i Bioassay Guided Extraction, Isolation and Characterization of Phytoconstituents from Datura innoxia Mill. PhD thesis by HUMAIRA FATIMA CHUGHTAI Department of Pharmacy Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2018
i
Bioassay Guided Extraction, Isolation and
Characterization of Phytoconstituents from
Datura innoxia Mill.
PhD thesis
by
HUMAIRA FATIMA CHUGHTAI
Department of Pharmacy
Faculty of Biological Sciences
Quaid-i-Azam University
Islamabad, Pakistan
2018
Bioassay Guided Extraction, Isolation and
Characterization of Phytoconstituents from
Datura innoxia Mill.
Thesis submitted by
HUMAIRA FATIMA CHUGHTAI
Registration No. 03331411006
to
Department of Pharmacy
In partial fulfillment of the requirements for degree of
Doctor of Philosophy
in
Pharmacy (Pharmacognosy)
Department of Pharmacy
Faculty of Biological Sciences
Quaid-i-Azam University
Islamabad, Pakistan
2018
DEDICATED TO
MY BELOVED ABU, AMMI, HUSBAND AND
CHILDREN
Contents
No. Title Page no.
Acknowledgements i
List of tables iii
List of figures iv
List of abbreviations vi
Abstract ix
Chapter 1 Introduction
1.1 Genus Datura 2
1.2 Datura innoxia 2
1.3 Extraction, fractionation, isolation and purification 3
1.3.1 Extraction 3
1.3.2 Fractionation, isolation and purification 4
1.3.2.1 Solvent-solvent extraction 4
1.3.2.2 Chromatography 4
1.4 Phytochemical analysis 8
1.4.1 Total phenolic content 9
1.4.2 Total flavonoid content 10
1.5 Biological evaluation 10
1.5.1 Antioxidant assays 11
1.5.1.1 Free radical scavenging assay 13
1.5.1.2 Total antioxidant capacity (TAC) 13
1.5.1.3 Total reducing power (TRP) 13
1.5.2 Antimicrobial assays 13
1.5.2.1 Antileishmanial assay 13
1.5.2.2 Antibacterial assay 14
1.5.2.3 Antifungal assay 16
1.5.3 Enzyme inhibition assays 16
1.5.3.1 α-amylase inhibition assay 16
1.5.3.2 Protein kinase inhibition assay 17
1.5.4 Cytotoxicity and cancer chemopreventive assays 18
1.5.4.1 Brine shrimp lethality assay 18
1.5.4.2 Cytotoxicity against cell lines 19
Contents
No. Title Page no.
1.5.4.3 Inhibition of TNF-α activated nuclear factor-kappa B (NF-ĸB) 23
1.5.4.4 Inhibition of lipopolysaccharide (LPS)-activated nitric oxide
(NO) production in murine macrophage RAW 264.7 cells (iNOs) 23
1.6 Techniques for structure elucidation of pure compounds 25
1.6.1 Nuclear magnetic resonance (NMR) spectroscopy 25
1.6.1.1 One dimensional NMR (1D-NMR) 25
1.6.1.2 Two Dimensional NMR (2D-NMR) 26
1.6.2 Other spectroscopic methods 27
1.6.3 Crystallography 27
1.6.4 Determination of molecular weight-Mass spectrometry (MS) 28
1.7 Aims and Objectives 29
Chapter 2 Materials and methods
2.1 Materials 30
2.1.1 Chemicals and reagents 30
2.1.2 Apparatus and equipment 30
2.1.3 Cultures and cell lines 31
2.2 Methods 32
Section-1 Extraction optimization from D. innoxia 32
2.2.1 Collection and identification 32
2.2.2 Preparation of crude extract 32
2.2.3 Phytochemical analysis 33
2.2.3.1 Total phenolic content determination 33
2.2.3.2 Total flavonoid content determination 33
2.2.3.3 RP-HPLC quantitative analysis 33
2.2.4 Biological evaluation 33
2.2.4.1 Antioxidant assays 33
2.2.4.2 Antimicrobial assays 34
2.2.4.3 Enzyme inhibition assays 34
2.2.4.4 Cytotoxicity assays 35
Section-2 Preparative extraction, biological evaluation and isolation 36
2.2.5 Preparative extraction 36
Contents
No. Title Page no.
2.2.6 Fractionation by Solid Phase Extraction (SPE) 36
2.2.7 Biological evaluation of D. innoxia fractions 38
2.2.7.1 Antioxidant assays 38
2.2.7.2 Antimicrobial activity 40
2.2.7.3 Enzyme inhibition assay 40
2.2.7.4 Cytotoxicity assays 40
2.2.8 Isolation from D. innoxia fractions 41
2.2.8.1 Isolation from D. innoxia leaf fractions 43
2.2.8.2 Isolation from D. innoxia fruit fractions 43
Section-3 Biological evaluation and characterization of compounds 48
2.2.9 Biological evaluation of isolated compounds 48
2.2.9.1 Antileishmanial assay 48
2.2.9.2 Protein kinase inhibition assay 48
2.2.9.3 Cytotoxicity assays 48
2.2.9.4 Cancer chemopreventive assays 48
2.2.10 Structure elucidation of isolated compounds 49
2.2.10.1 Nuclear magnetic resonance (NMR) spectroscopy 49
2.2.10.2 X-ray crystallography (XRD) 49
Chapter 3 Results
Section-1 Extraction optimization from D. innoxia 50
3.1 Extraction yield 50
3.2 Phytochemical analysis 50
3.2.1 Total phenolic content 50
3.2.2 Total flavonoid content 51
3.2.3 RP-HPLC analysis 51
3.3 Biological evaluation 60
3.3.1 Antioxidant assays 60
3.3.1.1 %RSA 60
3.3.1.2 TAC 60
3.3.1.3 TRP 60
3.3.2 Antimicrobial assays 60
Contents
No. Title Page no.
3.3.2.1 Antileishmanial activity 60
3.3.2.2 Antibacterial activity 62
3.3.2.3 Antifungal activity 62
3.3.3 Enzyme inhibition assays 63
3.3.3.1 α-amylase inhibition assay 63
3.3.3.2 Protein kinase inhibition assay 63
3.3.4 Assays for the determination of cytotoxic potential 63
3.3.4.1 Brine shrimp lethality assay 63
3.3.4.2 Cytotoxicity against cell lines 71
3.4 Summary 71
Section-2 Preparative extraction, biological evaluation and isolation 72
3.5 Fraction yield 72
3.6 Biological evaluation 72
3.6.1 Antioxidant assays 72
3.6.1.1 %RSA 72
3.6.1.2 TAC 73
3.6.1.3 TRP 74
3.6.2 Antimicrobial assays 74
3.6.2.1 Antileishmanial assay 74
3.6.2.2 Antibacterial activity 77
3.6.2.3 Antifungal activity 77
3.6.3 Enzyme inhibition assay 77
3.6.3.1 α-amylase inhibition assay 77
3.6.3.2 Protein kinase inhibition assay 78
3.6.4 Cytotoxicity assays 78
3.6.4.1 Brine shrimp lethality assay 78
3.6.4.2 Cytotoxicity against Hep G2 cell line 78
3.7 Summary 83
Section-3 Biological evaluation and characterization of compounds 84
3.8 Biological evaluation 84
Contents
No. Title Page no.
3.8.1 Antileishmanial assay 84
3.8.2 Protein kinase inhibition assay 85
3.8.3 Cytotoxicity against cell lines 85
3.8.4 Cancer chemopreventive assays 85
3.8.4.1 Inhibition of TNF-α activated nuclear factor-kappa B (NFĸB)
assay 85
3.8.4.2 Inhibition of nitric oxide (NO) production in lipopolysaccharide
(LPS)-activated murine macrophage RAW 264.7 cells (iNOs)
assay
86
3.9 Characterization of lead compounds 87
3.9.1 Structure elucidation of CL-1 87
3.9.2 Structure elucidation of CL-3 93
3.9.3 Structure elucidation of CF-5 96
3.10 Summary 97
Chapter 4 Discussion
Section-1 Preliminary phytochemical and in vitro biological evaluation 100
4.1 Effect of extraction solvent on the extract yields 102
4.2 Phytochemical analysis 103
4.3 Biological evaluation 105
4.3.1 Antioxidant potential 105
4.3.2 Antimicrobial activities 106
4.3.3 Enzyme inhibition assays 108
4.3.3.1 α-amylase inhibitory activity 108
4.3.3.1 Protein kinase inhibition potential 109
4.3.4 Cytotoxicity determination 109
Section-2 Preparative extraction, biological evaluation and isolation 111
4.4 Preparative extraction 111
4.5 Fractionation 112
4.6 Biological evaluation of fractions 112
Section-3 Biological evaluation and characterization of compounds 115
Contents
No. Title Page no.
Research outcomes 118
Conclusions 118
Study limitations 119
Future prospects 119
References 133
Annexure A
List of publications
Appendices
Turnitin originality report
i
ACKNOWLEDGEMENTS
I humbly thank Allah Almighty, Who is most Beneficent and the most Merciful, Whose
blessings are abundant and favors are unlimited. I offer humble durood-o-salam to the
Holy Prophet Muhammad صلى الله عليه وسلم .
First and foremost, enormous gratitude is to be paid to my PhD supervisor Dr. Ihsan-
ul-Haq, Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam
University (FBS-QAU) Islamabad, Pakistan who had been unstinting in his
constructive critique, support and mentorship throughout this project. Thank you for
shaping our character, caliber and future.
I am grateful to Prof. Dr. Gul Majid, Chairman, Department of Pharmacy, FBS-QAU,
Pakistan and Prof. Dr. Muhammad Shahab, Dean, FBS-QAU, Pakistan for providing
the research facilities to accomplish this work. I am also thankful to Prof. Dr. Bushra
Mirza, Department of Biochemistry, FBS-QAU, Pakistan for her kind cooperation to
provide the facility of HPLC. I am thankful to the Higher Education Commission
(HEC) Pakistan for providing me Indigenous scholarship.
I am extremely grateful to Dr. Paul Groundwater, Professor of Medicinal Chemistry,
Faculty of Pharmacy, University of Sydney, Australia for providing facilities for NMR
spectroscopy and structure elucidation. I am also indebted to Dr. Tamara P.
Kondratyuk, Assistant Specialist and Laboratory Manager, CoP, UHH, USA for
helping in cancer chemopreventive assays. I am also thankful to Dr. Nawaz Tahir,
Professor, Department of Physics, University of Sargodha, Sargodha, Pakistan for his
kind cooperation to provide the facility for crystallography.
I would like to extend my appreciation to all my friends who have helped me in any
possible way. I wish to express my heartfelt appreciation to my friends and colleagues
specially Madiha Ahmed, Durdana Waseem, Saira Tabassum, Syeda Saniya Zahra,
Bakht Nasir, Muhammad Majid, Attarad Ali, Hira Zafar, Joham Sarfaraz Ali, Maria
Zafar, Waleed Baig, Zafar Irshad, Masooma Ali and Komal Khan.
Special thanks to Ammi and Abu. I have no words to acknowledge the sacrifices they
made and the dreams they had to let go, just to give me a shot at achieving mine. I am
highly indebted to my husband Zaheer Shaukat Khan, no matter how badly I failed, he
ii
always treated me like a winner, thanks for being so supportive. I am grateful to my
children Mustafa Yousaf Khan and Ibrahim Yousaf Khan for providing me the joy in
my soul, and the love of my life. Thanks to my sisters (Sumayya, Qudsia and Maria),
Bhabhis (Shafaq and Tayiba) and brothers (Khalid and Saad) for supporting me
throughout the whole journey.
Humaira Fatima
iii
List of Tables
No. Title Page no.
3.1 Percent extract recoveries of leaf, stem and fruit of D. innoxia. 50
3.2(a) Calibration curve parameters for the standards 52
3.2(b) RP-HPLC profiling of phenols in D. innoxia extracts 55
3.3 Antibacterial activity of D. innoxia extracts 65
3.4 Antifungal activity of D. innoxia extracts. 67
3.5 Brine shrimp cytotoxicity, THP-1, Hep G2 and protein kinase
inhibitory potential of D. innoxia extracts 69
3.6 Fractionation scheme of D. innoxia leaf and fruit. 72
3.7 Antibacterial activity of D. innoxia fractions 79
3.8 Antifungal spectrum of D. innoxia fractions 80
3.9 α-amylase inhibitory activity of D. innoxia fractions 81
3.10 Protein kinase inhibitory potential of D. innoxia fractions 81
3.11 Cytotoxicity assessment of D. innoxia fractions using brine
shrimp lethality assay 82
3.12 Hep G2 cytotoxicity of D. innoxia fractions 83
3.13 Antileishmanial potential of compounds isolated from D.
innoxia 84
3.14 Protein kinase inhibitory potential of compounds isolated from
D. innoxia 85
3.15 Cytotoxicity assessment of compounds isolated from D. innoxia
against MCF-7, LU-1, THP-1 and Hep G2 cell lines 85
3.16
Results of cancer chemopreventive assays; TNF-α activated
NFĸB inhibition and inhibition of NO production by the
compounds isolated from D. innoxia.
86
3.17 Crystal data and structure refinement for CL-1. 88
3.18 1H and 13C data of CL-1 dissolved in CDCl3 88
3.19 Crystal data and structure refinement for CL-3 98
3.20 Crystal data and structure refinement for CF-5 99
iv
List of Figures
No. Figure Page no.
1.1 Taxonomical classification and pictorial presentation of D.
innoxia 4
1.2 Mechanism of α-amylase activity 18
1.3 Conversion of MTT to formazan 22
1.4 Model for the activation of nuclear factor (NF)-κB by a variety
of inducers and for the nuclear response controlled by NF-κB 24
1.5 NO production by various mechanisms in cancer 25
2.1 Schematic presentation of preparative extraction of D. innoxia
leaf part to obtain crude extract (DCL) 37
2.2 Schematic presentation of preparative extraction process of D.
innoxia fruit part to obtain crude extract (DCF) 37
2.3 SPE scheme for the fractionation of DCL 39
2.4 SPE scheme for the fractionation of DCF 39
2.5 Schematic representation of isolation from of DFL-2 44
2.6 Schematic representation of isolation from DFL-4 46
2.7 Schematic representation of isolation from DFF-2 47
3.1(a) Chromatograms of standard phenols 52
3.1(b) Chromatograms of phenols detected in M: Methanol extracts of
D. innoxia leaf 53
3.1(c) Chromatograms of phenols detected in D: Distilled water
extracts of D. innoxia stem 53
3.1(d) Chromatograms of phenols detected in E: Ethanol extracts of D.
innoxia fruit 54
3.2(a) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia
leaf extracts 57
3.2(b) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia
stem extracts 58
3.2(c) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia
fruit extracts 59
3.3 Inhibitory effects of D. innoxia extracts on in vitro growth of L.
tropica promastigotes 61
v
List of Figures
No. Figure Page no.
3.4 α-amylase inhibition by D. innoxia extracts 64
3.5 %RSA and IC50 of D. innoxia DCF and leaf fractions 73
3.6 %RSA and IC50 of D. innoxia DCL and fruit fractions 74
3.7 TAC and TRP potential of D. innoxia DCL and leaf fractions 75
3.8 TAC and TRP potential of D. innoxia DCF and fruit fractions 75
3.9 Antileishmanial potential of D. innoxia leaf fractions 76
3.10 Antileishmanial potential of D. innoxia fruit fractions 76
3.11 13C (100 MHz, in CDCl3) spectrum of CL-1 89
3.12 Enlarged 13C spectra (100 MHz, in CDCl3) of CL-1, 0-70 ppm 90
3.13 1H spectrum (400 MHz, in CDCl3) of CL-1 91
3.14 (a) 3D structural model of CL-1 as proposed by XRD (hydrogen
atoms have been removed for clarity (b) Elucidated structure of
CL-1
92
3.15 1H spectrum (400 MHz, in CDCl3) of CL-3 94
3.16 13C (100 MHz, in CDCl3) spectrum of CL-3 95
3.17 (a) 3D structural model of CL-3 as proposed by XRD (hydrogen
atoms have been removed for clarity (b) Elucidated structure of
CL-3
96
3.18 (a) 3D structural model of CL-3 as proposed by XRD (b)
Elucidated structure of CL-3 97
vi
List of Abbreviations
%RSA Percent radical scavenging activity
13C NMR 13Carbon nuclear magnetic resonance
1D-NMR One dimensional nuclear magnetic resonance
2D-NMR Two dimensional nuclear magnetic resonance
AAE Ascorbic acid equivalent
A Acetone
AlCl3 Aluminum chloride
ALT Alanine transferase
AST Aspartate transferase
ATCC American type culture collection
API Apigenin
CC Column chromatography
CA Caffeic acid
Catec Catechin
CDK2 Cycline dependent kinase 2
CHCl3 Chloroform
CHCl3-d4 Chloroform with four deuterium atoms
COSY Correlation spectroscopy
COX Cyclooxygenase
DCL Datura crude leaf
DCF Datura crude fruit
DFF Datura fraction fruit
DFL Datura fraction leaf
DMEM Dulbecco’s modified eagle medium
DMSO-d6 Dimethyl sulfoxide with six deuterium atoms
DPPH 2,2-diphenyl-1-picrylhydrazyl
D Distilled water
ePK eukaryotic protein kinases
Eth Ethyl acetate
E Ethanol
FBS Fetal bovine serum
FC Folin ciocalteu
vii
List of Abbreviations
FCBP Fungal culture bank of Pakistan
FCC Flash column chromatography
FeCl3 Ferric chloride
GA Gallic acid
GAE Gallic acid equivalent
GC-MS Gas chromatography-Mass spectrometry
h Hour
H2O2 Hydrogen per oxide
H2SO4 Sulfuric acid
HMBC Heteronuclear multiple bond correlation
HPLC-DAD High performance liquid chromatography coupled with
diode array detector
HSQC Heteronuclear single quantum coherence
IC50 Median inhibitory concentration
Kaemp Kaempferol
LCC Liquid column chromatography
M Methanol
MeOH-d4 Methanol with four deuterium atoms
mg Milligram
MIC Minimum inhibitory concentration
MPLC Medium pressure liquid chromatography
MS Mass spectrometry
MTT 3-(4,5 dimethylthiazo-2-yl)-2,5- diphenyl tetrazolium
bromide
Myr Myrecitin
NA Not active
NaH2PO4 Monosodium dihydrogen phosphate
NB Nutrient broth
NF-κB Nuclear factor kappa B
nh n-Hexane
NO Nitric oxide
PBS Phosphate buffer saline
viii
List of Abbreviations
QE Quercetin equivalent
Quer Quercitin
Rf Relative flow
ROS Reactive oxygen species
RPK Receptor protein kinases
RP-LCC Reverse phase liquid column chromatography
RPMI Roswell park memorial institute medium
RP-TLC Reverse phase thin layer chromatography
SD Standard deviation
SDA Sabouraud dextrose agar
SPE Solid phase extraction
SRB Sulforhodamine B
STPKs Serine-threonine specific protein kinase
TAC Total antioxidant capacity
TCA Trichloroacetic acid
TFC Total flavonoid content
TLC Thin layer chromatography
TMRE Tetra methyl rhodomine ethyl ester
TNF-α Tumor necrosis factor-alpha
TPC Total phenolic content
TPCK Nα-tosyl-L-phenylalanine chloromethyl ketone
TPKs tyrosine specific protein kinases
TRAF 3 TNF receptor-associated factor
TRP Total reducing power
TSB Trypton soy broth
WHO World Health Organization
Abstract
ix
Abstract
Natural products have been used to treat human disease since the dawn of medicine.
The present study aimed to isolate and characterize bioactive lead compounds from an
underexplored medicinal folklore D. innoxia Mill. Total 12 extracts from each of the
leaf, stem and fruit part were prepared by sonication aided maceration as the extraction
technique. The extracts were subjected to a range of phytochemical and in vitro
biological assays in order to optimize most operative plant part and extraction solvent
for preparative extraction. Phytochemical analysis included standard colorimetric
assays to determine phenolic and flavonoid contents whereas, RP-HPLC was
performed to quantify specific polyphenolic compounds. MTT assay was used to
determine antipromastigote activity against L. tropica while disc diffusion assay was
used to establish antibacterial, antifungal as well as protein kinase inhibitory spectrum.
Starch-iodine chromogenic assay elucidated the α-amylase inhibitory potential
whereas, brine shrimp lethality, MTT and SRB assays were employed to determine
cytotoxic potential of the subject plant. Highest amount of gallic acid equivalent
phenolic and quercetin equivalent flavonoid contents were obtained in the distilled
water and ethyl acetate-ethanol extracts of leaf i.e. 29.91 ± 0.12 and 15.68 ± 0.18 mg/mg
dry weight (DW), respectively. RP-HPLC detected significant amounts of catechin,
caffiec acid, apigenin and rutin ranging from 0.16–5.41 µg/mg DW. Highest DPPH
radical scavenging activity (IC50 16.14 µg/ml) was recorded in the ethyl acetate-acetone
stem extract. Maximum total antioxidant capacity and reducing power potential were
obtained in the aqueous leaf and ethyl acetate stem extracts i.e. 46.98 ± 0.24 and 15.35
± 0.61 mg ascorbic acid equivalent/g DW respectively. Ethanol-chloroform leaf extract
manifested a noteworthy antileishmanial activity (IC50 3.98 ± 0.12 µg/ml). A significant
antimicrobial activity was exhibited by leaf extracts against Micrococcus luteus and n-
hexane fruit extract against Aspergillus niger (MIC 3.70 and 12.5 µg/ml, respectively).
Moderate inhibition of α-amylase enzyme activity was observed in all the three plant
parts whereas, ethyl acetate and methanol-chloroform extracts of leaf exhibited
conspicuous protein kinase inhibitory activity with 22 mm bald phenotype. Significant
cytotoxicity against brine shrimps (IC50 85.94 ± 0.16 µg/ml), THP-1 (IC50 3.49 ± 0.17
µg/ml) and Hep G2 (6.54 ± 0.10 µg/ml) cell lines was manifested by the methanol-
chloroform leaf extract, n-hexane fruit extract and chloroform leaf extract, respectively.
In view of the aforementioned results, leaf and fruit parts were selected for preparative
Abstract
x
extraction with ethyl acetate-methanol (1:1) and chloroform as their extraction solvents,
respectively. The preparative extracts of leaf (DCL) and fruit (DCF) were then
partitioned using solid phase extraction and the resulting fractions were biologically
evaluated to prospect fraction hits for lead development. In case of leaf fractions,
moderately polar and polar fractions were found to be potential candidates for the
isolation of antileishmanial compounds whereas polar fractions exhibited profound
protein kinase inhibitory and cytotoxic activities. In case of fruit fractions nonpolar
fractions were identified as potential hits for cytotoxic leads isolation. Total three
compounds (CL-1, CL-3 and CF-5) were isolated using normal phase vacuum and
medium pressure column chromatography as the isolation technique. Compound CL-3
demonstrated noteworthy antileishmanial activity (IC50 8.34 ± 1.21 µg/ml) and
cytotoxic potential against MCF-7 (IC50 4.3 ± 0.93 µg/ml), LU-1 (6.9 ± 1.3 µg/ml) and
PC3 (0.01 ± 0.001 µg/ml) cancer cell lines. In protein kinase inhibition assay,
maximum bald growth inhibition zone of 11 ± 2.21 mm was formed around the CL-
1 loaded disc whereas, CF-5 demonstrated remarkable cancer chemopreventive
activity through inhibition of NFκB and NO production with IC50 1.1 ± 0.9 and 3.3 ±
0.6 µg/ml, respectively. Crystallography and NMR spectroscopy characterized the
structure of CL-1, CL-3 and CF-5 as β-sitosterol, isowithametelin and (4a, 4b, 6b, 8b,
10b, 14a) 7, 10 dimethyl dinoroleanan-12 en-3-one (new terpenoid), respectively. In
principle, the results of the current study endorses D. innoxia as a substantial source of
bioactive lead compounds.
