Protective Effects of Yuzu and Its Component against Cardiovascular Disease

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Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

by

Hye Yon Yu

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

by

Hye Yon Yu

A Dissertation Submitted to The Graduate School of

Ajou University in Partial Fulfillment of the Requirements for

The Degree of Master of Biomedical Sciences

Supervised by

Soo Hwan Lee, Ph.D.

Yi-Sook Jung, Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

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This certifies that

of Hye Yoⅱ

Yu

the dissertation

is approved●

The Gradμ

ate

December,

S UPERV工

SORY COMMITTEE

Schoo1; Aj˚

u

19th, 2014

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-ABSTRACT-

Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

Left ventricular (LV) remodeling, which includes ventricular dilatation and increased

interstitial fibrosis after myocardial infarction (MI), is the critical process underlying the

progression to heart failure (HF). Therefore, a novel approach for preventing LV remodeling

after MI is highly desirable. Yuzu is a citrus plant originating in East Asia, and has a number

of cardioprotective ingredients such as hesperidin. However, no study has proved whether

yuzu can prevent LV remodeling. The aim of this study was to determine the effects of yuzu

on HF and its potential impact on the LV remodeling process after MI and platelet

aggregation. In this study, it was investigated whether yuzu and its components hesperidin

and naringin, have anti-platelet activities. Yuzu and hesperidin inhibited collagen-,

arachidonic acid (AA)-, ADP- and thrombin-induced rat platelet aggregation in vitro and ex

vivo. Naringin also inhibited platelet aggregation induced by collagen, AA, or thrombin, but

not by ADP. The oral administration of yuzu or hesperidin prolonged mouse tail vein

bleeding time in a dose-dependent manner. LV remodeling in vivo study using the

permanent left anterior descending coronary artery (LAD) occlusion model demonstrated

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occlusion significantly attenuated cardiac dysfunction, myocyte apoptosis and inflammation.

Not only yuzu but also hesperidin inhibited caspase-3 activity, myeloperoxidase expression,

α-smooth muscle actin expression, and matrix metalloproteinase-2 activity in a permanent LAD occlusion rat model. These results suggest that yuzu and hesperidin have anti-platelet

activity, and that intake of yuzu, which contains various flavonoids such as hesperidin, may

be beneficial for individuals at high risk of cardiovascular diseases. These findings provide

the first evidence that yuzu and hesperidin prevent MI-induced ventricular dysfunction and

structural remodeling of myocardium.

Key words : yuzu, hesperidin, platelet aggregation, cardiomyocyte, ischemic heart disease,

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LIST OF FIGURES

Fig. 1. Dose-dependent inhibitory effect of yuzu on in vitro platelet aggregation ... 15

Fig. 2. Chromatographic result for yuzu with detection at 280 nm. ... 19

Fig. 3. Dose-dependent inhibitory effect of hesperidin on in vitro platelet aggregation ... 21

Fig. 4. Dose-dependent inhibitory effect of naringin on in vitro platelet aggregation ... 22

Fig. 5. Effect of yuzu, hesperidin, and naringin on collagen- and thrombin-induced TXB2 formation in rat platelets ... 26

Fig. 6. Effects of yuzu and hesperidin on mouse tail bleeding times ... 30

Fig. 7. Experimental schedule ... 32

Fig. 8. Effect of pre-treatment with yuzu or hesperidin on cardiac dysfunction ... 33

Fig. 9. Effect of pre-treatment with yuzu or hesperidin on LV dysfunction and LV remodeling ... 34

Fig. 10. Effect of pre-treatment with yuzu or hesperidin on cardiac fibrosis ... 36

Fig. 11. Effect of pre-treatment with yuzu or hesperidin on myofibroblast ... 37

Fig. 12. Effect of pre-treatment with yuzu or hesperidin on inflammatory reaction ... 39

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Fig. 14. Effect of pre-treatment with yuzu or hesperidin on cardiomyocyte apoptosis ... 42

Fig. 15. Effect of pre-treatment with yuzu or hesperidin on caspase-3 activity ... 43

Fig. 16. Effect of pre-treatment with yuzu or hesperidin on cardiomyocyte preservation ... 45

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LIST OF TABLES

Table 1. IC50 of yuzu, hesperidin, and naringin for platelet aggregation ... 16

Table 2. Contents of hesperidin and naringin in extract of yuzu ... 18

Table 3. Effects of yuzu, hesperidin, and naringin on LDH release by platelets ... 24

Table 4. Inhibitory effects (%) of orally administered yuzu and hesperidin on ex vivo platelet

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ABBREVIATION

AA arachidonic acid

ADP adenosine diphosphate

BSA bovine serum albumin

cTnI cardiac troponin I

CVD cardiovascular disease

DMSO dimethylsulfoxide,

HF heart failure

LAD left anterior descending

LV left ventricular

MI myocardial infarction

MMP matrix metalloproteinase

MPO myeloperoxidase

PEG polyethylene glycol

PO permanent LAD occlusion

SMA smooth muscle actin

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I. INTRODUCTION

A. Antiplatelet therapy in cardiovascular diseases

Platelets are essential for primary hemostasis and for repair of the endothelium, but they

are also responsible for the formation of pathogenic thrombi that cause cardiovascular

diseases (CVDs), such as acute coronary syndrome, ischemic stroke and symptomatic

peripheral artery disease. Platelets are also a source of inflammatory mediators, and their

activation by inflammatory triggers may be a critical component of atherothrombosis

(Jennings, 2009). Accordingly, the inhibition of platelet hyperactivation has been adopted as

a strategy to treat these disorders, and several anti-platelet drugs, including aspirin, are used

clinically. Since these anti-platelet drugs have all been reported to have adverse side effects,

the development of safe new therapeutic agents with anti-platelet activity remains a critical

issue. Interestingly, several natural compounds from plant sources, such as, spinach and

tomato, have been reported to have preventive effects against CVDs by inhibiting platelet

aggregation (O’Kennedy et al, 2006).

B. Left ventricular (LV) remodeling and heart failure (HF)

LV remodeling is a pathologic change in the architecture of the LV that occur due to

various CVDs including myocardial infarction (MI) and hypertension (Yi et al, 2012). Of

these, MI is caused by the partial interruption or occlusion of the blood supply to a part of

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the rupture of a vulnerable atherosclerotic plaque (Gao et al, 2010) LV remodeling after MI

is associated with a combination of pathologic conditions, including myocyte hypertrophy,

myocyte apoptosis, myofibroblast proliferation, inflammatory reaction, and interstitial

fibrosis, which ultimately lead to the loss of systolic and diastolic function (Konstam et al,

2011). Cardiac hypertrophy is a compensatory process in response to increased

hemodynamic overload, characterized by an increase in the size of individual cardiac

myocytes and wall thickness. On the other hand, in chronic MI following left anterior

descending coronary artery (LAD) occlusion, a transition occurs from compensatory cardiac

hypertrophy to decompensatory hypertrophy, characterized by a chamber dilation and wall

thinning. In this chronic condition, processes such as extracellular matrix turnover, fibrosis,

inflammation and apoptosis are crucial determinants (Eapen and Rogers, 2009 ; . Yi et al,

2012)

LV remodeling after MI is a key contributor to HF, which is one of the most common

causes of cardiovascular morbidity and mortality worldwide (González et al, 2011). HF is

defined as a clinical syndrome because of its complex pathologic mechanism changes that

contribute to myocardial dysfunction following LV remodeling (Nagarajan and Tang, 2011).

