDL which enhance the activity of the

protein lipase (Prince et al., 2008; Shatoor et al., 2014).


implies the significance of quadratic model (Table 4.17). Closer the R2 value (0.9243) to unity,

better the empirical model fits the actual data (Fan et al., 2008). The Adequate Precision

measures the signal to noise ratio, should above 4 so that it can be considered desirable (Loong

et al., 2014). In this case, the Adequate Precision obtained was 20.970 and this indicated this

model is significant. Moreover, the accuracy and reliability of the experiment was confirmed by

the coefficient of variance (6.82 %) for TGs. This suggested that the predicted quadratic model

defined well the effect of the different concentrations on level of TGs.

Table. 4.17 Analysis of variance (ANOVA) for response surface methodology of TGs


Source SS Df MS F Value

Prob > F

Model 10094.08

14 721.01 29.64 < 0.0001 Significant

A-Conc. 6160.01 1 6160.01 253.25 < 0.0001 B-Plant 2478.53 6 413.09 16.98 < 0.0001 AB 716.83 6 119.47 4.91 <0.0010 A2 738.72 1 738.72 30.37 < 0.0001Residual 827.02 34 24.32Lack of Fit 697.02 20 34.85 3.75 <0.0773 Non significant Pure Error 130.00 14 9.29R-Squared 0.9243 Standard Deviation 4.93Adj R-Squared 0.8931 Mean 72.35Pred R-Squared 0.8549 Coefficient of Variation (CV) % 6.82Adeq Precision 20.970 Prediction Error Sum of Squares (PRESS) 1585.06

The optimized doses of selected medicinal plants recommended by RSM have been given

in Table. 4.18. The RSM suggested the concentration of 176 mg/kg of T. arjuna to maintain the

TGs level in normal range that is near to our experimental value (170 mg/kg). RSM predicted the

possible concentration of 160 mg/kg of C.oxyacantha, to keep the level of TGs near to normal

against salbutamol induced MI that lies in our experimental therapeutic range from 110 to 170

mg/kg.b.wt. For P. nigrum and C.sativum, the RSM suggested the concentration of 193 and 175

111

mg/kg respectively that approaches to our experimental range (170-200 mg/kg). The RSM

recommended 200 and 195 mg/kg for A. sativum and R. serpentine respectively which is in

accordance to our experimental value 200 mg/kg. In case of E. cardamom, the dose suggested by

RSM was 160 mg/kg while in our experimental analysis the concentration of 200 mg/kg was

able to control the level of TG.

Table. 4.18 Optimized concentrations of medicinal plants for triglycerides (mg/dL) against

salbutamol induced myocardial infarction

Sr.No. Plants name Optimum concentration of selected

medicinal plants

Triglycerides

(mg/dL)

Based upon

biochemical evaluation

Suggested by

RSM

1 T. arjuna 170 176 62

2 C.oxyacanth

a

110-170 160 60

3 P.nigrum 170-200 193 50

4 C.sativum 170-200 175 60

5 A. sativum 200 200 54

6 R. serpentine 200 195 74

7 E. cardamom 200 160 64

Polyphenols are potent antioxidant that not only neutralize the lipid free radicals but also

prevent the decomposition of hydroperoxides into free radicals (Li et al., 2009; Rohman et al.,

2010). Medicinal plants significantly ameliorated the elevated lipid profile in rats to which MI

was induced by salbutamol due to presence of polyphenols. The cardioprotective potential of

medicinal plants may also be due to scavenging of highly oxidized metabolites produced by

salbutamol and stabilization of integrity of heart membrane with a consequent decrease in the

leakage of these markers (Beaulah et al., 2014; Zafar et al., 2015).4.6.1.1.2.4 Total Cholesterol:

High levels of total blood cholesterol are associated with the onset of coronary heart

disease (Carol and Merrily, 1984). Different herbs and natural products are highly effective in

lowering the cholesterol levels (Chand et al., 2007).

112

In normal control group Total Cholesterol (TC) level was 43 mg/dL and the positive

control group that was treated with salbutamol only, depicted the elevated TC level (196 mg/dL).

Different groups of rats were treated with various concentrations of selected medicinal plants

prior to salbutamol administration and their effects against MI have been given in Fig. 4.43. The

first group treated with T. arjuna illustrated that the increase in concentrations from 80 to 170

mg/kg b.wt. consequently decreased the level of TC from 109 to 56 mg/dL. The rats pretreated

with the concentration of 200 mg/kg of this plant sustained the level of T. cholesterol at the value

of 67 mg/dL. Thus the concentration of 170 mg/kg was considered as the concentration which

may use to maintain the level of T. cholesterol within range during MI.

The second group was pretreated with C. oxyacantha showed the considerable

maintenance of TC at 60 and 59 mg/dL when treated with corresponding concentrations of 170

and 140 mg/kg b. wt. Therefore the concentration of 140 mg/kg b.wt being the least

concentration would be rcommended to maintain the level of TC near to normal.

The concentration of 80 mg/kg of P. nigrum did not maintain the level of TC (119

mg/dL) but further increase in concentration from 110 to 170 mg/kg b.wt. resulted in decrease

the level of TC from 81 to 41 mg/dL. The concentration of 170 mg/kg b. wt. being most effective

dose and would be considered as therapeutic dose.

C. sativum considerably illustrated the dose dependant response as the level of TC was 92

to 59 mg/dL with increase in concentrations from 80 to 170 mg/kg b.wt. The concentration of

200 mg/kg showed the slight deviation from the dose dependant response as the level of TC was

68 mg/dL. Hence the concentration of 170 mg/kg would be considered as therapeutic dose.

The fifth group of rats pretreated with concentration of 80 to 170 mg/kg of A. sativum

depicted decline in TC level (101 to 65 mg/dL) against salbutamol induced MI. The

concentrations of 170 and 140 mg/kg presented the same effect on the level of T. cholesterol

therefore, the concentration of 140 mg/kg b.wt. being the least concentration might be considered

as appropriate dose to overcome the elevated level of TC during salbutamol intoxication.

113


Chol.



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

Cho

l.

41

60.5

80

99.5

119

Fig. 4.43 Graphical presentation of optimized concentration of medicinal plants for T.

cholesterol (mg/dL) against salbutamol induced Myocardial infarction

The preventive treatment of R. serpentina showed the dose dependent response in order

to decrease the level of TC from 102 to 58 mg/dL with corresponding concentrations of 80 to

200 mg/kg b.wt. The concentration of 200 mg/kg of R. serpentina proved as effective dose to

keep the TC near to normal range. The group of rats treated with various concentrations of E.

cardamom prior to salbutamol intoxication did not maintain the level of T. cholesterol near to

normal control group. However the concentration of 170 mg/kg b. wt. revealed the maximum

potential to control the level of TC with value of 72 mg/dL.


implies the significance of quadratic model (Table 4.19). The value of R2 (0.9451) for TC was

closer to unity, indicating better the empirical model fits the actual data (Fan et al., 2008).