Ch.1: Introduction
1
1. Introduction
Natural products from animals, plants and microorganisms have been utilized to treat
human ailments since the dawn of medicine. It has long been accepted that structures
derived from natural products possess the properties of biochemical specificity, high
chemical diversity and other molecular characteristics that make them promising as
lead structures for drug development, and this differentiates them from synthetic and
combinatorial compound libraries (Newman and Cragg, 2016). For improved
efficiency, numerous selection standards may be employed to decrease the number of
plant species to be evaluated including traditional use (ethnopharmacology),
chemotaxonomy or plant ecological observations (Tabassum et al., 2017). Plants have
been benefitting mankind since their origin. The active molecules derived from the
plants have served as the basis of new drug discovery. Plants not only have fulfilled the
nutritive requirements of human beings but also the curative one. They are a source of
plentiful molecules possessing biological activities. They synthesis a wide variety of
biochemicals called as secondary metabolites which are not indispensable for the
reproduction and growth of plants but when administered in the human body, they exert
multiple pharmacological effects (Rout et al., 2009). Nature is the best pharmacy and
has the potential to treat various ailments of human beings. Plant based medicines are
still the mainstay for the treatment of many complex ailments like inflammatory
disorders, neoplasia, diabetes and oxidative-stress induced disorders.
Drug development from plants in its contemporary understanding relies on pure
chemical moieties for which traditional knowledge has always played a fundamental
role. Morphine, taxol, vincristine, artemisinin, triptlide, celastrol, and capsaicin are
among prime compounds that demonstrated the potential of turning traditional remedies
into modern drugs (Heinrich, 2010). Thus ethnopharmacological approach provides
ideal prospects to limit the gigantic multiplicity of possible leads to more valuable hits.
One such ethnomedicinally important genus is Datura.
Empirical or systematic screening of plant extracts and pure compounds to explore
novel leads plays a focal role in drug development process. Recent advancements in
drug discovery research from medicinal plants encompass a multidimensional approach
combining phytochemical, botanical, biological and molecular techniques. Thus,
natural product drug discovery requires a persistent progress in the pace of screening,
Ch.1: Introduction
2
purification and structure determination processes in order to be competitive with the
other drug discovery methods.
1.1 Genus Datura
All over the world, Datura (family: Solanaceae) is well recognised as a medicinal and
hallucinogenic genus and comprises mostly of wild weeds. The word “Datura” has been
derived from Sanskrit word “Dhutra” meaning divine inebriation and is named so
because of its healing properties. Various species of Datura such as D. wrightii, D.
innoxia, D. metel and D. stramonium are well recognized and extensively utilized for
the toxic and medicinal attributes owing to the presence of more than 30 alkaloids as
their secondary metabolites (Vermillion et al., 2011). Some of the biological activities
of various Datura species as reported in different studies include antiviral, anticancer,
antiquorum sensing, antibacterial, antifungal, antiperspirant, immunomodulatory,
antiulcer, hypoglycemic, wound healing, and antistress activities (Maheshwari et al.,
2012). Keeping in view, the ethnomedicinal and pharmacological importance of the
subject genus; D. innoxia, one of its underexplored folklore was carefully chosen in the
current study for the investigation of therapeutic potential.
1.2 Datura innoxia
Downy Thorn Apple, Angel's-trumpet, Indian Apple or Thorn Apple with the scientific
name of Datura innoxia Mill. (Solanaceae), is a shrub that grows in United States,
China, Caribbean Islands, Mexico and Asia. It is amongst the popular medicinal plants
which has been traditionally used (Maheshwari et al., 2013). In Pakistan, it is common
to roadsides and weedy places and is locally named as Dhatura. It typically reaches a
height of 0.6-1.5 m. Soft short grayish hairs surrounds the leaves and stem rendering
the plant a grayish look. The flowers are trumpet-shaped, white and are 12–19 cm long
while the fruit spiny capsule which is egg shaped (5 cm in diameter). The fruit splits
open when it is fully ripped.
D. innoxia surmounts a distinct stature in Ayurveda as all of its parts namely roots,
flowers, leaves, stems, seeds and fruits have been used in the treatment of insanity,
rabies, leprosy, etc. Nevertheless, the higher doses of the extract may result in delirium,
acute poisoning and may cause death. The bioactive principles in various plant parts of
D. innoxia include hyoscyamine, withanolides, tropanes, atropine and scopolamine
(Vermillion et al., 2011).
Ch.1: Introduction
3
In a study, D. innoxia extracts were tested for antibacterial potential by preparing crude
aqueous and organic extracts. It was found that methanol extract of leaves was most
potent against almost all of the tested strains (Kaushik and Goyal, 2008). Eftekhar et
al. (2005) also described the antimicrobial activity of methanolic extract from its aerial
parts. A comparative analysis of scavenging capacity of seed extracts of several Datura
species towards the stable DPPH radical established that D. innoxia extracts possess
the highest antioxidant potential (Ramadan et al., 2007). A withanolide named dinoxin
B was isolated from of D. innoxia methanol extract of leaves which exhibited a sub
micromolar IC50 values and demonstrated substantial cytotoxicity against the tested
human cancer cell lines (Vermillion et al., 2011). Arulvasu et al. (2010) described that
methanol extract of D. innoxia leaves impedes the human larynx (Hep-2) cell
proliferation and human colon adenocarcinoma (HCT 15) cancer cell lines through
induction of apoptosis.
1.3 Extraction, Fractionation, Isolation and Purification
1.3.1 Extraction
Extraction being a significant step in the expedition of phytochemical screening for the
discovery of bioactive components from plants takes in to account the separation of
therapeutically active components of living tissues from the inactive components by
employing various solvents. The ultimate goal of extraction optimization of crude
extracts is to attain the desired bioactive constituents and to exclude the inert
components using a range of selective solvents termed as menstruum. The resulting
extract obtained in crude form may be ready for use as a medicinal cocktail or it may
be further partitioned to isolate distinct bioactive moieties. Numerous conventional
extraction techniques that are most often used include; percolation, infusion,
maceration as well as phytonic extraction and hot continuous extraction (soxhlet),
decoction, aqueous alcoholic extraction by fermentation are used. In the current study,
maceration accompanied with occasional use of high frequency and high intensity
sound waves (ultrasonication) was used as the extraction technique to recover desirable
compounds from D. innoxia. Ultrasonication aided maceration was employed because
chemical and physical characteristics of the materials are transformed due to the
interaction and dissemination of ultrasound waves disrupting the cell walls, thereby,
augmenting solvent’s mass transport across the plant cells (Dhanani et al., 2013).
Ch.1: Introduction
4
Figure 1.1 Taxonomical classification and pictorial presentation of D. innoxia.
1.3.2 Fractionation, isolation and purification
The process by which components are extracted on the basis of variances in their
physical and chemical characteristics is termed as fractionation. Most common
fractionation techniques include chromatographic methods and solvent-solvent
extraction.
Solvent-solvent partitioning/extraction also acknowledged as liquid–liquid
partitioning/extraction is the transfer of solute(s) from a feed solution to another
immiscible liquid (solvent). The solute(s) enriched solvent is termed as the extract
whereas, the feed solution which gets exhausted from solute(s) is known as raffinate.
The components are separated due to variable relative solubility in the immiscible
liquids.
1.3.2.2 Chromatography
Chromatography is a collective terminology used to describe laboratory procedures for
the separation of various mixtures that are run in a liquid termed as mobile phase, which
carries it over a backing material, the stationary phase. The different speeds with which
1.3.2.1 Solvent-solvent extraction
Ch.1: Introduction
5
various components of the mixture travel results in their separation. Thus, the
phenomenon of separation is due to the selective partitioning between the stationary
and mobile phases. Chromatography may be categorized as analytical or preparative.
Preparative chromatography is performed to isolate individual mixture components for
further advanced use and is consequently a procedure used for purification. Analytical
chromatography is performed generally with lesser quantities of material and is used
for assessing the comparative proportions of components in a mixture (Li and Vederas,
2009).
Among the several chemical methods of investigation, chromatography has always
played a substantial role and has therefore, been introduced to all the modern
pharmacopoeias. Because of the numerous advantages of chromatographic procedures
such as application for qualitative as well as quantitative analysis and their specificity,
they execute a fundamental part in the analysis of medicinal plants. The
chromatographic techniques exploited in the present study are as follows.
a) Solid phase extraction (SPE)
For the natural product purification, extraction methods taking benefit of the adsorption
of undesirable impurities or analytes on to a solid phase have got a central part. Most
of the applications make use of SPE utilizing a wide variation of stationary phases
having varied chemical properties such as reverse phase material, silica gel, ion
exchange resins in plastic or glass columns are employed and most commonly vacuum
is applied for a forced flow technique. Various strategies may be used in SPE such as
removal of undesired impurities such as chlorophyll. Elution of the substances under
study are obtained step by step through application of an increasing elution power
gradient (Li et al., 2014). In the present study, crude extract acquired after preparative
extraction was additionally fractionated by utilizing SPE technique. Extract loaded
silica packed in glass column was eluted with organic solvents of escalating polarity
and the fractions thus obtained comprising compounds of similar polarities were
grouped together for further purification.
b) Thin layer chromatography (TLC)
TLC is a rapid, easy, economical and the most extensively utilized technique for the
investigation and purification of small organic products of synthetic and natural origin.
TLC based separation is affected by applying an extract or a mixture in the form of a
Ch.1: Introduction
6
line or a spot on to a sorbent material containing a backing plate. Afterwards, the plate
is placed in a developing chamber of glass with adequate solvent for wetting the lower
end of the sorbent plate taking care of not to wet that part of the plate where the spots
were marked i.e. the origin. TLC chromatogram is developed as the solvent front moves
up as a result of capillary action and the process is named as development. Rf value is
a factor that quantifies the relative movement of a component through a particular
sorbent and solvent system. In order to achieve pure compounds, effective detection or
visualization is critical at both the analytical and preparative types of TLC. Among the
several methods, UV light detection and use of reagent spray are the two most widely
used visualization procedures. Detection by UV light involves absorption of exposed
light by compounds at 254 or 366 nm wavelength that would give the impression of
dark or glowing spots contrary to a light background when UV light is exposed over
the plate. In case of visualization by spraying with reagent, a color reaction between the
reagent which is sprayed as fine mist onto the developed plate and the compound results
in the detection of compounds of the mixture. Some of the universal spray reagents
react with most of the natural product classes e.g. phosphomolybdic acid, vanillin-
sulfuric acid and ammonium molybdate (VI) sprays. Analytical TLC is run for
“tracking” natural products by performing analytical TLC after other separation
processes, such as HPLC or column chromatography and every time TLC separation
should be performed by using more than one solvent system, as seemingly pure spots
may contain numerous compounds with identical Rf values (Gibbons, 2012).
c) Column chromatography (CC)
Column chromatography comprises of a packed bed of some material e.g. alumina or
silica through which solvent is moved at atmospheric, low or medium pressure. The
subsequent separation may be liquid-solid (adsorption etc.) or liquid-liquid
(partitioning). Analyte mixture is then dissolved in appropriate solvent and is packed in
the column which is termed as wet packing, or otherwise it is adsorbed on a coarse
silica gel called dry packing. The mobile phase then carries the sample mixture through
the column, resulting in the separation of various components. Stepwise or gradient
elution is usually performed and the fractions are collected in accordance with the
separation desired, with the eluting products typically monitored by TLC. CC offers
benefits such as no need of any expensive equipment and the method could be scaled
Ch.1: Introduction
7
up to handle varied sample sizes ranging quantities in grams to kilograms. Depending
on the polarity of mobile and stationary phases, CC can be categorized in as under.
Normal phase liquid column chromatography (NP-CC)
NP-CC employs use of a relatively polar stationary phase such as silica gel as compared
to the mobile phase. In gradient NP-CC, the mobile phase is changed in escalating
polarity order i.e. initiating the procedure from mobile phase which is least polar and
completing it with the mobile phase which is most polar.
Reverse phase liquid column chromatography (RP-CC)
A less polar stationeary phase including octylsilane and octadecylsilane are used in RP-
CC, while, a more polar mobile phase such as acetonitrile, methanol and water are
employed. Based on the pressure applied to force the mobile phase across the stationary
bed and stationary phase efficiency, column chromatography may be sub-categorized
as:
Vacuum liquid chromatography (VLC)
Vacuum liquid chromatography (VLC), incorporates vacuum instead of pressure is
applied to enhance the flow rate of mobile and thus fasten up the process of
fractionation. Lower end of the column is attached with vacuum of suitable power to
increase the mobile phase flow rate. Simplicity in use and high sample capacity makes
VLC a popular fractionation technique of crude extracts. Co-TLC monitoring of the
eluted fractions is usually performed to analyze their composition.
Flash chromatography (FC)
Similar to VLC, FC is mostly utilized for the fast fractionation of extracts that are crude
or semi-purified fractions. Compressed air or nitrogen at a pressure of 1-2 bars is
applied (Li and Vederas, 2009).
Medium pressure liquid chromatography (MPLC)
MPLC can be employed as a supplement to flash chromatography. Usually a piston
pump with variable flow rate is used to generate a pressure that ranges from 5-20 bars.
Fractions of crude plant extracts prepared by FC could be further purified by using
MPLC to give pure compounds. Approximately 50 g of partially purified fraction can
be easily chromatographed in a single run. Gradient elution is used and the polarity of
Ch.1: Introduction
8
mobile phase is slowly increased to ensure better separation. One of the examples of
natural product isolation employing
High performance liquid chromatography
HPLC being a multipurpose, robust and widely used chromatographic procedure for
natural product isolation and is extensively used in analytical chemistry and
phytochemical analysis for the identification, quantification and purification of
individual components of the mixture. Presently, among the various analytical
procedures, HPLC has gained popularity as the chief option for fingerprinting analysis
for the quality regulation of herbal extracts. NPs are often isolated following the
assessment of a crude extract in a bioassay in order to fully illustrate its properties. The
HPLC’s resolving power is appropriate to the fast handling and assessment of
multicomponent samples such as herbal extracts at analytical as well as preparative
scale. A number of authors describe the use of HPLC for classification and
quantification of plant secondary metabolites, mainly phenolic compounds, flavonoids,
steroids and alkaloids (Boligon and Athayde, 2014). Celighini et al. (2001) established
an HPLC process for the estimation of coumarin in the hydroalcoholic extracts of guaco
(Mikania glomerata) and described that HPLC method proved to be sensitive and
reproducible. Fatima et al. (2015) quantified various polyphenols in the crude extracts
of leaf, stem and fruit parts of D. innoxia using RP-HPLC as the chromatographic
technique.
1.4 Phytochemical analysis
Phytochemical analysis refers to the qualitative or quantitative assessment of the
medicinally active secondary metabolites from plant matrices. The phytochemical
screening is an effective tool for the extrapolation of chemical profile of plants for their
possible therapeutic implications. Medicinal plants have been known for centuries for
their numerous phytochemicals like tannins, glycosides, alkaloids, flavonoids,
polyphenols and many other. Nature is considered as an abundant pharmaceutical store
existing on this planet owing to their ability to produce various secondary metabolites
with a broad spectrum of bioactivities. Employing various phytochemical analysis
techniques many plant based chemicals have been characterized. Chemotaxonomy of
therapeutically important plants can be made with the help of phytochemical analysis
procedures. Phytochemicals are non-nutritive plant constituents which play a
Ch.1: Introduction
9
prophylactic and defensive job against health complications both in animals and plants.
Thousands of chemicals proven to be active against pests and diseases have been
discovered (Fatima et al., 2015). In qualitative screening, various phytoconstituents
such as reducing sugars, steroids, alkaloids, flavonoids, saponins, tannins, glycosides,
anthraquinones, amino acids, triterpenoids etc. are tested for their presence or absence.
Quantitaive phytochemical analysis involve total content determination of various
phytoconstituents and include assays such as total alkaloid, total phenolic content, total
flavonoid, total saponin and total tannins contents determination etc. In the current
analysis, polyphenolic and flavonoid contents were appraised by using colorimetric
assays and therapeutically significant polyphenolic compounds were quantified using
HPLC method against various external standards.
1.4.1 Total phenolic content
Phenolic compounds are ubiquitous and most abundant plant secondary metabolites
containing with hydroxyl groups attached to one or more aromatic rings. They can be
categorized as phenolic acids, flavonoids such as anthocyanins, flavonols, flavanols,
flavanones, isoflavones, and flavones), tannins (hydrolysable tannins; gallotannins and
ellagitannins and condensed tannins), stilbenes and lignins. Polyphenols are a part of
plant secondary metabolites capable of playing effective part in free radicals
neutralization as well as satiating singlet oxygen. These compounds have antioxidant
capability and are able to reduce the oxidative stress (Djeridane et al., 2006). Difference
between phenol and phenolics is that the former is an aromatic organic compound while
latter one is secondary metabolite of natural origin (plants, animals and
microorganisms). They are responsible for variations in appearance, taste, smell, and
oxidative stability of the plants (Stevanato et al., 2014). Polyphenols are the most
abundant and ubiquitous molecules occurring in plants. Their molecular weight ranges
from simple phenol moiety to complex polymerized molecules having molecular
weight greater than 30,000 Da (Bravo, 1998). Mode of action of phenolics involves the
inactivation of free radicals by donating hydrogen specie which inhibits lipid
peroxidation reaction and as a result prevention against oxidative degradation takes
place (Vauzour et al., 2010). Functional moieties e.g. ketonic, hydroxyl and methoxy
may be considered responsible for antioxidant potential of phenolics (Pandey and Rizvi,
2009). Phenolics are also a weapon of plant defense systems as they have proven to be
active against varied pathogenic microorganisms. Toxicity to microorganisms rely on
Ch.1: Introduction
10
the location and site of hydroxyl groups in these compounds (Parmar et al., 2008).
Various Scientific reports recommend that long term intake of foods abundant in
polyphenolic compounds offer fortification against cardiovascular diseases, cancers,
diabetes, neurodegenerative diseases and osteoporosis (Dai and Mumper, 2010) which
justifies the current interest in determination of phenolic profile of the subject plant.
Folin-ciocalteu reagent colorimetric assay is the most frequently employed technique
for the estimation of phenolic content (Haq et al., 2013).
1.4.2 Total flavonoid content
Flavonoids, the abundant plant metabolites and in addition to their phenomenal
antioxidant characteristics, flavonoids are also reported to be associated with several
therapeutic outcomes such as antimicrobial, vasodilatory, anti-inflammatory, anti-
ischemic and anticancer. Furthermore, they can also deter platelet aggregation, lipid
peroxidation and are able to improve elevated capillary fragility and permeability (Khan
et al., 2015). Some of the mechanisms by which flavonoids prevent free radical
mediated injury are reactive oxygen species (ROS) scavenging, antioxidant enzymes
activation, metal chelating activity, lessening of α-tocopheryl radicals, oxidase enzyme
inhibition, alleviation of nitric oxide (NO) mediated oxidative stress. The current
interest in flavonoid determination was stimulated by health promoting properties
owing to their remarkable antioxidant potential (Tabbassum et al., 2017).
1.5 Biological evaluation
Bioassay or biological standardization is a form of scientific experimentation that
involves the use of a living plant or animal (in vivo) or tissue or cell (in vitro) so that
biological potential of a test substance could be established. Imperative components of
bioassays comprise of a stimulus (test sample, drug candidate or agrochemical etc.), a
subject (subcellular organelles, cells, tissues, animals etc.) and a response (response of
the subject to various dosages of stimulus) (Vlietinck, 1999). Besides the versatile
requirements of validity, precision, easiness, reproducibility, lack of obscurity and
rational cost, the bioassays should also be extremely selective in order to minimize the
quantity of possible leads for secondary testing, extremely accurate to exclude false
positives and particularly sensitive to identify even minute concentrations of active
compound. Besides giving indication in research of novel active constituents, bioassays
also provide evidence about particular make up of complex bioactive mixtures.
Ch.1: Introduction
11
Accordingly, these procedures serve as premium biomarkers in diagnosis of multiple
health conditions and industrial scale manufacturing of drugs, particularly when
chemical standardization tools are inaccessible.
Besides giving indication in research of novel active constituents, bioassays also
provide evidence about particular make up of complex bioactive mixtures.
Accordingly, these procedures serve as premium biomarkers in diagnosis of multiple
health conditions and industrial scale manufacturing of drugs, particularly when
chemical standardization tools are inaccessible.
Two types of screening in bioassays are:
• Bench top (primary) bioassay screening: these are used to evaluate biological
potential of large number of samples. They are preferred because of their cost
effectiveness, reproducibility and production of fast results.
• High throughput (secondary) screening: used for further testing of highly active
samples selected from primary screening. Thorough investigation of bioactive
constituents on numerous model systems and to choose a chemical entity for clinical
trials. They have low capacity, expensive and give late results.
Bioassay procedure should have the following fundamental features; sensitivity and
reproducibility, simple and easy, rapid, high sample throughput and cost effective. The
level of complexity of any bioassay must be defined in accordance with the available
laboratory facilities and skill of personnel (Hadacek and Greger, 2000). The biological
potential of D. innoxia was elucidated in the present study by employing following
bioassays.
1.5.1 Antioxidant assays
ROS i.e. Reactive oxygen species and RNS i.e. reactive nitrogen species is an umbrella
term which covers alkoxyl (RO•) and oxygen free radicals. These free radicals are a
portion of normal biochemical processes but if get uncontrolled these free radicals are
involved in various pathogenesis of multiple diseases like cardiovascular, diabetes and
cancer.
It is assumed that ROS accepts electron and induce DNA mutations, lipid peroxidation,
oxidative inactivation of enzymes and protein damage. As a result, progression of
cancer, muscular dystrophy, liver injuries and cardiovascular diseases takes place
Ch.1: Introduction
12
(Apak et al., 2016). Free radicals are extremely reactive species and are capable of
damaging cellular proteins, membrane lipids and in the nucleus DNA (Cadet et al.,
1999).
Humans have protective mechanisms for combating these free radicals but the
imbalance between antioxidant defense mechanisms and free radical generation causes
defined as oxidative stress. Natural sources serve as a storehouse of different kinds of
antioxidants which have been separated and used continually without any harmful
effect (Moon and Shibamoto, 2009). According to an estimation, oxygen free radicals
(•OH) and other ROS attack each human cell 10,000 times per day. This oxidative stress
modifies genetic makeup and serves as the basis of development of mutagenesis
(Charles, 2012).
No single antioxidant assay is capable of evaluating complete antioxidant potential of
a sample. Variety of bioassays are executed to determine antioxidant potential. So,
different types of antioxidant assays were performed, each type signifies different
mechanism e.g. disintegration of peroxide, scavenging of free radical and prevention of
chain initiation.
Antioxidants are molecules that substantially avert or delay the oxidation of oxidizable
substrates when present at lower concentrations than the substrate (Halliwell, 2007).
Oxidative stress has been recognized as the mainstay in the progress and advancement
of many anomalies including cardiovascular and neurodegenerative disorders.
Augmentation with exogenous antioxidants or enhancing endogenous antioxidant
defenses of the body is a propitious way to combat the detrimental effects of reactive
oxygen species (ROS) induced oxidative injury. Plants have long been utilized as a
source of exogenous (i.e., dietary) antioxidants owing to their innate capacity to
biosynthesize an array of non-enzymatic antioxidants that are agile of mitigating ROS
induced oxidative stress. There are numerous in vitro bioassays designed to measure
the antioxidant potential of plants; nevertheless, each of them has its own limitations
with respect to applicability. Consequently, multiple assay approaches are generally
adopted to confer antioxidant stature of plants by assessing their role as hydrogen
donors, reducing agents, singlet oxygen scavengers or metal ion chelators (Kasote et
al., 2015). The antioxidant assays used to assess the antioxidant activity of D. innoxia
in the current investigation are described as follows.
Ch.1: Introduction
13
1.5.1.1 Free radical scavenging assay
A precise, cost effective and easily accessible method to observe such changes is DPPH
assay. 2,2-diphenyl-1-picrylhydrazyl known as DPPH is a stable and cell permeable
free radical. Free radical extinguishing capability of extracts or samples is assessed by
DPPH reagent based assay. A change in absorbance values is detected because
antioxidants in test samples cause production of hydrazine which renders DPPH reagent
to decolorize (Khan et al., 2015, Fatima et al., 2015).