Conventional HF therapy is still largely based on targeting the causes and neurohumoral

activation of HF, and includes agents such as angiotensin-converting enzyme inhibitors,

angiotensin-receptor antagonists, beta-blockers, and aldosterone antagonist (Jessup and

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C. Yuzu and its components

Recently, natural products have become popular worldwide and have gained wide

acceptance as adjuncts to conventional therapy. Various studies have shown natural products

such as citrus fruits, grape, broccoli, and cacao are rich sources of phytochemicals such as

polyphenols that are well known for their cardiovascular protective effects (Akhlaghi and

Bandy, 2010; Yamazaki et al, 2010; Kim et al, 2014). Furthermore, numerous researches

indicate that the consumption of flavonoid-rich foods decrease the incidence of CVDs

(Yamazaki et al, 2008).

In particular, yuzu (Citrus junos sieb ex Tanaka) is one of the most famous natural

products. Yuzu is small tree that produces yellow-golden colored citrus fruits native to

northeast Asia, including Korea, China, and Japan. Yuzu is used in traditional Chinese

medicine and yuzu tea is known to improve a cold in Korea. Several studies have shown that

components of yuzu such as limonene, vitamin C, phenolic substances exhibit an antioxidant

and anti-inflammatory activities (Hirota et al, 2010).

Like other citrus fruits, yuzu contains many biofunctional components, such as,

flavonoids, carotenoids and ascorbic acid (Yoo et al., 2009). Furthermore, numerous

experimental studies have shown flavonoids inhibit primary hemostasis and many pathways

which are associated with platelet activation and aggregation (Rein et al., 2000). Flavonoids,

which occur in the free form and as glycosides, are a large group of low molecular weight

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active compounds. Hesperidin and naringin are known to be major flavonoid components in

yuzu (Yoo et al, 2009). Hesperidin, a glycosidic form of hesperetin, is encountered

extensively in the plant kingdom, especially in citrus fruits, such as, grapefruits and oranges,

which are also commonly used in traditional medicines (Garg et al, 2001). Naringin is a

glycosidic form of naringenin, and the major flavanone found in citrus fruits. In animal

experiments, naringenin consumption was found to be associated with lipid-lowering effects,

reduced plasma markers of endothelial dysfunction, and improved insulin sensitivity (Chanet

et al, 2012). However, no report has been issued on its inhibitory effect on platelet

aggregation and LV remodeling.

D. Aims of study

1. In the present study, it was investigated the anti-platelet activities of the yuzu and its

major flavonoid components, in order to explain their possible contributions to the

attenuation of platelet hyperactivity.

2. The goal of the present study was to evaluate the effects of yuzu in a rat model of LV

remodeling induced by permanent LAD occlusion. Considering that hesperidin is

well-known major functional component of yuzu, it has also evaluated whether hesperidin

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II. MATERIALS AND METHODS

A. Materials

1. Materials

Collagen, ADP, thrombin, arachidonic acid (AA), and luciferin/luciferase reagent were

purchased from Chrono-Log Co. (Harvertown, PA, USA). Dimethylsulfoxide (DMSO),

polyethylene glycol (PEG), bovine serum albumin (BSA), β-nicotinamide adenine dinucleotide (reduced disodium salt hydrate, β-NADH), pyruvic acid, and histone H1-IIIS

were purchased from Sigma (St. Louis, MO, USA). Thromboxane B2 (TXB2) enzyme

immunosaasy (EIA) kit was purchased from Cayman Chemical Company (Ann Arbor, MI,

USA).

B. Methods

1. Plant material and preparation of samples

Extract of yuzu were obtained from Konkuk University (Seoul, Republic of Korea).

Briefly, minced yuzu fruits were extracted with ethanol and lyophilized to remove solvent.

Yuzu extract was dissolved in saline (0.9% NaCl) for the in vitro, ex-vivo and in vivo study.

Yuzu major components, hesperidin and naringin are water-insoluble, they were dissolved in

DMSO to make final concentration of 0.1% DMSO for the in vitro platelet aggregation

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a widely used solvent for water-insoluble compounds for in vivo study.

2. Experimental protocol

PART I: For platelet aggregation experiments, yuzu and its component, hesperidin and

narigin were administered with oral gavage at a volume of 0.5 ml/kg per mouse or rat 2 h

before experiments. Control animals received the same volume of saline or 70% PEG,

respectively. All agents were prepared just before use.

PART II: For LV remodeling experiments, all experimental procedures conformed to the

Guide for the Care and Use of Laboratory Animals published by the US National Institutes

of Health (NIH Publication No. 85-23, revised 1996), and the Committee on Animal

Research at Ajou Medical Center, Ajou University (Suwon, Republic of Korea), approved

the study. Male Sprague-Dawley rats (weight, 250–300 g) were anaesthetized with an

intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before surgery.

The body temperature of the rats was maintained at 37 ± 0.5°C during surgery by using a

thermostatically controlled warming plate as described previously (Kim et al., 2010) In the

loss-of-function study, ischaemia-induced myocardial injury was induced by ligating the

LAD artery as described previously (Kim et al., 2010;

Yamazaki

et al. 2010).

Sham-operated control group (sham) underwent the same surgical procedures except that the suture

placed under the left anterior descending was not tied. At 4 weeks after LAD occlusion, rats

were euthanized by CO2 inhalation for heart isolation. LV was used for staining experiments.

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blot analysis. Peri-infarct zone was defined as the area within 2 mm of the visible edge of

infarction (Kido et al, 2005).

The rats were randomly distributed into experimental pre-treatment groups with similar

body weights. Yuzu (100 mg/kg/day, n=20), hesperidin (30 mg/kg/day, n=20), vehicle (PEG

0.3 ml/day, n=23) or sham (PEG 0.3 ml/day, n=20) was administered by oral gavage once

daily beginning 7 days before LAD occlusion and continuously administered until the time

of the terminal study, using oral gavage around 10:00 A.M. every morning (for total 5 weeks,

Fig. 7.).

