XANTHINE OXIDASE INHIBITION AND ANTIOXIDANT ACTIVITY … · 2013-05-31 · 1 xanthine oxidase inhibition and antioxidant activity of an artichoke leaf extract (cynara scolymus l.)

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XANTHINE OXIDASE INHIBITION AND ANTIOXIDANT ACTIVITY OF AN ARTICHOKE LEAF EXTRACT (Cynara scolymus L.) AND ITS COMPOUNDS

By

SASIPORN SARAWEK

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

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© 2007 by Sasiporn Sarawek

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To my parents

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ACKNOWLEDGMENTS

I would like to express my appreciation and very grateful thanks to Dr. Veronika

Butterweck for accepting me into her group and for her encouragement and support during my

Ph.D. program. My special thanks go to Dr. Hartmut Derendorf for his guidance and helpful

advice. Many thanks also go to the members of my supervisory committee, Dr. Günther

Hochhaus, Dr. Jeffrey Hughes and Dr. Saeed Khan, for their helpful advice throughout the years.

I would like to thank friends and staff in the Department of Pharmaceutics for their

friendship and support, especially to Whocely Victor De Castro for his suggestions during the

validation of the analytical methods. I also would like to thank Pattaraporn Vanachayangkul for

her friendship.

I would like to extend my thanks to the program assistants of the Department of

Pharmaceutics Mr. James Ketcham, Mrs. Patricia Khan, Ms. Michelle Griffin, Mrs. Andrea

Tucker, and Mrs. Penny Canino for their technical support. I also would like to thank all my

assistants: Carmen Michalski, Sandra Weiss, Eva Kremser, and Christine Haefele and the post-

doc fellows, especially Dr. Vipul Kumar and Dr. Jie Wang for their friendship and technical

support.

My personal thanks go to my mother and my father for their love, friendship, support,

guidance and encouragement throughout my life.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES...........................................................................................................................9

LIST OF FIGURES .......................................................................................................................11

ABSTRACT...................................................................................................................................13

CHAPTER

1 INTRODUCTION ..................................................................................................................15

Artichoke (Cynara scolymus L.).............................................................................................15 Pharmacological Actions.................................................................................................15 Constituents .....................................................................................................................16 Dosage .............................................................................................................................17

Absorption and Metabolism of Caffeolyquinic Acids............................................................17 Absorption and Metabolism of Flavonoids ............................................................................17 Biological Effects of Flavonoids ............................................................................................19

Antioxidant Activity........................................................................................................19 Xanthine Oxidase Inhibitors............................................................................................20

Uric Acid, Hyperuricemia, and Gout......................................................................................22 Enzyme Inhibition ..................................................................................................................23

Competitive Inhibition.....................................................................................................24 Uncompetitive Inhibitions ...............................................................................................24 Mixed Inhibitions or Non Competitive Inhibitions .........................................................24

Pharmacokinetics....................................................................................................................24 Hypothesis and Objectives .....................................................................................................25

2 IDENTIFICATION AND QUANTIFICATION OF COMPOUNDS IN ARTICHOKE EXTRACT..............................................................................................................................40

Background.............................................................................................................................40 Specific Aim ...........................................................................................................................40 Materials and Methods ...........................................................................................................40

Materials ..........................................................................................................................40 Sample Preparation..........................................................................................................41 HPLC/DAD Analysis ......................................................................................................41 Work Solutions and the Preparation of Calibration Standards........................................41 Quantification ..................................................................................................................42 Validation ........................................................................................................................42

Results.....................................................................................................................................43 Linearity ..........................................................................................................................43 Sensitivity ........................................................................................................................43

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Specificity........................................................................................................................43 Precision and Accuracy ...................................................................................................44 Stability............................................................................................................................44 Quantification of Caffeoylquinic Acids (Chlorogenic Acid, Cynarin) and Luteolin

Derivatives (Luteolin-7-O-glucoside, Luteolin-7-O-glucuronide) in Artichoke Leaf Extract..................................................................................................................44

Discussion and Conclusion.....................................................................................................44

3 EFFECT OF ARTICHOKE LEAF EXTRACT, CAFFEIC ACID DERIVATIVES AND FLAVONOIDS ON XANTHINE OXIDASE INHIBITORY ACTIVITY............................54


Materials ..........................................................................................................................54 Preparation of Working Solutions and Test Solutions ....................................................55 Assay Procedure for Xanthine Oxidase Inhibitions ........................................................56 Lineweaver- Burk Plot ....................................................................................................57

Results.....................................................................................................................................57 Xanthine Oxidase Inhibitory Activity of Artichoke Extract ...........................................57 Xanthine Oxidase Inhibitory Activity of Various Flavonoids and Compounds in

Artichoke......................................................................................................................57 Inhibition Mechanism......................................................................................................58


4 EFFECTS OF ARTICHOKE LEAF EXTRACT AND VARIOUS FLAVONOIDS ON SERUM URIC ACID LEVELS IN OXONATE-INDUCED RATS .....................................67


Materials ..........................................................................................................................67 Stock Solutions and Preparation of Calibration Standards..............................................68 Animals and Experimental Protocols ..............................................................................68

Animals ....................................................................................................................68 Animal model of hyperuricemia in rats....................................................................69

Drug Administration:.......................................................................................................69 1. Oral administration...............................................................................................69 2. Intraperitoneal administration ..............................................................................70

Uric Acid Assay ..............................................................................................................70 Preparation of Rat Serum ................................................................................................70 Statistical Analysis ..........................................................................................................71 Validation ........................................................................................................................71

Results.....................................................................................................................................72 Validation of Analytical Method to Measure Uric Acid in Rat Serum. ..........................72

Linearity ...................................................................................................................72 Sensitivity.................................................................................................................72

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Specificity.................................................................................................................72 Precision, accuracy and recovery .............................................................................72 Stability ....................................................................................................................72

Effect of Artichoke Extract and Its Compounds on Serum Urate Levels in Hyperuricemic Rats .....................................................................................................73

Oral administration of artichoke in acute treatment.................................................73 Oral administration of artichoke in chronic treatment .............................................73 Oral administration of compounds in artichoke and various flavonoids in acute

treatment ...............................................................................................................73 Intraperitoneal administration of artichoke, compounds in artichoke and

various flavonoids in acute treatment ...................................................................74 Discussion and Conclusion.....................................................................................................74

5 THE EFFECT OF ARTICHOKE LEAF EXTRACT AND ITS COMPOUNDS ON ANTIOXIDANT ACTIVITY IN VITRO AND IN RATS ....................................................92

Background.............................................................................................................................92 Specific Aims..........................................................................................................................93 Materials and Methods ...........................................................................................................93

Materials ..........................................................................................................................93 Animals............................................................................................................................94

Acute treatment ........................................................................................................94 Chronic treatment .....................................................................................................94

Assessment of Antioxidative Capacity in Vitro and Plasma Antioxidant Status ............95 Assessment of Uric Acid in Plasma ................................................................................95 Assessment of Glutathione Peroxidase (GPx) in Plasma ................................................96 Statistical Analysis ..........................................................................................................97

Results.....................................................................................................................................97 Antioxidant Activity in Vitro...........................................................................................97 Plasma Antioxidant Activity in Vivo ...............................................................................97

Acute treatment ........................................................................................................97 Chronic treatment .....................................................................................................97

Plasma Urate Concentrations and Plasma Glutathione Peroxidase Activity after The Treatment with Artichoke Extract and Phenolic Compounds .....................................98


6 PHARMACOKINETICS OF LUTEOLIN AND ITS METABOLITES IN RATS.............108

Background...........................................................................................................................108 Specific Aims........................................................................................................................108 Materials and Methods .........................................................................................................108

Materials ........................................................................................................................108 Stock, Work Solutions, and Preparation of Calibration Standards ...............................109 Animals and Experimental Protocols ............................................................................110

Animals ..................................................................................................................110 Methods..................................................................................................................110

Analytical Methods .......................................................................................................111

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Data Analysis.................................................................................................................112 Statistical Analysis ........................................................................................................114 Validation ......................................................................................................................114

Results...................................................................................................................................114 Validation of Analytical Method to Measure Luteolin in Rat Plasma ..........................114

Linearity .................................................................................................................114 Sensitivity...............................................................................................................115 Specificity...............................................................................................................115 Precision, accuracy and recovery ...........................................................................115 Stability ..................................................................................................................115

Validation of Analytical Method to Measure Luteolin in Rat Urine.............................116 Linearity .................................................................................................................116 Sensitivity...............................................................................................................116 Specificity...............................................................................................................116 Precision, accuracy and recovery ...........................................................................116 Stability ..................................................................................................................116

Pharmacokinetic Study of Luteolin ...............................................................................117 Non-compartmental analysis .........................................................................................117 Compartmental Analysis ...............................................................................................118

Discussion and Conclusion...................................................................................................118

7 CONCLUSION.....................................................................................................................139

LIST OF REFERENCES.............................................................................................................141

BIOGRAPHICAL SKETCH .......................................................................................................151

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LIST OF TABLES

Table page 1-1 Annual incidence of gouty arthritis according to the serum urate concentration ..............27

1-2 Drugs used in the management of gout..............................................................................28

2-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of chlorogenic acid, cynarin, luteolin-7-O-glucoside and luteolin-7-O-glucuronide ....................................................................................................................46

2-2 The stability test of chlorogenic acid, cynarin and luteolin-7-O-glucoside after 24 hours on autosampler at 20oC ............................................................................................47

2-3 The stability test of luteolin-7-O-glucuronide after 24 hours on autosampler at 20oC. Data represents the percentage remaining of all test compounds ......................................48

2-4 Intra-day (n = 3) and inter-day (n = 9) assay parameters of caffeoylquinic acid (chlorogenic acid and cynarin) and luteolin derivatives (luteolin-7-O-glucoside and luteolin-7-O-glucuronide) ..................................................................................................49

2-5 Amounts of caffeoylquinic acids and luteolin derivatives expressed as milligram per gram of dried extract..........................................................................................................50

3-1 Structures of various flavonoids ........................................................................................61

3-2 Results of the % XO inhibition screening of artichoke extract .........................................62

3-3 The IC50 values (μM) of test samples on xanthine oxidase inhibition...............................63

3-4 Vmax and Km of flavonoids on xanthine oxidase inhibition................................................64

4-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of uric acid.................................................................................................77

4-2 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of uric acid in rat serum.................................................................................................................78

4-3 The stability test after 24 hours on autosampler at 20oC ...................................................79

4-4 Hypouricemic effects of allopurinol, water extract of artichoke on plasma urate levels (μg/mL) in oxonate-pretreated rats in acute treatment ............................................80

4-5 Hypouricemic effects of allopurinol and artichoke extract on plasma urate levels (μg/mL) in oxonate-pretreated rats after chronic treatment...............................................81

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4-6 Hypouricemic effects of allopurinol, apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin, quercetin on plasma urate levels (μg/mL) in oxonate-pretreated rats after oral administration .............................................................................82

4-7 Hypouricemic effects of allopurinol, apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin, quercetin on plasma urate levels (μg/mL) in oxonate-pretreated rats after i.p injection ........................................................................................83

5-1 ORAC values of artichoke extract ...................................................................................101

5-2 Relative ORAC values of pure chemicals with antioxidant activity ...............................102

5-3 ORAC values of plasma samples.....................................................................................103

5-4 ORAC values of plasma samples.....................................................................................104

5-5 Plasma urate concentrations in rats after administration of artichoke extract and phenolic compounds ........................................................................................................105

5-6 Plasma glutathione peroxidase activity in rats after administration of artichoke extract and phenolic compounds......................................................................................106

6-1 Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in plasma................................................................................122

6-2 Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in urine ...................................................................................123

6-3 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat plasma.....................................................................................................................124

6-4 The stability test after 48 hours on autosampler at 18oC .................................................125

6-5 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat urine ........................................................................................................................126

6-6 The stability test of luteolin in urine after 48 hours on autosampler at 18oC ..................127

6-7 Pharmacokinetic parameters of luteolin after oral and iv administration of luteolin at dose 50 mg/kg ..................................................................................................................128

6-8 Pharmacokinetic parameters of luteolin conjugates after oral and iv administration of luteolin at dose 50 mg/kg.................................................................................................129

6-9 Pharmacokinetic parameters of luteolin after oral and i.v. administration of luteolin 50 mg/kg ..........................................................................................................................130

6-10 The excretory recovery for 24 h of luteolin and luteolin conjugates in urine after oral and i.v administration of luteolin at dose 50 mg/kg.........................................................131

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LIST OF FIGURES

Figure page 1-1 Structures of caffeoylquinic acids and flavonoids detected in artichoke ..........................29

1-2 Hypothetical metabolic pathway of caffeoylquinic acids..................................................30

1-3 Proposed metabolic pathway of caffeic acid in isolated rat hepatocytes...........................31

1-4 Proposed recycling of flavonoids through sequential metabolism and/or secretion involving intestinal microflora, intestine, and liver ...........................................................32

1-5 The enzymatic process catalyzed by xanthine oxidase......................................................33

1-6 Purine degradation pathway in animals .............................................................................34

1-7 The mechanism of uricase and uricase inhibitors ..............................................................35

1-8 Enzyme inhibition..............................................................................................................36

1-9 Competitive inhibition .......................................................................................................37

1-10 Uncompetitive inhibition ...................................................................................................38

1-11 Mixed inhibition.................................................................................................................39

2-1 Mean calibration curves of compounds in artichoke .........................................................51

2-2 HPLC separation and absorbance-wavelength spectra of chlorogenic acid, cynarin, dihydrocaffeic acid, luteolin-7-O-glucoside and luteolin-7-O-glucuronide. .....................52

2-3 Absorbance-wavelength spectras.......................................................................................53

3-1 Inhibition dose-response effects ........................................................................................65

3-2 Lineweaver-Burk plots in the absence (control, ■-■) and in the presence of luteolin (0.5 μM, ◆-◆), apigenin (0.5 μM, ●-●), kaempferol (0.5 μM, ▲-▲) and quecetin (0.5 μM, ▼-▼) with xanthine as the substrate. ................................................................66

4-1 Mean calibration curves (n = 9) of uric in serum ..............................................................84

4-2 HPLC chromatogram of uric acid in serum.......................................................................85

4-3 Acute effects of allopurinol, artichoke extract on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate.....................................................................86

4-4 Chronic effects of allopurinol, artichoke extratc on serum urate levels in oxonate-treated rats..........................................................................................................................87

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4-5 Effects of allopurinol and luteolin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate...................................................................................88

4-6 Effects of allopurinol, apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, and quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate...............................................................................................................................89

4-7 Effects of allopurinol, apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate...............................................................................................................................90

4-8 Effects of artichoke extract, allopurinol, caffeic acid, chlorogenic acid, cynarin, luteolin, apigenin and quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate...................................................................................91

5-1 Structures of caffeic acid derivatives and flavonoids. .....................................................107

6-1 Two-compartment models after intravenous injection ....................................................132

6-2 Mean calibration curves (n = 9) of luteolin in plasma .....................................................133

6-3 The HPLC chromatogram of luteolin and naringenin (IS) in plasma..............................134

6-4 Mean calibration curves (n = 9) of luteolin in urine ........................................................135

6-5 The HPLC chromatogram of luteolin and naringenin (IS) in urine.................................136

6-6 Plasma concentration-time curves ...................................................................................137

6-7 Fitted luteolin concentrations after i.v. injection. ............................................................138

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

XANTHINE OXIDASE INHIBITION AND ANTIOXIDANT ACTIVITY OF ARTICHOKE LEAF EXTRACT (Cynara scolymus L.) AND ITS COMPOUNDS

By

Sasiporn Sarawek

August 2007

Chair: Veronika Butterweck Cochair: Hartmut Derendorf Major: Pharmaceutical Sciences

Gout is a disease characterized by elevated levels of uric acid in body fluids. This

hyperuricemia results in the deposition of urate crystals in tissue, especially joints. The uric acid

deposition initiates an inflammation process involving the release of reactive oxygen species.

The common treatments of gout are the use of anti-inflammatory agents to relieve the symptoms

of the disease and xanthine oxidase (XO) inhibitors to block the synthesis of uric acid. The most

common xanthine oxidase inhibitor is allopurinol. However, its use is limited by unwanted side

effects such as hypersensitivity problems. Therefore, alternatives are required.

Artichoke leaves (Cynara scolymus L.) have been used traditionally by the Eclectic

physicians as a diuretic and depurative for the treatment of gout. The major compounds in

artichoke leaves are phenolic compounds such as caffeoylquinic acids and flavonoids. These

phenolic compounds have shown xanthine oxidase inhibitory activity and antioxidant activity in

vitro and in vivo. Therefore, the goal of the present study was to examine the xanthine oxidase

inhibitory activity and antioxidant activity of the artichoke extract, and its main compounds in

vitro and in vivo.

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The in vitro study showed that the extract as well as caffeoylquinic acids showed only a

weak XO inhibition, whereas flavonoids (flavone and flavonols) had a highly inhibitory effect on

XO. Luteolin had the highest XO inhibition effect. This significant inhibition of XO by the

flavonoids in vitro suggested that they may suppress the production of uric acid in vivo.

However, the in vivo study showed that oral administration of the artichoke extract,

caffeoylquinic acids, and flavonoids could not decrease the serum urate levels in oxonate-treated

rats.

The antioxidant activities of the artichoke extract and its phenolic compounds were

determined using the oxygen radical absorbance capacity assay (ORAC). The results showed that

the artichoke extract and its compounds elicited an antioxidant activity in vitro, however, the

compounds again showed no antioxidant activity in vivo.

It was speculated that this lack of effect in vivo from both studies might be due to the

absorption, the high first pass effect through intestine and liver, the excretion into urine and bile

and the degradation in large intestine. Therefore, the pharmacokinetic of a compound in

artichoke was performed in order to explain the in vivo activity.

Pharmacokinetic study of luteolin, the compound which showed the highest XO

inhibition in vitro, showed that after oral administration of luteolin, luteolin rapidly absorbed and

metabolized in plasma. Additionally, plasma-concentration-time curves of luteolin metabolites

revealed secondary peaks. The bioavailability of luteolin was low and the urinary excretion of

luteolin and its conjugates did not dominate. This study could explain the lack of XO inhibitory

activity and antioxidant activity in vivo. Therefore, it can be concluded that artichoke might be

not a useful alternative treatment of gout.

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CHAPTER 1 INTRODUCTION

Artichoke (Cynara scolymus L.)

Artichoke or globe Artichoke (Cynara scolymus L.), a member of the Compositae (daisy)

family, is native to the Mediteranean area. The leaves are the commonly used part.

Pharmacological Actions

Traditionally artichoke leaves have been used by the Eclectic physicians as a diuretic and

depurative, for the treatment of rheumatism, gout, jaundice and especially for dropsies. For the

modern use, the leaf of artichoke is reported to process choleretic, hypocholesterolaemic [1, 2],

hypolipidaemic [3], hepatoprotective [4], anticarcinogenic [5] and antioxidative [6-9]. Diuretic

effect of artichoke helps the elimination of water and the consequent toxin and specially the uric

acid.

Hypolipidaemic, hypocholesterolaemic and choleretic activities are well documented for

artichoke leaf extracts and particularly for the constituent cynarin [10]. Artichoke leaves not only

increases choleresis and, therefore, cholesterol elimination, but also has been shown to inhibit

cholesterol biosynthesis. Clinical trials investigating the use of globe artichoke and cynarin in the

treatment of hyperlipidaemia report positive results [10]. However, studies in animals and

humans by Saenz et al. [11] have suggested that these effects may be due to the

monocaffeoylquinic acids present in artichoke extract (eg. chlorogenic acid) [11]. In vitro studies

on cultured hepatocytes suggested that artichoke extract inhibits the incorporation of 14C-

labelled acetate into the non-saponifiable lipid fraction and thus reduce cholesterol biosynthesis

[12, 13]. Other studies suggested indirect inhibitory effects exerted at the level of HMGCoA

reductase, a key enzyme in cholesterol biosynthesis [13-15].

