A chemical and pharmacological study of a commercial Andrographis paniculata extract and andrographolide, to determine their
anti-inflammatory drug potential
A thesis submitted for the degree of
Doctor of Philosophy
By Mitchell Nolan Low
Student number 16120280
Principal Supervisor:
Dr Cheang Khoo
Co-Supervisor: Professor Gerald Muench
School of Health and Science
Western Sydney University
STATEMENT OF AUTHENTICITY
This thesis is submitted in fulfilment of the requirements for the Doctor of Philosophy at the
Western Sydney University; School of Health and Science. The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not previously submitted this material, either in full or in part, for a degree at this or any other institution. Unless otherwise stated, all of the data and observations presented here are the results of my own work.
Mitchell Nolan Low
August 2015
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Abstract
There is increasing evidence that chronic inflammation is a contributing factor to many prevalent ageing-related diseases, such as acute and chronic neurodegenerative diseases, degenerative musculoskeletal diseases, cardiovascular diseases, diabetes, and cancer. To date, pharmacotherapy of inflammatory conditions is based mainly on the use of non- steroidal anti-inflammatory drugs (NSAIDs). However, the prolonged use of NSAIDs can cause serious side effects. There is a need to develop novel and safe anti-inflammatory medicines. Andrographis paniculata (Acanthaceae), has been used as an herbal medicine in both traditional Indian and Chinese medicine for inflammation related ailments. A. paniculata anti-inflammatory activity is commonly attributed to the ent-labdane diterpenoid andrographolide, A. paniculata’s characteristic and main secondary metabolite. This compound may be suitable as an alternative treatment for inflammation, with alternate pharmacological targets to that of NSAIDs with reduced side effects.
The commercial A. paniculata extract was fractionated based on polarity (by chloroform- aqueous partitioning), and characterised by HPLC-PDA to quantify the andrographolide. A sulphonation metabolite of andrographolide was synthesised, purified by preparative HPLC and characterised by MS and 1HNMR. The commercial A. paniculata extract, andrographolide, sulphonation metabolite and NSAIDs (Diclofenac, Ibuprofen, Aspirin,
Paracetamol) were tested in a number of anti-inflammatory in vitro cell based assays, nitric oxide (Griess reagent) , TNF-α and PGE2 (ELISA), 27 simultaneous cytokines (bead based
ELISA) and NF-κB activation (flow cytometry).
The andrographolide content accounted for the in vitro anti-inflammatory activity of A. paniculata. Andrographolide is a potent inhibitor of many mediators of inflammation.
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NSAIDs were not effective inhibitors of any of the mediators tested except PGE2.
Andrographolide showed comparable inhibition of PGE2 release to that of the weak NSAIDs aspirin and paracetamol. Andrographolide’s IC50 for most mediators was below the highest reported Cmax’s in rats, but above those achieve in humans. The sulphonation metabolite of andrographolide had comparable activity to andrographolide against most inflammatory mediators and has greater activity against TNF-α and IL-6. The sulphonation metabolite’s
IC50 for the majority of mediators was well below (47 times) which had the only reported
Cmax in rats.
The sulphonation metabolite shows promise as a treatment for inflammation as it inhibited a wide range of inflammatory mediators below the reported in vivo levels. Pharmacokinetics studies in humans are needed to determine its pharmacokinetics. Further animal studies should be conducted to assess its safety.
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Dedication
I would like to dedicate this work to my grandmother, Reta Low who solemnly passed away in the weeks leading to submission and was unable to see the first member of our family complete a doctoral thesis. Thank you for your great bravery in bringing your young family from a small Scottish fishing village across the world so that we might enjoy a better future.
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Acknowledgements
I am greatly appreciative and indebted to my primary supervisor Dr Cheang Soo Khoo (The
Compassionate One) who has provided invaluable expertise and guidance throughout the project. Not disregarding your well esteemed academic career you have inspired me to be a more considerate academic. Your humour, realism, patience and understanding nature have ensured the wheels continued turning.
I am thankful to my secondary supervisor Professor Gerald Munch, who kindly made himself available to join the project and provided timely expertise. Your pharmacological proficiency and energetic pursuit of academic success ensured the project’s success.
I am grateful to Professor Nickolas Sucher who laid the foundations for a strong start to the project. I am still in admiration of your tenacity, academic commitment and passion for research. Your insatiable appetite for data and commitment to cutting edge research ensured the project’s success from an early stage.
Many thanks go to the National Institute of Complementary Medicine (NICM) and the
University of Western Sydney for supporting this research. I am very grateful to the directory of NICM, Professor Alan Bensoussan who has always provided gentle encouragement and facilitated part-time studies while engaged in full time employment. To the NICM HDR director Professor Caroline Smith thank you for all your assistance. Thank you to Narelle,
Natalie and Micki who have provided fantastic support during the study; to A/Prof. Dennis
Chang and A/Prof. Chun Guang Li, who have been extremely understanding as I completed this thesis while working under their supervision; to all members of NICM and especially those in the labs.
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Thank you to Dr. Tara Robertson from University of Queensland who provided the
RAW264.7 ELAM9 cells used in the study.
Thank you to Dr Narelle Sales who made it possible to complete the multicykoines assays by providing access to equipment at the Department of Primary Industries, Menangle.
Thanks to Jarryd my friend and colleague who has shared the joys of completing the bachelors, honours and now working full time and completing a Ph.D. together.
A loving thank you to all my family and friends who have provided lots of love and support over the years and endured my academic pursuit. Thank you for providing many meals, assistance and babysitting. To my parents David and Michelle, who supported me materially and with love, guidance and encouragement throughout my whole life. To my father in law
Joe for extending our home so that our new daughter had her own room and we could enjoy sleep. To my daughter Penelope, who has made e the last year much more enjoyable but also challenging. To my beautiful and intelligent wife for all her love, support and editing.
To everyone who has helped me in some way throughout my Ph.D. candidature, I will always be grateful.
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Table of content
1. The inflammatory response and herbal medicine...... 1
1.1. Inflammation ...... 2
1.1.1. Pathogen associated molecular pattern’s acute inflammatory response ...... 3
1.1.2. Chronic inflammation ...... 4
1.1.3. Para-inflammation...... 5
1.1.4. Predicted increasing incidence of inflammation ...... 6
1.1.5. Limitations of current treatment for inflammation ...... 7
1.1.6. Possible alternatives to NSAIDs ...... 8
1.2. Principles of traditional herbal medicine ...... 8
1.2.1. Andrographis paniculata for the treatment of inflammation ...... 10
1.2.2. Herbal medicine’s safety and efficacy ...... 10
1.3. Biological targets for investigating inflammation ...... 11
1.3.1. Role of macrophages in inflammation ...... 11
1.3.2. Lipopolysaccharide’s stimulation of macrophages ...... 12
1.3.3. Interferon gamma stimulation of macrophages ...... 13
1.3.4. Role of nitric oxide in inflammation ...... 14
1.3.5. Prostaglandin’s role in inflammation ...... 17
1.3.6. Cytokine’s role in inflammation ...... 18
1.3.7. The role of NF-κB in inflammation ...... 26
1.4. Hypotheses and aims...... 28
1.4.1. Hypotheses ...... 28
1.4.2. The major specific aims of the thesis ...... 28
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2. Methods ...... 30
2.1. Reagents ...... 31
2.1.1. Solvents/chemicals ...... 31
2.1.2. Standards and other reagents ...... 31
2.1.3. Cell culture reagents ...... 32
2.2. Equipment ...... 33
2.2.1. General ...... 33
2.2.2. FACS Canto II flow cytometer ...... 33
2.2.3. Multiplex assay system ...... 34
2.2.4. Cell culture consumables ...... 34
2.3. Cell culture methods ...... 35
2.3.1. Cell culture ...... 35
2.3.1.1. Murine Macrophages RAW264.7 cells ...... 35
2.3.1.2. Cell maintenance- RAW264.7 ...... 35
2.3.1.3. Human THP-1 cells...... 36
2.3.1.4. Cell maintenance- THP-1 ...... 36
2.3.2. Cell counting ...... 37
2.3.2.1. RAW264.7 Vi-CELL XR cell viability analyser analysis parameters ...... 37
2.3.2.2. THP-1 Vi-CELL XR cell viability analyser analysis parameters ...... 37
2.3.3. Cell plating ...... 38
2.3.3.1. Plating of RAW264.7 cells ...... 38
2.3.3.2. Plating and differentiation of THP-1 cells ...... 38
2.3.4. Compound addition ...... 39
2.3.4.1. Compound preparation...... 39
2.3.4.2. Compound pre-incubation ...... 40
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2.3.5. Cell activation ...... 40
2.4. Cell viability by MTT ...... 41
2.4.1. Principles of MTT assay ...... 41
2.4.2. Protocol for MTT viability determination ...... 41
2.5. Determination of NO production ...... 42
2.5.1. Principles of NO assay - Griess method ...... 42
2.5.2. Nitric oxide quantification ...... 42
2.6. Determination of TNF-α release ...... 43
2.6.1. Principles of TNF-α ELISA ...... 43
2.6.1.1. Reagent preparation ...... 44
2.6.1.2. Peprotech TNF-α protocol for RAW264.7 ...... 45
2.7. Determination of PGE2 release ...... 46
2.7.1. Principles of COX ELISA ...... 46
2.7.2. Protocol for PGE2 quantification release by RAW264.7 and THP-1 cells ...... 48
2.8. Determination of NF-kB activation ...... 49
2.8.1. Principle of NF-κB assay ...... 49
2.8.2. Protocol for NF-kB activation assay ...... 49
2.8.3. Flow cytometer setup for analysis of GFP in ELAM9 RAW264.7 cells...... 50
2.8.4. NF-kB data analysis ...... 50
2.9. THP-1 multicytokine assay ...... 52
2.9.1. Principles of the multicytokine bead based assay ...... 52
2.9.2. Protocol for THP-1 multicytokine assay...... 53
2.10. Statistical data analysis ...... 54
3. Identification of the major active component of A. paniculata ...... 55
3.1. Introduction ...... 56
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3.1.1. Complexity of characterising herbal medicine ...... 56
3.1.2. Standardisation as a means to control variation ...... 56
3.1.3. Importance of correct plant identification...... 57
3.1.4. Physical description of A. paniculata ...... 58
3.1.5. Traditional uses of A. paniculata ...... 59
3.1.6. Chemical substances in A. paniculata ...... 59
3.1.7. A. paniculata’s inhibition of prostaglandin and nitric oxide production ...... 61
3.1.8. A. paniculata’s inhibition of inflammatory cytokines ...... 66
3.1.9. NF-κB activity of andrographolide ...... 73
3.2. Aims of the chapter 3 ...... 78
3.3. Methods...... 79
3.3.1. Materials ...... 79
3.3.1.1. Standards used in analysis ...... 79
3.3.1.2. Source of the A. paniculata extract ...... 79
3.3.2. Method used to partition and prepare extracts ...... 79
3.3.3. Preparation of the mixed standard solution ...... 80
3.3.4. HPLC system ...... 80
3.3.5. HPLC analysis of extracts ...... 81
3.3.6. HPLC method used to quantify andrographolide ...... 81
3.3.7. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells. 82
3.3.8. Determination of TNF-α release in LPS and IFN-γ stimulated RAW264.7 cells. 82
3.3.9. Determination of PGE2 release in LPS and IFN-γ stimulated RAW264.7 cells. .. 82
3.3.10. Determination of 17 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay...... 83
3.4. Results and discussion ...... 84
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3.4.1. Partition of the mixed standard ...... 88
3.4.2. Partition of A. paniculata commercial extract ...... 91
3.4.3. Quantification of andrographolide ...... 95
3.4.4. Nitric oxide production in RAW264.7 cells ...... 100
3.4.5. PGE2 release by RAW264.7 cells ...... 105
3.4.6. RAW264.7 TNF-α assay...... 107
3.4.7. THP-1 TNF-α results ...... 110
3.4.8. Multiplex results ...... 112
3.4.9. NF-κB results ...... 134
3.4.10. Summary of results ...... 136
3.5. Conclusions of chapter 3 ...... 143
4. In vitro comparison of Andrographolide to common NSAIDs ...... 144
4.1. Introduction to chapter 4 ...... 145
4.1.1. The use of NSAIDs ...... 145
4.1.2. Increasing use of NSAIDs ...... 146
4.1.3. Side effects of NSAIDs ...... 147
4.1.4. NSAID mechanism of action ...... 147
4.1.5. Strategies to reduce NSAIDs’ adverse effects ...... 148
4.1.6. Alternatives to NSAIDs ...... 149
4.1.7. Andrographolide as an alternative to NSAIDs ...... 150
4.2. Aims of chapter 4 ...... 151
4.3. Methods...... 152
4.3.1. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells.
...... 152
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4.3.2. Determination of TNF-α release in LPS and IFN-γ stimulated RAW264.7 cells.
...... 153
4.3.3. Determination of PGE2 release in LPS and IFN-γ stimulated RAW264.7 cells. 153
4.3.4. Determination of 27 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay...... 153
4.4. Results and discussion ...... 154
4.4.1. Nitric oxide production by RAW264.7 cells ...... 154
4.4.2. PGE2 release by RAW264.7 cells ...... 155
4.4.3. TNF-α release by RAW264.7 cells ...... 157
4.4.4. Multiplex assay ...... 159
4.4.5. NF-κB activation assay ...... 164
4.4.6. Summary ...... 166
4.5. Conclusions of chapter 4 ...... 170
5. Synthesis and in vitro assessment of the sulphonation metabolite of andrographolide ...... 171
5.1. Pharmacokinetics of herbal medicines ...... 172
5.1.1. Andrographolide’s Cmax in human plasma ...... 173
5.1.2. Andrographolide’s Cmax in animal plasma ...... 174
5.1.3. Andrographolide’s Cmax after formulation modification ...... 176
5.1.4. A. paniculata pharmacokinetics ...... 178
5.1.5. Biological significance of andrographolide’s sulphonation metabolite ...... 186
5.2. Aims of chapter 5 ...... 188
5.3. Methods...... 189
5.3.1. Materials ...... 189
5.3.2. LC-MS reagents ...... 189
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5.3.3. HPLC system ...... 189
5.3.4. Preparative HPLC system ...... 189
5.3.5. LC-MS instrumentation ...... 190
5.3.6. Proton nuclear magnetic resonance (1HNMR) spectrometer ...... 190
5.3.7. Synthesis of andrographolide sulfonate metabolite ...... 190
5.3.8. HPLC-PDA analysis of sulphonation products ...... 191
5.3.9. Preparative HPLC to partition the sulphonate product ...... 191
5.3.10. The characterisation of the sulphonation metabolite of andrographolide by
1HNMR ...... 192
5.3.11. Characterisation of the sulphonation metabolite by LC-MS ...... 193
5.3.12. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells.
...... 194
5.3.13. Determination of TNF-α release in LPS and IFN-γ stimulated THP-1 cells. .... 194
5.3.14. Determination of 27 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay...... 194
5.4. Results and discussion ...... 195
5.4.1. Analysis of synthesised product...... 195
5.4.2. Purification by preparative HPLC ...... 196
5.4.3. Characterisation of the sulphonation product of andrographolide by LC-MS .... 197
5.4.4. Characterisation of the sulphonation product of andrographolide by 1HNMR ... 198
5.4.5. Nitric oxide production in RAW264.7 cells ...... 201
5.4.6. TNF-α release by THP-1cells ...... 202
5.4.7. Multiplex assay for 27 cytokines released from THP-1 cells ...... 204
5.4.8. NF-κB activation assay in ELAM9 cells RAW264.7 cells ...... 212
5.4.9. Summary of results ...... 213
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5.5. Conclusions from chapter 5 ...... 218
______...... 219
6. Conclusions and future work ...... 219
6.1.1. Conclusion ...... 220
6.1.2. Future work ...... 220
7. References ...... 224
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List of figures
Figure 1: DAMPs and PAMPs role in inflammation [5]...... 3
Figure 2: Physiological purposes of inflammation dependent upon the trigger [15]...... 6
Figure 3: Schematic diagram of LPS induced inflammation in macrophage cells [51]...... 13
Figure 4: Schematic diagram of canonical activation of IFN-γ receptor involving activation
[56]...... 14
Figure 5: Formation of Nitric Oxide [61]...... 15
Figure 6: Superoxide and NO form peroxynitrite [66]...... 16
Figure 7: Diagram showing the activation pathway of NF-κB by LPS stimulation [129] ...... 27
Figure 8: Cell viability quantification with MTT [136]...... 41
Figure 9: Griess-reagent principle in nitrite quantification [139]...... 42
Figure 10: An illustration of the TNF-α assay [141]...... 44
Figure 11: TMB colour development (modified from [142])...... 44
Figure 12: An illustration of the principles of the competitive PGE2 ELISA [143]...... 47
Figure 13: Ellman reagent acetylcholinesterase enzymatic reaction [143]...... 48
Figure 14: Gating of normal cells by FSC and SSC...... 51
Figure 15: Gating of stimulated cells (stimulated control)...... 51
Figure 16: Gating of stimulated cells (unstimulated control)...... 52
Figure 18: A photograph of A. paniculata from the Botanical Gardens, Berlin.[153] ...... 58
Figure 19: The structure of some compounds identified in A. paniculata...... 60
Figure 20: HPLC chromatogram of A. paniculata commercial extract at 254 nm...... 84
Figure 21: Heat map contour chromatogram of A. paniculata commercial extract. The colours indicate the absorbance intensity with, red= 200+ mAu, yellow= 150 mAu, green= 100 mAu,
Blue= 0 mAu, purple= -50 mAu, white> -50 mAu...... 85
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Figure 22: HPLC chromatogram of A. paniculata commercial extract (zoomed to 200 mAu) at 254 nm...... 86
Figure 23: UV spectrum comparison between standards and peaks tentatively identified as corresponding peaks in A. paniculata commercial extract...... 87
Figure 24: HPLC chromatogram of aqueous fraction of mixed standards at 254 nm...... 88
Figure 25: HPLC chromatogram of chloroform fraction of mixed standards at 254 nm...... 90
Figure 26: HPLC chromatogram of the polar fraction of A. paniculata commercial extract at
254 nm...... 91
Figure 27: HPLC chromatogram of non-polar fraction of A. paniculata commercial extract at
254 nm...... 92
Figure 28: HPLC chromatogram of non-polar fraction of A. paniculata overlaid with the A. paniculata commercial extract at 254 nm...... 93
Figure 29: HPLC chromatogram of aqueous and chloroform fraction of the A. paniculata commercial extract at 254 nm...... 94
Figure 30: HPLC chromatogram comparing andrographolide standard to the A. paniculata commercial extract and non-polar fraction at 227 nm...... 96
Figure 31: UV spectrum of reference standard andrographolide and the A. paniculata commercial extract...... 96
Figure 32: HPLC chromatogram of aqueous fraction and andrographolide at 227 nm...... 97
Figure 33: UV spectrum of polar fraction peak at 5.7 min (green) and pure andrographolide
(black), normalised...... 98
Figure 34: Andrographolide standard curve (n=3)...... 99
Figure 35: NO dose response curve for andrographolide (red, circles), A. paniculata commercial extract (green, inverted triangles), the non-polar fraction (blue, squares) and the polar fraction (black, triangles), by LPS and IFN-γ stimulated RAW264.7 cells...... 101
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Figure 36: NO dose response curve for andrographolide (red, circles), A. paniculata commercial extract (green inverted, triangles) and the non-polar fraction (blue, squares), by
LPS and IFN-γ stimulated RAW264.7 cells...... 102
Figure 37: NO dose response curve combined F-test for pure andrographolide, A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
...... 104
Figure 38: PGE2 dose response curve for andrographolide (red, circles) and the A. paniculata commercial extract (green, squares), by LPS and IFN-γ stimulated RAW264.7 cells...... 105
Figure 39: PGE2 dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 107
Figure 40: TNF-α dose response curve for andrographolide (red, circles) and the A. paniculata commercial (green, squares), by LPS and IFN-γ stimulate RAW264.7 cells. .... 108
Figure 41: TNF-α dose response curve combined F-test for andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 109
Figure 42: TNF-α dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, squares) and the non-polar extract (blue, triangles), by
LPS stimulated THP-1 cells...... 110
Figure 43: TNF-α dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 112
Figure 44: IFN-γ dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 114
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Figure 45: IFN-γ dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 115
Figure 46: IL-1β dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 115
Figure 47: IL-1β dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 116
Figure 48: IL-1ra dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 116
Figure 49: IL-2 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 117
Figure 50: IL-2 dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 118
Figure 51: IL-4 dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 119
Figure 52: IL-4 dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 119
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Figure 53: IL-6 dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 120
Figure 54: IL-6 dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 120
Figure 55: BFGF dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 121
Figure 56: BFGF dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 121
Figure 57: G-CFS dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 122
Figure 58: G-CFs dose response curve combined F-test for pure andrographolide and the non- polar fraction normalised to andrographolide concentration...... 123
Figure 59: GM-CFS dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 124
Figure 60: GM-CFS dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 124
Figure 61: VEGF dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 125
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Figure 62: VEGF dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 126
Figure 63: IP-10 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 127
Figure 64: IP-10 dose response curve combined F-test for pure andrographolide and the non- polar fraction normalised to andrographolide concentration...... 128
Figure 65: MCP-1 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 129
Figure 66: Eotaxin dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 130
Figure 67: Eotaxin dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration...... 131
Figure 68: RANTES dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells...... 132
Figure 69: IL-9 released by LPS stimulated THP-1 cells. (n=2) ...... 133
Figure 70: %IL-17 release by LPS stimulated THP-1 cells. (n=2) ...... 133
Figure 71: NF-κB dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, squares) and the non-polar fraction (blue, inverted triangles) by LPS and IFN-γ stimulated ELAM9 RAW264.7 cells...... 135
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Figure 72: NF-κB dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration...... 136
Figure 73: NO dose response curve for andrographolide (red circles), diclofenac (green triangles), aspirin (brown squares), paracetamol (blue inverted triangles), ibuprofen (grey circles) and naproxen (pink circles) by LPS and IFN-γ by stimulated RAW264.7 cells...... 154
Figure 74: PGE2 dose response curve for andrographolide (red circles), diclofenac (green inverted triangles), aspirin (brown triangles), paracetamol (blue diamond) and ibuprofen
(grey circles) by LPS and IFN-γ by stimulated RAW264.7 cells...... 156
Figure 75: TNF-α dose response curve for andrographolide (red circles), diclofenac (green diamond), aspirin (brown triangles), paracetamol (blue circles) and ibuprofen (grey inverted triangles) by LPS and IFN-γ by stimulated RAW264.7 cells...... 158
Figure 76: TNF-α dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 159
Figure 77: IFN-γ dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 160
Figure 78: IL-1β dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 160
Figure 79: IL-2 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 161
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Figure 80: IL-4 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 161
Figure 81: IL-6 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 162
Figure 82: G-CSF dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 162
Figure 83: GM-CSF dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 163
Figure 84: MCP-1 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells...... 163
Figure 85: NF-κB activation dose response curve for andrographolide (red circles), diclofenac
(green squares), aspirin (brown crosses), paracetamol (blue circles) and ibuprofen (grey crosses) by LPS and IFN-γ stimulated ELAM9 RAW264.7 cells...... 165
Figure 86: Human plasma Cmax of andrographolide...... 173
Figure 87: Cmax of andrographolide in animals...... 175
Figure 88: Cmax for modified andrographolide formulations...... 177
Figure 89: Molecular structure of the sulphonation metabolite of andrographolide and andrographolide ...... 187
Figure 90: HPLC chromatogram of the sulphonation product overlaid with andrographolide standard at 210 nm...... 195
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Figure 91: Preparative HPLC chromatogram of the sulphonation product at 226 nm...... 196
Figure 92: HPLC chromatogram of the metabolite overlaid with andrographolide at 210 nm.
...... 197
Figure 93: LC-MS TIC of the andrographolide sulphonate metabolite...... 198
Figure 94: MS spectrum of the major peak in the andrographolide sulphonate metabolite TIC
(Rt. 11.2 min)...... 198
Figure 95: 1HNMR of the sulphonation product of andrographolide...... 199
Figure 96: Structure of sulphonation product with carbons numbered...... 200
Figure 97: NO dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated RAW264.7 cells...... 202
Figure 98: TNF-α dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated
THP-1 cells...... 203
Figure 99: IFN-γ dose response curve for andrographolide (red circle) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 205
Figure 100: IL-1β dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown diamonds), by LPS stimulated THP-1 cells...... 205
Figure 101: IL-1ra dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 206
Figure 102: IL-2 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 206
Figure 103: IL-4 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 207
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Figure 104: IL-6 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 207
Figure 105: IL-17 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 208
Figure 106: BFGF dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS stimulated THP-1 cells.
...... 208
Figure 107: G-CFS dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown inverted triangles), by LPS stimulated
THP-1 cells...... 209
Figure 108: GM-CFS dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown inverted triangles), by LPS stimulated
THP-1 cells...... 209
Figure 109: VEGF dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
...... 210
Figure 110: IP-10 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells...... 210
Figure 111: MCP-1 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
...... 211
Figure 112: Eotaxin dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
...... 211
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Figure 113: RANTES dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
...... 212
Figure 114: NF-κB dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated
ELAM9 RAW264.7 cells...... 213
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List of tables
Table 1: Cytokine's roles in inflammation ...... 19
Table 2: The function of inflammatory chemokines...... 24
Table 3: Anti-pro-inflammatory cytokines and chemokines [79, 127]...... 26
Table 4: Taxonomical classification of A. paniculata [152] ...... 58
Table 5: The effects of A. paniculata and andrographolide on inflammatory enzymes...... 61
Table 6: Andrographolide’s effect on NO and PGE2 production ...... 65
Table 7: Effects of andrographolide and A. paniculata on release of pro-anti-inflammatory cytokines and chemokines...... 66
Table 8: Andrographolide’s effect on inflammatory cytokines and chemokines...... 72
Table 9: The effects of A. paniculata and andrographolide on NF-κB...... 74
Table 10: Concentration of andrographolide in the A. paniculata commercial extract, non- polar fraction and polar fractions...... 100
Table 11: NO IC50's ...... 103
Table 12: PGE2 inhibition IC50's ...... 106
Table 13: IC50s for TNF-α inhibition by andrographolide and the andrographolide in the A. paniculata commercial extract...... 108
Table 14: IC50s for TNF-α inhibition by pure andrographolide and andrographolide in the A. paniculata commercial extract on THP-1 cells...... 111
Table 15: IP-10 IC50 comparison between extracts of the A. paniculata commercial extract and pure andrographolide ...... 113
Table 16: Summary of anti-inflammatory results ...... 137
Table 17: IC50's in order of activity of andrographolide activity ...... 140
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Table 18: Summary of IC50s for andrographolide, diclofenac, aspirin, paracetamol and ibuprofen...... 166
Table 19: PGE2 IC50s for andrographolide and NSAIDs, compared to reported plasma Cmaxs.
...... 168
Table 20 Pharmacokinetic studies of andrographolide ...... 179
Table 21: Comparison between 1HNMR for synthesised and isolated sulphonation product of andrographolide and the biologically significant metabolite reported by He et al. [15]...... 199
Table 22: IC50s for andrographolide and sulphonation metabolite TNF-α release inhibition203
Table 23: Summary of pure andrographolide and sulphonation metabolite IC50s ...... 214
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Table of abbreviation and acronyms
A. paniculata Andrographis paniculata
1HNMR Proton nuclear magnetic resonance
AChE PGE2-acetylcholinesterae
AGE Advanced glycation end-products
ATP Adenosine triphosphate
ATRA All trans retinoic acid
Aβ Amyloid-β
BFGF Basic fibroblast growth factor
BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate
CAY 10602 1-(4-fluorophenyl)-3-(phenylsulfonyl)-1H- pyrrolo(2,3-b)quinoxalin-2-amine
CD Cluster of differentiation cGMP Cyclic guanosine monophosphate
Cmax Concentration maximum
COX Cyclooxygenase
COX-1 Isoform-1 of cyclooxygenase
COX-2 Isoform-2 of cyclooxygenase
CTL Cytotoxic T lymphocyte
CTX Cyclophosphamide
DAMPs Damage-associated molecular patterns db-cAMP Dibutyryl cyclic adenosine monophosphate
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethyl sulfoxide
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DNA Deoxyribonucleic acid
ELAM Endothelial-leukocyte adhesion molecule
ELISA Enzyme-linked immunosorbent assay
EMSA Electrophoretic mobility shift assay
EP E prostanoid
ERK Extracellular-signal-regulated kinases
FBS Foetal bovine serum
G-CSF Granulocyte colony-stimulating factor
GFP Green fluorescent protein
GM-CSF Granulocyte-macrophage colony- stimulating factor gp130 Glycoprotein 130; also known as IL-6beta
GSH Glutathione
HIV-1 Human immunodeficiency virus type 1
HL-60 Human promyelocytic leukemia cells
HM Herbal medicine
HPLC High performance liquid chromatography
HRP Horseradish peroxidase i.g Intragastrically i.v Intravenously
IC50 Half maximal inhibitory concentration
IFN-γ Interferon gamma
IKκβ Inhibitory kinase of κB
IL Interleukin
IL-1ra Interleukin-1 receptor antagonist
IL-1β Interleukin-1 beta
XXX
iNOS Inducible nitric oxide synthase
IP Interferon gamma-induced protein
IP-10 Interferon gamma-induced protein 10
IRF1 Interferon regulatory factor one
IRF3 Interferon regulatory factor three
IκBα Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
JAK Janus kinase
JNK c-Jun N-terminal kinases
JSH-23 4-methyl-N1-(3-phenylpropyl)-1,2- benzenediamine
KO Knockout
LC Liquid chromatography
LD50 50% lethal dose
LPS Lipopolysaccharide
LTA Lipoteichoic acid
LTB4 Leukotriene B4
MAPK Mitogen-activated protein kinases
MCP Monocyte chemotactic protein
MD-2 Lymphocyte antigen 96
MHC Major histocompatibility complex
MIP Macrophage inflammatory protein mRNA Messenger ribonucleic acid
MS Mass spectroscopy
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
NED N-1-napthyl-etylenediamine
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NED Napthyethylene-diamine
NFAT Nuclear factor of activated T cells
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NK Natural killer
NKT Natural killer T-cell
NLRs NOD like receptors
NO Nitric oxide
NOD Nucleotide-binding oligomerization- domain
NOS Nitric oxide synthase
NSAIDs Non-steroidal anti-inflammatory drugs
ONOO- Peroxynitrite anion
OTC Over the counter
OVA Ovalbumin
PAF Platelet activation factor
PAMPs Pathogen associated molecular patterns
PBS Phosphate buffer saline
PC Phosphate citric acid
PDA Photodiode array detection
PDGF Platelet-derived growth factor
Pen Strep Penicillin and Streptomycin
PG Prostaglandin
PGE2 Prostaglandin E2
PMA Phorbol-12-myristate-13-acetate pMCAO Permanent middle cerebral artery occlusion
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PP2 Protein phosphatase 2
PRs Progesterone receptors
QC Quality control
RAGE Receptor for advanced glycation endproducts
RNA Ribonucleic acid
RNOS Reactive nitrogen oxide species
ROS Reactive oxygen species
RPMI Roswell Park Memorial Institute
SRs Scavenger receptors
STAT Signal transducer and activator of transcription
TCM Traditional Chinese Medicine
TFA Trifluroacetic acid
TGF-β Transforming growth factor beta
Th T helper
TIC Total ion count
TLR Toll-like receptor
TMB 3,3,5,5- tetramethylbenzidine
TNF-α Tumour necrosis factor alpha
TXA2 Thromboxane A2
TXB2 Thromboxane B2
UPLC Ultra high performance liquid chromatography
VEGF Vascular endothelial growth factor
WT Wild type
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CHAPTER 1
______1. The inflammatory response and herbal medicine
1
1.1. Inflammation
Inflammation is the body’s natural adaptive response to noxious stimuli such as infection and injury [1]. The role of inflammation is to resolve disruptions to homeostasis and return the body’s systems to balance [2]. A controlled inflammatory response is beneficial (for example, destroying infection), however, if dysregulated or persistent can be detrimental (for example, inflammatory bowel disease or asthma) [3, 4]. Inflammation can be broadly classified into two types based on the cause of the inflammation: pathogen associated molecular patterns
(PAMPs) and damage-associated molecular patterns (DAMPs) [5]. PAMPs inflammatory response typically invokes a series of events designed to destroy pathogens. PAMPs include lipopolysaccharide, lipoteichoic acid, peptidoglycan and viral or bacterial RNA/DNA. This type of inflammatory response is well understood and rationalised due to its large magnitude, clear inducers and it achieves a clear end goal. DAMPs induced inflammation covers a broad range of inflammatory conditions that are characterised by cell signaling molecules initiating a response specific to the stimulation. For example, in Alzheimer’s disease, neuronal and vascular damage is commonly caused by DAMPs including advanced glycation end-products
(AGE), amyloid-β (Aβ), and hyper-phosphorylated tau [5]. Unlike a response to PAMPs,
DAMPs responses are usually of a lower magnitude and often do not resolve the initial cause
(which is not always apparent), this can result in chronic or long-term inflammation. Due to the characteristically subtle inflammatory response and less clarity around the beneficial role of inflammation in many DAMPs cases, understanding is limited and the study of their mechanisms is challenging. Although the role of inflammation in PAMPs and DAMPs is different, many common mediators and mechanisms are shared, this is shown in Figure 1 [2].
Due to its larger magnitude, PAMPs inflammation is better suited to in vivo assessment for determining compounds that may elicit an anti-inflammatory response.
2
Figure 1: DAMPs and PAMPs role in inflammation [5].
PAMPs= pathogen associated molecular patterns, LPS= lippopolysacaride, LTA= lipoteichoic acid, RNA= ribonucleic acid, DNA= deoxyribonucleic acid, TLRs= toll like receptors, NOD= nucleotide-binding oligomerization domain protein, NLRs= NOD like receptors, DAMPs= damage associated molecular patterns, AGE= advanced glycation end products, ATP= adenosine triphosphate, Aβ= amyloid beta, RAGE= receptor for advanced glycation endproducts, PRs= progesterone receptors, SRs= scavenger receptors, iNOS= inducable nitric oxide synthase, COX-2= cycloxygenase 2.
1.1.1. Pathogen associated molecular pattern’s acute inflammatory response
Despite the complexity of the inflammatory response, the PAMPs’ induced inflammatory response is well understood and rationalised. The inflammatory response is complex, coordinated by a range of mediators working in an intertwined network. Conceptually, the inflammatory response can be divided into different phases that form a cycle: 1) inflammation is initiated by inducers, 2) inducers act on specific receptors, 3) receptor binding triggers the production and release of inflammatory mediators, 4) these mediators, in turn, act on and alter the functionality of inflamed tissues and organs, which then become the effectors of the acute inflammatory response [2].
The acute inflammatory response results in the increased transfer of immune blood components to the site of abnormality. Inducers are recognised by various receptors, for example, toll-like receptors (TLR) and nucleotide-binding oligomerization-domain (NOD)
3
proteins [6]. Mast cells and resident macrophages then produce inflammatory mediators, including cytokines, chemokines, eicosanoids, vasoactive amines and products of proteolytic cascades. These inflammatory mediators locally activate endothelium blood vessels, allowing neutrophils to enter the site of disturbance. The neutrophils are activated either by the pathogen or the activation cytokines released by resident macrophages. The activated neutrophils indiscriminately release toxins to destroy the invading cells, simultaneously damaging surrounding host tissue [7, 8]. A successful acute phase response is proceeded by a repair phase mediated by resident and recruited macrophages [9]. If, however, the stimulus is not resolved, this cycle continues to escalate in severity until the instigator is resolved, leading to chronic inflammation.