3. Animals

Sprague-Dawley (SD) rats and ICR mice were purchased from the Samtako Laboratory

Animal Center (Republic of Korea), and housed in a conventional animal facility with free

access to food and water in a temperature and relative humidity monitored and controlled

environment under artificial lighting (12 h of light per day). Animals were allowed to

acclimatize for at least 7 days before experiments. All animals related study protocols were

conducted in accordance with the guidelines for the Care and Use of Laboratory Animals

published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996),

and were approved by the Committee on Animal Research at Ajou Medical Center, Ajou

University.

4. Preparation of platelets

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Briefly, Sprague–Dawley (SD) rats, weighing 200–250 g, were lightly anesthetized with

ethyl ether and 8–10 ml of blood was collected from abdominal aorta into sodium citrate

(3.8%, 1:9 v/v) containing tubes. After centrifugation at 150g for 10 min at room temperature,

supernatants (PRP) were used for the aggregation study. PRP was centrifuged at 1200g for

10 min at room temperature and supernatant was obtained as platelet poor plasma (PPP)

which was used to adjust PRP. All experiments were conducted at least four times.

5. In vitro platelet aggregation study

Platelet aggregation studies were performed under the experimental setting described by

previous studies using the turbidimetric method (Seo et al., 2011). Briefly, PRP was

stimulated with different aggregating agents at the following final concentrations; collagen 2

μg/mL, thrombin 0.4 U/mL, AA 100 μM, or ADP 10 μM. Platelet aggregation was recorded 5 min after platelet stimulation. Aggregations were measured by a Lumi-aggregometer

(Chrono-Log Co., Harvertown, PA, USA) connected to computer and expressed as percent

changes in light transmission, taking the value of a blank sample (buffer without platelets) to

be 100%. For in vitro studies, PRP was preincubated with different concentrations of the four

anthraquinone derivatives for 5 min in the cuvette of an aggregometer before being

stimulated with the aggregating agents described above.

6. Determination of cytotoxicity

The cytotoxic effects of samples on platelets were determined by measuring lactate

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PRP was incubated at 37 °C for 5 min with vehicle or samples, and centrifuged at room

temperature for 1 min at 10,000 g. Aliquots of supernatant (25 μL) were then placed into a

96-well plate and mixed with 100 μL of NADH solution (0.03% β-NADH in phosphate

buffer) and 25 μL of pyruvate solution (22.7 mM pyruvic acid in phosphate buffer) at room temperature. Reductions in absorbance at 340 nm due to the conversion of NADH to NAD+

were used as measures of LDH activity. LDH leakages were expressed as percentages of

total enzyme activity measured in platelets completely lysed with 0.2% Triton X-100.

7. Ex vivo platelet aggregation study

Two hours after the oral administration of yuzu (100 mg/kg), hesperidin (10 mg/kg), or

aspirin (50 mg/kg), rat blood samples were collected and the platelet aggregation

experiments were performed as described above.

8. In vivo mice tail bleeding times

Bleeding times were determined as previously described (Cho et al, 2008). Male ICR

mice weighing 35–40 g were used in this experiment. Mice were fasted overnight

beforehand. Two hours after the oral administration of yuzu (3, 10, 30 mg/kg), hesperidin (1,

3, 10 mg/kg) or aspirin (50 mg/kg), mice were anesthetized with sodium pentobarbital (75

mg/kg, i.p.). Mice were then placed individually on a hotplate to control body temperature at

37 °C and tails were transected 3 mm from their tips with a razor blade and then immersed

in a 15 ml clear conical tube containing normal saline prewarmed to 37 °C. Times to blood

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stopped for 15 s. Bleeding times exceeding 15 min were recorded as 15 min for the purposes

of statistical analysis.

9. Measurement of thromboxane A2 (TXA2) formation

The formation of TXA2 in platelets was measured by determining TXB2 using a TXB2

EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA), because TXA2 is unstable and

quickly converted to TXB2. A suspension of rat PRP was preincubated for 5 min in the

presence or absence of DMSO, yuzu, hesperidin, naringin, or aspirin before adding collagen

(2 mg/ml) or thrombin (0.4 U/ml). After incubation at 37 °C for 5 min with collagen or

thrombin, EDTA (10 mM) was added to stop TXA2 formation. TXB2 in supernatant was

obtained by centrifuging at 12,000g for 1 min, and the amount of TXB2 in medium was

determined by using the TXB2 EIA kit, according to the procedure described by the

manufacturer (Cayman Chemical Co.).

10. Echocardiography

The rats were subjected to transthoracic echocardiography. In brief, the rats were

anaesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10

mg/kg) and were examined with non-invasive echocardiography (echocardiograph IE33

ultrasound, S12-4 probe, Philips). Ventricular remodeling in the vehicle-, yuzu-, or

hesperidin-treated groups was assessed weekly with serial echocardiography (beginning 0

week prior to LAD occlusion until 4 weeks after LAD occlusion). Cardiac ventricular

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of 5 consecutive cardiac cycles of each animal is reported.

11. Histological analysis

LV tissue was fixed with 4% paraformaldehyde for 24 hours, dehydrated with

increasing concentrations of ethanol, and then embedded in paraffin. LV sections (5 μm)

were stained with hematoxylin and eosin (H&E, Sigma-Aldrich) or Masson trichrome

(polysciences) as previously described (Akhlaghi and Bandy, 2010; Lal et al, 2012). Images

were captured by utilizing a Zeiss Axioplot Vision-series microscope and software (Carl

Zeiss, Oberkochen, Germany) and were quantified using the NIH Image J analysis program

(NIH, Bethesda, MD, USA).

12. Immunohistochemistry

Immunostaining was performed using a streptavidin–biotin-immunoperoxidase

complex method with 5 μm thick sections, which had been deparaffinized and heated in 0.01 M citrate buffer solution (pH ¼ 6.0) for 15 min for antigen retrieval. Rabbit polyclonal

antibody against, myeloperoxidase (MPO), or cardiac tronponin I (cTnI) purchased from

Abcam (Cambridge, UK) was used. The slides were examined using a light microscope

(Olympus CX21, Japan) and was evaluated with reference to the optical density of the stain

by using a computer-assisted image analysis system, ImageJ1.45F (NIH, USA).

13. Gelatin zymography

Gelatin zymography was performed utilizing the Novex In-gel Zymography System

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were homogenized in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 5 mM CaCl2.

After centrifugation, the supernatants were harvested and 25 µg of protein was mixed with

Tris-glycine sodium dodecyl sulfate (SDS) sample buffer. The samples were run on a Novex

10% Zymogram Gelatin Gel, followed by incubation with Zymogram Renaturing Buffer and

subsequent incubation with Zymogram Developing Buffer. After an overnight reaction, the

gel was stained with Simply Blue Safe Stain (Invitrogen).

