ISOLATION AND IDENTIFICATION OF COMPONENTS
FROM IXERIS SONCHIFOLIA HANCE AS POTENTIAL
ANTI-STROKE AGENTS
ZHANG YAOCHUN
B.Sc. (Pharm.), Shandong University
M.Sc. (Pharm.), Peking Union Medical College
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHARMACY
NATIONAL UNIVERSITY OF SINGAPORE
2010 Acknowledgement
I would like to express my sincere gratitude to my dear supervisor Dr Chew Eng-Hui for her continuous encouragement, wise advice and kind support. Her support, understanding, advice and mentoring have helped guide me through my doctoral program and my dissertation. Without her, none of this would have been possible. I know that I will continue to benefit from her advice and the skills that she has helped me achieve for the rest of my career. Deep appreciation also goes to my co-supervisor Dr Ng Ka-yun for his great help throughout the Ph.D project.
Special thanks to Dr Chui Wai Keung for his kind agreement for me to use his HPLC equipment, and Dr Koh Hwee Ling for her kind agreement for me to use her Blood Coagulation Analyzer. Their expertise advice on equipment techniques and analyzing methods accelerated my research progress. A big thank-you owned to Dr Keith Rogers from Institute of Molecular and Cell Biology, who helped me process the tissue immunohistological staining.
I would like to thank Dr Chan Lai Wah and Dr Go Mei Lin for their valuable guidelines on my Ph.D candidature and graduate study. Thanks to the department lab technicians namely, Ms Ng Sek Eng, Ms Ng Swee Eng, Mdm Oh Tang Booy, Ms Wong Meiyin, Ms Lye Peypey and Mr Goh Minwei. Their hard work provided great technical support for my research project. In addition, I wish to express my deep gratitude to National University of Singapore for offering the research scholarship that supported my candidature.
A sincere thank to my dear friends in NUS, who never failed to give me great suggestions in numerous ways. They are Dr Zhou Liang, Dr Lin Haishu, Dr Ling Hui, Dr Gapter Leslie A, Dr Surajit Das, Dr Yang Hong, Dr Hu Zeping, Dr Tian Quan, Dr Huang Meng, Dr He Chunnian, Dr Wu Zhenlong, Miss Gan Feifei, Miss Amrita Abhay Nagle, Miss Shridhivya Reddy Amarr, Miss Jin Jing, Miss Zhang Caiyu, Miss Yang Shili, Mr Shelar Sandeep Balu, Mr Sun Feng, Mr Yue Bingde, Mr Wang Zhe, Mr Wang Likun, Mr Tan Jing, Mr Si Chengtao, Mr Sun Lingyi and Mr Li Jian. The precious time spent with them will be preserved in my memory forever.
Last but not least, I would like to extend my appreciation to my families. My respected grandmother, industrious parents and kind sisters, though separated by an ocean, have never stopped their care. My dear wife, Li Guijun, has fulfilled my life outside the lab. My lovely daughter, Zhang Xiao, has rendered me a new angle to observe, feel and understand the world. I’m writing this thesis to share with all of them.
I
Table of contents
Acknowledgements I
Table of contents II
Abstract VII
List of tables X
List of figures XI
Abbreviations XIV
Chapter 1 Introduction 1
1.1 Stroke 3
1.1.1 Pathophysiology 3
1.1.2 Epidemiology 7
1.1.3 Progress in stroke treatment 10
1.1.3.1 Thrombolysis 10
1.1.3.2 Anticoagulants and antiplatelet drugs 11
1.1.3.3 Neuroprotectants 13
1.1.3.4 Combination therapy 21
1.1.3.5 Traditional Chinese Medicine 23
1.2 Studies on Ixeris sonchifolia Hance 24
1.3 Rationale and aims 28
Chapter 2 Preparation and bioactivity screening of fractions from Ixeris 30 sonchifolia Hance extract
2.1 Introduction 31
2.2 Materials and methods 32
2.2.1 Preparation of Ixeris sonchifolia Hance extract 32
2.2.1.1 Reagents and plant material 32
II
2.2.1.2 Extraction procedures 32
2.2.2 Screening assays to evaluate the bioactivities of Ixeris 32 sonchifolia Hance extract
2.2.2.1 Anticoagulation activity assay 33
2.2.2.2 In vitro MTT cell viability assay 34
2.2.2.3 Evaluation of antioxidant activities of 36 fractions 1 to 4
2.2.2.3a Determination of free radical scavenging 37 activity
2.2.2.3b Determination of inducibility of cellular 37 antioxidant enzymes and molecules
ARE-dependent luciferase reporter assay 37
Western blot analysis of cellular Nrf2 levels 38
GSH measurement 40
2.2.3 Statistical analysis 41
2.3 Results 42
2.3.1 Possible anti-coagulation activities of fractions 1 to 4 42 isolated from Ixeris sonchifolia Hance extract
2.3.2 Effects of fractions 1 to 4 isolated from Ixeris 42 sonchifolia Hance extract on the viability of H2O2-exposed PC12 cells
2.3.3 Evaluation of antioxidant activities of fractions 1 to 4 44 isolated from Ixeris sonchifolia Hance extract
2.4 Discussion and conclusions 48
Chapter 3 Isolation and characterization of components from ethyl 55 acetate fraction of Ixeris sonchifolia Hance
3.1 Introduction 56
3.2 Materials and methods 56
3.2.1 Isolation of the fractions from Ixeris Sonchifolia 56 Hance extract
III
3.2.1.1 Materials and general analytical procedures 56
3.2.1.2 Isolation procedures 57
3.2.1.3 Reactions 58
3.2.2 Quantitative HPLC analysis of compounds 1 to 5 and 59 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract
3.1.2.1 HPLC system and mobile phase 59
3.1.2.2 Sample preparations 60
3.1.2.3 Calibration graphs 60
3.1.2.4 Quality control 60
3.3 Results 61
3.3.1 Characterization of purified compounds isolated from 61 Ixeris sonchifolia Hance
3.3.2 Quantitative HPLC analysis of compounds 1 to 5 and 72 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance
3.4 Discussion and conclusions 75
Chapter 4 Cytoprotective effects of flavonoid compounds isolated from 78 ethyl acetate fraction of Ixeris sonchifolia Hance extract
4.1 Introduction 79
4.2 Materials and methods 79
4.2.1Preparation of stock solutions of flavonoid 79 compounds
4.2.2 In vitro MTT cell viability assay 80
4.2.3 Determination of LDH leakage 80
4.2.4 Flow cytometric analysis of cell death 80
4.2.5 Determination of free radical scavenging activity 81
4.2.6 ARE-dependent luciferase reporter assay 81
Western blot analysis 82
IV
GSH measurement 82
4.2.7 Statistical analysis 82
4.3 Results 82
4.3.1 Effects of flavonoid compounds 1 to 5 on the 82 viability of H2O2-exposed SH-SY5Y cells
4.3.2 Antioxidant activities of flavonoid compounds 1 to 5 88 isolated from Ixeris sonchifolia Hance extract
4.4 Discussion and conclusions 93
Chapter 5 Anti-inflammatory effects of flavonoid compounds isolated 98 from the ethyl acetate fraction of Ixeris sonchifolia Hance extract
5.1 Introduction 99
5.2 Materials and methods 99
5.2.1Preparation of stock solutions of flavonoid 99 compounds
5.2.2 Cell culture and treatments 100
5.2.3 In vitro MTT cell viability assay 101
5.2.4 Measurement of nitric oxide release 101
5.2.5 RNA extraction and reverse transcription-polymerase 102 chain reaction
5.2.6 Western blot analysis 104
5.2.7 Statistical analysis 105
5.3 Results 105
5.3.1 Effects of flavonoid compounds 1 to 5 on the 105 viability of RAW 264.7 cells and BV-2 cells
5.3.2 Effects of flavonoid compounds 1 to 5 on nitric oxide 105 production in LPS-activated RAW 264.7 cells.
5.3.3 Effects of flavonoid compounds 1 to 5 on LPS- 107 induced mRNA expression of cytokines in BV-2 cells
5.3.4 Effects of flavonoid compounds 1 to 5 on the 110 expression of Cox-2 and cPLA2 in LPS-stimulated BV-2
V
cells
5.3.5 Effects of flavonoid compounds 1 to 5 on the 111 expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 cells
5.4 Discussion and conclusions 113
Chapter 6 Possible protective effects of luteolin in rats suffering from 122 cerebral ischemia induced by middle cerebral artery occlusion
6.1 Introduction 123
6.2 Materials and methods 123
6.2.1 Experimental animals 123
6.2.2 Surgical procedures 124
6.2.3 Evaluation of neurobehavior 125
6.2.4 Measurement of infarct area 126
6.2.5 Immunohistological analysis 126
6.2.6 Statistical analysis 127
6.3 Results 127
6.3.1 Effect of luteolin on mortality and neurobehavioral 127 recovery of MCA-occluded rats
6.3.2 Effect of luteolin on infarct area in the brains of rats 128 suffering from cerebral ischemia induced by MCA occlusion
6.3.3 Immunohistochemistry staining of brain slices 129
6.4 Discussion and conclusions 132
Chapter 7 General discussion and conclusions 137
References 147
VI
Abstract
Ixeris sonchifolia Hance is a small and bitter perennial herb widely
distributed in the northeastern part of China. Its raw extract had recently been
developed into an injectable formulation showing considerable therapeutic
efficacy in stroke management. But the biological targets and the underlying
chemical components remained mostly unknown. Accordingly, (1) the
effectiveness of the herbal preparation may suffer from batch-to-batch
variations since it is difficult to ensure the presence of similar amounts of
active ingredients, and (2) some of the components present in the herbal
formation may pose certain side effects that potentially limit the therapeutic
benefits. This thesis aimed to isolate and identify constituents from Ixeris
sonchifolia Hance that display anti-stroke activities and to elucidate the
possible mechanism(s) through which the identified compound(s) exerted the
neuroprotective effects.
To accomplish this objective, the aerial part of Ixeris sonchifolia
Hance was extracted and the crude extract was partitioned into fractions. The
fractions were then subjected to screening using both cell-based in vitro
bioassays and biochemical reactions. The results had revealed that the ethyl acetate fraction, although failed to exhibit anticoagulant activities, dose- dependently protected cells from H2O2-induced cytotoxicity, scavenged DPPH free radicals, induced ARE-dependent transcriptional activity and caused upregulation of Nrf2 protein levels.
The follow-up isolation work focused on the ethyl acetate fraction revealed the presence of two classes of compounds: flavonoids and
VII
sesquiterpene lactones. In vitro bioassays conducted on the isolated flavonoids,
which were identified to be Apigenin (1), Luteolin (2), Apigenin-7-O-β-
glucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4), Luteolin-7-O-β-
glucruonopyranoside (5) and Luteolin-7-O-β-galacturonide (6) respectively,
revealed that these compounds could protect cells from H2O2-induced
cytotoxicity by scavenging free radical directly and inducing phase Ⅱ
enzymes, suggesting that the purified flavonoid compounds might exert
neuroprotective effects during the early response, characterized by excitotoxic
damage and oxidative stress, to stroke occurrence.
Considering that the second wave of cell death after a stroke incident
stems from the neuroinflammatory response, the anti-inflammatory effects of
these isolated flavonoids on the LPS-stimulated cells were next determined.
The results suggested that these flavonoid compounds might exert anti-
inflammatory activities by regulating cytokine secretion, inhibiting Cox-2
expression, as well as reducing NO release and iNOS expression.
As validation, the potential anti-stroke effects of Luteolin, a
representative of these isolated flavonoid compounds, were investigated using
rats suffering from cerebral ischemia induced by MCA occlusion. The results
revealed that while treatment with Luteolin failed to neither reduce MCAO-
induced mortality nor improve neurobehavioral recovery, it significantly
reduced the infarct area, decreased the number of cells positively stained with
anti-cleaved caspase-3 and anti-Cox-2 antibodies, suggesting that Luteolin might exert neuroprotective effects in an in vivo stroke model.
VIII
In conclusion, extraction and isolation works focused on Ixeris sonchifolia Hance revealed the presence of two categories of compounds in the ethyl acetate fraction of its raw extract: flavonoids and sesquiterpene lactones. The isolated bioactive flavonoids were found to possess potential anti-stroke effects, which at least in part were attributed to their antioxidant and anti-inflammatory activities.
IX
List of tables
Table Title Page
1.1.3.1 Summary of anti-stroke agents in preclinical studies and in 19 clinical applications
2.3.1.1 Anticoagulation activity of fractions 1 to 4 extracted from 42 Ixeris sonchifolia Hance
3.3.1.1 13C-NMR spectral data for compounds 1 to 7 65
3.3.1.2 13C-NMR spectral data for compounds 8 to 13 70
3.3.2.1 Linear regression, r2 values, detection limits and quantity 73 measurement of compounds 1 to 5 and 7 in fractions 1 to 4 by HPLC analysis.
5.3.3.1 Effects of flavonoid compounds 1 to 5 on the mRNA 109 expression of cytokines in BV-2 cells
X
List of figures
Figure Title Page
1.2.1 The aerial part of Ixeris sonchifolia Hance 25
2.3.2.1 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance 43 extract on the viability of H2O2-exposed PC12 cells.
2.3.3.1 DPPH free radical scavenging activity of fractions 1 to 4 isolated 45 from Ixeris sonchifolia Hance extract.
2.3.3.2 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance 45 extract on ARE-dependent transcriptional activity.
2.3.3.3 Representative Western blot analysis of protein levels of 46 transcription factor Nrf2 in SH-SY5Y cells treated with fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract.
2.3.3.4 Total cellular GSH levels in SH-SY5Y cells exposed to fractions 47 1 to 4 isolated from Ixeris sonchifolia Hance extract.
2.4.1 The coagulation cascade under physiological conditions and 50 experimental laboratory conditions.
2.4.2 Oxidative/electrophilic stress and Nrf2:Keap1 signaling pathway. 52
2.4.3 The glutathione system. 54
3.3.1.1 Chemical structures of compounds 1 to 7. 64
3.3.1.2 Chemical structure and important HMBC and NOESY 66 correlations of compound 8.
3.3.1.3 Chemical structures of compound 9 and 10. 68
3.3.1.4 Chemical structures of compounds 11 to 13. 71
3.3.1.5 Chemical structures of compounds 14 to 18. 71
3.3.2.1 Representative HPLC profiles for purified compounds 1 to 5 and 74 7 and fractions 1 to 4.
3.4.1 Structures of sesquiterpene lactones. 75
3.4.2 Skeletal structures of flavonoids, isoflavonoids and 76 neoflavonoids.
3.4.3 Skeletal structures of subgroups of flavonoids. 76
XI
4.3.1.1 Effects of flavonoid compounds 1 to 5 on the viability of H2O2- 85 exposed SH-SY5Y cells.
4.3.1.2 Effect of 24 h-treatment with flavonoid compounds 1 to 5 on 86 LDH release from H2O2-exposed SH-SY5Y cells.
4.3.1.3 Effect of 24 h-treatment with flavonoid compounds 1 to 5 on the 87 subG0/G1 DNA content in H2O2-exposed SH-SY5Y cells.
4.3.2.1 DPPH free radical scavenging activity of flavonoid compounds 1 88 to 5 isolated from Ixeris sonchifolia Hance extract.
4.3.2.2 Representative Western blot analysis of protein levels of 89 transcription factor Nrf2 in SH-SY5Y cells treated with flavonoid compounds 1 to 5.
4.3.2.3 Effects of flavonoid compounds 1 to 5 isolated from Ixeris 90 sonchifolia Hance extract on ARE-dependent transcriptional activity.
4.3.2.4 Representative Western blot analysis of protein levels of HO-1 91 and NQO1 in SH-SY5Y cells treated with flavonoid compounds 1 to 5.
4.3.2.5 Total cellular GSH levels in SH-SY5Y cells exposed to flavonoid 92 compounds 1 to 5 for 24 h.
4.4.1 Chemical view on the oxidoreductive activation of quercetin and 95 its possible reaction consequences.
5.3.1.1 Effects of flavonoid compounds 1 to 5 on the viability of RAW 106 264.7 cells and BV-2 cells
5.3.2.1 Effects of flavonoid compounds 1 to 5 on NO production in LPS- 107 activated RAW 264.7 cells.
5.3.3.1 Effects of flavonoid compounds 1 to 5 on LPS-induced mRNA 108 expression of cytokines in BV-2 cells.
5.3.4.1 Effects of flavonoid compounds 1 to 5 on the expression of Cox-2 111 and cPLA2 in LPS-stimulated BV-2 cells.
5.3.5.1 Effects of flavonoid compounds 1 to 5 on the expression of iNOS 112 in LPS-stimulated BV-2 cells.
5.4.1 The inflammatory cascade following a stroke event. 115
6.2.2.1 Schematic drawing of the suture placed into ECA and ICA of rat 125 brains to induce middle cerebral artery occlusion (MCAO).
XII
6.3.2.1 Analysis of the infarct area in rat brains by TTC staining. 129
6.3.3.1 Hematoxylin-Eosin staining of brain slices of rats subjected to 130 MCA occlusion and reperfusion.
6.3.3.2 Cleaved caspase-3 immunohistochemical staining of brain tissue 131 sections of rats subjected to MCA occlusion and reperfusion.
6.3.3.3 Cox-2 immunohistochemical staining of brain tissue sections of 132 rats subjected to MCA occlusion and reperfusion.
XIII
Abbreviations
AA arachidonic acid ACA anterior cerebral artery AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid AP-1 activator protein-1 aPTT activated partial thromboplastin time ARE antioxidant-responsive element ASICs acid-sensing ion channels ATP adenosine triphosphate BBB blood-brain barrier BCA bicinchoninic acid bFGF basic fibroblast growth factor BSA bovine serum albumin CCA common carotid artery cDNA cloning DNA Cox cyclooxygenase cPLA2 cytosolic phospholipase A2 Da Daltons DAMPs damage-asssociated molecular patterns DMEM Dulbecco’s modified Eagle’s medium DMSO dimethylsulphoxide DNA deoxyribonucleic acids dNTP deoxyribonucleoside triphosphate DPPH 2,2-diphenyl-1-picrylhydrazyl DTNB 5,5'-dithiobis(2-nitrobenzoic acid) ECA external carotid artery ECM extracellular matrix EDTA ehylenediamine teraacetic acid eNOS endothelial NOS EpRE electrophile-responsive element ESI electron spray ionization
XIV
FBS foetal bovine serum GABA γ-amino-butyric acid GADPH glyceraldehyde-3-phosphate dehydrogenase GPx glutathione peroxidase GR glutathione reductase GSH glutathione GSH-Px glutathione peroxidase GSK-3β glycogen synthase kinase-3 beta GSSG glutathione, oxidised form/glutathione disulfide GST glutathione S-transferase GT γ-glutamyltranspeptidase
H2O2 hydrogen peroxide HMGB-1 high mobility group box-1 HPLC high performance liquid chromatography HRP horseradish peroxidase HS heparan sulfate Hsp heat-shock proteins ICA internal carotid artery IL-1 interleukin-1 IL-10 interleukin-10 IL-6 interleukin-6 iNOS inducible NOS IR infrared spectroscopy JNK c-jun-N-terminal kinase Keap1 Kelch-like ECH-associated protein 1 LDH lactate dehydrogenase Lox lipoxygenase LPS lipopolysaccharides m.p. melting point MAPK mitogen-activated protein kinase MCA middle cerebral artery
XV
MCAO middle cerebral artery occlusion MMPs matrix metalloproteinases mRNA messenger ribonucleic acid MS mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylterazolium bromide NADPH nicotinamide adenine dinucleotide phosphate reduced form n-BuOH/HOAc/H2O normal butyl alcohol/acetic acid/water NF-κB nuclear factor kappa-B NMDA N-methyl-D-aspartate NMR nuclear magnetic resonance nNOS neuronal NOS NO nitric oxide NOS nitric oxide synthase Nrf2 Nuclear factor-erythroid 2 p45-related factor 2 -. O2 superoxide anion OH. hydroxyl radical PBS phosphate buffered saline PCA posterior cerebral artery PCR polymerase chain reaction PI propidium iodide PKC protein kinase C PLA2 phospholipase A2 PT prothrombin time RAGE receptor for advanced glycosylation end- product Redox reduction/oxidantion RNA ribonucleic acid RNS reactive nitrogen species ROS reactive oxygen species Rt retention time
XVI
RT-PCR reverse transcription polymerase chain reaction S.D. standard deviation SCA superior cerebellar artery SDS sodium dodecyl sulphate/lauryl sulphate SOD superoxide dismutase sPLA2 secreted phospholipase A2 TAE tir-acetate-EDTA buffer TBS-T tris-buffed saline with 0.05% Tween 20 TCM Traditional Chinese Medicine TEMED N,N,N',N'-tetramethyleneethyldiamine TGF-β transforming growth factor-β
XVII
Chapter 1
Introduction
1
Over 2,400 years ago, Hippocrates (460 to 370 BC) was first to describe the phenomenon of sudden paralysis, which is now known to be a consequence of stroke. Stroke, also known as cerebrovascular accident (CVA), is defined by WHO as a clinical syndrome characterized by “rapidly developing clinical signs of focal (or global) disturbance of cerebral function, with symptoms lasting 24 hours or longer or leading to death, and with no apparent cause other than a vascular one (1989; Sudlow et al., 1996; Kwan,
2001; Warlow et al., 2003).” Based on WHO’s definition, stroke includes ischemic stroke, primary intracerebral haemorrhage, and subarachnoid haemorrhage, but excludes transient ischemic attacks (TIA, which last for less than 24 hours), subdural or extradural haemorrhage, and infarction or haemorrhage secondary to infection or malignancy. Among all stroke incidents, ischemic stroke, which may be due to thrombosis, embolism, or systemic hypoperfusion, accounts for the majority of incidence (about 80%), followed by primary intracerebral (about 10%), subarachnoid haemorrhage
(about 5%) and uncertain type (about 5%).
When a stroke occurs, the perfusion-disturbed brain no longer receives adequate amounts of oxygen and glucose. This initiates the ischemic cascade and causes brain cells to die or be seriously damaged, impairing local brain function. Stroke is a medical emergency and can cause permanent neurologic damage or even death if not promptly diagnosed and treated. It is responsible for 10 - 12% of all deaths in industrialized countries and ranks the third leading cause (Kwan, 2001; Rosamond et al., 2007; Rosamond et al., 2008;
Lloyd-Jones et al., 2009, 2009). Along with the emergence of aging populations in increasing number of countries, the occurrence of stroke and
2
the associated medical and social burdens are on the rise. The principles in stroke treatment involve the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as to reduce ischemia-caused tissue damage. Unfortunately, despite decades of research, alteplase (a tissue plasminogen activator that catalyzes the conversion of plasminogen to plasmin) is still the only drug approved by FDA for stroke treatment. Therefore, finding new effective reagents efficient in stroke treatment and management has become crucial.
The following sections will give a short review on the physiopathology and epidemiology associated with stroke, followed by a summary of advancements in anti-stroke drug therapy. In addition, the application of
Traditional Chinese Medicine (TCM) in stroke management will be discussed.
Recently, the raw extract of Ixeris sonchifolia Hance was developed for use in the prevention and treatment of cardio- and cerebral-vascular diseases. As the main focus of this Ph.D study, the chemical constituents of Ixeris sonchifolia
Hance and their biological activities will also be addressed.
1.1 Stroke
1.1.1 Physiopathology
The brain cells have a high demand for energy to maintain their physiological functions such as synthesis and transport of enzymes and neurotransmitters to the every end of their nerve branches, as well as production of bioelectric signals responsible for communication throughout the nervous system. Although the brain represents only 1/40 of the body weight, it receives 15% of the cardiac output, consumes 20% of total body
3
oxygen and utilizes 25% of total body glucose (Pierre et al., 2000). Therefore, it is especially vulnerable to energy supply failure. When a stroke occurs, the perfusion-disturbed brain no longer receives adequate amount of oxygen and glucose. Aerobic respiration in these areas fails and oxygen-deprived neurons become unable to generate sufficient adenosine triphosphate (ATP) to maintain energy dependent cellular ion homeostasis (Rashidian et al., 2007).
High intracellular calcium levels increase cellular permeability and excitatory neurotransmitters (glutamate) release (Bouchelouche et al., 1989; Murphy et al., 1999; Fiskum, 2000). This in turn leads to over activation of glutamate- gated channels such as N-methyl-D-aspartate (NMDA) and α-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) channels. Excessive calcium influx is promoted, which triggers cell death by activating enzymes such as phospholipases and proteases that degrade membranes and proteins that are essential for cellular integrity (Lo et al., 2003). Furthermore, high levels of calcium, sodium and ADP in ischemic cells stimulate excessive mitochondrial oxygen radical production (Bouchelouche et al., 1989; Fiskum,
2000). Upon reperfusion, enzymatic reactions such as cyclooxygenase- dependent conversion of arachidonic acid (AA) to prostanoids and degradation of hypoxanthine will also lead to production of excessive oxygen radicals. The superoxide and hydroxyl radicals not only directly damage proteins, lipids and nucleic acids, but also cause mitochondrial membrane permealization that entails cell death (Kroemer et al., 2000). Thus, the early response to stroke is characterized by neuronal necrosis resulting from excitotoxic damage and oxidative damage.
4
The second wave of cell death in response to stroke involves the neuroinflammatory response. The surrounding microglial cells are activated and migrate to the injured site to remove cellular debris from the interstitial space (Lai et al., 2006). Activated astrocytes migrate to necrotic regions (a process known as “reactive astrogliosis”) where they upregulate expression of glial fibrillary acidic protein. At the same time, inflammatory cells within and around the injured site exhibit increased expression of proteoglycans, where in turn deposited in the extracellular space (Leonardo et al., 2008). Thus, a tissue barrier referred to as a “glial scar”, which comprises proteoglycans, and infiltrating astrocytes and microglial cells, is formed (Silver et al., 2004).
Formation of the glial scar is believed to be an innate protective mechanism so as to isolate the injured cells from the surrounding tissue. However, as time progresses, the glial scar will prevent neuroplasticity and regeneration (Chew et al., 2006). The activated microglial cells release several pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, as well as other potential cytotoxic molecules including NO, ROS and prostanoids (Lucas et al., 2006). The astrocytes are also capable of secreting inflammatory factors such as cytokines, chemokines, and NO (Swanson et al., 2004). The released proinflammatory cytokines and chemokines will further enhance recruitment of microglial cells and macrophages to the injured site, leading to a vicious cycle of excessive inflammatory events that promote cell death (Alvarez-Diaz et al., 2007).
Excessive oxygen radical formation, protease activation and DNA damage also trigger apoptotic pathways to induce cell death (Budd et al., 2000;
Yamashima, 2000; Salvesen, 2001). In the early stages of reperfusion, cysteine protease caspase 3 is cleaved and the active form in turn degrades numerous
5
substrate proteins, leading to cell demise (Namura et al., 1998). The stress- activated protein kinase, c-jun-N-terminal kinase (JNK), phosphorylates Bax and enhances its mitochondrial translocation to augment pro-apoptotic caspase activation (Lo et al., 2003). The stress-activated protein kinase p38 also mediates neuronal death (Lee et al., 2003). Cytosolic Bid facilitates cytochrome c release and promotes apoptosome formation (Wei et al., 2001).
In spinal cord ischemia, the formation of death-inducing signaling complex is involved in cell death (Qiu et al., 2002). The activation of acid-sensing ion channels (ASICs) by hydrogen ion, oxygen and glucose deprivation opens
ASIC1a channels and provides a pathway for Ca2+ entry to exacerbate the injury (Simon, 2006). These apoptotic pathways crosstalk with one another, of which, numerous molecules serve as attractive targets in the management of stroke.