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Antioxidant and hepatoprotective activity of artichoke leaves have been studied. In vitro, a

luteolin-rich artichoke leaf extract (flavonoid content around 0.4% w/w), the pure aglycone

luteolin, and luteolin-7-O-glucoside demonstrated a concentration-dependent reduction in low

density lipoprotein (LDL) oxidation [9]. The effects of artichoke extract and its constituents have

also been investigated for activity against oxidative stress in studies using human leucocytes. The

extract demonstrated a concentration-dependent inhibition of oxidative stress induced by several

agents, such as hydrogen peroxide, that generate reactive oxygen species. The constituents

cynarin, caffeic acid, chlorogenic acid and luteolin also showed concentration-dependent

oxidative stress inhibitory activity [16]. In addition, artichoke extract has marked protective

properties against oxidative stess induced by inflammatory mediators and ox-LDL in cultured

endothelial cells and monocytes [7]. In vivo, the administration of an edible artichoke in rats has

shown that artichoke extract increased the level of glutathione peroxidase activity in erythrocyte

and decreased the level of 2-Aminoadipic semialdehyde (a protein oxidation biomarker) [8].

Hepatoprotective and hepatoregenerating activities have been documented for cynarin in

vitro [4] and in rats [10, 17].

Artichoke extract has been reported to alleviated symptoms and improving the disease-

specific quality of life in patients with functional dyspepsia [18] and concomitant dyspepsia [19].

Constituents

The major chemical components of artichoke leaves include up to 2% phenolic acids with

mono-and dicaffeoylquinic acids, primarily chlorogenic acid, cynarin, and caffeic acid. Also up

to 0.1-1% flavonoids (Figure 1-1) [20-24].

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Dosage

The German Commission E recommends an average daily dose of 6 g drug, or an

equivalent dose of extract (based on the herb-to-extract ratio) or other preparations, for dyspeptic

problems. A recommended dosage regimen for liquid extract (1: 2) is 3-8 ml daily. Dosage used

in clinical trials of globe artichoke leaf extract have assessed the effects of dosages of up to 1.92

g daily in divided doses for up to six months [25].

Absorption and Metabolism of Caffeolyquinic Acids

The mechanism and site of absorption of caffeoylquinic acids is still unclear. There is no

published evidence for enzymatic hydrolysis of chlogenic acid by intestine, liver or plasma

extracts [26, 27]. Moreover, chlorogenic acid has been reported to be stable in the digestive or

intestinal juice [28, 29]. However, Wittemer et al. [30] suggested absorption and de-esterification

of caffeoylquinic acids may occur somewhere in the upper gut. After the release of caffeic acid

(CA) from caffeoylquinic acids, CA may be conjugated with glucuronic acid in the enterocytes

[30]. After entering the systemic circulation, CA conjugates were most likely methylated by

catechol-O-methyltransferase [31] during the first liver passage to methylation products of

ferulic acid (FA) and isoferulic acid (IFA).They also suggested that CA may be metabolized by

the colonic microflora to dihydrocaffeic acid (DHCA) prior to absorption. In enterocyte, DHCA

was metabolited to dihydroferulic acid (DHFA) using catechol-O-methyltransferase, and then

FA was formed by the dehydrogenation of DHFA [30, 31]. The hypothetical metabolic pathways

of caffeoylquinic acids and caffeic acid are shown in Figure 1-2, and Figure 1-3.

Absorption and Metabolism of Flavonoids

Most of flavonoids presented in plants are attached to sugar moieties thus tending to be

water-soluble, although, occasionally, they are found as aglycones. Absorption of flavonoid

glycosides was considered negligible. Only flavonoid aglycones were be able to pass the gut

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wall, and no enzymes that can split these β-glycosidic bonds are secreted into the gut or

presented in the intestinal wall [32]. Hydrolysis occurs in the colon by intestinal microflora,

which could be further metabolized by intestinal microflora to various single-ring aromatic

compounds [33, 34]. Hydrolytic enzymes of intestinal microflora could convert certain flavonoid

glycosides to their corresponding aglycones [33, 35]. Recently, it has been reported that enzymes

that are able to hydrolyse flavonoid glycosides are located in the cells (cytosolic beta-

glucosidase, CBG) and on the apical membrane (lactase-phlorizin hydrolase, LPH) [36].

Therefore, flavonoid glycosides may be hydrolysed by LPH and then the aglycone may diffuse

passively into the cell [37]. Alternatively, flavonoid glycosides may be enter the cell by sodium

dependent glucose transporter (SGTL1) [38] and then be cleaved inside the cell by CBG.

Absorbed flavonoids could undergo phase I (e.g., oxidation such as hydroxylation) and phase II

(e.g., conjugation such as glucuronidation) metabolisms in human intestine and liver. Phase I

metabolisms commonly attach a hydroxyl group to the molecule or break down a molecule so

that the compound can be further processed by the body. Phase II metabolisms can occur after

phase I metabolisms or simultaneously as phase I. Normally, phase II metabolisms convert

compounds and their phase I metabolites into hydrophilic and excretable conjugates which could

be eliminated by the urine or via the bile. In case of flavonoids, conjugated metabolites are

finally excreted into the intestinal lumen and eliminated or be hydrolysed by microbial

hydrolases (e.g., glucuronidases and sulfatase) at the intestinal lumen to aglycones, and then

transported into systemic circulation. (Figure 1-2) The low bioavailability of flavonoids may be

explained by duoenteric and enterohepatic recirculation [39].

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Biological Effects of Flavonoids

Flavonoids have been shown to exert protective effects against many diseases, in particular

cardiovascular diseases and cancer. The health benefit of flavonoids is usually linked to two

properties: (1) antioxidant activity and (2) inhibition of certain enzymes such as xanthine oxidase

[40, 41].

Antioxidant Activity

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide radical

anion (O2-•), hydroxyl radical (OH•), alkylperoxyl radical (ROO•), nitric oxide (NO•), singlet

oxygen (1O2) and hypochlorous acid (HOCl) react with biological molecules causing cell and

tissue injury. The ROS are considered to contribute to a wide variety of degenerative processes

and diseases such as atherosclerosis, Parkinson’s disease, Alzheimer’s dementia and reperfusion

injury of brain or heart [42]. Many studies have suggested that flavonoids exhibit biological

activities, including antiallergenic, antiviral, anti-inflammatory, vasodilating actions. These

pharmacological effects are linked to the antioxidant properties of flavonoids. Flavonoids can

express these properties by: (1) suppressing ROS formation by inhibiting some enzymes or

chelating trace elements involved in free radical production, (2) scavenging radical species and

more specially the ROS, or (3) up-regulating or protecting antioxidant defense [40].

Flavonoids can inhibit enzymes which are responsible for superoxide anion production

such as xanthine oxidase. Most of flavonoids can chelate trace metals, which play an important

role in oxygen metabolism, and therefore inhibit the initiation of the lipoxygenase reaction [43].

The possible metal-complexing sites within flavonoids are located between the C3 hydroxyl and

the carbonyl, the C5 hydroxyl and the carbonyl and between the ortho-hydroxyls on the B-ring

[40]. The radical scavenging activity of flavonoids depend on the structure and the substituents

of the heterocyclic and B-ring. The major determinants for radical-scavenging capacity are: (1)

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the otho-dihydroxy structure on the B-ring, which has the best electron-donating properties, (2)

the 2, 3-double bond in conjugation with a 4-oxofunction in the C-ring is responsible for electron

delocalization from the B-ring, and (3) the 3-and 5-hydroxyl group with a 4-oxofunction in the A

and C-ring for maximum radical scavenging potential [40]. Some flavonoids, such as qurcetin,

myricetin, and fisetin, were shown to alleviate oxidative stress by inducing glutathione S-

transferase (GST), an enzyme used to protect cells against free-radical damage [44]. Studies have

indicated that flavonoid aglycones, including quercetin, luteolin, myricetin, and kaempferol have

greater antioxidant capacity than their glycosides such as quercetin-3-glucoside [45]. Noroozi et

al. [45] reported that, at equimolar concentration, most flavonoids showed greater antioxidant

capacity than vitamin C.

Currently, the relevance of in vitro studies to the in vivo situation is unclear. Terao et al.

[46] found that oral administration of (-) epicatechin and quercetin enhanced the antioxidant

capacity of rat plasma, although both flavonoids accumulated mainly as glucuronide and sulfate

conjugates in blood plasma. Morand et al. [47] had reported that the conjugate metabolites of

quercetin could inhibit the oxidation of LDL catalyzed with Cu+2.[47] Janisch et al.[48] found

that flavonoid intestinal and hepatic metabolism had an ability to inhibit LDL oxidation. These

finding suggests that conjugated metabolites of flavonoids may play a role in the antioxidant

defenses of blood plasma. In human, Arai et al. [49] found total intake of flavonoids among

women to be inversely correlated with plasma total cholesterol and low density lipoprotein

concentrations, after adjustment for age, body mass index, and total energy intake. Further in

vivo experiments are needed to explore.

Xanthine Oxidase Inhibitors

Xanthine oxidase has a role in the generation of ROS in various pathologies such as viral

infection [50], inflammation [51], brain tumors [52] or the process of ischemia/reperfusion [53,

21

54] has been studied. Xanthine oxidase belongs to the molybdenum-protein family containing

one molybdenum, one of the flavin adenine dinucleotide (FAD) and two iron-sulfur (2Fe - 2S)

centers of the ferredoxine type in each of its two independent subunits. Xanthine oxidase is a

cytosolic enzyme found in many species such as bacteria, higher plants, invertebrates and

vertebrates [55]. It is present in the liver, intestine, kidney, lungs, myocardium, brain, plasma,

and erythrocytes, and other tissues of several mammalian species including human [56]. In all

mammals, the liver and intestine have the highest xanthine oxidase activity [55]. This enzyme

catalyzes the conversion of both hypoxanthine to xanthine and xanthine to uric acid while

reducing O2 to O2-• and H2O2 according to Figure 1-5 [57].

The enzyme contains two separated substrate-binding sites. Xanthine oxidase inhibitors

can act either at the purine binding site such as allopurinol [58, 59] or at the FAD cofactor site

such as benzimidazole [60]. Allopurinol is a potent inhibitor of xanthine oxidase which has been

widely used to treat gout and hyperuricemia [61, 62]. However, severe toxicity of allopurinol

such as vasculitis, rash, eosinophilia, hepatitis has been reported [63]. Currently, no clinically

effective xanthine oxidase inhibitor for the treatment of hyperuricemia has been developed since

allopurinol. Therefore, new inhibitor devoid of undesired side effects has been investigated.

Many studies of natural polyphenols, especially flavonoids, in the form of plants or purified

extracts show that they could be used as xanthine oxidase inhibitors [64-66]. The essential

structural characteristics for the inhibition of xanthine oxidase are (1) the presence of the benzo-

γ-pyrone structure (2) the presence of free hydroxyl groups at positions 7, 3 and /or 5 in the

flavonoid structure [40] and (3) an α, β-unsaturated carbonyl group that helps π electronic

delocalization of phenyl ring B [40, 56].

22

Different types of inhibition are found concerning xanthine and flavonoids as substrates:

competitive, non competitive and mixed type inhibition. Different modes of inhibition were

demonstrated at steady state measurements using Lineweaver-Burk plots. In the mixed

inhibition, the inhibitor can bind to the free enzyme and to the enzyme-substrate complex [40].

Uric Acid, Hyperuricemia, and Gout

Uric acid is produced by the degradation of purine compounds either from exogenous

(dietary) or endogenous origin (Figure 1-6).

Most species, except humans, some apes and the dalmatian dogs have rather low blood

levels of uric acid because of the presence of the uric acid catabolizing enzyme uricase in the

plasma and liver [67]. Uricase transforms uric acid to allantoin, which is water soluble and can

be excreted. Thus, in rat experiment, we have to use an uricase inhibitor such as potassium

oxonate to increase endogenously synthesized uric acid (Figure.1-7).

At physiological pH almost all uric acid is ionized to urate since the pKa of uric acid is

around 5.4. Urate has limited solubility in water. Therefore, the excess production of uric acid

can lead to the deposition of urate crystals in various locals, particulay in the joints, the

connective tissues, and the kidneys [68]. Hyperuricemia is generally the cause for gout which is

characterized by a serum uric acid level of above 7.5 mg per 100 mL for males and 6.6 mg per

100 mL for females [69].

Gout occurs when urate monohydrate crystals deposit in the joint space between two bones

or in both. These depositions lead to inflammatory arthritis, which causes swelling, redness, heat,

pain, and stiffness in the joints. The inflammatory response involves local infiltration of

granulocytes, which phagocyte the urate crystals. This process generates oxygen metabolites,

which damage tissue, and results in the release of lysosomal enzymes that inducing an

inflammatory response. Moreover, lactate production is high in synovial tissues and in the

23

leukocytes associated with the inflammatory process. The high level of lactate leads to a local

decrease in pH that fosters further deposition of uric acid. In fact, the major risk factor for the

development of gout is sustained asymptomatic hyperuricemia (Table 1-1) [70].The optimal

diagnosis of gout is the demonstrating urate crystals in synovial fluid or a tophus (a nodular

collection of urate crystals in soft tissue) [70, 71].

The commonly report of gout is 6 per 1000 population in men and 1 per 1000 population

for women [71]. The incidence of gout has been found to be increasing [72, 73]. With the

Rochester Epidemiology Project computerized medical record system, the incidence rate

increase more than twofold from 1977-1978 to 1986-1987 in Rochester, MN [73].

The goal of antihyperuricemic therapy is to reduce serum uric acid level below the

threshold required for supersaturation of extracellular fluid, to prevent or reverse tissue damage

resulting from uric acid deposition, and to decrease the incidence of recurrent attacks of gout

arthritis [69, 74]. Drugs used to reduce uric acid levels can be either uricosuric drugs or xanthine

oxidase inhibitors [74].

All the synthetic drugs used in the treatment of gout (Table 1-2) have some side effects,

therefore an alternative are required.

Enzyme Inhibition

The basic equation of enzyme kinetics is Michaelis-Menten equation (V = Vmax [S]/ Km +

[S]). This equation has the same form as the equation for a rectangular hyperbola; the reaction

rate (V) versus substrate concentration [S] produces a hyperbolic rate plot (Figure 1-8). To avoid

dealing with curvilinear plots of enzyme catalyzed reactions, the Lineweaver-Burk plot was

introduced (Figure 1-8).The equation of Lineweaver-Burk is [1/V] = [Km (1)/ Vmax[S] + (1)/Vmax]

[75].

24

Enzyme inhibitors are substances that reduce an enzyme activity and have similar structure

to their enzyme’s substrate but either does not react or react very slowly compared to substrate.

The mechanisms of inhibition are described as follow.

Competitive Inhibition

A substance that competes directly with a normal substrate for an enzymatic binding site is

known as a competitive inhibitor. These inhibitors usually resemble the substrate and act by

reducing the concentration of free enzyme available for substrate binding. The general model for

competitive inhibition and the Lineweaver-Burk plot are showed in Figure 1-9 [75].

Uncompetitive Inhibitions

The inhibitor binds directly to the enzyme–substrate complex but not to the free enzyme as

shown in Figure 1-10.

Mixed Inhibitions or Non Competitive Inhibitions

The inhibitors bind to both the enzyme and enzyme-substrate complex bind inhibitor as

shown in Figure 1-11.

Pharmacokinetics

Pharmacokinetics (PK) is defined as the study of the time course of drug absorption,

distribution, metabolism and excretion. Absorption describes the process of drug molecules

moving from the site of administration to systemic circulation. Distribution describes the

movement of drug molecules from systemic circulation to extravascular sites. Metabolism

describes the enzymatic breakdown of drugs. It is frequently a primary defense mechanism used

by the body to avoid exposure to xenobiotics. Drugs molecules are converted to more

hydrophilic metabolites and excreted from the body. Metabolites can be inactive, active or toxic.

Therefore, understanding the pathway where a compound is metabolized and PK of its

25

metabolites is essential. Finally, excretion describes passive or active transport of drug molecules

into urine or bile [76].

Pharmacokinetics studies rely on the measurement of the active drugs and/or its

metabolites in biological fluid such as blood, plasma or urine. From this information,

concentration-time curves may be constructed and pharmacokinetic parameters such as area

under the curve (AUC), maximum concentration (Cmax), clearance (Cl), volume of

distribution(Vd) and elimination half-life ( t 1/2) may be calculated [77].

Pharmacokinetics is also applied to therapeutic drug monitoring (TDM) for very potent

drugs such as those with a narrow therapeutic range, in order to optimize efficacy and to prevent

any adverse toxicities [78].

Hypothesis and Objectives

Gout is a common disease with a worldwide distribution and continues to be a health

problem. It is often associated with elevated serum levels of uric acid. The most common

symptom in gout is painful arthritis joint inflammation, caused by deposition of insoluble

crystals of sodium urate. Nowadays, it seems to be accepted that the key factor to control this

disease is the prevention and the treatment. The treatment of gout includes the use of anti-

inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs) for symptomatic

relief and xanthine oxidase inhibitors to block the endogenous production of uric acid. However,

NSAIDs produce side effects such as naturopathy, nitrogen retention, and, hyperkalemia.

Allopurinol, the most common xanthine oxidase inhibitor, also has unwanted side effects such as

hypersensitivity problems. Therefore, alternative treatments are required.

The leaves of artichoke have been used traditionally by the Eclectic physicians as a diuretic

and depurative, for treatments of rheumatism, gout, jaundice and especially for dropsies. The

major compounds of artichoke are phenolic compounds such as caffeoylquinic acids and

26

flavonoids. The phenolic compounds have shown xanthine oxidase inhibition and antioxidant

activity in vitro and in vivo. Therefore, artichoke leaves containing polyphenolic compounds may

show xanthine oxidase inhibitory activity and antioxidant activity. In the present study the

xanthine oxidase inhibitory activity and antioxidant activity of artichoke extract, and its main

constituents were investigated in vitro and in vivo.

Furthermore, the pharmacokinetic of an active compound in artichoke extract was studied

in male Sprague-Dawley rats in order to assess the in vivo efficacy and obtain more information

about absorption and disposition. The concentration of a single compound and its metabolites

will be detected in plasma and urine and pharmacokinetic parameters will be calculated.

Therefore, to test the hypothesis of this study the following specific aims were purposed:

Specific aim#1: Phytochemical investigation of compounds in artichoke extract.

Specific aim#2: Determine whether artichoke extract and its compounds show the inhibition of

xanthine oxidase in vitro.

Specific aim#3: Investigate whether artichoke extract and its compounds can decrease uric acid

in rat serum.

Specific aim#4: Determine whether artichoke extract and its compounds show antioxidant

activity in vitro and in vivo.

Specific aim#5: Pharmacokinetic analysis of an active compound in artichoke extract.

27

Table 1-1. Annual incidence of gouty arthritis according to the serum urate concentration [70]. Serum Urate Concentration (mg/dl) Annual Incidence of Gout (%)

<7.0 0.1-0.57.0 - 8.9 0.5-1.2

≥9.0 4.9-5.7

28

Table 1-2. Drugs used in the management of gout [79, 80]. Drug Comment To treat acute gouty arthritis

Colchicine Inhibits crystal phagocytosis; no effect on urate metabolism; increased toxicity in patients who have renal or hepatic dysfunction or are receiving concomitant therapy with P-450 enzyme inhibitors such as cimetidine, erythromycin, and tolbutamide [79]; current treatment is an intravenous dose of 2 mg, diluted in 10 to 20 mL of 0.9% sodium chloride solution; a total dose of 4 mg should not be exceeded. To avoid cumulative toxicity, treatment with colchicines should not be repeated within 7 days [80].

NSAIDs Effective in relieving pain and reducing inflammation in patients with acute gout but use limited by side effects (naturopathy, nitrogen retention, reduced creatinine clearance, hyperkalemia, abnormal liver-function values, and headache); greater risk of side effects in patients with renal dysfunction [79, 80].