1.5.1.2 Total antioxidant capacity (TAC)
Antioxidant potential of sample under investigation is also determined by
phosphomolybdenum based assay which involves the production of green
phosphomolybdate complex in acidic medium. Test samples which show higher
absorbance at 645 nm have higher ability to reduce Mo (VI) to Mo (V) and thus higher
antioxidant potential (Prieto et al., 1999). This is an important technique as it quantifies
the total antioxidant capableness of test samples and results are expressed as ascorbic
acid equivalent (Prieto et al., 1999).
1.5.1.3 Total reducing power (TRP)
Total reducing power is a simple colorimetric test for the quantitative estimation of
antioxidant capacity of samples. The elementary principle of TRP is the reduction of a
ferric tripyridyltriazine (Fe+3-TPTZ) complex to the ferrous form (Fe+2) at low pH
which generates an intense blue color that can be quantified by measuring the change
in absorption at 593 nm (Gulcin, 2015). There is no requirement of any specialized
equipment, or astringent control of reaction conditions or timing and could be easily
performed using automated, semi-automated, and manual versions (Benzie and Strain,
1999).
1.5.2 Antimicrobial assays
1.5.2.1 Antileishmanial assay
Leishmaniasis, a group of tropical infections, is caused by various protozoan parasite
species that belongs to the Leishmania genus. It is widespread in Africa, Europe,
Americas and Asia. It is a source of significant morbidity and mortality and thus
constitutes a serious public health problem. Every year, the leishmanial parasite effects
thousands and debilitates millions of people around the globe; about 2 million new
Ch.1: Introduction
14
cases are reported annually. Leishmaniasis is endemic in 98 countries of which 82%
are low income countries. Leishmania is an obligatory intracellular parasite of
monocytes and macrophages that shows a digenetic life cycle that alternates between
two life stages: flagellated promastigotes, which develops in the midgut of the insect
vector, and amastigotes, that proliferate in the host macrophages. Nearly 21 Leishmania
species are causative agents of the disease that manifests itself as a range of clinical
signs, including inflammation of oropharyngeal mucosa, cutaneous lesions and visceral
infection. The current antileishmanial agents most commonly used are mostly
antimonial drugs which are toxic, and tested vaccines have presented relatively less
protection under field conditions. Pentavalent antimonials such as meglumine
antimoniate and sodium stibogluconate are the foremost choice for the treatment of
leishmaniasis. Regardless of their widespread clinical use for several decades, their
mechanism of action still remains unclear. Other therapies available for leishmaniasis
include amphotericin B and pentamidine, but their use has been restricted because of
their high toxicity and cost. Therefore, the adverse effects of the treatment regimens
advocates prompt attention directed towards the discovery and development of novel
and less toxic chemotherapeutic agents. These days, significant consideration has been
specified to plant derived secondary compounds, in an effort to pursuit for new
antileishmanials. In the present investigation different plant parts of D. innoxia were
tested for their antipromastigote activity by using standard MTT protocol (Tasdemir et
al., 2006).
1.5.2.2 Antibacterial assay
Fungi, bacteria and algae are the pathogens responsible for most of the human diseases
that were prehistorically treated by using various folk remedies of plant origin. Despite
of the quick growth in the field of medicine, the undiscriminating usage of antibiotics
against countless infectious ailments, the emergence of multidrug resistance in
microbes and global emergence and dispersion of more deadly infections necessitates
progress of progressive scientific approaches and added research for the establishment
of innovative anti-infective agents. Curative potential of plants against infectious
diseases has been confirmed by ethnobotanical data where it is reported that 40-80% of
antimicrobials have been acquired from medicinal plants (Khan et al., 2015). A number
of evaluation techniques are presently utilized for the analysis of plant metabolites as a
source of novel antimicrobial agents. The most frequently utilized antimicrobial
Ch.1: Introduction
15
screening methods include; dilution and bioautographic diffusion methods. The
diffusion techniques are qualitative as they only perform a qualitative analysis of the
test samples and give information on the absence or presence of antimicrobial
substances. Dilution methods, on the other hand, are the quantitative assessments of
antimicrobial activity. Disc diffusion and broth microdilution methods as described
below were employed in the present study to analyze the antimicrobial potential of D.
innoxia.
Disc diffusion technique relies on the development of zones of growth inhibition around
sample infused discs. The degree of development is proportional to the activity of the
sample against the pre-seeded fungal and bacterial lawns. Sensitivity or susceptibility
of microorganisms to the analyte is analyzed in accordance with the diameter of zone
of growth inhibition which in turn is related with the minimal inhibitory concentration
(MIC). The diameter of growth inhibition zone depends on factors such as specie under
test and the potency of the test sample. Lower dose of antimicrobial agent with greater
zones of inhibition indicates superior susceptibility of microorganisms to the test
antimicrobial agent. The disc diffusion technique is comparatively economical, easy
and less time consuming which allows concurrent analysis of enormous number of
antimicrobials quite easily (Kaushik and Goyal, 2008).
Dilution methods are most frequently employed to assess the MIC of antimicrobials
that either destroy (bactericidal) or deter the growth (bacteriostatic) of microbes.
Various types of dilution tests are broth macrodilution, broth microdilution and agar
dilution methods. The final volume of test procedure outlines whether the method is
designated macrodilution i.e. when a total assay volume of 2 ml is utilized, or
microdilution, if performed in microtiter plates utilizing ≤ 500 µl/well. In both agar and
the broth dilution techniques, the lowest concentration of test sample that inhibits
visible growth of test microorganism under specific conditions is defined as MIC.
Dilution techniques often have certain advantages over diffusion methods such as;
improved sensitivity for lesser extract volumes, quantitative investigation and
capability to differentiate bactericidal and bacteriostatic effects of test samples. It is a
cost-effective assay procedure that can be employed on a large number of microbes and
it generates equitably reproducible results. Dilution procedures are well-thought-out as
reference protocol for in vitro susceptibility testing and are also utilized to assess the
efficiency of various other antimicrobial susceptibility testing (AST) techniques. Broth
Ch.1: Introduction
16
microdilution assay used in the present study, also known as microwell or microtiter
plate method is an alteration of the standard broth dilution assay in which the tests are
performed by using small volumes of test samples and permits a large number of
bacteria to be assessed quite rapidly (Nasir et al., 2015).
1.5.2.3 Antifungal assay
Infections caused by human pathogenic fungi are increasing day by day due to an
increase in the incidence of AIDS, cancer, and immunocompromised patients. The
upsurge in the consumption of antifungal agents has ensued in the development of
resistant strains to the currently available treatments. Therefore, novel classes of
antifungal compounds need to be discovered for the effective management of fungal
infections. Plants being a plentiful repository of bioactive phytochemicals such as
terpenoids, alkaloids, tannins, flavonoids, saponins and other compounds are stated to
possess antifungal characteristics (Arif et al., 2009). Consequently, the research
antifungal metabolites derived from natural sources has increased due to their
significance in drug discovery (Lavault et al., 2005). Disc diffusion method as
described above was used in the current analysis to evaluate the antifungal spectrum of
D. innoxia.
1.5.3 Enzyme inhibition assays
1.5.3.1 α-amylase inhibition assay
Diabetes mellitus (DM) a chronic metabolic disorder is characterized by hyperglycemia
with disturbances in carbohydrate, protein and fat metabolism being a consequence of
relative or absolute absence of insulin secretion. The incidence of diabetic anomalies is
on the upsurge globally and is expected to rise to 300 million by 2025 (Sudha et al.,
2011). The rising prevalence, chronic course and associated complications makes
diabetes as one of the major problems to human health. The contemporary regimes for
diabetes management are; endogenous insulin secretion stimulation, augmentation of
insulin action at the target sites, oral hypoglycemics (sulfonylureas and biguanides) and
inhibition or delay of breakdown of dietary starch by the glycosidases such as α-
glucosidase and α-amylase.
α-amylase is an important pancreatic enzyme in the digestive tract which catalyses the
preliminary stage of starch hydrolysis and converts it to a mixture of smaller
oligosaccharides and oligoglucans. The oligoglucans are then acted then on by α-
Ch.1: Introduction
17
glucosidases that cleave them to glucose sub units which enters the blood stream after
absorption. The breakdown of dietary starch ensues quickly resulting in elevated levels
of blood glucose level known as post prandial hyperglycemia (PPHG). It has been well
established that the action of pancreatic α-amylase in the small intestine is
proportionally linked with an increase in PPHG, the regulation of which is thus an
imperative aspect in type 2 diabetes management. Therefore, delay or inhibition of
starch hydrolysis via inhibition of α-amylase enzyme plays a vital role in the diabetes
control. Several inhibitors that are currently in clinical use are miglitol and acarbose.
These are capable of inhibiting glycosidases such as α-amylase and α-glucosidase
whereas others such as voglibose deter α-glucosidase activity only. Nevertheless, many
of these currently available hypoglycemics have their own limits, are nonspecific, cause
severe adverse effects and fail to address most of the diabetic complications. The chief
side effects associated with these inhibitors are gastrointestinal including abdominal
discomfort, diarrhea, bloating and flatulence. Herbal preparations are receiving more
prominence in the management of diabetes since they have fewer adverse effects and
have less cost in comparison to the contemporary synthetic hypoglycaemic drugs. A
broad range of plant principles from various classes of secondary metabolites such as
gums, glycosides, galactomannan polysaccharides, alkaloids, hypoglycans,
peptidoglycans, guanidine, terpenoids, steroids and glycopeptides have proved
therapeutic proficiency against hyperglycaemia (Sudha et al., 2011). Standard starch-
iodine colorimetric assay using α-amylase enzyme was performed in the present study
to evaluate the antidiabetic prospective of D. innoxia.
1.5.3.2 Protein kinase inhibition assay
In the current years, there has been a significant rush for the development of inhibitors
of protein kinases from natural products especially plants. Protein phosphorylation by
protein kinases at serine/threonine and tyrosine residues is a significant governing
mechanism in many biological processes i.e. cell proliferation, apoptosis, metabolism
and cell differentiation.
Around 518 protein kinases have been discovered in humans so far and are categorized
in 20 families based on the amino acid sequence (Ventura and Nebreda, 2006).
Research has proved that dysregulation of protein kinases hinders the cellular processes
relevant during disorders like neoplasia, therefore they hold pro-oncogenic potential
(Jung et al., 2009).
Ch.1: Introduction
18
Figure 1.2 Mechanism of α-amylase activity (Tormo et al., 2004).
Several molecules which inhibit the protein kinases are under investigation for their
potential role in cancer therapeutics. In Streptomyces hyphae formation, protein kinase
plays a critical role, it is especially sensitive to inhibition by the protein kinase
inhibitors. This aspect has been taken advantage of in our current investigation to check
the likelihood of our test extracts for protein kinase inhibitory effects (Yao et al., 2011).
A benefit of the whole cell Streptomycete test is that it effectively recognizes cytotoxic
activity of the samples being analysed. This simple assay permits the identification of
signal transduction inhibitors for a variety of applications including anti-infective,
antitumor agents and several of the inhibitors of mycobacteria (Barbara et al., 2002).
Imitinib (Gleevec, Novartis) is one of the first small molecule tyrosine kinase inhibitors
that has been successfully progressed to the pharmaceutical market. It has affectedly
improved the prognosis of chronic myeloid leukaemia sufferers.
1.5.4 Cytotoxicity and cancer chemopreventive assays
1.5.4.1 Brine shrimp lethality assay
Brine shrimps (Artemia salina) also known as sea monkeys are marine invertebrates.
Brine shrimp lethality assay is employed to evaluate a broad spectrum of biological
activities considering that pharmacology is toxicology at high doses. Positive
Ch.1: Introduction
19
correlation might be evident between cytotoxicity to human cells and fatality to nauplii
of brine shrimps (Qureshi et al., 2014). The basis of this assay is the ability of test
samples to kill the laboratory cultured larvae of brine shrimp. Safety profile of plant
extract can be inferred by results of this test moreover trends in biological activity can
also be illustrated. Substances which are considered toxic may act medicinally
advantageous in living systems when taken at lower doses used this assay for the first
time to determine cytotoxicity profile of various active constituents (Ajoy and Padma,
2013).
It is considered as an alternate approach to cell line bioassays. Researchers are using
this assay model for the last thirty years to evaluate if a plant is generally toxic or not.
It is advised to use fresh nauplii for toxicity evaluation which can be freshly harvested
using brine shrimp eggs (Carballo et al., 2002).
Non-compulsion of aseptic handling makes it simple to perform. Other advantages
include cost effectiveness and quick results. Afore mentioned plus points make this
assay a preliminary technique for other activities e.g. antitumor, antifungal,
antimalarial, antibacterial and insecticidal assays. Previously published data reports
have proposed a good relationship between the cytotoxic activity in the brine shrimp
assay and the cytotoxicity against some tumor cell lines (Anderson et al., 1991). In a
study, Phyllanthus engleri was evaluated to determine its cytotoxic profile using brine
shrimp lethality assay and an LC50 0.47 μg/mL was obtained (Moshi et al., 2004).
Englerin A, a selective anticancer compound has been recently isolated from the P.
engleri that has shown substantial cytotoxicity against kidney cancer cells (Ratnayake
et al., 2009). It gives a corroborative indication of the prospective of the brine shrimp
lethality assay for the prediction of anticancer potential of plant extracts. Brine shrimp
assay is typically carried out to draw interpretations from the safety profile of plant
extracts and it also describe trends of their pharmacological activities (Karchesy et al.,
2016).
1.5.4.2 Cytotoxicity against cell lines
Cancer, is a complex disease involving a plethora of changes in cell metabolism and
behaviour which results in excessive cell proliferation, escape from surveillance by
immune system and invasion to distant tissues to form metastases. Cancer remains one
of the foremost causes of morbidity and mortality universally. Amongst the non-
Ch.1: Introduction
20
communicable diseases, it is the main reason of death, after cardiovascular disease and
is the cause of one in eight deaths worldwide, more than tuberculosis, malaria and AIDS
altogether (Mathers and Loncar, 2006). Generally, the figure of cancer associated
mortality is predicted to rise from 7.1 million in 2002 to 11.5 million in 2030 (Sener
and Grey, 2005). Carcinogen is a substance, radiation or an agent which is directly
involved in causing cancer and has an ability to impair genomic material or disrupt
cellular metabolic processes. Oncogenesis or carcinogenesis or tumorigenesis is the
development cancerous tissue whereby normal cells are transformed into cancer cells.
Carcinogenesis is characterized by physiologic alterations at cellular, epigenetic and
genetic levels culminating in an atypical cell division and in some cancers forming a
malignant mass. Some of the most common causes of cancer deaths include tobacco
use (25-30% of deaths), obesity and diet (30-35%), radiations (including ionizing and
non-ionizing, up to 10%), infection (15-20%), sedentary lifestyle, stress, environmental
contaminants and hereditary predisposition to cancer (5-10%). It has been estimated
that in the developing countries approximately 20% of cancers are caused by infections
such as hepatitis B, hepatitis C, and human papillomavirus (Sener and Grey, 2005).
Chemoprevention is the employment of natural, synthetic or biological agents that are
capable to postpone, inverse, or deter tumour progression. Chemotherapy that makes
use of one or more anticancer drugs as part of treatment regimen is most commonly
used for cancer treatment. As cancer cells are devoid of several regulatory functions
existent in normal cells so they divide continuously when normal cells do not; the
strategic feature that makes cancer cells vulnerable to chemotherapeutic agents.
Nevertheless, chemotherapeutic drugs have their own intrinsic limitations such as
treatment failure and prolonged toxicity. For instance, 5-fluorouracil, a commonly used
anticancer agent causes cardiotoxicity and myelotoxicity. Doxorubicin, another
extensively employed chemotherapeutic agent is known to cause renal toxicity, cardiac
toxicity and myelotoxicity. Similarly, bleomycin a well-recognised anticancer drug,
causes cutaneous and pulmonary toxicity. Cyclophosphamide, a drug to treat many
malignant conditions, has been shown to cause bladder toxicity, immunosuppression,
haemorrhagic cystitis, alopecia and cardiotoxicity at higher doses (Desai et al., 2008).
One of the paramount clinical challenges is the therapeutic control of cancer and
nowadays naturopathy is being widely searched to have integrated method for cancer
treatment. The contemporary chemotherapeutic drugs in the market today using plant
derived products include four classes of anticancer agents; the bisindole or vinca
Ch.1: Introduction
21
alkaloids, the taxanes, the epipodophyllotoxins and the camptothecin derivatives. Vinca
alkaloids and their derivatives, isolated from Catharanthus roseus (Madagascar
periwinkle) are extensively employed in various combination therapies for the
management of leukemias, lymphomas, advanced testicular cancer, lung and breast
cancers. Taxanes (docetaxel and paclitaxel) previously extracted from the bark of Taxus
brevifolia and camptothecin derivatives purified from Camptotheca acuminate are
employed for the management of several forms of cancers. Podophyllotoxin was
originally isolated from the root of Pododphyllu peltatum and Podophyllum emodi.
Teniposide and etoposide are Podophyllotoxin derivatives and are widely used for the
management of lymphomas, testicular and bronchial cancers (Shukla and Mehta, 2015).
Plants still have great prospective to deliver novel anticancer drugs and contains a
plethora of bioactive phytoconstituents with chemopreventive as well as
chemotherapeutic efficacy. Analysis of the basic pharmacokinetic mechanisms through
which bioactive phytochemicals induce their antineoplastic effect reveal that a
multimode panel of molecular targets is involved including protein kinases (MAPK,
PKA, PKC, and TYK2), apoptotic proteins (bax and caspases), antiapoptotic proteins
(TRAF1, bcl2 and survivin), transcription factors (Nrf2, Ap1, p53 and NF-κB), growth
factors (EGF, TNF, FGF, and PDGF), cell cycle proteins (Cyclin D, CDK1, CDK2, p21
and p27) and cell adhesion molecules (VCAM and ICAM-1). Interference with
different steps in cell signalling pathways has also been associated as possible
anticancer mechanism of phytochemicals (Aggarwal and Shishodia, 2006).
Dysregulated cell death is the hall mark of cancer and inflection of this particular
response of cell has demonstrated itself as an operative anticancer modality.
Impairment of cellular apoptosis has been frequently allied with conditions of
hyperproliferation such as cancer and autoimmune diseases; therefore, cytotoxicity and
in vitro whole cell viability assays that quantify phenomena related to the death of cells
are extensively used for anticancer drug discovery. Some of the chemopreventive and
cytotoxic assays employed in the current study are as follows.
a) MTT assay
Cell-based assays are frequently employed for evaluating a library of compounds to
check whether the test samples have substantial effects on proliferation of cell or
evaluate direct cytotoxic effect that ultimately proceed to cellular death. Furthermore,
as dead cells stop all functions of cell, a decline in metabolic activity is also a marker
Ch.1: Introduction
22
of cell death in a population. There are several assays that quantify particular aspects
of cellular metabolism and are widely employed to gauge viability, including the
standard MTT protocol that has been utilized in the present study to assess the cytotoxic
potential of various extracts, fractions and compounds from D. innoxia against THP-1
human leukemia cell line. Living cells that are actively metabolising convert MTT to
formazan which is a purple coloured product and gives maximum absorbance at 570
nm. When the cells become dead, they are no moreable to convert MTT into purple
formazan, consequently development of a formazan sediments offers as an appropriate
and expedient marker of only the living cells. The formazan generated as end product
from the MTT tetrazolium dye remain there as an insoluble precipitate within and
accumulates near the cell surface as well as in the culture medium. This insoluble
formazan must be solubilized before measuring absorbance. A number of procedures
are employed for solubilizing the formazan precipitates, colour stability, preventing
evaporation and to decrease intervention by phenol red.
(Kepp et al., 2011).
MTT Formazan
Figure 1.3 Conversion of MTT to formazan.
b) Sulphorhodamine B (SRB) assay
The principle of sulforhodamine B (SRB) test relies on the capability of the protein dye
SRB to bind electrostatically and pH dependent on basic amino acid residues of cellular
proteins that are prefixed by trichloroacetic acid (TCA) (Vichai and Kirtikara, 2006).
The SRB assay has a colorimetric end point and is indefinitely stable and
nondestructive. These expedient merits make it a sensitive and appropriate test to
evaluate drug induced cytotoxicity. Furthermore, SRB method is economical and rapid.
In comparison with other cell viability assays for cytotoxicity assessment such as
tetrazolium assays or the clonogenic assay, SRB assay gave similarly when data were
Ch.1: Introduction
23
restricted to the inhibitory 50% concentration (IC50). Since the endpoint measurement
is not time limits, SRB procedure retains practical advantages over the tetrazolium
assays. Once automated using a microplate reader and microplate washer, it is
appropriate for high throughput screening assays (Voigt, 2005). SRB colorimetric assay
was used in the present study to assess the cytotoxic potential of crude extracts,
fractions and compounds of D. innoxia against cancer cell lines.
1.5.4.3 Inhibition of TNF-α activated nuclear factor-kappa B (NF-κB)
There are various types of in vitro bioassays for the identification of potential cancer
chemopreventive botanicals such as aromatase inhibition, inhibition of TNF-α activated
nuclear factor kappa-B (NFκB), inhibition of lipopolysaccharide (LPS)-activated nitric
oxide (NO) production in murine macrophage RAW 264.7 cells (iNOs assay),
interaction with retinoid X receptor responsive elements (RXRE), induction of quinone
reductase 1 (QR1) etc. A brief description of the chemopreventive assays employed in
the present study are as under.
Widespread exploration over the last few years has revealed that NF-κB is an important
transcription factor and is involved in the regulation of gene expression related to cell
survival, adhesion growth, differentiation and inflammation and is therefore, involved
in tumorigenesis. NF-κB could be induced and it is also expressed ubiquitously. It also
controls the gene expression involved in angiogenesis and invasion of cancer cells
embracing cell adhesion molecules, iNOS cyclooxygenase 2, inflammatory chemokines
and cytokines. Subsequently, NF-κB inhibition is prospected to inhibit the expression
of these genes and accordingly, deter tumour metastasis.
The step by step progression of neoplasia starting from initiation and promotion is
trailed by the progression stage and ultimately culminates in metastasis leading to
uncontrolled spread all over the body. Nevertheless, the initiation and promotion phases
are imperative, recent studies now reveal inflammation to be a thoughtful section of
tumor progression. Several types of neoplasms initiate from areas of obstinate irritation,
inflammation and infection. Several researches acclaim that the tumor
microenvironment being bounded mainly by inflammatory cells, is an important part of
the neoplastic process that promotes survival, multiplication, and migration of cancer
cells (Cheenpracha et al., 2010).
Ch.1: Introduction
24
Adopted with permission from Elsevier license terms and conditions under license no.
4081810327707 (Orlowski and Baldwin Jr, 2002).
A defensive mechanism of microcirculation in host against various injuries resulting
from irradiation, high temperature, physical force, irritants and commonly
communicable infectious agents is inflammation. However continuous dysregulated
inflammatory conditions may lead to several pathophysiological disorders including
hepatitis, esophagitis, gastritis, atherosclerosis and cancer (Kondratyuk et al., 2012). A
number of inflammatory mediators are released by macrophages during chronic course
of inflammation including interferons, cytokines, chemokines, colony stimulating
factors, proteases, eicosanoids, growth factors, lysozymes and nitric oxide (NO).
Amongst these mediators, NO is exceptionally produced from L-arginine endogenously
by one of proinflammatory enzymes; inducible nitric oxide synthase (iNOS) and
subsequently results in various diseases including psoriasis, asthma, arthritis, colitis,
multiple sclerosis, transplant rejection of septic shock and tumor development. NO can
easily diffuse into the surrounding tissues affecting targets at various locations via
covalent bonding, causing direct chemical alterations (Janakiram and Rao, 2012). Thus,
the discovery of novel iNOS and NO production inhibitors will open a novel avenue in
chemoprevenion and anticancer therapy.