Within minutes from the stroke onset, while cells in the center (core) of the ischemic region undergo progressive death (Dirnagl et al., 1999), the penumbra, a large volume of brain tissue surrounding this core, suffers a mild damage due to a residual perfusion from the collateral blood vessels
(Mergenthaler et al., 2004). These cells are viable, though functionally impaired; they can repolarize at the expense of further energy consumption and depolarize again in response to elevated levels of extracellular glutamate and potassium ions (Hossmann, 1996). Thus, the penumbra is the region responsive to therapeutic interventions, and its pathological revival, being associated with neurological improvement and recovery, forms the basis for neuroprotective therapies (Fisher, 2004).
6
1.1.2 Epidemiology
Ranked after coronary heart disease (CHD) and cancers of all types, stroke is the third leading cause of death worldwide, accounting for 10 - 12% of deaths. It is estimated that the annual incidence of stroke occurrence in the
United States is 700,000 among all races; for every 45 seconds, a stroke incident occurs and for every 3 minutes, a death incident results from stroke
(Sacco et al., 2001; Rosamond et al., 2007; Rosamond et al., 2008; Lloyd-
Jones et al., 2009, 2009). In the UK, about 130,000 people suffer a stroke each year; almost 40% of the survivors are disabled (Kwan, 2001). In India, the age-adjusted prevalence rate of stroke in 2006 was between 250 to 350 in
100,000. Specifically, the age-adjusted annual incidence rate in the urban and rural community was 105 in 100,000 and 262 in 100,000 respectively
(Banerjee et al., 2006). In China, stroke is the most frequent cause of death with an incidence of 219 in 100,000 people, which is more than 5-fold the incidence rate of myocardial infarction (Shi et al., 1989; Liu et al., 2007; Hu et al., 2008).
Strong geographic disparities in stroke incidence have been observed between countries or even in the same nations, with several countries of
Eastern Europe clocking the highest rates. Upon normalization to the
European population aged 45 to 84 years, it is reported that stroke incidence ranges from 240 in 100,000 people in Dijon, France to about 600 in 100,000 people in Novosibirsk, Russia, suggesting that environmental and genetic factors play important roles in determining stroke incidences (Warlow et al.,
2003; Donnan et al., 2008). In the United States, the incidence of stroke occurrence incidence is highest in the southeastern regions, commonly called
7
the “Stroke Belt”, and lowest in the southwestern regions (Donnan et al., 2008;
Rosamond et al., 2008; Lloyd-Jones et al., 2009). In China, the stroke incidence and prevalence follows a north-south gradient, with stroke rates almost 3-fold higher in northern China cities, suggesting environmental or dietary factors play important roles, whereas difference in racial background is unlikely to account for this observed difference on the basis that approximately 94% of Chinese are of Han ancestry (Shi et al., 1989).
Venketasubramanian et al compared the stroke prevalence among three Asian races in Singapore, the results, published in 2005, showed that Chinese had higher prevalence of stroke when compared to Indians and Malay
Singaporeans, although the difference bore no statistical significance
(Venketasubramanian N et al., 2005).
When considering the influence of gender on the epidemiology of stroke, a recent study by Appelros et al shows that a mismatch between the two genders exits. The incidence rate and prevalence is 33% and 41% higher respectively in males than in females. Among the female stroke patients, the first stroke occurs on average 4.3 years later than their male counterparts. In addition, while the incidence rates of brain infarction and intracerebral hemorrhage are higher among men, the rate of subarachnoidal hemorrhage is higher among women. Taken together, as women have longer life-span, the average age at which they suffer from stroke attacks is higher and thus the stroke is more severe, with a 1-month case fatality of 24.7% as compared to
19.7% for men (Appelros et al., 2009).
Among all the risk factors for stroke, increased age is a noncontroversial predictor. Although stroke can occur at any age, including in 8
fetus, the incidence increases exponentially from 30 years of age with 95% of strokes occurring in people aged 45 and older, and two-thirds of stroke incidents occurring in those over the age of 65 (Rosamond et al., 2007; Hu et al., 2008; Rosamond et al., 2008; Lloyd-Jones et al., 2009). In addition to age, hypertension is another undisputable factor for high risk of stroke. Subjects with hypertension have approximately two times the lifetime risk of stroke than those with blood pressure less than 120/80 mm Hg (Seshadri et al., 2006).
Other stroke risk factors include diabetes mellitus, smoking, previous stroke, heart disease, lack of exercise, low concentration of high-density lipoprotein and pregnancy. Moderate alcohol intake has been reported to provide possible protection against ischemic stroke (Hajat et al., 2001; Thom et al., 2006;
Rosamond et al., 2007; Rosamond et al., 2008; Lloyd-Jones et al., 2009).
The stroke incidence and mortality have been declining since the 1960s, with a relative reduction of about 1% per year until the late 1960s, followed by a more steeper fall of as much as 5% per year (Bonita, 1992). Recently, the rate of this decline has decreased greatly (Feigin et al., 2003). This decline in incidence and mortality is likely related to the improved control of stroke risk factors such as high blood pressure and cigarette smoking in particular, as well as an improvement in standards of living (Donnan et al., 2008). However, stroke occurrence and the number of stroke-related deaths have stably increased due to an increasingly aging population. If no more interventions are adopted, the number of global deaths is expected to rise to 6.5 million in 2015 and to 7.8 million in 2030. The burden of stroke, especially in low-income and middle-income countries, will continue to rise in the near future (Strong et al.,
2007; Donnan et al., 2008).
9
1.1.3 Progress in stroke treatment
The principles underlying stroke therapy are to restore blood supply to the affected brain area as soon as possible, and to reduce ischemia-induced tissue damage. In order to resume blood supply, thrombolytic agents or surgical procedures are adopted to remove the thrombus formed in ischemic stroke (Fernandes et al., 2000). Anticoagulants and antiplatelet agents will be administrated to prevent the recurrence of clot. Carotid endarterectomy (a surgical procedure to correct stenosis in the common carotid artery by removing thrombus formed in the blood vessel) in patients with TIA or minor stroke is another effective prevention strategy (1991; 1996; 1998). While for haemorrhage stroke, surgical occlusion of ruptured blood vessel is necessary
(van Gijn et al., 2007). In order to reduce tissue damage caused by ischemia, numerous neuroprotectants such as free radical scavengers, α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D- aspartic acid (NMDA) receptor antagonists, matrix metalloproteinase (MMP) inhibitors and anti-inflammatory reagents, are under active investigation. This section will focus on discussing the progress of drug development in stroke treatment.
1.1.3.1 Thrombolysis
Thrombolysis is the breakdown of blood clots and it may occur by pharmacological methods. By clearing the cross-linked fibrin mesh (the backbone of a clot) through enzyme-mediated proteolysis, blood flow in the occluded blood vessel can be restored. Commonly studied thromolytic agents include alteplase (rtPA) (2009), streptokinase (Lopez-Saura, 2003), urokinase
10
(Shrivastava et al., 2007), reteplase (Khatri et al., 2008), tenecteplase (Burkart et al., 2003) and desmoteplase (Hacke et al., 2009; Liebeskind, 2009;
Paciaroni et al., 2009; Tebbe et al., 2009). Despite decades of research, alteplase is still the only FDA approved drug for acute ischemic stroke treatment (2009).
Tissue plasminogen activator (tPA) is a serine protease catalyzing the conversion of plasminogen to plasmin. The latter is the major enzyme responsible for clot breakdown. When administered within 3 hours of onset, tPA can significantly reduce the neurological damage and permanent disability of stroke (2009). Nevertheless, the stringent inclusion criteria requiring tPA administration with a 3-hour post-stroke window period and presentation of no apparent hemorrhagic complications, has limited tPA’s wide application for treatment among stroke patients. Although some clinical trials extend the therapy window to 6 hours, there is an observed net increase in deaths during the first 10 days due to occurrence of secondary cerebral haemorrhage (Hacke et al., 2008; Wahlgren et al., 2008; 2009; Bluhmki et al., 2009). tPA may also damage the basal lamina of the blood vessels, resulting in edema and disruption of the blood-brain barrier (BBB) (Lo et al., 2004). Furthermore, tPA’s short half-life of 5 to 10 min has restricted its action on subsequent and continued vessel occlusions caused by newly-formed thrombus (Gravanis et al., 2008).
1.1.3.2 Anticoagulants and antiplatelet drugs
Anticoagulants are not able to re-canalize the occluded arteries, but they are useful in preventing progression of thrombosis and early recurrence
11
of embolic stroke. Intravenous administration of high doses of heparin (20,000
U/24 h) followed by oral administration of an anticoagulant is well accepted in the treatment of ischemic stroke in patients with a cardiac source of embolism
(1997). Low molecular weight heparin (Sandercock et al., 2008), aspirin
(Warlow et al., 2003; Chairangsarit et al., 2005) and warfarin (Goldstein et al.,
2006; Genovesi et al., 2009; Singer et al., 2009) are other widely investigated potential anticoagulants.
As an essential part of secondary prevention of ischemic stroke, antiplatelet treatment enrolls three commonly used drugs with independent mechanism of action; (1) the irreversible cyclo-oxygenase (Cox) inhibitor aspirin (Warlow et al., 2003; Chairangsarit et al., 2005), (2) the indirect antagonist of the adenosine diphosphate (ADP) receptor clopidogrel (Levine et al., 2003; Hassan et al., 2007; Belvis et al., 2008), and (3) the adenosine
(possesses antiplatelet activities) uptake preventer dipyridamole (Goldstein et al., 2006; De Schryver et al., 2007; Sudlow, 2007; De Schryver et al., 2008).
Among them, aspirin is the most widely studied antiplatelet drug, with daily dose of 75 – 150 mg. Higher doses have been found to exhibit no added effectiveness, and on the contrary, increase occurrence of adverse events
(Collaboration, 2002). Clopidogrel is slightly more effective than aspirin alone, but it is not cost-effective and is used only as a substitute drug for patients intolerant to aspirin (Chen et al., 2009). Glycoprotein IIb/IIIa inhibitors, such as abciximab, exert antithrombotic effect through inhibition of GpIIb/IIIa receptors on the surface of the platelets, which in turn prevent platelet aggregation and thrombus formation (Mandava et al., 2005; Mandava et al.,
2006; Puetz et al., 2006; Velat et al., 2006).
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1.1.3.3 Neuroprotectants
As discussed in Section 1.1, in contrast to the cells in the core of the ischemic region that progress to irreversible death within minutes of the stroke onset, cells in the penumbra are viable although they are functionally impaired.
They can repolarize at the expense of further energy consumption and depolarize again in response to elevated levels of extracellular glutamate and potassium ions. Upon therapeutic interventions, the penumbra’s pathological revival, being associated with neurological improvement and recovery, forms the basis for neuroprotective therapies (Fisher, 2004). Neuroprotectants refer to medications that reduce brain injury through their action on neuronal cells and tissues (Sacco et al., 2007). Numerous strategies and agents with neuroprotective potential are currently under investigation and development.
Excitotoxicity
Excitotoxicity is the pathological process caused by overactivation of glutamate receptors such as the NMDA receptor and AMPA receptor. Binding of excitotoxins like NMDA and kainic acid, as well as pathologically elevated levels of glutamate released from dying cells to these receptors results in abnormal calcium ions influx into cells. This in turn activates phospholipases, endonucleases and proteases, which catalyze the damage to cell structures. As such, strategies that interfere with this process are expected to reduce the excitotoxicity damage.
The earlier trials of NMDA receptor antagonists such as selfotel (Davis et al., 1997), aptiganel hydrochloride (Albers et al., 2001) and dextrorphan
(Albers et al., 1995) failed in part because of safety concern or poor
13
risk:benefit ratio. Two phase Ⅲ trials of eliprodil, a NMDA antagonist that binds to the polyamine modulatory site, were also stopped due to lack of clear beneficial effects (Devuyst et al., 1999, 2001). The AMPM receptor antagonist
YM872 had exhibited neuroprotective properties in animal models of acute stroke but failed an interim futility analysis in phase Ⅱtrials (Kawasaki-
Yatsugi et al., 2000; Suzuki et al., 2003; Hara et al., 2006). The calcium channel blocker nimodipine, the most widely tested neuroprotector, demonstrated a significant improvement in functional outcome for those who received nimodipine within 12 hours of stroke onset (Mohr et al., 1994).
However, an increased mortality, directly correlated with a fall in blood pressure, has cast safety concerns on its application (Wahlgren et al., 1994).
Another NMDA receptor blocker, magnesium, also blocks voltage-gated calcium channels. Its low cost, ease of use, wide experience with low risk of adverse effects and ability to penetrate the blood-brain barrier qualities make it a good neuroprotectant candidate (Sacco et al., 2007). However, a phase-Ⅲ trial conducted with magnesium given within 12 hours of ischemic stroke had disappointingly failed to demonstrate benefit (Muir et al., 2004). Attempts to reduce excitotoxicity by Gavestinel (GV150526), which acts at the glycine site of the NMDA receptor (Lees et al., 2000; Warach et al., 2006), and lubeluzole, which is a sodium channel blocker and reduces glutamate release from nerves
(Ashton et al., 1997; Scheller et al., 1997; Gandolfo et al., 2002), have similarly been unsuccessful in definitive studies (Diener et al., 2000).
As γ -amino-butyric acid (GABA) is the major inhibitory neurotransmitter in the brain and can balance the excitatory effects of
14
glutamate, developing GABA agonists is another approach to reduce excitotoxicity. Results obtained from the Early GABA-Ergic Activation Study
In Stroke trial (EGASIS) had favored diazepam treatment in various analysis
(Lodder et al., 2006). In the Clomethiazole Acute Stroke Study (CLASS), clomethiazole showed no overall benefit on stroke patients, but there was a significant improvement in functional outcome in the subgroup with large or cortical strokes (Wahlgren et al., 1999; Wahlgren et al., 1999; Lyden et al.,
2002). Further data suggests that 5-clomethiazole is safe even in patients with haemorrhagic stroke (Devuyst et al., 2001). A combination of caffeine and ethanol-caffeinol has been found to be neuroprotective through effects on the adenosine and NMDA receptors and GABAergic systems. In addition, caffeinol alone or combined with intravenous tPA is well tolerated and can be administered safely (Aronowski et al., 2003; Piriyawat et al., 2003; Martin-
Schild et al., 2009).
Repinotan (BAYx3702), a serotonin agonist at the 5HT1A receptor
(Berends et al., 2005; Lutsep, 2005), and ONO-2506, an agent that modulates the uptake capacity of glutamate transporters and expression of GABA receptors (de Paulis, 2003; Asano et al., 2005), had also failed in clinical trials despite their promise outcomes in previous animal models.
Free radical scavengers
Mitochondria utilize the vast majority of O2 to generate energy in the form of ATP. During cellular respiration, 1 to 3% of total reduced O2 leaks to be consumed by the NAD(P)H oxidase complexes to form reactive oxygen
.- .- species superoxide anion O2 . The cellular enzymatic systems involved in O2
15
elimination include superoxide dismutase (SOD), which catalyzes the
.- conversion of O2 to H2O2, and catalase, which in turn coverts H2O2 to H2O.
Glutathione (GSH) and its affiliated enzymes, glutathione peroxidase (GPx) and glutathione reductase (GR), are also involved in the reduction of oxidative stress. Under normal physiological circumstances, the free radical production and elimination rates are almost equal to maintain a balanced redox potential.
During cerebral ischemia, elevated intracellular Ca2+ activates phospholipase
A2 (cPLA2), which releases arachidonic acid and thus initiates the formation of free radicals via the cyclo-oxygenase and lipoxygenase pathways (Handlogten et al., 2001). Increased level of free radicals disrupts redox homeostasis and cause damages to the cells through several mechanisms, including lipid peroxidation, tyrosine nitration, sulfhydryl oxidation, nitrosylation and DNA breakage. Anti-stroke strategies that involve inhibiting the production, increasing the removal or fostering the degradation of free radicals, have been developed to reduce damage processes induced by free radicals. Ebselen, a mimic of glutathione peroxidase, possesses glutathione peroxidase-like activity and can react with peroxynitrite. Treatment with ebselen had been previously found to delay ischemia-induced injury processes in experimental models when the animals were sacrificed between 4 - 24 h after ischemia, but not if the animals were sacrificed at a later time point (Lynch et al., 2004;
Salom et al., 2004). Clinical trials have also suggested limited neuroprotective efficacy of Ebselen on stroke patients (Yamaguchi et al., 1998; Yamagata et al., 2008). Despite the benefit acquired from previous animal experiments, development of another free radical scavenger, Tirilazad, was also stopped because of safety concerns and limited efficacy on clinical trials (1994; Haley,
16
1998; Bath et al., 2001; Sena et al., 2007). Edaravone shows strong antioxidant actions by trapping hydroxyl radicals. In animal models, it can prevent brain edema after ischemia and reperfusion injury. Moreover, edaravone improves endothelial function in smokers through an increase in nitric oxide. The drug has been approved by the regulatory authority in Japan for the treatment of stroke patients. However, the development status of this compound outside Japan is still unclear (Toyoda et al., 2004; Kano et al., 2005;
Imai et al., 2006; Kitagawa, 2006). Nitrone-derived free radical trapping agents, first developed as tools for studying free radical chemistry, have also been tested for their efficacies in ischemic injury. As the most widely studied free radical trapping agent, NXY-059 had been shown to improve functional outcomes in small trials conducted in both animals and humans. However, the results of this trial could not be replicated in the follow-up trial; its development has since been terminated by its drug company AstraZeneca and it seems likely that there will be no further clinical study on this compound
(Marshall et al., 2001; Zhao et al., 2001; Lees et al., 2003; Green et al., 2005;
Koziol et al., 2006; Lyden et al., 2007).
Anti-inflammatory
The early response to stroke is characterized by excitotoxic damage and free radical attack, while the second wave of cell death comes from the neuroinflammatory response. Inflammatory processes are initiated by cytokines, adhesion molecules, prostanoid mediators of inflammation and nitric oxide (NO) and may progress for days and weeks. Inflammatory intervention serves an attractive therapeutic strategy in stroke treatment
(Michael et al., 2004; Sacco et al., 2007). 17
Cytokines have received most attention in relation to ischemic attack.
However, the biological signaling pathways are extremely complicated and certain cytokines can have different effects depending on the context
(Rothwell, 1997; Allan et al., 2001). Poly(ADP-ribose)polymerase inhibitor
PJ34 brings about blockade of ischemia-induced increase of TNF-α in mice, with a sustained effect for up to 24 h (Haddad et al., 2006). Modulators that target adhesion molecules have been tested in human trials. Enlimomab, a murine antibody directed against intercellular adhesion molecule-1, was previously found to yield worse outcomes in human trials (2001; Furuya et al.,
2001). A subsequent study of the humanized antibody Hu23F2G directed against CD18 had been terminated in a previous study due to unfavorable interim results (Yenari et al., 1998; Becker, 2002; Sacco et al., 2007). Non antibody-based small molecule antagonists of cell adhesion are now emerging
(Sacco et al., 2007). Immunosuppressive agent, Tacrolimus (FK506), has been shown to ameliorate the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells, as well as to limit attachment of granulocytes and platelets to blood vessels by inhibiting the expression of adhesion molecules (Furuichi et al., 2007; Maeda et al., 2009). Nevertheless, corticosteroids, to date, have not been demonstrated to exert effective neuroprotection (Sacco et al., 2007).
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Table 1.1.3.1 Summary of anti-stroke agents in preclinical studies and in clinical applications Objective Mechanism Anti-stroke agents in preclinical studies or in clinical use
Thrombolysis alteplase (rtPA), urokinase, streptokinase, reteplase, tenecteplase, desmoteplase
Anticoagulants heparin, warfarin
To restore the blood Cyclo-oxygenase inhibitor: aspirin supply Indirect antagonist of the adenosine diphosphate receptor: clopidogrel Antiplatelet drugs Adenosine uptake preventer: dipyridamole
Glycoprotein IIb/IIIa inhibitor: abciximab
NMDA receptor antagonist: selfotel, aptiganel hydrochloride, dextrorphan
AMPM receptor antagonist: YM872, magnesium, gavestinel
Excitotoxicity antagonist Calcium channel blocker: nimodipine
Sodium channel blocker: lubeluzole
To reduce the GABA agonist: diazepam, clomethiazole ischemia-induced Free radical scavengers ebselen, tirilazad, NXY-059, edaravone brain tissue damage Poly (ADP-ribose) polymerase inhibitor: PJ34 Anti-inflammatory agents Intercellular adhesion molecule-1 inhibitor: enlimomab, Hu23F2G
Immuno-suppressive agent: tacrolimus (FK506)
hydroxymethylglutaryl coenzyme A reductase inhibitors (statins), albumin, piracetam, bFGF, Others insulin-like growth factor, brain-derived neurotrophic factor and osteogenic protein 1
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Others
Since multiple pathways are activated resulting in neuron injury following stroke occurrence, it is rational to develop “broad spectrum neuroprotective agents” which activate multiple pathways to achieve the best therapeutic outcome. Hydroxymethylglutaryl coenzyme A reductase inhibitors
(statins) have gained increasing attention recently. The neuroprotective effects of statins are believed to be related to improved endothelial function, increased cerebral blood flow and reduced inflammation (2006; Amarenco et al., 2006;
Sacco et al., 2007; Zhang et al., 2007; Zhang et al., 2009). Results from preclinical studies and phase І trial have demonstrated the potential neuroprotective effects of albumin mediated through mechanisms including haemodilution, binding to free fatty acid, inhibition of free radical production, inhibition of platelet activation and maintenance of microvascular patency
(Ginsberg, 2003). Nootropic drugs, a class of memory enhancers that are purported to improve mental functions such as cognition, memory, intelligence, motivation, attention and concentration, are supposed to improve membrane bound cell functions, including ATP production, neurotransmission, and secondary messenger activity. In a phase Ⅲ trial, piracetam had been shown to improve the neurological scores of stroke patients, particularly in patients with moderate and severe degree damage (De Deyn et al., 1998).
Growth factors, such as basic fibroblast growth factor (bFGF), insulin-like growth factor, brain-derived neurotrophic factor and osteogenic protein 1, have also been found to possess both acute phase effects and an action on recovery by reinforcing plasticity phenomena (Greenberg et al., 2006; Kalluri et al., 2008; Lanfranconi et al., 2009). Earlier observational studies had
20
suggested a protective effect of estrogens on cardiovascular risk among women who received postmenopausal hormone replacement therapy
(Mendelsohn et al., 1999; Mendelsohn, 2002; Cho et al., 2003). However, there is now strong evidence that hormone replacement therapy increases the risk of stroke and venous thromboembolism. Thus, such treatments are not recommended in stroke treatmen (Beral et al., 2002).
1.1.3.4 Combination therapy Ischemia in stroke leads to excitotoxic cell death, apoptosis, complex cascades of local inflammatory response, and remodeling of neuronal function
(Chaudhuri, 2007). Since several pathways leading to cell death are activated in cerebral ischemia, in anti-stroke treatment, it is logical to combine a cocktail of drugs each targeting at distinct pathways.
In consideration that the platelet aggregation within the clot might resist the dissolution ability of fibrinolytic agents, glycoprotein IIb/IIIa inhibitors have been adopted in combination with tPA in the treatment of myocardial infarction (Ohman et al., 1997). To date, results from studies have demonstrated improved reperfusion without increasing bleeding rates. For instance, a study involving the combination use of intravenous abciximab followed by intra-arterial urokinase has found the recanalisation rate was increased by 2-fold as compared to the use of intra-arterial urokinase alone in stroke patients (Lee et al., 2002). In another study involving intravenous administration of tPA followed by argatroban, a direct thrombin inhibitor, the combination treatment has also resulted in improved recanalisation rates (Sugg et al., 2006). Although independent treatments may not necessarily yield additive results, combination therapies can render reduction in dosages for
21
each agent, thereby reducing the occurrence of adverse events (Lo et al., 2003).
In the Emergency Management of Stroke Bridging Trial, intravenous administration of two-thirds dose of tPA, followed by intra-arterial administration of tPA had resulted in improvement in the recanalisation rates as compared to intra-arterial administration of tPA alone. Nevertheless, the risk of symptomatic intracerebral haemorrhage did not differ between these two groups (Swarnkar et al., 1999). The efficacy of aspirin in combination with clopidogrel in the secondary prevention of vascular events in patients with stroke or systemic arterial embolism is still currently under investigation
(2008).
The combination of neuroprotective therapy with clot-lysing drugs reduces reperfusion injury and inhibits downstream targets in cell death cascades (Lo et al., 2003). Synergy is also observed between neuroprotectants
(Schmid-Elsaesser et al., 1999; Lo et al., 2003). Co-administration of an
NMDA receptor antagonist with GABA receptor agonists (Lyden et al., 2000), free radical scavengers (Barth et al., 1996), protein synthesis inhibitor cycloheximide (Du et al., 1996), caspase inhibitors (Ma et al., 1998) or growth factors such as bFGF (Barth et al., 1996), has demonstrated some success in animal models. In the combination treatments with caspase inhibitors, doses of bFGF or NMDA antagonists required for effective therapy are found to be reduced, and the therapeutic window extended (Ma et al., 1998; Ma et al.,
2001).
It can be anticipated that the combination approaches that encompass reperfusion, anti-inflammation, possibly neuroprotection, and reversal of excitotoxicity, is superior as compared to a single specific treatment. The main 22
challenge will be to find the right combination which is safe, efficacious, cost- effective, and exerts its therapeutic effects on both a short-term and long-term basis (Chaudhuri, 2007).
1.1.3.5 Traditional Chinese Medicine
Traditional Chinese Medicine (TCM) is a system of medical thought and practice that is distinctly different from Western Medicine. It postulates an unique and inextricable relationship between the human body and its environment. A disease state, where the body loses the ability to maintain normal physiological function, often ensues when this relationship is compromised. When disease occurs, Chinese medicine practitioners use herbal medicine derived from plant, animal and mineral sources to restore the compromised relationship in a sick individual.
The use of TCM in stroke therepy has a long history that can be dated back to as early as 2000 years ago. Scientific literature available in database contains a large body of articles devoted to the clinical evaluation of the use of
TCM in stroke therapy (Gong et al., 1999, 2002; Mattson et al., 2002).
Clinical data from these studies indicate that anti-stroke TCMs are effective and thus provide a basis for the pharmacological use of TCM in stroke management. However, as neither the biological targets nor the chemical compounds acting at these targets in most of the TCMs are known, it is challenging to ensure that a given herbal preparation used in a patient actually contains the active ingredient(s) underlying the purported therapeutic effect.
Hence, evidence-based evaluation of the effectiveness of herbal anti-stroke medicines in practice is usually difficult.
23
In an attempt to address some of these issues, a great deal of pharmacological research has been undertaken to review and establish reliable composite formulae of natural products in TCM such as “Hu Xin Dan”,
“Puerarin injection” and “Shen Mai injection” (Wang et al., 1995; Lee et al.,
2004; Fattorusso et al., 2006; Ichikawa et al., 2006). In addition, research aimed at the characterization of molecular targets and effects of herbal medicine using either crude extracts of TCM natural products or isolated compounds possessing bioactivities, has also been conducted (Sucher, 2006).
It is reasoned that the elucidation of molecular mechanisms underlying the anti-stroke effects of herbal anti-stroke medications, and the identification of chemical compounds responsible for these effects would be useful for the hypothesis-guided evaluation of their therapeutic efficacies.
In summary, the long history of use of anti-stroke TCM in China indicates that TCM preparations are to a certain extent effective in the management of stroke. There are now more than 100 traditional medicines for use in stroke therapy (Wu, 1989). Identification of molecular targets and subsequent identification of chemical compounds that bind to these targets in traditional Chinese stroke medicine therefore are pivotal in the discovery of natural products that may serve as leads for the development of safe and effective anti-stroke medications.
1.2 Studies on Ixeris sonchifolia Hance
Ixeris sonchifolia Hance (Fig.1.2.1), which belongs to the Compositae family, is a small and bitter perennial herb widely distributed in the northeastern part of China (Feng et al., 2001; He et al., 2006; Na et al., 2007).
