Studies on Antihyperlipidemic and Vasomodulating Effects

Studies on Antihyperlipidemic and

Vasomodulating Effects of Eruca sativa, Erucin,

Hedera helix and Hederacoside C

By

Umme Salma

CIIT/SP13-R60-008/ATD

PhD Thesis

In

Pharmacy

COMSATS University Islamabad

Abbottabad Campus - Pakistan

Spring, 2018

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COMSATS University Islamabad




A Thesis Presented to

COMSATS University Islamabad, Abbottabad Campus

In partial fulfillment

of the requirement for the degree of

PhD (Pharmacy)

By

Umme Salma


Spring, 2018

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A Post Graduate Thesis submitted to the Department of Pharmacy as partial

fulfillment of the requirement for the award of Degree of Ph.D in Pharmacy.

Name Registration Number

Umme Salma CIIT/SP13-R60-008/ATD

Supervisor

Dr. Abdul Jabbar Shah

Associate Professor

Department of Pharmacy

COMSATS University Islamabad,

Abbottabad Campus

Co-Supervisor

Dr. Taous Khan

Professor

Department of Pharmacy

COMSATS University Islamabad,

Abbottabad Campus

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DEDICATION

Dedicated to my beloved parents whose prayers have

encouraged me throughout my life. To my siblings who

have always looked up to me and gave me courage in

each and every step of my life which made me more

dedicated to work hard and fulfill this wonderful journey

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ACKNOWLEDGEMENTS

All praise and glory to Almighty Allah (SWT), the beneficent, the most

compassionate and the most merciful and to his messenger, the Holy Prophet

Muhammad (SAW) the greatest educator and the perfect example to mankind.

My sincere and deepest appreciation goes to my supervisor Associate Professor Dr.

Abdul Jabbar Shah (HoD), Department of Pharmacy, CUI, Abbottabad Campus,

without his help, comments and critique this research would not have been possible.

To my Co-supervisor Professor Dr. Taous Khan, Chairman Department of Pharmacy,

whose expertise and involvement in my research was immensely helpful in

completing my work. It was my greatest privilege to have accomplished my work

under their keen supervision.

I would also like to thank Dr. Nisar-Ur-Rehman (Ex-Chairman), Department of

Pharmacy, CUI, Abbottabad Campus and all the faculty members for their support

and guidance. I owe special thanks to Dr. Fiaz Ahmad (Ayub Medical College

Abbottabad) for his help. I am thankful to members of the cardiovascular research

group (Rahila Qayyum, Misbah-Ud-Din Qamar, Shamim Khan, Mubeen Kousar) and

my lab fellows (Kahif Bashir, Anam Saeed, Zainab, Hafiz Majid) who helped me

throughout the agonizing process of research.

I take this opportunity to pay my deepest and dearest appreciation to my whole

family, my parents who have always prayed for my success and have always taught

me to do my best. I also acknowledge my siblings (Hafsa, Asma, Ayesha, Sidra,

Salman and Nouman) who were always a source of inspiration and their prayers

encouraged me to be immensely dedicated to fulfill my work.

Umme Salma


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ABSTRACT

Studies on Antihyperlipidemic and Vasomodulating Effects of

Eruca sativa, Erucin, Hedera helix and Hederacoside C

Aerial parts of Eruca sativa (E. sativa) and leaves of Hedera helix (H. helix) are

popular remedies for the treatment of cardiovascular diseases in humans. Erucin and

hederacoside C (HDC) are the important constituents of E. sativa and H. helix,

respectively. Literature lacks pharmacological investigation on these plants and the

constituents in hyperlipidemia and hypertension. This study aimed to investigate the

E. sativa and H. helix, erucin and HDC effect in hyperlipidemia, hyperlipidemia-

induced vascular dysfunction and hypertension. Crude extracts of both plants (30, 100

and 300 mg/kg), erucin (1 and 3 mg/kg) and HDC (2.5 and 5 mg/kg) were tested in

tyloxapol and high fat diet (HFD)-induced hyperlipidemic Sprague-Dawley (SD) rats.

Biochemical evaluation of lipid profile was carried out on blood collected from all

groups. Histopathological and vascular dysfunction studies were performed on aortae

isolated from normal, hyperlipidemic and treated rats. The antihypertensive effect was

investigated in both normotensive and hypertensive rats. The mean arterial pressure

(MAP) was measured in both groups. The mechanisms were investigated using

isolated rat aorta and atria. Both extracts and compounds significantly reduced total

cholesterol and triglycerides (p < 0.001), compared to lovastatin in tyloxapol-induced

hyperlipidemia. In high fat diet-induced hyperlipidemia, both extracts significantly (p

< 0.001) reduced TC, LDL and increased (p < 0.05) HDL levels at higher dose.

Erucin and HDC also significantly (p < 0.001) decreased TC and LDL levels. Extract

of H. helix was more potent (p < 0.001) in decreasing the atherogenic index in both

hyperlipidemic models, compared to E. sativa. The data thus shows that extracts of

both plants and compounds are antihyperlipidemic agents. Further in-vitro studies

were carried out to explore the role of these agents on vascular endothelium

disruption (dysfunction). The thoracic aortae from HFD rats were used for

histopathological and vascular reactivity studies. Extract of E. sativa reversed

endothelial dysfunction in HFD-induced hyperlipidemic rats in-vitro by inhibiting

macrophages infiltration and reducing endothelial disruption. Extract of H. helix

markedly preserved endothelial dysfunction by improving the architecture of vascular

wall. Both compounds also improved endothelial disruption. The vascular dysfunction

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study in the aortic rings from hyperlipidemic rats treated with both extracts and

compounds showed that acetylcholine caused complete relaxation against

phenylephrine (PE) precontractions. This indicates that extract and compounds are

effective remedies in improving disrupted vascular architecture due to hyperlipidemia.

To see effect on blood pressure, extracts, fractions and compounds were tested in

normotensive, normotensive atropinized and hypertensive rats. Extract of E. sativa

and fractions, dose-dependently decreased mean arterial pressure (MAP) that was

significantly (p < 0.001) reduced with atropine (1 mg/kg) pretreatment in

normotensive rats. Extract of E. sativa and fractions also decreased MAP in

hypertensive rats. The effects of H. helix extract on MAP in both normotensive and

hypertensive rats were greater than E. sativa. The antihypertensive effect of extract

and fractions of H. helix remained unchanged in the presence of atropine in

normotensive rats excluding the involvement of muscarinic receptors. Erucin and

HDC also induced antihypertensive effect in normotensive rats (unaffected by

atropine) and hypertensive rats in-vivo. The underlying mechanisms of

antihypertensive effect of extracts and compounds were further investigated in in-

vitro experiments in rat aorta and atria. In rat aorta, extract and fractions of E. sativa

produced vasorelaxant effect that was partially inhibited with L-NAME and atropine

pretreatment indicating role of muscarinic receptor-linked nitric oxide (NO) pathway.

This effect of extract and fractions was also partially eliminated with denudation of

endothelium and aortic rings from hypertensive rats, also suggesting role of vascular

endothelium. The vasorelaxant effect of n-hexane fraction was least, indicating that it

might be due to presence of vasocontractile constituents, which may have role in

vasomodulation. Erucin also produced incomplete relaxation in normotensive rat

aorta, suggesting that it may be one of the constituents involved in vasomodulation.

The vasorelaxant effect of H. helix and HDC was inhibited with L-NAME

pretreatment and denudation but did not change with atropine pretreatment excluding

role of muscarinic receptors. The extracts of both plants, erucin and HDC produced

vasorelaxant effect against high K+ precontractions like verapamil. Extract of E.

sativa and H. helix, fractions and compounds suppressed PE peak formation; erucin

was less potent than HDC. In isolated at atrial strips, E. sativa and erucin induced

negative inotropic and chronotropic effects with a positive inotropic effect by the n-

hexane fraction, which was not affected by atropine pretreatment, suggesting that

cardiac muscarinic receptors are not involved. The extract, fractions of H. helix and

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HDC caused depression of force and rate of atrial contraction which remained

unchanged in the presence of atropine. To have possible chemical profile of the

extract, spectrophotometric and HPLC analysis were carried out that showed the

presence of quercetin and erucin in crude extract of E. sativa and HDC in H. helix.

According to acute toxicity test, crude extract of E. sativa and H. helix were safe at 3

and 5 g/kg, respectively. In conclusion, the findings of present study indicated that E.

sativa and H. helix are effective antihyperlipidemic and antihypertensive remedies.

Both extracts and important constituent’s erucin and hederacoside C significantly

reduced TC and LDL and preserved the endothelial disruption evident by

histopathological and vascular dysfunction studies in-vitro. The preservation of

endothelial dysfunction is due to decrease in LDL. The antihypertensive effect of E.

sativa and H. helix extracts is possibly due to vasodilatory and cardiac effects. The

endothelium-independent mechanisms involved inhibitory effect on calcium influx

and release. Endothelium-dependent mechanisms involved muscarinic receptor linked

NO mediated pathway. Erucin acted through endothelium-independent mechanism

mediated by calcium antagonism. E. sativa and erucin showed negative inotropic and

chronotropic effects, possible due to calcium channel blockade. Antihypertensive

effect of H. helix extract and HDC are mediated through NO release inhibiting

calcium release from stores and entry via VDCs also decrease cardiac rate and force

of contractions. This data provide pharmacological base to medicinal use of E. sativa

and H. helix in hyperlipidemia and hypertension. The presence of erucin in E. sativa

and HDC in H. helix further support the findings and this study identified erucin and

HDC as important constituents for the management of hyperlipidemia and

hypertension.

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TABLE OF CONTENTS

1 Introduction ................................................................................................. 1

1.1 Hyperlipidemia .................................................................................... 2

1.2 Hyperlipidemia and importance of regulation of cholesterol ............. 2

1.2.1 Metabolism of cholesterol............................................................. 2

1.2.2 Metabolism of chylomicron and very low density lipoprotein .... 3

1.2.3 Metabolism of low density lipoprotein ........................................ 3

1.2.4 Metabolism of high density lipoprotein ....................................... 3

1.2.5 Atherosclerotic plaque formation due to elevation of LDL ......... 5

1.3 Relation of cholesterol to endothelial dysfunction in hyperlipidemia..7

1.4 Hyperlipidemia and hypertension ....................................................... 8

1.5 Treatment of hyperlipidemia and hypertension ................................... 8

1.6 Use of medicinal plants in cardiovascular diseases ............................ 9

1.7 The medicinal plants and chemical constituents selected ................. 10

1.7.1 Eruca sativa Mill. ...................................................................... 10

1.7.2 Erucin ......................................................................................... 13

1.7.3 Hedera helix L. ........................................................................... 14

1.7.4 Hederacoside C (HDC) .............................................................. 17

1.8 Aims and objectives ........................................................................ 19

2 Materials and methods ............................................................................... 20

2.1 Drugs and standards ....................................................................... 21

2.2 Collection and identification of plants ........................................... 21

2.3 Crude extract ................................................................................. 21

2.4 Fractionation of crude extract ......................................................... 21

2.5 Phytochemical screening ................................................................ 22

2.5.1 Preliminary screening .............................................................. 22

2.5.2 Total phenolic content ............................................................. 22

2.5.3 Total flavonoid content ........................................................... 22

2.5.4 HPLC analysis ......................................................................... 22

2.6 Animals........................................................................................... 23

2.7 Pharmacological investigation ....................................................... 23

2.7.1 Safety study ............................................................................... 23

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2.7.2 Tyloxapol-induced model of hyperlipidemia .............................. 23

2.7.3 High fat diet (HFD)-induced model of hyperlipidemia ………..24

2.7.4 In-vivo recording and measurement of blood pressure ............... 25

2.7.5 In-vitro studies in isolated aorta from SD rats ............................ 25

2.7.6 Effect on intracellular Ca+2

stores ............................................... 26

2.7.7 Isolated right atrial preparations ................................................ 26

2.8 Statistics ........................................................................................... 26

3 Results ...................................................................................................... 27

3.1 Phytochemical screening of Eruca sativa ................................... 28

3.1.1 Preliminary screening …………………………………... .28

3.1.2 Total phenolic content ............................................................. 29

3.1.3 Total flavonoid content ........................................................... 29

3.1.4 HPLC analysis ........................................................................ 30

3.2 Pharmacological investigation of Eruca sativa .............................. 32

3.2.1 Safety study ............................................................................. 32

3.2.2 Antihyperlipidemic activities ................................................. 32

3.2.2.1 Serum lipid profile of hyperlipidemic (TI) SD rats ..... 32

3.2.2.2 Serum lipid profile of hyperlipidemic (HFD) SD rats . 39

3.2.2.3 Histopathological examination of hyperlipidemic

(HFD) rats…………………………………………………….46

3.2.2.4 Vascular function study of hyperlipidemic (HFD) SD

rats ........................................................................................... .50

3.2.3 Antihypertensive activities ....................................................... 59

3.2.3.1 Mean arterial pressure (MAP) in normotensive rats ... 59

3.2.3.2 Effect on blood pressure (MAP) in hypertensive

anaesthetized rats .................................................................... 68

3.2.3.3 Effect of erucin on MAP in normotensive and high salt

hypertensive rats ...................................................................... 73

3.2.4 Vascular reactivity studies ....................................................... 75

3.2.4.1 Eruca sativa extract and fractions effect in rat aortic

tissues of normal and hypertensive rats ................................... 75

3.2.4.2 Eruca sativa and fractions effect on intracellular Ca+2

stores ....................................................................................... 82

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3.2.4.3 Erucin effect in rat aorta from normotensive and

hypertensive rats ...................................................................... 83

3.2.5 Cardiac reactivity studies .......................................................... 87

3.2.5.1 Effect of Eruca sativa and fractions on rat right atrial

rhythmic contractions............................................................... 87

3.2.5.2 Effect of erucin on rat atrial rhythmic contractions ..... 93

3.3 Phytochemical screening of Hedera helix ................................................ 94

3.3.1 Analysis of preliminary phytochemicals .................................. 94

3.3.2 Determination of total phenolic content ................................... 95

3.3.3 Determination of total flavonoid content .................................. 95

3.3.4 HPLC analysis ........................................................................... 96

3.4 Pharmacological investigation of Hedera helix ........................................ 97

3.4.1 Safety study ................................................................................ 97

3.4.2 Antihyperlipidemic activities .................................................... 97

3.4.2.1 Serum lipid profile of hyperlipidemic (TI) SD rats` .... 97

3.4.2.2 Serum lipid profile of hyperlipidemic (HFD) SD

rats .......................................................................................... 104

3.4.2.3 Histopathological examination of hyperlipidemic (HFD)

SD rats ................................................................................... 111

3.4.2.4 Vascular function study of hyperlipidemic (HFD) SD

rats ....................................................................................... 114

3.4.3 Antihypertensive activities .................................................. 123

3.4.3.1 Mean arterial pressure (MAP) of normotensive rats 123

3.4.3.2 Hypertensive rats ..................................................... 130

3.4.3.3Hederacoside C (HDC) response on mean arterial

pressure (MAP) of normotensive and hypertensive (high salt)

rats ........................................................................................ 137

3.4.4 Vascular reactivity studies .................................................. 139

3.4.4.1Response in aortic tissues of normotensive and

hypertensive rats ................................................................... 139

3.4.4.2 Effect of Hedera helix and fractions on intracellular

Ca+2

stores ........................................................................... 145

3.4.4.3 Effect of hederacoside C (HDC) ............................. 146

3.4.5 Cardiac reactivity studies ................................................... 150

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3.4.5.1 Effect of Hedera helix and fractions on rat right atrial

rhythmic contractions............................................................. 150

3.4.5.2 Effect of hederacoside C (HDC) on rat atrial rhythmic

contractions ............................................................................ 156

4 Discussion ......................................................................................................... 157

5 Conclusion ......................................................................................................... 168

6 References ......................................................................................................... 169

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LIST OF FIGURES

Figure.1.1 Exogenous and endogenous pathways of lipid metabolism……………….4

Figure.1.2 Atherosclerotic plaque formation………………………………………….6

Figure.1.3 Structures of glucoraphanin (A), quercetin (B) and sulforaphane (C)…..12

Figure.1.4 Structure of erucin………………………………………………………..13

Figure.1.5 Structures of α-hederin (A), isoquercetin (B) and scopolin (C)…………16

Figure.1.6 Structure of hederacoside C……………………………………………...17

Figure.3.1 HPLC chromatogram of quercetin standard (A) and crude extract of Eruca

sativa (B) at wavelength of 280 nm. X-axis represents retention time (min) and y-axis

intensity (mV) (mobile phase: Solvent A (acetonitrile) and solvent B (0.03%

phosphoric acid in water). (Shim-pack C18, 150 mm × 4.6 mm I.D., 5 µm)………..31

Figure.3.2 HPLC chromatogram of erucin standard (A) and crude extract of Eruca

sativa (B) at wavelength of 365 nm. Retention time (min) is on the x-axis and

intensity (mV) on the y-axis. (mobile phase: Solvent A (1% formic acid in water) and

solvent B (methanol). (Shim-pack C18, 150 mm × 4.6 mm I.D., 5 µm).……………31

Figure.3.3 Bar diagrams (A+B) show lipid profile in normal control and tyloxapol-

induced (TI) hyperlipidemic SD rats respectively (mean±SEM, n=5-7) ***p < 0.001

vs normal control.....………………………………………………………………….34

Figure.3.4 Bar diagrams show effect of Eruca sativa (Es.Cr) at doses (mg/kg) of 30

(A), 100 (B), 300 (C) mg/kg and lovastatin (D) 10 mg/kg on total cholesterol (TC),

triglycerides (TG), low density lipoproteins (LDL) and high density lipoproteins

(HDL) in tyloxapol-induced hyperlipidemic rats. Values were expressed as mean ±

SEM (n=6-7) **p < 0.01, ***p < 0.001 vs hyperlipidemic control...… …………….35

Figure.3.5 Bar diagrams show erucin effect, at doses (mg/kg), 1 (A), 3 (B) on serum

total cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL) and high

density lipoproteins (HDL) (TC, TG, LDL, HDL) of tyloxapol-induced

hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) ***p < 0.001

vs hyperlipidemic control.……………………………………………………………36

Figure.3.6 Bar diagrams show serum atherogenic index of normal control, tyloxapol-

induced (TI) hyperlipidemic group, Eruca sativa extract (Es.Cr) and lovastatin (A),

and erucin at doses of 1, 3 mg/kg (B) and lovastatin 10 mg/kg treated tyloxapol-

xviii

induced hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) **p

< 0.01, ***p < 0.001 vs hyperlipidemic control…………………………………..…37

Figure.3.7 Bar diagrams show serum lipid profile of control (A) and high fat diet

(HFD)-induced hyperlipidemic (B) rats (mean±SEM, n=5-7) ***p < 0.001 vs normal

control.………………………………………………………………………………..41

Figure.3.8 Bar diagrams show Es.Cr effect at doses of 30 (A), 100 (B), 300 (C)

mg/kg and lovastatin (D) 10 mg/kg in high fat diet-induced hyperlipidemic SD rats on

TC, TG, LDL and HDL (mean±SEM, n=5-7) **p < 0.01, ***p < 0.001 vs

hyperlipidemic control……………………………………………………………….42

Figure.3.9 Bar diagrams show erucin 1 (A), 3 (B) mg/kg effect in the hyperlipidemic

(high fat diet) SD rats lipid profile (mean±SEM, n=5-7) ***p < 0.001 vs

hyperlipidemic control…………………………………...…………………………..43

Figure.3.10 Bar diagrams show serum atherogenic index of normal control, high fat

diet (HFD)-induced hyperlipidemic group, Eruca sativa extract (Es.Cr) and lovastatin

(A), and erucin at doses of 1, 3 mg/kg (B) and lovastatin 10 mg/kg treated HFD-

induced hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) ***p

< 0.001 vs hyperlipidemic control.…………………………………………………...44

Figure.3.11 Photomicrographs show histological examination of aortae from normal

control (A), hyperlipidemic (HFD) (B), Eruca sativa extract at 30 (C) and 100 mg/kg

(D) treated SD rats. Macrophages appear as large nucleus with vacoulated cytoplasm

while fat deposits appear as fat droplets with no distinct membrane..……………….48

Figure.3.12 Photomicrographs show histological examination of aortae from crude

extract of Eruca sativa (Es.Cr) at 300 mg/kg (E), lovastatin (10 mg/kg) (F) and erucin

at doses of 1 (G), 3 mg/kg (H) treated SD rats. Macrophages appear as large nucleus

with vacoulated cytoplasm while fat deposits appear as fat droplets with no distinct

membrane……………………………………….....…………………………………49

Figure.3.13 Tracing (A) and graph (B) show the acetylcholine response on

phenylephrine (PE; 1 µM)-induced contraction in normal control isolated rat aortic

rings (mean±SEM, n=5-7)……………………………………………………. ……..51

Figure.3.14 Tracing (A) and graph (B) show the acetylcholine response in isolated

aorta of high fat diet treated hyperlipidemic rats (mean±SEM, n=5-7). …………….52

Figure.3.15 Tracing (A) and graph (B) show acetylcholine effect against

phenylephrine (1 µM) precontraction in aorta of high fat diet (HFD) + crude extract of

Eruca sativa (Es.Cr; 30 mg/kg) treated group (mean±SEM, n=5-7)………………..53

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Figure.3.16 Tracing (A) and graph (B) show acetylcholine response against

phenylephrine (PE; 1 µM)-induced contraction in aortic rings from high fat diet

(HFD) + crude extract of Eruca sativa (Es.Cr; 100 mg/kg) treated group

(mean±SEM, n=5-7)………………………………………………………………….54

Figure.3.17 Tracing (A) and graph (B) show the acetylcholine response against

phenylephrine (1 µM) contraction in aorta of high fat diet (HFD) + crude extract of

Eruca sativa (Es.Cr; 300 mg/kg/day) treated group (mean±SEM, n=5-7)... ………..55


phenylephrine (PE; 1 µM)-induced contraction from the hyperlipidemic high fat diet

(HFD) with Lovastatin (10 mg/kg/day) treated rat aorta (mean±SEM, n=5-7)……56


phenylephrine (PE; 1 µM)-induced contraction from high fat diet (HFD) + Erucin (1

mg/kg) treated rat (mean±SEM, n=5-7).. ……………………………………………57


phenylephrine (PE; 1 µM)-induced contraction in aorta of high fat diet (HFD) +

Erucin (3 mg/kg) treated group….…………………………………………………...58

Figure.3.21 Tracings show norepinephrine (NE), acetylcholine (Ach) and Eruca

sativa (Es.Cr) response on mean arterial pressure (MAP) without (A) and with

atropine (1 mg/kg) (B) in the normotensive group under anaesthesia……………….61

Figure.3.22 Tracings show the effect of Eruca sativa n-hexane fraction (Es.n-hexane)

on mean arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B)

pretreatment in normotensive rats, under anaesthesia………………………………..62

Figure.3.23 Tracings show the Eruca sativa chloroform fraction (Es.Chlor) effect on

mean arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B) in

normotensive rats, under anaesthesia..……………….………………………...…….63

Figure.3.24 Tracings show Eruca sativa ethyl acetate fraction (Es.EtAc) effect on

mean arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B) in normal

rats……………………………………………………………………………………64

Figure.3.25 Tracings show MAP of aqueous fraction of Eruca sativa (Es.Aq) without

(A) and with atropine (1 mg/kg) (B) in normotensive rats under

anaesthesia…………………………………………………………………….. …….65

Figure.3.26 Graph (A) shows the arterial blood pressure of normotensive and

hypertensive (8% NaCl) groups. Tracing (B) shows crude extract (Es.Cr) response on

high salt-induced hypertensive rats mean arterial pressure under anaesthesia………69

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Figure.3.27 Tracings showing response of n-hexane (A) and chloroform (B) fractions

on hypertensive rats mean arterial pressure, under anaesthesia...……………………70

Figure.3.28 Tracings show mean arterial pressure of Eruca sativa ethyl acetate (A),

aqueous fraction (B) on hypertensive rats under anaesthesia………………………...71

Figure.3.29 Tracing (A) showing mean arterial pressure (MAP) of erucin in

normotensive rats and graph (B) showing the effect of erucin on MAP in normal and

hypertensive rats (mean±SEM, n=5-7). *p < 0.05, **p < 0.01 vs hypertensive. ……74

Figure.3.30 Response of acetylcholine (A), (Es.Cr) (B) response precontracted with

phenylephrine (PE; 1 µM) and L-NAME (10 µM) atropine (1 µM) treated intact and

denuded aortic tissues (mean±SEM, n=5-7). ANOVA (Two-way) Bonferroni’s post

hoc analysis. *p < 0.05, ***p < 0.001 vs control. …………………………………...76

Figure.3.31 Response of fractions of Eruca sativa in rat aorta with PE (1 µM)

precontractions and treatment of L-NAME (10 µM), atropine (1 µM) in intact and

denuded tissues (mean±SEM, n=5-7). [ANOVA (Two-way) analysis Bonferroni’s

post hoc] *p < 0.05, **p < 0.01, ***p < 0.001 vs control……………………………77

Figure.3.32 A and B show Eruca sativa crude extract, fractions and verapamil effect

to high K+ precontractions, in normal Kreb’s solution (mean±SEM, n=5-7).………78

Figure.3.33 Acetylcholine and (Es.Cr) effect precontracted with phenylephrine (1

µM) and pretreated [L-NAME (10 µM), atropine (1 µM)] in intact aortic and denuded

rings from hypertensive rats (mean±SEM, n=5-7)…………………………………...80

Figure.3.34 Eruca sativa fractions response with phenylephrine and L-NAME (10

µM), atropine (1 µM) pretreatment in aorta of high salt-induced hypertensive rats

(mean±SEM, n=5-7)………………………………………………………………….81

Figure.3.35 Tracing (A) showing crude extract effect and graph (B) represents (crude

extract and fractions) on phenylephrine (PE) responses in Ca+2

-free/EGTA medium

(mean±SEM, n=5-7)...………………………………………………………………..82

Figure.3.36 Response of erucin with phenylephrine (1 µM) precontraction and

pretreated by L-NAME (10 µM) and atropine (1 µM) in normotensive (A) and high

salt-induced hypertensive (B) rat aorta..…………….……………………………….84

Figure.3.37 Erucin and verapamil response against K+ in normotensive rats aorta

(mean±SEM, n=5-7)..………………………………………………………………...85

Figure.3.38 Tracing and graph show the effect of different concentrations of erucin

on phenylephrine (PE) in calcium free medium..……………………...……………..86

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Figure.3.39 Tracing (A) and graphs (B, C) show acetylcholine and Es.Cr effect on

spontaneous rhythmic force and rate in rat right atria with and without atropine (1

µM) (mean±SEM, n=5-7)……………………………………………………………88

Figure.3.40 Tracing (A) and graph (B) show n-hexane fraction of Eruca sativa (Es.n-

hexane) effect without and with atropine (1 µM) on spontaneous rhythmic force and

rate in isolated SD rat right atria (mean±SEM, n=5-7). . ..…....................................89

Figure.3.41 Tracing (A) and graph (B) show chloroform fraction of Eruca sativa

(Es.Chlor) response on spontaneous rhythmic force and rate in isolated SD rat right

atria... .. ………………………………………………………………………………90

Figure.3.42 Tracing (A) and graph (B) show Eruca sativa ethyl acetate fraction

(Es.EtAc) effect without and with atropine (1 µM) on spontaneous rhythmic force and

rate in isolated SD rat right atria (mean±SEM, n=5-7).. . .…………………………..91

Figure.3.43 Tracing (A) and graph (B) show the aqueous fraction of Eruca sativa

(Es.Aq) effect with and without atropine (1 µM) on spontaneous rhythmic force and

rate in isolated rat atria………………………………..............................................92

Figure.3.44 Tracing (A) and graph (B) show erucin response in the absence and

presence of atropine (1 µM) in SD rat right atria on spontaneous rhythmic rate and

force of contraction (mean±SEM, n=5-7).…………………………………………...93

Figure.3.45 Chromatogram of hederacoside C (A) and Hedera helix (B) detected at

wavelength 210 nm..……………………....................................................................96

Figure.3.46 Bar diagrams show lipid profile of normal control (A) and (tyloxapol-

induced) (TI) hyperlipidemic (B) SD rats (mean±SEM, n=5-7). ***p < 0.001 vs

normal control…….……………………….................................................................99

Figure.3.47 Bar diagrams show Hedera helix (Hh.Cr) effect of 30 (A), 100 (B), 300

mg/kg (C) and lovastatin 10 mg/kg (D) on lipid profile in tyloxapol-induced

hyperlipidemic SD rats (mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs

hyperlipidemic control.…………………………………………………………………..100

Figure.3.48 Bar diagrams show effect of hederacoside C (HDC) at doses of 2.5 (A)

and 5 (B) mg/kg on TC, LDL, TG and HDL in tyloxapol-induced hyperlipidemic SD

rats (mean±SEM, n=5-7). **p < 0.01, ***p < 0.001 vs hyperlipidemic

control..………………………………………...........................................................101

Figure.3.49 Bar diagrams show atherogenic index of normal control, hyperlipidemic

(tyloxapol-induced) (TI) group and crude extract of Hedera helix (Hh.Cr) at doses of

30, 100, 300 mg/kg (A) and hederacoside C (HDC) at doses of 2.5, 5 mg/kg (B) and

xxii

lovastatin 10 mg/kg treated TI hyperlipidemics SD rats (mean ± SEM, n=6-7) **p <

0.01, ***p < 0.001 vs hyperlipidemic control... …………………..………………..102

Figure.3.50 Bar diagrams show lipid profile of control (A) and high fat diet (HFD)-

induced hyperlipidemic (B) rats (mean±SEM, n=5-7). ***p < 0.001 vs normal

control…………………………………………………….........................................106

Figure.3.51 Bar diagrams show Hh.Cr effect at doses of 30 (A), 100 (B), 300 (C)

mg/kg and lovastatin (D) 10 mg/kg on high fat diet-induced hyperlipidemic SD rats

lipid profile (mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs

hyperlipidemic control.……………………………………………………………..107

Figure.3.52 Bar diagrams show effect of hederacoside C (HDC) at doses of 2.5 (A)

and 5 (B) mg/kg on the lipid profile of high fat diet-induced hyperlipidemic SD rats

(mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic

control.………………………………………………………………………………108

Figure.3.53 Bar diagrams show atherogenic index of normal control, high fat diet

(HFD)-induced hyperlipidemic and crude extract of Hedera helix (Hh.Cr) at doses of

30, 100, 300 mg/kg (A), hederacoside C (HDC) at doses of 2.5, 5 mg/kg (B) and

lovastatin 10 mg/kg treated high fat diet-induced hyperlipidemic SD rats

(mean±SEM, n=5-7)*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic

control……………………………………………………………………………….109

Figure.3.54 Photomicrographs show histological examination of normal control (A),

hyperlipidemic (B), Hedera helix crude extract (Hh.Cr) 30 (C) and 100 mg/kg (D)

treated SD rats aortae. Macrophages appear as large nucleus with vacoulated

cytoplasm while fat deposits appear as fat droplets with no distinct membrane…...112

Figure.3.55 Photomicrographs histological examination of Hedera helix crude extract

(Hh.Cr) 300 (E) and lovastatin (10 mg/kg) (F) and hederacoside C at doses of 2.5 (G),

5 mg/kg (H) treated SD rats aortae. Macrophages appear as large nucleus with

vacoulated cytoplasm while fat deposits appear as fat droplets with no distinct

membrane………………………...…………………………………………………113


phenylephrine (1 µM) contraction from the normal rat aorta (mean±SEM, n=5-7)..115


phenylephrine (PE; 1µM) in rat aortic rings from hyperlipidemic (high fat diet)

control (mean±SEM, n=5-7)..……………………………………………….……...116

xxiii

Figure.3.58 Tracing (A) and graph (B) show acetylcholine effect to phenylephrine

(PE; 1µM) precontraction in rat aorta isolated from Hh.Cr; 30 mg/kg treated group