14. Western blot analysis

Heart tissues were homogenized in a buffer containing 50 mmol/L Tris-HCl pH 7.4,

1% NP-40, 150 mmol/L NaCl, 0.25% Na-deoxycholate, 2 mmol/L EDTA, 1 mmol/L NaF, 1

mmol/L Na3VO4, 1 mmol/L PMSF, 10 μg/mL aprotinin, and 10 μmol/L leupeptin.

Homogenates were centrifuged at 14,681 g for 15 min and the supernatants were collected as

previously described (Kim et al, 2010). Equal amounts of protein were then separated by

SDS-polyacrylamide gel electrophoresis (PAGE) and reacted with antibodies specific for

caspase-3 (Cell Signaling, Danvers, MA, USA), α-SMA (Abcam) and α-tubulin

(Sigma-Aldrich). After probing with an HRP-conjugated secondary antibody, the proteins were

visualized using LAS 1000 (Fuji Photo Film, Tokyo, Japan). Densitometric analyses were

performed using Quantity One software,ImageJ1.45F (NIH, USA).

15. Terminal dUTP nick end-labeling (TUNEL) staining

In situ labeling of fragmented DNA was performed using the Apop Taq Plus Kit

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digoxigenin nucleotide and reacted with peroxidase-conjugated anti-digoxigenin antibody

and 3,3′-diaminobenzidine as previously described (Kim et al, 2010). The percent cell death

was calculated by expressing the number of TUNEL-positive cells as a percentage of total

cell counts.

16. Statistical analysis

All data are expressed as means ± SDs. The results were analyzed using 2-way ANOVA

and the differences between groups were compared by using the Student’s t-test. A p value of <0.05 was considered statistically significant. All experiments were repeated at least 4 times.

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III. RESULTS

A. PART I. Anti-platelet effects of yuzu extract and its component

1. Effects of yuzu on platelet aggregations in vitro

Yuzu (3–10 mg/ml) was found to inhibit collagen-, ADP-, AA- and thrombin-induced

aggregations in a dose-dependent manner (Fig. 1). Yuzu at 5 mg/ml inhibited collagen-,

ADP-, AA- and thrombin-induced platelet aggregations by 98.4 ± 1.6%, 41.5 ± 2.7%, 65.3 ±

21.7% and 31.1 ± 3.4% compared to vehicle, respectively. The IC50 values (half inhibitory

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Fig. 1. Dose-dependent inhibitory effect of yuzu on in vitro platelet aggregation. Platelet

were preincubated for 5 min with various concentrations of yuzu at 37 °C before being aggregated with collagen 2 µg/ml (A), ADP 10 µM (B), or arachidonic acid (AA) 100µM (C) and thrombin 0.4 U/ml (D). Data were expressed as means ± SEMs (n = 4–5). * P < 0.05

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Table 1. IC50 of yuzu, hesperidin, and naringin for platelet aggregation.

Results are expressed as means ± SDs. * Less than 50% inhibition at 1 mg/ml.

Concentrations of agonists were as follows: collagen (2 μg/ml), AA (100 μM), ADP (10 μM), thrombin (0.4 U/ml).

Agonists

IC50 (mg/ml)

Yuzu Hesperidin Naringin

Collagen 3.44 ± 0.02 0.55 ± 0. 01 0.59 ± 0. 01

ADP 6.37 ± 0. 04 0.73 ± 0. 02 > 1*

AA 4.75 ± 0. 03 0.25 ± 0. 01 0.30 ± 0. 01

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2. Contents of hesperidin and naringin in yuzu extracts

The high content of hesperidin and naringin in yuzu might be associated with significant

health benefits. The contents of hesperidin and naringin in yuzu extract are shown in Table 2.

Mean total amounts of hesperidin and naringin present in yuzu extract (mg/100 g of F.wt

yuzu) were 413 ± 15.06 and 1191.0 ± 17.19 mg/100 g fresh weight, respectively. HPLC

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Table 2. Contents of hesperidin and naringin in extract of yuzu.

Hesperidin Naringin

Contents

(mg/100 g of F.wt yuzu )

413.79 ± 15.06 1191.0 ± 17.19

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3. Effects of hesperidin and naringin on platelet aggregations in vitro

Hesperidin (0.1–1.0 mg/ml) was found to inhibit collagen-, ADP-, AA-, and

thrombin-induced aggregations in a dose-dependent manner (Fig. 3). As shown in Table 1, IC50 values

of hesperidin for collagen-, ADP-, AA- and thrombin-induced platelet aggregations were

0.55 ± 0.01, 0.73 ± 0.02, 0.25 ± 0.01, and >1 mg/ml, respectively.

Naringin at 1 mg/ml inhibited collagen-, AA-, and thrombin-induced platelet

aggregation by 82.4 ± 8.9%, 75.6 ± 24.4%, and 45.7 ± 8.9%, respectively (Fig. 4). However,

naringin showed little effect on ADP-induced platelet aggregation. IC50 values of naringin for

collagen-, ADP-, and AA- and thrombin-induced platelet aggregations were 0.59 ± 0.01, >1,

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Fig. 3. Dose-dependent inhibitory effect of hesperidin on in vitro platelet aggregation.

Platelets were preincubated for 5 min with various concentrations of hesperidin at 37 °C before being aggregated with collagen 2 µg/ml (A), ADP 10 µM (B), or arachidonic acid (AA) 100 µM (C) and thrombin 0.4 U/ml (D). Data were expressed as means ± SEMs (n =

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Fig. 4. Dose-dependent inhibitory effect of naringin on in vitro platelet aggregation.

Platelets were preincubated for 5 min with various concentrations of naringin at 37 °C before being aggregated with collagen 2 µg/ml (A), ADP 10 µM (B), or arachidonic acid (AA) 100 µM (C) and thrombin 0.4 U/ml (D). Data were expressed as means ± SEMs (n =

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4. Cytotoxic effects of yuzu, hesperidin, and naringin on platelets

To examine the cytotoxicities of yuzu, hesperidin, and naringin, LDH release from

platelets was measured. LDH released from platelets treated with DMSO, yuzu (10 mg/ml),

hesperidin (1 mg/ml), or naringin (1 mg/ml) were not significantly changed when compared

to non-treated controls, while that from platelets treated with digitonin (50 µM), used as a

positive control, were significantly increased. These results suggest that the anti-platelet

effects of yuzu, hesperidin, and naringin are unlikely to be associated with cytotoxicity

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Table 3. Effects of yuzu, hesperidin, and naringin on LDH release by platelets.

LDH release was measured after incubating rat platelet-rich plasma (2×108 cells/ml) with

vehicle or samples for 5 min. Data represent means ± SEMs (n=4~6). * P<0.05 vs.

control(no- treatment)s.