24
It is a well-known folk medicine and its whole plant has been used for many years in China to invigorate blood circulation, normalize menstruation and eliminate blood stasis to relieve pain (Feng et al., 2001; He et al., 2006; Na et al., 2007). Extensive phytochemical investigations on this herb have been carried out, and many components isolated from this plant exhibit significant bioactivities. Compounds isolated from Ixeris sonchifolia Hance can be classified into sesquiterpene lactones, triterpenes, steroids, phenylpropanoids, phenols, amino acids and fatty alcohols/acids.
Fig.1.2.1 The aerial part of Ixeris sonchifolia Hance
In previous studies, the extract of Ixeris sonchifolia Hance was reported to be able to decrease apoptosis of cardiomyocytes in overtrained rats by regulating the expression of Bax and Bcl-2, decreasing Bax/Bcl-2 ratio
(Rong et al., 2007). There are also studies reporting the use of Ixeris sonchifolia Hance extract in the preparation of medical agents for treating coronary heart diseases (Ma et al., 2009). In another study, cumulation administration of the water extract of Ixeris sonchifolia Hance was not observed to affect the vasomotion of aortic rings in basal tension, but the extract could induce a significant concentration-dependent relaxation of the intact endothelium rings preconstricted by phenylephrine (Lin et al., 2007).
The relaxation was inhibited by preincubation with N-nitro-L-arginine methyl
25
ester, methylene blue and indomethacin. The bioactivities were therefore deduced to be mediated through the nitric oxide-guanylyl cyclase and cyclooxygenase pathway (Lin et al., 2007). Based on these preliminary results demonstrating potential therapeutic benefits of Ixeris sonchifolia Hance, development of this herb into a promising medicinal preparation for the prevention and treatment of cardio- and cerebral-vascular diseases is warranted.
Capitalizing on the unique properties of this herb, an injectible formulation containing a raw extract of Ixeris sonchifolia Hance (Diemailing
Injection) was recently developed and launched by Liaoning Institute of
Pharmaceutical Industry, China. The product is reported to have effects such as vascular smooth muscle relaxation, enhancement of cerebral blood flow, improvement of the activity of fibrinolytic enzyme activity and inhibition of thrombus formation. In an animal study involving anesthetized open-chest dogs, the Diemailing injection was found to decrease myocardial infarct size, activity of serum creatine kinase and lactate dehydrogenase, contents of serum free fatty acid and lipid peroxide, as well as increase the activity of serum
SOD. In addition, myocardial blood flow was increased with an accompanying decrease in coronary vascular resistance (Wang et al., 2002). In another animal studies involving acute myocardial ischemic rats, the Diemailing
Injection was also demonstrated to decrease the activities of serum creatine kinase and lactate dehydrogenase, as well as diminish the myocardial ischemia size and ST segment abnormalities in the electrocardiography (ECG) (Wang et al., 2006; Zhou et al., 2007).
26
The therapeutic efficacy of Diemailing Injection in the management of stroke was investigated in clinical trials conducted by the Xiyuan Hospital of the Chinese Academy of Traditional Chinese Medicine, and five other hospital sites in China. In these studies, Diemailing Injection was shown to exhibit better therapeutic efficiency than the control medicine (standard stroke therapy proposal, 2000) in restoring stroke patients’ neural functions and recovery of daily vasomotor activities. Based on these favorable outcomes, the product has since been added onto to The National Basic Medical Insurance and Workman
Compensation Insurance Pharmaceutical List.
Based on the findings of the conducted clinical trials, Diemailing
Injection can be explored as an alternative treatment option in stroke management. Unfortunately, the biological targets and the chemical components underlying the activities of Ixeris sonchifolia Hance extract remains mostly unknown. Administration of a heterogenous herbal formulation does have its drawbacks. Firstly, the effectiveness of the herbal preparation may suffer from batch-to-batch variations since it is usually difficult to ensure the presence of similar amounts of active ingredients.
Secondly, some of the components present in the herbal formation may pose certain side effects that potentially limit the therapeutic benefits. For example,
Zhang et al had previously reported the occurrence of asthma (Zhang, 2007) and Guan et al had reported the occurrence of lactation incidence during the treatment with Diemailing injection (Guan et al., 2004). Therefore, elucidation of molecular targets responsible for the effects of Ixeris sonchifolia Hance and the subsequent identification of the chemical compounds contributing to the observed molecular effects, will not only serve as surrogate markers in the
27
evaluation of the therapeutic efficacy of Ixeris sonchifolia Hance, but also provide a basis for development of a more defined anti-stroke product with assured therapeutic effects and controllable formulation quality.
1.3 Rationale and aims
Based on the therapeutic outcomes of Diemailing Injection, which contains a raw extract of Ixeris sonchifolia Hance, it was hypothesized that the phytochemical constituents in this herb would possess anti-stroke potential with anticoagulation, antioxidant and anti-inflammatory activities. As such, the study aimed to elucidate the molecular targets responsible for the effects of
Ixeris sonchifolia Hance, followed by the identification of components contributing to these molecular effects. The specific objectives of this Ph.D study were:
(1) To isolate and identify compounds with potential anti-stroke properties from Ixeris sonchifolia Hance through conducting bioassay-guided extraction, fractionation and purification experiments.
(2) To elucidate the molecular mechanism(s) underlying the potential anti-stroke properties of these compounds. Particularly, the potential antioxidant and anti-inflammatory activities of the compounds would be explored.
(3) To validate the anti-stroke activity of these compounds using well- established animal stroke model.
To accomplish these objectives, crude extracts of Ixeris sonchifolia
Hance would be subjected to screening using both cell-based and biochemical reactions in vitro bioassays. Extracts that display desirable bioactivity would
28
be subjected to further fractionation until finally pure bioactive compounds were obtained. The selection of assays to elucidate the molecular mechanism(s) of potential anti-stroke compounds would be based on the current understanding of the pathophysiology of neuronal injury during the reperfusion phase following an acute ischemic stroke attack. Currently, five key mechanisms with well-defined molecular targets are known to be involved in the cascade of events leading to neuronal injury and death in acute stroke.
These mechanisms include (1) activation of receptors for excitatory amino acids, (2) calcium influx, (3) generation of free radicals, (4) programmed cell death, and (5) inflammation. It is plausible that Ixeris sonchifolia Hance may contain neuroprotective compounds with the ability to abate any of the five mechanisms. But to ensure the feasibility of the current proposal, efforts would be mainly directed towards the isolation of compounds that could exhibit either antioxidant, anti-apoptotic or anti-inflammatory activities. As
Ixeris sonchifolia Hance is known for its fibrinolytic and thrombolytic activity, another objective of this thesis was to test if the potential anti-stroke effect of
Ixeris sonchifolia Hance was partly attributed to the ability of its components to improve cerebral blood flow via their fibrinolytic and thrombolytic activity.
Finally, candidate compounds identified above would be evaluated in a rat model of transient forebrain ischemia. This model, where occlusion of the middle cerebral artery is performed on the rats, closely resembles clinical stroke due to focal ischemic attacks and has been shown to be sensitive to treatment with neuroprotective compounds that function via a number of mechanisms. The results of investigations undertaken to achieve each aim highlighted are discussed in Chapter 2, 3, 4, 5 and 6.
29
Chapter 2
Preparation and bioactivity screening of fractions from Ixeris
sonchifolia Hance extract
30
2.1 Introduction
Ixeris sonchifolia Hance is a well-known folk medicine and its whole plant has been used for many years in China to invigorate blood circulation, normalize menstruation and eliminate blood stasis to relieve pain (Feng et al.,
2001; He et al., 2006; Na et al., 2007). Previous studies had also provided preliminary indication of the potential of developing Ixeris sonchifolia Hance into a medicinal preparation for the treatment of cerebro-vascular diseases.
However, the chemical components accounting for the corresponding bioactivities have not been identified. This chapter would employ an approach involving extraction, fractionation and bioactivity screening on Ixeris sonchifolia Hance with the ultimate goal to isolate and identify the chemical components underlying the potential anti-stroke effects of this herb. Initiation of the isolation work involved preparation of an extract from the aerial part of
Ixeris sonchifolia Hance and further partitioning of the extract into fractions according to their polarities. The fractions would be then subjected to screening using both cell-based in vitro bioassays and biochemical reactions.
The selection of assays to screen the fractions was based on current understanding of the pathophysiology of neuronal injury during the reperfusion phase subsequent to acute ischemic stroke. In summary, the anticoagulation activities of the fractions, which would relate to the ability to prevent the progression of thrombosis and early recurrence of embolic stroke, and the antioxidant activities of the fractions, which would relate to the ability to reduce ischemia-caused tissue damage, were evaluated with in vitro bioassays. The cytoprotective effect of these fractions on H2O2-exposed cells was also evaluated using the MTT cell viability assay.
31
2.2 Materials and methods
2.2.1 Preparation of Ixeris sonchifolia Hance extract
2.2.1.1 Reagents and plant materials
The aerial part of Ixeris sonchifolia Hance was purchased from Beijing
DDLD Medicinal Technology Development Co. Ltd (Beijing, China) in
September 2006 and the identity was verified by Dr. Zhou Liang (Department of Pharmacy, National University of Singapore). The voucher specimen had been deposited in the laboratory of Dr. Chew Eng-Hui, Department of
Pharmacy, National University of Singapore (No. 060902). All chemicals used in this section were purchased from Tedia Company Inc. (OH, USA) unless otherwise noted.
2.2.1.2 Extraction procedures
The aerial part of Ixeris sonchifolia Hance (5 kg) was cut into small pieces (3 cm) and extracted three times with 95% C2H5OH at 60 °C for 3 h.
The extract was evaporated to dryness under reduced pressure to yield a dried residue. The dried crude extract (420 g) was dissolved in 800 ml of distilled water and subjected to liquid-liquid extraction with equal volumes of n-hexane, chloroform and ethyl acetate in a successive order. In all, four extracts were obtained, namely n-hexane fraction (Fr. 1, 100 g), chloroform fraction (Fr. 2,
80 g), ethyl acetate fraction (Fr. 3, 50 g) and water fraction (Fr. 4, 190 g). Each fraction was accurately weighed and dissolved in DMSO to constitute 20 mg/ml stock solutions that were stored at -20 ℃.
2.2.2 Screening assays to evaluate the bioactivities of Ixeris sonchifolia Hance extract 32
The principles in stroke treatment involve the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as to reduce ischemia-caused tissue damage. To identify the fraction(s) possessing significant bioactivities for potential stroke treatment, fractions 1 to 4 were assayed for their possible anticoagulation and antioxidant efficacies. The fraction(s) identified to exhibit the strongest bioactivities was then subjected to further fractionation and purification experiments, which would be the focus of discussion in Chapter 3.
2.2.2.1 Anticoagulation activity assay
Considering that anticoagulant therapy showed significant clinical advantage in preventing progression of thrombosis and early recurrence of embolic stroke (see Section 1.1.3.2), and Diemailing Injection could inhibit thrombus formation (see Section 1.2), the anticoagulant effects of Fr. 1 to 4 were tested by assaying the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. PT is the time taken for plasma to clot after addition of tissue factor; it measures the coagulation efficacy of the extrinsic pathway. On the other hand, aPTT measures the efficacy of the intrinsic coagulation pathway (also known as the contact activation pathway).
Thromborel® S, Actin® FSL, Ci-Trol® coagulation Control Level 1 and Calcium chloride solution were all obtained from Dade Behring (Marburg,
Germany). Clotting time was detected with a Sysmex CA-500 Automated
Blood Coagulation Analyzer (Sysmex Corporation, Japan). Briefly, once the coagulation cascade was initiated, the test tube was irradiated with light at 660 nm and the change in scattered light due to turbidity change during conversion
33
of fibrinogen to fibrin was measured. The clotting time was defined as the time taken to reach 50% change in scattered light.
All procedures were carried out in accordance to the manufacturer’s protocols. Briefly, the lyophilized pooled human plasma (Ci-Trol® level 1) was freshly reconstituted with distilled water. Different concentrations of Fr. 1 to 4 (50 µl) were then added to the reconstituted plasma (100 µl) to obtain the plasma mixture. PBS and Heparin (Mayne Pharma Pty Ltd, Australia) were added to the reconstituted plasma to serve as blank control and positive control respectively. For determination of PT, to initiate the coagulation process, 100
µl of thromboplastin (Thromborel® S) was added to 50 µl of the plasma mixture, which had already been incubated at 37 ℃ for 180 s. The reaction system was monitored with the Automated Blood Coagulation Analyzer for
120 s. For determination of aPTT, 50 µl of plasma mixture was incubated at
37 ℃ for 60 s before the addition of 50 µl of soy and rabbit brain phosphatides
(Actin® FSL). The mixture was incubated at 37 ℃ for another 180 s, after which 50 µl of calcium chloride solution supplied within the assay kits was added to initiate the coagulation cascade and the reaction system was monitored for 190 s (Lau et al., 2009).
2.2.2.2 In vitro MTT cell viability assay
PC12 rat pheochromocytoma cells were obtained from Dr. Liou Yih
Cherng (Department of Biological Sciences, National University of Singapore) and grown in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% horse serum (Hyclone, UT, USA) and 5% bovine serum (Hyclone, UT, USA), at 37 ℃ in a humidified 5% CO2 atmosphere.
34
Cells were harvested from maintenance cultures when they reach 70 -
80% confluence. By gently syringing the cell suspension 3 to 4 times through a 23 G needle, a single cell suspension was obtained. Cell counts were obtained using a haemocytometer. The cell suspension was diluted appropriately to achieve the desired cell density in 100 µl of culture medium for inoculation into each well of a 96-well plate. The seeding cell density for
PC12 cells was 5000 cells/well. In order to minimize medium evaporation from the plate, wells in the 2 peripheral lanes were left cell-free (blank wells) and filled with 200 µl of medium. The plates were then incubated for approximately 48 h at 37 ℃ to allow cell attachment. Serial drug dilutions of the fractions 1 to 4 DMSO stock solutions were prepared in medium immediately before each assay, of which an aliquot of 100 µl was added into each well to achieve final concentration of 1 µM, 10 µM, 20 µM, 50 µM and
100 µM (n = 8). To the control wells, 100 µl of medium was added. Previous assays had been carried out to verify that cell viability was not affected by the amount of DMSO added into the drug treated wells. To produce oxidative stress, H2O2, freshly prepared from 30% stock solution with PBS, was introduced (final concentration 150 µM) 30 min after the drug treatment. The drug treated plates were incubated for another 12 h at 37 ℃, after which 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT; purchased from Sigma-
Aldrich Pte Ltd, MO, USA) solution (5 mg/ml in PBS) was added into each well. Following MTT addition, the plates were reincubated for 4 h at 37℃ to allow metabolic conversion of MTT by dehydrogenases in viable cells to insoluble formazan crystals. The medium and any unconverted MTT was aspirated, 150 µl of DMSO:glycine buffer (pH 10.5, 4:1) was added to each
35
well and the plates were shaken to ensure complete formazan solubilization.
Absorbance was then read at 550 nm on an Infinite® M200 PRO (Tecan
Group Ltd, Switzerland) plate reader and data was automatically transferred to a computer, where the absorbance readings (corrected to background absorbance in blank wells) were displayed. The intensity of absorbance was directly proportional to cell viability. The assay for each fraction treatment was performed at least three times and the mean absorbance value at each drug concentration was recorded for plotting the dose-response graph (Hong et al.,
2004; Sun et al., 2005; Liu et al., 2007).
2.2.2.3 Evaluation of antioxidant activities of fractions 1 to 4
Redox homeostasis is critical for cell survival and growth, and under normal physiologic conditions, this is achieved by a number of cellular antioxidant systems including the two major thiol redox systems: the glutathione and thioredoxin systems (Bjornstedt et al., 1994; Mulder et al.,
1997; Dixon et al., 1998; Casagrande et al., 2002; Holmgren et al., 2005;
Koharyova et al., 2008). During cerebral ischemia, elevated intracellular Ca2+ activates a series of reaction pathways and initiates the formation of free radicals. The increase in free radical levels disrupts the redox homeostasis and cause cellular damage through lipid peroxidation, tyrosine nitration, sulfhydryl oxidation and nitrosylation, as well as DNA strand breakage. Strategies to re- establish redox homeostasis by inhibiting the production, increasing the removal or fostering the degradation of free radicals, have been investigated to reduce free radical-induced damage processes in stroke. This chapter had adopted two approaches to assess the antioxidant activities of Fr. 1 to 4: (1)
36
direct measurement of free radical scavenging activities and (2) detection of the induction on cellular antioxidant enzymes and molecules.
2.2.2.3a Determination of free radical scavenging activity
To measure the direct free radical scavenging activity, Fr. 1 to 4 were dissolved in 100 µl acetonitrile and added to an equal volume of acetonitrile solution containing 100 µM 2,2-diphenyl-1-picrylhydrazyl (DPPH; purchased from Sigma-Aldrich Pte Ltd, USA). The mixture were agitated vigorously and then kept in the dark at room temperature for 30 min. Absorbance (A) of residual DPPH was read at 516 nm and compared to a positive control solution of DPPH with Trolox (Sigma-Aldrich Pte Ltd, USA) and a solution containing only DPPH (Ono et al., 2008; Sugiyama et al., 2009). The DPPH free radical scavenging rate was calculated using the following formula: Scavenging Rate
= (Abs DPPH-only solution – Abs compound-treated DPPH solution) / Abs DPPH-only solution
×100% (Awika et al., 2003; Chung et al., 2007; Obied et al., 2007).
2.2.2.3b Determination of inducibility of cellular antioxidant enzymes and molecules
ARE-dependent luciferase reporter assay
Human-derived SH-SY5Y neuroblastoma cells (CRL-2266) were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured at 37 ℃, 5% CO2 in Dulbecco’s Modified Eagle’s Medium/Nutrient
Mixture F-12 Ham (1:1) culture medium supplemented with 10 % fetal bovine serum (Hyclone, UT, USA), 100 µg/ml streptomycin, 100 U/ml penicillin G and 1.5 g/L sodium bicarbonate. Cells were seeded in 12-well culture plates and at approximately 60% confluency, they were cotransfected with 1.0 μg 37
pGL3-ARE plasmid (gift from Professor Alan Porter, IMCB, Singapore) and
0.05 μg pRL-CMV plasmid (Promega, Madison, WI) per well for 24 h. After which the cells were treated with different concentrations of Fr. 1 to 4, as well as 10 µM sulforaphane (used as a positive control in this study) for 10 h.
Firefly and renilla luciferase activities were assayed using the Dual
Luciferase® Assay System (Promega, Madison, WI) and results were expressed as firefly luciferase activity normalized to renilla luciferase activity.
Each drug treatment was carried out in duplicates and means from three independent experiments (± SEM) were obtained. Statistical significance between treatment control groups were analyzed using Student’s t - test.
Western blot analysis of cellular Nrf2 levels
Drug treatment SH-SY5Y cells were seeded in 6-well culture plates and allowed to grow to 60 - 70% confluency before addition of Fr. 1 to 4 at 10 and 50 µg/ml for 8, 12 and 24 h. Cell treatment with 10 µM sulforaphane
(used as a positive control in this study), as well as 25 µM MG-132 (a proteasome inhibitor that causes accumulation of Nrf2 through preventing its degradation) was also carried out.
Preparation of cell lysates Following desired treatment, medium containing detached cells was collected. Attached cells were collected using a cell scraper. Both attached and floating cells were pooled and pelleted by centrifugation (1,200 rpm, 4 ℃, 5 min). The pellet was washed twice with cold PBS, and resuspended in an appropriate volume (0.1 - 0.5 ml, depending on size of cell pellet) of ice-cold lysis buffer (Cell Signaling Technology Inc.,
MA, USA) containing freshly added protease inhibitors. The lysis buffer was
38
composed of 20 mM Tris-HCl (PH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta- glycerophosphate, 1 mM Na3VO4 and 1 µg/ml leupeptin. The protease inhibitors added were 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and 5 µg/ml pepstatin A. After brief sonication on ice, cell lysates were centrifuged at 14,000 × g for 15 min at 4 ℃ to remove cell debris. The supernatant was collected and protein content was quantified using a Pierce®
BCA Protien Assay Kit (Pierce, Rockford, IL) according to the manufacturer’s instruction. This protein assay was conducted on a 96-well plate by adding
200 µl of BCA reaction solution to each well containing 20 µl of protein samples or different concentration of BSA. After incubation of the plate in
37 ℃ for 30 min, absorbance values at 562 nm were measured using an
Infinite® M200 PRO (Tecan Group Ltd, Switzerland) microplate reader.
Protein concentration of cell lysates was calculated against BSA standard calibration curve.
Western blot analysis Protein mixture in cell lysates was separated using SDS-PAGE based on their molecular size. Resolving gel (10%; solution formula for two gels: 9.6 ml distilled water, 5 ml 40% acrylamide, 5 ml 1.5 M
Tris pH 8.8, 200 µl 10% SDS, 200 µl 10% ammonium persulfate and 8 µl
TEMED) and stacking gel (5%; solution formula for two gels: 7.25 ml distilled water, 1.25 ml 40% acrylamide, 1.25 ml 1 M Tris pH 6.3, 100 µl 10%
SDS, 100 µl 10% ammonium persulfate and 8 µl TEMED) were used for the separation process. A 10- or 15-well comb was used to create wells for sample loading. Cell lysates containing equal amount of proteins were diluted with 1:1 laemmli sample buffer [62.5 mM Tris HCl (pH 6.8), 2% SDS, 25% glycerol,
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0.01% (w/v) bromophenol blue, 5% 2-mercaptoethanol] and boiled at 100 ℃ for 10 min. The samples were then loaded into wells in the stacking gel. The proteins were separated by 10% resolving gel under constant current of 40 mA using running buffer containing 25 mM Tris, 192 mM glycine and 0.1% SDS.
After separation of proteins by SDS-PAGE, the proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, CA, USA) under constant voltage of 25 V at 4 ℃ for overnight using transfer buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS and 20% methanol. The transblotted membrane was blocked with 5% (w/v) non-fat milk in TBS-T
(113 mM NaCl, 25 mM Tris HCl with 0.05% Tween 20) at room temperature for 1 h, and then incubated with the primary antibodies anti-Nrf2 (Santa Cruz
Biotechnology Inc., CA, USA) and anti-β-actin (Santa Cruz Biotechnology
Inc., CA, USA) for 2 h at room temperature. After washing the membrane with TBS-T for three times, appropriate secondary antibody (anti-rabbit IgG
HRP-linked antibody, purchased from Cell Signaling Technology Inc., MA,
USA) was added on to the membrane to incubate at room temperature for 1 h.
Prior to antibody visualization using the enhanced chemiluminescence system, the membrane was rinsed in TBS-T for three times to remove excess secondary antibody. The antibody/protein complexes were visualized using the enhanced chemiluminescence system (Perkin-Elmer, MA, USA).
GSH measurement
SH-SY5Y cells were seeded onto 60 mm diameter plates at a density of 5 × 104 cells/plate and allowed to grow to 60 - 70% confluency before addition of Fr. 1 to 4 at 10 and 50 µg/ml. Each treatment was performed in
40
duplicates (2 plates of cells for each treatment). After 24 h of treatment, detached cells floating in medium were then collected and the attached cells trypsinized. Attached and floating cells were pooled, pelleted by centrifugation (1,200 rpm, 4 ℃, 5 min), washed once in ice-cold PBS and the obtained cell pellet was frozen at -20 ℃. Cell pellets from one of the duplicate samples (per treatment) were thawed. To each pellet, 1 ml of 3% w/v 5- sulfosalicylic acid was added to extract GSH. The mixture was centrifuged to remove precipitated proteins, total GSH content was determined by the enzymatic method of Tietze with modification (Tietze, 1969; Chew et al.,
2006). Briefly, total GSH content was assayed by measuring the change in absorbance at 412 nm over 6 min in a 96-well plate containing 0.1 M sodium phosphate, 5 mM EDTA buffer (pH 7.5), 0.12 mM 5,5’-dithio-bis(2- nitrobenzoic acid), 0.2 IU yeast GSH reductase (Sigma-Aldrich Pte Ltd, USA),
0.04 mM NADPH, and 20 µl of sample in a final volume of 200 µl. GSH values were calculated from a standard curve that was generated for each experiment using known amounts of GSH (1×10-5-5×10-7 M). Total GSH levels were then normalized to total protein content that was determined from the cell lysates prepared from the remaining duplicate sample using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions.
2.2.3 Statistical analysis
All data were expressed as means ± S.D. (or means ± SEM) and
Student’s t - test was used for comparison between treatment groups.
Difference was considered significant at p < 0.05.
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2.3 Results
2.3.1 Possible anti-coagulation activities of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract
As shown in Table 2.3.1.1, results revealed that Heparin (1 U/ml), used
in this study as a positive control, could markedly prolong both PT and aPTT
to > 120 and > 190 s respectively. As compared to untreated control, while Fr.
1 at 100 µg/ml could prolong PT marginally, none of the other fractions
showed significant effects, indicating that at least in this in vitro
anticoagulation activity assay employed, the fractions isolated from Ixeris
sonchifolia Hance extract were found not to possess significant anticoagulant
activities.
Table 2.3.1.1 Anticoagulation activity of the fractions 1 to 4 extracted from Ixeris sonchifolia Hance
Prothrombin Time (s) Activated Partial Thromboplastin Time (s)
10 µg/ml 50 µg /ml 100 µg /ml 10 µg /ml 50 µg /ml 100 µg /ml
Unteated 15.7 ± 0.3 39.5 ± 0.9 Control
Heparin >120** >190** (1 U/ml)
Fr. 1 15.9 ± 0.2 15.9 ± 0.2 16.2 ± 0.1* 39.2 ± 0.4 40.7 ± 0.5 40.6 ± 1.8
Fr. 2 15.5 ± 0.2 15.0 ± 0.2 14.8 ± 0 37.8 ± 3.6 41.3 ± 1.4 40.1 ± 0.6
Fr. 3 15.2 ± 0.2 15.2 ± 0.2 15.2 ± 0.2 39.9 ± 1.5 40.7 ± 1.1 40.6 ± 1.0
Fr. 4 16.0 ± 0.3 15.8 ± 0.2 16.0 ± 0.3 39.7 ± 0.5 40.0 ± 0.7 39.8 ± 0.5
* p < 0.05 versus untreated control group; ** p < 0.01 versus untreated control group
2.3.2 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract on the viability of H2O2-exposed PC12 cells
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The fractions isolated from Ixeris sonchifolia Hance extract were evaluated for their possible cytoprotective effects against cells exposed to oxidative stress. MTT cell viability assays were carried out where PC12 cells
Fig 2.3.2.1 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract on the viability of H2O2-exposed PC12 cells. Cells were pre-treated with different concentrations of fractions 1 to 4 for 30 min, followed by H2O2 addition to a final concentration of 150 µM. Cell viability was determined using MTT assay after a 12 h-treatment. ## p < 0.01 versus control group; ** p < 0.01 versus H2O2 group.
pretreated with different concentrations of Fr. 1 to 4 for 30 min were treated with 150 µM H2O2 for another 12 h. As shown in Fig 2.3.2.1, cell viability was significantly decreased to around 70% after a 12 h exposure to H2O2. Pre- treatment of cells with the different fractions obtained from Ixeris sonchifolia
Hance extract yielded varied results; H2O2-induced cytotoxic effect was significantly attenuated in cells that were pre-exposed to Fr. 1 and 3 at concentrations more than 20 µg/ml, whereas cells pre-treated with Fr. 2 and 4
43
suffered an accentuated cytotoxic effect. Of note, the cytoprotective effects of
Fr. 1 and particularly Fr. 3 were dose-dependent, suggesting that these fractions contained components capable of scavenging the cytotoxic effect of
H2O2. Based on the results obtained from this preliminary screening assay, Fr.