(mean±SEM, n=5-7)……………………………………….. ………………………117

Figure.3.59 Tracing (A) and graph (B) show acetylcholine effect in rat aorta from

high fat diet treated with Hedera helix crude extract (Hh.Cr; 100 mg/kg) (mean±SEM,

n=5-7)...………………………………………………..............................................118


(PE; 1µM)-contraction in rat aortic tissues from high fat diet treated with Hedera

helix crude extract (Hh.Cr; 300 mg/kg/day) (mean±SEM, n=5-7)…........................119

Figure.3.61 Tracing (A) and graph (B) show acetylcholine response in isolated aortic

rings from high fat diet treated with lovastatin (10 mg/kg/day) (mean±SEM, n=5-

7)…………………………………………………………………………………….120


phenylephrine contraction in isolated aortic rings from high fat diet treated with

hederacoside C (HDC) (2.5 mg/kg) (mean±SEM, n=5-7).……………………........121

Figure.3.63 Tracing (A) and graph (B) show acetylcholine effect in aortic rings from

high fat diet treated with hederacoside C (HDC) (5 mg/kg) (mean±SEM, n=5-7)…122

Figure.3.64 Tracing (A) and graph (B) showing norepinephrine (NE) and

acetylcholine (Ach) response on mean arterial pressure (MAP) from normotensive

rats (mean±SEM, n=5-7).…………………………………………………………...124

Figure.3.65 Tracing (A) and graph (B) represent the Hedera helix crude extract

(Hh.Cr) response on normotensive rats MAP. Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001……………………………………………………..125

Figure.3.66 Tracing (A) and graph (B) showing mean arterial pressure of n-hexane

fraction in normotensive rats. Compared with pretreated values, *p < 0.05, **p <

0.01, ***p < 0.001.………………………………………………………………….126

Figure.3.67 Tracing (A) and graph (B) chloroform fraction (Hh.Chlor) on fall of

mean arterial pressure. Compared with pretreated values, *p < 0.05, **p < 0.01, ***p

< 0.001.……………………………………………………………………………...127

Figure.3.68 Tracing (A) and graph (B) show an effect on mean arterial pressure by

Hedera helix ethyl acetate fraction (Hh.EtAc). Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001..……………………………………........................128

xxiv

Figure.3.69 Tracing (A) and graph (B) represent MAP of Hedera helix aqueous

fraction (Hh.Aq). Compared with pretreated values, *p < 0.05, **p < 0.01, ***p <

0.001...………………………………………………………………………………129

Figure.3.70 Graph shows the MAP of normal and hypertensive rats (mean±SEM,

n=5-7) **p < 0.01 vs hypertensive……………………...……………………..........131

Figure.3.71 Tracing (A) and graph (B) showing Hedera helix crude extract (Hh.Cr)

effect on MAP in hypertensive (high salt) rats. Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001.………………….. ……….....…………….............132

Figure.3.72 Tracing (A) and graph (B) showing effect of Hh.n-hexane on MAP in

hypertensive rats under anaesthesia. Compared with pretreated values, *p < 0.05, **p

< 0.01, ***p < 0.001..………………………………………………………………133

Figure.3.73 Tracing (A) and graph (B) showing chloroform fraction of Hedera helix

(Hh.Chlor) effect on MAP in hypertensive rats under anaesthesia. Compared with

pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.……………………………134

Figure.3.74 Tracing (A) and graph (B) show Hedera helix ethyl acetate fraction

(Hh.EtAc) effect on MAP in hypertensive rats under anaesthesia. Compared with

pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.……………….. ………….135

Figure.3.75 Tracing (A) and graph (B) showing an aqueous fraction of Hedera helix

(Hh.Aq) effect on MAP in hypertensive rats under anaesthesia. Compared with

pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001…………………………….136

Figure.3.76 Tracing (A) and graph (B) represent effect of hederacoside C (HDC) on

MAP in normotensive and hypertensive rats.*p < 0.05, **p < 0.01 vs

hypertensive…………………………………………………………………………138

Figure.3.77 Vasorelaxant effect of Hedera helix crude extract (Hh.Cr) in rat aorta

precontracted with phenylephrine (1 μM) in intact and denuded tissues. [ANOVA

(Two-way) Bonferroni’s post hoc] *p < 0.05, ***p < 0.001.………………………140

Figure.3.78 Vasorelaxant effect of fractions of Hedera helix (A-D), in rat aorta with

phenylephrine (1 μM) contractions. [ANOVA (Two-way) Bonferroni’s post hoc] **p

< 0.01, ***p < 0.001 vs control..………………….................................................141

Figure.3.79 A and B show Hedera helix extracts and verapamil response on high K+

precontractions, in normal Kreb’s solution.………………………………………...142

Figure.3.80 Vasorelaxant effect of Hedera helix crude extract precontracted with

phenylephrine (1 µM) in hypertensive rat aorta (mean±SEM, n=5-7)......................143

xxv

Figure.3.81 Response of Hedera helix fractions (A-D) against phenylephrine (1 µM)

in rat aorta from hypertensive rats.....……………………………………………….144

Figure.3.82 Shows Hedera helix crude extract (Hh.Cr) and its fractions on

phenylephrine (PE) peak formation in Ca+2

-free/EGTA medium in isolated rat aorta

from normal rats (mean±SEM, n=5-7).……………………………………………..145

Figure.3.83 Endothelium-independent vasorelaxant response of hederacoside C

(HDC) precontracted with phenylephrine (1 µM) in aorta from normotensive (A) and

hypertensive (B) rats (mean±SEM, n=5-7)..…………………………………..........147

Figure.3.84 Hederacoside C (HDC) (A) and verapamil (B) effect against K+ in aortic

tissues from normal rats (mean±SEM, n=5-7)...……………………………............148

Figure.3.85 Tracing and graph show hederacoside C (HDC) effect on phenylephrine

(PE) peak formation in Ca+2

-free/EGTA medium in normal rat aorta (mean±SEM,

n=5-7).…………………............................................................................................149

Figure.3.86 Tracing (A) and concentration response curves (B) show the effect of

Hedera helix crude extract (Hh.Cr) on spontaneous rhythmic force and rate in SD rat

right atria in atropine (1 µM) absence and presence (mean±SEM, n=5-7)…………151

Figure.3.87 Tracing (A) and graph (B) show n-hexane fraction of Hedera helix

(Hh.n-hexane) effect on spontaneous rhythmic rate and force of contraction in the

absence and presence of atropine (1 µM) in isolated SD rat right atria (mean±SEM,

n=5-7). ………………………………………………………………………..........152

Figure.3.88 Tracing (A) and concentration response curves (B) show the effect of the

chloroform fraction of Hedera helix (Hh.Chlor) on spontaneous rhythmic rate and

force in SD rat right atria in atropine (1 µM) absence and presence (mean±SEM, n=5-

7)…………………………………………………………………………………….153

Figure.3.89 Tracing and graph show Hedera helix ethyl acetate fraction (Hh.EtAc)

response on spontaneous rhythmic rate and force of contraction in the absence and

presence of atropine (1 µM) in isolated SD rat right atrial preparations (mean±SEM,

n=5-7)…………………………………………………………………...…………..154

Figure.3.90 Tracing (A) and concentration response curves (B) show the Hedera

helix aqueous fraction (Hh.Aq) effect on force and rate in rat right atria in atropine (1

µM) absence and presence………………………………………………………….155

Figure.3.91Tracing and graph represent the hederacoside C (HDC) effect in the

absence and presence of atropine (1 µM) from isolated SD rats (mean±SEM, n=5-7).

………………………………………………………………………………………156

xxviii

LIST OF TABLES

Table.3.1 Analysis of phytochemicals of Eruca sativa crude extract ……………....28

Table.3.2 Total phenols and flavonoid content of Eruca sativa extracts …………...29

Table.3.3 Effect of Eruca sativa (Es.Cr) and erucin on serum lipid profile in

tyloxapol-induced (TI) hyperlipidemic Sprague-Dawley rats.………………………38

Table.3.4 Effect of Eruca sativa (Es.Cr) and erucin on serum lipid profile in high fat

diet-induced hyperlipidemic Sprague-Dawley rats.………………………………....45

Table.3.5 Shows percent fall in mean arterial pressure (MAP) induced by the crude

extract (Es.Cr) and its fractions (mg/kg) in normotensive rats under

anaesthesia.…………………………………………………………………………..66

Table.3.6 Shows percent fall of MAP of E. sativa extracts (mg/kg) in normotensive

rats pretreated with atropine (1 mg/kg)..……………………………………………..67

Table.3.7 Shows percent fall mean arterial pressure (MAP) in response to Eruca

sativa crude extract and fractions (mg/kg) in high salt hypertensive rats, administered

orally..………………………………………………………………………………..72

Table.3.8 The analysis of phytochemicals in Hedera helix crude extract ……..........94

Table.3.9 Total phenolic and flavonoid content of Hedera helix extracts.………….95

Table.3.10 Response by Hedera helix (Hh.Cr) and hederacoside C (HDC) on serum

lipid profile in tyloxapol-induced (TI) hyperlipidemic Sprague-Dawley rats……...103

Table.3.11 Response by Hedera helix (Hh.Cr) and hederacoside C on serum lipid

profile in hyperlipidemic Sprague-Dawley rats.……………………....……………110

xxix

LIST OF ABBREVIATIONS

°C Degree centigrade

µ Micron

µg Microgram

µM Micromolar

Ach Acetylcholine

Aq Aqueous

Ca+2

Calcium ion

CaCl2 Calcium chloride

CCB Calcium channel blocker

Chlor Chloroform fraction

CO2 Carbon dioxide

Cr Crude extract

CRCs Concentration response curves

CVDs Cardiovascular diseases

EDHF Endothelial-derived hyprerpolarizing factor

EGTA Ethylene Glycol-bis (β-Aminoethylether)-N-N-N-N-

Tetracetic acid

EtAc Ethyl acetate fraction

Es Eruca sativa

Es.Cr Crude extract of Eruca sativa

Es.n-hexane n-hexane fraction of Eruca sativa

Es.Chlor Chloroform fraction of Eruca sativa

Es.EtAc Ethyl acetate fraction of Eruca sativa

Es.Aq Aqueous fraction of Eruca sativa

g Gram

HDC Hederacoside C

xxx

HFD High fat diet

Hh.Cr Crude extract of Hedera helix

Hh.n-hexane n-hexane fraction of Hedera helix

Hh.Chlor Chloroform fraction of Hedera helix

Hh.EtAc Ethyl acetate fraction of Hedera helix

Hh.Aq Aqueous fraction of Hedera helix

i.p Intraperitoneal

K+

Potassium ion

KCl Potassium chloride

kg Kilogram

KH2PO4 Potassium dihydrogen phosphate

MAP Mean arterial pressure

mg Milligram

MgCl2.6H2O Magnesium chloride hexahydrate

MgSO4.7H2O Magnesium sulphate heptahydrate

min Minute

mL Milliliter

mm Millimeter

mM Millimolar

mmHg Millimeter of mercury

n Number of observations

NaCl Sodium chloride

NaH2PO4 Sodium dihydrogen phosphate

NaHCO3 Sodium bicarbonate

NE Norepinephrine

NO Nitric oxide

O2 Oxygen

xxxi

p.o. Per oral

PE Phenylephrine

ROCs Receptor operated channels

SEM Standard error mean

VDCs Voltage-dependent calcium channels

xxxii

LIST OF PUBLICATIONS

1. Salma, U., Khan, T. and Shah, A.J., 2018. Antihypertensive effect of the

methanolic extract from Eruca sativa Mill., (Brassicaceae) in rats: Muscarinic

receptor-linked vasorelaxant and cardiotonic effects. Journal of Ethnopharmacology.

224 (17), 409-420.

2. Salma, U., Khan, T. and Shah, A.J., 2018. Antihypertensive efficacy of the extract

of Hedera helix in high salt-induced hypertensive Sprague-Dawley rats. Asian Pacific

Journal of Tropical Medicine. 11 (8), 473-479.

3. Qamar, H.M.U.D., Qayyum, R., Salma, U., Khan, S., Khan, T. and Shah, A.J.,

2018. Vascular mechanisms underlying the hypotensive effect of Rumex acetosa.

Pharmaceutical Biology, 56 (1), 225-234.

4. Qayyum, R., Qamar, H.M.U.D., Khan, S., Salma, U., Khan, T. and Shah, A.J.,

2016. Mechanisms underlying the antihypertensive properties of Urtica

dioica. Journal of Translational Medicine, 14 (1), 254.

5. Qayyum, R., Qamar, H.M.U.D., Salma, U., Khan, S., Khan, T. and Shah, A.J.,

2019. Insight into the cardiovascular activities of Elaeagnus umbellate. FARMACIA,

67 (1), 133-139.

1

Chapter 1

Introduction

2

1.1 Hyperlipidemia

Hyperlipidemia is a medical condition manifested by the elevation of all lipid profiles

and/lipoproteins in the blood. It consists of elevated plasma cholesterol, low density

lipoproteins (LDL) (hypercholesterolemia), triglycerides (TG) (hypertriglyceridemia)

and low high density lipoproteins (HDL) (Grundy and Vega, 1988). The prevalence of

hyperlipidemia is high in developing countries, including Pakistan (63%) compared to

the developed countries such as America 53% (Karr, 2017: Zaid and Hasnain, 2018).

Hyperlipidemia leads to cardiovascular complications (Nelson, 2013). The main risk

factors for hyperlipidemia are unhealthy lifestyle, including high fat diet (cholesterol),

obesity, smoking, use of alcohol and lack of exercise. These factors contribute

towards an increase in cardiovascular diseases (CVD) in Asian population (Turin et

al., 2013). According to WHO, the estimated cardiovascular death rate will be 23.3

million by 2030 (Boutayeb et al., 2014). The cardiovascular disease can be prevented

by focusing on the control of hyperlipidemia and its associated complications

(Mensah et al., 2017).

1.2 Hyperlipidemia and importance of regulation of cholesterol

Cholesterol has been identified as one of the risk factors of coronary artery disease

due to the presence of cholesterol crystals in atherosclerotic lesion (Virchow and

Thrombose, 1856). In addition to elevated levels of cholesterol, triglycerides and LDL

are the leading causes of atherosclerosis, hypertension and coronary heart disease

(Shulman, 2014: Fordyce et al., 2015). The regulation of plasma cholesterol occurs

through biosynthesis, removal, absorption of dietary cholesterol and excretion through

the bile (Goldstein and Brown, 2015; Mozaffarian et al., 2016). However, in order to

counter the increasing CVD prevalence, there is a dire need for better understanding

of the pathways that govern the disease progression and thus to identify novel

therapeutic targets.

1.2.1 Metabolism of cholesterol

In humans, the cholesterol pool consists of two pathways. One is the absorption of

dietary cholesterol and biosynthesis of cholesterol in the liver (Fig.1.1). Both

pathways are equally important in the production of cholesterol (Marinetti, 1990). In

the small intestine, dietary cholesteryl esters are hydrolyzed into free cholesterol by

pancreatic cholesterol esterase. In the cytosol of liver cells, the biosynthesis of

cholesterol starts from acetyl-CoA. Three molecules of acetyl-CoA are converted into

3

mevalonate. This reaction is catalyzed by HMG-CoA reductase (rate-limiting step).

Cholesterol is converted to bile acids for excretion into feces (Mayes, 1993; Goldstein

and Brown, 2015).

1.2.2 Metabolism of chylomicron and very low density lipoprotein

Chylomicron (exogenous triacylglycerol rich lipoprotein) is biosynthesized in the

small intestine. It is synthesized from dietary cholesterol, triacylglycerol and

phospholipids in the small intestine. Lipoprotein lipase is responsible for degradation

of chylomicron to the chylomicron remnant (Fig.1.1). It also hydrolyzes the

triacylglycerol (Marinetti, 1990).

VLDL biosynthesized in rough endoplasmic reticulum, then assembled, packaged and

transported to plasma. It transports triacylglycerol phospholipids and cholesterol from

the liver to the other tissues of the body (Champe and Harvey, 1994; Mozaffarian et

al., 2016).

1.2.3 Metabolism of low density lipoprotein

VLDL is converted into LDL by lipoprotein lipase (Fig.1.1). The cholesterol is

mainly transported in LDL. It is composed of cholesterol, phospholipids,

triacylglycerol, cholesteryl esters and apoB-100. LDL is removed from plasma by

receptor-mediated endocytosis (Glew, 1992), with low-density lipoprotein receptor

(LDLR) and LDLR-related protein 1 (LRP1) (Van De Sluis et al., 2017).

1.2.4 Metabolism of high density lipoprotein

The biosynthesis of HDL takes place in the liver. It is released into the blood by

exocytosis. Free cholesterol in HDL is esterified by lecithin: cholesterol

acyltransferase (LCAT) (Fig.1.1). HDL serves as a circulating reservoir of apoC and

apoE for VLDL and chylomicron. It transfers cholesteryl esters to VLDL and LDL. It

also accumulates cholesterol from peripheral tissues and transports it to the liver for

elimination of excess cholesterol (Mayes, 1993). It has been demonstrated that reverse

cholesterol transport can proceed through a non-biliary pathway known as

transintestinal cholesterol excretion (TICE) (Temel and Brown, 2015). HDL is

atheroprotective which is contributed by its antiinflammatory and antioxidative

activities (Gordon and Remaley, 2017).

4

Figure.1.1 Exogenous and endogenous pathways of lipid metabolism (Karam et al.,

2017). Cholesterol in the circulation originates from endogenous or exogenous

pathway. In the exogenous pathway, cholesterol from dietary sources is absorbed in

the intestine and ultimately enters the circulation as a component of chylomicrons. In

endogenous pathway, the liver is responsible for the packaging of VLDL particles

which are hydrolyzed to IDL, returned to the liver so that they may be repackaged as

LDL then taken from the circulation by peripheral tissues.

5

1.2.5 Atherosclerotic plaque formation due to elevation of LDL

The arterial wall is composed of three layers; tunica intima, tunica media and tunica

adventitia. The initial injury takes place in the endothelium of the arterial wall due to

a toxin, chemical or physical stress (Favero et al., 2014). As a result, LDL enters the

arterial wall and then accumulates between the endothelium and tunica intima. The

high level of LDL in the plasma for long time results in its oxidation (Gao and Liu,

2017).

Oxidized LDL acts as an immunogen and triggers an immune response. The

monocytes migrate from the bloodstream and ingest oxidized LDL and then develop

into macrophages. Macrophages enlarge after becoming filled with oxidized lipids

and appear as foamy, referred to as “foam cells”. The cholesterol builds up in the

arterial wall leads to the formation of structures called “plaques” (Fig.1.2). The stable

plaque formation results in narrowing and occlusions of the artery. Thus, high levels

of LDL cholesterol lead to the impairment of endothelial function (Bui et al., 2009;

Chistiakov et al., 2017).

https://www.ncbi.nlm.nih.gov/pubmed/?term=Gao%20S%5BAuthor%5D&cauthor=true&cauthor_uid=29063061

https://www.ncbi.nlm.nih.gov/pubmed/?term=Liu%20J%5BAuthor%5D&cauthor=true&cauthor_uid=29063061

6

Figure.1.2 Atherosclerotic plaque formation. Atherosclerotic plaque formation

involves low density lipoprotein (LDL) accumulation, recruitment of monocytes-

macrophages, uptake of oxidized LDL by macrophage scavenger receptors and

transformation of macrophages into foam cells, leading to the formation of a fibrous

cap containing smooth muscle cells, which permits stabilization of the plaque.

This figure was taken and modified from Rochester Institute of Technology

(http://cias.rit.edu/faculty-staff/101/student/287)

7

1.3 Relation of cholesterol to endothelial dysfunction in

hyperlipidemia

Animal experiments and clinical data have provided evidence that hyperlipidemia

causes damage to endothelial function. It has been shown that impairment in

endothelium-dependent relaxation takes place in animals with experimentally-induced

hypercholesterolemia (Habib et al., 1986; Freiman et al., 1986; Jayakody et al.,

1987). Increase blood lipids induce endothelial damage and lipid deposition in the

vascular wall, leading to the formation of plaques and atherosclerosis (Huang et al.,

2002).

Vascular tone is regulated by the endothelium by maintaining vasodilation and

vasoconstriction. It is also involved in smooth muscle cell proliferation, migration and

thrombogenesis (Bonetti et al., 2003). The imbalance between vasodilation and

vasoconstriction leads to endothelial dysfunction. Intracellular binding of

acetylcholine (Ach) to muscarinic receptors (M3) activates phospholipase C (PLC).

PLC is responsible for production of IP3, binding to IP3 receptors stimulate the release

of endothelial Ca+2

(Clifford and Hellsten, 2004).

Endothelial cells increase nitric oxide (NO) synthase that catalyzes L-arginine and

oxygen (O2) into NO and L-citrulline (Palmer et al., 1987). NO activates its

intracellular receptor both in endothelial cells and in the adjacent smooth muscle cells.

NO binds to guanylyl cyclase leading to formation of cyclic GMP (cGMP) (Martin et

al., 1988). In smooth muscle cells, cGMP activates the cGMP-dependent kinase

leading to the phosphorylation of proteins. This activation results in reduction of the

amount of Ca+2

available for contraction and subsequent relaxation of smooth muscle

(Kobayashi et al., 1985; Kai et al., 1987).

Increased LDL is considered as the primary risk factor for endothelial dysfunction

(Bonetti et al., 2003). Lipid peroxidation takes place leads to atherogenesis (Drechsler

et al., 2010).

Decrease cholesterol levels leads to reduce the chances of myocardial infarction

disease, which might be linked to improved endothelial function (Casino et al., 1993).

Lysophosphatidylcholine and oxidized lipoproteins, are considered as atherosclerosis

mediators that stop NO and EDHF (endothelium derived hyperpolarizing actor)

production (Eizawa et al., 1995). Oxidized LDL (OxLDL) suppress the activity of

endothelial NO synthase (Vidal et al., 1998), increasing free radicals (Fleming et al.,

8

2005), and leading to the atherosclerosis (Maiolino et al., 2013; Pirillo and Catapano,

2013; Xu et al., 2013; Le, 2015). Antiaggregative and antiadhesive properties of

endothelium decrease due to less production of NO leading to atherosclerosis. In this

context, assessment of the regulation of endothelium-dependent vascular responses

becomes an important tool for early detection of preclinical dysfunction, diagnosis,

monitoring of treatment and possibly even prevention of cardiovascular diseases.

1.4 Hyperlipidemia and hypertension

Hypertension is highly prevalent in hyperlipidemic patients with increased level of

cholesterol (Thomas et al., 2001; O’meara et al., 2004). Hyperlipidemia can also

affect blood pressure by increasing the effects of vasoconstrictors in the endothelium

(Cardillo et al., 2000). Thus, less production of nitric oxide in addition to increased

vasoconstriction result in hypertension with hyperlipidemia. Patients with

hyperlipidemia and hypertension are at increased risk of developing atherosclerosis

(Thomas et al., 2002; Liao et al., 2004). The normal functions of the endothelium are

impaired in hyperlipidemia and hypertension (Drexler and Horning, 1999; Felmeden

et al., 2003).

Structural alterations in the endothelium cause adhesion of platelets and leukocytes.

Inflammation, loss of vasomotor tone and smooth muscle cell proliferation accelerate

formation of atheroma (Cai and Harrison, 2000). Hypertension impairs the

endothelium function as a result of shear and oxidative stress. This leads to increased

synthesis of collagen and fibronectin, resulting in reduced production of nitric oxide

and increased permeability to lipoproteins (Ross, 1990; Mason and Jacob, 2003).

Hypertension also increases the expression of lipid oxidation enzymes (O’ Donnell,

2003). Therefore, an effective therapy is needed to manage both hyperlipidemia and

hypertension.

1.5 Treatment of hyperlipidemia and hypertension

Treatment for the management of hyperlipidemia includes statins, fibrates, nicotinic

acids and bile acid sequestrants (Drexel, 2009). Statins are the most widely available

drugs used for hyperlipidemia is associated with myopathy (Ito, 2012). ACE

(angiotensin converting enzyme) inhibitors, angiotensin receptor blockers, calcium

channel blockers (CCB), beta adrenoceptor blockers, diuretics and direct vasodilators

are used in the management of hypertension (Hoffman, 2006).

9

The side effects of most commonly used antihypertensive drugs, CCB: reflux

tachycardia, flushing, headache and ACEI’s: dry cough, renal dysfunction and

angioedema. Statins, CCB and ACEI’s have high cost and result in rebound

phenomenon (Teixeira, 2013; Park et al., 2017). Therefore, additional therapies for

controlling lipid levels and blood pressure required to counteract the cardiovascular

disease risk. Herbal drugs are described as alternative by the World Health

Organization (WHO) (Goleniowski et al., 2006). Herbal drugs are often used for the

management and prevention of hyperlipidemia and hypertension, as recommended by

the WHO (Dhaliya et al., 2013: Rouhi-Boroujeni et al., 2015: Anwar, 2016).

1.6 Use of medicinal plants in cardiovascular diseases

Hyperlipidemia and hypertension identified as the risk factors of atherosclerosis.

Therefore, therapies used for the management of hyperlipidemia and hypertension

considered as the best approaches (Rhoads et al., 1986). Diet plays an important role

in the control of normal homeostasis of cholesterol biosynthesis and blood pressure

regulation. In this context, medicinal plants have been used for the management of

hyperlipidemia and hypertension offering reduced risk of cardiovascular diseases

(Craig, 1999).

Over the years, various natural resources proved as effective in the management of

hyperlipidemia and hypertension. The omega-3 polyunsaturated fatty acids, such as

eicosapentanoic acid and docosahexaenoic acid used as an adjunct therapy for

hyperlipidemia, are found in tuna, halibut and salmon (Simopoulos, 1999). Lovastatin

which is one of the efficacious statins derived from Aspergillus terreus (fungus)

(Hendrickson et al., 1999). In addition, nicotinic acid obtained from the dried leaves

of Nicotiana tobccum Linn. (Sinclair et al., 2000) used in the treatment of

hyperlipidemia. Garlic (Stevinson et al., 2000), red yeast rice (Nies et al., 2006) and

artichoke leaf extract (Wider et al., 2009) were reported in reducing lipid levels.

Antidislipidemic and antihypertensive activities of polyherbal formulation (POL-10)

(Aziz et al., 2009) and Orchis mascula (Aziz et al., 2009) were investigated. The

fruit, leaves and root extract of Morinda citrofolia were found to be antidislipidemic

(Mandukhail et al., 2010). Antihyperlipidemic and antihypertensive effects of Viola

adorata leaves extract (Siddiqi et al., 2012) and two polyherbal formulation (ZPTO

and ZTO) (Aziz et al., 2013) were also reported. Extract of Garcinia cambogia

10

(Sripradha et al., 2015), Moringa stenopetala (Geleta et al., 2016) and polyherbal

formulation (POL4) (Malik et al., 2017) are effective antihyperlipidemic agents.

The antidislipidemic activity of furano flavonoids from Indigofera tinctoria (Narendar

et al., 2006) and pyrenol from Coccinia grandis (Singh et al., 2007) was reported.

The alfalfa saponin extract involved in hepatic cholesterol metabolism in

hyperlipidemic rats (Shi et al., 2014). Antcin K, a triterpenoid compound from

Antrodia camphorate (Kuo et al., 2016) and XH601, synthesized derivative of

formononetin (Zhao et al., 2017) displayed antihyperlipidemic effects. This suggests

that medicinal plants and their constituents could serve as important sources in the

search for effective antihyperlipidemic and antihypertensive agents.

1.7 The medicinal plants and chemical constituents selected

Following medicinal plants and their chemical constituents were selected for the study

based on their traditional uses and reported activities.

1.7.1 Eruca sativa Mill.

Eruca sativa Mill. (Synonyms: Eruca vesicaria) belongs to the family Brassicaceae,

commonly called as Salad rocket, Rugula, Rucola, Colewort and locally as Tara mira

(Yaniv et al., 1998; Sabeen and Ahmed, 2009). Aerial parts of E. sativa used for the

present study.

1.7.1.1 Description

Eruca sativa is an edible annual plant. It is a leafy vegetable with flowers in typical

Brassicaceae fashion (Huxley, 1992). It is mentioned in the Bible as "Oroth means

light” (Yaniv et al., 1998). It is widely distributed around the world, including

Pakistan (Lamy et al., 2008; Sabeen and Ahmed, 2009).

1.7.1.2 Medicinal uses

Soaked seeds of E. sativa used as an aphrodisiac and for cleaning intestine (Yaniv et

al., 1998). It is available in super and farmers markets worldwide (Lamy et al., 2008)

and used as vegetable and salad. In Pakistan, local people use the whole plant of E.

sativa except roots for the treatment of hypertension, hyperlipidemia and diabetes

(Afifi and Irmaileh, 2000; Sabeen and Ahmed, 2009; Amjad, 2015). Fresh aerial parts

of E. sativa are cooked or as raw used to treat hypertensive patients (Ali-Shtayeh et

al., 2013). A clinical study demonstrated that cruciferous vegetables including E.

sativa associated with the prevention and treatment of cardiovascular diseases,

http://en.wikipedia.org/wiki/Flower

http://en.wikipedia.org/wiki/Brassicaceae

11

including hypertension (Mithen, 2015). Fresh leaves and shoots are known to manage

abdominal discomfort, digestion and constipation (Rehman et al., 2015).

1.7.1.3 Reported pharmacological activities

In a previous study, thirty two medicinal plants were tested for antihypertensive effect

in conscious rats. It was found that the aqueous ethanolic extract of E. sativa in 40

mL/kg oral solution resulted a fall in mean blood pressure after six hours (Ribeiro et

al., 1986). However, the underlying mechanism responsible for the decrease in blood

pressure was not investigated. Seeds of E. sativa were reported as antidiabetic through

its antioxidant and increased hepatic glutathione properties (El-Missiry and El-Gindy,

2000b). Leaf extracts of E. sativa plant possess stimulant, stomachic, diuretic and

antiscorbutic (Jaradat, 2005). The ethanolic extract from seeds of E. sativa was found

to be protective against mercuric chloride renal damage (Alam et al., 2007). Extract

from the leaves of E. sativa possess antigenotoxic activity (Lamy et al., 2008),

protective in ulcer and liver damage through its potent antioxidant activity

(Alqasoumi et al., 2009; Alqasoumi, 2010). The extract from fruits and oil of E.

sativa have antimicrobial activity (Darwish and Aburjai, 2010; Khoobchandani et al.,

2010). The extract from aerial parts, roots and seed oil of E. sativa inhibited tumor

growth observed in different cancer cell lines including colon, liver, breast and larynx

(Khoobchandani et al., 2011; Michael et al., 2011). E. sativa has anticholinesterase

(Boga et al., 2011), antiinflammatory (Kim et al., 2014) and antithrombotic (Fuentes

et al., 2014) activities. The hydroalcohol and petroleum ether extract of E. sativa

provide protection against diabetic neuropathy in rats by modulation of oxidative and

nitrosative stress (Kishore et al., 2017). The fatty acid rich fraction of E. sativa leaf

extract has also stimulated the glucose uptake and has been reported as antidiabetic

(Hetta et al., 2017).

1.7.1.4 Reported chemical constituents

Glucosinolates, flavonoids and isothiocyanates have been reported as the major

constituents of E. sativa leaves extract (Bennett, 2002; Lamy et al., 2008; Pasini et al.,

2012; Villatoro-Pulido et al., 2013; Bell et al., 2015). Glucoraphanin, mercaptobutyl

glucosinolate together with glucopyranosyldisulfanyl and glucoerucin are the major

glucosinolates (Pasini et al., 2012). The main flavonoids are kaempferol, isorhamnetin

and quercetin (Pasini et al., 2012). Isothiocyanates such as sulforaphane and erucin

are also present in E. sativa leaves extract (Melchini et al., 2009; Villatoro-Pulido et

al., 2013).

http://www.ncbi.nlm.nih.gov/pubmed?term=Darwish%20RM%5BAuthor%5D&cauthor=true&cauthor_uid=20187978

12

A

B

C

Figure.1.3 Structures of glucoraphanin (A), quercetin (B) and sulforaphane (C).