Control DMSO Yuzu Hesperidin Naringin Digitonin LDH

release

(% of max)

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5. Effects of yuzu, hesperidin, and naringin on TXB

2

formation

To investigate the possible mechanism responsible for the antiplatelet effects of yuzu,

hesperidin, and naringin, their effects on TXB2 formation were evaluated. As shown in Fig. 5,

pretreatment of platelets with 1 mg/ml of hesperidin significantly inhibited collagen-induced

TXB2 formation, but had little effect on thrombin induced TXB2 formation. In the case of

yuzu (10 mg/ml) and naringin (1 mg/ml) treatment, collagen- and thrombin-induced TXB2

formation was inhibited. Aspirin, a positive control, completely blocked TXB2 formation at a

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Fig. 5. Effect of yuzu, hesperidin, and naringin on collagen- and thrombin-induced

TXB2 formation in rat platelets. After preincubating platelet suspension with DMSO, yuzu (10 mg/ml) , hesperidin (1 mg/ml), naringin (1 mg/ml) or aspirin for 5 min, 2 µg/ml of

collagen or 0.4 U/ml of thrombin was added. After incubation at 37 °C for 5 min with collagen or thrombin, EDTA (10 mM) was added to stop TXB2 formation, and supernatant

was obtained by centrifugation at 12,000g for 1 min (A, B). TXB2 concentrations were

determined using an EIA kit. Data are expressed as means ± SEMs (n = 3–5). *P < 0.05 vs. controls. # P < 0.05 vs. vehicle.

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6. Effects of yuzu and hesperidin on platelet aggregation ex vivo

To determine the concentration required for the inhibitory effects of yuzu and hesperidin

on ex vivo platelet aggregation, the effects of various concentrations of yuzu (10, 30, 100

mg/kg) and hesperidin (1, 3, 10 mg/kg) were evaluated in a preliminary experiment (data not

shown); 100 mg/kg and 10 mg/kg were chosen as optimum concentrations for yuzu and

hesperidin, respectively, for the ex vivo platelet aggregation experiments. Hesperidin has

more inhibitory effects on 4 agonist-induced platelet aggregation than naringin, and thereby

it was focused on hesperidin in later experiments.

As shown in Table 4, yuzu administered at 100 mg/kg (p.o.) 2 h before blood collection

significantly inhibited collagen-, ADP-, AA-, and thrombin-induced platelet aggregations, by

32.0 ± 8.6%, 17.3 ± 2.5%, 37.5 ± 10.6%, 18.4 ± 4.8% compared to control, respectively.

Similarly, hesperidin administered at 10 mg/kg (p.o.) significantly inhibited collagen-, ADP-,

AA-, and thrombin-induced platelet aggregations by 23.6 ± 6.5%, 19.9 ± 4.2%, 41.0 ± 13.8%,

13.5 ± 2.1% compared to control, respectively. Aspirin (50 mg/kg), a reference anti-platelet

drug, also significantly inhibited collagen-, AA-, ADP-, and thrombin-induced platelet

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Table 4. Inhibitory effects (%) of orally administered yuzu and hesperidin on ex vivo

platelet aggregation.

Inhibition of platelet aggregation (% of control)

Agonists Yuzu (100 mg/kg, P.O.) Hesperidin (10 mg/kg, P.O.) Aspirin (50 mg/kg, P.O.) Collagen 32.0 ± 8.6%* 23.6 ± 6.5%* 32.5 ± 10.4%* AA 37.5 ± 10.6%* 41.0 ± 13.8%* 99.6 ± 0.3%* ADP 17.3 ± 2.5%* 19.9 ± 4.2%* 21.6 ± 6.6%* Thrombin 18.4 ± 4.8%* 13.5 ± 2.1%* 11.2 ± 3.0%*

Concentrations of agonists were as follows: collagen (2 μg/ml), AA (100 μM), ADP (10 μM),

and thrombin (0.4 U/ml). Data are expressed as means ± SEMs (n = 5~7). *P<0.05 vs.

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7. Effects of yuzu and hesperidin on tail bleeding times in mice

Because the mouse tail bleeding time assay is an easily accessed, reliable measure of

platelet function, it has been widely used to evaluate anti-platelet effects (Gadi et al., 2009).

Therefore, it was performed bleeding time assays to examine the effects of yuzu and

hesperidin on platelet function in vivo. As shown in Fig. 6, the mean bleeding time of

untreated control mice were 92.8 ± 13.4 sec, and this was significantly prolonged by 30

mg/kg (p.o.) of yuzu or 10 mg/kg (p.o.) of hesperidin to 442.3 ± 95.6 sec and 422.2 ± 94.9

sec, respectively. Aspirin at 50 mg/kg (p.o.) also significantly prolonged bleeding time (to

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Fig. 6. Effects of yuzu and hesperidin on mouse tail bleeding times. Yuzu (3, 10, or 30

mg/kg), hesperidin (1, 3, or 10 mg/kg) or aspirin (50 mg/kg) were orally administered to mice 2 h before the experiments. Data were expressed as means ± SEMs (n = 7–9). *P < 0.05 vs. control (no-treatment).

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B. PART II. Preventive effect of yuzu and hesperidin on LV remodeling and

dysfunction in rat permanent LAD occlusion model

1. Yuzu and hesperidin prevented LV remodeling and functional

deterioration following MI

In this study, it was evaluated the effects of yuzu and hesperidin on LV remodeling in a

rat model of permanent LAD occlusion (PO). As shown in Fig. 7, cardiac function

measurement time

was examined by non-invasive echocardiography.

Ventricular

remodeling in the vehicle-, yuzu-, or hesperidin-treated groups was assessed weekly

with serial M-mode echocardiogram (Fig. 8).

The vehicle-treated group showed a

significantly greater increase in end-diastolic and end-systolic dimensions (LVIDd & LVIDs)

compared to sham group. This was associated with marked LV dysfunction as reflected by

reduced LV ejection fraction (EF) (Fig. 9A-C). LV function was preserved more markedly in

the yuzu- or hesperidin-treated groups up to the termination of the study. However, yuzu and

hesperidin treatments for 4 week after LAD occlusion did not significantly attenuated

cardiac hypertrophy, as measured by the heart weight to body weight (HW/BW) ratio. (Fig.

