Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum
By
Kashif Maqbool Khan
CIIT/FA12-R60-004/ATD
PhD Thesis
In
Pharmacy
COMSATS University Islamabad, Abbottabad Campus - Pakistan
Fall, 2018 COMSATS University Islamabad
Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum
A Thesis Presented to
COMSATS University Islamabad, Abbottabad Campus
In partial fulfillment
of the requirement for the degree of
PhD (Pharmacy)
By
Kashif Maqbool Khan
CIIT/FA12-R60-004/ATD
Fall, 2018
ii Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum
A Post Graduate Thesis submitted to the Department of Pharmacy as partial fulfillment of the requirement for the award of Degree of Ph.D in Pharmacy.
Name Registration Number Kashif Maqbool Khan CIIT/FA12-R60-004/ATD
Supervisor
Dr. Abdul Manann Associate Professor Department of Pharmacy COMSATS University Islamabad, Abbottabad Campus
Co-Supervisor
Dr. Muhammad Arfan Associate Professor Department of Chemistry, School of Natural Sciences (SNS) National University of Sciences & Technology (NUST), Islamabad
iii
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vii
DEDICATION
Dedicated to my family and friends who were the pillars
of support during my PhD study
viii ACKNOWLEDGEMENTS
I bow my head before Almighty Allah, The omnipotent, The omnipresent, The merciful, The most gracious, The compassionate, The beneficent, who is the entire and only source of every knowledge and wisdom endowed to mankind and who blessed me with the ability to do this work. It is the blessing of Almighty Allah and His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal. . I would like to take this opportunity to convey my cordial gratitude and appreciation to my worthy, reverently and zealot supervisor Dr. Abdul Mannan, Associate Professor, Department of Pharmacy, COMSATS University Islamabad, Abbottabad campus, Pakistan. Without whose constant help, deep interest and vigilant guidance, the completion of this thesis was not possible. I am really indebted to him for his accommodative attitude, thought provoking guidance, immense intellectual input, patience and sympathetic behavior. I would like to pay my deepest gratitude and appreciation to one of the member of my supervisory committee and co-supervisor Dr. Muhammad Arfan, Associate Professor, School of Natural Sciences, National University of Sciences and Technology, Islamabad, Pakistan, for his generous cooperation and kind assistance and providing valuable suggestions during accomplishment of my Ph.D.
I am thankful to foreign supervisors Professor Dr. Satyajit D. Sarker and Dr. Lutfun Nahar, Medicinal Chemistry and Natural Products Research Group, School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, England, UK for their kind guidance and research assistance towards completing a part of my Ph. D. research work in UK. I am also extremely grateful to Dr. Ihsan ul Haq, Assistant Professor, Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan, for his valuable assistance, technical suggestions and kind help during the course of my Ph.D studies.
I am also very much grateful to Higher Education Commission (HEC), Pakistan, for awarding me six months foreign scholarship under International Research Support Initiative Program (IRSIP) to carry out a part of my research work in Liverpool John Moores University, England, United Kingdom. I am also highly indebted to my best ix friends and fellows especially, Kashif Bashir, Misbah-ud-Din Qamar, Muhammad Ubaid , Jawad Akbar Khan, and rest of my fellows for their assistance, good company, marvelous behavior and friendly attitude. I am also thankful to all the administrative and laboratory staff of the Department of Pharmacy, COMSTAS University Islamabad- Abbottabad campus for their kind support.
Last but not least, I really acknowledge and offer my heartiest gratitude to all members of my family especially, father, mother, wife, elder and younger brothers, sister and sweet daughter, Bareera Khan and son Saad Khan, for their huge sacrifice, moral support, cooperation, encouragement, patience, tolerance and prayers for my health and success which enabled me to achieve this excellent goal.
Kashif Maqbool Khan CIIT/FA12-R60-004/ATD
x ABSTRACT
Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum
This PhD thesis envisages the phytochemical and biological investigation of two important indigenous species of Pakistan. The main objective behind this investigation was to authenticate the folkloric history of these species. Asparagus adscendens Roxb. (A. adscendens), is native to the Himalayas. This plant has been used in the prevention and effective treatment of various forms of cancers. Trillium govanianum Wall. (T. govanianum), is a native species of the Himalayas. In folk medicine the plant has been reported for the treatment of wound healing, sepsis and in various sexual disorder
Finely ground roots of A. adscendens and T. govanianum were macerated in methanol and extracted through solid-phase extraction by using gradient solvent system (water: methanol) It was further proceeded for analysis of fingerprint high performance liquid chromatography - photodiode array and highly sensitive liquid chromatography- electrospray ionization-quadrupole time of flight- mass spectrometry to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain phenolic, flavonoids, saponins or spogenin as possible contributors to the significant antioxidant, antimicrobial, antileishmanial and cytotoxic activities of the extracts and its fractions.
Reverse phase HPLC-PDA based quantification revealed the presence of significant amount of quercetin, myricetin and kaempferol ranging from 23.31 to 234.23 & 0.221to 0.528 μg/mg DW for A. adscendens and T. govanianum, respectively. Moreover, in this study about 154 compounds have been identified by using both positive and negative ion mode liquid chromatography - mass spectrometry and gas chromatography - mass spectrometry analysis. LCMS analysis of A. adscendens, revealed compounds (4-29) and (30-37) and most of them are biological active e.g. Levoglucosan (C-10), Brugine (C-14) and (C-20) Bergenin. LCMS analysis of T. govanianum identified various biological active saponins and sapogenins e.g. Digoxigenin (C-52), Alliospiroside D (C-53), Hovenoside D(C-55), Pisumsaponin I (C-57), Fistuloside A (C-58), Pitheduloside F (C-60), Durupcoside B (C-61),
xi Cyclopassifloside I (C-68), Ophiopogonin D (C-72), Crosatoside B (C-99), Yayoisaponin B (C-107), Protodioscin (C-109), Isoeruboside B (C-111), Phytolaccasaponin B (C-114), Calendasaponin C (C-115), Calendasaponin D (C-116), Azukisaponin IV (C-119), Pseudoprotodioscin (C-121), Polypodoside A (C-122), Agavasaponin C (C-123), Schidigerasaponin B1 (C-124), Dioscin (C-125), Pitheduloside K (C-127), Fistuloside B (C-129) and Ophiopogonin B (C-130). The GC/MS analysis of n-hexane fraction of MeOH extract of the roots of A. adscendens and T. govanianum was performed to get the fatty profile of both extracts. GC/MS analysis revealed the presence of twelve components (C-141 to 155).
In current study, all the phytochemical and biological assays were performed on methanolic extract and SPE of A. adscendens and T. govanianum. The total phenolic and flavonoid contents of A. adscendens and T. govanianum in terms of gallic acid and quercetin equivalent per gram dry weight exhibited different levels of significant phenolic and flavonoid contents. Antioxidant assays, including DPPH scavenging activity, total antioxidant capacity and ferric reducing antioxidant power of A. adscendens and T. govanianum exhibited different levels activity, which might be attributed to the presence of phenolic compounds and possible saponins. The MeOH extract and SPE of A. adscendens and T. govanianum exhibited mild antibacterial activity determined by the zone of inhibition (mm diameter) ranges from 7 to 13 mm, against Staphylococcus aureus (NCTC 7508); Bacillus subtilis (NCTC 1604); M. luteus (NCTC 75080); Escherichia coli (ATCC 25922). The antimicrobial potential of both A. adscendens and T. govanianum was further accessed to determine the minimum inhibitory concentration (MIC) values by using resazurin microtiter assay (REMA), which exhibited considerable level of antibacterial potential against gram-positive bacteria (MIC: 2.5-0.009 mg/mL) than against gram-negative bacteria (MIC: 1.25-2.5 mg/mL). The antifungal potential of A. adscendens and T. govanianum were established against four strains of filamentous fungi. i.e Aspergillus fumigatus FCBP- 66; Mucor species (FCBP-0300); Aspergillus niger (FCBP-0198) and Aspergillus flavus (FCBP- 0064) showed mild to moderate or weak antifungal activity. Antileishmanial capability of A. adscendens and T. govanianum against Leishmania tropica KWH23 strain were manifested mild to moderate results.
xii Cytotoxicity potential of A. adscendens and T. govanianum were accessed by using brine shrimp lethality assay, protein kinase inhibition assay and in vitro 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. The distinguishable protein kinase inhibitory activity against Streptomyces 85E strain with 19± 1.06 mm bald, 9± 0.45mm clear phenotype was observed around the MeOH extract. The MeOH extract of the roots and SPE fractions of A. adscendens and T. govanianum displayed considerable levels of cytotoxicity (79 µg/mL & 14-29 µg/mL respectively) against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549), urinary bladder (EJ138) and one non-carcinoma vero (CL81) in the MTT cytotoxicity/viability assay. It is reasonable to assume that the cytotoxicity of MeOH extract and SPE fractions of the roots of A. adscendens and T. govanianum might be, at least partly, owing to the presence of saponins and their aglycones, suggest that these species could be exploited as a potential source of cytotoxic compounds with putative anticancer potential.
Among all the SPE of methanolic extracts of both species, which shows promising results against several bio-assays, were subjected to preparative-HPLC for isolation and characterization. Several compounds were isolated and collected through prep-HPLC but only three flavonoids C-156-C-158 (Epimedium C, Basohuoside I and Chrysin) were managed for their full characterization. The structures of all isolated compounds (C-156-C-158) were elucidated by spectroscopic analysis. This is the first report on the occurrence of compounds Epimedium C, Basohuoside I and Chrysin in the genus Asparagus. All isolated compounds exhibited different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138) and one non-carcinogenic vero (CL81) cell line, using the in vitro MTT cytotoxicity/viability assay. By keeping in view the IC50 values of isolated compounds ranges from 22-325 (µg/mL) against four human cancer cell lines, molecular docking studies were initiated to identify the binding modes of isolated compounds (C-156 and C-158) and selected identified compounds (Calendasaponin C (C-115), Yayoisaponin B (C-107), Agavasaponin C (C-123), Azukisaponin IV (C-119) and Protodioscin (C-109) with targeted receptors. The selected compounds have shown promising docking result against epithelium growth factor receptor protein. To our best knowledge, this is the first report showing significant phytochemical and biological potential of Asparagus adscendens and Trillium govanianum indigenous to Pakistan. xiii TABLE OF CONTENTS
Chapter 1 ...... 1
Introduction ...... 1
1.1 Natural product drug discovery ...... 2
1.1.1 Importance of medicinal plants in drug discovery ...... 2
1.1.2 Challenges in drug discovery from medicinal plants ...... 3
1.2 The Family Asparagaceae ...... 4
1.2.1 The genus Asparagus ...... 4
Phytochemistry of the genus Asparagus ...... 5
1.2.2 Asparagus adscendens ...... 14
Distribution and habitat ...... 15
Chemical constituents of Asparagus adscendens...... 16
Ethnopharmacological uses of Asparagus adscendens . 18
1.3 The Family Trilliaceae ...... 19
1.3.1 The genus Trillium ...... 20
Phytochemistry of the genus Trillium ...... 20
1.3.2 Trillium govanianum ...... 29
Distribution and habitat ...... 30
Chemical constituents...... 30
Ethanopharmacological uses of Trillium govanianum .. 32
1.4 Analytical techniques for natural product research ...... 33
1.4.1 Chromatography ...... 34
Thin-layer chromatography (TLC) ...... 34
Liquid chromatography (LC) & high performance liquid
chromatography (HPLC) ...... 35
1.4.2 Mass spectrometry ...... 36
Electron ionization (EI) ...... 37
Electrospray ionization (ESI) ...... 38
Collision-induced dissociation (CID) and tandem mass
spectrometry (MS/MS) ...... 40
xiv HPLC-MS, HPLC-MS/MS and matrix effects ...... 40
1.4.3 Fourier transform infrared spectroscopy (FTIR)...... 42
1.5 Phytochemical analysis ...... 43
1.5.1 Phenolic content assay ...... 43
1.5.2 Flavonoids content assay ...... 44
1.6 Bioassays ...... 45
1.6.1 Antioxidant activities ...... 45
Measurement of antioxidant activity ...... 45
In Vitro assays for antioxidant activities ...... 45
1.6.1.2.1 DPPH assay ...... 45
1.6.1.2.2 Total antioxidant capacity ...... 46
1.6.1.2.3 Reducing power assay ...... 46
1.6.2 Antimicrobial activities ...... 47
Antibacterial assay ...... 47
Resazurin assay ...... 47
Antifungal assay ...... 48
1.6.3 Antileishmanial assay ...... 48
1.6.4 Cytotoxicity assay ...... 49
Brine shrimp lethality bioassay ...... 49
Protein kinase inhibition assay ...... 49
MTT assay ...... 50
1.7 Molecular docking ...... 51
1.7.1 Advent of Molecular Docking ...... 51
1.7.2 Epithelial growth factor receptor (EGFR) ...... 51
Chapter 2 ...... 54
Material & Methods ...... 54
2.1 Chemical and standard compounds ...... 55
2.1.1 Cell lines used to evaluate the cytotoxicity of plants ...... 55
2.1.2 Strains of microorganism to evaluate the antimicrobial potential
of plants ...... 55
xv 2.2 Instruments ...... 56
2.3 Collection of plant materials ...... 57
2.3.1 Extraction and preparation of plant samples ...... 57
2.3.2 Solid-phase extraction (SPE) and sample purification ...... 58
2.4 Methods ...... 59
2.5 Phytochemical analysis ...... 59
2.5.1 Determination of total phenolic contents ...... 59
2.5.2 Determination of total flavonoid contents ...... 59
2.5.3 Chromatographic and spectroscopic analysis ...... 60
High performance liquid chromatography–photodiode
array detection (HPLC-PDA) ...... 60
2.5.3.1.1 Optimization of method for fingerprint analysis of SPE
fractions ...... 60
LC-ESI-QTOF-Mass spectrometry analysis ...... 61
2.5.3.2.1 Optimization of Method ...... 61
Gas chromatography/mass spectrometry analysis...... 61
2.5.3.3.1 Sample preparation for GC/MS analysis: ...... 61
2.5.3.3.2 Gas chromatography/mass spectrometry analysis: ...... 61
2.5.3.3.3 GC/MS identification of components ...... 62
2.6 Biological evaluation...... 62
2.6.1 Antioxidant assay ...... 62
DPPH assay ...... 62
Total antioxidant capacity assay ...... 63
Total reducing power assay ...... 63
2.6.2 Antimicrobial analysis...... 63
Antibacterial assay ...... 63
Resazurin microtiter assay (REMA) ...... 64
2.6.2.2.1 Minimum inhibitory concentration (MIC) determination . 64
Antifungal assay ...... 65
2.6.3 In vitro antileishmanial analysis ...... 65
2.6.4 Cytotoxicity assays ...... 66
xvi Brine shrimp lethality assay ...... 66
Protein kinase inhibition assay ...... 66
MTT assay ...... 67
2.6.5 Isolation and characterization ...... 69
Preparative HPLC-PDA ...... 69
2.6.5.1.1 Optimization of method ...... 69
Spectroscopic analysis:...... 70
2.6.6 Computational methods...... 70
Molecular docking simulations ...... 70
2.6.6.1.1 Preparation of protein structure ...... 70
2.6.6.1.2 Preparation of ligand structures ...... 70
2.6.6.1.3 Molecular docking protocol ...... 70
2.6.7 Statistical analysis ...... 71
Chapter 3 ...... 72
Results ...... 72
3.1 Phytochemical analysis ...... 73
3.1.1 Total phenolic contents in Asparagus adscendens...... 73
3.1.2 Total phenolic contents in Trillium govanianum ...... 74
3.1.3 Total flavonoid contents of Asparagus adscendens ...... 75
Total flavonoid contents of Trillium govanianum...... 77
3.1.4 Chromatographic and spectroscopic analysis ...... 78
HPLC-PDA analysis of Asparagus adscendens...... 78
3.1.4.1.1 Optimization of method for fingerprint analysis of SPE
fractions: ...... 78
HPLC-PDA analysis of Trillium govanianum ...... 91
3.1.4.2.1 Optimization of Method for fingerprint analysis of SPE
fractions ...... 91
LC-ESI-QTOF-Mass spectrometry analysis ...... 99
3.1.4.3.1 LC-MS analysis of Asparagus adscendens ...... 99
3.1.4.3.2 LC-MS analysis of Trillium govanianum ...... 112
xvii Gas chromatography/mass spectrometry analysis...... 152
3.1.4.4.1 GC/MS identification of components Asparagus
adscendens...... 152
3.1.4.4.2 GC/MS identification of components of Trillium
govanianum ...... 156
3.2 Biological evaluation...... 159
3.2.1 Antioxidant assay ...... 159
DPPH assay of Asparagus adscendens ...... 159
DPPH assay of Trillium govanianum ...... 160
Total antioxidant capacity assay of Asparagus
adscendens ...... 161
Total antioxidant capacity assay of Trillium govanianum
...... 162
Total reducing power assay of Asparagus adscendens .....
...... 164
Total reducing power assay of Trillium govanianum . 165
3.2.2 Antimicrobial analysis...... 166
Antibacterial assay of Asparagus adscendens ...... 166
Antibacterial assay of Trillium govanianum ...... 169
Resazurin microtiter assay (REMA) of Asparagus
adscendens ...... 171
3.2.2.3.1 Resazurin microtiter assay (REMA) of Trillium
govanianum ...... 173
Antifungal assay of Asparagus adscendens ...... 175
Antifungal assay of Trillium govanianum ...... 177
3.2.3 In vitro antileishmanial analysis of Asparagus adscendens ...... 179
3.2.4 In vitro antileishmanial analysis of Trillium govanianum ...... 179
3.2.5 Cytotoxicity assays ...... 180
Brine shrimp lethality assay of Asparagus adscendens
……………………………………………………………180
Brine shrimp lethality assay of Trillium govanianum . 181
xviii Protein kinase inhibition assay of Asparagus adscendens
……………………………………………………………...182
Protein kinase inhibition assay of Trillium govanianum ...
...... 183
MTT assay of Asparagus adscendens ...... 184
MTT assay of Trillium govanianum ...... 192
3.2.6 Isolation and characterization ...... 200
Spectroscopic analysis:...... 201
3.2.7 Cytotoxicity of isolated compound ...... 204
3.2.8 Computational methods...... 205
Molecular docking studies of compounds (C-156 and
C-157)………………………………………………………205
Molecular docking studies of compound (C-158)...... 207
Molecular docking studies of selected identified
compounds (saponins) ...... 208
Chapter 4 ...... 215
Discussion ...... 215
4.1 Phytochemical analysis ...... 216
4.1.1 Determination of total phenolic contents A. adscendens and T.
govanianum ...... 216
4.1.2 Determination of total flavonoid contents A. adscendens and T.
govanianum ...... 216
4.1.3 Chromatographic and spectroscopic analysis ...... 217
4.1.4 HPLC-PDA analysis of A. adscendens and T. govanianum ..... 217
4.1.5 LC-ESI-QTOF-Mass spectrometry analysis of A. adscendens and
T. govanianum ...... 218
4.1.6 GC/MS analysis of A. adscendens and T. govanianum ...... 219
4.2 Biological evaluation...... 220
4.2.1 Antioxidant assay ...... 220
4.2.2 DPPH assay of A. adscendens and T. govanianum ...... 220
xix 4.2.3 Total antioxidant capacity assay of A. adscendens and T.
govanianum ...... 220
4.2.4 Total reducing power assay of A. adscendens and T. govanianum.
...... 221
4.2.5 Antimicrobial analysis...... 222
Antibacterial assay of A. adscendens and T. govanianum ...... 222
Resazurin microtiter assay (REMA) of A. adscendens and
T. govanianum ...... 223
Antifungal assay of A. adscendens and T. govanianum
……………………………………………………….224
4.2.6 In vitro antileishmanial analysis of A. adscendens and T.
govanianum ...... 224
4.2.7 Cytotoxicity assays ...... 225
Brine shrimp lethality assay of A. adscendens and T.
govanianum ...... 225
Protein kinase inhibition assay of A. adscendens and T.
govanianum ...... 225
MTT assay of A. adscendens and T. govanianum ...... 226
4.2.8 Isolation and characterization ...... 228
4.2.9 Computational methods...... 229
Molecular docking studies ...... 229
4.3 Conclusion ...... 230
4.4 Future Strategies ...... 231
Chapter 5 ...... 232
References ...... 233
xx LIST OF FIGURES Fig 1.1 Asparagus adscendens (A) whole plant (B) roots………………………………………………………………… 16 Fig 1.2 Major saponins and sapogenins previously isolated from Asparagus adscendens ………………………………………………………… 18 Fig 1.3 Traditional uses of Asparagus adscendens ………………...... 19 Fig 1.4 Isolated compounds form Trillium govanianum……………………. 32 Fig 1.5 Traditional uses of Trillium govanianum ………………………… 33 Fig 1.6 Trillium govanianum (A) whole plant (B) roots …………………… 33 Fig 1.7 Illustration of oxidative stress and antioxidant mechanism………… 44 Fig 1.8 Reduction of resazurin to resorufin by oxidoreductases from viable cells ………………………………………………………………... 48 Fig 1.9 Illustration of MTT assay ………………………………………… 50 Fig 2.1 General research methodology scheme ……………………………. 58 Fig 2.2 Solid Phase Extraction (SPE) fractionation of A. asparagus and T. govanianum …...……………………………………………………. 59 Fig 2.3 Typical Plate, after 24 h in resazurin ……………………………….. 65 Fig 2.4 MTT assay protocol …..……………………………………………. 68 Fig 2.5 Isolation of compounds from A. asparagus………………………… 69 Fig 3.1 Regression line of Galli acid ………………………………………. 73 Fig 3.2 Total phenolic contents of Asparagus adscendens ……………….... 74 Fig 3.3 Total phenolic contents of Trillium govanianum ………………….. 75 Fig 3.4 Regression line for flavonoid contents …………………………...... 76 Fig 3.5 Total flavonoid contents of Asparagus adscendens ……………….. 76 Fig 3.6 Total flavonoid contents of Trillium govanianum …………………. 77 Fig 3.7 Identified compounds from SPE fractions of MeOH Extract of the roots of A. adscendens using HPLC-PDA …………………………. 79 Fig 3.8 HPLC-PDA Chromatogram of standard phenols monitored at 360nm………………………………………………………………. 80 Fig 3.9 Calibration curve of standard (A) Quercetin, (B) Myricetin, (C) Kaempferol ………………………………………………………… 81 Fig 3.10 HPLC-PDA Chromatogram of AAMF1 of Asparagus adscendens extract ……………………………………………………………… 82
xxi Fig 3.11 Corresponding UV-vis absorbance (AAMF1) at multiple wavelengths of the peaks separated by HPLC……………………… 83 Fig 3.12 HPLC-PDA Chromatogram of AAMF2 of Asparagus adscendens extract ……………………………………………...... 84 Fig 3.13 HPLC-PDA Chromatogram of AAMF3 of Asparagus adscendens extract ………………………………………………………………. 85 Fig 3.14 HPLC-PDA Chromatogram of AAMF4 of Asparagus adscendens extract ………………………………………………………………. 86 Fig 3.15 HPLC-PDA Chromatogram of detected compound from TGMF1 of Trillium govanianum extract………………………………………... 92 Fig 3.16 HPLC-PDA Chromatogram of detected compound from TGMF2 of Trillium govanianum extract.………………………………………. 93 Fig 3.17 Comparison of UV-vis spectra of a reference standard and detected compound.(A) Quercetin, (B) Myricetin, (C) Kaempferol ………… 94 Fig 3.18 HPLC-PDA Chromatogram of detected compound from TGMF3 of Trillium govanianum extract ……………………………………….. 95 Fig 3.19 HPLC-PDA Chromatogram of detected compound from TGMF4 of Trillium govanianum extract ……………………………………… 96 Fig 3.20 Corresponding UV-VIS. absorbance (A) and (B) of TGMF4 and (C) and (D) of TGMF3 at multiple wavelengths of the peaks separated by HPLC-PDA……………………………………………………… 97 Fig 3.21 Total Ion Chromatogram of Asparagus adscendens showing separation of chemical components ………………………………... 100 Fig 3.22 Compound (4-29) detected from Asparagus adscendens by LCMS (Positive Ion mode)…………………………………………………. 107 Fig 3.23 Compound (30-37) detected from Asparagus adscendens by LCMS (Negative Ion mode)………………………………………………... 110 Fig 3.24 Total Ion Chromatogram of Trillium govanianum showing separation of chemical components………………………………… 112 Fig 3.25 Compound (38-89) detected from Trillium govanianum by LCMS (Positive mode)……………………………………………………... 129 Fig 3.26 Compound (90-140) detected from Trillium govanianum by LCMS (Negative Ion mode)………………………………………………... 151
xxii Fig 3.27 Typical Gas Chromatogram of Asparagus adscendens showing separation of chemical components………………………………… 153 Fig 3.28 Compound identified by GC/MS from Asparagus adscendens……... 155 Fig 3.29 Typical Gas Chromatogram of Trillium govanianum showing separation of chemical components………………………………… 156 Fig 3.30 Compound identified by GC/MS from Trillium govanianum……… 159 Fig 3.31 DPPH assay of Asparagus adscendens …………………………...... 160 Fig 3.32 DPPH assay of Trillium govanianum …………………………...... 161 Fig 3.33 Regression curve of Ascorbic Acid………………………………… 162 Fig 3.34 Total antioxidant capacity assay of Asparagus adscendens ………. 162 Fig 3.35 Total antioxidant capacity assay of Trillium govanianum ………… 163 Fig 3.36 Regression curve of Ascorbic Acid………………………………… 164 Fig 3.37 Total Reducing capacity assay of Asparagus adscendens ………… 165 Fig 3.38 Total Reducing capacity assay of Trillium govanianum ………….. 166 Fig 3.39 The MeOH extract and its SPE of Asparagus adscendens exhibited different levels of antibacterial activity…………………………….. 168 Fig 3.40 The MeOH extract and its SPE of Trillium govanianum exhibited different levels of antibacterial activity…………………………….. 170 Fig 3.41 Typical 96-well plates shows the result after 24 hours……………... 172 Fig 3.42 Typical 96-well plates shows the result after 24 hours……………... 174 Fig 3.43 MeOH extract and its SPE fractions antifungal potential of Asparagus adscendens……………………………………………… 176 Fig 3.44 MeOH extract and its SPE fractions antifungal potential of Trillium govanianum………………………………………………………… 178 Fig 3.45 Antileishmanial results exhibited by Asparagus adscendens……… 179 Fig 3.46 Antileishmanial results exhibited by Trillium govanianum………... 180 Fig 3.47 Typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of Asparagus adscendens against HepG2 cell line……………………………………………………………… 186
Fig 3.48 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against breast (MCF-7)…………………………………………….. 187
Fig 3.49 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against hepato celluar carcinoma (HepG2)………………………… 188
xxiii Fig 3.50 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens .against lung carcinoma (A549)……………………………………. 189
Fig 3.51 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against urinary bladder (EF 138)………………………………….. 190
Fig 3.52 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against vero (CL 81)………………………………………………. 191 Fig 3.53 Typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of Trillium govanianum against HepG2 cell line …………………………………………………………….. 194
Fig 3.54 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against breast (MCF7)……………………………………………… 195
Fig 3.55 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against hepatocellular carcinoma (HepG2)………………………… 196
Fig 3.56 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against lung carcinoma (A549)……………………………………. 197
Fig 3.57 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against urinary bladder (EJ138)…………………………………….. 198
Fig 3.58 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against vero (CL 81)………………………………………………... 199 Fig 3.59 Chromatograms of isolated compounds (A) C-156,(B) C-157 (C) C- 158………………………………………………………………….. 200 Fig 3.60 Obtained binding modes of ligands in the ATP binding domain of 205 EGFR.………………………………………………………………. Fig 3.61 Obtained binding modes of ligands (C-158) in the ATP binding domain of EGFR……………………………………………………. 207 Fig 3.62 3D binding interactions of Calendasaponin C with EGFR (PDB ID:4P3R) …………………………………………………….……... 211 Fig 3.63 3D binding interactions of Yayoisaponin B with EGFR (PDB ID:4P3R) ……………………………………………………..…….. 212 Fig 3.64 3D binding interactions of Agavasaponin C with EGFR (PDB 212 ID:4P3R) .…….. Fig 3.65 3D binding interactions of Azukisaponin IV with EGFR (PDB ID : 4P3R) …………………………………………………….……... 213
xxiv Fig 3.66 3D binding interactions of Protodioscin with EGFR (PDB ID : 4P3R) ……………………………………………………..…….. 213 Fig 3.67 Etoposide binding interactions with EGFR………………………… 214
xxv LIST OF TABLES Table 1.1 Phytochemistry of the genus Asparagus ...... 5 Table 1.2 Taxonomic description of Asparagus adscendens ...... 15 Table 1.3 Photochemistry of the genus Trillium ...... 21 Table 1.4 Taxonomic description of the Trillium govanianum ...... 30 Table 2.1 Instruments used with their model and manufacturing company ...... 56 Table 2.2 SPE fractions analytical HPLC-PDA screening method ...... 60 Table 3.1 Retention time (tR), calibration curve parameters, limit of detection (LOD), limit of quantification (LOQ) for the standards...... 79 Table 3.2 Chemical profiling of SPE fractions of MeOH Extract of the roots of A. adscendens using HPLC-PDA monitored at 220nm ...... 80 Table 3.3 Retention times (tR) and corresponding UV-vis absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF1) of the MeOH extract of the roots of Asparagus adscendens ...... 87 Table 3.4 Retention times (tR) and corresponding UV-vis absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF2) of the MeOH extract of the roots of Asparagus adscendens ...... 88 Table 3.5. Retention times (tR) and corresponding UV-vis absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF3) of the MeOH extract of the roots of Asparagus adscendens ...... 89 Table 3.6. Retention times (tR) and corresponding UV-vis absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF4) of the MeOH extract of the roots of Asparagus adscendens ...... 90 Table 3.7 Chemical profiling of SPE fractions of MeOH Extract of the roots of T. govanianum using HPLC-PDA monitored at 360 nm ...... 92 Table 3.8 Retention times (tR) and corresponding UV-vis absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fractions of the MeOH extract of the roots of Trillium govanianum ...... 98 Table 3.9 Compound detected from Asparagus adscendens by LCMS (Positive ion) ...... 100 Table 3.10 Compound detected from Asparagus adscendens by LCMS (Negative ion) ...... 108
xxvi Table 3.11 Retention times (tR) and corresponding pseudomolecular ions [M+H]+ of Asparagus adscendens ...... 111 Table 3.12 Compound detected from Trillium govanianum by LCMS (Positive ion mode) ...... 113 Table 3.13 Compound detected from Trillium govanianum by LCMS (Negative mode) ...... 130 Table 3.14 Retention times (tR) and corresponding pseudomolecular ions [M+H]+ of Trillium govanianum ...... 152 Table 3.15 Compound identified by GC/MS from Asparagus adscendens...... 154 Table 3.16 Compound identified by GC/MS Trillium govanianum ...... 157 Table 3.17. The MIC (mg/mL) values of the MeOH extract of the roots of A. adscendens and its SPE fractions by using the Resazurin assay ...... 172 Table 3.18. The MIC (mg/mL) values of the MeOH extract of the roots of T. govanianum and its SPE fractions by using the resazurin assay ...... 174 Table 3.19 Brine shrimp lethality assay of MeOH Extract of the roots of A. asparagus and its SPE fractions...... 181 Table 3.20 Table 1. Brine shrimp lethality assay of MeOH Extract of the roots of T. govanianum and its SPE fractions...... 182 Table 3.21 Table. Streptomyces hyphae formation inhibition potential of MeOH extract of the roots of A. asparagus and its SPE fractions...... 183 Table 3.22 Streptomyces hyphae formation inhibition potential of MeOH extract of the roots of T. govanianum and its SPE fractions ...... 184 Table 3.23. The IC50 values of the MeOH extract of the roots of A. adscendens and its SPE fractions against four carcinoma cell lines ...... 185
Table 3.24 The IC50 values of the MeOH extract of the roots of T. govanianum and its SPE fractions against five carcinoma cell lines ...... 193 Table 3.25 13C NMR of compounds (C-156, C-157, C-158) chemical shift δ in ppm ...... 203 Table 3.26 MTT assay of isolated compounds ...... 205 Table 3.27 CDocker interaction energies of best pose of each compound ...... 209 Table 3.28 Detail binding interaction of compounds with amino acid residues of EGFR...... 209
xxvii LIST OF ABBREVIATIONS 13C-NMR Carbon Nuclear Magnetic Resonance 1H-NMR Proton Nuclear Magnetic Resonance 2D-NMR Two Dimensional Nuclear Magnetic Resonance A. flavus Aspergillus flavus A549 lung liver cancer cell line AA Arachidonic acid ADME Absorption, distribution, metabolism, elimination ATCC American tissue culture collection B. subtilus Bacillus subtilus BB Broad Band C. albicans Candida albicans CC Column chromatography CCL-81 Vero Cell Line CI Chemical Ionization CID Collison-induced dissociation DCM Dichloromethane DEPT Distortionless Enhancement by Polarization Transfer DMSO Dimethyl Sulfoxide DW Dry weight E. coli Escherichia coli EI Electron Impact EI-MS Electron Impact Mass Spectrum EJ138 urinary bladder cancer cell line ESI Electrospray ionization FAB Fast Atom Bombardment FD Field Desorption GC Gas chromatography GC-MS Gas chromatography-mass spectrometry GIT Gastrointestinal tract HEPG2 liver cancer cell line HPLC High performance liquid chromatography
IC50 Inhibitory concentration 50
xxviii IEM Ion evaporation model FTIR Fourier transform infrared spectroscopy
LD50 Lethal Dose 50 MALDI Matrix-assisted laser desorption ionization MCF7 Breast cancer cell line MDR Multi drug resistant MeOH Methanol MIC Minimum inhibitory concentration MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MS Mass Spectroscopy NCTC National culture tissue collection ppm Parts Per Million QSAR quantitative structure–activity relationship Rf Relative flow ROS Reative oxygen species RNA Reative nitrogen species S. aureus Staphylococcus aureus SPE Solid Phase Extraction TLC Thin Layer Chromatography UV Ultra violet UPLC Ultra performance liquid chromatography WHO World Health Organization
xxix LIST OF PUBLICATIONS
1- Kashif Maqbool Khan, Lutfun Nahar, Afaf Al-Groshi, Alexandra G. Zavoianu, Andrew Evans, Nicola M. Dempster, Jean D. Wansi, Fyaz M. D. Ismail, Abdul Mannan and Satyajit D. Sarker *2016. Cytotoxicity of the roots of Trillium govanianum against breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138) carcinoma cells. Phytotherapy Research 30: 1716–1720 (2016). 2- Kashif Maqbool Khan, Lutfun Nahar, Abdul Mannan, Muhammad Arfan, Ghazanfar Ali Khan, Glyn Hoobs, Satyajit D. Sarker, 2017. Evaluation of Resazurin Microtiter Plate Assay and HPLC- Photodiode Array Analysis of the Roots of Asparagus adscendens. Natural Products Research. 32:3, 346-349. 3- Kashif Maqbool Khan, Lutfun Nahar, Abdul Mannan, Ihsan-ul-Haq, Muhammad Arfan, Ghazanfar Ali Khan, Izhar Hussain and Satyajit D. Sarker, 2107. Cytotoxicity, In vitro anti-Leishmanial and fingerprint HPLC- photodiode array analysis of the roots of Trillium govanianum. Natural Products Research 32:18, 2193-2201. 4- Kashif Maqbool Khan, Lutfun Nahar, Abdul Mannan, Muhammad Arfan, Ghazanfar Ali Khan, Afaf Al-Groshi, Andrew Evans, Nicola M. Dempster, Fyaz M. D. Ismail and Satyajit D. Sarker 2018. Liquid Chromatography Mass Spectrometry Analysis and Cytotoxicity of the Roots of Asparagus adscendens Against Human Cancer Cell Lines. Pharmacognosy Magazine. 13, S890 5- Kashif Maqbool Khan, Satyajit D. Sarker , Ghazanfar Ali Khan , Hammad Saleem, Shujaat Ali Khan and Abdul Mannan 2019. Phytochemical profiling and evaluation of modified resazurin microtiter plate assay of the roots of Trillium govanianum.