13

1.7.2. Erucin

1.7.2.1 Natural sources of erucin

Erucin (isothiocyanate) is obtained by enzymatic hydrolysis of glucoerucin, isolated

for the first time in the 1970s from E. sativa Mill. (Gmelin and Schluter, 1970).

Erucin and other isothiocyanates are sulfur-containing compounds exist in various

cruciferous vegetables (Halkier and Gershenzon, 2006). Cruciferous vegetables

provide health benefits against cancer, cardiovascular diseases, diabetes, asthma and

neurologic diseases (Higdon et al., 2007; Manchali et al., 2012). Recently, essential

oils of Eruca vesicaria subsp. Longirostris have been identified as another source of

erucin (Omri Hichri et al., 2016).

1.7.2.2 Synthesis of erucin

Erucin produced during consumption of various cabbages and broccoli in animals and

humans (Melchini and Taraka, 2010; Saha et al., 2012). Sulforaphane reported as

antioxidant, antihypertensive and antiinflammatory (Wu et al., 2004; Angeloni et al.,

2009). The structural similarity between erucin and sulforaphane, and conversion of

sulforaphane to erucin in the body led to select erucin for studying its effects in

cardiovascular diseases.

1.7.2.3 Chemical structure of erucin

4. Methylthiobutyl isothiocyanate

Figure.1.4 Structure of erucin.

1.7.2.4 Reported pharmacological activities of erucin

Erucin has antioxidant (Barillari et al., 2005) activity. It is also reported as

antiinflammatory (Yehuda et al., 2009; Cho et al., 2013). Erucin suppresses 6-

OHDA-induced neurotoxicity and neuronal apoptosis in cellular models of

Parkinson’s disease (Tarozzi et al., 2012). Erucin also has a potential role in inhibition

of tumor growth in breast cancer in mouse models by mitochondrial translocation (Li

14

et al., 2015). Erucin also found protective against phenobarbital-induced hepatic

damage (Arora et al., 2016).

Current literature present knowledge of the potential antiproliferative activity of

erucin in several human cancer cell lines, such as lung (Jakubikova et al., 2005;

Melchini et al., 2009), liver, colon (Harris and Jaffery, 2008; Lamy et al., 2008),

bladder (Abbaoui et al., 2012), ovary (Lamy et al., 2013), prostate (Melchini et al.,

2013) and breast cancer (Azarenko et al., 2014; Prełowska et al., 2017). Erucin has

the potential to provide protection against carcinogen (DMBA)-induced oxidative

stress (Arora et al., 2017; Mannan et al., 2017). Erucin is the major contributor to the

neuroprotective effects in Parkinson’s disease models (Morroni et al., 2018).

1.7.3 Hedera helix L.

Hedera helix L. belongs to the family Araliaceae. It is commonly known as Common

ivy, English ivy and locally as Lablab (Valnet, 1983; Rehman et al., 2017). H. helix

leaves were used for the present study.

1.7.3.1 Description

Ivy is a winding plant with clinging roots. Its evergreen leaves on the non-flowering

branches are triangularly to pentagonally lobed and have white veins (Valnet, 1983).

Ivy and its various subspecies are spread throughout the world, including Pakistan

(Lutensko et al., 2010; Rehman et al., 2017).

1.7.3.2 Medicinal uses

It has been reported that H. helix leaves decrease blood pressure in hypertensive

patients (Steinmetz, 1961). It is also effective in gout, diabetes, cancer and parasitic

infections (Valnet, 1983; Duke, 2002; Lutensko et al., 2010).

1.7.3.3 Reported pharmacological activities

H. helix reported as an antimicrobial (Cioaca et al., 1978), spasmolytic (Trute et al.,

1997), antimutagenic (Elias et al., 1990; Villani et al., 2001), hepatoprotective (Jeong

and Park, 1998) antioxidant (Gulcin et al., 2004) and antiinflammatory (Gepdiremen

et al., 2005). H. helix leaf extract has effective bronchodilator activity (Fazio et al.,

2009; Wolf et al., 2011; Greunke et al., 2015). H. helix inhibits CYP2C8 and

CYP2C19 (Rehman et al., 2017). The H. helix extract also showed antifungal activity

(Parvu et al., 2015; Roşca-Casian et al., 2017). Recently the antibacterial activity of

silver nanoparticles using H. helix extracts have been reported (Abbasifar et al.,

2017).

15

1.7.3.4 Reported chemical constituents

Saponins, flavonoids, polyacetylenes and phenolic compounds are the reported

constituents of H. helix (Christensen et al., 1991; Crespin et al., 1995; Bedir et al.,

2000). Saponins include alpha hederin, hedarosaponins B, hederacoside C,

hederacoside D, E, F, G, H, I and hederagenin (Lutsenko et al., 2010). Flavonoids

include rutin, isoquercitin, astragalin and kaempferol (Trute and Nahrstedt, 1997).

Coumarin glycosides (scopolin), polyacetylenes (falcarinone and falcarinol, 11, 12-

dehydrofalcarinol), anthocyanin (cyanidine 3-monoside), aminoacids, vitamin (E, C,

pro-vitamin A), carbohydrates (hamamelitol) are also reported as important

constituents (Lutensko et al., 2010).

The saponins contributes to antimicrobial (Cioaca et al., 1978), antimutagenic (Elias

et al., 1990) , antispasmodic (Trute et al., 1997), hepatoprotective (Jeong and Park,

1998), antimutagenic (Villani et al., 2001), antioxidant (Gulcin et al., 2004),

antiinflammatory (Gepdiremen et al., 2005), anthelmintic (Eguale et al., 2007) and

bronchodilator (Sieben et al., 2009) activities.

16

A

B

C

Figure.1.5 Structures of α-hederin (A), isoquercetin (B) and scopolin (C).

17

1.7.4 Hederacoside C (HDC)

1.7.4.1 Natural source of hederacoside C

HDC (saponin) is one of the important constituent isolated from ivy leaf extract and is

responsible for spasmolytic and bronchodilatory activities of H. helix (Trute et al.,

1997; Sieben et al., 2009).

1.7.4.2 Synthesis of hederacoside C

HDC (prodrug) converted to α-hederin into the blood exhibits various

pharmacological effects (Trute et al., 1997; Yakovishin and Grishkovets, 2003;

Khdair et al., 2010).

1.7.4.3 Chemical structure of hederacoside C

Hederacoside C (HDC; 3-[{2-O-(α-L-rhamnopyranosyl)-α-Larabinopyranosyl} oxy]-

23 hydroxyolean-12-en-28-oicacid 6-O-{4-O-(α-L-rhamnopyranosyl)-β-D-

glucopyranosyl}-β-D glucopyranosyl ester.

Figure.1.6 Structure of hederacoside C.

18

1.7.4.4 Reported pharmacological activities of hederacoside C

HDC tested in the past having different biological activities. HDC inhibited

hyaluronidase providing evidence to be efficacious in the treatment of venous

insufficiency (Facino et al., 1995). HDC showed antioxidant (Gulcin et al., 2004) and

antiinflammatory (Gepdiremen et al., 2005) and antispasmodic (Trute et al., 1997)

activities. Whereas according to Mendel et al., (2011) HDC showed contractile effect.

HDC involved in regulation of β2-adrenergic receptors (Sieben et al., 2009). HDC is

also effective in respiratory disorders (Fazio et al., 2009; Cwientzek et al., 2011;

Sheikh et al., 2015).

19

1.8 Aims and objectives

E. sativa and H. helix have traditional use in cardiovascular disorders including

hyperlipidemia and hypertension. The important constituents from these plants erucin

(E. sativa) and hederacoside C (H. helix) respectively reported as cardioprotective and

smooth muscle relaxant. However the literature lacks investigation of these plants and

the active constituents in cardiovascular disorders particularly hyperlipidemia and

hypertension. The aims of the current study were to find out the antihyperlipidemic

and vasomodulating effects in aorta from normotensive and hypertensive rats and

different possible underlying mechanisms of Eruca sativa, erucin, Hedera helix and

hederacoside C. These aims were achieved through the following objectives:

To evaluate phytochemical analyis

To explore the antihyperlipidemic and antihypertensive effects

To find out the effect on lipid profile in hyperlipidemic rats

To investigate vascular histopathological changes in response to

hyperlipidemia

To evaluate the effect on blood pressure in normotensive and hypertensive

(high salt) rats

To investigate underlying antihyperlipidemic and antihypertensive effects

To explore the vascular endothelium-dependent and independent reactivity in

sufficient detail

To investigate the effect on cardiac performance in sufficient detail

To evaluate toxicity in mice

To investigate the vasomodulating effects

20

Chapter 2

Materials and methods

21

2.1 Drugs and standards

Atropine phosphate, verapamil hydrochloride, potassium chloride, dimethyl sulfoxide

(DMSO), acetylcholine chloride, Tween 80, norepinephrine bitartrate, cholesterol,

phenylephrine hydrochloride, cholic acid and hederacoside C (Sigma Chemicals,

USA), erucin (Santa Cruz Biotechnology, Germany) and heparin (Leo Pharmaceutical

Products, Denmark), were purchased.

2.2 Collection and identification of plants

Aerial parts of E. sativa and H. helix leaves were collected (Abbottabad, Pakistan)

March and August, 2014 respectively. E. sativa was authenticated by Dr. Zafar Iqbal,

Post Graduate College, Department of Botany, Abbottabad, Pakistan and H. helix by

Dr. Hassan Sher, University of Swat, Swat, Pakistan. The voucher specimens of E.

sativa (Es-A-03/14) and H. helix (Hh-L-08/14) were kept in COMSATS University

Islamabad (CUI), Abbottabad Campus, Pakistan.

2.3 Crude extract

Aerial parts of E. sativa and fresh leaves of H. helix were dried and homogenized into

powder. The dried powder E. sativa (7 kg) and H. helix (2 kg) was macerated in

methanol (12.0 L) and (7.0 L) respectively for15 days, separately. The materials were

filtered through eight folds of muslin cloth and filter paper. Rotary evaporator at 37°C

temperature and pressure (¯760 mm Hg) used for evaporation (Williamson et al.,

1996). E. sativa (Es.Cr) and H. helix (Hh.Cr) yield was 4.28% (w/w) and 12% (w/w)

respectively.

2.4 Fractionation of crude extract

Crude extract of both plants were fractionated according to previously reported

methods (Williamson et al., 1996; Bala et al., 1999) by using solvents in increasing

order of polarity in separating funnel. Crude extract of E. sativa (Es.Cr) (200 g)

dissolved and final volume of the solution was 500 mL with distilled water. Same

volume of n-hexane (500 mL) was added to this solution. After vigorous shaking in

separating funnel the solution was stabilized for 30-40 min. The upper organic layer

(n-hexane) was collected. The solution was successively extracted with n-hexane (500

mL × 2), then dried on rotary evaporator (-760 mmHg, 37°C), the yield of n-hexane

fraction (Es.n-hexane) was 20.0% (w/w). The recovered layer was considered as

aqueous, which was further extracted with chloroform (500 mL × 3). The lower

organic layer (chloroform) was dried to obtain chloroform fraction (Es.Chlor) with

22

approximate yield of 6.0% (w/w). Similarly, the remaining aqueous layer was

fractioned with ethyl acetate (500 mL × 3). In this case the upper organic layer (ethyl

acetate) was obtained under similar conditions, yielded approximately 9.5% (w/w).

The remaining layer was considered aqueous (Es.Aq) layer with approximately yield

of 64.5% (w/w). For H. helix same procedure was adopted as described for E. sativa.

The percent yield of H. helix was 10% (n-hexane), 7.5% (chloroform), 5% (ethyl

acetate) and 55.5% (aqueous).

2.5 Phytochemical screening

2.5.1 Preliminary screening

Preliminary screening of both plants was performed for chemical constituents such as

alkaloids, glycosides, phenols, saponins, tannins and terpenoids using standard

chemical tests (Edeoga et al., 2005).

2.5.2 Total phenolic content

Phenolic contents were analyzed according to the Folin-Ciocalteu method (Alam et

al., 2007). Each extract/gallic acid (0.5 mL) mixed with Folin-Ciocalteu reagent and

20% (w/v) sodium carbonate. Absorbance calculated (765 nm) with UV/VIS

spectrophotometer.

2.5.3 Total flavonoid content

The sample of quercetin standard/extract was made with methanol and aluminium

chloride (0.1 mL). Then potassium acetate and distilled water was added. Absorbance

read (415 nm) with UV/VIS spectrophotometer (Chang et al., 2002).

2.5.4 HPLC analysis

Extract E. sativa and H. helix, quercetin, erucin and hederacoside C were analyzed

with high performance liquid chromatography Perkin Elmer (Series 200) (Shim-pack

C18, 150 mm × 4.6 mm I.D., 5 µm).

2.5.4.1 Estimation of quercetin in crude extract of E. sativa

Quercetin standard solution 5 mg/mL (final volume 10 mL) was prepared with

methanol. The extract of E. sativa was also dissolved in methanol (100 mg/mL). The

mobile phase comprised of acetonitrile (solvent A) and 0.03% phosphoric acid in

water (solvent B). The samples were eluted at wavelength of 280 nm with flow rate of

0.8 mL/min (Kim et al., 2014).

23

2.5.4.2 Estimation of erucin in crude extract of E. sativa

The solution of erucin (5 mg/mL) and extract of E. sativa (100 mg/mL) were prepared

with methanol. The analysis was carried out with mobile phase consisting of solvent

A (1% formic acid in water) and solvent B (methanol). The gradient elution was

performed with 80% of solvent B in 0-7 min and 100% solvent B in 7-15 min. The

monitoring wavelength was 365 nm with flow rate of 1 mL/min (De Nicola et al.,

2013).

2.5.4.3 Estimation of HDC in H. helix crude extract

Hederacoside C (5 mg/mL) and H. helix extract (100 mg/mL) were dissolved in

methanol. Gradient elution programme was used by an increase of solvent B

(acetonitrile) from 20-100% with 80-20% of solvent A for 30 min at 210 nm. The

composition of solvent A is water (90%), acetonitrile (10%) and 85%H3PO4 (0.5%)

(Demirci et al., 2004).

2.6 Animals

All the experiments were performed following protocols (NRC, 2011) and with

approval from Pharmacy Department ethical committee, COMSATS University

Islamabad, Abbottabad Campus. Sprague-Dawley (SD) rats (200–250 g), BalbC mice

(18-23 g) kept in animal house (Pharmacy Department, COMSATS, Abbottabad).

2.7 Pharmacological investigation

2.7.1 Safety study

The test has been done as earlier described (Walum, 1998) . BalbC mice were divided

into different groups (n=5-7). The groups were administered different doses of the

plant extracts p.o. The mice were allowed water and food ad libitum. The mice were

regularly observed for mortality and toxic effects for 24 h.

2.7.2 Tyloxapol-induced model of hyperlipidemia

The protocols described earlier (Kuroda et al., 1977; de Sousa et al., 2017) were

followed. Male SD rats (180-220 g) were used. The rats were divided into 12 groups.

Group 1 served as control group while group 2 as hyperlipidemic group. Group 3 was

given lovastatin (10 mg/kg/day) p.o. Group 4, 5 and 6 with different doses of E. sativa

extract p.o. Erucin 1 and 3 mg/kg were administered to group 7 and 8. Group 9, 10, 11

were given H. helix crude extract 30, 100 and 300 mg/kg oral doses respectively.

Group 12 and 13 were given HDC at doses of 2.5 and 5 mg/kg respectively. At 11th

day animals fasted. Group 1 was given saline i.p (10 mL/kg), all other groups (2-13)

24

received tyloxapol (i.p) 500 mg/kg dose. Rats were anaesthetized after 24 h. The

blood was collected through cardiac puncture for analysis of serum lipid profile.

2.7.3 High fat diet (HFD)-induced model of hyperlipidemia

The protocols earlier described were used (Akiyama et al., 1996; Berroughui et al.,

2003; Buettner et al., 2006) with slight modifications. The adult SD rats (180-220 g)

were divided into different groups (n=5-7). Group 1 as control group and group 2 as

hyperlipidemic control given high fat diet. High fat diet composed of cholesterol (2%

w/w), cholic acid (0.5% w/w) and butter fat (5% w/w) added to normal diet. Group 3

was treated with high fat diet + lovastatin (10 mg/kg/day) p.o. Group 4, 5 and 6 were

given high fat diet + crude extract of E. sativa at doses of 30, 100 and 300 mg/kg p.o.

Group 7 and 8 were given high fat diet + erucin 1 and 3 mg/kg. Group 9, 10, 11 were

given high fat diet + H. helix crude extract in 30, 100 and 300 mg/kg oral doses

respectively. Group 12, 13 were given high fat diet + HDC at doses of 2.5 and 5

mg/kg p.o respectively. After 8 weeks of treatment, rats were fasted, anaesthetized

and blood was collected through cardiac puncture for analysis of lipid profile.

2.7.3.1 Biochemical study

Lipid profile of all groups was determined. Test samples (serum), standard and blank

were poured into eppendorf tube. The reaction mixtures were mixed and incubated at

20-25°C. After that, 0.25 mL of each sample was poured in 96 well plates.

Absorbance calculated (490 nm) with micro plate reader. Atherogenic index (A.I)

were calculated (Friedewald et al., 1972).

Atherogenic index= TC-HDL/HDL

2.7.3.2 Histopathological study

Thoracic aortae were isolated from high fat diet hyperlipidemic and treated rats. The

aortae were divided into two sections. One section used for histopathological study.

The other sections of rat aortae from all groups were used for vascular dysfunction

study. Aortic tissue samples were made with surgical blade and kept in perforated jars

in 10% formo-saline. Fixed tissues were dehydrated in absolute ethanol. In order to

remove alcohol tissue samples were placed in pure xylene (6 h). Paraffin waxing was

used for embedding of tissues (58 ± 5°C). Microtome was used for cutting 5μm aortic

tissues. The tissues were placed in warm water bath fixed at 55 ± 5°C for embedding

of tissues on slides. The tissues were transferred to precision mechanical conviction

incubator for 2 h. Tissues were stained with haematoxylin-eosin dye. Stained slides

25

were photographed under light microscope (Galigher and Kozloff, 1971; Naruse et

al., 1994).

2.7.3.3 Evaluation of vascular dysfunction

For vascular dysfunction study, the other sections of thoracic aortae from all groups

were used. Aortic rings hanged in bath were equilibrated at resting tension of 2 g. The

aortic rings were contracted with phenylephrine (1 µM) and exposed to acetylcholine

to assess the endothelium dysfunction (Furchgott and Zawadaski, 1980; Shah and

Gilani, 2009; Garjani et al., 2009).

2.7.4 In-vivo recording and measurement of blood pressure

2.7.4.1 High salt (8% NaCl)-induced hypertensive rats

Protocols of Lawler et al. (1987) and Vasdev et al. (2003) were followed with some

modifications. Male SD rats (n=60) received 8% NaCl in diet and water for 8 weeks.

At the end of 8 weeks, rats with blood pressure at range of 150-190 mmHg were

considered as hypertensive. Effect of extract and fractions was determined on MAP.

Each dose was injected intravenously.

2.7.4.2 Experimental protocol

For in-vivo study both normotensive (n=60) and hypertensive (high salt) (n=60) SD

rats were used (Shah and Gilani, 2011). Sodium thiopental i.p used for anesthesia

(Pentothal, 40-90 mg/kg body weight). Trachea was cannulated using polyethylene

tubing (PE-20). Polyethylene tubing (PE-50) used to cannulate carotid artery attached

with pressure transducer (MLT 0699). Extracts and standards were administered to

left jugular vein after cannulation. Injection of each dose of extract or standard was

followed by a 0.1 mL flush of normal saline. To see involvement of muscarinic

receptor stimulation, SD rats were pretreated with atropine (1 mg/kg). Extract and

fractions were administered intravenously. After intravenous injection of each dose,

the normal blood pressure pattern was achieved in 10-15 min.

2.7.5 In-vitro studies in isolated aorta from SD rats

As described previously (Furchgott and Zawaski, 1980; Shah and Gilani, 2011),

thoracic aortae were isolated from all groups and transferred to petri dish containing

normal Kreb’s solution. Rings were made 2-3 mm in width. In some aortic rings of

normotensive rats endothelium was deliberately removed by rubbing the luminal

surface with forceps and were considered denuded when acetylcholine failed to

induce relaxation <80%.

26


Rings equilibrated (60-90 min) at tension of 2 g. Bath solution was changed every 15

min. All tissues were stabilized with repeated administration of phenylephrine (1

µM). Sustained contractions were induced with phenylephrine and cumulative

additions of samples were made to determine effect on vascular tone. Interval

between each concentration was 10-15 min. Effect of crude extract and fractions on

vascular tension was calculated as percent of the phenylephrine control.

2.7.6 Effect on intracellular Ca+2

stores

As described previously (Jiang et al., 2005; Shah and Gilani, 2011), experiments were

conducted to see whether the relaxations induced by extracts involved inhibition of

intracellular calcium release. Some tissues of aorta were placed in Ca+2

-free/EGTA

(15-20 min). Normal Kreb’s solution was used for washing (3-4 times) and tissues

again incubated (40 min) in order to refill intracellular Ca+2

stores. Subsequently, the

normal Kreb’s solution was changed with Ca+2

-free/EGTA and further incubated (15-

20 min). Aortic tissues were now preincubated with extract and fractions for 30 min.

Phenylephrine was added in the preincubated aortic tissues and the contraction

produced was compared with and without extract and fractions.

2.7.7 Isolated right atrial preparations

As described previously (Yoshihisa et al., 1992), right atria from SD rats were used to

see effect of extracts on force of contraction and heart rate.


Right atria from SD rats were dissected out, cleaned of fatty tissue. The tissues were

allowed to beat spontaneously (due to the presence of pacemaker activity) under the

resting tension of 1 g and an equilibrium period of 30 min was given (Yoshihisa et al.,

1992). To see the involvement of muscarinic receptor activation some rings were

pretreated with atropine (1 µM).

2.8 Statistics

Data given in mean and standard error mean. Median effective concentrations (EC50)

represented (SPSS software, version 21, USA). Data were taken at significance level

of *p < 0.05.

27

Chapter 3

Results

28

3.1 Phytochemical screening of Eruca sativa

3.1.1 Preliminary screening

E. sativa indicated the presence of alkaloids, flavonoids, glycosides, phenols,

saponins, tannins and terpenoids (Table.3.1).

Table.3.1 Analysis of phytochemicals of Eruca sativa crude extract.

Phytochemical constituents

Results

Alkaloids +++

Flavonoids +++

Glycosides +++

Phenols +++

Saponins +

Tannins +

Terpenoids +

+ = Mild, ++ = Moderate and +++ = Strong presence of phytochemicals

On the basis of intensity of color

29

3.1.2 Total phenolic content

Es.EtAc fraction represented highest content. The aqueous, chloroform and n-hexane

respectively, followed by crude extract (Table.3.2).

3.1.3 Total flavonoid content

Es.EtAc was potent, then aqueous fraction. The n-hexane fraction represented the low

content (Table.3.2).

Table.3.2 Total phenols and flavonoid content of Eruca sativa extracts.

Values tabulated as mean±SEM (n=3).

Sample

Total phenolic content

(mg GAE/g)

Total flavonoid content

(mg QCE/g)

Crude extract (Es.Cr)

122.22±0.73 3.42±0.35

n-hexane ( Es.n-hexane)

20.83±2.29 3.28±0.38

Chloroform ( Es.Chlor)

65.27±1.21 3.61±0.22

Ethyl acetate ( Es.EtAc)

265.83±0.48 6.74±0.45

Aqueous ( Es.Aq)

101.11±1.94 4.11±0.10

30

3.1.4 HPLC analysis

HPLC analysis (Shim-pack C18, 150 mm × 4.6 mm I.D., 5 µm) of E. sativa crude

extract was performed to confirm the compounds quercetin (major flavonoid) (mobile

phase: Solvent A (acetonitrile) and solvent B (0.03% phosphoric acid in water) and

erucin (major isothiocyanate) (mobile phase: Solvent A (1% formic acid in water) and

solvent B (methanol).

3.1.4.1 Estimation of quercetin in crude extract of E. sativa

Quercetin identified at a retention time of 3.64 min by comparison with the standard

quercetin sample. Minor peaks at retention time of 11.67, 12.63, 14.41 and 17.35 min

were observed (Fig.3.1).

3.1.4.2 Estimation of erucin in crude extract of E. sativa

Erucin was observed at a retention time of 3.07 min by comparing with standard

sample of erucin (Fig.3.2).

31

Figure.3.1 HPLC chromatogram of quercetin standard (A) and crude extract of Eruca

sativa (B) at wavelength of 280 nm. X-axis represents retention time (min) and y-axis

intensity (mV) (mobile phase: Solvent A (acetonitrile) and solvent B (0.03%

phosphoric acid in water). (Shim-pack C18, 150 mm × 4.6 mm I.D., 5 µm).

Figure.3.2 HPLC chromatogram of erucin standard (A) and crude extract of Eruca

sativa (B) at wavelength of 365 nm. Retention time (min) is on the x-axis and

intensity (mV) on the y-axis. (mobile phase: Solvent A (1% formic acid in water) and

solvent B (methanol). (Shim-pack C18, 150 mm × 4.6 mm I.D., 5 µm).

32

3.2 Pharmacological investigation of Eruca sativa

Two types of pharmacological studies were conducted; in-vivo and in-vitro. In the in-

vivo studies, safety study, antihyperlipidemic and antihypertensive evaluation was

carried out. While in the in-vitro studies, rat aorta and atria were used.

3.2.1 Safety study

Es.Cr was investigated to be safe at 3 g/kg in mice.

3.2.2 Antihyperlipidemic activities

3.2.2.1 Serum lipid profile of hyperlipidemic tyloxapol induced (TI) SD rats

After the treatment with Es.Cr at different doses and lovastatin (10 mg/kg), blood

from SD rats was collected for analysis of serum lipid profile.

Normal control group

Control group received normal diet. The lipid profile determined was serum total

cholesterol (TC); 99.52±2.33, triglyceride (TG); 83.75±4.09, low density lipoprotein

(LDL); 39.50±2.10, high density lipoprotein (HDL); 60.25±2.78 mg/dL (Fig.3.3A).

Atherogenic index (A.I) was calculated and found 0.66±0.06 (Fig.3.6 and Table.3.3).

Hyperlipidemic control group (tyloxapol 500 mg/kg)

Tyloxapol resulted a significant (p < 0.001) increase of TC to 350.02±17.77 and TG

721.75±11.34 mg/dL, LDL; 69.75±5.02 (p < 0.01), HDL; 66.01±4.31 (Fig.3.3B).

Atherogenic index (A.I) was calculated and found 4.40±0.54 (p < 0.001) as compared

to untreated rats (Fig.3.6 and Table.3.3).

Es.Cr (30 mg/kg) treated group

There was a decrease in TC and TG with values of 200.33±7.31, 246.10±3.46, LDL;

65.33±4.25, HDL; 58.83±2.80 mg/dL (Fig.3.4A), A.I; 2.42±0.25 (p < 0.01), compared

with hyperlipidemic control (Fig.3.6A and Table.3.3).


The lipid profile found was TC; 175.93±3.52 and TG; 214.33±3.48 (p < 0.01), LDL;

61.16±3.81, HDL; 63.03±4.73 mg/dL (Fig.3.4B), A.I; 1.82±0.21 (p < 0.01) compared

to hyperlipidemic rats (Fig.3.6A and Table.3.3).


The lipid profile determined: TC; 156.02±5.58 and TG; 197.25±5.61 (p < 0.01), LDL;

55.25±2.49, HDL; 65.25±2.32 mg/dL (Fig.3.4C), A.I; 1.39±0.04 (p < 0.01) in

comparison to hyperlipidemic group (Fig.3.6A and Table.3.3).

33

Lovastatin (10 mg/kg) treated group

Lovastatin (10 mg/kg) resulted in a significant decrease in TC (151.75±8.04) and TG

(176.07±5.79) (p < 0.001), LDL (55.02±1.92), HDL (60.02±5.78) mg/dL (Fig.3.4D)

and A.I was 1.57±0.27 (p < 0.001) (Fig.3.6A and Table.3.3).

Erucin (1 mg/kg) treated group

In this group the lipid profile found was; TC; 146.10±4.18 and TG; 201.16±7.15 (p <

0.001), LDL; 68.33±6.11, HDL; 62.33±3.18 mg/dL (Fig.3.5A), A.I; 1.36±0.18 (p <

0.001) when compared to hyperlipidemic (Fig.3.6B and Table.3.3).


The lipid profile determined after treatment with erucin 3 mg/kg was; TC;

121.40±6.90 and TG; 185.53±4.07 (p < 0.01), LDL; 61.33±4.41, HDL; 64.06±2.65

mg/dL (Fig.3.5B), A.I; 0.89±0.08 (p < 0.001) (Fig.3.6B and Table.3.3).

34

A

B

Figure.3.3 Bar diagrams (A+B) show lipid profile in normal control and tyloxapol-

induced (TI) hyperlipidemic SD rats respectively (mean±SEM, n=5-7) ***p < 0.001

vs normal control.

0

100

200

300

400

TC

TGLDLHDL

Normal control

Co

nce

ntr

atio

n (

mg/d

L)

0

200

400

600

800

TC

TGLDLHDL

Hyperlipidemic (TI)

Co

nce

ntr

atio

n (

mg/d

L) ***

***

35

A B

C D

Figure.3.4 Bar diagrams show effect of Eruca sativa (Es.Cr) at doses (mg/kg) of 30

(A), 100 (B), 300 (C) mg/kg and lovastatin (D) 10 mg/kg on total cholesterol (TC),

triglycerides (TG), low density lipoproteins (LDL) and high density lipoproteins

(HDL) in tyloxapol-induced hyperlipidemic rats. Values were expressed as mean ±

SEM (n=6-7) **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

0

200

400

600

800

TC

TG

LDL

HDL

Es.Cr (30 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

*****

0

200

400

600

800

TC

TG

LDL

HDL

Es.Cr (100 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

******

0

200

400

600

800TC

TG

LDL

HDL

Es.Cr (300 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

******

0

200

400

600

800TC

TG

LDLHDL

Lovastatin (10 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

*** ***

36

A

B

Figure.3.5 Bar diagrams show erucin effect, at doses (mg/kg), 1 (A), 3 (B) on serum

total cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL) and high

density lipoproteins (HDL) (TC, TG, LDL, HDL) of tyloxapol-induced

hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) ***p <

0.001 vs hyperlipidemic control.

0

200

400

600

800

TC

TG

LDL

HDL

Erucin (1 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

******

0

200

400

600

800

TC

TG

LDL

HDL

Erucin (3 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

***

37

Nor

mal

con

trol

Hyp

erlip

idem

ic (T

I)

Es.Cr 3

0

Es.Cr 1

00

Es.Cr 3

00

Lovas

tatin

10

0

2

4

6

******

***

**

Ath

ero

genic

Ind

ex (

A.I

)

A B

Figure.3.6 Bar diagrams show serum atherogenic index of normal control, tyloxapol-

induced (TI) hyperlipidemic group, Eruca sativa extract (Es.Cr) and lovastatin (A),

and erucin at doses of 1, 3 mg/kg (B) and lovastatin 10 mg/kg treated tyloxapol-

induced hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) **p

< 0.01, ***p < 0.001 vs hyperlipidemic control.

Nor

mal

con

trol

Hyp

erlip

idem

ic (T

I)

Eruci

n 1

Eruci

n 3

Lovas

tatin

10

0

2

4

6

******

***

Ath

ero

genic

Ind

ex (

A.I

)

38

Table.3.3 Effect of Eruca sativa (Es.Cr) and erucin on serum lipid profile in tyloxapol-induced (TI) hyperlipidemic Sprague-Dawley

rats.