Corticosteroids Effective either by intraarticular (single joint) or systemic route (intramuscular, intravenous, or oral); potential for rebound inflammation and side effects; administered only when NSAIDs and colchicines have been ineffective or are contraindicated [79, 80].

To prevent acute attacks

Colchicine Effective in an oral dose (0.5-1.8 mg per day) adjusted so as not to cause diarrhea [80].

NSAIDs Useful if colchicine alone is insufficient and acute attacks recur frequently; usual dose is 150 to 300 mg of indomethacin per day or its equivalent [79].

To lower serum urate concentrations

Probenecid Increases urate excretion by inhibits urate reabsorption at renal tubule; interferes with excretion of many drugs; serious toxic effects rare, although nausea and rash reported in up to 10 % of patients [79]; effective in an oral dose of 250 mg twice daily for 1 week, following with 500 mg twice daily for chronic treatment [80].

Allopurinol Inhibits xanthine oxidase; common side effect are hypersensitivity reactions [80]

29

OH

OH

O

OH

OR4OR3

OR2HOOC

OR1 Caffeic acid Quinic acid Chlorogenic acid: R1=H, R2=H, R3=H, R4=caffeoyl 3,5-di-O-caffeoylquinic acid (Cynarin): R1=caffeoyl, R2=caffeoyl, R3=H, R4=H 3,5-di-O-caffeoylquinic acid: R1=H, R2=caffeoyl, R3=H, R4=caffeoyl 4,5-di-caffeoylquinic acid: R1=H, R2=H, R3=caffeoyl, R4=caffeoy

O

OOH

R1O

R2

OH

O

OOH

R1O

OH

luteolin-7-O-glucoside: R1=glc, R2=OH narirutin: R1=rutinose luteolin-7-O-rutinoside: R1=rut, R2=OH naringenin-7-O-glucoside: R1=glucose apigenin-7-O-glucoside: R1=glc, R2=H apigenin-7-O-rutinoside: R1=rut, R2=H Figure 1-1. Structures of caffeoylquinic acids and flavonoids detected in artichoke [22].

30

OHOH

OH

O

OHOMe

OH

OOMe

OH

OH

O

OMeOH

OH

O

OHOH

OH

O

OROR

HOOC OR

OR

CCA

IFA, IFA-Conj.

CA, CA-Conj.

DHFA, DHFA-Conj.

DHCA

DHCA-Conj.

LIV

ER

CO

LON

SMA

LL

INT

EST

INE

CO

LO

N

Figure 1-2. Hypothetical metabolic pathway of caffeoylquinic acids [30].

31

OO

COOH

OHOH

COOH

OHOH

COOH

OO

COOH

COOH

OHOCH3

OHOCH3

COOH

GS

COOH

OHOH

GS

COOH

OHOH

CYP 2E1

O2 or O2-

Acyl Co A dehydrogenase

(ATP, CoA)

Hydrogenase ?

CYP 2E1

o-quinone

GS-CA conjugate

Hydrogenase?

Acyl Co A dehydrogenase

(ATP, CoA)

GSH

CO

MT

CY

P 1 A1/ 2

CY

P 1A1 /2

CO

MT GSH

O 2 or O 2 -

FADHFA

Figure 1-3. Proposed metabolic pathway of caffeic acid in isolated rat hepatocytes [31].

32

Figure 1-4. Proposed recycling of flavonoids through sequential metabolism and/or secretion

involving intestinal microflora, intestine, and liver. In this scheme, flavonoids are assumed to be given orally. This recycling scheme involves dual loops: one is the classical enterohepatic recycling and the other is enteric recycling, where phase II metabolites formed and excreted by the small intestine could be reconverted to their aglycones again in the large intestine by the bacteria and reenter the blood via the colon. In this figure, SGLT1 refers to a glucose transporter and MRP refers to multidrug resistant related protein. SGLT1 could participate in the absorptive transport of glycosides [81], whereas MRP could act as a gatekeeper that prevents the absorption of glycosides [39, 81].

33

N

NH N

NH

O

NH

NH N

NH

O

O

NH

NH NH

NH

O

O

OH

Hypoxanthine Xanthine Uric acid

Figure 1-5. The enzymatic process catalyzed by xanthine oxidase [57].

Xanthine oxidase Xanthine oxidase

O2, H2O O2, H2O O2-•, H2O2 O2

-•, H2O2

34

Ficture 1-6. Purine degradation pathway in animals [67].

IMP

Inosine

AMP

Adenosine deaminase

AMP deaminase GMP

Guanosine

nucleotidases

Purine nucleoside phosphorylase

(PNP)

NH4+

N

NH N

NH

O

Xanthine oxidase

Hypoxanthine

O2, H2O H2O2

H2O2

O2, H2O xanthine oxidase

Adenosine

Guanine

NH4

NH

NH N

NH

O

O

NH

NH N

NH

O

O

O-

N

NH N

NH

O

NH2

NH4+

xanthine urate

35

NH

NH NH

NH

O

O

O

O

NH

NH2NH

NH

O

O

Figure 1-7. The mechanism of uricase and uricase inhibitors [67].

Uricase

O2 CO2 Uric acid Uricase Inhibitors Allantoin

36

A

B

Figure 1-8. Enzyme inhibition. A) Michaelis-Menten plot. B) Lineweaver-burk plot. V is defines as a intial velocity, [S] is the substrate concentration, Vmax is a maximum velocity and Km is a substrate concentration at ½ of Tmax [75].

37

A B

Figure 1-9. Competitive inhibition. A) The model for competitive inhibition. B) Lineweaver-

Burk plot of the competitively inhibited Michaelis-Menten enzyme. E is defined as enzyme, S is substrate, I is inhibitor; EI is enzyme-inhibitor complex and P is product. Note that Vmax, as defined as the maximum velocity of a reaction, is unchanged; Km, as defined by [S] required for ½ maximal activity, is increase [75].

38

A

B

Figure 1-10. Uncompetitive inhibition. A) The model for uncompetitive inhibition. B) Lineweaver-Burk plot of a single Michaelis Menten enzyme in the presence of uncompetitive inhibitor. Note that Vmax is decreased; Km, as defined by [S] required for ½ maximal activity, is decreased [75].

39

A B

Figure 1-11. Mixed inhibition. A) The model for mixed inhibition. B) Lineweaver-burk plot of a simple Michaelis Menton enzyme in the presence of a mixed inhibitor. Note that Vmax is decreased; Km appears unaltered [75].

40

CHAPTER 2 IDENTIFICATION AND QUANTIFICATION OF COMPOUNDS IN ARTICHOKE

EXTRACT

Background

The variation of the content of mono-and dicaffeoylquinic acids and flavonoids in

artichoke extracts has been reported [23, 82]. For example, the content of luteolin-7-O-glucoside

and 1, 3-O-dicaffeoylquinic acid were reported to vary from 1002 to 1616 mg/kg of dried

extracts and from 1292 to 30985 mg/kg of dried extracts, respectively [23]. This deviation of

phenolic compounds might affect the pharmacological activities of artichoke extracts.

Specific Aim

The objective of this study was to identify and quantify marker compounds in artichoke

extract.

Materials and Methods

Materials

Water extract of artichoke leaf (Cynara scolymus L.) was obtained from a German extract

manufacturing company (Finzelberg, Andernach, Germany). Dihydrocaffeic acid (90-95%) and

luteolin-7-O-glucoside (>90%) were purchased from Indofine Chemical Company, Inc.

(Somerville, NJ, USA). Chlorogenic acid (≥95%) was purchased Sigma Chemical Company (St.

Louis, MO, USA). Cynarin was purchased from Carl Roth GmbH+Co. (Germany). Acetonitrile

(CH3CN) and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Fair Lawn, NJ,

USA). Luteolin-7-O-glucuronide used in this study was a kind gift from Prof. Dr. A. Nahrstedt,

Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Germany. All

aqueous solutions were prepared with deionized water obtained from a NANOPure® system

from Barnstead (Dubuque, IA, USA).

41

Sample Preparation

500 mg of powdered extract of Cynara scolymus L.was dissolved in 20.0 mL of

MeOH/H2O (3:7) at 25 °C for 5 min. The solutions were filtered (0.45 μm) and were directly

analyzed by HPLC/DAD.

HPLC/DAD Analysis

Samples were analyzed using a reverse-phase partition mode of HPLC with diode array

detector. A Shimadzu VP series HPLC system (Kyoto, Japan) equipped with an SPD-M10Avp

diode array detector was used for this work. The column used was a 250- 4.0 mm i.d.(5μm )

Lichrospher® 100 RP-18e (Merck KgaA, Germany).The column temperature was kept at 25oC.

The eluents were (A) 0.3% TFA and (B) CH3CN. The following solvent gradient was applied:

5% B (5 min), 5-20% B (50 min), 20-5%B (15 min), total run time was 70 min. The injection

volume for all samples was 10 μL. Flow elution was 1 mLmin-1. Chromatograms were acquired

at 330 nm for the caffeoylquinic acid and 350 nm for the luteolin derivatives. UV-Vis spectra

were recorded in the range 200-400 nm.

Work Solutions and the Preparation of Calibration Standards

Chlorogenic acid, cynarin, and luteolin-7-O-glucoside work solutions (400 μg/mL):

The amount of 10.0 mg of chlorogenic acid, cynarin and luteolin-7-O-glucoside were accurately

weighed, and transferred to a 25.0 mL volumetric flask. The standards were then dissolved in

and brought to volume with methanol.

Luteolin-7-O-glucuronide work solution (500 μg/mL): The amount of 1.0 mg of

luteolin-7-O-glucuronide was weighted, and transferred to a 2.0 mL volumetric flask. The

standard was then dissolved in methanol to obtain a final concentration of 500 μg/mL. The

volume was completed with same solvent, and the final solution mixed thoroughly.

42

Standard solutions for chlorogenic acid, cynarin, and luteolin-7-O-glucoside: From the

chlorogenic acid, cynarin, and luteolin-7-O-glucoside work solutions, five different

concentrations of standard solutions of chlorogenic acid, cynarin, and luteolin-7-O-glucoside

and three quality controls (QC) were prepared in methanol according (Table 2-1). All solutions

were filtered through a 0.45 μm PVDF membrane filter (Millipore Corp.) before analysis.

Standard solutions for luteolin-7-O-glucuronide: From the luteolin-7-O-glucuronide

work solution, six different concentrations of standard solutions of -7-O-glucuronide and three

quality controls (QC) were prepared according to Table 2-1. The final volume was filled up with

methanol in 2.0 mL volumetric flask. All solutions were filtered through a 0.45 μm PVDF

membrane filter (Millipore Corp.) before analysis.

Quantification

Calibration was carried out by an external standardization method. Calculation was

performed using Microsoft Excel ®. The calibration curves were obtained by plotting the mean

area versus the corresponding concentration of the each standard solution. The calibration was

considered suitable if not more than 1/3 of the quality control standards showed a deviation from

the theoretical values equal or greater than 15%, except at the lower limit of quantification

(LLOQ), where it should not exceed 20%.

Validation

The method was validated over the range of concentration of the target compounds present

in the artichoke extracts. The validation parameters of linearity, sensitivity, specificity, precision,

accuracy and stability were determined.

The linearity of the calibration curves was determined by least-squares linear regression

method and expressed in terms of coefficient of determination (r2). The intra- and inter-day

43

precision and accuracy were measured by triplicate analyses of three different concentration

levels (low, medium and high) of quality control standards on the same day and on different

days. The precision was based on the calculation coefficient of variation (CV %), and the

accuracy was defined as the percent difference between the theoretical and measured values. The

limit of quantification for the assay was defined as the minimum concentration of quality

controls.

Results

Linearity

Calibration curves (n = 9) operating in the range of 5-500 μg/mL for all four artichoke

components were linear (r2 > 0.999) (Figure 2-1).

Sensitivity

In this study, the limit of quantification (LLOQ) is defined as the lowest concentration for

quality control with an accuracy and precision better than 20 %. The LLOQ of chlorogenic acid,

cynarin, luteolin-7-O-glucoside and luteolin-7-O-glucuronide were 0.5, 0.5, 1 and 5 μg/mL,

respectively.

Specificity

The methods provided good resolutions between chlorogenic acid, cynarin, luteolin-7-O-

glucoside and luteolin-7-O-glucuronide. Peaks of all test compounds had similar retention times

and the UV spectra (200- 400 nm) when compared to the standards. The wavelengths 350 and

330 nm used to quantify caffeolyquinic acids and luteolin derivatives at their maximum

absorption, respectively, were confirmed by their UV spectra (Figure 2-2). There was no

endogenous interference from artichoke extract (Figure 2-3) in this assay, indicating specificity

of the methods to the tested compounds. Additionally, The UV spectra of all tested compounds

44

showed more than 99% of similarity with those obtained using the respective standard

compounds (Figure 2-3).

Precision and Accuracy

The precision intra- and inter-day for chlorogenic acid, cynarin, luteolin-7-O-glucoside and

luteolin-7-O-glucuronide were satisfactory with CV values between 0.73 and 12.35%. Similarly

the accuracy of the assay was between 94.34 and 107.32% for all compounds tested at three

different concentrations. The results are summarized in Table 2-4.

Stability

The standard solutions of caffeoylquinic acids and luteolin derivatives were found stable

on autosampler at 20oC within 24 hours (Table 2-2 and Table 2-3). The shifting of the areas of

each sample tested was less than 15 % of those obtained from a fresh solution at the same level

of concentrations.

Quantification of Caffeoylquinic Acids (Chlorogenic Acid, Cynarin) and Luteolin Derivatives (Luteolin-7-O-glucoside, Luteolin-7-O-glucuronide) in Artichoke Leaf Extract

The results from Table 2-5 showed that the caffeoylquinic acids were the predominant

phenolic compounds of the artichoke extract, with 5-O-caffeoylquinic acid showing the highest

amount. The predominant flavonoid was luteolin-7-O-glucoside, followed by luteolin-7-O-

glucuronide.

Discussion and Conclusion

This study reported a quantitative evaluation of phenolic marker compounds of artichoke

extract using a HPLC with photodiode array detector (HPLC/DAD). The identification of each

compound was performed by a comparison with available standards and by UV evaluation. This

approach made it possible to rapidly discriminate between caffeoyl derivatives and flavonoids.

The main chemical structures of the identified compounds are showed in Figure 2-2 as

45

chlorogenic acid, cynarin, dihydrocaffeic acid, luteolin-7-O-glucoside and luteolin-7-O-

glucuronide.

The HPLC profiles of the extract are shown in Figure 2-2 with a profile of the caffeoyl

derivatives at 330 nm and profiles of flavonoids at 350 nm. The quantitative HPLC/DAD

findings of caffeoylquinic ester and flavonoid are summarized in Table 2-4

The developed method is appropriate to completely characterize and quantify phenolic

marker compounds in artichoke extract.

46

Table 2-1. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of chlorogenic acid, cynarin, luteolin-7-O-glucoside and luteolin-7-O-glucuronide

Compounds Standard solutions (μg/mL) QC (μg/mL) Chlorogenic acid 5, 10, 50, 100, 400 10, 25, 200Cynarin 5, 10, 50, 100, 400 10, 25, 200Luteolin-7-O-glucoside 5, 10, 50, 100, 400 10, 25, 200Luteolin-7-O-glucucronide 5, 10, 50, 100, 250, 500 8, 75, 200

47

Table 2-2. The stability test of chlorogenic acid, cynarin and luteolin-7-O-glucoside after 24 hours on autosampler at 20oC. Data represents the percentage remaining of all test compounds

% Remaining on autosampler at 20 oC within 24 hours Compound QC1-10 μg/mL QC2- 25 μg/mL QC3-200 μg/mL

Chlorogenic acid 90.41 ± 2.35 100.75 ± 2.33 104.58 ± 3.19Cynarin 95.83 ± 10.45 96.35 ± 0.73 98.58 ± 6.10Luteolin-7-O-glucoside 94.07 ± 5.72 98.77 ± 5.61 100.50 ± 6.23

48

Table 2-3. The stability test of luteolin-7-O-glucuronide after 24 hours on autosampler at 20oC. Data represents the percentage remaining of all test compounds

% Remaining on autosampler at 20 oC within 24 hours Compound QC1-8 μg/mL QC2-75 μg/mL QC3-200 μg/mL

Luteolin-7-O-glucuronide 90.43 ± 7.81 96.21 ± 3.45 101.53 ± 3.52

49

Table 2-4. Intra-day (n = 3) and inter-day (n = 9) assay parameters of caffeoylquinic acid (chlorogenic acid and cynarin) and luteolin derivatives (luteolin-7-O-glucoside and luteolin-7-O-glucuronide). Accuracy expressed as % of the theoretical concentration and precision expressed as %CV

Chlorogenic acid QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 5.92 12.52 11.84 1.83 10.25 2.35 2.36 3.86 5.54Accuracy 102.41 95.38 100.15 102.24 103.92 100.32 105.22 107.16 102.78Inter-day QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Precision 14.67 13.16 7.07 Accuracy 95.21 100.56 107.32

Cynarin QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 6.11 9.13 10.53 2.14 11.52 6.10 2.19 4.54 0.73Accuracy 92.11 100.25 93.57 98.23 102.21 102.46 100.81 107.68 105.53Inter-day QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Precision 13.31 8.73 8.59 Accuracy 94.34 98.90 105.22

Luteolin-7-O-glucoside QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 3.49 12.21 10.19 2.70 11.77 6.23 1.42 6.67 4.52Accuracy 102.26 97.31 106.69 97.34 102.65 107.71 106.88 104.38 109.81Inter-day QC1-10 μg/mL QC2–25 μg/mL QC3–200 μg/mL Precision 11.90 7.04 2.54 Accuracy 98.51 97.21 100.53

Luteolin-7-O-glucuronide QC1-8 μg/mL QC2–75 μg/mL QC3–200 μg/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 10.54 12.35 7.83 7.98 8.45 2.65 2.18 3.53 0.29 Accuracy 96.32 100.21 99.13 98.51 102.21 104.26 100.75 104.12 109.07 Inter-day QC1-8 μg/mL QC2–75 μg/mL QC3–200 μg/mL Precision 10.19 8.72 5.76 Accuracy 95.54 97.19 100.98

50

Table 2-5. Amounts of caffeoylquinic acids and luteolin derivatives expressed as milligram per gram of dried extract

Compound Amount of compounds in artichoke extract (mg/g) mean ± SEM

5-O-caffeoylquinic acid (chlorogenic acid) 8.71 ± 0.591,3-di-O-caffeoylquinic acid (cynarin) 2.47 ± 0.54luteolin-7-O-glucoside 3.60 ± 0.62luteolin-7-O-glucuronide 2.08 ± 0.73

51

A

0 100 200 300 400 5000

3.0×106

6.0×106

9.0×106

1.2×107

1.5×107

chlorogenic acid[ug/mL]

Are

a

B

0 100 200 300 400 5000

5.0×106

1.0×107

1.5×107

2.0×107

cynarin[ug/mL]

Are

a

C

0 100 200 300 400 5000

1.0×106

2.0×106

3.0×106

4.0×106

5.0×106

6.0×106

7.0×106

8.0×106

Luteolin-7-0-glucoside[ug/mL]

Are

a

D

0 100 200 300 400 500 6000

1.0×106

2.0×106

3.0×106

4.0×106

5.0×106

6.0×106

7.0×106

Luteolin-7-0-glucuronide[ug/mL]A

rea

Figure 2-1. Mean calibration curves of compounds in artichoke (n = 9). A) Chlorogenic acid. B) Cynarin. C) Luteolin-7-O-glucoside. D) Luteolin-7-O-glucuronide in methanol. Vertical bars represent the standard deviations (SD) of the means.

Y = 37766 X - 209223 R2 = 0.999

Y = 30030 X - 45570 R2 = 0.9992

Y = 11951 X - 86968 R2 = 0.9992

Y = 12680 X - 7939.7 R2 = 0.9997

52

Figure 2-2. HPLC separation and absorbance-wavelength spectra of chlorogenic acid, cynarin,

dihydrocaffeic acid, luteolin-7-O-glucoside and luteolin-7-O-glucuronide.