Natural Products Research. https://doi.org/10.1080/14786419.2019.1590716 CONFERENCE PROCEEDINGS FROM PRESENT WORK 1- Abdul Mannan, Kashif Maqbool Khan, Izhar Hussain, 2015. Phytochemical and biological investigations of Asparagus adscendens growing in Himalayas Region of Pakistan. In Vitro Cell Dev Biol Anim 51: S58-S58. 2- Abdul Mannan, Kashif Maqbool Khan, Muhammad Arfan, Ghazanfar Ali Khan, Satyajit D. Sarker*2016 .Fingerprint Analysis of Methanol Extract of Trillium govanianum by HPLC-Photodiode Array Coupled with ESI Quadrupole Time-of- Flight Mass Spectrometry 2016. In Vitro Cell Dev Biol Anim. DOI 10.1007/s11626- 016-00
xxx
Chapter 1
Introduction
1
Introduction
1.1 Natural product drug discovery The man has taken benefit from the plant by different ways like shadow; food and medicine even from his birth. Today, the supreme creature, the man, is not only busy in research of some particular herbs to explore the secrets of nature kept in herbs and their effective use such as morphine from opium poppy and cocaine form coca but he is also discovering the modern techniques for the concentration of the medicine to make it more effective (Balunas & Kinghorn, 2005).
The man chooses the plants according to his interest and starts exploring it. He is known as the specific name linked with his interest like botanist, ethno botanist and ecologist etc. The man has created several techniques and guides to study the plant deeply. According to the peculiar knowledge, the man has, every person uses it according to his turn. In this way, the particular plant is processed from the experts (Samuelsson & Bohlin, 2017). Molecular biology helps Pharmacognosy to encapsulate all of these advanced fields of biosciences into a major interdisciplinary science.
1.1.1 Importance of medicinal plants in drug discovery Several experts (Ley & Baxendale, 2002) discovered the various ways to get compounds for the drug discovery by its isolation from plants and many natural resources. Furthermore, several funding organizations and pharmaceutical companies have invented synthetic chemistry techniques through which an innovative lead compounds and new chemical entities are produced from plants.
According to Newman et al. (2000) view, natural chemical entities were either natural products or obtained from natural product up to 28% between 1981
2
and 2002. Several synthetic and medicinal scientists are searching new ways to get natural product and natural-product-like libraries that may have structural features of natural products due to the existence of appreciation of interest in consequent realization and combinatorial chemistry. Medicinal and synthetic chemists have made new drugs from plants and drug leads suitable for optimization from the medicinal plants. If the experts do not find any new entity from medicinal important herbs, many acknowledged compounds from the herbs are provided.
1.1.2 Challenges in drug discovery from medicinal plants In future, the search leads to many challenges in spite of the obvious accomplishment of new drug entity from the under-experienced plants. According to Butler, the experts linked with this research must improve the quantity and quality of compounds which enter the development phase of drug for keeping pace with the other discovery efforts of drug. No doubt, the experts have spent much time and a lot of money on the various leads that were given up during the process of discoveries. According to the given figures and facts, only the one in 5000 lead compounds will successfully progress through the clinical trials and can be approved for the use (Kinghorn et al., 2003).
Do et al. (2004) explain that more and effective techniques are required in the discovery of drug from the medicinal plants so that the time, expenditure and methodologies for this process may be better. The design and accomplishment of suitable, clinically relevant, high-throughput bioassays is not an easy process. Butler et al. (2004) tells us the new techniques can improve some of these issues. The sprint of biological active compound isolation may require the amalgamation of new technologies. The compound development of the drugs discovered from medicinal plants also faces sole challenges (Pezzuto et al., 1999).
3
The natural products produced from the plants, are isolated in less amount that are inadequate for drug design and drug discovery. According to Ley et al. (2002), research collaboration with synthetic and medicinal scientist is very important to establish if synthesis or semi-synthesis could be possible. Drug discovery from natural product can also be enhanced by making natural products data libraries linked with combinatorial chemistry. (Lee & Schneider, 2001, Hall et al., 2001, Eldridge et al., 2002, Feher & Schmidt, 2003, Burke et al., 2004, Ganesan, 2004, Piggott & Karuso, 2004, Koehn & Carter, 2005).
The crux of the discussion is that the natural products revealed from medicinal plants have furnished several clinically important drugs. In spite of all the hurdles in front of drug discovery from natural products, the natural products which are isolated from medicinal plants can be predicted to stay a vital module in the research for new medicines.
1.2 The Family Asparagaceae The family Asparagaceae contain several genus but our region have only one genus having remarkable medicinal values. It is widely distributed through Himalayan ranges of India and Pakistan. It is classified as perennial herb, leaves are small bracts, whereas stems are in green color, primarily responsible for photosynthesis process. Flowers are small in size, bell-shaped and usually grow on stalks at the junction of leaf and stem.
1.2.1 The genus Asparagus The genus Asparagus comprises about 300 species, out of which most of the European species are used as vegetables (Goyal et al., 2003a). Among the species that grow in the Himalayan ranges of Pakistan, A. adscendens and A. racemose, are the most commonly used species of traditional medicines. About 300 species of asparagus are identified uptill now by the taxonomists, most of them are native to Europe, Africa and western part of Asia. Other then, medicinal values of Asparagus, it is also extensively cultivated as a vegetable crop. Among so many species, few like A. acutifolius A. officinalis and A. sprengerin are reported to have dietary use, native to Europe (Goyal 4
et al., 2003b). A. adscendens, A. gonaclades and A. racemosus species of Asparagus, indigenous to Indian and Pakistani northern areas are believed to have medicinal values in their folklore system (Hayes et al., 2008). Asparagus officinalis is assumed to be used as a medicinal herb along with dietary source since 2000 years ago in Egypt, majorly due to its remarkable diuretic properties (Chauhan, 1999).
Phytochemistry of the genus Asparagus The genus Asparagus comprise more than 300 species, mostly widely distrusted throughout the world. Some species are very rich in medical constituents and a series of novel compounds has been isolated and characterized by using advanced chromatographic and spectroscopic techniques. Phytochemical investigations of genus revealed the presence of alkaloids, Saponins and sapogenins as a major bioactive constituent (Alok et al., 2013). Two new alkaloids, aspastipuline and 5- hydroxyaspastipuline have been reported from roots of A. stipularis (Galala et al., 2015). From A. filicinus two new steroidal saponins, filiasparosides A and aspafiiosides E have been reported (Zhou et al., 2007). However a brief phytochemisrty of the genus Asparagus is summarized in Table 1.1.
Table 1.1 Phytochemistry of the genus Asparagus Mol. S/ Formula Structure Reference No & Mol. Mass, m/z
C39H64O15 Zue 1 etal; 2014 772.93
5
C40H66O15 Zue 2 etal; 2014 786.95
C32H52O9 Zue 3 etal; 2014 580.76
C39H64O16 Zue 4 etal; 2014 788.93
C39H64O15 Zue 5 etal; 2014 772.93
6
C33H54O8 Zue 6 etal; 2014 578.79
C33H54O9 Zue 7 etal; 2014 594.79
C53H90O20 Pactricia 8 etal; 2006 1047.28
7
C54H52O19 Pactricia 9 etal; 2006 1045.31
C54H92O20 Pactricia 10 etal; 2006 1061.31
8
C56H98O18 Pactricia 11 etal; 2006 1057.37
C48H84O13 Pactricia 12 etal; 2006 869.19
C47H80O16 Pactricia 13 etal; 2006 901.14
9
C49H78O21
14 Corrina etal; 1003.14 2009
C50H80O21 15 1017.17 Corrina etal; 2009
C43H68O17
16 857.00 Corrina etal; 2009
C44H70O17 17 871.03 Corrina etal; 2009
C44H70O17
18
871.03 Corrina etal; 2009
10
C44H72O17
19 873.04 Corrina etal;
2009
C43H70O17
20 859.02 Corrina etal;
2009
C44H72O17
21
873.04 Corrina etal;
2009
C44H70O15
22 839.03
Corrina etal;
2009
11
C43H68O15
23 825.00
Corrina etal; 2009
C44H70O15
24 839.03 Corrina etal; 2009
C44H64O17
25 816.94 Rui jain et al; 2012
C34H54O12
26 654.79 Rui jain et al; 2012
12
C39H62O16
27 786.91 Rui jain et al; 2012
C52H88O23
28 1081.25 Rui jain et
al; 2012
C53H90O23
29 1095.28 Rui jain et
al; 2012
13
C47H80O18
30 933.14 Rui jain et
al; 2012
1.2.2 Asparagus adscendens Asparagus adscendens Roxb. (Asparagaceae), commonly known as “safed musli” in Pakistan and various common names in India i.e. Shatawari, Shatavar, Shatamuli, Sahasrapal, Sainsarbuti, is native to Himalayas ranges (Mannan et al., 2015). Asparagus adscendens belongs to a large genus of herbs, under class of shrubs with tuberous roots. Internally it was class categorized in Lilly family, later on it was separated and classified under family Asparagaceae (Thakur & Sharma, 2015a). Table 1.2 describes the taxonomic description of Asparagus adscendens.
14
Table 1.2 Taxonomic description of Asparagus adscendens
Kingdom Plantae
Family Asparagaceae
Clade Monocots
Order Asparagales
Clade Angiosperms
Subfamily Asparagoideae
Genus Asparagus
Specie A. adscendens
Distribution and habitat This plant grows upto one to two meters tall and prefers to take root in gritty rocky soils high up in piedmont plains, at 1300-1500 meters above the sea level (Thakur & Sharma, 2015b). is the example of medicinal herbs, which are being used from centuries to mitigate the various diseases and have established the alternative medicine system, which have proven its worth and importance. The natural habitat of Asparagus adscendens is in thick forest; is a herb tuberous roots (Figure 1.1), can grow up to 10 inches depth. This plant is a form of shrub with woody stem and sub-erect lanceolate leaves. Flowers are small, white, 3-4 cm across, solitary or fascicled with copious racemes (Alok et al., 2013).. Fruits are 0.8 cm in diameter, globes, 3 lobed berries with only one seed. Normally this plant is distributed throughout in India, specifically Himalayan mountain ranges of Pakistani region including Galyat and Sawat valley.
15
Fig 1.1 Asparagus adscendens (A) whole plant (B) roots
Chemical constituents of Asparagus adscendens Aliphatic, nitrogenous and phenolic compounds, saponins, steroids and triterpenoids have been reported from A. adscendens of Indian origin (Mannan et al., 2015) ; β-sitosterol gluoside, spirostanol glycosides (asparanin C and asparanin D) and furostanol glycosides (asparoside C and asparoside D) were also isolated by (Sharma et al., 1982); steroidal saponins, glycosides, and various lipophilic compounds were found in the tuberous roots and leaves (Thakur & Sharma, 2015b); sarsasapogenin, diosgenin, β-sitosterol gluoside, spirostanol glycosides (asparanin A and B) and furostanol glycosides (asparoside A and B) were also isolated from this plant (Tandon &Shukla, 1992, Jadhav & Bhutani, 2006, Sharma et al., 1980). Major saponins and sapogenins previously isolated from Asparagus adscendens can be seen in Figure 1.2
16
O
O H
H H
HO Diosgenin
O
O H H H
H H
HO H Sarsasapogenin
H3C O H3C H HO H C 3 O HO O H3C HO O OH O HO O O O HO H HO OH H Asparanin C (2)
17
H3C O H H3C HO H C 3 O HO O H3C HO O OH O HO O O O HO H HO O O H HOH2C
HO OH HO Asparanin D (3)
Fig 1.2 Major saponins and sapogenins previously isolated from Asparagus adscendens
Ethnopharmacological uses of Asparagus adscendens Owing to its aphrodisiac and immunomodulatory properties, Asparagus adscendens has become one of the most commercially exploited species in India and Pakistan (Gautam et al., 2009). Recently, this plant is being used to treat the various lethal disease conditions including various carcinomas, infectious diseases, symptomatic treatment and have effective hepatoprotective outcomes. On contrary, the genus Asparagus is known to its medicinal uses like immunomodulatory activity (Gautam et al., 2009), antistress (Alok et al., 2013), anti-secretory and antiulcer activity (Goyal et al., 2003a, Bhatnagar & Sisodia, 2006) (Figure 1.3).
This plant is very valuable in Ayurvedic system of medicines, can be used in different part used depending upon different health conditions. For example, its powdered dried roots can be used to treat various sexual weakness, as a nutritive tonic, also exhibits galactogogic properties. Roots bark are believed to have aphrodisiac properties, due to which it is hot selling product to gulf and cold countries. It also contain various trace elements, which exhibits important nutritious values and also a good source of vitamins. Its important constituents have also been used in the commercial production of estrogen (Umashanker & Shruti, 2011).
18
However, with the only exception of the report on the isolation and identification of a compound Conypododiol from A. adscendens (Khan et al., 2010) exhibited significant inhibition of both acetylcholinesterase and butyrlcholinesterase and was found inactive against LCMK-2 monkey kidney epithelial cell and mice hepatocytes (Mannan et al., 2015), to the best of our knowledge, there has been no systematic pharmacological and phytochemical work performed on A. adscendens native to Pakistan.
Improve fertility Regulate Digestive blood sugar support
Anticancer Asparagus Antioxidant potential adscendens potential
Prevent Anti- nervous inflammatory disorders agent Natural diuretic
Fig 1.3 Traditional uses of Asparagus adscendens
1.3 The Family Trilliaceae Trillium govanianum belongs to the family Trilliaceae and is mainly distributed in South Asia, from Pakistan to Bhutan, at an altitude of 2700-3800 m (Ismail et al., 2015).
19
1.3.1 The genus Trillium Herbs of this genus are perennial, growing from rhizomes. Most of the species contain large bracts leaves, which also serves the photosynthesis purpose. Usually flowers has three green sepals and three petals having red, pink, white coloration. These plant species are largely disseminated throughout the world, from Japan to north western America. Asian species of Trillium, like T. govanianum are reported to exploited in various cancer treatments (Huang et al., 2011). Species from North America are reported for their antifungal and antibacterial use.(Yokosuka & Mimaki, 2008). Approximately 45 species are documented uptill now, out which nine belongs from Japan (Ur Rahman et al., 2015).
Phytochemistry of the genus Trillium The genus Trillium comprise about forty eight medicinal rich species, distributed widely from western north America to Himalayan ranges of Asia. Up till now, about 40 steroidal saponins have been reported from this genus Their structures was determined by advanced chromatographic and spectroscopic techniques. The publish data strongly depicted that the species belongs to genus Trillium are very rich sourse of sterodila saponins and flavanoids (Ur Rahman et al., 2017). However briefly phytochemistry of the genus Triliium reported in Table 1.3.
20
Table 1.3 Photochemistry of the genus Trillium
Mol. S/ Formula Structure Reference No & Mol. Mass, m/z
Man
1 et al., C27H44O9 2010; 513.3063
OH OH
CH3
OH HO Man C27H44O7 2 et al., 481.3165 OH HO 2010; H O
OH OH
CH3
OH Man C27H44O8 3 HO et al., 497.3114 OH 2010; HO OH O
OH
O
HO OH Kang O O OH O O C H O CH 33 54 12 3 4 HO et al., O 625.3588 HO OH OH OH OH 2012; OH
HO H O
21
OH
O
HO OH Kang O O OH O O C H O CH 33 54 13 3 5 HO et al., O 659.3646 HO OH OH OH OH 2012; OH
HO OH O
HO OH HO O
HO
O HO
O Nakano OH C57H92O27 6 et al., 1209.5905 OH O O 1983 O O OH O O
OH O O
OH OH OH OH
OH OH
HO OH HO O
HO
O HO
O OH Nakano C51H82O23 7 et al., 1063.5325 O OH 1983 O
O OH
O
OH O O
OH OH
OH OH
22
HO OH HO O
HO
O HO Nakano C45H72O19 O 8 OH et al., 917.4746 1983
O
HO HO OH
O HO O OH O OH OH O O O OH O HO HO O HO O O OH O Fukuda C47H70O24 HO HO 9 et al., OH 1019.4335 O OH O 1981 OH
O
O O O OH O HO OH HO
O OH O Ono C42H62O20 10 HO HO et al., 887.3913 OH O OH 2007a O OH
O
23
O O OH O O HO OH HO HO OH O Ono C37H54O16 OH 11 O et al., 755.3490 OH 2007a O
O
CH2OH
O H
OH
OH Li C51H82O22 12 O O et al., 1047.5376 O O OH 2013 O O
OH O O
OH OH OH OH
OH OH O
CH2OH
OH OH O H
OH OH OH Li
C45H72O18 O O 13 OH et al., O 901.4797 O OH O 2013 O
HO
HO HO O
HO HO
OH OH O O O Fukuda OH O C42H62O19 OH 14 OH et al., 871.3964 O O 1981 O OH
24
OH OH
O
HO
O O OH OH O O Fukuda C37H54O15 15 OH et al., 739.3941 OH 1981 O OH
O
CH3 OH OH
O H OH OH Li C45H72O18 O O 16 OH et al., 901.4797 O OH O OH O 2013 O HO
HO
O
O
O
Xie OH C51H80O21 17 et al., 1029.527 O O O O OH 2009 O O
OH O O
OH OH OH OH
OH OH
25
O
O
O OH OH
OH OH Xie C45H70O17 O 18 O OH et al., 883.4691 O O 2009 OH O
O
HO
O
CH3
O H
OH
OH Zhang C51H82O21 19 O O et al.,
1031.5427 O O OH O 2011 O
OH O O
OH OH OH OH
OH OH O
CH3
OH OH O H
OH OH OH
C45H72O17 O Wei et al., 20 O OH 885.4848 O 2012; O OH O
O
HO
26
O
CH3
O H
HO HO OH
C39H62O13 Wei et al., 21 O 739.4269 O O 2012; HO
OH
O
HO
O
CH3
O H
HO OH C33H52O9 Ono et al., 22 593.3690 2007b OH O OHO
HO
O
O
C45H72O16 O Ono et al., 23 OH 869.4899 O 1986 O OH
O
OH O O
OH OH
OH OH
27
O
O
OH OH
C39H62O12 Ono et al., 24 O 723.4319 O O 2007b OH
OH
O
HO
O
O
OH C33H52O8 Yu et al., 25 577.3740 2008b OH O OHO
HO
O
CH3
O H Zhang C27H42O4 26 et al., 431.3161 OH 2011
HO
O
CH3
O OH Hayes C27H40O4 27 et al., 429.3005 OH 2009
HO
28
O
O Zhang C27H42O3 28 et al., 415.3212 2011
H
1.3.2 Trillium govanianum Trillium govanianum Wall. (Melanthiaceae alt. Trilliaceae), frequently known as “nagchhatry” in India and “teen patra” or “matar zela” in Pakistan, distributed from Pakistan to Bhutan about 2500-3800 m altitude is indigenous to Himalayas ranges (Ismail et al., 2015). Since it is reported the presence of the steroid, trillarin, in this species (Chauhan, 1999), it has been enormously used in numerous traditional medicinal preparations that contain steroids and sex hormones, and now, it is one of the hot-selling herbal products in the Indo-Pak subcontinent. In folk medicine the plant has been reported for the cure of wound healing, sepsis and in various sexual problems (Pant & Samant, 2010). Taxonomic description of T. govanianum is summarized in Table 1.4.
29
Table 1.4 Taxonomic description of the Trillium govanianum
Kingdom Plantae
Family Melanthiaceae
Clade Monocots
Order Liliales
Clade Angiosperms
Subfamily Asparagoideae
Genus Trillum
Species T. govanianum
Distribution and habitat
The genus Trillium is embraced of long lived herbaceous flowering plants. Their many species are largely disseminated whole the world, and majorly the medicinal important species reported from Pakistan is T. govanianum (Khan et al., 2016).
Chemical constituents The reported studies on different species of the genus Trillium have revealed that this genus is ample in steroidal saponins, e.g., steroidal saponins were found in T. erectum L. (Yokosuka & Mimaki, 2008, Hayes et al., 2009), T. kamtschaticum Pall. (Ono et al., 2003, Yokosuka & Mimaki, 2008, Wei et al., 2012) and T. tschonoskii Maxim. (Nakano et al., 1983, Man et al., 2010, Wei et al., 2012, Wang et al., 2013a). Previously isolated compounds from Trillium govanianum are shown in Figure 1.4.
30
O
O H HO
H H
HO Pennogenin E
O
O H
H H
HO Diosgenin
O HO OH O HO O O
H O H HO HO O
O
HO O
HO
OH Govanoside
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Borassoside E
H3C O CH3 CH3
O H3C
OH
HO O O O OH O O O
OH O OH O
HO HO
OH OH Polyphyllin VII Fig 1.4 Isolated compounds from Trillium govanianum
Ethanopharmacological uses of Trillium govanianum Ttraditionally Trillium. govanianum (Figure 1. 6) have been reported to used as immuno-regulation, to cure sepsis, inflammation, wound healing, dysentry, menstrual, antiaging agent skin boils and sexual disorders (Figure 1.5). The powder of this plant has also been used to treat anthelmintic and were found to have antitumor property (Luo et al., 2006, Wang et al., 2013a, Khan et al., 2016).
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Boils
Antiseptic Dysentery
Anti- Immuno Trillium inflammatory regulation govanianum agent
Wound Menstrual healing disorders Improve fertility
Fig 1.5 Traditional uses of Trillium govanianum
Fig 1.6 Trillium govanianum (A) whole plant (B) roots
1.4 Analytical techniques for natural product research Analytical techniques in chemistry involve the separation, identification and quantification of the chemical compounds. It is fundamental to various fields of science that works with chemical substances such as medicinal chemistry, materials science, forensic science, etc. Separation is commonly achieved by chromatography. It is often
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coupled with an identification tool such as mass spectrometry to provide a powerful tool to separate and identify each components of a mixture. Alternatively, chromatography is also coupled with a spectroscopic detector such as ultra-violet (UV) detector. Other powerful identification techniques include nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). Identification of an unknown usually requires approaches from various perspectives and a combination of spectrometric (MS) and spectroscopic (IR, UV, NMR) is essential.
1.4.1 Chromatography The term chromatography was first used in 1906 (Braithwaite & Smith, 1999) and was derived from the Greek word chroma meaning “colour” and graphein meaning “to write” (Borror, 1960). The term was coined perhaps due to the ability of separating a homogenous mobile phase into bands of different colours in paper chromatography, one of the earliest forms of the technique. It is now mostly replaced by thin-layer chromatography (TLC), which using the same principle (planar chromatography) but with higher efficiency. There are many types of chromatographic techniques such as gas chromatography (GC), liquid chromatography (LC), supercritical fluid chromatography (SFC), ion exchange chromatography (IEC), gel permeation chromatography (GPC), etc. Each of the techniques has its special uses and thus chromatography is employed vastly across many fields of science (Braithwaite & Smith, 1999).
Thin-layer chromatography (TLC) Paper chromatography, now replaced by thin-layer chromatography. It is one of the first forms of separation technique, probably due to its simple principle. The TLC plate composes of an inert supporting plate made of aluminum or plastic with a thin layer of silica coat as the stationary phase. The procedure is performed in a small closed bottle or a closed tank containing the mobile phase. Sample is applied onto the TLC plate as a tiny spot, approximately 1cm above this mobile phase level. As the mobile phase travels up the plate due to capillary action, different components in the sample are separated due to intermolecular interactions, i.e. with the mobile phase and the stationary phase. The procedure is finished when the mobile phase attains within 1.5–2 cm of the TLC plate and the solvent front is marked using a pencil. Separate spots on
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the TLC plate could be sampled and identified using mass spectrometry. Retardation factor (Rf) of each components can be calculated from the result . Rf = da / ds where da is the length from the sample A spot at time t when the procedure was terminated to the starting point and ds is length from the solvent front at time t to the starting point (Dolan & Snyder, 1989).
Liquid chromatography (LC) & high performance liquid chromatography (HPLC) Liquid chromatography (LC) has many advantages in comparison with other chromatographic techniques developed earlier such as TLC and GC. One of its improvements is the shortened time required for separation, which was further shortened in HPLC and UPLC. The relative difference between LC, HPLC, and UPLC is in the time required for satisfactory separation of samples. HPLC has replaced LC in the past and will be replaced by UPLC and superior systems as technology advanced. Currently, HPLC is widely employed in various fields of natural sciences that require mixture separation and analysis.
The main principle of separation is actually very similar between TLC and
HPLC. A liquid mobile phase is used to transport the analytes (sample) through a column packed with a desired material (a stationary phase). However, HPLC provides much more convenient control on many aspects of the separation process including mobile phase ratio and gradient, flow, temperature, sample injection and type of detector. This is because this technique was developed based on the instrumentation that was originally developed for gas chromatography (Lindsay, 1992).
Separation of different components in the sample was achieved due to interaction between those molecules in the mobile phase with the stationary phase. The choice of mobile and stationary phase depends on the polarity of the components targeted for separation. Normal phase HPLC consisting of a polar stationary phase packed with polar materials such as unmodified silica is suitable for separation of polar compounds or those that can form hydrogen bonds. The mobile phase in this case should be relatively low in polarity, only sufficient to dissolve the sample. Conversely, a reverse phase HPLC consisting of hydrocarbon-type stationary phase is commonly used for separation of organic compounds including antimalarial. Characterization of 35
organic metabolites could be achieved from the HPLC chromatogram. More polar metabolites elude faster, have shorter retention time and their corresponding peaks appear more to the left of a chromatogram. Polar solvents such as acetonitrile, methanol or water are commonly used in various combination of ratio. Successful separation greatly depends on the choice of the mobile phase although there are other contributing factors such as column packing, dispersion or salt formation. Snyder (1974) had classified a range of solvent based on complex factors and suggested a very useful method development strategy.
1.4.2 Mass spectrometry It provides a powerful technique in analytical chemistry to determine molecular mass and structure with high accuracy and sensitivity. The MS is faster and provides better results than other conventional methods in this field such as chromatographic techniques (gel filtration) or gel electrophoresis (Meyers, 2008). Some exceptional advantages of MS are its unmatched selectivity, detection limits, speed and flexibility.
The basic principle of this technique is simple although complexity comes with more advanced configurations and strategies to gain more valuable pieces of information. Basically, atoms or molecules from a sample, usually in gas phase, are ionized. Those newly formed ions which can be fragmented, depending on the conditions, are distinctive according to their mass-to-charge ratio (m/z) and separated in the mass analyzer. The mass spectrometer scans the defined range of m/z, records any detected ions and produces the m/z spectrum of the analyzed sample. In most cases, the ions only bear the charge of +1 (Barker, 1999), thus m/z values are also the masses of these ions. An m/z spectrum then generally becomes a mass spectrum.
Different types of ionization source and condition sets produce different spectra, each providing unique information about the same analyte. Exploiting these advantages, one can fulfil various purposes by not only determining the molecular mass and structure but also the isomerism and further characteristics of the target molecule (Watson & Sparkman, 2007).
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Electron ionization (EI)
Electron ionization was one of the first ionization techniques developed for mass spectrometry. Traditionally, extensive use has led to the development of common fragmentation mechanism as well as the huge database of spectra, i.e. more than 200,000 mass spectra at the National Institute of Science and Technology (NIST) database. Any spectrum of an unknown sample can be compared with the database and this makes identification process rapid. This method has a good sensitivity and can generate unique fragmentation data. However, due to its design in which the sample must be thermally transferred into the gas phase, thermal decomposition can occur within biomolecules. This requirement also puts an upper limit on the mass range of molecules to be analyzed (up to approx. 400 Da) because heavier species are involatile. Therefore, EI is mostly used for typical analysis of small, hydrophobic, thermally stable molecules, usually whilst coupled with GC.
The principle of this technique utilizes a beam of electron that is constantly fired at the flow of the sample gas through an electric field of 70 V from a heated filament. Every electron, carrying 70 electron volts (eV), transfers some of its kinetic energy to the molecule it impacts. Positively charged radicals are usually formed as a result of electron ejection. The excess energy causes the ion to fragment. In some cases of compounds having high electron affinity, electron capture can occur, leading to the formation of negatively charged ions (Mark & Dunn, 2013).
Although EI is a robust and established technique, it does not allow chemists and biochemists to analyze the vast majority of bio-organic compound due to the mass limit of ionization. This limitation had driven the development of the new generation of ionization techniques, including fast atom/ion bombardment (FAB) (Barber et al., 1981a, Barber et al., 1981b), matrix-assisted laser desorption/ionization (MALDI) (Karas & Hillenkamp, 1988, Tanaka et al., 1988), and electrospray ionization (ESI) (Whitehouse et al., 1985, Fenn et al., 1989, Mann et al., 1989). With the capability of analyzing much bigger molecules (more than 100,000 Da) in hand, these techniques have revolutionized science especially proteomics and xenobiotic metabolism studies.
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Electrospray ionization (ESI) Many review articles attribute the first foundation for electrospray ionization development in mass spectrometry to the work of Dole et al. (1968). It then took approximately 20 years of development until the seminal contributions of Fenn et al. (1989) and Mann et al. (1989) to enable ESI to become a routine mass spectrometry tool. Despite this delay, its applications have successfully revolutionized the field of mass spectrometry in numerous ways, especially in conjunction with high performance liquid chromatography.
