1 XANTHINE OXIDASE INHIBITION AND ANTIOXIDANT ACTIVITY OF AN ARTICHOKE LEAF EXTREACT (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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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
Absorption and Metabolism of Caffeolyquinic Acids............................................................17 Absorption and Metabolism of Flavonoids ............................................................................17 Biological Effects of Flavonoids ............................................................................................19
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
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
Background.............................................................................................................................54 Specific Aim ...........................................................................................................................54 Materials and Methods ...........................................................................................................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
Discussion and Conclusion.....................................................................................................58
4 EFFECTS OF ARTICHOKE LEAF EXTRACT AND VARIOUS FLAVONOIDS ON SERUM URIC ACID LEVELS IN OXONATE-INDUCED RATS .....................................67
Background.............................................................................................................................67 Specific Aim ...........................................................................................................................67 Materials and Methods ...........................................................................................................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
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
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
Plasma Urate Concentrations and Plasma Glutathione Peroxidase Activity after The Treatment with Artichoke Extract and Phenolic Compounds .....................................98
Discussion and Conclusion.....................................................................................................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
Data Analysis.................................................................................................................112 Statistical Analysis ........................................................................................................114 Validation ......................................................................................................................114
Results...................................................................................................................................114 Validation of Analytical Method to Measure Luteolin in Rat Plasma ..........................114
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
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
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
6-7 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
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,
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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].
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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
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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].
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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
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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
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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.
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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
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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]
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].
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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
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
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
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
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.
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 and Methods
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,
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 and Methods
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,
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
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
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
Treatment groups Animals Dosage of drugs (mg/kg)
Serum Urate levels (ug/mL) mean ± SEM
Normal rats 8 - 14.58 ± 0.97Hyperuricemia rats dosed with vehicle (PG:water,50:50)
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
Treatment groups Animals Dosage of drugs (mg/kg)
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.
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.
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
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 and Methods
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,
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
96
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
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
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.
98
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.
Discussion and Conclusion
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
99
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.
101
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.
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
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
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
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
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
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