Moreover, the accuracy and reliability of the experiment was also confirmed by the coefficient of

114

variation (5.66) for TC. This suggested that the predicted quadratic model defined well the

behavior of the studied markers.

Table. 4.19 Analysis of variance (ANOVA) for response surface methodology of TC (mg/dL) as a function of independent variablesSource SS Df MS F Value Prob > F

Model 16582.63 14 1184.47 59.98 < 0.0001 Significant

A-Conc. 10496.03 1 10496.03 531.54 < 0.0001

B-Plant 1347.14 6 224.52 11.37 < 0.0001

AB 1677.86 6 279.64 14.16 < 0.0001

A2 3061.59 1 3061.59 155.05 < 0.0001

Residual 671.37 34 19.75

Lack of Fit 577.87 20 28.89 4.33 0.0937 Non signifcant

Pure Error 93.50 14 6.68

Cor Total 17254.00 48

R-Squared 0.9611 Standard Deviation 4.44

Adj R-Squared 0.9451 Mean 78.57

Pred R-Squared

0.9259 Coefficient of Variation (CV) % 5.66

Adeq Precision 27.116 Prediction Error Sum of Squares (PRESS) 1278.23

The Response Surface Methodology suggested the optimal concentrations of selected

medicinal plants given in Table. 4.20. The RSM suggested the concentration of 175, 163, 159

and 161 mg/kg of T. arjuna, C.oxyacantha, C. sativum and E. cardamom to maintain the TC

level that was related to our experimental ranged from 140-170 mg/kg. RSM predicted the

possible concentration of 193 and 174 mg/kg for P. nigrum and R. serpentine respectively which

is in accordance to our experimental range 170-200 mg/kg.

Table. 4.20 Optimized concentrations of medicinal plants for Total Cholesterol (mg/dL) against salbutamol induced myocardial infarction

115

Sr. No. Plants

extract

Optimum Conc. (mg/kg) of selected medicinal

plants

T.Cholesterol

(mg/dL)


evaluation

Suggested by RSM

1 T. arjuna 140-170 175 632 C.oxyacantha 140-170 163 613 P.nigrum 170-200 193 464 C.sativum 140-170 159 585 A. sativum 140-170 159 676 R. serpentina 170-200 174 607 E. cardamom 140-170 161 74

The observed elevated levels of TC, TGs and LDL in salbutamol induced control group

indicated the presence of hyperlipidemia. The oxidative stress produced by salbutamol is mainly

responsible for damage to myocardial membrane, lipids leakage and prominent decrease in HDL

cholesterol levels (Fravin et al., 2004). The highly oxidative metabolites of salbutamol lead lipid

peroxidation responsible for the destructive reactions in cellular mechanism of myocardial

infarction (Sivakumar et al., 2007). These oxidative metabolites also also accelerate the rate of

peroxidation in membrane phospholipids that lead to hyperlipidemia (Panda and Naik 2009). The

mechanism involved in increase in lipids level by salbutamol was due to increase in adenylate

cyclase action causes to enhance cAMP production, which lead to increase of lipid accumulation

in myocardium (Adi et al., 2013).

The treatment of experimental animals with selected medicinal plants at different doses

decreased salbutamol induced hyperlipidemia. With preventive mode of treatment the levels of

lipid profile reduced closer to the normal level because of the remedial action of medicinal

plants. The group which was treated with C. oxyacantha, T. ajuna and R. serpentina showed the

maximum potential against MI and minimized the elevated enzymatic level considerably as

compared to other selected plants. The presence of terminoarjunoside I and other antioxidants are

responsible for the efficacy of T. arjuna. In our findings the LC-MS analysis of these selected

plants showed the presences of many phytochemicals responsible for its cardioprotective

potential. Quercetin present in C. oxyacantha, T. arjuna and A. sativum may be responsible to

116

cure the impairment of cardiac functions, possibly via a mechanism involving the improvment in

mitochondrial function during ischemia (Agnihotri et al., 2008). In case of lipid profile the

preventive treatment of coriander decreased the LDL-c and triglycerides level but increase the

HDL-c level considerably.

4.6.1.1.2 Antioxidant profiling:

Endogenous antioxidative defense is a very important source to neutralize the free radical

mediated tissue injuries. Superoxide dismutase and Catalase, the primary free radical scavenging

enzymes, are the first line of cellular defense against oxidative injury, decomposing O2 and H2O2

before their interaction to form more reactive hydroxyl radical (Kumar and Gurusamy, 2014).

The antioxidant profiling of the entire treatment groups has been discussed as follows.

4.6.1.1.2.1 Superoxide dismutase:

SOD, a free radical scavenging enzyme, is the first line of cellular defense against

oxidative injury decomposing superoxide and hydrogen peroxide before interacting to form the

more reactive hydroxyl radical. The equilibrium of enzyme is an important process for the

effective removal of oxygen free radicals (Dormandy, 1978; Bhattacharya et al., 2000).

The antioxidative strength of selected medicinal plants at various concentrations against

salbutamol oxidative stress has presented graphically in Fig. 4.44. The normal control group

showed the SOD with value of 12 IU/mg while the positive control group that was treated with

salbutamol resulted in decrease in SOD level (5 IU/mg). Reductions in myocardial SOD activity

strongly suggested the overwhelming superoxide radical generation and hydrogen peroxide

formation following catecholamine administration (Rathore et al., 2000).

The first group of rats treated with T. arjuna prior to salbutamol intoxication showed the

increase in maintenance of SOD with increase in its concentrations. However the concentration

of 200 mg/kg b. wt. of T. arjuna showed good maintenance of SOD level (10 IU/mg). The rats

treated with concentration of 170 mg/kg also showed maintenance in the level of SOD (9 IU/mg)

almost similar to the concentration of 200 mg/kg b.wt. Hence the concentration of 170 mg/kg

b.wt. of T. arjuna could be recommended as effective dose to kept the level of SOD within

normal range.

In second group, the rats treated with concentration of 110 and 140 mg/kg b. wt. of C.

oxyacantha did not show the maintenance of SOD level (5 and 6 IU/mg) and these

concentrations were unable to cope the complications related to salbutamol intoxication. Further

117

increased in concentration up to 200 mg/kg b.wt. showed the effective maintenance of SOD (11

IU/mg). The increase in endogenous antioxidants contributes to hawthorn’s cardioprotective

effect during MI and this property is primarily correlated with the oligomeric procyanidins

(Verma et al., 2007).

The third group pretreated with different concentrations of P. nigrum demonstrated the

dose dependant response as the antioxidative power increased with increase in concentration.

The concentration of 200 mg/kg showed the good maintenance of SOD level (10 IU/mg) and

could be considered as the dose of choice. Ahmad et al. (2012) reported the prevention of

oxidative stress by arresting free radicals and maintaining the level of SOD. The ability of P.

nigrum to maintain the level of antioxidant may be due to presence of flavonoids and phenolic

contents present in it.