NC: Normal control, TI: Tyloxapol-induced (One-way ANOVA Dunnet’s multiple comparison)

Values were expressed as mean ± SEM. (n=6-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

Lipid Profile

Groups

Total cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Low density

lipoproteins

(mg/dL)

High density

lipoproteins

(mg/dL)

Atherogenic

index

NC 99.52±2.33

83.75±4.09

39.50±2.10

60.25±2.78

0.66±0.06

Hyperlipidemic (TI) 350.02±17.77

721.75±11.34

69.75±5.02 66.01±4.31

4.40±0.54

TI+ Es.Cr 30 mg/kg 200.33±7.31**

246.10±3.46*** 65.33±4.25

58.83±2.80

2.42±0.25**

TI+ Es.Cr 100 mg/kg 175.93±3.52*** 214.33±3.48*** 61.16±3.81

63.03±4.73

1.82±0.21***

TI+ Es.Cr 300 mg/kg 156.02±5.58*** 197.25±5.61*** 55.25±2.49

65.25±2.32

1.39±0.04***

TI+Lovastatin10 mg/kg 151.75±8.04*** 176.07±5.79*** 55.02±1.92 60.02±5.78

1.57±0.27***

TI+ Erucin 1 mg/kg 146.10±4.18*** 201.16±7.15*** 68.33±6.11

62.33±3.18

1.36±0.18***

TI+ Erucin 3 mg/kg 121.40±6.90*** 185.53±4.07*** 61.33±4.41

64.06±2.65

0.89±0.08***

39

3.2.2.2 Serum lipid profile of hyperlipidemic (HFD) SD rats

After 8 weeks of treatment with HFD, blood samples were analyzed for lipid profile

group wise, as stated below.


The lipid profile determined was; serum total cholesterol (TC); 100.02±2.68, triglyceride

(TG); 62.50±6.23, low density lipoprotein (LDL); 36.50±1.70, high density lipoprotein

(HDL) 52.50±3.96 mg/dL and atherogenic index (A.I); 0.94±0.17 as shown in Fig.3.7A;

3.10 and Table.3.4.

High fat diet (HFD) Hyperlipidemic control group

The lipid profile determined was; TC; 358.02±6.58, TG; 80.50±4.99, LDL: 192.25±4.97,

HDL; 25.25±2.86 mg/dL and A.I; 13.86±2.05 as compared with normal control

(Fig.3.7B; 3.10 and Table.3.4).


The results of lipid profile were TC; 210.06±7.68, TG; 80.33±1.85, and LDL; 89.33±2.96

(p <0.001), HDL; 27.06±2.25 mg/dL, A.I; 6.88±0.80 (p < 0.01) compared to

hyperlipidemic (Fig.3.8A; 3.10A and Table.3.4).


The results were TC; 175.03±5.30, TG; 67.33±4.63 and LDL; 73.66±1.45 (p < 0.001),

HDL; 40.33±4.84 mg/dL, A.I; 3.45±0.51 (p < 0.001) in comparison with hyperlipidemic

rats (Fig.3.8B; 3.10A and Table.3.4).


Lipid profile was TC; 105.40±4.90, TG; 63.02±3.34 and LDL; 66.25±2.17 (p < 0.001),

HDL; 45.02±2.52 (p < 0.05) mg/dL, A.I; 1.35±0.09 (p < 0.001) (Fig.3.8C; 3.10A and

Table.3.4).


Lovastatin (10 mg/kg) resulted in TC; 90.75±3.94, TG; 61.50±4.05 and LDL; 50.50±3.47

(p < 0.001), HDL; 43.75±4.95 (p < 0.05) mg/dL, A.I; 1.13±0.19 (p < 0.001) (Fig.3.8D;

3.10A and Table.3.4).

40


Lipid profile of this group was; TC; 154.26±5.10, TG; 80.33±3.71, LDL; 75.70±2.36 (p <

0.001), HDL; 31.40±2.03 mg/dL, A.I; 3.96±0.42 (p < 0.001) (Fig.3.9A; 3.10B and

Table.3.4).


Lipid profile of erucin treated rats was; TC; 124.50±4.10, TG; 64.66±4.48, LDL;

59.33±5.60 (p < 0.001), HDL; 38.33±2.02 mg/dL, A.I; 2.26±0.17 (p < 0.001) (Fig.3.9B;

3.10B and Table.3.4).

41

A

B

Figure.3.7 Bar diagrams show serum lipid profile of control (A) and high fat diet (HFD)-

induced hyperlipidemic (B) rats (mean±SEM, n=5-7) ***p < 0.001 vs normal control.

0

100

200

300

400

TC

TGLDLHDL

Normal control

Co

nce

ntr

atio

n (

mg/d

L)

0

100

200

300

400 TC

TGLDLHDL

***

***

Hyperlipidemic (HFD)

Co

nce

ntr

atio

n (

mg/d

L)

42

A B

C D

Figure.3.8 Bar diagrams show Es.Cr effect at doses of 30 (A), 100 (B), 300 (C) mg/kg

and lovastatin (D) 10 mg/kg in high fat diet-induced hyperlipidemic SD rats on TC, TG,

LDL and HDL (mean±SEM, n=5-7) **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

0

200

400

TC

TG

LDL

HDL

Es.Cr (30 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

**

***

0

200

400

TC

TG

LDL

HDL

Es.Cr (100 mg/kg)C

on

cen

trat

ion

(m

g/d

L)

***

***

0

200

400TC

TG

LDL

HDL

***

***

Es.Cr (300 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

0

200

400TC

TG

LDLHDL

***

***


Co

nce

ntr

atio

n (

mg/d

L)

43

0

100

200

300

400

TC

TG

LDL

HDL

***

***

Erucin (3 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

0

100

200

300

400

TC

TG

LDL

HDL

Erucin (1 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

***

A

B

Figure.3.9 Bar diagrams show erucin 1 (A), 3 (B) mg/kg effect in the hyperlipidemic

(high fat diet) SD rats lipid profile (mean±SEM, n=5-7) ***p < 0.001 vs hyperlipidemic

control.

44

Nor

mal

con

trol

Hyp

erlip

idem

ic (H

FD)

Es.Cr 3

0

Es.Cr 1

00

Es.Cr 3

00

Lovas

tatin

10

0

5

10

15

20

***

***

*** ***

Ath

ero

genic

Ind

ex (

A.I

)

Nor

mal

con

trol

Hyp

erlip

idem

ic (H

FD)

Eruci

n 1

Eruci

n 3

Lovas

tatin

10

0

5

10

15

20

***

******

Ath

ero

genic

Ind

ex (

A.I

)

A B

Figure.3.10 Bar diagrams show serum atherogenic index of normal control, high fat diet

(HFD)-induced hyperlipidemic group, Eruca sativa extract (Es.Cr) and lovastatin (A),

and erucin at doses of 1, 3 mg/kg (B) and lovastatin 10 mg/kg treated HFD-induced

hyperlipidemic SD rats. Values were expressed as mean ± SEM (n=6-7) ***p < 0.001 vs

hyperlipidemic control.

45

Table.3.4 Effect of Eruca sativa (Es.Cr) and erucin on serum lipid profile in high fat diet-induced hyperlipidemic Sprague-Dawley

rats.

NC: Normal control, HFD: High fat diet, (Analysis by One-way ANOVA, Dunnet’s multiple comparison)

Values were expressed as mean ± SEM (n=6-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

Lipid Profile

Groups

Total cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Low density

lipoproteins

(mg/dL)

High density

lipoproteins

(mg/dL)

Atherogenic

index

NC

100.02±2.68

62.50±6.23

36.50±1.70

52.50±3.96

0.94±0.17

Hyperlipidemic (HFD) 358.02±6.58

80.50±4.99 192.25±4.97

25.25±2.86

13.86±2.05

HFD + Es.Cr 30 mg/kg 210.06±7.68**

80.33±1.85

89.33±2.96*** 27.06±2.25

6.88±0.80**

HFD + Es.Cr 100 mg/kg 175.03±5.30*** 67.33±4.63

73.66±1.45*** 40.33±4.84 3.45±0.51***

HFD + Es.Cr 300 mg/kg 105.40±4.90*** 63.02±3.34

66.25±2.17*** 45.02±2.52* 1.35±0.09***

HFD + Lovastatin10 mg/kg 90.75±3.94*** 61.50±4.05

50.50±3.47*** 43.75±4.95*

1.13±0.19***

HFD + Erucin 1 mg/kg 154.26±5.10*** 80.33±3.71

75.70±2.36*** 31.40±2.03

3.96±0.42***

HFD + Erucin 3 mg/kg 124.50±4.10*** 64.66±4.48

59.33±5.60*** 38.33±2.02

2.26±0.17***

46

3.2.2.1 Histopathological examination of aorta from hyperlipidemic (HFD) SD

rats

Normal, hyperlipidemic and treated rats aortae used for histological examination.

Normal control

Aorta showed the normal structure of the three tunics; tunica intima, tunica media and

tunica adventitia. Endothelial cells round in shape were present in tunica intima. The

endothelium was found to be smooth and regular. Tunica media comprised of smooth

muscle cells and elastic lamina. Smooth muscle cells were elongated. Tunica

adventitia comprised of connective tissues. The thickness of aorta was normal

(Fig.3.11A).

Hyperlipidemic control group (High fat diet)

Aorta from the high fat diet group showed marked structural changes in the three

tunics. The endothelial cells were elongated with increased thickness of tunica media.

Smooth muscle cells were flattened with irregular shape. There were lipid droplets

with infiltration of macrophages, vacuoles and foam cells. Macrophages appear as

large round cells with central round nucleus and vacuolated cytoplasm. Some

macrophages were filled with lipids giving them foamy appearance and transformed

into foam cells. Tunica media did not reveal the intimal plaque formation. The elastic

membranes had a loose arrangement of connective tissues, resulting in an increase in

matrix. The fatty degeneration and cytoplasmic displacement were evident

(Fig.3.11B).


Photomicrograph of the aorta from hyperlipidemic rats treated with Es.Cr 30

mg/kg/day showed proliferation of smooth muscle cells in the tunica media. The lipid

accumulation was reduced, but there was an increase in the thickness of aorta

(Fig.3.11C).


In this group, smooth muscle cells proliferation decreased. The thickness of the aorta

and fatty deposits also reduced (Fig.3.11D).


Photomicrograph of the aorta from hyperlipidemic rats showed almost regular

morphology of aortic intima, media and adventitia. The fatty degeneration was

improved. The thickness of aorta was also decreased (Fig.3.12E).

47


Photomicrograph of the aorta showed reduced spaces between tunica media and

tunica intima with no macrophages infiltration. The lipid deposits were also reduced

in the aorta of lovastatin-treated group (Fig.3.12F).


In aorta from animals treated with erucin 1 mg/kg showed vacoulation in the tunica

media with infiltration of macrophages. The thickness of aorta was also increased

(Fig.3.12G).


Aorta from treated animals showed that aorta thickness was decreased. The lipid

deposits were also reduced (Fig.3.12H).

48

Figure.3.11 Photomicrographs show histological examination of aortae from normal

control (A), hyperlipidemic (HFD) (B), Eruca sativa extract at 30 (C) and 100 mg/kg

(D) treated SD rats. Macrophages appear as large nucleus with vacoulated cytoplasm

while fat deposits appear as fat droplets with no distinct membrane.

A

B

C

D

49

Figure.3.12 Photomicrographs show histological examination of aortae from crude

extract of Eruca sativa (Es.Cr) at 300 mg/kg (E), lovastatin (10 mg/kg) (F) and erucin

at doses of 1 (G), 3 mg/kg (H) treated SD rats. Macrophages appear as large nucleus

with vacoulated cytoplasm while fat deposits appear as fat droplets with no distinct

membrane.

E

F

G

H

50

3.2.2.4 Vascular function study of hyperlipidemic (HFD) SD rats

Thoracic aortae were isolated from normal control, hyperlipidemic control (HFD),

extracts and compounds treated rats for vascular dysfunction study.


Isolated rat aorta of normal control group pre-contracted with phenylephrine (1 µM)

showed relaxation to Ach [EC50 value; 0.08 µg/mL (0.03-0.13)] (Fig.3.13B).


The maximum relaxation induced with 1 µg/mL of acetylcholine was < 20% compared to

100% in the normal control group (Fig.3.14B).


Aorta isolated from 30 mg/kg treated E. sativa, showed a partial response to Ach with

33% relaxation (Fig.3.15).


Ach induced relaxation was further accentuated in 100 mg /kg treated rats up to 54%

(Fig.3.16).


Aorta showed complete relaxation to acetylcholine with EC50 value; 0.15 µg/mL (0.08-

0.22)] (Fig.3.17).


Aorta isolated from lovastatin treated rats, showed 49% relaxation to Ach (Fig.3.18).


Ach induced relaxation in erucin (1 mg/kg) treated rats was 24% (Fig.3.19).


Aorta isolated from this group showed complete relaxation comparable to normal rat

aorta (Fig.3.20).

51

0.003 0.03 0.3 3

0

50

100

Normal control

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

.

B

Figure.3.13 Tracing (A) and graph (B) show the acetylcholine response on

phenylephrine (PE; 1 µM)-induced contraction in normal control isolated rat aortic rings

(mean±SEM, n=5-7).

52

0.003 0.03 0.3 3

0

25

50

75

100

Hyperlipidemic (high fat diet)

[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B

Figure.3.14 Tracing (A) and graph (B) show the acetylcholine response in isolated aorta

of high fat diet treated hyperlipidemic rats (mean±SEM, n=5-7).

53

0.003 0.03 0.3 3

0

25

50

75

100

HFD + Es.Cr (30 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B

Figure.3.15 Tracing (A) and graph (B) show acetylcholine effect against phenylephrine

(1 µM) precontraction in aorta of high fat diet (HFD) + crude extract of Eruca sativa

(Es.Cr; 30 mg/kg) treated group (mean±SEM, n=5-7).

54

0.003 0.03 0.3 3

0

25

50

75

100


[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


phenylephrine (PE; 1 µM)-induced contraction in aortic rings from high fat diet (HFD) +

crude extract of Eruca sativa (Es.Cr; 100 mg/kg) treated group (mean±SEM, n=5-7).

55

0.003 0.03 0.3 3

0

25

50

75

100


[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


phenylephrine (1 µM) contraction in aorta of high fat diet (HFD) + crude extract of Eruca

sativa (Es.Cr; 300 mg/kg/day) treated group (mean±SEM, n=5-7).

56

0.003 0.03 0.3 3

0

25

50

75

100

HFD + Lovastatin (10 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


(PE; 1 µM)-induced contraction from the hyperlipidemic high fat diet (HFD) with

Lovastatin (10 mg/kg/day) treated rat aorta (mean±SEM, n=5-7).

57

0.003 0.03 0.3 3

0

25

50

75

100

HFD+ Erucin (1 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


(PE; 1 µM)-induced contraction from high fat diet (HFD) + Erucin (1 mg/kg) treated rat

(mean±SEM, n=5-7).

58

0.003 0.03 0.3 3

0

25

50

75

100

HFD + Erucin (3 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B


(PE; 1 µM)-induced contraction in aorta of high fat diet (HFD) + Erucin (3 mg/kg)

treated group.

59

3.2.3 Antihypertensive activities

3.2.3.1 Mean arterial pressure (MAP) in normotensive rats

Before injecting extracts of E. sativa, effect of norepinephrine (1 μg/kg) and

acetylcholine (1 μg/kg) was monitored and determined (Fig.3.21).

Crude extract of E. sativa (Es.Cr)

The MAP at 1, 3, 10 and 30 mg/kg was calculated as 10.09±0.55, 25.55±6.05,

31.88±2.78 and 41.79±1.55 mmHg, respectively. The effect was statistically significant

(p < 0.001), especially at 30 mg/kg vs control values (Fig.3.21 and Table.3.5). Similarly,

acetylcholine also reduced MAP: 26.81±1.10, 39.26±0.81, 48.42±1.99 and 61.83±4.21

mmHg, respectively (Table.3.5). To see if muscarinic receptor stimulation played a role

in blood pressure lowering effect, rats were pretreated with atropine (1 mg/kg). The effect

of Es.Cr was ablated at the same doses with percent fall of 2.4±0.08, 5.33±3.12,

7.41±3.67 and 10.88±5.19 mmHg, respectively (Fig.3.21 and Table.3.6). Similarly,

acetylcholine response was also ablated in atropine (1 mg/kg) treated rats with percent

fall of 2.37±1.08, 5.62±1.50, 8.43±3.95 and 9.48±4.06 mmHg, respectively (Table.3.6).

Following fractions of E. sativa were tested parallel.

n-hexane fraction (Es.n-hexane)

The percent fall of MAP observed at same doses was 12.18±4.60, 21.94±3.13,

26.05±0.08 and 39.49±1.28 mmHg, respectively (Fig.3.22 and Table.3.5). Atropine (1

mg/kg) pretreatment decreased MAP with percent fall of 3.37±0.88, 4.57±0.92,

8.32±0.94 and 11.60±0.9 mmHg, respectively (Fig.3.22 and Table.3.6).

Chloroform fraction (Es.Chlor)

Chloroform fraction also decreased MAP (Fig.3.23) with percent fall of 3.67±1.14,

6.81±0.25, 12.73±0.24 and 28.05±0.27 mmHg, respectively. The chloroform fraction was

the least potent fraction (p < 0.05) (Fig.3.23 and Table 3.5). MAP was reduced with

atropine (1 mg/kg) with respective values of 1.15±0.07, 2.63±0.03, 2.63±0.03 and

4.18±0.05 mmHg (Fig.3.23 and Table.3.6).

Ethyl acetate fraction (Es.EtAc)

The MAP was 4.32±1.04, 18.76±1.21, 25.51±3.53 and 34.35±2.62 mmHg, respectively.

The percent fall was statistically significant at 30 mg/kg (p < 0.001) (Fig.3.24 and

Table.3.5). The MAP was reduced with atropine (1 mg/kg) with respective values of

60

3.42±1.55, 4.10±1.15, 4.74±1.52 and 6.65±1.50 mmHg, respectively (Fig.3.24 and

Table.3.6).

Aqueous fraction (Es.Aq)

The aqueous fraction resulted in fall in MAP of 21.11±1.53, 30.45±2.13, 42.58±4.27 and

53.14±0.15 mmHg (Fig.3.25 and Table 3.5). The percent fall with atropine incubation

was 2.39±0.02, 7.45±1.90, 11.37±1.97 and 16.66±0.67 mmHg (Fig.3.25 and Table.3.6).

61

A

B

Figure.3.21 Tracings show norepinephrine (NE), acetylcholine (Ach) and Eruca sativa

(Es.Cr) response on mean arterial pressure (MAP) without (A) and with atropine (1

mg/kg) (B) in the normotensive group under anaesthesia.

62

A

B

Figure.3.22 Tracings show the effect of Eruca sativa n-hexane fraction (Es.n-hexane) on

mean arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B) pretreatment

in normotensive rats, under anaesthesia.

63

A

B

Figure.3.23 Tracings show the Eruca sativa chloroform fraction (Es.Chlor) effect on

mean arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B) in

normotensive rats, under anaesthesia.

64

A

B

Figure.3.24 Tracings show Eruca sativa ethyl acetate fraction (Es.EtAc) effect on mean

arterial pressure (MAP) without (A) and with atropine (1 mg/kg) (B) in normal rats.

65

A

B

Figure.3.25 Tracings show MAP of aqueous fraction of Eruca sativa (Es.Aq) without

(A) and with atropine (1 mg/kg) (B) in normotensive rats under anaesthesia.

66

Table.3.5 Shows percent fall in mean arterial pressure (MAP) induced by the crude

extract (Es.Cr) and its fractions (mg/kg) in normotensive rats under anaesthesia.

Values tabulated, mean±SEM (n=5-7).

Compared with pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.

[ANOVA (One-way) post hoc Tukey’s HSD analysis]

% Fall in mean arterial pressure (mmHg)

Dose

Ach

(µg/kg)

Crude

extract

n-hexane

Chloroform

Ethyl

acetate

Aqueous

1

26.81±1.10*

10.09±0.55

12.18±4.60

3.67±1.14

4.32±1.04

21.11±1.53*

3

39.26±0.81**

25.55±6.05*

21.94±3.13*

6.81±0.25

18.76±1.21

30.45±2.13*

10

48.42±1.99***

31.88±2.78**

26.05±0.08*

12.73±0.24

25.51±3.53*

42.58±4.27***

30

61.83±4.21***

41.79±1.55**

39.49±1.28**

28.05±0.27*

34.35±2.62**

53.14±0.15***

67

Table.3.6 Shows percent fall of MAP of E. sativa extracts (mg/kg) in normotensive rats

pretreated with atropine (1 mg/kg).


*p < 0.05, **p < 0.01, ***p < 0.001 without atropine vs with atropine (1 mg/kg)



Dose

Ach

(µg/kg)

Crude

n-hexane

Chloroform

Ethyl acetate

Aqueous

1

2.37±1.08*

2.4±0.08

3.37±0.88*

1.15±0.07

3.42±1.55

2.39±0.02**

3

5.62±1.50*

5.33±3.12**

4.57±0.92*

2.63±0.03

4.10±1.15**

7.45±1.90**

10

8.43±3.95**

7.41±3.67**

8.32±0.94**

2.63±0.03*

4.74±1.52**

11.37±1.97***

30

9.48±4.06**

10.88±5.19**

11.60±0.9***

4.18±0.05**

6.65±1.50***

16.66±0.67***

68

3.2.3.2 Effect on blood pressure (MAP) in hypertensive anaesthetized rats

After feeding high salt (8% NaCl) in water and diet to normotensive rats resulted a

significant increase in MAP after 8 weeks (Fig.3.26).

E. sativa crude extract (Es.Cr)

The mean arterial pressure decreased at 1, 3, 10 and 30 mg/kg: 25.41±3.85, 39.20±1.84,

46.84±3.61 and 58.25±0.91 mmHg, respectively. The effect was statistically significant

at all doses, especially at 30 mg/kg (p < 0.001) vs control values (Fig.3.26 and Table.3.7).

Similarly with acetylcholine at 1, 3, 10 and 30 µg/kg MAP was 38.75±0.90, 52.69±0.31,

67.25±1.95 and 78.25±1.24 mmHg, respectively (Table.3.7). Following fractions were

tested parallel.

n-hexane fraction (Es.n-hexane)

The MAP was 20.68±1.67, 32.53±1.00, 41.07±2.88 and 53.69±1.18 mmHg, respectively

(Fig.3.27 and Table.3.7).

Chloroform fraction (Es.Chlor)

The percent fall in MAP with chloroform fraction was 6.67±1.53, 14.98±0.64,


Ethyl acetate fraction (Es.EtAc)

Ethyl acetate fraction (Es.EtAc) resulted percent fall in MAP was 9.84±0.95, 23.34±3.87,


Aqueous fraction (Es.Aq)

Aqueous fraction caused a fall in MAP was 29.66±2.61, 39.82±2.29, 53.33±0.95 and

67.89±1.67 mmHg (Fig.3.28 and Table.3.7).

69

0

50

100

150

200

250Normotensive (n=5-7)

Hypertensive (n=5-7) **

Groups

MA

P (

mm

Hg)

A

B

Figure.3.26 Graph (A) shows the arterial blood pressure of normotensive and

hypertensive (8% NaCl) groups. Tracing (B) shows crude extract (Es.Cr) response on

high salt-induced hypertensive rats mean arterial pressure under anaesthesia.

70

A

B

Figure.3.27 Tracings showing response of n-hexane (A) and chloroform (B) fractions on

hypertensive rats mean arterial pressure, under anaesthesia.

71

A

B

Figure.3.28 Tracings show mean arterial pressure of Eruca sativa ethyl acetate (A),

aqueous fraction (B) on hypertensive rats under anaesthesia.

72

Table.3.7 Shows percent fall mean arterial pressure (MAP) in response to Eruca sativa

crude extract and fractions (mg/kg) in high salt hypertensive rats, administered orally.


Compared with pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.



Dose

Ach

(µg/kg)

Crude

n-hexane

Chloroform

Ethyl

acetate

Aqueous

1

38.75±0.90*

25.41±3.85*

20.68±1.67*

6.67±1.53

9.84±0.95

29.66±2.61*

3

52.69±0.31**

39.20±1.84*

32.53±1.00*

14.98±0.64*

23.34±3.87

39.82±2.29*

10

67.25±1.95***

46.84±3.61**

41.07±2.88**

21.53±2.52**

33.93±3.26*

53.33±0.95**

30

78.25±1.24***

58.25±0.91**

53.69±1.18**

42.50±4.21**

51.41±2.50**

67.89±1.67***

73

3.2.3.3 Effect of erucin on MAP in normotensive and high salt hypertensive rats

Erucin, effect on MAP was measured in both rats. Different doses of erucin were used.

The respective fall in MAP of erucin observed was 10.50±1.50, 21.01±2.01, 26.50±1.50,

35.03±1.20, 40.20±2.50 and 50.01±5.03 mmHg (Fig.3.29A).

The effect of erucin on MAP was also evaluated in normotensive rats with atropine (1

mg/kg). The effect of erucin on MAP remains same (data not shown).

In hypertensive rats MAP decreased at same doses with respective values of 20.10±4.01,

30.02±1.10, 36.50±1.50, 45.50±0.50, 53.50±3.50 and 66.01±6.20 mmHg was observed

(Fig.3.29B).

74

A

B

Fig.3.29 Tracing (A) showing mean arterial pressure (MAP) of erucin in normotensive

rats and graph (B) showing the effect of erucin on MAP in normal and hypertensive rats

(mean±SEM, n=5-7). *p < 0.05, **p < 0.01 vs hypertensive.

1 3 10 30 100 300

0

40


Hypertensive (n=5-7)**

*

[Erucin] g/kg

% F

all

in M

AP

75

3.2.4 Vascular reactivity studies

Effect of crude extracts, fractions and erucin was further evaluated in isolated vascular

preparations.

3.2.4.1 Eruca sativa extract and fractions effect in rat aortic tissues of normal and

hypertensive rats

Aortae from both normal and hypertensive rats were used for vascular reactivity studies.

Normotensive rats

In aorta rings from normotensive rats, with PE (1 µM) precontraction, acetylcholine

produced a concentration-dependent relaxation. This relaxation to Ach was reduced by

removal of endothelium. Pretreatment with atropine (1 µM) and L-NAME (10 µM)

ablated this relaxation to acetylcholine (Fig.3.30A), confirming the validity of the

protocol. The crude extract also induced relaxation against phenylephrine precontractions

(Fig.3.30). The vasorelaxation was significantly (p < 0.01) ablated with atropine and L-

NAME. The EC50 values were 3.0 (1.0-5.0) and 3.3 mg/mL (1.3-5.3) (Fig.3.30B)

respectively. The crude extract vasorelaxant effect was also significantly decreased in

aortic rings with endothelium denudation (Fig.3.30B). All the fractions induced

endothelium-dependent and atropine and L-NAME sensitive vasorelaxation (Fig.3.31).

Crude and aqueous fractions have similar potency while other fractions were less potent.

High K+ used for sustained induction of contractions. Aortic ring incubated in normal

Kreb’s solution, extract also produced relaxation to the high K+ precontractions, similar

to verapamil (Fig.3.32).

76

0.0003 0.003 0.03 0.3 3

0

50

100

*

***

***

Intact (PE)

L-NAME

Atropine

Denuded

[Ach] g/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

A

B

Figure.3.30 Response of acetylcholine (A), (Es.Cr) (B) response precontracted with

phenylephrine (PE; 1 µM) and L-NAME (10 µM) atropine (1 µM) treated intact and

denuded aortic tissues (mean±SEM, n=5-7). ANOVA (Two-way) Bonferroni’s post hoc

analysis. *p < 0.05, ***p < 0.001 vs control.

0.003 0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded ***

*

***

10

[Es.Cr] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

77

A B

C D

Figure.3.31 Response of fractions of Eruca sativa in rat aorta with PE (1 µM)

precontractions and treatment of L-NAME (10 µM), atropine (1 µM) in intact and

denuded tissues (mean±SEM, n=5-7). [ANOVA (Two-way) analysis Bonferroni’s post

hoc] *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

0.003 0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded ***

***

***

***

*

10

[Es.n-hexane] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.003 0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded

**

***

***

***

10

[Es.Chlor] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.003 0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded

*

***

***

***

10

[Es.EtAc] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.001 0.01 0.1 1 10

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded ***

***

***

[Es.Aq] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

78

0.003 0.03 0.3 3

0

20

40

60

80

100

Es.Cr

Es.Chlor

Es.EtAc

Es.Aq

Es.n-hexane

10

[Concentration] mg/mL

% o

f K

+ (

80 m

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

20

40

60

80

100

Verapamil

[Concentration] M

% o

f K

+ (

80

mM

)

Co

ntr

acti

on

A

B

Figure.3.32 A and B show Eruca sativa crude extract, fractions and verapamil effect to

high K+ precontractions, in normal Kreb’s solution (mean±SEM, n=5-7).

79

Hypertensive rats

To have a good comparison, the extracts were tested parallel in hypertensive rats.

According to previous studies, high salt induces endothelial damage (Ni and Vaziri,

2001). This finding was confirmed when acetylcholine failed to induce relaxation

(Fig.3.33). The extracts induced relaxation of the phenylephrine precontraction at

comparatively higher concentration. This relaxation was not affected by atropine (1 µM)

and L-NAME (10 µM) (Fig.3.33; 34).

80

A

B

Figure.3.33 Acetylcholine and (Es.Cr) effect precontracted with phenylephrine (1 µM)

and pretreated [L-NAME (10 µM), atropine (1 µM)] in intact aortic and denuded rings

from hypertensive rats (mean±SEM, n=5-7).

0.003 0.03 0.3 3

0

50

100

Hypertensive

L-NAME

Atropine

10

[Es.Cr] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.0003 0.003 0.03 0.3 3

0

50

100

Hypertensive rat aorta

Atropine

L-NAME

[Ach] g/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

81

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAME

Atropine

0.3 3 50.03

[Es.n-hexane] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAME

Atropine

0.03 0.03 3 5

[Es.Chlor] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAME

Atropine

0.3 30.03 5

[Es.EtAc] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAME

Atropine

0.3 3 50.03

[Es.Aq] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

A B

C D

Fig.3.34 Eruca sativa fractions response with phenylephrine and L-NAME (10 µM),

atropine (1 µM) pretreatment in aorta of high salt-induced hypertensive rats (mean±SEM,

n=5-7).

82

0.001 0.01 0.1 1

0

20

40

60

80

100

Es.Cr

Es.n-hexane

Es.Chlor

Es.EtAc

Es.Aq

0.003 0.03 0.3


% o

f P

E (

1

M)

Co

ntr

ol

3.2.4.2 Eruca sativa and fractions effect on intracellular Ca+2

stores

To test the effect on intracellular Ca+2

stores, rat aorta incubated in Ca+2

-free/EGTA

medium. Es.Cr suppressed the phenylephrine individual peak formation (Fig.3.35A).

Fractions were also tested. The chloroform fraction (Es.Chlor) was the potent while crude

extract, n-hexane and aqueous fractions were comparable (Fig.3.35B).

A

B

Figure.3.35 Tracing (A) showing crude extract effect and graph (B) represents (crude

extract and fractions) on phenylephrine (PE) responses in Ca+2

-free/EGTA medium

(mean±SEM, n=5-7).

83

3.2.4.3 Erucin effect in aorta from normotensive and hypertensive rats

Erucin produced endothelium-independent vasorelaxation. In the normotensive rat aorta,

erucin produced 25% vasorelaxation against phenylephrine-induced contraction. This

relaxation was not significantly affected with L-NAME (10 µM), atropine (1 µM) and

denudation of aortic rings (Fig.3.36A).

Erucin produced a vasorelaxant effect to high K+. EC50 value was 1.68 µg/mL (1.06-2.30)

like verapamil (Fig.3.37).

Erucin was also tested parallel in rat aorta from high salt-induced hypertensive rats.

Erucin did not produce a significant vasorelaxation as shown in Fig.3.36B.