Ch.1: Introduction
25
Figure 1.5 NO production by various mechanisms in cancer. Adopted with permission
from Nature publishing group license no. 4081810060239.
1.6 Techniques for structure elucidation of pure compounds
1.6.1 Nuclear magnetic resonance (NMR) spectroscopy
NMR procedures are normally employed by chemists to investigate molecules having
simple chemical structure by usng one dimensional spectroscopy (1D-NMR), while two
dimensional techniques (2D-NMR) typically elucidate structure of comparatively more
complex molecules.
1.6.1.1 One dimensional NMR (1D-NMR)
a) Proton NMR (1H-NMR)
Proton NMR refers to the plot of signals generated as a result of absorption of radio
frequency waves in the course of an NMR experiment. Information about the number
Ch.1: Introduction
26
of protons in a molecule is determined by area under the plots, whereas the electronic
and chemical environment of the protons is revealed by the position of signals (the
chemical shift). The number of vicinal or geminal protons is determined by the splitting
pattern of protons (Lambert et al., 2013).
b) Carbon NMR (13C-NMR)
Conforming to proton NMR, 13C NMR is a plot of signals originated from the variations
in the type of carbons as a function of chemical shifts. Several procedures have been
developed to record the 1D 13C-NMR in such a way that carbons of different types e.g.
primary, secondary, tertiary and quaternary could be differentially identified using the
1D 13C-NMR plot. The range of values for the chemical shifts are dissimilar for the 1H
NMR (generally 0-10) and 13C (generally 0-230) NMR (Lambert et al., 2013).
1.6.1.2 Two Dimensional NMR (2D-NMR)
a) 1H, 1H-COSY (Correlated spectroscopy)
1H, 1H-COSY is one of the most advantageous spectroscopic techniques. It delivers
data about the connectivity of various groups within a molecule (Lambert et al., 2013).
b) Nuclear overhauser enhancement spectroscopy (NOESY)
2D NOESY is a homonuclear association through coupling of dipoles; dipolar coupling
may be because of chemical exchange or NOE. It is amongst the expedient methods as
it permits correlation between nuclei through space and allows the designation of
relative configurations of various substituents at chiral centers (Kessler et al., 1988).
c) HMQC (Heteronuclear multiple quantum correlation)
The HMQC testing delivers the association among various protons and the hetero nuclei
connected to them by means of scalar coupling of heteronuclei. The elementary
principle underlying this testing is the echo differences that are exploited to exclude
proton signals of only those protons that do not couple to the hetero nuclei. From this
experimentation vital data about the chemical shifts as well as number of methane,
methyl and methylene and groups might be deduced (Kessler et al., 1988).
d) HMBC (Heteronuclear Multiple Bond Correlation)
HMBC experiment in complement with 1H, 1H-COSY allows the interpretation of
skeleton of the compound under investigation (Kessler et al., 1988).
Ch.1: Introduction
27
1.6.2 Other spectroscopic methods
Other spectroscopic techniques most often employed for structure elucidation includes
the infrared (IR) spectroscopy and the ultraviolet (UV) spectroscopy. IR spectroscopy
delivers information about the functional groups, whereas the UV spectroscopy
provides information regarding the occurrence of unsaturated sites in a structure. The
aforementioned techniques are becoming not as much significant in structure
illustration analysis of products of natural origin owing to the supremacy of data
developed from the experiments based on NMR spectroscopy that also require much
less sample quantities (Lambert et al., 2013).
1.6.3 Crystallography
X-ray crystallography refers to a microscopic procedure, the details of which can be
simplified by comparing to a microscope i.e. substance of microscopic dimensions
interacts with the EMR resulting in elastic scattering of a small segment of incoming
light. The subsequent scattered EMR moves through an optical system of lenses which
recollects the scattered light generating an enlarged image of the object under
investigation. Crystallography is amongst the most fundamental approaches for the
three-dimensional structure determination of molecular assemblies or molecules at the
level of atomic resolution, principally of natural products. Contemporary
crystallography can be categorized further into two main sub disciplines; small
molecules crystallography dealing with molecular masses only up to few thousand
Daltons and the crystallography of macromolecules handling with molecular masses of
> 5 kDa. Though these sub groups rely on similar physical phenomenon i.e. diffraction
of X-rays by crystals however they vary distinctly depending upon the technology
requisite for each phase of structure determination, and noticeably vary in terms of
effort and time required to assess a structure. NMR and crystallography being the most
operative structure elucidation techniques are largely complementary, each having its
own specific merits and weaknesses. Accordingly, the applications of NMR
spectroscopy are still limited owing to the molecular sizes under investigation. Such
limitations are not that much severe for crystallography. On the other hand,
crystallographers solely rely on the crystals availability in contrast to the NMR
spectroscopists that merely require adequately concentrated sample solution for their
investigations. It is therefore, obvious that for biological crystallography, the
Ch.1: Introduction
28
crystallization is the critical limiting step in the crystallographic structure interpretation
(Wagner and Kratky, 2015).
1.6.4 Determination of molecular weight
Mass spectrometry is an investigative procedure in which ionization of sample is
followed by the sorting of these ions on the basis of charge to mass ratio. In general MS
measures the various masses within a sample. The resultant spectrum is plotted between
charge to mass ratio and ion signal and are used to find the isotopic or elemental name
of a sample, the masses of molecules and particles and to characterize other molecular
structures such as peptides and several chemical compounds (Lambert et al., 2013).
Ch.1: Introduction
29
1.7 Aims and objectives
The aims and objectives of the current study are:
1. Polarity guided optimization of extraction efficiency of D. innoxia in terms of
bioactivity in order to identify most operative solvent system and plant part for
preparative extraction.
2. To pursue with bioactivity guided fractionation and preparative isolation of D.
innoxia in order to get the purified compounds and determination of their
structure by employing modern characterization techniques.
3. Comparative evaluation of the biological potential of purified compounds from
D. innoxia.
Ch.2: Material and Methods
30
2. Materials and Methods
2.1 Materials
2.1.1 Chemicals and reagents
Dimethyl sulfoxide (DMSO), n-hexane, chloroform, acetone, ethyl acetate, methanol
and ethanol were purchased from Sigma (Sigma-Aldrich, Germany). Folin–Ciocalteu
reagent and phosphate buffer were acquired from (Riedel-de Haen, Germany). Gallic
acid, quercetin, potassium acetate, aluminium chloride, 2,2-diphenyl-1-picryhydrazyl
(DPPH), ascorbic acid, sulfuric acid, ammonium molybdate, potassium ferricyanide,
trichloroacetic acid (TCA), ferric chloride, trypton soy broth (TSB), nutrient agar and
sea salt were purchased from Sigma (Sigma Aldrich, USA). Tween-20 (Merck-
Schuchardt, USA). Sabouraud dextrose agar (Oxoid, England). Medium 199 and heat
inactivated fetal bovine serum (Biowest, South America). Brine shrimp (Artemia
salina) eggs (Ocean star Int, USA). Dried instant yeast (Fermipan BDH, England).
RPMI-1640 culture media (Gibco BRL, Life Technologies, Inc). Dulbecco’s Modified
Eagle Medium (DMEM), DMEM/F1 supplemented with L-glutamine and 2.438 g/l
sodium bicarbonate (gibco® by life technologies), 3-(4,5-Dimethyl thiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) powder, tumour necrosis factor-α (TNF-α,
Calbiochem catalog number 654205), phosphate saline buffer (PBS), 1X reporter lysis
buffer (Promega catalog number E3971), lipopolysaccharide, Griess reagent, SRB
(sulphorhodamine B), acetic acid, Nα-tosyl-L-phenylalanine chloromethyl ketone
(TPCK) were purchased from Sigma (Sigma-Aldrich, Germany). Pre-coated silica gel
60 F254 TLC plates, normal phase silica gel 60 (70-230 mesh) and silica gel 60 (230-
400 mesh) and chromatography columns were purchased from Merck, Germany.
Medium ISP4 (prepared in lab). Standard antibacterials (roxithromycin and cefixime),
standard antifungals (clotrimazole and amphotericin B), doxorubicin, 5- florouracil,
vincristine and surfactin were purchased from Sigma (Sigma Aldrich, USA).
2.1.2 Apparatus and equipment
Erlenmeyer flask, muslin cloth, whatman filter paper, beaker, funnel, tripod stand, petri
plates, micropipette (Sartorius, France), Vernier calliper (Tailin, Japan), pasteur pipette,
bi-compartment perforated tray, sterile transparent 96 well plate (SPL life science,
Korea), sonicator (Sweepzone technology, USA), incubator (Memmert, Germany),
microplate reader (Elx 800, Biotek, USA), PDA spectrophotometer (8354 Agilent
Ch.2: Material and Methods
31
Technologies, Germany), HPLC-DAD (1200 series, Agilent Technologies, Germany),
rotary evaporator (Buchi, Switzerland), centrifuge (B. Bran, Germany), compound light
microscope (Irmeco, Germany), neubaeur chamber (Marien, Germany) and 5% CO2
incubator (Sanyo MCO-17AIC, Japan). Refrigerator -80°C, Sterile white walled 96
well plates, sterile transparent 96 well plates, Forma series II water jacketed CO2
incubator, LUMIstar galaxy luminometer (BMG Labtechnologies, Durham, NC),
gyratory shaker. 1D and 2D NMR spectroscopic data were recorded using a Varion
Gemini 2000, 400 MR with a SMS autosampler (Palo Alto, California, USA). XRD
analysis was carried out using STOE-IPDS II fitted with low temperature unit of a
Bruker and Kappa APEXII CCD diffractometer having Mo-Kα radiation (λ = 0.71073
Å) and graphite-monochromator at room temperature. Crystal structure was refined by
SHELXL97 (Sheldrick, 2008) and WinGX (Farrugia, 1999) soft wares.
2.1.3 Cultures and cell lines
Fungal strains
Fungal strains employed in biological assays were Aspergillus niger (FCBP-0198),
Aspergillus fumigatus (FCBP-66), Aspergillus flavus (FCBP 0064), Mucor species
(FCBP 0300) and Fusarium solani (FCBP-0291).
Bacterial strains
The gram positive bacterial strains included were Staphylococcus aureus (ATCC-6538)
and Bacillus subtilis (ATCC-6633) while gram negative strains used were Escherichia
coli (ATCC-25922), Klebsiella pneumoniae (ATCC-1705) and Pseudomonas
aeruginosa (ATCC-15442), Streptomyces 85E.
Protozoal strain
Leishmania tropica kwh 23
Invertebrates Artemia salina eggs
Cell lines
Human leukaemia (THP-1) cell line (ATCC TIB-202), Hep G2 cancer cell line (RBRC-
RCB1648), Hormone responsive breast cancer cell line MCF-7(ATCC number HTB-
22), human lung carcinoma cells LU-1 (established from department of Surgical
Oncology University of Illinois, College of Medicine at Chicago), Human prostate
Ch.2: Material and Methods
32
adenocarcinoma (PC-3) cell line (ATCC® CRL-1435), 293/NFĸB-Luc HEK cell
(Panomics catalog number RC0014), Murine macrophage RAW 264.7 cells.
2.2 Methods
Section-1: Extraction optimization from D. innoxia Mill.
Extraction is the primary critical step in novel drug finding process from plants.
Extraction efficiency in terms of extract yield and biological potential of herbal extracts
can be augmented by changing polarity of extraction solvent. Solvent and sample
composition are the most important parameters that effects the extraction efficiency
under constant conditions of temperature and time of extraction (Xu and Chang, 2007).
Therefore, in this section, a wide range of extraction solvent polarity was employed as
a variable to demonstrate, correlate and optimize its effects on extraction efficiency and
bioactivity of various plant parts of D. innoxia.
2.2.1 Collection and identification
D. innoxia was collected in September 2013 from Quaid-i-Azam University Islamabad.
Credentials of the field gathered plant was authenticated as D. innoxia by Prof. Dr.
Rizwana A. Qureshi, Department of Plant Sciences, Faculty of Biological Sciences,
Quaid-i-Azam University Islamabad, Pakistan. Dried voucher specimen was submitted
in the Herbarium of medicinal plants, Quaid-i-Azam University Islamabad under
herbarium number PHM-487.
2.2.2 Preparation of crude extract
D. innoxia was washed carefully under running water to remove contamination and was
dried under shade with active aeriation at ambient temperature for about 3 weeks. After
drying the stem, fruit and leaves were ground separately to fine powder using electric
knife mill and stored in air-tight containers. The powdered plant parts were extracted
by adopting the following procedure.
Maceration
The indivisual ground plant parts were extracted by using sonication aided maceration.
Analytical grade solvents i.e. n-hexane (Nh), chloroform (C), acetone (A), ethyl
acetate-acetone (EthA), ethyl acetate (Eth), ethanol-chloroform (EC), methanol-
chloroform (MC), ethyl acetate-ethanol (EthE), methanol-ethyl acetate (MEth),
methanol (M), ethanol (E) and distilled water (D) were used. A ratio of 1:1 was used
Ch.2: Material and Methods
33
for preparing extraction solvent having various solvent combinations as described
above. Accurately weighed (40 g) plant material was macerated with 120 ml solvent in
250 ml Erlenmeyer flask and were macerated for twenty four hours at room temperature
followed by sonication (ultrasonic bath, room temperature, 30 min). The marc was
extracted twice using same procedure and the extracts were combined which were then
filtered through muslin cloth followed by filtration through whatman No. 1 filter paper.
The extracts were concentrated with vacuum evaporation in rotary evaporator, dried in
vacuum oven at 45oC and were stored at -20°C till further use.
The dried extracts were weighed to calculate the percent recovery of crude extract by
the following formula.
%Extract recovery = (d / b)*100
d = Weight of extract obtained after drying.
b = Weight of ground plant material.
2.2.3 Phytochemical analysis
2.2.3.1 Total phenolic content determination
The total phenolic contents were estimated according to slightly modified procedure
as described previously using Folin–Ciocalteu reagent (Fatima et al., 2015) and is
attached as annexure A.
2.2.3.2 Total flavonoid content determination
For total flavonoids content determination, aluminum chloride colorimetric method was
employed as described by Haq et al. (2012).
2.2.3.3 RP-HPLC quantitative analysis
RP-HPLC analysis was performed in accordance with the protocol previously described
and is attached as annexure A (Fatima et al., 2015).
2.2.4 Biological evaluation
2.2.4.1 Antioxidant assays
a) Radical scavenging activity-DPPH assay
The antioxidant potential of the crude extracts was gauged by standard DPPH free
radical assay and is attached as annexure A (Fatima et al., 2015).
Ch.2: Material and Methods
34
b) Total antioxidant capacity estimation
Phosphomolybdenum dependent antioxidant capacity of the extracts was appraised by
using standard protocol as described previously and is attached as annexure A (Fatima
et al., 2015).
c) Total reducing power assay
The reducing power of sample extracts was appraised in accordance with the method
described previously (Fatima et al., 2015) and is attached as annexure A.
2.2.4.2 Antimicrobial assays
a) Antileishmanial assay
Antileishmanial assay was performed in accordance with the previously described
protocol (Fatima et al., 2015).
b) Antibacterial assay
Antibacterial potential of each extract was determined by previously documented disc
diffusion protocol by Khan et al. (2015).
MIC determination
Microbroth dilution method previously described by Fatima et al. (2015) was used for
determination of minimum inhibitory concentration (MIC) of test samples with ≥ 10
mm zone of inhibition and is attached annexure A.
c) Antifungal assay
The sensitivity of test samples against fungal strains was evaluated through previously
described agar disc diffusion protocol by Fatima et al. (2015) attached as annexure A.
2.2.4.3 Enzyme inhibition assays
a) α-amylase inhibition assay
A slightly modified alpha amylase inhibition assay was used to assess antidiabetic
activity of test samples (Ahmed et al., 2017).
Ch.2: Material and Methods
35
b) Protein kinase inhibition assay
The protein kinase inhibition test was carried out in triplicate by recording hyphae
development in isolates of Streptomyces 85E strain and is attached as annexure A
(Fatima et al., 2015).
2.2.4.4 Cytotoxicity assays
a) Brine shrimp lethality assay
A twenty four hour lethality assay was executed using 96 well plate against brine
shrimp (A. salina) larvae according to the previously described protocol with minor
modifications and is attached as annexure A (Fatima et al., 2015).
b) Cytotoxicity against cell lines
MTT assay
The cytotoxicity assessment of D. innoxia extracts in vitro against THP-1 human
leukaemia cell line (ATCC # TIB-202) was carried out by adopting standard method as
described earlier attached as annexure A(Fatima et al., 2015).
SRB assay
To evaluate the cytotoxic effects of D. innoxia leaf, stem and fruit extracts on Hep G2
cells, SRB colorimetric cell viability assay was performed as described previously
(Tabassum et al., 2017).
Section-2: Preparative extraction, biological evaluation and isolation
In spite of the extensive advances in various extraction and separation methods,
isolation of natural products remains a tough challenge. The classical natural product
isolation techniques starts with plant identification, collection and effective extraction.
Maceration, the classical extraction technique used for preparative scale extraction
from plant material is most often employed and is carried out by soaking it in solvent
at room temperature with occasional stirring providing the benefit of temperate
extraction conditions. The extracts thus obtained are concentrated by removing excess
of solvent. Solid phase extraction (SPE), initially designed as a purification process
prior to chromatographic separation, is currently acknowledged as an efficient
fractionation technique of crude plant extracts. Moreover, determination of bioactivity
Ch.2: Material and Methods
36
should be preceded after each step of purification process so that interference with
inactive accompanying compounds could be excluded.
2.2.5 Preparative extraction
Based on the preliminary extraction optimization results described in detail in section-
1 of this chapter, leaf and fruit parts were selected as lead plant parts. For preparative
extraction of leaves, mixture of ethyl acetate-methanol (1:1) was selected as extraction
solvent. The collected plant parts were sorted to remove any deteriorated or diseased
parts and adulterations and shade dried in well ventilated room for 6 weeks until all of
the water content was removed and became crispy. The dried parts were comminute to
fine powder by using commercial miller. To prepare crude leaf extract, a total of 5 Kg
of grounded leaves were macerated with 15 L of ethyl acetate-methanol (1:1) for three
days with occasional stirring. Afterwards, the soaked material was strained and filtered
by using muslin cloth followed by filtration using whatman No. 1. filter paper and the
process was repeated two more times. The menstruum were combined and dried using
rotary evaporator at 45°C to obtain 678 g of final leaf crude extract (DCL) which was
stored at -20°C till further use (Fig 2.1).
For the preparative extraction of D. innoxia fruit part, 10 Kg of plant material was
soaked in 30 litres of chloroform for 3 days with occasional shaking which was then
strained and filtered using muslin cloth trailed by filtration using whatman No. 1 filter
paper. The marc was extracted three times using the same procedure and the menstruum
were combined and concentrated at 45°C using rotary evaporator. The final fruit crude
extract designated as DCF (900 g) was stored at -20°C till further use (Fig 2.2).
2.2.6 Fractionation by Solid Phase Extraction (SPE)
The technique of solid phase extraction (SPE) was used to fractionate DCL and DCF.
For the fractionation of DCL (678 g), it was dissolved in ethyl acetate and was adsorbed
on 2100 g of silica gel 60 (70-230 mesh) using 1:3 as sample to silica ratio. The
adsorbed silica was dried in vacuum oven at 45°C and was loaded in a glass column. A
protective layer of 2 cm was also added over the top of the loaded sample. The column
loaded with DCL was eluted with 2 L of each elution solvent with a gradient change in
mobile phase; starting from n-hexane with ethyl acetate (1:0 to 100% ethyl acetate)
followed by ethyl acetate with methanol (5:1 to 100% methanol) as shown in figure 2.3.
Ch.2: Material and Methods
37
Figure 2.1 Schematic presentation of preparative extraction of D. innoxia leaf part to
obtain preparative extract (DCL).
Figure 2.2 Schematic presentation of preparative extraction process of D. innoxia fruit
part to obtain preparative extract (DCF).
D. innoxia leaves
(5 Kg)
Macerated with Ethyl acetate- methanol (1:1)
15 L × 3 days
Menstruum fitered Process repeated twice
Menstruum combined, filtered and evaporated
Extract dried
(DCL)
(678 g)
Dried D. innoxia fruit (10 Kg)
Macerated with Chloroform
30 L × 3 days
Menstruum fitered Process repeated
twice
Menstruum combined, filtered
and evaporated
Extract dried
(DCF)
(900 g)
Ch.2: Material and Methods
38
Each fraction (2 L) was collected and dried in rotary evaporator at 35°C to obtain a total
of 8 fractions (DFL-1 to DFL-8). In case of D. innoxia crude extract of fruit (900 g), it
was dissolved in a mixture of n-hexane and ethyl acetate (2:1) which was then adsorbed
on 2700 g of silica gel 60 (70-230 mesh) using 1:3 as sample to silica ratio. The
adsorbed silica was dried in vacuum oven at 45°C and was loaded in a glass column. A
protective layer of 2 cm was also added over the top of the loaded sample. The column
loaded with DCF was eluted with 2 L of each elution solvent with a gradient change in
mobile phase; starting from n-hexane with chloroform (1:0 to 100% chloroform)
followed by chloroform with ethyl acetate (1:1 to 100% ethyl acetate) and ethyl acetate
with methanol (1:1 to 100% methanol) as shown in figure 2.4. Each fraction of 2 L was
collected and dried in rotary evaporator at 35°C and a total of 7 fractions (DFF-1 to
DFF-7) were obtained.
2.2.7 Biological evaluation of D. innoxia fractions
The fractions obtained from DCL and DCF were subjected to the following
phytochemical and biological investigations in order to extrapolate their therapeutic
perspective.
2.2.7.1 Antioxidant assays
a) Radical scavenging assay (RSA)
The antioxidant potential of various D. innoxia leaf and fruit fractions was gauged by
monitoring their capacity to quench the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical as described in detail in chapter 2, section-1, page no. 40 (Khan et al., 2015).
b) Total antioxidant capacity (TAC) estimation
Phosphomolybdenum based total antioxidant capacity (TAC) was determined by using
standard protocol (Fatima et al., 2015).
c) Total reducing power (TRP) estimation
The reductive power of various D. innoxia leaf and fruit fractions was determined
calorimetrically by using standard protocol (Fatima et al., 2015).
Ch.2: Material and Methods
39
Figure 2.3 SPE scheme for the fractionation of DCL.
Figure 2.4 SPE scheme for the fractionation of DCF.
DCL (678 g) dissolved in ethyl acetate
Adsorbed on 2100 g of silica gel 60 (70 - 230
mesh)
Dried in vacuum oven at 45°C
Dry column packing
Elution with
a) n-hexane and ethyl acetate (1:0 to 0:1)
b) Ethyl acetate and methanol (5:1 to 0:1)
2 L of each elution solvent dried to yeild 8
fractions
(DFL-1 to DFL-8)
Dried fruit extract (900 g) dissolved in a mixture
of n-hexane and ethyl acetate
Adsorbed on 1500 g of silica gel 60 (70 - 230
mesh)
Dried in vacuum oven at 45°C
Dry column packing
Elution with a) n-hexane and chloroform (1:0 to 0:10 b)
Chloroform and ethyl acetate (1:1 to 0:1) c) Ethyl acetate and methanol (1:1 to 0:1)
2 L of each elution solvent dried to yeild 8
fractions
(DFF-1 to DFF-7)
Ch.2: Material and Methods
40
2.2.7.2 Antimicrobial activity
a) Antileishmanial assay
The in vitro activity of leaf and fruit fractions of D. innoxia against L. tropica axenic
promastigotes was determined using standard MTT assay (Khan et al., 2015).
b) Antibacterial assay
Table 3.6 shows the antibacterial activity in terms of zone of inhibition (ZOI) of various
leaf and fruit fractions of D. innoxia. Standard disc diffusion assay (Fatima et al., 2015).
c) Antifungal assay
The antifungal potential of test extracts was evaluated as triplicate analysis by agar disc
diffusion method (Khan et al., 2015).