Except for a few instances described by Whitehouse et al. (1985) and Berkel et al. (1992), electrospray in fact is not an ionization process that forms radical cations (Gaskell, 1997), but mostly generates anions or cations depending on the ionization conditions employed. The principles of electrospray mainly involve the transfer of analyte molecule from its condensed phase into gas phase (Kebarle & Tang, 1993, Kebarle & Verkerk, 2009).
The analyte solution is passed through a capillary under high potential of 3-6kV influenced by an electric field (Hoffmann & Stroobant, 2011). Ions in the solution are pulled toward the capillary tip where the field is highest and destabilize the meniscus into a shape named a “Taylor cone” (after the work of Taylor & Mcewan (1965)). The surface tension of the liquid is on the other side of the balance to resist cone development. Once the applied electric field exceeds the surface tension, the tip of the cone breaks out of the rest, forming a droplet with excessive charge on its surface (Wilm & Mann, 1994). The charged droplets drift through the hot desolvation gas toward the opposing electrode, shrinking on its way due to solvent evaporation. This leads to a continuous increase in the abhorrence between the charges on the droplet surface. When the droplet radius shrinks down to the Rayleigh limit, a phenomenon called Coulomb fission or Coulomb explosion occurs (Kebarle & Verkerk, 2009). The droplet surface becomes unstable and small, charged progeny droplets are released. The fission continues on the progeny droplets and their subsequent droplets until fully desolvated ions are produced.
Currently, two mechanisms for gas phase ions formation have been proposed and there is good evidence backing up both proposals. The first mechanism was 38
proposed by Dole et al. (1968), now known as charged residue model (CRM). This model assumes that as fission of droplets occurs, there will be smaller droplets that contain only one analyte molecule with an ionic charge on the droplet surface. When the solvent from these droplets completely evaporates, there remains a gas phase analyte ions with the charge originating from the droplet surface. This mechanism is mostly suited for the formation of gas phase ions of macromolecules such as proteins (Winger et al., 1993). On the other hand, the Ion Evaporation Model (IEM) suggested by Iribarne & Thomson (1976) and Thomson & Iribarne (1979) is more plausible for small organic and inorganic ions. Based on calculations, the IEM predicts that when the droplet radius shrinks to less than 10nm, ion emission will occur instead of Coulomb fission. The process involves ions escape directly from the surface of ‘parent’ droplets, removing the charges. Perhaps both mechanisms operate, depending on the condition and the type of molecules, and they complement rather than contradict each other.
A modified version of electrospray is nanoelectrospray which was developed by Wilm & Mann (1996). It has the same principles of working as electrospray but with much smaller spray tip. This helps to conserve valuable analytes and therefore becomes important in the analysis of biochemical and biopharmaceutical samples. ESI has many advantages in comparison with other ionization techniques such as EI, FAB or MALDI. The ability to produce multiply charge ions from large molecules with several ionisable sites is the major advantage of ESI. This vastly increase the upper mass limit of detection to above 130 kDa (Hoffmann & Stroobant, 2011), making it a powerful tool for proteomic studies. Even ionization and identification of whole intact virus has been successfully achieved using ESI-MS (Fuerstenau et al., 2001). Importantly, its soft ionization process also preserves the non-covalently bound complexes in the gas phase (Daniel et al., 2002), providing a novel way to study complexes in terms of stoichiometry and energetics. Unlike in the solution phase, non-covalently bound complexes in gas phase can possess a very high activation energy and thus are strongly associated (Kebarle & Verkerk, 2009). Where necessary, collision induced dissociation can be employed in tandem mass spectrometry to get useful fragmentation data although this fragmentation mechanism is quite complicated and not standardized unlike EI processes. Furthermore, ESI can work with very low analyte concentration i.e. at 10-7 M (Kebarle & Verkerk, 2009). Methanol, acetonitrile and mixtures of these with water are commonly used as the preferred solvents for mass spectrometry. With 39
the greater sensitivity of ESI-MS, solvents that possess low solubility for electrolytes, such as toluene, can also be used.
Collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS)
Collision-induced dissociation involves the use of a collision cell filled with a non-reactive molecule, commonly noble gases especially helium or argon. When an ion is passed through a filled collision cell, depending on the gas pressure inside the cell, it generates fragments which are detected by the mass spectrometer. The collision energy can be controlled via the collision cell pressure and thus the degree of fragmentation. As a consequence, this can be applied to studying non-covalent binding of complexes based on the how the complex fragments under low energy collision. At high collision energy, intense fragmentation including radical formation similar to that in electron ionization (EI) had been observed (March, 1997).
Tandem mass spectrometry includes more than one mass analyzer with a collision cell either filled or empty in between each analyzer in order to perform a desire task. Depending on the configuration of the system, various purposes such as finding the daughter ion of a fragmented parent molecule or finding the molecules generating a neutral lost, etc. can be achieved. Usually, a triple quadrupole mass analyzer is used and three main configurations. Daughter ion scan is the most common mode of MS/MS. The first mass analyzer was set to only allowed the desired ion (at specific m/z) to pass to the collision cell. Fragment ions from the CID of the parent ion were all scanned by the second mass analyzer and recorded on the spectrum. Because generally, different molecules have specific fragmentation pathway, the daughter ion scan spectrum can serve as a unique identifier for individual molecules, except for enantiomers. Therefore, fragmentation data is valuable in identity clarification of a suspected metabolite by comparison with standards. In this work, we also examined the use of an “inverse approach” for metabolism prediction (Medzihradszky etal; 2000).
HPLC-MS, HPLC-MS/MS and matrix effects The combination of HPLC and MS provides one of the most powerful analytical techniques. It can effectively separate then identify individual components in a mixture 40
of unknown compounds. By coupling MS with HPLC, the difficulties of mobile phase choice and method development as well as various separation problems are alleviated. Ions with the m/z in the scan range are constantly recorded with respect to time to give the extracted chromatograms of the chosen m/z value. These extracted chromatograms can reveal the order in retention time of specific ions and confirmation can be made by comparison with standards or previous experiments. With tandem mass spectrometry available, unequivocal identification is made simple once efficient separation is achieved. Although powerful, HPLC-MS Often encounters some difficulties, most notably the matrix effect.
Matrix effects arise during the process of transferring the sample in the liquid phase post-HPLC into the gas phase for analysis by a mass spectrometer (Mei, 2005). Of the two effects, ion suppression is more problematic and its causes have been thoroughly investigated. Several mechanisms are suggested directly based on how electrospray ionization (ESI) works. There are numerous factors affecting the ionization process and thus contributing to matrix effects.
The principal factors to be considered are properties of the analyte. The ion evaporation model (IEM) based on the link between ion intensity and its concentration proposes that ions with higher charge density and lower desolvation will give higher signals. This model correctly predicts the ion suppression caused by NH4Cl (Kebarle & Peschke, 2000) but it fails when applied to more complex organic molecules. In those molecules, the signals are influenced more by surface activity, which is described in the surface activity and partitioning-equilibrium model (Enke, 1997). This model looks at the problem from the aspect of an ESI droplet having two different components: the charged surface phase and the neutral interior phase. It proposes that ions partitioning more into the charged surface phase will be expected to give better ESI signals and vice versa. As a consequence, high surface active ionic contaminants such as SDS or CTAB (present in glassware detergents) or Tween 80 (present as excipients in medicine manufacture) will suppress the signals of other important analytes. Both of the above- mentioned models rather than conflicting with one another work in concert as each model can be used depending on the properties of the analyte under consideration.
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One common cause of matrix effect is incomplete evaporation, mostly due to the formation of the analyte as non-volatile phosphate or sulphate salts (Constantopoulos et al., 1999, King et al., 2000). These salts can change the physical properties of the sprayed solution and cause weak Taylor cone emission, consequently decreasing ionization efficiency and therefore the detected signals. Furthermore, involatile salts can often cause blockage of the ESI probe capillary and permanent damage require laborious repair. Analytical planning and sample preparation must carefully consider this point.
1.4.3 Fourier transform infrared spectroscopy (FTIR) The history of infrared spectroscopy (IR) was initiated in 1686 when observations on behavior of heat radiation. It was subsequently confirmed that this invisible irradiation was beyond the red end of the solar spectrum, hence its name infrared (Schrader, 1995). This region includes wavelengths in the range of approx. 800 nm to 1 mm, in which the vibrational portion of 2.5 m to 25 m (middle infrared) is particularly of chemical interest. Commonly, the radiation in the vibrational infrared region is referred to in terms of wavenumber rather than wavelength. The unit of wavenumbers is reciprocal centimeter (cm-1). Conversion from wavelength to wavenumber is done by taking the reciprocal of the wavelength expressed in the same unit i.e. cm-1 and vice versa. The main reason that wavenumbers are used instead of wavelength is that they are directly proportional to energy (Pavia et al., 2001).
The principle of IR involves the use of radiation to excite the bonds of a molecule and record the vibration pattern via absorption. For convenience, a bond between two atoms is considered to possess spring-like elasticity. Each bond has several resonant frequencies at which it vibrates more strongly when irradiated. This increase in amplitude of the vibrational motions is observed and assigned to the corresponding bonds (Griffiths & De Haseth, 2007).
One of the best features of IR is that it is very sensitive to slight changes in the bond’s nature. Even two identical bonds in slightly different environment cannot generate identical spectra, although most the absorption bands are similar. The ‘fingerprint’ region in IR is in the range of approx. 1500 to 500 cm-1, consisting of a
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complexity of bands with unique pattern of absorption. Consequently, it serves as a powerful tool for identity confirmation, corresponds to fingerprint check in human. Other regions of the IR spectrum provide information on the vibrations of different groups. Each group of bonds usually exhibit absorption in a distinct, confined region and thus detection and general assignment are relatively simple with a correlation chart. For example, a broad absorption band with medium intensity in the region of 3500 cm- 1 is often attributed to the presence of an alcoholic hydroxyl group (O-H). Similarly, an absorption in the range of 1700 ± 100 cm-1 often confirms the presence of a carbonyl group (C=O). Individual assignment of each peak to the corresponding bond in a molecule for the purpose of structural clarification during synthesis requires an experienced expert. However, this is not often necessary for the purpose of identity confirmation via comparison (Smith, 2011).
In general, IR provides valuable information on the structure of a molecule, especially the presence of any functional group. Although difficulty in spectrum interpretation is its drawback, IR is a simple and robust technique. When used in conjunction with other techniques such as mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy, it will provide an additional perspective to the insight of a molecular structure and aid identification.
1.5 Phytochemical analysis 1.5.1 Phenolic content assay Determination of phenolic contents in plants has vital importance, as its linked with the antioxidant phenomenon of plants. Shikimic acid produced by plants are responsible for the production of phenolic compounds, which are basically secondary metabolites and are being regulated by phenylpropanoid metaboilzation. Structure wise they may form complex polymerized compounds, but at least they must contain one benzene ring (Velderrain-Rodríguez et al., 2014). Normally phenolic compounds are believed to be linked with defense responses in plants. Plants contain verity of phenolic or polyphenolic compounds, comprises phenolic acid, flavonoids and colored anthocyanins (Babbar et al., 2014). Animal body tends to produced highly reactive oxidized molecules like reactive oxygen (ROS) and reactive nitrogen species (RNS), which leads to damage and hinder the certain biological mechanism at cellular level
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(Maria Cova etal, 2015), (Figure 1.7). Body needs verity of oxidants like phenolic compounds to deal with such situation and preventing cellular injury. Reported literature revealed the advantages of phenolic compounds, such as antioxidant, anticancerous and anti-inflammatory agent (Lin et al., 2016).
Fig 1.7 Illustration of oxidative stress and antioxidant mechanism
1.5.2 Flavonoids content assay Flavonoids are categorized among larger group of phenolic compounds, widely distributed in plant species, especially citrus family. About 6500 flavonoids having different classes and biosynthesis routes have been identified uptill now. Flavonoids exhibited verity of biological function against biotic and abiotic stresses. Flavonoids are key compounds to mask the effect of UV radiations and hence reduced the cellular damage. It is believed that flavonoids are responsible for plant hormone transportation and initiates the coloring phenomenon in flowers. Flavonoids have good antioxidant activity and its depends upon the position of hydroxyl groups in molecular structure. Isflavanoids are also famous as a detoxifying agent which diffuses the free radicals and hinders the peroxidation (Buer et al., 2010).
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1.6 Bioassays Numerous phytochemical and biological assays are being used to estimate the medicinal characteristics of plants. These assays are more reliable and specific towards their reproducibility (Singh et al., 2012, Da Silva et al., 2000). Aromatic ring with one or more hydroxyl group in phenolic compounds are present in most of the plant species. They depicted more desirable physiological, biological outcomes and reflects the basic aspects of bioactive chemical entities of natural products like antioxidant potential. Compound like Phenols and Polyphenols are considered to be a vital secondary metabolites of natural products. Various studies revealed the importance of antioxidant properties of secondary metabolites of plant based diets against many disease. Crude extract and their isolated compounds have been enormously screened to estimate their cytotoxicity (Meyer et al., 1982b).
1.6.1 Antioxidant activities Measurement of antioxidant activity It would be nice to know the antioxidant capacity and responsible compounds, which we consume in our daily life. It is very difficult to relay on single antioxidant capacity assay, which determines the quantitative amount of antioxidant agent. Literature reveals the serval methods which claims the in vitro measurement of antioxidant capacity. Phenolic compounds are believed to associated with very strong scavenging activity and has also been correlated with reduced risk of certain disease conditions. Separation of antioxidant compounds from complex natural products is very tedious and time consuming. In this area, researcher are trying to established a validated methods, which could easily be used to determine the antioxidant capacity of plants and food stuff (Natella et al., 1999). Moreover, from previous published reports (Siquet et al., 2006), it is easily assumed that, there is no single accurately assay is established which could authenticates the quantitatively measurement of antioxidant capacity of a compound or extract.
In Vitro assays for antioxidant activities 1.6.1.2.1 DPPH assay Assay used to quantify the antioxidant capacity of selected plant was slightly modified from previously established protocol by Frankel et al., 1994. DPPH assay is
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widely used to determine the in vitro antioxidant capacity of compounds and extracts. DPPH● is most stable organic nitrogen radicals, which is being extensively used to measure the reducing ability of antioxidant. DPPH is colorimetric methods, which depends upon the change of its radical form DPPH● absorbs at 515 nm, when reacted with sample compounds. Deep violet color of DPPH dye is changed to colorless, when received a hydrogen atom from antioxidant. A simple and rapid, spectrophotometric methods can be used to determine the free radical scavenging ability of test compounds.
DPPH●+ AH → DPPH-H + A● DPPH●+ R● → DPPH-R
1.6.1.2.2 Total antioxidant capacity An adequate amount of antioxidant is necessary to prevent any cellular damage. In either case ,if antioxidant are deficient in body, then the body cells and tissues are more prone to develop any mal functioning or disease. Total antioxidant capacity is also used as a biomarker in various diseases in the field of medicines and nutritional sciences. TAC assay principle based upon the reduction of Molybdenum (VI) to Molybdnem (V) by the test sample. At acidic pH, the formation of green phosphate/Mo (V) complex is observed (Prieto et al., 1999).
1.6.1.2.3 Reducing power assay Reducing power assay principle based upon the formation of complex with metal atoms like iron and copper. As a result of this reaction mixture between test sample and metallic ions, a colored complex of potassium ferricynanide and ferric chloride is formed, which can be measure at 700 nm by UV-spectrophotometer. If phenolic or flavonoids are present in a reasonable quantity then, yellow color changes to green and blue color. During reaction when more elections are transferred by antioxidant compound, then fe3+ are reduced to ferrous form and resulting in increased absorbance (Jayanthi & Lalitha, 2011).
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1.6.2 Antimicrobial activities Antibacterial assay Antibiotics are the front line therapy against bacterial infections and benefited by large to humanity, since from their discovery. However, from last few decades, due to the emergence of drug resistant bacteria, certain groups of antibiotics become less effective or ineffective (Burt, 2004). Natural products based drugs played a significance role in combatting different diseases. Tradition uses of natural products against certain disease conditions has been well documented (Balouiri et al., 2016). Various plants secondary metabolites like phenolic compounds, tannins, glycosides, alkaloids have been reported as good source of anti- microbial agent (Davidson & Parish, 1989). Due to the emergence of drug resistance, it is extremely important to discover new antibiotic from natural source. For this purpose several methods have been developed and reported to evaluate the antibacterial potential of plants against different pathogenic bacterial strains (Khan et al., 2013).
Resazurin assay Resazurin based on dye, which has been extensively used as an indicator in various cytotoxic assay. Resazurin dye based on colorimetric method, which determined the cellular metabolic reaction with reduced cell toxicity. Mitochondria and cellular cytosol is the main site for reduction reactions (Ertürk, 2006). Resazurin dye will oxidized to reduced form (resorufin) and in return color changes blue to red. This reduction of resazurin to resorufin is associated with live organism both prokaryotic and eukaryotic. Mitochondrial group enzyme dehydrogenases are supposed to be responsible for this cellular reduction (Pfaller et al., 1994, Pital et al., 1958, Boum et al., 2013). The spectrum of reduction can be quantified by using spectrophotometer on wavelength 600 nm and 500 nm, by using appropriate filters. Resazurin assay is reported as simple, rapid, reproducible assay, extensively utilized to access natural products toxicity profile. Sarker et al., has modified classic REMA to simple and more authentic result oriented method (Sarker et al., 2007). Antimicrobial activities from different natural products like higher plants have been reported against different pathogenic strains (Palomino et al., 2002). Due to the bacterial resistance against certain antibiotic groups, there is need to develop consolidated drug testing methods against
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resistant bacteria to achieve a target of novel scaffolds in the field of medicine (Rates, 2001).
Fig 1.8 Reduction of resazurin to resorufin by oxidoreductases from viable cells
Antifungal assay The surge in fungal infection has been observed since last few decades. Most of the cases, it appears as a systemic infection or as co-infections align with other disease like cancer or viral infections (Dellavalle et al., 2011). It is evident that fungal infections are show higher mortality rates than bacterial infection due to resistance develop against limited antifungal drugs. This tends to increase the demand of antifungal agents development along with their rapid and accurate screening methods. Higher plants are a big source of potential naturally antifungal agent. Cylinder plate method and paper disc methods are extensively been used to evaluate the antifungal potential of both natural origin products and synthetic products (Fieira et al., 2013).
1.6.3 Antileishmanial assay The emergance in the endemicity of genus Leishmania has become worldwide health issue, caused by the protozoa. Due to the lack of vaccine, there is a drastic increase in disease condition.Since the emergance of AIDS, the occurance of this diease has been
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reported significantly. This is fatal tropical disease and second most lethal protozoan diease as per World Health Organization (WHO). Domestic and wild animals such as dogs, are the main carriers of the parasite. According to WHO reports, plants are the biggest and potential sourse of Leishmaniasis treatment (Brito et al., 2013)
1.6.4 Cytotoxicity assay Brine shrimp lethality bioassay Brine shrimp lethality assay is widely employed to evaluate the toxicity profile of heavy metals, drugs especially from natural product origins. It is also a preliminary cytotoxic test for natural products (Pisutthanan et al., 2013). This assay is based upon the mortality rate of brine shrimp organism (Atremia salina). Initially it was proposed by Michael etal, which was further developed by different researcher. Another very important aspect of this assay is use in design of experiments. This assay can be use as a drug vehicles or carries in order to established the toxicity profile of tested compounds (Meyer et al., 1982a). Effect of solvent is very crucial in this assay, for this both positive and negative control should be designed (Colegate & Molyneux, 2007). The low cost and availability of commercial brine shrimps eggs, makes this assay a reliable tool for isolation of secondary metabolites from natural products (Mclaughlin et al., 1991).
Protein kinase inhibition assay Protein kinases are considered to be as one of the most promising group drug targets, which relates with the various pathological conditions, like various cancers, inflammation and metabolism problems (Shchemelinin et al., 2006). By keeping in view its importance, lot of work has been done to discover and establish Protein kinases inhibition assay by using HTP screening. In recent past, various assay has been optimized to deal with the challenges of discovering Protein kinases inhibitors. After series of phosphorylation of protein kinases, the enzyme kinases transfers the phosphoryl group onto the target protein, which plays important role by initiating the various biological process. Protein kinases inhibition turned into promising tool to access the anti-proliferating activity (Ma et al., 2008).
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MTT assay Previous literature revealed the cytotoxic potential of natural products and their major bioactive consitituents by using dyes (fluoresent) peculiar staining methods particular MTT assay (Manosroi et al., 2006). Some other metal based tests like propidium iodide, resazurin and trypan Blue tests were also been used (Horvathova et al., 2006). Among all the assays MTT assay has been tremendously used because of its accurate and reliable results for the screening of cytotoxic agents (Manosroi et al., 2006, Hou et al., 2007).
Measurement of cell viability depends upon the intensity of viable cell intact to mitochondrial membrane and to respiratory chain of the mitochondria. The toxicity profile of the bioactive constituents of natural products can be assessed by using the mitochondrial dehydrogenases from viable cells. In MTT assay the mitochondrial succinate dehydrogenase system is supposed to potentiates the viable cells, which convert MMT dye i-e salt of tetrazolium to formazan dye. Mitochondrial succinate dehydrogenase enzyme system reduced the yellow tetrazolium salt to purple formazan water insoluble dye (Figure 1.9).
Fig 1.9 Illustration of MTT assay
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1.7 Molecular docking Molecular docking is an advanced and novel technique in drug design and discovery. Docking is a technique which anticipates the potential orientation of one molecule to another, to get a stable complex. The bond orientation between two molecules predicts the binding affinity or strength. Molecular docking is widely used to predict the bond ordination of drug molecule to their protein targets, which is a reliable tool for drug design and analysis. There a series of software are available to access the structural data base of medicinal scientist. Molecular docking works on phenomenon of “lock and key”, in terms of protein and targeted ligand. The aim of molecular docking is to achieve an optimized conformation for both protein of interest and targeted ligands (Kellenberger et al., 2004).
1.7.1 Advent of Molecular Docking There are mainly two types of techniques being employed to access the molecular docking outcomes. One technique relays on the binding affinity of the ligand and targeted molecule and know as computer simulations studies. This technique requires more time for the energy profiling and it is more fit to accept the ligand flexibility, which triggers the interpretation of molecular perception among ligand and receptor. Second technique which demonstrate the calculation of complementarity among ligand and receptor. This technique have much faster outcomes in terms of scanning ligands and their binding sites. Moreover this techniques entertains the both flexible and rapid docking (Agarwal & Mehrotra, 2016).
1.7.2 Epithelial growth factor receptor (EGFR) Epithelial growth factor receptor, an important protein which gives the insight of cancer cell mechanism especially against lung cancer cell lines. This protein is present on both cancer cells and as well on normal cells. The mutation in EGFR is directly linked with lung carcinoma. The abnormal growth or gene expression of this protein result in the abnormal growth of lung cell, which eventually leads to lung carcinoma. There are several drug therapy available, like tyrosine kinase inhibitor, which potentially inhibits this protein and restrict the abnormal growth of cancer cells. In this scenario we need such inhibitors, which suppress the protein expression. Therefore in the current study several isolated and identified compounds along with
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control (Etoposide) were dock against EGFR protein to predict the binding affinity towards EGFR (Ferguson, 2008).
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AIMS AND OBJECTIVE To collect the under ground part of Asparagus adscendens Roxb. and Trillium govanianum Wall. To prepare MeOH extract by maceration followed by solid phase extraction fractions, eluting with a step gradient of MeOH-water mixtures. To carry out the phytochemical analysis of solid phase extraction fractions by using advanced chromatographic and spectroscopic techniques to get better insight of chemical constitutes of Asparagus adscendens Roxb. and Trillium govanianum Wall. To isolate and characterized phytochemical constituents by using advanced chromatographic and spectroscopic techniques. To carry out antioxidant, antimicrobial, in vitro antileishmanial and cytotoxicity analysis of MeOH extract, SPE fractions and possible isolated compounds of Asparagus adscendens Roxb. and Trillium govanianum Wall. To carry out the molecular docking studies of isolated and identified compounds of Asparagus adscendens Roxb. and Trillium govanianum Wall.
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Chapter 2
2 Material & Methods
54
Material & Methods
2.1 Chemical and standard compounds Solvents used, triluoroacetic acid and formic acid were purchased from Sigma –Aldrich (Dorest, UK) and Folin–Ciocalteu reagent, 2, 2-diphenyl, 1-picrylhydrazine (DPPH), quercetin, myricetin, Kaempferol, ascorbic acid, gallic acid , sodium hydroxide, ferrous chloride, dipotassium hydrogen phosphate, dihydrogen phosphate were purchased from Sigma–Aldrich (Steinheim, Germany), sterile resazurin tablets were purchased from Fischer Chemicals. Regents and standard drugs used in cytotoxity assay (MTT reagent,RPMI-1640 medium, phosphate buffer saline, etoposide, surfactin, doxorubicin, eggs for Artemia salina were purchased from Sigma–Aldrich (Steinheim, Germany) ; unless stated otherwise.
2.1.1 Cell lines used to evaluate the cytotoxicity of plants (i) Breast cancer cell line- MCF7 (ATCC-HTB-22) (ii) Liver cancer cell line- HEPG2 (ATCC-HB-8065) (iii) Lung liver cancer cell line-A549 (ATCC-CLL 185) (iv) Urinary bladder cancer cell line-EJ138 (ATCC-CRL-10708) (v) Vero Cell Line-SF (ATCC-CCL-81)
All these cancer cell line were procured from School of Pharmacy and Bimolecular Sciences, Liverpool John Moores University, UK.
2.1.2 Strains of microorganism to evaluate the antimicrobial potential of plants Bacterial Strains (Bacterial strains were from National collection of type culture (NCTC) and American type culture collection (ATCC)) (i) Staphylococcus aureus (NCTC 7508) (ii) Bacillus subtilis (NCTC 1604) (iii) Micrococcus luteus (NCTC 7508) (iv) Klebsiella oxytoca (NCTC 8017) 55
(v) Escherichia coli (ATCC 25922)
Fungal Strains (Fungal strains were obtained from First culture bank of Pakistan (FCBP), Punjab University, Lahore). (i) Aspergillus fumigatus (FCBP- 66) (ii) Mucor species (FCBP-0300) (iii) Aspergillus niger (FCBP-0198) (iv) Aspergillus flavus (FCBP-0064)
2.2 Instruments Major instruments along with their company identification, which were used in current study are mentioned in Table 2.1
Table 2.1 Instruments used in present study
S No Instrument Origin 1 Analytical Balance (PA214C) Ohaus corporation USA 2 Water bath (TW2O) Julabo, GmbH Germany 3 Rotatory vacuum evaporator (Stuart RE301) Cole-parmer, UK 4 Vacuum pump (2G6) Telstar, Spain 5 Oven (30 S) Sci Quip UK 6 Lyophilizer Telstar Lyo Quest, Spain 7 Sonicator (U400D) Scientific lab, UK 8 Vortex mixer (S0100) VX 100 Labnet, USA 9 Micro pipette Rainin, USA 10 P H meter (LE409) Metter Toledo, USA 11 Glass tubes (B7815-13) Lined caps, USA 12 CENTRIFUGE (5810R) EPPENDROF, Germany 13 Ultra-low freezer Bio Cold, UK 14 Dish washer (G7804) Miele Professional, Germany 15 Autoclave (F150 OPT.7) Touch clave Lab, UK 16 Milli-Q (MPGP04001) MILLIPORE , USA
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17 EISA PALTE READER (430-0037) CLARIO STAR, BMG LABTECH 18 StrataTM Cartridge Phenomenex, USA 19 Incubator (13-13900) Binder Lab mode Germany 20 Laminar hood Kojair Blue Series Tech., Finland 21 Inverted Microscope (DMIL LED) Leica Microsystem CMS GmbH Germany 22 Laminar safety cabinets Samber limited, England 23 Analytical HPLC (1206 Infinity) Agilent tech., GERMANY 24 Preparative HPLC (1206 Infinity) Agilent tech., GERMANY 25 LC/MS (QUATTRO VAA001) WATERS UK 26 GC/MS (DSQ II 11152) Thermo Scientific Co., USA 27 NMR JEOL, USA 28 Orbitrary shaker Gallenkamp, England 29 Magnet shaker Gallenkamp, England
2.3 Collection of plant materials Asparagus adscendens was collected from Nathia Gali, region of Khyber Pakhtunkhwa, Pakistan in the month of September, 2014 and identified as Asparagus adscendens Roxb. whereas Trillium govanianum was collected from Muzaffarabad district of Pakistan-controlled Azad Kashmir in the month of October, 2014 and identified as Trillium govanianum Wall. by Dr Muhammad Zafar, Herbarium Botanist, Department of Plant Sciences, Quaid-I-Azam University, Islamabad, Pakistan. A herbarium specimen for these collections Asparagus adscendens Roxb. and Trillium govanianum Wall. (voucher number: Acc no. PAC1001 and Acc no.128085 respectively) have been deposited and retained in the above herbarium.
2.3.1 Extraction and preparation of plant samples Shade-dried and finely ground roots (2.5 kg) of Asparagus adscendens and Trillium govanianum were macerated in MeOH (5 L) for 10 days at room temperature, filtered, and the solvent was evaporated under vacuum using a rotatory evaporator (<45oC) to obtain concentrated gummy crude extract and carefully sealed with
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aluminium foil and stored in -40 oC refrigerator. Figure 2.1 presents general research methodology schem
Fig 2.1 General research methodology scheme
2.3.2 Solid-phase extraction (SPE) and sample purification A portion of the dried MeOH extract (2 g) was suspended in 20 mL of HPLC grade water and loaded on to a Strata C-18 cartridge (20 g), previously washed with MeOH (50 mL) followed by equilibration with water (100 mL). The cartridge was eluted with MeOH-water mixture of decreasing polarity to obtain four fractions: 20, 50, 80 and 100% MeOH in water (250 mL each), coded A. asparagus and T. govanianum SPE fractions AAMF1, AAMF2, AAMF3, AAMF4 and TGMF1, TGMF2, TGMF3 and TGMF4 respectively (Figure 2.2). All four fractions were evaporated to dryness using a combination of rotary evaporator and freeze-dryer, re- dissolved in MeOH (10 mg/mL), centrifuged at 12,000 rpm for 3 min, filtered through 0.20 µm sterile syringe filter for injection (10 µL) into the liquid chromatography mass spectrometry (LCMS) system and into the HPLC-PDA system (Sarker & Nahar, 2012).
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Fig 2.2 Solid Phase Extraction (SPE) fractionation of A. asparagus and T. govanianum
2.4 Methods 2.5 Phytochemical analysis 2.5.1 Determination of total phenolic contents Total phenolic contents of MeOH extract of the roots of A. asparagus and T. govanianum and its SPE were estimated by following the procedures of (Clarke et al., 2013) with slight modifications. Each test sample of 20 μl from 4 mg/mL DMSO stock solution was applied in respective well (96 well plate) and subsequently proceeded for further addition of 90 µl of diluted folinciocalteu reagent followed by 5 min incubation. Further addition of 90 µl of 6% sodium carbonate to each well of the plate and incubated for 1hr. The absorbance of each sample was taken at 630nm.
2.5.2 Determination of total flavonoid contents Aluminum chloride colorimetric method reported by Ul-Haq et al., 2012, with slight modification were used to estimate the total flavonoid contents of MeOH extract. From stock solution of (4.0 mg/mL) DMSO, 20 µl of sample were accurately applied to each well (96 wells plate), subsequently followed by the addition of 10 µl 10% 59
aluminum chloride, 10 µl 1M potassium acetate and 160 µl distilled water. The incubation time of reaction mixture was 30 minutes and absorbance was measured at 415nm.
2.5.3 Chromatographic and spectroscopic analysis High performance liquid chromatography–photodiode array detection (HPLC-PDA) 2.5.3.1.1 Optimization of method for fingerprint analysis of SPE fractions An analytical Agilent 1260 Infinity equipment was used. Reversed-phase chromatography was performed on a Phenomenex Gemini-NX 5 U C18 column (250 x 4.6 mm). The chromatographic conditions were optimized by method development with slight modification reported by (Nazemiyeh et al., 2008). A linear gradient elution with water (solvent A) and MeOH (solvent B) containing 1% TFA as the mobile phase offered the best resolution. The column temperature was set at 25oC. A variable wavelength UV-Vis detector was set at 220 nm, 254nm and 360nm. The initial mobile phase composition was 70% of A and 30% B at 0 min, then linear gradient to 100% of B over 30 min and held at that composition for 5 min before to returning to start conditions and column equilibration at flow rate of 0.800 mL/min (Table 2.2). The chromatograms were monitored as 220 nm, 254 nm and 360 nm. Quantitative estimation of bioactive constituents of plant extract are extensively being used by Reverse phase HPLC-PDA. It was done by matching the standard compounds with the test sample in term of their retention time and UV spectra.