The effect of erucin was also studied on intracellular Ca+2

stores. Erucin suppressed the

phenylephrine individual peak formation up to 30% (Fig.3.38).

84

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAMEAtropine

0.03 0.3 3

[Erucin] g/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

A

B

Fig.3.36 Response of erucin with phenylephrine (1 µM) precontraction and pretreated by

L-NAME (10 µM) and atropine (1 µM) in normotensive (A) and high salt-induced

hypertensive (B) rat aorta.

0.01 0.1 1 10

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded

0.03 0.3 3

[Erucin] g/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

85

0.01 0.1 1 10

0

20

40

60

80

100

0.03 0.3 3

[Erucin] g/mL

% o

f K

+(8

0 m

M)

Co

ntr

ol

0.01 0.1 1 10

0

20

40

60

80

100

0.03 0.3 3

[Verapamil] g/mL

% o

f K

+(8

0 m

M)

Co

ntr

ol

A

B

Fig.3.37 Erucin and verapamil response against K+ in normotensive rats aorta

(mean±SEM, n=5-7).

86

0.001 0.01 0.1 1

0

20

40

60

80

100

Erucin

0.003 0.03 0.3

[Concentration] g/mL

% o

f P

E (

1

M)

Co

ntr

ol

A

B

Fig.3.38 Tracing and graph show the effect of different concentrations of erucin on

phenylephrine (PE) in calcium free medium.

87

3.2.5 Cardiac reactivity studies

Effect of crude, fractions and erucin was further evaluated in cardiac preparations.

3.2.5.1 Effect of Eruca sativa and fractions on rat right atrial rhythmic contractions

Right atria strips from normotensive rats, exhibited rhythmic contraction and relaxation,

where the cumulative addition of acetylcholine (0.01-10 µg/mL) suppressed rate

(negative chronotropic) and force (negative inotropic) of contraction. These effects were

abolished when atrial tissues were pretreated with atropine (1 µM) as shown in Fig.3.39,

confirming the validity of protocol. This was followed by testing the extract and

fractions. Crude extract decreased rate and force of spontaneously beating atria with EC50

values of 3.57 (1.10-6.04) and 7.39 mg/mL (5.34-9.44) as shown in Fig.3.49. However,

these effects remained same with atropine (1 µM) (Fig.3.39C). The n-hexane fraction

partially (70%) suppressed the rate of atrial contractions while induced an increase in

force of contraction (positive inotropic). Atropine (1 µM) pretreatment did not affect

either of these effects (Fig.3.40). Ethyl acetate and chloroform fractions induced partial

(Fig.3.41; 3.42) while the aqueous fraction produced complete suppression that remain

unchanged with atropine (1 µM) as shown in Fig.3.43.

88

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine

10

[Es.Cr] mg/mL

% o

f C

on

tro

l

A

B C

Figure.3.39 Tracing (A) and graphs (B, C) show acetylcholine and Es.Cr effect on

spontaneous rhythmic force and rate in rat right atria with and without atropine (1 µM)

(mean±SEM, n=5-7).

0.003 0.03 0.3 3

0

25

50

75

100

ForceRate

Force

Rate

Without atropine

With atropine

10

[Ach] g/mL

% o

f C

on

tro

l

89

0.003 0.03 0.3 3

0

25

50

75

100

125

Force

Rate

Force

Without atropine

With atropine (1 M)

Rate

10

[Es.n-hexane] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.40 Tracing (A) and graph (B) show n-hexane fraction of Eruca sativa (Es.n-

hexane) effect without and with atropine (1 µM) on spontaneous rhythmic force and rate

in isolated SD rat right atria (mean±SEM, n=5-7).

90

A

B

Figure.3.41 Tracing (A) and graph (B) show chloroform fraction of Eruca sativa

(Es.Chlor) response on spontaneous rhythmic force and rate in isolated SD rat right atria.

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine

10

[Es.Chlor] mg/mL

% o

f C

on

tro

l

91

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10

[Es.EtAc] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.42 Tracing (A) and graph (B) show Eruca sativa ethyl acetate fraction

(Es.EtAc) effect without and with atropine (1 µM) on spontaneous rhythmic force and

rate in isolated SD rat right atria (mean±SEM, n=5-7).

92

A

B

Figure.3.43 Tracing (A) and graph (B) show the aqueous fraction of Eruca sativa

(Es.Aq) effect with and without atropine (1 µM) on spontaneous rhythmic force and rate

in isolated rat atria.

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine

10

[Es.Aq] mg/mL

% o

f C

on

tro

l

93

0.001 0.01 0.1 1 10

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

[Erucin] g/mL

% o

f C

on

tro

l

3.2.5.2 Effect of erucin on rat atrial rhythmic contractions

Erucin decreased rate and force of spontaneously beating atria with EC50 values of 2.73

(1.03-4.43) and 3.16 (1.30-5.02) µg/mL as shown in Fig.3.44. However, these effects

remained same with atropine (1 µM) pretreatment (Fig.3.44).

A

B

94

Figure.3.44 Tracing (A) and graph (B) show erucin response in the absence and presence

of atropine (1 µM) in SD rat right atria on spontaneous rhythmic rate and force of

contraction (mean±SEM, n=5-7).

3.3 Phytochemical screening of Hedera helix

3.3.1 Analysis of preliminary phytochemicals

Following phytochemicals were identified (Table.3.8).

Table.3.8 The analysis of phytochemicals in Hedera helix crude extract.

Phytochemicals

Results

Alkaloids +++

Flavonoids +++

Glycosides ++

Phenols +++

Saponins +++

Tannins +

Terpenoids +

+ = Mild, ++ = Moderate and +++ = Strong presence of phytochemicals

95

3.3.2 Determination of total phenolic content

Hh.EtAc produced the highest yield of total phenolic content [(74.44 ± 2.65) mg GAE/g],

followed by Hh.Cholr, Hh.Aq, Hh.Cr and Hh.n-hexane fraction (Table.3.9).

3.3.3 Determination of total flavonoid content

Hh.EtAc produced (13.61 ± 0.08) mg QCE/g the highest yield, followed by Hh.Cholr,

Hh.Cr, Hh.Aq and Hh.n-hexane (Table.3.9).

Table.3.9 Total phenolic and flavonoid content of Hedera helix extracts.

Values expressed as mean±SEM (n=3).

Sample

Total phenolic content

(mg GAE/g)

Total flavonoid content

(mg QCE/g)

Crude extract (Hh.Cr) 26.66±2.20 2.82±0.05

n-hexane (Hh.n-hexane)

25.00±1.39 2.06±0.02

Chloroform fraction (Hh.Chlor)

42.50±2.93 7.56±0.10

Ethyl acetate fraction (Hh.EtAc)

74.44±2.65 13.61±0.08

Aqueous fraction (Hh.Aq)

39.16±0.48 2.25±0.05

96

3.3.4 HPLC analysis

3.3.4.1 Estimation of hederacoside C (HDC) in H. helix

Hederacoside C was detected in H. helix crude extract at retention time of 23 min when

compared to standard hederacoside C (Fig.3.45).

Figure.3.45 Chromatogram of hederacoside C (A) and Hedera helix (B) detected at

wavelength 210 nm.

97

3.4 Pharmacological investigation of Hedera helix

Two types of pharmacological studies were conducted; in-vivo and in-vitro. In the in-vivo

studies, safety study, antihyperlipidemic and antihypertensive evaluation was carried out.

While in the in-vitro studies, rat aorta and atria were used.

3.4.1 Safety study

The safety of Hh.Cr was up to 5 g/kg.

3.4.2 Antihyperlipidemic activities

3.4.2.1 Serum lipid profile of hyperlipidemic (TI) SD rats

After the treatment with Hh.Cr at different doses and lovastatin (10 mg/kg), SD rats were

anaesthetized and blood was analyzed for serum lipid profile.


The control group received normal diet. The lipid profile determined was; serum total

cholesterol (TC); 100.40±3.13, triglyceride (TG); 85.25±2.25, low density lipoprotein

(LDL); 38.80±1.37, high density lipoprotein (HDL); 59.50±3.13 mg/dL (Fig.3.46A).

Atherogenic index (A.I) was calculated and found 0.68±0.05 (Fig.3.49 and Table.3.10).

Hyperlipidemic control group (tyloxapol 500 mg/kg)

Lipid profile determined was TC; 355.33±8.31, TG; 715.66±9.54 (p < 0.001), LDL;

71.16±4.28, HDL; 68.25±3.12 (Fig.3.46B). Atherogenic index (A.I) was calculated and

found 4.20±0.62 (p < 0.001) as compared to untreated rats (Fig.3.49 and Table.3.10).

Hh.Cr (30 mg/kg) treated group

Lipid profile was TC; 247.36±3.43, TG; 412.33±9.73, LDL; 60.48±3.25, HDL;

65.43±2.22 mg/dL, A.I; 2.79±0.71 (p < 0.01) compared with hyperlipidemic control

(Fig.3.47A; Fig.3.49A; Table.3.10).


The lipid profile was found TC; 180.54±5.74, TG; 362.36±4.05, LDL; 51.81±1.48, HDL;

66.06±1.36 mg/dL (Fig.3.47B), A.I; 1.73±0.07 (p < 0.01) compared with hyperlipidemic

control (Fig.3.49A and Table.3.10).


Hh.Cr 300 mg/kg treatment caused TC; 140.18±2.86, TG; 312.34±5.97 (p < 0.001), LDL;

40.51±3.24, HDL; 67.19±2.44 mg/dL (Fig.3.47C), A.I; 1.09±0.10 (p < 0.001) in

comparison to hyperlipidemic group (Fig.3.49A and Table.3.10).

98


Lovastatin (10 mg/kg) resulted in a significant decrease in TC; 154.26±3.95 and TG;

180.43±4.50 (p < 0.001), LDL; 50.300±3.30, HDL; 61.36±4.31 mg/dL (Fig.3.47D), A.I;

1.51±0.30 (p < 0.001) (Fig.3.49 and Table.3.10).

HDC (2.5 mg/kg) treated group

The results of lipid profile of HDC at 2.5 mg/kg treatment were TC; 175.33±2.33, TG;

214.33±7.44 (p < 0.001), LDL; 52.23±1.62, HDL; 61.16±0.88 mg/dL (Fig.3.48A), A.I;

1.84±0.07 (p < 0.01) (Fig.3.49B and Table.3.10).

HDC (5 mg/kg) treated group

HDC treated group showed TC; 162.50±2.29, TG; 190.11±7.67 (p < 0.001), LDL;

43.07±1.79, HDL; 62.33±1.20 mg/dL (Fig.3.48B), A.I; 1.60±0.02 (p < 0.001) (Fig.3.49B

and Table.3.10).

99

0

200

400

600

800

TCTGLDLHDL

Normal control

Co

nce

ntr

atio

n (

mg/d

L)

A

B

Figure.3.46 Bar diagrams show lipid profile of normal control (A) and (tyloxapol-

induced) (TI) hyperlipidemic (B) SD rats (mean±SEM, n=5-7). ***p < 0.001 vs normal

control.

0

200

400

600

800

TC

TGLDLHDL

***

***

Hyperlipidemic (TI)

Co

nce

ntr

atio

n (

mg/d

L)

100

A B

C D

Figure.3.47 Bar diagrams show Hedera helix (Hh.Cr) effect of 30 (A), 100 (B), 300

mg/kg (C) and lovastatin 10 mg/kg (D) on lipid profile in tyloxapol-induced

hyperlipidemic SD rats (mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs

hyperlipidemic control.

0

200

400

600

800

TC

TG

LDL

HDL

Hh.Cr (30 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

*

**

0

200

400

600

800TC

TG

LDL

HDL

Hh.Cr (100 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

**

***

0

200

400

600

800

TC

TG

LDL

HDL

***

***

Hh.Cr (300 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

0

200

400

600

800TC

TG

LDLHDL

******


Co

nce

ntr

atio

n (

mg/d

L)

101

A

B

Figure.3.48 Bar diagrams show effect of hederacoside C (HDC) at doses of 2.5 (A) and 5

(B) mg/kg on TC, LDL, TG and HDL in tyloxapol-induced hyperlipidemic SD rats

(mean±SEM, n=5-7). **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

0

200

400

600

800

TC

TG

LDL

HDL

HDC (2.5 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

*****

0

200

400

600

800

TC

TG

LDL

HDL

******

HDC (5 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

102

A B

Figure.3.49 Bar diagrams show atherogenic index of normal control, hyperlipidemic

(tyloxapol-induced) (TI) group and crude extract of Hedera helix (Hh.Cr) at doses of 30,

100, 300 mg/kg (A) and hederacoside C (HDC) at doses of 2.5, 5 mg/kg (B) and

lovastatin 10 mg/kg treated TI hyperlipidemics SD rats (mean ± SEM, n=6-7) **p < 0.01,

***p < 0.001 vs hyperlipidemic control.

Nor

mal

con

trol

Hyp

erlip

idem

ic (T

I)

HD

C 2

.5

HD

C 5

Lovas

tatin

10

0

2

4

6

*****

***

Ath

ero

genic

Ind

ex (

A.I

)

Nor

mal

con

trol

Hyp

erlip

idem

ic (T

I)

Hh.

Cr 3

0

Hh.

Cr 1

00

Hh.

Cr 3

00

Lovas

tatin

10

0

2

4

6

*** ***

***

**

Ath

ero

gen

ic I

nd

ex (

A.I

)

103

Table.3.10 Response by Hedera helix (Hh.Cr) and hederacoside C (HDC) on serum lipid profile in tyloxapol-induced (TI)

hyperlipidemic Sprague-Dawley rats.

NC: Normal control, TI: Tyloxapol-induced (One-way ANOVA Dunnet’s multiple comparison)

Values were expressed as mean ± SEM. (n=6-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

Lipid Profile

Groups

Total cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Low density

lipoproteins

(mg/dL)

High density

lipoproteins

(mg/dL)

Atherogenic

index

NC 100.40±3.13

85.25±2.25

38.80±1.37

59.50±3.13

0.68±0.05

Hyperlipidemic (TI) 355.33±8.31

715.66±9.54

71.16±4.28

68.25±3.12

4.20±0.62

TI+ Hh.Cr 30 mg/kg 247.36±3.43*

412.33±9.73**

60.48±3.25

65.43±2.22

2.79±0.71**

TI+ Hh.Cr 100 mg/kg 180.54±5.74**

362.36±4.05***

51.81±1.48

66.06±1.36

1.73±0.07***

TI+ Hh.Cr 300 mg/kg 140.18±2.86***

312.34±5.97***

40.51±3.24

67.19±2.44

1.09±0.10***

TI+Lovastatin10 mg/kg 154.26±3.95***

180.43±4.50***

50.30±3.30

61.36±4.31

1.51±0.30***

TI+ HDC 2.5 mg/kg 175.33±2.33** 214.33±7.44***

52.23±1.62

61.16±0.88

1.84±0.07**

TI+ HDC 5 mg/kg 162.50±2.29***

190.11±7.67*** 43.07±1.79

62.33±1.20

1.60±0.02***

104

3.4.2.2 Serum lipid profile of hyperlipidemic (HFD) SD rats

After 8 weeks of treatment with high fat diet, SD rats were anaesthetized; blood was

analyzed for lipid profile group wise as stated below.


The lipid profile of normal rats was serum total cholesterol (TC); 104.03±3.21,

triglyceride (TG); 60.30±3.14, low density lipoprotein (LDL); 38.30±2.12, high density

lipoprotein (HDL) 50.36±1.71 mg/dL and atherogenic index (A.I); 1.06±0.21 (Fig.3.50A;

3.53 and Table.3.11).


The lipid profile determined; TC; 361.33±2.54, TG; 81.25±3.30, LDL: 195.37±3.90,

HDL; 24.25±1.82 mg/dL and atherogenic index (A.I); 13.90±3.21 (Fig.3.50B; 3.53 and

Table.3.11).


Lipid profile was; TC; 135.80±2.57, TG; 79.33±4.05 and LDL; 78.16±5.37 (p <0.001),

HDL; 29.46±1.77 mg/dL, A.I; 3.64±0.28 (p < 0.001) compared with hyperlipidemic

control (Fig.3.51A; 3.53A and Table.3.11).


H. helix treatment caused decrease in TC; 115.70±3.05, TG; 76.43±5.16, LDL;

65.93±3.33 (p <0.001), HDL; 30.06±1.73 mg/dL, A.I; 2.86±0.19 (p < 0.001) vs

hyperlipidemic group (Fig.3.51B; 3.53A and Table.3.11).


Lipid profile was TC; 100.20±2.83, TG; 67.53±1.33, LDL; 56.16±2.40 (p <0.001), HDL;

46.26±2.11 (p < 0.01) mg/dL, A.I; 1.17±0.15 (p < 0.001) compared to hyperlipidemic rats

(Fig.3.51C; 3.53A and Table.3.11).


Treatment with lovastatin (10 mg/kg) resulted in a significant decrease in TC;

93.60±3.54, TG; 62.25±2.60, LDL; 48.63±2.10 (p <0.001), HDL; 40.23±3.15 (p < 0.05)

mg/dL, A.I; 1.32±1.51 (p < 0.001) in comparison with hyperlipidemic rats (Fig.3.51D;

3.53A and Table.3.11).

105


Lipid profile was TC; 149.20± 3.87, TG; 70.90±2.08, LDL; 51.50±3.41 (p <0.001), HDL;

29.66±1.76 mg/dL, A.I; 4.05±0.17 (p < 0.01) compared with hyperlipidemic control

(Fig.3.52A; 3.53B and Table.3.11).


Treatment with HDC 5 mg/kg caused TC; 120.30±2.77, TG; 62.16±2.57, LDL;

40.60±2.73 (p <0.001), HDL; 36.06±2.06 mg/dL, A.I; 2.36±0.23 (p < 0.001) compared to

hyperlipidemic (Fig.3.52B; 3.53B and Table.3.11).

106

A

B

Figure.3.50 Bar diagrams show lipid profile of control (A) and high fat diet (HFD)-

induced hyperlipidemic (B) rats (mean±SEM, n=5-7). ***p < 0.001 vs normal control.

0

100

200

300

400

TC

TGLDLHDL

Normal control

Co

nce

ntr

atio

n (

mg/d

L)

0

100

200

300

400 TC

TGLDLHDL

***

***

Hyperlipidemic (HFD)

Co

nce

ntr

atio

n (

mg/d

L)

107

A B

C D

Figure.3.51 Bar diagrams show Hh.Cr effect at doses of 30 (A), 100 (B), 300 (C) mg/kg

and lovastatin (D) 10 mg/kg on high fat diet-induced hyperlipidemic SD rats lipid profile

(mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

0

50

100

150

200

250

300

350

400

TC

TG

LDL

HDL

Hh.Cr (30 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

***

0

50

100

150

200

250

300

350

400

TC

TG

LDL

HDL

Hh.Cr (100 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

***

0

50

100

150

200

250

300

350

400

TC

TG

LDL

HDL

Hh.Cr (300 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

*** *

0

50

100

150

200

250

300

350

400TC

TG

LDLHDL

***

*** *


Co

nce

ntr

atio

n (

mg/d

L)

108

0

100

200

300

400

TC

TG

LDL

HDL

HDC (2.5 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

***

***

A

B

Figure.3.52 Bar diagrams show effect of hederacoside C (HDC) at doses of 2.5 (A) and 5

(B) mg/kg on the lipid profile of high fat diet-induced hyperlipidemic SD rats

(mean±SEM, n=5-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

0

100

200

300

400

TC

TG

LDL

HDL

***

***

HDC (5 mg/kg)

Co

nce

ntr

atio

n (

mg/d

L)

109

A B

Figure.3.53 Bar diagrams show atherogenic index of normal control, high fat diet

(HFD)-induced hyperlipidemic and crude extract of Hedera helix (Hh.Cr) at doses of

30, 100, 300 mg/kg (A), hederacoside C (HDC) at doses of 2.5, 5 mg/kg (B) and

lovastatin 10 mg/kg treated high fat diet-induced hyperlipidemic SD rats (mean±SEM,

n=5-7)*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

Nor

mal

con

trol

Hyp

erlip

idem

ic (H

FD)

Hh.

Cr 3

0

Hh.

Cr 1

00

Hh.

Cr 3

00

Lovas

tatin

10

0

5

10

15

20

******

*** ***

Ath

ero

genic

Ind

ex (

A.I

)

Nor

mal

con

trol

Hyp

erlip

idem

ic (H

FD)

HD

C 2

.5

HD

C 5

Lovas

tatin

10

0

5

10

15

20

***

***

***

Ath

ero

genic

Ind

ex (

A.I

)

110

Table.3.11 Response by Hedera helix (Hh.Cr) and hederacoside C on serum lipid profile in hyperlipidemic Sprague-Dawley rats.

NC: Normal control, HFD: High fat diet (One-way ANOVA analysis followed by Dunnet’s multiple comparison)

Values were expressed as mean ± SEM (n=6-7).*p < 0.05, **p < 0.01, ***p < 0.001 vs hyperlipidemic control.

Lipid Profile

Groups

Total cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Low density

lipoproteins

(mg/dL)

High density

lipoproteins

(mg/dL)

Atherogenic

index

NC

104.03±3.21

60.30±3.14

38.30±2.12

50.36±1.71

1.06±0.21

Hyperlipidemic (HFD) 361.33±2.54

81.25±3.30 195.37±3.90

24.25±1.82

13.90±3.25

HFD+Hh.Cr 30 mg/kg 135.80±2.57***

79.33±4.05

78.16±5.37***

29.46±1.77

3.64±0.28***

HFD+Hh.Cr 100 mg/kg 115.70±3.05***

76.43±5.16

65.93±3.33***

30.06±1.73

2.86±0.19***

HFD+Hh.Cr 300 mg/kg 100.20±2.83*** 67.53±1.33

56.16±2.40***

46.26±2.11* 1.17±0.15***

HFD+Lovastatin10 mg/kg 93.60±3.54*** 62.25±2.60

48.63±2.10*** 40.23±3.15*

1.32±1.51***

HFD+ HDC 2.5 mg/kg 149.20± 3.87***

70.90±2.08

51.50±3.41***

29.66±1.76

4.05±0.17***

HFD+ HDC 5 mg/kg 120.30±2.77***

62.16±2.57

40.60±2.73***

36.06±2.06 2.36±0.23***

111

3.4.2.3 Histopathological examination of hyperlipidemic (HFD) SD rats

Thoracic aortae from all groups were studied for any histopathological changes.

Normal control

Aorta of normal control group composed of tunica intima, tunica media and tunica

adventitia. In tunica intima endothelium was found intact. Smooth muscle cell and

elastic lamina in tunica media and connective tissues in tunica adventitia had a normal

arrangement giving normal thickness of the aorta (Fig.3.54A).


Photomicrograph of the aortae of rats showed infiltration of macrophages. Lipid

droplets, vacuoles and foam cells were observed under the tunica intima. The elastic

membranes had a loose arrangement with an increase in matrix, leading to increase

thickness of aorta (Fig.3.54B).


Photomicrograph of the aorta showed decreased smooth muscle cells proliferation in

the tunica media. There was a reduction in thickness of aorta (Fig.3.54C) compared to

the hyperlipidemic rat aorta.


Photomicrograph of the aorta showed no macrophages infiltrations. The thickness of

aorta was near to normal (Fig.3.54D).


Aorta from hyperlipidemic rats with Hh.Cr 300 mg/kg/day treatment showed almost

normal structure of aortic intima, media and adventitia with no fatty degeneration.

The thickness of aorta was normal (Fig.3.55E).


Photomicrograph of lovastatin treated hyperlipidemic rat aorta showed reduced lipid

deposits and macrophages infiltration. The tunica media and tunica intima had normal

arrangement (Fig.3.55F).

Hederacoside C (2.5 mg/kg) treated group

In aorta from animals with HDC 2.5 mg/kg treatment showed reduced thickness of

aorta with no macrophages infiltration (Fig.3.55G).

Hederacoside C (5 mg/kg) treated group

In aorta from animals treated with HDC 5 mg/kg showed improved thickness of aorta

with small lipid deposits (Fig.3.55H).

112

Figure.3.54 Photomicrographs show histological examination of normal control (A),

hyperlipidemic (B), Hedera helix crude extract (Hh.Cr) 30 (C) and 100 mg/kg (D)

treated SD rats aortae. Macrophages appear as large nucleus with vacoulated

cytoplasm while fat deposits appear as fat droplets with no distinct membrane.

A

B

C

D

113

Figure.3.55 Photomicrographs histological examination of Hedera helix crude extract

(Hh.Cr) 300 (E) and lovastatin (10 mg/kg) (F) and hederacoside C at doses of 2.5 (G),

5 mg/kg (H) treated SD rats aortae. Macrophages appear as large nucleus with

vacoulated cytoplasm while fat deposits appear as fat droplets with no distinct

membrane.

E

F

G

H

114

3.4.2.4 Vascular function study of hyperlipidemic (HFD) SD rats

For vascular function study, thoracic aortae from all groups extracts, standard and

HDC treated were used.


Isolated rat aorta of normal control group pre-contracted with phenylephrine showed

complete relaxation to Ach (Fig.3.72A). The EC50 value is 0.07 µg/mL (0.03-0.12)

(Fig.3.56B).


There was no relaxation compared to 100% in the normal control group (Fig.3.57).


Aorta isolated from this group showed concentration-dependent relaxant effect that

reached to 45% at the maximum 3 mg/mL (Fig.3.58).


In the 100 mg/kg/day dose treated HFD rats, isolated rat aortic rings relaxed to about

75% in response to different concentrations of acetylcholine (Fig.3.59).


Aorta isolated showed relaxation to phenylephrine (1 µM) precontraction with EC50

value; 0.12 µg/mL (0.08-0.16) (Fig.3.60).


Aorta isolated from HFD rats treated with lovastatin, showed 51% of relaxation

(Fig.3.61).


In aorta from the HFD rats with HDC 2.5 mg/kg treatment, Ach induced relaxation

was 30% (Fig.3.62).


Aorta isolated from HDC 5 mg/kg treated HFD rats, showed complete relaxation

comparable to normal rat aorta (Fig.3.63).

115

0.003 0.03 0.3 3

0

50

100

Normal control

[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B


phenylephrine (1 µM) contraction from the normal rat aorta (mean±SEM, n=5-7).

116

0.003 0.03 0.3 3

0

25

50

75

100

Hyperlipidemic (high fat diet)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


phenylephrine (PE; 1µM) in rat aortic rings from hyperlipidemic (high fat diet)

control (mean±SEM, n=5-7).

117

0.003 0.03 0.3 3

0

25

50

75

100

HFD + Hh.Cr (30 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


(PE; 1µM) precontraction in rat aorta isolated from Hh.Cr; 30 mg/kg treated group

(mean±SEM, n=5-7).

118

0.003 0.03 0.3 3

0

25

50

75

100


[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B

Figure.3.59 Tracing (A) and graph (B) show acetylcholine effect in rat aorta from

high fat diet treated with Hedera helix crude extract (Hh.Cr; 100 mg/kg) (mean±SEM,

n=5-7).

119

0.003 0.03 0.3 3

0

25

50

75

100


[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B


(PE; 1µM)-contraction in rat aortic tissues from high fat diet treated with Hedera

helix crude extract (Hh.Cr; 300 mg/kg/day) (mean±SEM, n=5-7).

120

0.003 0.03 0.3 3

0

25

50

75

100

HFD + Lovastatin (10 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B

Figure.3.61 Tracing (A) and graph (B) show acetylcholine response in isolated aortic

rings from high fat diet treated with lovastatin (10 mg/kg/day) (mean±SEM, n=5-7).

121

0.003 0.03 0.3 3

0

25

50

75

100

HFD+ HDC (2.5 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Contr

ol

A

B


phenylephrine contraction in isolated aortic rings from high fat diet treated with

hederacoside C (HDC) (2.5 mg/kg) (mean±SEM, n=5-7).

122

0.003 0.03 0.3 3

0

25

50

75

100

HFD + HDC (5 mg/kg)

[Ach] g/mL

% o

f P

E (

1

M)

Con

tro

l

A

B

Figure.3.63 Tracing (A) and graph (B) show acetylcholine effect in aortic rings from

high fat diet treated with hederacoside C (HDC) (5 mg/kg) (mean±SEM, n=5-7).

123

3.4.3 Antihypertensive activities

3.4.3.1 Mean arterial pressure (MAP) of normotensive rats

Before injecting crude extract, effect of norepinephrine and acetylcholine was

observed (Fig.3.64).

Crude extract of H. helix (Hh.Cr)

The percent fall in MAP with Hh.Cr at 1, 3, 10 and 30 mg/kg was 13.41±1.11,

33.47±1.87, 42.83±2.22 and 58.59±0.02 mmHg, respectively (Fig.3.65).

Following fractions of H. helix were tested.

n-hexane fraction (Hh.n-hexane)

The percent fall in MAP with n-hexane fraction at same doses was 6.05±2.74,



The percent fall in MAP with chloroform fraction (Hh.Chlor) was 12.68±1.33,



The percent fall in MAP with ethyl acetate fraction (Hh.EtAc) was 11.55±2.42,



The percent fall in MAP with aqueous fraction (Hh.Aq) was 19.80±5.19, 37.59±2.16,

59.48±3.36 and 62.11±4.45 mmHg (Fig.3.69).

To see if muscarinic receptor stimulation played a role in blood pressure lowering

effect, rats were pretreated with atropine (1 mg/kg). However, it did not change effect

on MAP (data not shown).

124

-80

-60

-40

-20

0

20

40

60

80NE (1 g/kg)

Ach (1 g/kg)% F

all

in M

AP

%

Ris

e in

MA

P

A B

Figure.3.64 Tracing (A) and graph (B) showing norepinephrine (NE) and

acetylcholine (Ach) response on mean arterial pressure (MAP) from normotensive

rats (mean±SEM, n=5-7).

125

A

B

Figure.3.65 Tracing (A) and graph (B) represent the Hedera helix crude extract

(Hh.Cr) response on normotensive rats MAP. Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001.

1 3 10 30

0

25

50

75

Hh.Cr [mg/kg]

% F

all

in M

AP

*

**

**

***

126

1 3 10 30

0

25

50

75

Hh.n-hexane [mg/kg]

% F

all

in M

AP

*

**

**

A

B

Figure.3.66 Tracing (A) and graph (B) showing mean arterial pressure of n-hexane

fraction in normotensive rats. Compared with pretreated values, *p < 0.05, **p <

0.01, ***p < 0.001.

127

1 3 10 30

0

25

50

75

Hh.Chlor [mg/kg]

% F

all

in M

AP

*

*

**

**

A

B

Fig.3.67 Tracing (A) and graph (B) chloroform fraction (Hh.Chlor) on fall of mean

arterial pressure. Compared with pretreated values, *p < 0.05, **p < 0.01, ***p <

0.001.

128

1 3 10 30

0

25

50

75

Hh.EtAc [mg/kg]

% F

all

in M

AP

*

**

**

***

A

B

Fig.3.68 Tracing (A) and graph (B) show an effect on mean arterial pressure by

Hedera helix ethyl acetate fraction (Hh.EtAc). Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001.

129

1 3 10 30

0

25

50

75

100

Hh.Aq [mg/kg]

% F

all

in M

AP

*

**

******

A

B

Fig.3.69 Tracing (A) and graph (B) represent MAP of Hedera helix aqueous fraction

(Hh.Aq). Compared with pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.

130

3.4.3.2 Hypertensive rats

High salt resulted in a significant increase in MAP (Fig.3.70).

H. helix extract (Hh.Cr)

MAP with Hh.Cr (1, 3, 10 and 30 mg/kg) was 24.45±0.86, 47.84±4.41, 57.05± 2.78

and 67.53±3.07 mmHg, respectively. The effect was statistically significant,

especially at 30 mg/kg (p < 0.001) vs control values (Fig.3.71). Following fractions

were also tested parallel.

n-hexane fraction (Hh.n-hexane)

The percent fall in MAP was 17.96±2.03, 24.03±4.52, 34.55±6.35 and 43.29±1.38

mmHg, respectively (Fig.3.72).