Minutes0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

mVo

lts

0

50

100

150

200

250

300

350

400

450Chlorogenic acid

Cynarin

nm200 220 240 260 280 300 320 340 360 380 400

mA

u

0

500

1000

1500

mA

u

0

500

1000

1500

25.20 Min / Smooth

nm

200 220 240 260 280 300 320 340 360 380 400

mAu

0

500

1000

1500

2000

mAu

0

500

1000

1500

200038.35 Min / Smooth

nm200 220 240 260 280 300 320 340 360 380 400

mAu

0

250

500

750

1000

mAu

0

250

500

750

1000

52.88 Min / Smooth

Luteolin-7-O-glucuronide

n m2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0

mA

u

- 6

- 4

- 2

0

2

4

6

8

1 0

mAu

- 6

- 4

- 2

0

2

4

6

8

1 02 5 . 6 3 M i n / S m o o t h

Dihydrocaffeic acid

Luteolin-7-O-glucoside

nm

200 220 240 260 280 300 320 340 360 380 400

mA

u

0

200

400

600

mA

u

0

200

400

600

51.33 Min / Smooth

53

A Similarity: 0.9998

nm200 250 300 350 400

mAu

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

mAu

-20

0

20

40

60

80

100

120

140

160

180

200

220

240

Unknown Known

B Similarity: 0.9963

nm200 250 300 350 400

mAu

0

10

20

30

40

50

60

70

80

mAu

0

10

20

30

40

50

60

70

80

Unknown Known

C Similarity: 0.9807

nm200 250 300 350 400

mAu

0

10

20

30

40

50

60

70

80

90

mAu

0

10

20

30

40

50

60

70

80

90Unknown Known

D Similarity: 0.9753

nm200 250 300 350 400

mAu

0

5

10

15

20

25

30

35

40

45

50

mAu

0

5

10

15

20

25

30

35

40

45

50Unknown Known

Figure 2-3. Absorbance-wavelength spectras. A) Chlorogenic acid. B) Cynarin. C) Luteolin-7-O-glucoside. D) Luteolin-7-O-glucuronide. (1) Represents the spectra of the standard compound and (2) represents the spectra of the peak with same retention time of the corresponding standard but obtained after injection of artichoke extract.

1

2

1 2

1

2

1

2

54

CHAPTER 3 EFFECT OF ARTICHOKE LEAF EXTRACT, CAFFEIC ACID DERIVATIVES AND

FLAVONOIDS ON XANTHINE OXIDASE INHIBITORY ACTIVITY

Background.

Xanthine oxidase (XO) is a key enzyme that catalyses the oxidation of oxypurines

(hypoxanthine and xanthine) to uric acid in the purine metabolic pathway [67]. The uric acid

plays a vital role in producing hyperuricemia and gout. Allopurinol is a clinically used XO

inhibitor in the treatment of gout. However, due to the unwanted side effect of allopurinol such

as hypersensitivity problem [74], Steven-Johnson syndrome [83], renal toxicity [84], and fatal

liver necrosis [85] the alternative treatment with increased therapeutic activity and less side

effects is necessary.

The leaves of artichoke consists of many chemical components such as caffeoylquinic

acids and flavonoids and one or more of these components may be effective agents as XO

inhibitors. Flavonoids have been shown to be inhibitors of XO activity in vitro [65]. In this aim,

the efficacy of artichoke leaf extract and its main components in inhibiting XO was performed.

Additionally, various flavonoids such as flavones, flavonols and flavanones have been

investigated as XO inhibitors. The results are shown in Table 3-1.

Specific Aim

Determine the in vitro XO inhibition of Cynara scolymus L., its compounds and some

flavonoids.


Materials

Water extract of artichoke leaf (Cynara scolymus L.) were obtained from a German extract

manufacturing company (Finzelberg, Andernach, Germany). Allopurinol, chlorogenic acid (≥

95%), quercetin dihydrate (> 98%) and xanthine oxidase from bovine milk (25 units/1.3 mL),

55

NaH2PO4.H2O, Na2HPO4.12H2O were purchased from Sigma Chemical Company (St. Louis,

MO, USA). Apigenin (98%), eriodictyol (≥95%), dihydrocaffeic acid (90-95%), luteolin (99%),

luteolin-7-O-glucoside (> 90%) were purchased from Indofine Chemical Company, Inc.

(Somerville, NJ, USA). Cynarin and naringenin (≥ 96%) were purchased from Carl Roth

GmbH+Co. (Germany). Kaempferol (RG) was purchased from Chromadex (Santa Ana, CA,

USA). Xanthine was purchased from Carl MP Biomedicals (Solon, OH, USA). All buffers and

aqueous solutions were prepared with deionized water obtained from a NANOPure® system

from Barnstead (Dubuque, IA, USA).

Preparation of Working Solutions and Test Solutions

Phosphate buffer solution: (A). 0.2 M NaH2PO4 solutions: NaH2PO4.H2O (2.78 g) was

dissolved in distilled water to make 100.0 mL solution. (B). 0.2 M Na2HPO4 solution:

Na2HPO4.12H2O (71.50 g) was dissolved in distilled water to make 100.0 mL solution. 85 mL of

A solution and 915 mL of B solution were added to 1000.0 mL of distilled water to make 0.1 M

phosphate buffer solution, pH 7.8.

Xanthine buffer solution: Xanthine (12.20 mg.) was initially dissolved in 0.25 N NaOH

and then diluted with 0.1 mM phosphate buffer to obtain a 400 μM solution.

Xanthine oxidase: The xanthine oxidase solution was prepared by diluting xanthine

oxidase from cow’s milk to a final concentration of 0.4 U/mL in cold 0.1 mM phosphate buffer

(pH 7.8). (Enzyme 200.0 μL filled up to 10.0 mL)

Test solution: 1.36 mg of Allopurinol (M.W. = 136.1) was dissolved in 2000 μL of

DMSO to make a concentration of 50 mM solution, which was then diluted with 0.1 mM

phosphate buffer to obtain a 400, 200, 100, 50, 25, 10, 5, and 1 μM solution. Apigenin, caffeic

acid, chlorogenic acid, cynarin, dihydrocaffeic acid, eriodictyol, kaempferol, luteolin, luteolin-7-

56

O-glucoside, luteolin-7-O-glucuronide, quercetin, naringenin were prepared in the same way as

allopurinol. The final concentration of DMSO was less than 2%.

Artichoke extract. was dissolved in 1 mM phosphate buffer to make a concentration of

1000, 500, 300, 100 μg extracts/ mL phosphate buffer.

Assay Procedure for Xanthine Oxidase Inhibitions

The inhibitory activity of each compound was determined using a slight modification of

the reference methods [64, 86-88].

Control: 7.0 μL of xanthine oxidase buffer solution (0.4 U/mL) were added to 0.1 M

phosphate buffer (127.0 μL) and the mixture was incubated at 37°C for 10 minute Then 66.0 μL

of 400 μM xanthine buffer solution were added to the mixture and the absorbance at 295 nm of

the reaction mixture was measured at 37° C for 10 min by multi-detection microplate reader

(Synergy HT). The blank solution was prepared in an analogous way, but instead of the enzyme,

it contained 7 μL of phosphate buffer solution. The test was performed in triplicate.

Sample test: 7.0 μL of xanthine oxidase buffer solution (0.4 U/mL) was added to a solution

consisting of 0.1 M phosphate buffer pH 7.8 (77.0 μL) and 50.0 μL each of test samples which

was treated in the same way as the control. 3.5 μL of phosphate buffer solution were used instead

of xanthine oxidase solution (0.4 U) for blank tests.

Xanthine oxidase activity was expressed as percent inhibition of xanthine oxidase,

calculate as (1-B/A) x 100, where A is the change in absorbance of the assay without the test

samples. (∆ abs with enzyme -∆ abs without enzyme), and B is the change in absobance of the

assay with the test sample (∆ abs with enzyme - ∆ abs without enzyme). IC50 was calculated

using Graphpad Prism® (version 4.0, Sandiego, CA).

57

Lineweaver- Burk Plot

Enzyme kinetics was similar to xanthine oxidase assay methodology with varying

concentrations of xanthine as the substrate as 200, 150, 110, 100, 90, 80, 70, 60, and 50 μM.

Lineweaver-Burk plots were generated in Graphpad Prism® (version 4.0, Sandiego, CA). Vmax

(a maximal velocity) and Km (a concentration at 50% Vmax) were calculated.

Results

Xanthine Oxidase Inhibitory Activity of Artichoke Extract

The artichoke extract was assayed for xanthine oxidase inhibitory activity at 1000, 500,

300, 100 μg water extracts / mL (Table 3-2). Artichoke extracts showed a dose dependence XO

inhibitory effect with minimal XO inhibitory activity (< 5%) at 100 μg / ml.

Xanthine Oxidase Inhibitory Activity of Various Flavonoids and Compounds in Artichoke

The inhibition of xanthine oxidase results in a decreased production of uric acid was

measured spectrophotometrically at 295 nm. The IC50 values (50% inhibitory concentrations) of

caffeic acid derivatives and flavonoids were calculated and listed in Table 3-3 and Figure 3-1

Caffeic acid and caffeic acid derivatives such as dihydrocaffeic acid, chlorogenicacid and

cynarin showed weak xanthine oxidase inhibitory effect with IC50 > 100 μM. For flavonoids,

only flavones (luteolin, apigenin) and flavonols (kaempferol, quercetin) were shown to have

potent xanthine oxidase inhibitory activities, with IC50 values 1.49, 2.37, 3.35, and 2.34 μM,

respectively. Flavanones such as naringenin and eriodictyol showed weak xanthine oxidase

inhibitory activity with IC50 > 50μM. Flavonoid glycosides such as luteolin-7-O-glucoside

showed weaker activities with IC50 value 19.90 μM.

58

Inhibition Mechanism

Kinetic analysis using Lineweaver-Burk plots (Figure 3-2 and Table 3-4) revealed that

apigenin, luteolin and kaempferol had a linear mixed-type mode of inhibition, as can be seen

from different Vmax and different Km value. Quercetin showed a competitive type inhibition, as

can be concluded from similar Vmax and different Km values.


Artichoke leaf extract inhibited XO in vitro in a dose-dependent manner with minimal XO

inhibitory activity (< 5%) at 100 μg/mL as shown in Table 3-1. To our knowledge , this is the

first time that the XO activity of artichoke had been observed.

The XO inhibitory of caffeoylquinic acids and flavonoids were shown in Table 3-3. and

Figure 3-1. Caffeic acid and caffeic acid derivatives such as dihydrocaffeic acid, chlorogenic

acid and cynarin showed weak XO inhibitory effect with IC50 > 100 μM. Our results were

similar to the previous study. Chan et al. [86] found hydrocaffeic acid was inactive, caffeic acid

and chlorogenic acid had IC50 values about 74.6 ± 11.04 μM and 126.28 ± 2.86 μM,

respectively. Nguyen et al.[89] reported IC50 value of 85.4 μM by caffeic acid.

The activity of flavonoids as inhibitors of xanthine oxidase in vitro has been reported to be

largely determined by the double bond between C-2 and C-3. Additionally, the absence of a

hydroxyl group at C-3 enhances slightly the inhibition effect on xanthine oxidase [65, 87, 90].

Our results are in agreement with these observations (Figure 3-1). Flavonoid aglycons, only

flavones and flavonols showed potent XO inhibitory activities. The IC50 values of luteolin,

apigenin, kaempferol and quercetin were 1.49, 2.37, 3.35, and 2.34 μM, respectively. Flavanones

such as naringenin and eriodictyol showed weak XO inhibitory activity with IC50 > 50 μM.

59

Flavonoid glycosides showed a much lower inhibition of xanthine oxidase than flavonoid

aglycons, such as luteolin-7-O-glucoside (IC50 = 19.90 μM). This result might be due to the steric

interactions of glycosides on xanthine oxidase [65, 90, 91].

The Lineweaver-Burk plot of apigenin, luteolin and kaempferol showed mixed-type

inhibition. Quercetin showed a competitive type inhibition. The different types of inhibition by

flavonoids have been reported in the previous studies. Lin et al. [92] reported competitive

inhibition by apigenin and quercetin. Cotelle et al. [40] reported competitive inhibition by

luteolin. Nguyen et al. [89] reported competitive inhibition by luteolin and apigenin. The

differences observed between these studies could be explained by the different reaction mixtures,

the different concentrations of enzyme and the different methods. However, Nagao et al. [65]

reported mixed-typed inhibition by luteolin and kaempferol. Van Hoorn et al. [87] and Chang et

al. [93] reported competitive inhibition by quercetin. Noro et al. [94] reported mixed-typed

inhibition by luteolin and apigenin. These reports are consistent with our results. The results

suggest that luteolin, kaempferol and apigenin inhibit XO activity not only by competitive mode,

but also by interaction with the enzyme at a site other than the active center.

The significant inhibition of XO by the flavonoids in vitro suggested that they may

suppress the production of active oxygen species in vivo under the conditions that XO works.

Additionally, their IC50 values are comparable to that of allopurinol (3.65 μM), a therapeutic

drug for treating gout, which indicated a possibility of flavonoids for treating gout. However,

recent studies have shown that after orally administration of quercetin in human, most of

quercetin was found in a form of metabolites in plasma [95]. Our study had shown that luteolin-

7-O-glucuronide, one of the metabolites of luteolin, showed weaker XO inhibitory (IC50 = 20.24

μM) comparing with luteolin (IC50 = 1.49 μM). This result presumably indicates a weaker XO

60

inhibitory activity of metabolites in vivo. However luteolin-7-O-glucuronide is not the only

metabolites found in plasma after incubation of luteolin with microsomal samples from human

intestine [96]. Therefore, the in vitro data obtained from the study does not necessarily predict

the in vivo effects of flavonoids on XO, and further study of the inhibitory effects by flavonoids

in vivo will be required.

In this study, artichoke extract, caffeoylquinic acids and various flavonoids were evaluated

for the inhibition of XO activity. The extract and caffeoylquinic acids showed weak XO

inhibition. Flavone and flavonols had a highly inhibitory effect on XO. The in vivo effect of

these compounds on urate accumulation by XO remains to be studied to clarify the roles of these

compounds in human health.

61

Table 3-1. Structures of various flavonoids Flavones Flavonols Flavanones

4

8

5

7

6

2

3

O

OOH

OH

4'

5'

3'

6'

2'OH

O

OOH

OH

OH

OH

O

OOH

OH

OHOH

Apigenin Kaempferol

Eriodictyol

O

OOH

OH

OH

OH

OH

R=H, Luteolin R=Glc, Luteolin-7-O-glucoside

Quercetin Naringenin

O

OOH

OH

OH

RO

O

OOH

OH

OH

A

B

C

62

Table 3-2. Results of the % XO inhibition screening of artichoke extract XO inhibition (%)

Test Sample 100μg/mL 300μg/mL 500μg/mL 1000μg/mL

Artichoke extract 5.11 ± 1.77 9.83 ± 2.07 19.76 ± 0.89 26.02 ± 1.81

Control 1 μM 10 μM 50 μM 100 μM

Allopurinol 5.35 ± 0.77 33.50 ± 1.18 93.22 ± 0.19 97.192 ± 0.28

Note: Data are expressed as mean ± SEM.

63

Table 3-3. The IC50 values (μM) of test samples on xanthine oxidase inhibition Compounds IC50 (μM) C.I. Caffeic acid and Caffeoylquinic acid Caffeic acid > 100 Cynarin > 100 Chlorogenic acid > 100 Dihydrocaffeic acid > 100 Flavonoids Flavone Apigenin 2.37 1.51 to 3.70Luteolin 1.49 1.23 to 1.83Luteolin-7-O-glucoside 19.90 17.97 to 22.09Luteolin-7-O-glucuronide 20.24 18.47 to 22.17Flavonol Quercetin 2.34 2.11 to 2.59Kaempferol 3.35 2.71 to 4.14Flavanone Naringenin > 50Eriodictyol > 50Control Allopurinol 3.65 3.38 to 3.72Note: Data are expressed as mean with 95% of confidence interval (C.I.).

64

Table 3-4. Vmax and Km of flavonoids on xanthine oxidase inhibition Compounds Vmax (μM / min)

Mean ± SEM Km (μM)

Mean ± SEM Type of Inhibition

Control 6.27 ± 0.35 8.18 ± 2.05 -Apigenin 0.5 μM 3.78 ± 0.14 17.84 ± 1.87 MixedLuteolin 0.5 μM 2.78 ± 0.07 21.10 ± 1.37 MixedQuercetin 0.5 μM 6.22 ± 0.34 21.38 ± 2.81 CompetitiveKaempferol 0.5 μM 4.50 ± 0.34 23.26 ± 4.04 MixedNote: Vmax is a maximum velocity; Km is a concentration at 50% Vmax.

65

10 -1 10 0 10 1 10 20

25

50

75

100

125

Apigenin (μM)

%ur

ic a

cid

Form

atio

n

10 -1 10 0 10 1 10 20

25

50

75

100

125

Luteolin (μM)

%ur

ic a

cid

Form

atio

n

10 -1 10 0 10 1 10 2 10 30

25

50

75

100

125

Luteolin-7-0-glucoside (μM)

%U

ric

Aci

d Fo

ram

atio

n

10 0 10 1 10 2 10 30

25

50

75

100

125

Luteolin-7-0-glucuronide (μM)

%ur

ic a

cid

Form

atio

n

10 -1 10 0 10 1 10 2 10 30

25

50

75

100

125

Quercetin (μM)

%ur

ic a

cid

Form

atio

n

10 -1 10 0 10 1 10 20

25

50

75

100

125

Kaempferol (μM)

%ur

ic a

cid

Form

atio

n

10 -1 10 0 10 1 10 2 10 30

25

50

75

100

125

Allopurinol (μM)

%ur

ic a

cid

Form

atio

n

Figure 3-1. Inhibition dose-response effects. A) Apigenin. B) Luteolin. C) Luteolin-7-O-glucoside. D) Quercetin. E) Kaempferol. F) Allopurinol. Data are expressed as mean ± SEM (n = 3). The IC50 values of each compound and their respective 95% of confidence interval (C.I.) were estimated by nonlinear regression using GraphPad Prism 4.0 as described in “Material and Methods”.

G

F

A B

C D

E

IC50 = 2.37 C.I. = 1.51-3.70

IC50 = 2.34 C.I. = 2.11-2.59

IC50 = 1.49 C.I. = 1.23-1.83

IC50 = 20.24 C.I. = 18.47-22.17

IC50 = 3.65 C.I. = 3.38-3.72

IC50 = 3.35 C.I. = 2.71-4.14

IC50 = 19.90 C.I. = 17.97-22.09

66

-0.15 -0.10 -0.05 -0.00 0.05 0.10 0.15

0.10.20.30.40.50.60.70.80.91.01.11.21.31.4

kaempferol 0.5 μM

Luteolin 0.5 μM

control

apigenin 0.5 μM

quercetin 0.5μM

1/[xanthine]μM

1/v

( μM

/min

)

Figure 3-2. Lineweaver-Burk plots in the absence (control, ■-■) and in the presence of luteolin

(0.5 μM, ◆-◆), apigenin (0.5 μM, ●-●), kaempferol (0.5 μM, ▲-▲) and quecetin (0.5 μM, ▼-▼) with xanthine as the substrate.

67

CHAPTER 4 EFFECTS OF ARTICHOKE LEAF EXTRACT AND VARIOUS FLAVONOIDS ON SERUM

URIC ACID LEVELS IN OXONATE-INDUCED RATS

Background

The previous in vitro study of XO inhibitory activity of artichoke and its components has

shown that the extract and caffeic acid derivatives had weak inhibitory activity on XO.

Flavonoids such as flavone and flavonols showed a highly inhibitory effect with IC50 < 20 μM.