Table 2.2 SPE fractions analytical HPLC-PDA
Time (min) A (Water %) B (MeOH %)
0.00 70.00 30.00
10.00 30.00 70.00
30.00 0.00 100.00
40.00 70.00 30.00
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LC-ESI-QTOF-Mass spectrometry analysis 2.5.3.2.1 Optimization of Method
An Alliance HPLC System 2695 (Waters) was used. Reversed-phase chromatography was performed on a Phenomenex Gemini-NX 5m C18 column (250 x 4.6 mm) with slight modification reported by (Felipe et al., 2014). The column temperature was set at 25oC. A variable wavelength UV-Vis detector was set at 220 nm. An elution gradient was used with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in MeOH). The initial mobile phase composition was 70% of A and 30% B at 0 min, then linear gradient to 100% of B over 30 min and held at that composition for 5 min at a flow rate of 1mL/min.
The LC system was coupled to a quadrupole TOF mass spectrometer (Waters Micromass LCT) attached with an electrospray ion source. Real time by the mass spectrometer data system recorded responses. The tuning parameters were set as follows: electrospray interface 3000 V, rangefinder lens 250 V, extraction cone 3 V, desolvation temperature 20oC, source temperature 100oC, nebulizer gas flow 20 L/hour, whereas desolvation gas flow 760 L/hour, and TOF tube 4687 V. Data acquisition method was set as follows: cycle time 1 sec, scan duration 0.9 sec, inter scan delay 0.1 sec, mass range 100 to 1600, centroid mode. Cone voltage were set at 40V. to get better results in positive ion mode.
Gas chromatography/mass spectrometry analysis 2.5.3.3.1 Sample preparation for GC/MS analysis: The MeOH extract (5g) was fractionized through solvent-solvent extraction. Dried MeOH extract was dissolved in 250 mL of water and then resuspended in 250 mL of n-hexane in separating funnel. The n- hexane layer was filtered and evaporated under vacuum using rotary evaporated (<40°C) to get concentrated non-polar (n- hexane) fraction.
2.5.3.3.2 Gas chromatography/mass spectrometry analysis:
The n-hexane fraction of MeOH extract of the roots of A. adscendens and Trillium govanianum were analyzed using Thermo Scientific DSC-II gas chromatography-mass spectrometer (Thermo Scientific Co.) equipped with capillary
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column by slight modification reported by (Al Hashmi et al., 2013). Experimental conditions were as fellow: DB 5-MS capillary standard non-polar column (15m x 0.25mm x 0.25μm film thickness). The stationary phase is DB5 (95% methyl siloxane and 5% phenyl siloxane). Flow rate of mobile phase (carrier gas: He) was set at 1.0 mL/min. The gas chromatography part was fitted with a programmable temperature vaporizing injector and the analysis was carried out in EI mode at 70eV beam energy. The GC temperature was ramped from 50 to 300°C at a rate of 25°C per minute. Injection volume was 1.0 μl and mass scanning range was 50 –550 m/z. Total elution time was 14 mins.
2.5.3.3.3 GC/MS identification of components GC/MS is mostly used to identify the fatty profile of non-polar fraction of plant extracts. The spectra of GC/MS analysis of tested compounds was compared with the inbuilt library of National institute standard and technology (NIST), which have approximately more then 62,000 patterns. On the basis of retention time and molecular weight, the sample components were identified.
2.6 Biological evaluation 2.6.1 Antioxidant assay DPPH assay Clarke et al., 2013, method with slight modifications were used to access the sscavenging property of MeOH extract and SPE of A. asparagus and T. govanianum. From stock solution of 4 mg/mL sample dissolved in DMSO, 20µl from each tested sample were poured accurately to each well of 96 wells plate followed by 180µl of the DPPH reagent. The incubation time of reaction mixture was 60 minutes and absorbance was measured at 517nm. Following equation was used to determine the sscavenging activity in percent.
% RSA = (1-Abs / Abc) x 100
Where Abs is the absorbance of DPPH regent with sample, whereas Abc is the absorbance of negative control. Ascorbic acid was exert as a positive control in current assay.
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Total antioxidant capacity assay
The total antioxidant capacity of MeOH extract and SPE of A. asparagus and T. govanianum was estimated by using phosphomolybdenum procedures reported by (Ullah et al., 2013). The 1mL (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) reagent and 0.1mL of each test sample (4mg/mL DMSO) were mixed thoroughly and incubated for 1.5hr in eppendorf tube at 95ºC. Absorbance was taken at 695nm. TAC was evaluated as mg ascorbic acid AAE/g DW.
Total reducing power assay The reducing power of MeOH extract and SPE of A. asparagus and T. govanianum were estimated as reported by (Ullah et al., 2013). From stock solution of 4.0 mg/mL DMSO, 200µl from each tested sample were taken in eppendorf tube followed by the addition of 400µl buffer and 500µl potassium ferricyanide. The incubation time of reaction mixture was 20 minutes at 50ºC and absorbance was measured at 517nm. After the addition and centrifugation of 500µl trichloroacetic acid to the mixture about 100µl supernatant was drawn and added to the respective well along with ferric chloride and distilled water (100µl 0.1% and 500µl respectively) and absorbance was measured at 700 nm. The TRP of each test sample was evaluated as mg ascorbic acid AAE/g DW.
2.6.2 Antimicrobial analysis Antibacterial assay The antibacterial activity of MeOH extract and SPE (AAMF1, AAMF2, AAMF3, AAMF4 and TGMF1, TGMF2, TGMF3, TGMF4) of A. asparagus and T. govanianum were measured by disc diffusion method (Islam et al., 2013). A fresh bacterial nutrient agar plates were prepared with appropriate seeding density [Staphylococcus aureus NCTC 7508; Bacillus subtilis NCTC 1604; Micrococus luteus NCTC 7508; Escherichia coli ATCC 25922]. From 20mg/mL DMSO stock solution of test sample, 5µL was poured accurately on sterile filter paper discs and were placed for incubation for 24 hours at 37°C. The average zones of inhibition of both test samples and controlled were noted., as experiment were performed in triplicate. Cefotaxime was used as positive control whereas, DMSO served as negative control. 63
Resazurin microtiter assay (REMA) 2.6.2.2.1 Minimum inhibitory concentration (MIC) determination The in vitro susceptibility testing was performed using a 96-well microtiter plate with resazurin. A stock solution having concentration of 128 µg/mL were prepared by dissolving in sterile distilled water. Plant extracts stock solution having concentration of 10 mg/mL were prepared with 10% DMSO (Sarker et al., 2007). Sterile Distilled water was used to dissolve resazurin dye to obtain 0.02 % and the solution was then sterilized by filtration. The MIC assay was carried out in according to CLSI guideline for microdilution test (Clinical Laboratory Standards Institute, 2012). Briefly, the stock antibiotics and plant extracts were serially twofold diluted with cation-adjusted Mueller hinton broth (CAMHB). The additional 60 µL of the CAMHB and 20 µL of 0.02% resazurin were added to all wells. An overnight culture of test bacteria, after centrifugation at 4,000 rpm for 10 min, was harvested with with NaCl, which were centrifugation at 4,000 rpm for 5 min each. The pellet collected was then adjusted approximately 0.5 standard McFarland equivalent ( 1 x 108 CFU/mL), diluted to give 5 x 106 CFU/mL, and then 20 µL will be transferred to the well so that the final concentration of inoculum was approximately 5 x 105 CFU/mL. The total volume in each well was 200 µL and the final concentration of antibiotics and the extracts were 0.06-64 µg/mL and 0.005-5 mg/mL, respectively. Wells without antibacterial agents and bacterial strain were used as controls. The 96-well microplate was then incubated at 37 °C for 24 h. The lowest concentration showing no colorimetric change from blue (resazurin) to pink (resorufin) was noted as the MIC. Each test was carried out in triplicate. The average values were calculated for the MIC of test material.
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Fig 2.3 Typical Plate, after 24 h in resazurin assay
Antifungal assay Antifungal activity based on disc diffusion protocol, were employed to access the antifugal potential of MeOH extract and SPE of A. asparagus and T. govanianum (Islam et al., 2013). Four (4) fungal strains were used [Aspergillus niger (FCBP-0198), Aspergillus flavus (FCBP-0064), Mucor species (FCBP-0300) and Aspergillus fumigatus (FCBP- 66)] and harvested in solution of Tween 20 (0.02 %) and McFarland (0.5) turbidity standard was adjusted. Sabouraud Dextrose Agar media was poured in petri dish and Inoculum of each fungus was swabbed. From stock solution of 20mg/mL DMSO, impregnated sterile filter paper disc with 5µl of each test sample was placed on media and kept for incubation for 24 hours and 28°C. Vernier scale was used to measure the zones of inhibition. For positive control and negative control, Clotrimazole and DMSO were used respectively.
2.6.3 In vitro antileishmanial analysis The in vitro antileishmanial activity of MeOH extract and SPE of A. asparagus and T. govanianum were estimated by the method reported by (Ullah et al., 2014). A medium containing 10% fetal bovine serum was used to kept Leishmania tropica KWH23 strain for 24 hours incubation. Stock solution was prepared in DMSO (10 mg/mL DMSO) and 96 wells plate was serially diluted. Each well of 96 wells of plate
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contained 5x106 promastigotes and samples were tested at three different concentrations (1000, 100, 10 µg/mL). For positive control and negative control Amphotericin B and 1% DMSO in PBS were used respectively. The incubation temperature for 96 wells plates were at 24oC for 72 hours. Neubauer counting chamber were used for the count of survived promastigotes For this purpose about 15 µl of test sample were poured.
2.6.4 Cytotoxicity assays Brine shrimp lethality assay The cytotoxic effect of MeOH extract and SPE of A. asparagus and T. govanianum were measured in wells (96 well plate) by using brine shrimp lethality bioassay (Ul-Haq et al., 2012). Eggs of test organism Artemia salina (Ocean 90, USA), were retained for (24–48 hours) in hatching period, by using simulated sterile sea water with constant supply of oxygen. Each fraction was tested at three graded concentrations (1000µg/mL, 500µg/mL and 250µg/mL). Doxorubicin (4mg/mL) and DMSO were used as positive control and negative control respectively. In each well of 96 wells plate, the mature phototropic nauplii were then harvested and fraction were dissolved by the addition of sea water (200µl) and subsequently volume was make up to 300µl. The incubation time was 24 hours and the degree of lethality exhibited was estimated by counting the number of shrimps per well for each test samples. Table curve (2D v5.01) software was used to calculate the median lethal concentration (LC50) of each test sample with ≥ 50 % mortality.
Protein kinase inhibition assay The protein kinase inhibition assay of MeOH extract and its SPE of A. asparagus and T. govanianum were performed by using purified isolates of Streptomyces (85E strain) and hyphae formation were observed (Yao et al., 2011). Fortified Streptomyces culture were used on sterile plates to develop a bacterial lawn. From stock solution (20 mg/mL) of DMSO, 5 μl of each sample was poured onto the 6mm sterile filter paper discs. The sterile plates were seeded with Streptomyces 85E and soaked paper discs having concentration (100 μg/disc) were placed directly on the surface. The incubation time for plates was 72 hours at room temperature (25°C) and
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results were depicted as bald zone of inhibition around test samples and soaked discs. Positive control (surfactin) and negative control (DMSO) soaked discs were placed.
MTT assay The potential cytotoxicity of the MeOH extract and SPE fractions of A. asparagus and T. govanianum were studied against four human carcinoma cell lines: breast (MCF7), liver (HepG2), lung (A549), urinary bladder (EJ138) and Vero (CL- 81) using the MTT assay (Mosmann, 1983; Basar et al., 2015; Khan et al., 2016). The cells were washed by phosphate buffer saline (PBS) and harvested by tripsinization. All cell lines were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum. All cells were cultured at 37 °C in 95% air and 5% CO2. For the MTT assay, cells were seeded into 24 well plates at density 1.2 x 104 cells/well in a working volume of 1 mL/well and allowed to grow for 24 hours before the commencement of each experiment.
The cells were treated for 24hours with different concentrations of test samples (the MeOH extract and SPE fractions; 0, 5, 10, 50, 100 and 500 µg/mL), presented in Figure 2.4. Dilution of stock solutions was made in culture medium yielding final sample concentrations with a final dimethyl sulfoxide concentration of 0.1%, including the control. Each sample was used to treat four wells of cells in each 24-well plate. After 24-hours treatment period, the toxicity of the samples on each carcinoma cell line was quantified. To achieve this, the medium in each well was replaced by MTT solution (500 µg/mL) and incubated for 2hours. Toxicity was assessed by the ability of the cells to reduce the yellow dye MTT to a blue formazan product (Popescu et al., 2015). MTT reagent was removed and the formazan crystals produced by viable cells were dissolved in isopropanol and OD560 was determined with the microplate reader (CLARIO Star Microplate reader, BMG Labtech, UK). The average OD560 obtained from all the control wells (without test sample) on each plate was arbitrarily set at 100% and the OD560 value for the average of wells of cells treated with each test samples were expressed as a percentage of this control. Each assay was performed on a minimum of three separate occasions, and the IC50 values were for each sample, on each cell line were calculated using Microsoft Excel version 2013.
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Fig 2.4 MTT assay protocol
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2.6.5 Isolation and characterization Figure 2.5 presents the isolation scheme of compounds by using analytical and preparative Reverse phase- HPLC from solid phase extraction fractions of A. adscendens.
Fig 2.5 . Isolation of compounds from A. asparagus
Preparative HPLC-PDA 2.6.5.1.1 Optimization of method Preparative Agilent 1260 Infinity was used. Reversed-phase chromatography was performed on a ACE-5 C18 column (150 x 21 mm). The column temperature was set at 25oC. A variable wavelength UV-Vis detector was set at 220 nm, 254nm and 360nm. An elution gradient was used with solvent A (1% trifluoroacetic acid in water) and solvent B (1% trifluoroacetic acid in MeOH). The initial mobile phase composition was 70% of A and 30% B at 0 min, then linear gradient to 100% of B over 30 min and held at that composition for 5 min before to returning to start conditions and column equilibration at flow rate of 0.800 mL/min. The chromatograms were monitored as 220 nm, 254 nm and 360 nm.
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Spectroscopic analysis: 1H-NMR (600 MHz), 13C-NMR (150 MHz), DEPT-135 and 2D NMR (HMBC, HSQC) were recorded Jeol NMR spectrometer. Chemical shifts of puirifed compounds are expressed in ppm and coupling constant (J) are expressed in Herts (Hz). For NMR anaylsis, deuterated solvents CDCl3, CD3OD, Pyridine-d6 or DMSO-d6 were used. Mass spectra were recorded Electron spray ionization (ESI) on Waters MassLynx version 4.1 system.
2.6.6 Computational methods Molecular docking simulations 2.6.6.1.1 Preparation of protein structure Three Dimensional structure of the target protein Epidermal growth factor receptor (EGFR) with PDB ID: 4R3P was retrieved from RCSB database. Preparation of protein was carried out by Protein preparation utilities of Discovery Studio Client 16.1.0. In protein preparation to achieve correct ionization and tautomeric state, all water molecules were removed and hydrogen atoms were added to protein and missing loop were also added into protein structure.
2.6.6.1.2 Preparation of ligand structures Structures of all selected compounds were generated using the tool ChemDraw profeesional v15 and then 3D optimizations and minimization of the compounds were carried using ligand preparation utilities of Discovery Studio Client 16.1.0.
2.6.6.1.3 Molecular docking protocol After preparation of protein and compounds docking protocol was validated by redocking the crystal ligand present in 4R3P and was found as having RMSD lower than 2A. After validation of docking protocol, all compounds were docked into the active site of EGFR and minimum 30 poses were generated. After completion of docking calculation, the best poses of each compounds were selected based on lowest CDocker Interaction Energies (CDIE). Visualization of binding interaction of all compounds were also carried out using Discovery Studio.
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In order to predict the binding affinity of control (Etoposide) towards EGFR, the compound was dock to the active site of EGFR. The bioactive confirmation of Etoposide was retrived from PDB (PDB-ID=3QX3) and wasdock into EGFR, following the aforementionted docking procedure and parameter.
2.6.7 Statistical analysis ANOVA (One way analysis of variance) was used for the statistical analysis of phytochemical, antimicrobial assays (antibacterial and antifungal), antileishmanial and cytotoxic results followed by Tukey and Duncan’s test.
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Chapter 3
3 Results
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Results
3.1 Phytochemical analysis 3.1.1 Total phenolic contents in Asparagus adscendens The total phenolic content of MeOH extract and SPE fractions in terms of gallic acid equivalent per gram dry weight are presented in Figure 3.2 and error bars represents the standard deviation. Significant content of gallic acid was shown in AAMF2, equivalent phenols (29.648± 1.55 mg GAE/ g DW), followed by the SPE fractions AAMF4, AAM (MeOH extract) AAMF1, and AAMF3 (21.37 ±2.66, 19.78 ± 2.55, 17.09 ±1.45, and 12.97 ±2.93 mg GAE/ g DW respectively). The SPE fraction AAMF2, which had the polar components of the parent MeOH extract, showed most promising total phenolic contents among other SPE fractions. On the contrary, the lowest total phenolic contents were shown by AAMF3, due to the presence of semi polar components of the parent MeOH extract. The SPE fraction AAMF4, which contained the least polar components of the parent MeOH extract, also exhibited notable content of gallic acid equivalent phenols. Gallic acid (5–25.00 μg /mL, positive control) was utilized to obtained a calibration curve (y = 0.01471x + 0.3097, R2 = 0.9877) and significant correlation was found at 0.05 level. (Figure 3.1).
4.5 y = 0.1471x + 0.3097 3.805 4 R² = 0.9877 3.519 3.5
3 2.439 2.5
2 1.788
OD of standard of OD 1.5 1.098 1
0.5 0.24 0 0 5 10 15 20 25 30 conc (µg/ml)
Fig 3.1 Regression line of Gallic acid
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40
30
20
TPC (µg TPC GAE/mg) 10
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.2 Total phenolic contents of Asparagus adscendens
3.1.2 Total phenolic contents in Trillium govanianum The total phenolic content of MeOH extract and SPE fractions in terms of gallic acid equivalent per gram dry weight are presented in Figure 3.3 and error bars represents the standard deviation. Significant content of gallic acid was shown by MeOH extract equivalent phenols (20.27± 3.03 mg GAE/ g DW), followed by the SPE fractions TGMF1, TGMF2, TGMF4 and TGMF3 (19.07±2.53, 16.70± 0.56, 15.768±1.44, and 13.35±3.43 mg GAE/ g DW, respectively). The SPE fraction TGMF1, which had the most polar components of the parent MeOH extract, showed most promising total phenolic contents among other SPE fractions. On the contrary, the lowest total phenolic contents were shown by TGMF3, due to the presence of semi polar components of the parent MeOH extract. The SPE fraction TGMF4, which contained the least polar components of the parent MeOH extract, also exhibited notable content of gallic acid equivalent phenols.
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25
20
15
10
TPC (µg TPC GAE/mg) 5
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.3 Total phenolic contents of Trillium govanianum
3.1.3 Total flavonoid contents of Asparagus adscendens The total flavonoid content of MeOH extract and SPE in terms of quercetin equivalent per gram dry weight (QE/ g DW) exhibited different levels of significant flavonoid contents (Figure 3.5) and error bars represents the standard deviation. Significant content of gallic acid was shown by fraction AAMF2 equivalent phenols (14.34 ± 1.44 mg QE/ g DW), followed by the SPE fractions AAMF4, AAMF3, AAM (MeOH extract), and AAMF1 (10.27 ± .233, 8.67 ± 0.34, 8.84 ± 1.34, and 7.53 ± 2.33 mg QE/ g DW respectively).The SPE fraction AAMF2, which had the polar components of the parent MeOH extract, showed most promising total flavonoid contents among other SPE fractions in term of mg quercetin equivalent per gram dry weight. On the contrary, the lowest total flavonoid contents were shown by AAMF1. The SPE fraction AAMF4, which contained the least polar components of the parent MeOH extract, also exhibited notable content of flavonoid. A positive correlation was observed between phenolic and flavonoid contents (correlation coefficient; R2 = 0.9992 and 0.9877 respectively, Figure 3.4), suggesting that the antioxidant prospective of phenols might be caused by the manifestation of flavonoids.
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3 2.833 y = 0.0572x - 0.0443 R² = 0.9992 2.5
2
1.5
OD of Standard of OD 1 0.855
0.5 0.281 0.09 0 0 10 20 30 40 50 60 conc (µg/ml)
Fig 3.4 Regression line for flavonoid contents
20
15
10
TFC (µg TFC QE/mg) 5
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.5 Total flavonoid contents of Asparagus adscendens
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Total flavonoid contents of Trillium govanianum The total flavonoid content of MeOH extract and SPE in terms of QE/ g DW exhibited different levels of significant flavonoid contents. The total flavonoid content of MeOH extract of roots of T. govanianum and SPE are presented in Figure 3.6 and error bars represents the standard deviation. The TGM (MeOH extract) showed highest flavonoid content (9.250 ± 0.50 mg QE/ g DW), followed by the SPE fractions TGMF1, TGMF2, TGMF4 and TGMF3 (8.882 ± 0.79, 7.038 ± 1.23, 6.684 ± 1.23, and 6.407 ± 1.14 GAE/ g DW respectively). The SPE fraction TGMF1, which had the most polar components of the parent MeOH extract, showed most promising total flavonoid contents among other SPE fractions. This fractions was almost as potent as its parent MeOH extract in term of mg quercetin equivalent per gram dry weight. On the contrary, the lowest total flavonoid contents were shown by TGMF3, due to the presence of semi polar components of the parent MeOH extract. The SPE fraction TGMF4, which contained the least polar components of the parent MeOH extract, also exhibited notable content of flavonoid. A positive correlation was observed between the phenolic and flavonoid contents (correlation coefficient; R2 = 0.9992 and 0.9877 respectively, Figure 3.4) suggesting that the antioxidant capacity of phenols might be caused by the apparition of flavonoids.
15
10
5
TFC (µg TFC QE/mg)
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.6 Total flavonoid contents of Trillium govanianum
77
3.1.4 Chromatographic and spectroscopic analysis HPLC-PDA analysis of Asparagus adscendens 3.1.4.1.1 Optimization of method for fingerprint analysis of SPE fractions: HPLC-PDA analysis on the SPE fractions of the MeOH extract of the roots of A. adscendens was performed to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain phenolic and flavonoids as possible contributors to the significant cytotoxic and antileishmanialactivity of the extract and its fractions. The chromatographic conditions were optimized by a analytical method A linear gradient elution with water and MeOH containing 1% TFA as the mobile phase offered the best resolution. Chromatographic finger printing analysis was done by using RP-HPLC-PDA for the estimation of proposed phenolic constituents of SPE fractions. (Figure 3.7). The results were interpreted by the comparison of respective retention time and UV-visible spectra of the reference standard compounds (Table 3.2).
A significant amount of quercetin, myricetin and kaemferol (68.90 ± 0.05, 27.78 ± 0.07 and 234.23 ± 0.14 μg/mg DW, respectively) were quantified from AAMF1. SFE fraction AAMF2 has shown significant amount of quercetin, myricetin and Kaemferol (170.11 ± 0.03, 175.27 ± 0.5 and 25.46 ± 0.16 μg/mg DW, respectively), where as only quercetin and myricetin (23.31 ± 0.01 and 73.77 ± 0.08 μg/mg DW) were quantified from AAMF4. Typical chromatograms of fractions (Figure 310, 3.11, 3.12,3.13 & 3.14.) were recorded by using PDA detector at 220, 254, 360 nm to provide a real time chromatograms and on-line Ultraviolet (UV) spectra from 200-500 nm were recorded (Table 3.3, 3.4,3.5 & 3.6) for identification of different groups and classes of compounds. The possible presence of compounds such as flavonoids, quercetin, myricetin and kaemferol in SPE fractions leads towards the biological potential of plant (Figure 3.8).
It was observed that the most compounds in the chromatograms (Figure
3.10,3,12, 3.13 & 3.14) possessed strong UV absorption at different retention time (tR in min.). The SPE fraction AAMF1 (Table 3.3) possessed 12 distinct peaks at different retention times (tR in min.), however peaks 1 (tR : 8.36 ), 2 (tR: 11.93), 6 (tR : 23.12 ) and 10 (tR : 28.34) showed UV- ʎ max 400,345,445 and 440nm respectively at 220nm.
78
The SPE fraction AAMF2 (Table 3.4) possessed 15 distinct peaks at various retention time (tR in min.), where as peaks 10 (tR : 18.78), 12 (tR : 23.11) and 18 (tR : 32.31) showed UV- ʎ max 400, 340 and 485nm respectively at 220nm. The SPE fraction
AAMF3 (Table 3.5) possessed 12 distinct peaks at different retention time (tR in min.), peak 7 (tR : 19.95) and 8 (tR : 20.96) showed UV- ʎ max 455 and 480 nm respectively at 220nm. The SPE fraction AAMF4 (Table 3.6), which is least polar fraction possessed 5 distinct peaks at various retention time, peak 1(tR : 19.80) and 2 (tR : 20.29) showed UV-ʎ max 365 and 360 nm respectively at 220nm. Regression curve of standard quercetin, myricetin and Kaempferol are expressed in Figure 3.8.
Table 3.1 Chemical Profiling of standards.
Correlation tR Calibration curve LOD LOQ Standard coefficient (min) equation (μg/mL) (μg/mL) (r2)
Quercetin 4.162 y = 20.26x + 10.207 0.9989 3.64 11.04
Myricetin 5.921 y= 17.796x + 3.0343 0.9988 3.85 11.66
Kaempferol 8.997 y = 18.395x - 58.817 0.9963 6.7 20.31
Quercetin Myricetin Kaempferol
Fig 3.7 Identified compounds from SPE fractions of A. adscendens using HPLC- PDA
79
Table 3.2 Chemical profiling of SPE fractions of A. adscendens using HPLC-PDA monitored at 220nm
Flavonol flavonoid (μg/mg DW)
SPE Quercetin Myricetin Kaempferol Fractions
Quantified Quantified Quantified tR (min) tR (min) tR (min) Amount (μg/mg) Amount (μg/mg) Amount (μg/mg)
AAMF1 4.5 68.90 ± 0.05 5.9 27.78 ± 0.07 9.2 234.23 ± 0.14
AAMF2 4.1 170.11 ± 0.03 5.5 175.27 ± 0.5 9.5 25.46 ± 0.16
AAMF3 ------
AAMF4 3.9 23.31 ± 0.01 5.1 73.77 ± 0.08 - -
Fig 3.8 HPLC-PDA Chromatogram of standard phenols monitored at 360nm
80
1800 1600 y = 20.26x + 10.207 A R² = 0.9989 1400 1200 1000 800 600 400 200 0 0 20 40 60 80 100
1600 y = 17.796x + 3.0343 1400 R² = 0.9988 B 1200
1000
800
600
400 200 0 0 10 20 30 40 50 60 70 80 90
1600 y = 18.395x - 58.817 1400 C R² = 0.9963 1200 1000 800 600 400
200 0 0 20 40 60 80 100 -200
Fig 3.9 Calibration curve of standard (A) Quercetin, (B) Myricetin, (C) Kaempferol
81
Fig 3.10 HPLC-PDA Chromatogram of AAMF1 of Asparagus adscendens
82
Fig 3.11 Corresponding UV-vis absorbance (AAMF1) at multiple wavelengths
83
Fig 3.12 HPLC-PDA Chromatogram of AAMF2 of Asparagus adscendens
84
Fig 3.13 HPLC-PDA Chromatogram of AAMF3 of Asparagus adscendens extract
85
Fig 3.14 HPLC-PDA Chromatogram of AAMF4 of Asparagus adscendens extract
86
Table 3.3 Retention times (tR) and corresponding UV-VIS. absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF1) of Asparagus adscendens
AAMF1 220nm 254nm 360nm
tR in tR in tR in Peaks (min) (min) (min)
1 8.36 225 270 295 320 400 7.47 220 280 12.85 240 260 290 310 2 11.93 240 270 290 310 345 8.42 225 270 320 400 16.66 285 325 360 3 17.4 220 265 12.91 245 260 295 310 340 28.34 265 295 385 440 4 20.53 230 290 26.44 240 290 5 22.38 235 270 28.34 230 265 290 390 440 6 23.12 230 265 290 285 445 32.76 230 260 7 25.24 230 280 34.61 240 290 8 26.22 230 295 36.21 245 295 375 390 440 9 27.48 230 245 10 28.34 230 265 295 390 440 11 33.35 235 285 12 37.65 220 270
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Table 3.4 Retention times (tR) and corresponding UV-VIS. absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF2) of A. adscendens
AAMF2 220nm 254nm 360nm
tR in tR in tR in Peaks (min) (min) (min)
1 5.29 220 245 275 7.47 220 280 12.85 225 240 260 290 310
2 5.3 210 245 270 8.42 225 270 320 400 16.66 220 285 295 325 360
3 9.99 220 275 290 320 12.9 260 295 310 340 28.34 225 265 295 385 440
4 13.77 220 230 240 250 295 26.4 240 290
5 14.54 230 260 275 310 28.3 265 290 390 440
6 15.27 230 260 310 370 32.8 230 260
7 16.17 220 270 290 390 34.6 240 290
8 16.11 220 270 290 390 36.2 295 375 390 440
9 17.52 215 230 250 300
10 18.78 220 230 265 310 400
11 21.73 235 280 380
12 23.11 240 260 290 310 340
13 26.16 220 260
14 28.83 230 280
15 29.29 230 260 280
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Table 3.5. Retention times (tR) and corresponding UV-VIS. absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF3) of A. adscendens
AAMF3 220nm 254nm 360nm
tR in tR in tR in Peaks (min) (min) (min) 1 11.68 225 275 10.74 260 11.68 225 280 2 15.74 220 240 15.72 220 245 15.39 225 245 260 335 3 16.94 245 285 16.92 245 285 17.91 260 280 320 4 17.34 230 275 17.89 260 280 330 18.87 240 255 265 275 5 17.93 260 280 320 18.89 250 255 280 335 19.92 255 290 430 460 6 19.44 235 265 280 19.92 260 280 290 430 460 20.94 265 345 420 480 7 19.95 260 290 430 455 20.94 265 320 335 415 480 21.7 280 8 20.96 265 335 410 480 23.54 230 270 285 9 21.41 235 285 350 10 22.62 230 285 11 23.54 230 270 285 12 23.83 220 255 285
89
Table 3.6. Retention times (tR) and corresponding UV-VIS absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fraction (AAMF4) of A. adscendens
AAMF4 220nm 254nm 360nm
tR in tR in tR in Peaks (min) (min) (min) 1 19.80 224 270 300 325 365 19.80 225 275 300 320 370 19.80 224 275 320 365 2 20.29 225 270 295 320 360 20.29 227 270 295 322 365 20.29 226 270 324 370 3 22.00 230 275 315 360 22.00 224 275 325 360 22.00 225 272 320 367 4 26.56 226 265 320 365 26.56 225 272 320 365 26.56 225 268 322 355 5 27.28 225 270 320 370 27.28 222 270 310 360 27.28 228 270 325 368
90
HPLC-PDA analysis of Trillium govanianum 3.1.4.2.1 Optimization of Method for fingerprint analysis of SPE fractions HPLC-PDA analysis on the SPE fractions of the MeOH extract of the roots of T. govanianum was performed to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain phenolic and flavonoids as possible contributors to the significant cytotoxic and antileishmanialactivity of the extract and its fractions. The chromatographic conditions were optimized by analytical method . A linear gradient elution with water and MeOH containing 1% TFA as the mobile phase offered the best resolution. A significant amount of quercetin (0.221 μg/mg DW), myricetin (0.09 μg/mg DW) and Kaempferol (0.528 μg/mg DW) were quantified from SPE fraction TGMF1 and TGMF2 (Table 3.7). Typical chromatograms and UV spectra of fractions (Figure 3.18, 3.19 & 3.20) were recorded by using PDA detector at 220, 254, 360 nm (Table 3.8) for the identification of different groups and classes of compounds. The possible presence of compounds such as flavonoids, quercetin, myricetin and kaemferol in SPE fractions, strengthen the notion that, such phenolic compounds are responsible for the biological profile of the plant (Figure 3.15, 3.16 & 3.17).