The percent fall in MAP with chloroform fraction (Hh.Chlor) was 15.18±0.31,



The percent fall in MAP with ethyl acetate fraction (Hh.EtAc) was 9.44±1.35,



The percent fall in MAP with aqueous fraction (Hh.Aq) was 23.97±1.37, 46.69±5.14,

60.53±2.96 and 71.93±1.21 mmHg (Fig.3.75).

131

0

50

100

150

200


Hypertensive (n=5-7) **

Groups

MA

P (

mm

Hg)

Figure.3.70 Graph shows the MAP of normal and hypertensive rats (mean±SEM,

n=5-7) **p < 0.01 vs hypertensive.

132

1 3 10 30

0

30

60

90

Hh.Cr [mg/kg]

% F

all

in M

AP

*

**

**

***

A

B

Figure.3.71 Tracing (A) and graph (B) showing Hedera helix crude extract (Hh.Cr)

effect on MAP in hypertensive (high salt) rats. Compared with pretreated values, *p <

0.05, **p < 0.01, ***p < 0.001.

133

1 3 10 30

0

30

60

90

Hh.n-hexane [mg/kg]

% F

all

in M

AP

*

*

*

**

A

B

Figure.3.72 Tracing (A) and graph (B) showing effect of Hh.n-hexane on MAP in

hypertensive rats under anaesthesia. Compared with pretreated values, *p < 0.05, **p

< 0.01, ***p < 0.001.

134

1 3 10 30

0

30

60

90

Hh.Chlor [mg/kg]

% F

all

in M

AP

*

*

**

***

A

B

Fig.3.73 Tracing (A) and graph (B) showing chloroform fraction of Hedera helix

(Hh.Chlor) effect on MAP in hypertensive rats under anaesthesia. Compared with

pretreated values, *p < 0.05, **p < 0.01, ***p < 0.001.

135

1 3 10 30

0

30

60

90

Hh.EtAc [mg/kg]

% F

all

in M

AP

*

**

***

A

B

Fig.3.74 Tracing (A) and graph (B) show Hedera helix ethyl acetate fraction

(Hh.EtAc) effect on MAP in hypertensive rats under anaesthesia. Compared with


136

1 3 10 30

0

30

60

90

Hh.Aq [mg/kg]

% F

all

in M

AP

*

**

**

***

A

B

Fig.3.75 Tracing (A) and graph (B) showing an aqueous fraction of Hedera helix

(Hh.Aq) effect on MAP in hypertensive rats under anaesthesia. Compared with


3.2.3.5 Hederacoside C (HDC) response on mean arterial pressure (MAP) of

normotensive and hypertensive (high salt) rats

137

Hederacoside C, a triterpenoid saponin of Hedera helix, response on MAP was

measured. Fall in MAP at 1, 3, 10, 30 µg/kg with respective values; 26.75±1.25,

39.10±1.10, 45.65±0.35 and 57.85±2.85 mmHg (Fig.3.76).

Pretreatment with atropine (1 mg/kg) did not change effect of hederacoside C on

MAP in normotensive rats.

The MAP in hypertensive rats was 39.55±1.45, 49.20±2.20, 57.10±1.90, 68.65±3.65

mmHg (Fig.3.76).

138

A

B

Figure.3.76 Tracing (A) and graph (B) represent effect of hederacoside C (HDC) on

MAP in normotensive and hypertensive rats.*p < 0.05, **p < 0.01 vs hypertensive.

1 3 10 30

0

20

40

60

80Normotensive

Hypertensive

**

*

*

*

Hederacoside C [g/kg]

% F

all

in M

AP

(m

mH

g)

139

3.2.4 Vascular reactivity studies

Hedera helix crude extract, fractions and hederacoside C was further evaluated in

vascular preparations.

3.2.4.1 Response in aortic tissues of normotensive and hypertensive rats

Normotensive rats

Crude extract induced relaxation against phenylephrine precontractions with EC50

value of 0.30 mg/mL (0.12-0.48). This relaxation was significantly (p < 0.01) ablated

by L-NAME and denudation of aortic rings unaffected by pretreatment with atropine

(Fig.3.77). All the fractions induced endothelium-dependent and L-NAME sensitive

vasorelaxation with varying potencies (Fig.3.78).

It also produced vasorelaxation to K+, as verapamil (Fig.3.79).

Hypertensive rats

All extracts induced relaxation of the phenylephrine precontractions at a

comparatively higher concentration (Fig.3.80; 81). This relaxation was unchanged by

atropine and L-NAME (Fig.3.80; 81).

140

Fig.3.77 Vasorelaxant effect of Hedera helix crude extract (Hh.Cr) in rat aorta

precontracted with phenylephrine (1 μM) in intact and denuded tissues. [ANOVA

(Two-way) Bonferroni’s post hoc] *p < 0.05, ***p < 0.001.

0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded***

***

***

*

0.003 10

[Hh.Cr] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

141

0.003 0.03 0.3 3

0

50

100

Intact

L-NAME

Atropine

Denuded

***

***

**

***

10

[Hh.Chlor] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s0.003 0.03 0.3 3

0

50

100

Intact

L-NAME

Atropine

Denuded***

***

***

10

[Hh.n-hexane] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

A B

C D

Figure.3.78 Vasorelaxant effect of fractions of Hedera helix (A-D), in rat aorta with

phenylephrine (1 μM) contractions. [ANOVA (Two-way) Bonferroni’s post hoc] **p

< 0.01, ***p < 0.001 vs control.

0.003 0.03 0.3 3

0

50

100

Intact

L-NAME

Atropine

Denuded

10

[Hh.EtAc] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.001 0.01 0.1 1 10

0

20

40

60

80

100

Intact

L-NAME

Atropine

Denuded***

***

***

[Hh.Aq] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

142

0.01 0.1 1 10

0

20

40

60

80

100

Verapamil

[Concentration] M

% o

f K

+ (

80

mM

)

Co

ntr

acti

on

0.001 0.01 0.1 1 10

0

25

50

75

100

Hh.Cr

Hh.n-hexane

Hh.EtAc

Hh.Aq

Hh.Chlor


% o

f K

+ (

80

mM

)-in

du

ced

Co

ntr

acti

on

s

A

B

Figure.3.79 A and B show Hedera helix extracts and verapamil response on high K+

precontractions, in normal Kreb’s solution.

143

Figure.3.80 Vasorelaxant effect of Hedera helix crude extract precontracted with

phenylephrine (1 µM) in hypertensive rat aorta (mean±SEM, n=5-7).

0.01 0.1 1 10

0

20

40

60

80

100

Hypertensive

L-NAME

Atropine

0.03 0.3 3 5

[Hh.Cr] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

144

A B

C D

Figure.3.81 Response of Hedera helix fractions (A-D) against phenylephrine (1 µM)

in rat aorta from hypertensive rats.

0.01 0.1 1 10

0

20

40

60

80

100

Hypertensive

L-NAMEAtropine

0.03 0.3 3 5

[Hh.n-hexane] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

20

40

60

80

100

Hypertensive

L-NAMEAtropine

0.03 0.3 3 5

[Hh.Chlor] mg/mL%

of

PE

(1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

20

40

60

80

100

Hypertensive

L-NAMEAtropine

0.03 0.3 3 5

[Hh.EtAc] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

20

40

60

80

100

L-NAMEAtropine

Hypertensive

0.03 0.3 3 5

[Hh.Aq] mg/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

145

0.001 0.01 0.1 1

0

25

50

75

100

Hh.Cr

Hh.n-hexane

Hh.EtAc

Hh.Aq

Hh.Chlor


% o

f P

E (

1

M)

Co

ntr

ol

3.2.4.2 Effect of Hedera helix and fractions on intracellular Ca+2

stores

To test the intracellular Ca+2

stores effect, aortic rings were incubated in Ca+2

-

free/EGTA medium. Crude extract completely suppressed the phenylephrine

individual peak formation (Fig.3.82). Fractions were also tested. The ethyl acetate

(Hh.EtAc) was the least potent while the chloroform and n-hexane were comparable

(Fig.3.82).

A

B

Figure.3.82 Shows Hedera helix crude extract (Hh.Cr) and its fractions on

phenylephrine (PE) peak formation in Ca+2

-free/EGTA medium in isolated rat aorta

from normal rats (mean±SEM, n=5-7).

146

3.2.4.3 Effect of hederacoside C (HDC)

Hederacoside C produced vasorelaxation against phenylephrine-induced contraction

[EC50 was 0.28 µg/mL (0.12-0.44)]. This relaxation was significantly affected with L-

NAME (10 µM) and the denudation while remaining unchanged with atropine (1 µM)

(Fig.3.83). Hederacoside C also produced a vasorelaxant effect [EC50:1.68 µg/mL

(1.00-2.40)] like verapamil (Fig.3.84). Hederacoside C was also tested parallel in rat

aorta from hypertensive rats, produced endothelium independent relaxation

(Fig.3.83B).

Hederacoside C effect was also studied on intracellular Ca+2

stores. Hederacoside C

completely suppressed the phenylephrine peak formation at a concentration of 5

µg/mL (Fig.3.85).

147

A

B

Fig.3.83 Endothelium-independent vasorelaxant response of hederacoside C (HDC)

precontracted with phenylephrine (1 µM) in aorta from normotensive (A) and

hypertensive (B) rats (mean±SEM, n=5-7).

0.003 0.03 0.3 3

0

50

100

Intact (PE)

L-NAME

Atropine

Denuded

10

[Hederacoside C] g/mL

% o

f P

E (

1

M)

Co

ntr

acti

on

s

0.01 0.1 1 10

0

50

100

Hypertensive

L-NAME

Atropine

0.03 0.3 0.3


% o

f P

E (

1

M)

Co

ntr

acti

on

s

148

0.01 0.1 1 10

0

20

40

60

80

100

0.03 0.3 3

[Verapamil] g/mL

% o

f K

+ (

80

mM

) C

on

tro

l

0.01 0.1 1 10

0

20

40

60

80

100

0.03 0.3 3


% o

f K

+ (

80 m

M)

Co

ntr

ol

A

B

Figure.3.84 Hederacoside C (HDC) (A) and verapamil (B) effect against K+ in aortic

tissues from normal rats (mean±SEM, n=5-7).

149

A

B

Figure.3.85 Tracing and graph show hederacoside C (HDC) effect on phenylephrine

(PE) peak formation in Ca+2

-free/EGTA medium in normal rat aorta (mean±SEM,

n=5-7).

0.001 0.01 0.1 1

0

25

50

75

100

Hederacoside C

[Concentration] g/mL

% o

f P

E (

1

M)

Co

ntr

ol

150

3.2.5 Cardiac reactivity studies

Effect of crude, fractions and hederacoside C was further evaluated in cardiac

preparations.

3.2.5.1 Effect of Hedera helix and fractions on rat right atrial rhythmic

contractions

Right atria strips from normotensive rats, exhibited rhythmic contraction and

relaxation. Crude extract decreased both rate and force of spontaneously beating right

atria [EC50 values was 0.15 (0.05-0.25) and 0.56 (0.34-0.78) mg/mL] as shown in

Fig.3.86. However, these effects were not affected by atropine (1 µM) pretreatment.

All fractions decreased force and rate of contractions. All fractions effect remained

unchanged with atropine (1 µM) (Fig.3.87; 3.88; 3.89; 3.90).

151

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10

[Hh.Cr] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.86 Tracing (A) and concentration response curves (B) show the effect of

Hedera helix crude extract (Hh.Cr) on spontaneous rhythmic force and rate in SD rat

right atria in atropine (1 µM) absence and presence (mean±SEM, n=5-7).

152

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10

[Hh.n-hexane] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.87 Tracing (A) and graph (B) show n-hexane fraction of Hedera helix

(Hh.n-hexane) effect on spontaneous rhythmic rate and force of contraction in the

absence and presence of atropine (1 µM) in isolated SD rat right atria (mean±SEM,

n=5-7).

153

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine

10

[Hh.Chlor] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.88 Tracing (A) and concentration response curves (B) show the effect of the

chloroform fraction of Hedera helix (Hh.Chlor) on spontaneous rhythmic rate and

force in SD rat right atria in atropine (1 µM) absence and presence (mean±SEM, n=5-

7).

154

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10

[Hh.EtAc] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.89 Tracing and graph show Hedera helix ethyl acetate fraction (Hh.EtAc)

response on spontaneous rhythmic rate and force of contraction in the absence and

presence of atropine (1 µM) in isolated SD rat right atrial preparations (mean±SEM,

n=5-7).

155

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10

[Hh.Aq] mg/mL

% o

f C

on

tro

l

A

B

Figure.3.90 Tracing (A) and concentration response curves (B) show the Hedera

helix aqueous fraction (Hh.Aq) effect on force and rate in rat right atria in atropine (1

µM) absence and presence.

156

0.003 0.03 0.3 3

0

25

50

75

100

Force

Rate

Force

Rate

Without atropine

With atropine (1 M)

10


% o

f C

on

tro

l

3.2.5.2 Effect of hederacoside C (HDC) on rat atrial rhythmic contractions

Hederacoside C decreased both rate and force of spontaneously beating atria with

EC50 values of 0.25 (0.13-0.37) and 1.04 (0.03-2.05) µg/mL as shown in Fig.3.91.

However, these effects were unaffected by atropine (1 µM) pretreatment (Fig.3.91).

A

B

Figure.3.91 Tracing and graph represent the hederacoside C (HDC) effect in the

absence and presence of atropine (1 µM) from isolated SD rats (mean±SEM, n=5-7).

157

Chapter 4

Discussion

158

Discussion

Based on the medicinal use of aerial parts of Eruca sativa and leaves of Hedera helix

in the treatment of cardiovascular diseases in humans (Steinmetz, 1961; Duke, 2002;

Sabeen and Ahmed, 2009; Ali-Shtayeh et al., 2013; Amjad, 2015; Mithen, 2015),

extracts and fractions of these plants were pharmacologically evaluated in the

hyperlipidemia and hypertension with aims to explore the possible underlying

mechanisms of action. Important constituents, erucin and hederacoside C (HDC)

derived from these plants, respectively, were also evaluated under similar

experimental conditions. Two models, tyloxapol (acute) and high fat diet (HFD)-

induced (chronic) hyperlipidemia in rats were used. Tyloxapol is a nonionic

surfactant, causes acute hyperlipidemia in animals that acts through activation of

hydroxy-methyl glutaryl coenzyme A (HMG-CoA) reductase. However, tyloxapol

does not cause vascular endothelial disruption. Alternatively, high fat diet-induced

hyperlipidemia model was used to study the effects of these agents on hyperlipidemia-

induced vascular dysfunction/disruption.

The extract of E. sativa and H. helix were first tested in tyloxapol-induced

hyperlipidemic rats at different doses. Intraperitoneal injection of tyloxapol (500

mg/kg) to SD rats resulted a significant (p < 0.001) increase in serum total cholesterol

(3.5 fold), triglycerides (7.25 fold) and low density lipoprotein (1.8 fold), compared to

normal control. High density lipoprotein (HDL) remained unchanged. The extract of

E. sativa and H. helix, in tyloxapol-induced hyperlipidemic rats, decreased dose-

dependently, TC and TG levels. This effect was significant (p < 0.001) at 300 mg/kg

dose that resulted 55% and 60% respective reduction in TC by both extracts,

comparable to lovastatin treated hyperlipidemic rats. The effect of E. sativa extract on

TG levels was 17 times more potent than H. helix at same dose. These initial findings

indicate that both extracts are effective in hyperlipidemias such as

hypercholesterolemia and hypertriglyceridemia. However the extract of E. sativa is

more promising in the management of hypertriglyceridemia. As tyloxapol does not

affect the levels of LDL and HDL (Istvan and Deisenhofer, 2001), that’s why the

extracts of E. sativa and H. helix have no effect on these lipids.

To have insight into the chemical constituents involved in lipid lowering effects,

extracts were analyzed for phytochemicals presence. Erucin, which is one of the

important constituents (isothiocyanate) of E. sativa (Gmelin and Schluter, 1970) was

159

confirmed through HPLC analysis in crude extract of E. sativa. Erucin was

investigated also in hyperlipidemic model. Erucin at all doses, decreased TC and TG

in tyloxapol induced model. Similarly, HDC is one of the active constituents of H.

helix (Trute et al., 1997; Sieben et al., 2009) confirmed through HPLC analysis in the

extract of H. helix, also significantly (p < 0.001) decreased TC and TG levels at the

dose of 5 mg/kg. Erucin at both doses was more effective in reducing TC and TG

levels compared to HDC. Erucin is considered one of the constituents of E. sativa as a

candidate to treat hypertriglyceridemia. Tyloxapol induces hyperlipidemia through

inhibition of triglyceride hydrolysis by deactivation of lipoprotein lipase (Kellner et

al., 1951). Initially, mobilization of cholesterol from the liver into plasma takes place,

which increases synthesis of hepatic cholesterol. The increase in synthesis is also

supported by an increase in HMG-CoA reductase activity (Kuroda et al., 1977),

which control cholesterol biosynthesis ((Istvan and Deisenhofer, 2001). It is suggested

that reduction in TC and TG levels by both extracts might have had inhibited

biosynthesis of cholesterol through HMG-CoA pathway. These data indicate that

extracts and constituents of both plants are effective in hypercholesterolemia and

hypertriglyceridemia. However, this model does not allow to investigate effect on

vascular disruption as tyloxapol-induced model is acute and endothelium

disruption/dysfunction does not occur, therefore chronic high fat diet model was used

to study the effect of extracts on vascular function. For this purpose, HFD model of

hyperlipidemia was used, which is a chronic model leads to vascular disruption and

dysfunction.

The high fat diet results in vascular endothelial disruption and marked impairment in

endothelium-dependent vasorelaxation. HFD constitutes many ingredients such as

cholesterol, cholic acid and butter fat. Cholesterol present in the high fat diet blocks

receptor mediated uptake of LDL and thus results in increase concentration of plasma

LDL. Similarly, cholic acid increases the intestinal absorption of cholesterol that

disrupts the synthesis of cholesterol in the liver (Chen et al., 2005). HFD resulted

hyperlipidemia in 8 weeks which was followed by oral administration of E. sativa and

H. helix extracts to the rats that significantly (p < 0.001) reduced TC to 70% and 72%.

The E. sativa and H. helix extract also produced a decline in LDL that was 65% and

71%, respectively. The extracts of both plants significantly decreased TC and LDL to

same levels. Both extracts at high dose increased HDL (p < 0.05). Both extracts also

decreased the TG levels to normal baseline values. In comparison to tyloxapol model,

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extracts have significantly decreased TC, LDL and increased HDL in the HFD rats,

which indicate that these extracts are effective antihyperlipidemic agents. Erucin and

HDC were also tested in HFD model parallel.

Erucin at dose of 3 mg/kg significantly (p < 0.001) decreased TC and LDL levels with

no significant effect on HDL and TG when compared to crude extract. These results

indicate that effect on HDL may be contributed by the other phytochemical

constituents present in E. sativa that are not yet identified and tested and erucin may

not be the only constituent responsible for effects of E. sativa on all lipids. HDC at

2.5 and 5 mg/kg significantly (p < 0.001) reduced TC, LDL levels and increased HDL

(p < 0.05). These results on the crude extract and compounds of E. sativa and H. helix

in tyloxapol and HFD models indicated that these are effective antihyperlipidemic

agents with differential effect on lipid profile.

Hyperlipidemia leads to atherosclerosis, marked and characterized by elevated

atherogenic index that predicts risk of CVDs (Dobiasova and Frohlich, 2001; Daniels

et al., 2008). In this study, high fat diet significantly increased atherogenic index in

the hyperlipidemic rats. Based on the lipid profile of extracts and compounds,

atherogenic index was significantly decreased in treated groups. H. helix extract was

more potent in reducing the atherogenic index compared to E. sativa at all doses.

Erucin was more potent than HDC. These interesting findings on the lipid profile of

extracts and compounds indicate that the decrease in the atherogenic index reveals the

therapeutic significance of the extracts, erucin and HDC.

High fat diet causes increase in LDL level that accumulates between vascular

endothelium and tunica intima and later oxidized leading to monocyte adhesion. This

results in endothelial disruption (Norata et al., 2013). To see effect of extracts and

compounds in hyperlipidemia-induced endothelial disruption, hitopathological

investigation was carried out. Histological examination of the aorta of hyperlipidemic

rats revealed changes such as macrophages, vacuoles and foam cell formation

compared to normal architecture of control group. The normal structure of aorta

comprised of tunica intima with regular endothelium lining, tunica media with smooth

muscle cells and elastic lamina and tunica adventitia comprised of connective tissues.

Histopathological microscopic examination of the aortic slices of the groups, treated

with different doses of E. sativa extract, showed gradual improvement of endothelium

disruption, thickness of the aorta and lipid accumulation. This effect was more

significant at 300 mg/kg, which showed almost regular morphology of aortic intima,

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media and adventitia. Erucin at a dose of 3 mg/kg also reduced endothelium

disruption, aortic thickness and macrophages infiltration in hyperlipidemic rats. The

effect of extract of H. helix revealed that endothelium lining was intact at the high

doses, and macrophages and smooth muscle infiltration decreased dose dependently.

There was minor increase in thickness of aorta in hyperlipidemic rats treated with 30

mg/kg, while the groups with higher dose treatment showed normal structure. HDC at

2.5 and 5 mg/kg showed normal thickness of aorta without endothelium disruption

and macrophages infiltration. These results indicate that extracts and compounds

improved architecture of the vascular wall. However the effect of H. helix and HDC

was more prominent. These include beneficial effects on endothelial damage and

inhibition of vascular smooth muscle cell proliferation compared to lovastatin, which

improves endothelial function by inhibiting oxidized-LDL in endothelial cells (Li and

Mehta, 2009).

Vascular studies in hyperlipidemia and atherosclerotic animals and humans indicate

that endothelial function is impaired (d’Uscio et al., 2001; Lee et al., 2002; Bourgoin

et al., 2008), due to increase in cholesterol, leading to endothelial dysfunction in the

aorta. In the endothelial dysfunction, free radicals inactivate NO (Ohara et al., 1993;

Li et al., 2007; Gradinaru et al., 2015). Aortae isolated from such animals do not

response well to acetylcholine. To see endothelial dysfunction in-vitro, aortae were

isolated from the normal, hyperlipidemic control and hyperlipidemic rats treated with

different doses of the extracts and compounds. Aortic rings made from the normal

control and hyperlipidemic control showed complete and incomplete relaxation to

acetylcholine on phenylephrine precontractions, respectively. This indicates that

endothelium is intact and partially damaged, respectively. Same protocol was

followed in aortic rings from different hyperlipidemic rats treated with different doses

of extracts and compounds. Results show that relaxation to acetylcholine increased

with increase in dose and at dose of 300 mg/kg treated rats, relaxation to acetylcholine

was complete. This indicates that at this particular dose vascular endothelial

disruption was reversed induced by hyperlipidemia. Erucin, also reversed endothelial

disruption at dose of 3 mg/kg, which suggests that erucin is important therapeutic

agent of the E. sativa extract that shares this property of the extract and supports its

role in vascular dysfunction. These findings were similar to the lovastatin treated

group in which acetylcholine produced about 50% relaxation on PE precontractions.

In the same way, in isolated aortic rings from different hyperlipidemia rats treated

162

with doses of H. helix, also produced relaxation to acetylcholine that completed at 300

mg/kg dose. Similar to crude extract of H. helix, HDC also provided protection to the

endothelium and aortic rings completely relaxed to acetylcholine at 5 mg/kg dose.

Thus the findings clearly showed that the extracts and the respective compounds are

the potential antihyperlipidemic agents with therapeutic beneficial effect on

endothelium disruption and subsequently dysfunction. These results also indicated

that extracts and compounds, dose-dependently preserved the endothelial dysfunction

through improving relaxation to acetylcholine. Increased LDL is oxidized in

hyperlipidemia resulting in decrease level of nitric oxide (Ryoo et al., 2006;

Schalkwijk et al., 2007) due to which relaxation to acetylcholine is decreased. The

extracts and constituents effect on acetylcholine-induced relaxation in hyperlipidemic

rats might be the result on nitric oxide synthase, or reducing inflammation of vascular

smooth muscles.

Hyperlipidemia leads to many complications including hypertension (Thomas et al.,

2001), which is due to endothelial dysfunction, a hallmark of hypertension (LaRosa,

2001). Hypertension and hyperlipidemia contribute to atherosclerosis and are

important vascular risks. On these bases, it was hypothesized that plant extracts and

constituents, which decreased cholesterol level and improved endothelial function

could also have beneficial effects on high blood pressure. In addition, one of the

previous studies showed that aqueous extract of E. sativa was found reducing MAP in

rats (Ribeiro et al., 1986). To test this hypothesis, a hypertensive rat model was used

and results were compared with normotensive rats. When tested in high salt-induced

hypertension, E. sativa extract decreased MAP. This was exciting finding that extract

of E. sativa also possesses antihypertensive effect. Previously, E. sativa possess

anticholinesterase (Boga et al., 2011) activity. Substances having anticholinesterase

activity are cholinomimetic resulting a decrease in vascular resistance and blood

pressure (Ally et al., 1993; Lazartigues et al., 1999). Crude extract of E. sativa

significantly (p < 0.01) decreased the MAP (42%) in normotensive rats. It was

hypothesized that the fall in MAP induced by the crude extract of E. sativa may

involve the anticholinesterase constituents. To test this possibility, atropine a

muscarinic receptor antagonist (Arunlakhshana and Schild, 1959), was administered

to normotensive rats in-vivo. To confirm that atropine has occupied muscarinic

receptors in-vivo, a sub-maximal dose of acetylcholine was tested. Interestingly the

effect of acetylcholine on blood pressure was attenuated in the atropinized rats, thus

163

allow to test effect of extracts and compounds on muscarinic receptors in-vivo, if any.

In the atropinized rats, effect of intravenous injection of E. sativa extract on MAP was

significantly (p < 0.001) blocked, indicating that muscarinic receptor stimulation is

partly involved in the antihypertensive effect of E. sativa extract. Under similar

experimental conditions, the effect of extract of H. helix on MAP did not change in

the atropine treated rats, ruling out muscarinic receptors involvement.

To identify the constituents responsible for the blood pressure lowering effect and

segregation of activity to any specific fraction, fractionation of both extracts was

carried out further. All the fractions of E. sativa and H. helix were also tested parallel

in hypertensive, normotensive and normotensive atropinized rats in-vivo. In high salt-

induced hypertensive and normotensive rats, all fractions of both extracts caused fall

in the MAP. The potency order of decreasing MAP of fractions of E. sativa was,

aqueous > crude > n-hexane > ethyl acetate > chloroform. The potency order of

fractions of H. helix was, aqueous > crude > ethyl acetate > chloroform > n-hexane.

All the fractions were comparatively more potent as antihypertensive in the

hypertensive rats than the normotensive.

Fractions of both plants were also tested parallel in atropinized rats. The effect of all

the fractions of E. sativa was significantly blocked with atropine (p < 0.001).

However the aqueous fraction was the most sensitive to the effect of atropine, which

indicates that this fraction contains the highest content of cholinesterase inhibitory

constituents. These results also indicate that fall in MAP may be due to stimulation of

muscarinic receptors. All fractions, like the parent crude extract of H. helix, remained

same in the presence of atropine again suggesting that muscarinic receptors

stimulation are not involved.

Compounds were tested parallel; erucin induced a fall in MAP in both groups with a

maximum fall of 50 and 66%, respectively that remained same in the atropinized rats

ruling out the muscarinic receptors stimulation. This finding suggests that erucin is

not like the parent extract of E. sativa and further indicates that the underlying

antihypertensive mechanism might be different. HDC also caused a fall in MAP in

both models; 58 and 69%, respectively which did not change with atropine

pretreatment. Both extracts and compounds decrease blood pressure in normotensive

and hypertensive rats. The antihypertensive effect of E. sativa is through muscarinic

receptors while H. helix, HDC and erucin do not involve muscarinic receptor

involvement. In order to determine the underlying mechanisms responsible for the

164

antihypertensive effect of E. sativa, H. helix and compounds, further studies were

carried out. As vascular resistance and cardiac output are important determinants for

blood pressure (Lund-Johansen, 1985), therefore further studies were important in

isolated vascular and cardiac tissues. The extracts and fractions were tested against

phenylephrine-induced precontraction. E. sativa extract and fractions produced a

vasodilatory effect. The endothelium denudation ablated this response indicating role

of endothelial derived factors. The endothelium synthesizes varieties of vasoactive

substances that regulate vascular tone and blood pressure (Campbell et al., 1996). The

vascular muscarinic receptors activate the Gq-PLC-IP3 pathway, leading to activation

of endothelial NO synthase and production of NO (Moncada and Higgs, 1995). Aortic

rings with intact endothelium were pretreated with L-NAME, a nitric oxide synthase

inhibitor (Li et al., 1998). This pretreatment ablated the vasorelaxant effect indicating

that NO is the mediator responsible for the vasorelaxation. As the E. sativa is reported

having cholinomimetic (anticholinesterase) activity. It was hypothesized that the

constituents in the crude extract might have had activated endothelial muscarinic

receptors and led to the liberation of NO. To test this hypothesis, aortic rings with

intact endothelium, were pretreated with atropine. This pretreatment with atropine,

decreased the response, indicating that vasodilation is via muscarinic receptors-linked

nitric oxide pathway. However, this relaxation was not completely inhibited with

denudation of endothelium, L-NAME and atropine pretreatment at higher

concentrations, suggesting that extract has a direct effect on vascular smooth muscle

cells also. In the vascular reactivity studies in rat aortic rings, all the fractions of E.

sativa were also tested. The fractions showed endothelium-dependent atropine and L-

NAME sensitive vasodilator effect. The attenuation of the vasorelaxant effect of

fractions indicates that acetylcholine mediated nitric oxide pathway is involved. The

n-hexane fraction was the least potent fraction, indicating that this fraction might have

constituents, which produce vasocontractile effect and thus play a role in

vasomodulation. Unlike the parent crude extract of E. sativa, erucin caused

endothelial-independent vasorelaxation in normotensive rat aortic rings. Erucin

produced 25% vasorelaxation against phenylephrine-induced contraction unaffected

by L-NAME, atropine pretreatment or denudation. It also indicated that erucin may be

one of the constituents responsible for regulation of vasomodulation because it

produced only 25% relaxation and it may have vascontractile effect, which is not

explored in the current study. However, erucin showed endothelium-independent

165

response, which indicated that constituents in the extract of E. sativa also act on the

vascular smooth muscle cells.

Crude extract of H. helix also produced vasorelaxant effect on phenylephrine

precontractions, ablated by endothelium denudation and pre-incubation with L-

NAME. However pretreatment with atropine did not affect the vasorelaxant response

to the crude extract of H. helix which shows that vascular muscarinic receptors are not

involved. This finding strengthened the results in in-vivo where the effect did not

change with atropine pretreatment. All fractions of H. helix produced endothelium-

dependent relaxation that was reduced with denudation and L-NAME pretreatment.

The vasorelaxant response of all the fractions remained same in the presence of

atropine, suggesting that vasorelaxation induced by the fractions is directly mediated

by nitric oxide. HDC produced vasorelaxation against phenylephrine-induced

contraction, which was significantly affected by the L-NAME and denudation,

however it remained same in the presence of atropine. These results indicate that

vasorelaxant effect of H. helix extract and HDC is endothelium-dependent and

mediated through nitric oxide.

For comparison, the extracts and fractions were further evaluated in aortae from

hypertensive rats in-vitro. High salt attenuates NO-mediated vascular response in

hypertensive rats. This happens through suppression of endothelial nitric oxide

synthase (Li et al., 2009). Moreover, the endothelium-dependent vasorelaxation

decreases solely due to an altered release of endothelium-derived relaxing factor

(Luscher and Vanhoutte, 1986). Interestingly, both extracts and fractions induced

relaxation of phenylephrine precontraction in hypertensive rats at higher

concentrations, superimposed to that observed with L-NAME, atropine or denudation.