However, the in vivo effect of artichoke extract and its components on urates accumulation by

XO is limited. Therefore, in this study, the effect of artichoke extract, and various flavonoids on

serum uric acid levels in oxonate induced rats was performed.

Most species, except humans, some apes and the dalmatian dogs have rather low blood

levels of uric acid because of the presence of the uric acid catabolizing enzyme uricase in the

plasma and liver. Uricase transforms uric acid to allantoin, which is water soluble and can be

excreted [67]. Thus, in rat experiment, an uricase inhibitor such as potassium oxonate was used

in order to increase endogenously synthesized uric acid.

Specific Aim

Investigate the hypouricemic activity of artichoke leaf extract, and various flavonoids.


Materials

Water extract of artichoke leaf (Cynara scolymus L.) were obtained from a German extract

manufacturing company (Finzelberg, Andernach, Germany). Allopurinol, CMC-Na,

NaH2PO4.H2O and propylene glycol were purchased from Sigma Chemical Company (St. Louis,

MO, USA). Apigenin (98%), eriodictyol (≥95%), dihydrocaffeic acid (90-95%), luteolin (99%),

luteolin-7-O-glucoside (> 90%) were purchased from Indofine Chemical Company, Inc.

(Somerville, NJ, USA). Naringenin (≥ 96%) were purchased from Carl Roth GmbH+Co.

68

(Germany). Kaempferol (RG) was purchased from Chromadex (Santa Ana, CA, USA). All

buffers and aqueous solutions were prepared with deionized water obtained from a NANOPure®

system from Barnstead (Dubuque, IA, USA).

Stock Solutions and Preparation of Calibration Standards

Uric acid stock solution: The amount of 20.0 mg of uric acid (MW = 580.53 g/mol) was

accurately weighed, and transferred quantitatively to a 200.0 mL volumetric flask. The standard

was then dissolved in 20.0 mL of 0.25 N NaOH, the volume was completed with phosphate

buffer (pH 2.3), and the final solution mixed thoroughly. The final concentration of uric acid was

100 μg/mL.

Standard solutions of uric acid: From the uric acid stock solution, six different

concentrations of standard solutions of uric acid and three quality controls (QC) were prepared in

phosphate buffer pH 2.3 according Table 4-1. All solutions were filtered through a 0.45 μm

PVDF membrane filter (Millipore Corp.) before analysis.

Animals and Experimental Protocols

Animals

Male Sprague Dawley rats (250-350 g.) were purchased from Harlan (IN, USA) and

divided into the experimental groups; containing 8-10 rats per group. They were housed in

plastic cages. They were allowed one week to adapt to their environment before used for

experiments. All the animals were maintained on a 12hr/12hr light/dark cycle with light on at 6

am. They were given standard chow and water ad libitum during the course of the study. All

animal experiments were performed according to the policies and guidelines of the Institutional

Animal Care and Use Committee (IACUC) of the University of Florida, Gainesville, USA (NIH

publication # 85-23).

69

Animal model of hyperuricemia in rats

Potassium oxonate (250 mg/kg) was injected intraperitoneally as a suspension in 0.8 %

carboxymethyl cellulose sodium salt (CMC-Na) 1 h before orally or intraperitoneal

administration of tested compounds as described as follows. [97, 98] The animals were

anaesthetized with halothane and blood samples (1000 μL) were taken from the sublingual vein

1 h. after compound administration. After the blood collection, approximately 1000 μL of

isotonic saline were given intraperitoneally in order to maintain the blood fluid. The blood was

allowed to clot for approximately 1 h. at room temperature and then centrifuged at 2800 x g for

15 min. to obtain the serum which was stored at -20°C until use.

Drug Administration:

1. Oral administration

Artichoke leaf extract: artichoke extract (250, 500, 1000 mg/kg) and allopurinol (50

mg/kg) were dissolved in 0.8% CMC-Na. The control groups received 0.8% CMC-Na. The

volume of the drug administered to each rats was based on the body weight of the animal

measured immediately prior to each dose. The extract and allopurinol were administered orally 1

h after the administration of potassium oxonate. For chronic treatment, test samples were given

orally once daily for 1, 3, 5, and 7 days.

Flavonoids: Luteolin (16, 32, 50, 100 mg/kg: test the optimum dose), other flavonoids

(50, 100 mg/kg) and allopurinol (50 mg/kg) were administered orally at one hour after potassium

oxonate. Flavonoids and allopurinol were suspended in 1:1 (propylene glycol (PG): water). The

control groups received 1:1 (PG: water). The volume of the drug administered to each rats was

based on the body weight of the animal measured immediately prior to each dose.

70

2. Intraperitoneal administration

Artichoke (500 mg/kg), caffeoylquinic acids (50mg/kg) such as caffeic acid, chlorogenic

acid, cynarin, and flavonoids (50mg/kg) such as luteolin, apigenin, quercetin and allopurinol

(50mg/kg) were administered intraperitoneal injection (i.p.) at one hour after potassium oxonate.

Artichoke extract were dissolved in 0.8% CMC-Na. Other test samples were suspended in 1:1

(PG: water). The control groups received 1:1 (PG: water).

Uric Acid Assay

Uric acid in rat serum was determined by reversed-phase high performance liquid

chromatography and photodiode array detection (DAD) [99] [100].Standards were prepared by

diluting the stock uric acid solution with 200 mM phosphate buffer (0.1 mg/mL). The stock

solution was prepared as follows. Uric acid has low solubility in water. Therefore, an aliquot

(20.0 mL) of a 0.25 N NaOH was dropped into 20.0 mg of uric acid. Sonicate shortly and fill up

with buffer to 200.0 mL. Samples were analyzed using a reverse-phase partition mode of HPLC

with diode array detector A Shimadzu VP series HPLC system (Kyoto, Japan) equiped with an

SPD-M10Avp diode array detector was used for this work. A Lichrospher® 100 RP-18 (5μm.

Merck KgaA) was used for the separation of uric acid. The column temperature was kept at

25oC. The mobile phase was 200 mM phosphate buffer (NaH2PO4, pH 2.0). The flow rate was

0.5 mL/min. Ten micro liters of each sample was injected into the RP-HPLC system. Comparing

the respective peak area in the chromatogram with the value from a standard calibration curve

quantitated uric acid.

Preparation of Rat Serum

The proteins in rat plasma were precipitate by adding 150.0 μL of 10% trichloroacetic acid

to 150.0 μL of plasma and adding 750.0 μL of buffer to make 1 mL. The precipitates were

71

removed from the mixture by centrifugation at 3,000 g for 3 min. Supernatants were filtered

through 0.45 μm filters and ten micro liters of the plasma sample were injected into the RP-

HPLC with photodiode array detector system.

Statistical Analysis

All data are expressed as the mean ± SEM. Group mean differences were ascertained with

analysis of variance (ANOVA). Multiple comparisons among treatment means were checked

with the Tukey’s test. The results were considered significant if the probability of error was <

0.05

Validation

The method was validated over the range of concentration of uric acid present in serum.

The validation parameters of linearity, sensitivity, specificity, precision, accuracy and stability

were determined.







limit of quantification for the assay was defined as the minimum concentration of quality

controls. The calibration was considered suitable if not more than 1/3 of the quality control

standards showed a deviation from the theoretical values equal or greater than 15%, except at the

lower limit of quantification (LLOQ), where it should not exceed 20%.

72

Results

Validation of Analytical Method to Measure Uric Acid in Rat Serum.

Linearity

Calibration curves (n = 9) operating in the range of 2-50 μg/mL for uric acid were linear

(r2> 0.999) (Figure 4-1).

Sensitivity


quality control. This concentration would be acceptable with the precision (%CV < 20), and

accuracy (%error < 20).The LLOQ of uric acid in plasma was 0.1 μg/mL.

Specificity

The method provided good resolutions between uric acid and interference in serum. Peak

of uric acid had similar retention times to the standard. The method showed good specificity

since the chromatograms of the serum samples did not show any co-eluting peak with similar

retention time as uric acid as shown in Figure 4-2.

Precision, accuracy and recovery

The precisions intra- and inter-day for uric acid were satisfactory with CV values between

1.2 and 12.3%. Similarly, the accuracy of the assay obtained with quality control samples

containing 3, 12, and 35 μg/mL uric acid was between 91.9 and 109.1% of the nominal values.

The mean recovery assessed at three distinct levels of concentration (1, 10 and 20 μg/mL) ranged

from 92.8 to 94.7% of the expected values. The results are summarized in Table 4-2.

Stability

Uric acid was stable under the tested conditions. The mean % remaining in rat serum after

2 hours at room temperature was 98.1± 6.2, 100.4 ± 5.7, 100.3 ± 2.8 for the low, medium and

73

high concentrations, respectively. Uric acid was stable on autosampler at 20 oC within 24 hours

(Table 4-3).

Effect of Artichoke Extract and Its Compounds on Serum Urate Levels in Hyperuricemic Rats

Oral administration of artichoke in acute treatment

Uricase inhibitor potassium oxonate treatment showed hyperuricemia in rats, as indicated

by increased in serum uric acid levels from 9.76 to 33.40 μg/mL (Table 4-4 and Figure 4-3).

Artichoke water extracts did not affect the serum uric acid level after 1 h treatment. In contrast,

allopurinol (50 mg/kg) lowered the uric acid levels in hyperuricemic rats.

Oral administration of artichoke in chronic treatment

The hypouricemic effects of the orally administered artichoke water extracts on serum uric

acid levels in oxonate-petreated rats are shown in Table 4-5 and Figure 4-4. After day 1, 3, 5 and

7 treatment, when compared with that of the hyperuricemic control group, artichoke water

extract did not show effect on serum uric acid levels. In contrast, allopurinol (50 mg/kg) lowered

the uric acid levels in hyperuricemic rats after day 1, 3, 5 and 7 treatments.

Oral administration of compounds in artichoke and various flavonoids in acute treatment

Potassium oxonate treatment caused hyperuricemia in rats, as indicated by increase in

serum uric acid levels. As shown in Table 4-6, Figure 4-5, Figure 4-6 and Figure 4-7, luteolin at

dose 16 and 32 mg/kg did not show a decrease in serum urate levels compared with the control

group. Therefore, the higher doses such as 50 mg/kg and 100 mg/kg were performed for all

flavonoids. The results showed that at 50 and 100 mg/kg, apigenin, eriodictyol, luteolin, luteolin-

7-O-glucoside, naringenin, and quercetin did not affect the serum uric acid level as shown in

Table 4-6, Figure 4-6, and Figure 4-10. In contrast, the reference drug allopurinol (50 mg/kg)

significantly lowered the uric acid levels in hyperuricemic rats.

74

Intraperitoneal administration of artichoke, compounds in artichoke and various flavonoids in acute treatment

The hypouricemic effects of the intraperitoneal treatment of artichoke water extracts,

apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin and quercetin on serum uric

acid levels in oxonate-pretreated rats are shown in Table 4-7 and Figure 4-8. After 1 h treatment,

when compared with that of the hyperuricemic control group, none of the test samples showed

effect on serum uric acid levels. In contrast, allopurinol (50 mg/kg) was active in this

experiment.


Our previous data showed that flavonoids could inhibit the formation of uric acid from

xanthine by XO in vitro. Thus, there is a possibility that the flavonoids may inhibit the XO in

vivo which results in the decrease of uric acid levels in plasma. Uricase inhibitor such as

potassium oxonate is needed to perform the experiment in rats. Potassium oxonate is an inhibitor

of uricase. An i.p. injection of oxonate could partially block the conversion of uric acid to

allantion and thus artificially elevate the plasma uric acid level in rats to provide a hyperuricemic

animal model [101, 102]. However, in the present study, the in vivo experiments demonstrated

that artichoke leaf extract (250,500 and 1000 mg/kg) and flavonoids (50 and 100 mg/kg) could

not exert hypouricemic in the oxonate-induced rats after 1 h oral administration. This lack of

effect via the oral route might be due to the first pass metabolism through gut, intestine or liver

as reported in previous studies. In vitro experiments demonstrated that 74% of luteolin was

conjugated to glucuronic acid after incubation with microsomal samples from human intestine

[96]. Wittemer et al. [30] had shown that after oral administration of artichoke leaf extract in

human, none of the genuine extract constituents such as caffeoylquinic acids (e.g. chlorogenic

acid, cynarin), caffeic acid and flavonoids (e.g. luteolin-7-O-glucosides) could be detected in

75

plasma and urine, however, the metabolites in the form of glucuronides and sulfates were

observed instead. Chen et al. [95] reported the systemic bioavailability of quercetin and quercetin

conjugates as 5.3% and 55.8%, respectively in rats after administration of quercetin.

Additionally, about 93.3% of quercetin was metabolized in the gut, with only 3.1% metabolized

in the liver.

Beside the first pass effect, the absorption of the compound should also be considered. In

humans, peak plasma concentrations of total luteolin and total caffeic acid were reached within

0.5 h and 1 h , respectively, after a single oral dose of artichoke leaf extract (153.8 mg) [30].

After the flavonol administration in human, the peak in blood occurred at approximately 2.9 h

[103]. In rats, flavonoids seemed to appear more rapidly. Luteolin appeared in plasma 15 min

after given via gastric intubation [104]. Apigenin occurred in plasma 30 min after intraperitoneal

administration in rats [105]. These results from the literature suggested a relatively rapid

absorption of flavonoids. Thus, in our study, most of flavonoids might be metabolized and

excreted after 1 h oral administration of flavonoids.

It has been reported that intraperitoneal administration of some flavonoids such as

apigenin, kaempferol, naringenin and rutin significantly reduced small and large intestinal transit

in mice [106]. The compounds via oral route have both intestinal absorption and first pass effect

through the liver. Thus, the amount of active compounds that appear via oral route may be higher

than those via intraperitoneal route because of different absorption. In our study, after i.p.

injection of artichoke leaf extracts (500 mg/kg), flavonoids (50 mg/kg) and caffeoyl quinic acids

(50 mg/kg) to the hyperuricemic rats, they did not elicit any significant hypouricemic effect. This

lack of effect via intraperitoneal route might be due to small and large intestinal transit reduction

and first pass metabolism through liver.

76

Jiménex-Escrig et al. [8] found a 55 % reduction of plasma urate levels in rats fed with a

diet containing the edible part of artichoke (∼138 g/kg diet) for 3 weeks. The differences

observed between our study and this study could be explained by the different part of artichoke

(heads of artichoke), the different species of rats (Wistar rats) and the different formulation of

artichoke (a diet containing artichoke). Zhu et al. [66] reported a hypouricemic effect in oxonate-

pretreated mice after a three-time pretreatment of quercetin and rutin (100 mg/kg). The test

compounds were dissolved in propyleneglycol/water (50/50). Yu et al. [107] showed a decrease

of plasma urate levels after 5 h administration of morin (80 mg/kg) in hyperuricemic rats. Morin

was prepared in 0.3% Tween 20. The differences observed between these studies are the species

of animals and the solvent used to prepare test samples.

In conclusion, the data reported in this study indicated that oral and intraperitoneal

administration of artichoke leaf extract, flavonoids and caffeolyquinic acids could not reduce

serum urate levels of hyperuricemic rats induced by oxonate. This lacks of effect might be due to

first pass metabolism through gut, intestine or liver for the oral route and due to small and large

intestinal transit reduction and first pass metabolism through liver for intraperitoneal route.

Additionally, blood collection after 1 h treatment may not be a right time point since

caffeoylquinic acids and flavonoids have been reported to have rapid absorption and metabolite.

Therefore, the pharmacokinetics of flavonoids and caffeoylquinic acids should be further

studied.

77

Table 4-1. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of uric acid

Standard Uric acid stock solution (mL)

Fill volume up in volumetric flask (mL) Concentration (μg/mL)

1 0.20 10 22 0.25 5 53 1 10 104 0.75 5 155 1 5 206 5 10 50QC1 0.3 10 3QC2 1.2 10 12QC3 3.5 10 35

78

Table 4-2. Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of uric acid in rat serum. Precision expressed as CV%, accuracy and recovery as % of the theoretical concentration

Intra-day QC1 – 3 μg/mL QC2 – 12 μg/mL QC3 – 35 μg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Precision 11.4 12.3 10.1 5.7 1.2 8.7 3.5 5.4 4.6Accuracy 94.7 91.9 94.5 100.8 95.3 98.7 109.1 100.6 102.3Inter-day QC1 – 3 μg/mL QC2 – 12 μg/mL QC3 – 35 μg/mL Precision 6.5 3.4 3.1 Accuracy 94.3 100.4 108.8 Recovery Uric acid – 1 μg/mL Uric acid – 10 μg/mL Uric acid – 20 μg/mL % 92.8 94.1 94.7 CV% 10.2 5.4 5.5

79

Table 4-3. The stability test after 24 hours on autosampler at 20oC. Data represents the percentage remaining of uric acid

% Remaining on autosampler Luteolin concentration 12 hours 24 hours

QC1-3 μg/mL 95.3 ± 6.5 98.6 ± 10.2QC2-12 μg/mL 101.5 ± 3.6 100.5 ± 5.2QC3-35 μg/mL 106.0 ± 2.4 106.7 ± 3.1

80

Table 4-4. Hypouricemic effects of allopurinol, water extract of artichoke on plasma urate levels (μg/mL) in oxonate-pretreated rats in acute treatment

Treatment groups Animals Dosage of drugs (mg/kg)

Serum Urate levels ( ug/ml) ± SEM

Normal rats 10 - 9.76 ± 0.95

Hyperuricemia rats dosed with vehicle (0.8% CMC-Na)

10 - 33.40 ± 1.64

Hyperuricemia rats dosed with artichoke extract

10 250 30.56 ± 0.77

10 500 32.02 ± 1.25

10 1000 32.34 ± 1.90

Hyperuricemia rats dosed with allopurinol

10 50 6.26 ± 0.35*

Note: Hyperuricemia was induced by injecting potassium oxonate. They were then orally given artichoke extract, or allopurinol at different doses. Data represent mean value (± SEM) of plasma urate level (μg/mL) in animals groups (n = 10). For statistical significant,* indicates P < 0.001 when the compounds-treated animals were compared with the hyperuricemic rats without drug treatment (vehicle controls).

81

Table 4-5. Hypouricemic effects of allopurinol and artichoke extract on plasma urate levels (μg/mL) in oxonate-pretreated rats after chronic treatment

Duration of drug treatment (days) Treatment groups N Dosage (mg/kg) 1 3 5 7

Normal rats 8 - 15.06 ± 0.77 18.95 ± 0.83

11.52 ± 0.83

14.82 ± 1.49

Hyperuricemia rats dosed with vehicle

8 - 29.94 ± 0.87 28.73 ± 1.38 26.22 ± 1.39 28.75 ± 0.73

Hyperuricemia rats dosed with artichoke extract

8 500 33.38 ± 1.19 27.87 ± 0.65 25.94 ± 1.34 29.67 ± 1.37

8 1000 31.35 ± 1.05 26.00 ± 1.53 24.57 ± 2.08 26.93 ± 1.22

Hyperuricemia rats dosed with allopurinol

8 50 9.02 ± 0.37* 9.31 ± 0.70* 5.89 ± 0.34* 6.10 ± 0.8*

Note: Hyperuricemia was induced by injecting potassium oxonate. They were then orally given artichoke or allopurinol at different doses. Data represent mean value (± SEM) of plasma urate level (μg/mL) in animals groups (n = 8). For statistical significant, * indicates P < 0.001 when the compounds-treated animals were compared with the hyperuricemic rats without drug treatment (vehicle controls).