The group of rats treated with C. sativum also showed dose dependant response against

oxidative stress induced by salbutamol. The rats treated with concentration of 200 mg/kg showed

effective maintenance of SOD with value of 10 IU/mg. In case of the group of rats treated with

different concentrations of A. sativum, the concentration of 80 and 110 mg/kg showed the equal

maintenance of SOD level (8 IU/mg) against salbutamol induced oxidative stress. However the

concentration of 200 mg/kg b. wt. depicted the considerable maintenance of SOD level (11

IU/mg). The group of rats treated with varying concentrations of R. serpentina also showed dose

dependant response. The rats pretreated with concentration of 110 and 140 mg/kg sustained the

SOD at the level of 7 IU/mg. While the further increase in concentration up to 200 mg/kg

b.wt.resulted in increase in the maintenance of SOD. This may be due to the presence of

therapeutic phytochemicals.

118


SOD



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

SO

D

4.2

6.15

8.1

10.05

12

Fig. 4.44 Graphical representation of dose optimization of medicinal plants for superoxide

dismutase

The rats pretreated with different concentrations of E. cardamom depicted the dose

dependant response. The maintenance of SOD level increased with increase in concentration

from 80 to 110 mg/kg but the level of SOD decreased with further increase in concentration up

to 140 mg/kg. The rats pretreated with concentration of 200 mg/kg prominently maintained the

level of SOD (12 IU/mg).

The ANOVA depicted the F-value 12.97 which implied the significance of cubic model

(Table. 4.21). The linear terms of concentration (A) and plant (B) are also significant which

showed that both have important impact on the level of SOD. In addition, the coupling term AB

is significant which indicated a positive interaction between the two variables including

concentrations and plants. This suggested that the predicted cubic equation defined well the real

behavior of the interaction. In addition, the closer the adjusted R2 value (0.8397) to the R2

(0.9098) depicted the significance of model (Hismath et al., 2011). It has also proved that the

cubic model is the best model as it showed the characteristic of a good model.

119

Table. 4.21 Analysis of variance (ANOVA) for response surface methodology of SOD (IU/mg) as a function of independent variablesSource SS Df MS F Value Prob > FModel 161.02 21 7.67 12.97 < 0.0001 Significant A-Conc. 120.07 1 120.07 203.13 < 0.0001 B-Plant 13.27 6 2.21 3.74 0.0077 AB 6.76 6 1.13 1.91 0.1162A2 9.62 1 9.62 16.27 0.0004Residual 15.96 27 0.59Lack of Fit 12.46 13 0.96 3.83 0.0090Pure Error 3.50 14 0.25Cor Total 176.98 48R-Squared 0.9098 Standard Deviation 0.77Adj R-Squared

0.8397 Mean 8.02

Pred R-Squared


Adeq Precision

11.951 Prediction Error Sum of Squares (PRESS) 50.90

4.6.1.1.2.2 Glutathione peroxidase:

GPX is implicated in cellular defence against xenobiotics and not only protects cell

membrane from oxidative damage, but also help to maintain the sulphydryl groups of many

proteins in reduced form, required for their normal function (Saiprasanna et al., 2012).

The normal control group of rats showed the 1.44 IU/mg of GPX while the group of rats

merely treated with salbutamol depicted the decrease in GPX level (0.531 IU/mg). Reduction in

myocardial GPx activities strongly suggested the overwhelming superoxide radical generation

and hydrogen peroxide formation following catecholamine administration (Rathore et al., 2000).

The rats pretreated with various concentrations of selected medicinal plants were divided into

seven groups and their response against oxidative stress was presented graphically (Fig. 4.45).

Although all the concentrations of selected medicinal plants showed dose dependant response as

the increase in concentration from 80 to 200 mg/kg of all the plants depicted increase in the

maintenance of the level of glutathione peroxidase however the concentration of 200 mg/kg

showed the prominent protection of GPX. The increase in antioxidant potential may be due to

increase in phenolic components such as flavonoids, phenolic acids and phenolic diterpenes.

120

These phenolic components possess many hydroxyl groups including O-dihydroxy group which

have very strong antioxidant power (Soni and Sosa, 2013). Among all the selected medicinal

plants, T. arjuna, P. nigrum and C. sativum showed good maintenance of GPX even at the

concentration of 80 mg/kg. T. arjuna has significant antioxidant properties and proved as a good

heart tonic (Jahan et al., 2012).


GSH



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

GS

H

0.4

0.7

1

1.3

1.6

Fig. 4.45 Graphical representation of dose optimization of medicinal plants for glutathione

peroxidase

The endogenous antioxidant enzyme level increased the synthesis of glutathione

peroxidase and attributed to free radical scavenging capacity of antioxidant polyphenols in

medicinal plants (Karthikeyan et al., 2007). Piper nigrum being a very good antioxidant is used

to treat cardiac diseases (Jahan et al., 2011). C. sativum has also been reported to increase in

catalase and Glutathione (GSH) content considerably in rabbits. This revealed the protective

antioxidant action of C. sativum on cells suffering from oxidative stress induced by free radicals

(Joshi et al., 2012).

121

The F value of 63.57 in ANOVA suggested the significance of model (Table. 4.22). The

value of R2 (0.9832) for GPX gave an idea that closer the R2 value to unity, better the empirical

model fits the actual data (Fan et al., 2007). Additionally, the accuracy and reliability of the

experiment was confirmed by the coefficient of variance (4.87 %) for GPX. The model was

deemed appropriate in this study based on the significance of model F-value, good agreement

between adjusted and predicted R2. Adequate Precision measures the signal to noise ratio should

above 4 so that it can be considered desirable. In this case, the Adequate Precision obtained was

31.061 and this indicated the significance of model.

Table. 4.22 Analysis of variance (ANOVA) for response surface methodology of GPX (IU/mg) as a function of independent variablesSource SS Df MS F Value Prob > F Model 3.23 21 0.15 63.57 < 0.0001 Significant A-Conc. 2.38 1 2.38 981.90 < 0.0001 B-Plant 0.43 6 0.072 29.72 < 0.0001 AB 0.057 6 9.542E-003 3.94 0.0059 A2 0.16 1 0.16 67.73 < 0.0001Residual 0.065 27 2.420E-003Lack of Fit 0.065 13 5.021E-003 1124.63 < 0.0001Pure Error 6.250E-005 14 4.464E-006Cor Total 3.30 48R-Squared 0.9802 Standard Deviation 0.049Adj R-Squared

0.9648 Mean 1.01

Pred R-Squared


Adeq Precision


4.6.1.1.2.3 Catalase:

Catalase, the primary free radical scavenging enzyme, is the first line of cellular defense

against oxidative injury, decomposing O2 and H2O2 before their interaction to form the more

reactive hydroxyl radical (Kumar and Gurusamy, 2014). Catalase is also responsible for

breakdown of H2O2, formed during the reaction catalyzed by SOD (Lobo et al., 2010).