This finding confirms that high salt impairs NO production in the hypertensive rats

and provides evidence that the extracts have endothelium-dependent NO mediated

vasodilatory effect in-vitro. L-NAME and atropine pretreatment or denudation of

endothelium did not modify the maximum vasodilatory effect of the both extracts at

the higher concentration, suggesting that additional mechanisms may also be

involved. HDC produced the vasorelaxant effect at higher concentration in

hypertensive rat aorta, compared to normotensive rats, suggesting that endothelium-

independent mechanisms are also involved.

Phenylephrine, is an adrenergic agonist, and increases intracellular calcium level via

membrane bound receptors and also via release from internal stores (Karaki et al.,

166

1997). The inhibitory effect of all tested agents against phenylephrine precontractions,

also suggests a possible inhibitory effect on calcium release from internal calcium

stores. It was hypothesized that this effect on vascular smooth muscle might be due to

a decrease in the intracellular calcium, as phenylephrine is known to induce vascular

contraction through the release of intracellular stored calcium (Godfraind et al.,

1986). To test this possibility, aortic rings were incubated in Ca+2

free/EGTA

medium. Pretreatment of the aortic rings from normotensive rats with different

concentrations of extract of E. sativa, concentration-dependently suppressed

phenylephrine peak formation, thus indicating an inhibitory effect on calcium release

also, an indirect vasodilatory mechanism. Crude extract of H. helix completely

suppressed the phenylephrine peak formation at 0.3 mg/mL, compared to 50%

suppression of E. sativa extract. Fractions of both extracts were also tested under

similar experimental conditions. The chloroform fraction of E. sativa was more potent

while the n-hexane and aqueous fraction were comparable to crude extract. The

aqueous fraction of H. helix was more potent. Erucin suppressed phenylephrine peak

formation up to 30% while HDC suppressed phenylephrine peak formation

completely. These findings indicate that extract of E. sativa and erucin have less

potent than the extract of H. helix and HDC on intracellular calcium stores. To have

further insight into the effect of extracts and compounds on calcium influx through

membrane channels particularly calcium channels, additional protocols were

followed. Aortic rings from the normotensive rats were precontracted with high K+.

Interestingly, the extracts of E. sativa and H. helix inhibited high K+

precontractions,

similar to verapamil, which blocks calcium channels (Godfraind et al., 1986). Thus

the inhibitory effect of extracts to K+ precontractions, similar to verapamil, indicates

Ca+2

entry blocking activity. High K+

depolarizes the smooth muscles and opens

voltage dependent calcium channels (VDCs), increases Ca+2

influx and produced

sustained contractions (Karaki et al., 1997). The inhibitory effect of the extracts

against high K+

precontractions suggests inhibitory effect on calcium movements. All

the fractions of E. sativa were also tested against high K+ precontractions that caused

relaxation with crude extract the most potent and chloroform the least. H. helix

aqueous fraction found least potent while crude was more potent. Erucin and HDC

produced a vasorelaxant effect against high K+ precontractions like verapamil. These

findings indicated that extract of E. sativa and H. helix mediates vasodilation through

dual inhibitory effect on release from internal stores and voltage dependent calcium

167

channels. These findings on the vascular effects of the crude extracts, fractions and

compounds of E. sativa and H. helix further provides evidence that the vasodilatory/

vasorelaxant effect is by both endothelium-dependent pathway and independent

pathway. The synergistic effect is responsible for the decrease in vascular resistance

and fall in blood pressure observed in in-vivo.

To have insight into the effects of extracts and compounds on cardiac mechanisms,

extract and fractions and compounds were further tested on isolated rat atrial strips.

Crud extract and aqueous fraction of E. sativa completely suppressed rate (negative

chronotropic) and force (negative inotropic) of atrial contractions. However, the ethyl

acetate and chloroform fractions induced partial suppression of the rate and force of

contractions. The n-hexane fraction partially inhibited the rate of contraction with

increase in force of contraction (positive inotropic). It was hypothesized that the effect

on the rate and force of contraction might involve cardiac muscarinic receptor

activation, so atrial strips were pretreated with atropine. This pretreatment did not

change the effect of E. sativa extract and fractions on the rate and force of atrial

contraction, indicating that cardiac muscarinic receptors activation are not involved.

This is an interesting finding indicating that the possible anticholinesterase

constituents of E. sativa might be selective for vascular M3 muscarinic receptors

without having effect on cardiac M2 receptors. This also shows that the decrease in

rate and force of contractions might be due to inhibitory effect on calcium movements

in the cardiac muscles. The positive inotropic effect of the n-hexane fraction of E.

sativa, suggests presence of constituents which activate atrial tissues. This might be of

therapeutic importance in patients having heart failure as a consequence of

hyperlipidemia and or hypertension. Additionally, the cardiotonic effect, particularly

positive inotropic effect might be of clinical importance in situation to counteract

severe fall in blood pressure associated with use of vasodilators and thus contribute to

maintenance of vascular tone (vasomodulation). Erucin also completely suppressed

rate and force of atrial contractions that was remained unchange with atropine

pretreatment. The extract of H. helix, all fractions and HDC also suppressed both rate

and force of contractions that was not affected by pretreatment with atropine, ruling

out the involvement of muscarinic receptors. H. helix extract, fractions and HDC

possibly decrease rate and force of contractions by inhibition of calcium movements

in the cardiac muscles. Thus it is suggested that the fall in blood pressure observed in

in-vivo experiments by both extracts and compounds might be due to negative

168

inotropic and chronotropic effects, which are responsible for decrease in cardiac

output and hence blood pressure. The aqueous fraction of both plant extracts were

potent in lowering blood pressure, endothelium-dependent vasorelaxation and

decrease rate and force of contractions, which indicate that blood pressure lowering

constituents are more concentrated in this particular fraction of both plants.

In order to have an overview on the possible phytochemical constituent of both plants

that might have role in the cardiovascular effects, phytochemical analysis of the

extracts was carried out in sufficient detail. Spectrophotometric and HPLC analysis of

both E. sativa and H. helix indicated high amount of phenols and flavonoids. Phenols

and flavonoids contribute to cardiovascular protection (Han et al., 2007; Torres-

Piedra et al., 2011; Szwajgier, 2015; Fu et al., 2005). Therefore, the phenols and

flavonoids present in extracts might be responsible for the antihypertensive activities.

Previous studies also reported that saponins decrease serum cholesterol (Francis et al.,

2002) by enhancing the excretion of bile (Oakenfull and Sidhu, 1990; Han et al.,

2000). These facts suggest that saponins may also be responsible for the

antihyperlipidemic and antihypertensive effects of the extract.

169

Conclusion

These findings indicate that E. sativa and H. helix are effective antihyperlipidemic

agents. Both extracts and important constituents erucin and hederacoside C

significantly reduced total cholesterol and triglycerides in tyloxapol-induced

hyperlipidemia compared to lovastatin. In high fat diet-induced hyperlipidemia both

extracts significantly reduced TC, LDL and increased HDL levels. Erucin and HDC

also significantly reduced TC, TG and LDL. Both extracts and compounds preserved

endothelial function in high fat diet-induced hyperlipidemia by decreasing LDL levels

and macrophages infiltration and improving endothelium disruption. E. sativa extract

is also an effective antihypertensive, which is the outcome of vasodilatory

(endothelium-dependent and independent) and cardiotonic effects. Vasodilatory

mediators involved muscarinic receptor (vascular selective) mediated NO and dual

inhibitory effect on calcium influx and release from store. Erucin is also effective

antihypertensive, acts through endothelium-independent mechanism. E. sativa and

erucin also produced negative inotropic and chronotropic effect without activation of

cardiac muscarinic receptors. Vasodilatory effect of H. helix extract and HDC is

mediated through NO release and by inhibiting calcium release and decreasing

cardiac rate and force of contractions. The vasodilatory and cardiac mechanisms

explain the respective decrease in mean arterial pressure. This finding supports the

use of E. sativa and H. helix as antihyperlipidemic and antihypertensive remedies.

Erucin and HDC are the important constituents of E. sativa and H. helix, respectively

as potential antihyperlipidemic and antihypertensive agents. The extracts were found

safe in mice. As the antihyperlipidemic effect is possibly mediated by HMG-CoA

reductase inhibition so the molecular study targeting the enzyme will be helpful to

further evaluate this mechanism. Calcium signaling at the molecular level is also

required. The use of mesenteric arteries would be a good approach to have more

specific insight into the mechanisms, related to peripheral vascular resistance. There

is need to explore these herbal drugs and important constituents for clinical trials in

hyperlipidemia and hypertension.

170

Chapter 5

References

171

References

Abbaoui, B., Riedl, K.M., Ralston, R.A., Thomas‐Ahner, J.M., Schwartz, S.J.,

Clinton, S.K. & Mortazavi, A. (2012). Inhibition of bladder cancer by broccoli

isothiocyanates sulforaphane and erucin: characterization, metabolismand

interconversion. Mol. Nut. Food Res. 56, 1675-1687.

Abbasifar, A., Ghani, S., Irvani, M.A., Rafiee, B., Kaji, B.V. & Akbari, A. (2017).

Antibacterial activity of silver nanoparticles synthesized by using extracts of

Hedera helix. Zahedan J. Res. Med. Sci. 19, 1-6.

Afifi, F. U., & Abu-Irmaileh, B. (2000). Herbal medicine in Jordan with special

emphasis on less commonly used medicinal herbs. J. Ethnopharmacol. 72,

101-110.

Akiyama, T., Tachibana, I., Shirohara, H., Watanabe, N. & Otsuki M. (1996). High-

fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia

in normal adult male Wistar rat. Diabetes Res. Clin. Pract. 31, 27-35.

Alam, M.S., Kaur, G., Jabbar, Z., Javed, K, & Athar, M. (2007). Eruca sativa seeds

possess antioxidant activity and exert a protective effect on mercuric chloride

induced renal toxicity. Food Chem. Toxicol. 45, 910-920.

Ali-Shtayeh, M.S., Jamous, R.M., Jamous, R.M. & Salameh, N.M. (2013).

Complementary and alternative medicine (CAM) use among hypertensive

patients in Palestine. Complement. Ther. Clin. Pract. 19, 256-263.

Alqasoumi, S. (2010). Carbon tetrachloride-induced hepatotoxicity: Protective effect

of 'Rocket' Eruca sativa L. in rats. Am. J. Chin. Med. 38, 75-88.

Alqasoumi, S., Al-Sohaibani, M., Al-Howiriny, T., Al-Yahya, M. & Rafatullah, S.

(2009). Rocket “Eruca sativa”: A salad herb with potential gastric anti-ulcer

activity. World J. Gastroenterol. 15, 1958-1965.

Ally, A., Hara, Y. & Murayama, S. (1993). Cardiovascular effects of central

administration of cholinomimetics in anesthetized cats. Neuropharmacology

32, 185-193.

Amjad, M.S. (2015). Ethnobotanical profiling and floristic diversity of Bana Valley,

Kotli (Azad Jammu and Kashmir), Pakistan. Asian Pac. J. Trop. Biomed. 5,

292-299.

172

Angeloni, C., Leoncini, E., Malaguti, M., Angelini, S., Hrelia, P. & Hrelia, S. (2009).

Modulation of phase II enzymes by sulforaphane: implications for its

cardioprotective potential. J. Agric. Food Chem. 57, 5615-5622.

Anwar, M.A., Al Disi, S.S. & Eid, A.H. (2016). Anti-hypertensive herbs and their

mechanisms of action: part II. Front. Pharmacol. 7, 50.

Arora, R., Bhushan, S., Kumar, R., Mannan, R., Kaur, P., Singh, B. & Arora, S.

(2016). To analyze the amelioration of phenobarbital induced oxidative stress

by erucin, as indicated by biochemical and histological alterations. Anticancer

Agents Med. Chem.16, 1445-1454.

Arora, R., Kukreja, S., Mannan, R., Bhushan, S. & Arora, S. (2017). Evaluating the

renoprotective activity of 4-methylthiobutyl isothiocyanate against 7, 12

dimethylbenz (α) anthracene generated radical stress in male Wistar rats.

CTDT 1, 10-14.

Arunlakhshana, O. Schild, H.O. (1959). Some quantitative uses of drug antagonists.

Br. J. Pharmacol. 14, 48-58.

Azarenko, O., Jordan, M.A. & Wilson, L. (2014). Erucin, the major isothiocyanate in

arugula (Eruca sativa), inhibits proliferation of MCF7 tumor cells by

suppressing microtubule dynamics. PloS One 9, e100599.

Aziz, N., Mehmood, M.H., Mandukhal, S.R., Bashir, S., Raoof, S. & Gilani, A.H.

(2009). Antihypertensive, antioxidant, antidyslipidemic and endothelial

modulating activities of a polyherbal formulation (POL-10). Vasc. Pharmacol.

50, 57-64.

Aziz, N., Mehmood, M.H., Siddiqi, H.S., Sadiq, F., Maan, W. & Gilani, A.H. (2009).

Antihypertensive, antidyslipidemic and endothelial modulating activities of

Orchis mascula. Hypertens. Res. 32, 997-1003

Aziz, N., Mehmood, M.H. & Gilani, A.H. (2013). Studies on two polyherbal

formulations (ZPTO and ZTO) for comparison of their antidyslipidemic,

antihypertensive and endothelial modulating activities. BMC Complement.

Altern. Med. 13, 371.

Bala, S., Uniyal, G.C., Chattopadhyay, S.K., Tripathi, V., Sashidhara, K.V.,

Kulshrestha, M., Sharma, R.P., Sharma, R.P., Jain, S.P., Kukreja, A.K., &

Kumar, S. (1999). Analysis of taxol and major taxoids in Himalayan yew,

Taxus wallichiana. J. Chromatogr. A 858, 239-244.

173

Barillari, J., Canistro, D., Paolini, M., Ferroni, F., Pedulli, G. F., Iori, R. &

Valgimigli, L. (2005). Direct antioxidant activity of purified glucoerucin, the

dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds

and sprouts. J. Agric. Food Chem. 53, 2475-2482.

Bedir, E., Kirmızipekmez, H., Sticher, O. Calis, I. (2000). Triterpene saponins from

the fruits of Hedera helix. Phytochemistry 53, 905-909.

Bell, L., Oruna, M.J. & Wagstaff, C. (2015). Identification and quantification of

glucosinolate and flavonol compounds in rocket salad (Eruca sativa, Eruca

vesicaria and Diplotaxis tenuifolia) by LC–MS: Highlighting the potential for

improving nutritional value of rocket crops. Food Chem. 172, 852-861.

Bennett, R.N., Mellon, F.A., Botting, N.P., Eagles, J., Rosa, E.A. & Williamson, G.

(2002). Identification of the major glucosinolate (4-mercaptobutyl

glucosinolate) in leaves of Eruca sativa L. (salad rocket). Phytochemistry 61,

25-30.

Berroughui, H., Ettaib, A., Herrera Gonzalez, M.D., Alvarez de Sotomayor, M.,

Bennari-Kabchi, N. & Hmamouchi M. (2003). Hypolipidemic and

hypocholesterolemic effect of argan oil (Argan spinosa L.) in Meriones Shawi

rats. J. Ethnopharmacol. 89, 15-18.

Boga, M., Hacıbekiroglu, I. & Kolak, U. (2011). Antioxidant and anticholinesterase

activities of eleven edible plants. Pharm. Biol. 49, 290-295.

Bonetti, P.O., Lerman, L.O. & Lerman, A. (2003). Endothelial dysfunction: a marker

of atherosclerotic risk. Arterioscler. Thromb. Vasc. Biol. 23, 168-175.

Boutayeb, A., Derouich, M., Boutayeb, W. & Lamlili, M.E.N. (2014).

Cerebrovascular diseases and associated risk factors in WHO Eastern

Mediterranean countries. Cardiol. Angiol. 2, 62-75.

Bourgoin, F., Bachelard, H., Badeau, M., Melancon, S., Pitre, M., Lariviere, R. &

Nadeau, A. (2008). Endothelial and vascular dysfunction and insulin

resistance in rats fed a high fat high sucrose diet. Am. J. Physiol. 295, 1044-

1055.

Buettner, R., Parhofer, K.G., Woenckhaus, M., Wrede, C.E., Kunz-Schughart, L.A.,

Scholmerich, J. & Bollheimer, L.C. (2006). Defining high-fat-diet rat models:

metabolic and molecular effects of different fat types. J. Mol. Endocrinol. 36,

485-501.

174

Bui, Q.T., Prempeh, M. & Wilensky, R.L. (2009). Atherosclerotic plaque

development. Int. J. Biochem. Cell Biol. 41, 2109-2113.

Campbell, W.B., Gebremedhin, D., Pratt, P.F. & Harder, D.R. (1996). Identification

of epoxyeicosatrienoic acids as endothelium-derived hyperpolrizing factor.

Circ. Res. 78, 415-423.

Cai, H. & Harrison, D.G. (2000). Endothelial dysfunction in cardiovascular diseases:

the role of oxidant stress. Circ. Res. 87, 840-844.

Cardillo, C., Kilcoyne, C.M., Cannon, R.O. & Panza, J.A. (2000). Increased activity

of endogenous endothelin in patients with hypercholesterolemia. J. Am. Coll.

Cardiol. 36, 1483-1488.

Casino, P.R., Kilcoyne, C.M., Quyyumi, A.A., Hoeg, J.M. & Panza, J.A. (1993). The

role of nitric oxide in endothelium-dependent vasodilation of

hypercholesterolemic patients. Circulation 88, 2541-2547.

Champe, P.C. & Harvey, R.A. Metabolism of dietary lipids. In “Champe, P.C.

Harvey, R.A. Lippincott's illustrated reviews: biochemistry”. 2nd

eds. JB

Lippincott Company, Philadelphia, PA. pp.163 (1994).

Chang, C.C., Yang, M.H., Wen, H.M. & Chern, J.C. (2002). Estimation of total

flavonoid content in propolis by two complementary colorimetric methods. J.

Food Drug Anal. 10, 178-182.

Chen, W., Suruga, K., Nishimura, N., Gouda, T., Lam, V. N. & Yokogoshi, H.

(2005). Comparative regulation of major enzymes in the bile acid biosynthesis

pathway by cholesterol, cholate and taurine in mice and rats. Life Sci. 77, 746-

757.

Chistiakov, D.A., Melnichenko, A.A., Myasoedova, V.A., Grechko, A.V. & Orekhov,

A.N. (2017). Mechanisms of foam cell formation in atherosclerosis. J. Mol.

Med. 95, 1153-1165.

Cho, H.J., Lee, K.W. & Park, J.H.Y. (2013). Erucin exerts anti-inflammatory

properties in murine macrophages and mouse skin: Possible mediation through

the inhibition of NFκB signaling. Int. J. Mol. Sci. 14, 20564-20577.

Christensen, L.P., Lam, J. & Thomasen, T. (1991). Polyacetylenes from the fruits of

Hedera helix. Phytochemistry 30, 4151-4152.

Cioaca, C., Margineanu, C. & Cucu, V. (1978). The saponins of Hedera helix with

antibacterial activity. Pharmazie 33, 609.

175

Clifford, P.S. & Hellsten, Y. (2004). Vasodilatory mechanisms in contracting skeletal

muscle. J. Appl. Physiol. 97, 393-403.

Craig, W.J. (1999). Health-promoting properties of common herbs. Am. J. Clin. Nutr.

70, 491-499.

Crespin, F., Elias, R., Morice, C., Ollivier, E., Balansard, G. & Faure, R. (1995).

Identification of 3-O-β-D-glucopyranosyl-hederagenin from the leaves of

Hedera helix. Fitoterapia 66, 477.

Cwientzek, U., Ottillinger, B. & Arenberger, P. (2011). Acute bronchitis therapy with

ivy leaves extracts in a two-arm study. A double-blind, randomised study vs.

another ivy leaves extract. Phytomedicine 18, 1105-1109.

Daniels, L. B., Laughlin, G. & Sarno, M.J. (2008). Lp- PLA2 is an independent

predictor of incident coronary heart disease in apparently healthy older

population. J. Am. Coll. Cardiol. 51, 913-919.

Darwish, R. M. & Aburjai, T.A. (2010). Effect of ethnomedicinal plants used in

folklore medicine in Jordan as antibiotic resistant inhibitors on Escherichia

coli. BMC Complement. Altern. Med. 10, 9.

Demirci, B., Goppel, M., Demirci, F. & Franz, G. (2004). HPLC profiling and

quantification of active principles in leaves of Hedera helix L. Pharmazie 59,

770-774.

De Nicola, G.R., Bagatta, M., Pagnotta, E., Angelino, D., Gennari, L., Ninfali, P.,

Rollin, P. & Iori, R. (2013). Comparison of bioactive phytochemical content

and release of isothiocyanates in selected brassica sprouts. Food Chem. 141,

297-303.

de Sousa, J.A., Pereira, P., da Costa, Allgayer.M., Marroni, N.P., Ferraz, A.D. &

Picada, J.N. (2017). Evaluation of DNA damage in Wistar rat tissues with

hyperlipidemia induced by tyloxapol. Exp. Mol. Pathol. 103, 51-55.

Dhaliya, S.A., Surya, A.S., Dawn, V.T., Betty, C., Arun, K. & Sunil, C. (2013). A

review of hyperlipidemia and medicinal plants. Int. J.A. PS. 2, 219-237.

Dobiasova, M. & Frohlich, J. (2001). The plasma parameter log (TG/HDL-C) as an

atherogenic index: correlation with lipoprotein particle size and esterification

rate in apo B- lipoprotein-depleted plasma (FERHDL). Clin. Biochem. 34,

583-588.

176

Drechsler, M., Megens, R.T., van Zandvoort, M., Weber, C. & Soehnlein, O. (2010).

Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis clinical

perspective. Circ. Res. 122, 1837-1845.

Drexel, H. (2009). Statins, fibrates, nicotinic acid, cholesterol absorption inhibitors,

anion exchange resins, omega 3 fatty acids: which drugs for which patients?.

Fundam. Clin. Pharmacol. 23, 687-692.

Drexler, H. & Hornig, B. (1999). Endothelial dysfunction in human disease. J. Mol.

Cell. Cardiol. 31, 51-60.

Duke, J.A. (2002). Handbook of Medicinal Herbs. (2nd

eds), pp. 412. Boca Raton:

CRC Press.

d’Uscio, L.V., Smith, L.A. Katusic, Z.S. (2001). Hypercholesterolemia impairs

endothelium-dependent relaxations in common carotid arteries of

apolipoprotein E-deficient mice. Stroke 32, 2658-2664.

Edeoga, H., Okwu, D. & Mbaebie, B. (2005). Phytochemical constituents of some

Nigerian medicinal plants. Afr. J. Biotechnol. 4, 685-688.

Elias, R., De Meo, M., Vidal, E., Laget, M., Balansard, G. & Dumenil, G. (1990).

Antimutagenic activity of some saponins isolated from Calendula officinalis

L., C. arvensis L. and Hedera helix L. Mutagenesis 5, 327-332.

El-Missiry, M.A. & El-Gindy, A.M. (2000). Amelioration of alloxan induced diabetes

mellitus and oxidative stress in rats by oil of Eruca sativa seeds. Ann. Nutr.

Metab. 44, 97-100.

Eguale, T., Tilahun, G., Debella, A., Feleke, A. & Makonnen, E. (2007). Haemonchus

contortus: in vitro and in vivo anthelmintic activity of aqueous and hydro-

alcoholic extracts of Hedera helix. Exp. Parasitol. 116, 340-345.

Eizawa, H., Yui, Y., Inoue, R., Kosuga K., Hattori, R., Aoyama, T. & Sasayama, S.

(1995). Lysophosphatidylcholine inhibits endothelium-dependent

hyperpolarization and N ω-nitro-l-arginine/indomethacin resistant

endothelium-dependent relaxation in the porcine coronary artery. Circulation

92, 3520-3526.

Facino, R.M., Carini, M., Stefani, R., Aldini, G. & Saibene, L. (1995). Anti‐elastase

and anti‐hyaluronidase activities of saponins and sapogenins from Hedera

helix, Aesculus hippocastanum and Ruscus aculeatus: factors contributing to

their efficacy in the treatment of venous insufficiency. Arch. Pharm. 328, 720-

724.

177

Favero, G., Paganelli, C., Buffoli, B., Rodella, L.F. & Rezzani, R. (2014).

Endothelium and its alterations in cardiovascular diseases: life style

intervention. Biomed. Res. Int. 2014. 1-28.

Fazio, S., Pouso, J., Dolinsky, D., Fernandez, A., Hernandez, M., Clavier, G. &

Hecker, M. (2009). Tolerance, safety and efficacy of Hedera helix extract in

inflammatory bronchial diseases under clinical practice conditions: A

prospective, open, multicentre postmarketing study in 9657 patients.

Phytomedicine 16, 17-24.

Felmeden, D.C., Spencer, C.G.C., Blann, A.D., Beevers, D.G. & Lip G.Y.H. (2003).

Low-density lipoprotein subfractions and cardiovascular risk in hypertension:

relationship to endothelial dysfunction and effects of treatment. Hypertension

41, 528-533.

Fleming, I., Mohamed, A., Galle, J., Turchanowa, L., Brandes, R.P., Fisslthaler, B. &

Busse, R. (2005). Oxidized low-density lipoprotein increases superoxide

production by endothelial nitric oxide synthase by inhibiting

PKCα. Cardiovasc. Res. 65, 897-906.

Fordyce, C.B., Roe, M.T., Ahmad, T., Libby, P., Borer, J.S., Hiatt, W.R. & Pitt, B.

(2015). Cardiovascular drug development: is it dead or just hibernating? J.

Am. Coll. Cardiol. 65, 1567-1582.

Francis, G., Kerem, Z., Makkar, H.P.S. & Becker, K. (2002). The biological action of

saponins in animal systems: a review. Br. J. Nutr. 88, 587-605.

Freiman, P.C., Mitchell, G.G., Heistad, D.D., Armstrong, M.L. & Harrison, D.G.

(1986). Atherosclerosis impairs endothelium-dependent vascular relaxation to

acetylcholine and thrombin in primates. Circ. Res. 58, 783-789.

Friedewald, W.T., Levy, R.I. & Fredrickson, D.S. (1972). Estimation of the

concentration of low-density lipoprotein cholesterol in plasma, without use of

the preparative ultracentrifuge. Clin. Chem. 18, 499-502.

Fu, Z., Bo, H., Chun-yan, H., Qing-yuan, J., Zhen-guo, L., Ming, L., Mei-hua, W.,

Min, G., Chun-ping, Q., Wei, C. & Pan-hua, H. (2005). Effects of total

flavonoids of Hippophae rhamnoides L. on intracellular free calcium in

cultured vascular smooth muscle cells of spontaneously hypertensive rats and

Wistar-Kyoto rats. Chin. J. Integr. Med. 11, 287-292.

178

Fuentes, E., Alarcon, M., Fuentes, M., Carrasco, G. & Palomo, I. (2014). A novel role

of Eruca sativa Mill. (rocket) extract: Antiplatelet (NF-κB Inhibition) and

antithrombotic activities. Nutrients 6, 5839-5852.

Furchgott, R.F. & Zawadski, J.V. (1980). The obligatory role of endothelial cells in

the relaxation of arterial smooth muscle by acetylcholine. Nature 228, 373-

376.

Galigher A.E. & Kozloff E.N. (1971). Essential of practical microtechnique. (2nd

eds), pp.77. Lea and Fediger: Philadelphia.

Gao, S. & Liu J. (2017). Association between circulating oxidized low-density

lipoprotein and atherosclerotic cardiovascular disease. Chronic Dis. Transl.

Med. 3, 89-94.

Garjani, A., Fathiazad, F., Zakheri, A., Akbari, N.A., Azarmie, Y., Fakhrjoo,

A.andalib, S. & Maleki-Dizaji, N. (2009). The effect of total extract of

Securigera securidaca L. seeds on serum lipid profiles, antioxidant status and

vascular function in hypercholesterolemic rats. J. Ethnopharmacol. 126, 525-

532.

Geleta, B., Makonnen, E., Debella, A. & Tadele, A. (2016). In-vivo antihypertensive

and antihyperlipidemic effects of the crude extracts and fractions of Moringa

stenopetala (Baker f.) Cufod. leaves in rats. Front. Pharmacol. 7, 97.

Gepdiremen, A., Mshvildadze, V., Süleyman, H. & Elias, R. (2005). Acute anti-

inflammatory activity of four saponins isolated from ivy: alpha-hederin,

hederasaponin-C, hederacolchiside-E and hederacolchiside-F in carrageenan-

induced rat paw edema. Phytomedicine 12, 440-444.

Glew, R.H. Lipid rnetabolism II: pathways of metabolism of special lipids. In “Text

Book of Biochemistry with CIinical Correlations”.3rd

eds, Devlin TM, ed.

Wiley-Liss New York. pp. 438-448 (1992).

Gmelin, R. & Schluter, M. (1970). Isolierung von 4-Methylthiobutylglucosinolat

(Glucoerucin) aus Samen von Eruca sativa Mill. Arch. Pharm. 303, 330-334.

Godfraind, T., Miller, R. & Wibo, M. (1986). Calcium antagonism and calcium entry

blockade. Pharmacol. Rev. 38, 321- 416.

Goldstein, J.L. & Brown, M.S. (2015). A century of cholesterol and coronaries: from

plaques to genes to statins. Cell 161, 161-172.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Gao%20S%5BAuthor%5D&cauthor=true&cauthor_uid=29063061

https://www.ncbi.nlm.nih.gov/pubmed/?term=Liu%20J%5BAuthor%5D&cauthor=true&cauthor_uid=29063061

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627698/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627698/

179

Goleniowski, M.E., Bongiovanni, G.A., Palacio, L., Nunez, C.O. & Cantero, J.J.

(2006). Medicinal plants from the “Sierra de Comechingones”. Argentina. J.

Ethnopharmacol. 107, 324-341.

Gordon, S.M. & Remaley, A.T. (2017). High density lipoproteins are modulators of

protease activity: Implications in inflammation, complement activation and

atherothrombosis. Atherosclerosis 259, 104-113.

Gradinaru, D., Borsa, C., Ionescu, C. & Prada, G.I. (2015). Oxidized LDL and NO

synthesis-biomarkers of endothelial dysfunction and ageing. Mech. Ageing

Dev. 151, 101-113.

Greunke, C., Hage-Hülsmann, A., Sorkalla, T., Keksel, N., Haberlein, F. & Haberlein,

H. (2015). A systematic study on the influence of the main ingredients of an

ivy leaves dry extract on the β2-adrenergic responsiveness of human airway

smooth muscle cells. Pulm. Pharmacol. Ther. 31, 92-98.

Grundy, S.M. & Vega, G.L. (1988). Hypertriglyceridemia: causes and relation to

coronary heart disease. In: “Seminars in Thrombosis and Hemostasis”. 14,

149-164.

Gulcin, I., Mshvildadze, V., Gepdiremen, A. & Elias, R. (2004). Antioxidant activity

of saponins isolated from ivy: alpha-hederin, hederasaponin-C,

hederacolchiside-E and hederacolchiside-F. Planta Med. 70, 561-563.

Habib, J.B., Bossaller, C., Wells, S., Williams, C., Morrisett, J.D. & Henry, P.D.

(1986). Preservation of endothelium-dependent vascular relaxation in

cholesterol-fed rabbit by treatment with the calcium blocker PN 200110. Circ.

Res. 58, 305-309.

Halkier, B.A. & Gershenzon, J. (2006). Biology and biochemistry of glucosinolates.

Annu. Rev. Plant Biol. 57, 303-333.

Han, X., Shen, T. & Lou, H. (2007). Dietary polyphenols and their biological

significance. Int. J. Mol. Sci. 8, 950-988.

Han, L.K., Xu, B.J., Kimura, Y., Zheng, Y.N. & Okuda, H. (2000). Platycodi radix

affects lipid metabolism in mice with high fat diet induced obesity. J. Nutr.