82

Table 4-6. Hypouricemic effects of allopurinol, apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin, quercetin on plasma urate levels (μg/mL) in oxonate-pretreated rats after oral administration


Serum Urate levels (ug/mL) mean ± SEM

Normal rats 8 - 14.58 ± 0.97Hyperuricemia rats dosed with vehicle (PG:water,50:50)

8 - 28.87 ± 1.22

Luteolin 8 16 26.21 ± 1.28 8 32 33.14 ± 3.42 8 50 27.93 ± 1.55 8 100 27.09 ± 0.81Luteolin-7-O-glucoside 8 50 30.54 ± 1.34 8 100 29.75 ± 2.12Apigenin 8 50 32.90 ± 2.56 8 100 33.99 ± 1.72Eriodictyol 8 50 27.50 ± 0.64 8 100 33.51 ± 2.05Kaempferol 8 50 29.77 ± 0.91 8 100 27.54 ± 2.17Naringenin 8 50 27.82 ± 1.14 8 100 30.45 ± 1.80Quercetin 8 50 27.28 ± 0.84 8 100 27.84 ± 2.74Allopurinol 8 50 8.53 ± 2.091*Note: Data represent mean value (± SEM) of plasma urate level (μg/mL) in animals groups (n =

8). For statistical significant, * indicates P < 0.001 when the compounds-treated animals were compared with the hyperuricemic rats without drug treatment (vehicle controls)

83

Table 4-7. Hypouricemic effects of allopurinol, apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin, quercetin on plasma urate levels (μg/mL) in oxonate-pretreated rats after i.p injection


Serum Urate levels (ug/mlL) mean ± SEM

Normal rats 5 - 11.06 ± 0.84 Hyperuricemia rats 5 - 31.14 ±1.65 Artichoke 5 500 27.32 ± 2.11 Caffeic acid 5 50 35.21 ± 3.02 Chlorogenic acid 5 50 33.86 ± 3.15 Cynarin 5 50 35.11 ± 4.05 Apigenin 5 50 34.06 ± 2.89 Quercetin 5 50 26.39 ± 0.92 Luteolin 5 50 27.60 ± 2.17 Allopurinol 5 50 9.12 ± 1.39*Note: The hyperuricemic rats were produced by potassium oxonate pretreatment. They were then

intraperitoneally given of artichoke leaf extracts (500 mg/kg), 50mg/kg of allopurinol, apigenin, eriodictyol, luteolin, luteolin-7-O-glucoside, naringenin and quercetin. Data represent (mean ± SEM) of plasma urate level (μg/ml) in animals groups (n = 5). For statistical significant, * indicates P < 0.001 when the compounds-treated animals were compared with the hyperuricemic rats without drug treatment (vehicle controls)

84

0 10 20 30 40 50 600

5.0×105

1.0×106

1.5×106

2.0×106

2.5×106

3.0×106

3.5×106

4.0×106

4.5×106

Uric acid [μg/mL]

Are

a

Figure 4-1. Mean calibration curves (n = 9) of uric in serum. Vertical bars represent the standard deviations (SD) of the means.

Y = 82480 X + 6314 r2 = 0.9990

85

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mVo

lts

-1

0

1

2

3

4

5

6

7

8

9

10

Figure 4-2. HPLC chromatogram of uric acid in serum.

Uric acid

86

Normal Control 250 500 1000 Allopurinol0

10

20

30

40

*

Uri

c ac

id in

pla

sma

(ug/

ml)

Figure 4-3. Acute effects of allopurinol, artichoke extract on serum urate levels in rats pretreated

with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before administration of artichoke and allopurinol (50 mg/kg).The data represent the mean ± SEM for 10 animals. * P < 0.001; significant from the control.

87

Day1

0

5

10

15

20

25

30

35

*

Uri

c ac

id in

seru

m(u

g/m

l)Day 3

0

5

10

15

20

25

30

35

*

NormalControlAllopurinol 50 mg/kgArtichoke500mg/kgArtichoke 1000 mg/kg

Uri

c aic

d in

pla

sma

(ug/

ml)

Day 5

0

5

10

15

20

25

30

35

*

Uri

c aci

d in

seru

m(u

g/m

l)

Day 7

0

5

10

15

20

25

30

35

*

NormalControlAllopurinol 50 mg/kgArtichoke500mg/kgArtichoke 1000 mg/kg

Uri

c aci

d in

seru

m (u

g/m

l)

Figure 4-4. Chronic effects of allopurinol, artichoke extratc on serum urate levels in oxonate-treated rats. Rats were treated with potassium oxonate (250 mg/kg) before administration of artichoke (500, 1000 mg/kg) and allopurinol (50 mg/kg) for 1, 3, 5 and 7 days. The data represent the mean ± SEM for 8 animals. * P < 0.001: significantly from the control.

88

Normal Control Allopurinol 16 32 50 1000

10

20

30

40

Luteolin (mg/k g)

*

Uri

c ac

id le

vels

in se

rum

(μg/

ml)

Figure 4-5. Effects of allopurinol and luteolin on serum urate levels in rats pretreated with the

uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before oral administration of luteolin (16, 32, 50, 100 mg/kg) and allopurinol.The data represent the mean ± SEM for 8 animals. *P < 0.001: significantly from the control.

89

Normal Control Allopurinol Apigenin L-glc Kaempferol Quercetin Eriodictyol Naringenin0

10

20

30

40

*

Uri

c ac

id le

vels

in se

rum

(μg/

ml)

Figure 4-6. Effects of allopurinol, apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, and quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before oral administration of 50 mg/kg of apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, quercetin and allopurinol. The data represent the mean ± SEM for 8 animals. * P < 0.001: significantly from the control.

90

Normal Control Allopurinol Apigenin L-glc Kaempferol Quercetin Eriodictyol Naringenin0

10

20

30

40

*

Uri

c ac

id le

vels

in se

rum

(μg/

ml)

Figure 4-7. Effects of allopurinol, apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before oral administration of 100 mg/kg of apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, quercetin and allopurinol (50 mg/kg).The data represent the mean ± SEM for 8 animals. * P < 0.001: significantly from the control.

91

Normal Control Artichoke Allopurinol Caffeic acid Chlorogenic Cynarin Apigenin Quercetin Luteolin0

10

20

30

40

*

Uri

c ac

id le

vels

in se

rum

( μg/

ml)

Figure 4-8. Effects of artichoke extract, allopurinol, caffeic acid, chlorogenic acid, cynarin,

luteolin, apigenin and quercetin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before i.p. injection of artichoke (500 mg/kg), and 50 mg/kg of allopurinol, caffeic acid, chlorogenic acid, cynarin, luteolin, apigenin and quercetin. The data represent the mean ± SEM for 8 animals. * P < 0.001: significantly from the control.

92

CHAPTER 5 THE EFFECT OF ARTICHOKE LEAF EXTRACT AND ITS COMPOUNDS ON

ANTIOXIDANT ACTIVITY IN VITRO AND IN RATS

Background

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide and singlet

oxygen are implicated in some diseases such as inflammation, cancer, ageing, and degenative

diseases [108]. ROS are common products of several oxidative systems, especially the xanthine-

xanthine oxidase system. Xanthine oxidase is an important enzyme which catalyses the oxidation

of hypoxanthine to xanthine and then to uric acid in man. The accumulation of uric acid can not

only lead to gout and hyperuricemia, but can also provoke inflammation by various mechanisms

such as an neutrophil recruitment and the release of leukotriene B4, interleukin-1 (IL-1),

interleukin-2 (IL-2) and superoxide [109, 110]. Therefore, the compound that can scavenge free

radicals could have a beneficial effect not only in the treating of gout and hypreuricemia, but also

in the alleviation of inflammation.

Artichoke leaves (Cynara scolymus L.) is a good source of natural antioxidants since major

compounds in artichoke leaves are polyphenolic compounds with mono- and dicaffeoylquinic

acids and flavonoids. Artichoke leaf extract has been reported to show antioxidative and

protective properties against hydroperoxide-induced oxidative stress in cultured rat hepatocytes

[6], to protect low density lipoprotein from oxidation in vitro [9], to inhibit hemolysis induced by

hydrogen peroxide and to inhibit oxidative stress when human leucocytes are stimulated with

agents that generate reactive oxygen species such as hydrogen peroxide [16]. The phenolic

compounds in artichoke have been reported to show antioxidant activity in vitro [24]. However,

only one study reported an effect of the edible part of artichoke on biomarkers of antioxidants in

rats [8]. Therefore, in vivo studies of artichoke leaves on antioxidant activity should be

performed.

93

In this study, the in vitro antioxidant properties of major compounds in artichoke extract

(caffeoylquinic acids and flavonoids) and some reference flavonoids (quercetin) as shown in

Figure 5-1 were investigated. Moreover, the effect of the intake of artichoke extract, and its

components for 2 h and 3 weeks on total antioxidant activity and antioxidant enzyme glutathione

peroxidase in plasma of male rats were evaluated.

Specific Aims

Investigate whether artichoke extract and its compounds show antioxidant activity in vitro

and in rats.


Materials

Artichoke leaf extract was obtained from a German extract manufacturing company

(Finzelberg, Andernach, Germany). Dihydrocaffeic acid (90-95%), luteolin (99%), and luteolin-

7-O-glucoside (> 90%) were purchased from Indofine Chemical Company, Inc. (Somerville, NJ,

USA). Chlorogenic acid (≥ 95%), dihydrate (> 98%), dipotassium hydrogenphosphate

(K2HPO4), sodium dihydrogenphosphate (NaH2PO4), and AAPH (2, 2’-Azobis (2-

amidinopropane) dihydrochloride were purchased from Sigma Chemical Company (St. Louis,

MO, USA). Cynarin was purchased from Carl Roth GmbH+Co. (Germany). Fluorescein was

purchased from Fluka (Milwaukee, WI, USA). Acetonitrile (CH3CN), methanol, perchlorogenic

acid (PCA), and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Fair Lawn,

NJ, USA). Trolox 97% (6-Hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) (Biomol,

PA, USA), were used. All aqueous solutions were prepared with purified water obtained from a

NANOPure® system from Barnstead (Dubuque, IA, USA). Fluorescence filters with an

excitation wavelength of 485 nm and emission wavelength of 538nm were used. The microplate

94

reader (Synergy HT) was purchased from Bio-TEK® (Wincoski, VT, USA). The 96 well-plates

(Corning) were puchased from Fisher Scientific (Fair Lawn, NJ, USA).

Animals

Male Sprague Dawley rats (250-350 g) were purchased from Harlan (IN, USA) and

divided into the experimental groups; containing 8 rats per group. They were housed in plastic

cages. They were allowed one week to adapt to their environment before used for experiments.

All the animals were maintained on a 12h/12h light/dark cycle. They were given standard chow

and water ad libitum during the course of the study. All animal experiments were performed

according to the policies and guidelines of the Institutional Animal Care and Use Committee

(IACUC) of the University of Florida, Gainesville, USA (NIH publication # 85-23).

Acute treatment

Artichoke extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg), and quercetin (25 mg/kg)

was administered orally. Artichoke was suspended in 0.8% carboxyl methylcellulose sodium salt

(CMC-Na). The control groups received 0.8% CMC-Na. The volume of the drug administered to

each rats was based on the body weight of the animal measured immediately prior to each dose.

Rats were anaesthetized with halothane and 500 μL blood samples were taken from sublingual

vein at 2 h into tubes containing sodium heparin 900 units. Each tube was centrifuged at 2800 x

g for 15 min to obtain the plasma which was stored at -80°C until use for antioxidant analysis.

Chronic treatment

Artichoke extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg), and quercetin (25 mg/kg)

were administered orally once a day for 21 days. Artichoke and flavonoids were suspended in

0.8% carboxyl methylcellulose sodium salt (CMC-Na). The control groups received 0.8% CMC-

Na. 1000 μL blood samples were taken from sublingual vein on day 0, 7, 14 and 21. Prior to

95

blood collection, rats were anaesthetized with halothane and blood loss was replaced with an

equal volume of normal saline. Blood samples were treated in the same way as acute treatment.

Plasma samples were used for antioxidant activity, uric acid concentration and glutathione

peroxidase activity.

Assessment of Antioxidative Capacity in Vitro and Plasma Antioxidant Status

ORAC assays were carried out on a synergy HT plate reader (Biotex, USA) with

fluorescence filters (excitation wavelength: 485 nm, and emission filter: 538 nm).The

temperature of the incubator was set to 37 oC. Procedures were based on the previous report by

Ou et al.[111]. Briefly; AAPH was used as peroxyl generator and Trolox as a control standard.

50.0 μL of sample, blank, and Trolox calibration solutions were transformed to 98-well

microplates in triplicate. 100.0 μL of fluorescene solution were added and then 50 μL of AAPH

solution were added immediately before reading in microplate reader. Fluorescence reading were

taken every 10 min for a duration of 70 min. Final results were calculated based on the

difference in the area under the fluorescein decay curve between the blank and each sample.

Artichoke extracts 10.0 mg were dissolved in 10.0 mL of phosphate buffer pH 7.0 and then

dilute in a ratio of 1 to 100 with phosphate buffer; phenolic compounds were dissolved in DMSO

and then diluted with phosphate buffer pH 7.0. The concentration of DMSO was less than 0.1 %

for in vitro study. ORAC values were expressed as relative Trolox equivalents in respect to 25

μM of phenolic compounds and expressed as μmol Trolox quivalent (TE)/ g of artichoke

extracts.

Assessment of Uric Acid in Plasma

Uric acid was measured by reversed-phase high performance liquid chromatography and

photodiode array detection (DAD). Standards were prepared by diluting the stock uric acid

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solution with 200 mM phosphate buffer (0.1 mg/mL). The stock solution was prepared as

follows. Uric acid has low solubility in water. Therefore, a small amount of 0.25 N NaOH was

added into uric acid. Sonicated shortly and filled up with buffer. The column temperature was

kept at 25 oC. The mobile phase was 200 mM phosphate buffer (NaH2PO4, pH 2.0). The flow

rate was 0.5 mL/min. Ten micro liters of each sample was injected into the RP-HPLC system.

Comparing the respective peak area in the chromatogram with the value from a standard

calibration curve quantitated uric acid.

The proteins in rat plasma were precipitated by adding 150.0 μL of 10% tricholoacetic acid

to 150.0 μL of plasma and adding 700.0 μL of buffer to make 1 mL. The precipitates were

removed from the mixture by centrifugation at 3,000 g for 3 min. Supernatants were filtered

through 0.45 μm filters and ten micro liters of the plasma sample were injected into the RP-

HPLC with photodiode array detector system and measured at a wavelength value 285 nm.

The plasma nonprotein fraction was prepared by diluting plasma with 0.5 M perchloric

acid (PCA) (1:1, v/v). The samples were vortexed for 15 sec and centrifuged at 4000 rpm for 10

min at 4° C. Then, the supernatant was removed as the plasma nonprotein fraction, and diluted in

a ratio of 1 to 20 with phosphate buffer pH 7.0 for the analysis. All plasma samples were

assessed within 1 week after blood drawing. ORAC values were expressed as mmol Trolox

equivalents per liter.

Assessment of Glutathione Peroxidase (GPx) in Plasma

GPx activity was determined by using a glutathione peroxidase assay kit. (Cayman

chemical company, Ann Arbor, MI, USA)

97


All data are expressed as the mean ± SEM Group mean differences were ascertained with

analysis of variance (ANOVA). Multiple comparisons among treatment means were checked

with the Tukey’s test. The results were considered significant if the probability of error was <

0.05

Results

Antioxidant Activity in Vitro

The antioxidant activities of artichoke extract and phenolic compounds were estimated by

ORAC assay as shown in Table 5-1. One gram of artichoke extract had 1623.35 μmol of Trolox

equivalent. 1, 3-di-O-caffeoylquinic acid (cynarin), quercetin and luteolin showed the strongest

antioxidant activity with 6.73, 5.30 and 5.16 relative Trolox equivalent in vitro, respectively

(Table 5-2).

Plasma Antioxidant Activity in Vivo

Acute treatment

There was no significant difference in the plasma antioxidant activity between artichoke

group and the control group after orally treatment of artichoke (500, 1000 mg/kg), luteolin (25,

50 mg/kg) and quercetin (25 mg/kg) for 2 h as shown in Table 5-3.

Chronic treatment

After orally administration of 500 and 1000 mg/kg of artichoke extract for 21 days,

there was no significant difference in the plasma antioxidant activity between the artichoke

groups and the control groups. 25 and 50 mg/kg of luteolin and 25 mg of quercetin also did not

showed antioxidant activity in vivo as shown in Table 5-4.

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Plasma Urate Concentrations and Plasma Glutathione Peroxidase Activity after The Treatment with Artichoke Extract and Phenolic Compounds

There were no significant difference in the plasma urate concentrations or glutathione

peroxidase activity between the experiment groups and the control group after orally

administration of 500 and 1000 mg/kg of artichoke extract, 25 and 50 mg/kg of luteolin and 25

mg of quercetin over a 21 days period as shown in Table5-5 and Table 5-6.


The in vitro antioxidant activites were tested by using the ORAC assay, which evaluates

the radical scavenging activity of the test samples towards peroxyl radicals generated through the

thermal decomposition of a radical initiator (AAPH). Table 5-1 and Table 5-2 summarized the

results expressed as μmol of Trolox equivalents/ g of arichoke extract and relative Trolox

equivalents for caffeic acid derivatives and flavonoids. It showed that the extract and all the

compounds were found to be more active than Trolox.This result is consistant with previous

studies. Ou et al. [111] found that caffeic acid, chlorogenic acid and quercetin showed high

relative ORAC values. Wang et al. [24] measured the relative antioxidant activities (% inhibition

of DPPH free radicals) of phenolic compounds and found that cynarin, cynaroside, luteolin-7-

rutinoside and chlorogenic acid showed high antioxidant activities.

The antioxidant activities of phenolic compounds have been reported to be largely

determined by the number of hydroxyl groups on the aromatic ring and the position of the

substituents. The higher the number of hydroxyl groups, the greater the antioxidant activity. In

addition, the presence of a catechol group in phenolic ring also increases the antioxidant activity

[24]. Our results are in agreement with this report (Figure 5-1). Cynarin, with two adjacent

hydroxyl groups on both phenolic rings showed the highest antioxidant activity. Quercetin,

luteolin and luteolin-7-O-glucoside with two adjacent hydroxyl groups on one ring and only a

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single hydroxyl group on the other ring showed less antioxidant activity, which was still higher

than chlorogenic acid, caffeic acid and dihydrocaffeic acid with two adjacent hydroxyl groups on

one ring. Artichoke leaf extract contains caffeoylqunic acids and flavonoids, thus, the antioxidant

activity of the extract was high (1623.36 ± 2.84 μmol TE/ g of dry extracts).

In vivo antioxidant activity showed that the acute and chronic treatment of artichoke

extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg) and quercetin (25 mg/kg) could not increase

the total antioxidant activity in rats plasma. In addition, the chronic treatment did not lead to an

increase in the value of glutathione peroxidase (a marker of antioxidative defense), and in the

uric acid levels (endogenous antioxidant compound) in plasma. This lack of effect might be due

to the low absorption of caffeoylquinic acids and flavonoids. In human study, the maximal

plasma concentrations of flavonoids, reached between 1 and 3 h after consumption of flavonoid

–rich food, is between 0.06 and 7.6 μM for flavonols, flavanols and flavanones [112]. In rats, the

concentration of luteolin at 30 min was 15.5 ± 3.8 nmol/mL after administration of one single

dose of luteolin [104]. The maximum concentration of total caffeic acids and luteolin, reached at

0.83 and 0.36 h, respectively, were 59.07 and 6.51 ng/mL after consumption of artichoke leaf

extract (107 mg) [30]. In addition, the half-lives of flavonoids in human plasma are short, usually

in a range of a few hours [112]. In rats, after administration of artichoke extract (107 mg/kg), the

half-lives of total caffeic acid and luteolin were 3.08 and 2.50 h. These factors limit the

capability of dietary flavonoids to act as antioxidant in plasma in vivo. Chronic consumption of

flavonoid-rich foods does not result in the significant increase of amounts of flavonoids in

plasma. For example, the concentrations of quercetin at the steady-state in human plasma are less

than 1 μM [113].