1 and 3 could be selected for further isolation and purification work to identify the components accountable for the possible cytoprotective effect.
2.3.3 Evaluation of antioxidant activities of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract
As preliminary results presented in Section 2.3.2 were suggestive of possible antioxidant activities of the fractions isolated from the Ixeris sonchifolia Hance extract, the free radical scavenging activity of these fractions were determined in an in vitro assay using DPPH as substrate. As shown in Fig 2.3.3.1, while the low concentrations (10 - 100 µg/ml) of Fr. 1, 2 and 4 exhibited little activity, a high concentration (1 mg/ml) of all fractions could scavenge DPPH free radical effectively. Among the fractions, it was observed that Fr. 3 possessed the most promising and effective free radical scavenging activity; elimination of 90% of DPPH free radical could be achieved at a low dose of 50 µg/ml.
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Fig 2.3.3.1 DPPH free radical scavenging activity of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract. The DPPH scavenging rate for each sample was calculated as percentage of (Abs DPPH-only solution – Abs compound-treated DPPH solution)/ Abs DPPH-only solution. Trolox at the concentration of 100 µM was used as positive control. * p < 0.05, **p < 0.01 versus solution containing only DPPH.
Fig 2.3.3.2 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract on ARE-dependent transcriptional activity. SH-SY5Y cells cotransfected with pGL3-ARE and pRL-CMV were treated with fractions 1 to 4 or 10 µM sulforaphane (SF) for 10 h. Ratios of firefly luciferase activities normalized to renilla luciferase activities were determined. * p < 0.05, ** p< 0.01 versus DMSO control group.
45
Fr. 1 Fr. 2 Fr. 3 Fr. 4
Nrf2 8 h β -actin
12 h Nrf2 β -actin
Nrf2 24 h β -actin
Fig 2.3.3.3 Representative Western blot analysis of protein levels of transcription factor Nrf2 in SH-SY5Y cells treated with fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract. Cells were treated with indicated concentrations of fractions 1 to 4 and 10 µM of sulforaphane for 8, 12 and 24 h and whole cell lysates were collected for SDS-PAGE and Western blot analysis of Nrf2 protein levels. β-actin immunodetection was carried out to verify equal protein loading. The use of proteasome inhibitor MG-132 served the purpose of prevention Nrf2 degradation so as to aid in locating the Nrf2 protein band on the Western blot films.
To further assess the antioxidant activities of Fr. 1 to 4, their ability to induce ARE-dependent transcriptional activity was determined. Results revealed that as compared to DMSO control, Fr. 1 to 3 brought about an induction in ARE-dependent transcriptional activity in a dose-dependent manner. In particular, Fr. 3 registered the greatest fold of induction that was comparable to that of positive control sulforaphane (SF) (Fig 2.3.3.2). In view that the upregulation of ARE-dependent transcriptional activity could be due
46
to an enhancement in the activity of the transcription factor Nrf2, the effects of
Fr. 1 to 4 on the cellular levels of Nrf2 protein were next determined. As depicted in the representative Western blot in Fig 2.3.3.3, all fractions 1 to 4 at a low concentration of 10 µg/ml were found to increase Nrf2 protein levels at a time point of 8 h. Among the fractions, Fr. 3 displayed remarkably and sustainable Nrf2 induction at all time points (8, 12 and 24 h) and at both concentrations of 10 and 50 µg/ml. Nrf2 induction was not maintained for Fr.
1, 2 and 4 at later time points of 12 and 24 h.
Fig 2.3.3.4 Total cellular GSH levels in SH-SY5Y cells exposed to fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract. Cells were treated with 10 and 50 µg/ml of fractions 1 to 4 and whole cell lysates were collected for GSH measurement after 24 h treatment. Each data were obtained from the average of three independent experiments. ** p < 0.01 versus DMSO control
GSH is an abundant thiol redox molecular whose levels could be elevated in times of oxidative stress in order to restore redox homeostasis.
Measurement of total GSH content in SH-SY5Y cells treated with 10 and 50
µg/ml Fr. 1 to 4 for 24 h was conducted, and it was found that among the
47
fractions tested, only Fr. 3 at 50 µg/ml was able to result in a significant increase in total GSH content (Fig 2.3.3.4). The other fractions, on the contrary, showed little effects.
2.4 Discussion and conclusions
In this chapter, the aerial part of Ixeris sonchifolia Hance was extracted by 95% ethanol and the crude extract was partitioned into four fractions (Fr. 1 to 4) according to their polarity. The fractions were then subjected to screening using both cell-based in vitro bioassays and biochemical reactions.
The screening assays were selected based on current understanding of the pathophysiology of neuronal injury during the reperfusion phase subsequent to acute ischemic stroke.
The principles in stroke treatment involve the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as to reduce ischemia-caused tissue damage. In order to restore the blood supply, thrombolytic reagents or surgical intervention are adopted to remove the thrombus in ischemic stroke (Fernandes et al., 2000).
Anticoagulants and antiplatelet agents will be administrated to prevent the progression of thrombosis and early recurrence of embolic stroke. Coagulation is a complex process with several coagulation factors activated one after the other, and at last resulting in the formation of blood clots (Fig 2.4.1). The clots block the injured vessel until it heals. Of the anticoagulants, warfarin affects the vitamin K-dependent clotting factors (II, VII, IX, X) (Salte et al., 1991), whereas heparin increases the action of antithrombin on thrombin and Factor
Xa. In this chapter, the anticoagulant effects of Fr. 1 to 4 were tested by
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assaying the prothrombin time (PT) and activated partial thromboplastin time
(aPTT) in vitro. PT is the time it takes for plasma to clot after addition of tissue factor. It assessed Factors VII, X, V, II and fibrinogen that are involved in the extrinsic pathway. On the other hand, aPTT assesses factors that take part in the intrinsic pathway: XII, XI, IX, VIII, X, V, II. Results obtained from the in vitro anticoagulation activity assay had revealed that fractions 1 to
4 isolated from Ixeris sonchifolia Hance extract possess negligible anticoagulant activities. However, significant differences exist between the physiological cascade and the in vitro experimental set-up. For instance, under physiological conditions, the speed of the extrinsic pathway is greatly affected by levels of Factor VII, which has a short half-life and its synthesis is dependent on Vitamin K. Therefore, the deficiencies in vitamin K, poor factor
VII synthesis or increased consumption may prolong the PT in the physiological cascade (Godsafe et al., 1992). However, under experimental laboratory conditions, interfering factors that do not exert direct and immediate inhibitory effects on clotting factors would probably failed to yield a similar inhibitory outcome. Therefore, possibilities that fractions 1 to 4 isolated from Ixeris sonchifolia Hance might exert anti-coagulation activity in an in vivo setting could not be excluded.
It was further found that Fr. 1 and 3 at concentrations more than 20
µg/ml could bring about a reduction in H2O2-induced cytotoxic effect on PC12 cells (refer to Fig 2.3.2.1). PC12 is a cell line derived from pheochromocytoma of the rat adrenal medulla. It responds reversibly to nerve growth factor by induction of the neuronal phenotype, which makes it useful as a cell model for screening of neuroprotectants. The cytoprotective effects of
49
Fr. 1 and particularly Fr. 3 were dose-dependent, suggesting that these fractions contained components capable of scavenging the cytotoxic effect of
H2O2.
Damaged Contact activation Tissue factor surface (intrinsic) pathway (extrinsic ) pathway Trauma TFPI XII XIIa VIIa VII XI XIa VIII Tissue factor Trauma IX Ixa VIIIa Antithrombin X X Xa Prothrombin Thrombin Common (II) Va (IIa) pathway V Fibrinogen Fibrin (I) (Ia) Active Protein C XIIIa XIII Protein S Cross‐linked Protein S fibrin clot thrombomodulin (A)
Partial Thromboplastin Time (Intrinsic Pathway) Prothrombin Time (Extrinsic Pathway) Contact Activation Tissue Factor/ Phospholipid + Calcium
XIIa + Calcium
VIIa VII XI XIa VIII IX Ixa VIIIa
X X Xa Prothrombin (II) Thrombin (IIa) Va V Fibrinogen Fibrin (I) (B) (Ia)
Fig 2.4.1 The coagulation cascade under physiological conditions (A), and experimental laboratory conditions (B). Solid arrows indicate activation of a coagulation factor by the factor above it in the cascade; dotted arrows indicate activation of factors by thrombin.
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Free radical insult accounts for the early response following a stroke episode and strategies to inhibit the production, increase the removal or foster the degradation of free radicals have been investigated to reduce these detrimental processes. Medicines under investigation such as Tirilazad,
Edaravone and NXY-059 are all free radical trapping reagents and most of them register significant efficiency in pre-clinical experiments. In the DPPH free radical scavenging assay, it was observed that among the four fractions,
Fr. 3 exhibited the most efficient free radical scavenging effect, thus indicating that this fraction contained components that indeed possessed direct free radical scavenging activity.
Another strategy to protect cells against oxidative stress is to stimulate the production of cellular antioxidant enzymes and molecules. The Keap1-
Nrf2-ARE pathway is presumably the most important modulator to coordinate the phase II response. Under basal conditions, the actin-associated and cysteine-rich Kelch-like ECH-associated protein 1 (Keap1) serves as an adaptor to link Nrf2 to the ubiquitin E3 ligase Cul3-Rbx1 complex that ubiquitinates Nrf2 and leads to proteasomal degradation of Nrf2. When oxidative/electrophilic stress is introduced, modification of critical cysteine residues in Keap1 causes Keap1 to lose the ability to negatively regulated Nrf2.
Accumulation of Nrf2 therefore results, which upon translocation into the nucleus, Nrf2 binding to the antioxidant response element (ARE), the promoter region of a battery of genes encoding Phase Ⅱ enzymes, leads to induction of expression of these cytoprotective enzymes. As a delayed response to oxidative/electrophilic stress, Glycogen synthase kinase-3 beta
(GSK-3β) is activated and phosphorylates Fyn, followed by nuclear
51
uclear clear
Nrf2 Nrf2 Keap1 Nrf2 degradation Keap1 Basal RbxI E2 RbxI E2 Cul3 Cul3 Delayed Cellular protection Electrophiles Response & cell survival Reactive Oxygen Species
Nrf2 Nrf2 Keap1 Nrf2 Keap1 GSK‐3β RbxI E2 Keap1 C151 Nrf2 P Cul3 Fyn Antioxidant * Proteins P Nrf2 Cytoplasm Fyn Keap1
Export Nrf2‐P Nrf2 Maf
ARE Phase II genes Nucleus (GTGACNNNGCN) (NQO1, HO‐1, γ‐GCS, GCS)
Fig 2.4.2 Oxidative/electrophilic stress and Nrf2:Keap1 signaling pathway. Under basal conditions, Keap1 (INrf2) serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex and leads to proteasomal degradation of Nrf2. When oxidative/electrophilic stress is introduced, modification of critical cysteine residues in Keap1 causes Keap1 to lose the ability to negatively regulated Nrf2. Accumulation of Nrf2 therefore results, which upon translocation into the nucleus, Nrf2 binding to the antioxidant response element (ARE), leads to induction of expression of phase Ⅱ enzymes. As a delayed response to oxidative/electrophilic stress, GSK-3β is activated and phosphorylates Fyn. The following nuclear translocation of phosphorylated Fyn phosphorylates Tyr568 in Nrf2, which results in nuclear exporting and degradation of Nrf2. Nrf2 is also degraded by prothymosin-α-mediated nuclear translocation of INrf2. Nrf2 degradation both in the cytosolic and nuclear compartments thus rapidly returns Nrf2 to normal levels. Adapted from Zhang, Li et al and Kaspar et al’s publications (Zhang, 2006; Kaspar et al., 2009; Li et al., 2009).
translocation of phosphorylated Fyn. Phosphorylated Fyn in turn phosphorylates Tyr568 in Nrf2, which results in nuclear exporting and
52
degradation of Nrf2. Nrf2 is also degraded by prothymosin-α-mediated nuclear translocation of INrf2. Nrf2 degradation both in the cytosolic and nuclear compartments thus rapidly returns Nrf2 to normal levels (Fig 2.4.2)
(Zhang, 2006; Li et al., 2009). A broad spectrum of electrophiles has been found to inhibit Keap1-mediated Nrf2 degradation. In this chapter, the inducibility of cellular antioxidant enzymes and molecules was determined.
Among the fractions evaluated, Fr. 3 was found to induce markedly ARE- dependent transcriptional activity. Consistent with these results, Fr. 3 brought about accumulation of Nrf2 protein whereas the other fractions failed to display significant induction of Nrf2 levels.
Redox homeostasis is achieved by a number of antioxidant systems including the two major thiol redox systems: glutathione and thioredoxin systems (Bjornstedt et al., 1994; Holmgren et al., 2005; Koharyova et al.,
2008). The thioredoxin system comprises thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. TrxR catalyzes the NADPH-dependent reduction of the active-site disulfide of oxidized Trx. The glutathione system consists of reduced glutathione (GSH), oxidized glutathione (GSSG), peroxidase (GSH-Px) and glutathione reductase (GR). The thiol group in the cysteine residue within a GSH molecule is able to donate a reducing equivalent (H+ + e-) to ROS. In donating an electron, glutathione readily reacts with another reactive glutathione to form glutathione disulfide (GSSG). The
NADPH-dependent enzyme glutathione reductase reduces GSSG to GSH (Fig
2.4.3). The ratio of GSSG-to-GSH is considered indicative of the redox status
(Bjornstedt et al., 1994; Holmgren et al., 2005; Koharyova et al., 2008).
Measurement of intracellular total GSH content in SH-SY5Y cells treated with
53
Fr. 1 to 4 had revealed that only Fr. 3 at 50 µg/ml caused an increase in total
GSH content, while the other fractions showed little effects. This suggested that Fr. 3 contained components capable of inducing production of GSH.
Fig 2.4.3 The glutathione system. The Grx system consists of Grx, GSH and NADPH-dependent-GSH-reductase. Grxs are able to catalyse reactions not only via a dithiol mechanism, but also via a monothiol mechanism, which is required for the reduction of protein GSH-mixed disulphides. Both these oxidized forms can be catalytically reduced back to GSH by the NADPH- dependent glutathione reductase.
In conclusion, while the ethyl acetate fraction (Fr. 3) isolated from
Iexris sonchifolia Hance extract was found not to possess anticoagulant activities, it dose-dependently protected cells from H2O2-induced cytotoxicity, scavenged DPPH free radicals, induced ARE-dependent transcriptional activity and caused upregulation of Nrf2 protein levels. Based on these findings indicating the potential neuroprotective properties of Fr. 3, further fractionation and purification experiments were performed (to be highlighted in Chapter 3) with the ultimate goal of identifying the underlying components that could play key roles in the prevention and treatment of ischemia-related diseases.
54
Chapter 3
Isolation and characterization of components from ethyl
acetate fraction of Ixeris sonchifolia Hance
55
3.1 Introduction
In the previous chapter, ethyl acetate fraction (Fr. 3) isolated from
Ixeris sonchifolia Hance extract had been proven to protect cells from H2O2- induced cytotoxicity dose-dependently, scavenged DPPH free radicals, induced ARE-dependent transcriptional activity and caused upregulation of
Nrf2 protein levels, indicating the potential neuroprotective properties of Fr. 3.
In this chapter, further fractionation and purification experiments was performed by repeated chromatography with the ultimate goal of identifying the underlying components that could play key roles in the prevention and treatment of ischemia-related diseases. The quantities of the isolated flavonoid compounds, which were identified by spectral analysis and postulated to be the underlying components exerting cytoprotective effects, was quantitatively determined with an Agilent 1100 series HPLC system.
3.2 Materials and methods
3.2.1 Isolation of the fractions from Ixeris Sonchifolia Hance extract
3.2.1.1 Materials and general analytical procedures
All chemicals were purchased from Tedia Company Inc. (OH, USA) unless otherwise stated. The deuteriated solvents for NMR analysis, pyridine- d5 and dimethyl sulfoxide-d6, were purchased from Sigma-Aldrich Inc. (MO,
USA). The sorbents for chromatographic isolation, such as silica gel 60 (40 -
63 μm) and precoated silica gel 60 TLC plates were purchased from Merck
KGaA (Darmstadt, Germany). Sephadex LH-20 was obtained from
Pharmacia (Uppsala, Sweden) and Chromolith® SemiPrep 100-10 mm RP-18 endcapped column was purchased from Merck KGaA.
56
Melting points were determined in Pyrex capillaries using a
Gallenkamp Griffin MPA350.BM2.5 melting point apparatus. IR spectra were recorded on the Perkin-Elmer 983G spectrometer. 1H and 13C NMR spectra were obtained using a Bruker ACF300 spectrometer (Karlsruhe, Germany) operating at 300 MHz and 75 MHz respectively. Mass spectra and ESI-TOF-
MS analysis was performed on a QTrap® 2000 ESI mass spectrometer
(Applied Biosystems Inc., USA) and a Waters Q-TOF premierTM mass spectrometer respectively. Data were acquired in continuum mode until acceptable averaged data were obtained.
3.2.1.2 Isolation procedures
Fr. 3, the ethyl acetate fraction isolated from Ixeris sonchifolia Hance extract (as discussed in Chapter 2) was further separated by a sephadex LH-20 column using CHCl3/MeOH in a 1:1 volume ratio as the mobile phase. Nine fractions (Fr. 3.1 - 3.9) were obtained. Fr. 3.2 was separated by silica gel column chromatography with solvents of increasing polarity (CHCl3/MeOH,
95:5 → 85:15 → 80:20, with methanol added over a 1% gradient) to yield four fractions (Fr. 3.2.1 - 3.2.4). Fr. 3.2.3 was subjected to further separation with semi-preparative reversed phase HPLC using H2O/MeOH [flow rate: 4ml/min,
H2O:MeOH 88:12 (0 min)→ 40:60 (25 min)] as eluting solvent to yield compound 13 (Rt = 10.6 min, 10 mg) and compound 11 (Rt = 14.0 min, 25 mg).
Fr. 3.3 was separated by reverse-phase silica gel column chromatography with a mobile phase system of H2O/MeOH (90:10→10:90) to afford six fractions
(Fr. 3.3.1 - 3.3.6). Fr. 3.3.3 and 3.3.5 were further separated by reverse-phase silica gel column chromatography to yield compound 2 (88 mg) and 1 (116
57
mg) respectively. Fr. 3.4 was separated using the same semi-preparative
HPLC system applied to Fr. 3.2 to yield compound 8 (Rt = 9.8 min, 5 mg).
Similarly, separation of Fr. 3.5 isolated compound 9 (Rt = 12.8 min, 12 mg) and 12 (Rt = 14.2 min, 10 mg) and Fr. 3.6 yielded compound 10 (Rt = 15.1 min,
12 mg). The waste elution solvents of Fr. 3.4 to 3.6 were pooled and concentrated. The residue was subjected to silica gel column chromatography with CHCl3/MeOH (70:30→25:75) as elution system to afford compound 14
(29 mg). When separated with the preparative HPLC system using
H2O/MeOH [flow rate: 4ml/min, H2O:MeOH 90:10 (0 min)→ 72:28 (20 min)] as mobile phase, Fr. 3.7 yielded compound 5 (Rt = 4.9 min, 22 mg), 4 (Rt =
12.6 min, 33 mg) and 3 (Rt = 15.5 min, 20 mg), and Fr. 3.8 yielded compound
6 (Rt = 4.9 min, 4 mg).
In addition, Fr. 2, the chloroform fraction isolated from Ixeris sonchifolia Hance extract, was further separated on a silica gel column with
Hexane/EtOAc (85:15→10:90) as elution system to yield compound 15 (8 mg) and 18 (19 mg). Fr. 4, the water fraction, was separated on a reverse-phase silica gel column with H2O/MeOH (90:10→30:70) as elution system to yield compound 7 (28 mg), 16 (6 mg) and 17 (4 mg).
3.2.1.3 Reactions
Molisch’s reaction Samples were dissolved in 0.5 ml ethanol, and 1 ml
α-naphthol (10%) dissolved in ethanol was added. After mixing, 0.5 ml concentrated sulfuric acid was slowly added down the sides of the sloping test- tube, without mixing, to form a bottom layer. The appearance of a purple ring
58
at the interface between the acid and test layers was referred as positive result of the Molisch’s reaction.
Acid hydrolysis Samples were refluxed with 2 M HCl for 2 h. After cooling to room temperature, the reaction mixture was neutralized with
AgNO3 and centrifuged. The supernatant was evaporated to dryness on a water bath and subjected to TLC analysis on precoated silica gel 60 TLC plates with a elution system of n-BuOH/HOAc/H2O (4:1:5 v/v/v). An authentic sample of glucose was adopted for comparison.
3.2.2 Quantitative HPLC analysis of compounds 1 to 5 and 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract
3.2.2.1 HPLC system and mobile phase
An Agilent 1100 series HPLC system consisting of autosampler, quartpump, on-line degasser and DAD-detector (Palo Alto, USA) was adopted to assay the contents of the compounds that had been identified as flavonoids in the fractions. The separations were performed on a Waters XBridgeTM C18
3.5 μm 150 × 4.6 mm column with solvent A (H2O + 0.8% Acetic Acid): solvent B (Methanol + 0.8% Acetic Acid) as mobile phase. The gradient elution was set at a flow rate of 1 ml/min. The injection volume and column temperature was set at 5 µl and 36 ℃ respectively. The detector wavelength was 350 nm with 410 nm as reference. The optimized linear gradient elution conditions were: 26% to 40% to 95% B over 28 min (0→18 min→28 min), then followed by an isocratic hold at 95% B for another 10 min. At 38 min, B was returned to 26% in 2 min and the column was equilibrated for 10 min before the next injection. The total run time was 50 min. The HP ChemStation
Software was used for data analysis.
59
3.2.2.2 Sample preparations
The purified compounds 1 to 5 and 7 were accurately weighed and dissolved in DMSO to constitute 20 mM stock solutions. Fr. 1 to 4 obtained from Ixeris sonchifolia Hance extract were dissolved in DMSO to make stock solution of 20 mg/ml. All samples were filtered with a 0.22 µm nylon membrane filter before being loaded into the HPLC system.
3.2.2.3 Calibration graphs
Standard curve for each flavonoid was obtained over a range of eight different concentrations (0.5, 1, 10, 50, 100, 250, 500 and 1000 µM) diluted from stock solution. Calibration curves (y = ax + b) were represented by plotting peak area (y) of the compounds versus the concentrations (x) of the calibration standards.
3.2.2.4 Quality control
Following 72 h post-preparation of samples, the stability profiles of compounds 1 to 5 and 7 in DMSO were investigated in one sample run in the autosampler at room temperature. The intra-day precision was calculated on a six-time continuous injection of standard solutions with a 4 h interval; the inter-day precision was calculated on a four-time continuous injection of the same solutions with a 24 h interval. The recovery rates for each compound in
Fr. 1 to 4 were calculated based on the addition of three different concentrations of standard solutions (5, 50 and 500 µM) for each fraction.
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3.3 Results
3.3.1 Characterization of purified compounds isolated from Ixeris sonchifolia Hance
Two main categories of compounds were isolated from the ethyl acetate fraction (Fr.3) of Ixeris sonchifolia Hance extract: flavonoids and sesquiterpene lactones. The isolated flavonoids were identified to be Apigenin
(1), Luteolin (2), Apigenin-7-O-β-glucopyranoside (3), Luteolin-7-O-β- glucopyranoside (4), Luteolin-7-O-β-glucruonopyranoside (5) and Luteolin-7-
O-β-galacturonide (6). The identified sesquiterpene lactones included 11,13α-
Dihydroixerinoside (8), Ixerinoside (9), 3-Hydroxydehydroleucodin (10),
Ixerin Z (11), 11,13α-Dihydroixerin Z (12) and Ixerin Z1 (13). In addition to these two categories of compounds, Oleanolic Acid (14), a triterpenoid, was also isolated from this fraction.
Compounds 15 and 18, which were isolated from the chloroform fraction (Fr. 2), were elucidated as β-sitosterol and Ceryl Alcohol respectively.
In addition to the six flavonoids isolated from the ethyl acetate fraction (Fr.3), one more flavonoid glycoside, Luteolin-7-O-gentibioside (7), together with two phenols, Vanillin (16) and Protocatechuic aldehyde (17), were obtained from the water fraction (Fr.4).
Compound 1 Apigenin
Pale yellow powder, positive Mg-HCl reaction; mp. > 300℃; UV
1 (MeOH) λmax: 260 nm, 360 nm; H NMR (300 MHz, DMSO-d6): δ 6.20 (1H, d, J = 2.0 Hz, H-8), δ 6.49 (1H, d, J = 2.0 Hz, H-6), δ 6.77 (1H, s, H-3), δ
61
6.94 (2H, d, J = 9.0 Hz, H-3', 5'), δ 7.92 (2H, d, J = 9.0 Hz, H-2', 6'); 13C
- NMR (75 MHz, DMSO-d6) (Table 3.3.1.1); negative ESI-MS: m/z 269 [M-H] .
Compound 2 Luteolin
Yellow powder, positive Mg-HCl reaction; mp. > 300℃; UV (MeOH)
1 λmax: 260 nm, 370 nm; H NMR (300 MHz, DMSO-d6): δ 6.13 (1H, d, J = 2.0
Hz, H-6), δ 6.39 (1H, d, J = 2.0 Hz, H-8), δ 6.62 (1H, s, H-3), δ 6.86 (1H, d,
J = 9.0 Hz, H-5'), δ 7.38 (1H, br s, H-2'), δ 7.41 (1H, br d, H-6'); 13C NMR
- (75 MHz, DMSO-d6) (Table 3.3.1.1); negative ESI-MS: m/z 285 [M-H] .
Compound 3 Apigenin-7-O-β-glucopyranoside
Pale yellow powder, positive Mg-HCl reaction, positive Molisch’s
1 reaction; H NMR (300 MHz, DMSO-d6): δ 5.07 (1H, d, J = 7.5 Hz, H-1''), δ
6.45 (1H, d, J = 2.0 Hz, H-6), δ 6.83 (1H, d, J = 2.0 Hz, H-8), δ 6.87 (1H, s,
H-3), δ 6.94 (2H, d, J = 8.5 Hz, H-3', 5'), δ 7.96 (2H, d, J = 8.5 Hz, H-2', 6');
13 C NMR (75 MHz, DMSO-d6) (Table 3.3.1.1); negative ESI-MS: m/z 431
[M-H]-, 269 [M-162-H]-.
Compound 4 Luteolin-7-O-β-glucopyranoside
Yellow powder, positive Mg-HCl reaction, positive Molisch’s reaction;
1 H NMR (300 MHz, DMSO-d6): δ 5.09 (1H, d, J = 7.5 Hz, H-1''), δ 6.45 (1H, d, J = 2.0 Hz, H-6), δ 6.76 (1H, s, H-3), δ 6.79 (1H, d, J = 2.0 Hz, H-8), δ
6.91 (1H, d, J = 9.0 Hz, H-5'), δ 7.43 (1H, br s, H-2'), δ 7.47 (1H, d, J = 2 Hz,
62
13 H-6'); C NMR (75 MHz, DMSO-d6) (Table 3.3.1.1); negative ESI-MS: m/z
447 [M-H]-, 285 [M-162-H]-.
Compound 5 Luteolin-7-O-β-glucruonopyranoside
Yellow amorphous powder, positive Mg-HCl reaction, positive
1 Molisch’s reaction; H NMR (300 MHz, DMSO-d6): δ 5.29 (1H, d, J = 6.0 Hz,
H-1''), δ 6.48 (1H, br s, H-6), δ 6.76 (1H, s, H-3), δ 6.83 (1H, br s, H-8), δ
6.91 (1H, d, J = 8.0 Hz, H-5'), δ 7.44 (1H,br s, H-2'), δ 7.47 (1H, br s, H-6'), δ
13 13.01 (-OH); C NMR (75 MHz, DMSO-d6) (Table 3.3.1.1); negative ESI-
MS: m/z 461 [M-H]-, 285 [M-176-H]-.