2.2.7.3 Enzyme inhibition assay
a) α-amylase inhibition assay
Standard chromogenic starch-iodine assay was performed to assess the α-amylase
inhibitory potential of test fractions in accordance with the protocol (Xiao et al., 2006).
b) Protein kinase inhibition assay
Standard protocol was followed for the assessment of protein kinase inhibitory
potential of samples (Fatima et al., 2015).
2.2.7.4 Cytotoxicity assays
a) Brine shrimp lethality assay
Cytotoxicity potential of the plant was tested against brine shrimp (Artemia salina)
larvae to reveal its lethality profile using protocol (Fatima et al., 2015).
b) Cytotoxicity against cell lines
Sulforhodamine B (SRB) assay
The cytotoxicity of D. innoxia leaf and fruit fractions towards Hep G2 cancer cell line
was evaluated by employing SRB assay as previously reported (Haq et al., 2012).
Ch.2: Material and Methods
41
2.2.8 Isolation from D. innoxia fractions
Bioactivity focused isolation approaches involving data on the chemical fingerprints of
extracts and fractions with their corresponding bioactivity profile has significantly
condensed the time for hit finding. For the isolation of momentous quantities (mg to g)
of pure compounds a wide variety of liquid chromatographic techniques like medium
pressure liquid chromatography (MPLC), vacuum liquid chromatography (VLC) and
high-performance liquid chromatography (HPLC) are most commonly employed.
These chromatographic procedures take benefit of enhanced separation abilities due to
variable selectivity and smaller particle size. However, the choice of separation
technique is mainly dependent on the stage of extract or fraction purity and the ultimate
purpose of isolated compounds.
The fractions of D. innoxia leaf and fruit were subjected to isolation and purification
process using normal phase liquid column chromatography in order to get single
purified chemical entities. The compounds thus obtained were biologically evaluated
using various high through put screening assays and afterwards structure of compounds
having substantial bioactivity profile were characterized for structure elucidation. In
this section only isolation schemes of pure compounds from those fractions have been
described that fulfilled the following three criteria:
• Isolated in quantities sufficient for biological and structure determination
studies.
• Pure enough to pursue for structure elucidation.
• Bioactive in high through put screening assays employed in the current study.
2.2.8.1 Isolation from D. innoxia leaf fractions
a) Isolation from DFL-2
Keeping in view the antimicrobial and cytotoxic potential of DFL-2 fraction determined
through biological evaluation (Chapter 3, section 2), it was selected for isolation of
bioactive compounds. Briefly, TLC analysis of DFL-2 was performed to select a
suitable mobile phase for column chromatographic elution by using various solvent
combinations of n-hexane, chloroform, ethyl acetate and methanol. Among all the
solvent combinations, n-hexane with ethyl acetate combinations gave best resolution of
Ch.2: Material and Methods
42
DFL-2 components and was therefore, used in various combinations as mobile phase
for its purification using VLC. Briefly, DFL-2 (38.5 g) was dissolved in ethyl acetate
and was loaded on silica gel 60 (70-230 mesh; 80 g). The adsorbed fraction was dried
in vacuum oven at 45°C and was then packed (dry packing) in glass column containing
blank silica gel 60 (230-400 mesh; 600 g). A protective layer of 2 cm of silica was also
added at the top of the loaded sample. The elution solvent consisted of a mixture of n-
hexane with ethyl acetate (1:0-0:1) to obtain a total of 73 fractions each having an
approximate volume of 10 ml. After monitoring of TLC resolution, fractions 34-73 of
DFL-2 were combined and were designated as DFL-2I. Briefly, DFL-2I (10.5 g) was
dissolved in ethyl acetate and was loaded on silica gel 60 (70-230 mesh; 21 g). The
adsorbed fraction was dried in vacuum oven at 45°C and was then packed (dry packing)
in glass column containing blank silica gel 60 (230-400 mesh; 150 g). A protective
layer of 2 cm of silica was also added at the top of the loaded sample. The elution
solvent consisted of a mixture of n-hexane with ethyl acetate (1:0-1:5) using MPLC to
obtain a total of 123 fractions each having an approximate volume of 10 ml. Precipitates
in fractions 22 till 33 were washed with n-hexane to get the compound CL-1 (550 mg)
(Figure 2.5).
b) Isolation and purification from DFL-4
DFL-4 was selected for isolation and purification of compounds due to the noteworthy
antileishmanial and cytotoxic activities observed when it was subjected to a panel of
bioassays, the results of which have been described in detail in Chapter 3, section-2).
Concisely, TLC analysis of DFL-4 was performed to select a suitable mobile phase for
column chromatographic elution by using various solvent combinations of n-hexane,
chloroform, ethyl acetate and methanol.
Among all the solvent combinations, n-hexane with ethyl acetate and ethyl acetate with
methanol combinations gave best resolution of DFL-4 components and were therefore,
used in various ratios as mobile phase for its purification. Briefly, DFL-4 (42.9 g) was
dissolved in ethyl acetate and was loaded on silica gel 60 (70-230 mesh; 85 g). The
adsorbed fraction was dried in vacuum oven at 45°C and was then packed (dry packing)
in glass column containing blank silica gel 60 (230-400 mesh; 500 g). A protective
layer of silica (2 cm) was also added over the top of the sample. The elution solvent
consisted of a mixture of n-hexane with ethyl acetate (1:0-0:1) followed by elution with
Ch.2: Material and Methods
43
a combination of ethyl acetate with chloroform (1:0-0:1) using VLC to obtain a total of
48 sub fractions each having an approximate volume of 10 ml. Based on co-TLC
resolution of components of sub fractions, three main sub fractions of DFL-4 were
identified i.e. sub fractions 1-15 of DFL-4 were combined to yield DFL-4a, sub
fractions 16-29 were combined to yield DFL-4b while sub fractions 30-48 were
combined to yield DFL-4c.
TLC monitoring of DFL-4a was performed to select a suitable mobile phase for column
chromatographic elution by using various solvent combinations of n-hexane,
chloroform, ethyl acetate and methanol. Among all the solvent combinations, n-hexane:
ethyl acetate and ethyl acetate: methanol combinations gave best resolution of DFL-4a
components and were therefore, used in various ratios as mobile phase for its
purification using normal phase medium pressure column chromatography. Briefly,
DFL-4a (9.50 g) was dissolved in ethyl acetate and was loaded on silica gel 60 (70-230
mesh; 20 g). The adsorbed fraction was dried in vacuum oven at 45°C and was then
packed (dry packing) in glass column containing blank silica gel 60 (230-400 mesh;
150 g). A protective layer of silica (2 cm) was also added over the top of the sample.
The elution solvent consisted of a mixture of n-hexane with ethyl acetate (1:0-0:1)
followed by elution with a mobile phase of ethyl acetate with methanol (1:0-0:1) using
MPCC to obtain a total of 98 fractions each having an approximate volume of 10 ml.
Crystals in fractions 29-34 were washed again and again with a mixture of n-hexane
and ethyl acetate to yield compound CL-3 (43 mg) (Figure 2.6).
a) Isolation and purification from DFF-2
DFF-2 was found to be bioactive in the cytotoxicity assays against cell lines and was
therefore, subjected to chromatographic resolution for the isolation of bioactive
compounds. TLC analysis of DFF-2 was performed to select a suitable mobile phase.
2.2.8.1 Isolation from D. innoxia fruit fractions
for column chromatographic elution by using various solvent combinations of n-
hexane, chloroform, ethyl acetate and methanol. Among all the solvent combinations,
n-hexane: chloroform and chloroform: ethyl acetate combinations gave best resolution
of DFF-2 components and were therefore, used in various ratios as mobile phases for
its purification using VLC.
Ch.2: Material and Methods
44
Figure 2.5 Schematic representation of isolation from of DFL-2. n-hex: n-hexane, EA:
ethyl acetate.
DFL-2 (38.5 g)Adsorbed on Silica gel 60 (70-230 mesh; 80 g)
Dry packing
Column chromatography (CC)-Silica gel 60 (230-400
mesh; 600 g)
Eluted with n-hex and EA (1:0-0:1)
73 fractions collected
(10 ml each)
34-73 vials of DFL-2 designated as DFL-2I
DFL-2I (10.5 g)Adsorbed on Silica gel 60 (70-230 mesh; 21 g)
Dry packing
Column chromatography (CC)-Silica gel 60 (230-400 mesh; 150 g) Eluted with n-
hex and EA (1:0-1:5)
123 fractions collected (10 ml each).
Precipitates in vials 22-33 washed with n-hex
Compound obtained
CL-1 (550 mg)
Ch.2: Material and Methods
45
Briefly, DFF-2 (41 g) was completely dissolved in a mixture of n-hexane and ethyl
acetate (1:1) and was loaded on silica gel 60 (70-230 mesh; 80 g). The adsorbed fraction
was dried in vacuum oven at 45°C and was then loaded (dry loading) in glass column
containing blank silica gel 60 (70-230 mesh; 500 g). A protective layer of silica (2 cm)
was also added over the top of the sample. The elution solvent consisted of a mixture
of n-hexane with chloroform (1:0-0:1) followed by elution with a combination of
chloroform with ethyl acetate (1:0-0:1) to obtain a total of 133 sub fractions each having
an approximate volume of 10 ml.
Based on co-TLC monitoring of sub fractions, three main sub fractions of DFF-2 were
identified i.e. sub fractions 1-70 of DFF-2 were combined to yield DFF-2a, sub
fractions 71-89 were combined to yield DFF-2b while sub fractions 90-133 were
combined to yield DFF-2c. TLC monitoring of DFF-2a was performed to select a
suitable mobile phase for column chromatographic elution by using various solvent
combinations of n-hexane, chloroform, ethyl acetate and methanol. Among all the
solvent combinations, n-hexane: ethyl acetate combinations gave best resolution of
DFL-2a components and were therefore, used in various ratios as mobile phase for its
purification using VLC. Briefly, DFL-2a (21.35 g) was completely dissolved in a
mixture of n-hexane and ethyl acetate (1:1) and was loaded on silica gel 60 (70-230
mesh; 50 g). The adsorbed fraction was dried in vacuum oven at 45°C and was then
packed (dry packing) in glass column containing blank silica gel 60 (230-400 mesh;
400 g). A protective layer of silica (2 cm) was also added over the top of the sample.
The elution solvent consisted of a mixture of n-hexane: ethyl acetate:: 30:1, 25:1, 15:1,
10:1 and 5:1 to obtain a total of 210 fractions each having an approximate volume of
10 ml. Crystals in vials 44-51 were washed with n-hexane to give CF-5 (125 mg)
(Figure 2.7).
Ch.2: Material and Methods
46
Figure 2.6 Schematic representation of isolation from DFL-4. n-hex: n-hexane, EA:
ethyl acetate, MEOH: methanol, CHCL3: chloroform.
DFL-4 (42.9 g)Adsorbed on Silica gel 60 (70-230 mesh; 85 g)
Dry packing
Column chromatography (CC)-Silica gel 60 (230-400 mesh; 500 g). Eluted with
a) n-hex and EA (1:0-0:1)
b) EA and CHCL3 (1:0-0:1)
48 fractions collected
(10 ml each)
1-15 vials of DFL-4 combined designated
as DFL-4a
DFL-4a
(9.50 g)
Adsorbed on silica gel 60
(70-230 mesh; 20 g)
Dry packing
Column chromatography (CC)-silica gel 60 (230-400
mesh; 150 g)
n-hex: EA ::1:0-0:1 and EA:MEOH::1:0-0:1
98 fractions collected
(10 ml each)
Crystals in fractions 29-34 to yield CL-3
(43 g)
Ch.2: Material and Methods
47
Figure 2.7 Schematic representation of isolation from DFF-2. n-hex: n-hexane, EA:
ethyl acetate, CHCL3: chloroform.
DFF-2 (41 g)Adsorbed on Silica
gel 60 (70-230 mesh; 80 g)
Dry packing
Column chromatography (CC)-Silica gel 60 (230-
400 mesh; 500 g).
Eluted with
a) n-hex and CHCL3 (1:0-0:1)
b) CHCL3 with EA (1:0-0:1)
133 fractions collected
(10 ml each)
1-70 vials of DFL-4 combined designated
as DFL-4aDFF-2a (21.35 g)
Adsorbed on silica gel 60 (70-230
mesh; 50 g)Dry packing
Column chromatography
(CC)-silica gel 60 (230-400 mesh; 400
g) n-hexane and ethyl acetate (30:1,
25:1, 15:1, 10:1, 5:1)
210 fractions collected
(10 ml each)
Crystals in vials 44-51 were washed with n-hexane to give CF-
5 (125 mg)
Ch.2: Material and Methods
48
Section-3: Biological evaluation and characterization of isolated compounds
2.2.9 Biological evaluation of isolated compounds
The compounds purified by aforementioned isolation and purification procedures were
then biologically evaluated. The following bioassays were used in the current study to
determine the bioactivity of isolated compounds.
2.2.9.1 Antileishmanial assay
The antileishmanial activity of compounds isolated from D. innoxia in comparison to
amphotericin B was assessed in vitro against the promastigote form of L. tropica using
MTT based microassay as an indicator of cell viability (Khan et al., 2015).
2.2.9.2 Protein kinase inhibition assay
The protein kinase inhibition assay was performed using standard protocol and is
attached as annexure A (Fatima et al., 2015).
2.2.9.3 Cytotoxicity assays
a) Sulforhodamine B (SRB) assay
The cytotoxic effects of compounds isolated from D. innoxia leaf and fruit fractions
towards Hep G2, LU-1 and MCF-7 cancer cell line was evaluated by employing
standard SRB protocol (Haq et al., 2012).
b) MTT assay
The in vitro cytotoxicity evaluation of compounds isolated from D. innoxia leaf and
fruit fractions towards human leukemia (THP-1) cell line (ATCC # TIB-202) was
performed using standard protocol (Fatima et al., 2015).
2.2.9.4 Cancer chemopreventive assays
a) Inhibition of TNF-α activated nuclear factor-kappa B (NFĸB) assay
The inhibitory potential of D. innoxia samples in TNF-α activated NFĸB assay was
assessed by using 293/NFĸB-Luc HEK cells as described previously (Haq et al., 2013).
Ch.2: Material and Methods
49
b) Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-
activated murine macrophage RAW 264.7 cells (iNOs) assay
The inhibitory potential of D. innoxia test samples on the production of nitric oxide
(NO) was assessed by using iNOs assay as described previously by Haq et al. (2013).
2.2.10 Structure elucidation of isolated compounds
2.2.10.1 Nuclear magnetic resonance (NMR) spectroscopy
Both 1H and 13C spectra were recorded using Varion Gemini 2000, 400 MR with a SMS
autosampler (Palo Alto, California, USA) at 400 and 100 MHz respectively in NMR
facility, Faculty of Pharmacy, University of Sydney, Australia. Data was interpreted
with the auspicious support of Prof. Dr. Paul Groundwater. Deuterated chloroform
(CDCl3) was used as solvent for the dissolution of solvents. 5 mm (20 cm length) NMR
tubes were used to carry out the experiment. Coupling constants (J) and chemical shifts
(δ) were expressed in Hertz (Hz) and parts per million (ppm), reported relative to
residual peaks.
2.2.10.2 X-ray crystallography (XRD)
Single crystals were sorted by an experiment under polarizing microscope for the
performance of x-ray crystallography. XRD analysis was carried out using STOE-IPDS
II fitted with low temperature unit of a Bruker and Kappa APEXII CCD diffractometer
using Mo-Kα radiation (λ = 0.71073 Å) and graphite-monochromator at room
temperature. Crystal structure was refined by SHELXL97 (Sheldrick, 2008) and
WinGX (Farrugia, 1999) software. The experiment was performed in University of
Sargodha, Sargodha, Pakistan and the data was interpreted under the guidance of Dr.
Muhammad Nawaz Tahir.
Ch.3: Results
50
3. Results
Section-1: Extraction Optimization from D. innoxia Mill.
3.1 Extraction yield
Percentage of the extract recovery determined for different plant parts, using sonication
followed by maceration as extraction technique is presented in Table 3.1. Highest
quantity of extract was obtained when distilled water was utilized as the extraction
solvent with percentage yield of 33.28 ± 0.87%, 18.88 ± 1.18% and 16.83 ± 0.99% for
leaf, stem and fruit respectively.
Table 3.1 %Extract recovery of leaf, stem and fruit of D. innoxia.
Extract
names
%Extract recovery
Leaf Stem Fruit
Nh 0.75 ± 0.20ba 3.90 ± 0.87a
b 4.05 ± 0.11ba
C 6.23 ± 0.47cb 8.83 ± 0.64c
b 11.28 ± 0.10ca
A 15.07 ± 0.94ac 13.08 ± 0.22a
b 5.53 ± 0.49ac
EthA 15.66 ± 0.76a 11.96 ± 0.62ac 2.49 ± 0.33b
b
Eth 11.10 ± 0.74bc 13.62 ± 0.56a
b 4.37 ± 0.48bc
EC 7.63 ± 1.66bb 6.97 ± 0.44b
c 6.30 ± 0.16ab
MC 8.01 ± 0.40ba 9.18 ± 2.88b
c 9.63 ± 0.71ab
EthE 11.33 ± 0.85cb 5.79 ± 0.46c
a 7.90 ± 1.41ba
MEth 8.55 ± 0.73c 10.45 ± 1.96ca 9.38 ± 0.34a
c
E 8.82 ± 0.33bc 6.16 ± 0.72c
c 7.03 ± 1.94ad
M 8.79 ± 0.93cb 3.43 ± 1.45b
b 11.61 ± 1.53ac
D 33.28 ± 0.87aa 18.88 ± 1.18b
a 16.83 ± 0.99ba
3.2 Phytochemical analysis
3.2.1 Total phenolic content
The total phenolic content of leaf, stem and fruit of D. innoxia are presented in figure
3.2 a, b and c respectively. Highest content of gallic acid equivalent phenols i.e. 29.91
Ch.3: Results
51
± 0.12 and 21.86 ± 0.45 mg/g DW was documented in the aqueous extract of leaf and
fruit extracts respectively while in case of stem, it was highest in the ethyl acetate-
acetone extract (25.06 ± 0.45 mg GAE/g DW). Among leaf extracts the phenolic
content declined according to the following order of extraction solvent polarity; D ˃
EthA ˃ EthE ˃ Eth ˃ A ˃ M ˃ E ˃ EC ˃ MC ˃ MEth ˃ C ˃ Nh. The stem extracts
demonstrated decreasing phenolic content in the following order: EthA ˃ D ˃ Eth ˃
MC ˃ MEth ˃ EthE ˃ A ˃ EC ˃ E ˃ C ˃ M ˃ Nh while it was D ˃ M ˃ EthE ˃ MC ˃
MEth ˃ C ˃ E ˃ EC ˃ Eth ˃ EthA ˃ A ˃ Nh in case of fruit extracts. The total phenols
in various plant parts ranged from 29.91 ± 0.12 mg GAE/g DW for the highly polar
aqueous to 2.5 ± 0.12 mg GAE/g DW for non-polar n-hexane.
3.2.2 Total flavonoid content
The total flavonoid content of leaf, stem and fruit in terms of mg QE/g DW are
presented in figure 3.2 a, b and c respectively. Among all the leaf extracts the highest
TFC of 15.68 ± 0.18 mg QE/g DW was quantified in the ethyl acetate-acetone extract
followed by D ˃ A ˃ E ˃ Eth ˃ M ˃ MC = E ˃ EC ˃ C ˃ MEth ˃ Nh while in case of
stem extracts, the maximum flavonoids were quantified in the ethyl acetate extract
yielding about 5.29 ± 1.22 mg QE/g DW. The aqueous fruit extract unveiled highest
total flavonoid content of 15.28 ± 1.132 mg QE/g DW as compared to other plant parts
analysed.
3.2.3 RP-HPLC analysis
Reverse phase HPLC-DAD based profiling was used for quantitative analysis of
selected plant phenolics and the chromatographic finger printing was done by
comparison of the retention time and UV spectrum of reference compounds with those
of the test sample, the outcomes of which are given in table 3.2. A significant amount
of catechin, myricetin, quercetin, rutin and caffeic acid were quantified in some of the
analyzed extracts. Among the leaf extracts a substantial amount of catechin and
apigenin was present in the methanol (5.41 and 2.11 µg/mg DW respectively) and
methanol-chloroform (1.28 and 1.78 µg/mg DW respectively) extracts. Significant
amount of catechin and apigenin were also quantified in ethanolic extract of fruit (2.65
and 2.46 µg/mg DW respectively). The chromatograms of standards as well as
compounds quantified in various plant parts are presented in Figure 3.1.
Ch.3: Results
52
Table 3.2(a) Calibration curve parameters for the standards.
Standard Retention
time (min)
Calibration curve
equation
Correlation
coefficient
(r2)
Gallic Acid 4.19 y = 24.857x - 45.174 0.9979
Catechin 7.10 y = 7.9854x - 17.565 0.9995
Caffiec Acid 9.66 y = 26.097x + 95.435 0.9924
Rutin 3.12 y = 8.3367x + 22.217 0.9966
Myrisitin 15.44 y = 5.2278x - 6.3043 0.9988
Quercitin 18.52 y = 12.21x - 20.348 0.9978
Kaempherol 21.32 y = 9.9944x + 15.261 0.9998
Apigenin 22.15 y = 18.111x + 25.565 0.9970
Figure 3.1(a) Chromatograms of standard phenolics.
Ch.3: Results
53
Figure 3.1(b) Chromatograms of phenolics detected in M: Methanol extracts of D.
innoxia leaf.
Figure 3.1(c) Chromatograms of phenolics detected in D: Distilled water extracts of D.
innoxia.
Ch.3: Results
54
.
Figure 3.1(d) Chromatograms of phenolics detected in E: Ethanol extracts of D.
innoxia fruit.
Ch.3: Results
55
Table 3.2(b) RP-HPLC profiling of phenolics in D. innoxia extracts.
Extracts
Polyphenols (µg/mg DW)
Phenolic
acid
Flavonol
glycoside
Hydroxy
cinnamate Flavan-3-ol
Flavone
aglycone Flavonol flavonoid
GA Rutin CA Catec Api Myr Quer Kaemp
Leaf
Nh -- -- -- -- -- -- -- --
C -- -- -- -- -- -- -- --
A -- -- -- -- -- -- -- --
EthA -- -- -- -- -- -- -- --
Eth -- -- -- -- -- -- -- --
EC -- -- -- -- -- -- -- --
MC -- -- -- 1.28 ± 0.01 1.78 ± 0.02 -- -- --
EthE -- -- -- -- -- -- -- --
MEth -- -- -- -- -- -- -- --
E -- -- -- -- -- -- -- --
M -- -- -- 5.41 ± 0.03 2.11 ± 0.01 -- 0.84 ± 0.02 --
D -- -- -- -- -- -- -- --
Stem
Nh -- -- -- -- -- -- -- --
C -- -- -- -- -- -- -- --
A -- -- -- -- -- -- 1.33 ± 0.01 --
EthA -- -- -- -- -- -- -- --
Eth -- -- -- -- -- -- -- --
EC -- -- -- -- -- -- -- --
MC -- -- -- -- -- -- -- --
EthE -- -- -- -- -- -- -- --
MEth -- -- -- -- -- -- -- --
Ch.3: Results
56
Extracts
Polyphenols (µg/mg DW)
Phenolic
acid
Flavonol
glycoside
Hydroxy
cinnamate Flavan-3-ol
Flavone
aglycone Flavonol flavonoid
GA Rutin CA Catec Api Myr Quer Kaemp
Leaf
E -- -- -- 0.18 ± 0.01 0.16 ± 0.03 -- -- --
M -- -- -- -- -- -- -- --
D -- 1.58 ± 0.02 1.69 ± 0.01 0.51 ± 0.01 -- 1.67 ± 0.03 0.67 ± 0.01 --
Fruit
Nh -- -- -- -- -- -- -- --
C -- -- -- -- -- -- -- --
A -- -- -- 1.75 ± 0.01 -- -- -- --
EthA -- -- -- -- -- -- -- --
Eth -- -- -- -- -- -- -- --
EC -- -- -- -- -- -- -- --
MC -- -- -- -- -- -- -- --
EthE -- -- -- -- -- -- -- --
MEth -- -- -- -- -- -- -- --
E -- -- -- 2.65 ± 0.02 2.46 ± 0.02 1.74 ± 0.01 -- 0.85 ± 0.01
M -- -- -- -- -- -- -- --
D -- -- -- -- -- -- -- --
Ch.3: Results
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Figure 3.2(a) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia leaf
extracts. Values are given as mean ± Standard error from investigation performed
thrice.