1.1.2. Chronic inflammation
Chronic inflammation is long-term inflammation that ensues when the acute phase response fails to return the system to homeostasis [1]. When the acute inflammatory response fails to eliminate the source of disturbance, the inflammation persists and evolves. In response to persistent pathogens, macrophages and T cells replace the neutrophils, escalating the response. If these cells fail to resolve the disturbance, a chronic inflammatory state ensues potentially resulting in the formation of granulomas in a final attempt to protect the host [1].
In addition to unresolved infections, chronic inflammation can result from inert foreign bodies or autoimmune responses [1]. Chronic inflammatory processes are less understood than acute inflammation, due to their discreet mechanisms, often developing over long periods of time and resulting from no apparent stimulus [2].
4
1.1.3. Para-inflammation
Para-inflammation is the term given to less intense inflammatory responses that result in the persistence of a comparatively mild state of chronic inflammation (compared to the acute phase response) [2]. Although the physiological purpose of para-inflammation processes is to preserve homeostasis, when a tissue is exposed to stress and/or malfunction for a prolonged period, para-inflammation is implicated in both initiation and progression of many human age-related disorders (such as Alzheimer’s disease and macular degeneration [10, 11]) and modern diseases (such as metabolic syndrome and cardiovascular disease [12, 13]). The persisting causes of para-inflammation are not always understood. When there is a shift in homeostasis the system can either be induced into an acute stress response (that affords a transient adaptation to the condition) or a sustained change causing a slight redefinition to the homeostatic parameters [2]. A physiological illustration of this is observed in insulin resistance, where sustained insulin release leads to desensitisation to insulin, resulting in type-2 diabetes [14]. What begins as a minor adaptive change causes a long-term shift to homeostatic parameters with maladaptive consequences. This may be the cause of para- inflammation when there is no apparent inducer. The different roles of inflammation are summarised in Figure 2.
5
Figure 2: Physiological purposes of inflammation dependent upon the trigger [15].
Para-inflammation may be implicated in the progression of many chronic inflammatory diseases. Many chronic conditions progress over time and age is the leading risk factor for diseases such as chronic neurodegenerative disease, cardiovascular disease, diabetes, cancer and degenerative musculoskeletal disease [16-20].
1.1.4. Predicted increasing incidence of inflammation
The number of people aged 80 and over will increase significantly in the coming decades, and it is predicted that the proportion of elderly citizens above the age of 60 will double between now and the year 2025 [21]. Consequently, age-related diseases are projected to be the leading cause of disability by the year 2020, imposing a significant burden on affected individuals and on society in general [22]. Age is the leading risk factor for many chronic inflammatory diseases. To date, pharmacotherapy of inflammatory conditions is based on the use of non-steroidal anti-inflammatory drugs (NSAIDs). Considering the prevalence of
6
degenerative and inflammatory disease, it is therefore not surprising that NSAIDs are presently among the most commonly used drugs [23].
1.1.5. Limitations of current treatment for inflammation
With the increasing prevalence of degenerative and inflammatory conditions as the global population ages, NSAIDs have become one of the most commonly prescribed and used drugs
[23]. However, there are many side effects associated with their use. NSAIDs can cause serious gastrointestinal toxicity in 0.7-1.3% of patients who take the drugs for one year or longer and 5-15% of patients with rheumatoid arthritis have been reported to discontinue
NSAID therapy due to dyspepsia [24]. Other side effects included nervous system impairment, renal impairment and allergic responses [25]. NSAIDs target the prostaglandin
G/H synthase enzyme that is responsible for the production of prostaglandins (PGs) which are implemented in inflammation [26]. There are two major isoforms of cyclooxygenase
(COX), namely COX-1 and COX-2. COX-1 is expressed constitutively in most tissue and its inhibition was theorised to be the major cause of the side effects, whereas COX-2 is subject to rapid induction by inflammatory cytokines and mitogens, so was thought to be exclusively linked to PG production at sites of inflammation [27, 28]. When COX-2 selective drugs were discovered, it was believed that a new era in NSAID pharmacology had been launched. COX-
2 inhibitors were quickly established as important therapeutic medications as it was assumed that they would have fewer side effects than traditional NSAIDs [28]. Unfortunately, some
COX-2 inhibitors were found to increase the risk for thrombosis, heart attack and stroke. One such drug, Rofecoxib (commonly known as Vioxx), was removed from the US market in
2004 because of these concerns. It has been reported recently, however, that while complicated gastrointestinal events appeared to occur more frequently with NSAIDs than
7
with selective COX-2 inhibitors, serious cardiovascular events appeared to occur at approximately equal rates [29]. It is estimated that approximately 30% of hospital admissions related to adverse drug effects result from the gastrointestinal, nervous system, renal, or allergic effects of NSAIDs [25]. Thus, there is a need to develop anti-inflammatory drugs with minimal adverse effects.
1.1.6. Possible alternatives to NSAIDs
Natural products or derivatives of natural products have long formed “the backbone of modern pharmacopoeias” [30, 31]. There is an increasing realisation that purely synthetic approaches to drug development have not lived up to their promise thus renewing interest in natural products as sources of drug discovery [32]. Plants have long been an important source for the discovery of new drugs. Herbal medicines derived from plants rich in the secondary metabolite salicylic acid, such as the bark of the willow tree (Salix alba), have been used for the treatment of diseases with prominent inflammatory components for thousands of years.
The development of acetylsalicylic acid (aspirin) by the German drug and dye firm Bayer at the end of the 19th century, was motivated by the desire to find a less-irritating replacement for the traditional salicylate-based anti-inflammatory medicines of the time. Medicinal plants, the foundation material of traditional herbal medicines, constitute a particularly rich source of pharmacologically active compounds.
1.2. Principles of traditional herbal medicine
Traditional Chinese Medicine (TCM) and Ayruvedic (Indian) medicine are both empirical systems of medicine developed over thousands of years, utilising natural products to treat
8
disease and illness [33, 34]. TCM and Ayruvedia are the largest, best preserved and embraced remaining traditional medicine systems in the world, together utilising thousands of plant derived products. Like many other forms of alternative medicine they are holistic in approach and focus more on the patient than the disease [35]. Individually tailored mixtures containing plant and animal-derived products, minerals and metals are prescribed based on traditional practice [36]. Both systems of medicine were developed through clinical use with no understanding of the microbiology involved. They are based on the world being composed of five elements that, in Ayruveda, interact to give three forces referred to as ‘tridosha’ which is believed to regulate every physiological and psychological process [37]. In TCM ‘yin’ and
‘yang’ are the two forces that are balanced by bodily humours (‘qi’ energy, blood, moisture and essence). Imbalance in the forces are corrected with the administration of herbs or other remedies [38]. These conceptual frameworks for treating disease have the ultimate goal of returning the system to homeostasis. The forces are not associated with target pathogens, enzymes or pathways, which is in stark contrast to western medicine which has a mechanistic view of disease. The unique origins of these traditional medicines, historically showing efficacy through clinical use, may present an alternate approach to resolving diseases that modern western pharmacotherapies fail to treat. This may be particularly relevant to inflammation, where western medicines focus on COX inhibition by NSAIDs, yielding adverse side effects. Herbal medicine was developed to clinically treat inflammation but not necessarily by COX inhibition, it may have novel effective targets that create less side effects.
9
1.2.1. Andrographis paniculata for the treatment of inflammation
One plant that has traditionally been used for the treatment of conditions characterised by inflammation is Andrographis paniculata (A. paniculata) (Burm. f.) Wall. ex Nees. A. paniculata extracts are commonly standardised to andrographolide concentration, the most abundant and researched secondary metabolite. A. paniculata belongs to the family of
Acanthaceae and is endogenous to South India and South East Asia. This plant has been used in herbal medicine in traditional Indian and Chinese medicine since ancient times; many of its uses are associated with inflammation or treatment of diseases that cause inflammation. Its use has been described in various pharmacopeias and traditional texts as a treatment for intestinal inflammation, kidney inflammation, lung inflammation, common colds, swelling, skin diseases, headaches, diabetes and colon inflammation [39-43]. The use of A.paniculata over thousands of years for treating inflammation related ailments strongly supports its anti- inflammatory activity and safety. However, western medicine is yet to accept the safety and efficacy of TCM and Ayruvedia.
1.2.2. Herbal medicine’s safety and efficacy
There are a number of concerns surrounding traditional herbal medicine’s safety and efficacy.
Significant variations in the chemical composition of herbal medicines must be resolved before they can be accepted as safe and consistently effective. Many factors influence the chemical composition of herbs and inconsistencies in final herbal products have been widely observed. As the scientific understanding and evaluation of herbal medicines increases, it is evident that the variations in the chemical components of herbal medicines are significant and require quality control to ensure safety and efficacy [44-47]. Standardisation is the process whereby the dried extract is prepared in a way that a consistent concentration of one or more
10
analytes (usually a bioactive component found in relatively high concentration) is ensured, even though there may be variations in the concentration of these substances in the raw herb from which the dried extract is prepared. If standardisation can be achieved, the herbal extracts can be administered at consistent doses of phytochemicals, increasing product safety and efficacy. The standardised components of the herbs are often based on abundance of one or two target analyte/s and not necessarily pharmacological activity. The components that are active in the herb should be determined in a system that is linked to the ailment the medicine is administered for. In complex conditions like inflammation, it is essential to assess efficacy using a number of mediators that adequately represent the system, this is exemplified by the failing of NSAIDs that simply focus on COX inhibition. Inflammation is a complex biological response involving a web of cell types and interacting through a number of cellular signals at various stages of inflammation.
1.3. Biological targets for investigating inflammation
1.3.1. Role of macrophages in inflammation
Macrophages are a major cell type involved in inflammation and are implemented in the acute phase response and chronic inflammation as well as repair. Macrophages are specialised white blood cells, which constitute 10–15% of most tissues. Under conditions of stress (disease, infection or injury), resident macrophages are activated by tissue-derived inflammation inducers and produce inflammatory mediators. These mediators have been classified into seven groups according to their biochemical properties [2]: vasoactive amines, vasoactive peptides, fragments of complement components, lipid mediators, cytokines, chemokines and proteolytic enzymes. Macrophages are integral to the inflammatory response and influence the levels of a majority of inflammatory mediators. They are a crucial cell type
11
to assess when investigating inflammation. There are a number of macrophage cell lines that have been developed and used in in vivo studies of inflammation.
1.3.2. Lipopolysaccharide’s stimulation of macrophages
Lipopolysaccharide (LPS) is a heat resistant toxin that is located in the outer membrane of nearly all gram negative pathogens. As LPS is common to many pathogens it is not surprising that immune cells have developed a receptor mechanism that detects its presence and elicits a pathogenic inflammatory response. Sensitivity to LPS has been demonstrated in human whole blood with adverse effects being detected with doses as low as 0.1μg/ml [48].
Although LPS is associated with a PAMP inducer of inflammation, due to the cross talking nature of the inflammatory response, some downstream inflammatory effectors are common to both PAMPs and DAMPs responses, namely nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) [49]. Therefore,
LPS is commonly used to not only mimic inflammation pathogen induced inflammation, but is also useful in the study of inflammation induced by non-pathogenic stimuli in vitro.
LPS binds to lipopolysaccharide binding protein, which aids the binding of mCD14
(membrane bound CD14) to form a tertiary complex, this enables transfer to the LPS receptor complex composed of TLR4 and lymphocyte antigen 96 (MD-2) [50], as shown in Figure 3.
LPS signaling leads to the activation of NF-κB via the interferon regulatory factor 3 (IRF3) and MAPK kinase pathways. This amplifies lipid mediator’s production, increases oxidative stress by superoxide, hydroxyl radicals and nitric oxide (NO) and up regulates cytokines and chemokines including tumour necrosis factor alpha (TNF-α), interleukin (IL) -1 beta, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-γ),
12
which feed back into the signal to enhance activation. IFN-γ is used in vitro as a co- stimulator to LPS as it enhances activation.
Figure 3: Schematic diagram of LPS induced inflammation in macrophage cells [51].
1.3.3. Interferon gamma stimulation of macrophages
IFN-γ is a well-accepted and long recognised host-derived factor in response to PAMPs. In an inflammatory response IFN-γ is released by cluster of differentiation (CD) 4+ T helper
(Th) cell type 1 lymphocytes, CD8+ cytotoxic lymphocytes and natural killer T-cell (NKT).
However, macrophages and microglia can also produce IFN-γ upon activation [52, 53]. IFN-γ can enhance innate pro-inflammatory responses of immune cells if combined with TLR pathways [52]. IFN-γ was extensively studied, leading to the important finding of a common
Janus kinase (JAK) signal transducer and activator of transcription (STAT) cytokine signaling pathway [54]. The binding of IFN-γ leads to the translocation of STAT1 and interferon regulatory factor one (IRF1) to the nucleus and deoxyribonucleic acid (DNA)
13
binding activity of phosphorylated STAT1 that initiates transcription of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS), as shown in Figure 4, leading to the production of the important anti-inflammatory molecule NO [54, 55].
Figure 4: Schematic diagram of canonical activation of IFN-γ receptor involving activation [56].
1.3.4. Role of nitric oxide in inflammation
NO is a radical metabolite, which has been shown to have numerous physiological functions both as a signaling molecule and as a toxic agent in inflammation [57]. NO is derived from the oxidation of L-arginine (Figure 5) by three types of nitric oxide synthases (NOS); the constitutive forms, neuronal NOS and endothelial NOS and the inducible form, iNOS, originally described in murine macrophages [58, 59]. NOS is well conserved across mammalian species with 90% primary amino acid homology [58]. The constitutive forms are 14
activated in response to Ca2+ generated, for example, by an action potential, which initiates a rapid and short interval low concentration (pM) release of NO [60]. However, the inducible form is continually activated once expressed, and is therefore regulated at the transcription level by NF-κB, stimulated by inflammatory molecules like LPS and IFN-γ. The production of NO by iNOS (due to the time taken for messenger ribonucleic acid (mRNA) and protein synthesis) experiences hours of lag time before NO is produced in much higher (nM) sustained levels [58]. The inducible form of NOS is most likely implicated in inflammation and due to the higher levels of NO produced, it is more easily assessed in-vitro.
Figure 5: Formation of Nitric Oxide [61].
NO is an unusual signaling molecule. As there is no specific cell surface receptor for NO it enters cells indiscriminately, where the effect is dependent on cell type and NO concentration, thus producing a wide range of physiological responses. NO causes increased vascular permeability, vasodilatation and generation of radicals which causes tissue damage and eliminates pathogens [62]. These physiological changes are associated with inflammation, increasing blood flow and allowing immune cells to enter affected tissue thereby destroying the pathogen. At high concentrations and under aerobic conditions NO is rapidly oxidised to form various reactive nitrogen oxide species (RNOS), N2O3 being the major product. RNOS can induce cell toxicity by nitrosating tyrosine and DNA residues, and
15
inducing lipid peroxidation [63]. Furthermore, in the presence of oxidative stress, as can occur at the site of inflammation and as iNOS can also produce superoxides, NO and superoxides interact to generate the highly reactive oxidant peroxynitrite anion (ONOO-)
(Figure 6) which is associated with more severe toxic effects [63]. The collateral damaging effect of these RNOS has been implicated in chronic inflammatory diseases like Alzheimer’s
[64, 65].
Figure 6: Superoxide and NO form peroxynitrite [66].
NO may also play a regulatory role in immunity and inflammation. In animal models iNOS deficient mice were shown to be more susceptible to inflammatory damage and tumours, but more resistant to septic shock [67]. There is evidence that NO inhibits NF-κB and JAK/STAT by S-nitrosylation, which suggest a controlling mechanism [68]. However, TNF-α expression is upregulated by NO via cyclic guanosine monophosphate (cGMP), protein kinase G and subsequently activates NF-κB [69]. In asthma NO accumulation has been observed in human lungs, this may be beneficial by means of its muscle relaxing effect or it could exasperate inflammation [70]. In an iNOS knockout (KO) mice asthma model IFN-γ production was increased, but not IL-4 or IL-5, suggesting a pro-inflammatory role [71]. Although the role of
NO in inflammation is not fully understood (much like inflammation itself) it plays an
16
important role in inflammation. Many mediators of inflammation seem to play this dual role of antagonist and protagonist like prostaglandin, produced by the COX enzyme.
1.3.5. Prostaglandin’s role in inflammation
PGs play a major role in the generation of the inflammatory response. Their biosynthesis is significantly increased at sites of inflammation and they contribute to an escalation in inflammation. PGs are produced from arachidonic acid by prostaglandin G/H synthases, a bi- functional enzyme that contains both COX and peroxidase activity. There are four major bioactive PGs produced in-vivo; prostaglandin E2 (PGE2), prostacyclin, prostaglandin D2 and prostaglandin F2α. PGE2 is the most abundant prostaglandin in the body [72]. The prolife of
PGs’ production at the site of inflammation is determined by the immune cell’s activation state and cells types present. For example, macrophages favour the production of PGE2 when activated by LPS, however, when resting produce thromboxane A2 (TXA2) in excess of
PGE2 [73].
There are two major isoforms of COX, COX-1 and COX-2. COX-1 is expressed constitutively in most tissue and is the dominant source of PGs that serve housekeeping functions, such as gastric epithelial cytoprotection and homeostasis [74]. COX-2 is subject to rapid induction by inflammatory cytokines and mitogens, so is thought to be exclusively linked to PGs’ production at sites of inflammation [27, 28]. However, more recent evidence suggests that both isoforms play a role, albeit COX-1 the lesser. An early stage inflammation in air pouch study in COX KO mice found that PGE2 production was reduced by 75% in
COX-2 KO mice and 25% in COX-1 KO mice compared to the wild type (WT) [75]. COX-2
17
was also observed to have a pivotal role in resolution of inflammation with the COX-2 KO failing to recover as well as the WT and COX-1 KO mice after 7 days.
After PGE2 is formed it diffuses across the plasma-membrane or is actively transported by multidrug resistant protein-4 [76]. PGE2 exerts its effect by activating rhodopsin-like transmembrane spanning G protein couple receptors; E prostanoid (EP) receptor 1, EP2, EP3 and EP4. EP1 increases intracellular Ca2+ and inositol trisphosphate, EP2 and EP4 increase cyclic adenosine monophosphate (cAMP), EP3 elevates intracellular Ca2+ and inhibits cAMP
[77].
PGE2 increases blood flow to sites of inflammation by augmenting arterial dilation and increased microvascular permeability. Pain also results from PGE2 action on sensory neurons and central sites within the spinal cord and brain [78]. Although COX inhibition is the major target of current anti-inflammatories it is only one of many mediators of inflammation.
1.3.6. Cytokine’s role in inflammation
Cytokines are released by nearly all cells in response to various stimuli. Cytokines are inflammatory mediators that act on and alter the functionality of inflamed tissues and organs.
Cytokines bind to specific receptors located on the surface of cells resulting in the activation of intracellular signaling cascades, which in turn alter the functional state of the cells. There are approximately 150 cytokines produced by human cells. Cytokines are pleiotropic, for example, IL-1 which affects body temperature, liver protein synthesis, and T cells’ response to antigens. Many cytokines have similar or the same function in some systems and this is
18
how they are classified - for example, the IL-1 family has 11 members including IL-18 and
IL-33, each is a separate gene, however, their products overlap in functioning as pro- inflammatory cytokines [79]. Many cytokines appear redundant, their roles are performed by other cytokines. The cytokine signaling system is complex and influenced by many factors. It has evolved this way to maximise cell survival and resistance to antigens, i.e. the more complex and redundant a system, the more difficult it is to block or manipulate. Table 1 shows some cytokines and their functions.
Table 1: Cytokine's roles in inflammation TNF-α TNF-α is a cell signaling protein (cytokine) involved mainly in the acute phase inflammatory response. Macrophages are the major source of TNF-α, although it can be released by many other cell types such as CD4+ lymphocytes, natural killer (NK) cells, neutrophils, mast cells, eosinophils, and neurons. TNF-α is produced by activation of MAPK and NF-κB. It acts to increase its own production and that of other pyrogens such as interleukin-1 beta (IL-1β). Intracellular calcium levels mediate cellular contraction, enzymatic activity and mitochondrial function. TNF-α depresses cellular calcium transients. TNF-α induces fever, apoptotic cell death, cachexia, inflammation and inhibits tumourgenesis and viral replication. TNF-α is implicated in many disease states, including, sepsis, traumatic injury, ischemia, asthma, burns, irritable bowel syndrome, Alzheimer's disease, cancer, major depression, arthritis and multiple sclerosis [80-82].
IFN-γ IFN-γ is an inflammatory cytokine. IFN-γ is produced predominantly by NK and NKT cells as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells, once antigen-specific immunity develops. IFN-γ is an important activator of macrophages and inducer of Class I major histocompatibility complex (MHC) molecule expression. IFN-γ stimulates phagocytosis, oxidative burst, intracellular killing on microbes, supresses IL-4 (anti-inflammatory) secretion and inhibits viral replication directly. IFN-γ expression is associated with a number of auto inflammatory and autoimmune diseases [83, 84].
IL-1β IL-1β is one of the most significant cytokines in acute and chronic inflammation. IL-1β is a pro-inflammatory cytokine involved in the generation of pain and inflammation at multiple levels, both peripherally and centrally. It is implicated in a wide range of disease states, including gout, type 2 diabetes, osteoarthritis, rheumatoid arthritis, neuropathic pain, inflammatory bowel disease, vascular disease and Alzheimer’s disease [85-87]. The IL receptor
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domain is present in the TLR, which is a major inflammatory receptor responding to microbial products (LPS) and viruses and is present on many cell types. IL-1β binding triggers the activation of MAPK, translocation of NF-κB to the nucleus is then observed [88], resulting in increased expression of many mediators including COX2, iNOS, IL-1, IL-6, TNF-α, interleukin-1 receptor antagonist (IL-1ra) to name a few [89]. The IL-1β promoter region contains a TATA box, a typical motif of inducible genes. LPS triggers transient transcription and steady state levels of IL-1β mRNA, which after 4 h rapidly decreases due to transcriptional repressors [90].
IL-1ra IL-1ra is considered an anti-inflammatory cytokine as it regulates IL-1α and IL-1β pro-inflammatory activity by competing with them for binding sites of the IL-1 receptor, but does not activate downstream signaling [87].
IL-2 IL-2 is considered an anti-inflammatory cytokine [91]. It affects the differentiation of T cells as it directs the differentiation of CD4+ T cells into regulatory T cells, which supresses the immune response. IL-2 promotes the differentiation of CD8+ T cells into effector and memory T cells, thus helping to further regulate the immune response. IL-2 does not directly affect T cell differentiation but influences the expression of the many receptors and transcription factors that control the differentiation [92].
IL-4 IL-4 is considered an anti-inflammatory cytokine due to its role in repair. However, overproduction of IL-4 is associated with allergies and chronic inflammation such as asthma. IL-4 decreases the production of Th1 cells, M1 macrophages, IFN-γ, and dendritic cell IL-12. IL-4 triggers activated B cells’ differentiation into antibody secreting plasma cells. IL-4 increases the proportion of Th2 cells, which produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 and evokes strong antibody responses and eosinophil accumulation [93]. Macrophages play an important role in chronic inflammation but also repair. The presence of IL-4 in extravascular tissues promotes activation of macrophages into repair macrophages, this causes the secretion of IL-10 and transforming growth factor beta (TGF-β) that result in a diminution of pathological inflammation [94]. IL-4 has two receptors, one specific and the other shared with IL-13, which induces similar signaling [93].
IL-5 IL-5 stimulates B cell growth and differentiation into antibody secreting plasma cells. It is a key mediator in eosinophil activation, increasing proliferation, maturation and survival. IL-5 is associated with allergies and chronic inflammation such as asthma [95].
IL-6 IL-6 is mainly considered a pro-inflammatory cytokine but also acts as an anti- inflammatory cytokine. IL-6 is secreted by T cells and macrophages to stimulate immune response, playing a role in fighting infection. IL-6 is responsible for increased production of neutrophils in bone marrow. It supports the growth of B cells and is antagonistic to differentiation of T cells into regulatory T cells. It is capable of crossing the blood-brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature set point [96]. In muscle and fatty tissue, IL-6 stimulates energy
20
mobilisation that leads to increased body temperature [97].
IL-6 signals through a membrane bound specific receptor which upon ligand binding associates with a signal receptor glycoprotein 130 (gp130). The specific IL-6 receptor is expressed in a few cells, however, gp130, is present on all cell surfaces. IL-6 is capable of trans signaling where free soluble IL-6 receptor protein forms a complex with IL-6 and binds to gp130, initiating a signal transduction cascade leading to c-Jun N-terminal kinases (JNK) activation and activation of MAPK pathways. The inflammatory activities of IL-6 are mediated by this trans-signaling, whereas the anti-inflammatory or regenerative activities are mediated by the classical signaling (binding to the specific receptor) [98].
IL-6 acts as an anti-inflammatory cytokine through its inhibitory effects on TNF-α and IL-1, and activation of IL-1ra and IL-10. In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation in bone repair and maintenance. IL-6 acts as a muscular repair signal (myokine) during exercise [97].
IL-7 IL-7 is a growth factor for various blood cells (including macrophages and monocytes) secreted by stromal cells [99].
IL-9 IL-9 is produced mainly by CD4+ T cells in response to TGF-β and IL-4. This cytokine is a growth factor for mast and T cells and has an effect on B cells’ growth and function. It functions through the IL-9 receptor that has 2 sub units, one exclusive and the other shared with IL-2, IL-4 and IL-7, binding activates the STAT1, 3, 5 proteins leading to further effects. IL-9 has been linked to asthma parthenogenesis, in KO mice models and with transgenic expression of IL-9 in lungs resulting in allergic inflammation. IFN-γ inhibits IL-9 production [100, 101].
IL-10 Anti-inflammatory cytokine IL-10 binding induces STAT3 signaling and can block NF-κB activity. IL-10 down regulates the production of pro- inflammatory cytokines (IL-1α and β, IL-6, IL-12, IL-18 and TNF-α) and pro- inflammatory chemokines (monocyte chemotactic protein (MCP) 5, MCP1, regulated on activation, normal T expressed and secreted (RANTES), IL-8, interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein (MIP) 2) in macrophages. It limits T cell activation and differentiation by inhibiting production of IL-2, IFN-γ, IL-4, IL-5 and TNF-α by CD4+T cells. A number of pathogens have evolved to stimulate IL-10 production to avoid an immune response [102].
IL-12 (p70) IL-12 (p70) is the bioactive form of IL-12 made when two sub units, p35 and p40 combine. IL-12 is produced mainly by activated monocytes, macrophages, dendritic cells and B cells. IL-12’s major function is increasing the production of IFN-γ by NK cells and T cells via STAT4 activation, in a positive feedback loop producing more IL-12. This feedback loop greatly increases the cytotoxicity of T and NK cells. It also plays a role in the differentiation of naïve T cells to Th1 effector cells. IL-12 has an anti-inflammatory effect on Th1 memory cells causing them to produce anti-inflammatory IL-10 [103, 104].
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IL-13 Anti-inflammatory cytokine IL-13 was originally described as a T cell derived cytokine that inhibits inflammatory cytokine production [105]. Its roles are similar to IL-4, however, have been shown to differ slightly. It is produced by Type-2 producing macrophages, Th2 T cells, NK cells, eosinophils, basophils and mast cells. It acts to promote tissue healing and repair and inhibits further inflammation. Its production is inhibited by IL-12, IL-18 and IFN-γ [106].
IL-15 IL-15 is an anti-inflammatory and pro-inflammatory cytokine [107], which plays a role in acquired immunity. IL-15 has a specific binding protein IL- 15Rα and signals through a β and γ chain complex, which it shares with IL-2, as it does some functionality. When IL-15 binds to its receptor, JAK, STAT3, STAT5, and STAT6 transcription factors are activated. IL-15 plays a crucial role in the development, survival and differentiation of NK cells. It also plays an integral role in the development of immunity in memory T cells and co administration with vaccines has been shown to enhance vaccine effectiveness. IL-15 mRNA is present in many cell types, however, is only excreted in detectable amounts by monocytes, epithelial cells, bone marrow stromal cells and fibroblasts [108].
IL-17 IL-17 acts as a potent mediator in delayed-type inflammatory reactions by increasing chemokine production in many cells to recruit monocytes and neutrophils to the site of inflammation. IL-17 is produced mainly by specialised Th17 cells. IL-17 induces the production of many other cytokines (such as IL-6, granulocyte-macrophage colony-stimulating factor (G-CSF), GM-CSF, IL-1β, TGF-β, TNF-α), chemokines (including IL-8, MCP-1, MCP- 3, MIP3A and RANTES), and prostaglandins (e.g. PGE2) from many cell types. As a result of these roles, the IL-17 family has been linked to many immune/autoimmune related diseases including multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease [109].
Basic In normal blood vessels BFGF is present in basement membranes and in the fibroblast sub-endothelial extracellular matrix. When signalled, heparan sulfate- growth degrading enzymes activate BFGF. BFGF promotes angiogenesis and factor endothelial cell mitogen. BFGF is increased at sites of chronic inflammation in (BFGF) rheumatoid arthritis, inflammatory bowel disease and asthma. BFGF synergistically promotes leukocyte recruitment [110].
G-CSF G-CSF’s major role is increasing the survival, proliferation and differentiation of all cells in the neutrophil linage. It also mobilises haemopoietic stem cells from bone marrow into the blood stream. G-CSF has been shown to have an anti-inflammatory effect in T-cells reducing Th1 cytokine (IFN-γ), cytokine release and increasing Th2 (IL-10) release. It is released by cells including macrophages, endothelial and fibroblast cells when stimulated by IL-1, LPS, TNF-α and IL-17. JAK1, JAK2 and non-receptor tyrosine-protein kinase (Tyk2) are phosphorylated after receptor binding leading to the activation of STAT1, STAT3 and STAT5. In mice G-CSF pre-treatment reduced Th1 cytokine levels and increased survival of septic shock, however, G-CSF in humans has been shown to exasperate arthritis [111].
GM-CSF GM-CSF is a hematopoietic growth factor which stimulates the survival,
22
proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. It is produced upon stimulation (IL-6, IL-1, TNF-α) by T cells, macrophages, endothelial cells and fibroblasts recruiting neutrophils, monocytes and lymphocytes to the site of inflammation. Its receptor is associated with JAK2, activating STAT5 and MAPK. Over expression leads to macrophage accumulation. GM-CSF is found in asthmatic lungs, arthritic synovial fluid and allergic reactions on skin [112].
Platelet- PDGF is secreted by platelet cells and macrophages, it promotes collagen derived repair by acting as a chemoattractant to fibroblast cells, but also, neutrophils, growth macrophages and smooth muscle cells. When an injury occurs, blood factor components rush to the site, platelets are then exposed to collagen and other (PDGF) extracellular components stimulating them to release PDGF, to recruit cells for the immune response. Macrophages release more PDGF, which actives, attracts and stimulates mitogenesis of fibroblast and smooth muscle cells to accelerate extracellular matrix and collagen formation and thus reducing the time for the healing process to occur. PDGF’s role of increasing collagen repair and the formation of scar tissue is responsible for its association with atherosclerosis and fibrotic disorders. PDGF is also involved in angiogenesis [113].
Vascular VEGF stimulates vasculogenesis and promotes angiogenesis. VEGF enhances endothelial the permeability of the blood brain barrier. VEGF further promotes growth inflammation by up-regulation of CD54 and MIP-1α and monocyte factor extravasation. Low doses elicit the inflammatory and permeability effect, while (VEGF) higher doses induce vascular proliferation. High concentrations of VEGF are reported in asthma, diabetes, cerebral ischemia, trauma, pre-eclampsia, ovarian hyper stimulation syndrome and status epilepticus [114].
Chemokines (chemotactic cytokines) direct the movement of circulating leukocytes to the site of inflammation or injury causing an escalation in the response. There are around 50 chemokines segregated into four families based on structure and function. The nomenclature is based on the structure. The largest family is the CC chemokines, so named as the first two cysteine molecules are adjacent to one another. The second largest family is the CXC family, which has a single amino acid group between the first cysteines. Chemokines affect cells by activating both specific and shared surface transmembrane-domain G-protein coupled receptors. The leukocytes’ response to the chemokine binding is specific to the complement of receptors expressed. The binding of chemokines to the receptor activates a signaling
23
cascade that causes a change in the shape and movement of the actin. Table 2 describes the function of a number of major inflammatory chemokines.
Table 2: The function of inflammatory chemokines IL-8, (also Inflammatory chemokine IL-8 is a potent promoter of angiogenesis and known as induces chemotaxis in neutrophils, causing them to migrate toward the site CXCL-8) of infection. IL-8 is secreted by cells with TLR that are stimulated in the innate immune response. Local tissue macrophages are usually the first to detect an antigen and thus are the first cells to release IL-8 in large amounts to recruit other cells [115, 116].
Interferon IP-10 is a chemokine also referred to as CXCL10. IP-10 is secreted by gamma- several cell types in response to IFN-γ, but most abundantly by endothelial induced protein cells. IP-10 is involved in chemotaxis for monocytes/macrophages, T cells, (IP) 10 (IP-10), NK cells, and dendritic cells. It is an important inflammatory target as it (also known as increases mononuclear cellular infiltration in immune-inflammatory CXCL10) demyelination diseases [117]. It also has an anti-inflammatory action as it attenuates angiogenesis [118].
MCP-1, (also MCP-1 recruits monocytes, memory T cells, and dendritic cells to the sites known as of inflammation. It is released by many cell types, but mainly monocytes CCL2) and macrophages in response to stimulation with IFN-γ, TNF-α, IL-1 and endotoxins. Its release is inhibited by IL-4, IL-10 and IL-13. MCP-1 is implicated in the pathogenesis of several diseases characterised by monocytic infiltrates, such as psoriasis, rheumatoid arthritis and atherosclerosis. It has been shown to be a potential intervention point in multiple sclerosis, rheumatoid arthritis, atherosclerosis and diabetes. MCP-1 is involved in neuro-inflammatory diseases as it induces an increase in the brain endothelial permeability [109]. MCP-1 expression in glial cells is increased in epilepsy, brain ischemia, Alzheimer’s disease and traumatic brain injury [119, 120].
MIP-1α, (also MIP-1α is a major inflammatory factor produced by macrophages after known as stimulation with bacterial endotoxins. MIP-1α is best known for its CCL3) chemotactic and pro-inflammatory effects. MIP-1α activates human granulocytes (neutrophils, eosinophils and basophils) which can lead to acute neutrophilic inflammation. It induces the synthesis and release of other pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α from fibroblasts and macrophages [121].
MIP-1β, (also MIP-1β is a chemoattractant for NK cells, macrophages and monocytes. It known as has been connected to human immunodeficiency virus type 1 (HIV-1) and CCL4) transplant rejection [122]. MIP-1β is a major HIV-1 suppressive factor produced by CD8+ T cells and monocytes [121].