The normal control group showed 34 IU/mg of catalase and the positive control group to

which the optimized dose of salbutamol was given depicted the 12 IU/mg of catalase.

Presumably, a decrease in CAT activity could be attributed to cross-linking and inactivation of

122

the enzyme protein in the lipid peroxides (Prabha et al., 2014). The preventive effect of selected

medicinal plants has been presented graphically in Fig. 4.46. Although all the plants showed

maximum protection against salbutamol induced oxidative stress but the C. sativum, R.

serpentina and C. oxyacantha showed good maintenance of catalase level even at the lower

concentration of 80 mg/kg b. wt.

The groups treated with various concentrations (80, 110, 140, 170 and 200 mg/kg b.wt.)

of T. arjuna, C. sativum and E. cardamom illustrated the dose dependent response. The increased

in concentrations resulted in increase in antioxidant activity of these selected medicinal plants.

Joshi et al. (2012) examined the antioxidant potential of C. sativum and reported the considerable

increased in catalase level in rabbits against oxidative stress induced by free radicals. The

cardioprotective potential of T. arjuna extracts in terms of preventive mode can be correlated

with polyphenolic fraction and antioxidant activity (Karthikeyan et al., 2007).

Although the group of rats pretreated with various concentrations of P. nigrum showed

the increase in the antioxidative strength with increase in concentration but the rats pretreated

with concentration of 170 and 200 mg/kg of P. nigrum showed almost equal effect on catalase

level 27 and 26 IU/mg respectively.


CAT



Plant species

80.00 110.00 140.00 170.00 200.00

Conc.

CA

T

14

20.75

27.5

34.25

41

Fig. 4.46 Graphical representation of dose optimization of medicinal plants for catalase

123

The maintenance of antioxidants level near to normal can be correlated to the free radical

scavenging potential of the medicinal plants during oxidative stress generated by myocardial

infarction (Mohanty et al., 2013; Goyal et al., 2010). Medicinal Plants are noticed to generate a

prominent effect on functional revival and improvement in the tissue defense antioxidant

network (Aslam et al., 2015).

The ANOVA indicated the model F value 29.95 for CAT which suggested the

significance of model (Table. 4.23). The accuracy and reliability of the experiment was

confirmed by the coefficient of variance (6.11) for catalase. The model was considered

appropriate in this study based on the good agreement between adjusted and predicted R2. In

addition, the closer the adjusted R2 value (0.9268) to the R2 (0.9588) showed the significance of

the model. It has also proved that the cubic model is the best model as it showed the

characteristic of a good model.

Table. 4.23 Analysis of variance (ANOVA) for response surface methodology of Catalase as a function of independent variablesSource SS Df MS F Value Prob > F Model 1669.13 21 79.48 29.95 < 0.0001 Significant A-Conc. 1104.20 1 1104.20 416.14 < 0.0001 B-Plant 417.63 6 69.61 26.23 < 0.0001 AB 66.86 6 11.14 4.20 0.0041 A2 12.87 1 12.87 4.85 0.0364Residual 71.64 27 2.65Lack of Fit 68.64 13 5.28 24.64 < 0.0001Pure Error 3.00 14 0.21Cor Total 1740.78 48R-Squared 0.9588 Standard Deviation 1.63Adj R-Squared

0.9268 Mean 26.67

Pred R-Squared


Adeq Precision


The presence of polyphenols in medicinal plants is responsible for their antioxidant

potential, neutralization of lipid free radicals and also prevents decomposition of hydroperoxides

into free radicals. The cardioprotective potential may also be due to scavenging of highly

124

oxidized metabolites produced by salbutamol and stabilization of heart membrane by herbal

medicines with a consequent decrease in the leakage of cardiac markers.

4.6.1.1.3 Hematological analysis:

The hematological analysis of various groups of rats treated with different concentrations

of T. arjuna, C. oxyacantha, C. sativum, P. nigrum, E. cardamom, A. sativum and R. serpentina

against salbutamol induced MI was executed (Table. 4.24). The higher concentration 200 mg/kg

b.wt. of T. arjuna, P. nigrum and R. serpentina depicted the significant decreased in the count of

RBC along with Hb contents. However, no effect could be observed on PCV, MCH, MCHC and

platelet count also remained unaffected. All other concentrations did not showed any negative

effect on hematological parameters. While the group that merely treated with salbutamol showed

significant changes in hematological parameters.

Table. 4.24 Hematological analysis of different groups of rats treated with various

concentrations of selected medicinal plants

Runs

Plplant ant Cco WBCs (×10^3/uL)

LYM(×10^3/uL)

GRA(×10^3/uL)

RBC×10^12/L

HGB g/dl

MCHC g/dl

MCV fl

PLT×10^3

Control 9.84 5.35 1.74 6.72 13.7 32.04 68.78 1144

Positive control

2.92 3.54 1.84 3.21 6.54 22.98 79.83 845

1 T. arjuna 80 10.47 7.70 0.42 5.22 11.0 33.27 63.36 1038 2 T. arjuna 80 9.63 7.38 0.37 6.49 13.5 31.16 66.71 1219

3 T. arjuna 110 8.43 6.79 0.20 4.55 10.22 35.17 63.78 1249 4 T. arjuna 170 6.86 4.54 0.69 8.34 17.1 30.67 66.83 1222

5 T. arjuna 200 9.75 5.40 0.20 3.79 7.5 31.82 62.22 988 6 T. arjuna 200 9.9 8.86 0.61 3.88 7.3 31.39 66.21 997 7 T. arjuna 140 10.86 6.68 1.32 6.20 12.4 31.68 64.23 14428 C. oxyacantha 80 11.84 7.35 1.04 8.72 15.7 34.04 65.85 1244