Extract names
jkjkkjjj((Leaf//0
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58
Figure 3.2(b) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia stem
extracts. Values are given as mean ± Standard error from investigation performed
thrice.
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59
Figure 3.2(c) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia fruit
extracts. Values are given as mean ± Standard error from investigation performed
thrice.
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60
3.3 Biological evaluation
3.3.1 Antioxidant assays
3.3.1.1 %RSA
The percent radical scavenging activity (%RSA) of the test samples was assessed by
the decrease in the colour intensity of the MEOH solution of DPPH, the results of which
are summarized in figure 3.2. The best antioxidant activity in DPPH assay was
demonstrated by the aqueous extract of leaf (IC50 = 22.83 µg/ml) while it was lowest
for n-hexane (IC50 = 383.31 µg/ml). Ethyl acetate-acetone stem extract exhibited
maximum quenching activity with an IC50 of 16.14 µg/ml trailed by ethyl acetate (IC50
= 19.34 µg/ml) and aqueous (IC50 = 23.22 µg/ml) extracts. Among all the fruit extracts
the most potent radical scavenging potential was shown by the Dw extract with IC50 of
19.22 µg/ml.
3.3.1.2 TAC
The total antioxidant capacity of different leaf, stem and fruit extracts are summarized
in figure 3.2 a, b, c respectively. Maximum TAC was displayed by the Dw extract of
the leaf (46.98 ± 0.14 mg AAE/g DW), ethyl acetate extract of the stem (42.90 ± 1.25
mg AAE/g DW) and ethanol-chloroform extract of the fruit (43.86 ± 1.18 mg AAE/g).
3.3.1.3 TRP
Figure 3.2 (a, b, c) shows the reducing potential of various extracts of D. innoxia. In
our present study, the supreme extraction efficiency was documented in the aqueous
extract of leaf (9.46 ± 1.12 mg AAE/g DW), ethyl acetate extract of stem (15.35 ± 0.61
mg AAE/g DW) and methanol-ethyl acetate extract (13.90 ± 0.87 mg AAE/g DW) of
fruit.
3.3.2 Antimicrobial assays
3.3.2.1 Antileishmanial activity
The in vitro activities of extracts from leaf, stem and fruit of D. innoxia, using MTT
assay against L. tropica axenic promastigotes are reported in figure 3.3. It was observed
that 66.66% of the leaf extracts were substantially leishmanicidic exhibiting more than
90% inhibition at 100 µg/ml. Among the leaf extracts EC, MC and EthA extracts
demonstrated a noteworthy antiprotozoal activity as compared to Amphotericin B (IC50
Ch.3: Results
61
= 0.01 µg/ml) with 50% mortality at 3.98 ± 0.12, 5.59 ± 0.11 and 5.62 ± 0.13 µg/ml
respectively followed by MEth > Eth > C = M > D > A > E > EthE > Nh. It was found
that inhibitory potential decreased when ethanol either alone or in combination was
employed as extraction system; therefore, retrieval of secondary metabolites
antagonizing the antileishmanial activity by ethanol might be ascribed for the observed
decrease in leishmanicidal potential. Among all the stem and fruit extracts only EthE
(46.30 ± 1.75%) and A (39.73 ± 1.95%) extracts of stem exhibited mild leishmanicidic
activity.
Figure 3.3 Inhibitory effects of D. innoxia extracts on in vitro growth of L. tropica
promastigotes. The IC50 of Amphotericin B (positive control) = 0.01 µg/ml. *IC50 >100
µg/ml.
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3.3.2.2 Antibacterial activity
Table 3.3 represents the results of activity against tested bacteria in terms of ZOI (mm
diameter) of extracts from plant parts (leaves, stem and fruit) of D. innoxia. In the
current research, the extracts showing a ZOI ≥ 10 mm in standard disc diffusion test
were further tested at lower concentration to determine their MIC by using broth micro
dilution method. A better antibacterial spectrum against Gram +ive bacteria as
compared to Gram -ve was observed. Among all the extracts 39% of leaf, 16% of stem
and 29% of fruit extracts were found to be active (zone ≥ 10 mm). Among all the
bacterial strains tested, Microcococcus luteus was found most susceptible with
maximum inhibition by the n-hexane fruit extract producing zone of inhibition of 24
mm (MIC = 3.70 µg/ml). Data indicated that extracts prepared from leaves possess
better antibacterial activity than those prepared from stem and fruit. Among all the leaf
extracts a maximum zone of growth inhibition was displayed by the ethyl acetate,
ethanol and acetone extracts against K. pneumonae, S. typhi, M. luteus and S. aureus
respectively. Among all the stem extracts the maximum growth inhibition zone of 20
mm was produced by the ethanolic extract having MIC of 3.70 µg/ml. The n-hexane
fruit extract presented antibacterial activity with an inhibition zone ranging between 7
mm to 24 mm. The absence of growth inhibition zones confirmed the non-toxic effect
of DMSO (negative control) while cefixime served as positive control.
3.3.2.3 Antifungal activity
Antifungal potential of extracts was evaluated against four strains of filamentous fungi.
The antifungal growth inhibitory activities of extracts of various plant parts are
represented in table 3.4. The data indicate that acetone extract of leaf and stem while n-
hexane extract of fruit showed a prominent growth inhibition zone of 22, 20 and 24 mm
against A. niger respectively. Among all the samples, least MIC of 12.5 µg/disc against
A. niger was noted for the n-hexane fruit extract. A reasonable antifungal activity was
shown by most of the test extracts against Mucor sp. with an average diameter of ZOI
ranging between 7-14 mm. It was observed that mostly the antifungal activity increased
as the polarity decreased. Thus the chloroform and n-hexane extracts showed better
antifungal activity than aqueous or ethanolic extracts.
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3.3.3 Enzyme inhibition assays
3.3.3.1 α-amylase inhibition assay
Standard chromogenic starch-iodine assay was executed to assess the α-amylase
inhibitory activity of test samples (Figure 3.4). Acarbose was used as positive control
in the assay which inhibited 80.34 ± 1.12% of α-amylase enzyme’s activity and
exhibited an IC50 of 33.73 ± 0.12 µg/ml. Among the leaf extracts, E and Eth exhibited
a noteworthy inhibition of 40.09 ± 1.54 and 38.34 ± 2.25% followed by MC > EC > C
= M > EthA > A > EthE > D > Meth > Nh. Among the stem extracts, EthA, MEth and
A extracts demonstrated a significant inhibition of 49.76 ± 2.12, 45.06 ± 1.23 and 44.59
± 2.02% of amylase enzyme’s activity respectively lagged by E > EthE > C > MC >
Eth > M > EC = D > Nh. A mild to moderate enzyme inhibition was exhibited by the
fruit extracts with C and EC being the lead extracts demonstrated an enzyme inhibition
of 39.75 ± 1.43 and 24.17 ± 1.67% respectively.
3.3.3.2 Protein kinase inhibition assay
Table 3.5 shows the results of protein kinase inhibitory potential recorded as zone of
inhibition around the test samples. Amongst all the test extracts, a noteworthy ZOI of
22 mm bald, 11 mm clear was formed around the ethyl acetate extract of both leaf and
stem, while the most prominent hyphae formation inhibition in case of fruit was
presented by the ethanol extract (20 mm bald, 11 mm clear) lagged by acetone (18 mm
bald, 11 mm clear) and acetone-ethyl acetate extract (17 mm bald, 11 mm clear).
Surfactin, the positive control established a 30 mm bald growth inhibition zone.
3.3.4 Assays for the determination of cytotoxic potential
3.3.4.1 Brine shrimp lethality assay
Cytotoxic proficiency of the test extracts was evaluated against brine shrimps to
determine its lethality profile. Out of total 36 D. innoxia extracts screened for cytotoxic
activity against brine shrimp larvae, 25% of the leaf, 16% of the stem and 8.3% of the
fruit extracts demonstrated activity at or below 100 μg/ml and were categorized as
highly cytotoxic. The remaining 75% of the leaf, 84% of the stem and 91.7% of the
fruit extracts had LC50 values ≤ 250 μg/ml and were categorized as moderately
cytotoxic. The results from screening of the organic extracts of different plant organs
against A. salina larvae are shown in Table 3.5. Doxorubicin which was used as positive
Ch.3: Results
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control in the assay demonstrated an LC50 value 5.930 µg/ml. It was noticed that
amongst all the individual plant part extracts, methanol-chloroform was the most
cytotoxic exhibiting an LC50 of 85.94 μg/ml for leaf and stem while 54.07 μg/ml for
fruit.
Figure 3.4 α-amylase inhibition by D. innoxia extracts. The IC50 of acarbose (positive
control) = 33.73 ± 0.12 µg/ml.
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Table 3.3: Antibacterial activity of D. innoxia extracts.
Extracts
*Diameter of growth inhibition zone (mm ± SD) at 100 µg/disc
K. pneumoniae MIC
(µg/ml) S. typhimurium
MIC
(µg/ml) M. luteus
MIC
(µg/ml) S. aureus
MIC
(µg/ml)
Leaf
Nh 8.5 ± 0.76 c -- 10 ± 2.85 a 33.33 7 ± 1.84 c -- 9 ± 1.89 b --
C 10 ± 0.96 b 33.33 6.5 ± 1.32 c -- 9 ± 1.45 c -- 9 ± 2.31 b --
A 10 ± 0.76b 33.33 7.5 ± 1.33 c -- 14.5 ± 1.1 a 3.70 10 ± 1.63 a 100
EthA 10 ± 0.98 b 100 7 ± 1.54 c -- 12 ± 1.17 b 33.33 9 ± 1.78 b --
Eth 12 ± 0.22 b 100 8 ± 1.24 b -- 18 ± 1.34 a 3.70 8 ± 1.49 c --
EC 10 ± 0.48 b 100 7.5 ± 1.34 c -- 12 ± 1.22 b 33.33 8.5 ± 1.87 c --
MC 7 ± 0.45 c -- 6 ± 2.21 c -- 16 ± 1.45 a 3.70 6 ± 1.89 c --
EthE 12 ± 0.18a 33.33 7.5 ± 1.76 c -- 14 ± 1.45 b 33.33 7 ± 2.21 c --
Meth 9.5 ± 0.34 b -- 7 ± 1.32 c -- 10 ± 1.34 b 100.00 8. 5± 1.38c --
E 8 ± 0.67 c -- 17 ± 1.45 c 11.11 13 ± 1.45 b 11.11 9.5 ± 1.22a --
M 7 ± 0.34 c -- 8 ± 1.24 b -- 16 ± 1.67 a 3.70 7 ± 1.23 c --
D 7 ± 0.56c -- 7 ± 1.23 c -- 14 ± 1.13 b 11.11 6 ± 1.43 c --
Stem
Nh -- -- 10 ± 1.67 b 33.33 8 ± 2.34 b -- 10 ± 1.83 a 100
C -- -- 14 ± 0.88 a 11.11 8 ± 1.45 b -- 9 ± 1.21 b --
A -- -- 7 ± 1.21 c -- 8 ± 2.2 a -- -- --
EthA -- -- 10 ± 2.67 b 100 7 ± 1.22 c -- -- --
Eth -- -- 7 ± 2.34 c -- -- -- -- --
EC -- -- 7 ± 1.23 c -- 13 ± 1.3 a 100 -- --
MC 9 ± 1.87 b -- 9 ± 1.56 b -- -- -- 7 ± 2.21 c --
EthE 8 ± 2.14 b -- -- 7 ± 1.45 c -- -- --
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Meth -- -- -- 7 ± 0.77 c -- -- --
E -- -- 10 ± 2.13 b 100 20 ± 1.82 b 3.70 -- --
M 9 ± 2.32 b -- 6 ± 1.43 c -- 7 ± 1.23 c -- 8 ± 1.21 b --
D -- -- 10 ± 1.56 b 100 0 -- -- --
Fruit
Nh -- -- 12 ± 1.21a 100 24 ± 1.32 a 3.70 -- --
C 8 ± 1.23 b -- 8 ± 2.45 c -- 9 ± 1.22 b -- 9 ± 1.32 c --
A 8 ± 1.45 b -- 7 ± 1.13 c -- 8 ± 1.45 c -- -- --
EthA -- -- 13 ± 2.21 a 33.33 10 ± 3.12 b 100 10 ± 1.21 b 100
EC -- -- 8 ± 1.56 c -- -- -- -- --
MC -- -- 10 ± 1.56 b 100 9 ± 1.82 b -- -- --
EthE -- -- 11 ± 1.65 b 100 8 ± 1.21 c -- -- --
Meth 9 ± 1.76 b -- 10 ± 1.67 b 100 8 ± 2.13 c -- -- --
E 10 ± 1.12 a 100 12 ± 2.21 a 100 8 ± 1.23 c -- 12 ± 1.28 a 33.33
M 7 ± 1.23 c -- 12 ± 1.54 a 33.33 9 ± 1.51 a -- -- --
D -- -- -- -- 10 ± 1.21 b 100 -- --
DMSO -- -- -- -- -- -- -- --
Cefixime 28 ± 0.07 3.33 26 ± 0.21 3.33 18 ± 0.44 1.11 16 ± 1.22 1.11
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Table 3.4 Antifungal activity of D. innoxia extracts.
Extracts *Diameter of growth inhibition zone (mm ± SD) at 100 µg/disc
A. fumigatus MIC
µg/disc
Mucor sp. MIC
µg/disc
A. niger MIC
µg/disc
A. flavus MIC
µg/disc
Leaf
Nh 8 ± 3.24b 100 10 ± 2.85 a 100 7 ± 2.21 c 100 -- --
C 8 ± 1.43b 100 -- -- 9 ± 1.32 c 100 -- --
A 7 ± 1.56c 100 8 ± 1.34 b 100 22 ± 1.5 a 25 -- --
EthA 7 ± 2.45 c 100 12 ±3.45 a 50 10 ± 1.78 b -- -- --
Eth -- -- 7 ± 1.67 c 100 -- -- -- --
EC -- -- -- -- -- -- -- --
MC -- -- -- -- -- -- -- --
EthE 7 ±1.22 c 100 10 ± 3.27a 100 -- -- -- --
Meth -- -- 9 ± 1.65 b 100 -- -- -- --
E -- -- 7 ± 2.43 c 100 10 ± 1.45 b 100 -- --
M -- -- 9 ± 2.56 b 100 7 ± 1.67 c 100 -- --
D -- -- 8 ± 1.54 b 100 -- -- --
Stem
Nh -- -- 10 ± 1.67 b 50 8 ± 2.34 b 100 10 ± 1.8 b 100
C -- -- 14 ± 0.88 a 50 8 ± 1.45 b 100 9 ± 1.21 b 100
A -- -- 7 ± 1.21 c 100 20 ± 2.23 a 25 --
EthA -- -- 10 ± 2.67 b 50 7 ± 1.22 c 100 --
Eth -- -- 7 ± 2.34 c 100 -- --
EC -- -- 7 ± 1.23 c 100 13 ± 1.32 b 100 --
MC 9 ± 1.87 b 100 9 ± 1.56 b 100 -- 7 ± 2.21 c 100
EthE 8 ± 2.14 c 100 -- 7 ± 1.45 c 100 --
Meth -- -- -- 7 ± 0.77 c 100 --
E -- -- 10 ± 2.13 b 100 8 ± 1.82 c 100 --
M 9 ± 2.32 b 100 6 ± 1.43 c 100 7 ± 1.23 c 100 8 ± 1.21 b 100
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Table 3.5 Brine shrimp cytotoxicity, THP-1, Hep G2 and protein kinase inhibitory potential of D. innoxia extracts.
D -- -- 10 ± 1.56 b 100 -- --
Fruit
Nh -- -- 12 ± 1.21 100 24 ± 1.32 a 12.5 -- --
C 8±1.23 c 100 8 ± 2.45 c 100 9 ± 1.22 b 100 9 ± 1.32 b 100
A 8 ± 1.45 c 100 7 ± 1.13 c 100 8 ± 1.45 c 100 -- --
EthA -- -- 13 ± 2.21 50 10 ± 3.12 a 100 10 ± 1.21 a 100
Eth -- -- -- -- 16 ± 1.67 a 12.5 -- --
EC -- -- 8 ± 1.56 c 100 -- -- -- --
MC -- -- 10 ± 1.56 b 50 9 ± 1.82 b 100 -- --
EthE -- -- 11 ±1 .65 b 100 8 ± 1.21c 100 -- --
Meth 9 ± 1.76 b 100 10 ± 1.67 b 100 8 ± 2.13 c 100 -- --
E 10 ± 1.12 b 100 12 ± 2.21 50 8 ± 1.23 c 100 12 ± 1.28 a 100
M 7 ± 1.23 c 100 12 ± 1.54 50 9 ± 1.51 b 100 -- --
D -- -- -- -- 10 ± 1.21 a 100 -- --
Extracts
Brine shrimp cytotoxicity
(µg/ml)
THP-1 cytotoxicity
(µg/ml)
Hep G2 cytotoxicity
(µg/ml)
Protein kinase inhibition
%Mortality LC50 %Inhibition IC50 %Inhibition IC50 *Diameter (mm ± SD)
1000 10 20 Clear zone
Bald zone
Leaf
Nh 100.0 ± 0.00 a 235.44 ± 1.12 22.32 ± 2.41 c ˃10 9.00 ± 0.52 > 20 -- 7 ± 0.38
C 90.00 ± 0.49 b 199.91 ± 0.98 25.32 ± 2.41 5.91 ± 0.78 90.73 ± 2.34 6.54 ± 0.10 13 ± 0.18 b 21 ± 0.29
A 96.60 ± 0.94 b 226.67 ± 1.34 18.22 ± 1.68 ˃10 60.87 ± 1.54 18.81 ± 0.92 7 ± 0.15 11 ± 0.47
EthA 98.30 ± 0.47 150.48 ± 1.14 23.22 ± 2.13 b ˃10 60.81 ± 3.23 13.36 ± 0.32 7 ± 0.33 11 ± 0.39
Ch.3: Results
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Eth 78.30 ± 1.25 133.26 ± 0.43 80.24 ± 2.99a 5.11 ± 0.98 90.74 ± 2.57 8.98 ± 0.14 11 ± 0.43 22 ± 0.47 a
EC 90.0 ± 0.94 c 154.90 ± 0.22 55.23 ± 3.22 8.9 ± 0.53 62.66 ± 1.43 11.89 ± 0.22 14 ± .41 b 21 ± 0.52 b
MC 81.60 ±1.70 a 85.94 ± 0.16 52.38 ± 2.87 9.8 ± 0.15 41.23 ± 0.67 > 20 7 ± 0.32 10 ± 0.21
EthE 86.60 ± 0.94 186.95 ± 0.17 16.33 ± 3.18 ˃10 42.00 ± 0.15 > 20 7 ± 0.62 10 ± 0.15
MEth 88.30 ± 2.36 117.69 ± 0.18 18.32 ± 4.11 ˃10 55.19 ± 1.21 17.24 ± 1.12 12 ± 0.51 18 ± 0.41 b
E 78.30 ± 0.94 a 97.08 ± 1.35 25.34 ± 1.45 b ˃10 96.63 ± 3.26 9.72 ± 0.32 7 ± 0.13 9 ± 0.42
M 85.00 ± 1.25 97.06 ± 1.18 16.21 ± 2.45 ˃10 79.24 ± 2.31 7.99 ± 0.21 6 ± 0.22 b 19 ± 0.35
D 67.00 ± 1.70 247.27 ± 0.74 12.85 ± 0.83 a ˃10 3.00 ± 0.21 > 20 -- --
Stem
Nh 100.45 ± 0.00 a 194.95 ± 0.34 -- -- 3.00 ± 0.14 > 20 6 ± 0.44 --
C 90.50 ± 0.00 a 125.53 ± 0.22 -- -- 4.00 ± 0.21 > 20 6 ± 0.58 --
A 96.60 ± 0.94 b 155.33 ± 0.98 -- -- 4.00 ± 0.34 > 20 7 ± 0.42 13 ± 0.42 b
EthA 98.35 ± 0.58 b 153.38 ± 0.54 -- -- 1.18 ± 0.47 > 20 7±0.22 a 11 ± 0.22 a
Eth 78.35 ± 0.82 b 250.00 ± 0.34 -- -- 11.00 ± 0.76 > 20 9 ± 0.35 a 18 ± 0.35 a
EC 90.45 ± 0.94 154.90 ± 0.16 -- -- 15.79 ± 1.43 > 20 6 ± 0.28 9 ± 0.28 a
MC 85.40 ± 1.25 c 97.06 ± 0.17 -- -- 3.56 ± 0.28 > 20 11 ± 0.21 a 22 ± 0.21 a
EthE 86.60 ± 0.47 b 117.69 ± 0.16 -- -- 28.35 ± 2.43 > 20 6 ± 0.45 b 8 ± 0.45
MEth 88.30 ± 0.95 b 117.69 ± 1.46 -- -- 2.00 ± 0.15 > 20 6 ± 0.27 9 ± 0.27
E 78.30 ± 0.47 a 137.29 ± 1.65 -- -- 12.00 ± 1.12 > 20 8 ± 0.33 13 ± 0.33 b
M 81.60 ± 0.82 b 87.94 ± 0.76 -- -- 2.00 ± 0.11 > 20 9 ± 0.51 b 14 ± 0.51 c
D 65.56 ± 0.82 b 250.00 ± 0.17 -- -- 10.00 ± 0.87 > 20 6 ± 0.47 8 ± 0.47
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Extracts Brine shrimp cytotoxicity
(µg/ml)
THP-1 cytotoxicity
(µg/ml)
Hep G2 cytotoxicity
(µg/ml)
Protein kinase inhibition
%Mortality LC50 %Inhibition IC50 %Inhibition IC50 Diameter (mm ± SD)
1000 10 20 Clear zone
Bald zone
Fruit
Nh 78.30 ± 1.25 a 359.12 ± 0.18 76.19 ± 1.92 a 3.49 ± 0.17 2.00 ± 0.12 > 20 -- 10 ± 0.81 b
C 91.60 ± 1.25 a 141.68 ± 1.34 76.66 ± 2.22 a 4.52 ± 1.23 95.57 ± 0.13 10.93 ± 0.42 -- 21 ± 0.28 a
A 93.30 ± 0.47 a 240.91 ± 1.56 16.23 ± 2.23 ˃10 11.00 ± 1.32 > 20 11 ± 0.23 b 18 ± 0.54 a
EthA 91.60 ± 1.25 b 114.40 ± 0.78 16.88 ± 2.23 ˃10 2.00 ± 0.08 > 20 11 ± 0.47 b 17 ± 0.22 a
Eth 93.30 ± 1.25 a 201.96 ± 0.17 10.32 ± 1.45 ˃10 8.32 ± 0.53 > 20 -- 19 ± 0.22
EC 91.65 ± 1.25 a 234.92 ± 0.27 18.3 ± 2.55 b ˃10 2.00 ± 0.12 > 20 -- 10 ± 0.24
MC 85.00 ± 2.83 b 54.07 ± 0.16 18.3 ± 2.66 a ˃10 10.00 ± 1.13 > 20 -- 9 ± 0.32
EthE 98.35 ± 0.47 a 218.64 ± 0.34 61.91 ± 1.22 b 8.88 ± 1.16 65.57 ± 2.43 12.76 ± 0.34 8 ± 0.38 b 14 ± 0.38 a
MEth 90.00 ± 1.63 a 152.61 ± 1.14 15.62 ± 1.87 ˃10 45.57 ± 3.45 > 20 -- 13 ± 0.62 b
E 90.00 ± 0.82 a 229.21 ± 1.56 18.23 ± 1.27 ˃10 2.00 ± 0.16 > 20 11 ± 0.22 a 20 ± 0.67
M 85.00 ± 1.41 b 164.39 ± 1.78 16.57 ± 3.24 ˃10 23 ± 0.16 > 20 7 ± 0.43 c
D 65.00 ± 0.82 351.71 ± 1.76 10.34 ± 1.28 ˃10 10.00 ± 1.01 > 20 -- 7 ± 0.56
Doxorubicin 100 5.93 98 ± 0.18 5.1
5-Florouracil 100 5
Vincristine 100 8.1
Surfactin 30 ± 1.02
DMSO -- -- --
1% DMSO in
PBS/sea water
-- -- --
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3.3.4.2 Cytotoxicity against cell lines
a) THP-1 human leukaemia cell line
the substantial cytotoxic activity observed in the brine shrimp lethality assay was
further extended and the plant extracts were screened for an in vitro cytotoxicity assay
using human leukaemia cell line (Table 3.5). Among all the leaf extracts, the
chloroform extract was most potent as it considerably inhibited the cell line
proliferation exhibiting 80.95 ± 1.77% cell mortality at 10 µg/ml concentration and an
IC50 5.91 µg/ml. In the present study the stem extracts did not display any cytotoxic
potential while in case of fruit, the most prominent lethality was shown by the C and
nh extracts with an LC50 of 4.52 and 3.49 µg/ml respectively as compaired to the
positive controls i.e. 5 florouracil and vincristine with 50% lethality of 5.0 µg/ml and
8.10 µg/ml respectively.
b) Hep G2 cell line
The cytotoxic potential of D. innoxia test extracts against Hep G2 human hepatocellular
carcinoma cell line was articulated as %inhibition at a concentration of 20 µg/ml
concentration (Table 3.5). Among all the leaf extracts, 25% of the test samples
demonstrated a significant cytotoxic activity (> 90% inhibition), 33.33% exhibited a
moderate inhibition (> 60%) whereas 8.30% possessed a mild (> 50%) cytotoxicity
against the tested cell line. C, EthA and E leaf extracts are suggested as promising leads
exhibiting > 90% inhibition at 20 µg/ml with IC50 6.54 ± 0.10, 8.36 ± 0.32 and 9.72 ±
0.32 µg/ml respectively. The stem extracts were not found to be cytotoxic whereas EthE
fruit extract demonstrated a noteworthy inhibition with 50% mortality at 10.93 ± 0.42
µg/ml. The IC50 obtained in case of doxorubicin (positive control) was 5.93 ± 0.01
µg/ml.