RANTES, (also RANTES is chemotactic for T cells, dendritic cells, eosinophils, NK cells,
24
known as mast cells and basophils. RANTES is not T-cell specific and is excreted by CCL5) activated platelets, macrophages, eosinophils, fibroblasts, endothelium, epithelial and endometrial cells. RANTES binding causes the activation of NF-κB, resulting in the transcription of a number of inflammatory mediators. RANTES also promotes angiogenesis. RANTES has been implemented in disease progression of inflammatory diseases like asthma, rheumatoid arthritis and multiple sclerosis. Mutation of CCR5 (receptor) or blockage of RANTES by small molecules has a positive effect in the treatment of HIV [123]. RANTES has been shown to increase the permeability of the blood brain barrier [124].
Eotaxin-1, Eotaxin-1 is an inflammatory chemokine that primarily recruits eosinophil (also known as cells. It is excreted by eosinophils, macrophages, lymphocytes, fibroblasts, CCL-11) smooth muscle cells, epithelial cells and chondrocytes. Its production is stimulated by IL-17, IL-1β and TNF-α. It is present in high levels in adipose tissue of diet induced obesity. High eotaxin-1 levels are linked to allergic inflammation, inflammatory bowel disease, rheumatoid arthritis, ulcerative colitis and eosinophilic oesophagitis. In ulcerative colitis and inflammatory bowel disease a direct correlation was established between the disease severity and the eosinophil recruitment [125, 126].
25
Table 3 categorises the cytokines discussed in Table 1 and Table 2, as either pro, secondary pro or anti-inflammatory cytokines.
26
Table 3: Anti-pro-inflammatory cytokines and chemokines [79, 127].
Pro-inflammatory cytokines Secondary pro-inflammatory Anti-inflammatory cytokines
effects
TNF-α IL-5 IL-1ra
IFN-γ IL-7 IL-2
IL-1β IL-9 IL-4
IL-6 IL-15 IL-6 (by specific binding)
IL-8 BFGF IL-10
IL-12 (p70) G-CSF IL-13
IP-10 GM-CSF IL-15
RANTES PDGF TGF-β
Eotaxin-1 VEGF
MCP-1
MIP-1
1.3.7. The role of NF-κB in inflammation
Transcription factors control the expression of genes. NF-κB has been shown to increase the expression of a number of genes including cytokines, chemokines, and adhesion molecules, which are known to play a role in the maintenance of chronic inflammatory diseases. The
NF-κB dimers bind to specific DNA sequences referred to as “κB” sites. In most cells,
NF-κB is present in an inactive form in the cytoplasm, bound to an inhibitor referred to as
IκB. NF-κB can be activated by various stimuli, cytokines (TNF-α and IL-1), T and B cell mitogens, viral proteins, UV and ROS. Activation of certain signaling cascades by inflammatory stimuli results in the phosphorylation, ubiquitination and subsequent degradation of IκB, which frees NF-κB to then move from the cytoplasm to the cell nucleus.
The most common NF-κB dimer contains p50/p65 heterodimers, this specific heterodimer is
27
referred to as NF-κB [128]. The importance of NF-κB in inflammation and related diseases makes it a potential target for the development of anti-inflammatory drugs. Figure 7 shows the activation pathways of NF-κB by LPS.
Figure 7: Diagram showing the activation pathway of NF-κB by LPS stimulation [129]
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1.4. Hypotheses and aims
1.4.1. Hypotheses
1. The A. paniculata commercial extract with its complex mixture of phytochemicals
will exhibit more potent anti-inflammatory activity than the pure andrographolide.
2. The A. paniculata commercial extract and andrographolide will have a broader range
of anti-inflammatory activity on key mediators in model cell lines of inflammation
compared to current NSAIDs.
3. The metabolites of andrographolide will retain some anti-inflammatory activity of the
parent.
1.4.2. The major specific aims of the thesis
1. To characterise the effect of andrographolide and standardized A. paniculata extracts
on the release of pro- anti-inflammatory cytokines in LPS and IFN-γ stimulated
murine macrophages and human monocyte-derived macrophages.
2. To measure the effect of andrographolide and A. paniculata extracts on the activity of
key enzymes involved in the production of mediators of inflammation in LPS and
IFN-γ stimulated murine macrophages and human monocyte-derived macrophages.
3. To determine the effect of andrographolide and A. paniculata extracts on activation of
the transcription factor NF-κB, a key regulator of genes for pro-inflammatory
cytokines, chemokines and enzymes that generate mediators of inflammation in LPS
and IFN-γ stimulated murine macrophages.
4. To synthesize and determine the activity of the metabolites of andrographolide in
cellular models of inflammation and compare them to the parent compound.
29
5. To determine the activity of NSAIDs in cellular models of inflammation and compare
them to andrographolide.
30
Chapter 2
______2. Methods
31
2.1. Reagents
2.1.1. Solvents/chemicals
The reagents used in this study have reported purity and were purchased from reputable suppliers. The acids used as modifiers in the liquid chromatography (LC) mobile phase, formic acid (99%) and trifluroacetic acid (99.5%) (TFA) and the sulphuric acid (95%) used in synthetic work were obtained from Ajax (Sydney, Australia). The acetonitrile used in high performance liquid chromatography (HPLC) and Prep-HPLC was of certified HPLC grade from Chemsupply (Sydney, Australia). The nitrogen gas (99.9%) used to evaporate down samples was 4.0 grade from Core gas (Sydney, Australia). The Milli-Q water (>18 MΩ cm) was obtained from a Purite300 Milli-Q system supplied by MicroAnalytix (Sydney,
Australia). The sodium hydroxide (NaOH, 98%) used in synthesis and the ammonia (30%) used to neutralise the mobile phase was from Sigma-Aldrich (Sydney, Australia).
2.1.2. Standards and other reagents
The standards and reagents used are from reputable sources with claimed purity.
Acetylsalicylic acid (aspirin, 99%), apigenin (98%), chlorogenic acid (99%), ibuprofen sodium salt (98%), diclofenac (99%), acetaminophen (paracetamol, 99%), prednisone (98%), dexamethasone (97%) and 4-methyl-N1-(3-phenylpropyl)-1,2-benzenediamine (JSH-23,
98%) were purchased from Sigma (Sydney, Australia). 1-(4-fluorophenyl)-3-
(phenylsulfonyl)-1H-pyrrolo(2,3-b)quinoxalin-2-amine (CAY 10602, 95%), Zileuton (98%),
Ro 106-9920 (95%) and phenethyl caffeiate (CAPE, 98%) were purchased from Sapphire
Bioscience (Sydney, Australia). The Xiyanping Zhusheye sulfonated andrographolide injection was purchased from the Jiangxi Qingfeng Pharmacy Co. Ltd. (Ganzhou, China).
32
Andrographolide (98%), 14-deoxy-11,12-didehydroandrographolide (98%), wogonoside
(98%), isoquercetin (98%) were purchased from Biopurify (Chengdu, China).
2.1.3. Cell culture reagents
The cell culture reagents used were certified for use in cellular applications. Dulbecco’s
Modified Eagle Medium (DMEM), Phorbol-12-myristate-13-acetate (PMA), Penicillin and
Streptomycin (Pen Strep), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and GlutaMax® (Glutamine) were obtained from Invitrogen (Carlsbad, USA). The
Roswell Park Memorial Institute (RPMI) medium, napthyethylene-diamine (NED), sulphanilamide, all trans retinoic acid (ATRA), dibutyryl cyclic adenosine monophosphate
(db-cAMP), albumin and bovine serum albumin (BSA), monobasic phosphate (Na2HPO4), dibasic phosphate (NaH2PO4) resazurin, LPS from E. Coli strain 0111:B4, 3,3,5,5- tetramethylbenzidine (TMB), and citric acid were purchased from Sigma-Aldrich (Sydney,
Australia). The murine IFN-γ and murine TNF-α enzyme-linked immunosorbent assay (ELISA) kit were from Peprotech, supplied by Lonza (Sydney, Austalia). The foetal bovine serum (FBS) (French origin) was purchased from Bovogen Biologicals (Keilor East,
Australia). The strep avadin horse radish peroxidase used in the TNF-α ELISA was purchased from BD (Sydney, Australia). The Bio-Plex Pro cytokine, chemokine and growth factors assay kits, human cytokine 17 and 27-plex were purchased from Bio-Rad (Sydney,
Australia). The phosphate buffer saline (PBS) used in the study was prepared from NaH2PO4
(1.9 mM), Na2HPO4 (8.1 mM), and NaCl (154 mM) in Milli-Q water and adjusted to pH 7.2.
33
2.2. Equipment
2.2.1. General
The general equipment used in this study was of scientific quality and maintained and calibrated regularly. The balance used for weighing samples and reagents from 10 mg - 50 g was a Mettler Toledo ML52 analytical balance (Mettler Toledo, Australia) and for <10 mg a
Sartorius SE-2 micro Sartorius analytical balance (Sartorius, Australia) was used. A
Powersonic 420 ultrasonic bath (Thermoline Scientific, Australia) was used to dissolve the samples. The pH meter used was a Jenway 3505 (VWR, Australia).
Automatic pipettes P10 (0.5 - 10 μL), P100 (10 - 100 μL) and P1000 (100 - 1000 μL) were from Eppendorf (Eppendorf South Pacific, Australia). The multichannel pipette 20 - 300 μL was from Biohit (Sartorius, Australia) and the 0.5 - 10 μL was from Thermofisher
(Thermofisher Scientific, Australia).
The cells were counted using a Vi-CELL XR cell viability analyser (Beckman Coulter,
Australia). The plate shaker (ThermoStar) and plate reader (FLUOstar Optima) were purchased from BMG (BMG Labtech, Australia).
2.2.2. FACS Canto II flow cytometer
The ELAM9 RAW264.7 cells used in the NF-κB activation assay were analysed using a BD
FACS Canto II flow cytometer (Becton, Dickinson and Company, Australia and New
34
Zealand). The flow cytometer was equipped with a high throughput Facs flow auto sampler.
The flow cytometer is equipped with a 405, 488, 635 laser set and a corresponding filter set.
2.2.3. Multiplex assay system
The human cytokine 17 and 27-plex assays were analysed using a BioPlex 100 Bio-Plex system from Bio-Rad (Sydney, Australia). The software used to operate the system and interpret the data was Bio-Plex Manager 3.0.
The plates were washed using a 96 well plate magnetic hand held washer from Bio-Rad
(Sydney, Australia).
2.2.4. Cell culture consumables
The RAW 264.7 murine macrophage cell line and the THP-1 human leukemic monocyte cell line were purchased from American Type Culture Collection (Manassas, USA). All the
Greiner Bio-One GmbH general plastic cell culture consumables used were supplied by
Interpath (Sydney, Australia). The Corning 96 well EIA/RIA flat bottom high binding
(polystyrene) plates used with the Peprotech ELISA were purchased from Sigma (Sydney,
Australia).
35
2.3. Cell culture methods
2.3.1. Cell culture
2.3.1.1. Murine Macrophages RAW264.7 cells
The murine macrophages cell line RAW264.7 is a cell line that has been used widely in the study of inflammation. The cell line was established from a tumour induced by Abelson murine leukaemia virus in 1978 [130]. The cell line maintains many of the properties of macrophages including NO production, phagocytosis, extreme sensitivity to TLR agonists, cytokine release and motility [131, 132]. These traits make RAW264.7 cells one of the most widely used cell types for the study of inflammation. Whilst RAW264.7 cells are an excellent platform to study the inflammation response, they may be considered limited in human applicability as they are a murine cell line.
2.3.1.2. Cell maintenance- RAW264.7
The RAW264.7 murine macrophages were maintained in DMEM media containing 4.5 g/l
D-glucose and supplemented with 2 mM l- GlutaMax, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS. The cells were incubated at 37˚C, 5% CO2 in 95% air in 75 ml vented flasks. Every 3-4 days the culture was split to maintain a manageable number of cells.
To split the cells they were aspirated with DMEM. Complete DMEM was then added to the cells and they were removed from adhesion to the bottom of the flask using a rubber cell scraper. The cells were diluted in fresh complete DMEM by gentle up and down pipetting, counted and used for experimentation or split according to cell count. The cells were transferred to a new 75 ml flask and made to 10 ml with fresh complete DMEM. The cells were incubated with the flask placed horizontally.
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2.3.1.3. Human THP-1 cells
The human monocyte cell line THP-1 has been used widely in the study of inflammation.
THP-1 is a human monocyte like cell line derived from the peripheral blood of a 1 year old human male with acute monocytic leukemia. It was isolated in 1980 by Tsuchiya et al. as single round suspension cells with distinct monocytic markers [133]. THP-1 cells can be differentiated to macrophages like cells when exposed to phorbol-12-myristat-13-acetate
(PMA) which activates protein kinase C. They adhere and show other morphological changes consistent with macrophage phenotypes; flat and amoeboid in shape with a well-developed rough endoplasmic reticulum with abundant free ribosomes and golgi apparatus [134] and expression of surface markers associated with macrophage differentiation [135]. Importantly when stimulated with inducers of inflammation like bacterial LPS they excrete a number of inflammatory proteins.
2.3.1.4. Cell maintenance- THP-1
The THP-1 cells were cultured in RPMI media containing 4.5 g/l D-glucose and supplemented with 2 mM GlutaMax, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10%
2 FBS at 37˚C, 5% CO2 in 95% air in 75 cm vented flasks. Every 3-4 days the culture was split as required to maintain a manageable number of cells. To split the cells they were transferred to a 15 ml Falcon tube, this was then centrifuged at 200 RCF for 5 min to form a cell pellet.
The expired medium was tipped off. The cells were re-suspended in fresh complete warm
RPMI medium by gentle up and down pipetting and counted. The cells were then split according to cell count or used for experimentation. The cells were transferred to a new 75 ml
37
flask and made to 15 ml with fresh complete medium. The cells were incubated with the flask placed vertically.
2.3.2. Cell counting
Cell counting was performed using the Vi-CELL XR cell viability analyser. The cells
(passage number between 10 and 25) were counted after being suspended (well mixed) in complete medium according to cell splitting protocols 2.3.1. 1 ml of the cell suspension was transferred to a counting vial, the cells were diluted accordingly if high density was found or suspected. The cell suspension was then subjected to analysis according to the cell type parameters. The culture was then diluted to desired density and recounted to ensure correct dilution.
2.3.2.1. RAW264.7 Vi-CELL XR cell viability analyser analysis parameters
The counting parameters for RAW264.7 cells were optimised. The cells were counted according to the following parameters; minimum diameter 5 μm, maximum diameter 20 μm, the number of images 50, aspiration cycles 1, trypan blue mix cycles 3, cell brightness 90%, cell sharpness 120, viable spot brightness 70%, viable cell spot area 15%, minimum circularity 0 and de-cluster degree high.
2.3.2.2. THP-1 Vi-CELL XR cell viability analyser analysis parameters
The counting parameters for THP-1 cells were optimised. The cells were counted according to the following parameters; minimum diameter 5 μm, maximum diameter 50 μm, the number of images 50, aspiration cycles 1, trypan blue mix cycles 3, cell brightness 85%, cell
38
sharpness 100, viable spot brightness 65%, viable cell spot area 5%, minimum circularity 0 and de-cluster degree medium
2.3.3. Cell plating
The cells were plated at high density to limit the impact of proliferation on cell number and to maximise the readout amplitude.
2.3.3.1. Plating of RAW264.7 cells
After initial counting of the Raw264.7 cells the cells were diluted to 1 × 106 cells/ml in complete DMEM. These were then recounted to ensure dilution and correct counting, if inconsistency was found the cell dilution was corrected. The cells (1x10^5/well, 100 μl) were
o then transferred to a 96 well plate and incubated for 48 h at 37 C (5% CO2) to allow the cells fully attach and reach consistent confluence.
2.3.3.2. Plating and differentiation of THP-1 cells
After initial counting of the THP-1 cells the cells were diluted to 1 × 106 cells/ml in complete
RPMI. These were then recounted to ensure dilution and correct counting. If any inconsistency was found the cell dilution was corrected. PMA was prepared in dimethyl sulfoxide (DMSO) and stored in 100 μl frozen aliquots at 100 μM. The PMA stock (10 μl) was then added to the complete medium (10 ml, 1:1000) to produce a final concentration of
100 nM .The cells (100 μl) were then transferred to a fresh 96 well plate and incubated for 48
o h at 37 C (5% CO2) to allow the cells to differentiate. The cells were then washed in FBS free
39
RPMI and rested for 24 h in complete RPMI. Prior to compound addition the cells were washed again.
2.3.4. Compound addition
The cells were pre-incubated with the compounds being tested prior to activation unless stated otherwise. Pre-incubation was performed to allow the compounds to interact with the cells prior to activation. This may allow sufficient time for the compound to enter the cells and affect transcription and translational modifications. The compounds were either prepared in DMSO or PBS.
2.3.4.1. Compound preparation
The compounds were prepared either in PBS or DMSO according to their solubility. The PBS soluble compounds were either weighed (≥ 5 mg) out into a 15 ml or 2 ml centrifuge tube and made to concentration in PBS, typically at 20 times the desired final concentration, unless further dilution was required. The DMSO soluble compounds were weighed (≥ 5 mg) into a 2 ml centrifuge tube and made to concentration in DMSO, typically at 1000 times the desired final concentration, unless further dilution was required. After the solvent was added the compounds were vortexed and sonicated for ≥ 10 min or until fully dissolved. The stock solutions were then serially diluted to create the desired dose response curve. The DMSO stock solutions were diluted (1:10) in PBS prior to adding to the plate.
40
2.3.4.2. Compound pre-incubation
After cells had been incubated post plating for the recommended time, the cells were washed with fresh medium (100 μl) and the medium was replaced with new complete medium (180 -
188 μl, to produce a final volume of 200 μl). The compound solution was then added and the
o plate incubated for 1 h at 37 C (5% CO2) prior to activation. Compounds that were prepared in PBS were added to the cells at 1:20 dilution (10 μl per well) and compounds that were prepared in diluted DMSO were added at 1:100 final dilution (2 μl per well) (0.1% DMSO).
2.3.5. Cell activation
After the cells were pre-incubation with the compounds being tested, they were activated to stimulate inflammation. The cells were activated by the addition of LPS and murine or human IFN-γ (unless otherwise stated) according to the cell type. LPS was prepared in sterile
PBS to a concentration of 2 μg/ml and stored (-20oC) in 1 ml aliquots until use. IFN-γ (both human and murine) was prepared to 20 μg/ml in PBS and stored (-20oC) in 100 μl aliquots until use. The activation solution was prepared by adding 1 ml LPS to 10 μl IFN-γ and 990μl complete medium. Unless otherwise stated the cells were activated, by the addition of 10μl of this solution to make a final volume of 200 μL, giving a final concentration of 50 ng/ml
LPS and 5 ng/ml IFN-γ in the well. The cells were then returned to the incubator for typically
18 h, depending on the assay being performed.
41
2.4. Cell viability by MTT
2.4.1. Principles of MTT assay
The effect of the compounds on the viability of the cells was determined by MTT. Activity was determined spectophotometrically as mitochondrial dehydrogenase present in viable cells cleaves the tetrazolium ring of MTT to yield a purple MTT formazan. The reaction is shown in Figure 8. The greater the number of viable cells present the more purple MTT formazan will be formed and therefore the higher the absorbance.
Figure 8: Cell viability quantification with MTT [136].
2.4.2. Protocol for MTT viability determination
To determine proliferation, 100 µl of MTT solution (0.2 mg/ml MTT in complete medium)
o was added and incubated for 2 h at 37 C (5% CO2). MTT solution was removed and 150 µl of DMSO was added to dissolve the formazan crystals. The plate was shaken for 5 min before absorbance was measured at 595 nm on the Fluostar plate reader.
42
2.5. Determination of NO production
2.5.1. Principles of NO assay - Griess method
The Griess method is a colorimetric method for the quantification of nitrites. Cells produce
NO in response to stress or stimulation with LPS. NO is a free radical and is spontaneously oxidised to nitrite when expose to oxygen. NO was indirectly quantified by nitrites dissolved in the cell culture medium. The Griess method quantifies nitrites based on the principle of chromophore formation from diazotization of sulphanilamide by nitrite in acidic conditions followed by coupling with bicyclic amines, i.e.N-1-napthyl-etylenediamine (NEDD) [137,
138]. The reaction is shown in Figure 9. The diazonium product has strong absorbance at
543 nm. The NEDD is supplied in excess so that the avalible nitirate in solution limits the production of the diazonium product, thus the stronger the absorbance at 543 nm the more nitrate is present in the solution.
Figure 9: Griess-reagent principle in nitrite quantification [139].
2.5.2. Nitric oxide quantification
Nitric oxide concentration in the cell culture medium was determined by the Griess reagent quantification of nitrite. The supernatent (100 μl) collected after activation with LPS and 43
IFN-γ (18 h) was transferred to a new 96 well plate. To this 50 μl of 1% sulfanilamide in
2.5% phosphoric acid was added followed by 50 μl of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride to complete the Griess reagent. The Griess reagent and cell supernatant was incubated for 5 min at room temperature to let the reaction go to completion. In the presence of nitrite the Griess reagent forms a violet colour measured by absorbance at 540 nm on a
Flurostar plate reader. The amount of nitrite produced was normalised to the control wells, so that non stimulated cells were 0 % NO production and untreated stimulated cells were 100 %
NO production.
2.6. Determination of TNF-α release
2.6.1. Principles of TNF-α ELISA
The TNF-α released by the cells after activation was quantified using an enzyme linked immunosorbent assay (ELISA). A specific TNF-α antibody is coated onto a 96 well plate.
Free TNF-α present in the cell culture medium binds to this. A TNF-α specific antibody bound to a biotin group is then added which binds to the TNF-α. Strepavadin HPR is added which binds to the biotin group. After each step the plate is washed so that no unbound antibodies are left. A tetramethylbenzidine (TMB) solution is then added to the plate which is enzymatically oxidised by the HPR, to produce a strong blue colour. The blue colour can be measured by absorbance at 655 nm. Additionally, stop solution (H2PO4) turns the colour from blue to yellow, which is detectable at 450 nm as illustrated in Figure 11 [140]. The higher the absorbance at 450 nm the more TNF-α is present in the cell supernatant. An illustration of an
ELISA is shown in Figure 10.
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Figure 10: An illustration of the TNF-α assay [141].
420 nm 655 nm
Figure 11: TMB colour development (modified from [142]).
The concentrations of TNF-α released into the cell medium, following 18 h incubation with
LPS and IFN-γ, was determined by ELISA. The protocol accompanying the ELISA kit from
Peprotech was altered, to the following procedure:
2.6.1.1. Reagent preparation
The reagents were prepared according to manufacturer’s protocol. Antibodies were reconstituted in Milli-Q water and aliquoted into 10 tubes each and refrigerated (-20oC). The rest of the solutions were prepared daily or as needed. The diluent buffer for analysis was prepared by diluting 0.05% (w/v) Tween-20 and 0.1% (w/v) BSA in PBS. Block buffer was
45
prepared by addition of 2% BSA (w/v) in PBS and filtered through vacuum driven 0.22 μm polyvinylidene fluoride filter. The wash buffer was prepared to contain 0.05% (w/v) Tween-
20 in PBS. The coating buffer (carbonate-bicarbonate) was prepared by mixing NaHCO3 (4 g) and Na2CO3 (1.5 g) in 1l Milli-Q water and adjusting to pH 9.5. Phosphate citric acid (PC) buffer was produced by diluting 25.7 ml of 0.2 M dibasic sodium phosphate (NaH2PO4), 24.3 ml of 0.1 M citric acid, to 100 ml with Milli-Q water. Stock solution of TMB for colour development was prepared by diluting 1 ml DMSO (1mg/ml TMB) in 9.5 ml PC buffer.
2.6.1.2. Peprotech TNF-α protocol for RAW264.7
The captured antibody (100 µl) was diluted with carbonate-bicarbonate buffer (9.9 ml) and added (100 µl) to each well in the ELISA plate and incubated overnight. The plate was washed 4 times with wash buffer. The plate was blocked by adding block buffer (200 μl) and incubated for 1 h on shaker at 250 rpm and 1 h still. The plate was then washed 2 times. The standard curve was prepared and added in duplicate as per instruction to give a range from
2000-39 pg/ml. The supernatant was diluted in diluent buffer and added to the plate (100 ul).
For the RAW264.76 cells a 1:100 dilution of the supernatant was necessary. For the THP-1 cells a 1 in 10 dilution of the supernatant was necessary. The plates were incubated 2 h at room temperature and then washed twice before the detection antibody was added and incubated for a further 2 h. The plates were again washed twice, before the addition of the strep avadin horseradish peroxidase (HRP) which was purchased separately from the kit (BD,
USA), 20μl was diluted in 10 ml diluent and 100 μl was added to each well and incubated for
45 min. The TMB solution was prepared in this time and 1 ul 30% H2O2 prior to use. The plates were washed 4 times before adding the TMB solution (100 μl per well). The colour development was monitored at 655 nm, with readings taken after every 5 min. After the plates were developed the reaction was stopped using 0.5 M sulphuric acid, and the
46
absorbance of the resultant yellow colour was measured at 450 nm. The standard curve for
TNF-α was produced by plotting respective data in GraphPad Prism version 5, where the standard curve was fitted to a 4 parameter logistic curve fit and used to calculate the concentration of the TNF-α in each sample. The TNF-α concentration was then normalised to the unstimulated and stimulated untreated control and expressed as a percentage of stimulated untreated TNF-α release. The dose response curves were constructed in Graphpad prism by plotting the log of the dose concentration against the percentage release, to this a non-linear 4 parameters variable slope dose response curve was fit, to calculate the half maximal inhibitory concentration (IC50) for each sample tested.
2.7. Determination of PGE2 release
2.7.1. Principles of COX ELISA
The PGE2 released by the activated cells was quantified by competitive ELISA. The quantification of PGE2 in the supernatant is based on the competition between PGE2 and a
PGE2-acetylcholinesterae (AChE) conjugate for a limited number of PGE2 monoclonal antibodies. As the monoclonal binding sites are limited and the amount of the AChE conjugate is kept uniform in all wells, the amount of free PGE2 present limits the amount of
AChE conjugate that can bind to the monoclonal antibody, thus the level of free PGE2 is inversely proportional to the AChE that is able to bind to the monoclonal antibody. The PGE2 complexes then bind to the goat polyclonal anti-mouse IgG that is attached to the 96 well plate. The plate is washed to remove all unbound AChE conjugate that has not been able to bind to a monoclonal antibody and then to the polyclonal attached antibody. After washing the plate, the Ellman reagent which contains the substrate to AChE is added to the wells and an enzymatic reaction occurs. An illustration of the competitive PGE2 ELISA is shown in
47
Figure 12. The enzymatic reaction produces 5-thio-2-Nitrobenzoic acid which absorbs strongly at 412 nm. The mechanism of this reaction is shown in Figure 13. The intensity of the absorbance is inversely proportional to the amount of free PGE2 in the supernatant. Thus compared to the standard curve, PGE2 was quantified in the supernatant.
Figure 12: An illustration of the principles of the competitive PGE2 ELISA [143].
48
Figure 13: Ellman reagent acetylcholinesterase enzymatic reaction [143].
2.7.2. Protocol for PGE2 quantification release by RAW264.7 and THP-1 cells
The competitive ELISA was run as per the manufacturer’s instructions. Briefly, the standard
PGE2, AChE conjugate and monoclonal antibody were resuspended according to the manufacturer’s instructions. To each well of the supplied ELISA plate 50 μl of supernatant or diluted standard was added, followed by PGE2 AChE conugate (50 µl) and PGE2 monoclonal antibody (50 μl). The plate was then incubated for 60 min at room temperature on an orbital shaker, while the Ellman reagent was prepared. The plate was washed 5 times before the
Ellman reagent (200 μl) was added. The plate was then incubated in the dark on the plate shaker for 90 min for the colour to develop. The absorbance of each well was then read on the BMG plate reader at 420 nm. The results were exported to Graphpad prism5, where the standard curve was fitted to a 4 parameter logistic curve fit and used to calculate the concentration of PGE2 in each sample. The PGE2 concentration was then normalised to the unstimulated and stimulated untreated control and expressed as a percentage of stimulated
49
untreated PGE2 release. The dose response curves were constructed in Graphpad prism5 by plotting the log of the dose concentration against percentage release, to this a non-linear 4 parameters variable slope dose response curve was fitted, to calculate the IC50 for each sample tested.
2.8. Determination of NF-kB activation
2.8.1. Principle of NF-κB assay
The NF-κB assay was performed to determine the compound’s potency to inhibit NF-κB activation. The ELAM9 RAW 264.7 cells used in this study were modified to produce green florescence protein (GFP) when activated with LPS and IFN-γ. These cells were originally created by Dr. Tara Robertson from University of Queensland. The murine macrophage-like cell line was stably transfected with the human endothelial-leukocyte adhesion molecule
(ELAM9) (E-selectin) promoter (-760 to +60) driving destabilised enhanced GFP. Activated
NF-ĸB can induce the transcription of many genes such as cytokines, chemokines, growth factors, adhesion molecules in macrophage and microglial cells and GFP in the ELAM9 macrophage cell line used in this study. The number of activated cells was counted by a flow cytometer setup to detect the florescence of the GFP protein. The percentage of activated cells was used as the readout.
2.8.2. Protocol for NF-kB activation assay
The cells were plated (1x105cells/well), compounds added (1h preincubation 0.1% DMSO) and stimulated (50 μg/ml LPS, 50 Units/ml IFγ) according to method described in section 2.3
Cell culture methods, however, were only incubated for 5.5 h after stimulation. The cells
50
were then washed twice with ice cold PBS and detached by trypsin (40 μl) taking 10-15 min until they were all detached. PBS containing 10% FBS was then added to the wells and mixed well by pipetting to break apart cell clusters and dislodge loosely attached cells. The cells were then filtered through a 50 μm Nylon filter into a new 96 well plate for flow cytometer analysis. The plates were kept on ice until run and mixed on the plate shaker just prior to running.
2.8.3. Flow cytometer setup for analysis of GFP in ELAM9 RAW264.7 cells
The plates were analysed on the flow cytometer using the high through-put auto sampler, directly from the 96 well plates. The blue layer was used for the analysis of GFP in the
ELAM9 RAW264.7 cells. The FSC and SSC were determined based on the size and shape of the control cells plated in each separate experiment. The readout of the laser intensity was normalised in each experiment to the fluorescence of the normal RAW264.7 cells.
2.8.4. NF-kB data analysis
The data was analysed to obtain the percentage of cells where NF-kB had been activated to calculate an IC50 for each compound. The data was analysed using FlowJo v10. The scatter plots were gated by FSC-A (40K-110K) and SSC-A (20K-120K) to disregard abnormal cells from the analysis as shown in Figure 14. The normal population was then separated by florescence at 530 nm with stimulated cells being selected above 104 units as shown in Figure
15 and Figure 16, based on experimental data. The percentage of stimulated cells was then normalised to the unstimulated and stimulated untreated control and expressed as a percentage of stimulated untreated NF-kB activation. The dose response curves were constructed in Graphpad prism5 by plotting the log of the dose concentration against the
51
percentage release, to this a non-linear 4 parameters variable slope dose response curve was fitted, to calculate the IC50 for each sample tested.
Figure 14: Gating of normal cells by FSC and SSC.
Figure 15: Gating of stimulated cells (stimulated control).
52
Figure 16: Gating of stimulated cells (unstimulated control).
2.9. THP-1 multicytokine assay
The 17 and 27 multiple cytokines, chemokines and growth factors released by the cells after activation were quantified using bead based ELISA assay (Bio-Rad, Philadelphia, USA).
2.9.1. Principles of the multicytokine bead based assay
The principle behind the bead based ELISA assay are similar to the sandwich ELISA described in section 2.6.1. An antibody specific to a desired cytokine, chemokine or growth factor is covalently coupled to a uniquely dyed bead. These beads are allowed to react with the cell supernatant and bind the release target biomolecules. They are washed and a 53
biotinylated detection antibody specific to an epitope different from the captured antibody is added and allowed to bind. The beads are again washed and a streptavidin-phycoerythrin reporter complex is added and allowed to bind to the biotinylated detection antibodies, sandwiched to the dyed bead where a released biomolecule had bound. The more biomolecules that bind from the cell supernatant, the more biotinylated detection antibody is able to bind to the bead, so the greater the intensity of fluorescence. This allows quantification in comparison to a standard curve. The correct biomolecule is identified by the uniquely dyed bead it is coupled to. Using this approach up to 100 unique biomolecules can be quantified in a single assay, with less incubation time and a larger dynamic range of detection concentrations (due to the much increased surface area of the beads compared to a flat surface).
2.9.2. Protocol for THP-1 multicytokine assay
The THP-1 cells were plated according to section 2.3.3.2 (1x105/well, 48 h PMA, 24 h rest) and compounds added according to section 2.3.4 (0.1% DMSO). The THP-1 cells were stimulated with 1μg/ml LPS and incubated for 6 h before the supernatant was harvested and stored (-80oC) for analysis using the bead based assay. Due to the high cost of the kit the supernatant was assayed for TNF-α content (according to the protocol outlined in section 2.6) prior to being tested in the multicytokine assays, to ensure the cytokine levels were correct.
Cytotoxicity was then assessed on the remaining cells by MTT as described in section 2.4.2.
The 17 and 27 Bio-plex assays were performed according to the manufacturer’s protocol. The supernatant (50 μl) was used without dilution. There were no alterations made to the standard curve or instrumental parameters. [144]
54
2.10. Statistical data analysis
All data is reported as mean ± standard deviation, with the standard error of the mean displayed in figure error bars. The in vitro experiments were performed in triplicates and the entire experiments repeated on three or more separate days, 3 repeat experiments is expressed as n=9 in the dose response curves figures. For the multicytokine assays they were only performed once and in duplicate, due to the expense of the assay, this is expressed as n=2 in the dose response curves. The dose response data were fitted with a log (inhibitor) vs. normalised response with variable slope model using Prism 5 for Mac OSX (GraphPad
Software, La Jolla, CA). IC50 values were calculated from the fitted curves. The curves were constrained to a minimum of 0 and a maximum of 100 unless this did not fit the experimental results. The uncertainly in the IC50 values is expressed as the 95 % confidence interval (CI).
55
Chapter 3
______3. Identification of the major active component of
A. paniculata
56
3.1. Introduction
3.1.1. Complexity of characterising herbal medicine
Herbal medicine (HM) refers to medicinal products containing exclusively herbal materials or herbal drug preparations [145]. HMs can be composed of a single herb or a combination of herbs or added non-herbal material. In contrast to chemically defined drugs, HMs typically contain complex mixtures of chemicals that may have active, synergistic, complementary, antagonistic or even toxic effects. Even single herb HMs contain complex mixtures of chemicals, for example, A. paniculata has been reported to contain 55 ent-ladbane diterpenoids, 30 flavonoids, 8 quinic acids, 4 xanthones and 5 rare noriridoids [43]. HMs’ active components are secondary plant metabolites which are compounds, that while not essential for plant growth, are thought to play a role in plant defence [146]. They are the component of the plant typically extracted and used as medicines, flavourings and illicit drugs [147]. The amount of secondary metabolites in HMs can vary depending on growing conditions (soil, climate), harvest season, and post-harvest treatment [148, 149]. Furthermore, once ingested, some secondary plant metabolites may be metabolised into active compounds, a process termed as bioactivation [150]. With the large number of compounds present and the possibility of interactions, variable compositions and the possibility of bioactivation, it is challenging to assess and ensure the efficacy and consistency of HMs.