Compound 6 Luteolin-7-O-β-galacturonide
Yellow powder, positive Mg-HCl reaction, positive Molisch’s reaction;
1 H NMR (300 MHz, DMSO-d6): δ 5.11 (1H, d, J = 6.0 Hz, H-1''), δ 6.80 (1H, d, J = 2.0 Hz, H-6), δ 6.69 (1H, s, H-3), δ 6.85 (1H, d, J = 2.0 Hz, H-8), δ
6.86 (1H, d, J = 8.0 Hz, H-5'), δ 7.37 (1H, dd, J = 2.0, 2.0 Hz, H-6'), δ 7.43
13 (1H, J = 2.0, H-2'); C NMR (75 MHz, DMSO-d6) (Table 3.3.1.1); negative
- - - ESI-MS: m/z 475[M+CH3OH-H2O-H] , 461 [M-H] , 285 [M-176-H] .
Compound 7 Luteolin-7-O-gentibioside
Yellow amorphous powder, positive Mg-HCl reaction, positive
1 Molisch’s reaction; H NMR (300 MHz, DMSO-d6): δ 5.11 (1H, d, J = 7.0 Hz,
H-1''), δ 6.53 (1H, br s, H-6), δ 6.75 (1H, br s, H-8), δ 6.83 (1H, s, H-3), δ
6.94 (1H, d, J = 8.5 Hz, H-5'), δ 7.48 (1H, br s, H-6'); 13C NMR (75 MHz,
63
- DMSO-d6) (Table 3.3.1.1); negative ESI-MS: m/z 609 [M-H] , 447 [M-162-
H]-.
R1 OH R1 R2 R2 O 1 H OH
2 OH OH OH O
OH 3 H OGlc O OH O HO A= O B= O 4 OH OGlc HO O O HO OH HO OH 5 OH A HO O C= 6 OH B HO O HO OH O HO O 7 OH C HO OH
Fig 3.3.1.1 Chemical structures of compounds 1 to 7. Compounds 1 to 7 were identified as Apigenin (1), Luteolin (2), Apigenin-7-O-β-glucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4), Luteolin-7-O-β-glucuronopyranoside (5), Luteolin-7-O-β-galacturonide (6) and Luteolin-7-O-gentibioside (7) respectively.
64
Table 3.3.1.1 13C-NMR spectral data for compounds 1 to 7 which were identified as Apigenin (1), Luteolin (2), Apigenin-7-O-β-glucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4), Luteolin-7-O-β-glucruonopyranoside (5), Luteolin-7-O-β-galacturonide (6) and Luteolin-7-O-gentibioside (7) respectively. (75 MHz; compounds 1 to 7 in DMSO-d6; δ in ppm).
Position 1 2 3 4 5 6 7 2 164.1 165.9 164.2164.7 164.6 164.4 164.6 3 102.8 102.4 103.0103.4 103.1 102.7 103.1 4 181.7 181.3 181.9182.1 181.9 181.7 181.9 5 161.4 157.3 161.3157.1 161.2 160.9 157.0 6 98.8 99.2 99.899.7 99.4 99.4 99.6 7 163.7 163.6 162.9163.1 162.5 162.8 162.9 8 93.9 94.0 94.794.9 94.6 94.4 94.8 9 157.2 161.3 156.8161.3 157.0 156.8 161.2 10 103.6 102.9 105.2105.5 105.5 105.1 105.4
1′ 121.1 120.8 120.9 121.6 121.3 120.9 121.4
2′ 128.4 112.9 128.5 113.8 113.7 113.4 113.7
3′ 115.9 145.9 115.9 145.9 145.8 145.9 145.8
4′ 161.1 150.5 161.0 150.1 150.0 150.2 150.0
5′ 115.9 115.9 115.9 116.2 116.5 116.0 116.1
6′ 128.4 118.8 128.5 119.4 119.1 118.8 119.2 7-Glc II
1′′ - - 99.4100.1 99.2 99.3 99.8 103.5
2′′ - - 73.0 73.3 71.3 73.9 73.5 73.1
3′′ - - 77.1 76.6 72.8 72.8 77.0 76.3
4′′ - - 69.4 69.7 75.7 71.8 70.1 68.6
5′′ - - 76.3 77.4 75.4 76.2 76.8 75.4
6′′ - - 60.5 60.8 170.0 172.1 69.4 61.2
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Compound 8 11, 13-Dihydroixerinoside
White amorphous powder; positive Molisch’s reaction; mp: 187 -
189 ℃; UV (MeOH) λmax: 226 nm; IR (KBr) νmax: 3400 (OH), 1760, 1678,
-1 1 1640 cm ; H NMR (300 MHz, Pyridine-d5) δ 0.67 (3H, d, J = 6.0 Hz, H-14),
1.25 ( 3H, d, J = 6.0 Hz, H-13), 2.11 (3H, s, H-15), 2.41 (1H, m, H-10), 3.62
(1H, overlapped with H-6′, H-9), 4.96 (1H, d, J = 6.0 Hz, H-1′), 6.07 (1H, s,
13 H-3); C NMR (75 MHz, Pyridine-d5) (Table 3.3.1.2); negative ESI-MS: m/z
425 [M-H]－; negative ESI-TOF-MS m/z: 425.1805 [M-H]－ (calculated for
C21H29O9, 425.1812).
14 O O O OGlu 2 10 O O 4 5 8 HO HO 6 15 11 O O O O O 12 13 OH OH O O O OH OH (A) (B) OH (C) OH
Fig 3.3.1.2 Chemical structure (A) and important HMBC (B) and NOESY (C) correlations of compound 8 which was elucidated as 11,13-dihydroixerinoside.
The molecular formula for compound 8 was deduced as C21H30O9 on the basis of its ESI-TOF-MS ([M-H]－, m/z 425.1812) in tandem with the 13C and 1H NMR spectra. IR spectrum of compound 8 revealed absorption bands
-1 -1 of a hydroxyl group (υOH 3400 cm ), a γ-lactone group (υC=O 1760 cm ), and an α,β-unsaturated five member ring carbonyl moiety (υC=O 1678, υC=C 1640 cm-1). On acid hydrolysis, 8 gave glucose that was identified by comparison
66
with authentic glucose samples on co-TLC and by 13C NMR signals [δ 103.9
(C-1′), 75.1 (C-2′), 79.7 (C-3′), 71.6 (C-4′), 79.0 (C-5′) and 63.1 (C-6′)]. The
13C NMR spectrum displayed signals of an α,β-unsaturated ketone at δ 210.8
(C-2), 132.3 (C-3), and 179.9 (C-4). Furthermore, the 13C NMR spectrum revealed a lactone carbonyl at δ 178.3 (C-12), and two carbons bearing oxygen at δ 81.6 (C-6) and 79.7 (C-9). The 1H NMR spectrum displayed the characteristic signals of three methyl groups at δ 0.67 (3H, d, J = 6.0 Hz, H-
14), 1.22 (3H, d, J = 6.0 Hz, H-13), and 2.11 (3H, s, H-15), of which the latter clearly depicted a vinyl methyl group. The spectrum also revealed an anomeric proton of a sugar moiety at δ 4.96 (1H, d, J = 6.0 Hz, H-1′), whose coupling constant affirmed the presence of a β-glycosidic linkage. The linkage position of the sugar moiety to the aglycone was determined and confirmed by HMBC experiment (Fig 3.3.1.2B). The 1H NMR spectral data of 8 and 9 showed close similarities, suggesting similarities between the configurations at the C-5, C-6, C-7 and C-9 positions for the two compounds.
1 However, H NMR spectrum of compound 8 revealed a methyl proton (CH3-
13) at δ 1.22 (3H, d, J = 6.0 Hz, H-13) that was missing in compound 9. In contrast, the methylene proton of a terminal olefinic double bond at δ 5.38 and
6.22 (each 1H, d, J = 3.0 Hz) in the spectrum of compound 9 was clearly missing in the spectrum of compound 8. In addition, the 13C NMR signal of
C-13 showed a significant upfield shift from δ 119.3 (compound 9) to 12.8
(compound 8). These shifts could be explained by a hydrogenation at C-13 of the α-methylene-γ-lactone unit in compound 9. The stereochemistry of the aglycone part was determined by NOESY experiments (Fig 3.3.1.2C). Based
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on these spectral analyses, the structure of 8 was elucidated to be 11,13- dihydroixerinoside.
Compound 9 Ixerinoside
1 Yellow amorphous powder, H NMR (300 MHz, Pyr-d5) δ 0.66 (3H, d,
J = 6.0 Hz, H-14), δ 2.14 (3H, s, H-15), δ 2.46 (1H, s, H-10), δ 3.65 (1H, overlapped with H-6′, H-9), δ 5.01 (1H, d, J = 9.0 Hz, H-1′), δ 6.09 (1H, s, H-
13 3), δ 5.38, 6.22 (e.a. 1H, d, J = 3.0 Hz, H-13); C NMR (75 MHz, Pyr-d5)
(Table 3.3.1.2); negative ESI-MS: m/z 423 [M-H]-.
14 O O 14 OGlu 2 10 2 HO 10 4 5 4 8 5 8
6 15 15 6 O 11 O 11 13 12 13 12
O 9 O 10
Fig 3.3.1.3 Chemical structures of compound 9 and 10 which were identified as Ixerinoside and 3-Hydroxydehydroleucodin respectively.
Compound 10 3-Hydroxydehydroleucodin
1 Yellow amorphous powder, H NMR (300 MHz, DMSO-d6) δ 2.03
(3H, s, H-14), δ 2.50 (3H, s, H-15), δ 3.05 (1H, m, H-7), δ 3.50 (1H, d, J =
10.0 Hz, H-5), δ 3.58 (1H, t, J = 10.0 Hz, H-6), δ 5.58, 6.00 ( e.a. 1H, d, J =
13 3.0 Hz, H-13); C NMR (75 MHz, DMSO-d6) (Table 3.3.1.2); negative ESI-
MS: m/z 259 [M-H]-.
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Compound 11 Ixerin Z
1 Amorphous powder, H NMR (300 MHz, Pyr-d5) δ 2.47 (3H, s, H-14),
δ 2.35 (3H, s, H-15), δ 2.75 (1H, br. t, J =12.0 Hz, H-7), δ 1.89 (1H, br. d, J =
12.0 Hz, H-9), δ 2.10 (1H, m, H-9), δ 3.23 (1H, d, J = 9.0 Hz, H-5), δ 3.18
(1H, dd, J = 9.0, 12.0 Hz, H-6), δ 1.06 (1H, dd, J = 12.0, 8.0 Hz, H-8), δ 2.31
(1H, overlapped, H-8), δ 6.33 (1H, d, J = 6.0 Hz, H-1′), δ 5.38, 6.18 ( e.a. 1H,
13 d, J = 3.0 Hz, H-13); C NMR (75 MHz, Pyr-d5) (Table 3.3.1.2); negative
ESI-MS: m/z 421 [M-H]-.
Compound 12 11,13α-Dihydroixerin Z
1 Amorphous powder, H NMR (300 MHz, Pyr-d5) δ 1.17 ( 3H, d, J =
9.0 Hz, H-13), δ 2.45 (3H, s, H-14), δ 2.32 (3H, s, H-15), δ 3.15 (1H, m, H-5),
δ 3.24 (1H, m, H-6), δ 6.35 (1H, d, J = 9.0 Hz, H-1′); 13C NMR (75 MHz, Pyr-
- d5) (Table 3.3.1.2); negative ESI-MS: m/z 423 [M-H] .
Compound 13 Ixerin Z1
1 Amorphous powder, H NMR (300 MHz, Pyr-d5) δ 2.47 (3H, s, H-14),
δ 2.33 (3H, s, H-15), δ 2.77 (1H, br. s, H-7), δ 3.28 (1H, H-5, overlapped with
H-6), δ 3.30 (1H, H-6), δ 6.17 (1H, d, J = 6.0 Hz, H-1′), δ 5.36, 6.18 ( e.a. 1H, d, J = 3.0 Hz, H-13), δ 3.70 (2H, s, H-2″), δ 7.32 (2H, d, J = 8.0 Hz, H-4″, 8″),
13 δ 7.13 (2H, d, J = 8.0 Hz, H-5″, 7″); C NMR (75 MHz, Pyr-d5) (Table
3.3.1.2); negative ESI-MS: m/z 555 [M-H]-.
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Table 3.3.1.2 13C-NMR spectral data for compounds 8 to 13 which were elucidated as 11,13-dihydroixerinoside (8), Ixerinoside (9), 3- Hydroxydehydro-leucodin (10), Ixerin Z (11), 11,13α-Dihydroixerin Z (12) and Ixerin Z1 (13) respectively. (75 MHz; compounds 8, 9, 11, 12 and 13 in C5D5N and compound 10 in DMSO-d6; δ in ppm).
Position 8 9 10 11 12 13 a)
1 47.5 47.4 153.2 153.1 153.3 152.6
2 210.8 210.8 188.8 189.0 189.2 188.6
3 132.2 132.5 152.1 153.7 153.8 153.2
4 179.9 179.7 135.1 146.0 146.7 146.5
5 53.2 53.0 51.2 52.4 47.6 46.5
6 81.6 81.8 85.8 85.1 84.9 84.6
7 44.2 41.0 46.7 47.9 55.6 51.9
8 30.2 29.9 23.7 24.1 25.7 23.6
9 79.7 80.1 36.1 36.9 37.3 36.4
10 40.0 40.2 128.8 129.0 129.6 129.1
11 41.1 139.8 139.0 139.5 41.0 139.1
12 178.3 169.8 169.0 169.1 177.5 168.7
13 12.8 119.3 118.2 118.0 12.3 117.5
14 13.7 13.6 21.3 21.7 21.6 21.2
15 18.6 18.8 13.9 14.9 15.0 14.5
1′ 103.9 104.6 101.7 101.8 101.4
2′ 75.1 75.1 75.4 75.5 74.8
3′ 79.7 79.1 78.3 78.4 77.6
4′ 71.6 71.7 71.1 71.2 70.7
5′ 79.0 78.4 78.7 78.8 75.1
6′ 63.1 63.2 62.3 62.4 64.2
a ) p-Hydroxyphenylacetyl resonances of 13 at δC 171.6 (C-1′′), 39.8 (C-2′′), 124.6 (C-3′′), 130.5 (C-4′′, 8′′), 115.7 (C-5′′, 7′′), and 157.4 (C-6′′).
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14 O RO
2 O O 10 4 4' OH 5 8 11 R=H 1' OH 15 6 12 R=H 11,13-2H 11 HO O O 5' 3' 12 13 13 R= OH 1' O
Fig 3.3.1.4 Chemical structures of compounds 11 to 13 which were identified as Ixerin Z (11), 11,13α-Dihydroixerin Z (12) and Ixerin Z1 (13) respectively.
The remaining compounds 14 to 18 were identified as Oleanolic Acid,
β-sitosterol, Vanillin, Protocatechuic aldehyde and Ceryl Alcohol respectively.
As they were prototypical compounds commonly found in natural products, they were not the focus in this Ph.D project.
OH
O
HO 14 15
CHO CHO
CH3-(CH2)26-CH2OH
OCH3 OH HO OH 16 17 18
Fig 3.3.1.5 Chemical structures of compounds 14 to 18 which were identified as Oleanolic Acid (14), β-sitosterol (15), Vanillin (16), Protocatechuic aldehyde (17) and Ceryl Alcohol (18) respectively.
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3.3.2 Quantitative HPLC analysis of compounds 1 to 5 and 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract The retention times for compounds 1 to 5 and 7 were 26.13, 25.20,
22.07, 17.69, 17.06 and 13.34 min respectively. Typical chromatograms are presented in Fig 3.3.2.1A. These profiles showed that no interferences existed under the conditions adopted for HPLC analysis. The linear relationship, correlation coefficients r2 and limits of detection were listed in Table 3.3.2.1.
The regression equations for calibration curves at the range of 0.5-1000 µM display a good linearity with the r2 values ranging from 0.9998 to 0.9999. The stock solutions of compounds 1 to 5 and 7 were found to be stable for at least
72 h under room temperature. The precision of the method was evaluated by the inter- and intra-day assays at three different concentrations of these compounds. The repeatability for inter-day and intra-day was 0.45 - 2.97%
R.S.D. The accuracy of the method, which was studied by calculating the mean recovery rate of the compounds by adding standards to the fractions had also met the acceptable criteria. Contents of compounds 1 to 5 and 7 in Fr. 1 to
4 are displayed in Table 3.3.2.1. While none or minor of compounds 1 to 5 and 7 were detected in Fr. 1 and 2, all of them presented in Fr. 3 with compound 1 and 7 registering the highest (3.7%, w/w) and the lowest (0.37%, w/w) content respectively. High quantity of compound 7 (0.45%, w/w) was also found in Fr.4.
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Table 3.3.2.1 Linear regression, r2 values, detection limits and quantity measurement of compounds 1 to 5 and 7 in fractions 1 to 4 by HPLC analysis.
Compounds 1 2 3 4 5 7
y = 5.0159x y = 4.827x y = 5.6499x y = 3.8521x y = 4.6622x y = 3.0819x
Calibration Curve + 0.9429 - 2.5915 - 6.1079 + 9.0277 - 12.614 + 0.1152
Linear 2 regression r 0.9999 0.9998 0.9999 0.9999 0.9999 0.9998
Detection Limit (ng) 0.135 0.160 0.27 0.56 0.577 1.525
Fr. 1 (w/w, %) ------5 to 1
Fr. 2 (w/w, %) 0.018 ± 0.001 0.014 ± 0.005 - - - - 7
and Fr. 3 (w/w, %) 3.679 ± 0.045 0.675 ± 0.008 0.732 ± 0.011 0.794 ± 0.014 0.448 ± 0.006 0.369 ± 0.002 Contents of of Contents Fr. 4 (w/w, %) - 0.010 ± 0.000 0.057 ± 0.002 0.285 ± 0.008 - 0.446 ± 0.002 Compounds Compounds
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Fig 3.3.2.1 Representative HPLC profiles for purified compounds 1 to 5 and 7 (A) and fractions 1 to 4 (B-E). The separations and analysis were performed on an Agilent 1100 series HPLC system with solvent A (H2O + 0.8% Acetic Acid): solvent B (Methanol + 0.8% Acetic Acid) as mobile phase. The
74
gradient elution was set at a flow rate of 1 ml/min and the optimized linear gradient elution conditions were: 26% to 40% to 95% B over 28 min (0→18 min→28 min), then followed by an isocratic hold at 95% B for another 10 min. At 38 min, B was returned to 26% in 2 min and the column was equilibrated for 10 min before the next injection. The injection volume and column temperature was set at 5 µl and 36 respectively. The detector wavelength was 350 nm with 410 nm as reference.
3.4 Discussion and conclusions
In this chapter, isolation work focused on the ethyl acetate fraction (Fr.
3) of Ixeris sonchifolia Hance had revealed the presence of two classes of compounds: flavonoids and sesquiterpene lactones. Sesquiterpene lactones are a class of terpenes with a molecular formula C15H24 that comprises three isoprene units. They can be classified into six categories based on their skeletal structure: Guaianolides, Germacranolides, Heliangolides,
Pseudoguaianolides, Hypocretenolides and Eudesmanolides (Fig 3.4.1). All the sesquiterpene lactones isolated from the ethyl acetate fraction of Ixeris sonchifolia Hance extract were found to be Guaianolides.
O O O O O O CD A B O O
O O O
O O
EF G O
Fig 3.4.1 Structures of sesquiterpene lactones. A and B: Guaianolides; C: Germacranolides; D: Heliangolides; E: Pseudoguaianolides; F: Hypocretenolides; G: Eudesmanolides.
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Flavonoids are a class of plant secondary metabolites and can be classified into flavonoids, isoflavonoids and neoflavonoids (Fig 3.4.2). Based on the presence of a 3-hydroxyl or/and 2,3-dihydro substitution, flavonoids are subdivided into four subgroups, namely flavone, flavonol, flavanone and flavanonol (Fig 3.4.3). The six flavonoids (Apigenin, Luteolin, Apigenin-7-O-
β-glucopyranoside, Luteolin-7-O-β-glucopyranoside, Luteolin-7-O-β- glucruonopyranoside and Luteolin-7-O-β-galacturonide) isolated from Fr. 3, as well as the flavonoid glycoside (Luteolin-7-O-gentibioside) isolated from Fr. 4, are categorized under the flavones subgroup.
OO O
O
A O BC
Fig 3.4.2 Skeletal structures of flavonoids (A), isoflavonoids (B) and neoflavonoids (C).
R
A H O 8 1 7 2 B OH 6 3 5 4 R C H 2, 3-dihydro
O D OH 2,3-dihydro Fig 3.4.3 Skeletal structures of subgroups of flavonoids. (A) flavones; (B) flavonol; (C) flavanone and (D) flavanonol
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Pharmacological studies on sesquiterpene lactones have recently demonstrated variety of biological activities such as cytotoxicity (Kim et al.,
2007; Suzuki et al., 2007; Tabopda et al., 2007; Yang et al., 2007; Yang et al.,
2007), antimalarial (Bischoff et al., 2004; Pedersen et al., 2009), antibacterial
(Theodori et al., 2006) and herbicidal (Macias et al., 2000) effects. If overdosed, sesquiterpene lactones can cause allergic reactions and toxicities
(Stampf et al., 1978). On the contrary, flavonoids are found ubiquitously in plants and among the most common group of polyphenolic compounds in the diet of humans and animals. Biological studies on this category of compounds revealed health-modulating effects, such as anti-allergic (Muthian et al., 2004), anti-inflammatory (Kao et al., 2005; Lim et al., 2006; Zhou et al., 2009), anti- microbial (Cushnie et al., 2005; Alvarez et al., 2008; Pistelli et al., 2009) and anti-cancer (Choi et al., 2009; Guo et al., 2009; Jung et al., 2009; Rajkapoor et al., 2009) activities. In addition, bioflavonoids, such as rutin, has been found to exhibit potential vasoprotective proprieties and significantly reduce the myocardial antioxidant status and infarct size on MI/R rats (Ali et al., 2009).
Studies on the cytoprotective effects of compounds 1 to 5 on H2O2-exposed
SH-SY5Y cells also revealed that the H2O2-induced cytotoxicity could be significantly attenuated by the isolated flavonoid compounds (the research focus of Chapter 4). To move the project forward, the next chapters would focus on studying the potential protective effects of these isolated flavonoid compounds on oxidative injury and inflammatory reactions, which are the two main pathological responses after stroke.
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Chapter 4
Cytoprotective effects of flavonoid compounds isolated from
ethyl acetate fraction of Ixeris sonchifolia Hance extract
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4.1 Introduction
As discussed in Chapter 3, isolation work performed on the ethyl acetate fraction (Fr. 3) from Ixeris sonchifolia Hance extract had resulted in the identification of two classes of compounds, flavonoids and sesquiterpene lactones, to be present in the fraction. Flavonoids belong to a class of plant secondary metabolites and are among the most common group of polyphenolic compounds present in human diet. Biological studies conducted on this category of compounds have revealed their beneficial effects on cardio- and cerebral-vascular related diseases (Ali et al., 2009). This chapter would investigate the cytoprotective effects of these isolated flavonoids on H2O2- exposed SH-SY5Y cells with the final goal to find chemical basis for potential stroke therapy effects of Ixeris sonchifolia Hance. Cell viabilities would be evaluated by MTT assay, LDH release and flow cytometery. The capacities to scavenge free radicals directly, as well as to induce phase Ⅱdetoxifying enzymes, would also be measured to see whether these anti-oxidant abilities contribute to their cytoprotective effects. Overall 6 flavonoids had been isolated from the ethyl acetate fraction of Ixeris sonchifolia Hance extract, which were Apigenin (1), Luteolin (2), Apigenin-7-O-β-glucopyranoside (3),
Luteolin-7-O-β-glucopyranoside (4), Luteolin-7-O-β-glucruonopyranoside (5) and Luteolin-7-O-β-galacturonide (6), respectively. For lack of enough flavonoid 6, the bioactivity assays would be performed on flavonoids 1 to 5 only.
4.2 Materials and methods
4.2.1 Preparation of stock solutions of flavonoid compounds
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Purified compounds 1 to 5 were accurately weighed and dissolved in
DMSO to constitute 20 µM stock solutions that were stored at -20 ℃ before use.
4.2.2 In vitro MTT cell viability assay
See Section 2.2.2.2 for experimental procedures.
4.2.3 Determination of LDH leakage
SH-SY5Y cells were seeded into 6-well culture plates at a density of 1
× 105 cells per well. The plates were then incubated for approximately 48 h at
37 to allow cell attachment and exponential cell growth. When cells reached 60 – 70% confluency, compounds 1 to 5 diluted from DMSO stock solutions were added to attain a final concentration of 20 µM. Cells were also treated with 200 µM Vitamin E (served as positive control; Sigma-Aldrich Pte
Ltd, MO, USA). To produce oxidative stress, H2O2, freshly prepared from
30 % stock solution with PBS, was introduced (final concentration 200 µM)
30 min following drug treatment. The cells were incubated for another 24 h at
37 ℃, after which the culture medium was gently collected and centrifuged
(4,000 g, 5 min, 4 ) to obtain the cell-free supernatant for measurement of lactate adhydrogenase (LDH) levels. The release of LDH, an in vitro marker for cellular toxicity, was determined using an assay kit (Nanjing Jiancheng
Bioengineering Institute, China) according to the manufacturer’s protocol.
4.2.4 Flow cytometric analysis of cell death
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SH-SY5Y cells were seeded into 6-well plates at a density of 1 × 105 cells per well. Upon cell attachment and exponential cell growth to 60 – 70% confluency, the cells were treated with compounds 1 to 5 for 24 h at indicated concentrations. Attached and detached cells were then pooled together and centrifuged at 1,200 rpm, 4 ℃ for 5 min to obtain cell pellet. After washing twice with ice-cold PBS, the cell pellets were fixed with 1 ml of 70% ice-cold ethanol for a minimum of 4 h at 4 ℃. The fixed cells were washed with ice- cold PBS, and resuspended in 100 µl of PBS containing 100 μg/ml ribonuclease A (Sigma-Aldrich Pte Ltd, MO, USA). Finally, 400 µl of propidium iodide (PI, 50 μg/ml, purchased from Sigma-Aldrich Pte Ltd, USA) was added and the cell samples were left in darkness at room temperature for
30 min. The tubes were shaken occasionally to maintain the cells in suspension (Zhao et al., 2010). PI-stained cells were then analyzed by a BD
FACSCalibur™ flow cytometer (BD Biosciences, CA, USA). Cell death was measured as the percentage of cell population with a sub-G0/G1 DNA content in the PI intensity-area histogram plot.
4.2.5 Determination of free radical scavenging activity
See Section 2.2.2.3a for experimental procedures.
4.2.6 ARE-dependent luciferase reporter assay
See Section 2.2.2.3b for experimental procedures. Briefly, SH-SY5Y cells contransfected with pGL3-ARE and pRL-CMV plasmids were treated with 5, 10 and 20 µM of compounds 1 to 5, as well as 10 µM of sulforaphane
(used as a positive control in this assay) for 14 h before firefly and renilla
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luciferase activities were assayed using the Dual Luciferase® Assaay System
(Promega, WI, USA).
Western blot analysis
See Section 2.2.2.3b for experimental procedures. Briefly, SH-SY5Y cells were treated with 5 and 20 µM of compounds 1 to 5 for 12 h and 24 h, and the collected cell lysates were subjected to SDS-PAGE. Antibodies against HO-1 (Stressgen Biotechnologies Corporation, CA, USA), Nrf2,
NQO1 and β-actin (Santa Cruz Biotechnology Inc., CA, USA) were used to probe for the cellular levels of the respective proteins.