3.4 Summary
The preliminary optimization studies on extraction efficiency of D. innoxia, in terms of
most efficacious plant part for bioactivity revealed that among the leaf, stem and fruit
parts, leaf and fruit are most proficient in terms of their bioactivity profile. Therefore,
these plant parts were selected to proceed for preparative extraction and isolation of
lead compounds. Keeping in view the efficiency of ethyl acetate leaf extract of D.
innoxia as a substantial source of phytochemicals harbouring extensive antioxidant
capability, cytotoxic, kinase inhibitors and antibacterial compounds, it was selected as
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one of the solvents for preparative extraction. Similarly, methanolic extracts of leaf
were remarkably effective against brine shrimps, THP-1 and Hep G2 cell proliferation.
Therefore, ethyl acetate and methanol (1:1) binary solvent combination was selected as
the extraction solvent system for preparative extraction of D. innoxia leaf part. In case
of fruit, nonpolar solvents showed phenomenal cytotoxic, antimicrobial and
leishmanicidic potential; therefore, chloroform was selected for the preparative
extraction of fruit part.
Section-2: Preparative Extraction, Biological Evaluation and Isolation
3.5 Fraction yield
The fractions yields obtained by fractionation of preparative leaf and fruit extracts along
with their elution solvents is summarized in table 3.6. Maximum fraction yield was
obtained in case of DFL-7 and DFF-1 i.e. 83.35 and 310 g respectively.
Table 3.6 Fractionation scheme of D. innoxia leaf and fruit.
Fraction name Elution solvent fraction yield (g)
Leaf
DFL-1 n-hexane 55.4 g
DFL-2 n-hexane: Ethyl acetate (5:1) 35 g
DFL-3 n-hexane: Ethyl acetate (1:1) 48 g
DFL-4 n-hexane: Ethyl acetate (1:5) 42.9 g
DFL-5 Ethyl acetate (0:1) 24.65 g
DFL-6 Ethyl acetate: Methanol (5:1) 59.02 g
DFL-7 Ethyl acetate: Methanol (1:1) 83.35 g
DFL-8 Methanol 60.75 g
Fruit
DFF-1 n-hexane 310 g
DFF-2 n-hexane: chloroform (1:1) 41 g
DFF-3 Chloroform 82 g
DFF-4 Chloroform: ethyl acetate
(1:1)
62 g
DFF-5 Ethyl acetate 57.95 g
DFF-6 Ethyl acetate: methanol (1:1) 42.35 g
DFF-7 Methanol 70.90 g
3.6 Biological evaluation
3.6.1 Antioxidant assays
3.6.1.1 %RSA
Among the leaf fractions, maximum antioxidant activity in DPPH assay was
demonstrated by DFL-8 and DFL-5 leaf fractions i.e. IC50 = 22.83 ± 0.49 and 51.23 ±
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0.87 µg/ml respectively followed by DFL-6 (52.02 ± 1.22 µg/ml) while it was lowest
in case of DFL-1 (Fig 3.5).
In case of D. innoxia fruit fractions, the most potent radical scavenging potential was
exhibited by the DFF-7 (IC50 = 23.22 ± 1.03 µg/ml) followed by DCF-7 i.e. 36.75 ±
2.13 µg/ml while the least quenching effect was manifested in case of DFF-1 (Fig 3.6).
3.6.1.2 TAC
TAC of the D. innoxia leaf fractions was expressed as equivalent of ascorbic acid (µg
AAE/mg) with highest capability exhibited by DFL-7 (65.23 ± 1.05 µg AAE/mg) and
DFL-8 (62.67 ± 1.01 µg AAE/mg) followed by DFL-5 with 59.86 ± 0.72 µg AAE/mg
respectively. The others followed the following trend: DFL-7 > DFL-8 > DFL-5 > DFL-
6 > DCL > DFL-2 > DFL-3 > DFL-2 > DFL-1 with 32.17 ± 0.96 µg AAE/mg
antioxidant potential (Fig 3.7). In case of D. innoxia fruit fractions, highest antioxidant
capability was exhibited by DFF-7 (58.14 ± 2.16 µg AAE/mg) and DFF-6 (54.32 ± 2.22
µg AAE/mg) extracts followed by DCF with 48.54 ± 1.21 µg AAE/mg respectively
whereas, least antioxidant potential was obtained in its DFF-2 fraction (Fig 3.8).
Figure 3.5 %RSA and IC50 of D. innoxia DCL and leaf fractions. Values represent
mean ± standard deviation performed thrice.
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Figure 3.6 %RSA and IC50 of D. innoxia DCF and fruit fractions. Values represent
mean ± standard deviation performed thrice.
3.6.1.3 TRP
The maximum extraction efficiency in terms of highest reducing power when expressed
as equivalent of ascorbic acid was achieved in DFL-7 and DFL-8 i.e. 16.30 ± 0.36 and
15.64 ± 0.23 µg AAE/mg respectively followed by the others in decreasing order as
DFL-5 > DFL-6 > DCL > DFL-2> DFL-3 while the least activity was observed in DFL-
1 with 11.38 ± 0.43 µg AAE/mg reduction potential (Fig 3.7). In case of D. innoxia
fruit fractions, highest ascorbic acid equivalent reductive potential was expressed by
DFF-7 as 14.53 ± 1.21 µg AAE/mg while it was least in case of DFF-2 i.e. 8.04 ± 0.65
µg AAE/mg followed by the others in decreasing order as DFF-6 > DCF > DFF-5 >
DFF-4 > DFF-3> DFL-1> DFF-2 (Fig 3.8).
3.6.2 Antimicrobial assays
3.6.2.1 Antileishmanial assay
Among the leaf fractions prepared from DCL, DFL-4 revealed most potent
antileishmanial activity as compared to Amphotericin B (IC50 = 0.01 µg/ml) possessing
94.00 ± 1.34% inhibition at 100 µg/ml (IC50 = 11.21 ± 0.87 µg/ml) followed by DFL-
8 with an inhibition percent of 85.54 ± 1.34% and IC50 of 13.45 ± 1.11 µg/ml. The
antileishmanial spectrum of leaf fractions decreased in the following order DFL-4 >
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DFL-8 > DFL-7 > DFL-6 > DFL-5 (Fig 3.9). Among the fruit fractions, none of the test
samples exhibited more than 50% growth inhibition of L. tropica promastigotes (Fig
3.10).
Figure 3.7 TAC (µg AAE/mg) and TRP (µg AAE/mg) of D. innoxia DCL and leaf
fractions. Values represent mean ± standard deviation performed thrice.
.
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Figure 3.8 TAC and TRP of D. innoxia DCF and fruit fractions. Values represent
mean ± standard deviation performed thrice.
Figure 3.9 Antileishmanial potential of D. innoxia leaf fractions. Values represent
mean ± standard deviation performed thrice. *IC50 >100 µg/ml.
Figure 3.10 Antileishmanial potential of D. innoxia fruit fractions. Values represent
mean ± standard deviation performed thrice.
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3.6.2.2 Antibacterial activity
In the current analysis, the fractions exhibiting a growth inhibitory zone ≥ 10 mm in
agar disc diffusion assay were considered active and were further evaluated for MIC
determination (Table 3.7). Among all the samples tested, DCL exhibited substantial
activity against B. subtilis with a growth inhibition zone of 21 ± 0.45 mm (MIC = 3.33
µg/disc). Most of the leaf fractions demonstrated substantial antibacterial activity
against B. subtilis with maximum inhibition zone of 22 ± 0.58 mm (MIC = 3.33 µg/disc)
being formed around the DFL-5 loaded disc. Among the leaf fractions, 62.5% were
active (zone ≥ 10 mm) against K. pneumoniae, 37.5% against P. aeruginosa, 62.5%
against B. subtilis, while none of the fractions was active against S. aureus. A
noteworthy inhibition zone against B. subtilis was demonstrated by DFL-5 (ZOI = 19
± 0.58 mm, MIC = 33.33 µg/disc) and DFL-6 (ZOI = 18 ± 0.58 mm, MIC = 33.33
µg/disc). Among the fruit fractions, a moderate antibacterial activity was observed only
against K. pneumoniae.
3.6.2.3 Antifungal activity
The plant’s antifungal potential was assessed against four strains of filamentous fungi,
the results of which have been summarized in table 3.8. The fractions exhibiting a
growth inhibitory zone ≥ 10 mm in agar disc diffusion assay were considered active.
The crude DCL leaf extract exhibited a 9 ± 0.45 mm ZOI against only one strain i.e. A.
flavus whereas, the crude DCF fraction of fruit did not exhibit any antifungal activity.
The data indicate that 37.50% of the leaf fractions were active against A. flavus, 25%
against A. niger whereas, none of the leaf fractions were active against F. solani, A.
fumigatus or Mucor sp. The maximum ZOI of 13 ± 0.65 mm around A. flavus was
demonstrated by DFL-7. Among the fruit fractions a moderate antifungal activity was
observed only against A. flavus while there was little or no activity against the other
tested strains.
3.6.3 Enzyme inhibition assay
3.6.3.1 α-amylase inhibition assay
Acarbose, the positive control inhibited 80.34 ± 1.12% of α-amylase enzyme’s activity
and demonstrated an IC50 of 33.73 ± 0.12 µg/ml. Among the leaf fractions, a maximum
inhibition of 18.41 ± 1.54% was demonstrated by DFL-7 while in case of fruit fractions
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a maximum inhibition of amylase enzyme’s activity was manifested by DFF-4 i.e.
52.72 ± 1.34% (Table 3.9).
3.6.3.2 Protein kinase inhibition assay
The results of protein kinase inhibition zones calculated for the test extracts are shown
in table 3.10. Among all the leaf fractions, a noteworthy inhibition zones of 13 ± 1.21
mm clear, 18 ± 1.45 mm bald and 11 ± 1.21 mm clear, 16 ± 2.23 mm bald phenotype
were observed around DCL and DFL-7 extract loaded discs lagged by DFL-6 (9 ± 0.96
mm clear, 16 ± 0.76 mm bald zone) while, the most prominent hyphae formation
inhibition in case of fruit fractions was presented by DFF-3 (8 ± 0.25 mm clear, 10 ±
1.12 mm bald zone). Surfactin, the positive control established a 27 mm bald growth
inhibition zone.
3.6.4 Cytotoxicity assays
3.6.4.1 Brine shrimp lethality assay
Among the leaf and fruit fractions screened for cytotoxicity, most of the leaf fractions
exhibited a promising cytotoxic profile (Table 3.11). Among the leaf fractions, most
potent sample being DFL-6 exhibited 50% mortality (LC50) at 18.87 ± 0.87 µg/ml
followed by DFL-5, DFL-7 and DFL-4 with LC50 of 24.72 ± 0.76, 32.59 ± 0.85 and
33.07 ± 0.76 µg/ml respectively. The cytotoxicity level of the extracts was observed to
be concentration dependent as the mortality rate of brine shrimps decreased with the
increase concentration of the test sample. Among the fruit fractions, DFF-1 (LC50 =
67.43 ± 2.54 µg/ml), DFF-2 (LC50 = 74.12 ± 3.12 µg/ml) and DCF (LC50 = 158.12 ±
3.43 µg/ml) exhibited substantial cytotoxicity against the shrimp larvae.
3.6.4.2 Cytotoxicity against Hep G2 cell line
Among all the leaf samples, DFL-5 was most potent as it considerably inhibited the cell
line proliferation exhibiting 88.56 ± 3.45% cell inhibition at 20 µg/ml concentration
and an IC50 of 9.76 ± 0.87 µg/ml followed by DFL-8 exhibiting 79.24 ± 2.31%
inhibition (IC50 = 7.99 ± 0.21 µg/ml). Least cytotoxic potential was demonstrated by
the DFL-1 fraction exhibiting an inhibition of 12.00 ± 0.52%. In case of fruit fractions,
DFF-2 and DFF-3 demonstrated an inhibition of 65.00 ± 1.32 and 62.40 ± 2.81% with
an IC50 of 12.54 ± 1.10 and 21.85 ± 2.18 µg/ml respectively (Table 3.12).
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Table 3.7 Antibacterial activity of D. innoxia fractions.
Fractions
*Diameter of growth inhibition zone (mm) at 100 µg/disc
Gram negative Gram positive
K. pneumoniae MIC
µg/ml
P. aeruginosa MIC
µg/ml
B. subtilis MIC
µg/ml
S. aureus MIC
µg/ml
Leaf
DCL 14 ± 0.52# 100 12 ± 0.55 100 21 ± 0.45 3.33 -- --
DFL-1 7 ± 0.58 -- 7 ± 0.00 -- 8 ± 0.58 -- 8 ± 0.76 --
DFL-2 8 ± 0.58 -- 8 ± 0.58 -- -- --
DFL-3 11 ± 0.58# 100 11 ± 0.00 100 7 ± 0.50 --
DFL-4 10 ± 0.29 100 10 ± 0.58 100 7 ± 0.58 -- -- --
DFL-5 8 ± 0.58 -- -- -- 19 ± 0.58* 33.33 -- --
DFL-6 10 ± 0.58 100 -- -- 18 ± 0.58* 33.33 8 ± 0.58 --
DFL-7 12 ± 0.50# 100 -- -- 16 ± 0.76 33.33 7 ± 0.58 --
DFL-8 12 ± 0.58# 100 -- -- 12 ± 0.58 100 -- --
Fruit
DCF 12 ± 0.58 100 -- -- -- -- -- --
DFF-1 7 ± 0.58 -- 9 ± 0.58 -- 8 ± 0.58 -- -- --
DFF-2 9 ± 0.58 -- -- -- 7 ± 0.76 7 ± 1.00 100
DFF-2 7 ± 0.58# 100 -- -- 7 ± 0.58 -- 8 ± 0.58 --
DFF-4 7 ± 0.00 -- 9 ± 0.58 -- 8 ± 0.58 -- 7 ± 0.58 --
DFF-5 8 ± 0.58 -- 8 ± 0.58 -- -- -- -- --
DFF-6 10 ± 0.29 100 -- -- -- -- 7 ± 1.15 --
DFF-7 7 ± 0.58 -- -- -- -- -- 7 ± 1.00 --
Ciprofloxacin
(20µg/disc)
17 ± 1.6 0.06 10 ± 0.07 0.06 17 ± 0.95 0.8 15 ± 0.85 0.125
Cefixime
(20µg/disc)
19.5 ± 1.3 0.2 21 ± 0.85 0.02 24.6 ± 0.69 0.8 22.5 ± 0.11 0.25
DMSO -- -- -- --
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Table 3.8 Antifungal spectrum of D. innoxia fractions.
Fractions *Diameter of growth inhibition zone (mm) at 100 µg/disc
A. flavus A. niger F. solani A. fumigatus Mucor sp.
1. Leaf
DCL 9 ± 0.45 -- -- -- --
DFL-1 8.5 ± 0.54 -- -- -- --
DFL-2 10 ± 0.43# -- 7 ± 0.58 -- 9 ± 0.57#
DFL-3 8 ± 0.45 -- -- -- --
DFL-4 7 ± 0.53 -- -- -- 9 ± 0.54#
DFL-5 8 ± 0.56 -- -- -- --
DFL-6 10 ± 0.45# 10 ± 0.54 6 ± 0.58 -- --
DFL-7 13 ± 0.65* 9 ± 0.57 8 ± 0.58 -- 7 ± 0.56
DFL-8 8 ± 0.56 12 ± 0.43* 9 ± 0.58 -- --
2. Fruit
DCF -- -- -- -- --
DFF-1 -- -- -- -- --
DFF-2 -- -- -- -- --
DFF-3 -- -- -- -- 9 ± 0.58
DFF-4 9 ± 0.58# -- -- -- --
DFF-5 9 ± 0.58# 8 ± 0.57 -- -- --
DFF-6 7 ± 0.00 -- -- -- --
DFF-7 7 ± 1.73 -- -- -- --
Clotrimazole 30 ± 0.58 35 ± 2.30 28 ± 0.00 34 ± 0.58 --
DMSO -- -- -- -- --
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Table 3.9 α-amylase inhibitory activity of D. innoxia fractions.
%α-amylase inhibition at 200 µg/ml
Leaf Fruit
Fractions %inhibition Fractions %inhibition
DCL 15.32 ± 1.21 DCF 35.43 ± 2.13
DFL-1 10.48 ± 0.65 DFF-1 2.01 ± 0.03
DFL-2 10.61 ± 0.97 DFF-2 17.80 ± 0.13
DFL-3 1.21 ± 0.01 DFF-3 42.78 ± 1.21
DFL-4 2.22 ± 0.01 DFF-4 52.72 ± 1.34
DFL-5 13.03 ± 1.01 DFF-5 38.01 ± 1.21
DFL-6 16.49 ± 1.01 DFF-6 27.80 ± 1.11
DFL-7 18.41 ± 1.54 DFF-7 29.56 ± 1.21
DFL-8 13.30 ± 1.24
Values are represent mean ± standard deviation from triplicate analysis. The IC50 of acarbose (positive
control) was 33.73 ± 0.12 µg/ml.
Table 3.10 Protein kinase inhibitory potential of D. innoxia fractions.
Protein kinase inhibition
Leaf Fruit
Fractions *Diameter (mm) at 100
µg/disc
MIC
(µg)
Fractions Diameter (mm) at 100
µg/disc
MIC
(µg)
Clear zone Bald zone Clear zone Bald zone
DCL 13 ± 1.21 18 ± 1.45 50 DCF -- -- --
DFL-1 -- -- -- DFF-1 7 ± 0.34 12 ± 1.56 --
DFL-2 8 ± 0.56 11 ± 1.25 100 DFF-2 -- -- --
DFL-3 8 ± 1.56 10 ± 1.87 100 DFF-3 8 ± 0.25 10 ± 1.12 100
DFL-4 -- 11 ± 2.16 100 DFF-4 -- -- --
DFL-5 7 ± 0.45 12 ± 1.13 100 DFF-5 -- -- --
DFL-6 9 ± 0.96 16 ± 0.76£ 100 DFF-6 -- -- --
DFL-7 11 ± 1.21 16 ± 2.23£ 50 DFF-7 7 ± 0.21 9 ± 0.56 --
DFL-8 8 ± 0.14 14 ± 1.76£ 100
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Table 3.11 Cytotoxicity assessment of D. innoxia fractions using brine shrimp lethality assay.
Values represent mean ± standard deviation of triplicate analysis. --: no activity. Fractions with statistically significant results are indicated by a superscript. In all the results *
indicate (P < 0.01) and # indicate (P < 0.05). IC50 of Doxorubicin (positive control) = 5.93.± 0.23 µg/ml.
Brine shrimp cytotoxicity
Leaf Fruit
Fractions %Mortality LC50
(µg/ml)
Fractions %Mortality LC50
(µg/ml) 200 100 50 25 200 100 50 25
DCL 100 ± 0.00 88.87 ± 2.45 82.12 ± 2.34 62.21 ± 0.93 70.44 ± 1.21 DCF 72.65 ± 1.34 56.00 ± 3.45 -- -- 158.12 ± 3.43
DFL-1 -- -- -- -- > 200 DFF-1 85.76 ± 3.21 72.34 ± 2.12 56.13± 2.12 -- 67.43 ± 2.54
DFL-2 -- -- -- -- > 200 DFF-2 80.12 ± 1.21 74.87 ± 1.65 -- -- 74.12 ± 3.12
DFL-3 -- -- -- -- > 200 DFF-3 -- -- -- -- > 200
DFL-4 90.90 ± 2.23 77.77 ± 1.54 72.72 ± 2.45 20 ± 1.01 33.07 ± 0.76# DFF-4 -- -- -- -- > 200
DFL-5 100 ± 0.00 90 ± 0.93 70 ± 2.13 50 ± 2.34 24.72 ± 0.76* DFF-5 -- -- -- -- > 200
DFL-6 100 ± 0.00 70 ± 1.76 30 ± 1.87 0 18.87 ± 0.87 DFF-6 -- -- -- -- > 200
DFL-7 70 ± 1.43 63.63 ± 1.21 50 ± 1.22 50 ± 1.87 42.59 ± 0.85# DFF-7 -- -- -- -- > 200
DFL-8 80 ± 3.21 70 ± 2.54 50 ± 1.65 10 ± 1.21 45 ± 0.87
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Table 3.12 Hep G2 cytotoxicity of D. innoxia fractions.
Hep G2 cytotoxicity (µg/ml)
Fractions
%Inhibition IC50
Fractions
%Inhibition IC50 20 20
Leaf Fruit
DCL 80.87 ± 4.54 10.81 ± 0.92£ DCF 60.45 ± 2.34 18.98 ± 2.12
DFL-1 12.00 ± 0.52 > 20 DFF-1 5.00 ± 0.12 > 20
DFL-2 16.73 ± 2.34 > 20 DFF-2 65.00 ± 1.32 12.54 ± 1.10
DFL-3 30.87 ± 1.54 > 20 DFF-3 62.40 ± 2.08 21.85 ± 2.18
DFL-4 46.81 ± 3.23 > 20 DFF-4 15.34 ± 4.32 > 20
DFL-5 88.56 ± 3.45 9.76 ± 0.87 DFF-5 17.32 ± 0.53 > 20
DFL-6 86.81 ± 3.23 12.36 ± 0.32£ DFF-6 11.00 ± 0.12 > 20
DFL-7 71.23 ± 0.67 18.23 ± 1.34 DFF-7 18.00 ± 1.13 > 20
DFL-8 79.24 ± 2.31 7.99 ± 0.21£
Values are represented as mean ± standard deviation of triplicate analysis. £Means difference is
significant at P < 0.05.