3.1.2. Standardisation as a means to control variation
As the scientific understanding and evaluation of HM increases, it has become evident that variation in the putative bioactive components of HMs is often significant and requires quality control (QC) to improve safety and efficacy [44-47]. Standardisation of the herbal extract is a method to address this variation. Extract standardisation is applicable because
57
herbs are typically consumed as an extract (often aqueous, sometimes aqueous ethanol) rather than being consumed directly. Standardisation is the process whereby an extract is prepared in a way that ensures a consistent concentration of one or more analytes (usually a bioactive compound found in high concentration) regardless of variation in the concentration of these substances in the raw material from which the extract is prepared. While the extract may be standardised to contain a predetermined concentration of a particular compound(s), other compounds in the herb which may or may not have an impact on its efficacy are ignored, [47,
151] often for reasons of practicality. In the case of A. paniculata, the extract is generally standardised to contain 10% or 30% w/w andrographolide which is the major secondary metabolite [42]. Prior to extraction, the correct identification of the starting material is required.
3.1.3. Importance of correct plant identification
To ensure that the correct plant is used to prepare the extract, the starting material needs to be authenticated by a systematic botanist. Due to their established traditional use, many herbs have different names based on locale – for example, A. paniculata is known as: Chuan Xin
Lian (Chinese), Kirayat (Hindi), Green chirayta, Creat, King of bitters, India echinacea, Se- ga-gyi (Burmese) and Sambiroto (Indonesian), to name a few. Due to the large number of local names, taxonomical classification is the preferential method of nomenclature. The taxonomical classification for A. paniculata is given in Table 4.
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Table 4: Taxonomical classification of A. paniculata [152] Kingdom: Plantae Sub kingdom: Tracheobionta Super division: Spermatophyta Division: Angiosperma Class: Dicotyledonae Sub class: Gamopetalae Series: Bicarpellatae Order: Personales Tribe: Justicieae Family: Acanthaceae Genus: Andrographis Species: paniculata
3.1.4. Physical description of A. paniculata
A. paniculata is an annual, branched, herbaceous plant which grows to a height of 30-110 cm.
The leaves are green and taper from a rounded base toward an apex. The leaves are hairless and typically 2-12 cm long by 1-3 cm wide. The plant has small white flowers 2.5-10 cm long with pink-purple spots and slender stems. Error! Reference source not found. and
Figure 17 show a sketch and photograph, respectively, of A. paniculata. The leaves and stems are the most commonly used parts of the plant for medicinal purposes [152].
Figure 17: A photograph of A. paniculata from the Botanical Gardens, Berlin.[153]
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3.1.5. Traditional uses of A. paniculata
A. paniculata is used in herbal formulations in traditional Indian, Chinese, Thai, Malayan,
Japanese and Scandanavian medicines. Its use has been described in various pharmacopeia and traditional texts as a treatment for inflammatory conditions including dysentery
(intestinal inflammation), enteritis (intestinal inflammation), pyelonephritis (kidney inflammation), respiratory infection, fever, pneumonia (lung inflammation), coughs, bronchitis (lung inflammation), excess mucus, sinusitis, common colds, fever, pharyngotonsillitis (inflammation of tonsils and pharynx), tonsillitis, pharyngitis, laryngitis
(inflammation of the larynx), swelling, wounds, ulcers, skin diseases, eczema (inflamed skin), pruritus (itching skin), headaches, encephalitis B (viral inflammation of the brain), diabetes, and colitis (inflamed colon) [39-43]. A. paniculata achieved official status as a medicine in the Indian pharmacopeia in 1998 [154]. Most of its uses and pharmacological activity can be attributed to its major chemical component, andrographolide [155], however, there are many other compounds that have been identified in A. paniculata.
3.1.6. Chemical substances in A. paniculata
A. paniculata has been reported to contain 55 ent-ladbane diterpenoids, 30 flavonoids, 8 quinic acids, 4 xanthones and 5 rare noriridoids [43]. Some of the major diterpenoids present are andrographolide, neoandrographolide, andropanoside, deoxyandrographolide, 14-deoxy-
11,12-didehydroandrographolide and dehydroandrographoside. A. paniculata has also been reported to contain quinic acids, for example, chlorogenic acid and flavonoids including wogonoside, isoquercetin (glucoside) and apigenin (flavone) [152, 155-158]. The molecular structures of these major components are shown in Figure 18.
60
Andrographolide Andropanoside O O
O O OH
HO HO H OH GluO Neoandrographolide Deoxyandrographolide O O
O O
HO
GluO HO 14-deoxy-11,12-didehydroandrographolide Iso quercetin O OH OH O
HO O
O HO O OH O
HO HO OH HO OH Apigenin Wogonoside OH
HO O
H3C O O
O OH O HO O O OH OH O HO OH Chlorogenic acid OH
HO O O O OH
HO
HO OH Figure 18: The structure of some compounds identified in A. paniculata.
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Many of the compounds present in A. paniculata show anti-inflammatory activity. However, it remains unclear which compounds or combination of compounds contribute to the herb’s anti-inflammatory activity.
3.1.7. A. paniculata’s inhibition of prostaglandin and nitric oxide production
Prostaglandins play a major role in inflammation. Inhibition of the COX enzyme that produces prostaglandins is the current drug target for NSAIDs, the main modern treatment for inflammation. NO is similarly produced by an enzyme (NOS) and can be affected at a transcriptional or protein level. NO is also implicated in inflammation as discussed in section
1.3.4. Table 5 summarises research related to A. paniculata and its components on the modulation of inflammatory enzymes COX and NOS and their products.
Table 5: The effects of A. paniculata and andrographolide on inflammatory enzymes. Reference Method Effect Comment 2004 Wang BV-2 microglia, Andrographolide reduced Pre-treatment of the cells [159] LPS stimulated. COX and iNOS activity, with andrographolide as determined by ELISA significantly and dose (PGE2 levels) and Greiss dependently attenuated reagent (NO the LPS-induced concentration) microglial activation. respectively. Reactive oxygen species (ROS), which may be a Andrographolide secondary activator, were attenuated iNOS and also reduced. COX-2 protein In the presence of expression. andrographolide, COX-2 showed increased protein Andrographolide did not degradation and reduced significantly affect iNOS activity. There was an mRNA expression but effect observed in slightly attenuated COX-2 suppressing protein mRNA (30%) and TNF-α expression. mRNA (20%). 2005 Hidalgo Human Andrographolide reduced The reduced expression
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[160] promyelocytic COX-2 expression. of COX-2 could be leukemia cells attributed to reduced NF- (HL-60) cells Andrographolide reduced κB DNA binding activity. differentiated to the DNA binding of NF- neutrophils. κB. 2007 Liu [161] Murine Neoandrographolide This study uses a similar macrophages inhibited TNF-α and NO inflammation model to RAW264.7, LPS production. our study. stimulated. 2009 Boa [162] BALB/c mice Andrographolide reduced The reduced expression sensitised and NO levels and release of of iNOS could be challenged with IL-13 in the lungs. attributed to reduced NF- ovalbumin κB activity. (OVA) develope Andrographolide reduced d airway expression of iNOS by inflammation. reducing the transcription factor NF-κB’s activity. 2010 Chan Cerebral Andrographolide reduced The results indicate [163] ischaemia rat the production of TNF-α, reduced NF-κB activation model with IL-1β and PGE2. in activated microglial. permanent NF-κB inhibition could middle cerebral The translocation of the account for the reduced artery occlusion. NF-κB subunit, p65 from production of the cytosol to the nucleus was cytokines and eicosanoid. suppressed by It may not be the only andrographolide. effect. 2009 Chao Murine The A. paniculata acetate The suppression of NO [164] macrophages fraction, caused decreased and PGE2 release may be RAW 264.7, NF-κB linked (by caused by reduced NF-κB LPS/IFN-γ transfections) luciferase activity. stimulated. production and the secretion of NO and PGE2 The acetate extract was Mouse in the RAW264.7 cells. shown to contain splenocytes, Con andrographolide and there A-activated. The fractions did not were at least two other affect IFN-γ linked (by HPLC peaks but no other transfection) luciferase compounds were production or IFN-γ identified and none production in Con A quantified. activated mouse splenocytes. 2010 J774A.1 murine A. paniculata reduced the The reduced production Chandrasekaran macrophages, release of pro- of NO may be due to [165] LPS stimulated. inflammatory (NO, IL-1 inhibition of iNOS. The beta and IL-6), reduced production of Differentiated inflammatory (PGE2 and PGE2 and TXB2 showed HL-60 thromboxane B2 (TXB2)) the inhibition of COX-1 promyelocytic and allergic (LTB4) and/or 2. leukemic cells, mediators. calcimycin
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induced. No inhibition observed against histamine release. Histamine measured in RBL-2H3 rat basophilic leukemia cells. 2010 Levita Energy Andrographolide to COX- Both andrographolide and [166] minimisation was 2 binding was favoured in neoandrographolide simulated using a computerised binding showed selective MMFF94x. modelling. inhibition of COX-2. Their selectivity is due to Stochastic The docking simulation their specific interaction conformational indicates that both with Arg 513 in the search using andrographolide and binding pocket of COX-2, molecular neoandrographolide are which is also shown by mechanics able to be located in the SC-558, a COX-2 method of MOE- COX-2’s binding pocket selective inhibitor. 2007.09.02. but not in the COX-1’s. Andrographolide had a similar binding energy Human fibroblast The production of PGE2 requirement to SC-558 (a cell culture LPS was reduced in LPS known COX-2 inhibitor), stimulated. stimulated fibroblast cells. of -11.8 and -10.8 kcal/mol respectively.
Andrographolide shown to potentially directly inhibit COX-2. 2010 Human whole COX-1/2 activity in Andrographolide was Parichatikanon blood, LPS human blood was down- most active out of d [167] stimulated regulated by dehydro- and neo- (platelet andrographolide, andrographolide, the two suspension for neoandrographolide and next most abundant COX-1). 14-deoxy-11,12- diterpenes in A. didehydroandrographolide paniculata. . Andrographolide was the best COX-1 and COX-2 inhibitor, 14-deoxy- 11,12- didehydroandrographolid e was similar but slightly less active. Neoandrographolide showed little COX-1 activity but inhibited COX-2 as strongly as andrographolide and celecoxib (selective COX-2 inhibitor). This study showed that
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andrographolide is not the only active component of the herb. 2011 J774A.1 murine Andrographolide, The study shows that Chandrasekaran macrophage cell isoandrographolide, andrographolide and [168] line, LPS skullcapflavone-I and 7- other components of A. stimulated. O-methylwogonin paniculata, exhibited significant dose- isoandrographolide, dependent inhibition of skullcapflavone-I and 7- NO and PGE2 release. O-methylwogonin have anti-inflammatory activity against different HL-60 human Significant reduction in pathways. promyelocytic TXB2 levels was leukaemia cell displayed by Some of the effect may be line, A23187 andrographolide, associated with inhibition stimulated. isoandrographolide and of NF-κB. However, the skullcapflavone-I. inhibition of IL-1 by 7-O- methylwogonin but not of IL-6 and the inhibition of IL-6 by skullcapflavone-I but not IL-1 would not be explained by this theory. There may be a complementary effect present.
7-O-methylwogonin inhibited PGE2 but not TXB2 which are both part of the same pathway from COX enzymes. The inhibition may be due to inhibition at the PG synthases level and not the COX level.
Skullcapflavone-I showed inhibition of PGE2, TXB2 and LTB4 which may be due to its inhibition of phospholipaseA2 that is responsible for the synthesis of arachidonic acid the median of COX and lipoxygenase 5 (Figure 1).
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A. paniculata has been shown to exhibit inhibitory effects on COX-1, COX-2 and iNOS at both a transcriptional and protein level. Andrographolide is the most studied component of the herb and has shown this activity. Table 6 summarises the reported effects of andrographolide on the activity of COX and NOS enzymes, at a transcription and protein level.
Table 6: Andrographolide’s effect on NO and PGE2 production 2004 Wang Microglia, LPS stimulation NO levels↓ PGE2 levels ↓ iNOS protein ↓ COX-2 protein ↓ iNOS mRNA ↔ COX-2 mRNA ↓ 2005 Hidalgo Murine Neutrophils COX-2 protein ↓ NF-κB activity ↓ 2009 Boa Mice OVA NO levels ↓ NO expression ↓ NF-κB activity ↓ 2009 Chao Macrophages NO levels ↓ PGE2 levels ↓ NF-κB activity ↓ 2010 Levita Computer model COX-2 biding 2010 Parichatikan Human whole blood COX-1 activity ↓ COX-2 activity ↓ 2011 Chandrasekaran Murine Macrophages NO levels ↓ PGE2 levels ↓
It is clear from the literature that andrographolide exhibits an inhibitory effect on COX-1,
COX-2 and iNOS at both a transcriptional and protein level. Neoandrographolide isoandrographolide, skullcapflavone-I and 7-O-methylwogonin have been shown to affect these enzymes at some level. Other components of A. paniculata may have a similar effect but the quantification of their contribution is lacking. Although other components of the herb have been shown to be effective at inhibiting COX and NOS function, it is unclear if in a
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typical herbal extract these other components are present in high enough concentrations to elicit a measurable effect. Whilst inflammatory enzymes play a role in the inflammatory response, they are only one part of a complex system, cytokines are another major anti- inflammatory target.
3.1.8. A. paniculata’s inhibition of inflammatory cytokines
Cytokines are cell signaling molecules that play a major role in inflammation, acting on cells to alter their functionality. A. paniculata and its components have been shown to inhibit a variety of cytokines in many models, as summarised in Table 7.
Table 7: Effects of andrographolide and A. paniculata on release of pro-anti-inflammatory cytokines and chemokines. Reference Method Cytokine effect Comments 2005 Burgos Murine T cells, Andrographolide Inhibition of extracellular- [169] Concanavaline A inhibited IFN-γ and IL- signal-regulated kinases (ConA) 2. (ERK) 1/2 by reduced stimulated. Deoxyandrographolide phosphorylation was only inhibited IFN-γ attributed to the inhibition and showed protection of IFN-γ and IL-2. from PMA and Andrographolide hydrocortisone induced suppressed T-cell immune apoptosis in thymocyte response and potentially cells (T cell precursor). macrophage stimulation, deoxyandrographolide showed a similar effect. The other diterpenoids in the herb may also be active and contribute to these effects. 2004 Wang BV-2 Microglia, Andrographolide Pre and post treatment [159] LPS stimulated inhibited TNF-α, ROS, were both effective. The NO and PGE2 pre- treatment effect was production. attributed to the reduction in pro-inflammatory Attenuated iNOS and mediators (TNF-α) by COX-2 protein possible reduction in LPS translation and/or induced ROS. The post stability (western blot). treatment of
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andrographolide attenuated No effect was found on iNOS and COX-2 stability. transcription (PCR Andrographolide was mRNA). shown to inhibit inflammation by at least two related but separate mechanisms. 2006 Qin [170] Murine Andrographolide Andrographolide inhibited macrophages, decreased TNF-α, IL- the activation of ERK1/2, LPS stimulated. 12a and IL-12b at MAPK, which could mRNA level, and reduce TNF-α transcription reduced the production but not IL-12a/b. of TNF-α and IL-12p70 Suppression of IL-12a/b proteins in a transcription must be due concentration to another effect of dependent manner. andrographolide. Andrographolide was shown not to affect the activation of JNK, p38 or NF-κB.
2006 Sheeja Swiss albino mice A. paniculata and CTX metabolite acrolein [171] against andrographolide induced ROS production. cyclophosphamide inhibited TNF-α Glutathione (GSH) (body (CTX)– production. antioxidant) was shown to induced urothelial be depleted in CTX treated (urinary) toxicity. IL-2 and IFN-γ levels mice. Andrographolide and during CTX treatment A. paniculata preserved were elevated. normal bladder morphology and GSH levels. It is not clear whether the anti- inflammatory effect was due to antioxidant activity reducing inflammatory response stimulation (TNF-α) or if it reduced inflammation and prevented further free radical production (NO). CTX in therapeutic doses suppressed immune response, increased IL-2 and IFN-γ shows andrographolide may attenuate this effect (mechanisms unknown). 2007 Liu [161] Murine Neoandrographolide This study uses a similar Macrophage inhibited TNF-α and inflammation model to our sRAW264.7, LPS NO production. study.
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stimulated. 2007 Li [172] Murine Andrographolide This study synthesised and Macrophages inhibited TNF-α and tested a number of J774A.1, LPS IL-6 production. andrographolide stimulated. derivatives. Two isoandrographolide derivatives showed up to double the inhibition of andrographolide in TNF-α suppression but most were lower. Five andrographolide derivatives showed greater inhibition of IL-6. The study indicated that many of the diterpenes in A. paniculata may have an anti-inflammatory effect. 2007 Sheeja B16F-10 A. paniculata and The herbal extract [173] melanoma cell andrographolide containing both flavonoids line- in mice. increased IL-2 and terpenoids showed decreased IL-1β, IL-6, similar activity to the pure TNF-α and GM-CSF andrographolide at 20 levels. times the dose, but the andrographolide in the extract was not quantified. The reduction over 24 h was less than half but over 9 days there was up to 6 times less TNF-α. NO production was inhibited and may have contributed to reduced release of cytokines. Results show that although there is a significant initial effect, the longer term effect is even greater. 2008 Liu [174] L-929 Andrograpanin Andrograpanin is a macrophage cells, (diterpene in A. diterpene in A. paniculata. LPS stimulated. paniculata) reduced It is naturally much less NO, TNF-α, IL-6 and abundant than IL-12p70 production. andrographolide, the focus This was determined by of most research. However, western blotting and this study illustrated that reverse transcription andrograpanin has anti- polymerase chain inflammatory activity by reaction, showing reduced phosphorylation of reduced gene p38 MAPKs, which is expression levels. different to the mechanism
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of andrographolide Andrograpanin also reported by Burgos et. al caused down regulation [169]. of LPS induced Other diterpenoids in A. phosphorylation of p38 paniculata may contribute MAPKs. However, to the anti-inflammatory phosphorylation of effect by the same ERK 1/2, SAPK/ JNK mechanisms as MAPKs and andrographolide or degradation of nuclear complementary factor of kappa light mechanisms as with polypeptide gene andrograpanin. It is unclear enhancer in B-cells if andrograpanin is in high inhibitor, alpha (IκBα) enough concentration in the (NF-κB activator) were herb to contribute to this not affected by effect when A. paniculata andrograpanin. is administered. 2009 Abu- Mouse peritoneal Andrographolide Andrographolide was as Ghefreh [175] macrophages and suppressed the release effective as the steroid drug in vitro mice with of TNF-α and GM-CSF – dexamethasone, and like OVA airway both in vivo and in steroids, it appears to act inflammation. vitro. primarily by inhibiting the expression of mRNA for Andrographolide inflammatory cytokines. suppressed LPS- This effect may be due to induced expression of reduced NF-κB activity. mRNA for the two Anti-inflammatory effects cytokines. were conserved in vivo, showing the effect is The accumulation of independent of whole lymphocytes and immune system eosinophils in vitro was stimulation, although this abolished. may have complementary effects in vitro. 2009 Boa [162] Balb/c mice with Andrographolide It was proposed that the OVA airway reduced IL-4, IL-5, inhibition of NF-κB, was a inflammation. IL-13, eotaxin and result of reduced eosinophil count, phosphorylation activating TNF-α induced despite increased IFN-γ inhibitory kB kinase-b activation in in OVA inflamed mice. (IkBKb) (western blott). normal human bronchial Andrographolide The reduced eosinphil epithelial cells. blocked TNF-α, count may have been induced up regulation caused by the reduced of IL-6, IL-8 and CCL5 release of cytokines due to mRNA expression. reduced NF-κB activity or may be due to another effect of andrographolide. The reduced number of eosinophils may have
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significantly reduced all inflammatory indicators at the site of inflammation. 2010 Chan Cerebral Andrographolide The results indicated [163] ischaemia rat reduced the production reduced NF-κB activation model with of TNF-α, IL-1β and in activated microglial. NF- permanent middle PGE2. κB inhibition could account cerebral artery for the reduced production occlusion. Suppressed the of the cytokines and translocation of the NF- eicosanoid. It may not be κB subunit, p65 from the only effect. cytosol to nucleus. 2010 Chao RAW 264.7 cells, Andrographolide and Several other compounds [176] LPS/IFN-γ components of A. in the herb (flavonoids, stimulated. paniculata reduced the phytosterols and production of TNF-α, diterpenes) were more IL-6 and macrophage active than andrographolide inflammatory protein-2. and 4 derivatives were up to 3 times more active. Andrographolide and This suggests that the anti- components of A. inflammatory effect by paniculata inhibited the reduced NF-κB activity is transcriptional activity caused by many of NF-κB. components of the herb. It is unclear if the active compounds are present in high enough concentration to contribute to the activity of A. paniculata.
The increased activity of the derivatives also highlights the potential for drug development from andrographolide. 2010 Human whole Andrographolide Down regulation was Parichatikanond blood, LPS reduced the production observed in nearly all 76 [167] stimulated of TNF-α, IL-6, IL-1β gene expressions measured. (platelet and IL-10. Most were minor (<1.2 suspension for fold) and could be COX-1). Andrographolide attributed to caused the down inconsistencies in blood regulation of cytokines samples. However, and cytokine receptors andrographolide down (TNFSF14, TNF, regulation of NF-κB (2 TNFRSF6, and IL1A), fold) could cause some of chemokines (MCP-2 the observed effects. and CXCL11), JAK/STAT signaling Andrographolide down (JAK3 and STAT5A), regulated
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TLRs family (TLR4 TNF ligands and their and TLR8) and NF-κB. receptor superfamilies up to 12 fold. This is likely due to a separate effect to that on NF-κB. 2010 Wang Human peripheral Andrographolide In this study [177] blood reduced expression and andrographolide showed mononuclear cells production of IL-18, IL- regulation at a (PBMC), LPS 1β and IL-1Rα. IL- transcriptional level. This stimulated. 18BP increased which could be due to modulation is an IL-18 inhibitor. of NF-κB or another transcription factor. 2010 Sheeja Metastatic tumour A. paniculata and The mechanism was not [178] bearing animals. andrographolide explored. Andrographolide reduced production of and A. paniculata extracts IL-1, IL-6, GM-CSF produced similar effect on and TNF-α. inflammatory mediators, but the extract was not phytochemically characterised making comparison difficult. 2011 J774A.1 murine Andrographolide, This study indicated that Chandrasekaran macrophage cell isoandrographolide and some of these [168] line, LPS 7-O-methylwogonin phytoconstituents exhibit stimulated. inhibited IL-1. potent anti-inflammatory/anti- Andrographolide, allergic effects by isoandrographolide and modulating different skullcapflavone-I inflammatory/allergic inhibited IL-6. mediators. Many of the components in A. paniculata are active and may contribute to the anti-inflammatory effect.
A. paniculata has been shown to suppress the release of a number of different inflammatory cytokines. Cytokines are potent signallers and cells are very sensitive to cytokine levels. The concentration of the cytokine IL-1 that induces gene expression and synthesis of COX-2 is 10 pM, so that minute changes in cytokine levels can cause large responses [79]. Most of the research has focused on a small number of cytokines. Comparing different models and stimulations makes analysing results difficult. Many cytokines are interrelated, for example
Burges et al. [169] shows andrographolide reduced IFN-γ release by T cells. IL-12p70
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induces Th1 response which causes the release of IFN-γ from T cells. Qin et al. [170] shows that IL-12p70 is inhibited by andrographolide which may contribute to the suppression in
IFN-γ release - in contrast, Sheeja et al. [173] showed in cancer models that IFN-γ levels were increased by andrographolide. Andrographolide was reported to reduce the levels of many cytokines in a variety of models. A summary of andrographolide’s effects is shown in
Table 8.
Table 8: Andrographolide’s effect on inflammatory cytokines and chemokines. 2005 Burgos Murine T cells IFN-γ levels ↓ IL-2 levels ↓ 2004 Wang Microglia TNF-α production ↓ 2006 Qin Murine macrophages TNF-α mRNA ↓ IL-12a mRNA ↓ IL-12b mRNA ↓ TNF-α production ↓ IL-12p70 production ↓ 2006 Sheeja Mice, CTX TNF-α levels ↓ IL-2 levels ↑ IFN-γ levels ↑ 2007 Li Murine macrophages TNF-α production ↓ IL-6 production ↓ 2007 Sheeja Murine melanoma IL-2 levels ↑ IL-1β levels ↓ IL-6 levels ↓ TNF-α levels ↓ GM-CSF levels ↓ 2009 Abu-Ghefreh Murine macrophages TNF-α release ↓ GM-CSF release ↓ TNF-α mRNA ↓ GM-CSF mRNA ↓ Mice, OVA TNF-α release ↓ GM-CSF release ↓ 2009 Boa Mice, OVA IL-4 levels ↓ IL-5 leves ↓ IL-13 levels ↓ Eotaxin levels ↓ IFN-γ levels ↑ Bronchial epithelial cells IL-6 mRNA ↓ IL-8 mRNA ↓ RANTES mRNA ↓ 2010 Chan Rat pMCAO TNF-α levels ↓
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IL-1β levels ↓ 2010 Chao Murine macrophages TNF-receptor release ↓ IL-6 release ↓ MIP-2 release ↓ 2010 Parichatikanond Human whole blood TNF-α production ↓ IL-6 production ↓ IL-1β production ↓ IL-10 production ↓ 2010 Wang Human mononuclear cells IL-18 expression ↓ IL-18 production ↓ IL-18BP expression ↓ IL-18BP production ↓ IL-1β production ↓ IL-1β expression ↓ IL-1Rα expression ↓ IL-1Rα production ↓ 2010 Sheeja Metastatic tumour bearing IL-1 levels ↓ animals IL-6 levels ↓ GM-CSF levels ↓ TNF-α levels ↓ 2011 Chandrasekaran Murine macrophages IL-1 release ↓ IL-6 release ↓
Andrographolide clearly plays a major role in the reduction of cytokine levels, both in vitro and in vivo. Neoandrographolide and andrograpin were shown to be active at inhibiting cytokine release, however, it remains unknown if they or other components contribute to the activity of A. paniculata.
3.1.9. NF-κB activity of andrographolide
NF-κB is the controlling transcription factor for mediators of inflammation. The reduction in the expression of many cytokines and reduced expression of iNOS, COX-1 and 2 may occur due to NF-κB inhibition. Andrographolide is shown to inhibit NF-κB activity through a number of mechanisms as shown in Table 9.
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Table 9: The effects of A. paniculata and andrographolide on NF-κB. Reference Method Comments 2004 Xia [179] Human embryonic NF-κB activity was reduced. Andrographolide kidney 293 cells and formed a covalent adduct with reduced cysteine promyeloid cells HL-60, 62 of p50, supported by KO study on p50 C62S cells transfected with an mutants and mass spectroscopy showing NF-κB-luciferase increase in mass of the p50 subunit. reporter. Andrographolide was shown to bind to p50 as a mechanism of inhibiting NF-κB. 2005 Hidalgo HL-60 cells Andrographolide inhibited the NF-κB- [180] differentiated to luciferase production induced by PAF. It did neutrophils, platelet not reduce phosphorylation of p38 MAPK or activation factor (PAF) ERK1/2 and did not change IκBα degradation. stimulated. Reduced NF-κB DNA binding was inferred Action of from lack of degradation of IκBα, and directly andrographolide on NF- observed in EMSA. κB determined by IκBα in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 2006 Female C57BL/6 mice Andrographolide NF-κB-blockade was Iruretagoyena bone marrow dendritic observed. It can benefit by interfering with [181] cells transfected with an unwanted T cell responses by inhibiting NF-κB-luciferase maturation. reporter. 2007 Wang Isolated primary murine Andrographolide attenuated the up regulation [182, 183] pulmonary vein of NF-κB target genes detected within the endothelial cells and p50 injured arterial walls. Genetic deletions of p50 null mice, with carotid caused the same effect. No effect on -50p artery restriction. mutant with andrographolide was observed so no other complementary effect in this pathway assumed. However, this only confirms p50 is necessary for transcription. In this model, inhibition of NF-κB by andrographolide binding to p50 is supported. 2009 Bao [184] BALB/c mice sensitised In normal human bronchial epithelial cells, and challenged with andrographolide blocked TNF-α induced OVA developed airway phosphorylation of IκBα kinase-b, and inflammation. downstream IκBα degradation, p65 subunit of Bronchoalveolar lavage NF-κB phosphorylation, and p65 nuclear fluid was assessed for translocation and DNA-binding activity. total and differential cell Similarly, andrographolide blocked p65 nuclear counts, and cytokine and translocation and DNA-binding activity in the chemokine levels. nuclear extracts from lung tissues of OVA- challenged mice. TNF-α induced
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activation in normal Inhibiting the NF-κB pathway was proposed to human bronchial occur at the level of IκBα kinase-b activation epithelial cells. by reduced phosphorylation (western blott).
2009 Li [185] p50 null mice and C57 Andrographolide attenuated PAF, E-selectin murine macrophages (cell adhesion), and vascular cell adhesion situated with PMA or protein (Vcam-1) (cell adhesion) activity in TNF-α stimulated endothelial cells and human monocytic THP-1 monocytes/macrophages similar to deletion of cells transfected with an p50. Results of the electrophoretic mobility NF-κB-luciferase shift assay and chromatin immunoprecipitation reporter and stimulated demonstrated the direct interaction of the with TNF-α. p50/p65 heterodimer with the NF-κB site interrupted by andrographolide. 2010 Chang Rat model with pMCAO was found to induce activation of [163] permanent middle microglia and TNF-α, IL-1β and PGE2, in the cerebral artery occlusion ischaemic brain areas. Andrographolide (pMCAO). significantly attenuated or abolished these effects. In addition, andrographolide suppressed the translocation of p65 from cytosol to nucleus, indicating reduced NF-κB activation. 2010 Chao Murine macrophages Andrographolide was shown to inhibit NF-κB. [176] RAW 264.7, LPS/IFN-γ Andrographolide was part of the most active stimulated. fractions, other fractions showed lesser activity. Andrographolide was one of the less active NF-κB-promoted compounds in its fraction of flavonoids, luciferase reporter was phytosterols and other diterpenes. Greater used to assay the activity activity of andrographolide was achieved by of NF-κB hydrogenation, oxidation, or acetylation transactivation. thereby showing the potential of andrographolide as a lead. This study shows that other components of the herb contribute to NF-κB inhibition. Andrographolide may not be the major contributor to NF-κB activity. 2010 Levita Energy minimisation Andrographolide fulfils Lipinkis rule of 5 as a [166] was simulated using drug and has favourable van der Wall’s and MMFF94x software. electrostatic interactions with the proposed binding site on the p50 subunit of NF-κB. Human fibroblast cell Scoring values of binding was 10 kcal/mol culture, LPS stimulated. similar in binding strength and mechanism to gallic acid and tricarboxylic acid (12 kcal/mol). This study supports andrographolide’s inhibition of NF-κB by binding to the p50 subunit. 2010 Human blood levels of Andrographolide showed the most inhibition of Parichatikanond mRNA measured using COX-1 and 2 out of neo- and dehydro- [167] human cDNA andrographolide. microarrays, stimulated with LPS. Andrographolide attenuated the expression of
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TLRs leading to modulation of inflammatory responses through the inhibition of NF-κB. Andrographolide affected many transcription factors other than NF-κB which have been shown to participate in the induction of chemokine gene transcription by cytokines and engagement of TLRs. Most of the genes in the micro assay were slightly down regulated. This could be due to inconsistencies in blood samples. NK-kB was 2 fold down regulated, more than other transcription factors. 2010 Ren [186] Human lung Andrographolide inhibited TNF-α induced adenocarcinoma (cancer) phosphorylation of IKκβ in A549 cells, cell line A549, reduced phosphorylation of IκBα, blocked the stimulated with TNF-α subsequent degradation of IκBα, and had no to study the expression effect on the expression of IKκβ. of NF-κB signaling Andrographolide decreased the DNA binding proteins by western activity of NF-κB p65 in the nucleus of A549 blotting and NF-κB cells, inhibiting their growth. This may have DNA binding activity been due to the inhibition of activation or using ELISA. previously shown p50 binding. 2011 Hsieh Rats vascular smooth Pre-treatment with andrographolide decreased [187] muscle cells, LPS/IFN-γ iNOS and MMP-9 expressions and markedly stimulated. impaired p65 translocation from cytosol to nucleus in cells exposed to LPS/IFN-γ.
Andrographolide inhibited p65 nuclear translocation, DNA binding activity, p65 Ser536 phosphorylation, and NF-κB reporter activity. However, IKκβ phosphorylation and downstream inhibitory IκBα phosphorylation and degradation were not altered by the presence of andrographolide. Andrographolide did not affect NF-κB regulators activation.
Andrographolide inhibited NF-κB by preventing p65 Ser536 phosphorylation. Andrographolide activates protein phosphatase 2 (PP2) that dephosphorylates Ser536, leading to NF-κB inactivation. This was supported by Okadaic acid (a selective inhibitor of PP2A) pre-treatment, which prevented dephosphorylation of p65 Ser536 leading to no inhibition of NF-κB. Additionally pPP2a siRNAs, which silenced PP2A-C, also attenuated andrographolide dephosphorylation of p65 Ser536. Andrographolide was also shown to induce PP2A activation inhibiting NF-κB.
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PP2 activation has been shown to inhibit NF- κB. (Li, S., Wang, L., Berman, M. A., Zhang, Y., and Dorf, M. E. (2006) Mol. Cell 24, 497– 509) Inhibition of PP2 was shown to attenuate andrographolide NF-κB suppression.
Other pathways have been shown to be regulated by PP2, for example, JNK-AP-1 cascade and p65 cascade. This may culminate in decreasing iNOS and MMP-9 expressions and other anti-inflammatory effects.
There is a large body of research (Table 9) supporting andrographolide’s activity on NF-κB.
There are three main mechanisms whereby activity has been shown, namely p50 binding, reduced phosphorylation of IκBα kinase-b and PP2 activation. Andrographolide’s effect on
NF-κB appears to be due to a number of possible interactions. Many compounds in A. paniculata are structurally similar and could also elicit an effect.
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3.2. Aims of the chapter 3
The aims of this chapter are to identify if andrographolide is solely accountable for the activity of the commercial A. paniculata commercial extract on:
The activity of inflammatory enzymes, iNOS and COX
The release of inflammatory cytokines
The activation of NF-κB
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3.3. Methods
3.3.1. Materials
3.3.1.1. Standards used in analysis
The standards and reagents used are from reputable sources and with claimed purity.
Apigenin (98%) and chlorogenic acid (99%) were purchased from Sigma (Sydney,
Australia). Andrographolide (98%), 14-deoxy-11,12-didehydroandrographolide (98%), wogonoside (98%), isoquercetin (98%) were purchased from Biopurify (Chengdu, China).
3.3.1.2. Source of the A. paniculata extract
The A. paniculata commercial extract (extract ratio 14:1) standardised to ≥30% andrographolide was donated by LIPA Pharmaceuticals Ltd (NSW, Australia). The sample was re-analysed by HPLC with photodiode array detection (PDA) for andrographolide content to confirm the manufacturer’s certificate of analysis. The extracts comply with the
Therapeutic Goods Administration’s guidelines for incorporation in herbal medicines manufactured in Australia. The plant material used to prepare the extract was sourced from and manufactured in southern India. The plant material was authenticated by a systematic botanist and extract traceability documents were provided by the manufacturer.