GSH measurement
See Section 2.2.2.3b for experimental procedures.
4.2.7 Statistical analysis
Data were expressed as means ± S.D. (or means ± SEM) and the
Student’s t - test was used for comparison between treatment groups.
Difference was considered significant at p < 0.05.
4.3 Results
4.3.1 Effects of flavonoid compounds 1 to 5 on the viability of H2O2- exposed SH-SY5Y cells
MTT cell viability assays were carried out where SH-SY5Y cells were exposed to a range of concentrations of H2O2 (50 - 500 µM) for 24 h. As shown in Fig 4.3.1.1A, 24 h exposure of 150 - 500 µM of H2O2 dose-
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dependently caused a decrease in cell viability. Cell viability was reduced to around 70% and 20% after a 24 h-exposure to 200 and 500 µM H2O2 respectively. When the cells were treated with 30 µM of the purified flavonoid compounds 1 to 5 alone, viability remained largely unaffected (Fig 4.3.1.1B).
In contrast, pre-treatment of the cells with different concentrations of 1 to 5 before H2O2 challenge brought about varied results. It was observed that in a dose-dependent manner, pre-treatment with compounds 2, 4 and 5 between 2 to 25 µM for durations 12 to 72 h resulted in significant attenuation of H2O2- induced cytotoxicity (Fig 4.3.1.1C-F). However, these flavonoids at higher concentrations, particularly at 100 µM, displayed a marked reduction in cytoprotective abilities (Fig 4.3.1.1C-F). Compound 3 at all doses failed to display impressive cytoprotective effects as compared to 2, 4 and 5.
Interestingly, among the isolated flavonoids, combined addition of compound
1 apigenin and H2O2 produced obvious cytotoxicity instead of cytoprotective actions; cell viability had dropped to less than 20% of the viability of untreated control cells upon cell exposure to 100 µM compound 1 and 200 µM
H2O2 for 48 and 72 h (p < 0.01).
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Fig 4.3.1.1 Effects of flavonoid compounds 1 to 5 on the viability of H2O2- exposed SH-SY5Y cells. (A) Effects of different concentrations of H2O2 on cell viability. Cell viability was assessed using MTT assay following 24 h- exposure to H2O2. (B) Effect of 24 h-treatment with flavonoid compounds 1 to 5 (30 µM) on cell viability. (C-F) Effects of co-administration of flavonoid compounds 1 to 5 and H2O2 on SH-SY5Y cells. Cells were pre-treated with different concentrations of flavonoid compounds 1 to 5 for 30 min, followed by H2O2 addition to a final concentration of 200 µM. Cell viability was determined at time points 12 h (C), 24 h (D), 48 h (E) and 72 h (F). # p < 0.05, ## p < 0.01 versus untreated control; * p < 0.05, ** p < 0.01 versus H2O2 group.
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Fig 4.3.1.2 Effect of 24 h-treatment with flavonoid compounds 1 to 5 on LDH release from H2O2-exposed SH-SY5Y cells. Cells were pre-treated with 30 µM flavonoid compounds 1 to 5 for 30 min, followed by H2O2 addition to a final concentration of 200 µM. LDH levels in the culture medium were quantified after a 24 h-treatment. # p < 0.05, ## p < 0.01 versus untreated control; * p < 0.05 versus H2O2 group.
To further investigate the possible protective effect of flavonoid compounds 1 to 5 against H2O2-induced toxicity on SH-SY5Y cells, the release of lactate dehydrogenase into the culture medium, an in vitro marker for cellular toxicity, was measured. As depicted in Fig 4.3.1.2, 24 h exposure of 200 µM of H2O2 resulted in an increase in LDH release from SH-SY5Y cells into the culture medium. While pre-treatment of the cells with 30 µM of compound 1 before H2O2 challenge exacerbated the H2O2-induced cytotoxicity, pre-treatment of the cells with compounds 2, 4, 5 at 30 µM and vitamin E (Vit.
E) at 200 µM brought about moderate attenuation in the H2O2-induced LDH release, with compound 4 showing the greatest cytoprotective effect. The activities of LDH released from the compounds 4 and Vit. E pre-treated cells
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were reduced to a low level that was comparable to that of DMSO control
(compared with DMSO control, p > 0.05).
G0/G1 G0/G1 S S subG0/G1 subG0/G1 G2/m G /m
counts 2 counts
(A) (B) 0 40 80 120 160 200 160 120 80 40 0 0 40 80 120 160 200 160 120 80 40 0 0 200 400 600 800 1000 0 200 400 600 800 1000
30 H O H O + 30 µM flavonoid compounds 25 2 2 2 2 ## 20 * 15 * ** DNA content (%) DNA 1 10 /G 0 5 Sub-G
0 Control H2O2 Comp.1 Comp.2 Comp.3 Comp.4 Comp.5
(C)
Fig 4.3.1.3 Effect of 24 h-treatment with flavonoid compounds 1 to 5 on the subG0/G1 DNA content in H2O2-exposed SH-SY5Y cells. Untreated and treated cells were harvested and labeled with PI, and analyzed by flow cytometry. Representative flow cytometric histograms of untreated cells (A) and cells exposed to 200 µM H2O2 for 24 h (B) showed an accumulation of subG0/G1 DNA content in the H2O2-treated cells. (C) Cells were treated with 30 µM of flavonoid compounds 1 to 5 for 30 min prior to 24 h exposure of H2O2 (200 µM) and percentage of cells with subG0/G1 DNA content was analyzed by flow cytometry. ## p < 0.01 versus untreated control; * p < 0.05, ** p < 0.01 versus H2O2 group.
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Furthermore, flow cytometry analysis of PI-stained SH-SY5Y cells treated or untreated with compounds 1 to 5 (30 µM) and H2O2 (200 µM) revealed that compounds 2, 4 and 5 brought about a statistically significant reduction in the population of cells with subG0/G1 DNA content (Fig 4.3.1.3).
As detection of subG0/G1 DNA content was indicative of cell death, these findings, in agreement with results obtained from the MTT and LDH release assay, suggested that the isolated flavonoids, in particular compounds 2, 4 and
5, exerted a cytoprotective effect.
4.3.2 Antioxidant activities of flavonoid compounds 1 to 5 isolated from Ixeris sonchifolia Hance extract
Fig 4.3.2.1 DPPH free radical scavenging activity of flavonoid compounds 1 to 5 isolated from Ixeris sonchifolia Hance extract. The DPPH scavenging rate for each sample was calculated as percentage of (Abs DPPH-only solution – Abs compound-treated DPPH solution) / Abs DPPH-only solution. * p < 0.05, **p < 0.01 versus solution containing only DPPH.
In the in vitro free radical scavenging assay, while flavonoids 1 and 3 exhibited little activity, flavonoid compounds 2, 4 and 5, even at a
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concentration of 10 µM, were found to scavenge DPPH free radical effectively
(p < 0.01). Compounds 2 and 4 from 25 to 100 µM, as well as compound 5 between 50 to 100 µM displayed antioxidant capacity comparable to that of
100 µM of positive control Trolox (Fig 4.3.2.1).
Comp. 1 Comp. 2 Comp. 3 Comp. 4 Comp. 5
Nrf2
β-actin
12 h
Nrf2
β-actin
24 h
Fig 4.3.2.2 Representative Western blot analysis of protein levels of transcription factor Nrf2 in SH-SY5Y cells treated with flavonoid compounds 1 to 5. Cells were treated with 5 and 20 µM of compounds 1 to 5 for 12 and 24 h and whole cell lysates were collected for SDS-PAGE and Western blot analysis of Nrf2 protein levels. β-actin immunodetection was carried out to verify equal protein loading.
The antioxidant activity of a compound could be a result of the compound’s direct and indirect effect. While the free radical scavenging activity would be a measure of the compound’s direct antioxidant effect, the compound might exert indirect antioxidant activity by inducing the expression of antioxidant and cytoprotective enzymes through upregulation of Nrf2- mediated ARE-dependent transactivation. To determine whether the isolated flavonoid compounds 1 to 5 possessed the latter biological effect, protein
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levels of transcription factor Nrf2 in lysates of SH-SY5Y cells treated with compounds 1 to 5 were analyzed by Western blotting. As depicted in the representative Western blot in Fig 4.3.2.2, all flavonoid compounds 1 to 5, at concentrations of 5 and 20 µM were found to upregulate Nrf2 protein levels at both time points (12 and 24 h). The use of proteasome inhibitor MG-132 served the purpose of prevention Nrf2 degradation so as to aid in locating the
Nrf2 protein band on the Western blot films.
Fig 4.3.2.3 Effects of flavonoid compounds 1 to 5 isolated from Ixeris sonchifolia Hance extract on ARE-dependent transcriptional activity. SH- SY5Y cells cotransfected with pGL3-ARE and pRL-CMV were treated with compounds 1 to 5 or 10 µM solforaphane (SF) for 14 h. Ratios of firefly luciferase activities normalized to renilla luciferase activities were determined. * p < 0.05, ** p < 0.01 versus DMSO control group.
The effects of these compounds on ARE-dependent transcriptional activity were next determined. Results revealed that in agreement with the observed enhanced accumulation of Nrf2 (Fig 4.3.2.2), compounds 1 to 5 at 5
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and 10 µM brought about an upregulation in ARE-dependent transcriptional activity, with compound 2 registering the greatest fold of induction of around
5-fold at 10 µM (Fig 4.3.2.3). Nevertheless, this induction was not comparable to that brought about by 10 µM sulforaphane (~ 9-fold). Notably, it was also observed that the addition of these isolated compounds at a higher concentration of 20 µM generally failed to elevate significantly ARE- dependent transcriptional activity (Fig 4.3.2.3).
Comp. 1 Comp. 2 Comp. 3 Comp. 4 Comp. 5
HO-1
NQO1
β -actin
12 h
NQO1
β -actin
24 h
Fig 4.3.2.4 Representative Western blot analysis of protein levels of HO-1 and NQO1 in SH-SY5Y cells treated with flavonoid compounds 1 to 5. Cells were treated with 5 and 20 µM of compounds 1 to 5 for 12 and 24 h and whole cell lysates were collected for SDS-PAGE and Western blot analysis of HO-1 and NQO1 protein levels. β-actin immunodetection was carried out to verify equal protein loading.
Finally, the expression levels of HO-1 and NQO1, which were cytoprotective enzymes whose expression were under the transcriptional activity of Nrf2, were determined. As shown in Fig 4.3.2.4, the protein level of
HO-1 was increased within 12 h of treatment, while NQO1 level was only
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increased marginally. The obtained results, taken together, therefore suggested that the isolated flavonoids from Ixeris sonchifolia Hance extract, particularly compound 2 luteolin, when used at low concentration of 10 µM, exhibited potential cytoprotective effect through its free radical scavenging activity and induction of ARE-dependent transcriptional activity.
Fig 4.3.2.5 Total cellular GSH levels in SH-SY5Y cells exposed to flavonoid compounds 1 to 5 for 24 h. Cells were treated with 5 and 20 µM of compounds 1 to 5 and whole cell lysates were collected for GSH measurement after 24 h treatment. * p < 0.05, ** p < 0.01 versus DMSO control
The effects of flavonoids 1 to 5 on the cellular total GSH content in
SH-SY5Y cells were also measured. As shown in Fig 4.3.2.5, treatment of
SH-SY5Y cells with 5 µM of compounds 2, 4 and 5 for 24 h caused a slight increase in total GSH levels, with only compound 2 registering a statistically significant elevation as compared to that of DMSO control. At higher concentration of 20 µM, compounds 1 to 4 surprisingly caused a decline in
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total GSH content (Fig 4.3.2.5). In particular, compounds 1 and 2 at 20 µM had significantly resulted in depletion of GSH by almost 50% of that in the
DMSO control cells.
4.4 Discussion and conclusions
Flavonoids are naturally occurring polyphenolic compounds found ubiquitously in vegetables, herbs, nuts and teas. Together with other dietary reducing agents, such as vitamin C, vitamin E and carotenoids, flavonoids protect the body's tissues against oxidative stress and associated pathologies
(Jan et al., 2010). The in vitro antioxidant activities offer biological foundations for flavonoids’ health-modulating effects, such as anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities (Muthian et al.,
2004; Cushnie et al., 2005; Kao et al., 2005; Lim et al., 2006; Alvarez et al.,
2008; Pistelli et al., 2009; Zhou et al., 2009). In this chapter, the antioxidant activities of flavonoid compounds 1 to 5 were assayed by the in vitro DPPH free radical scavenging assay. The results revealed that while compounds 1 and 3 exhibited negligible activity, compounds 2, 4 and 5 at low concentration of 10 µM were able to scavenge DPPH free radical effectively.
In addition to the direct free radical scavenging activities, the other important mechanism contributing to the protective effects of flavonoids is the capacity to induce detoxifying enzymes, such as glutathione S-transferases
(GST), UDP-glucuronosyltransferases (UGTs), γ-glutamylcysteine synthetase
(γ-GCS), NAD(P)H-quinone oxidoreductase 1 (NQO1) and heme oxygenase-1
(HO-1) (Lee et al., 2005). These enzymes play a pivotal role in maintaining the redox homeostasis. The expression of these phase II detoxifying enzymes
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is mediated by the antioxidant-responsive element (ARE), also known as electrophile-responsive element (EpRE) (Moinova et al., 1998). In this chapter, it was found that 12 h and 24 h treatment with 5 and 20 µM of flavonoid compounds 1 to 5 could upregulate Nrf2 levels in SH-SY5Y cells. The cellular levels of phase II enzymes HO-1 and NQO1 were also elevated, although the increase for NQO1 was only marginal. ARE-dependent luciferase reporter assays conducted on contransfected SH-SY5Y cells exposed to low doses (5 and 10 µM) of compounds 1 to 5 had revealed a dose-dependent enhancement in ARE-dependent transcriptional activity, with compound 2 luteolin displaying the greatest fold of induction at 10 µM. However, when a higher concentration (20 µM) was used for all compounds, although Nrf2 protein levels remained elevated, the upregulation in ARE-dependent transcriptional activity was drastically reduced. The extract mechanism accounting for this decline in ARE transcriptional activity induction was not well clarified.
Flavonoids have been demonstrated to behave as both antioxidant and prooxidant agents depending on the concentrations used and the free radical source (Liu et al., 2010). Therefore, it was postulated that at high concentrations of 20 µM, the predominated prooxidant action of flavonoids caused depletion of cellular GSH, as evident from the obtained results that
GSH content had markedly decreased (Fig 4.3.2.5).
Under basal conditions, Keap1 serves as an adaptor to link Nrf2 to the ubiquitin E3 ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2
(Kaspar et al.). When a cell encounters oxidative/electrophilic stress, Keap1 loses the ability to negatively regulate Nrf2 such that Nrf2 accumulation leads to increased ARE activation to transcribe and translate a myriad of genes
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encoding phase II enzymes (see Section 2.4). Accumulation of Nrf2 was proposed to be due to either a direct interaction of the drug inducer with
Keap1 that weakened the Nrf2-Keap1 interaction or by protein kinase C
(PKC)-mediated phosphorylation of Nrf2. The study by Lee-Hilz et al using
EpRE-LUX cells had revealed that PKC was not involved in flavonoids induction of ARE-mediated gene transcription (Lee-Hilz et al., 2006). As such, the direct oxidant reaction with the Keap1-Nrf2 complex was proposed to be the mechanism underlying flavonoids’ action. The dimeric Keap1 protein contains multiple cysteine residues in each monomer which would likely offer potential sites of oxidative interaction with flavonoids. The mechanistic model
- Prooxidant effects OH O . OH OH O O H HO O HO O H2O2 HO O
OH OH OH OH O OH O OH O quercetin o-semiquinone O Aryloxyl (phenoxyl) racidal GSH 2 .- O2 NADPH
O H2O2 O
HO O
OH Prooxidant effects OH O
o-quinone
Fig 4.4.1 Chemical view on the oxidoreductive activation of quercetin and its possible reaction consequences. Quercetin is oxidized by the classic peroxidase mechanism to form a free radical intermediate, o-semiquinone. The reaction would be followed by radical-radical comproportionation reaction to give o-quinone and parent molecule.
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by which flavonoids would oxidatively interact with Keap1 is currently not clearly elucidated. It was proposed that electrophilic flavonoid metabolites could be the interacting species with Keap1’s cysteine. Metodiewa et al proposed the formation of free radical intermediate, o-semiquinone, during quercetin oxidative by the classic peroxidase mechanism (Fig 4.4.1)
(Metodiewa D et al., 1999). The reaction would be followed by radical-radical comproportionation reaction to give o-quinone and parent molecule. The prooxidant reactive metabolites would possess electrophilic properties to oxidize the cysteine residues in Keap1 protein.
The prooxidant effects of flavonoids cause cell damage by lipid peroxidation, tyrosine nitration, sulfhydryl oxidation and nitrosylation and
DNA breakage, while the anti-oxidant activities of these compounds would neutralize these harmful effects and restore redox homeostasis. The biological activities of flavonoids on cells should be the combination effects of their anti-
/prooxidant property. As demonstrated in the performed experiments, the
H2O2-induced cytotoxicity on SH-SY5Y cells was attenuated when the cells were pre-treated with compounds 2, 4 and 5. The protective effect was in a dose-dependent manner from 2 µM to 25 µM. But when the dose exceeded 50
µM, the cytoprotective effects decreased drastically. When analyzing the protective effects of compounds 1 to 5 on H2O2-induced cell death by flow cytometry, the results were in concert with that determined by MTT and LDH release assay. It could be deduced from the results that the anti-oxidant effects took priority over the prooxidant effects when the flavonoids were in low concentrations, which could reduce the H2O2-induced cytotoxicity by scavenging free radical directly and inducing the expression of detoxifying
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enzymes. But when the concentration was allowed to exceed a “threshold”, the induced detoxifying enzymes could no longer scavenge efficiently the free radicals produced by the flavonoids. In doing so, flavonoids’ prooxidant effects took priority over the anti-oxidant effects and thus reacted with the antioxidant molecule GSH to produce reduced cytoprotective effects.
In conclusion, flavonoids 1 to 5 were found to protect cells from H2O2- induced cytotoxiciy by scavenging free radical directly and inducing phase Ⅱ detoxifying enzymes, suggesting that these compounds might exert neuroprotective effects during the early response to stroke occurrence. Since the second wave of cell death after stroke stems from the neuroinflammatory response, the next chapter will be focused on investigating whether these isolated flavonoid compounds would be able to exert any anti-inflammatory effects on the LPS-stimulated cells.
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Chapter 5
Anti-inflammatory effects of flavonoid compounds isolated
from the ethyl acetate fraction of Ixeris sonchifolia Hance
extract
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5.1 Introduction
While the early response to stroke is characterized by excitotoxic damage and oxidative damage, the second wave of cell death after stroke stems from the neuroinflammatory response. An inflammatory reaction characterized by leukocyte influx into the cerebral parenchyma and activation of endogenous microglia occurs following ischemic stroke and hemorrhage stroke (Hallenbeck, 1996; Becker, 1998; Zheng et al., 2004), and leads to inflammatory injury to the neural tissues. The activated inflammatory cells release a variety of agents including cytokines, matrix metalloproteinases
(MMPs), nitric oxide (NO) and reactive oxygen species (ROS). These substances may trigger more cell damage as well as disruption of the blood- brain barrier (BBB) and extracellular matrix (ECM) (Danton et al., 2003). As a consequence of BBB disruption, which permits serum elements and blood to enter the brain (Rosenberg, 1999), secondary neural damage develops through activation of microglia and brain infiltration of peripheral leukocytes that produce cascades of inflammatory events leading to exacerbation of the inflammatory damage (Dirnagl et al., 1999). Blockade of various aspects of the inflammatory cascades has been shown to ameliorate injury resulting from experimental stroke (Han et al., 2003). This chapter was focused on investigating whether the isolated flavonoid compounds from Ixeris sonchifolia Hance extract could exert potential anti-inflammatory activities on the lipopolysaccharides (LPS)-stimulated cells.
5.2 Materials and methods
5.2.1 Preparation of stock solutions of flavonoid compounds
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See Section 4.2.1 for preparation of DMSO stock solutions of flavonoid compounds 1 to 5, namely Apigenin (1), Luteolin (2), Apigenin-7-
O-β-glucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4) and Luteolin-7-
O-β-glucruonopyranoside (5) respectively.
5.2.2 Cell culture and treatment
Mouse macrophage cell line RAW 264.7 (TIB-71) was purchased from the American Type Culture Collection (Rockville, MD, USA) and grown in
Dulbecco’s Modified Eagle’s Medium (DMEM; purchased from Sigma-
Aldrich Pte Ltd, MO, USA) supplemented with 10% fetal bovine serum
(Hyclone, UT, USA), 3.7 g/L sodium bicarbonate, 100 µg/ml streptomycin and 100 U/ml penicillin G. The cells were incubated at 37 °C in a 5 % CO2 humidified environment.
BV-2 cells, a murine microglial cell line, were obtained from Dr. Dong
Yiyu (Department of Anatomy, National University of Singapore) and cultured in DMEM supplemented with 10% fetal bovine serum, at 37 °C in a humidified 5% CO2 atmosphere. When BV-2 cells reached 70 - 80% confluence in the maintenance cultures, they were trypsinized by 0.25% trypsin-EDTA to obtain a single cell suspension. The cell suspension was diluted appropriately to achieve the desired cell density in 2 ml of culture medium for inoculation into each well of a 6-well plate (20,000 cells/well).
The plates were then incubated for approximately 24 h at 37 ℃ to allow cell attachment.
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Usually, the culture medium was changed to fresh serum-free medium
(for RAW 264.7 cells) or fresh medium with 2% FBS (for BV-2 cells) before any treatment to reduce the serum effect. In all experiments, cells were pre- treated with flavonoids 1 to 5 for 30 min before the addition of lipopolysaccharides (LPS; purchased from Sigma-Aldrich Pte Ltd, MO, USA).
5.2.3 In vitro MTT cell viability assay
See Section 2.2.2.2 for experimental procedures. The macrophage
RAW 264.7 cells and microglial BV-2 cells (5 × 103 cells) were seeded in 96- well plates and incubated for 24 h. After replacing the culture medium with serum free medium (for RAW 264.7 cells) or DMEM containing 2% fetal bovine serum (for BV-2 cells), various concentrations of flavonoid compounds
1 to 5, freshly prepared by diluting DMSO stock solutions in each assay, were then added to the wells (final concentrations 2, 5, 10 and 20 µM). The plates were incubated for another 24 h at 37 ℃, after which 10 µl of MTT solution (5 mg/ml in PBS) was added into each well to evaluate the effect of compounds on cell viability.
5.2.4 Measurement of nitric oxide release
The production of nitric oxide (NO) was measured by the accumulation of nitrite in culture media. Briefly, RAW 264.7 cells were seeded and cultured in 24-well plates till 80% confluence. Culture medium was replaced by serum-free and phenol red-free DMEM before addition of 1,
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5 or 10 µM of flavonoid compounds 1 to 5. Following 30 min of flavonoids addition, 1 µg/ml LPS was added and the cells were further incubated for 24 h.
The medium was gently collected and centrifuged (4,000 g, 5 min, 4 ℃), after which 100 μl of the cleared medium was added to an equal volume of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) (Fluka BioChemika, Switzerland). The mixture was agitated and kept in darkness at room temperature for 15 min. The absorbance was read at 540 nm using a plate reader. Absorbance readings of a range of known nitrite concentrations derived from sodium nitrite (Sigma-
Aldrich Pte Ltd, MO, USA) were predetermined to obtain a standard plot, from which the nitrite concentrations detected in the collected medium samples were determined and expressed in micromoles per liter (Madrigal-
Carballo et al., 2009; Sae-wong et al., 2009; Yoo et al., 2009).
5.2.5 RNA extraction and reverse transcription-polymerase chain reaction After BV-2 cells were pre-treated with 2 and 10 µM of flavonoid compounds 1 to 5 for 30 min, LPS dissolved in PBS was introduced (final concentration 1 µg/ml). The drug treated plates were incubated for another 4 h at 37 ℃, after which total RNA from each sample was isolated using an
RNeasy® Mini Kit (Qiagen, Hilden, Germany). Following the manufacturer’s instructions, cells were scraped in lysis buffer provided in this kit, and homogenized with a QIAshredder® homogenizer. Ethanol (70%) was mixed with the homogenized lysate, and the mixture was applied on a RNeasy spin column to remove ethanol. After washing the RNeasy spin column with wash
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buffers, RNA was finally eluted in RNase-free water. The concentration of extracted RNA was determined by measurement of absorbance at 260 nm on an UV spectrophotometer (Ultro Spec 2100 Pro UV/visible spectrophotometer,
Amersham Biosciences, Sweden).
Single-stranded cDNA was synthesized from 2 µg of RNA by reverse transcription using a RevertAidTM First Strand cDNA Synthesis Kit
(Fermentas, MD, USA). An appropriate volume containing 2 µg of total RNA was mixed with 1 µl Oligo18 primer and made up finally to 12 µl with nuclease-free water. The mixture was incubated in 70 ℃ for 5 min, and subsequently mixed with reaction buffer, RiboLockTM Ribonuclease inhibitor and deoxyribonucleoside triphosphate (dNTP) for 5 min at 37 ℃.
RevertAidTM M-MuLV Reverse Transcriptase was next added to the above mixture and incubated at 42 ℃ for 60 min for reverse transcription to occur.
The reverse transcription was stopped by heating at 70 ℃ for 10 min. The resulting cDNA was stored at -20 ℃ until use.
Fragments specific to genes to be examined were amplified in reaction solution (20 µl) containing 2 µl of generated cDNA, 2 µl of dNTP, 0.2 µl of
Taq DNA Polymerase (New England BioLabs® Inc., UK) and 1 µl of each specific sense and anti-sense primers (5 nM). PCR was run for 26 cycles of
94 ℃ for 15 sec (denaturation), 55 ℃ for 15 sec (annealing) and 72 ℃ for 30 sec (extension). The nucleotide sequences of the PCR primers were 5’- GGA
GAG ACT ATC AAG ATA GTG ATC - 3’ (forward) and 5’- ATG GTC
AGT AGA CTT TTA CAG CTC - 3’ (reverse) for Cyclooxygenase-2 (Cox-2,
861 bp), 5’- TTG ACC TCA GCG CTG AGT TG- 3’ (forward) and 5’- CCT
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GTA GCC CAC GTC GTA GC - 3’ (reverse) for tumor necrosis factor (TNF-
α, 374 bp), 5’- GTG TTC CAC CAG GAG ATG TTG - 3’ (forward) and 5’-
CTC CTG CCC ACT GAG TTC GTC - 3’ (reverse) for inducible nitric oxide synthase (iNOS, 576 bp), 5’- CAG GAT GAG GAC ATG AGC ACC- 3’
(forward) and 5’- CTC TGC AGA CTC AAA CTC CAC- 3’ (reverse) for interleukin-1 beta (IL-1β, 447 bp), 5’- GTA CTC CAG AAG ACC AGA GG -
3’ (forward) and 5’- TGC TGG TGA CAA CCA CGG CC - 3’ (reverse) for interleukin-6 (IL-6, 308 bp), and 5’- CAC TGC TAT GCT GCC TGC TC - 3’
(forward) and 5’- TCT TCA CCT GCT CCA CTG CC - 3’ (reverse) for interleukin-10 (IL-10, 417 bp). Samples of each single-stranded cDNA were also treated with primers that amplify a 452 bp fragment of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) house keeping gene
[5’- GGT GCT GAG TAT GTC GTG GA - 3’ (forward), 5’- GAC ACA TTG
GGG GTA GGA AC - 3’ (reverse)] to allow for quantitative comparison of loading. The PCR products were subjected to electrophoresis on 1.2% agarose gels containing ethidium bromide and images were captured under UV light in a Bio-Rad ChemiDoc imaging system (Bio-Rad Laboratories, CA, USA).