100

Besides the poor absorption, caffeolyquinic acids and flavonoids are highly metabolized in

the intestine and liver. Flavonoids and caffeic acid are good substrates of phase II enzymes and

can be metabolized to glucuronidation, methylation and sulfation [31, 96, 114, 115]. These

biotransformations affect the physical properties of flavonoids, making them more water soluble

and may affect their antioxidant activity. Flavonoid metabolites generally are less potent

antioxidants than their parent compounds because of the modification of their catechol and

phenol group [47, 48, 116, 117]. Furthermore, the major part of ingested flavonoids is not

absorbed and is largely degraded by the intestinal microflora [118]. The breakdown products

may have antioxidant or non-antioxidant activities [119, 120].

More over, our in vitro preliminary study demonstrated that a plasma concentration higher

than 1 μg/mL of luteolin and quercetin was required to increase the antioxidant activity in

plasma above base line using the ORAC assay. Therefore, it might be possible that the maximum

plasma concentrations of luteolin and quercetin are below the plasma concentration necessary to

expect an increase in the antioxidant activity.

In conclusion, the in vitro antioxidant activity of artichoke and its compounds could not be

confirmed in a rat model. This lack of effect might be due to the low bioavailabilty of

caffeoylquinic acids and flavonoids. Therefore, the pharmacokinetics study of these compounds

should be performed.

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Table 5-1. ORAC values of artichoke extract Sample μmol TE/g artichoke extract mean ± SEMArtichoke extract 1623.36 ± 2.84Note: ORAC values are expressed as micromole of Trolox equivalent per gram.

102

Table 5-2. Relative ORAC values of pure chemicals with antioxidant activity Compound Relative Trolox Equivalent mean ± SEMCaffeic acid 3.48 ± 0.08Dihydrocaffeic acid 2.83 ± 0.06Chlorogenic acid 1.83 ± 0.22Cynarin 6.73 ± 0.06Luteolin 5.16 ± 0.05Luteolin-7-O-glucoside 4.35 ± 0.13Quercetin 5.30 ± 0.03Note: ORAC values are expressed as relative Trolox equivalent.

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Table 5-3. ORAC values of plasma samples Test samples Dose (mg/kg) ORAC (mmol trolox equivalent/L) mean ± SEMcontrol - 0.27 ± 0.04Artichoke 500 0.22 ± 0.03 1000 0.21 ± 0.04Luteolin 25 0.38 ± 0.05 50 0.31 ± 0.03Quercetin 25 0.31 ± 0.05

Note: Rats were orally given artichoke extract, luteolin and quercetin at the different doses indicated for 2 h. Data represent mean value ± SEM (n = 8).

104

Table 5-4. ORAC values of plasma samples ORAC (mmol trolox equivalent/L) mean ± SEM

Compounds Day 0 Day7 Day14 Day21 Control 0.44 ± 0.03 0.50 ± 0.07 0.43 ± 0.05 0.40 ± 0.03Artichoke 500 mg/kg 0.34 ± 0.04 0.35 ± 0.03 0.37 ± 0.03 0.33 ± 0.04Artichoke 1000 mg/kg 0.49 ± 0.03 0.41 ± 0.02 0.43 ± 0.03 0.43 ± 0.02Luteolin 25 mg/kg 0.44 ± 0.03 0.38 ± 0.03 0.45 ± 0.04 0.39 ± 0.03Luteolin 50 mg/kg 0.46 ± 0.02 0.37 ± 0.03 0.37 ± 0.03 0.45 ± 0.02Quercetin 25 mg/kg 0.42 ± 0.01 0.33 ± 0.02 0.31 ± 0.04 0.39 ± 0.03Note: Rats were orally given artichoke extract, luteolin and quercetin at the different doses

indicated for 21 days. Data represent mean value ± SEM. (n = 8).

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Table 5-5. Plasma urate concentrations in rats after administration of artichoke extract and phenolic compounds

Uric acid levels (ug/ml) Treatment Dose (mg/kg)

NDay 0 Day 7 Day 14 Day 21

Control - 8 4.82 ± 0.28 3.37 ± 0.38 4.05 ± 0.40 4.34 ± 0.67Artichoke 500 8 3.37 ± 0.57 3.04 ± 0.31 3.11 ± 0.18 3.68 ± 0.53 1000 8 3.79 ± 0.33 3.98 ± 0.54 4.19 ± 0.48 4.64 ± 0.56Luteolin 25 8 2.65 ± 0.15 3.09 ± 0.22 3.86 ± 0.51 4.01 ± 0.63 50 8 3.37 ± 0.29 3.36 ± 0.35 3.49 ± 0.27 3.69 ± 0.31Quercetin 25 8 2.41 ± 0.17 2.33 ± 0.13 3.16 ± 0.15 3.40 ± 0.34Note: Rats were orally given artichoke extract, luteolin and quercetin at the different doses

indicated for 21 days. Data represent mean value ± SEM (n = 8).

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Table 5-6. Plasma glutathione peroxidase activity in rats after administration of artichoke extract and phenolic compounds

GPx Activity (nmol/min/ml) mean ± SEM Treatment

Day 0 Day 21Control 5436 ± 417.1 7279 ± 513.5Artichoke 500 mg/kg 5356 ± 281.9 6970 ± 249.8Artichoke 1000 mg/kg 7263 ± 271.6 7080 ± 852.5Luteolin 25 mg/kg 5501 ± 166.4 6529 ± 424.8Luteolin 50 mg/kg 6282 ± 454.2 5692 ± 490.2Quercetin 25 mg/kg 6418 ± 460.7 6282 ± 537.3Note: Rats were orally given artichoke extract, luteolin and quercetin at the different doses

indicated for 21 days. Data represent mean value ± SEM. (n = 8).

107

OH

OH

O

OH

OH

OH

O

OH

Caffeic acid Dihydrocaffeic acid

OH

O

OH

O

HOOC

O

OH

OH

O

OH

OH

OH

OH

O

OH

HOOC

C

O

OH

OH

Cynarin Chlorogenic acid

O

OOH

OH

OH

OH

OH

Quercetin R=H, Luteolin R=Glc,Luteolin-7-O-glucoside

Figure 5-1. Structures of caffeic acid derivatives and flavonoids.

O

OOH

OH

OH

RO

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CHAPTER 6 PHARMACOKINETICS OF LUTEOLIN AND ITS METABOLITES IN RATS

Background

Luteolin, one of the active components in artichoke leaves (Cynara scolymus L.), had

strong xanthine oxidase inhibitory and antioxidant activity in vitro as shown in our previous

study. Luteolin also has been reported to be non-mutagenic [121], antitumorigenic [122], and has

been recognized as an inhibitor of protein kinase C [123]. Therefore, in view of the potential of

luteolin as a pharmacological agent, the pharmacokinetics should be carefully studied.

At present, the pharmacokinetics of luteolin has not been fully characterized, although a

number of studies have been reported in animals and humans. These studies showed that the

concentration of luteolin in plasma is low after oral administration. However high amount of

metabolites, for example, luteolin conjugates were found in systemic circulation [30, 104]. The

bioavailability of luteolin is unknown. Therefore, to obtain more information about absorption

and disposition, the pharmacokinetics of luteolin in rats treated with oral and intravenous

administration of luteolin should be performed.

Specific Aims

Pharmacokinetic analysis of luteolin and its metabolites in rats.


Materials

Luteolin (99%) was purchased from Indofine Chemical Company, Inc. (Somerville, NJ,

USA). Naringenin (≥96%) (internal standard) was purchased from Roth Carl Roth GmbH+Co.

(Germany). Acetone, acetonitrile (CH3CN), acetic acid, Dimethyl sulfoxide (DMSO), methanol,

orthophosphoric acid (85% p.a.) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). L

(+)-ascorbic acid (≥ 99.9%) was obtained from Acros organics (New Jersey, USA).

109

Trifluoroacetic acid was obtained from Fluka (Milwaukee, WI, USA). β-glucuronidase/sulfatase

(type HP-2, Helix pomatia), polyethylene glycol 200, and sodium dihydrogenphosphate

monohydrate (NaH2PO4.H2O), were obtained from Sigma Chemical Company (St. Louis, MO,

USA). All buffers and aqueous solutions were prepared with purified water obtained from a

NANOPure® system from Barnstead (Dubuque, IA, USA).

Stock, Work Solutions, and Preparation of Calibration Standards

The stock solutions of luteolin 10.0 mg/mL and naringenin (internal standard) 22.05

mg/mL were prepared in DMSO and kept at -80oC.

Luteolin stock solution: (10 mg/mL): Luteolin 20.0 mg was accurately weighed and

transferred to a 2.0 mL volumetric flask. The standard was then dissolved in DMSO and the

volume was completed with the same solvent.

Naringenin stock solution: 22.05 mg/mL: Naringenin 110.25 mg was weighed and

transferred to a 5 mL volumetric flask and then dissolved in DMSO. The volume was completed

with the same solvent.

Luteolin work solution: 500 μg/mL: Volume of 100 μL from luteolin stock solution was

accurately transferred to a 2 mL volumetric flask. The volume was completed with methanol and

mixed thoroughly. The final concentration of luteolin was 500 μg/mL.

Naringenin work solution (internal standard): 1.05 mg/mL: Volume of 95.0 μL from

naringenin stock solution was transferred to a 2.0 mL volumetric flask and then completed the

volume with methanol. The final concentration of naringenin was 1.05 mg/mL.

Standard solutions of luteolin in plasma: From the luteolin work solution, five different

concentrations of standard solutions and three quality controls (QC) were prepared in methanol.

Then 10 μL of standard solutions was added to 200 μL plasma according Table 6-1.

110

Standard solutions of luteolin in urine: From the luteolin stock solution and work

solution, five different concentrations of standard solutions and three QC were prepared in

methanol and 10 μL of standard solutions was added to 200 μL urine according Table 6-2.

Animals and Experimental Protocols

Animals

Male Sprague-Dawley rats, weighing 250-350 g. were purchased from Harlan (IN, USA)

and divided into the experimental groups; containing 11 rats per group. They were housed in

plastic cages. They were allowed one week to adapt to their environment before used for

experiments. All the animals were maintained on a 12hr/12hr light/dark cycle. They were given

standard chow and water ad libitum during the course of the study. All animal experiments were

performed according to the policies and guidelines of the Institutional Animal Care and Use

Committee (IACUC) of the University of Florida, Gainesville, USA (NIH publication # 85-23).

Methods

The pharmacokinetic studies were carried out by the sparse sampling approach wherein

blood samples were collected from 8-11 different rats. Luteolin was administered in two groups

of rats (n = 8-11 in each group). Group one received a single i.v. dose of 50 mg/kg of luteolin via

a tail vein. Group two received the same dose orally by gavage. Luteolin was dissolved in 30%

DMSO and 70% PEG200. For luteolin analysis, plasma samples (500 μL per blood sample) were

collected from sublingual vein into heparinized tubes at 3, 5, 10, 30 min, and 1, 2, 4, 6, 12, 24 h

for i.v. injection and at 5, 10, 15, 30, 45 min, and 1, 2, 4, 6, 12, 24 h for oral administration. The

blood collections were separated into two different days apart by one week of wash out period

(5-6 blood collections per day per animal). For luteolin conjugates, plasma samples (1000 μL per

blood sample) were collected at 5, 10, 30 min and 1, 2, 4, 6, 12, 24 h for i.v. and oral

111

administration. The blood collections were separated into two different days apart by one week

of wash out period (3 blood collections per day per animal). The variability of the weight of each

animal on both periods was not higher than 20%. Prior to blood collection, the rats were

anaesthetized with halothane and the blood loss was replaced with an equal volume of normal

saline. The blood sample was centrifuged for 15 min at 4,000 rpm at 4oC. The supernatant in

aliquots of 200.0 μL was transferred into tubes and 10.0 μL of 0.58 M acetic acid was added to

each aliquot for stabilization. The plasma samples were stored at -80oC until analysis. Urine was

collected over 24 h and an aliquot of 50.0 mL was mixed with 1g ascorbic acid as antioxidant

and stored at -80oC until analysis.

Analytical Methods

The plasma concentrations of unchanged free and conjugated luteolin in rat plasma and

urine were determined by the method published earlier with a slight modification [124] Plasma

samples and urine samples were analyzed using a reverse-phase partition mode of HPLC with

diode array detector. A Shimadzu VP series HPLC system (Kyoto, Japan) equipped with an

SPD-M10Avp diode array detector was used for this work. A Lichrospher® 100 RP-18 (5μm.

Merck KgaA) was used for the separation of luteolin. The column temperature was kept at 25oC.

The eluents were (A) 50 mM phosphate buffer (NaH2PO4, pH 2.1) and (B) CH3CN.The

following solvent gradient was applied: 20% B (6 min) and 20-50% B (21 min).The gradient was

followed by 10 min column flushing and post-run equilibration, respectively. Total run time was

40 min. The flow rate was 1 mL/min. 40 μL of each sample was injected into the RP-HPLC

system. Chromatograms were acquired at 330nm.

For the determination of total luteolin, 10.0 μL of internal standard (naringenin, 1.05

mg/ml), 10.0 μL of 0.5% (m/v) ascorbic acid and 20.0 μL of acetic acid (0.58 M) were added to

112

200.0 μL of plasma sample; followed by the addition of 12.0 μL of β-glucuronidase/sulfatase

solution. The mixture was incubated at 37oC for 1 h. Protein was precipitated by adding 240 μL

of acetone. The mixture was vortexed for 1 min and centrifuged for 15 min at 4000 rpm at 4oC.

Then the supernatant was transferred to tubes containing 4.0 μL of 0.5% (m/v) ascorbic acid and

8.0 μL of 1 M trifluoroacetic acid and evaporated to dryness in a vacuum centrifuge. The residue

was reconstituted in 60 μL of methanol: water (1:1, v/v), centrifuged for 10 min at 13200 rpm,

and 40 μL was injected into HPLC.

For the determination of unchanged luteolin in plasma, the sample was extracted in the

same manner as described above without adding the enzyme.

The concentrations in urine was measured using the same method as plasma, except urine

samples were centrifuged for 15 min at 13200 rpm after adding 240.0 μL of acetone and then

40.0 μL of the supernatant was injected into HPLC.

The conjugates (glucuronides or sulfates) of luteolin were calculated by subtracting total

luteolin with unchanged luteolin.

Data Analysis

Plasma samples showed measurable concentrations for luteolin before administration of

luteolin. Therefore, plasma concentrations at each time points were subtracted with baseline level

before pharmacokinetic data analysis. Mean concentration of luteolin and its conjugates versus

time curves were generated in Grapad Prism® (version 4.0, San Diego, CA). The

pharmacokinetic parameters were determined by non-compartmental analysis and compartmental

analysis using WinNonlin software package, version 3.1, (Pharsight Corporation, USA). The

mean data was used for both analyses.

113

Non-compartmental PK analysis: The PK parameters determined were the areas under

the concentration time curve (AUC), maximum concentration in plasma (Cmax), time to reach

Cmax (Tmax), the elimination rate constant (ke), the elimination half life (t1/2), the volume of

distribution (Vd) and the clearance (CL). AUC 0→last was calculated using linear/log trapezoidal

method from time zero to last sampling point equal to or above the lower limit of quantification.

AUC 0→∞ was calculated as AUC 0→last + AUCextra, and AUCextra was determined as the

calculated last concentration (Clast)/ke. Both Cmax and Tmax were obtained from the plots of

plasma concentration versus time. The ke was obtained by linear regression of the terminal log

linear phase of the concentration-time curve. The elimination half-life (t1/2) was determined as

0.693/ke. The volume of distribution of central compartment (V) was calculated as D/C0, where

D is the dose. The clearance (Cl) was calculated as D/AUC. The systemic bioavailability (F %)

was calculated as F % = (AUC p.o. × Dose i.v. /AUC i.v. × Dose p.o.) × 100.

Compartmental PK analysis: Luteolin concentrations showed better fit in a two

compartment body model compared with one compartment body model. The equation for two

compartment model (Figure 6-1.) is as followed:

C = A.e-αt + B.e-βt (i.v.)

Where C is the concentration of drug in plasma at time t; A and B are mathematical

coefficient; α is the distribution rate constant; β is the elimination rate constant; and t is time.

After i.v. administration, AUC 0-∞ was calculated using following equation: AUC 0-∞ i.v. =

A/α + B/β. The elimination half life was calculated as ln (2/Ke). The volume of distribution of

central compartment (Vc) was calculated as Dose/Ke × AUC. The volume of distribution of

peripheral compartment (Vt) was calculated as Vc × k12/k21. The clearance (Cl) was calculated

as Dose/AUC.

114

Goodness of fit was determined by the AIC (Akaike Criteria) and SC (Schwartz Criteria).

The lower the AIC and SC, the more appropriate the selected model.


WinNonlin software package, version 3.1, (Pharsight Corporation, USA) was used for

statistical analysis. Data are given as mean with corresponding standard deviation.

Validation

The method was validated over the range of concentration of luteolin present in plasma.

The validation parameters of linearity, sensitivity, specificity, precision, accuracy and stability

were determined.







calibration was considered suitable if not more than 1/3 of the quality control standards showed a

deviation from the theoretical values equal or greater than 15%, except at the lower limit of

quantification (LLOQ), where it should not exceed 20%.

Results

Validation of Analytical Method to Measure Luteolin in Rat Plasma

Linearity

Calibration curve (n = 9) operating in the range of 100-10000 ng/mL for luteolin in rat

plasma was linear (r2 > 0.99) (Figure 6-2).

115

Sensitivity


quality control. This concentration would be acceptable with the precision (%CV < 20), and

accuracy (%error < 20).The LLOQ of luteolin in plasma was 100 ng/mL.

Specificity

The methods provided good resolutions between luteolin, β-glucuronidase and interference

in plasma and there was no endogenous interference from plasma (Figure 6-3) in this assay,

indicating the specificity of this method.


The precisions intra- and inter-day for luteolin were satisfactory with CV values between


containing 300, 800, and 3000 ng/mL luteolin was between 94.2 and 106.3 % of the nominal

values. The mean recovery assessed at three distinct levels of concentration (100, 500 and 10000

ng/mL) ranged from 95.7 to 106.4 % of the expected values. The results are summarized in

Table 6-3.

Stability

Luteolin was stable under the tested conditions. The mean % remainings in rat plasma after

2 hours at room temperature were 98.78 ± 5.34, 96.29 ± 1.47, 102.4 ± 7.76 for the low, medium

and high concentrations, respectively. The mean % remainings of luteolin after an evaporation

and keep at -20 °C for 24 hours were 106.68 ± 8.97, 102.40 ± 3.11 and 100.07 ± 0.50 for the

low, medium and high concentrations, respectively. Luteolin was stable on autosampler at 18 oC

within 48 hours (Table 6-4).

116

Validation of Analytical Method to Measure Luteolin in Rat Urine

Linearity

Calibration curve (n = 9) operating in the range of 500-50000 ng/mL for luteolin in rat

urine was linear (r2 > 0.99) (Figure 6-4).

Sensitivity

The limit of quantification (LLOQ) of luteolin in urine was 500 ng/mL.

Specificity

Good resolutions between luteolin, β-glucuronidase and interference in urine and no

endogenous interference from urine (Figure 6-5) indicated the specificity of this method.


The precisions intra- and inter-day for luteolin were satisfactory with CV values between


containing 500, 3000, and 10000 ng/mL luteolin was between 98.21 and 109.28 % of the

nominal values. The mean recovery assessed at three distinct levels of concentration (500, 3000

and 10000 ng/mL) ranged from 99.56 to 112.23 % of the expected values. The results are

summarized in Table 6-5.

Stability

Luteolin was stable under the tested conditions. The mean % remainings in rat urine after 2

hours at room temperature were 98.78 ± 5.34, 96.29 ± 1.47, 102.4 ± 7.76 for the low, medium

and high concentrations, respectively. Luteolin was stable on autosampler at 18 oC within 48

hours (Table 6-6).