3.3.2. Method used to partition and prepare extracts
The standardised A. paniculata commercial extract was separated by solvent partitioning to produce an extract with increased diterpene concentration (non-polar fraction) and an extract free from andrographolide (polar fraction). The standardised A. paniculata commercial
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extract (40 g) was dissolved in chloroform (500 ml), it was then partitioned between water
(200 ml) and acetonitrile (200 ml). The mixture was shaken and sonicated for 10 min and left to settle. The top aqueous layer was removed and washed twice with chloroform 200 ml, to yield the “Polar fraction”. The original organic layer was washed twice with water/acetonitrile (50/50, 200 ml) to yield the “Non-polar fraction”. Both extracts were gravity filtered and evaporated to dryness by rotary evaporator. The dry extracts were then resuspended in minimal water and freeze dried to yield an extract powder.
3.3.3. Preparation of the mixed standard solution
A mixed standard solution containing compounds identified in A. paniculata was used to assess the performance of the partition system. The mixed standard solution contained andrographolide (0.73 mg/ml), apigenin (0.27 mg/ml), chlorogenic acid (0.83 mg/ml), 14- deoxy-11,12-didehydroandrographolide (0.21 mg/ml), wogonoside (0.22 mg/ml) and isoquercetin (5.6 mg/ml) in 50% aqueous acetonitrile. The mixed standards were prepared at concentrations that produced comparable peak sizes.
3.3.4. HPLC system
HPLC-PDA was used to profile the phytochemical composition of the A. paniculata extracts.
The HPLC-PDA analysis was performed on Shimadzu UFLC system (Shimadzu, Australia) comprising of a LC-30AD pumps, SIL-30ACHT auto sampler, SPD-M20A PDA detector and DGU-20A5 inline solvent degasser. The system was controlled using Class-VP 7.4SP4 software.
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3.3.5. HPLC analysis of extracts
This method was used to detect the minor components of the herb but not for quantification, therefore, the samples were run at high concentrations. The A. paniculata commercial extract
(30 mg/ml), Non-polar fraction (15 mg/ml) and Polar fraction (50 mg/ml) were dissolved by sonication in 50 % aqueous acetonitrile. The HPLC-PDA analysis of the extracts was performed using an Alltech Alltima (Alltech, Australia) reverse phase C18 column (46 × 150 mm I.D., 5 µm) with a Phenomenex (California, USA) Security C18 guard column (20 mm ×
4 mm, 5 μm).
HPLC-PDA profiles were generated by 5 µl injection of fractionated samples. The mobile phase consisted of 0.1 % (v/v) aqueous formic acid (mobile phase A) and 0.1 % (v/v) formic acid in acetonitrile (mobile phase B). The gradient program was 10% B for 10 min with a linear increase; to 50% B at 63 min, 70% B at 72 min and then 100% B (wash) for 8 min before equilibrating at the starting composition for 5 min. Mobile phase flow rate was maintained at 1 ml/min. The PDA was set to acquire absorbance data from 200 to 500 nm.
3.3.6. HPLC method used to quantify andrographolide
HPLC-PDA was used to quantify the andrographolide in the A. paniculata extract, non-polar fraction and polar fraction. The extracts (A. paniculata 1.5mg/ml, Non-polar fraction
0.8mg/ml and Polar fraction 1.7 mg/ml) were dissolved by sonication in 50% aqueous acetonitrile. HPLC analysis of the extracts was performed using an Alltech Alltima (Alltech
Australia) reverse phase C18 column (46 × 150 mm I.D., 5 µm) with a Phenomenex
(California, USA) Security C18 guard column (20 mm × 4 mm, 5 μm).
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HPLC-PDA profiles were generated by 10 µl injection of extracted samples. The mobile phase was isocratic 30% aqueous acetonitrile with 0.1% formic acid at 1 ml/min. The PDA
(200-500 nm) was recorded and 227 nm was used to quantify the andrographolide peak.
3.3.7. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells.
NO release was quantified by Griess reagent as described in section 2.5. Briefly the
RAW264.7 cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was removed (180 μl) and reacted with Griess reagent (100 μl) to calorimetrically quantify dissolved nitrates. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
3.3.8. Determination of TNF-α release in LPS and IFN-γ stimulated RAW264.7 cells.
TNF-α was quantified by ELISA as explained in section 2.6. Briefly the cells were seeded at
1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO
(final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A MTT solution (60 μl) was used to assess the viability of the remaining cells.
3.3.9. Determination of PGE2 release in LPS and IFN-γ stimulated RAW264.7 cells.
PGE2 release was quantified by competitive ELISA as described in section 2.7. Briefly the cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS
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and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was tested (180 μl) in the PGE2
ELISA. A MTT solution (60 μl) was used to assess the viability of the remaining cells.
3.3.10. Determination of 17 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay
The release of 17 cytokines was quantified by multiplex assay as described in section 2.9.
5 Briefly the cells were seeded at 1×10 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ. After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
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3.4. Results and discussion
The chemical characterisation of A. paniculata has been performed by a number of groups. It
is outside the scope of this project to repeat the work and identify all peaks present in the A.
paniculata commercial extract and the non-polar and polar fractions. Instead a mixture of
standards was composed to assess if known components of A. paniculata were detectable in
the commercial extract using the HPLC-PDA system and using mixed standards to determine
the family of compounds being divided by the solvent partitioning.
1400
λdetection = 254 nm Andrographolide
700 Absorbance (mAu)
0
0 20 40 60 80 Retention time (min)
Figure 19: HPLC chromatogram of A. paniculata commercial extract at 254 nm.
Figure 19 is a chromatogram of the A. paniculata commercial extract used in the study. It is
standardised to contain 30% w/w andrographolide, which is the largest peak (retention time
35.9 min) in the chromatogram with other compounds also present. To simplify the
determination of the compounds responsible for activity in the in vitro anti-inflammatory
testing, the complexity of the extract was reduced by separation into crude fractions by
solvent partitioning based on polarity.
85
Compounds have different absorption peaks (λmax) and molar absorptivities (ε) at different
wavelengths, depending on the chromophore in the molecule. As the A. paniculata
commercial extract contains different compounds with different structures, a wavelength that
detects most of the compounds was selected for visualisation.
200
300 Wavelength
400
0 20 40 60 80 Retention time (min)
Figure 20: Heat map contour chromatogram of A. paniculata commercial extract. The colours indicate the absorbance intensity with, red= 200+ mAu, yellow= 150 mAu, green= 100 mAu, Blue= 0 mAu, purple= -50 mAu, white> -50 mAu.
Figure 20 shows a contour heat map chromatogram of the A. paniculata commercial extract
where the colour from red (the hotter (red) the colour the higher the absorbance) to white
shows the relative absorbance as a function of retention time (X axis) and wavelength (Y
axis). The line drawn at 254 nm shows the wavelength selected for visualisation of the
chromatograms. λdetection = 254 nm gave a balance for detecting the most number of
compounds, whilst not showing the absorbance from the increasing % acetonitrile in the
mobile phase. As this chromatogram was run over a steep gradient, this is more of a factor
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than when it runs at shallow gradient where the normalised absorbance reduces the
acetonitrile visibility.
λdetection = 254 200 nm
Isoquercetin Wogonoside
Apigenin 100
Chlorogenic acid Andrographolide
Absorbance (mAu) 14-deoxy-11,12- didehydroandrographolide
0
0 20 40 60 80
Retention time (min) Figure 21: HPLC chromatogram of A. paniculata commercial extract (zoomed to 200 mAu) at 254 nm.
A. paniculata commercial extract (green), mixed standard (black). The overlay (black) was normalised to the absorbance of A. paniculata commercial extract, with an offset of 10 mAu.
Figure 21 is a chromatogram of the A. paniculata commercial extract overlaid with the mixed
standard. Comparison of the UV spectrum of the sample and standard peaks allow the
tentative identity confirmation for some peaks.
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20 20 10 10
8 8 15 15
6 6
10 10
mAu mAu
mAu mAu
4 4
5 5 2 2
0 0 0 0 250 275 300 325 350 375 400 425 450 250 275 300 325 350 375 400 425 450 nm nm The chlorogenic acid peak was Rt 14.7 min The isoquercetin peak was Rt 25.9 min in the in the mixed standard and 14.8 min A. mixed standard and 25.8 min A. paniculata paniculata extract (green). extract (green).
1500 1500
40 40
1250 1250
1000 1000 30 30
750 750
mAu mAu mAu mAu 20 20
500 500
10 10
250 250
0 0 0 0 250 275 300 325 350 375 400 425 450 250 275 300 325 350 375 400 425 450 nm nm The andrographolide peak Rt was 35.9 min The apigenin peak was Rt 41.5 min in the for both the mixed standard and A. mixed standard and 41.6 min A. paniculata paniculata extract (green). extract (green).
150 150
125 125
100 100 mAu mAu 75 75
50 50
25 25
0 0 250 275 300 325 350 375 400 425 450 nm The 14-deoxy-11,12- didehydroandrographolide peak Rt was 49.1 min for both the mixed standard and A. paniculata extract (green).
Figure 22: UV spectrum comparison between standards and peaks tentatively identified as corresponding peaks in A. paniculata commercial extract.
It can be seen in Figure 22 that the peaks with the matching retention times to the mixed standards have similar UV spectrum. The lower concentration of the compounds in the A.
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paniculata commercial extract make it more difficult to compare the UV spectrum as at low
absorbance the absorbance of the mobile phase dominates. In any case there is a good
retention time match between sample and standard peaks and there is a fair match between
their UV spectrums. There was no matching peak identified for wogonoside, despite it being
reported in A. paniculata, as it may be below the detection limit. The identification of the
small peaks is considered only tentative as some are close to detection limit.
3.4.1. Partition of the mixed standard
The mixed standard was subjected to the solvent partitioning described in section 3.3.2 to
assess its suitability for fractionating the A. paniculata commercial extract. Figure 23 shows
an overlay of the mixed standard and the aqueous partition of the mixed standard.
150 λdetection = 254 nm
Isoquercetin Wogonoside
Apigenin
14-deoxy-11,12- 75 Chlorogenic acid didehydroandrographolide
Andrographolide Absorbance (mAu)
0
0 15 30 45 60 Retention time (min)
Figure 23: HPLC chromatogram of aqueous fraction of mixed standards at 254 nm.
Polar fraction of mixed standard (green), mixed standard (black). The overlay (black) was normalised to the absorbance of the polar fraction of mixed standard with an offset of 20 mAu.
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It can be seen in Figure 23 that only chlorogenic acid, isoquercetin and wogonoside were extracted into the aqueous fraction. There is no detectable andrographolide, apigenin or 14- deoxy-11,12-didehydroandrographolide. This can be explained by the chemical structure and hence polarity of the analytes. As can be seen in Figure 18, wogonoside and isoquercetin, both contain a polar sugar group making them water soluble. Chlorogenic acid is composed of a polar quinic acid and caffeic acid, making it polar and therefore water soluble. In contrast andrographolide, apigenin and 14-deoxy-11,12-didehydroandrographolide are mostly non-polar and would partition into chloroform. The partitioning results are supported by the predicted pH dependent partition coefficient (log D) by ACD/Labs Percepta Platform -
PhysChem Module. The compounds that were present in the aqueous fraction are chlorogenic acid (log D (pH 7.4) = -3.91) wogonoside (log D (pH 7.4) = -3.42), isoquercetin (log D (pH
7.4)= -1.19). In contrast the compounds that were not in the aqueous fraction are andrographolide (log D (pH 7.4) = 1.90), apigenin (log D (pH 7.4) = 1.26) and 14-deoxy-
11,12-didehydroandrographolide (log D (pH 7.4) = 2.48).
Figure 24 shows the chromatogram of the chloroform fraction of the mixed standard, overlaid with the non-partitioned mixed standard.
90
170 λdetection = 254 nm
Isoquercetin Wogonoside
Apigenin
14-deoxy-11,12- 85 Chlorogenic acid didehydroandrographolide
Andrographolide Absorbance (mAu)
0
0 15 30 45 60 Retention time (min)
Figure 24: HPLC chromatogram of chloroform fraction of mixed standards at 254 nm.
Non-polar fraction of mixed standard (green), mixed standard (black). The overlay (black) was normalised to the absorbance of the non-polar fraction of mixed standard with an offset of 20 mAu.
It can be seen in Figure 24 that the remaining analytes, andrographolide, apigenin and
dehydroandrograpohlide are present in the non-polar extract, whereas chlorogenic acid and
isoquercetin are not detected. Wogonoside may also be present in small quantities, so there
may be some overlap in the extraction of mid-polarity molecules. This overlap is likely due to
the non-polar wogonin (o-methylated flavone) group that is attached to a sugar to form
wogonside.
Based on the separation achieved with the mixed standard, this method of partition is suitable
to reduce the complexity of the extract and produce chemically fractionated extracts useful in
locating the major active component(s) of the A. paniculata commercial extract.
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3.4.2. Partition of A. paniculata commercial extract
As the partition method described in section 3.3.2 was able to resolve compounds present in
the mixed standard solution into different fractions, it was applied to the A. paniculata
commercial extract. Figure 25 shows the chromatogram of the aqueous fraction of the A.
paniculata commercial extract overlaid with the mixed standard. It can be seen that
andrographolide is not detected in this fraction.
400 λdetection = 254 nm
Isoquercetin Wogonoside
Apigenin
14-deoxy-11,12- 200 Chlorogenic acid didehydroandrographolide
Andrographolide Absorbance (mAu)
0
0 20 40 60 80 Retention time (min)
Figure 25: HPLC chromatogram of the polar fraction of A. paniculata commercial extract at 254 nm.
Polar fraction of A. paniculata commercial extract (green), mixed standard (black). The overlay (black) was normalised to the absorbance of the non-polar fraction of mixed standard with an offset of 50 mAu.
92
Figure 26 shows an overlay of the mixed standard and the chloroform fraction of the A.
paniculata commercial extract. It can be seen that andrographolide and 14-deoxy-11,12-
didehydroandrographolide are present in this fraction.
1400 λdetection = 254 nm
Isoquercetin
Wogonoside Apigenin
14-deoxy-11,12- 700 Chlorogenic acid didehydroandrographolide
Andrographolide Absorbance (mAu)
0
0 20 40 60 80 Retention time (min)
Figure 26: HPLC chromatogram of non-polar fraction of A. paniculata commercial extract at 254 nm.
Non-polar fraction of A. paniculata commercial extract (green), mixed standard (black). The overlay (black) is normalised to the absorbance of the non-polar fraction of mixed standard with an offset of 50 mAu.
93
Figure 27 shows an overlay of the A. paniculata commercial extract and the non-polar
fraction. This overlay allows the observation that the less retained compounds before the
andrographolide peak are excluded from the non-polar extract, to the enrichment of
andrographolide and possible other diterpenes. The enrichment of the non-polar compounds
in the A. paniculata non-polar extract is difficult to observe in Figure 27 as A. paniculata (30
mg/ml, 5 μl) was injected at twice the concentration of the non-polar fraction (15 mg/ml, 5
μl). The enrichment of andrographolide from 32.1 mg/ml to 65.1 mg/ml in the non-polar
extract is quantified in section 3.4.3.
1400
λdetection = 254
nm
14-deoxy-11,12- 700 didehydroandrographolide
Andrographolide Absorbance (mAu)
0
0 20 40 60 80 Retention time (min)
Figure 27: HPLC chromatogram of non-polar fraction of A. paniculata overlaid with the A. paniculata commercial extract at 254 nm.
Non-polar fraction of the A. paniculata commercial extract (green), the A. paniculata commercial extract (black). The overlay (black) was normalised to the absorbance of the non-polar fraction of mixed standard with an offset of 50 mAu.
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Figure 28 shows an overlay of the aqueous and chloroform extracts. It can be seen that some
peaks are common to both fractions, however, andrographolide is only detected in the non-
polar (chloroform) fraction.
200 λdetection = 254 nm
100 Absorbance (mAu) Absorbance
0
0 20 40 60 80 Retention time (min)
Figure 28: HPLC chromatogram of aqueous and chloroform fraction of the A. paniculata commercial extract at 254 nm.
A. paniculata commercial extract (green), polar fraction (black). The overlay (black) was normalised to the absorbance of the A. paniculata with an offset of 10 mAu.
The solvent partitioning procedure, developed and applied to the mixed standard containing
compounds present in A. paniculata, was also applied to the A. paniculata commercial
extract. The resulting polar (or aqueous) extract contained no detectable compounds eluting
before andrographolide. In the mixed standard this correlated to the polyphenolic acid
(chlorogenic acid) and flavonol glycoside (isoquercetin). The non-polar (or chloroform)
extract showed a lower concentration of other glycosides, correlating to wogonoside in the
mixed standard and has enrichment of andrographolide and likely enrichment of other more
hydrophobic compounds. The polar fraction had no detectable andrographolide or 14-deoxy-
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11,12-didehydroandrographolide. These extracts will assist in narrowing down the major
active components of the A. paniculata commercial extract and assessing the contribution of
andrographolide to the herb’s total activity. The disadvantage of this crude extraction is that
there is some overlap with compounds being in both the polar and non-polar fractions. There
are also multiple components in each fraction so we cannot determine the compound
responsible for activity without further fractionation.
3.4.3. Quantification of andrographolide
The concentration of the andrographolide in the standardised A. paniculata commercial
extract as well as the non-polar fraction created by the solvent partition was determined using
the HPLC method described in section 3.3.6. Figure 29 shows an overlay of the
chromatograms of the andrographolide standard, the andrographolide present in the A.
paniculata commercial extract and the non-polar fraction.
200 λdetection = 227 nm
Reference standard Andrographolide
Non-polar extract 100
A. paniculata Absorbance (mAu)
0
0 2.5 5 7.5 10 Retention time (min)
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Figure 29: HPLC chromatogram comparing andrographolide standard to the A. paniculata commercial extract and non-polar fraction at 227 nm.
The retention time for the peak identified as andrographolide in the A. paniculata commercial extract and the non-polar fraction are almost the same. This identity of this peak is further supported by the UV spectrum of these peaks shown in Figure 30.
600 600
400 400
mAu mAu
200 200
0 0 200 225 250 275 300 nm
Figure 30: UV spectrum of reference standard andrographolide and the A. paniculata commercial extract.
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Figure 31 shows the chromatogram of the aqueous extract with an overlay of the
andrographolide standard.
30 λdetection = 227 nm Andrographolide
Non-polar fraction
0
Standard Absorbance (mAu)
-30
0 2.5 5 7.5 10 Retention time (min)
Figure 31: HPLC chromatogram of aqueous fraction and andrographolide at 227 nm.
Polar fraction (green), andrographolide (black). The overlay (black) was normalised to the absorbance of the A. paniculata with an offset of -15 mAu.
In Figure 31 there is a peak of similar retention time (5.8 min) to that of andrographolide. The
UV spectrum for this peak is shown overlaid with the reference andrographolide in Figure 32.
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20 20
10 10
mAu mAu
0 0 200 225 250 275 300 nm
Figure 32: UV spectrum of polar fraction peak at 5.7 min (green) and pure andrographolide (black), normalised.
In Figure 32 it can be seen that the UV spectrum of the peak at 5.8 min in the polar extract does not match that of andrographolide and their retention times are different by 0.2 min. It is therefore likely that it is not andrographolide so was not quantified as andrographolide.
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Figure 33 shows the linear relationship between the peak area versus concentration produced by a range of known andrographolide concentrations standards. The calibration curve is linear (R2 = 0.9996) over the range relevant to this study.
Andrographolide standard curve
800
700
600
500
400
Peak area Peak 300 y = 968.95x + 6.7907 200 R² = 0.9996
100
0 0 0.2 0.4 0.6 0.8 1 Andrographolide concentration (mg/ml)
Figure 33: Andrographolide standard curve (n=3).
Table 10 shows the results for the analysis of andrographolide in the A. paniculata commercial extract in the non-polar fraction and polar fraction. The absence of andrographolide in the polar fraction shows the effectiveness of the partitioning, as does enriching of andrographolide in the non-polar fraction. The calculated concentrations of andrographolide were used to normalise the in vitro doses to the andrographolide concentration of the extracts, to determine if the activity observed was accounted for by andrographolide concentration alone.
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Table 10: Concentration of andrographolide in the A. paniculata commercial extract, non-polar fraction and polar fractions.
RSD % w/w (%) A. paniculata un-partitioned 32.1 0.1 commercial extract Non-polar fraction 65.1 0.1 Not Polar fraction detected
3.4.4. Nitric oxide production in RAW264.7 cells
The A. paniculata commercial extract, non-polar fraction, polar fraction and pure andrographolide were tested in the NO assay to determine their IC50 for inhibiting NO production by LPS and IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 34. It can be seen that andrographolide is the most potent by weight (μg/ml) at inhibiting NO production, followed by the non-polar fraction, the A. paniculata commercial extract and then the polar fraction.
101
Nitric Oxide production by RAW264.7 cells 110
100
90
80
70
60
50
40 NO production (%) NO
30 Andrographolide Non-polar fraction 20 Polar fraction A.paniculata 10
0 1 10 100 1000 Concentration (g/ml) -10
Figure 34: NO dose response curve for andrographolide (red, circles), A. paniculata commercial extract (green, inverted triangles), the non-polar fraction (blue, squares) and the polar fraction (black, triangles), by LPS and IFN-γ stimulated RAW264.7 cells.
The data was fitted with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
To determine if the activity observed in the NO production assay can be attributed to andrographolide in the extract and non-polar fraction, the doses were normalised to andrographolide concentration (μM andrographolide) so that the dose of the non-polar fraction and the A. paniculata commercial extract reflected the calculated concentration of andrographolide present. The dose response curve when normalised to andrographolide concentration (μM andrographolide) is shown in Figure 35.
102
Nitric Oxide production by RAW264.7 cells (normalised) 110
100
90
80
70
60
50 Andrographolide Non-polar fraction
40 A.paniculata NO production (%) NO 30
20
10
0 1 10 100 M andrographolide -10
Figure 35: NO dose response curve for andrographolide (red, circles), A. paniculata commercial extract (green inverted, triangles) and the non-polar fraction (blue, squares), by LPS and IFN-γ stimulated RAW264.7 cells.
The doses have been normalised to andrographolide content (μM) for A. paniculata commercial extract and the non-polar fraction. The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
In Figure 35 it can be observed that the dose response curves for the andrographolide, the A. paniculata commercial extract and non-polar fraction, when normalised to andrographolide concentration are very similar. A comparison of the resulting IC50s and confidence intervals is given in Table 11.
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Table 11: NO IC50's 95% CI IC μM 50 95% CI range of range of normalised to IC μg/ml IC μM 50 IC andrographolide 50 50 andrographolide μg/ml content Pure 2.6 2.4 to 2.9 7.4 6.7 to 8.1 andrographolide
A. paniculata 6.2 5.6 to 6.9 6.3 5.6 to 6.9 commercial extract
Non-polar fraction 3.5 3.2 to 3.7 7.2 6.7 to 7.7
285.8 to Polar fraction 330.6 NA 382.5
The IC50 is calculated from the fitted curves in Figure 34 and Figure 35.
Table 11 shows that per gram of andrographolide the A. paniculata commercial extract is the most active. This would suggest that other components of the herb contribute to the NO inhibition, however, this contribution can also be attributed to the uncertainty in the assay, as the results for the A. paniculata commercial extract and pure andrographolide overlap when considering the 95% confidence interval. It is probable that andrographolide is the exclusive contributor to the effect.
The NO inhibition dose response curves were compared for andrographolide and the normalised (to andrographolide concentration) A. paniculata commercial extract and non- polar fraction. An F-test was used to compare the curves, fitting the three curves to one curve and giving a p-value that reflects if the model fit is statistically significantly. The dose response curve comparison is shown in Figure 36.
104
Nitric Oxide production by RAW264.7 cells (normalised) F-test 110
100
90
80
70
60
50 Andrographolide Non-polar fraction
40 A.paniculata NO production (%) NO 30
20
10
0 1 10 100 M andrographolide -10
Figure 36: NO dose response curve combined F-test for pure andrographolide, A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range. p= 0.1278.
Andrographolide, the A. paniculata commercial extract and the non-polar fraction were potent inhibitors of NO in RAW264.7 cells. The polar fraction containing no detectable andrographolide was considerably less active. The dose response curves when normalised to andrographolide content are similar. The IC50s of the andrographolide content in each extract are within statistical agreement and the dose response curves when fitted to a single curve showed statistical agreement with the fit. Thus, at this level of analysis, there is no evidence for antagonistic, additive or synergistic effects between andrographolide and other phytochemical constituents of the A. paniculata commercial extract. The extract’s activity was almost entirely accounted for by the andrographolide content, and the contribution of the other compounds is not evident, probably due to their low concentration.
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3.4.5. PGE2 release by RAW264.7 cells
To determine if the results observed for NO inhibition are applicable to other mediators of inflammation, pure andrographolide and the A. paniculata commercial extract (normalised to andrographolide concentration) were tested for their inhibition of PGE2 release by LPS and
IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 37.
PGE2 released by RAW264.7 cells 110
100
90
80
70
60 Andrographolide release
2 A.paniculata
50 % PGE % 40
30
20
10
0 1 10 100 1000 M andrographolide
Figure 37: PGE2 dose response curve for andrographolide (red, circles) and the A. paniculata commercial extract (green, squares), by LPS and IFN-γ stimulated RAW264.7 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract. The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
In Figure 37 it can be observed that the PGE2 release dose response curves for the andrographolide and A. paniculata commercial extract (normalised to andrographolide concentration) are very similar. A comparison of the resulting IC50s and confidence intervals is given in Table 12.
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Table 12: PGE2 inhibition IC50's IC μM normalised to 95% CI* range of IC μM 50 50 andrographolide content andrographolide Pure andrographolide 8.8 7.4 to 10.4 Andrographolide in A. paniculata commercial 6.1 4.4 to 8.4 extract * CI, confidence interval
It can be seen in Table 12 that the A. paniculata commercial extract normalised to andrographolide content is slightly more potent at inhibition of PGE2 release from
RAW264.7 cells than andrographolide. The IC50s are in agreement as to the difference between andrographolide and the A. paniculata commercial extract but again the results are not outside the range of the uncertainty for the assay.
The PGE2 inhibition dose response curves were compared to pure andrographolide and the normalised (to andrographolide concentration) A. paniculata commercial extract. An F-test was used to compare the curves, fitting the two curves to one curve and giving a p-value that reflect if the model fits are statistically significant. The dose response curve comparison is shown in Figure 38.
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PGE2 released by RAW264.7 cells F-test 110
100
90
80
70
60 Andrographolide release
2 A.paniculata
50 % PGE % 40
30
20
10
0 1 10 100 1000 M andrographolide
Figure 38: PGE2 dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range; p= 0.0849
Andrographolide and the A. paniculata commercial extract are both potent inhibitors of PGE2 release from RAW264.7 cells. The dose response curves when normalised to andrographolide content are similar. The IC50s of the andrographolide content in the extract are within statistical agreement and the dose response curves when fitted to a single curve showed statistical agreement with the fit. Thus, at this level of analysis, the A. paniculata commercial extract’s activity appears to be almost entirely accounted for by the andrographolide content.
3.4.6. RAW264.7 TNF-α assay
To determine if the andrographolide concentration accounts for the activity of the A. paniculata commercial extract on the release of TNF-α, pure andrographolide and the A. paniculata commercial extract were tested for their inhibition of TNF-α release by LPS and
IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 39. 108
TNF- released by RAW264.7 cells 110
100
90
80
70 Andrographolide 60 A.paniculata
released 50
40 % TNF %
30
20
10
0 1 10 100 1000 M andrographolide -10
Figure 39: TNF-α dose response curve for andrographolide (red, circles) and the A. paniculata commercial (green, squares), by LPS and IFN-γ stimulate RAW264.7 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata. The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=12)
Table 13: IC50s for TNF-α inhibition by andrographolide and the andrographolide in the A. paniculata commercial extract.
IC μM normalised to 95% CI* range of IC μM 50 50 andrographolide content andrographolide Pure Andrographolide 23.3 20.1 to 27 Andrographolide in A. paniculata commercial 22.3 19.5 to 25.5 extract *CI – confidence interval
It can be seen in Table 13 that A. paniculata commercial extract normalised to andrographolide content was slightly more potent at inhibition of PGE2 release from
RAW264.7 cells than pure andrographolide. However, the IC50s are in agreement and the
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difference between pure andrographolide and the A. paniculata commercial extract is not outside the range of the uncertainty for the assay.
The TNF-α inhibition dose response curves were compared for pure andrographolide and the
A. paniculata commercial extract (normalised to andrographolide concentration). An F-test was used to compare the curves, fitting the two curves to one curve and giving a p-value that reflects if the model fits are statistically significant. The dose response curve comparison is shown in Figure 38.
TNF- released by RAW264.7 cells F-test 110
100
90
80
70 Andrographolide 60 A.paniculata
released 50
40 % TNF %
30
20
10
0 1 10 100 1000 M andrographolide -10
Figure 40: TNF-α dose response curve combined F-test for andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
Pure andrographolide and the A. paniculata commercial extract are both potent inhibitors of
TNF-α release from RAW264.7 cells. The dose response curves when normalised to andrographolide content are similar. The IC50s of the andrographolide content in each extract 110
are within statistical agreement and the dose response curves when fitted to a single curve showed statistical agreement with the fit. Thus, at this level of analysis, the extract’s activity against LPS and IFN-γ induced TNF-α release from RAW264.7 cells appears to be almost entirely due to andrographolide content.
3.4.7. THP-1 TNF-α results
The results observed so far indicate that in the LPS and IFN-γ stimulated murine RAW264.7 cell line, inhibition of the release of NO, PGE2 and TNF-α is due to the andrographolide in the extract. If the other components of the herb were contributing to the inhibition, it is not evident in the assays. To see if the effect was conserved in human THP-1 cells the inhibitory effect of pure andrographolide was compared to the A. paniculata commercial extract and the non-polar fraction.
TNF- released by THP-1 120
110
100
90
80
70 Andrographolide
release 60 A.paniculata Non-polar fraction
50 % TNF % 40
30
20
10
0 1 10 100 1000 M andrographolide
Figure 41: TNF-α dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, squares) and the non-polar extract (blue, triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction. The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=12)
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Table 14: IC50s for TNF-α inhibition by pure andrographolide and andrographolide in the A. paniculata commercial extract on THP-1 cells.
IC μM normalised to 95% CI range of IC μM 50 50 andrographolide content andrographolide Pure andrographolide 16.5 13.1 to 21.0 Andrographolide in A. paniculata commercial 17.5 14.7 to 20.1 extract Andrographolide in non- 22.3 19.5 to 25.5 polar fraction
It can be seen in Table 14 that pure andrographolide is slightly more potent at inhibition of
TNF-α release from THP-1 cells than the A. paniculata commercial extract and the non-polar fraction. However, the IC50s are in agreement.
The TNF-α inhibition dose response curves were compared for pure andrographolide and the normalised (to andrographolide concentration) A. paniculata commercial extract. An F-test was used to compare the curves, fitting the three curves to one curve and giving a p-value that reflects if the model fit is statistically significant. The dose response curve comparison is shown in Figure 42.
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TNF- released by THP-1 F-test 120
110
100
90
80
70 Andrographolide
release 60 A.paniculata Non-polar fraction
50 % TNF % 40
30
20
10
0 1 10 100 1000 M andrographolide
Figure 42: TNF-α dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range; p= 0.0981
Pure andrographolide, the A. paniculata commercial extract and the non-polar fraction are all potent inhibitors of TNF-α release from LPS stimulated THP-1 cells. The dose response curves when normalised to andrographolide content are similar. The IC50s of the andrographolide content in each extract are within statistical agreement and the dose response curves when fitted to a single curve showed statistical agreement with the fit. Thus, at this level of analysis, the extract’s activity against LPS and IFN-γ induced TNF-α release from
THP-1 cells is almost entirely accounted for by the andrographolide content.
3.4.8. Multiplex results
The release of 27 cytokines by a multiplex bead based ELISA was performed on the supernatant of LPS stimulated THP-1 cells. The experiment was to determine if
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andrographolide has an inhibitory effect on the 27 cytokines and chemokines and to see if the effect of the A. paniculata commercial extract could be accounted for by the andrographolide concentration alone.
The commercially available kit was expensive, limiting the number of replicates to 2. This limitation means that the statistical significance of the results is also limited. Figures 44 to 68 show the dose response curves and the F-test for pure andrographolide, the commercial extract of A. paniculata and the non-polar fraction, normalised for andrographolide content.
A summary of the IC50 values and p-tests is presented in Table 16, after the figures. The polar fraction is also shown in the figures, however, as it contained no quantifiable andrographolide content, the dose cannot be converted to μM andrographolide concentration, so is presented as μg/ml. This makes comparison from the dose response curves difficult. In the vast majority of the cytokines quantified the polar fraction is significantly less active. The polar fraction displayed the most activity at IP-10 inhibition. The IC50 for the polar fraction was less than but comparable to andrographolide in this assay. The IC50s converted to μg/ml are shown in
Table 15.
Table 15: IP-10 IC50 comparison between extracts of the A. paniculata commercial extract and pure andrographolide A. paniculata Pure Non- polar Assay commercial Polar fraction andrographolide fraction extract IP-10 IC- (μM 50 12.8 15.6 17.9 andrographolide)
CI 95% IC50 6.8 to 24.1 7.4 to 32.9 11.2 to 28.8 IP-10 IC- 50 4.5 8.4 19.7 9.1 (μg/ml)
CI 95% IC50 2.4 to 8.5 4.0 to 17.8 12.3 to 31.6 5.2 to 16.1
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As can be seen in Table 15, the IC50 when converted to μg/ml for pure andrographolide, decreased by about a third, the non-polar extract decreased about a half and the A. paniculata commercial extract remained comparable. In virtually all other cytokine assays the IC50 of the polar fraction was greater than the others tested, indicating less activity. This would be exaggerated further in by presenting all the data in μg/ml.
Figures 44 to 68 show the dose response curves and the F-test for pure andrographolide, the
A. paniculata commercial extract and the non-polar fraction, normalised to andrographolide content and the polar fraction in μg/ml.
INF- release by THP-1 cells 120
100
80
60
release
INF % 40
Androgragholide Non-polar fraction 20 A.paniculata
0 1 10 100 Dose (M andrographolide)
Figure 43: IFN-γ dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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INF- release by THP-1 cells F-test 120
100
80
60
release
INF % 40
Androgragholide Non-polar fraction 20 A.paniculata
0 1 10 100 Dose (M andrographolide)
Figure 44: IFN-γ dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
IL-1 release by THP-1 cells 140
120
100
80 release
60 %IL-1
40
Androgragholide 20 Non-polar Fraction A.paniculata Polar Fraction 0 10 100 1000 Dose (M andrographolide)
Figure 45: IL-1β dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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IL-1 release by THP-1 cells F-test 140
120
100
80 release
60 %IL-1
40
Androgragholide 20 Non-polar Fraction A.paniculata
0 10 100 1000 Dose (M andrographolide)
Figure 46: IL-1β dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
IL-1ra release by THP-1 cells 150
125
100
75
Andrographolide A.paniculata 50
%IL-1ra release%IL-1ra Polar fraction
25
0 1 10 100 Dose (M andrographolide)
-25
Figure 47: IL-1ra dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The curve fit did not converge with the non-polar data. (n=2)
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There was no F-test fit possible for IL-1ra release inhibition by andrographolide, the A. paniculata commercial extract and the non-polar fraction dose response curve had a wide range of error and insufficient activity at the doses tested.