5.2.6 Western blot analysis
See Section 2.2.2.3b for experimental procedures. Briefly, BV-2 cells in DMEM containing 2% FBS were pre-treated with 1, 5 and 10 µM flavonoid compounds 1 to 5 for 30 min before the addition of 1 µg/ml LPS. The cells were cultured for another 24 h at 37 ℃, then lysed and collected for SDS-
PAGE. Protein levels in cell lysates were determined using specific antibodies against Cox-2 (Santa Cruz Biotechnology Inc., CA, USA), cPLA2 (Cell
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Signaling Technology Inc., MA, USA), iNOS (Santa Cruz Biotechnology Inc.,
CA, USA) and β-actin (Santa Cruz Biotechnology Inc., CA Biotechnology
Inc., USA).
5.2.7 Statistical analysis
Numerical data were presented as means ± S.D. of different determinations. Statistical comparisons between groups were performed using the Student’s t - test. Values of p < 0.05 were considered statistical significant.
5.3 Results
5.3.1 Effects of flavonoid compounds 1 to 5 on the viability of RAW 264.7 cells and BV-2 cells
MTT cell viability assays were carried out where RAW 264.7 and BV-
2 cells were exposed to a range of concentrations of flavonoid compounds 1 to
5 (2 - 20 µM) for 24 h. As shown in Fig 5.3.1.1, viabilities of RAW 264.7 cells and BV-2 cells remained unaffected upon treatment with the flavonoid compounds across all tested concentrations.
5.3.2 Effects of flavonoid compounds 1 to 5 on nitric oxide production in LPS-activated RAW 264.7 cells.
To determine the effects of flavonoid compounds 1 to 5 on NO production, RAW 264.7 cells were pretreated with compounds 1 to 5 (1, 5 and
10 µM) for 30 min before the addition of LPS (1 µg/ml). The cells were cultured for another 24 h and the nitrite concentrations in culture medium were
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Fig 5.3.1.1 Effects of flavonoid compounds 1 to 5 on the viability of RAW 264.7 cells and BV-2 cells. (A) RAW 264.7 cells cultured in serum free medium were treated with 1, 5, 10 and 20 µM of flavonoid compounds 1 to 5 for 24 h at 37 ℃, after which 10 µl of MTT solution (5 mg/ml in PBS) was added to evaluate cell viabilities. (B) BV-2 cells were cultured in medium containing 2% fetal bovine serum. Effects of flavonoids on the cell viability were assessed using MTT assay following 24 h treatment with 1, 5, 10 and 20 µM of compounds 1 to 5.
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mesured. As shown in Fig 5.3.2.1, the concentration of NO released into the culture medium was significantly enhanced when the cells were treated with 1
µg/ml of LPS, whereas pre-treatment with compounds 1 to 5 caused a reduction in NO release in a dose-dependent manner. Among all the flavonoids, compound 1 apigenin displayed the greatest reduction in NO release, where a dose of 10 µM was observed to reduce NO release by more than 50% of that released by LPS-stimulated RAW 264.7 cells.
Fig 5.3.2.1 Effects of flavonoid compounds 1 to 5 on NO production in LPS- activated RAW 264.7 cells. RAW 264.7 cells were pretreated with 1, 5 and 10 µM of flavonoid compounds 1 to 5 for 30 min before addition of LPS (1 µg/ml). Following 24 h of LPS treatment, nitrite levels in the culture medium were determined by Griess reaction. # p < 0.01 versus DMSO control; * p < 0.01 versus LPS alone.
5.3.3 Effects of flavonoid compounds 1 to 5 on LPS-induced mRNA expression of cytokines in BV-2 cells
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The effects of flavonoid compounds 1 to 5 on mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-10 in LPS-activated
BV-2 cells were next determined (Fig 5.3.3.1). The results revealed that there was a basal expression of TNF-α, IL-1β, IL-6 and IL-10 mRNA in the untreated cells. When the BV-2 cells were stimulated by 1 µg/ml of LPS for
4 h, while the mRNA level of IL-1β was moderately increased, TNF-α and IL-
6 mRNA levels remained largely unaffected. On the contrary, mRNA expression of IL-10 was reduced to undetectable level after LPS treatment.
IL-1β
TNF-α
IL-6
IL-10
GADPH
LPS - + + + + + + + + + + + --2 10 2 10 2 10 2 10 2 10 (µM) Compounds 1 2 3 4 5
Fig 5.3.3.1 Effects of flavonoid compounds 1 to 5 on LPS-induced mRNA expression of cytokines in BV-2 cells. BV-2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ for 24 h to allow cell attachment, after which the culture medium was replaced with low-serum DMEM containing 2% fetal bovine serum. Flavonoid compounds 1 to 5 were introduced at indicated concentrations 30 min before LPS (1 µg/ml) stimulation. The cells were cultured for another 4 h for collection of total RNA. The mRNA levels of TNF-α, IL-1β, IL-6 and IL-10 were determined by RT- PCR. A house keeping gene GADPH was also amplified to allow for quantitative comparison of loading. Results are representative of three independent experiments.
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Pre-treatment of the cells with 2 and 10 µM of flavonoid compounds 1 to 5 yielded varied results. The enhanced IL-1β mRNA levels in the cells were suppressed upon treatment with the higher concentration of compounds 1 to 4
(10 µM), whereas a low concentration of these compounds at 2 µM exerted no effect. Interestingly, compound 5 failed to suppress IL-1β induction at both concentrations. TNF-α expression was not induced by LPS and it was found that this basal expression was generally not suppressed by the compounds at both concentrations, except for compound 2 luteolin at 10 µM. Similarly, cells pre-treated with 10 µM of compounds 1, 2 and 3 also produced less IL-6 mRNA than the DMSO control group. On the contrary, 2 µM of compounds 1,
2 and 5 promoted the expression of IL-6 mRNA to a higher level than the untreated and LPS-stimulated cells. The LPS-inhibited mRNA expression of
IL-10 was restored by pre-treatment of the cells with 2 µM of compounds 1, 2 and 3. Compound 2 at concentration of 10 µM and compound 5 at 2 µM also attenuated the inhibition effects of LPS, but the mRNA expression was not restored to the normal level.
Table 5.3.3.1 Effects of flavonoid compounds 1 to 5 on the mRNA expression of cytokines in BV-2 cells
Compound Compound Compound Compound Compound 1 2 3 4 5 Cytokines LPS 2 10 2 10 2 10 2 10 2 10 (µM)
IL-1β ↑ − ↓ − ↓ − ↓ − ↓ − −
TNF-α − − − − ↓ − − − − − −
IL-6 − ↑ ↓ ↑ ↓ − ↓ − − ↑ −
IL-10 ↓ ↑ − ↑ ↑ ↑ − − − ↑ −
Note: “↑” promote; “↓” inhibit and “−” almost do not affect the expression level
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5.3.4 Effects of flavonoid compounds 1 to 5 on the expression of Cox-2 and cPLA2 in LPS-stimulated BV-2 cells
The effects of flavonoid compounds 1 to 5 on the expression of Cox-2 and cPLA2 in LPS-stimulated BV-2 cells were next investigated. As shown in
Fig 5.3.3.1A, treatment with 1 µg/ml of LPS moderately elevated the expression of Cox-2 mRNA. It could be observed that while compound 1 at 10
µM and compounds 2 to 5 at 2 and 10 µM display no inhibitory effects on
LPS-induced Cox-2 expression, lower concentration of compound 1 apigenin
(2 µM) caused a reduction in the induced levels (Fig 5.3.4.1A). In agreement with the RT-PCR results, Western blot analysis of Cox-2 protein levels revealed that compound 1 brought about a reduction in the induced expression of the Cox-2 enzyme in a dose-dependent manner, although the protein expression was not reduced to the normal level. In addition to compound 1, compound 3 also showed considerable bioactivity in reducing the expression of the Cox-2 enzyme in a dose-dependent manner. Results from Western blot analysis of cPLA2 suggested that the protein level of cPLA2 in BV-2 cells was regulated neither by the LPS stimulation nor by pretreatment with the flavonoid compounds (Fig 5.3.4.1B).
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Fig 5.3.4.1 Effects of flavonoid compounds 1 to 5 on the expression of Cox-2 and cPLA2 in LPS-stimulated BV-2 cells. BV-2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ for 24 h to allow cell attachment, after which the culture medium was replaced with low-serum DMEM containing 2% fetal bovine serum. Different concentrations of flavonoid compounds 1 to 5 were introduced 30 min before LPS (1 µg/ml) stimulation. The cells were cultured for another 4 h or 24 h for collection of total RNA and whole cell lysates respectively. The mRNA levels of Cox-2 were determined by RT-PCR. A house keeping gene GADPH was also amplified to allow for quantitative comparison of loading (A). The whole cell lystaes were collected for SDS-PAGE and Western blot analysis of Cox-2 and cPLA2 protein levels. β-actin immunodetection was carried out to verify equal protein loading (B). Results are representative of three independent experiments.
5.3.5 Effects of flavonoid compounds 1 to 5 on the expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 cells
The effects of flavonoid compounds 1 to 5 on iNOS expression in
LPS-activated BV-2 cells were also investigated. Firstly, RT-PCR was performed to determine mRNA expression levels in the flavonoids-treated
BV-2 cells. The results revealed that treatment with LPS (1 µg/ml) for 4 h produced a marked enhancement in iNOS mRNA expression (Fig 5.3.5.1A).
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Fig 5.3.5.1 Effects of flavonoid compounds 1 to 5 on the expression of iNOS in LPS-stimulated BV-2 cells. BV-2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ for 24 h to allow cell attachment, after which the culture medium was replaced with low-serum DMEM containing 2% fetal bovine serum. Different concentrations of flavonoid compounds 1 to 5 were introduced 30 min before LPS (1 µg/ml) stimulation. The cells were cultured for another 4 h or 24 h for collection of total RNA and whole cell lysates respectively. The mRNA levels of iNOS were determined by RT-PCR. A house keeping gene GADPH was also amplified to allow quantitative comparison of loading (A). The whole cell lysates were collected for SDS-PAGE and Western blot analysis of iNOS protein levels. β-actin immunodetection was carried out to verify equal protein loading (B). Results are representative of three independent experiments.
While the enhanced iNOS mRNA expression remained largely unaffected when the cells were pre-treated with 2 µM of compounds 2 to 5, compounds 1 to 5 suppressed the expression at a higher concentration of 10 µM, with compound 1 registering the greatest activity (Fig 5.3.5.1A). Pretreatment of the cells with 10 µM of compound 1 30 min before LPS stimulation effectively suppressed the mRNA level of iNOS to a level comparable to that of DMSO control. Western blot analysis was also performed to determine the
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effects of flavonoid compounds 1 to 5 on iNOS protein levels. Consistent with the RT-PCR results, LPS-induced enhanced expression of iNOS protein level was attenuated by pre-treatment of the cells with higher concentration (10 µM) of compounds 1 to 5 (Fig 5.3.5.1B). Among the compounds, compounds 1, 3,
4 and 5 (5 and 10 µM) produced a strong suppressive effect such that the iNOS protein levels were reduced to levels lower than that of DMSO control
(Fig 5.3.5.1B).
5.4 Discussion and conclusions
Following an episode of stroke that results in a state of oxygen-glucose deprivation, ROS, activated proteases as well as complements and factors subsequently released from necrotic cells rapidly lead to a robust inflammatory reaction characterized by leukocyte influx into the cerebral parenchyma and microglia activation (Hallenbeck, 1996; Becker, 1998; Zheng et al., 2004). The activated microglial cells produce more cytokines that cause upregulated production of adhesion molecules in the cerebral vasculature, which in turn mediate adhesion of circulating leukocytes to the vascular endothelia and infiltration into the brain parenchyma. Once gaining entry into the brain, the activated leukocytes and microglia produce a variety of inflammatory mediators such as inducible nitric oxide synthase (iNOS)
(Yamagata et al., 2008) that generates nitric oxide (NO), matrix metalloproteinases (MMPs), cytokines and more ROS. The vicious cycles of inflammatory responses will lead to brain edema, hemorrhage and ultimately, cell death. Notably, the microvascular injury provides a positive feedback loop which will amplify the inflammatory cascade (Fig 5.4.1).
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Several so-called damage-asssociated molecular patterns (DAMPs) such as heat-shock proteins (Hsp), heparan sulfate (HS) that are released during the early onset of cerebral ischemia bind to Toll-like receptors (TLRs) and subsequently activate the nuclear factor kappa-B (NF-κB) and mitogen- activated protein kinase (MAPK) pathways in various brain cell types.
Synthesis and secretion of proinflammatory cytokines and chemokines, as well as expression of leucocyte adhesion molecules therefore set in (Kleinig et al.,
2009). LPS, employed in the experiments described in this chapter, had been reported to induce inflammatory reaction via binding to TLR-4 and activating the NF-κB signaling pathway (Beutler, 2000; Triantafilou et al., 2005).
Through binding to high mobility group box-1 (HMGB-1) protein, receptor for advanced glycosylation end-product (RAGE) has also been found to potently up-regulate NFκB signaling pathway (Qiu et al., 2008). CD36 may also serve as “danger sensors” responding to early post-stroke formation of oxidized low-density lipoproteins and diacylglycerides; it forms signaling complexes with TLRs to activate NFκB, MAPK and AP-1 signaling pathways to play a deleterious role in ischemic stroke (Cho et al., 2005; Bjorkbacka,
2006). The major proinflammatory cytokine pro-IL-1β is inactive unless it is cleaved by the so-called “inflammasome”, a protein assembly incorporating caspase-1 (Ogura et al., 2006). While the TLRs sense predominantly extracellular DAMPs, upstream components of the inflammasome sense intracellular danger signals such as uric acid and low potassium
(Trendelenburg, 2008).
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Fig 5.4.1 The inflammatory cascade following a stroke event. Initiation of the inflammatory cascade by a variety of factors leads to systemic inflammation or activation of cellular proinflammatory pathways in a typical cell (neuron, astrocyte, endothelial cell, etc.). Chemokines and cytokines are consequently secreted, microglial cells activated and endothelial adhesion molecules expressed. These initiators lead to microglial activation which produces more cytokines causing upregulation of adhesion molecules in the cerebral vasculature. Adhesion molecules mediate adhesion of circulating leukocytes to vascular endothelia and infiltration into the brain parenchyma. Once in the brain, activated leukocytes and microglia produce a variety of inflammatory mediators such as iNOS, MMPs, cytokines and ROS which lead to brain edema, hemorrhage and ultimately, cell death. The microvascular injury provides a positive feedback loop which will amplify the inflammatory cascade. AP-1, activator protein-1; MAPK, mitogenactivated protein kinase; RAGE, receptor for advanced glycosylation end-product; MMP, matrix metalloproteinase; NFκB, nuclear factor kappa-B; RAGE, receptor for advanced glycosylation end-products; TLR, toll-like receptor. Adapted from Wang et al (2007) and Kleinig et al’s (2009) publications with modification.
Adhesion molecules are proteins located on the cell surface; they are involved in the binding with other cells or with the extracellular matrix in the
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process called cell adhesion. These proteins are typically transmembrane receptors and are composed of three domains: an intracellular domain that interacts with the cytoskeleton, a transmembrane domain, and an extracellular domain that interacts either with other adhesion molecules or the extracellular matrix (Mousa, 2008; Yilmaz et al., 2008). Adhesion molecules play a pivotal role in the infiltration of leukocytes into the brain parenchyma after stroke.
Three main groups of cell adhesion molecules: (1) selectins (P-selectin, E- selectin, and L-selectin), (2) the immunoglobulin superfamily of intercellular adhesion molecules (e.g. ICAM-1, 2) and vascular cell adhesion molecule-1
(VCAM-1), and (3) integrins (CD11a-c) are involved in the interaction between leukocytes and the vascular endothelium (DeGraba, 1998; Emsley et al., 2002). Targeting on these adhesion molecules to inhibit leukocyte adhesion has been reported to prevent leukocytes from entering the ischemic brain, thus resulting in reduced neurologic injury (Wang et al., 2007).
Furthermore, reduced infarct volumes have been observed in animals deficient in adhesion molecules after transient focal cerebral ischemia (Soriano et al.,
1999).
Cytokines are non-antibody proteins secreted not only by cells of the immune system, but also by resident brain cells, including the glial cells and neurons (Wang et al., 2007). They generally act locally as intercellular mediators in a paracrine or autocrine manner. Cytokines are upregulated in the brain after stroke attack. The most studied cytokines related to inflammation in stroke are interleukin-1 (IL-1), TNF-α, interleukin-6 (IL-6), interleukin-10
(IL-10) and transforming growth factor-β (TGF-β) (Han et al., 2003; Zheng et al., 2003). Among the cytokines, while IL-1 and TNF-α exacerbate cerebral
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injury, IL-6, IL-10 and TGF-β appear to be neuroprotective (Allan et al.,
2001). In this chapter, the LPS-enhanced IL-1β mRNA level was inhibited when the cells were pre-treated with compounds 1 to 4 at 10 µM. Pre- treatment of the LPS-stimulated cells with 10 µM of compound 2 luteolin brought about suppression of TNF-α mRNA expression to a level lower than that of the untreated cells. In addition, The LPS-reduced mRNA expression of
IL-10 was restored by pre-treatment of the cells with 2 µM of compounds 1, 2 and 3. Considering that IL-10 blocks NF-κB activity, down-regulates the expression of Th1 cytokines, MHC class II antigens and is involved in the regulation of the JAK-STAT signaling pathway (Schottelius et al., 1999;
Murray, 2005), through promoting IL-10 expression, flavonoid compounds 1,
2 and 3 isolated from Ixeris sonchifolia Hance might exert important cytoprotective effects in the inflammatory reactions after a stroke event.
The arachidonic acid (AA) cascade lies downstream of immune cell activation and is initiated via the release of phospholipase A2 (PLA2). PLA2s are from several unrelated protein families sharing common enzymatic activities. Among which, secreted and cytosolic phospholipase A2 (sPLA2 and cPLA2) are the two most notable families. High concentration of calcium due to energy failure after a stroke event activates PLA2 to hydrolyze glycerophospholipids to release AA (Kudo et al., 2002; Murakami et al.,
2002). AA is next metabolized through the cyclooxygenase (Cox) (Soriano et al.) or lipoxygenase (Lox) pathways (Saeed et al., 1997; Funk, 2006;
Gonzalez-Periz et al., 2007). There are two isoforms of cyclooxygenase, namely Cox-1 and Cox-2. Cox-1 is constitutively expressed in many cells types, whereas Cox-2 is upregulated in regions of ischemic injury, as well as
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in regions remote from the infarct area (Sairanen et al., 1998; Iadecola et al.,
1999; Toti et al., 2001; Schwab et al., 2002). While Cox-1 exacerbates or attenuates the inflammatory reaction depending on the differences in the immune response, upregulation of Cox-2 is deleterious. Treatment with Cox-2 inhibitors has been reported to improve neurological outcome after stroke
(Iadecola et al., 2001). In this chapter, investigative work on the effects of flavonoids on the LPS-stimulated microglial BV-2 cells had revealed that while the protein level of cPLA2 was not regulated by LPS stimulation and pretreatment with the flavonoid compounds, lower concentration (2 µM) of compound 1 apigenin attenuated the LPS-elevated expression of Cox-2 mRNA and Cox-2 protein, suggesting this compound might exert anti- inflammatory activity via inhibiting Cox-2 expression and subsequent removing downstream effector of PGE2.
Nitric oxide (NO), an important signaling molecule involved in physiological processes such as neuronal communication, host defense, and regulation of vascular tone, is a relatively stable gas that is water-soluble and can readily diffuse into cells and cell membranes where it reacts with molecular targets (Wang et al., 2007). The molecule is synthesized from L- arginine (L-Arg) by the enzyme nitric oxide synthase (Yamagata et al.). Three distinct isoforms of NOS have been isolated, namely neuronal NOS (nNOS,
NOS I, NOS1), endothelial NOS (eNOS, NOS III, NOS3) and inducible NOS
(iNOS, NOS II, NOS2). The first two isoforms, which are constitutively expressed under “basal” conditions, are calcium-dependent. In contrast, iNOS is a calcium-independent enzyme that is only produced by many cell types in response to endotoxins or cytokines. The main physiological effects of NO are
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mediated through activation of soluble guanylate cyclase (SGC) in smooth muscle cells, platelets, leucocytes, neurons and other cells to increase the secondary messenger cyclic GMP (cGMP) levels (Schmidt et al., 1993;
Denninger et al., 1999). In the cardiovascular system, NO acts as a mediator of vascular tone and blood flow. In addition, NO acts to protect the vascular endothelium against atherosclerosis through suppression of leukocyte migration and adhesion to endothelium (Bath, 1993), exerting antiproliferative effects on vascular smooth muscle cells (Garg et al., 1989), and reducing lipoprotein oxidation and absorption into blood vessels (Cardona-Sanclemente et al., 1995). Moreover, NO has antiplatelet properties, and protects against thrombosis by inhibiting platelet adhesion and aggregation. When released in controlled amounts, NO also plays a fundamental role in many biological processes outside of the cardiovascular system. However, when high concentrations of NO are secreted, in particular as derived from iNOS, it can react with ROS such as superoxide anion radical to produce reactive nitrogen species (RNS) like peroxynitrite. This can disrupt many cellular processes through direct oxidative effects and/or alteration of protein conformation via a process termed nitration (Landino et al., 1996; Packer et al., 1996; Xia et al.,
1996). Interestingly, NO release in stroke appears to be able to either promote or prevent cellular inflammation and death. It has been revealed that NO can be anti-inflammatory through (1) inhibition of the maturation of cytokines such as IL-1β and IL-18, (2) blockade of interferon γ, and (3) prevention of the expression of cellular adhesion molecules by acting on NF-κB pathway
(Murphy, 2000; Liu et al., 2002; Eynott et al., 2003; Zeidler et al., 2004).
Alternatively, NO can be pro-inflammatory by ptomoting the expression of
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certain cytokines like TNF-α and IL-6 (Liu et al., 2002; Eynott et al., 2003;
Zeidler et al., 2004). While transgenic mice which do not express eNOS develop larger infarcts after middle cerebral artery (MCA) occlusion and show decreased blood flow at the periphery of the ischemic lesion (Huang et al.,
1996; Huang, 1999), transgenic mice that are deficient in iNOS (Zhao et al.,
2000) or nNOS (Huang et al., 1994) have smaller infarcts and better outcomes than control mice when subjected to MCA occlusion. The protective effect of hypothermia in stroke management is associated with reduced generation of both NO and iNOS by microglial cells (Han et al., 2002), and ischemic brain protection by estrogen and progesterone is proven to be mediated through modulation of iNOS expression (Park et al., 2006). In this chapter, it had been revealed that the concentration of NO released into the culture medium and the iNOS expression was significantly enhanced when the cells were treated with
1 µg/ml of LPS, whereas pre-treatment of the cells with 1 to 10 µM of compounds 1 to 5, in particular compound 1 apigenin at 10 µM, resulted in a reduction in NO release and iNOS expression, thus indicating the potential anti-inflammatory activities of these flavonoid compounds.
In conclusion, compounds 1 to 4 at concentration of 10 µM inhibited
LPS-enhanced IL-1β mRNA level, and compound 2 luteolin at 10 µM depressed TNF-α mRNA expression. The LPS-inhibited mRNA expression of
IL-10 was restored by pre-treatment of the cells with 2 µM of compounds 1, 2 and 3. In addition, compound 1 apigenin at 2 µM attenuated the LPS-elevated gene and protein expression of Cox-2. Finally, 1 to 10 µM of compounds 1 to
5 at concentration of 10 µM reduced the NO release and iNOS expression.
The results suggest that flavonoid compounds 1 to 5 isolated from Ixeris
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sonchifolia Hance extract, in particular compound 1, might exert anti- inflammatory activities by regulating cytokine secretion, inhibiting Cox-2 expression, and reducing iNOS expression and NO release.
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Chapter 6
Possible protective effects of luteolin in rats suffering from cerebral ischemia induced by middle cerebral artery occlusion
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6.1 Introduction
In the previous studies, flavonoid compounds 1 to 5 isolated from
Ixeris sonchifolia Hance extract, in particular compounds 2, 4 and 5, were found to protect cells from H2O2-induced cytotoxiciy by scavenging free radical directly and inducing phase Ⅱdetoxifying enzymes (see Chapter 4). In addition, compounds 1 to 5 were revealed to regulate cytokine secretion and reduce NO release and iNOS expression (see Chapter 5). These results suggested that flavonoid compounds 1 to 5, namely Apigenin (1), Luteolin (2),
Apigenin-7-O-β-glucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4),
Luteolin-7-O-β-glucruonopyranoside (5), might exert neuroprotective effects during the early response to stroke occurrence (characterized by excitotoxicity and oxidative damage) and the second wave of cell death (characterized by neuroinflammation). To study the potential in vivo anti-stroke effects of these compounds, this chapter investigated the possible protective effects of a selected compound luteolin on rats suffering from cerebral ischemia induced by middle cerebral artery (MCA) occlusion. The mortality rates for each treatment group and the infarct area in the brain slices were analyzed. In addition, the expression of cleaved caspase-3 and Cox-2 in samples of brain slices was also assessed.
6.2 Materials and methods
6.2.1. Experimental animals
The animal experimentation described in this study was approved by the Institutional Animal Care and Use Committee (IACUC, Singapore)
(Protocol number 110/06). Male Sprague-Dawley rats weighing 280 to 320 g
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were purchased from the Animal Holding Unit, National University of
Singapore. They were provided with a standard diet and water ad libitum. The room was kept on a 12/12 h light/dark cycle at a temperature of 23 ± 1 °C and relative humidity of 50 ± 10%. An acclimatization period of at least 1 week was allowed for the rats prior to drug administration.
6.2.2 Surgical procedures
Following 12 h fasting of food, animals were neurologically evaluated before anesthesia to confirm normal neurological function. Anesthesia was induced with 12% chloral hydrate (350 mg/kg; intraperitoneal injection). The right common carotid artery was carefully exposed, the occipital branches of the external carotid artery were coagulated, and the internal carotid artery was isolated. A 4 cm length of φ0.24 mm monofilament, poly-L-lysine-coated nylon suture was inserted through the proximal external carotid artery into the internal carotid artery, and was advanced a distance of 18 to 20 mm to occlude the MCA (Fig 6.2.2.1). Incisions were closed, and rats were returned to their cages. After 2 h of MCA occlusion, animals were anesthetized again and the suture was withdrawn to achieve reperfusion injury (Ley et al., 2005). Sham occlusion animals underwent the same surgical procedures without inserting the MCA occlusion suture.
At time points 0, 24 and 48 h post MCA occlusion, luteolin dissolved in 0.5% NaHCO3 solution, sterile-filtered, was injected to the animals via the tail vein (4 mg/kg). For sham occlusion animals and vehicle control animals,
0.5% NaHCO3 sterile solution was injected instead.