91
Table 3.7 Chemical profiling of SPE fractions of MeOH Extract of the roots of T. govanianum using HPLC-PDA monitored at 360 nm
Flavonol flavonoid (μg/mg DW)
SPE Quercetin Myricetin Kaempferol Fractions
Quantified Quantified Quantified tR (min) tR (min) tR (min) Amount (μg/mg) Amount (μg/mg) Amount (μg/mg)
TGMF1 4.1 0.221 ± 0.011 - - - -
TGMF2 - - 6.5 0.09 ± 0.010 8.8 0.528 ± 0.011
TGMF3 ------
TGMF4 ------
Fig 3.15 HPLC-PDA Chromatogram of detected compound from TGMF1 of Trillium govanianum
92
Fig 3.16 HPLC-PDA Chromatogram of detected compounds from TGMF2 of Trillium govanianum
93
Fig 3.17 Comparison of UV-VIS. spectra of a reference standard and detected compound.(A) Quercetin, (B) Myricetin, (C) Kaemferol
94
Fig 3.18 HPLC-PDA Chromatogram of TGMF3 of Trillium govanianum
95
Fig 3.19 HPLC-PDA Chromatogram of TGMF4 of Trillium govanianum extract
96
Fig 3.20 Corresponding UV-VIS. absorbance (A) and (B) of TGMF4 and (C)and (D) of TGMF3 at multiple wavelengths
97
Table 3.8 Retention times (tR) and corresponding UV-VIS. absorbance at multiple wavelengths of the peaks separated by HPLC of SPE fractions of the MeOH extract of the roots of Trillium govanianum
TGMF1 220nm 254nm 360nm tR in tR in tR in Peaks (min) Peaks (min) Peaks (min) 1a 3.23 230 270 295 320 1 7.47 220 280 1a 12.85 240 260 290 310 2a 4.1 230 250 285 315 365 2a 8.42 225 270 320 2a 16.66 285 325 360 3 14.57 220 275 3a 12.91 260 295 310 4 15.60 230 290 4 26.44 240 290 5a 23.22 230 265 290 285 445 5a 28.34 265 290 390 6a 24.38 230 265 295 390 440 6 32.76 230 260 7 26.57 220 270 7a 36.21 295 375 390 TGMF2 1a 6.5 230 265 320 340 2a 8.42 225 270 320 1a 12.85 240 260 290 310 2a 7.72 240 260 290 310 340 5a 28.3 265 290 390 2a 16.66 285 295 325 360 3a 8.88 230 265 290 325 368 8a 36.2 295 375 390 3a 28.34 265 295 385 410 TGMF3 1a 6.25 235 285 320 1 6.56 225 265 290 1a 24.57 230 280 330 365 2 20.36 230 260 2a 17.18 250 285 355 4a 24.45 230 280 330 365 3a 24.89 235 280 330 365 5a 26.57 245 290 320 360 4a 29.47 240 335 360 380 TGMF4 a 1a 11.84 225 265 285 330 360 1a 11.41 225 270 290 335 1 11.52 220 270 310 390 a 2a 15.69 220 260 335 365 2a 15.36 222 260 310 360 2 13.54 225 255 310 380 3 18.57 225 255 290 3 18.57 225 250 285 3 20.21 235 260 290 4 20.25 235 265 4 20.67 230 260 280 aPossible Phenolic Compounds
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LC-ESI-QTOF-Mass spectrometry analysis 3.1.4.3.1 LC-MS analysis of Asparagus adscendens LC-MS studies on the SPE fractions of A. adscendens were carried out to get an insight into the possible chemical composition of the fractions, particularly, to have an indication whether they contain saponins and sapogenins as possible contributors to the significant cytotoxicity of the extract and fractions. The chromatographic conditions were optimized by analytical method. A linear gradient elution with water and MeOH containing 0.1% formic acid as the mobile phase offered the best resolution. Typical chromatograms of fractions with mass spectrometric detection in positive ion mode exhibited quite complex patterns of peaks (Table 3.11), and only the possible presence + of saponins ( e.g., Paris saponins VII at tR 18.64 (Fig. 2), [M+1] m/z 1032) and + spirostanol ( e.g., type VI daucosterol at tR 34.82, [M+1] m/z 576) in all SPE fractions could be suggested from the retention times and the MS spectral data of the separated peaks (Woldemichael &Wink, 2001, Madl et al., 2006, Zhang et al., 2014, Gao et al., 2015). Total Ion chromatogram (TIC) of MeOH extract of A. adscendens shown in Figure 3.21. Compound identified from both positive and negative ion mode along with
their retention time (tR) and molecular mass are presented in table 3.9 and 3.10, which were matched with NIST library of LCMS. Compound (4-29) and (30-37) were detected from positive and negative ion mode of MeOH extract respectively (Figure 3.22 & Figure 3.23). The presence of saponins and their aglycones was in agreement with that of other Asparagus species (Khan et al., 2018a).
99
Fig 3.21 Total Ion Chromatogram of Asparagus adscendens showing separation of chemical components
Table 3.9 Compound detected from Asparagus adscendens by LCMS (Positive ion mode) Chemical Compound Formula RT(min) Compound No & Mol. Mass
C6 H8 O3 4 0.561 Dihydrophloroglucinol 128.04742
C8 H10 O6 5 0.648 Ethyl aconitate 202.0467
C15 H16 N4 O7 6 0.669 His-Ser-OH 364.10179
C14 H18 O10 Methyl 6-O-galloyl-beta-D- 7 0.716 346.08964 glucopyranoside
C6 H8 O4 8 0.727 Methylitaconate 144.04246
C6 H6 O3 9 0.728 4-Hydroxy-6-methylpyran-2-one 126.03198
100
C6 H10 O5 10 0.731 Levoglucosan 162.05416
C6 H11 N O2 11 0.75 DL-pipecolic acid 129.07941
C6 H13 N O2 12 0.76 Aminocaproic acid 131.09466
C8 H10 N4 O2 13 0.775 Enprofylline 194.08009
C12 H19 N O2 S2 14 0.929 Brugine 273.08658
C7 H15 N O2 15 0.946 2-amino-heptanoic acid 145.11083
C6 H13 N O2 16 1.083 131.09447 L-Leucine
C7 H15 N O2 17 1.12 2-amino-heptanoic acid 145.11018
C9 H17 N O8 18 1.208 Neuraminic acid 267.09527
C8 H17 N O2 19 1.745 8-Amino Caprylic acid 159.12532
C14 H16 O9 20 7.293 Bergenin 328.08003
C16 H35 N O2 21 12.269 C16 Sphinganine 273.26798
C14 H31 N O 22 12.332 Xestoaminol C 229.24044
C21 H27 N O3 23 16.329 p-Hydroxynorpropoxyphene 341.20015
C16 H22 O4 24 17.169 Emmotin A 278.15278
C12 H12 O3 25 17.175 3-Butylidene-7-hydroxyphthalide 204.07919
101
C19 H32 O3 methyl 15,16-epoxy-9,12- 26 18.705 308.23549 octadecadienoate
C16 H33 N O 27 19.398 Palmitic amide 255.25664
C19 H38 O4 28 19.587 1-Monopalmitin 330.27782
C22 H39 N O 2,4,12-Octadecatrienoic acid 29 20.334 333.3044 isobutylamide
Dihydrophloroglucinol (4)
Ethyl aconitate (5)
His-Ser-OH (6)
102
Methyl 6-O-galloyl-beta-D-glucopyranoside (7)
Methylitaconate (8)
4-Hydroxy-6-methylpyran-2-one (9)
Levoglucosan (10)
DL-pipecolic acid (11) 103
Aminocaproic acid (12)
Enprofylline (13)
Brugine (14)
2-amino-heptanoic acid (15)
L-Leucine (16) 104
2-amino-heptanoic acid (17)
Neuraminic acid (18)
8-Amino Caprylic acid (19)
Bergenin (20)
C16 Sphinganine (21)
105
Xestoaminol C (22)
p-Hydroxynorpropoxyphene (23)
Emmotin A (24)
3-Butylidene-7-hydroxyphthalide (25)
106
methyl 15,16-epoxy-9,12-octadecadienoate (26)
Palmitic amide (27)
1-Monopalmitin (28)
2,4,12-Octadecatrienoic acid isobutylamide (29) Fig 3.22 Compound (4-29) detected from Asparagus adscendens by LCMS (Positive ion mode)
107
Table 3.10 Compound detected from Asparagus adscendens by LCMS (Negative ion mode) Chemical Compound Formula RT(min) Compound No & Mol. Mass C H N O 5-Hydroxy-4-methoxy-3-methyl-2,6- 30 0.731 16 12 2 4 296.07987 canthinedione C H O 31 0.873 24 42 21 Maltotetraose 666.22314 C H O 32 0.901 30 52 26 Maltopentaose 828.27268 C H O 33 7.296 14 16 9 Bergenin 328.08087 C H O 34 9.686 9 16 4 Nonic Acid 188.10552 C H O 35 11.558 18 34 5 5,8,12-trihydroxy-9-octadecenoic acid 330.24178 C H O 36 15.822 18 32 4 γ- 12,13-DiHODE 312.23133 C H N O 37 19.303 16 28 4 4 Pro Lys Pro 340.20945
5-Hydroxy-4-methoxy-3-methyl-2,6-canthinedione (30)
108
Maltotetraose (31)
Maltopentaose (32)
Bergenin (33)
109
Nonic Acid (34)
5,8,12-trihydroxy-9-octadecenoic acid (35)
γ- 12,13-DiHODE (36)
5-Hydroxy-4-methoxy-3-methyl-2,6-canthinedione (37)
Fig 3.23 Compound (30-37) detected from Asparagus adscendens by LCMS (Negative ion mode)
110
Table 3.11 Retention times (tR) and corresponding pseudomolecular ions [M+H]+ of Asparagus adscendens Peak SPE fractions no. AAMF1 AAMF2 AAMF3 AAMF4 + + + + tR in [M+H] tR in [M+H] tR in [M+H] tR in [M+H] min m/z min m/z min m/z min m/z 1 3.39 273 4.80 7011 18.91 4532 2.62 279 2 14.79 214 5.70 6791 24.96 6791 2.95 4312 3 18.47 288 18.64 10321 27.08 7021 3.85 275 4 18.91 296 34.07 12191 28.41 9271 10.65 336 5 23.68 8081 34.67 12451 28.86 7501 15.62 5792 6 28.18 5572 35.45 9801 35.82 9611 16.41 5672 7 29.93 5612 36.57 7011 36.88 8031 17.52 5392 8 31.56 319 37.12 8031 38.17 8371 19.71 4432 9 34.93 7011 38.38 8371 20.18 5712 10 38.62 8371 21.84 7411 11 22.86 214 12 23.39 4272 13 24.54 6621 14 24.86 6841 15 26.21 5932 16 29.11 6911 17 31.36 5572 18 32.35 6151 19 34.82 5762 20 36.78 8041 1Possible saponins. 2Possible sapogenins
111
3.1.4.3.2 LC-MS analysis of Trillium govanianum LC-MS analysis on the SPE fractions of the MeOH extract of the roots of T. govanianum was performed to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain saponins and sapogenins as possible contributors to the significant cytotoxicity of the extract and its fractions. Optimized chromatographic conditions were achieved through trial and error. A linear gradient elution with water and MeOH containing 1% formic acid as the mobile phase offered the best resolution. Typical chromatograms of fractions with mass spectrometric detection in positive ion mode exhibited quite complex patterns of peaks (Table 3.14), and only the possible presence of saponins and sapogenins in all SPE fractions could be suggested from the retention times and the MS spectral data of the separated peaks (Gao et al., 2015, Hayes et al., 2009). Total Ion chromatogram (TIC) of MeOH extract of T. govanianum shown in Fig. 3.25. Compound identified from both positive and negative ion mode along with their retention time (tR) and molecular mass are presented in table 3.12 and 3.13, which were matched with NIST library of LCMS. Compound (38-89) and (90-140) were detected from positive and negative ion mode of MeOH extract respectively (Fig 3.25 & Fig. 3.26). Presence of saponins and their aglycones in T. govanianum was in agreement with that of other Trillium species. This is the first report, on the preliminary LC-MS analysis on T. govanianum.
Fig 3.24 Total ion chromatogram of Trillium govanianum showing separation of chemical components
112
Table 3.12 Compound detected from Trillium govanianum by LCMS (Positive ion mode) Chemical Compound RT Formula Compound No (min) & Mol. Mass C H N 38 0.6 11 14 2 N-Methyltryptamine 174.11586 C H N O 39 0.601 10 15 4 Kainic Acid 213.09934 C H N 40 0.617 14 15 2-(3-Phenylpropyl)pyridine 197.11961 C H N O 41 0.633 12 17 5 4 N6,N6-Dimethyladenosine 295.12772 C H N O 42 0.638 4 9 2 2R-amino-butanoic acid 103.06345 C H N O 43 0.651 11 15 5 3 N6-Methyl-2'-deoxyadenosine 265.11857 C H N O 44 0.885 8 15 5 N-Acetyl-D-quinovosamine 205.09556 C H N O 45 1.056 6 13 2 Aminocaproic acid 131.09489 C H O 46 1.084 9 8 3 m-Coumaric acid 164.04685 C H N O 47 1.157 6 13 2 L-Leucine 131.09456 C H N O 48 5.066 11 9 2 3-Amino-2-naphthoic acid 187.06356 C H O (25S)-11alpha,20,26- 49 8.162 27 44 9 512.29769 trihydroxyecdysone C H O 50 8.766 27 42 6 2-dehydroecdysone 462.29952 C H O 51 8.77 27 44 7 Inokosterone 480.30978 C H O 52 8.933 23 34 5 Digoxigenin 390.24106 C H O 53 9.435 39 62 14 Alliospiroside D 754.41497 C H O 54 9.444 45 72 18 26-Desglucoavenacoside A 900.47276 C H O 55 9.458 57 92 26 Hovenoside D 1192.58954 C H O 56 9.834 26 26 11 2'',6''-Di-O-acetylononin 514.14758 C H O 57 9.907 51 80 21 Pisumsaponin I 1028.52239 58 9.983 C39 H62 O13 Fistuloside A 113
738.41905 C H O Nuatigenin 3-[rhamnosyl-(1->2)- 59 10.137 39 62 13 738.42062 glucoside] C H O 60 10.399 52 84 21 Pitheduloside F 1044.55053 C H O 61 11.819 47 74 18 Durupcoside B 926.48969 C H N O 62 12.17 16 35 2 C16 Sphinganine 273.26727 C H O 63 12.37 18 30 3 α-9(10)-EpODE 294.21934 C H O 64 12.374 9 14 2 2-Nonynoic acid 154.09889 C H O 65 12.38 9 16 3 8-oxo-nonanoic acid 172.10938 C H N O 66 13.421 18 39 3 Phytosphingosine 317.29374 C H O Hydroxy (3β)Isoallospirost-9 67 13.911 27 42 3 414.31451 (11)-Ene C H O 68 14.267 37 62 12 Cyclopassifloside I 698.42735 C H O 69 14.268 12 20 8 Pantoyllactone glucoside 292.11571 C H O 70 14.301 18 34 4 9,10-Epoxy-18-hydroxystearate 314.24532 C H O Yamogenin 3-O- 71 14.459 39 62 12 722.42495 neohesperidoside C H O 72 14.467 44 70 16 Ophiopogonin D 854.46668 C H O 73 14.67 21 38 4 1-Linoleoyl Glycerol 354.27774 C H O 74 14.676 33 58 14 Gingerglycolipid B 678.38352 C H O 75 15.209 19 38 4 1-Monopalmitin 330.27711 C H O 9Z,12Z,15E-octadecatrienoic 76 16.088 18 30 2 278.22526 acid C H O 77 17.146 16 22 4 Emmotin A 278.15277 C H O 78 17.242 18 32 3 12-oxo-10Z-octadecenoic acid 296.23576 C H N O P 79 17.266 24 50 7 PE(19:0/0:0) 495.33392 C H N O N-cis-octadec-9Z-enoyl-L- 80 17.788 22 39 3 365.29251 Homoserine lactone C H O 81 18.214 19 32 2 Pinolenic Acid methyl ester 292.24076 C H N O 82 18.581 20 39 3 C-2 Ceramide 341.29312
114
C H O methyl 15,16-epoxy-9,12- 83 18.705 19 32 3 308.23553 octadecadienoate C H O 84 19.19 16 30 2 8E-Tetradecenyl acetate 254.22601 C H N O 85 19.364 16 33 Palmitic amide 255.25578 C H O 86 19.514 18 32 2 6E,9E-octadecadienoic acid 280.24101 C H O 87 19.579 19 38 4 1-Monopalmitin 330.2772 C H N O 88 19.61 18 35 Oleamide 281.27229 C H N O 89 19.755 17 35 Capsi-amide 269.27236
N-Methyltryptamine (38)
Kainic Acid (39)
2-(3-Phenylpropyl)pyridine (40)
115
N6,N6-Dimethyladenosine (41)
2R-amino-butanoic acid (42)
N6-Methyl-2'-deoxyadenosine (43)
N-Acetyl-D-quinovosamine (44)
116
Aminocaproic acid (45)
m-Coumaric acid (46)
L-Leucine (47)
3-Amino-2-naphthoic acid (48)
117
(25S)-11alpha,20,26-trihydroxyecdysone (49)
2-dehydroecdysone (50)
Inokosterone (51)
118
Digoxigenin (52)
Alliospiroside D (53)
26-Desglucoavenacoside A (54)
119
Hovenoside D (55)
2'',6''-Di-O-acetylononin (56)
120
Pisumsaponin I (57)
Fistuloside A (58)
121
Nuatigenin 3-[rhamnosyl-(1->2)-glucoside] (59)
Pitheduloside F (60)
122
Durupcoside B (61)
C16 Sphinganine (62)
α-9(10)-EpODE (63)
2-Nonynoic acid (64)
8-oxo-nonanoic acid (65)
123
Phytosphingosine (66)
Hydroxy (3β)Isoallospirost-9 (11)-Ene (67)
Cyclopassifloside I (68)
124
Pantoyllactone glucoside (69)
9,10-Epoxy-18-hydroxystearate (70)
Yamogenin 3-O-neohesperidoside (71)
125
Ophiopogonin D (72)
1-Linoleoyl Glycerol (73)
Gingerglycolipid B (74)
126
1-Monopalmitin (75)
9Z,12Z,15E-octadecatrienoic acid (76)
Emmotin A (77)
12-oxo-10Z-octadecenoic acid (78)
PE(19:0/0:0) (79)
127
N-cis-octadec-9Z-enoyl-L-Homoserine lactone (80)
Pinolenic Acid methyl ester (81)
C-2 Ceramide (82)
methyl 15,16-epoxy-9,12-octadecadienoate (83)
8E-Tetradecenyl acetate (84)
128
Palmitic amide (85)
6E,9E-octadecadienoic acid (86)
1-Monopalmitin (87)
Oleamide (88)
Capsi-amide (89)
Fig 3.25 Compound (38-89) detected from Trillium govanianum by LCMS (Positive mode)
129
Table 3.13 Compound detected from Trillium govanianum by LCMS (Negative mode) Chemical Compound Formula RT(min) Compound No & Mol. Mass
C6 H14 N4 O2 90 0.599 173.10401 L-Arginine
C12 H25 N2 O10 P Fructoselysine 6- 91 0.691 447.13993 phosphate
C19 H23 N6 O10 P 3-Hydroxy-L-tyrosyl- 92 0.728 525.11297 AMP
C12 H13 N5 O4 93 0.93 290.08955 Toyocamycin
C9 H11 N O3 N-Hydroxy-L- 94 1.105 180.06721 phenylalanine
C4 H4 O4 95 1.137 115.00412 Fumaric acid
C22 H18 N2 O4 96 6.689 373.11857 Famoxadone
C8 H10 O5 S 97 7.323 217.0182 Tyrosol 4-sulfate
C14 H20 N2 O3 98 7.348 263.14101 Phe Val
C20 H30 O11 99 8.031 481.14923 Crosatoside B
C16 H18 N4 O4 100 8.509 329.12567 Pseudomonine
C27 H34 O15 101 8.92 Geniposide pentaacetate 597.18252 130
C55 H77 Co N15 O11 Adenosyl cobyrinate 102 8.972 1241.53799 hexaamide
C96 H168 N4 O47 Ganglioside GT1b 103 9.466 1063.53602 (d18:0/18:0)
C57 H94 O27 104 9.537 1209.59314 Convallamaroside
C9 H16 O4 (+/-)-Methyl 5- 105 9.685 187.09699 acetoxyhexanoate
C32 H38 O17 Sucrose 1',4'-(4,4'- 106 9.737 693.20537 dihydroxy-3,3'-dimethoxy- b-truxinate) C56 H90 O29 107 9.87 1225.55023 Yayoisaponin B
C51 H82 O22 108 9.964 1045.52494 Graecunin E
C51 H84 O22 109 10.094 1047.54126 Protodioscin
C54 H84 O24 28-Glucosyloleanolic acid 3-[rhamnosyl-(1->2)- 110 10.096 1161.53301 galactosyl-(1->3)- glucuronide]
131
C51 H84 O24 111 10.194 1079.5314 Isoeruboside B
C41 H76 O16 P2 112 10.244 945.47481 PIP(16:1(9Z)/16:1(9Z))
1alpha,3beta,22R- Trihydroxyergosta-5,24E- C46 H74 O20 dien-26-oic acid 3-O-b-D- 113 10.35 945.47206 glucoside 26-O-[b-D- glucosyl-(1->2)-b-D- glucosyl] ester C48 H76 O21 114 10.663 987.4824 Phytolaccasaponin B
C54 H86 O25 115 10.701 1133.53926 Calendasaponin C
C54 H86 O25 116 10.799 1133.5416 Calendasaponin D
C45 H72 O17 117 11.277 929.47633 beta-Chacotriosyllilagen
C18 H34 O5 5,8,12-trihydroxy-9- 118 11.568 329.23502 octadecenoic acid
C48 H76 O20 119 11.578 971.48782 Azukisaponin IV
C54 H76 N8 O15 120 12.42 1075.53718 Aeruginopeptin 917S-B
C51 H82 O21 121 12.421 1029.53005 Pseudoprotodioscin
C45 H72 O17 122 12.58 919.44612 Polypodoside A
C45 H72 O19 123 12.712 915.46201 Agavasaponin C
124 12.981 C44 H68 O18 Schidigerasaponin B1 132
943.45716
C45 H72 O16 125 13.791 913.48068 Dioscin
C50 H81 N O21 126 13.932 1076.53015 Dehydrotomatine
C52 H84 O22 127 13.936 1059.54127 Pitheduloside K
C46 H72 O18 Medicagenic acid 28-O-[b- D-xylosyl-(1->4)-a-L- 128 14.001 911.46642 rhamnosyl-(1->2)-a-L- arabinosyl] ester C45 H72 O18 129 14.515 899.46644 Fistuloside B
C39 H62 O12 130 14.887 767.42508 Ophiopogonin B
C23 H44 N O7 P 131 15.122 476.27952 LysoPE(0:0/18:2(9Z,12Z))
C18 H30 O3 132 15.304 293.21276 α-9(10)-EpODE
C34 H44 O9 133 15.792 595.29088 Salannin
C44 H82 N O10 P 134 16.078 814.56101 PS(18:0/20:2(11Z,14Z))
C18 H32 O3 12-oxo-10Z-octadecenoic 135 16.103 295.22871 acid
C18 H32 O4 136 16.358 311.2234 9(S)-HpODE
C32 H44 O9 137 16.891 571.2908 Ganoderic acid H
133
C17 H28 O3 S N- 138 17.1 311.16988 Undecylbenzenesulfonic acid C16 H28 N4 O4 139 19.542 339.20314 Pro Lys Pro
C16 H28 O10 3-Methyl-3-butenyl 140 19.586 379.16069 apiosyl-(1->6)-glucoside
134
L-Arginine (90)
Fructoselysine 6-phosphate (91)
3-Hydroxy-L-tyrosyl-AMP (92)
Toyocamycin (93)
135
N-Hydroxy-L-phenylalanine (94)
Fumaric acid (95)
Famoxadone (96)
Tyrosol 4-sulfate (97)
Phe Val (98) 136
Crosatoside B (99)
Pseudomonine (100)
Geniposide pentaacetate (101)
137
Adenosyl cobyrinate hexaamide (102)
Ganglioside GT1b (d18:0/18:0) (103)
138
Convallamaroside (104)
(+/-)-Methyl 5-acetoxyhexanoate (105)
Sucrose 1',4'-(4,4'-dihydroxy-3,3'-dimethoxy-b-truxinate) (106)
139
Yayoisaponin B (107)
Graecunin E (108)
140
Protodioscin (109)
28-Glucosyloleanolic acid 3-[rhamnosyl-(1->2)-galactosyl-(1->3)-glucuronide] (110)
Isoeruboside B (111) 141
PIP(16:1(9Z)/16:1(9Z)) (112)
1alpha,3beta,22R-Trihydroxyergosta-5,24E-dien-26-oic acid 3-O-b-D-glucoside 26-O-[b- D-glucosyl-(1->2)-b-D-glucosyl] ester (113)
Phytolaccasaponin B (114)
142
Calendasaponin C (115)
Calendasaponin D (116)
beta-Chacotriosyllilagen (117)
143
5,8,12-trihydroxy-9-octadecenoic acid (118)
Azukisaponin IV (119)
Aeruginopeptin 917S-B (120)
144
Pseudoprotodioscin (121)
Polypodoside A (122)
145
Agavasaponin C (123)
Schidigerasaponin B1 (124)
146
Dioscin (125)
Dehydrotomatine (126)
147
Pitheduloside K (127)
Medicagenic acid 28-O-[b-D-xylosyl-(1->4)-a-L-rhamnosyl-(1->2)-a-L-arabinosyl] ester (128)
Fistuloside B (129)
148
Ophiopogonin B (130)
LysoPE(0:0/18:2(9Z,12Z)) (131)
α-9(10)-EpODE (132)
149
Salannin (133)
PS(18:0/20:2(11Z,14Z)) (134)
12-oxo-10Z-octadecenoic acid (135)
9(S)-HpODE (136)
150
Ganoderic acid H (137)
N-Undecylbenzenesulfonic acid (138)
Pro Lys Pro (139)
3-Methyl-3-butenyl apiosyl-(1->6)-glucoside (140) Fig 3.26 Compound (90-140) detected from Trillium govanianum by LCMS (Negative ion mode)
151
Table 3.14 Retention times (tR) and corresponding pseudomolecular ions [M+H]+ of Trillium govanianum Peak SPE fractions no. TGMF1 TGMF2 TGMF3 TGMF4 + + + + tR in [M+H] tR in [M+H] tR in [M+H] tR in [M+H] min m/z min m/z min m/z min m/z 1 3.72 365 2.52 362 2.62 278 2.5 362 2 14.26 475b 2.82 158 3.02 226 3.78 205 3 19.51 702a 3.82 214 3.78 158 3.8 242 4 25.83 1031a 15.52 249 15.59 295 15.39 557b 5 29.68 702a 16.21 450b 20.34 274 17.26 702a 6 33.45 763a 18.02 221 24.38 353 19.06 471b 7 34.87 702a 18.32 317 26.46 351 19.44 928a 8 36.75 413b 21.79 273 29.85 417b 20.27 274 9 38.10 837a 22.16 235 34.8 702a 23.28 351 10 22.48 241 36.62 413b 24.46 353 11 22.79 214 38.05 837a 26.88 437b 12 25.01 353 38.95 702a 28.76 331 13 25.63 288 39.34 541b 29.58 702a 14 25.88 259 29.98 301 15 26.18 286 31.51 309 16 29.06 357 34.78 702a 17 32.9 683a 36.67 804a 18 37.93 837a 19 38.97 702a 20 39.29 541b aPossible saponins bPossible sapogenins
Gas chromatography/mass spectrometry analysis 3.1.4.4.1 GC/MS identification of components Asparagus adscendens The GC/MS analysis of n-hexane fraction of MeOH extract of the roots of A. adscendens revealed the presence of six components (C-141 to 146) (Table 3.15 & Fig.
152
3.28). The major constituents were n-Hexadecanoic acid methyl ester (tR: 6.65), n-
Hexadecanoic acid (tR: 6.87), 9,12-Octadecadienoic acid (Z,Z) (tR: 7.37), cis-13-
Eicosenoic acid (tR: 8.4), Diisooctyl phthalate(tR: 8.5) and β –Sitosterol (tR: 10.88). The GC/MS chromatogram (Fig. 3.27) shows the peak area separation of the components. The above mentioned identified components were reported biological active against bacterial strains.
C:\Users\...\LJMSAR040-GG-GCEIP 8/19/2015 1:49:05 PM AM-Hexane MW=?? GC-EI RT: 0.00 - 14.34 RT: 6.87 NL: 100 1.02E7 TIC MS 90 LJMSAR04 0-GG- 80 GCEIP
70
60
50
40
RelativeAbundance RT: 7.37 30 RT: 7.43 RT: 8.50 20 RT: 6.65
10 RT: 10.88 7.89 RT: 9.92 1.08 1.39 RT: 2.47 3.95 RT: 4.16 5.91 RT: 6.43 9.04 11.33 11.72 12.74 14.07 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (min)
Fig 3.27 Typical Gas Chromatogram of Asparagus adscendens showing separation of chemical components
153
Table 3.15 Compound identified by GC/MS from Asparagus adscendens
Chemical Formula Compound tR & Compound No (min) Mol. Mass
C17H34O2 141 6.65 n-Hexadecanoic acid methyl ester 270.46
C16H32O2 142 6.87 n-Hexadecanoic acid 256.43
C18H32O2 143 7.37 9,12-Octadecadienoic acid 280.24
C20H38O2 144 8.4 cis-13-Eicosenoic acid 310.29
C24H38O4 145 8.5 Diisooctyl phthalate 390.56
C29H50O 146 10.88 β-Sitosterol 414.72
n-Hexadecanoic acid methyl ester (141)
n-Hexadecanoic acid (142)
9,12-Octadecadienoic acid (143) 154
cis-13-Eicosenoic acid (144)
Diisooctyl phthalate (145)
β-Sitosterol (146)
Fig 3.28 Compound identified by GC/MS from Asparagus adscendens
155
3.1.4.4.2 GC/MS identification of components of Trillium govanianum The GC/MS analysis of n-hexane fraction of MeOH extract of the roots of T. govanianum revealed the presence of six components (C-147 to 155) (Table 3.16 &
Figure. 3.30). The major constituents were n-Hexadecanoic acid methyl ester (tR: 6.67),
n-Hexadecanoic acid (tR: 7.01), 9,12-Octadecadienoic acid (Z,Z) (tR: 7.48) and
Octadecanoic acid (tR: 7.53). The GC/MS chromatogram (Fig. 3.29) shows the peak area separation of the components. The above mentioned identified components were reported biological active against bacterial strains C:\Users\...\LJMSAR043-GG-GCEIP 8/19/2015 3:24:39 PM TG-1 MW=?? GC-EI RT: 0.00 - 14.35 RT: 7.01 NL: 100 3.06E7 TIC MS 90 LJMSAR04 3-GG- 80 GCEIP
70 RT: 6.67 60 RT: 7.48 50
40
RelativeAbundance RT: 7.53 30 RT: 4.82 RT: 6.61 RT: 7.60 20 5.58 8.26 6.14 8.52 10 RT: 4.44 8.57 RT: 2.69 8.84 1.40 RT: 1.74 9.47 10.16 11.44 12.81 13.83 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (min)
Fig 3.29 Typical Gas Chromatogram of Trillium govanianum showing separation of chemical components
156
Table 3.16 Compound identified by GC/MS Trillium govanianum
Chemical Formula Compound tR & Compound No (min) Mol. Mass
C12H22O3 147 5.21 Isopropyl 9-oxononanoate 214.31
C10H18O4 148 5.58 Nonanedioic acid, monomethyl ester 202.25
C10H20O3 149 6.14 10-Hydroxydecanoic acid 188.27
C17H32O2 150 6.61 9-Hexadecenoic acid, methyl ester 268.44
C17H34O2 151 6.67 Hexadecanoic acid, methyl ester 270.46
C16H32O2 152 7.01 Hexadecanoic acid 256.43
C19H36O2 153 7.25 6-Octadecenoic acid, methyl ester 296.50
C18H32O2 154 7.48 9,12-Octadecadienoic acid 280.45
C18H36O2 155 7.53 Octadecanoic acid 284.48
157
Isopropyl 9-oxononanoate (147)
Nonanedioic acid, monomethyl ester (148)
10-Hydroxydecanoic acid (149)
9-Hexadecenoic acid, methyl ester (150)
Hexadecanoic acid, methyl ester (151)
Hexadecanoic acid (152)
158
6-Octadecenoic acid, methyl ester (153)
9,12-Octadecadienoic acid (154)
Octadecanoic acid (155)
Fig 3.30 Compound identified by GC/MS from Trillium govanianum
3.2 Biological evaluation 3.2.1 Antioxidant assay DPPH assay of Asparagus adscendens The DPPH scavenging activity of MeOH extract of roots of T. govanianum and its SPE exhibited different levels of radical scavenging activity, ranged from 38.84 to 74.74 %. The % free radical scavenging of MeOH extract and its SPE fractions are shown in Figure 3.31 and error bars represents the standard deviation. The AAMF1 extract showed highest DPPH free radical scavenging (74.748 ± 1.14%), followed by the SPE fractions AAM4, AAM, AAMF2 and AAMF3 (% RSA= 60.75 ± 2.07, 48.74 ± 2.72, 43.74 ± 0.71 and 38.84 ± 5.51 % respectively).