IL-2 release by THP-1 cells 125
100
75
50 Androgragholide
%IL-2 release %IL-2 Non-polar fraction A.paniculata Polar fraction
25
0 1 10 100 1000 Dose (M andrographolide)
Figure 48: IL-2 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The fit was not possible for the non-polar extract. (n=2)
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IL-2 release by THP-1 cells F-test 125
100
75
50 Androgragholide
%IL-2 release %IL-2 A.paniculata
25
0 1 10 100 Dose (M andrographolide)
Figure 49: IL-2 dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
The non-polar extract could not be incorporated in the F-test in Figure 49, as its dose response did not fit the log (inhibitor) vs. normalised response with variable slope model.
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IL-4 released by THP-1 cells 100
80
60
40 %IL-4 released %IL-4
Androgragholide Non-polar fraction 20 A.paniculata Polar fraction
0 1 10 100 Dose (M andrographolide)
Figure 50: IL-4 dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-4 released by THP-1 cells F-test 100
80
60
40 %IL-4 released %IL-4
Androgragholide Non-polar fraction 20 A.paniculata
0 1 10 100 Dose (M andrographolide)
Figure 51: IL-4 dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
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IL-6 released by THP-1 cells
120
100
80
60
%IL-6 released %IL-6 Androgragholide 40 Non-polar fraction A.paniculata Polar fraction
20
0 1 10 100 Dose (M andrographolide)
Figure 52: IL-6 dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-6 released by THP-1 cells F-test
120
100
80
60
%IL-6 released %IL-6 Androgragholide 40 Non-polar fraction A.paniculata
20
0 1 10 100 Dose (M andrographolide)
Figure 53: IL-6 dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
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BFGF release by THP-1 cells 160
140
120
100
80
Androgragholide
%BFGF release Non-polar fraction 60 A.paniculata Polar fraction
40
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 54: BFGF dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
BFGF release by THP-1 cells F-test 160
140
120
100
80
Androgragholide
%BFGF release %BFGF A.paniculata 60
40
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 55: BFGF dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
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The dose response curve in Figure 55, for the non-polar extract did not fit with the F-test for
BFGF release. This may be due to a limited number of data points showing significant inhibition, as the extracts were less active at BFGF inhibition.
G-CFS released by THP-1 cells
120
100
80
60 %G-CFS released %G-CFS 40
Androgragholide Non-polar fraction 20 A.paniculata Polar fraction
0 10 100 1000 Dose (M andrographolide)
Figure 56: G-CFS dose response curve for pure andrographolide (red, squares), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, circles) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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G-CFS release by THP-1 cells F-test
120
100
80
60 %G-CFS release %G-CFS 40
Androgragholide Non-polar fraction 20
0 10 100 1000 Dose (M andrographolide)
Figure 57: G-CFs dose response curve combined F-test for pure andrographolide and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted line shows the 95% confidence interval range.
In Figure 57, the A. paniculata commercial extract was excluded from the F-test as its fit with a log (inhibitor) vs. normalised response with variable slope model was ambiguous. This is a result of the steep decline in G-CFS release observed in Figure 56, meaning there was only one point on the IC50 curve between near 100% and 0% inhibition. This makes the fit unreliable and the error in the IC50 too large to compare. More replicates with closer intervals in the doses tested might correct for this in future studies and make comparison possible.
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GM-CFS release by THP-1 cells 140
120
100
80
60 %G-CFS release %G-CFS
40 Androgragholide Non-polar fraction A.paniculata Polar fraction 20
0 1 10 100 1000 Dose (M andrographolide)
Figure 58: GM-CFS dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
GM-CFS released by THP-1 cells F-test 140
120
100
80
60 %G-CFS released %G-CFS
40 Androgragholide Non-polar fraction A.paniculata
20
0 1 10 100 1000 Dose (M andrographolide)
Figure 59: GM-CFS dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted line shows the 95% confidence interval range.
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VEGF released by THP-1 cells 140
120
100
80
60
%VEGF released%VEGF 40 Androgragholide Non-polar fraction A.paniculata 20 Polar fraction
0 1 10 100 Dose (M andrographolide) -20
Figure 60: VEGF dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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VEGF released by THP-1 cells F-test 140
120
100
80
60
%VEGF released%VEGF 40 Androgragholide A.paniculata 20
0 1 10 100 Dose (M andrographolide) -20
Figure 61: VEGF dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted line shows the 95% confidence interval range.
In Figure 61, the non-polar fraction was excluded from the F-test as the fit with a log
(inhibitor) vs. normalised response with variable slope model was ambiguous. This is a result of the steep decline in VEGF release observed in Figure 60, meaning there is only one point on the IC50 curve between near 100% and 0% inhibition. There were also large differences between duplicates in the assay when normalised to % release, complicating the analysis.
This makes the fit unreliable and the error in the IC50 too large for reliable comparison. More replicates with closer intervals in doses tested might correct for this in future studies and make comparison possible.
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IP-10 released by THP-1 cells 120
100
80
60 release
IP-10 40 %
Androgragholide 20 Non-polar fraction A.paniculata Polar fraction
0 1 10 100 Dose (M andrographolide)
-20
Figure 62: IP-10 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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IP-10 released by THP-1 cells F-test 120
100
80
60 release
IP-10 40 %
Androgragholide 20 Non-polar fraction
0 1 10 100 Dose (M andrographolide)
-20
Figure 63: IP-10 dose response curve combined F-test for pure andrographolide and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
The A. paniculata commercial extract was significantly less active and did not fit the F-test with pure andrographolide and the non-polar fraction, as shown in Figure 63.
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MCP-1 released by THP-1 cells
100
80
60
40 Androgragholide
%MCP-1released Non-polar fraction A.paniculata Polar fraction
20
0 10 100 1000 Dose (M andrographolide)
Figure 64: MCP-1 dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
An F-test curve fit was not possible for MCP-1 inhibition as the A. paniculata commercial extract and the nonpolar fraction were significantly more active than pure andrographolide.
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Eotaxin released by THP-1 cells 120
100
80
60
%Eotaxin released %Eotaxin Androgragholide 40 Non-polar fraction A.paniculata Polar fraction
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 65: Eotaxin dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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Eotaxin released by THP-1 cells F-test 120
100
80
60
%Eotaxin released %Eotaxin Androgragholide 40 A.paniculata
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 66: Eotaxin dose response curve combined F-test for pure andrographolide and the A. paniculata commercial extract normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted lines show the 95% confidence interval range.
The non-polar fraction did not fit the F-test in Figure 66. This may be due to a limited number of data points showing significant inhibition, as the non-polar extract appears to be less active at eotaxin inhibition.
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RANTES released by THP-1 cells 160
140
120
100
80
60 Androgragholide %RANTES released %RANTES Non-polar fraction A.paniculata Polar fraction 40
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 67: RANTES dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, triangles), the non-polar fraction (blue, squares) and the polar fraction (black, inverted triangles), by LPS stimulated THP-1 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction except for the polar fraction (μg/ml). The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
An F-test was not possible for RANTES inhibition as there is insufficient activity to fit a dose response curve. Higher doses may be needed to test RANTES inhibition.
There were a number of cytokines that showed no dose dependent inhibition by the A. paniculata commercial extract or its components. The effect of the tested extracts on IL-9 release is shown in Figure 68. It can be seen that although IL-9 was released at detectable levels, there was no observed dose dependent response with little difference between LPS stimulated and non-stimulated cells.
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IL-9 released by THP-1 cells
250 200 150
9 released 100 - 50
0
pg/ml pg/ml IL
H1 A3 A1 N3 N1 H3
A30 A10 N30 N10 H30 H10
P 10 P P 30 P
A0.3 N0.3 H0.3
LPS-
LPS+ P 100 P Dose (μM andrographolide)
Figure 68: IL-9 released by LPS stimulated THP-1 cells. (n=2) A= pure andrographolide, N= nonpolar fraction of A.panicualta, H= commercial extract of A.panicualta, P=polar extract of A. paniculata
In Figure 69 the % release of IL-17 is shown, normalised to LPS stimulated and non- stimulated. It can be seen that there was no dose dependent response to A. paniculata nor any of its components.
160 140 IL-17 release by THP-1 cells
120 100
80 17 release
- 60 40
% % IL 20
0
A1 N3 N1 H3 H1
-20 A3
P30 P10
H10 A30 A10 N30 N10 H30
A0.3 N0.3 H0.3
P100 LPS- LPS+ Dose (μM andrographolide)
Figure 69: %IL-17 release by LPS stimulated THP-1 cells. (n=2) A= pure andrographolide, N= nonpolar fraction of A.panicualta, H= commercial extract of A.panicualta, P=polar extract of A. paniculata
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Due to the high cost of the multicytokine assay, it was not possible to optimise the parameters for each cytokine, as a result of this not all of the 27 cytokines assayed produced usable results. Indeed, due to the limitations of the single cell and model not all cytokines were released by LPS stimulated THP-1 cells. These results are summarised in, Table 16.
3.4.9. NF-κB results
The results presented thus far, testing the A. paniculata commercial extract and its components’ effect on the release of a number of inflammatory mediators in murine
RAW264.7 and human THP-1 cells, suggest that in the vast majority of these mediators the effect can be attributed to andrographolide concentrations. This would strongly suggest an overall controlling factor. NF-κB is the transcription factor that controls the transcription of many inducible inflammatory mediators. It is plausible that its inhibition could account for the observed wide spectrum effect. The dose response for NF-κB testing is shown in Figure
70.
135
NF-B activation assay in ELAM9 RAW264.7 cells 120
110
100
90
80
70 Andrographolide 60
B activation B A.panicualta Non-polar fraction 50
% NF- % 40
30
20
10
0 0.1 1 10 100 M andrographolide
Figure 70: NF-κB dose response curve for pure andrographolide (red, circles), the A. paniculata commercial extract (green, squares) and the non-polar fraction (blue, inverted triangles) by LPS and IFN-γ stimulated ELAM9 RAW264.7 cells.
The doses have been normalised to andrographolide content (μM) for the A. paniculata commercial extract and the non-polar fraction. The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=12)
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NF-B activation assay in ELAM9 RAW264.7 cells F-test 120
110
100
90
80
70 Andrographolide 60
B activation B A.panicualta Non-polar fraction 50
% NF- % 40
30
20
10
0 0.1 1 10 100 M andrographolide
Figure 71: NF-κB dose response curve combined F-test for pure andrographolide, the A. paniculata commercial extract and the non-polar fraction normalised to andrographolide concentration.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The dotted line shows the 95% confidence interval range.
It can be seen in Figure 70 that andrographolide, the A. paniculata commercial extract and the non-polar extract all inhibited NF-κB activation in a similar dose dependent manner. This was confirmed with the F-test showing that they could all be fit to a single curve. The activity of the A. paniculata commercial extract on NF-κB activation inhibition is due to the andrographolide content in the extracts.
3.4.10. Summary of results
A summary of the IC50s for the in vitro anti-inflammatory assays is given in Table 16.
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Table 16: Summary of anti-inflammatory results A. Pure Non-polar paniculata t-test Polar Assay andrographolide fraction commercial combined fraction (µM) (µM*) extract (µM*) (µg/ml) (µM*) RAW 264.7
NO IC50 7.4 7.2 6.3 6.9 330.6 285.8 to CI 95% IC 6.7 to 8.1 6.7 to 7.7 5.6 to 6.9 6.6 to 7.3 50 382.5 p=0.1278
PGE2 IC50 8.8 6.1 7.5
CI 95% IC50 7.4 to 10.4 4.4 to 8.4 6.3 to 8.9 p=0.0849
TNF-α IC50 23.3 22.3 22.8
CI 95% IC50 20.1 to 27.0 19.5 to 25.5 20.7 to 25.2 p=0.8609 THP-1
TNF-α IC50 16.5 22.3 17.5 18.3 ~109.6 19.5 to CI 95% IC 13.1 to 21.0 14.7 to 20.1 16.5 to 20.4 v. wide 50 25.5 p=0.0981
INFγ IC50 11.6 15.5 16.8 13.9 ~31.04
CI 95% IC50 7.7 to 17.6 8.4 to 28.6 8.0 to 35.1 10.0 to 19.4 v. wide p=0.6212
IL-1β IC50 18.2 11.3 13 22.3 221.6
CI 95% IC50 11.1 to 29.8 6.0 to 21.4 9.4 to 17.8 14.9 to 33.5 v. wide p=0.6212 No fit IL-1ra IC 54.8 Not active 31.4 30.14 50 possible
CI 95% IC50 v. wide v. wide v. wide
No fit IL-2 IC 20.4 24.9 22.3 ~32.1 50 possible CI 95% IC50 8.9 to 46.5 14.6 to 42.6 14.9 to 33.5 v. wide p=0.6218
IL-4 IC50 23.2 31.5 28.0 27.4 48.1 19.0 to 22.84 to CI 95% IC 15.9 to 33.8 15.7 to 49.9 21.6 to 34.9 50 52.2 101.4 p=0.7281
IL-6 IC50 10.2 11.1 7.9 9.6 21.7
CI 95% IC50 7.7 to 13.6 7.1 to 17.5 6.0 to 10.3 8.1 to 11.4 18.6 to 25.4 p=0.5772
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BFGF IC50 21.4 Not active 22.9 22.0 44.2
CI 95% IC50 11.5 to 39.6 13.9 to 37.7 15.3 to 31.7 21.0 to 93.3 p=0.3789
G-CSF IC50 25.1 46.9 32.0 31.0 355.6 20 to 126.8 to CI 95% IC 15.9 to 39.5 v. wide 19.5 to 49.4 50 109.5 218.5 p=0.3458 GM-CSF 47.7 68.6 49.6 54.7 166.4 IC50 30.9 to 126.8 to CI 95% IC 35.3 to 63.5 31.45 to 78.3 42.2 to 70.9 50 152.1 218.5 p=0.4959
VGEF IC-50 6.0 ~10.5 7.1 6.5 ~31.97
CI 95% IC50 1.9 to 18.7 v. wide 3.7 to 13.5 3.6 to 11.6 v. wide p=0.9214
IP-10 IC-50 12.8 15.6 17.9 14.1 9.1
CI 95% IC50 6.8 to 24.1 7.4 to 32.9 11.2 to 28.8 9.3 to 21.4 5.2 to 16.1 p=0.9005 MCP-1 31.8 18.5 14.0 ~141.1
CI 95% IC50 27.3 to 37.1 7.4 to 32.9 11.2 to 28.8 v. wide p < 0.0001 Eotaxin 22.7 ~30.1 21.8 22.1 45.6
CI 95% IC50 14.5 to 35.4 v. wide 12.2 to 39.1 16.7 to 29.2 8.4 to 248.2 p=29.2 No fit RANTES Not active Not active Not active Not active possible RAW264.7
ELAM9 GFP NF-κB activation 26.0 21.4 23.7 24.6 625.2 IC50 19.5 to 584.1 to 23.4 to 29.0 21.3 to 26.3 23.1 to 26.1 23.5 669.2 p=0.1517 Failed IL-5 <2 pg/ml detection limit IL-7 < 0.8 pg/ml detection limit IL-9 There was no stimulation of release by LPS IL-10 < 1.5 pg/ml detection limit IL-12 < 2.4 pg/ml detection limit IL-13 < 1.8 pg/ml detection limit IL-15 < 1.3 pg/ml detection limit PDGF-bb < 32 pg/ml detection limit
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IL-8 > 6200 pg/ml detection limit MIP-1a > 5200 pg/ml detection limit MIP-1b > 4000 pg/ml detection limit *normalised to andrographolide content
In Table 16, it can be seen that for the vast majority of mediators tested in murine RAW264.7 and human THP-1 cells, the A. paniculata commercial extract and its components effect on the release of a number of inflammatory mediators, can be attributed to andrographolide concentration. This was tested by F-test curve fits that displayed the variation observed was within the uncertainty of the assay. Pure andrographolide showed inhibitory activity against
IL-1ra and MCP-1, however, is not the active component of the extract that the activity could be attributed to. In examples such as IL-6 and IP-10 where the polar extract containing no detectable andrographolide showed activity comparable to the A. paniculata commercial extract, it is likely that there is another highly active compound (or compounds) present.
However, in the A. paniculata commercial extract where this compound should also be present, the activity is masked by andrographolide’s activity.
The in-vitro anti-inflammatory results suggest that the pure andrographolide has equivalent anti-inflammatory activity to the extract. The pure compound has additional advantages such as decreased risk of side effects from other components and as it is pure, it is able to be administered practically at higher doses. However, the pharmacokinetics research suggest that 14-deoxy-11,12-didehydroandrographolide, deoxyandrographolide and neoandrographolide all play a role in reducing the rate of metabolism of andrographolide
[188], so the extract has justifiable favourable pharmacokinetics [189]. This might mean that clinically the pure andrographolide and A. paniculata may not be equivalent.
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Andrographolide shows strong inhibition of a large number of inflammatory mediators. This would strongly suggest an overall controlling factor. NF-κB is the transcription factor that controls the transcription of many inducible inflammatory mediators. Andrographolide’s effect on NF-κB activation was assayed in LPS and IFN-γ stimulated ELAM9 RAW264.7 cells to see if this was the controlling factor. The most commonly implicated molecular mechanism underpinning the anti-inflammatory and immunomodulatory effects of andrographolide is the inhibition of the mitogen-activated protein kinase/extracellular signal- regulated kinase (MAPK/ERK) signaling (specifically p38 MAPK/ERK1/2) pathway and downstream transcription factors such as NF-κB and nuclear factor of activated T cells
(NFAT) [190]. As the THP-1 and RAW264.7 macrophages models are not T cells, NFAT’s role is excluded, meaning, the role of NF-κB activation should be a major factor. In Table 16, it can be seen that the majority of the IC50s for the inhibition of mediators of inflammation within agreement of that of NF-κB activation. It is therefore likely that NF-κB activation inhibition is the major controlling effect of andrographolide for these cytokines. In a number of mediators the IC50 observed is significantly below that of NF-κB activation, this may be due to an undetermined secondary or specific mechanism of action. It is probable that inhibition is happening at both a transcriptional level and directly on the enzyme for nitric oxide and PGE2. Investigation of the mechanism for VEGF, NO, PGE2, IL-6, IFN-γ and
TNF-α is warranted.
The IC50 results for andrographolide in decreasing order are shown in Table 17.
Table 17: IC50's in order of activity of andrographolide activity Pure Non-polar A. paniculata Polar Combined Assay andrographolide fraction commercial fraction (µM*) (µM) (µM*) extract (µM*) (µg/ml)
VGEF IC-50 6 ~10.5 7.1 6.5 ~31.97
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NO IC50 in RAW264.7 7.4 7.2 6.3 6.9 330.6 cells PGE2 IC50 in RAW 264.7 8.8 6.1 7.5
cells IL-6 IC50 10.2 11.1 7.9 9.6 21.7
IFN-γ IC50 11.6 15.5 16.8 13.9 ~31.04
IP-10 IC-50 12.8 15.6 17.9 14.1 9.1
TNF-α IC50 16.5 22.3 17.5 18.3 ~109.6
IL-1β IC50 18.2 11.3 13 22.3 221.6 No fit IL-2 IC 20.4 24.9 22.3 ~32.1 50 possible
BFGF IC50 21.4 NA 22.9 NA 44.2 Eotaxin 22.7 ~30.1 21.8 22.1 45.6
IL-4 IC50 23.2 31.5 28 27.4 48.1 TNF-α IC50 in RAW 23.3 22.3 22.8
264.7 cells
G-CSF IC50 25.1 46.9 32 31 355.6 NF-κB activation IC50 in 26 21.4 23.7 24.6 625.2 RAW264.7 cells No fit MCP-1 31.8 18.5 14 ~141.1 possible GM-CSF 47.7 68.6 49.6 54.7 166.4 IC50 No fit IL-1ra IC 54.8 NA 31.4 30.14 50 possible No fit RANTES NA NA NA NA possible *normalised to andrographolide concentration
The inhibition of IL-1ra, IL-2 and IL-4 is not desirable for an anti-inflammatory drug as their roles as cytokines is considered anti-inflammatory. Andrographolide inhibited IL-1ra (IC50
54.8 μM), IL-2 (IC50 20.4 μM) and IL-4 (IC50 23.2 μM), however not as potently as major inflammatory cytokines. The IC50 confidence interval was within agreement with NF-κB activation and so the inhibition of these cytokines may occur due to this mechanism of action.
However further experiments are required to confirm this.
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3.5. Conclusions of chapter 3
Andrographolide is solely accountable for the detectable activity of the A. paniculata commercial extract on:
The activity of inflammatory enzymes, iNOS and COX in RAW264.7 cells.
The release of cytokines, VGEF, IL-6, IFN-γ, IP-10, TNF-α, IL-1β, IL-2,
BFGF, eotaxin, IL-4, G-CSF and GM-CSF which are linked to inflammatory
by THP-1 cells.
The activation of NF-κB in RAW264.7 cells.
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Chapter 4
______4. In vitro comparison of Andrographolide to
common NSAIDs
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4.1. Introduction to chapter 4
NSAIDs are the current pharmacotherapy of choice for inflammation related diseases. With inflammation being implicated in an increasing number of diseases and the ageing population, the use of NSAIDs is growing strongly. Their use comes at a cost, with NSAIDs being linked to serious adverse side effects including gastric toxicity and cardiovascular failure. The adverse effects are directly linked to NSAID’s mechanism of action, namely inhibition of prostaglandin production by the cyclooxygenase enzymes. Due to the widespread reliance on NSAIDs there are practices in place to control the impact of their side effects, however, an alternative medication is needed. Inflammation is a complex response and there are a number of mediators that control its severity. NSAIDs focus on reducing prostaglandin levels, however, prostaglandins are only one mediator in the complex inflammatory response mechanism. Additionally their reduction leads to negative side effects. In this chapter, andrographolide, a potential anti-inflammatory compound isolated from a traditionally used Ayruvedic herb A. paniculata, is compared to NSAIDs in a variety of in vitro cell based inflammatory assays. A broad array of inflammatory mediators was assayed, this reflecting the complex mechanism of inflammation, not just the prostaglandin reduction that NSAIDs are targeted to and is the cause of their side effects. By approaching inflammation in this way it is hoped that a more holistic treatment will emerge with reduced side effects but increased efficacy.
4.1.1. The use of NSAIDs
NSAIDs are one of the most widely used therapeutic agents [191]. They are used for relieving both pain and inflammation [192]. NSAIDs are often self-medicated for the treatment of short term inflammation and pain, such as headaches, injury, cold, flu, muscular
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pain and menstrual pain. Common over-the-counter (OTC) NSAIDs available in Australia include aspirin, ibuprofen (Neurophen), paracetamol (Panadol) and diclofenac (Voltaren). A
US poll of more than 2,000 adults found that approximately 30% used OTC NSAIDs for pain on a regular basis [193]. The most commonly used NSAIDs are diclofenac (27.8 %), ibuprofen (11 %) and naproxen (9.4 %) in Australia by sales and this is reflected globally
[194]. Intriguingly, diclofenac is by far the most popularly used NSAID despite having a risk to reward ratio identical to that of Rofecoxib, which was withdrawn from the market due to safety concerns [195]. NSAIDs are also the pharmacotherapy of choice for chronic inflammatory diseases including asthma, rheumatoid arthritis, and inflammatory bowel disease [196]. The use of NSAIDs, especially chronic use, increases with age with an estimate of up to 40% of people aged over 65 using NSAIDs daily [197, 198].
4.1.2. Increasing use of NSAIDs
With the high prevalence of chronic use among the elderly, society’s dependence on NSAIDs is growing. The number of people aged 80 and over will increase dramatically in the near future, and it is predicted that the proportion of citizens above the age of 60 will double between now (2015) and the year 2025 [21]. Consequently, age-related diseases are projected to be the leading cause of disability by the year 2020, imposing a significant burden on affected individuals and on society in general [22]. Age is identified as a leading risk factor for many diseases such as acute and chronic neurodegenerative disease, cardiovascular disease, diabetes, cancer and degenerative musculoskeletal disease. There is increasing evidence suggesting that chronic inflammation is a contributing factor in these age-related diseases [16-20]. With the increasing elderly population and the growing implication of inflammation in a wide range of age related diseases, chronic use of NSAIDs is set to rise
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rapidly. However, the widespread use of NSAIDs comes at a price - NSAIDs can cause serious gastrointestinal toxicity [24] and some have been linked to increased blood pressure, greatly increasing the risk of congestive heart failure and thrombosis [25].
4.1.3. Side effects of NSAIDs
NSAIDs are often naively perceived as a safe over the counter medicine without awareness of their potential adverse effects [196]. A combined US study of 9,062 adults found OTC
NSAIDs were used on an as-needed basis, with 40% using prescription and OTC NSAIDs at the same time and 26% using OTC NSAIDs above the recommended dose [199]. NSAIDs are responsible for the largest number of hospital admissions for adverse prescription drug reactions, with a UK study finding they are responsible for 29.6% of all hospital admissions for adverse drug reactions [25]. The risks of adverse events increase with long-term use but
NSAIDs remain the major pharmacotherapy for a number of chronic inflammatory conditions like asthma, rheumatoid arthritis and inflammatory bowel disease. The serious adverse effects of NSAIDs are well understood to be largely related to their mechanism of action [200].
4.1.4. NSAID mechanism of action
NSAIDs target the prostaglandin G/H synthase enzyme (COX) that is responsible for the production of PGs which are implicated in inflammation [26]. There are two major isoforms of COX, COX-1 and COX-2. Many NSAIDs inhibit both COX enzymes to different degrees.
COX-1 is expressed constitutively in most tissue, giving the impression its major role is housekeeping. This impression gave rise to the theory that COX-1 inhibition is the major cause of the side effect observed with NSAID treatment. COX-2 on the other hand is subject 148
to rapid induction by inflammatory cytokines and mitogens, so is thought to be exclusively linked to PG production at sites of inflammation [27, 28]. It was thought that COX-2 selective inhibitors would reduce the side-effects and increase the potency of NSAIDs.
COX-2 conformational protein studies revealed that COX-2 has a wider binding site compared to COX-1, favoring targeted inhibition [201]. The rapid development of selective
COX-2 inhibitors ensued which ultimately led to the development of selective COX-2 inhibitors. These, however, failed to mitigate the side effects and in some cases increased the cardiovascular side effects leading to the withdrawal of a selective COX-2 inhibitor,
Rofecoxib from the North American market. The major failure of NSAIDs is that their mechanism of action is directly linked to the adverse side effect [200]. Due to the prevalence of adverse events and an understanding of their mechanism of action, the side effects of
NSAIDs are well understood leading to a number of strategies to help mitigate them.
4.1.5. Strategies to reduce NSAIDs’ adverse effects
Despite the adverse effects of NSAIDs and selective COX-2 inhibitors, in the majority of cases there is no viable alternative. Recently TNF-α inhibitors have been developed that are appropriate alternatives in some cases and steroid based anti-inflammatory drugs are used in other cases [23, 202, 203]. The side effects of steroids limit their use and popularity- in fact it led to the coining of the term “Nonsteroidal” to differentiate NSAIDs from the adverse events of steroids used in the 1950s [204]. There are a number of strategies employed to reduce the adverse effects of NSAIDs, with gastrointestinal damage being reduced with the co- prescription of proton pump inhibitors [205] and lower risk NSAIDs are used with patients identified as having a high cardiovascular risk [206]. This has led to the continual increase in non-selective NSAID (as tested in this study), where selective COX-2 inhibitors is in decline
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[207, 208]. These efforts to mitigate the adverse events highlight the need for NSAIDs despite the cost which underpins the need to develop novel and safe anti-inflammatory medicines [209].
4.1.6. Alternatives to NSAIDs
In vivo inflammation is a complex interaction of different cells and signals [210]. Targeting just one major pathway (PG, NSAIDs) for inflammation, especially one linked to so many harsh side effects, is a very limited approach to solving a complex problem. The development of TNF-α inhibitors shows a step away from the traditional prostaglandin focused pathway.
However, it is still limited to a specific pathway where there are many other inflammatory mediators that also contribute.
NSAIDs were simplistically developed to target specific enzymes and have been linked to serious side effects. Traditional systems of medicine developed with no knowledge of enzymes are likely less targeted to COX inhibition than modern NSAIDs. Traditional medicines have been developed based on human studies and efficacy, with no in vitro bias toward COX inhibition. Herbs/components of herbs are theorised to act synergistically on separate related pathways and systems to produce a stronger but less focused effect than modern pharmaceuticals [211-214]. It is plausible that traditionally used anti-inflammatory drugs may target other mediators of inflammation and present a different approach to anti- inflammatory development that is not narrowly focused on a pathway that has been linked to many serious adverse effects.
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4.1.7. Andrographolide as an alternative to NSAIDs
Andrographolide is an ent-labdane diterpenoid and the major secondary metabolite isolated from the historically used medicinal herb, Andrographis paniculata (Acanthaceae) [42, 167,
168, 175, 215-227]. A. paniculata is used as a herbal medicine in both traditional Indian and
Chinese medicine (where it is known as Kalmegh and Chuanxinlian, respectively) to treat a wide variety of ailments linked to inflammation [228, 229]. Andrographolide has been shown to be the major active component and inhibits a wide range of inflammatory mediators and accounts for most of the activity of the herb. Andrographolide is a potential lead compound for the development of new anti-inflammatory compounds guided by historic use and not by specific COX inhibition. Intriguingly, andrographolide has been reported to exhibit gastro- protective and ulcer preventive effects, which, combined with its well documented anti- inflammatory effects could make it a safe alternative to traditional NSAIDs [230]. A proprietary A. paniculata extract (HMPL-004, Hutchison MediPharma) is under development for the treatment of inflammatory bowel disease [231, 232] and is currently being tested in a global phase III clinical trial (http://clinicaltrials.gov/show/NCT01805791).
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4.2. Aims of chapter 4
The aims of this chapter are to compare current NSAIDs to andrographolide in in vitro inflammation assays to assess their effect on:
The activity of inflammatory enzymes, iNOS and COX
The release of inflammatory cytokines
The activation of NF-κB
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4.3. Methods
The drugs and standards used in this study were of known purity and obtained from reputable sources. The acetylsalicylic acid (aspirin, 99%), Ibuprofen sodium salt (98%), diclofenac
(99%), acetaminophen (Paracetamol, 99%), prednisone (98%), dexamethasone (97%) were purchased from Sigma (St. Louis, USA). CAY 10602, 95%, Zileuton (98%), Ro 106-9920
(95%) and phenethyl caffeiate (CAPE, 98%) were purchased from Cayman Chemical
(Michigan, USA). Andrographolide (99%) was purchased from Biopurify (Chengdu, China).
The cell culture reagents used were certified suitable for in-cellular applications. DMEM,
Penicillin and Pen Strep, MTT and GlutaMax® (Glutamine) were obtained from Invitrogen
(Carlsbad, USA). The RPMI medium, NED, sulphanilamide, ATRA, dibutyryl cyclic adenosine monophosphate (db-cAMP), albumin and BSA, monobasic phosphate (Na2HPO4), dibasic phosphate (NaH2PO4) Resazurin, LPS from E. Coli strain 0111:B4, 3,3,5,5- TMB, and citric acid were purchased from Sigma-Aldrich (St. Louis, USA). The Murine IFN-γ and murine TNF-α ELISA kit were from Peprotech, (Rocky Hill, USA). PMA was purchased from Fluka (Steinheim, Germany). The FBS (French origin) was purchased from Bovogen
Biologicals (Keilor East, Australia).
4.3.1. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells.
NO release was quantified by Griess reagent as described in section 2.5. Briefly the
RAW264.7 cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was removed (180 μl) and reacted with Griess reagent (100 μl) to calorimetrically quantify dissolved nitrates. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
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4.3.2. Determination of TNF-α release in LPS and IFN-γ stimulated RAW264.7 cells.
TNF-α was quantified by ELISA as explained in section 2.6. Briefly the cells were seeded at
1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO
(final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A MTT solution (60 μl) was used to assess the viability of the remaining cells.
4.3.3. Determination of PGE2 release in LPS and IFN-γ stimulated RAW264.7 cells.
PGE2 release was quantified by competitive ELISA as described in section 2.7. Briefly the cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was tested (180 μl) in the PGE2
ELISA. A MTT solution (60 μl) was used to assess the viability of the remaining cells.
4.3.4. Determination of 27 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay
The release of 27 cytokines was quantified by multiplex assay as described in section 2.9.
5 Briefly the cells were seeded at 1×10 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ. After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
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4.4. Results and discussion
4.4.1. Nitric oxide production by RAW264.7 cells
The andrographolide and NSAIDs were tested in the NO assay to determine their IC50 for inhibiting NO production by LPS and IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 72. It can be seen that andrographolide is more potent at inhibition of NO production.
NO production by RAW 264.7 cells
110
100
90
80 Ibuprofen 70 Aspirin Diclofenalac 60 Paracetamol Naproxen 50 Andrographolide
40 %NO production %NO
30
20
10
0 1 10 100 1000 10000 M drug -10
Figure 72: NO dose response curve for andrographolide (red circles), diclofenac (green triangles), aspirin (brown squares), paracetamol (blue inverted triangles), ibuprofen (grey circles) and naproxen (pink circles) by LPS and IFN-γ by stimulated RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
NO is a mediator of inflammation. This experiment was carried out to compare andrographolide’s inhibition of NO production to that of the NSAIDs: diclofenac, aspirin, paracetamol, ibuprofen and naproxen. Andrographolide inhibited NO production at much
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lower doses than NSAIDs. As NSAIDs are not targeted at inhibition of iNOS it is not surprising that they show little to no activity. On the other hand andrographolide showed strong inhibition.
4.4.2. PGE2 release by RAW264.7 cells
PGE2 is a major inflammatory mediator and a product of the enzyme target by NSAIDs. The pure andrographolide and NSAIDs were tested in the PGE2 assay to determine their IC50 for inhibiting PGE2 release by LPS and IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 73. It can be seen that NSAIDs are more potent at inhibition of
PGE2 release.
.
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PGE2 release by RAW264.7 cells 110
100
90
80
70
60 Andrographolide Aspirin
release 50 Diclofenac 2 Paracetamol Ibuprofen
40 % PGE %
30
20
10
0 0.0001 0.001 0.01 0.1 1 10 100 1000
-10 M drug
Figure 73: PGE2 dose response curve for andrographolide (red circles), diclofenac (green inverted triangles), aspirin (brown triangles), paracetamol (blue diamond) and ibuprofen (grey circles) by LPS and IFN-γ by stimulated RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
In Figure 73, it can be seen that andrographolide is a weaker inhibitor of PGE2 than diclofenac and ibuprofen, but is similar to aspirin and paracetamol. The COX enzyme, which produces PGE2, is the pharmacological target for NSAIDs. This causes and anti-inflammatory effect and has been linked to side effects [200]. Andrographolide inhibits PGE2 production at about the same potency as weak non selective NSAIDs such as aspirin and paracetamol.
Diclofenac and ibuprofen are stronger PGE2 inhibitors but they are not considered selective
COX-2 inhibitors though they show some selectivity for COX-2 [196]. Compared to andrographolide, diclofenac and ibuprofen are much more potent at inhibiting COX. The long
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term use of diclofenac and ibuprofen is associated with side effects [196] while paracetamol and aspirin are considered comparatively safer [233]. Aspirin has even been shown to reduce the risk of cardiovascular disease (opposite to the side effect of selective COX-2 inhibitors)
[234].
Andrographolide displayed less potency than the semi selective COX-2 inhibitors, diclofenac and ibuprofen, making it appear inferior as a treatment for inflammation. However, PGE2 inhibition has been shown to be an undesirable pharmacological target, due to side effects.