9 C. oxyacantha 80 10.45 5.45 1.25 7.45 12.4 32.28 63.43 1398

10 C. oxyacantha 110 10.43 8.35 1.81 7.92 15.3 33.57 64.64 1352

11 C. oxyacantha 170 11.04 6.87 1.76 7.21 13.5 32.09 63.81 1265

12 C. oxyacantha 200 8.90 7.37 1.86 7.59 14.4 31.07 67.44 899

13 C. oxyacantha 200 8.99 7.65 1.67 6.1 10.3 32.04 63.12 863

14 C. oxyacantha 140 9.26 8.23 1.56 8.21 13.9 32.76 66.35 1344

125

15 P. nigrum 80 9.88 5.35 1.73 6.62 13.1 30.04 65.85 1122

16 P. nigrum 80 10.09 6.65 0.49 5.91 9.8 32.84 60.81 869

17 P. nigrum 110 10.43 6.15 1.81 7.35 14.3 30.57 63.64 1242

18 P. nigrum 170 15.68 6.23 0.93 5.16 10.6 34.01 60.46 997

19 P. nigrum 200 10.80 3.27 1.86 4.19 10.4 31.07 64.44 898

20 P. nigrum 200 12.17 3.16 0.61 4.68 9.6 32.88 62.41 895

21 P. nigrum 140 15.25 6.32 0.78 5.69 10.12 33.47 61.42 1029

22 C. Sativum 80 10.41 4.03 2.78 5.31 12.9 32.42 74.94 1142

23 C. Sativum 80 13.57 3.92 0.68 4.46 10.01 32.64 70.73 1145

24 C. Sativum 110 10.47 6.42 129 7.17 14.3 30.62 65.13 1229

25 C. Sativum 170 13.42 7.29 1.85 5.64 12.0 34.47 61.71 1035

26 C. Sativum 200 11.57 3.87 1.15 7.65 10.9 31.32 66.32 900

27 C. Sativum 200 12.13 2.41 1.06 7.71 9.4 31.13 64.10 911

28 C. Sativum 140 7.21 5.23 0.98 6.67 11.23 32.65 62.81 1327

29 A. sativum 80 10.84 6.35 1.14 7.72 14.7 33.04 64.85 1244

30 A. sativum 80 11.04 7.12 1.29 7.87 10.1 32.84 62.81 1201

31 A. sativum 110 11.43 7.35 1.21 7.42 14.3 31.57 62.64 1352

32 A. sativum 170 12.08 8.02 1.09 7.51 15.12 32.81 63.29 1269 33 A. sativum 200 11.80 6.77 1.36 5.59 5.4 32.07 64.44 799

34 A. sativum 200 12.88 7.88 1.28 5.34 5.98 31.07 66.23 798

35 A. sativum 140 10.26 8.21 0.95 7.98 15.21 32.90 66.12 1193

36 R. serpentina 80 12.92 7.15 1.5 7.70 15 30.84 63.17 1256

37 R. serpentina 80 7.8 7.33 6.37 5.51 12.5 31.83 61.66 1119

38 R. serpentina 110 13.50 7.04 2.65 7.18 14.1 30.7o 64.40 1128

39 R. serpentina 170 13.62 6.22 2.37 6.39 12.4 31.78 61.11 1103

40 R. serpentina 200 3.27 3.22 1.16 1.42 6.7 21.58 66.43 879

41 R. serpentina 200 4.70 4.78 1.63 1.52 6.4 33.90 61.35 836 42 R. serpentina 140 10.29 4.58 1.63 4.52 10.28 32..81 62.19 1328 43 E. cardamom 80 9.84 5.35 1.74 6.72 13.7 32.04 65.85 1144 44 E. cardamom 80 10.69 7.31 0.93 3.12 7.8 30.82 68.74 1504

45 E. cardamom 110 10.43 6.35 1.01 7.12 13.3 31.57` 64.64 1252

126

46 E. cardamom 170 10.06 7.94 0.34 5.20 10.5 32.54 62.01 1325 47 E. cardamom 200 8.80 6.27 1.86 8.19 15.4 31.07 62.44 999

48 E. cardamom 200 9.37 8.15 0.36 7.18 10.6 34.40 61.67 962

49 E. cardamom 140 11.32 8.39 1.98 6.78 10.34 32.67 62.63 1224

The assessments of haematological parameters are used to determine the extent of

decreased count of RBC that showed the suppression of erythrocytes. Reduction in RBC, Hb and

PCV is an indication of either the destruction of RBC or their decreased production, which may

lead to anemia (Adedapo et al., 2007; Onyeyilli et al., 1998). Excessive intake of variety of

medicinal plants or their active ingredients have been reported to generate hypo proliferative or

non regenerative anemia, which is a stem cell disorder distinguished by reduced bone marrow

production of all blood components in the absence of a primary disease process infiltrating the

bone marrow or suppressing haematopoiesis (Olson et al., 1984). It showed that regular intake of

extract of medicinal plants may also cause these disorders in experimental animals. It may also

compromise the principal function of white blood cells (Adedapo et al., 2007).

4.7 Phase-III:

The optimal concentration of each herbal extract obtained in Phase-II, was further used to

formulate four herbal combinations (HCs) against the extent of jeopardized myocardium. To

achieve better results for the development of therapeutic approaches, the synergestic

cardioprotective potential of herbal combinations was evaluated against surgically induced

myocardial infarction. The surgical model to induce myocardial infarction facilitates a precise

timing and location of coronary events consequently leading to more reproducible results

(Klocke et al., 2007). The MI was induced by ligating the left anterior descending coronary

artery (LADCA) in dogs. The dogs were selected as experimental animal due to the relevance of

large animal models to human physiological and pathophysiological processes which is very

important (Lukacs et al., 2012).

4.7.1 Herbal combination therapy:

It is usually expected that the medicinal plants when used in combination, impart

additional therapeutic benefits due to the interaction among various active ingredients (Che et

al., 2013). Although the therapeutic effects of various herbal combinations have been

documented but still there is need to explore the synergestic potential of medicinal plants.

127

Different herbal combination of Rauvolfia serpentina, Terminalia arjuna, Coriandrum sativum,

Piper nigrum, Eletaria cardamom, Allium sativum and Crataegus oxyacantha were formulated

by using their optimized concentrations and with consultation of Complementry and alternative

medical practitionars with varying ratios (Table. 4.25).

Table. 4.25 Formulation of different herbal combinations of selected medicinal plants

HC R.serpenti

na

E.cardamo

m

P.nigru

m

A.sativum T.arjun

a

C.oxyacant

ha

C.

sativum

Herbal ratio

HC

1

1 0.5 1 0.5 - - 0.5

HC

2

1 0.5 1 0.25 - 1 0.5

HC

3

1 1 0.5 - 1 - 0.5

HC

4

0.5 - - 0.5 1 0.5 1

4.7.2 Effect of herbal combinations on cardiac markers:

The dogs were administered with said herbal combinations for three weeks before

ligating left anterior descending coronory artery. To investigate whether the combinations of

herbal extracts under investigation would offer any added advantage over individual herbal

treatment, the effects of HCs were compared with normal and the surgically induced MI group.

The potential of herbal combinations was evaluated by analysing the cardiac markers (CK-MB,

SGOT and LDH) as described under following headings.

4.7.2.1 CK-MB:

CK-MB is restricted primarily in the heart and considered a valuable diagnostic tool for

myocardial infarction. The damage to myocardium during MI would result in increase in the CK-

MB level in serum (Loh et al., 2007). The effect of different herbal combinations on CK-MB

level against surgically induced MI has been presented graphically in Fig. 4.47. The normal

control group showed the CK-MB level 173±3.51 IU/L throughout the experimental period.

There was considerabrle increase in CK-MB level (274±2.08 IU/L) in surgically induced MI

128

group after 12 hr of left anterior descending coronary artery ligation while the level of enzyme

was further raised up to 294.3±1.53 IU/L after 24 hr. This prominent increase in level of CK-MB

in surgically induced MI group might be due to enhanced susceptibility of damage to myocardial

cell membrane because of ligation (Beaulah et al., 2014). Alterations in integrity, fluidity and

permeability of myocardial membrane have been believed to be a reason for the leakage of CK-

MB (Ojha et al., 2012).