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ACA
ACA MCA
MCA ICA
PCA ECA ICA SCA
ICA CCA Suture ECA ECA ICA
CCA CCA
Fig 6.2.2.1 Schematic drawing of the suture placed into ECA and ICA of rat brains to induce middle cerebral artery occlusion (MCAO). ACA: anterior cerebral artery; MCA: middle cerebral artery; PCA: posterior cerebral artery; ICA: internal carotid artery; ECA: external carotid artery; CCA: common carotid artery; SCA: superior cerebellar artery
6.2.3 Evaluation of neurobehavior
Neurological examinations were performed at 1 h prior to, as well as
24, 48 and 72 h post MCA occlusion. The neurological findings were scored on a 5- points scale as described by Longa et al with modifications (Longa et al., 1989; Zhang et al., 1997; Wei et al., 2005):
0 = no neurologic deficit
1 = failure to extend left forepaw fully
2 = circling to the left
3 = falling to the left
4 = no spontaneous walking with a depressed level of consciousness
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6.2.4 Measurement of infarct area
Following 72 h of MCA occlusion, the rats were neurologically assessed and killed by carbon dioxide euthanasia. The brains were removed and washed with cold PBS, and then cut using a vibrating-blade microtome
(Leica VT1000S; Leica microsystems, Germany) into 7 coronal sections each of 2 mm thick. The fourth slice (~2 to 4 mm posterior to the bregma) for each rat brain was quickly immersed in freshly prepared 1% 2,3,5- triphenyltetrazolium chloride (TTC; purchased from Sigma-Aldrich Pte Ltd,
USA) solution in PBS for 20 min at room temperature. The area of damaged parenchyma (unstained tissue) was measured on the posterior surface of each slice using Image-Pro® Analyzer software (Media Cybernetics, USA). The percentage of infarct area, corrected for edema, was expressed as {Infarct area
× [1 - (ipsilateral hemispheric area - contralateral hemispheric area)/contralateral hemispheric area]}/brain slice area × 100% (Swanson et al.,
1990; Lee et al., 2005). After measurement of infarct area, the brain slices were stored in 10% formaldehyde (Fluka BioChemika, Switzerland) solution in PBS at 4 ℃ before immunohistological analysis.
6.2.5 Immunohistological analysis
The formaldehyde-fixed brain slices were sent to Self Service
Histology Laboratory, Institute of Molecular and Cell Biology (IMCB,
Singapore) for embedding and sectioning. Hematoxylin-Eosin staining on the deparaffinized brain slices (5 µm) was performed according to the reported protocol (Kiernan, 2008). Immunohistological analysis on the brain slices was
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carried out by Dr. Keith Rogers (IMCB, Singapore). Briefly, antigen retrieval was performed on the deparaffinized coronal brain sections (5 µm) with 0.01
M sodium citrate buffer at 95 ℃ for 30 min. Following a rinse in PBS, endogenous peroxidase activity was blocked with 0.3% H2O2. Nonspecific binding sites were saturated with goat serum. The expression of cleaved caspase 3 and cyclooxygenase-2 (Cox-2) was detected by overnight incubation at 4 ℃ with the respective anti-cleaved caspase 3 (Cell Signaling Technology
Inc., MA, USA) and anti-Cox-2 (Santa Cruz Biotechnology Inc., CA, USA) antibody, followed by 60 min incubation at room temperature with biotinylated anti-rabbit IgG (BD Biosciences, CA, USA), and finally the processing for immunoperoxidase staining using 3,3-diaminobenzidine tetrahydrochloride as the substrate (BD Biosciences, CA, USA).
6.2.6 Statistical analysis
All results were presented as means ± S.D. and statistical analysis was performed using the software SPSS 13.0. Mortality comparisons between the sham occlusion group, vehicle control group and luteolin treatment group were analyzed one to one using the Chi-square test. The neurobehavioral scores, due to a lack of normality, were analyzed using the Mann-Whitney U test. The infarct area between vehicle control group and luteolin treatment group were compared with one-way ANOVA. Difference was considered significant at p < 0.05.
6.3 Results
6.3.1 Effect of luteolin on mortality and neurobehavioral recovery of MCA-occluded rats
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All the animals before MCA occlusion surgery, as well as sham occlusion animals at 72 h showed normal neurobehavior (neurobehavioral scores being 0). Three rats died during the surgery and 5 rats failed to show ischemic symptoms after MCA occlusion, thus these animals were excluded from statistical analysis. The 3-day survival rates for sham occlusion group, vehicle control group and luteolin treatment group were 100% (4/4), 59%
(16/27) and 60% (18/30) respectively, suggesting that treatment with luteolin did not reduce mortality induced by MCA occlusion (p > 0.05 between vehicle control group and luteolin treatment group). Animals exhibited moderate to severe neurological deficit 72 h after MCA occlusion, with neurobehavioral scores being 2.0 ± 0.4 (p < 0.01 between sham occlusion group and vehicle control group). Treatment with luteolin was found not to improve neurobehavioral recovery with neurobehavioral scores being 1.9 ± 0.5 for rats receiving luteolin treatment (p > 0.05 between vehicle control group and luteolin treatment group).
6.3.2 Effect of luteolin on infarct area in the brains of rats suffering from cerebral ischemia induced by MCA occlusion
For calculating of the infarct area, the rat brain slices were treated with
TTC staining, which due to reaction of the tetrazolium salts with viable dehydrogenases would result in viable tissues being stained “brick-red” while infracted tissues were stained “pale-white”. As shown in Fig 6.3.2.1, MCA occlusion gave rise to an infarct area of 33% when the rats were killed 72 h after surgery (Fig 6.3.2.1B, D), while treatment with luteolin had significantly reduced the infarct area to 25% (p < 0.01; Fig 6.3.2.1C, D), suggesting a neuroprotective effect of luteolin on the MCA-occluded rats.
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Fig 6.3.2.1 Analysis of the infarct area in rat brains by TTC staining. Representative images for brain slices of the sham occlusion rats (A), vehicle control rats (B) and luteolin treatment rats (C) are presented. The luteolin treatment group exhibited significantly reduced infarct area as compared with the vehicle control group (D); results were obtained from brain slices of 8 rats from each group. ** p < 0.01 versus vehicle control.
6.3.3 Immunohistochemistry staining of brain slices
MCA occlusion and the subsequent reperfusion injury resulted in obvious cell death and cell apoptosis in the rat brains, which were evidently characterized by eosinophilic neurons (red neurons, Fig 6.3.3.1A) and neurons with a loss of cellular integrity and affinity for hematoxylin staining (ghost neurons, Fig 6.3.3.1B), as well as an enhancement in the number of cells in the ischemic lesion that were positively stained with anti-cleaved caspase-3
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antibody (Fig 6.3.3.2B). Luteolin treatment resulted in the inhibition of caspase-3 cleavage (Fig 6.3.3.2C), which suggested the potential neuroprotective effects of luteolin on MCA occlusion-induced ischemic rats.
Fig 6.3.3.1 Hematoxylin-Eosin staining of brain slices of rats subjected to MCA occlusion and reperfusion. The middle cerebral arteries of male Sprague-Dawley rats were occluded by inserting nylon suture and the suture was withdrawn 2 h after ischemia to achieve reperfusion injury. The animals were killed 72 h after MCA occlusion and the brains were removed, embedded and sectioned for Hematoxylin-Eosin staining. Eosinophilic neurons (red neurons, A) and neurons with a loss of cellular integrity and affinity for hematoxylin staining (ghost neurons, B) were evident in the ischemic lesion at 72 h after MCA occlusion.
MCA occlusion and the subsequent reperfusion injury also resulted in enhanced expression of Cox-2 in the ischemic tissues (Fig 6.3.3.3B), indicating an activation of neuroinflammatory status in the rat brains following
MCA occlusion. Treatment of the animals with luteolin was found to reduce the number of cells positively stained with anti-Cox-2 antibody (Fig 6.3.3.3C), implying that the compound might exert a certain extent of neuroprotective effects by inhibiting inflammatory reactions.
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(A) (B)
1.4 ##
1.2
positive 1
3 0.8 (%)
0.6 cells caspase
‐ ** 0.4 0.2 Cleaved 0 (C) (D)
Fig 6.3.3.2 Cleaved caspase-3 immunohistochemical staining of brain tissue sections of rats subjected to MCA occlusion and reperfusion. At time points 0, 24 and 48 h post MCA occlusion, luteolin in 0.5% NaHCO3 solution, sterile- filtered, was injected to animals via the tail vein (4 mg/kg). For sham occlusion animals and vehicle control animals, 0.5% NaHCO3 sterile solution was injected instead. The animals were killed 72 h after MCA occlusion and the brains were removed, embedded and sectioned for immunohistochemical staining. Representative images for ischemic parietal cortex of the sham occlusion rats (A), vehicle control rats (B) and luteolin treatment rats (C) are presented. MCA occlusion resulted in a significant elevation in the number of cells positively stained with cleaved caspase-3 antibody, while treatment with luteolin inhibited the induction (D). Results were obtained from brain slices of 3 rats from each group. ## p < 0.01 versus sham occlusion control, ** p < 0.01 versus vehicle control.
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(A) (B)
0.18 0.16 ## (%)
0.14 0.12 cells *
0.1 0.08 positive
2 0.06 ‐ 0.04 Cox 0.02 0 (C) (D)
Fig 6.3.3.3 Cox-2 immunohistochemical staining of brain tissue sections of rats subjected to MCA occlusion and reperfusion. At time points 0, 24 and 48 h post MCA occlusion, luteolin in 0.5% NaHCO3 solution, sterile-filtered, was injected to animals via the tail vein (4 mg/kg). For sham occlusion animals and vehicle control animals, 0.5% NaHCO3 sterile solution was injected instead. The animals were killed 72 h after MCA occlusion and the brains were removed, embedded and sectioned for immunohistochemical staining. Representative images for ischemic parietal cortex of the sham occlusion rats (A), vehicle control rats (B) and luteolin treatment rats (C) are presented. MCA occlusion resulted in a significant elevation in the ratio of anti-Cox-2 positive stained cells, while treatment with luteolin inhibited the induction (D). Results were obtained from brain slices of 3 rats from each group. ## p < 0.01 versus sham occlusion control, * p < 0.05 versus vehicle control.
6.4 Discussion and conclusions
Ranked after coronary heart disease (CHD) and cancers of all types, stroke is the third leading cause of death. The principles in stroke therapy involve the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as to reduce ischemia-caused tissue damage (see Chapter 2). Being different from strategies targeted to restore the blood supply, which can be achieved by delivering thrombolytic
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reagents to the thrombus, strategies to reduce ischemia-caused tissue damage such as through scavenging of free radicals and through inhibition of inflammatory reactions can only be attained when the involved reagents can be transported to the ischemic tissues. Unfortunately, a physiological barrier, the blood-brain barrier (BBB), exists between the cerebral capillaries and the brain tissue. The BBB consists of specialized non-fenestrated tightly-joined endothelial cells around the capillaries and separates the circulatory blood system from the cerebrospinal fluid in the CNS; it restricts the diffusion of microscopic objects (e.g. bacteria) and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the free diffusion of a restricted class of small molecules which have a molecular mass less than 400 Da and are lipid soluble (O2, hormones, CO2). Less than 2% of all small molecules falls under this category. All large molecules with molecular mass higher than 400 Da either do not cross the BBB, or are transported across the BBB via certain transport systems (Pardridge, 2005). Under such a situation, among all the flavonoid compounds isolated from Ixeris sonchifolia Hance, only compound
1 (apigenin, with a molecular mass 270) and compound 2 (luteolin, with a molecular mass 286) possess the possibilities to pass through BBB via free diffusion. As presented in the findings in Chapter 4, compound 2 showed more potential than compound 1 in protecting cells from H2O2-induced cytotoxiciy by scavenging free radicals directly and inducing phase Ⅱ detoxifying enzymes. Furthermore, in anti-inflammatory studies, compound 1 showed almost the same effects with compound 2 in inhibiting cytokine secretion, NO release and iNOS expression (see Chapter 5). Taken together, in this chapter, compound 2 luteolin was selected as the representative compound to
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investigate the potential anti-stroke effects of flavonoids isolated from Ixeris sonchifolia Hance.
Animal models play pivotal roles in screening and developing of pharmacological agents for stroke treatment. Several animal models in different species are currently used to produce cerebral ischemia, while hemorrhagic animal models are less appropriate and less studied due to the relative low occurrence (~15% of all stroke occurrences; see Chapter 1).
Ischemic stroke animal models can be divided into global ischemic and focal ischemic models. Global models, both complete and incomplete, results in ischemia over a large area of the brain. Obvious advantage for global models is that it can be easily performed. However, they are of less immediate relevance to human stroke than the focal stroke models, and are now generally considered to model the cerebral consequences of cardiac arrest rather than stroke (Green et al., 1997). Focal models occlude a specific vessel, usually the middle cerebral artery (MCA) because the majority of human ischemic strokes result from an occlusion in the region of the MCA (Millikan,
1992; Richard Green et al., 2003; Traystman, 2003). MCA occlusion models, either permanent or transient (based on the removal of the blockade to allow reperfusion), can be achieved by (1) injection of particles like blood clots or artificial spheres into the carotid artery (to achieve thromboembolic MCAO),
(2) surgical occlusion (e.g. by electrocautery or ligation) of MCA (Millikan,
1992; Richard Green et al., 2003; Traystman, 2003), or by cerebrocortical photothrombosis (van Rossem et al., 1992). Another simple, relatively noninvasive method to produce either permanent or transient MCAO animal models is to insert a piece of surgical filament into the internal carotid artery
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and push the filament forward until the tip occludes the origin of the middle cerebral artery. If the filament is left in place, the procedure is suitable as a model of permanent MCAO. Reperfusion can be easily obtained by withdrawing the thread, and the animals may survive for days, weeks, and months, which will enable functional outcome measures to be recorded
(Longa et al., 1989). The experiment employed in this Ph.D study had used transient focal ischemic rats to study the potential anti-stroke effects of compound 2 luteolin. Ischemia was achieved by inserting a nylon suture 17 to
20 mm from the origin of the internal carotid artery to occlude collateral circulation from the anterior communicating arteries; reperfusion was performed 2 h after the occlusion by withdrawing the thread. The suture was coated with poly-L-lysine to afford a tighter adherence to the surrounding endothelium and a more consistent infarction. Previous observational studies had suggested a protective effect of estrogens on stroke risk among women who received postmenopausal hormone replacement therapy (Mendelsohn et al., 1999; Mendelsohn, 2002; Cho et al., 2003). Therefore, to avoid the possible influence of estrogens on ischemic outcomes, only male rats were used in this experiment to minimize individual variation.
In view that excitotoxicity and free radicals attack occurs in minutes after ischemia with a “burst” upon reperfusion, and neuroinflammation occurs after several hours, to investigate the neuroprotective effects of luteolin on
MCA-occluded rats, the compound, dissolved in 0.5% NaHCO3 sterile solution, was intravenously administrated immediately after the ischemia (4 mg/kg). The dose selection was as referenced from the study by Li et al (2006).
In their study, flavonoid glycoside nicotiflorin, at a concentration of 5 to 10
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mg/kg, was demonstrated to reduce the size of infarction and improve the behavioral deficits observed in permanent MCAO rats (Li et al., 2006). Upon normalization to molar concentration, 4 mg/kg of luteolin fell under the same concentration range (8.4 to 16.8 µM). In consideration that the maximum infarct size would have been established 24 to 36 h after stroke onset (Pantano et al., 1999), in this study, the ischemic rats were killed 72 h after MCA occlusion for infarct area calculation and immunohistological staining.
The 3-day survival rates for sham occlusion group, vehicle control group and luteolin treatment group were 100% (4/4), 59% (16/27) and 60%
(18/30) respectively. The high mortality rates for the latter two groups might be due to surgery-related complications, such as bleeding and infections during surgery, extended surgery duration or body temperature loss after ischemia induction. Another reason could be that severe ischemic rats had failed to consume food and water. In rats that survived 72 h post MCA occlusion had neurobehavioral scores less than 2; animals with scores higher than 2 (3: falling to the left; 4: no spontaneous walking with a depressed level of consciousness), though might be surviving at 24 h after MCA occlusion, had indeed lost their ability to ingest food and water and thus hardly survived at 72 h. Treatment of the MCA-occluded rats with intravenous injection of 4 mg/kg luteolin had reduced the infarct area, decreased the cell number positively stained with anti-cleaved caspase-3 antibody and anti-Cox-2 antibody. Taken together, the results obtained in this chapter suggest that luteolin might exert neuroprotective effects by inhibiting cell apoptosis and by inhibiting inflammatory reactions.
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Chapter 7
General discussion and conclusions
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Based on the therapeutic outcomes of Diemailing Injection, which contains a raw extract of Ixeris sonchifolia Hance, this Ph.D project had hypothesized that Ixeris sonchifolia Hance contained phytochemical constituents that possessed anti-stroke potential with anticoagulation, antioxidant and anti-inflammatory activities, and by conducting bioassay- guided isolation and purification would indentify the constituents. Therefore, the primary objective of this Ph.D study was to isolate and identify constituents from Ixeris sonchifolia Hance that display anti-stroke activities and to elucidate the possible mechanism(s) through which the identified compound(s) exerted the neuroprotective effects.
To accomplish this objective, the aerial part of Ixeris sonchifolia Hance was extracted and the crude extract was partitioned into fractions using different solvents. The fractions were then subjected to screening using both cell-based in vitro bioassays and biochemical reactions. Taking into consideration that the principles in stroke treatment would involve (1) the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as (2) to reduce ischemia-caused tissue damage, this study had employed an in vitro assay to evaluate the anticoagulation activities and two in vitro approaches to evaluate the antioxidant activities of these fractions. The cytoprotective effect of these fractions on H2O2-exposed cells was also evaluated using the MTT cell viability assay. Based on what has been reported so far, this was the first time that fractions isolated from Ixeris sonchifolia Hance extract were systematically screened with the objective to identify fraction(s) possessing desired neuroprotective bioactivities. The results had revealed that the ethyl
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acetate fraction (Fr. 3) isolated from Ixeris sonchifolia Hance extract protected cells dose-dependently from H2O2-induced cytotoxicity, scavenged DPPH free radicals, induced ARE-dependent transcriptional activity and caused upregulation of Nrf2 protein levels. However, it failed to exhibit anticoagulant activities. In vitro bioassays are adopted in drug discovery screening programs as they possess the advantages of high throughput, low cost, and operational convenience. Although they may successfully model one or two aspects of the interactions between the investigated agents and the pathological response, there exist significant differences between the physiological response and the in vitro experimental set-up. As such, as demonstrated in this study, although the fractions failed to exert direct and immediate inhibitory effects on clotting factors in the in vitro anti-coagulation assay, they might yield different outcome under physiological conditions. Possibilities that these fractions isolated from Ixeris sonchifolia Hance might exert anti-coagulation activity in an in vivo setting therefore could not be excluded. Similarly, possibilities that these fractions might exert beneficial effects in the treatment of stroke via pathways other than restoration of redox homeostasis also could not be excluded. Based on what had been obtained from the preliminary assays, the ethyl acetate fraction (Fr. 3) was identified as the fraction possessing potential neuroprotective properties and was subjected to further fractionation and purification experimental work with the ultimate goal of identifying components that could play key roles in the prevention and treatment of ischemia-related diseases.
The follow-up isolation work focused on the ethyl acetate fraction (Fr.
3) of Ixeris sonchifolia Hance revealed the presence of two classes of
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compounds: flavonoids and sesquiterpene lactones. Among all the purified compounds, compound 8, 11,13-Dihydroixerinoside, is a new sesquiterpene lactone which has not been reported previously. Sesquiterpene lactones demonstrate a variety of biological activities such as cytotoxicity (Kim et al.,
2007; Suzuki et al., 2007; Tabopda et al., 2007; Yang et al., 2007; Yang et al.,
2007), antimalarial (Bischoff et al., 2004; Pedersen et al., 2009), antibacterial
(Theodori et al., 2006) and herbicidal (Macias et al., 2000) effects. The isolation of this new compound and characterization of its structure and physical and chemical properties has enabled new information about the Ixeris genus to be generated. Given that thousands of compounds exist in a single herb, and that the chemical properties of these compounds may vary greatly, the efficiency of isolating natural pure components from the herb by traditional strategies is heavily challenged. In most cases, an optimized isolation method is only suited for the purification of one or one group of components with similar chemical properties. Therefore, compounds that possess distinct physical and chemical properties from these components of interest will be missed out. Such loss of compounds in the isolation procedures, especially those possessing desirable bioactivities, is one obvious shortcoming in the traditional approach in natural product research. In this thesis, HPLC analysis of the ethyl acetate fraction from Ixeris sonchifolia Hance extract had revealed the presence of more than twenty compounds with conjugated structure (compounds that gave signals when the detector wavelength was set at 350 nm with 410 nm as reference; Fig 3.3.2.1B). However, only six flavonoid compounds had been isolated and identified from this fraction, suggesting that the majority (at least in quantity) of compounds were lost
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during the isolation and purification process. In addition, the purity of samples isolated from natural products was directly related to the rigor of bioassay and the intensity of side effect reactions. In order to achieve a highly purified compound on column chromatography, the tails on both sides of the peak for the compound of interest, which might overlap partially with that of compounds of non-interest, would have to be discarded. The exclusion of the overlaping parts of the compound of interest would result in poor yield or poor recovery, which made up another obvious shortcoming in the traditional natural product isolation techniques. Fortunately, outstanding developments in the areas of separation science and spectroscopic techniques (e.g. GC-MS,
LC-PDA, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, CE-MS) over the last decade have allowed higher sensitivity and efficiency in the isolation analysis of crude extracts or fractions from different natural sources (Sarker et al.,
2005). Even so, to obtain adequate and highly purified compounds from natural products for bioactivity studies remains to be challenging.
Flavonoids are found ubiquitously in plants and possess multiple health-modulating effects, such as anti-allergic (Muthian et al., 2004), anti- inflammatory (Kao et al., 2005; Lim et al., 2006; Zhou et al., 2009), anti- microbial (Cushnie et al., 2005; Alvarez et al., 2008; Pistelli et al., 2009) and anti-cancer (Choi et al., 2009; Guo et al., 2009; Jung et al., 2009; Rajkapoor et al., 2009) activities. In this thesis, the in vitro bioassays conducted on the flavonoids isolated from the ethyl acetate fraction of Ixeris sonchifolia Hance extract had revealed that these compounds could protect cells from H2O2- induced cytotoxiciy by scavenging free radical directly and inducing phase Ⅱ detoxifying enzymes. This has suggested that the purified flavonoid
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compounds might exert neuroprotective effects during the early response to
stroke occurrence. To our knowledge so far, this is the first time to attribute
the chemical components being responsible for the neuroprotective effects of
Ixeris sonchifolia Hance. The findings provide important experimental data for developing potential anti-stroke agents derived from Ixeris sonchifolia Hance.
Although flavonoid compounds isolated from the ethyl acetate fraction of
Ixeris sonchifolia Hance extract showed considerable bioactivities in
protecting cells from H2O2-induced cytotoxiciy through induction of phase Ⅱ
detoxifying enzymes, the greatest fold of induction of ARE-dependent
transcriptional activity effected by the isolated flavonoid compounds was not
as high as the induction fold registered by the ethyl acetate fraction (the
induction fold being around 5 and 20 respectively; see Section 2.3.3 and 4.3.2).
Components in the fraction might exert synergistic effects with one another,
which could account for the higher phase Ⅱ enzymes inducing ability of the
fraction over that of each purified flavonoid compound. The isolation and
identification of compounds with higher phase Ⅱ enzymes induction abilities
from the ethyl acetate fraction, or to elucidate the possible synergistic
interactions between these compounds should be one of the research directions
in future studies.
Considering that the second wave of cell death after a stroke incident
stems from the neuroinflammatory response, the anti-inflammatory effects of
these isolated flavonoid compounds on the LPS-stimulated cells were next
determined. The results suggested that flavonoid compounds isolated from
Ixeris sonchifolia Hance extract might exert anti-inflammatory activities by
regulating cytokine secretion, inhibiting Cox-2 expression, as well as reducing
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NO release and iNOS expression. Inflammatory processes are initiated by cytokines, adhesion molecules, prostanoid mediators of inflammation and NO and may progress for days and weeks. The wide therapeutic window and the multiple targets have rendered inflammatory intervention an attractive therapeutic strategy in stroke treatment (Michael et al., 2004; Sacco et al.,
2007). The findings presented in this thesis had indicated that the isolated flavonoid compounds showed potential neuroprotective activities by inhibiting the inflammatory reactions. However, the biological signaling pathways in inflammatory reactions are extremely complicated and certain cytokines can have different effects depending on the context (Rothwell, 1997; Allan et al.,
2001). The investigation of the intricate interactions between the compounds and the inflammatory response, and the detailed relationship between the inflammatory response and the stroke injury, will be another arduous task in the future studies.
As validation, the potential anti-stroke effects of luteolin, a representative of these isolated flavonoid compounds, were investigated in vivo using rats suffering from cerebral ischemia induced by MCA occlusion.
This MCAO animal model closely resembles a clinical stroke incident caused by focal ischemic attack and has been shown to be sensitive to treatment with neuroprotective compounds that function via a number of mechanisms. The results presented in this study revealed that while treatment with luteolin failed to neither reduce MCAO-induced mortality nor improve neurobehavioral recovery in the treated rats, it significantly reduced the infarct area, decreased the number of cells positively stained with anti-cleaved caspase-3 and anti-
Cox-2 antibody, suggesting that luteolin might exert neuroprotective effects in
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an in vivo stroke model. Unfortunately, the development of neuroprotective therapies for acute ischemic stroke has proven to be a difficult and challenging endeavor. To date, more than 700 compounds of potential neuroprotective effects have been studied, but none of them has been accepted by regulatory authorities to be used for treatment of patients with acute stroke (Kaste, 2005).
The failure in developing neuroprotective agents into clinical application may be attributed to several factors. For example, it must be recognized that no animal stroke model will precisely mimick humans’ heterogeneous physiological reactions. In a laboratory study, the investigator can modify all known confounding factors. The age, body weight, body temperature, blood pressure, blood glucose, and acid-base balance of animals can be kept consistent and within normal physiological ranges, whereas in a hospital emergency room, stroke patients may be admitted with several severe concomitant diseases, e.g. fragile diabetes, untreated hypertension and recent myocardial infarction; their physiological parameters can hardly be unified. In addition, inappropriate time points to administer the experimental drug may be another reason contributing to the failure in developing neuroprotective agents.
Once the necrotic core has been formed in the brain tissue, which accounts for the largest proportion (~70%) of the final infarct volume, even if neuroprotectants can prevent the maturation of the ischemic penumbra to an infarct by half, it would only reduce the size of the final area of infarction by
15%, which implies a minor neuro-function recovery. Therefore, careful constitution of patient enrolment criterion and optimization of drug dose and administration timepoints play a crucial role in the successful development of neuroprotectants for the treatment of ischemic stroke.
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In conclusion, extraction and isolation works on Ixeris sonchifolia
Hance revealed the presence of two categories of compounds in the ethyl acetate fractions of its raw extract: flavonoids and sesquiterpene lactones. The isolated bioactive flavonoids were found to possess potential anti-stroke effects, which at least in part were attributed to their antioxidant and anti- inflammatory activities. These findings have provided important experimental data for developing potential anti-stroke agents from Ixeris sonchifolia Hance.
Considering that the ethyl acetate fraction of Ixersi sonchifolia Hance extract is also a rich source of sesquentepene lactones, screening of the bioactivities of this category of compounds are to be performed in the future. In view of the fact that the majority of components were discarded during the isolation procedure, this traditional natural product research strategy had only successfully isolated and identified a small percentage of all the compounds contained in the herb. For the future work, the use of tandem techniques (e.g.
GC-MS, LC-NMR-MS, CE-MS) should be employed in order to systematically draw a comprehensive “component mapping” of Ixeris sonchifolia Hance. Following “component mapping”, the bioactivities of these components need be studied, the quantity of active components present in the herb be determined and the possible interactions between the components are to be elucidated. Another research direction in the future work can be focused on the screening strategies. Since ischemia in stroke leads to the activation of several pathways such as excitotoxic cell death, apoptosis, complex cascades of local inflammatory response, and remodeling of neuronal function, anti- stroke effects exerted via pathway(s) other than anticoagulation activities and antioxidant activities may be missed. Thus, more bioassays that model
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different aspects of the pathophysiological responses following stroke can be adopted in the future screening studies. At last, with the aim to optimize dosing regimen, pharmacokinetics studies conducted on animals or human body are to be carried out. After which a “modern” herb preparation that consists of all the beneficial components with optimized ratios of each desired component and dose regimen can be ultimately developed.
146
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