Furthermore, the inflammatory response involves many other mediators that should also be considered in assessing anti-inflammatory potential.
4.4.3. TNF-α release by RAW264.7 cells
TNF-α is a mediator of inflammation. The pure andrographolide and NSAIDs were tested to determine their IC50 for inhibiting TNF-α release by LPS and IFN-γ stimulated RAW264.7 cells. This experiment was carried out to compare andrographolide’s inhibition of TNF-α to
NSAIDs. The dose response curve is shown in Figure 74. It can be seen that andrographolide is more potent at inhibition of TNF-α.
.
.
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TNF- released by RAW264.7 cells 110
100
90
80
70
60
release 50
Andrographolide 40 Aspirin
% TNF- % Ibuprofen 30 Diclofenac Paracetamol
20
10
0
-10 0.1 1 10 100 1000 10000 M drug
Figure 74: TNF-α dose response curve for andrographolide (red circles), diclofenac (green diamond), aspirin (brown triangles), paracetamol (blue circles) and ibuprofen (grey inverted triangles) by LPS and IFN-γ by stimulated RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
TNF-α is a cytokine which is a mediator of inflammation. This experiment was carried out to compare andrographolide’s inhibition of TNF-α release to that of the NSAIDs; diclofenac, aspirin, paracetamol and ibuprofen. Andrographolide inhibited TNF-α release at much lower doses than the NSAIDs, and the only NSAID that showed any detectable activity diclofenac.
As NSAIDs are not targeted at inhibition of TNF-α release, it is not surprising that they show little to no activity. On the other hand andrographolide displayed strong inhibition of TNF-α which is considered an alternative anti-inflammatory treatment to NSAIDs [203].
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4.4.4. Multiplex assay
The release of 17 cytokines by LPS stimulated THP-1 cells was determined by multiplex bead based ELISA. The experiment was performed to determine if andrographolide and the
NSAIDs had an inhibitory effect on the 17 cytokines and chemokines linked to inflammation.
The commercially available kit was expensive, limiting the number of replicates to 2 and the range of doses. This limitation means that the statistical significance of the results is also limited. The dose response curves are shown in figures 76 to 84 and IC50 summarised in
Table 18.
TNF- released by THP-1 160
140
120
100 Andrographolide Diclofeanac
release Aspirin 80 Paracetamol Ibuprofen
60 % TNF- %
40
20
0 1 10 100 1000 10000 Dose (M)
Figure 75: TNF-α dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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IFN- release by THP-1 cells 160
140
120
100
80
release
60 Andrographolide
% IFN- % Diclofeanac 40 Aspirin Paracetamol Ibuprofen 20
0
1 10 100 1000 10000 Dose (M)
Figure 76: IFN-γ dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-1 release by THP-1 cells 200
100
release 0 10 100 1000 10000 Dose (M) Andrographolide % IL-1 % Diclofeanac Aspirin -100 Paracetamol Ibuprofen
-200
Figure 77: IL-1β dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The data was not constrained to bottom equalling 0 as this did not reflect the data fit. (n=2)
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IL-2 release by THP-1 cells 160
140
120
100
80
60 Andrographolide
%IL-2 release %IL-2 Diclofeanac 40 Aspirin Paracetamol Ibuprofen 20
0
1 10 100 1000 10000 Dose (M)
Figure 78: IL-2 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-4 release by THP-1 cells 160
140
120
100
80
60 Andrographolide
%IL-4 release %IL-4 Diclofeanac 40 Aspirin Paracetamol Ibuprofen 20
0
1 10 100 1000 10000 Dose (M)
Figure 79: IL-4 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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IL-6 release by THP-1 cells 160
140
120
100
80
60 Andrographolide
%IL-6 release %IL-6 Diclofeanac 40 Aspirin Paracetamol Ibuprofen 20
0
1 10 100 1000 10000 Dose (M)
Figure 80: IL-6 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
G-CSF release by THP-1 cells 160
140
120
100
80
60 Andrographolide Diclofeanac %G-CSF release %G-CSF 40 Aspirin Paracetamol 20 Ibuprofen
0 10 100 1000 10000 Dose (M) -20
Figure 81: G-CSF dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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GM-CSF release by THP-1 cells 160
140
120
100
80
60 Andrographolide
Diclofeanac %GM-CSFrelease 40 Aspirin Paracetamol Ibuprofen 20
0
1 10 100 1000 10000 Dose (M)
Figure 82: GM-CSF dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
MCP-1 release by THP-1 cells 160
140
120
100
80
60
40 Andrographolide
20 Diclofeanac %MCP-1release Aspirin 0 10 100 1000 10000 Paracetamol -20 Dose (M) Ibuprofen
-40
-60
Figure 83: MCP-1 dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown triangles), paracetamol (blue inverted triangles) and ibuprofen (grey diamonds) by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. The data was not constrained to bottom equalling 0 as this did not reflect the data fit. (n=2)
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Due to the high cost of the multicytokine assay, it was not possible to optimise the parameters for each cytokine, as a result of this not all of the 17 cytokines assayed produced usable results. Indeed, due to the limitations of the single cell and model not all cytokines were released by LPS stimulated THP-1 cells. These results are summarised in, Table 18.
4.4.5. NF-κB activation assay
Andrographolide and diclofenac at high doses have shown activity against a number of inflammatory cytokines and chemokines, suggesting an overall controlling factor. In contrast, the other NSAIDs have not (unsurprisingly) but show activity exclusively to PGE2 inhibition.
As NF-κB is the transcription factor that controls the transcription of many inducible inflammatory mediators, if NSAIDs inhibited its activation, reductions in downstream inflammatory cytokines would have been observed in the multicytokine assay. It is therefore unlikely that NSAIDs inhibit the activation of NF-κB at doses below 100 μM. However, to complete the comparison, the dose response for NF-κB testing is shown in Figure 84.
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NF-B activation assay in ELAM9 RAW264.7 cells 120
110
100
90
80
70
60
50 % of GFP expressing cells % GFP expressing of 40 Andrographolide Aspirin 30 Ibuprofen Paracetamol Diclofenac 20 Prednisone
10
0 1 10 100 1000 10000 M drug
Figure 84: NF-κB activation dose response curve for andrographolide (red circles), diclofenac (green squares), aspirin (brown crosses), paracetamol (blue circles) and ibuprofen (grey crosses) by LPS and IFN-γ stimulated ELAM9 RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model.
It can be seen in Figure 84 that andrographolide and diclofenac (to some extent) both inhibit
NF-κB activation in a dose dependent manner. However, andrographolide is far more potent.
Diclofenac’s ability to inhibit NF-κB activation at high doses (>100 μM), could account for its inhibition of multiple cytokines at similarly high doses.
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4.4.6. Summary
A summary of the IC50s for the in vitro anti-inflammatory assays is given in Table 18.
Table 18: Summary of IC50s for andrographolide, diclofenac, aspirin, paracetamol and ibuprofen.
Andrographolide Diclofenac Aspirin Ibuprofen Assay Paracetamol (µM) (µM) (µM) (µM) (µM) RAW264.7
NO IC50 7.4 222 >1600 1058 2763
CI 95% IC50 6.7 to 8.1 v. wide 169 to 292 949 to 1180 2406 to 3174
PGE2 IC50 8.8 ~ 0.008 14.1 7.73 0.0925
CI 95% IC50 7.4 to 10.4 v. wide 10.1 to 19.7 6.14 to 9.73 0.0769 to 0.111
TNF-α IC50 23.3 >333 >1600 >6000 >1500
CI 95% IC50 20.1 to 27.0 THP-1
TNF-α IC50 29.3 ~469 >1000 >6000 ~1671
CI 95% IC50 24.7 to 34.7 v. wide v. wide
IFN-γ IC50 ~31.4 ~162 >1000 >6000 ~1574
CI 95% IC50 v.wide v. wide v. wide
IL-1β IC50 18.1 ~151 >1000 ~4362 ~1241
CI 95% IC50 5.1 to 63 v. wide v. wide v. wide
IL-2 IC50 35.7 ~365 >1000 ~6000 ~1663
CI 95% IC50 28.3 to 45.0 v. wide v. wide v. wide
IL-4 IC50 32.8 ~326 >1000 >6000 ~1519
CI 95% IC50 28.2 to 38.3 v. wide v. wide
IL-6 IC50 12.2 ~189 >1000 ~920 ~907
CI 95% IC50 9.1 to 16.2 v. wide v. wide v. wide
G-CSF IC50 ~32.2 ~213 >1000 ~6831 ~1800
CI 95% IC50 v.wide v. wide v. wide v. wide
GM-CSF IC50 65.2 ~405 >1000 >6000 >1500
CI 95% IC50 31.5 to 135.0 v. wide MCP-1 ~28.6 ~314 >1000 ~2936 ~872
CI 95% IC50 v.wide v. wide v. wide v. wide RAW264.7
ELAM9 GFP NF-κB 26.0 508.3 >1600 >6000 >1500 activation IC50 23.4 to 29.0 400.3 to 645.4 Failed IL-5 < 2 pg/ml detection limit IL-7 < 0.8 pg/ml detection limit
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IL-10 < 1.5 pg/ml detection limit IL-12 < 2.4 pg/ml detection limit IL-13 < 1.8 pg/ml detection limit IL-8 > 6200 pg/ml detection limit MIP-1b > 4000 pg/ml detection limit
As can be seen in Table 18, andrographolide inhibited multiple mediators of inflammation where most NSAIDs showed little or no activity. The inhibition of IL-2 and IL-4 is not desirable for an anti-inflammatory drug as their roles as cytokines are considered anti-inflammatory. Andrographolide inhibited IL-2 (IC50 35.7 μM) and IL-4 (IC50 32.8 μM), however, not as potently as major inflammatory cytokines such as IL-6 (IC50 12.2 μM) and
IL-1β (IC50 18.1 μM).
The mechanism of andrographolide action was not determined in this study, however, it would appear from the IC50 that andrographolide may be acting by inhibition of NF-κB activation, as the IC50 observed (23.4 to 29.0 μM) is in agreement or below most of the assayed mediators. The major inflammatory cytokines IL-6 (IC50 12.2 μM) and IL-1β (IC50
18.1 μM), showed IC50s below this, so it is possible that there is a specific undetermined mechanism for their inhibition, which warrants further study. For an estimation of the IC50s in vivo potential, the reported Cmax plasma concentrations for andrographolide and NSAIDs are compared in Table 19.
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Table 19: PGE2 IC50s for andrographolide and NSAIDs, compared to reported plasma Cmaxs.
Andrographolide Diclofenac Aspirin Ibuprofen Assay Paracetamol (µM) (µM) (µM) (µM) (µM)
PGE2 IC50 8.8 ~ 0.008 14.1 7.73 0.0925 *minimal IC 50 7.4 (NO) ~151 (IL-1β) >1000 ~920 (IL-6) ~872 (MCP-1) from Table 18 Reported 3.8 to 34.2*** 4.4 to 9.4 111 to 422 46 to 79 359 to 394 Plasma Cmax**
*excluding PGE2. ** the Cmax is typically reported as μg/ml however was converted to μM to enable comparison to IC50’s which are reported in μM. The literature references for the reported ranges are; diclofenac [235],aspirin [236], paracetamol [237] and ibuprofen [238]. *** up to 3.8 μM reported in human plasma [239] up to 34.2 μM in rat plasma [240].
Cmax plasma levels are only a guide used to assess pharmacological potential, and in vivo testing is needed to determine if there is a pharmacologically significant effect. In addition, the IC50 concentrations reported are nominal concentrations and the “actual” concentration at the site of interaction between andrographolide and their potential (extra- and/or intracellular) target proteins (receptor(s) or enzymes), have not been determined. NSAIDs have displayed
COX inhibitory activity and at pharmacological doses are specific to COX inhibition, which is supported by the PGE2 results in this study.
Andrographolide was shown to inhibit NO production, PGE2 and TNF-α release and NF-κB activation in RAW264.7 cells and TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, G-CSF, GM-CSF, and MCP-1 in human THP-1 cells, below or within (the uncertainty) the maximum reported
Cmax in rats [240]. Andrographolide has a greater widespread anti-inflammatory effect than
NSAIDs and may be better suited to the complex nature of the inflammatory immune response.
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NSAID’s defined mechanism of action makes understanding and rationalising their anti-inflammatory effect simpler. Their side effects are easily linked to the mechanism of action because as it is clearly understood. Andrographolide’s widespread action makes its safety and side effects more difficult to predict. However, A. paniculata has been used for thousands of years in Ayurvedic medicine and is considered safe [39-43]. Andrographolide has been shown to have a high safety margin with the 50% lethal dose (LD50) for andrographolide through intraperitoneal routes being 11.46 g/kg [241]. Andrographolide has also been reported to exhibit gastro-protective and ulcer preventive effects, which, combined with its anti-inflammatory effects could make it a safe alternative to traditional NSAIDs
[230]. Taking all evidence together andrographolide presents as a promising alternative to
NSAIDs for broad inflammatory aliments but further study is required to establish this.
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4.5. Conclusions of chapter 4
Pure andrographolide was tested in and compared to common NSAIDs, in in vitro anti- inflammatory assays where the NSAIDs diclofenac and ibuprofen were found to be more potent inhibitors of LPS and IFN-γ induced PGE2 production in RAW264.7 cells.
Andrographolide showed comparable potency to the NSAIDs, aspirin and paracetamol.
Andrographolide was found to be more potent than any NSAID tested at inhibition of:
The activity of inflammatory enzymes, iNOS and COX in RAW264.7 cells.
The release of cytokines linked to inflammation, TNF-α, IFN-γ, IL-1β, IL-2,
IL-4, IL-6, G-CSF, GM-CSF, and MCP-1 by THP-1 cells and TNF-α and by
RAW264.7 cells.
The activation of NF-κB in ELAM9 RAW264.7 cells.
Andrographolide may have pharmacological anti-inflammatory activity based on the inhibition of a wide range of inflammatory mediators.
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Chapter 5
______5. Synthesis and in vitro assessment of the
sulphonation metabolite of andrographolide
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5.1. Pharmacokinetics of herbal medicines
The use of HM has grown steadily over the two last decades [242, 243]. This may be due to an increasing view that natural products are safer and have fewer side effects. With the rapid growth in modern use of HM, there has been an increase in the in vitro and clinical evidence that supports supposed efficacy. Unlike synthetic pharmaceutical products, HMs can be marketed without strong supporting clinical evidence. Due to the time and cost of clinical studies, in vitro data is more abundant for most HMs. However there is often a disconnect between in vitro data and the clinical results. One of the major hurdles faced by many HMs is that the dose required to reach the pharmaceutical threshold of activity is not being reached due to poor bioavailability of the active components. For the more popular HMs, such as A. paniculata, processing techniques like forming liposomes and nanoparticles have been used to try to boost bioavailability. However if the concentration of an active constituent is too high, toxic effects may be observed. There are 5 main factors that are considered when assessing pharmacokinetics of a drug candidate:
Liberation - the process of release from the compounding of the drug. As traditional
medicines were often administered as a water decoction, liberation is a modern
dosage issue.
Absorption - the process of the drug crossing the lipophilic cell linings of the
digestive tract or skin and entering the blood stream.
Distribution - the transport of substances throughout the fluids and tissues of the body.
Some drugs are soluble in the blood stream and some interact with proteins in the
blood stream.
Metabolism - the irreversible transformation of parent compounds into daughter
metabolites. The metabolites can be active (biotransformation) or inactive.
Excretion - the removal of substances (parent and metabolites) from the body.
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There are many parameters that effect bioavailability. It can be estimated by molecular size, shape, functional groups and lipophilicity, however, administration of the substance and analysis of the blood remain the most widely accepted method.
5.1.1. Andrographolide’s Cmax in human plasma
The maximum plasma levels observed are a good indicator of the compound’s absorption and bioavailability, but there are other factors that play a role in the compound’s in vivo efficacy.
Despite the widespread use of A. paniculata, there are relatively few human pharmacokinetics studies. Figure 85 shows the Cmax for the human pharmacokinetics studies reported.
1.6
1.34 1.4
g/ml) 1.2 μ 1
0.8
0.6 0.39 0.4 0.34
Andrographolide Cmax ( 0.16 0.2 0.063 0.058 0.06 0 2000 2000 2006 2007 Jian-jun, 2007 Jian-jun, 2007 Gu, 2009 Xu, Panossian, sigle Panossian, Wangboonskul, tablet dispersion (~0.71mg/kg) (~2.86mg/kg) dose steady state (~1.27mg/kg) tablet (0.3mg/kg) (0.3mg/kg)
Figure 85: Human plasma Cmax of andrographolide.
The dose is given in mg (andrographolide) / kg (body weight).
As can be seen in Figure 85, the reported Cmax varies between 0.058 to 1.34 μg/ml. In humans andrographolide appears to be rapidly absorbed and excreted. Administration of
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andrographolide (1 mg/kg/day) produced maximal plasma levels (1.34 µg/ml) after 1.5-2 h, with a half-life of 6.6 h in humans [239]. The drug appears to be non-toxic and well tolerated by humans with no serious adverse effects at doses in the range of 1 to 2 mg/kg/day [244,
245].
5.1.2. Andrographolide’s Cmax in animal plasma
There are a greater number of animal pharmacokinetics studies that administer andrographolide at higher doses, as presented in Figure 86.
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8
7 Rats
Rabbits
6
Chickens g/ml)
μ 5
4
3 AndrograpohlideCmax( 2
1
0 2000 2000 2000 2014 2007 Chen 2007 Suo 2011 2009 Yu- 2014 Wu 2009 Maiti 2014 Bera 2012 Du 2014 Wu 2009 2014 2009 Liu 2014 Chen 2014 Chen 2014 Wu 2011 Ye Panossian Panossian Panossian Zhang (rabbits, (rats, 10 Chellapillai Fang (rats, 20 (rats, 25 (rats, 30 (rabbits, (rats, 40 Akowuah Wang (chickens, (rats, 50 (rats, ~50 (rats, 80 (rats, 120 (rats, 1 (rats, (rats, 3 (rats, 5 8.8 mg/kg) mg/kg) (rats, 10 (rabbits, mg/kg) mg/kg) mg/kg) 40 mg/kg) mg/kg) (rats, 44 (rats, 48.75 50 mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) steady 1 mg/kg) mg/kg) mg/kg) ~20 mg/kg) mg/kg) mg/kg mg/kg)
Increasing andrographolide dose
Figure 86: Cmax of andrographolide in animals.
The dose is given in mg (andrographolide) / kg (body weight).
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As can be seen in Figure 86, the plasma levels of andrographolide are higher in animals with higher doses being administered. However, in these animal studies, reported plasma Cmax is not always dependent on the dose, especially when comparing dosing levels between studies.
This is assuming that the observed variation in reported Cmax’s are not due to different procedures employed in the plasma extraction and analysis. However, it should be noted that the result obtained is significantly dependent on the analytical method employed. An extreme example is Panossian et al. [246], reporting plasma levels of 1.3 μg/ml with a dose of 1 mg/kg in rats, whereas at a dose 120 times higher Ye et al. [247] report a Cmax of 0.23 μg/ml.
The dosage form and preparation may play as important role as the dose level. The study by
Chen et al. [189] which compared pure andrographolide pharmacokinetics to that of equivalent andrographolide amounts administered as A. paniculata extract, found that the dosage form as a whole herb greatly improved pharmacokinetics. This would support the important role of dosage form in the final plasma levels. The reason(s) for this is likely to be a complex interplay of factors and requires further research.
5.1.3. Andrographolide’s Cmax after formulation modification
There have been a number of attempts to improve pharmacokinetics, these are presented in
Figure 87.
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14
12 12 Rats
10 Humans 9.64
g/ml) μ 8 6.79
6 5.37
AndrograpolideCmax ( 4 3.06 2.67 1.98 2 1.6 1.74 0.83 0.063 0.058 0.13 0.13 0 2011 2011 2012 Du 2012 Du 2007 Jian- 2007 Jian- 2009 Maiti 2009 Maiti 2007 Suo 2007 Suo 2009 Yu- 2009 Yu- 2014 2014 Chellapillai Chellapillai (rabbits) (rabbits, jun jun (rats) (rats, (rats, (rats, Fang Fang Zhang Zhang (rats) (rats, micro (humans, (humans, liposomes) tablet) liposomes) (rabbits) (rabbits, (rats) (rats, nano) emulsion) tablet) dispersion micro micelles) tablet) powder)
Figure 87: Cmax for modified andrographolide formulations.
Note: Figure organised according to principal author of study.
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Attempts to improve the pharmacokinetics by altering the dosage form, as shown in Figure
87, produced mixed results. Many of the studies found no improvement while increasing the
Cmax of andrographolide in the plasma. Chellapillia et al. prepared andrographolide in nanoparticles sensitised to release at pH 5.5, preserving them from degradation in the stomach and targeting the upper GI tract [248]. Micro emulsions were also shown to improve pharmacokinetics in rabbits [249]. The highest plasma levels reported for andrographolide were by Suo et. al. [240] and Matiti et al. [250] with their liposome preparations. These modifications may improve the pharmacokinetics of andrographolide in humans.
5.1.4. A. paniculata pharmacokinetics
Considering the popularity of A. paniculata as a medicine there is a surprising lack of high quality pharmacokinetic studies. Of the studies available, there are many inconsistencies making comparisons difficult. In some studies andrographolide dose is not reported, only the grams (or tablets) of A. paniculata administered. Sometimes the dose is reported but not the weight of the animals, so conversion to a comparable dose unit (dose/kg) is not possible. The pharmacokinetic studies of A. paniculata are summarised in Table 20.
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Table 20 Pharmacokinetic studies of andrographolide
Publication Subject Analytical method Dose equivalent Cmax plasma Tmax Comments andrographolide
1982 Zheng Mice 3H Labelled Intragastrically (i.g) 0.5 h The 3H-andrographolide administered i.g rapidly was absorbed and [251] andrographolide and intravenous distributed to organs in 30 min maximum. By 24 h 90 % was (i.v) excreted in urine and faeces, with only 10 % accounted for at 48 h as andrographolide, the rest was metabolised. 1995 Wang Rabbit Chemiluminescence 22.4 µg/ml The andrographolide rapidly reached maximum concentration, the [252] half-life of elimination is long and bioavailability high. The study lacked detail. 2000 Rats HPLC-UV/PDA 1 mg/kg i.v 5.6 µg/ml 0.25 h The bioavailability of andrographolide at 1 mg/kg and 10 mg/kg Panossian n=42 1 mg/kg i.g 1.3 µg/ml single 2 h was 91 % and 21 % respectively, 90 % and 99 % was reported to be [239] Methanol acetone 5.4 µg/ml steady metabolised. The pharmacokinetics in humans was highly variable extraction 10 mg/kg i.g 3 µg/ml 2 h but was explained by an open two-compartment model. In the two compartment model the concentration of the drug is first highest in organs with high blood flow i.e. brain, liver, lungs, then disperses Humans GC-MS ~0.3 mg/kg i.g 0.39 µg/ml (1.12 1.5 h to the rest of the body. Sites of inflammation often attract higher n=16 µM) single blood flow, so andrographolide may have a targeted effect, with Methanol and C18 Tablet higher concentrations at the site of inflammation. Up to 55 % of SPE extraction therapeutic dose 1.34 µg/ml (3.8 andrographolide was reported to be bound to protein. Renal regimen µM) steady excretion is not the main route for eliminating andrographolide. It is most likely intensely and dose dependently metabolised. All doses (Kan Jang tablets) were administered in Kan Jang tablets. Elimination halftime is 2-7 h in humans. The study uses a low dose, typical therapeutic dose is 60 mg. 2003 He Rats 1HNMR 13CNMR 120 mg/kg Metabolite of andrographolide was isolated and characterised as a [253] sulphonate metabolite.
2005 Wen- Mice HPLC-UV 14-deoxy-11,12- 14-deoxy-11,12- 0.22 h 14-deoxy-11,12-didehydroandrographolide was quantified in the tao [254] didehydroandrograp didehydroandrogr mice plasma. Methanol acetic acid holide apholide acetic ester extraction (Chuanxinlian 8 µg/ml preparation) i.g 2005 Dog Ion-pair HPLC-UV Sodium bisulphite Sodium bisulphite The andrographolide sodium bisulphite was administered by i.v so Jin [255] (beagle) i.v Initial, 0.5 h, 1 h, bioavailability was not a factor. There is a sulphonated
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Dichloromethane and 20 mg/kg 2 h andrographolide commercial injection XiYanPing that could be tetrabutyl ammonium similar to sodium bisulphite andrographolide studied, but bromine extraction 100, 66, 36,10 insufficient information was published to allow comparison. The µg/ml sodium bisulphite could also be a metabolite of andrographolide produced by its extensive metabolism when ingested. 40 mg/kg 180, 77, 36, 14 µg/ml
50 mg/kg 265, 113, 53, 22 µg/ml 2006 Human HPLC-UV 30 mg i.g (A. Nil Andrographolide was only detected in plasma for the highest dose. Wangboons n=6 paniculata tablet) Another peak was detected, assumed to be a metabolite that had a kul [256] C-18 SPE quicker elution time on the C18 column and is likely more polar. 59 mg i.g Nil
89 mg i.g 0.34 µg/ml 1 h 2007 Hong Human HPLC-MS 1 g Chuanxinlian (3 0.15 µg/ml 1.5 h 14-deoxy-11,12-didehydroandrographolide has rapid absorption [257] n=8 herbs combination) dehydro and long elimination rate after oral administration. i.g 2007 Suo Rats HPLC-UV 10 mg/kg liposomes 12 µg/ml max, The pharmacokinetic of liposome injection is explained by the two [258] i.v second peak 4 compartment model. Rapid absorption with concentrations up to 12 CHCl3 extraction μg/ml in whole ug/ml then second peak (250 min) as disperses from liver and blood spleen into blood. The tablet followed a one compartment model. The chromatogram showed there is a more polar peak that is larger 10 mg/kg 1.6 µg/ml in 1 h than andrographolide and not in the blank plasma, likely a Tablet i.g whole blood metabolite. 2007 Jian- Human LC-MS/MS Dispersion tablet 0.063 µg/ml 1.5 h The two preparations, standard and dispersion tablets, had jun [259] n=20 equivalent pharmacokinetics. Standard tablet i.g 0.058 µg/ml 1.6 h
2007 Gu Human HPLC-MS 50 mg/person 0.16 µg/ml 2 h The andrographolide follows a one compartment model in this [260] n=15 tablet study. Ethyl acetate extraction 2007 Chen Rabbit HPLC-UV online Aqueous ethanol 2.28 µg/ml The pharmacokinetics followed the one compartment model for [261] C18 SPE extract 8.8 mg /kg (6.5µM) both andrographolide and 14-deoxy-11,12- andrographolide andrographolide didehydroandrographolide. The chromatogram showed several
181
Plasma other peaks that may be metabolites or other diterpenes.
20.7 mg 14-deoxy- 1.33 µg/ml 14- 11,12- deoxy-11,12- didehydroandrograp didehydroandrogr holide apholide
2008 Rat HPLC-UV 44 mg/kg i.g (1 After administration 0.88% of the andrographolide was excreted in Akowuah g/kg A. paniculata the urine by 24 h. There was an increase in the total antioxidant [262] extract) status and reduction in lipid peroxidation of the urine collected at 24 h. 2009 Rat HPLC-UV 44 mg/kg i.g (1g/kg 1.42 µg/ml 3 h Rats were under anaesthesia for the whole pharmacokinetics study - Akowuah n=6 Acetonitrile A. paniculata this may have influenced the distribution rate and metabolism. [263] extraction extract)
2009 Xu Human HPLC-ESI-MS/MS 200 mg/person 0.06 µg/ml 1.6 h This was a high dose, triple the normal dose, with no reported side [264] N=20 (~3mg/kg) effects. The plasma level reported was extremely low. Panossian et Methanol extraction i.v al. [239] found that with a dose 10 times less they detected 7 times more andrographolide in plasma.
2009 Liu Chicken HPLC-UV 5 g/kg (~ 50 mg/kg 1.6 µg/ml 0.9 h Although the content of 14-deoxy-11,12-didehydroandrographolide [265] n=10 andrographolide) andrographolide is lower than that of andrographolide in A. paniculata, mean Ethyl acetate Ultra-fine A. residence time of 14-deoxy-11,12-didehydroandrographolide was extraction paniculata powder longer than andrographolide in chicken plasma. The 1.4 µg/ml 14- 0.6 h pharmacokinetics is described well by a two-compartment model deoxy-11,12- didehydroandrogr apholide 2009 Maiti Rats HPLC-UV 25 mg/kg 6.79 µg/ml 2.5 h Liposome complexed with andrographolide had improved [250] n=10 andrographolide (19.4 µm) pharmacokinetics, higher Cmax and longer retention. Centrifuged and evaporated
25 mg/kg 9.64 µg/ml (27.5 4 h andrographolide µM) liposomes
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2009 Yu- Rabbits HPLC-UV 2 g/kg (approx. 0.13 µg/ml 0.6 h Pharmacokinetics followed the two compartment model. The levels Fang [266] 20mg/kg andrographolide of andrographolide reported are likely wrong. In the chromatogram andrographolide) they appear to have quantified the wrong peak, possibly i.g powder 5.1 µg/ml 0.65 h isoandrographolide, as the andrographolide appears to co-elute with 14-deoxy-11,12- a peak in the blank plasma. This is supported by Liu. et al. [265], didehydroandrogr Chen et al. [261] and Wu et al. [267] showing andrographolide is apholide more abundant in the herb and comparable in the body when ingested.
2 g/kg (approx. 20 0.13 µg/ml 0.5 h mg/kg andrographolide andrographolide) i.g micro powder 9.7 µg/ml 14- 0.6 h deoxy-11,12- didehydroandrogr apholide
2010 Levita Mice Radionuclide The andrographolide was distributed to all organs with the highest [268] labelling of distribution in the stomach. andrographolide
2011 Ye Rats UPLC-MS/MS 120 mg/kg i.g 0.23 µg/ml 0.5 h The absolute bioavailably was reported to be very low (2.67%), this [247] n=5 may be due to the high dose used, as Panossian et al. [239] reported that as the dose was increased the bioavailability dropped significantly. Efflux at the terminal ileum and colon was found to contribute to low bioavailability at high doses by Ye et al. [247].
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24 mg/kg i.v 7.5 µg/ml (at This may have also been due to Ye’s sample preparation, dissolving 0.5h) andrographolide (not very water soluble) in ice cold water, then filtering through a 0.2 μm, likely losing large amounts of the andrographolide, so the dose delivered may not be lower than assumed. This would account for the low Cmax, even with the dose 10x-100x most other studies. Ye also reports that andrographolide has high permeability and that this is not the reason for its poor bioavailability, but its rapid and extensive metabolism to 14-deoxy- 12-sulfoandrographolide is. Ye et al. detected ~14x levels of the metabolite in the blood and organs. 2011 Rats HPLC-UV 10 mg/kg (nano) i.g 2.67 µg/ml (nano) 0.25 h The nanoparticle encapsulation greatly increased the uptake speed Chellapillai and maximum plasma concentration. It was sensitised to release [248] Chloroform below pH 5.5, targeting the upper GI tract. extraction 10 mg/kg i.g 0.83 µg/ml 1 h
2012 Du Rabbits HPLC-UV 40 mg/kg i.g 3.06 µg/ml 1 h The micro emulsions improved the rate of uptake and the amount of [249] n=5 andrographolide detected in the plasma. Du et al. [249] also Chloromethane 5.37 µg/ml Micro 0.5 h performed an in vivo anti-inflammatory experiment using an extraction emulsion albumin induced rat paw oedema model. Andrographolide (50 mg/kg) matched the positive control (aspirin, 150 mg/kg) and the micro emulsions outperformed the positive control at all doses (4, 8 and 16 mg/kg). The LD50 of the micro emulsion preparation was determined in mice to be 138.36 mg/kg, 266 times the daily oral dose (~0.5 mg/kg). 2012 Zhang Dog HPLC-MS/MS 50 mg/kg i.v 100 µg/ml max [269] (Beagle) andrographolide sodium bisulphite 2012 Chong Rats UPLC-ESI-MS/MS 1 mg/kg i.g 0.8 µg/ml 5 min The sulphonated form of 14-deoxy-11,12- [270] n=6 14-deoxy-11,12- didehydroandrographolide reached much higher plasma didehydroandrograp concentrations. holide
6 mg/kg i.g 14- 5 µg/ml 5 min deoxy-11,12- didehydroandrograp holide sulphonate
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2013 Liu Rats HPLC-UV Andrographolide 132.33 µg/ml 2 h The sulphonated form of andrographolide reached much higher [271] derivative i.g andrographolide plasma concentrations compared to other studies where the Cmax derivative reported for andrographolide i.g is 12 μg/ml [240]. 2013 Yang Rats UPLC-MS/MS 5 mg/kg i.v 48 µg/ml 14- This study reports that 14-deoxy-12-hydroxy-andrographolide is a [272] n=6 deoxy-12- phase one metabolite of andrographolide, but there was no hydroxy- experimental evidence presented in the paper or reference for the andrographolide claim. 0.6 µg/ml andrographolide 2014 Wu Rats HPLC-UV 20 mg/kg i.g 0.9 µg/ml 1 h Andrographolide was quantified in organs after i.g administration [273] n=5 of the 80mg/kg dose - the highest level of sulphonate metabolite HCl ethylacetate 40 mg/kg i.g 3.9 µg/ml 1.1 h was observed in the liver, which may be a main metabolising organ. extraction 80 mg/kg i.g 5.5 µg/ml 1.5 h On the contrary, increasing concentrations of sulphonate metabolite 80 mg/kg i.v 18 µg/ml in kidney over time indicated that renal excretion might be a major elimination route. Meanwhile, at 45 min the high levels in the pancreas demonstrated that it might be the target organ. The concentration of sulphonate metabolite in brain samples was consistently low at all the time points, indicating its poor penetration across the blood-brain barrier in the range of 1 μg/ml. 2014 Chen Rats n=3 LC-MS ESI 50 mg/kg i.g 0.35 µg/ml 0.5 h Andrographolide was absorbed more rapidly, to a higher [189] concentration and retained for longer when administered at the Acetonitrile 940 mg/kg A. 0.86 µg/ml 0.35 h same dose as in the herb as compared to the pure compound. There extraction paniculata was a 5x increase in bioavailability from 1 to 5% when andrographolide was administered as the whole herb extract. 2014 Zhang Rats n=5 LC-MS 5 mg/kg i.v 1.98 µg/ml The area under the curve and retention time was longer for the [274] 5 mg/kg micelles 1.74 µg/ml micelles. Methanol extraction i.v 2014 Bera Rats n=8 LC-MS/MS 30 mg/kg i.g 0.12 µg/ml 0.75 h More andrographolide was detected in the organs when [275] administered as the herb and it is retained for longer. Acetonitrile extraction 2014 Wang Rats LC-MS/MS 750 mg/kg i.g A. Andrographolide was reported to be unstable in plasma, but [267] paniculata stabilised by acidification. The metabolite had a molecular weight 48.75 mg/kg 0.18 µg/ml 0.3 h of 368, however, no structure was proposed. andrographolide 0.36 µg/ml 0.7 h metabolite 23.25 mg/kg 0.15 µg/ml 0.3 h neoandrographolide
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3 mg/kg 14- 0.06 µg/ml 0.3 h deoxyandrographoli de 25.5mg/kg 14- 0.39 µg/ml 0.3 h deoxy-11,12- didehydroandrograp holide 14.25 mg/kg 0.3 µg/ml 1.7 h chlorogenic acid 7.5 mg/kg apigenin- 0.1 µg/ml 6 h 7-O-β-D- glucuronopyranosid e
2015 Tian liver Glucironidation was the main metabolism pathway reported for [276] microso humans, pigs, dog and monkey. mes
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As can be seen in Table 20, there is wide variation in the reported pharmacokinetics of andrographolide. Taken together, the research presented in Table 20 sheds some light on its pharmacokinetics. It is clear that andrographolide is absorbed to some extent and passes into the blood stream with Tmax’s of between 0.5 to 2 h. Andrographolide is quantifiable in the μg to ng/mg range in the reported studies. The lower doses tested in humans may limit the plasma concentrations being observed and higher doses may be advisable. Promisingly, it is reported to follow a 2 compartment model, being distributed first to areas of high blood flow.