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Fig. 8. Effect of pre-treatment with yuzu (100 mg/kg/day) or hesperidin (HSP, 30

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mg/kg/day) on LV dysfunction and LV remodeling Echocardiographic measurement of

left ventricular (LV) internal dimensions at both systole and diastole (LVIDs and LVIDd,

respectively), LV ejection fraction, andheart weight (HW) to body weight (BW) ratio. Data

are expressed as mean ± SEM; *p<0.05 vs. sham; #p<0.05 vs. vehicle (permanent LAD

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2. Yuzu and hesperidin prevented myocardial fibrosis during LV remodeling after

chronic MI

To determine whether in vivo findings have pathological relevance, it was evaluated the

effects of yuzu and hesperidin on cardiac fibrosis in a rat MI model of permanent LAD

occlusion. The rats were treated with yuzu, hesperidin, or vehicle 1 week before LAD

occlusion. As observed upon Masson’s trichrome staining, increased interstitial fibrosis was observed in the rats treated with the vehicle after 4 weeks of LAD occlusion, which was

significantly reduced in the group that was pre-treated with yuzu or hesperidin (Fig. 10A and

B). Western blot analysis revealed upregulation of α-SMA expression in hearts from the vehicle group, which was reversed to control levels by pre-treatment with yuzu or hesperidin

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Fig. 10. Effect of pre-treatment with yuzu (100 mg/kg/day) or hesperidin (HSP, 30 g/kg/day) on cardiac fibrosis. (A) Representative Masson’s trichrome sections of the left

ventricle. Scale bar, 100 μm. n=6-7. (B) Quantitative analysis of fibrosis. Data are expressed as mean ± SEM; *p<0.05 vs. sham; #p<0.05 vs. vehicle. n=6-7. PO, permanent LAD occlusion.

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g/kg/day) on myofibroblast. Expression of myocardial α-smooth muscle actin (α-SMA)

protein. Representative image (upper part) and quantitative analysis (bottom part). PO, permanent LAD occlusion. Data are expressed as mean ± SEM; *p<0.05 vs. sham; #p<0.05

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3. Yuzu and hesperidin reduced the inflammatory reaction and MPO

expression during LV remodeling after chronic MI

To histologically confirm that myocardial injury reflected inflammation, it was

performed H&E staining and

myeloperoxidase

(MPO) immunohistochemical analysis to

detect neutrophil activity. H&E staining showed that the strongest inflammatory reactions

were observed in the vehicle group. Examination of heart sections of the yuzu- and

hesperidin-treated groups showed nearly normal cardiac cells with a well-preserved

cytoplasm and prominent nucleolus (Fig. 12). The number of MPO-expressing cells was

significantly higher in the vehicle groups than in the yuzu- and hesperidin-treated groups

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mg/kg/day) on inflammatory reaction Representative H & E staining sections of the left

ventricle (LV). PO, permanent LAD occlusion, H & E, hematoxylin & eosin Scale bar, 100

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Fig. 13. Effect of pre-treatment with yuzu (100 mg/kg/day) or hesperidin (HSP, 30 mg/kg/day) on MPO expression (A) Representative H & E staining sections of the left

ventricle (LV). Scale bar, 100 μm. n=6-7. (B) Representative examples of neutrophil activity expressed as myeloperoxidase (MPO) antibody activity (arrow) in the LV. Scale bar, 100 μm. n=6-7. (B) Quantitative analysis of MPO positive cells. Data are expressed as mean ± SEM;

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4. Yuzu and hesperidin block myocardial apoptosis through inhibition of

caspase-3 activation during LV remodeling after chronic MI

It was next investigated whether adverse LV remodeling contributed to increased cardiac

cell death in the rats with LV remodeling after MI. Apoptosis was evaluated using the

TUNEL assay. After 4 weeks of LAD occlusion, the number of TUNEL-positive myocytes

was significantly higher in the vehicle groups than in the yuzu- and hesperidin-treated groups

(Fig. 14A-B). Thus, yuzu and hesperidin attenuated MI-induced myocardial apoptosis. Next,

we compared the caspase-3 activity of the yuzu- and hesperidin-treated groups with the

vehicle group to determine whether caspase-3 is involved in myocardial apoptosis.

Treatment with yuzu or hesperidin significantly reduced caspase-3 activity to levels similar

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Fig. 14. Effect of pre-treatment with yuzu (100 mg/kg/day) or hesperidin (HSP, 30 mg/kg/day) on cardiomyocyte apoptosis. (A) Representative TUNEL staining and (B)

quantitative analysis of TUNEL positive cells. Data are expressed as mean ± SEM; *p<0.05

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mg/kg/day) on caspase-3 activity. The activity of myocardial caspase-3 protein.

Representative image (upper part) and quantitative analysis (bottom part). n=7-8. PO, permanent LAD occlusion

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5. Yuzu and hesperidin block LV wall thinning through inhibition of MMP-2

activation during LV remodeling after chronic MI

LAD occlusion for 4 weeks induced prominent cardiomyocyte loss in the LV mass, as demonstrated by decreased cardiac troponin I (cTnI) staining. Compared to vehicle treatment,

yuzu or hesperidin treatment significantly inhibited cardiomyocyte loss and expression of cTnI (Fig. 16). To gain a better understanding of the mechanisms leading to the prevention of

LV wall thinning in yuzu- and hesperidin-treated groups, it was examined matrix metalloproteinases-2 (MMP-2) activity and resident cardiomyocytes in the LV myocardium.

MMP-2 activity increased in the left ventricles of rats subjected to LAD occlusion, whereas treatment with yuzu significantly reduced MMP-2 activity to levels similar to those observed in sham-operated animals (Fig. 17). These effects were associated with a significant

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Fig. 16. Effect of pre-treatment with yuzu (100 mg/kg/day) or hesperidin (HSP, 30 mg/kg/day) on cardiomyocyte preservation. Representative cardiac Troponin I (cTnI, red

florescence, blue, neuclei) staining. Scale bar, 100 μm. n = 6-7. PO, permanent LAD

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mg/kg/day) on MMP-2 activation. The activity of MMP-2. Representative image by

zymography (upper part) and quantitative analysis (bottom part). n=7-8. PO, permanent LAD occlusion

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IV. DISCUSSION

A. PART I. Anti-platelet effects of yuzu extract and its component

Under pathologic conditions, vascular injury results in the rapid generation of thrombin

at sites of injury and the exposure of extracellular matrix, such as, collagen in vessel walls,

thereby triggering platelet aggregation and thrombus formation via collagen and thrombin

dependent mechanisms (Nieswandt et al, 2005). Collagen- and thrombin-induced platelet

aggregations are ADP and TXA2-dependent, because both agonists can induce the release of

ADP and TXA2 (Farndale et al, 2004). There are, however, differences between the signaling

pathways of collagen- and thrombin-induced platelet activation. Platelet responses to

collagen are mediated via glycoprotein VI (GPVI) and integrin 21 (Farndale et al, 2004),

while those to thrombin are largely mediated through G-protein coupled protease-activated

receptors (PARs) (Brass, 2003). Collagen induces platelet activation through a tyrosine

kinase-based signaling pathway that involves Syk kinase and PLC-2, which results in cytosolic Ca2+ increase, shape change, and the releases of ADP and TXA2. Thrombin bound

to PAR-1 on platelet surfaces causes the activation of the Gq/PLC-2pathways (Brass, 2003), which leads to generation of IP3, the mobilization of intracellular Ca

2+

, an increase in Src

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upon exposure to activating agonists, such as collagen and thrombin, platelets liberate AA,

which is then converted by cyclooxygenase and TXA2 synthase into TXA2 (Jennings, 2009).