159
100
80
60
% RSA % 40
20
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.31 DPPH assay of Asparagus adscendens
DPPH assay of Trillium govanianum The DPPH scavenging activity of MeOH extract of roots of T. govanianum and its SPE exhibited different levels of radical scavenging activity, ranged from 33.82 to 67.32 %. The % free radical scavenging of MeOH extract and its SPE fractions are shown in Figure 3.32 and error bars represents the standard deviation. The TGMF1 showed highest DPPH free radical scavenging (67.32 ± 4.96%), followed by the SPE fractions, TGMF2, TGM (MeOH extract), TGMF3 and TGMF4 (% RSA= 52.98 ± 2.72, 43.40 ± 2.93, 40.05 ± 1.03 and 33.82 ± 5.51 % respectively).
160
80
60
40
% RSA %
20
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.32 DPPH assay of Trillium govanianum
Total antioxidant capacity assay of Asparagus adscendens The total antioxidant capacity of AAM (MeOH extract) and its SPE fractions are shown in Figure 3.34 and error bars represents the standard deviation. In present study, total antioxidant capacity (TAC) was found maximum for AAMF2 (38.26±1.03 mg AAE/g DW), followed by the SPE fractions AAMF4, AAM (MeOH extract) AAMF3 and AAMF1. The SPE fraction AAMF1, which had the most polar components of the parent MeOH extract, showed the least significant total antioxidant capacity results among other SPE fractions.
161
2.5 2.27 y = 0.0206x + 0.1552 2 R² = 0.9927
1.5 1.089 1 0.612
OD of standard of OD 0.482 0.5 0.314 0.224 0 0 20 40 60 80 100 120 Conc. (µg/ml) Fig 3.33 Regression curve of Ascorbic Acid
50
40
30
20
TAC (mg AAE/g DW) (mg AAE/g TAC 10
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.34 Total antioxidant capacity assay of Asparagus adscendens
Total antioxidant capacity assay of Trillium govanianum The total antioxidant capacity of MeOH extract and its SPE fractions are shown in Figure 3.35 and error bars represents the standard deviation. In present study, total antioxidant capacity (TAC) was found maximum for TGMF1 (41.27 ± 5.98 mg AAE/g DW) followed by the SPE fractions, TGMF2, TGM (MeOH extract), TGMF4 and TGMF3 (30.80 ± 4.05, 22.978 ± 0.52, 22.09 ± 6.33 and18.32 ± 0.75 mg AAE/g DW
162
respectively). The SPE fraction TGMF2, which had the polar components of the parent MeOH extract, showed the significant total antioxidant capacity results among other SPE fractions.
50
40
30
20
TAC (mg AAE/g DW) (mg AAE/g TAC 10
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.35 Total antioxidant capacity assay of Trillium govanianum
163
Total reducing power assay of Asparagus adscendens The ferric reducing antioxidant power of MeOH extract and its SPE fractions are shown in Figure 3.37 and error bars represents the standard deviation. In present study, was found maximum for AAM (MeOH extract) (23.75 ± 1.99 mg AAE/g DW) followed by the SPE fractions, AAMF2, AAMF4, AAMF1 and AAMF3 (21.75 ± 1.20, 19.53 ± 0.29, 7.65 ± 2.32and 5.75 ± 0.54 mg AAE/g DW respectively).The SPE fraction AAMF2, which had the polar components of the parent MeOH extract, showed most promising total ferric reducing antioxidant capacity results among other SPE fractions.
5 4.644 4.5 y = 0.0392x + 0.8122 R² = 0.9765 4 3.5 3 2.765 2.5 2.229
2 OD of Standard of OD 1.5 1.25 0.986 1
0.5 0.716 0 0 20 40 60 80 100 120 conc (µg/ml)
Fig 3.36 Regression curve of Ascorbic Acid
164
30
20
10
TRP (mg AAE/g DW) (mg TRP AAE/g
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.37 Total Reducing capacity assay of Asparagus adscendens
Total reducing power assay of Trillium govanianum The ferric reducing antioxidant power of MeOH extract and its SPE fractions are shown in Figure 3.38 and error bars represents the standard deviation. In present study, ferric reducing antioxidant power was found maximum for TGMF1 (15.78 ± 2.32mg AAE/g DW) followed by the SPE fractions TGMF2, TGM (MeOH extract), TGMF4 and TGMF3 (14.73 ± 3.20, 11.78 ± 0.99, 2.80 ± 0.59 and 1.240 ± 1.54 and mg AAE/g DW respectively).The SPE fraction TGMF2, which had the polar components of the parent MeOH extract, also showed the promising total ferric reducing antioxidant capacity results among other SPE fractions.
165
20
15
10
5
TRP (mg AAE/g DW) (mg TRP AAE/g
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.38 Total Reducing capacity assay of Trillium govanianum
3.2.2 Antimicrobial analysis Antibacterial assay of Asparagus adscendens The MeOH extract and its SPE exhibited different levels of antibacterial activity determined by the zone of inhibition (mm diameter) ranges from 7 to 13 mm, against Staphylococcus aureus (NCTC 7508); Bacillus subtilis (NCTC 1604); Micrococcuss luteus (NCTC 75080; Escherichia coli (ATCC 25922). Zone of inhibition (mm diameter) of MeOH extract and its SPE fractions are shown in Figure 3.39 and error bars represents the standard deviation. AAM (MeOH extract) exhibited a significant antibacterial activity only against S. aureus (13± 0.3mm). Among SPE fractions, AAMF1, which had the most polar components of the parent MeOH extract has exhibited the highest level of antibacterial activity against B. subtilis (10± 1.33) and has shown the mild and week antibacterial activity against S. aureus, M. luteus and E. coli (9±0.7mm, 9±1.23mm and 7±0.80 mm respectively). AAMF2, which had polar components of the parent MeOH extract has exhibited the highest level of antibacterial activity against B. subtilis and E. coli (12± 1.34mm and 12± 1.44mm respectively), where as has shown mild antibacterial activity against S. aureus and M. luteus (11±0.5mm, 10±1.22mm respectively). AAMF3, which had semi polar components of the parent MeOH extract has exhibited the mild level of antibacterial activity against B.
166
subtilis (10 ± 0.92mm), where as has shown week antibacterial activity against M. luteus, S. aureus and E. coli, (9 ± 0.2mm ,8 ± 0.64mm and 8 ± 1.43mm respectively). AAMF4, which had least polar components of the parent MeOH extract has exhibited the significant level of antibacterial activity against B. subtilis (13 ± 0.8mm), where as has shown week antibacterial activity against M. luteus, S. aureus and E. coli (12 ± 0.68mm ,7 ± 1mm and 11 ± 1.55mm respectively). Data indicated (Figure 3.39) that the overall SPE fractions showed mild or low antibacterial activity in term of zone of inhibition in agar disc diffusion assay. The negative control (DMSO) showed no zone of inhibition, while Cefixime was used as positive control and exhibited a significant values against S. aureus, B. subtilis, M. luteus and E. coli (28 ± 0.6, 27 ± 1.342, 28 ± 0.92and 24 ± 1.2 mm respectively).
167
A B
40 30 *** SA BS 30 *** 20 20
IZ (mm) IZ
IZ (mm) IZ 10 10
0 0
AAM Control AAMF1 AAMF2 AAMF3 AAMF4 AAM Control AAMF1 AAMF2 AAMF3 AAMF4
C D
40 30 ML *** EC 30 *** 20 20
IZ (mm) IZ IZ (mm) IZ 10 10
0 0
AAM AAM Control AAMF1 AAMF2 AAMF3 AAMF4 Control AAMF1 AAMF2 AAMF3 AAMF4
Negative control: DMSO. Cefixime exhibited maximum activity (28 ± 0.07 mm) employed as positive control in antibacterial acvtivity (A) SA, Staphylococcus aureus NCTC 7508; (B) BS, Bacillus subtilis NCTC 1604;(C) ML, M. luteus NCTC 7508; (D) EC, Escherichia coli ATCC 25922. Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
Fig 3.39 The MeOH extract and its SPE of Asparagus adscendens exhibited different levels of antibacterial activity
168
Antibacterial assay of Trillium govanianum The MeOH extract and its SPE exhibited different levels of antibacterial activity determined by the zone of inhibition (mm diameter) against S. aureus (NCTC 7508); B. subtilis (NCTC 1604); M. luteus (NCTC 75080; E. coli (ATCC 25922). Zone of inhibition (mm diameter) of MeOH extract and its SPE fractions are shown in Figure 3.40 and error bars represents the standard deviation The MeOH extract displayed the highest level of antibacterial activity against M. luteus and E. coli (14 ± 1.5mm and 14 ± 0.5mm), and it has shown week antibacterial activity against S. aureus and B. subtilis (10 ± 0.95mm and 10 ± 1.87mm). The SPE TGMF1, which had the most polar components of the parent MeOH extract has shown similar and week antibacterial activity against S. aureus, B. subtilis, M. luteus and E. coli (13 ± 1.57mm,12 ± 1.33mm ,11 ± 0.75mm and 14 ± 0.79mm respectively). The SPE TGMF2, which also had the polar components of the parent MeOH extract has shown mild and week antibacterial activity against S. aureus, B. subtilis, M. luteus and E. coli (11 ± 0.5mm,13 ± 1.34mm ,12 ± 1.2mm and 13 ± 1.44mm respectively). The SPE TGMF3, which also had the semi polar components of the parent MeOH extract has shown week antibacterial activity against S. aureus, B. subtilis, M. luteus and E. coli (9.5 ± 0.86mm,10 ± 0.89mm ,9 ± 0.59mm and 9 ± 0.65mm respectively). The SPE TGMF4, which also had the least polar components of the parent MeOH extract has shown week antibacterial activity against S. aureus, B. subtilis, M. luteus and E. coli (7 ± 1.5mm,11 ± 1.64mm ,11 ± 1.45mm and 11 ± 1.98mm respectively). Data indicated (Figure 3.40) that the overall SPE fractions showed mild or low antibacterial activity in term of zone of inhibition in agar disc diffusion assay. The negative control (DMSO) showed no zone of inhibition, while Cefixime was used as positive control and exhibited a significant values against S. aureus, B. subtilis, M. luteus and E. coli (28 ± 1.56, 27 ± 0.43, 28 ± 1.5 and 24 ± 1.74 mm respectively).
169
A B
40 30 *** SA BS *** 30 20 20
IZ (mm) IZ IZ (mm) IZ 10 10
0 0
TGM TGM TGMF1 TGMF2 TGMF3 TGMF4 Control Control TGMF1 TGMF2 TGMF3 TGMF4
C D 40 30 ML *** EC 30 *** 20 20
IZ (mm) IZ IZ (mm) IZ 10 10
0 0
TGM TGM Control TGMF1 TGMF2 TGMF3 TGMF4 Control TGMF1 TGMF2 TGMF3 TGMF4
Negative control: DMSO. Cefixime exhibited maximum activity (28 ± 0.05 mm) employed as positive control in antibacterial activity (A) SA, Staphylococcus aureus NCTC 7508; (B) BS, Bacillus subtilis NCTC 1604;(C) ML, M. luteus NCTC 7508; (D) EC, Escherichia coli ATCC 25922. Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
Fig 3.40 The MeOH extract and its SPE of Trillium govanianum exhibited different levels of antibacterial activity
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Resazurin microtiter assay (REMA) of Asparagus adscendens The MeOH extract of the roots of Asparagus adscendens and its SPE fractions (AAMF1, AAMF2, AAMF3 & AAMF4) exhibited varying antibacterial activity by using Resazurin microtiter plate assay (REMA) as shown in (Table3.17). The MIC (mg/mL) values of the MeOH extract and its SPE fractions are shown in Table 3.16. The results from (Table 3.16) indicated that the MeOH extract (AAM) and four SPE fractions showed higher antibacterial activity against Gram-positive bacteria (MIC: 2.5- 0.009 mg/mL) than against Gram-negative bacteria (MIC: 1.25-2.5 mg/mL). The SPE fraction AAMF1, which had the most polar components of the parent MeOH extract, showed most significant antibacterial activity against M. luteus (MIC: 0.078 mg/mL), and considerable antibacterial activity against S. aureus, B. subtilis, E. coli and K. oxytoca (MIC: 2.5, 2.5, 2.5 and 2.5 mg/mL respectively).The SPE fraction AAMF2 showed most prominent antibacterial activity against B. subtilis and M. luteus (MIC: 0.31 and 0.31 mg/mL respectively), and showed mild activity against S. aureus, E. coli and K. oxytoca (MIC: 1.25, 2.5 and 2.5 mg/mL respectively). The SPE fraction AAMF3 showed most significant antibacterial activity against S. aureus, B. subtilis and M. luteus (MIC: 0.625, 0.0195 and 0.078 mg/mL respectively) and showed mild antibacterial activity against E. coli and K. oxytoca (MIC: 2.5 and 1.25 mg/mL respectively) was quite similar to that of the AAMF1, AAMF2 and the MeOH extract. The SPE fraction AAMF4 which contained the least polar components of the parent MeOH extract, exhibited notable antibacterial activity against S. aureus, B. subtilis and M. luteus (MIC: 0.156, 0.009 and 0.156 mg/mL) and showed no activity against E. coli and K. oxytoca (MIC: ≥10). Figure 3.41 shows result after 24 hours incubation.
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Table 3.17. The MIC (mg/mL) values of by using the Resazurin assay
Bacterial strains (MIC mg/mL) Test compounds SA BS ML EC KO
AAMa (mg/mL) 2.5 0.039 0.31 2.5 1.25 AAMF1 (mg/mL) 2.5 2.5 0.078 2.5 2.5 AAMF2 (mg/mL) 1.25 0.31 0.31 2.5 2.5 AAMF3 (mg/mL) 0.625 0.0195 0.078 2.5 1.25 AAMF4 (mg/mL) 0.156 0.009 0.156 ≥10 ≥10 CTXb (µg/mL) 2 0.25 0.25 ≤0.06 ≤0.06 AMPc (µg/mL) 0.125 ≤0.06 0.125 4 2 AMXd (µg/mL) 4 0.5 4 2 2 SA, Staphylococcus aureus NCTC 7508; BS, Bacillus subtilis NCTC 1604; ML, M. luteus NCTC 7508; EC, Escherichia coli ATCC 25922; KO, K. oxytoca NCTC 8017; AAMa (Asparagus adscendens methanol extract), CTXb (Cefotaxime), AMPc (Ampicillin), AMXd (Amoxicillin). Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
Fig 3.41 Typical 96-well plates shows the result after 24 hours
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3.2.2.3.1 Resazurin microtiter assay (REMA) of Trillium govanianum The MeOH extract of the roots of Trillium govanianum and its SPE fractions (TGMF1, TGMF2, TGMF3 & TGMF4) exhibited varying antibacterial activity by using Resazurin microtiter plate assay as shown in (Table 3.18). The MIC (mg/mL) values of the MeOH extract and its SPE fractions are shown in Table 3.17. The results from (Table 3.17) indicated that the MeOH extract (TGM) and four SPE fractions showed higher antibacterial activity against Gram-positive bacteria (MIC: 2.5-0.039 mg/mL) than against Gram-negative bacteria (MIC: 2.5-0.165 mg/mL). The SPE fraction TGMF1, which had the most polar components of the parent MeOH extract, showed most significant antibacterial activity against M. luteus (MIC: 0.156 mg/mL), and considerable antibacterial activity against S. aureus, B. subtilis, E. coli and K. oxytoca (MIC: 2.5, 2.5, 1.25 and 2.5 mg/mL respectively).The SPE fraction TGMF2 showed most prominent antibacterial activity against B. subtilis and M. luteus (MIC: 0.615 and 1.25 mg/mL respectively), and showed mild activity against S. aureus, E. coli and K. oxytoca (MIC: 2.5, 2.5 and 2.5 mg/mL respectively). The SPE fraction TGMF3 showed most significant antibacterial activity against B. subtilis, M. luteus ,S. aureus and (MIC: 0.0195, 0.039 and 0.31 mg/mL respectively) and showed mild antibacterial
The SPE fraction AAMF4 which contained the least polar components of the parent MeOH extract, exhibited notable antibacterial activity against S. aureus, B. subtilis and M. luteus (MIC: 0.615, 0.039 and 0.156 mg/mL) and showed no activity against E. coli and K. oxytoca (MIC: ≥10) activity against E. coli and K. oxytoca (MIC: 1.25 and 0.615 mg/mL respectively). Figure 3.42 shows result after 24 hours incubation.
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Fig 3.42 Typical 96-well plates shows the result after 24 hours
Table 3.18. The MIC (mg/mL) values of the MeOH extract of the roots of T. govanianum and its SPE fractions by using the resazurin assay Bacterial strains (MIC mg/mL) Test sample SA BS ML EC KO TGM (mg/mL) 2.5 0.078 0.156 2.5 0.165 TGMF1(mg/mL) 2.5 2.5 0.156 1.25 2.5 TGMF2 (mg/mL) 2.5 0.615 1.25 2.5 2.5 TGMF3 (mg/mL) 0.31 0.0195 0.039 1.25 0.615 TGMF4 (mg/mL) 0.615 0.039 0.156 ≥10 ≥10 CTXb(µg/mL) 2 0.25 0.25 ≤0.06 ≤0.06 AMPc (µg/mL) 0.125 ≤0.06 0.125 4 2 AMXd (µg/mL) 4 0.5 4 2 2 SA, Staphylococcus aureus NCTC 7508; BS, Bacillus subtilis NCTC 1604; ML, M. luteus NCTC 7508; EC, Escherichia coli ATCC 25922; KO, K. oxytoca NCTC 8017; AAMa (Asparagus adscendens methanol extract), CTXb (Cefotaxime), AMPc (Ampicillin), AMXd (Amoxicillin). Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
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Antifungal assay of Asparagus adscendens Anti fungal assay was established against MeOH extract and its SPE fractions by using four strains of fungi and results are summarized in Figure 3.43 and error bars represents the standard deviation. The MeOH extract displayed the highest level of antifungal activity against A. niger (14.50 ± 1.07) but it has also shown mild antifungal activity against A. fumigatus, Mucor species and A. flavus (13 ± 2.67, 3 ± 1.25 and 10 ± 1.07mm respectively). The SPE AAMF1, which had the most polar components of the parent MeOH extract has shown moderate and week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (13.5 ± 1.96, 13.5 ± 1.96, 12 ± 2.68 and 12 ± 2.68mm respectively).
The SPE AAMF2, which had the polar components of the parent MeOH extract has shown moderate and week antifungal activity against A. fumigatus,Mucor species, A. niger and A. flavus (10.5 ± 1.50, 10.5 ± 1.67, 11 ± 1.60 and 9 ± 0.8 mm respectively).The SPE AAMF3, which had the semi polar components of the parent MeOH extract has shown week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (7 ± 0.56, 7 ± 2.53, 8.5 ± 1.96 and 6.5 ± 0.83 mm respectively).The SPE AAMF4, which had the least polar components of the parent MeOH extract has shown moderate and week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (12 ± 2.56, 14 ± 1.08, 7.5 ± 1.4 and 8.5 ± 1.05mm respectively). It was noticed that the typical antifungal activity shown by SPE fraction were similar and had no impact of polarity decreased. The negative control (DMSO) showed no zone of inhibition, while Clotrimazole was (as positive control) and showed notable zone of inhibition A. fumigatus , Mucor species , A. niger and A. flavus (31 ± 1.67, 29.5 ± 0.56, 28 ± 1.43 and 32 ± 0.85 mm respectively).
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B A
40 40 AF MS *** *** 30 30
20 20
IZ (mm) IZ
IZ (mm) IZ 10 10
0 0
AAM AAM Control AAMF1 AAMF2 AAMF3 AAMF4 Control AAMF1 AAMF2 AAMF3 AAMF4
C D
40 40 AN *** *** AF 30 30
20 20
IZ (mm) IZ
IZ (mm) IZ 10 10
0 0
AAM AAM Control AAMF1 AAMF2 AAMF3 AAMF4 Control AAMF1 AAMF2 AAMF3 AAMF4
(A) AF, A. fumigatus FCBP- 66; (B) MS, Mucor species FCBP-0300; (C) AN, A. niger FCBP- 0198 and (D) AF, A. flavus FCBP-0064. Clotrimazole exhibited maximum activity (31 ± 1.25 mm) employed as positive control in antifungal activity. Comparison with control all SPE fractions have no significant values at P< 0.05.
Fig 3.43 MeOH extract and its SPE fractions antifungal potential of Asparagus adscendens
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Antifungal assay of Trillium govanianum Anti fungal assay was established against MeOH extract and its SPE fractions by using four strains of fungi and results are summarized in Figure 3.44 and error bars represents the standard deviation. The MeOH extract displayed the moderate of antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (12 ± 1.95, 9 ± 0.96, 12 ± 0.96 and 12 ± 0.58mm respectively). The SPE TGMF1, which had the most polar components of the parent MeOH extract has shown moderate antifungal activity and highest among the other SPE fractions against A. fumigatus, Mucor species, A niger and A. flavus (15 ± 1.57, 11 ± 1.24, 13.5 ± 1.45 and13.5 ± 1.44 mm respectively). The SPE TGMF2, which had the polar components of the parent MeOH extract has shown moderate and week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (13 ± 0.8, 11 ± 0.30, 10.5 ± 0.85 and 8.67 ± 1.6 mm respectively). The SPE AAMF3, which had the semi polar components of the parent MeOH extract has shown week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (11 ± 0.84, 10 ± 0.43, 10 ± 0.94 and 5.2 ± 0.57mm respectively). The SPE AAMF4, which had the least polar components of the parent MeOH extract has shown week antifungal activity against A. fumigatus, Mucor species, A. niger and A. flavus (8 ± 2.05, 7 ± 2.05, 6.55 ± 2.05 and 6.55 ± 2.56 mm respectively). The negative control (DMSO) showed no zone of inhibition, while Clotrimazole was used (as positive control) and showed notable zone of inhibition A. fumigatus, Mucor species , A. niger and A flavus (31 ± 1.96, 31 ± 0.76, 30 ± 0.97 and 33.5 ± 1.67 mm respectively).
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B A
40 40 *** AF *** MS 30 30
20 20
IZ (mm) IZ
IZ (mm) IZ 10 10
0 0
TGM TGM Control TGMF1 TGMF2 TGMF3 TGMF4 Control TGMF1 TGMF2 TGMF3 TGMF4
C D
40 40 *** *** AN AF 30 30
20 20
IZ (mm) IZ
IZ (mm) IZ 10 10
0 0
TGM TGM TGMF1 TGMF2 TGMF3 TGMF4 Control Control TGMF1 TGMF2 TGMF3 TGMF4
(A) AF, A. fumigatus FCBP- 66; (B) MS, Mucor species FCBP-0300; (C) AN, A. niger FCBP- 0198 and (D) AF, A. flavus FCBP-0064Clotrimazole exhibited maximum activity (31 ± 1.25 mm) employed as positive control in antifungal activity. Comparison with control all SPE fractions have no significant values at P< 0.05.
Fig 3.44 MeOH extract and its SPE fractions antifungal potential of Trillium govanianum
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3.2.3 In vitro antileishmanial analysis of Asparagus adscendens Antileishmanial capability of MeOH and its SPE fractions was evaluated for the first time in the present study (Figure 3.45) and error bars represents the standard deviation. The antileishmanial potential of MeOH 45.1 ± 6.1 µg/mL. The SPE fraction AAMF1, which had the most polar components of the parent MeOH exhibited as good result as its parent MeOH extract (LC50 38.5 ± 2.45 µg/mL). Rest of SPE fractions
AAMF2, AAMF3 and AAMF4 (LC50= 118.2 ± 2.65, 112.3 ± 2.83 and 78.3 ± 4.71µg/mL respectively) also showed considerable good results. The SPE fraction AAMF4, which contained the least polar components of the parent MeOH extract, exhibited the notable antileishmanial potential (LC50 78.3 ± 4.71 µg/mL). The antileishmanial potential of Ampotericin (control) manifested LC50= 21.78 ± 1.45, whereas DMSO (negative control) exhibited no effect.
% Mortality 100 140 LC50 µg/ml 90 120 80 70 100 ) 60 80
50 (µg/ml 60 40 50
30 40 LC
% mortality mortality µg/ml % 20 20 10 0 0
Fig 3.45 Antileishmanial results exhibited by Asparagus adscendens
3.2.4 In vitro antileishmanial analysis of Trillium govanianum Antileishmanial capability of MeOH and its SPE fractions was evaluated for the first time in the present study. The antileishmanial potential of MeOH extract of and its SPE fractions against Leishmania tropica KWH23 strain shown in Figure 3.46 and
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error bars represents the standard deviation. The MeOH extract exhibited the most significant results with 70% mortality at 38.5±6.02 µg/mL (control: 0.36 µg/mL). The SPE fraction TGMF1, which had the most polar components of the parent MeOH exhibited as good result as its parent MeOH extract (LC50 40.5 ± 2.51 µg/mL). Rest of
SPE fractions TGMF2, TGMF3 and TGMF4 (LC50= 189.5 ± 2.88, 105.6 ± 2.56 and 66.5 ± 4.50 µg/mL respectively) also showed considerable good results. The SPE fraction TGMF4, which contained the least polar components of the parent MeOH extract, exhibited the notable antileishmanial potential (LC50 66.5 ± 4.5 µg/mL). The antileishmanial potential of Ampotericin (control) manifested LC50= 23.56 ± 1.55, whereas DMSO (negative control) exhibited no effect.
% Mortality 100 200 90 180 LC50 µg/ml 80 160 70 140 ) 60 120
50 100 (µg/ml
40 80 50
30 60 LC
% mortality mortality µg/ml % 20 40 10 20 0 0
Fig 3.46 Antileishmanial results exhibited by Trillium govanianum
3.2.5 Cytotoxicity assays
Brine shrimp lethality assay of Asparagus adscendens
In present study, cytotoxicity potential of the MeOH extract and its SPE fractions were estimated against brine shrimp larvae (Artemia salina). Highly toxic and toxic LC50 values were considered to be below the 100 μg/mL and ≤ 250 μg/mL. The MeOH extract of roots of A. adscendens and its SPE exhibited different levels of Brine 180
shrimp lethality (Table 3.19). The LC50 value (μg/mL) of MeOH extract and its SPE fractions are shown in Table 3.18. The MeOH extract showed the most toxic exhibiting
LC50 of 75 ± 0.85 μg/mL followed by the SPE fractions AAMF1, AAMF2, AAMF3
and AAMF4 (LC50 = 70 ± 1.89, 70 ± 1.67, 75 ± 1.55 and 70 ± 0.95 μg/mL respectively). All most all the SPE fraction showed similar toxic effects as of the parent MeOH extract
(LC50 75 ± 0.85 μg/mL). The positive control, doxorubicin demonstrated an LC50 value 1.98 ± 0.58 μg/mL.
Table 3.19 Brine shrimp lethality assay of A. asparagus
Brine shrimp lethality assay
Sample % mortality µg/mL
1000 500 250 LC50 (µg/mL)
a b c AGM 75 ± 0.84 63 ± 0.21 58 ± 0.12 75 ± 0.85 a b c AAMF1 70 ± 0.95 65 ± 0.31 60 ± 0.32 70 ± 1.89 a b c AAMF2 70 ± 1.55 68 ± 0.12 63 ± 0.41 70± 1.67 a b c AAMF3 75 ± 2.05 65 ± 0.45 60 ± 0.51 75 ± 1.55 a b c AAMF4 70 ± 0.95 65 ± 0.35 60 ± 0.34 70 ± 0.95 Negative Control 0 0 0 0 Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
Brine shrimp lethality assay of Trillium govanianum In present study, cytotoxicity potential of the MeOH extract and its SPE fractions were estimated against brine shrimp larvae (Artemia salina). Highly toxic and
toxic LC50 values were considered to be below the 100 μg/mL and ≤ 250 μg/mL. The MeOH extract of roots of T. govanianum and its SPE exhibited different levels of Brine
shrimp lethality. The LC50 value (μg/mL) of MeOH extract and its SPE fractions are
shown in Table 3.20. The MeOH extract showed the most toxic exhibiting LC50 of 10.1 ± 0.55 μg/mL followed by the SPE fractions TGMF1, TGMF2, TGMF3 and TGMF4
(LC50=77.5 ± 0.95, 38.2 ± 1.6, 93.1 ± 0.5and 22.7 ± 1.4 μg/mL respectively). The
positive control, doxorubicin demonstrated an LC50 value 2.76 ± 0.65 μg/mL.
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Table 3.20 Table 1. Brine shrimp lethality assay of MeOH Extract of the roots of T. govanianum and its SPE fractions.
Brine shrimp lethality assay
Sample % mortality µg/mL
1000 500 250 LC50 (µg/mL) TGM 70 ± 0.55a 68 ± 2.5b 59 ± 1.1c 10.1 ± 0.55 TGMF1 70 ± 1.53a 63 ± 2.8b 58 ± 2.0c 77.5 ± 0.95 TGMF2 70± 1.80a 63 ± 1.5b 58 ± 1.5c 38.2 ± 1.6 TGMF3 70 ± 2.45a 63 ± 1.3b 60 ± 1.6c 93.1 ± 0.5 TGMF4 70 ± 1.70a 63 ± 0.5b 58 ± 1.2c 22.7 ± 1.4 Negative Control 0 0 0 0 Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
Protein kinase inhibition assay of Asparagus adscendens In current exploration, the zones recorded in protein kinase inhibition activity for the MeOH extract and its SPE fractions are summarized in Table 3.21. The MeOH extract of roots of A. asparagus and its SPE exhibited different levels of protein kinase inhibition zones. Among all the MeOH extracts and its SPE fractions, a significant inhibition zone of 19 ± 1.06 mm bald, 9 ± 0.45mm clear phenotype was observed around the MeOH extract, while the SPE fractions AAMF1 (17 ± 0.50 mm bald, 12 ± 0.53 mm clear) showed the most noteworthy hyphae formation inhibition straggled by AAMF2 (15 ± 0.78 mm bald, 9 ± 0.97 mm clear), AAMF3 (14 ± 1.04 mm bald, 8 ± 0.54 mm clear) and AAMF4 (10 ± 0.57 mm bald, 8 ± 0.75 mm clear). The positive control (surfactin) showed prominent 22 ± 1.01 mm bald growth inhibition zone, while negative control (DMSO) showed no zone of inhibition establishing its non-toxic effect. The results of the current study revealed that the most promising kinase inhibitory was shown by MeOH extract and SPE fraction AAMF1, which had the most polar components of the parent MeOH extract. Rest of SPE fractions (AAMF2, AAMF3 and AAMF3) shown mild kinase inhibitory activity.
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Table 3.21 Table. Streptomyces hyphae formation inhibition potential of A. asparagus
Streptomyces hyphae formation inhibition Diameter of growth inhibition zone Sample Clear Zone (mm ± SD ) Bald Zone (mm ± SD )
AAM 9 ± 0.45a 19 ± 1.06b
AAMF1 12 ± 0.53a 17 ± 0.50b
AAMF2 9 ± 0.97a 15 ± 0.78b
AAMF3 8 ± 0.54a 14 ± 1.04b
AAMF4 8 ± 0.75a 10 ± 0.57b Negative Control 0 0 Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05
Protein kinase inhibition assay of Trillium govanianum In current exploration, the zones recorded in protein kinase inhibition activity for the MeOH extract and its SPE fractions are summarized in Table 3.22. The MeOH extract of roots of T. govanianum and its SPE exhibited different levels of protein kinase inhibition zones. Among all the MeOH extracts and its SPE fractions, a significant inhibition zone of 18 ± 0.5 mm bald, 8 ± 0.71± mm clear phenotype was observed around the MeOH extract, while the SPE fractions TGMF1 (18 0.45 mm bald, 11 ± 0.5 mm clear) showed the most noteworthy hyphae formation inhibition straggled by TGMF2 (13 ± 1.0 mm bald, 7 ± 1.5 mm clear), TGMF3 (11 ± 1.1 mm bald, 7 ± 0.58 mm clear) and TGMF4 (11 ± 0.95 mm bald, 7 ± 0.88 mm clear). The positive control (surfactin) showed prominent 21 ± 0.95 mm bald growth inhibition zone, while negative control (DMSO) showed no zone of inhibition establishing its non-toxic effect. The results of the current study revealed that the most promising kinase inhibitory was shown by MeOH extract and SPE fraction TGMF1, which had the most polar components of the parent MeOH extract. Rest of SPE fractions (TGMF2, TGMF3 and TGMF3) shown mild kinase inhibitory activity.