The first herbal combination (HC1) did not show any considerable maintenance in CK-

MB level (253±1 and 224 ±3 IU/L) after 12 and 24 hr of ligation as compared to normal control

group. In comparision of HC1, the group pretreated with HC2 showed better maintenance of CK-

MB level, 215±2 and 201±2.65 IU/L after 12 and 24 hr of ligation respectively. This

improvement in efficacy of HC2 might be due to the addition of C. oxyacantha that possessed

more cardioprotective polyphenols (Verma et al., 2007). The antioxidant and lipid neutralizing

potential of polyphenols in combination may synergise the therapeutic effect against myocardial

infarction (Li et al., 2009; Rohman et al., 2010). The group treated with HC3 showed the level of

CK-MB 241±2 IU/L after 12 hr and 203±1 IU/L after 24 hr of ligation. A decrease in CK-MB

level (213±1.73 IU/L) was observed in group pretreated with HC4 after 12 hr of ligating left

anterior descending coronary artery. After 24 hr of ligation, this group showed considerable

decline in the level of CK-MB with value of 192±1.53 IU/L that was very close to the control

group. The prior administration of HC4 depicted the better maintenance of the serum CK-MB as

compared to other herbal combinations. The phytoconstituents and antioxidants present in this

herbal combination might be responsible for the cardioprotection (Aslam et al., 2015). The HC4

may render the myocytes less leaky by preventing myocardial membrane destruction and

disorganization (Aslam et al., 2015).

129

Treatment

Time_hr

350

300

250

200

150

100

50

0

CK-M

B le

vel (

IU/L

)

HC1HC2HC3HC4Normal controlSurg

Treatment

24

82

96

27

61

792

00

22

42

53

174

.667

18

32

03

24

11

66

169

20

121

51

71

17

21

922

13

168

17

31

73

173

174

Fig. 4.47 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on CK-MB level (IU/L) in the serum of experimental groups through the preventive mode of treatment4.7.2.2 SGOT:

The increase in level of SGOT usually results in hemolytic anemia, myocardial infarction

and cholestatic disease of the liver and may be a useful tool in assessing the extent of damage

(Gupta, 2004; Javanmardi et al., 2003).

The effect of different herbal combinations on the level of SGOT has been presented in

Fig. 4.48. In normal control group the SGOT level was 43±2 and 46±1.05 IU/L with time

intervals of 12 and 24 hr respectively. The SGOT level raised up to 115±1.527 IU/L and

130

123±1.154 IU/L after the corresponding time intervals of 12 and 24 hr of LADCA ligation in

surgically control group. The ligation of coronary artery imparts an additional workload on the

remaining viable myocytes that may be unbearable, resulting in pathological stimuli and death

signals. The treatment with HCs might salvage these viable myocytes, which are at risk of injury,

thus prevent cell loss induced by necrosis (Mohanty et al., 2013). The HC1 showed the SGOT

level with value of 94±1.53 IU/L after 12 hr and 74±1 IU/L after 24 hr of ligation. The

pretreatment of HC2 showed the SGOT level 75±1 IU/L after 12 hr of surgery and considerably

maintained at level of 62±0.57 IU/L after 24 hr of ligation in LADCA as compared to surgical

control group (123±1.154 IU/L). The preventive treatment of HC3 after 12 and 24 hr of LADCA

ligation indicated the corresponding 84±1.53 and 73±1.53 IU/L level of SGOT (Fig. 4.48). There

was no considerable variation in the outcomes of HC1 and HC3. However the pretreatment of

HC4 showed maximum potential against myocardial infarction as it uphold the SGOT level 73±1

IU/L after 12 hr and 53±1.53 IU/L after 24 hr of LADCA ligation. The HC4 gave better results

and modulated the SGOT level near to normal control, suggesting the beneficial action of HC4

as compared to other groups. Pretreatment of HC4 improved the status of antioxidants that

further contributes to its cardioprotective property (Orhue and Nawanze, 2004; Rafatullah et al.,

2008; Mahaswari et al., 2008). The cardioprotective potential of herbal combinations is because

of the correlation of the total contents of phytoconstituents and biological activities (Sapna et al.,

2007).

The depletion of cardiomyocytes specific enzymes CK-MB, SGOT and LDH in ischemic

reperfusion control group revealed the injury of heart. However treatment with I. racemosa

significantly restored the myocardial antioxidant status and prevented the leakage of

cardiomyocytes specific enzymes (Ojha et al., 2010).

131

Tre atme nt

Time_hr

140

120

100

80

60

40

20

0

SG

OT lev

el (IU

/L)


Treatment

13

91

23

11

545

687

494

45

677

384

44.3

33

3

566

275

444653

73

4546

44

44

43

Fig. 4.48 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on SGOT level (IU/L) in the serum of experimental groups through the preventive mode of treatment

4.7.2.3 LDH:

LDH has been used as traditional diagnostic tool for myocardial infarction and usually

raised within 6-12 hours of MI (Prabha et al., 2014). The preventive treatment of herbal

combinations against surgically induced MI on the level of LDH has been presented graphically

in Fig. 4.49. The serum analysis of normal control group revealed 223±1.15 to 235 IU/L of LDH

132

from 0 to 48 hr respectively. The LDH level in surgically induced MI group was considerably

higher (565.3±2.31 IU/L) as compared to normal control group. The group of dogs pretreated

with HC1 showed 382.33±1.53 IU/L of LDH after 12 hr and 283±1.15 IU/L after 24 hr of

ligation. In dogs treated with HC2, the LDH level was 291.67±1.15 IU/L and 264±2.08 IU/L at

corresponding time intervals of 12 and 24 hr after LADCA ligation. While the pretreatment of

HC3 showed 343±1.53 IU/L level of LDH after 12 hr and maintained at the level of 250±1 IU/L

after 24 hr of ligation. The preventive treatment of HC4 revealed considerable maintenance of

LDH level (285±2 IU/L) after 12 hr of ligation. The HC4 sustained the level of LDH up to

264±1.52 IU/L after 24 hr of ligating LADCA (Fig. 4.49).

Treatment

Time_hr

600

500

400

300

200

100

0

LD

H le

vel (

IU/L

)


Treatment

328

405

561

23427

728

338

222

7245

250

343

226.

667

24226

4.66

729

122

6

234.

333

26428

522

6

235

227

225

223

Fig. 4.49 Graphical representation of Cardioprotective effect of herbal combinations of plant extracts on LDH level (IU/L) in the serum of experimental groups through the preventive mode of treatment

133

The HC2 and HC4 showed the prominent cardioprotective potential by maintaining the

cardiospecific markers (CK-MB, SGOT and LDH) near the normal against surgically induced

myocardial infarction. The HC3 also showed relatively better maintenance of enzyme level after

24 hr of LADCA ligation. Although the precise mechanism of the cardioprotective effects of

HCs in surgically induced myocardial injury is not fully understood but the cardioprotection by

HC4 treatment may be attributed to its favorable myocardial adaptogenic properties.