ADP secreted from dense granules in activated platelets induces platelet aggregation via the

G protein-coupled purinergic receptors, P2Y1 and P2Y12, in an autocrine manner and this

promotes stable platelet aggregation. The current view of the relationship between these two

platelet ADP receptors is that P2Y1 initiates aggregation and that this is reinforced by P2Y12

(Davì et al, 2007). P2Y1 coupled to Gq regulates Ca 2+

-dependent signaling events, and

P2Y12 is Gi-linked and activates integrin IIb3 by a mechanism involving the inhibition of

cAMP production by adenyl cyclase (Hardy et al, 2004). Taken together, platelets are

activated by multiple physiological agonists that interact with their specific receptors and

trigger different signaling pathways, and thus, antiplatelet agents that act only at one site are

likely to be limited in terms of preventing the formation of pathogenic thrombi. Indeed, dual

anti-platelet therapy with aspirin plus clopidogrel, a P2Y12-receptor antagonist, has been

shown to be more effective at reducing ischemic events in patients with atherothrombotic

cardiovascular diseases than aspirin alone (Chen et al, 2005). Furthermore, previous

observations support the notion that a combination of antiplatelet drugs and preparations of

herbal food supplements may be beneficial in some clinical states and that a synergism exists

between flavonoids and aspirin in terms of their inhibitory effects on platelet function.

(Navarro-Núñez et al, 2008). Taken together, antiplatelet agents that act at multiple sites are

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has significant inhibitory effects on platelet aggregations induced by multiple agonists in in

vitro PRP and ex vivo models. However, it is important to note that after oral ingestion, the

bioavailability of active compounds present in the yuzu and hesperidin depended on their

gastrointestinal metabolization and absorption degree. Nevertheless, this result indicated that

yuzu may have therapeutic benefit in terms of preventing atherothrombotic disease due to its

potential to inhibit multiple sites of platelet aggregation.

Consistent with previous reports, the present study shows that hesperidin and naringin

are major flavonoid components in yuzu. Regarding their effects on platelet aggregation,

hesperidin and naringin showed limited inhibitory effect on thrombin-induced aggregation,

and naringin, in particular, was found to have no effect on ADP-induced aggregation,

whereas yuzu showed multiple inhibitory effects on collagen-, ADP, AA, and

thrombin-induced platelet effects. These results suggest that the anti-platelet activity of yuzu may be

attributable to the combined effects of its components, including those of hesperidin and

naringin.

Regarding platelet activation in response to various stimuli, TXA2 is an important

mediator of platelet activation and aggregation (Jennings, 2009). Accordingly, agents that

inhibit TXA2 formation are expected to play important roles in the pathogenesis of

platelet-linked cardiovascular disease. In this study, it was examined the effects of yuzu, hesperidin,

and naringin on TXA2 formation induced by collagen and thrombin. It was found that yuzu,

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However, when platelets were stimulated by thrombin, yuzu and naringin (but not

hesperidin) significantly inhibited TXB2 formation. In terms of collagen-induced platelet

aggregation, it has been reported that hesperetin inhibits platelet aggregation by inhibiting

PLC-γ2 phosphorylation (Jin et al, 2007). However, in case of thrombin-mediated platelet

aggregation, both Gαq and PLC-β2 play major roles in responses to activation by PAR1 or PAR4, and that PLC-β2 is required for the sustained Ca2+ increase that occurs after thrombin

activation (Vaidyula and Rao, 2003). Based on these results, it is likely that hesperidin has a

limited effect on the PLC-β2 signaling pathway, and thus, a limited inhibitory effect on

thrombin-induced platelet aggregation. The inhibitions of cyclooxygenase and TXA2

synthase are well known inhibitory mechanisms of TXA2 formation. Therefore, further study

is required to elucidate whether the anti-platelet effects of yuzu and hesperidin are associated

with the inhibitions of cyclooxygenase and/or TXA2 synthase.

In this study, yuzu and hesperidin increased bleeding time more than aspirin in in vivo

study. Actually, there are many reports showing that the administration of anti-platelet drug

causes an increase in the bleeding time as a side effect (Kim et al, 2004). However, even now,

no drug is used by a greater number of people worldwide than aspirin. Furthermore, its

combination therapy, such as aspirin-clopidogrel-cilostazol, provides incremental benefit,

becausemultiple pathways activate platelets (Goto, 2005; Han et al, 2009; Jennings, 2009).

In case of aspirin, given the chronicity of aspirin therapy used to treat CVD, optimal dosing

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Sweeny, 2011). Therefore, the use of aspirin for either primary or secondary prevention of

coronary artery disease is largely a risk-benefit calculation. Likewise, these results suggest

that optimal intake of yuzu to minimize its adverse effect should be emphasized and that

development of a novel combination approach with yuzu remains to be investigated.

B. PART II. Preventive effect of yuzu and hesperidin on LV remodeling and

dysfunction in rat permanent LAD occlusion model

Herein, this study provide the first evidence for the cardioprotective role of yuzu and its

major component, hesperidin in rat chronic MI model induced by permanent LAD occlusion,

by demonstrating that yuzu and hesperidin prevented heart from MI-induced dysfunction, LV

fibrosis, inflammatory reaction and cardiomyocyte apoptosis.

Chronic MI results in complex architectural alterations such as dilatation of the LV and

infarct thinning, which is called LV remodeling, in both infarct and non-infarct region.

Patients exhibiting extensive LV remodeling after MI are more likely to experience

complications such as HF and myocardial rupture, leading to an elevated risk of mortality

(Creemers et al, 2001). Although modern cardiology has made substantial advances in the

diagnosis and management of MI, it is necessary to design therapeutic strategies to attenuate

LV remodeling after MI by modulation of the molecular and cellular factors involved in the

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After chronic MI, LV fibrosis contributes to adverse structural remodeling, leading to

impaired contractile properties of the LV as well as deteriorated electrical conduction system

(van den Borne et al, 2010). LV fibrosis occurs as a result of the imbalance between

enhanced synthesis reduced degradation of collagen (González et al, 2011). As a critical

step in response to myocardial injury, fibroblasts are activated into α-SMA-positive myofibroblasts which can generate extracellular matrix proteins such as type I collagen.