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Table 3.22 Streptomyces hyphae formation inhibition potential of MeOH extract of the roots of T. govanianum and its SPE fractions
Streptomyces hyphae formation inhibition Diameter of growth inhibition zone Sample Clear Zone (mm ± SD) Bald Zone (mm ± SD )
TGM 8 ± 0.71a 18 ± 0.5b
TGMF1 11 ± 0.5a 18 ± 0.45b
TGMF2 7 ± 1.5a 13 ± 1.0b
TGMF3 7 ± 0.58a 11 ± 1.1b
TGMF4 7 ± 0.88a 11 ± 0.95b Negative Control 0 0 Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
MTT assay of Asparagus adscendens
The MeOH extract of the roots of A. adscendens and its SPE fractions (AAMF1, AAMF2, AAMF3 and AAMF4) displayed different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549), urinary bladder (EJ138) and one non-carcinoma vero African Green Monkey kidney cell lines (VERO-CL81, as control) in the in vitro MTT cytotoxicity/viability assay (Table 3.23).
Figures 3.48, 3.49, 3.50, 3.51 & 3.52 reveled the cell viability and IC50 µg/mL values of agaisnt four human carcinoma cell lines and one non-carcinoma vero cell line and error bars represents the standard error mean. The MeOH extract exhibited the highest level of cytotoxicity against the breast cancer cell line (MCF7; IC50 = 6.7 ± 2.2 µg/mL), but it was also active against three other cell lines, HepG2, EJ138, and A549 (IC50 = 13.8 ± 3.7, 30.6 ± 1.3 and 63.4 ± 2.4 µg/mL, respectively) (Table 1). The SPE fraction AAMF1, which had the most polar components of the parent MeOH extract, showed the most significant cytotoxicity against MCF7 (IC50 = 8.5 ± 3.8µg/mL), and considerable cytotoxicity against HepG2, EJ138, and A549 with the IC50 values of 17.5 ± 1.3, 34.6 ± 1.6and 65.3 ± 1.5 µg/mL, respectively. This SPE fraction was almost as potent as its parent MeOH extract in terms of cytotoxicity against the urinary bladder cell line EJ138. The SPE fraction AAMF2 showed most prominent cytotoxicity against
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the breast cancer cell line MCF7 (IC50 = 10.2 ± 1.8 µg/mL), and also activity against other three cell lines, HepG2, EJ138, and A549 (IC50 = 18.8 ± 2.4, 36.4 ± 2.5 and 67.7 ± 1.9 µg/mL, respectively). The cytotoxicity pattern of the SPE fraction AAMF3 was quite similar to that of the AAMF1 and the MeOH extract; it showed most significant cytotoxicity against the HepG2 cell line (IC50 = 15.7 ± 3.7 µg/mL). This fraction was also cytotoxic to MCF7, EJ138, and A549 cell lines with the IC50 values of 17.3 ± 3.7, 39.3 ± 2.8 and 73.4 ± 3.6 µg/mL, respectively. The SPE fraction AAMF4, which contained the least polar components of the parent MeOH extract, exhibited notable cytotoxicity against the HepG2 cell line (IC50 = 19.2 ± 1.2 µg/mL), and was also active against the MCF7, EJ138 and A549 cell lines (IC50 = 27.8 ± 2.6, 43.4 ± 3.7 and 79.6 ± 2.5 µg/mL, respectively). With the exception of AAMF3 and AAMF4, two other SPE fractions showed the highest level of cytotoxicity against the MCF7 cell line, as was observed with their parent MeOH extract. The parent MeOH extract and all SPE fraction has shown low and similar cytotoxicity against vero cell line (100.5 ± 6.7, 102.4 ± 4.8, 110.5 ± 2.7, 104.4 ± 5.3, 92.8 ± 3.3 µg/mL respectively) (Khan et al., 2018a). Etoposide (positive control) results has summerized in Table 3.23, whereas 1% DMSO used as negative control which has exhibited no effect. In Figure 3.23, typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of A. adscendens against HepG2 cell line.our findings are in aggrement with previous reported studies (Senthilraja & Kathiresan, 2015).
Table 3.23. The IC50 values of the A. adscendens
Cell IC50 ± S.E.M (µg/mL) lines MeOH SPE fractions extract AAMF1 AAMF2 AAMF3 AAMF4 Etoposide (µM/mL) A549 63.4±2.4 65.3±1.5 67.7±1.9 73.4±3.6 79.6±2.5 100.7±1.5 EJ138 30.6±1.3 34.6±1.6 36.4±2.5 39.3±2.8 43.4±3.7 115.4±1.8 HepG2 13.8±3.7 17.5±1.3 18.8±2.4 15.7±3.7 19.2±1.2 110.7±2.3 MCF-7 6.7±2.2 8.5±3.8 10.2±1.8 17.3±3.7 27.8±2.6 95.7±1.8 Vero 100.5±6.7 102.4±4.8 110.5±2.7 104.4±5.3 92.8±3.3 - Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
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Fig 3.47 Typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of Asparagus adscendens against HepG2 cell line
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Cytotoxic Activity of Asparagus adscendens Against Breast Carcinoma [MCF-7] Cell Line
120 A
100
80 AAM
AAMF1 60 AAMF2
Cell viability (%) AAMF3 40 AAMF4
20
0 10 -4 10-3 10 -2 10 -1 10 0 Log concentration (mg/ml)
MCF-7 B 40
30
µg/ml 20
50
IC
10
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.48 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against breast (MCF-7)
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Cytotoxic Activity of Asparagus adscendens Against Hepatocellular Carcinoma [HepG2] Cell Line
120 A 100
80 AAM
AAMF1 60 AAMF2
Cellviability (%) AAMF3 40 AAMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
HepG2 B 25
20
15
µg/ml
50 10
IC
5
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.49 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against hepato celluar carcinoma (HepG2)
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Cytotoxic Activity of Asparagus adscendens Against Lung Cancer [A-549] Cell Line
120 A
100
80 AAM
AAMF1 60 AAMF2
Cell viability (%) AAMF3 40 AAMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
B A549 100
80
60
µg/ml
50 40
IC
20
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.50 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against lung carcinoma (A549)
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Cytotoxic Activity of Asparagus adscendens Against Urinary Bladder Carcinoma [EI-138] Cell Line
A 120
100
80 AAM
AAMF1 60 AAMF2
Cell viability (%) AAMF3 40 AAMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
EJ138 B 50
40
30
µg/ml
50 20
IC
10
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.51 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against urinary bladder (EF 138)
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Cytotoxic Activity of Asparagus adscendens Against Vero
120 A
100
80 AAM
AAMF1 60 AAMF2
Cellviability (%) AAMF3 40 AAMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml) B Vero 150
100
µg/ml
50
IC 50
0
AAM AAMF1 AAMF2 AAMF3 AAMF4
Fig 3.52 (A) Cell viability % (B) IC50 µg/mL of Asparagus adscendens against vero (CL 81)
191
MTT assay of Trillium govanianum
The MeOH extract of the roots of T. govanianum and its SPE fractions (TGMF1, TGMF2, TGMF3 and TGMF4) exhibited different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138) and one non-carcinoma vero African Green Monkey kidney cell lines
(VERO-CL81) using the in vitro MTT cytotoxicity/viability assay (Table 3.24). The IC50 values of the MeOH extract and its SPE fractions are shown in Table 3.24. Figures 3.54,
3.55, 3.56, 3.57 & 3.58 reveled the cell viability and IC50 µg/mL values of agaisnt five human carcinoma cell lines and error bars represents the standard error mean. The MeOH extract displayed the highest level of cytotoxicity against the urinary bladder cell line (EJ138; IC50 = 5.4 ± 3.4 µg/mL), but it was also considerably active against three other cell lines, MCF7, HepG2 and A549 (IC50 = 5.4 ± 2.5, 7.5 ± 1.3 and 6.4 ± 1.4 µg/mL, respectively). The SPE fraction TGMF1, which had the most polar components of the parent MeOH extract, showed most significant cytotoxicity against EJ138 (IC50 = 6.3 ± 1.7 µg/mL), and considerable cytotoxicity against MCF7, HepG2 and A549 with the IC50 values of 9.1 ± 1.3, 11.4 ± 1.7 and 9.5 ± 3.5 µg/mL, respectively. This SPE fraction was almost as potent as its parent MeOH extract in terms of cytotoxicity against the urinary bladder cell line EJ138. The SPE fraction TGMF2 showed most prominent cytotoxicity against the lung cancer cell line A549 (IC50 = 5.7 ± 3.3 µg/mL), and was also active against other three cell lines, EJ138, MCF7 and HepG2 (IC50 = 11 ± 4.7, 11.6 ± 1.5 and 13.8 ± 2.8 µg/mL, respectively). The cytotoxicity pattern of the SPE fraction TGMF3 was quite similar to that of the TGMF1 and the MeOH extract, and showed most significant cytotoxicity against the EJ138 cell line (IC50 = 9 ± 2.8 µg/mL). This fraction was also cytotoxic to MCF7, A549 and HepG2 cell lines with the IC50 values of 11.7 ± 1.8, 13 ± 2.4 and 16.2 ± 1.6 µg/mL, respectively. The SPE fraction TGMF4, which contained the least polar components of the parent MeOH extract, exhibited notable cytotoxicity against the EJ138 cell line (IC50 = 13 ± 2.2 µg/mL), and was also considerably active against the HepG2, MCF7 and A549 cell lines (IC50 = 10.8 ± 2.9, 13.4 ± 2.4 and 13.5 ± 4.7 µg/mL, respectively). With the only exception of TGMF2, three other SPE fractions showed the highest level of cytotoxicity against the EJ138 cell line, as was observed with their parent MeOH extract. The parent MeOH extract and all SPE fraction has shown low and similar cytotoxicity against vero cell line (120.5 ± 3.4, 142.4±6.4, 127.8±3.6, 150.2±5.3, 117.45±4.5µg/mL respectively). Etoposide (positive
192
control) results has summerized in Table 3.24, whereas 1% DMSO used as negative control which has exhibited no effect. This is the first report on cytotoxicity of the the MeOH extract of the roots of T. govanianum and SPE fractions against any carcinoma cell lines. The current finding is in line with the findings of a few other previous studies on cytotoxicity of some other species of the genus Trillium (Hayes et al., 2009, Nooter & Herweijer, 1991, Yokosuka & Mimaki, 2008). In Figure 3.53, typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of Trillium govanianum against HepG2 cell line.
Table 3.24 The IC50 values of the MeOH extract of the roots of T. govanianum and its SPE fractions against five carcinoma cell lines
IC50 ± S.E.M (µg/mL)
Cell SPE fractions MeOH lines Etoposide extract TGMF1 TGMF2 TGMF3 TGMF4 (µM/mL) A549 6.4 ± 1.4 9.5 ±3.5 5.7±3.3 13±2.4 13.5±4.7 62.6±2.4
EJ138 5.4 ± 3.4 6.3 ± 1.7 11± 4.7 9± 2.8 13± 2.2 87.3±2.5
HepG2 7.5±1.3 11.4±1.7 13.8±2.8 16.2±1.6 10.8±2.9 103.3±2.1
MCF-7 5.4± 2.5 9.1±1.3 11.6±1.5 11.7±1.8 13.4±2.4 115.5±1.5
Vero 120.5±3.4 142.4±6.4 127.8±3.6 150.2±5.3 117.45±4.5 -
Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
193
Fig 3.53 Typical 96-well plate showing results after (A) 24 h, (B) 48 h and (C) 72h of MeOH extract of Trillium govanianum against HepG2 cell line
194
Cytotoxic Activity of Trillium govanianum Against Breast Carcinoma [MCF-7] Cell Line
120
A 100
80 TGM
TGMF1 60 TGMF2
Cellviability (%) TGMF3 40 TGMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
MCF-7 B 20
15
µg/ml 10
50
IC
5
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.54 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against breast (MCF-7) cell line
195
Cytotoxic Activity of Trillium govanianum Against Hepatocellular Carcinoma [HepG2] Cell Line
120
A 100
80 TGM
TGMF1 60 TGMF2
Cellviability (%) TGMF3 40 TGMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
HepG2 B 20
15
µg/ml 10
50
IC
5
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.55 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against hepatocellular carcinoma (HepG2)
196
Cytotoxic Activity of Trillium govanianum Against Lung Cancer [A-549] Cell Line
120
A 100
80 TGM
TGMF1 60 TGMF2
Cellviability (%) TGMF3 40 TGMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
A549 B 20
15
µg/ml 10
50
IC
5
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.56 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against lung carcinoma (A549)
197
Cytotoxic Activity of Trillium govanianum Against Urinary Bladder Carcinoma [EI-138] Cell Line
120
A 100
80 TGM
TGMF1 60 TGMF2
Cellviability (%) TGMF3 40 TGMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml)
EJ138 B 20
15
µg/ml 10
50
IC
5
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.57 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against urinary bladder(EJ138) cell line
198
Cytotoxic Activity of Trillium govanianum Against Vero
A 120
100
80 TGM
TGMF1 60 TGMF2
Cellviability (%) TGMF3 40 TGMF4
20
0 10 -4 10 -3 10 -2 10 -1 10 0 Log concentration (mg/ml) B Vero 200
150
µg/ml 100
50
IC
50
0
TGM TGMF1 TGMF2 TGMF3 TGMF4
Fig 3.58 (A) Cell viability % (B) IC50 µg/mL of Trillium govanianum against vero (CL 81)
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3.2.6 Isolation and characterization Among all the SPE of methanolic extracts of both species, which shows promising results against several bio-assays, were subjected to prep-HPLC for isolation and characterization. Several compounds were isolated and collected through prep- HPLC but only three compounds were managed for their full characterization.
The SPE fraction eluted with 50% MeOH (AAMF2) was analyzed by prep-
HPLC afforded compounds C-156 and C-157 (tR=24.5 and 26.7 min respectively), whilst 100% MeOH (AAMF4) SPE fraction produced compound C-158 (tR= 25 min). The structures of all isolated compounds (C-156-158) were elucidated by spectroscopic analysis. The chromatogram along with UV-spectra of all isolated compounds are presented in Figure 3.59. This is the first report on the occurrence of compounds Epimedium C, Basohuoside I and Chrysin in the genus Asparagus.
Fig 3.59 Chromatograms of isolated compounds (A) C-156,(B) C-157 (C) C-158
200
Spectroscopic analysis:
Compound (156): Epimedium C
The compound (C-156) was isolated through prep-HPLC from SPE fraction 50% MeOH (AAMF2) at 1mg/mL concentration. Injected on LCMS and show + moluceluar peaks at m/z 823.6 [M+H] corresponds to molecular formula C39H50O19 with degree of unsaturation as 16. High polarity of SPE fraction shows it must contain sugar moieties. While comparing the NMR and Mass data with literature, we easily found and matched isolated compound with Epimedium C (Chen et al., 2008, Li et al., 1 1 1996). HNMR: H NMR: δ 0.88 (3 H, d, J=5.5 Hz), 1.22 (3H,dd, CH3), 1.60 (6H, dd,
2 CH3), 2.86-2.87(m, 5H), 3.33-3.76 (m, 9H), 3.88-3.90 (m,2H), 4.26 (2H,d,J=6.6 Hz), 4.85-5.04 (m, OH), 6.60 (1H), 7.06 (2H,d,J=8.4 Hz), 7.82 (2H,d,J=8.4 Hz). 13C NMR spectrum together with DEPT 135 experiment (Table 3.25) showed 39 signals. UV λmax (MeOH) 270, 290and 410 (sh) nm.
Compound (157): Basohuoside I
201
The compound (C-157) was isolated through prep-HPLC from SPE fraction 50% MeOH (AAMF2) at 1mg/mL concentration. Injected on LCMS and show + molecular peaks at m/z 515.5 [M+H] corresponds to molecular formula C27H30O10 with degree of unsaturation as 16. High polarity of SPE fraction shows it must contain sugar moieties. While comparing the NMR and Mass data with literature, we easily found and matched isolated compound with Basohuoside I (Chen et al., 2008). 1H NMR: δ
1.63 (3H,s, 5’ CH3), 1.68 (3H, s, 5’ CH3), 3.3-3.45(m,2H,H7,7’), 3.7 (1H,dd), 3.86(3H),6.2 (1H,s), 7.07 (2H,d,J=8.22 Hz), 7.84 (2H,d,J=8.22). 13C NMR spectrum together with DEPT 135 experiment (Table 3.25) showed 27 signals. UV λmax (MeOH) 265, 354 and 490 (sh) nm.
Compound (158): Chrysin
The compound (C-158) was isolated through prep-HPLC from SPE fraction 100% MeOH (AAMF4) at 1mg/mL concentration. Injected on LCMS and show + molecular peaks at m/z 515.5 [M+H] corresponds to molecular formula C15H10O3 with degree of unsaturation as 16. While comparing the NMR and Mass data with literature, we easily found and matched isolated compound with a flavonoid Chrysin (Tsimogiannis et al., 2007). 1H NMR: δ 6.42 (1H, s), 5.96 (1H, s, H-3), 6.55-7.01 (4H, m, H- 5,6,7,8), 7.35 (2H, d, J = 8.0 Hz, H-2’,6’), 7.85 (2H, d, J = 8.0, Hz, H-3’,5’). 13C NMR spectrum together with DEPT 135 experiment (Table 3.25) showed 15 signals. UV λmax (MeOH) 240, 285 and 320 (sh) nm.
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Table 3.25 13C NMR of compounds (C-156, C-157, C-158) chemical shift δ in ppm Position C-156 C-157 C--158 1 162.1 154.4 123.8 2 108.5 102.3 122.4 3 - 157.2 163.6 4 102.1 102.3 128.0 5 153.4 - 128.0 6 - 104.6 177.2 7 178.7 178.5 131.3 8 - - 157.8 9 153.6 154.4 155.7 10 - - 118.0 11 122.4 - 123.3 12 113.5 - 115.7 13 160.6 162.0 115.7 14 113.8 113.8 107.3 15 122.4 130.4 125.66 16 54.7 54.7 17 24.5 21.09 18 129.3 16.5 19 130.5 - 20 16.4 16.8 21 21.4 24.5 22 100.8 72.15 23 76.9 70.7 24 61.0 70.3 25 70.7 98.0 26 - 70.5 27 - 16.5 28 - 29 68.8 32 65.4 33 16.3 35 70.7 36 73.5 38 70.54 39 18.5
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3.2.7 Cytotoxicity of isolated compound The SPE fraction eluted with 50% MeOH (AAMF2) afforded compounds Epimedium C (C-156) and Basohuoside I (C-157) whilst 100% MeOH (AAMF4) SPE fraction produced compound Chrysin (C-158). All isolated compounds exhibited different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138) and one non- carcinogenic vero (CL81) cell line, using the in vitro MTT cytotoxicity/viability assay
(Table 3.25). The IC50 values of the isolated compounds are shown in Table 3.26.
The compound Epimedium C (C-156) displayed the highest level of cytotoxicity against the breast cancer cell line (MCF7; IC50 = 35 ± 2.1µg/mL), but it was also considerably active against two other cell lines HepG2 and A549 (IC50 = 41 ± 2.8, 55 ± 3.1 µg/mL, respectively). Where as Epimedium C showed minor activity against urinary bladder (EJ138) and vero (CL81) cell lines (IC50 =325 ± 2.74, 301 ± 3.67).
The compound Basohuoside I (C-157) displayed the highest level of cytotoxicity against the breast cancer cell line (MCF7; IC50 = 41 ± 2.8 µg/mL), but it was also considerably active against two other cell lines HepG2 and A549 (IC50 = 78 ± 2.28, 50 ± 1.48 µg/mL, respectively). Where as Basohuoside I showed no activity against urinary bladder (EJ138) and minor activity against vero (CL81) cell line (IC50 =367 ± 7.57).
The compound Chrysin (C-158) displayed the highest level of cytotoxicity against the lungs cancer cell line (A549; IC50 = 22 ± 1.10 µg/mL), but it was also considerably active against two other cell lines HepG2 and MCF7 (IC50 = 75 ± 2.14, 56 ± 1.38 µg/mL, respectively). Where as Chrysin showed no activity against urinary bladder (EJ138) and minor activity against Vero (CL81) cell line (IC50 =299 ± 8.63). Etoposide is used as a standard drug which showed good results against all the four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138)( IC50 = 18 ± 1.04, 19 ± 1.2, 17 ± 1.04 and 58 ± 1.04 µg/mL, respectively) and showed no activity against non-carcinogenic viro cell line. Negative control (DMSO) has shown no activity against any cell line.
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Table 3.26 MTT assay of isolated compounds
IC50 ± S.E.M (µg/mL)
Cell lines Isolated Compounds
C-156 C-157 C-158 Etoposide A549 55 ± 3.10 50 ± 1.48 22 ± 1.10 17 ± 1.04
EJ138 325 ± 2.74 - - 58 ± 1.04
HepG2 41 ± 2.80 78 ± 2.28 75 ± 2.14 19 ± 1.2
MCF-7 35 ± 2.10 41 ± 2.8 56 ± 1.38 18 ± 1.04
Vero 301 ± 3.67 367 ± 7.57 299 ± 8.63 -
Data presented here were the outcome of study conducted in triplicate and mean difference is significant at at p< 0.05.
3.2.8 Computational methods
Molecular docking studies of compounds (C-156 and C-157)
Fig 3.60 Obtained binding modes of ligands in the ATP binding domain of EGFR. (A) Superimposing of compounds C-156 (magenta), C-157 (yellow) and 5B (cyan) docked to EGFR. (B) Binding mode of compound C-156 in EGFR ATP binding site. (C) Binding mode between compound C-157 and EGFR. 205
Top three ligand conformations obtained from docking analysis were found to be almost overlapped to the crystallographic conformation of donepezil in EGFR. The value of root-mean-square deviation (RMSD) between predicted and experimental confirmation has been observed to be less than 0.67 Å which further reinforces the reliability of docking results
Moreover, both inhibitors C-156 and C-157 (Figure 3.60), were scrutinized on the basis of their inhibitory potency for EGFR. C-156 and C-157 both have displayed appreciable activity against EGFR. However both compound have almost similar scaffold however C-157 differs from C-156 only by the absence of a glucose at 7 and rhamnose moiety at 14 positon aglycone moiety.
Only the top ranked ligand conformations scored by Cscore were preferred for further analysis. The selected (Surflex’s) docked poses were then critically investigated by graphically visualizing in MOLCAD (SYBYL-X) to identify the key ligand-protein interactions responsible for ligand-receptor complex formation. As depicted by molecular docking results (Figure 3.60A); both compounds acquire similar binding conformations in the main active site of EGFR to that of ligand conformation in 2W2S. Both ligands may establish H-bond interactions with Asp800 near water exposed region. Figure 3.60B demonstrate that the compound C-156 incorporates deeply and gain access into the subsites located in deep region of EGFR main binding site. The two subsites namely CAS and PAS are relatively deeper region and are connected by a hydrophobic gorge. The rhamnose ring of C-156 is able to establish hydrophobic interactions with residue Phe338 and sulfonyl oxygen accept an H-bond from TRP886 in the CAS. Meanwhile, the OH group of C-156 extends into pocket to form H-bod interaction with carbonyl oxygen atom located at backbone of residue TYR341 in PAS region. Moreover, the oxygen atom of -OH group substituted in aglycone ring can also make hydrogen bond with the –OH group of residues TYR72 and amino group of TRP286. These findings suggest that the docking results are in strong agreement with experimentally determined biological activity
Due to the absence of rahmnose and glucose moieties, as shown in Figure 3.60B and 3.60C, both compounds C-156 and C-157, respectively, obtained a similar binding pattern to C-156. However, C-157 is not able to establish H-bond interaction with 206
TYR72 and TRP286. The graphical (Figure 3.60C) analysis illustrate that substitution of these two rings group of C-156 with methyl group in C-157 is not suitable to be accommodated in solvent exposed region and TYR72 is pushed away in an upward direction. These substantial conformational changes in C-157-EGFR complex may results in impairing of overall binding interactions in C-157-EGFR complex.
Molecular docking studies of compound (C-158)
Fig 3.61 Obtained binding modes of ligands (C-158) in the ATP binding domain of EGFR
The docking results revealed that the C-158 occupied the major portion of ATP binding site of EGFR (Figure 3.61). As depicted in Fig. the carbonyl group at C3 of pyran ring extends towards solvent exposed region of EGFR, where it may accept H- bond attractions from –NH group of Met93.Where as, Oxygen atom pf pyran ring lies in close proximity to Thr 854 to accept an H-bond from side chain –OH group of Thr 207
854. The Phenol ring of C-158 extends towards a deep left to established vendor wall and hydrophobic interatctions with surrounding residues such as i-e Val 726, Leu 788, Lys 745 and Thr 790. In the mean while the Benzyl moetie of C-158 is able to established hydrophobic contact to side chain of Leu 844 and Thr 854.
Molecular docking studies of selected identified compounds (saponins)
Based on CDocker Interaction Energies all poses of docked compounds were sorted out and best one was selected. CDocker Interaction Energies of all selected compounds are tabulated in Table 3.27. Calendasaponin C shows highest binding interaction with EGFR having CDIE as -76.6 followed by Yayoisaponin B, Agavasaponin C, Azukisaponin IV, Protodioscin which are having CDIE -72.1, -63.4 - 62.1 -60.9 respectively. Detail binding interaction of compounds with EGFR are depicted in Table 3.28, while 3D binding interactions of each compound with EGFR are shown in Figure 3.62, 3.63, 3.64, 3.65 & 3.66. Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line.
As depicted in Table 3.28, the most active compound (C-115) was able to establish at least 15 Hydrogen Bond interaction and 4 Hydrophobic contacts with surrounding residues. Similarly second highest binding interaction (C-107) was able to establish 9 Hydrogen Bond interaction and 1 Hydrophobic contacts with surrounding residues. Third highest binding interaction with EGFR (C-123) has managed to establish 11 Hydrogen Bond interaction and 4 Hydrophobic contacts with surrounding residues. Where as the ligands (C-119) and (C-109) has shown comparatively low binding interaction with EGFR have establish 11 & 8 Hydrogen Bond interaction and 4 & 4 Hydrophobic contacts respectively.
In order to rationalized the difference in inhibitory potential of studied compounds, as compared to standard Etoposide was docked into EGFR (Figure 3.67). The molecular docking results have revealed that Etoposide has superior binding affinity towards EGFR in comparison to other studied compounds (Table 3.27). The graphical analysis of Etoposide bonded to EGFR has revealed that, the compound occupies the major area of EGFR binding side, where it may established at least five
208
H-bond interactions (Table 3.28), along with several vanderwaal and hydrophobic contacts. These results are consistant with the expermiantal findings.
Table 3.27 CDocker interaction energies of best pose of each compound
Compounds CDocker Interaction Energies
Etoposide -87.4 Calendasaponin C (C-115) -76.6 Yayoisaponin B (C-107) -72.1
Agavasaponin C (C-123) -63.4
Azukisaponin IV (C-119) -62.1 Protodioscin (C-109) -60.9
Table 3.28 Detail binding interaction of compounds with amino acid residues of EGFR. Compounds Detail binding interactions of compounds with different amino acid residue of EGFR Residue and atoms involved in forming Type of interaction interactions Etoposide Etoposide: O21-LYS745:HZ2 Hydrogen Bond Etoposide: OH23-PHE856:O7 Hydrogen Bond Etoposide: O26-LYS745:HZ2 Hydrogen Bond Etoposide: O18-ASP855:NH1 Hydrogen Bond Etoposide: O20-LEU744:NH2 Hydrogen Bond Calendasaponin C Calendasaponin C:H111 - LYS875:O Hydrogen Bond (C-115) Calendasaponin C:H112 - ASP837:OD2 Hydrogen Bond Calendasaponin C:H152 - ASP837:OD2 Hydrogen Bond Calendasaponin C:H160 - ILE886:O Hydrogen Bond Calendasaponin C:H166 - GLY874:O Hydrogen Bond ARG841:HE - Calendasaponin C:O65 Hydrogen Bond ARG841:HH22 - Calendasaponin Hydrogen Bond C:O65 LYS875:HZ2 - Calendasaponin C:O61 Hydrogen Bond ARG889:HH21 - Calendasaponin Hydrogen Bond C:O58 ARG889:HH22 - Calendasaponin Hydrogen Bond C:O57
209
Calendasaponin C:H108 - LYS875:O Hydrogen Bond ARG858:HD1 - Calendasaponin C:O32 Hydrogen Bond GLY873:HA2 - Calendasaponin C:O78 Hydrogen Bond PRO877:HD2 - Calendasaponin C:O32 Hydrogen Bond Calendasaponin C:C33 - ILE878 Hydrophobic Calendasaponin C:C33 - ALA920 Hydrophobic LYS875 - Calendasaponin C Hydrophobic ALA920 - Calendasaponin C Hydrophobic
Yayoisaponin B Yayoisaponin B:H125 - GLY874:O Hydrogen Bond (C-107) Yayoisaponin B:H168 - LYS875:O Hydrogen Bond Yayoisaponin B:H169 - ASP916:OD1 Hydrogen Bond Yayoisaponin B:H170 - PRO914:O Hydrogen Bond LYS879:HZ2 - Yayoisaponin B:O75 Hydrogen Bond Yayoisaponin B:H129 - VAL876:O Hydrogen Bond LYS875:HA - Yayoisaponin B:O31 Hydrogen Bond LYS879:HE1 - Yayoisaponin B:O50 Hydrogen Bond LYS879:HE1 - Yayoisaponin B:O79 Hydrogen Bond Yayoisaponin B:C27 - ILE878 Hydrophobic
Agavasaponin C Agavasaponin C:H112 - LEU887:O Hydrogen Bond (C-123) Agavasaponin C:H113 - LEU887:O Hydrogen Bond ARG841:HE - Agavasaponin C:O39 Hydrogen Bond VAL876:HN - Agavasaponin C:O15 Hydrogen Bond ARG889:HH12 - Agavasaponin C:O7 Hydrogen Bond ARG889:HH12 - Agavasaponin C:O51 Hydrogen Bond ARG889:HH22 - Agavasaponin C:O10 Hydrogen Bond Agavasaponin C:H67 - ILE886:O Hydrogen Bond Agavasaponin C:H79 - VAL876:O Hydrogen Bond LYS875:HA - Agavasaponin C:O15 Hydrogen Bond LYS875:HA - Agavasaponin C:O64 Hydrogen Bond Agavasaponin C:C59 - LYS879 Hydrophobic Agavasaponin C:C63 - PRO877 Hydrophobic LYS879 - Agavasaponin C Hydrophobic ALA920 - Agavasaponin C Hydrophobic
Azukisaponin IV Azukisaponin IV:H103 ILE886:O Hydrogen Bond (C-119) Azukisaponin IV:H142 - ASP837:OD2 Hydrogen Bond ARG841:HE - Azukisaponin IV:O67 Hydrogen Bond ARG841:HH22 - Azukisaponin IV:O64 Hydrogen Bond ARG841:HH22 - Azukisaponin IV:O67 Hydrogen Bond LYS879:HN - Azukisaponin IV:O63 Hydrogen Bond ARG889:HH12 - Azukisaponin IV:O32 Hydrogen Bond SER921:HG - Azukisaponin IV:O58 Hydrogen Bond Azukisaponin IV:H141 - ASP837:OD2 Hydrogen Bond PRO877:HA - Azukisaponin IV:O48 Hydrogen Bond
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SER921:HA - Azukisaponin IV:O58 Hydrogen Bond Azukisaponin IV - LYS875 Hydrophobic Azukisaponin IV:C35 - ILE878 Hydrophobic Azukisaponin IV:C35 - ALA920 Hydrophobic ALA920 - Azukisaponin IV Hydrophobic
Protodioscin Protodioscin:H87 - LYS875:O Hydrogen Bond (C-109) ARG841:HH22 - Protodioscin:O10 Hydrogen Bond ARG889:HH21 - Protodioscin:O13 Hydrogen Bond SER921:HG - Protodioscin:O25 Hydrogen Bond Protodioscin:H84 - ASP837:OD2 Hydrogen Bond VAL876:HA - Protodioscin:O9 Hydrogen Bond PRO877:HD2 - Protodioscin:O9 Hydrogen Bond SER921:HA - Protodioscin:O25 Hydrogen Bond Protodioscin:C19 - VAL876 Hydrophobic Protodioscin:C62 - LYS875 Hydrophobic LYS875 - Protodioscin Hydrophobic ALA920 - Protodioscin Hydrophobic
Fig 3.62 3D binding interactions of Calendasaponin C with EGFR (PDB ID:4P3R) Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line
211
Fig 3.63 3D binding interactions of Yayoisaponin B with EGFR (PDB ID:4P3R) Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line
Fig 3.64 3D binding interactions of Agavasaponin C with EGFR (PDB ID:4P3R) Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line
212
Fig 3.65 3D binding interactions of Azukisaponin IV with EGFR (PDB ID:4P3R) Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line
Fig 3.66 3D binding interactions of Protodioscin with EGFR (PDB ID:4P3R) Hydrogen bonds are shown as green dashed line while hydrophobic interactions are shown as light pink dashed line
213
Fig 3.67 3D binding interactions of Etoposide with EGFR (PDB ID:4P3R)
214
Chapter 4
4 Discussion
Discussion
4.1 Phytochemical analysis 4.1.1 Determination of total phenolic contents A. adscendens and T. govanianum The total phenolic content of MeOH extract and SPE fractions of A. adscendens are summarized in Figure 3.2. The SPE fraction AAMF2, which had the polar components of the parent MeOH extract, showed most promising total phenolic contents among other SPE fractions.The activity exhibited by AAMF2 might be attributed to the presence of quercetin, myricetin and Kaemferol (detected by HPLC fingerprint analysis). Where as total phenolic content of MeOH extract and SPE fractions of T. govanianum are summarized in Figure 3.3. The SPE fraction TGMF1, which had the most polar components of the parent MeOH extract, showed most promising total phenolic contents among other SPE fractions. The activity shown by TGMF1 might be attributed to the presence of quercetin (detected by HPLC fingerprint analysis).The phenolic contents present in plants revealed different physiological role by scavenging free radicals and chelating trace elements (Kumar et al., 2013, Afshar et al., 2012, Kim et al., 2006).