130, 2760-2764.

Harris, K.E. & Jeffery, E.H. (2008). Sulforaphane and erucin increase MRP1 and

MRP2 in human carcinoma cell lines. J. Nutr. Biochem. 19, 246-254.

Hendrickson, L., Davis, C.R., Roach, C., Aldrich, T., McAda, P.C. & Reeves, C.D.

(1999). Lovastatin biosynthesis in Aspergillus terreus: characterization of

180

blocked mutants, enzyme activities and a multifunctional polyketide synthase

gene. Chem. Biol. 6, 429-439.

Hetta, M.H., Owis, A.I., Haddad, P.S. & Eid, H.M. (2017). The fatty acid-rich

fraction of Eruca sativa (rocket salad) leaf extract exerts antidiabetic effects in

cultured skeletal muscle, adipocytes and liver cells. Pharm. Biol. 55, 810-818.

Higdon, J.V., Delage, B., Williams, D.E. & Dashwood, R.H. (2007). Cruciferous

vegetables and human cancer risk: epidemiologic evidence and mechanistic

basis. Pharmacol. Res. 55, 224-236.

Hoffman, B.B. Therapy of hypertension. In “Goodman and Gilman’s the

pharmacological basis of therapeutics”. Mc Graw-Hill, USA. pp.845-868

(2006).

Huang, K.C., Lin, W.Y., Lee, L.T., Chen, C.Y., Lo, H., Hsia, H.H., Liu, I.L., Shau,

W.Y. & Lin, R.S. (2002). Four anthropometric indices and cardiovascular risk

factors in Taiwan. Int. J. Obes. 26, 1060.

Huxley, A. (1992). New RHS Dictionary of Gardening. 3, pp.532. London:

MacMillan Press.

Istvan, E.S. & Deisenhofer, J. (2001). Structural mechanism for statin inhibition of

HMG CoA- reductase. Science 292, 1160-1164.

Ito, M.K. (2012). Dyslipidemia: management using optimal lipid-lowering

therapy. Ann. Pharmacother. 46, 1368-1381.

Jakubikova, J., Bao, Y. & Sedlak, J. (2005). Isothiocyanates induce cell cycle arrest,

apoptosis and mitochondrial potential depolarization in HL-60 and multidrug-

resistant cell lines. Anticancer Res. 25, 3375-3386.

Jaradat, N.A. (2005). Medical plants utilized in Palestinian folk medicine for

treatment of diabetes mellitus and cardiac diseases. J. Al-Aqsa Uni. 9, 1-28.

Jayakody, L., Senaratne, M., Thomson, A. & Kappagoda, T. (1987). Endothelium-

dependent relaxation in experimental atherosclerosis in the rabbit. Circ. Res.

60, 251-264.

Jeong, H.G. & Park, H.Y. (1998). The prevention of carbon tetrachloride-induced

hepatotoxicity in mice by α-hederin: Inhibiton of cytochrome P450 2E1

expression. IUBMB Life 45, 163-170.

Jiang, H.D., Jun, C., Juan-Hua, X., Xin-Mei, Z. & Qiang X. (2005). Endothelium-

dependent and direct relaxation induced by ethyl acetate extract from Flos

chrysanthemi in rat thoracic aorta. J. Ethnopharmacol. 101, 221-226.

181

Karaki, H., Ozaki, H., Hori, M., Mitsui-Saito, M., Amano, K.I., Harada, K.I.,

Miyamoto, S., Nakazawa, H., Won, K.J. & Sato, K. (1997). Calcium

movements, distribution, and functions in smooth muscle. Pharmacol. Rev.

49, 157-230.

Karam, I., Yang, Y.J. & Li, J.Y. (2017). Hyperlipidemia background and

progress. SM Atheroscler. J. 1, 1003.

Kai, H., Kanaide, H., Matsumoto, T. & Nakamura, M. (1987). 8-Bromoguanosine 3′:

5′-cyclic monophosphate decreases intracellular free calcium concentrations in

cultured vascular smooth muscle cells from rat aorta. FEBS Letters 221, 284-

288.

Karr, S. (2017). Epidemiology and management of hyperlipidemia. Am. J. Manag.

Care 23, S139-S148.

Kellner, A., Correll, J.W. & Ladd, A.T. (1951). The influence of intravenously

administered surface-active agents on the development of experimental

atherosclerosis in rabbits. J. Exp. Med. 93, 385-398.

Khdair, A., Mohammad, M.K., Tawaha, K., Al-Hamarsheh, E., AlKhatib, H.S., Al-

khalidi, B. & Hudaib, M. (2010). A validated RP HPLC-PAD method for the

determination of hederacoside C in Ivy-Thyme cough syrup. Int. J. Anal.

Chem. 2010, 1-5.

Khoobchandani, M., Ganesh, N., Gabbanini, S., Valgimigli, L. & Srivastava, M.M.

(2011). Phytochemical potential of Eruca sativa for inhibition of melanoma

tumor growth. Fitoterapia 82, 647-653.

Khoobchandani, M., Ojeswi, B.K., Ganesh, N., Srivastava, M.M., Gabbanini, S.,

Matera, R. & Valgimigli, L. (2010). Antimicrobial properties and analytical

profile of traditional Eruca sativa seed oil: Comparison with various aerial and

root plant extracts. Food Chem. 120, 217-224.

Kim, B., Choi, Y.E. & Kim, H.S. (2014). Eruca sativa and its flavonoid components,

quercetin and isorhamnetin, improve skin barrier function by activation of

peroxisome proliferator activated receptor (PPARα and suppression of

inflammatory cytokines. Phytother. Res. 28, 1359-1366.

Kishore, L., Kaur, N., Kajal, A. & Singh, R. (2017). Extraction, characterization and

evaluation of Eruca sativa against streptozotocin-induced diabetic

nephropathy in rat. Bangladesh J. Pharmacol. 12, 216-227.

182

Kobayashi, S., Kanaide, H. & Nakamura, M. (1985). Cytosolic-free calcium transients

in cultured vascular smooth muscle cells: microfluorometric measurements.

Science 229, 553-556.

Kuo, Y.H., Lin, C.H., Shih, C.C. & Yang, C.S. (2016). Antcin K, a triterpenoid

compound from Antrodia camphorata, displays antidiabetic and

antihyperlipidemic effects via glucose transporter 4 and AMP-activated

protein kinase phosphorylation in muscles. Evid. Based Complement. Altern.

Med. 2016, 1-16.

Kuroda, M., Tanzawa, K., Tsujita, Y. & Endo, A. (1977). Mechanism for elevation of

hepatic cholesterol synthesis and serum cholesterol levels in Triton WR-1339-

induced hyperlipidemia. Biochim. Biophys. Acta. (BBA)-Lipids and Lipid

Metabolism 489, 119-125.

LaRosa, J.C. (2001). Pleiotropic effects of statins and their clinical significance. Am.

J. Cardiol. 88, 291-293.

Lamy, E., Oey, D., Eißmann, F., Herz, C., Münstedt, K., Tinneberg, H.R. & Mersch-

Sundermann, V. (2013). Erucin and benzyl isothiocyanate suppress growth of

late stage primary human ovarian carcinoma cells and telomerase activity in-

vitro. Phytother. Res. 27, 1036-1041.

Lamy, E., Schroder, J., Paulus, S., Brenk, P., Stahl, T. & Mersch-Sundermann, V.

(2008). Antigenotoxic properties of Eruca sativa (rocket plant), erucin and

erysolin in human hepatoma (HepG2) cells towards benzo (a) pyrene and their

mode of action. Food Chem. Toxicol. 46, 2415-2421.

Lawler, J.E., Sanders, B.J., Chen, Y.F., Nagahama, S. & Oparil, S. (1987).

Hypertension produced by a high sodium diet in the borderline hypertensive

rat (BHR). Clin. Exp. Hypertens. Part A: Theory and Practice 9, 1713-1731.

Lazartigues, E., Brefel-Courbon, C., Tran, M.A., Montastruc, J.L. & Rascol, O.

(1999). Spontaneously hypertensive rats cholinergic hyperresponsiveness:

central and peripheral pharmacological mechanisms. Br. J. Pharmacol. 127,

1657-1665.

Le, N.A. (2015). Lipoprotein-associated oxidative stress: a new twist to the

postprandial hypothesis. Int. J. Mol. Sci. 16, 401-419.

Lee, I.K., Kim, H.S. & Bae, J.H. (2002). Endothelial dysfunction: its relationship with

acute hyperglycemia and hyperlipidemia. Int. J. Clin. Pract. Suppl. 129, 59-

64.

183

Li, W., Altura, B.T. & Altura, B.M. (1998). Differential effects of methanol on rat

aortic smooth muscle. Alcohol 16, 221-229.

Li, D. & Mehta, J.L. 2009. Intracellular signaling of LOX-1 in endothelial cell

apoptosis. Circ. Res. 104, 566-568.

Li, R., Wang, W., Zhang, H., Yang, X., Fan, Q., Christopher, T.A., Lopez, B.L., Tao,

L., Goldstein, B.J., Gao, F. & Ma, X.L. (2007). Adiponectin improves

endothelial function in hyperlipidemic rats by reducing oxidative/nitrative

stress and differential regulation of eNOS/iNOS activity. Am. J. Physiol.

Endocrinol. Metab. 293, 1703-1708.

Li, J., White, J., Guo, L., Zhao, X., Wang, J., Smart, E.J. & Lil, X.A. (2009). Salt

inactivates endothelial nitric oxide synthase in endothelial cells. J. Nutr. 108,

447-451.

Li, G., Zhou, J., Budhraja, A., Hu, X., Chen, Y., Cheng, Q. & Gao, N. (2015).

Mitochondrial translocation and interaction of cofilin and Drp1 are required

for erucin-induced mitochondrial fission and apoptosis. Oncotarget 6, 1834.

Liao, D., Mo, J., Duan, Y., Lin, H.M., Darnell, M. & Qian, Z. (2004). The joint effect

of hypertension and elevated LDL-cholesterol on CHD is beyond additive.

Eur. Heart J. 25, 235.

Lund-Johansen, P. (1985). Hemodynamic effects of calcium channel blockers at rest

and during exercise in essential hypertension. Am. J. Med. 79, 11-18.

Luscher, T.F. & Vanhoutte, P.M. (1986). Endothelium-dependent contractions to

acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension

8, 344-348.

Lutsenko, Y.U., Bylka, W.I., Matlawska, I. & Darmohray, R.O. (2010). Hedera helix

as a medicinal plant. Herba Pol. 56, 83-96.

Maiolino, G., Rossitto, G., Caielli, P., Bisogni, V., Rossi, G.P. & Calò, L.A. (2013).

The role of oxidized low-density lipoproteins in atherosclerosis: the myths and

the facts. Mediators Inflamm. 2013, 1-13.

Malik, A., Mehmood, M.H., Akhtar, M.S. & Gilani, A. (2017). Studies on

antihyperlipidemic and endothelium modulatory activities of polyherbal

formulation (POL4) and its ingredients in high fat diet-fed rats. Pak. J. Pharm.

Sci. 30, 295.

Manchali, S., Murthy, K.N.C. & Patil, B.S. (2012). Crucial facts about health benefits

of popular cruciferous vegetables. J. Funct. Foods 4, 94-106.

184

Mandukhail, S.U.R., Aziz, N. & Gilani, A.H. (2010). Studies on antidyslipidemic

effects of Morinda citrifolia (Noni) fruit, leaves and root extracts. Lipids

Health Dis. 9, 88.

Mannan, R., Arora, R., Bhushann, S., Sharma, S., Bhasin, T. & Arora, S. (2017).

Bioprotective efficacy of erucin against 7, 12-Dimethylbenz (α) anthracene-

induced microstructural changes in male Wistar rats.

Turk. Patoloji Derg. 33, 150-156.

Marinetti, G.V. Disorders of Lipoprotein Metabolism. In “Disorders of Lipid

Metabolism". Springer, Boston, MA. pp. 75-119 (1990).

Martin, W., White, D.G. & Henderson, A.H. (1988). Endothelium-derived relaxing

factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial

cells. Br. J. Pharmacol. 93, 229-239.

Mason, R.P. & Jacob, R.F. (2003). Membrane microdomains and vascular biology:

emerging role in atherogenesis. Circ. Res. 107, 2270-2273.

Mayes, P.A. Cholesterol Synthesis, Transportand Excretion. In “Harper 's

Biochemistry”. 23rd

eds, Murray, R.K., Granner, D.K., Mayes, P.A., RodweiI,

V.W., eds. Appleton & Lange, Norwalk. pp.266-278 (1993).

Melchini, A., Costa, C., Traka, M., Miceli, N., Mithen, R., De Pasquale, R. &

Trovato, A. (2009). Erucin, a new promising cancer chemopreventive agent

from rocket salads, shows anti-proliferative activity on human lung carcinoma

A549 cells. Food Chem. Toxicol. 47, 1430-1436.

Melchini, A. & Traka, M.H. (2010). Biological profile of erucin: A new promising

anticancer agent from cruciferous vegetables. Toxins 2, 593-612.

Melchini, A., Traka, M.H., Catania, S., Miceli, N., Taviano, M.F., Maimone, P. &

Costa, C. (2013). Antiproliferative activity of the dietary isothiocyanate

erucin, a bioactive compound from cruciferous vegetables, on human prostate

cancer cells. Nutr. Cancer 65, 132-138.

Mendel, M., Chłopecka, M., Dziekan, N. Wiechetek, M. (2011). The effect of the

whole extract of common ivy (Hedera helix) leaves and selected active

substances on the motoric activity of rat isolated stomach strips. J.

Ethnopharmacol. 134, 796-802.

Mensah, G.A., Wei, G.S., Sorlie, P.D., Fine, L.J., Rosenberg, Y., Kaufmann, P.G.,

Mussolino, M.E., Hsu, L.L., Addou, E., Engelgau, M.M. & Gordon, D.

185

(2017). Decline in cardiovascular mortality: possible causes and

implications. Circ. Res. 120, 366-380.

Michael, H.N., Shafik, R.E. & Rasmy, G.E. (2011). Studies on the chemical

constituents of fresh leaf of Eruca sativa extract and its biological activity as

anticancer agent in-vitro. J. Med. Plants Res. 5, 1184-1191.

Mithen, R. (2015). Method for treating a cardiovascular disease, hypercholesterolemia

or hypertension or improving serum cholesterol levels by administering

broccoli with a high level of glusosinolates. United States Patent US 9, 023,

811.

Moncada, S. & Higgs, E.A. (1995). Molecular mechanisms and therapeutic strategies

related to nitric oxide. FASEB J. 9, 1319-1330.

Morroni, F., Sita, G., Djemil, A., D’Amico, M., Pruccoli, L., Cantelli-Forti, G. &

Tarozzi, A. (2018). Comparison of adaptive neuroprotective mechanisms of

sulforaphane and its interconversion product erucin in in-vitro and in-vivo

models of Parkinson’s disease. J. Agric. Food Chem. 66, 856-865.

Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M.,

Das, S.R., De Ferranti, S., Després, J.P., Fullerton, H.J. & Howard, V.J.

(2016). Executive summary: heart disease and stroke statistics-2016 update: a

report from the American Heart Association. Circulation 133, 447-454.

National Research Council. (2011). Guide for the care and use of laboratory animals.

8th ed. Washington DC: National Academy Press.

Narender, T., Khaliq, T. & Puri, A. (2006). Antidyslipidemic activity of furano-

flavonoids isolated from Indigofera tinctoria. Bioorg. Med. Chem. Lett. 16,

3411-3414.

Naruse, K., Shimizu, K., Muramatsu, M., Toki, Y., Miyazaki, Y., Okumura, K.,

Hashimoto, H. & Ito, T. (1994). Long-term inhibition of NO synthesis

promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta.

PGH2 does not contribute to impaired endothelium-dependent

relaxation. Arterioscler. Thromb. 14, 746-752.

Nelson, R.H. (2013). Hyperlipidemia as a risk factor for cardiovascular disease. Prim.

Care 40, 195-211.

Ni, Z. & Vaziri, N.D. (2001). Effect of salt loading on nitric oxide synthase

expression in normotensive rats. Am. J. Hypertens. 14, 155-163.

186

Nies, L.K., Cymbala, A.A., Kasten, S.L., Lamprecht, D.G. & Olson, K.L. (2006).

Complementary and alternative therapies for the management of dyslipidemia.

Ann. Pharmacother. 40, 1984-1992.

Norata, G.D., Ballantyne, C.M. & Catapano, A.L. (2013). New therapeutic principles

in dyslipidaemia: focus on LDL and Lp (a) lowering drugs. Eur. Heart J. 34,

1783-1789.

Oakenfull, D.G. & Sidhu, G.S. (1990). Could saponins be a useful treatment for

hypercholesterolemia? Eur. J. Clin. Nutr. 44, 79-88.

O’Donnell, V.B. (2003). Free radicals and lipid signaling in endothelial cells.

Antioxid. Redox Signal. 5, 195-203.

Ohara, Y., Peterson, T.E. & Harrison, D.G. (1993). Hypercholesterolemia increases

endothelial superoxide anion production. J. Clin. Invest. 91, 2546-2551.

O'meara, J.G., Kardia, S.L., Armon, J.J., Brown, C.A., Boerwinkle, E. & Turner, S.T.

(2004). Ethnic and sex differences in the prevalence, treatment and control of

dyslipidemia among hypertensive adults in the GENOA study. Arch. Intern.

Med. 164, 1313-1318.

.Omri Hichri, A., Mosbah, H., Majouli, K., Besbes Hlila, M., Ben Jannet, H., Flamini,

G. & Selmi, B. (2016) Chemical composition and biological activities of

Eruca vesicaria subsp. longirostris essential oils. Pharm. Biol. 54, 2236-

2243.

Palmer, R.M., Ferrige, A.G. & Moncada, S. (1987). Nitric oxide release accounts for

the biological activity of endothelium-derived relaxing factor. Nature 327,

524.

Park, C., Wang, G., Durthaler, J.M. & Fang, J. (2017). Cost-effectiveness analyses of

antihypertensive medicines: a systematic review. Am. J. Prev. Med. 53, S131-

S142.

Parvu, M., Vlase, L., Parvu, A.E., Rosca-Casian, O., Gheldiu, A.M. & Parvu O.

(2015). Phenolic compounds and antifungal activity of Hedera helix L.(Ivy)

flowers and fruits. Not. Bot. Horti Agrobot. Cluj-Napoca 43, 53-58.

Pasini, F., Verardo, V., Caboni, M.F. D’Antuono, L.F. (2012). Determination of

glucosinolates and phenolic compounds in rocket salad by HPLC-DAD–MS:

Evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic

resources. Food Chem. 133, 1025-1033.

187

Pirillo, A. & Catapano, A.L. (2013). Soluble lectin-like oxidized low density

lipoprotein receptor-1 as a biochemical marker for atherosclerosis-related

diseases. Dis. Mark. 35, 413-418.

Prełowska, M., Kaczyńska, A. Herman-Antosiewicz, A. (2017). 4-(Methylthio)

butyl isothiocyanate inhibits the proliferation of breast cancer cells with

different receptor status. Pharmacol. Rep. 69, 1059-1066.

Rehman, K., Khan, M.A., Ullah, Z. & Chaudhary, H.J. (2015). An ethno botanical

perspective of traditional medicinal plants from the Khattak tribe of Chonthra

Karak, Pakistan. J. Ethnopharmacol. 165, 251-259.

Rehman, S.U., Kim, I.S., Choi, M.S., Kim, S.H., Zhang, Y. & Yoo, H.H. (2017).

Time-dependent Inhibition of CYP2C8 and CYP2C19 by Hedera helix

extracts, A traditional respiratory herbal medicine. Molecules 22, 1241.

Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E. & Dannenberg, A.L. (1986). Lp (a)

lipoprotein as a risk factor for myocardial infarction. JAMA 256, 2540-2544.

Ribeiro, R.D.A., De Melo, M.M.R.F., De Barros, F., Gomes, C. & Trolin, G. (1986).

Acute antihypertensive effect in conscious rats produced by some medicinal

plants used in the state of Sao Paulo. J. Ethnopharmacol. 15, 261-269.

Roşca-Casian, O., Mircea, C., Vlase, L., Gheldiu, A.M., Teuca, D.T. & Pârvu, M.

(2017). Chemical composition and antifungal activity of Hedera helix leaf

ethanolic extract. Acta Biol. Hung. 68, 196-207.

Ross, R. (1999). Atherosclerosis-an inflammatory disease. N. Engl. J. Med. 340, 115-

126.

Rouhi-Boroujeni, H., Rouhi-Boroujeni, H., Heidarian, E., Mohammadizadeh, F. &

Rafieian-Kopaei, M. (2015). Herbs with anti-lipid effects and their interactions

with statins as a chemical anti-hyperlipidemia group drugs: A systematic

review. ARYA Atheroscler. 11, 244.

Ryoo, S., Lemmon, C.A., Soucy, K.G., Gupta, G., White, A.R., Nyhan, D., Shoukas,

A., Romer, L.H. & Berkowitz, D.E. (2006). Oxidized low-density lipoprotein-

dependent endothelial arginase II activation contributes to impaired nitric

oxide signaling. Circ. Res. 99, 951-960.

Sabeen, M. & Ahmad, S.S. (2009). Exploring the folk medicinal flora of Abbotabad

city, Pakistan. Ethnobot. Leaflets 2009, 1.

188

Saha, S., Hollands, W., Teucher, B., Needs, P.W., Narbad, A., Ortori, C.A., Barrett,

D.A., Rossiter, J.T., Mithen, R.F. & Kroon, P.A. (2012). Isothiocyanate

concentrations and interconversion of sulforaphane to erucin in human

subjects after consumption of commercial frozen broccoli compared to fresh

broccoli. Mol. Nutr. Food Res. 56, 1906-1916.

Schalkwijk, C.G., van Dam, B., Stehouwer, C.D. & van Hinsbergh, V.W. (2007).

Mevastatin increases eNO synthase expression and inhibits lipid peroxidation

in human endothelial cells. Clin. Hemorheol. Microcirc. 37, 179-184.

Shah, A.J. & Gilani, A.H. (2009). Blood pressur-lowering and vascular modulator

effects of Acorus calamus extract are mediated through multiple pathways.

Cardiovasc. Pharmacol. 54, 38-46.

Shah, A.J. & Gilani, A.H. (2011). Blood pressure lowering effect of the extract of

aerial parts of Capparis aphylla is mediated through endothelium-dependent

and independent mechanisms. Clin. Exp. Hypertens. 33, 470-477.

Sheikh, Z.A., Zahoor, A., Khan, S.S., Naveed, S., Zaidi, S.F. & Usmanghani, K.

(2015). Ivy herbal products for cough and its validation for hederacoside c

biomarker determination with HPLC-PAD techniques. RADS J. Pharm.

Pharm. Sci. 3, 1-6.

Shi, Y., Guo, R., Wang, X., Yuan, D., Zhang, S., Wang, J., Yan, X. & Wang, C.

(2014). The regulation of alfalfa saponin extract on key genes involved in

hepatic cholesterol metabolism in hyperlipidemic rats. PloS One 9, 88282.

Shulman, G.I. (2014). Ectopic fat in insulin resistance, dyslipidemia and

cardiometabolic disease. N. Engl. J. Med. 371, 1131-1141.

Siddiqi, H.S., Mehmood, M.H., Rehman, N.U. & Gilani, A.H. (2012). Studies on the

antihypertensive and antidyslipidemic activities of Viola odorata leaves

extract. Lipids Health and Dis. 11, 6.

Sieben, A., Prenner, L., Sorkalla, T., Wolf, A., Jakobs, D., Runkel, F. & Haberlein, H.

(2009). α-Hederin, but not hederacoside C and hederagenin from Hedera

helix, affects the binding behavior, dynamicsand regulation of β2-adrenergic

receptors. Biochemistry 48, 3477-3482.

Simopoulos, A. P., Leaf, A. & Salem Jr, N. (1999). Essentiality of and recommended

dietary intakes for omega-6 and omega-3 fatty acids. Ann. Nutr. Metab. 43,

127-130.

189

Sinclair, S.J., Murphy, K.J., Birch, C.D. & Hamill, J.D. (2000). Molecular

characterization of quinolinate phosphoribosyltransferase (QPRTase) in

Nicotiana. Plant Mol. Biol. 44, 603-617.

Singh, G., Gupta, P., Rawat, P., Puri, A., Bhatia, G. & Maurya, R. (2007).

Antidyslipidemic activity of polyprenol from Coccinia grandis in high-fat

diet-fed hamster model. Phytomedicine 14, 792-798.

Sripradha, R., Sridhar, M.J. & Maithilikarpagaselvi, N. (2015). Antihyperlipidemic

and antioxidant activities of the ethanolic extract of Garcinia cambogia on

high fat diet-fed rats. J. Complement. Integr. Med. 2015, 1-8.

Steinmetz, E.F. (1961) Hederae helicis folia. Q. J. Crude Drug Res. 1, 93-95.

Stevinson, C., Pittler, M.H. & Ernst, E. (2000). Garlic for treating

hypercholesterolemia: a meta-analysis of randomized clinical trials. Ann.

Intern. Med. 133, 420-429.

Szwajgier, D. (2015). Anticholinesterase activity of selected phenolic acids and

flavonoids-interaction testing in model solutions. Ann. Agric. Environ. Med.

22, 690-694.

Tarozzi, A., Morroni, F., Bolondi, C., Sita, G., Hrelia, P., Djemil, A. & Cantelli-Forti,

G. (2012). Neuroprotective effects of erucin against 6-hydroxydopamine-

induced oxidative damage in a dopaminergic-like neuroblastoma cell line. Int.

J. Mol. Sci. 13, 10899-10910.

Teixeira, M.Z. (2013). Rebound effect of modern drugs: serious adverse event

unknown by health professionals. Rev. Assoc. Med. Bras. 59, 629-638.

Temel, R.E. & Brown, J.M. (2015). A new model of reverse cholesterol transport:

enTICEing strategies to stimulate intestinal cholesterol excretion. Trends

Pharmacol. Sci. 36, 440-451.

Thomas, F;, Rudnichi, A., Bacri, A.M., Bean, K., Guize, L. & Benetos, A. (2001).

Cardiovascular mortality in hypertensive men according to presence of

associated risk factors. Hypertension 37, 1256-1261.

Thomas, T.R., Pellechia, J., Rector, R.S., Sun, G.Y., Sturek, M.S. & Laughlin, M.H.

(2002). Exercise training does not reduce hyperlipidemia in pigs fed a high-fat

diet. Metab. Clin. Exp. 51, 1587-1595.

190

Torres-Piedra, M., Figueroa, M., Hernandez-Abreu, O., Ibarra-Barajas, M., Navarrete-

Varquez, G. & EstradaSoto, S. (2011). Vasorelaxant effect of flavonoids

through calmodulin inhibitor: ex vivo, in vitro and in silico approaches.

Bioorg. Med. Chem. 19, 542-546.

Trute, A., Gross, J., Mutschler, E. & Nahrstedt, A. (1997). In-vitro antispasmodic

compounds of the dry extract obtained from Hedera helix. Planta Med. 63,

125-129.

Trute, A. & Nahrstedt, A. (1997). Identification and quantitative analysis of phenolic

compounds from the dry extract of Hedera helix. Planta Med. 63, 177-179.

Turin, T.C., Shahana, N., Wangchuk, L.Z., Specogna, A.V., Al Mamun, M., Khan,

M.A. & Rumana, N. (2013). Burden of cardio-and cerebro-vascular diseases

and the conventional risk factors in South Asian population. Glob. Heart, 8,

121-130.

Valnet J. Lierre grimpant. In “Maloine (editor). Phytotherapie”. 5th

eds. Paris.

pp.506-509 (1983).

Van De Sluis, B., Wijers, M. & Herz, J. (2017). News on the molecular regulation and

function of hepatic LDLR and LRP1. Curr. Opin. Lipidol. 28, 241.

Vasdev, S., Gill, V., Longerich, L., Parai, S. & Gadag V. (2003). Salt-induced

hypertension in WKY rats: prevention by α-lipoic acid supplementation. Mol.

Cell. Biochem. 254, 319-326.

Vidal, F., Colome, C., Martínez-González, J. & Badimon, L. (1998). Atherogenic

concentrations of native low density lipoproteins down regulate nitric oxide

synthase mRNA and protein levels in endothelial cells. Eur. J. Biochem. 252,

378-384.

Villani, P., Orsiere, T., Sari-Minodier, I., Bouvenot, G. & Botta, A. (2001). In-vitro

study of the antimutagenic activity of alpha hederin. Ann. Boil. Clin. 59, 285-

289.

Villatoro-Pulido, M., PriegoCapote, F., Álvarez Sánchez, B., Saha, S., Philo, M.,

Obregón Cano, S. & Del RíoCelestino, M. (2013). An approach to the

phytochemical profiling of rocket [Eruca sativa (Mill.) Thell]. J. Sci. Food

Agric. 93, 3809-3819.

191

Virchow, R.P. & Thrombose, I.G. (1856). In: “Gesammelte Abhandlungen zur

Wissenschaftlichen Medicin. Frankfurt-am-Main”. pp. 458-564. Meidinger

Sohn & Company.

Walum, E. (1998). Acute oral toxicity. Environ. Health Perspect. 2, 497.

Wider, B., Pittler, M.H., Thompson-Coon, J. & Ernst, E. (2009). Artichoke leaf

extract for treating hypercholesterolaemia. Cochrane Database Syst. Rev. 4. 1-

28.

Williamson, E.M., Okpako, D.T., Evans, F.J. (1996). Pharmacological methods in

phytotherapy research: volume 1: Selection, preparation and pharmacological

evaluation of plant material. John Wiley & Sons Ltd. pp 15-23.

Wolf, A., Gosens, R., Meurs, H. & Haberlein, H. (2011). Pre-treatment with α-hederin

increases β-adrenoceptor mediated relaxation of airway smooth muscle.

Phytomedicine 18, 214-218.

Wu, L., Ashraf, M.H.N., Facci, M., Wang, R., Paterson, P.G., Ferrie, A. & Juurlink,

B.H. (2004). Dietary approach to attenuate oxidative stress, hypertension and

inflammation in the cardiovascular system. Proc. Natl. Acad. Sci. 01, 7094-

7099.

Xu, S., Ogura, S., Chen, J., Little, P.J., Moss, J. & Liu, P. (2013). LOX-1 in

atherosclerosis: biological functions and pharmacological modifiers. Cell.

Mol. Life Sci. 70, 2859-2872.

Yakovishin, L.A. & Grishkovets, V.I. (2003). Triterpene glycosides of the medicinal

preparation hedelix®. Chem. Nat. Compd. 39, 508-509.

Yaniv, Z., Schafferman, D. & Amar, Z. (1998). Tradition, uses and biodiversity of

rocket (Eruca sativa, Brassicaceae) in Israel. Econ. Bot. 52, 394-400.

Yehuda, H., Khatib, S., Sussan, I., Musa, R., Vaya, J. & Tamir, S. (2009). Potential

skin antiinflammatory effects of 4-methylthiobutylisothiocyanate (MTBI)

isolated from rocket (Eruca sativa) seeds. Biofactors 35, 295-305.

Yoshihisa, N., Ichihara, K., Yoshida, R. & Abiko, Y. (1992). Positive inotropic and

negative chronotropic effects of (−) cis-diltiazem in rat isolated atria. Br. J.

Pharmacol. 105, 696-702.

192

Zaid, M. & Hasnain, S. (2018). Plasma lipid abnormalities in Pakistani population:

trends, associated factors, and clinical implications. Braz. J. Med. Biol. Res.

51, 1-9.

Zhao, M.J., Wang, S.S., Jiang, Y., Wang, Y., Shen, H., Xu, P., Xiang, H. & Xiao, H.

(2017). Hypolipidemic effect of XH601 on hamsters of hyperlipidemia and its

potential mechanism. Lipids Health Dis. 16, 85.

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Studies on Antihyperlipidemic and Vasomodulating Effects