Furthermore, these herbal extracts might have the potential for the management of patients at

risk of myocardial infarction.

The increased in CK-MB, LDH and SGOT levels in surgically induced MI group as

compared to normal control group consistent with idea that development of degenerative changes

in myocardial cell membrane resulted in the leakage of myocardial enzymes from the tissue to

the plasma (Gokkusu et al., 2003). The therapeutic properties of plants have been widely

explored in the scientific researches owing to their powerful biological activities. Traditional

remedial system also documented several plants having cardioprotective properties. The

presence of flavonoids, glycosides and tannins are responsible for antioxidant, antihypertensive

and cardioprotective properties of Terminalia arjuna (Nema et al., 2012). The roots of Rauvolfia

serpentina are reported to enclose almost fifty indole alkaloids that attributed to its

cardioprotective and antihypertensive potential (Deshmukh et al., 2012). Seeds of Elettaria

cardamom are documented to use for the treatment of cardiac disorders (Verma et al., 2009). The

flowers, leaves and berries of Crataegus oxyacantha are reported to possess a diversity of

flavonoid compounds that minimize the risks of CVDs (Rasmussen, 2011). All these plants

showed sufficient therapeutic effects because of their synergetic potential and thus exhibited no

side effects (Tende et al., 2015). The results gave an idea that it is preferable to use herbal

combination instead of depending on single herb (Prince et al., 2008; Baber et al., 2012;

Rajalakshmy et al., 2011).

The herbal combinations (HC2 and HC4) considerably ameliorated cardiotoxicity by

keeping the levels of biochemical parameters towards the normal. Phytoconstituents and

antioxidants present in these herbal combinations might be responsible for their cardioprotection

against surgically induced myocardial necrosis, thus it can be optimally used in herbal

preparations to form novel herbal drugs for the treatment of cardiovascular diseases. In case of

HC4 the bioactive compounds present in different plants exert synergistic biofunctionalities in

134

combination rather than acting alone. This membrane stabilizing effect of plant mixture on the

myocardium secured the cardiac injury, and thus limiting the escape of these enzymes (Aslam et

al., 2015). Thus HC4 can be used as an alternative effective drug for the treatment of myocardial

infarction.

4.7.3 Effect of herbal combinations on heamodynamic variable:

4.7.3.1 The Mean arterial pressure:

Measurement of the hemodynamic variables was also incorporated into the experimental

design for better understanding and more precise information of the corelation between

biochemical and functional changes in the myocardium subjected to surgically induced damage.

The normal control group depicted the 85±6.81 mean arterial pressure (MAP) while the positive

control group showed the decline in MAP (33±4.35) after occlusion in LADCA (Fig. 4.50). The

pretreatment of HC1 tried to sustain the level of MAP up to 52±5.13. However the group treated

with HC2 and HC4 substantialy maintained the MAP 76 ±4.04 and 77± 5.13 respectively as

compared to other groups.

Similarly in the positive control group there was an abrupt increase in heart rate (HR)

(277±8.02) as compared to normal control group (186±4.04). It is well documented that a

considerable fall in MAP and increased HR indicated hemodynamic impairment and ventricular

dysfunction due to increased generation of ROS (Ojha et al., 2012). While the pretreatment of

herbal combinations to surgically induced MI group revealed considerable maintenance of HR

especially in HC4 (213±4.36) and HC2 group (224±3.61). A fall in MAP due to coronary

occlusion is expected to increase HR and myocardial contractility by activating the baroreceptor

reflex, which may subsequently result in reflex vasoconstriction and thus worsening the

imbalance, between myocardial oxygen demand and supply (Gupta et al., 2004).

Cardioprotective potential of inula racemosa in experimental model of ischemic reperfusion

injury also depicted the clear restoration of MAP and HR (Ojha et al., 2010).

4.7.3.2 Effect of herbal combinations on ventricular function:

A significant decline in Left Ventricular End Diastolic Pressure (LVEDP) (9±3.05)

marked the onset of myocardial infarction in surgically induced MI group which remained

decreased throughout the experimental period in comparison of normal control group (32±5.51)

(Fig. 4.50). The pretreatment with HC4 and HC2 effectively maintained the LVEDP level

25±2.52 and 18±1.53 respectively as compared to surgically induced ischemic group. The HC1

135

and HC3 also tried to sustain the LVEDP with corresponding values 12±4.04 and 08±1.53. The

increase in blood flow through the subendocardial region of the left ventricular muscle is the

major consequence of the reduction in LVEDP in surgically induced infarction group. Under

ischemic conditions, the disproportionate reduction in microcirculation and blood flow to the

subendocardial regions of the heart occour, which is subjected to the greatest extravascular

compression during systole. In addition, the therapeutic efficacy of HC4 against surgically

induced MI might be due to the improvement in both inotropic and lusitropic function of the

heart and considerable maintainenance of antioxidant defense capacity of the myocardium

(Mohanty et al., 2013).

Table. 4.26 Hemodynamic analysis of herbal combination against surgically induced myocardial infarction

Hemodynamic parameters

Control Surg HC1 HC2 HC3 HC4

MAP 85±6.81 33±4.35 52±5.13 76±4.04 57±4.16 77±5.13

HR 186±4.04 277±8.02 252±6.51 224±3.61 231±9.29 213±4.36

LVEDP 32±3.05 9±5.51 12±4.04 18±1.53 08±1.53 25±2.52

LVSP 120±4.58 79±5.56 91±8.33 107±3.21 102±4.04 112±2.08

The surgically induced myocardial infarction group showed the significant decrease in

left ventricular systolic pressure (LVSP) (79±5.56) as compared to normal control group

(120±4.58). The pretreatment of HC1 maintained the LVSP up to 91±8.33 while the HC2 tried to

sustain the level up to 107±3.21 (Table. 4.26). However the HC3 maintained the level of systolic

pressure 102±4.04 while the pretreatment of HC4 showed the marked restoration as compared to

other groups as it elevated the the level of LVSP (112±2.08) near to normal control group. It is

materialized that the HC4 are more potent in preventing the hemodynamic deteriorations

observed in the surgically induced MI group. The hydroalcoholic extract of Andrographis

paniculata was evaluated against MI induced by LADCA ligation. The LADCA ligation resulted

in significant cardiac dysfunction evidenced by reduced mean arterial pressure (MAP) and

increased heart rate (HR). The left ventricular contractile function was also altered (Ojha et al.,

2012).

136

Mohanty et al. (2009) also studied the efficacy of herbal combinations to limit the

myocardial injury in left anterior descending coronary artery ligation model. The animals treated

with herbal combination prior to left anterior descending coronary artery ligation maintained the

heamodynamic level near to normal as compared to positive control group. Many experimental

and clinical studies have observed that natural products could be used prophylatically in the

treatment of various disorders because of their antioxidants and adaptogenic properties (Nandave

et al., 2013).