4.1.2 Determination of total flavonoid contents A. adscendens and T. govanianum The total flavonoid content of MeOH extract and SPE of A. adscendens are summarized in Figure 3.5. The SPE fraction AAMF2, which had the polar components of the parent MeOH extract, showed most promising total flavonoid contents among other SPE fractions in term of mg quercetin equivalent per gram dry weight. The activity exhibited by AAMF2 might be attributed to the presence of quercetin, myricetin and Kaemferol (detected by HPLC fingerprint analysis). Where as total flavonoid content of MeOH extract and SPE fraction of T. govanianum in terms of quercetin equivalent per gram dry weight exhibited different levels of significant flavonoid contents. The total flavonoid content of MeOH extract of roots of T. govanianum and its SPE are presented in Figure 3.6. The SPE fraction TGMF1, which had the most polar components of the parent 216
MeOH extract, showed most promising total flavonoid contents among other SPE fractions. This fractions was almost as potent as its parent MeOH extract in term of mg QE/ g DW. A positive correlation was evident to be present between the phenolic and flavonoid contents of both A. adscendens and T. govanianum suggesting that the antioxidant potential of phenols might be caused by the presence of flavonoids (Afshar et al., 2012, Kim et al., 2006)
4.1.3 Chromatographic and spectroscopic analysis 4.1.4 HPLC-PDA analysis of A. adscendens and T. govanianum HPLC-PDA analysis on the SPE fractions of A. adscendens and T. govanianum were performed to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain phenolic and flavonoids as possible contributors to the significant cytotoxic, antibacterial and antileishmanial activity of the extract and its fractions. Previous studies revealed that different phytochemicals like steroids, triterpenoids, glycosides, saponins, phenolic compounds, aliphatic compounds, alkaloids, tannins and nitrogenous constituents reported in these plants (Manta et al., 1995, Thakur & Sharma, 2015b). Spirostanol glycosides (asparanin A and asparanin B) and two furostanol glycosides (asparoside A and asparoside B) have been isolated from the methanol extract of A. adscendens (Jadhav & Bhutani, 2006). From one of the previous published data (Liang tan et al), 280nm was chosen as the detection wavelength for epicatechin and 360nm for various flavonoids such as myricetin, hyperoside, quercitrin and quercetin. Presence of phenolic compounds like epicatechin and various flavonoids such as myricetin, hyperoside, quercitrin and quercetin in A. adscendens was in agreement with that of other Asparagus species.
It is reasonable to assume that the significant cytotoxic, anti bacterial and antileishmanialactivity of the MeOH extract and its SPE fractions of the roots of A. adscendens and T. govanianum might be, at least partly, owing to the presence of phenolic compounds (Khan et al., 2017). This is the first report, on the preliminary HPLC-PDA analysis on A. adscendens and T. govanianum.
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4.1.5 LC-ESI-QTOF-Mass spectrometry analysis of A. adscendens and T. govanianum LC-MS studies on the SPE fractions of the MeOH extract of the roots of A. adscendens and T. govanianum were carried out to get an insight into the possible chemical composition of the fractions, particularly, to have an indication whether they contain saponins and sapogenins as possible contributors to the significant cytotoxicity of the extract and its fractions. The bioactive components of the genus Asparagus belong predominantly to the chemical classes of sapogenins and saponins (Table 3.10), which are well known to exhibit cytotoxicity. The presence of saponins and their aglycones was in agreement with that of other Asparagus species (Khan et al., 2018a). Most of the compounds isolated previously from the genus Asparagus as described in the literature are steroidal sapogenins and saponins. Sarsasapogenin, diosgenin, β-sitosterol and its glucoside, spirostanol glycoside and furostanol glycoside were reported from A. adscendens (Sharma et al., 1980, Tandon & Shukla, 1992, Thakur & Sharma, 2015b). Compound (4-29) and (30-37) were detected from positive and negative ion mode of MeOH extract respectively (Fig 3.23 & Fig. 3.24). Various biological active compounds were identified e.g. Levoglucosan (C-10) at tR 0.731, Brugine (C-14) at tR 0.929 and (C-
20) Bergenin at tR 7.293 were previously isolated from different species with considerable cytotoxic activity against different human cancer cell lines (Das et al., 2015, Khan et al., 2015b, Abu-Reidah et al., 2015).
The reported studies on different species of the genus Trillium have revealed that this genus is ample in steroidal saponins, e.g., steroidal saponins were found in T. erectum L. (Yokosuka & Mimaki, 2008, Hayes et al., 2009), T. kamtschaticum Pall. (Ono et al., 2003, Yokosuka & Mimaki, 2008, Wei et al., 2012) and T. tschonoskii Maxim. (Nakano et al., 1983, Man et al., 2010, Wei et al., 2012, Wang et al., 2013a). Various biological active saponins and sapogenins were identified e.g. Digoxigenin (C-52) at tR 8.933,
Alliospiroside D (C-53) at tR 9.435, Hovenoside D(C-55) at tR 9.458, Pisumsaponin I (C-
57) at tR 9.907, Fistuloside A (C-58) at tR 9.983, Pitheduloside F (C-60) at tR 10.137,
Durupcoside B (C-61) at tR 11.819, Cyclopassifloside I (C-68) at tR 14.268 Ophiopogonin
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D (C-72) at tR 14.467, Crosatoside B (C-99) at tR 8.031, Yayoisaponin B (C-107) at tR
9.87, Protodioscin (C-109) at tR 10.094, Isoeruboside B (C-111) at tR 10.194,
Phytolaccasaponin B (C-114) at tR 10.663, Calendasaponin C (C-115) at tR 10.701,
Calendasaponin D (C-116) at tR 10.799, Azukisaponin IV (C-119) at tR 11.578,
Pseudoprotodioscin (C-121) at tR 12.421, Polypodoside A (C-122) at tR 12.58,
Agavasaponin C (C-123) at tR 12.712, Schidigerasaponin B1 (C-124) at tR 12.981, Dioscin
(C-125) at tR 13.791, Pitheduloside K (C-127) at tR 13.936, Fistuloside B (C-129) at tR
14.515 and Ophiopogonin B (C-130) at tR 14.887 (Zhang et al., 2014, Madl et al., 2006, Bonnin et al., 1996, Aumsuwan et al., 2016, Jesus et al., 2016, Liu et al., 2017, Chen et al., 2011, Sata et al., 1998, Wang et al., 2013b, Ohana et al., 1998). Presence of saponins and their aglycones in T. govanianum was in agreement with that of other Trillium species. This is the first report, on the preliminary LC-MS analysis on A. adscendens and T. govanianum.
4.1.6 GC/MS analysis of A. adscendens and T. govanianum The GC/MS analysis of n-hexane fraction of A. adscendens revealed the presence of six components (C-141 to 146) presented in Table 3.13 and Figure. 3.28. GC/MS analysis revealed the presence of saturated and unsaturated components. Previous investigation have demonstrated that fatty components like n-Hexadecanoic acid methyl ester, 9, 12-Octadecadienoic acid, Diisooctyl phthalate and β –Sitosterol showed strong biological activity (Asghar & Choudahry, 2011). Yeong et al., 1989 stated the anticarcinogenic activity of n-Hexadecanoic acid methyl ester. In another study, Yamada et al., 2009 elaborated the effect of hydroxy unsaturated fatty acid on the immunity. However, the present study aims to get an better idea about the fatty profile of n-hexane fraction of MeOH extract.
The GC/MS analysis of n-hexane fraction of MeOH extract of the roots of T. govanianum revealed the presence of six components (C-147 to 155) presented in Table 3.15 and Figure 3.30. GC/MS analysis revealed the presence of saturated and unsaturated components. These components are biologically active and well reported in literature
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specifically relevant to antibacterial, antifungal and cytotoxic actives (Hou, 2008, Xu et al., 2011, Ur Rahman et al., 2015).
4.2 Biological evaluation 4.2.1 Antioxidant assay 4.2.2 DPPH assay of A. adscendens and T. govanianum The DPPH scavenging activity of MeOH extract and SPE fractions of roots of A. adscendens and T. govanianum exhibited different levels of radical scavenging activity. The DPPH findings of MeOH extract and SPE fractions of A. adscendens are shown in Figure 3.31. Among the SPE fraction AAMF1 showed the highest scavenging activity, which might be attributed to the presence of phenolic compounds and possible saponins in AAMF1, identified by LCMS analysis (Singh & Geetanjali, 2016, Tan et al., 2014).
The SPE fraction of T. govanianum, TGMF1, which had the most polar components of the parent MeOH extract, showed most promising scavenging properties among other SPE fractions. The DPPH scavenging pattern of the TGMF2 and TGMF3 fractions were quite similar, due to the presence of semi polar components of the parent MeOH extract. The SPE fraction TGMF4, which contained the least polar components of the parent MeOH extract, also exhibited notable scavenging activity. From previous reported data the most polar extract showed remarkable scavenging activities than non- polar extracts and fraction. Our current finding strengthens the reported data, that significant scavenging effect was manifested by methanolic extract. (Khan et al., 2014).
4.2.3 Total antioxidant capacity assay of A. adscendens and T. govanianum The total antioxidant capacity of AAM (MeOH extract) and SPE fractions A. adscendens are presented in Figure 3.34. SPE fraction AAMF2 showed a maximum total antioxidant capacity (TAC), might be attributed to the presence of quercetin, myricetin and Kaempferol (detected by HPLC fingerprint analysis). The current findings are in line with the previous findings (Fatima et al., 2015, Khan et al., 2015a). Due to the oxidation in biological system, phenolic compounds quench these radicals to avoid the cell DNA
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damage, denaturing of proteins and phospholipids of cell membrane from the possible damage, which may leads to the diseases like cancer (Abdel-Hameed, 2009). The result of total antioxidant capacity of MeOH extract and SPE fractions of T. govanianum are shown in Figure 3.35. SPE fraction TGMF2 showed a maximum total antioxidant capacity (TAC), might be attributed to the presence of quercetin, myricetin and Kaemferol (detected by HPLC fingerprint analysis). The current finding is concordant with the finding of a few other previous studies.These outcomes are also matched and aligned with thoses reported by Chaovanalikit & Wrolstad, 2004 .The plants having good antioxidants are supposed to have excellent chemopreventive potential (Singh et al., 2011). Our findings are in concurrence with the previous published data, which revealed that increased antioxidant capacity directly related to the higher phenolic contents (Brusotti et al., 2014). A positive correlation was found to be present between the phenolic and antioxidant capacity (correlation coefficient; R2 = 0.9877 and 0.9927 respectively).
4.2.4 Total reducing power assay of A. adscendens and T. govanianum The results of ferric reducing antioxidant power of MeOH extract and SPE fractions of A. adscendens are shown in Figure 3.36. Among SPE fraction AAMF2 showed a maximum ferric reducing antioxidant power, might be attributed to the presence of quercetin, myricetin and Kaempferol (detected by HPLC fingerprint analysis). The ferric reducing activity are generally linked with the presence of reductones, which are supposed to be the responsible of antioxidant activity in plants (Abdel-Hameed, 2009). The results of ferric reducing antioxidant power of MeOH extract and SPE fractions of T. govanianum are shown in Figure 3.38. This fractions was almost as potent as its parent MeOH extract in term of mg ascorbic acid equivalent per gram dry weight. A direct relationship between reducing power and free radical scavenging activity has reported in certain plant extracts. By keeping in the view the importance of this assay, it has been used primarily to evaluate the antioxidant potential from both natural products and food industry (Pellegrini et al., 2003). In our current findings the reducing power and antioxidant potential showed excellent correlation coefficients (R2 = 0.9765and 0.9927 respectively).
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4.2.5 Antimicrobial analysis Antibacterial assay of A. adscendens and T. govanianum In current study, the MeOH extract and SPE of A. adscendens and T. govanianum exhibited different levels of antibacterial activity determined by the zone of inhibition (mm diameter) ranges from 7 to 13 mm, against Staphylococcus aureus (NCTC 7508); Bacillus subtilis (NCTC 1604); M. luteus (NCTC 75080; Escherichia coli (ATCC 25922) shown in Figure 3.39. The reported literature stated that anti bacterial activity of plant extracts and fractions might be attributed due to the hydroxylated phenolic compounds such as quercetin, myricetin and Kaemferol. Which were also quantified from HPLC fingerprint analysis. The possible mechanism which initiate the phenolic toxicity towards microorganism is thought to be the enzyme inhibition by the compounds which are oxidized by the reaction with sulfhydryl group or interactions with non specific proteins (Cowan, 1999, Sharma et al., 2009). AAMF4, which had least polar components of the parent MeOH extract has exhibited the significant level of antibacterial activity among rest of SPE fractions. This might be attributed due to the presence of phenolic compounds, as AAMF4 is active fraction, which also afforded a C-158 (Chrysin).
The result of T. govanianum are shown in Figure 3.40. The SPE TGMF1, which had the most polar components of the parent MeOH extract has shown mild to moderate antibacterial activity against most of tested bacterial strains, while other SPE fractions, TGMF2, TGMF3 and TGMF4 showed similar and mild activity against tested bacterial strains. SPE fraction TGMF1 showed a maximum total antioxidant capacity (TAC), might be attributed to the presence of quercetin, myricetin and Kaemferol (detected by HPLC fingerprint analysis). The presence of saponins and sapogenin in the T. govanianum is in agreement with the other Trillium species and with the reported data (Ismail et al., 2015, Khan et al., 2016). The saponins and sapogenins are well know for their cytotoxic activity, however some studies stated (Oyekunle et al., 2006) the antibacterial effect of saponins against gram-positive organism (S. aureus).
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Resazurin microtiter assay (REMA) of A. adscendens and T. govanianum The MeOH extract of the roots of Asparagus adscendens and SPE fractions (AAMF1, AAMF2, AAMF3 & AAMF4) exhibited varying antibacterial activity by using resazurin microtiter plate assay (REMA) as shown in (Table 3.16). The results from (Table 3.16) indicated that the MeOH extract (AAM) and four SPE fractions showed higher antibacterial activity against Gram-positive bacteria (MIC: 2.5-0.009 mg/mL) than against Gram-negative bacteria (MIC: 1.25-2.5 mg/mL). The SPE fraction AAMF1, which had the most polar components of the parent MeOH extract, showed most significant antibacterial activity among other SPE fractions. Presence of phenolic compounds like epicatechin and various flavonoids such as myricetin, hyperoside, quercitrin and quercetin in A. adscendens was in agreement with that of other Asparagus species. It is reasonable to assume that the antibacterial activity of the MeOH extract and its SPE fractions of the roots of A. adscendens might be, at least partly, owing to the presence of phenolic compounds and could be potential source of antimicrobial compounds (Singh & Geetanjali, 2016, Tan et al., 2014).
The MeOH extract of the roots of Trillium govanianum and SPE fractions (TGMF1, TGMF2, TGMF3 & TGMF4) exhibited varying antibacterial activity by using Resazurin microtiter plate assay as shown in (Table 3.17). Table 3.17 reveled that SPE fraction TGMF3 possessed most significant antibacterial activity against all the test bacterial strains, which had a moderate level of polar components of the parent MeOH extract. Literature reported that phenolic compounds and saponins are more sensitive to Gram positive than Gram negative bacteria (Sivropoulou et al., 1995, Gulluce et al., 2007), which is extended and confirmed in present studies. REMA assay was found to be an effective in terms of accuracy, precision, robustness and fast as compared to the conventional methods (Khan et al., 2018b). This is the first report to explore the antimicrobial potential to determine the minimum inhibitory concentration (MIC) values of the MeOH extract and SPE fractions of the roots of Asparagus adscendens and Trillium govanianum by using resazurin microtiter assay (REMA) against Gram positive and negative bacterial strains.
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Antifungal assay of A. adscendens and T. govanianum The antifungal potential of MeOH extract and SPE fractions of A. adscendens and T. govanianum were established and summarized in Figure 3.43. All the four SPE fractions showed mild to moderate or weak antifungal activity. The SPE fraction AAMF1, which had the most polar components of the parent MeOH extract, showed most significant antibacterial activity among other SPE fractions. Presence of phenolic compounds like epicatechin and various flavonoids such as myricetin, hyperoside, quercitrin and quercetin in A. adscendens was in agreement with that of other Asparagus species (Quiroga et al., 2001). Activity shown by AAMF1 might be attributed to the presence of phenolic compounds such as flavonoids.
The antifungal growth inhibitory activities of MeOH extract and SPE fractions of T. govanianum against are summarized in Figure 3.44. Among other SPE fractions, TGMF2, TGMF3 and TGMF4 showed similar and mild activity against tested fungal strains. It was noticed that the typical antifungal activity shown by SPE fraction were similar and had no impact of polarity decreased. The negative control (DMSO) showed no zone of inhibition, while Clotrimazole showed notable zone of inhibition (31 ± 1.25 mm) was used as positive control. The steroidal saponins have shown to possess antifungal potential (Ismail et al., 2015, Khan et al., 2016) which endorse the finding of this study.
4.2.6 In vitro antileishmanial analysis of A. adscendens and T. govanianum Antileishmanial capability of MeOH and its SPE fractions of A. adscendens and T. govanianum against Leishmania tropica KWH23 strain were exhibited in Figure 3.45 & 3.46 respectively. The SPE fraction AAMF1, which had the most polar components of the parent MeOH exhibited as good result as its parent MeOH extract. The SPE fraction TGMF1, which had the most polar components of the parent MeOH exhibited as good result as its parent MeOH extract (LC50 40.5 ± 2.51 µg/mL). SPE fraction TGMF1 showed a maximum Antileishmanial capacity, might be attributed to the presence of quercetin, myricetin and kaemferol (detected by HPLC fingerprint analysis) (Khan et al., 2017). The presence of saponins and sapogenin in the T. govanianum is in agreement with the other
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Trillium species and with the reported data (Ismail et al., 2015, Khan et al., 2016). In the previous findings flavonoids exhibited good antileishmanial activity by forming a complexes with the parasite cell wall (Wabwoba et al., 2010). To best of our knowledge, this is the first report, which represents the antileishmanial capability of MeOH and SPE fractions of A. adscendens and T. govanianum.
4.2.7 Cytotoxicity assays Brine shrimp lethality assay of A. adscendens and T. govanianum In present study, cytotoxicity potential of the MeOH extract and SPE fractions were estimated against brine shrimp larvae (Artemia salina) and data presented in Table 3.18. These results are in agreement with the cytotoxicity of MeOH extract and its SPE fractions against four human carcinoma cell lines (IC50 = 5-16 μg/mL) (Khan et al., 2016). The concentration of the extract determined the degree of lethality which was found directly proportional. The brine shrimp lethality assay determined the cytotoxicity of plant extract, if the LC50 value of less than 1000 μg/mL is observed. In present study, MeOH extract and its SPE fractions demonstrated LC50 values < 1000 μg/mL, indicated the presence of compounds having cytotoxic potential determined the observed cytotoxicity (Fatima et al., 2015). Table 3.19 presents the brine shrimp lethality profile of T. govanianum The MeOH extract showed the most toxic exhibiting LC50 of 10.1 ± 0.55 μg/mL followed by the SPE fractions TGMF1, TGMF2, TGMF3 and TGMF4. The brine shrimp lethality assay is considered to be an appropriate tool for the primary estimation of cytotoxicity (Khan et al., 2017).
Protein kinase inhibition assay of A. adscendens and T. govanianum In current exploration, the zones recorded in protein kinase inhibition activity for the MeOH extract and SPE fractions of A. adscendens are summarized in Table 3.20. Fraction AAMF1 (17 ± 0.50 mm bald, 12 ± 0.53 mm clear) showed the most noteworthy hyphae formation inhibition. The presence of quercetin has been reported to inhibit the multiple kinases like ABL1, CLK1, MET, NEK4, associated with cancer cell biology (Boly et al., 2011). The biological processes like apoptosis, cell differentiation and cell
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proliferation, which are regulated by the protein phosphorylation through protein kinases needs the surge for the development of protein kinases inhibitors especially from natural origin. In this regard, the protein kinases inhibitor are established as a potential target for the cancer treatment (Yao et al., 2011). Table 3.21 represents the finding of protein kinase inhibition activity of MeOH extract and SPE fractions of T. govanianum. The MeOH extract of roots of T. govanianum and its SPE exhibited different levels of protein kinase inhibition zones. Among SPE fractions, TGMF1, which had the most polar components of the parent MeOH extract showed noteworthy kinase inhibitory activity, might be attributed to the presence of quercetin, myricetin and kaemferol (detected by HPLC fingerprint analysis) (Khan et al., 2017).
MTT assay of A. adscendens and T. govanianum The SPE fractions of A. adscendens and T. govanianum displayed different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549), urinary bladder (EJ138) and one non-carcinoma vero (CL81) in the in vitro MTT cytotoxicity/viability assay Table 3.22 and Figure 3.48, 3.49, 3.50, 3.51 & 3.52 represents the A. adscendens findings. This is the preliminary report on cytotoxicity of A. adscendens against any carcinoma cell lines (Khan et al., 2018a). The current finding is in line with the findings of a few other previous studies on cytotoxicity of some other species of the genus Asparagus (Zhou et al., 2007, Wu et al., 2010, Bhutani et al., 2010, Zhao et al., 2012, Galala et al., 2015).
The bioactive components of the genus Asparagus belong predominantly to the chemical classes of sapogenins and saponins (Figure.1), which are well known to exhibit cytotoxicity. Most of the compounds isolated previously from the genus Asparagus as described in the literature are steroidal sapogenins and saponins. Sarsasapogenin, diosgenin, β-sitosterol and its glucoside, spirostanol glycoside and furostanol glycoside were reported from A. adscendens (Sharma et al., 1980, Tandon & Shukla, 1992, Thakur & Sharma, 2015b). In another study, the steroidal saponins from the roots of A. filicinus showed significant cytotoxic activities against human lung carcinoma (A549) and breast adenocarcinoma (MCF7) tumour cell lines (Zhou et al., 2007) compounds isolated from 226
this species were cytotoxic to human breast adenocarcinoma cell line MDA-MB-231 (IC50 3.4 to 6.6 μM) (Wu et al., 2010). A. officinalis displayed significant activity against HeLa and BEL-7404 cells in a dose dependant manner in vitro at 10 mg/mL (Zhao et al., 2012). The alkaloid, aspastiluine, isolated from A. stipularis was shown to be active against MCF7
(IC50 = 47.7 µM) (Galala et al., 2015). Other species of genus Asparagus also reinforced the current findings, that steroidal constituents from these species exhibited significant cytotoxicity against human cancer cell lines (Bhutani et al., 2010).
This is the first report on cytotoxicity of the the MeOH extract of the roots of T. govanianum and SPE fractions against any carcinoma cell lines. The current finding is in line with the findings of a few other previous studies on cytotoxicity of some other species of the genus Trillium (Hayes et al., 2009, Nooter & Herweijer, 1991, Yokosuka & Mimaki, 2008). The major bioactive components of the genus Trillium are saponins, which are well known to exhibit cytotoxicity (Hayes et al., 2009, Nooter & Herweijer, 1991, Yokosuka & Mimaki, 2008). Most of the compounds isolated from the genus Trillium described in the literature are saponins containing mono, di, tri- or tetrasacchride, commonly composed of apisoe, arabinose, glucose, rhamnose and xylose which are linked to a β-D-glucosyl moiety at C3 of the aglycone (Gao et al., 2015). Paris saponin VII isolated from T. tschonoskii Maxim. showed cytotoxicity against MCF7, human colorectal cancer cells-29 and SW-620
(IC50 = 9.547, IC50 = 1.02 ± 0.05 and 4.90 ± 0.23 µM, respectively) (Li et al., 2014). Paris VII induced cell apoptosis together with caspase-3-dependent manner and cell cycle arrest in G1 Phase. Two new saponins from the underground part of T. tschonoskii displayed strong cytotoxic activity against HepG2 cell line (IC50 = 0.499 mmol/L) (Chai et al., 2014). A steroidal saponin from the same species exhibited prominent potential in combating multi-drug-resistance (MDR) hepatocellular carcinoma (HCC) (Wang et al., 2013a). Six steroidal saponins isolated from the roots of T. erratum were shown to possess considerable cytotoxicity against the HL60 human promyelocytic leukemia cells. From the same species, spirostanol and furostanol saponins showed moderate level of cytotoxicity (IC50 =1.68-8.85 µg/mL) (Yokosuka & Mimaki, 2008). Diosgenin, isolated as a major compound from Trillium species, showed potent cytotoxic effect against HepG2 and HCT116 cells in the MTT assay (Eskander et al., 2013). 227
It is rational to suppose that the cytotoxicity of Asparagus adscendens and Trillium govanianum might be, at least partly, owing to the presence of saponins and their aglycones, as implicated in several previously published studies outlined earlier. This is the first report, on the preliminary LC-MS analysis on A. adscendens and T. govanianum. The significant cytotoxicity observed against four carcinoma cell lines in the current study, and the previously published data on the antitumour/anticancer potential of the genus Asparagus and Trillium as well as the presence of saponins in A. adscendens and T. govanianum, suggest that, these species could be used as a prospective origin of cytotoxic compounds with convincible anticancer potential.
4.2.8 Isolation and characterization Among all the SPE of methanolic extracts of both species, which shows promising results against several bio-assays, were subjected to prep-HPLC for isolation and characterization. The SPE fraction eluted with 50% MeOH (AAMF2) was analyzed by prep-HPLC afforded compounds Epimedium C (C-156) and Basohuoside I (C-157), whilst 100% MeOH (AAMF4) SPE fraction produced compound Chrysin (C-158). The structures of all isolated compounds (C-156-158) were elucidated by spectroscopic analysis. The chromatogram along with UV-spectra of all isolated compounds are presented in Figure 3.59. This is the first report on the occurrence of compounds Epimedium C, Basohuoside I and Chrysin in the genus Asparagus.
All isolated compounds exhibited different levels of cytotoxicity against four human carcinoma cell lines, e.g., breast (MCF7), liver (HepG2), lung (A549) and urinary bladder (EJ138) and one non-carcinogenic vero (CL81) cell line, using the in vitro MTT cytotoxicity/viability assay (Table 3.25). Epimedium C, Basohuoside I and Chrysin showed considerable level of cytotoxicity against human carcinoma cell lines and expressed slight activity against non-carcinogenic vero cell line.
Epimedium C and Basohuoside I are well known about their anticancer activity against different human cancer cell lines. Their anticancer action initiates through a variety
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of mechanism like apoptosis inducing effect, cell- cycle modulation, anti-angiogensis, anti- metastasis (Tan et al., 2016, Li et al., 2017, Gui et al., 2018, Kim et al., 2017). Chrysin is naturally occurring flavonoid, identified from different plat species and has been investigated and reported for anti-cancer potential. Primarily exhibited its anticancer activity by inhibiting the proliferation and induce apoptosis especially against leukemia cancer cells (Khoo et al., 2010, Kasala et al., 2015, Bahadori et al., 2016). Chrysin has been reported as potential agent against several human carcinoma cell lines. Salimi A etal; 2017 reported Chrysin cytotoxicity, intercellular reactive oxygen species and mitochondrial membrane potential collapse in detail (Salimi et al., 2017). Epimedium C, Basohuoside I and Chrysin have been first time isolated form genus Asparagus.
4.2.9 Computational methods Molecular docking studies Previous literature reveled that EGFR protein are involved in the regulation of cancer cell growth, due to the hyper expression of proteins. So in this scenario, we need potential inhibitors to suppress the protein expressions, which are responsible of cancer cell growth. By keeping in view the IC50 values of isolated compounds ranges from 22-325 (µg/mL) against four human cancer cell lines and one non-carcinogenic cell line, there is a need to identify a key therapeutic targets responsible for anticancer activity of selected compounds, specifically against Human lungs carcinoma. From Literature (Rho et al., 2009) EGFR have impact and responsible in cell growth of Human lungs cancer cell lines. Thus to neutralized the anticancer activities of selected compounds, Molecular docking studies were initiated to identify the binding modes of selected compounds with targeted receptors. The selected compounds have shown promising docking result against EGFR protein, hence we can conclude, that the said compounds exhibited strong anticancer activity and they may proceeded towards the lead anticancer agent.
Molecular docking findings of selected identified compounds (saponins) are tabulate in Table 3.26. All the ligands were able to establish several Hydrogen bond and Hydrophobic interactions with surrounding residues, However the Hydrogen bond found
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to be the key driving force for the binding affinity of the ligands towards EGFR. It was observed that the most active ligand was able to establish more Hydrogen bond interactions with key residues in the binding pocket of EGFR than moderately active and least EGFR inhibitors. While all of the ligands were able to establish at least 4 Hydrogen bond attractions with conserved residues. However due to the presence of rhamnose and glucose moieties in compound (C-115) was able to establish some additional Hydrogen bond interactions (ASP837, ILE886 & GLY874) as compare to least binding interaction compounds (C-109). These findings supports the hypothesis, that the binding affinity of compounds are mainly derived by Hydrogen bonding and electrostatic interaction rather than Hydrogen phobic interactions.
4.3 Conclusion This piece of research work was conducted in the laboratories of department of Pharmacy, COMSATS University Islamabad, Abbottabad campus, department of Pharmacy, Quaid-i- Azam University, Islamabad, Natural Product Chemistry labs of school of Pharmacy and Biosciences, Liverpool John Mooers University, England, UK. This PhD thesis envisages the phytochemical and biological investigation of two important indigenous species of Pakistan. The main objective behind this investigation was to authenticate the folkloric history of these species. Asparagus adscendens Roxb. (A. adscendens), is native to the Himalayas.
The findings of current study support the notion that use of different fractions on the basis of different solvent systems truly retrieves a complete phytochemical and biological profiling of plants. The current study concludes that MeOH extract and SPE fractions of roots of Asparagus adscendens and Trillium govanianum are potential source of phytochemicals provoking highly significant antioxidant capability, antimicrobial potential as well as effective against Leishmaniasis. Similarly MeOH extract and fractions of these plants are lethal to brine shrimps and depicted the oncogenic kinases inhibitor potential, which signify there cytotoxic potential. In current study, considering the significant cytotoxicity observed against four carcinoma cell and on the basis of the previously
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published data on the antitumour/anticancer potential of the genus Asparagus and Trillium as well as the presence of saponins and sapogenins (evident from LC/ESI/QTOF/MS analysis) in Asparagus adscendens and Trillium govanianum, it is reasonable to state that these plants could be exploited as a good source of cytotoxic compounds with probable anticancer potential. The present study may proceeded for further investigation to target the isolation of secondary metabolites responsible for the observed activity. The given effective MeOH extract and SPE fractions could serve as novel scaffolds in drug discovery. To our best knowledge, this is the prime outline showing significant phytochemical and biological potential of Asparagus adscendens and Trillium govanianum indigenous to Pakistan.
As is evident from the data presented in current study, this manifested the important and prime information about the composition, antioxidant, antimicrobial, antileishmanial and cytotoxic activities of the selected plants native to Pakistan.
4.4 Future Strategies The isolated compounds can be a potential source of antitumour/anticancer, so there is a need to evaluate these isolated compounds for further anti cancer assays In vivo and toxicity studies of isolated compounds could be initiated for preclinical trails. New bioactive secondary metabolites can be further isolated from these species. Several new proteins can dock to get the better understanding about binding energies and interaction pattern of isolated and identified compounds.
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Chapter 5
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