117

Pharmacokinetic Study of Luteolin

Non-compartmental analysis

Plasma levels of luteolin after oral and i.v. administration of luteolin: The

concentration-time profiles and the pharmacokinetic parameters of luteolin after oral and i.v.

administration are presented on Figure 6-6 and Table 6-7. For oral administration, plasma

concentrations of luteolin attained maximum level of 5.49 μg/ml at 0.08 h and decreased to

below LOQ (100 ng/ml) after 1 h. Ke could not be calculated because the elimination phase was

below LOQ. Our assumption was Ke after oral administration was similar to ke after i.v

injection. Therefore, the AUC 0-∞ p.o. was calculated using ke from i.v. The low bioavailability (F)

of luteolin, 4.10 % at dose 50 mg/kg are presumably due to the significant first pass effect. For

i.v. administration, the maximum concentration of luteolin was 23.42 μg/mL at 0 h. The plasma

concentration versus time profile of luteolin was biphasic, subdivided into a distribution phase

and a slow elimination phase for oral and intravenous administration.

Plasma levels of luteolin conjugates after oral and i.v. administration of luteolin: The

concentration-time profiles and the pharmacokinetic parameters of luteolin conjugates after oral

and i.v. administration are presented on Figure 6-6 and Table 6-8. Plasma concentration of

luteolin conjugates after oral and i.v administration of luteolin attained maximum level of 5.77

μg/mL at 0.25 h and 4.31 μg/ml at 0.08 h, respectively, and decreased to below LOQ at 24 h.

The double peaks were found in luteolin conjugates after oral and i.v. administration at 0.25 and

1 h, respectively, suggesting it might pass enterohepatic circulation.

Urinary excretion of luteolin and its metabolites after oral and i.v. administration:

Urinary excretion of luteolin and luteolin conjugates within 24 h after oral and intravenous

118

administration of luteolin were very low (0.98 - 4.97% of the dose), suggesting these compounds

are not primarily excreted via the urine (Table 6-10).

Compartmental Analysis

Figure 6-7 shows the fitted luteolin concentrations versus time profiles with the two

compartment body model. It can be seen that the model describes luteolin data very well. The

AIC and SC were -11.18 and -9.59, representing a good fit. The PK parameters obtained by

fitting the mean concentration versus time profiles of luteolin concentrations after i.v. treatment

are presented in Table 6-9.


Luteolin and luteolin conjugates were presented in rat plasma and urine after oral and

intravenous administration. However, the conjugates (glucuronides or sulfates) could not be

further identified in this study. The analytical methods were developed for the parent compound,

and the conjugates presumably coeluted with matrix compounds. The present of free luteolin

suggested that some luteolin can escape the intestinal and hepatic conjugation.

Pharmacokinetic profiles of luteolin and luteolin conjugates in rat plasma are shown in

Figure 6-6. When rats were given luteolin (50 mg/kg) in 30% DMSO: 70% PEG 200 orally, the

maximum concentration of luteolin and luteolin conjugates were 5.48 and 5.77 μg/mL at 5 min

and 15 min, respectively. The total concentration of luteolin in rat plasma at 5 min after dosing

was 9.25 μg/mL. Shimoi et al. [104] observed 15.5 ± 3.8 nmol/mL (∼4.4 ± 1.09 μg/mL) of total

luteolin concentration in rat plasma 30 min after administration of one single dose of luteolin (50

μmol/kg, ∼14.3 mg/kg) in propylene glycol. In dog, the maximum concentration of luteolin was

about 450 ng/mL at 3 h after a single oral dosing of Chrysanthemum morifolium Ramat extracts

(102 mg/kg containing 7.60% luteolin, ∼ 7.75 mg/kg luteolin) [125]. In human, peak plasma

119

concentrations of total luteolin were reached within 0.5 h with maximum level of 156.5 ± 92.29

ng/mL after a single oral dose of artichoke leaf extracts (153.8 mg containing luteolin-7-O-

glucosides; equivalent to 35.2 mg luteolin) [30]. The differences observed between these studies

could be explained by the different initial dose administration of flavonoids and the different

source of intake flavonoids.

The rapid absorption of flavonoids has been reported in previous literature. When diosmin

was administered to humans, a peak occurring 2 h after administration [126], whereas diosmetin

administered per os to rats appeared in blood after 6 h as unchanged and glucuronated compound

[127]. In pigs, after an oral dose of 50 mg/kg, only 17 % of the quercetin administered was

recovered in blood as free conjugate and derivative products within 8 h postadministration [128].

In humans, the peak in blood occurred more rapidly, approximately 2.9 h after the flavonol

administration [103]. In rats, flavonoids seemed to occur more rapidly. Luteolin given via gastric

intubation, appeared in plasma after 15 min [104]. Apigenin given to rats via the intraperitoneal

pathway appeared in plasma 30 min after administration [105]. From these literatures, the

presence of flavonoids in blood occurs within a few minutes to a few hours which are similar to

our result.

The low bioavailability of luteolin (F = 4.1%) and high metabolite concentrations indicate

first pass metabolism. Absorbed luteolin could undergo biotransformation (methylation,

glucuronidation or sulfation) as shown in previous literature. In vitro experiments demonstrated

that 74 % of luteolin was conjugated to glucuronic acid after incubation with microsomal

samples from human intestine. Most common binding sites of the molecule were the hydroxyl

groups in the 3′- and 4′- position (51% and 44%) [96]. Boersma et al. [96] found three

glucuronosyl conjugates of luteolin, the 7-O-, the 3′-O- and the 4′-O-glucuronosyl luteolin after

120

incubation with intestine microsomes and liver microsomes from rat and man. Shimoi et al. [104]

investigated the absorption of luteolin by rat everted small intestine. Luteolin was recovered in

rat plasma as two metabolites, glucuronidate or sulfate forms of O-methylate conjugate. Only a

small part of the compound remained unconjugated. Murota et al. [129] reported the uptake and

transport of flavonoids aglycones by human intestinal Caco-2 cells. The flavonoids, quercetin,

kaempferol, luteolin and apigenin, were converted to their glucuronide/sulfates by Caco-2 cells,

and the level of the intact aglycone form was less than those of the glucuronide/sulfates in the

basolateral solution. To our knowledge, this was the first study on bioavailability of luteolin, thus

a comparable data is lacking. However, the bioavailability of luteolin is similar to that of

quercetin. Chen et al. [95] reported the systemic bioavailability of quercetin and quercetin

conjugates as 5.3% and 55.8%, respectively in rats. Moreover after oral administration of

quercetin, about 93.3% of quercetin was metabolized in the gut, with only 3.1% metabolized in

the liver.

Only small amounts of luteolin and luteolin conjugates were found to be eliminated in the

urine in our study. This is consistent with the observations by others. Shimoi et al. [104] found

excretory recovery for 24 h as unmodified luteolin from the urine was about 4 % in rats. Luteolin

conjugates was recovered only 1.99 ± 1.50 % after intake of luteolin-7-O-glucoside (equivalent

to 35.2 mg luteolin) [30]. Only 0.58 % of apigenin was recovered in urine samples within 24 h

after parsley ingestion in human [130]. Gugler et al. [131] found that after intravenous

administration of a 100 mg quercetin, only 0.65 % of the dose was recovered in the form of

unchanged quercetin, while 7.4 % of the dose was excreted in the urine in the form of conjugated

metabolite of quercetin. In a phase I clinical trial of quercetin (in fifty-one cancer patients) at

121

dose of 60 to 2000 mg/m2, the percentage of quercetin in urine over 24 h ranged from 0.03% to

7.6% [132].Therefore, urinary elimination of luteolin is not the main excretion route in rats.

Our study found multiple peaks in a plasma concentration-time profile of luteolin

conjugates after oral and intravenous administration of luteolin suggesting an enterohepatic

recirculation of luteolin which is similar to other studies. Liu et al. [39] reported a rapidly

absorbed and rapidly metabolized of aglycones such as apigenin and quercetin into phase II

conjugates, which were then excreted back into the lumen; following and enteric and

enterohepatic recycling. Ma et al. [133] reported an enterohepatic recirculation of naringenin in

rat plasma. Our data did not show multiple peaks in a plasma concentration-time profile of free

luteolin after intravenous and oral administration presumably due to the limit of data time points.

In the present investigation, pharmacokinetics of luteolin and its metabolites in rats were

studied. After oral administration of luteolin, luteolin was rapidly absorbed and metabolized in

plasma; moreover, plasma-concentration-time curves of luteolin metabolites revealed secondary

peaks. The bioavailability of luteolin is low and the urinary excretion of luteolin and its

conjugates did not dominate. This study could explain a lack of in vivo activity of artichoke leaf

and its compounds on xanthine oxidase inhibitory and antioxidant activity. Moreover, it can be

used to predict the in vivo activity of other herbal products that contain this compound.

122

Table 6-1. Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in plasma

Standard Luteolin in methanol (μg/mL)

Luteolin in plasma (ng/mL)

1 10.50 5002 21.00 10003 105.00 50004 210.00 100005 1050.00 50000QC1 10.50 500QC2 63.00 3000QC3 210.00 10000

123

Table 6-2. Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in urine

Standard Luteolin in methanol (μg/mL)

Luteolin in plasma (ng/mL)

1 2.10 1002 10.50 5003 21.00 10004 105.00 50005 210.00 10000QC1 2.10 100QC2 16.80 800QC3 63.00 3000

124

Table 6-3. Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat plasma. Precision expressed as CV%, accuracy and recovery as % of the theoretical concentration

Intra-day QC1 100 ng/mL QC2 800 ng/mL QC3 3000 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 3.82 12.34 8.71 1.33 5.70 5.29 1.73 5.92 2.28Accuracy 94.20 98.72 100.21 102.57 104.06 104.30 106.25 104.68 104.12Inter-day QC1 100 ng/mL QC2 800 ng/mL QC3 3000 ng/mL Precision 9.65 2.82 3.02 Accuracy 98.17 104.69 104.21 Recovery Luteolin-100 ng/mL Luteolin-500 ng/mL Luteolin-10000 ng/mL % 95.78 96.42 106.40 CV% 10.94 8.46 5.48

125

Table 6-4. The stability test after 48 hours on autosampler at 18oC. Data represents the percentage remaining of luteolin in plasma ± SD

% Remaining on autosampler Luteolin concentration 12 hours 24 hours 48 hours

Low-100 ng/mL 108.81 ± 15.13 90.83 ± 11.37 91.28 ± 14.38Medium-500 ng/mL 100.10 ± 2.44 98.34 ± 2.98 114.21 ± 13.10High-10000 ng/mL 99.70 ± 0.66 99.89 ± 2.40 100.52 ± 1.33

126

Table 6-5. Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat urine. Precision expressed as CV%, accuracy and recovery as % of the theoretical concentration

Intra-day QC1 500 ng/mL QC2 3000 ng/mL QC3 10000 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Precision 6.48 7.24 0.81 0.30 0.73 1.52 2.96 0.61 1.66Accuracy 98.21 100.12 98.51 101.81 101.92 100.98 110.01 112.45 108.99Inter-day QC1 500 ng/mL QC2 3000 ng/mL QC3 10000 ng/mL Precision 13.25 3.24 1.37 Accuracy 98.72 103.49 109.28 Recovery Luteolin-500 ng/mL Luteolin-3000 ng/mL Luteolin-10000 ng/mL % 99.56 108.46 112.23 CV% 3.19 3.32 3.21

127

Table 6-6. The stability test of luteolin in urine after 48 hours on autosampler at 18oC. Data represents the percentage remaining of luteolin ± SD

% Remaining on autosampler Luteolin concentration 24 hours 48 hours

Low-500 ng/mL 97.44 ± 2.38 92.98 ± 0.61Medium-3000 ng/mL 98.77 ± 0.71 98.85 ± 1.48High-10000 ng/mL 100.10 ± 0.53 100.32 ± 1.65

128

Table 6-7. Pharmacokinetic parameters of luteolin after oral and iv administration of luteolin at dose 50 mg/kg

Parameter Luteolin oral Luteolin ivTmax (h) 0.08 0Cmax (μg/mL) 5.48 23.42Ke (1/h) ND 0.08t ½ (h) ND 8.94Cl/F (L/h/kg) NDVd/F (L/kg) NDCl (L/h/kg) 2.14Vd (L/kg) 27.58AUC0-last (h*μg/mL) 0.87 20.55AUC0-α (h*μg/mL) 0.96 23.39F (%) 4.10

Note: All Pk parameters are mean values calculated by a normalized dose (50 mg/kg)

129

Table 6-8. Pharmacokinetic parameters of luteolin conjugates after oral and iv administration of luteolin at dose 50 mg/kg

Parameter Luteolin conjugates oral Luteolin conjugates ivTmax (h) 0.25 0.08Cmax (μg/mL) 5.77 4.31Ke (1/h) 0.10 0.14t ½ (h) 6.57 4.98AUC0-last (h*μg/mL) 11.49 12.83AUC0-α (h*μg/mL) 15.68 15.26

Note: All pk parameters are mean values calculated by a normalized dose (50 mg/kg)

130

Table 6-9. Pharmacokinetic parameters of luteolin after i.v. administration of luteolin 50 mg/kg. Data was fitted to a two-compartment model.

Parameter Luteolin i.v.A (μg/mL) 9.66 ± 1.14B (μg/mL) 1.36 ± 0.16α (1/h) 1.95 ± 0.32β (1/h) 0.08 ± 0.01K12 (1/h) 1.24 ± 0.25K21 (1/h) 0.31 ± 0.06Ke (1/h) 0.48 ± 0.06Vc (L/kg) 4.54 ± 0.48Vt (L/kg) 18.26 ± 2.24Cl (L/h/kg) 2.18 ± 0.13 t1/2 α (h) 0.36 ± 0.06t1/2 β (h) 9.15 ± 1.15t1/2 Ke (h) 1.44 ± 0.17AUC (h μg /mL) 22.93 ± 1.39Cmax (μg /mL) 11.02 ± 1.15Note: All pk parameters are mean ± S.D.

131

Table 6-10. The excretory recovery for 24 h of luteolin and luteolin conjugates in urine after oral and i.v administration of luteolin at dose 50 mg/kg

Treatment % Luteolin % Luteolin conjugatesoral 0.98 ± 0.98 3.91 ± 0.52i.v. 2.05 ± 0.90 4.97 ± 1.68

Note: Data expressed as mean ± SD (n = 11)

132

bolus IV

1

2

K12 K21

Ke

Figure 6-1. Two-compartment models after Intravenous injection. 1 is the central compartment, 2 is the peripheral compartment, Ke is the first order elimination rate constant, K12 is the rate constant for transfer of drug from the central compartment to the peripheral compartment and K21 is the rate constant for transfer of drug from the peripheral compartment to the central compartment.

133

0 2500 5000 7500 10000 125000.0

0.5

1.0

1.5

2.0

2.5

Luteolin [ng/ml]

Rat

io( a

rea

lute

olin

: are

a IS

)

Figure 6-2. Mean calibration curves (n = 9) of luteolin in plasma. Vertical bars represent the

standard deviations (SD) of the means.

Y = 0.0002057 X - 0.003217 R = 0.9978

134

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

mVo

lts

0

20

40

60

80

100

120

140

160

180

200

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

mVo

lts

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

Figure 6-3. The HPLC chromatogram of luteolin and naringenin (IS) in plasma. A) With out β-

glucuronidase. B) With β-glucuronidase/sulfatase.

A B

Naringenin

Luteolin

Luteolin

Naringenin

135

0 10000 20000 30000 40000 50000 600000.0

2.5

5.0

7.5

10.0

12.5

Luteolin [ng/mL]

Rat

io (A

rea

Lute

olin

:Are

a IS

)

Figure 6-4. Mean calibration curves (n = 9) of luteolin in urine. Vertical bars represent the

standard deviations (SD) of the means.

Y = 0.0002X – 0.0703 R 2 = 0.9992

136

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

mVo

lts

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

mVo

lts

0

25

50

75

100

125

150

175

200

225

250

275

300

325

350

Figure 6-5. The HPLC chromatogram of luteolin and naringenin (IS) in urine. A) With out β-

glucuronidase/ sulfatse. B) With β-glucuronidase/ sulfatase.

A B

Naringenin

Luteolin

Naringenin

Luteolin

137

A

0 5 10 15 20 25 300.01

0.1

1

10

100Luteolin p.o.Luteolin i.v.

Time(h)

log

[lut

eolin

](ug

/mL

pla

sma)

B

0.0 2.5 5.0 7.5 10.0 12.5 15.00.01

0.1

1

10

100

Luteolin conjugates i.v.Luteolin conjugates p.o.

Time(h)

Log

[Lut

eolin

] (m

g/m

l)

Figure 6-6. Plasma concentration-time curves. A) Luteolin. B) Luteolin conjugates. After oral and intravenous administration of 50 mg/kg to rats (n = 8-11). Error bars refers to the standard deviation of concentration data at each sampling time point.

138

Figure 6-7. Fitted luteolin concentrations after i.v. injection. Experimental points represent the

means of 8-11 rats.

0.1

1.0

10.0

100.0

0 5 10 15 20 25

Time (h)

Observed

Predicted

139

CHAPTER 7 CONCLUSION

Gout is a common metabolic disorder in human. It results from deposits of needle-like

crystals of uric acid in connective tissue, in the joint space between two bones, or in both. These

depositions lead to inflammatory arthritis, which causes swelling, redness, heat, pain, and

stiffness in the joints. The common treatments for an acute attack of gout are colchicine, non-

steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. Allopurinol, a xanthine oxidase

inhibitor, is used for the prevention of chronic gout attacks. Its use is limited by unwanted side

effects such as hypersensitivity problems. Therefore, alternatives are required.

Leaf of Artichoke (Cynara scolymus L.) is a good source of polyphenolic compounds such

as mono- and dicaffeoylquinic acids and flavonoids. Polyphenolic compounds have a role in the

prevention of degenerative diseases such as cancer, cardiovascular disease and

neurodegenerative diseases, which is usually linked to two properties: antioxidant activity and

inhibition of certain enzymes such as xanthine oxidase. Therefore, artichoke leaves containing

polyphenolic compounds may show xanthine oxidase inhibitory activity and antioxidant activity.

In this study, artichoke leaf extract and caffeoylquinic acids showed weak or no XO

inhibitory activity in vitro; whereas, the inhibitions of most flavonoids on XO were stronger than

a standard compound, allopurinol. However, after oral and intraperitoneal administration of

different doses of artichoke and polyphenolic compounds in rats, none of the test compounds

could decrease serum urate levels. This result of the XO study was similar to that of the

antioxidant study. The study of antioxidant activity of artichoke and its components also showed

that although there was an antioxidant activity in vitro, the antioxidant activity in vivo was not

found after oral treatment. This lack of XO and antioxidant activity in vivo might be explained

by low absorption, high first pass effect through gut, intestine and liver, rapid excretion into

140

urine and bile, degradation and metabolization by the colonic microflora. In addition, the

metabolites may differ from the native substances in terms of biological activity. Therefore, the

further studies of bioavailability of polyphenolic compounds and the activity of metabolites are

essential.

The activity of metabolites such as luteolin-7-O-glucuronide has shown a weaker

inhibition on XO comparing to luteolin in vitro. Moreover the pharmacokinetics of luteolin

showed that luteolin has low bioavailability after oral administration. These results could explain

a lack of activity of artichoke leaf extract and its components on xanthine oxidase inhibitory

activity and antioxidant activity in vivo. Therefore, we can conclude artichoke leaf does not seem

to be an alternative for treating gout.

141

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BIOGRAPHICAL SKETCH

Sasiporn Sarawek was born in April 14th, 1978, in Chiangmai, Thailand. She obtained her

bachelor’s degree in Pharmacy in 2001 from Chiangmai University. She started her PhD

program in January 2003 in the Department of Pharmaceutics of the University of Florida under

supervision of Dr. Veronika Butterweck and Dr. Hartmut Derendorf. Sasiporn received her PhD

in Pharmaceutics in August 2007.

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XANTHINE OXIDASE INHIBITION AND ANTIOXIDANT ACTIVITY … · 2013-05-31 · 1 xanthine oxidase inhibition and antioxidant activity of an artichoke leaf extract (cynara scolymus l.)