Control Surgery HC1 HC2 HC3 HC40

50

100

150

200

250

300

MAPHRLVEDPLVSP

Fig. 4.50 Graphical representation of hemodynamic parameters of various groups treated

with different herbal combinations

137

The combination of herbal extracts significantly ameliorated the MI compromised

enzymatic status and hemodynamic alterations as compared to control ischemic group. The HCs

decreased the severity of pathological changes and significantly preserved the myocardial

markers confirming its salvaging effects (Tende et al., 2015).

4.7.4 Histopathological examination:

The histopathological findings of myocardial tissue in normal control group illustrated

clear integrity of the myocardial cell membrane. The myofibrillar structure was normal with no

inflammatory cell infiltration. The nuclei were also normal without any pyknotic changes (Fig.

4.51). The histopathological examination of surgically induced MI group showed extensive

myofibrillar degeneration related to infiltration and disruption of cardiac myofibers. There was

marked necrosis in the ventricular region. Pyknotic changes in nuclei were also observed (Fig.

4.52).

Fig. 4.51 The histopathological representation of cardiac tissue of normal control group

138

Fig. 4.52 The histopathological representation of cardiac tissue surgically induced MI control group

The treatment of HC1 prior to ligation showed myofibrilation (Fig. 4.53) while the

pretreatment with HC2 demonstrated marked improvement in surgically induced alterations but

there was cellular infilteration at few places. The nuclei were also normal (Fig. 4.54). The group

treated with HC3 did not protect the cardiac dysfunctions as compared to other groups.

Myocardial fibrillation as well as some pyknotic changes in nuclei were also seen in group

treated with HC3 (Fig. 4.55). The histopathological examination of group treated with HC4

showed that there was no myofibrilation and the cardiac parenchyma was also normal. This

confirmed the potential of herbal combination (HC4) over oxidative stress related to cardiac

ailment (Fig. 4.56).

139

Fig. 4.53 The histopathological representation of cardiac tissue of HC1 treated group


140



141

The cardioprotective and histopathological examination of animals treated with herbal

mixture (Terminalia arjuna, Rauvolfia serpentina, Elettaria cardamom and Crataegus

oxyacantha) considerably ameliorated the cardiotoxicity by bringing back the cardiospecific

enzymes towards the normal (Aslam et al., 2015). Therefore it can be optimally used in herbal

preparations for the treatment of cardiovascular diseases with no side effects. Histopathological

examination of heart proved the cardioprotective potential of herbal combination that was in line

with many previous studies (Hina et al., 2010; Fathiazad et al., 2012; Mohanty et al., 2013;

Sahreen et al., 2011; Yousefi et al., 2014).

Conclusively the HC4 prepared by optimized medicinal plants showed better synergistic

cardioprotective potential. The bioactive compounds present in medicinal plants exert synergistic

biofunctionalities in combination by interacting with one another, rather than acting alone. HC4

can be used as an alternative of synthetic drugs for the treatment and management of

cardiovascular diseases as it considerably maintained the cardiac markers as well as

hemodynamic parameters against surgically induced MI because of its synergistic

cardioprotective potential. In view of the safety, efficacy and traditional acceptability of these

plants extract, well controlled clinical trials should be contemplated to establish the efficacy of

herbal combination in the treatment of myocardial infarction.

142

Chapter # 5 SUMMARY

Myocardial infarction (MI) and the resulting complications in cardiac functions are

leading cause of morbidity and mortality in developed countries. MI is a complex phenomenon

affecting the mechanical, electrical, structural and biochemical properties of the cardiac system

(Ramadoss et al., 2012). The research was planned in two parts including in vitro and in vivo

analysis. In vitro analysis involved the screening of selected medicinal plants by Angiotensin

Converting Enzyme inhibition assay. Out of all selected medicinal plants, the methanolic extracts

of Terminalia arjuna (56.23±3.427), Piper nigrum (63.03±0.153), Coriandrum sativum

(63.033±0.153), Allium sativum (52.9±2.621), Rauvolfia serpentina (63.467±3.198), Eletaria

cardamom (53.467±0.55) and Crataegus oxyacantha (64.267±0.2) showed maximum ACE

inhibition. These selected medicinal plants were further subjected to LC-MS analysis which

confirmed the presence of important phytoconstituents and phenolic acids responsible for

antioxidative and cardioprotective potential of these plants. The antioxidant execution of these

selected medicinal plants at different concentrations was performed by DPPH and DNA

protection assay. The plants showed the antioxidant potential with descending order Terminalia

arjun> Crataegus oxycantha> Allium sativum> Coriandrum sativum > Piper nigrum>

Rauvolfia serpentine> Eletaria cardamom. Moreover all the said plants showed the dose

dependant response for free radical scavenging potential. This may be due to increase in phenolic

components such as flavonoids, phenolic acids and phenolic diterpenes. These phenolic

components possess many hydroxyl groups including o-dihydroxy group which have very strong

antioxidant power (Soni and Sosa, 2013). The toxicity assay was also performed to get relatively

safe product. The cytotoxicity findings showed that all the selected medicinal plants are safe and

may be used as herbal medicine for various therapeutic purposes. In vivo analysis was conducted

in three phases. The phase-I included the preliminary trial, where the different concentrations of

salbutamol were optimized by using the Response Surface Methodology. The analysis revealed

that the concentration of 80 mg/kg b.wt of salbutamol considerably elevated the level of cardiac

markers including CK-MB, SGOT and LDH. This elevated cardiac markers indicated the onset

of myocardial infarction. In phase-II, various concentrations (80, 110, 140, 170 and 200 mg/kg

b.wt) of selected medicinal plants suggested by RSM were optimized against salbutamol induced

myocardial infarction. The cardiac markers and lipid profile were analyzed to get the optimal

143

concentration of each medicinal plant that could provide maximum protection against MI.

Among all the studied medicinal plants T. arjuna, C. oxyacantha and C. sativum showed good

cardioprotective potential even at their least concentration. In Phase-III, the optimized doses of

selected medicinal plants were used to formulate four different herbal combinations with

appropriate ratio. These herbal combinations were further evaluated for their cardioprotective

potential against MI induced by ligating left anterior descending coronary artery in dogs. The

pretreatment of HC4 including R.serpentina, A.sativum, T.arjuna, C.oxyacantha, C. sativum with

corresponding ratio of 0.5:0.5:1:0.5:1 sustained the cardiac marker enzymes (CK-MB, SGOT

and LDH) near to normal. The HC4 also maintained the hemodynamic parameters (MAP, HR,

LVEDP) against surgically induced MI. The medicinal plants in herbal combination (HC4) with

appropriate ratio showed better synergistic cardioprotective potential by interacting with one

another, rather than acting alone. The combination of herbal extracts significantly ameliorated

the MI compromised cardiac markers because of its bioactive contents.

144

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