This is desirable for anti-inflammatory compounds, as areas of inflammation attract increased blood flow, meaning that andrographolide might be considered a targeted approach for treating inflammation.
A. paniculata appears to be non-toxic even at very high doses in animals [277] and is well tolerated. A. paniculata has a high safety margin with the LD50 of the alcohol extract is
1.8g/kg [278]. When administered as the pure andrographolide the safety LD50 is higher. The
LD50 for andrographolide through intraperitoneal routs is 11.46 g/kg [241]. Based on these findings, andrographolide and A. paniculata extracts show promise as potential leads for the development of novel safe anti-inflammatory drugs. Further investigations into their molecular effects would therefore be justified.
5.1.5. Biological significance of andrographolide’s sulphonation metabolite
One of the major drawbacks of andrographolide’s pharmacokinetics is it is highly and rapidly metabolised. It has been reported that even when andrographolide is administered
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intravenously, the plasma levels still decline rapidly. The major metabolite observed is a sulphonation product of andrographolide, shown in Figure 88.
O O
O O OH
SO3H
CH3 CH3 CH2 CH2
HO HO
H3C CH2OH H3C CH2OH
Sulphonation metabolite of andrographolide Andrographolide
Figure 88: Molecular structure of the sulphonation metabolite of andrographolide and andrographolide
As evident in Figure 88, much of the structure of andrographolide is conserved in the metabolite. The conserved structure might mean that the bioactivity is conserved. The sulphonation metabolite has been tested in vivo by Guo et al. [279] in a mouse sepsis model.
The sulphonated metabolite was shown to outperform andrographolide in preventing death from sepsis in rats.
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5.2. Aims of chapter 5
The aims of this chapter are to report on the synthesis of the sulphonation metabolite of andrographolide and test it in comparison to andrographolide in a number of in vitro anti- inflammatory assays, to determine if the anti-inflammatory activity is conserved on:
The activity of inflammatory enzymes iNOS
The release of inflammatory cytokines
The activation of NF-κB
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5.3. Methods
5.3.1. Materials
The andrographolide (98%) used for the synthesis of the metabolite was purchased from
Biopurify (Chengdu, China). The XiYanPing (Xiyanping Zhusheye) was purchased from
Jiangxi Qingfeng Pharmacy Co. Ltd (Ganzhou , China).
5.3.2. LC-MS reagents
The reagents used in mass spectroscopy (MS) analysis were of high purity and appropriate for MS analysis. Formic acid (~ 98%) and acetonitrile (MS grade) used for the chromatography were obtained from Sigma (Sydney, Australia). The nitrogen gas (HP grade) and argon gas (HP grade) were supplied by BOC gases (Sydney, Australia).
5.3.3. HPLC system
HPLC-PDA was used to profile the products of the sulphonation of andrographolide and the fraction that was identified as the metabolite. The HPLC-PDA analysis was performed on
Shimadzu UFLC system (Shimadzu, Australia) comprising of a LC-30AD pumps, SIL-
30ACHT auto sampler, SPD-M20A PDA detector and DGU-20A5 inline solvent degasser.
The system was controlled using the Shimadzu Class-VP 7.4SP4 software.
5.3.4. Preparative HPLC system
Isolation of the synthesised andrographolide sulphonation metabolite was performed on a
Shimadzu preparative HPLC system (Shimadzu, Australia) comprising of a 2 × LC-20AP 190
pumps, SIL-20AHT auto sampler, SPD-20A UV/Vis detector, DGU-20A3 inline solvent degasser and FRC-10A fraction collector. The system was controlled using Labsolutions software.
5.3.5. LC-MS instrumentation
Samples and standards were analysed using a Waters Inc. (Waters, Australia) Acquity ultra high performance liquid chromatography (UPLC) with a Xevo TQ MS tandem MS detector.
Data processing was performed using the Waters MassLynx V4.1 SCN 714 software.
5.3.6. Proton nuclear magnetic resonance (1HNMR) spectrometer
For the analysis of the andrographolide sulphonated metabolite we used a Varian 300 MHz spectrometer (Palo Alto, USA). The Varian Mercury 300 MHz spectrometer was equipped with a 5 mm 4-nuclei (1H, 13C, 31P and 19F) inverse probe.
5.3.7. Synthesis of andrographolide sulfonate metabolite
The method to convert andrographolide to its sulphonated form was based on that described by He et al. [253]. Andrographolide (196 mg) was dissolved in ethanol (15 ml), to which an aqueous solution of Na2SO4 (2 ml, 1 M), H2SO4 (2.4 ml, 2% v/v) and water (8 ml) was added.
The mixture was refluxed (40 min) and the pH adjusted to ~7 using Na2CO3 solution (1 M) and the solvent removed in vacuo. The residue was then dissolved in water (20 ml) and washed with chloroform (3 × 25 ml). The aqueous layer was collected and evaporated in
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vacuo. The residue was dissolved in methanol and filtered, yielding the andrographolide sulfonate upon evaporation (210 mg).
5.3.8. HPLC-PDA analysis of sulphonation products
This method was used to determine the sulphonation products, the fractions collected of the sulphonation products and the commercial XiYanPing. The sulphonation products and fractions were dissolved (200 μg/ml) by sonication in 50 % aqueous acetonitrile. The concentrated XiYanPing was dissolved in minimal 50 % aqueous acetonitrile by sonication.
The HPLC-PDA analysis of the extracts was performed using an Alltech Alltima (Alltech
Australia) reverse phase C18 column (46 × 150 mm I.D., 5 µm) with a Phenomenex
(California, USA) Security C18 guard column (20 mm × 4 mm, 5 μm).
HPLC-PDA profiles were generated by a 5 µl injection of samples. The mobile phase consisted of 0.1 % (v/v) aqueous formic acid (mobile phase A) and 0.1 % (v/v) formic acid in acetonitrile (mobile phase B). The gradient program was 10% B for 3 min with a linear increase to 40% B at 30 min, and then a 95% B (wash) for 5 min before equilibrating at the starting composition for 5 min. Mobile phase flow rate was maintained at 1.3 ml/min. The
PDA was set to read absorbance from 200 to 500 nm.
5.3.9. Preparative HPLC to partition the sulphonate product
The method of synthesising the andrographolide sulphonate produced a mixture of products which had to be resolved by preparative HPLC. The sulphonation products were dissolved in
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a minimal amount of 50 % aqueous acetonitrile by sonication. A Shimadzu Shimpack PRC-
005(H) (250 × 20 mm, 5μm) semi preparative column was used.
LC fractionation was performed with repeated 50 µl injections of the sample. The mobile phase consisted of 0.1 % (v/v) aqueous formic acid (mobile phase A) and 0.1 % (v/v) formic acid in acetonitrile (mobile phase B). The gradient program was 23% B with a linear increase to 56% B at 21.5 min, and then a 95% B (wash) for 5 min before equilibrating at the starting composition for 10 min. Mobile phase flow rate was maintained at 5 ml/min. The chromatogram was visualised at 226 nm.
The fractions collected were pooled and neutralised with ammonia (3%) solution and evaporated to dryness by rotary evaporation followed by drying under a stream of nitrogen.
The residue from each fraction was dissolved in a minimum volume of water by sonication and passed through a C18 SPE column. The column was washed with Milli-Q water and eluted with methanol. The eluate was filtered through a 0.2 μm syringe filter and evaporated to dryness to yield the dry fraction.
5.3.10. The characterisation of the sulphonation metabolite of andrographolide by 1HNMR
1HNMR spectroscopy was used to determine the structure of the andrographolide sulphonated metabolite in comparison to that reported by He et al. [253] . The sample was manually analysed at 298 K. A standard proton pulse sequence was used with the following parameters. Flip angle 60 º, relaxation delay 1.5 s, complex points 16 000, dummy scans = 4,
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number of scans =64, zero filling 64 K, line broadening 0.3 Hz. The 300 MHz Varian spectrometer was manually tuned before analysis and MeOD was used as the lock signal, the sample was manually shimmed.
5.3.11. Characterisation of the sulphonation metabolite by LC-MS
The sulphonation metabolite of andrographolide was characterised by LC-MS. The sulphonation product was dissolved (2 μg/ml) by sonication in 50 % aqueous acetonitrile.
The analytical column was a Waters ACQUITY UPLC BEH reversed phase C18 column (2.1 x 50 mm, 1.7 µm), with a Waters reversed phase C18 guard column.
LC-MS analysis was performed with a 1 µl injection of the sample. The mobile phase consisted of 0.1 % (v/v) aqueous formic acid (mobile phase A) and 0.1 % (v/v) formic acid in acetonitrile (mobile phase B). The gradient program was 10% B held for 1 min with a linear increase to 35% B at 15 min, and then a 35% B (wash) for 2 min before equilibrating at the starting composition for 3 min. Mobile phase flow rate was maintained at 0.1 ml/min.
The MS was operated using (-)-ESI with a desolvation temperature of 350 ºC at 650 l/h, capillary voltage of -2500 V, cone voltage of 60 V, collision energy of 2 units and collision gas flow of 0.15 ml/min. The m/z range of 290 to 450 was scanned with a scan time of 1 s.
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5.3.12. Determination of NO production in LPS and IFN-γ stimulated RAW264.7 cells.
NO release was quantified by Griess reagent as described in section 2.5. Briefly the
RAW264.7 cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was removed (180 μl) and reacted with Griess reagent (100 μl) to colourmetrically quantify dissolved nitrates. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
5.3.13. Determination of TNF-α release in LPS and IFN-γ stimulated THP-1 cells.
TNF-α was quantified by ELISA as explained in section 2.6. Briefly the THP-1 cells were seeded at 1×105 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ (50 ng/ml, 50 units/ml). After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
5.3.14. Determination of 27 cytokines released by LPS stimulated THP-1 cells using a
multiplex assay
The release of 27 cytokines was quantified by multiplex assay as described in section 2.9.
5 Briefly the cells were seeded at 1×10 cells/well in a 96 well plate for 48 h. The compounds of interest were added in DMSO (final concentration 0.1 % DMSO), 1 h before stimulation with LPS and IFN-γ. After 18 h the supernatant was tested (180 μl) in the TNF-α ELISA. A
MTT solution (60 μl) was used to assess the viability of the remaining cells.
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5.4. Results and discussion
5.4.1. Analysis of synthesised product
The andrographolide was successfully reacted with sulphuric acid by the method described in section 5.3.7 to yield a mixture of compounds as shown in Figure 89.
700 Sulphonation metabolite of andrographolide Andrographolide 600 standard
500
400
300 Absorbance (mAu) Absorbance
200
100
0 5 10 15 20 25 30 Retention time (min)
Figure 89: HPLC chromatogram of the sulphonation product overlaid with andrographolide standard at 210 nm.
Sulphonation product (black) and andrographolide standard (red). The overlay (red) was normalised to the absorbance of the largest peak in the sulphonation product with an offset of 100 mAu. One of the peaks eluting before andrographolide would be the sulphonated product because with a sulphate group attached, it would be more polar than andrographolide and hence elute earlier on a C18 column.
As can be seen in Figure 89 the andrographolide is nearly completely reacted but a mixture of products is formed. The reaction conditions were optimised, but a mixture was still left.
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5.4.2. Purification by preparative HPLC
The reaction mixture was separated by preparative HPLC. The chromatogram with the fractions collected is shown in Figure 90.
Datafile Name:sulf w3 b 004.lcd Sample Name:sulf005 Sample ID:sulf005 mV 250 UV VIS 226nm
225
200 Sulphonation metabolite of andrographolide
175
150
125
100
75
50
25
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
Figure 90: Preparative HPLC chromatogram of the sulphonation product at 226 nm.
The coloured sections (pink and purple) show the fractions that were collected for further analysis.
The collected fractions were rerun on analytical HPLC to assess their purity before characterisation. The HPLC for the sulphonation metabolite of andrographolide is shown in
Figure 91.
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1400 Sulphonation metabolite of andrographolide
1200
1000 Andrographolide 800
600 Absorbance (mAu) Absorbance 400
200
0 5 7 9 11 13 15 17 19 21 23 25 Retention time (min)
Figure 91: HPLC chromatogram of the metabolite overlaid with andrographolide at 210 nm.
Sulphonation product (brown) and andrographolide (red). The overlay (red) was traced from the andrographolide in Figure 89 with an offset of 100 mAu.
In Figure 91 it can be seen that the fraction collected for the sulphonation product of andrographolide contained a single detectable compound by HPLC-PDA analysis.
5.4.3. Characterisation of the sulphonation product of andrographolide by LC-MS
The sulphonation product of andrographolide was run on LC-MS to determine the likely molecular weight and detect other impurities that were not identified by HPLC-PDA. The
LC-MS total ion count (TIC) is shown in Figure 92, the m/z spectrum for the major peak at
11.2 min in the TIC is given in Figure 92.
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Figure 92: LC-MS TIC of the andrographolide sulphonate metabolite.
Figure 93: MS spectrum of the major peak in the andrographolide sulphonate metabolite TIC (Rt. 11.2 min).
5.4.4. Characterisation of the sulphonation product of andrographolide by 1HNMR
The sulphonation product of andrographolide was run on 1HNMR to determine if the compounds match the biologically significant metabolite reported by He et al. [253]. The 199
1HNMR is shown in Figure 94. A comparison between reported proton signals is given in
Table 21.
Figure 94: 1HNMR of the sulphonation product of andrographolide.
Table 21: Comparison between 1HNMR for synthesised and isolated sulphonation product of andrographolide and the biologically significant metabolite reported by He et al. [15]. Carbon No. Sulphonation metabolite Synthesised metabolite of identified by Yao [253] andrographolide 1 1.83 (H, m) 1.8 (H,m) 1.02 (H, m) 1.1 (H, m) 2 1.71 (2H, m) 1.7 (H, m) 3 3.30 (H, t, 9.8) 3.3 (H, t) 4 5 1.10 (H, dd, 12.6, 2.4) 1.1 (H, m) 6 1.28 (H, qd, 12.6, 4.2) 1.3 (H, m) 1.8 (H, m) 1.8 (H, m) 7 1.86 (H, m) 1.9 (H, m) 2.36 (H, m) 2.4 (H, m) 8 9 1.38 (H, Br.d, 11.8) 1.4 (H, m) 10 11 2.31 (H, dd, 12.2, 1.8) 2.4 (H, m) 2.088 (H, t, 12.6) 2.1 (H, t) 12 3.92 (H, dd, 12.2, 1.8) 3.9 (H, d)
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13 14 7.65 (H, t, 1.8) 7.7 (H, s) 15 4.95 (2H, o) 4.9 (H, m) 4.16 (H, dd, 10.2, 6.1) 4.2 (H, d) 16 17 4.87 (2H, o) 4.8 (H, m) 4.66 (H, br, s) 18 1.12 (3H, s) 1.1 (H, m) 19 4.05 (H, d, J = 11.4 Hz) 4 (H, d) 3.27 (H, d, J = 11.4 Hz 3.3 (H, m) 20 0.68 (3H, s) 0.7 (3H, s)
The numbered carbons shown in Table 21 are shown on the molecular structure in Figure 95.
O 15
16 14 O 13
12 SO H 11 3 20C H 1 3 9 CH2 2 17 10 8
3 5 7 6 HO 4 H C CH OH 3 19 2 18 Figure 95: Structure of sulphonation product with carbons numbered.
The 1HNMR of the isolated andrographolide sulphonation product matched that of the metabolite (in rats) of andrographolide’s spectrum reported by He et al. [253]. As a 300 MHz
NMR was used in this study and He et al. [253] were using a 600 MHz NMR their resolution could not be matched. However, the spectrum was a reasonable match with only the second
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signal for carbon number 17 not being detected. It is also not possible to confirm the stereo chemistry of carbon number 12 without further spectroscopic analysis.
It is likely that the sulphonation product of andrographolide is the biologically significant sulphonation metabolite of andrographolide reported by He et al. (in rats), synthesised in this study using a method adapted from the work of He et al. [253]. The molecular mass detected for the synthesised sulphonation derivative of andrographolide by LC-MS (414 m/z) and
1HNMR match that of the sulphonated product reported by He et al. [253]. It is therefore reasonable to conclude that the metabolite that was synthesised by sulphonation of andrographolide is the same as that reported by He et al. in rats [253].
5.4.5. Nitric oxide production in RAW264.7 cells
The pure andrographolide and synthesised andrographolide sulphonation metabolite were tested in the NO assay to determine their IC50 for inhibiting NO production by LPS and IFN-γ stimulated RAW264.7 cells. The dose response curve is shown in Figure 96. It can be seen that andrographolide is more potent at inhibition of NO than the sulphonation metabolite.
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NO production
100
80
60
%NO release 40
20 Metabolite Andrographolide
0 1 10 100 1000 10000 Dose (M)
Figure 96: NO dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
5.4.6. TNF-α release by THP-1cells
The pure andrographolide and synthesised andrographolide sulphonation metabolite were tested in the TNF-α assay to determine their IC50 for inhibiting TNF-α release by LPS and
IFN-γ stimulated THP-1 cells. The dose response curve is shown in Figure 97. It can be seen that andrographolide is more potent at inhibition of NO than the sulphonation metabolite.
The IC50s for andrographolide and the sulphonation metabolite are given in Table 22.
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TNF- released by THP-1
100
80
60
release
% TNF 40 Metabolite Andrographolide
20
0 1 10 100 1000 Dose (M)
Figure 97: TNF-α dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=9)
Table 22: IC50s for andrographolide and sulphonation metabolite TNF-α release inhibition
Sulphonation metabolite (μM) Andrographolide (μM)
IC50 6.9 23.3
95% confidence interval 4.1 to 11.5 20.1 to 27.0
As can be seen in Table 22 the sulphonation metabolite had significantly more potency at inhibiting TNF-α release. The biologically significant sulphonation metabolite of andrographolide is evidence of biotransformation for andrographolide, in terms of TNF-α release.
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5.4.7. Multiplex assay for 27 cytokines released from THP-1 cells
The sulphonation metabolite of andrographolide has shown improved anti-inflammatory activity reducing the release of TNF-α in THP-1 cells more potently than andrographolide.
Andrographolide was shown to be active at inhibiting a number of cytokines linked to inflammation (as discussed in chapters 3 and 4), the sulphonation metabolite could have maintained or enhanced similar broad activity.
The release of 27 cytokines by a multiplex bead based ELISA was performed on the supernatant of LPS stimulated THP-1 cells. The experiment was completed to determine if andrographolide and the sulphonation metabolite have an inhibitory effect on 27 cytokines and chemokines related to inflammation. The commercially available kit was expensive, limiting the number of replicates to 2. This limitation means that the statistical significance of the results is also limited. The dose response curves are shown in figures 99 to 114 and IC50s are summarised in Table 23.
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INF release by THP-1 cells 140
120
100
80
Androgragholide release
Metabolite
INF 60 %
40
20
0 0.1 1 10 100 Dose (M)
Figure 98: IFN-γ dose response curve for andrographolide (red circle) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-1 release by THP-1 cells
140
120
100
80 Androgragholide
Metabolite
release
60 %IL-1
40
20
0 1 10 100 1000 Dose (M)
Figure 99: IL-1β dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown diamonds), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 206
IL-1ra release by THP-1 cells
160
140
120
100
80
60
40 Andrographolide Metabolite
20 %IL-1ra release 0 1 10 100 -20 Dose (M)
-40
-60
-80
-100
Figure 100: IL-1ra dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-2 release by THP-1 cells
140
120
100
80 %IL-2 release 60
40 Androgragholide Metabolite
20
0 0.1 1 10 100 Dose (M)
Figure 101: IL-2 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 207
IL-4 release by THP-1 cells 120
100
80
60 %IL-4 release Androgragholide Metabolite 40
20
0 0.1 1 10 100 Dose (M andrographolide)
Figure 102: IL-4 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IL-6 release by THP-1 cells 120
100
80
60
Androgragholide
%IL-6 release 40 Metabolite
20
0 1 10 100 Dose (M)
-20
Figure 103: IL-6 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 208
IL-17 release by THP-1 cells 160
140
120
100
80 %IL-17 release 60
40 Androgragholide Metabolite 20
0 0.1 1 10 100 Dose (M)
Figure 104: IL-17 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
BFGF release by THP-1 cells
140
120
100
80
%BFGF release 60
40 Androgragholide Metabolite
20
0 0.1 1 10 100 Dose (M)
Figure 105: BFGF dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 209
G-CFS release by THP-1 cells
120
100
80
60 %G-CFS release
40
20
Metabolite Androgragholide 0
0.1 1 10 100 1000 Dose (M)
Figure 106: G-CFS dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown inverted triangles), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
GM-CFS release 120
100
80
60 %GM-CFS release Androgragholide 40 Metabolite
20
0 0.1 1 10 100 1000 Dose (M andrographolide)
Figure 107: GM-CFS dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown inverted triangles), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
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VEGF release by THP-1 cells 150
100 %VEGF release
50
Androgragholide Metabolite
0 0.1 1 10 100 Dose (M)
Figure 108: VEGF dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
IP-10 release by THP-1 cells 140
120
100
80
release 60
IP-10 % 40
Androgragholide Metabolite 20
0 1 10 100 Dose (M) -20
Figure 109: IP-10 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 211
MCP-1 release by THP-1 cells
100
80
60
MCP-1 releaseMCP-1 % 40
Androgragholide Metabolite
20
0 0.1 1 10 100 Dose (M)
Figure 110: MCP-1 dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
Eotaxin released by THP-1 cells
100
80
60
Androgragholide
Metabolite %Eotaxin release %Eotaxin 40
20
0 0.1 1 10 100 Dose (M)
Figure 111: Eotaxin dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2) 212
RANTES release by THP-1 cells 160
140
120
100
80 %RANTES release %RANTES 60
Androgragholide 40 Metabolite
20
0 0.1 1 10 100 Dose (M)
Figure 112: RANTES dose response curve for andrographolide (red circles) and the sulphonation metabolite of andrographolide (brown squares), by LPS stimulated THP-1 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
Due to the high cost of the multicytokine assay, it was not possible to optimise the parameters for each cytokine, as a result of this not all of the 27 cytokines assayed produced useable results. Indeed, due to the limitations of the cellular model (single cell type) not all cytokines were released by LPS stimulated THP-1 cells. These results are summarised in Table 23.
5.4.8. NF-κB activation assay in ELAM9 cells RAW264.7 cells
Andrographolide and the sulphonation metabolite have both shown activity against a number of inflammatory cytokines and chemokines, suggesting an overall controlling factor. NF-κB is the transcription factor that controls the transcription of many inducible inflammatory mediators. It is plausible that its inhibition could account for the observed wide spectrum effect. The dose response for NF-κB testing is shown in Figure 113.
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NF-B activation assay in ELAM9 RAW264.7 cells
100
80
60
B activation B
40 % NF- %
Metabolite Andrographolide 20
0 1 10 100 1000 Dose (M)
Figure 113: NF-κB dose response curve for andrographolide (red squares) and the sulphonation metabolite of andrographolide (brown circles), by LPS and IFN-γ stimulated ELAM9 RAW264.7 cells.
The data is fit with a log (inhibitor) vs. normalised response with variable slope model. (n=2)
It can be seen in Figure 113 that that andrographolide and the sulphonation metabolite both inhibit NF-κB activation in a dose dependent manner. However, andrographolide is far more potent.
5.4.9. Summary of results
The in vitro anti-inflammatory assay IC50s for andrographolide and the sulphonation metabolite are given in Table 23.
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Table 23: Summary of pure andrographolide and sulphonation metabolite IC50s
Assay* Andrographolide (µM) Sulphonation metabolite (µM) RAW 264.7
NO IC50 (n=9) 7.4 501.9
CI 95% IC50 6.7 to 8.1 376 to 669 THP-1
TNF-α IC50 (n=9) 16.5 6.8
CI 95% IC50 13.1 to 21.0 4.1 to 11.5
IFN-γ IC50 11.6 20.2
CI 95% IC50 7.7 to 17.6 2.5 to 162
IL-1β IC50 18.2 NA
CI 95% IC50 11.1 to 29.8
IL-1ra IC50 54.8 14.5
CI 95% IC50 v.wide 3.1 to 66.6
IL-2 IC50 20.4 26.1
CI 95% IC50 8.9 to 46.5 11.7 to 58.6
IL-4 IC50 23.2 34.3
CI 95% IC50 15.9 to 33.8 14.0 to 85.5
IL-6 IC50 10.2 6.6
CI 95% IC50 7.7 to 13.6 5.9 to 7.4
IL-17 IC50 37.7 3.1 to 453
BFGF IC50 21.4 30.8
CI 95% IC50 11.5 to 39.6 2 to 470
G-CSF IC50 25.1 Non convergent
CI 95% IC50 15.9 to 39.5
GM-CSF IC50 47.7 20.2
CI 95% IC50 35.3 to 63.5 10.2 to 40.0
VEGF IC50 6.0 97.2
CI 95% IC50 1.9 to 18.7 46.6 to 202.4
IP-10 IC50 12.8 17.8
CI 95% IC50 6.8 to 24.1 6.2 to 50.7 MCP-1 31.8 16.3
CI 95% IC50 27.3 to 37.1 9.3 to 28.4 Eotaxin 22.7 28.0
CI 95% IC50 14.5 to 35.4 11.9 to 65.6 RANTES NA NA RAW264.7 ELAM9 215
GFP NF-κB activation IC 834 50 26.0 (n=9) 23.4 to 29.0 764 to 910 Failed IL-5 < 2 pg/ml detection limit IL-7 < 0.8 pg/ml detection limit IL-9 There was no stimulation of release by LPS IL-10 < 1.5 pg/ml detection limit IL-12 < 2.4 pg/ml detection limit IL-13 < 1.8 pg/ml detection limit IL-15 < 1.3 pg/ml detection limit PDGF-bb < 32 pg/ml detection limit IL-8 > 6200 pg/ml detection limit MIP-1a > 5200 pg/ml detection limit MIP-1b > 4000 pg/ml detection limit *Where not defined n=2
As can be seen in Table 23 the IC50 for the sulphonation metabolite’s inhibition IC50 for
NF-κB activation is high (834 μM). The sulphonation metabolite showed much more potency at inhibiting the cytokines tested (>100 μM). It is therefore unlikely that activity of the sulphonate can be attributed to inhibiting NF-κB activation. However, it should be noted that both the assays, NF-κB and NO, where the sulphonation metabolite showed little activity were in a murine model. It is possible that the effect is species dependent or specific to either the THP-1 cells or RAW264.7 cells. Further experiments are required to determine this. The first experiment required would be testing the sulphonation metabolite’s activity on the release of cytokines like TNF-α and IL-6 in the RAW264.7 cells.
The inhibition of IL-1ra, IL-2 and IL-4 is not desirable for an anti-inflammatory drug as they have anti-inflammatory functions. Andrographolide inhibited IL-2 (IC50, 26.1 μM) and IL-4
216
(IC50, 34.3 μM), however, not as potently as major inflammatory cytokines such as TNF-α
(IC50, 16.5 μM) and IL-6 (IC50, 10.2 μM). The sulphonation metabolite’s inhibition of IL-1ra
(IC50, 14.5 μM) was potent, this may account for the sulphonation metabolite’s inability to inhibit IL-1β where the parent andrographolide was active, as IL-1ra is an agonist for IL-1β.
This warrants further study to determine the mechanism and confirmation of the effect.
In the majority of cytokines assayed (IFN-γ, IL-1ra, IL-2, IL-4, BFGF, GM-CSF, IP-10,
MCP-1 and Eotaxin), the sulphonation metabolite displayed similar activity to the parent andrographolide. It is therefore likely that for these cytokines the mechanism of action is conserved. This also suggests an additive effect may occur, where the metabolite and andrographolide equally contribute to the inhibition of the mediator. This would affect required in vivo doses to reach therapeutic effects. Dose response assays, where andrographolide and the sulphonate metabolite are added in combination, are needed to assess if this is occurring.
The sulphonation metabolite of andrographolide showed significantly greater activity than the parent compound andrographolide in the TNF-α and IL-6 assays. These are both major inflammatory mediators in the acute inflammatory response. This may be due to a specific undetermined mechanism. The significantly increased activity observed for the biological metabolite, over the parent compound, is evidence of biotransformation in rats.
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The sulphonation metabolite may be exclusively relevant to rats as a metabolite. A recent study by Tian et al. [276] showed that glucuronidation is the major metabolic pathway of andrographolide in humans, with the sulphonation pathway being species specific to rats.
This may limit the sulphonation metabolite’s biological significance to dose and pharmacokinetics to rats. Sulphonation metabolites are still reported in humans, however, are likely a minor metabolite, whereas they are the major metabolite reported in rats [272].
Despite the sulphonation metabolite’s biological significance to rats, in humans this appears to be minor. However, the sulphonation metabolite still showed high potency in the in-vitro assays and presents itself as a viable alternative anti-inflammatory drug to NSAIDs.
The pharmacokinetics of the sulphonation metabolite, reported in only a limited number of studies, were extremely promising. Ye et al. reported that the concentration for the metabolite in rat plasma and liver was ~14 times of that observed for the parent andrographolide [247].
Liu et al. reported that when the sulphonation metabolite of andrographolide was administered to rats as an i.g dose, the Tmax was reached after 2 h, with a Cmax plasma level of
132.3 μg/ml (319 μM). This reported Cmax is significantly (~47 times) higher than the IC50 value observed for TNF-α release inhibition in human THP-1 cells. The study is published in
Chinese, making assessment of the techniques used difficult. More pharmacokinetics studies of the andrographolide sulphonate are required to confirm this finding.
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5.5. Conclusions from chapter 5
The sulphonated metabolite of andrographolide was synthesised and tested in comparison to pure andrographolide in vitro. The sulphonation metabolite was found to have biological activity similar to andrographolide on the release of cytokines by THP-1 cells, cytokines include IFN-γ, IL-1ra, IL-2, IL-4, BFGF, GM-CSF, IP-10, MCP-1 and eotaxin which are linked to inflammation. The sulphonation metabolite was found to be less active on:
The activity of inflammatory enzymes, iNOS in RAW264.7 cells.
The activation of NF-κB in RAW264.7 cells.
The release of cytokines linked to inflammation, IL-1β, G-CSF and VEGF by
THP-1 cells.
The sulphonation metabolite was found to have higher potency than andrographolide on the release of cytokines linked to inflammation TNF-α, IL-6 and IL-17 by THP-1 cells.
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______6. Conclusions and future work
220
6.1.1. Conclusion
In contrary to the hypothesis, the A. paniculata commercial extract, with its complex
mixture of phytochemicals, exhibited less potent anti-inflammatory activity than the
pure andrographolide and the activity was attributed to andrographolide alone.
The A. paniculata commercial extract and andrographolide had a broader range of
anti-inflammatory activity on key mediators in cell line models of inflammation
compared to current NSAIDs.
The metabolites of andrographolide retained some anti-inflammatory activity of the
parent and showed improved inhibition of important inflammatory mediators TNF-α
and IL-6.
6.1.2. Future work
The commercial extract of A. paniculata was standardised to 30% w/w andrographolide concentration. It was shown that at this concentration andrographolide was solely accountable for the anti-inflammatory activity of the extract. Future work should be directed at the raw herb and lesser concentrated (for example, 5% or 10% w/w) standardised extract, to assess if the effect is conserved at lower andrographolide concentrations.
The andrographolide and A. paniculata were shown to be as effective as weak non selective
NSAIDs, paracetamol and aspirin. Further study should be directed to determining which specific enzyme is being inhibited, be it COX-1 or COX-2. This could be achieved by testing the activity of andrographolide, A. paniculata and NSAIDs in isolated preparations of COX-1
221
and COX-2. This would aid in assessing the mechanism of andrographolide’s inhibition of
PGE2 and determining if it is likely to have related side effects.
NF-κB activation inhibition was attributable to much of the observed inhibition of cytokines by andrographolide, this should be confirmed firstly with more replicates and concentrations.
In a number of mediators the IC50 observed is significantly below that of NF-κB activation and this may be due to an undetermined secondary or specific mechanism of action. An investigation of the mechanism for VEGF, NO, PGE2, IL-6, IL-1β, IFN-γ and TNF-α is warranted, firstly by confirming the result with more replicates and concentrations.
The reported Cmaxs for andrographolide in plasma, varied significantly between studies. It would be ideal to have human pharmacokinetics studies that:
test the pharmacokinetics of andrographolide, in known and increased doses, both at
steady repeated doses and single administration,
test different dosage forms of A. paniculata and andrographolide, for example,
liposomal enhancement in humans, which was reported to be effective in rat models,
quantify the metabolites of andrographolide in humans to aid future bioactivation
studies,
test the pharmacokinetics of andrographolide when administered as A. paniculata
tablets, tea, ethanol decoction and pure andrographolide, to determine what
components were reaching pharmacological levels of significance in traditional use
compared with modern use. It is suggested to confirm, in humans, if other
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components of the herb improve the pharmacokinetics of andrographolide as reported
in a rat model.
The andrographolide sulphonate had little activity in the NF-κB and NO assays. It is possible that the effect is species dependent or specific to either the THP-1 cells or RAW264.7 cells.
Further experiments are required to determine this. The first experiment required would be testing the sulphonation metabolite’s activity on the release of cytokines such as TNF-α and
IL-6 in the RAW264.7 cells.
The sulphonation metabolites inhibition of IL-1ra (IC50, 14.5 μM) was potent and this may account for the sulphonation metabolites inability to inhibit IL-1β where the parent andrographolide was active, as IL-1ra is an agonist for IL-1β. This warrants further study to determine the mechanism and confirm the effect with more replicates and concentrations.
The sulphonation metabolite of andrographolide showed significantly greater activity than the parent compound andrographolide in the TNF-α and IL-6 assays. These are both major inflammatory mediators in the acute inflammatory response. This may be due to a specific undetermined mechanism. Future experiments should be directed to confirm this observation with more replicates and concentrations before mechanistic studies.
There were impurities of salt and charcoal observed and low levels of comparable andrographolide sulphonation products in the commercial andrographolide sulphonation
223
product obtained from mainland China. This should be further investigate with confirmation of the salt and charcoal content and the sulphonation products. A wider study should investigate if these impurities are present in other similar commercial products.
The pharmacokinetics of the sulphonation metabolite is presented in a limited number of studies, however, was extremely promising. More pharmacokinetics studies of the andrographolide sulphonate are required to confirm these findings. As this preparation has not been traditionally used, safety of the drug should be determined, particularly as it can reach reported high plasma concentrations. These studies should not be conducted in rats if possible as they may have a yet undetermined specie specific resistance to the sulphonation metabolite. Pigs were reported to be the most appropriate model, by Tian et al. [276].
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