Therefore, the degree of fibroblast activation is a significant predictor of HF progression in

both experimental animal models and in human patients (Pchejetski et al, 2012). The

present study have demonstrated that yuzu and hesperidin exert anti-fibrotic remodeling

effects via the inhibition of excess collagen deposition and conversion of fibroblasts into

α-SMA-positive myofibroblasts during LV remodeling process following permanent LAD

occlusion. These results suggest that yuzu and hesperidin represent novel preventive natural

products against LV remodeling and cardiac dysfunction induced by chronic MI.

Regarding cardiac structure remodeling process after chronic MI, recent studies have

identified the importance of several inflammatory mediators that are released during this

process, such as neutrophils and various cytokines, and inflammatory cells are attracted to

the myocardial injury site (Paulus, 2000; Diwan et al, 2003; Campian et al, 2010). MPO is a

well-known enzyme that is released by activated neutrophils and has powerful pro-oxidative

and pro-inflammatory properties. Recent studies have suggested that regional MPO activity

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2010; Tang et al, 2011). Subsequent studies have provided quantitative support for this

observation by showing significant elevations in the systemic levels of MPO in a wide

spectrum of CVD scenarios, with chronic MI and HF being the most frequently studied

(Anatoliotakis et al, 2013). Accordingly, pre-clinical studies in experimental models suggest

a possible therapeutic role of MPO inhibition in HF. In this study, it was found that yuzu and

hesperidin have anti-inflammatory properties and participate in the control of the

inflammatory response through the inhibition of MPO expression during chronic MI.

Experimental models and clinical studies have shown that loss of functional

cardiomyocytes contributes to structural changes that underlie progressive LV remodeling

during chronic MI (González et al, 2011; Konstantinidis et al, 2012). Although the

significance of apoptosis in LV remodeling still remains debatable, cardiomyocyte apoptosis

leads to the loss of cardiomyocyte mass and the reduction of myocardial contractile function

(Dom, 2009). Therefore, elimination of pro-apoptotic signals may prevent the progression of

LV remodeling during chronic MI. Since the main apoptotic death pathways converge on

caspases, the most efficient approach for interrupting cardiomyocyte apoptosis might be the

targeting of these enzymes. Many animal studies have confirmed that the inhibition of

caspases, such as caspase-3, mitigates LV dysfunction and enables survival during the

progression to end-stage HF. Although specific caspase inhibitors are being developed and a

few have shown promising results for clinical therapy by targeting cell death in LV

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until very recently (Yang et al, 2013). The present study have demonstrated that yuzu and

hesperidin have anti-apoptotic properties and participate in the control of cell death by

inhibiting caspase-3 activity and expression.

Cardiomyocyte loss during ischemic damage can be replaced by non-contractile fibrotic

cells rather than by new cardiomyocytes (van Wijk et al, 2012). In humans, it is known that

extensive LV dilatation after chronic MI increases the risk of complications such as the HF,

aneurysm formation and cardiac rupture. In addition, the positive effects of MMP inhibition

on LV dilatation in animal models have led to the proposed use of MMP inhibitors as

potential therapies in patients at risk for the development of HF after MI (Creemers et al,

2001). MMP-2 is abundant and ubiquitously expressed in almost all of the cells that

comprise the heart. Activated MMP-2 degrades susceptible sarcomeric and cytoskeletal

proteins including troponin I (TnI), myosin light chain-1 (MLC-1), and α-actin, leading to

the acute contractile dysfunction observed in ischemia/reperfusion injury (Kandasamy et al,

2010). Interestingly, the present study have demonstrated that yuzu and hesperidin

effectively preserved cardiomyocyte mass during chronic MI, possibly through the inhibition

of MMP-2-induced wall thinning.

LV remodeling after chronic MI remains a major cause of morbidity and mortality

worldwide, leading to a dramatical increase of health care costs (Strauer et al, 2010).Despite

a number of pharmacological advances, mortality following MI remains still high. The

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prevented LV remodelling and LV dysfunction following chronic MI, and suggested a

potential use of yuzu or hesperidin as a cardioprotective strategy.

V. CONCLUSION

In conclusion, yuzu and its major compounds hesperidin and naringin were found to

have significant anti-platelet activity, possibly via the inhibition of TXA2 formation. These

findings suggest that yuzu-based foods may be especially beneficial in the prevention of

platelet-associated atherothrombotic disease

In addition, the present study has demonstrated that pre-treatment of yuzu or hesperidin

significantly prevented LV remodeling following chronic MI in rat LAD occlusion model.

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Protective Effects of Yuzu and Its Component against Cardiovascular Disease

Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

by

Hye Yon Yu

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

by

Hye Yon Yu

A Dissertation Submitted to The Graduate School of

Ajou University in Partial Fulfillment of the Requirements for

The Degree of Master of Biomedical Sciences

Supervised by

Soo Hwan Lee, Ph.D.

Yi-Sook Jung, Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

This certifies that

of Hye Yoⅱ

Yu

the dissertation

is approved●

The Gradμ

ate

December,

S UPERV工

SORY COMMITTEE

Schoo1; Aj˚

u

19th, 2014

Protective Effects of Yuzu and Its Component

against Cardiovascular Disease

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

ABBREVIATION

I. INTRODUCTION

II. MATERIALS AND METHODS

Yamazaki

III. RESULTS

A. PART I. Anti-platelet effects of yuzu extract and its component

1. Effects of yuzu on platelet aggregations in vitro

2. Contents of hesperidin and naringin in yuzu extracts

3. Effects of hesperidin and naringin on platelet aggregations in vitro

4. Cytotoxic effects of yuzu, hesperidin, and naringin on platelets

5. Effects of yuzu, hesperidin, and naringin on TXB

formation

6. Effects of yuzu and hesperidin on platelet aggregation ex vivo

7. Effects of yuzu and hesperidin on tail bleeding times in mice

B. PART II. Preventive effect of yuzu and hesperidin on LV remodeling and

dysfunction in rat permanent LAD occlusion model

1. Yuzu and hesperidin prevented LV remodeling and functional

deterioration following MI

was examined by non-invasive echocardiography.

Ventricular

remodeling in the vehicle-, yuzu-, or hesperidin-treated groups was assessed weekly

with serial M-mode echocardiogram (Fig. 8).

3. Yuzu and hesperidin reduced the inflammatory reaction and MPO

expression during LV remodeling after chronic MI

myeloperoxidase

4. Yuzu and hesperidin block myocardial apoptosis through inhibition of

caspase-3 activation during LV remodeling after chronic MI

5. Yuzu and hesperidin block LV wall thinning through inhibition of MMP-2

activation during LV remodeling after chronic MI

IV. DISCUSSION

A. PART I. Anti-platelet effects of yuzu extract and its component

B. PART II. Preventive effect of yuzu and hesperidin on LV remodeling and

dysfunction in rat permanent LAD occlusion model

V. CONCLUSION

REFERENCES