3.7 Summary
Among all the samples tested for scavenging potential, highest free radical quenching
potential was manifested by the most polar DFL-8 and DFF-7 fractions of leaf and fruit
respectively as compared to their preparative crude extracts which shows that polar
components are the major contributors to the radical scavenging potential of DCF and
DCL. The phosphomolybdenum based total antioxidant capacity was highest in case of
DFL-7 and DFL-8 fractions of leaf whereas, in case of fruit fractions it was highest in
DFF-7 and DFF-6 fractions. In case of TRP, maximum reducing potential was
manifested DFL-7 and DFL-8 fractions of leaf whereas, in case of fruit fractions it was
highest in DFF-7 fraction. Among the leaf samples, maximum activity was manifested
by the DFL-4 leaf fraction lagged by polar fractions while the nonpolar fractions did
not exhibit any leishmanicidal activity. The DFL-4 fraction manifested minimum IC50
value and was therefore, identified as the hit fraction for the isolation of lead
antileishmanial principles. Disc diffusion assay was employed to determine the
antibacterial spectrum of test samples. Among the leaf samples, the most prominent
antibacterial activity was observed against B. subtilis as compared to other tested strains
with maximum zone of growth inhibition being formed around the preparative DCL
extract loaded disc lagged by DFL-5 and DFL-6 indicating the antibacterial proficiency
of moderately polar fractions as compared to nonpolar and polar fractions. Among the
fruit samples, a moderate antibacterial activity against K. pneumoniae was exhibited by
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most of the samples with maximum ZOI being recorded around the DCF loaded disc.
The data indicate that 37.50% of the leaf fractions were active against A. flavus, 25%
against A. niger whereas, none of the leaf fractions were active against F. solani, A.
fumigatus and Mucor sp. The maximum ZOI of 13 ± 0.65 mm around A. flavus was
demonstrated by DFL-7. Among the fruit fractions a moderate antifungal activity was
observed only against A. flavus while there was little or no activity against the other
tested strains. Overall, the leaf samples manifested better kinase inhibitory prospective
as compared to the fruit fractions with maximum bald phenotype being observed around
the DCL preparative extract lagged by DFL-6 and DFL-7 fractions. Among the fruit
samples, only DFF-1, DFF-3 and DFF-7 displayed a moderate protein kinase inhibition.
Cytotoxic potential of the samples was evaluated using brine shrimp lethality assay and
in vitro cell viability assay against cell lines. Among all the leaf samples analysed,
minimum 50% inhibitory concentration was obtained in case of DFL-5, DFL-6 and
DFL-7. Among all the leaf samples, DFL-8 most considerably inhibited the Hep G2
cell line proliferation lagged by DCL and DFL-5. Overall, the results suggest that polar
fractions such as DFL-5 till DFL-8 are the hit fractions for the isolation of cytotoxic
principles.
Section-3: Biological Evaluation and Characterization of Isolated Compounds
3.8 Biological evaluation
In the present study the method of bioactivity directed fractionation trailed by random
isolation from bioactive fractions was adopted. Total three compounds was isolated and
purified from the bioactive fractions of D. innoxia leaf and fruit and were then subjected
to the following bioassays, the results of which have been presented as under:
3.8.1 Antileishmanial assay
The antipromastigote activity of the purified compounds against L. tropica has been
summarized in table 3.13. Among all the samples analyzed CL-3 exhibited a
noteworthy leishmanicidic potential of 95.76 ± 5.67% and an IC50 8.34 ± 1.21 µg/ml.
Amphotericin B (IC50 = 0.01 µg/ml) was used as positive control.
Table 3.13 Antileishmanial potential of compounds isolated from D. innoxia.
Antileismanial assay
Compound code % inhibition at 20 µg/ml IC50 (µg/ml)
CL-1 15.21 ± 4.22 > 20
CL-3 95.76 ± 5.67 8.34 ± 1.21
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CF-5 32.65 ± 3.85 > 20
Values are presented as mean ± standard deviation from triplicate investigation.
3.8.2 Protein kinase inhibition assay
The protein kinase inhibition potential of the compounds isolated from D. innoxia is
shown in table 3.14. Among all the samples analyzed, a moderate inhibition zone of 8
± 2.21 mm clear, 11 ± 1.21 mm bald phenotype was observed around the disc loaded
with CL-1 lagged by CL-3 (7 ± 0.52 mm clear, 10 ± 2.76 mm bald ZOI). The MIC for
both CL-1 and CL-3 was found to be 20 µg/disc.
Table 3.14 Protein kinase inhibitory potential of compounds isolated from D. innoxia.
Protein kinase inhibition assay
code
*Diameter of zone of inhibition (mm) at 20 µg/disc
Clear zone Bald zone MIC
(µg/disc)
CL-1 8 ± 1.21 11 ± 2.21 20
CL-3 7 ± 0.52 10 ± 2.76 20
CF-5 7 ± 1.43 8 ± 3.11 20
3.8.3 Cytotoxicity against cell lines
In the current study, the cytotoxic activity of the compounds was evaluated by using
SRB assay and MTT based in vitro cytotoxicity assays (table 3.15). Among all the
test samples, only CL-3 isolated from the D. innoxia leaf part exhibited substantial
cytotoxicity against the MCF-7, LU-1 and PC3 cell lines with and IC50 of 4.3 ± 0.93
and 6.9 ± 1.3 and 0.01 ± 0.001 µg/ml, respectively. Doxorubicin was used as positive
control.
Table 3.15 Cytotoxicity assessment of compounds isolated from D. innoxia against
MCF-7, LU-1 and PC3.
Compound
code
MCF-7 LU-1 PC3
% survival
at 20 µg/ml
IC50
(µg/ml)
% survival
at 20 µg/ml
IC50
(µg/ml)
% survival
at 20 µg/ml
IC50
(µg/ml)
CL-1 222.3 ± 23.30 > 20 149.0 ± 16.30 > 20 29.23 ± 3.31 18.97 ± 2.59
CL-3 10.5 ± 16.2 4.3 ± 0.93 24.35 ±0.5 6.9 ± 1.3 17.34 ± 2.87 0.01 ± 0.001
CF-5 221 ± 18.4 > 20 152.26 ± 19.5 > 20 77.39 ± 3.98 > 20
All the tests were executed in triplicate. IC50 = 50% inhibitory concentration. Doxorubicin was utilized
as positive control with IC50 values of 3.2 (MCF-7), 4.5 (LU-1) and 2.95 (PC3) μg/ml respectively.
3.8.4 Cancer chemopreventive assays
3.8.4.1 Inhibition of TNF-α activated nuclear factor-kappa B (NFĸB) assay
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Out of the three isolated compounds, CF-5 isolated from fruit part of D. innoxia
demonstrated remarkable cancer chemopreventive activity through inhibition of NFĸB
with an inhibition percent of 92.18 ± 5.1% at 20 µg/ml (IC50 1.1 ± 0.9 µg/ml) and
exhibited 98.9 ± 5.54% cell survival at 20 µg/ml concentration. None of the compounds
isolated from D. innoxia leaf part i.e. CL-1 and CL-3 exhibited any NFĸB inhibitory
activity (table 3.16). In this assay Nα-tosyl-L-phenylalanine chloromethyl ketone
(TPCK) was used as a positive control (IC50 = 5.09 µM).
3.8.4.2 Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-
activated murine macrophage RAW 264.7 cells (iNOs) assay
In the current study, inhibition of lipopolysaccharide (LPS)-activated nitric oxide (NO)
production in murine macrophage RAW 264.7 cells was used as an indirect indicator
of the inhibition of iNOS. Among all the compounds screened, CF-5 isolated from fruit
part of D. innoxia exhibited substantial potential for the inhibition of NO production
with an inhibition potential of 87 ± 4.1% at 20 µg/ml (IC50 = 3.3 ± 0.6 µg/ml) and
demonstrated 85.2 ± 5.8% cell survival at the same concentration whereas, CL-3
demonstrated a moderate inhibitory potential of 66 ± 3.4% and an IC50 of 18.5 ± 1.8
µg/ml (table 3.16). Na-L monomethyl arginine (L-NMMA) was used as positive control
in this assay (IC50 = 19.7µM).
Table 3.16 Results of TNF-α activated NFĸB inhibition and inhibition of NO
production assays.
Cancer chemopreventive assays
Compound TNF-α activated NFĸB inhibition Inhibition of NO production
% inhibition
(20 µg/ml)
% survival
(20 µg/ml)
IC50
(µg/ml)
% inhibition
(20 µg/ml)
% survival
(20 µg/ml)
IC50
(µg/ml)
CL-1 1.03 ± 8.1 82.5 ± 5.40 > 20 34.00 ± 5.12 76.4 ± 2.3 > 20
CL-3 50.00 ± 7.1 50.00 ± 8.60 > 20 66 ± 3.4 78.3 ± 5.2 18.5 ± 1.8
CF-5 92.18 ± 5.1 98.9 ± 5.40 1.1 ± 0.9 87 ± 4.1 85.2 ± 5.8 3.3 ± 0.6
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3.9 Characterization of isolated compounds
Various NMR techniques (1H and 13C) along with X-ray crystallography were
performed in order to determine the structures of isolated compounds. Resulting spectra
along with data of all compounds have been presented as follows.
3.9.1 Structure elucidation of CL-1
Compound CL-1 was isolated as mixture of white amorphous powder and crystals from
isolation of DFL-2 as described in detail in chapter 2, section-2, page no. 58. Single
crystal XRD studies led to identification of the compound revealed the presence of two
unit cells of compound conjugated with a water molecule. On the basis of 3D picture
of unit cell and after comparing its dimensions with previously reported structure, the
compound CL-1 was identified and elucidated. The molecular weight of CL-1 was
414.70. The crystallography data and structure refinement studies have been
summarized in table 3.17. The 3D structure of the molecule has been presented in figure
3.11 (a). Selected bond angles and lengths have been described in Appendix I. The
crystal system of the unit cell existed in orthorhombic space group. CL-1 possessed a
steroidal skeleton with a conjugated side chain. The main skeleton was composed of
three six-membered (A, B, C) and one five-membered ring (D). Ethyl group on position
24 was observed with β-orientation whereas methyl group at position 21 was present in
α-configuration. Further, the hydroxyl group at position 3 and methyl groups at
positions 10 and 13 in the steroidal nucleus were also observed with β-orientation. The
length of double bond present between carbons 5 and 6 was 1.290 (10) Å. The structure
was further confirmed by means of NMR spectroscopy. 1H spectrum presented that
hydrogen at position 3 showed peak at δH 3.53 (1H, m). The methyl groups at positions
18 and 19 showed characteristic single peaks at δH 0.68 and δH 1.01 respectively.
Several other peaks were also observed at δH 1.86 (m, H-1), δH 2.00 (m, H-8), δH 2.28
(2H, m, H-4) and δH 5.36 (1H, d, H-6). The 13C spectrum of CL-1 indicated the presence
of eight methyl peaks at 12.03, 12.15 19.15, 19.56, 18.87, 19.98, and 42.48 ppm. Two
olefinic carbon peaks were observed at δC 140.93 and δC 121.88. Overall, seven
methyls, ten methylenes, nine methines and three quaternary carbon peaks were
observed in the spectrum. The peaks were assigned on the basis of the comparison with
the reported literature (table 3.18). On the basis of spectroscopic and crystallographic
analyses, the name (24R) ethylcholest-5en-3β-ol or β-sitosterol was given to the
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compound CL-1. The 1H and 13C spectra have been presented in figures 3.12, 3.13 and
3.14 whereas, the structure has been shown in figure 3.11 (b).
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Figure 3.11 (a) 3D structural model of CL-1 as proposed by XRD (hydrogen atoms
have been removed for clarity (b) Elucidated structure of CL-1.
a
b
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Figure 3.12 13C (100 MHz, in CDCl3) spectrum of CL-1.
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Figure 3.13 Enlarged 13C spectra (100 MHz, in CDCl3) of CL-1, 0-70 ppm.
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Figure 3.14 1H spectrum (400 MHz, in CDCl3) of CL-1.
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3.9.2 Structure elucidation of CL-3
Compound CL-3 was isolated as mixture of white amorphous powder and crystals from
isolation of DFL-4 as described in detail in chapter 2, section-2, page no. 60. The
melting point of the isolated compound was observed at 277-281ºC. The molecular
weight of CL-3 was 436.57 as determined by x-ray crystallography. The
crystallography data and structure refinement studies have been summarized in table
3.19. The 3D structure of the molecule has been presented in figure 3.17(a). Selected
bond angles and lengths have been described in Appendix II. The unit cell of the
compound possessed monoclinic shape. Compound CL-3 was observed to possess α-
β-unsaturated steroidal δ-lactone skeleton. Main skeleton was composed of 3 six
membered and 1 five membered carbon containing rings, termed as A, B, C, and D
respectively. Methyl groups at positions 18, 19 and 28 were observed at β orientation.
Two double bonds with bond lengths 1.323 (6) Å and 1.327 (6) Å were observed in
between C-3 and C-4 as well as C-5 and C-6 respectively. Hydrogen atom at position
20 also existed in β orientation. The epoxide ring at position 21 showed β orientation
whereas methylene group at position 23 was observed in α-orientation. NMR
spectroscopic analysis was also performed to confirm the elucidated structure. 1H
spectrum of CL-3 showed the presence of a broad singlet at δH 4.65 (figure 3.16).
Protons on methyl groups also appeared as singlets at δH 0.71 (H-18), δH 1.23 (H-19)
and δH 1.44 (H-28). Out of five vinylic protons, two protons showed singlets with fine
splitting at δH 6.75 and δH 6.02. These peaks were assigned to terminal methylene
protons bound to side chain. The oxymethyl group (-CH2-O, H-21) gave signal at δH
3.73 (dd). The 13C spectrum of compound CL-3 showed the presence of olefinic carbons
which resonated at δC 145.12 and δC 124.61 (figure 3.15). Characteristic methyl peaks
were observed at δC 12.75 (C-18) and 25.63 (C-28). The carbon in methylene group at
C-23 resonated at δC 33.26 whereas, C-12, C-13, C-14, C-15, C-16, C-17 and C-24
showed peaks at δC 39.87, δC 42.86, δC 56.00, δC 24.09, δC 26.52, δC 47.62 and δC 69.34
respectively. On the basis of XRD and NMR analyses as well as after comparing the
results with reported literature, the name 21,24-epoxy-22-hydroxy-1-oxo, ergosta-3, δ-
lactone, (22R)-5, 25(27)-trien-26-oic acid and the trivial name isowithametelin was
given to CL-3. The 1H and 13C spectra have been presented in figures 3.15 and 3.16
respectively. The elucidated structure of CL-3 has been presented in figure 3.17(b).
Ch.3: Results
94
Figure 3.15 13C (100 MHz, in CDCl3) spectrum of CL-3
Ch.3: Results
95
Figure 3.16 1H spectrum (400 MHz, in CDCl3) of CL-3.
Ch.3: Results
96
``
Figure 3.17(a) 3D structural model of CL-3 as proposed by XRD (hydrogen atoms
have been removed for clarity (b) Elucidated structure of CL-3.
3.9.3 Structure elucidation of CF-5
Compound CF-5 was isolated as white crystals and its molecular weight was
determined as 438.71 by x-ray crystallography. It was isolated from DFF-2 as described
in detail in chapter 2, section-2, page no. 61. Melting point of the isolated compound
was 138ºC. The crystallography data and structure refinement studies have been
summarized in table 3.20. The 3D structure of the molecule has been presented in
figure 3.18. Selected bond angles and lengths have been described in appendix III. The
unit cell of compound CF5 was orthorhombic in shape. Main structure belonged to
pentacyclic triterpenoidal class. XRD studies showed methyl groups at positions 6, 8.
10 and 17 in β-orientation. On the other hand, methyl group at position 14 was observed
in α-orientation. The bond length of unsaturated bond at position 12 was 1.334(4) Å.
a
b
Ch.3: Results
97
On the basis of these studies, the name 4a, 4b, 6b, 8b, 10b, 14a) 7,10 dimethyl
dinoroleanan-12 en-3-one was given to the compound CF5.
3.10 Summary
Total three compounds (CL-1, CL-3 and CF-5) were isolated using normal phase
vacuum and medium pressure column chromatography as the isolation technique.
Compound CL-3 demonstrated noteworthy antileishmanial activity (IC50 8.34 ± 1.21
µg/ml) and cytotoxic potential against MCF-7 (IC50 4.3 ± 0.93 µg/ml), LU-1 (6.9 ±
1.3 µg/ml) and PC3 (0.01 ± 0.001 µg/ml) cancer cell lines. In protein kinase
inhibition assay, maximum bald growth inhibition zone of 11 ± 2.21 mm was
formed around the CL-1 loaded disc whereas, CF-5 demonstrated remarkable cancer
chemopreventive activity through inhibition of NFκB and NO production with IC50 1.1
± 0.9 and 3.3 ± 0.6 µg/ml, respectively. Crystallography and NMR spectroscopy
characterized the structure of CL-1, CL-3 and CF-5 as β-sitosterol, isowithametelin and
(4a, 4b, 6b, 8b, 10b, 14a) 7, 10 dimethyl dinoroleanan-12 en-3-one (new terpenoid),
respectively.
Figure 3.18 (a) 3D structural model of CF-5 as proposed by XRD Thermal ellipsoids
are drawn at 30 % probability level. The H-atoms are shown as small circles of arbitrary
radii (b) Elucidated structure of CF-5.
a
b
Ch.3: Results
98
Table 3.19 Crystal data and structure refinement for CL-3.
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99
Table 3.20 Crystal data and structure refinement for CF-5.
Ch.4: Discussion
100
4. Discussion
Natural products plants have undergone a revival in drug discovery programs, owing to
their inherent legitimacy in terms of chemical assortment over combinatorial compound
libraries as well as their drug like properties. Natural products embracing the
phenomenon of biodiversity have formulated a number of unique chemical entities for
the discovery of pharmaceutical compounds over the past century (Lee, 2010).
Plants have been benefitting mankind since their origin. The active molecules derived
from the plants have served as the basis of new drug discovery. Plants not only have
fulfilled the nutritive requirements of human beings but also the curative one. They are
a source of plentiful molecules possessing biological activities. They synthesis a wide
variety of biochemicals called as secondary metabolites which are not indispensable for
the reproduction and growth of plants but when administered in the human body, they
exert multiple pharmacological effects (Rout et al., 2009).
Nature is the best pharmacy and has the potential to treat various ailments of human
beings. Plant based medicines are still the mainstay for the treatment of many complex
ailments like inflammatory disorders, neoplasia, diabetes and oxidative-stress induced
disorders (Tabassum et al., 2017).
Drug development from plants in its contemporary understanding relies on pure
chemical moieties for which traditional knowledge has always played a fundamental
role. Morphine, taxol, vincristine, artemisinin, triptlide, celastrol, and capsaicin are
among prime compounds that demonstrated the potential of turning traditional remedies
into modern drugs (Heinrich, 2010). Therefore, ethnopharmacological approach
provides ideal prospects to limit the gigantic multiplicity of possible leads to more
valuable hits. Thus ethnopharmacological approach provides ideal prospects to limit
the gigantic multiplicity of possible leads to more valuable hits. One such
ethnomedicinally important genus is Datura.
Empirical or systematic screening of plant extracts and pure compounds to explore
novel leads plays a focal role in drug development process. Recent advancements in
drug discovery research from medicinal plants encompass a multidimensional approach
combining phytochemical, botanical, biological and molecular techniques. Thus,
natural product drug discovery requires a persistent progress in the pace of screening,
Ch.4: Discussion
101
purification and structure determination processes in order to be competitive with the
other drug discovery methods.
Downy Thorn Apple, Angel's-trumpet, Indian Apple or Thorn Apple with the scientific
name of Datura innoxia Mill. (Solanaceae), is a shrub that grows in United States,
China, Caribbean Islands, Mexico and Asia. It is amongst the popular medicinal plants
which has been traditionally used (Maheshwari et al., 2013). In Pakistan, it is common
to roadsides and weedy places and is locally named as Dhatura. Soft short greyish hairs
cover the leaves and stem rendering the plant a greyish look. The flowers are trumpet-
shaped, white and are 12–19 cm long while the fruit spiny capsule which is egg shaped
(5 cm in diameter). The fruit splits open when it is fully ripped.
D. innoxia surmounts a distinct stature in Ayurveda as all its parts namely roots,
flowers, leaves, stems, seeds and fruits have been used in the treatment of insanity,
rabies, leprosy, etc. Nevertheless, the higher doses of the extract may result in delirium,
acute poisoning and may cause death. The bioactive principles in various plant parts of
D. innoxia include hyoscyamine, withanolides, tropanes, atropine and scopolamine
(Vermillion et al., 2011).
In the present study Datura innoxia an ethnomedicinally important plant was selected
for lead compound identification. Extracts prepared from different plant parts of D.
innoxia were subjected to a battery of phytochemical and in vitro biological assays in
order to optimize extraction solvent and plant part for preparative scale extraction,
isolation and purification of lead compounds. The purified bioactive compounds were
then characterized for structure determination.
Section-1: Extraction Optimization from D. innoxia Mill.
Extraction being a significant step in the expedition of phytochemical screening for the
discovery of bioactive components from plants takes in to account the separation of
therapeutically active components of living tissues from the inactive components by
employing various solvents. The ultimate goal of extraction optimization of crude
extracts is to attain the desired bioactive constituents and to exclude the inert
components using a range of selective solvents termed as menstruum. The resulting
extract obtained in crude form may be ready for use as a medicinal cocktail or it may
be further partitioned to isolate distinct bioactive moieties. In the preliminary
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102
phytochemical and in vitro biological investigation studies, extracts of the medicinal
plant D. innoxia were subjected to standard bioassays to determine their possible
therapeutic proficiency. Phytochemical profile included determination of total phenolic
and flavonoid contents using standard colorimetric assays and specific phenol
quantification using RP-HPLC analysis. In vitro biological evaluation was performed
to establish antioxidant, antimicrobial, enzyme inhibitory and cytotoxic spectrum of
leaf, stem and fruit parts of D. innoxia.
4.1 Effect of extraction solvent on the extract yields
Maceration using a total of 12 solvent systems from polarity index of 0.1-10 was
accompanied with occasional use of high frequency and high intensity sound waves
(ultrasonication) as the extraction technique to recover desirable compounds from plant
matrices. Upon extraction a total of 36 extracts from leaf, stem and fruit were obtained.
Ultrasonication aided maceration was employed because chemical and physical
characteristics of the materials are transformed due to the interaction and dissemination
of ultrasound waves disrupting the cell walls, thereby, augmenting solvent’s mass
transport across the plant cells (Dhanani et al., 2013). A number of factors effects the
extraction efficiency such as type of method employed, particle size of plant material,
chemical characteristics of phytoconstituents in the matrix and solvent used. Extraction
yield also depends on a number of parameters including extraction solvent polarity,
temperature, time, pH, and the sample characteristics. Solvent and sample composition
are the most important parameters that effects the extract yield under constant
conditions of temperature and time of extraction (Xu and Chang, 2007). In the current
study, it was observed that as the polarity of extraction solvent changed from highly
polar water to nonpolar n-hexane, the extract yields decreased drastically. As extraction
is the first critical step in drug discovery process from plants; therefore, a wide range
of extraction solvent polarities and sonication followed by maceration as extraction
technique was employed. It has also been suggested from the extraction yield
optimization studies of herbal material that as a sample preparation method, maceration
under sonication is a superlative choice when considering the time/yield ratio
(Celeghini et al., 2001).
4.2 Phytochemical analysis
Ch.4: Discussion
103
Phytochemical analysis refers to the qualitative or quantitative assessment of the
medicinally active secondary metabolites from plant matrices. The phytochemical
screening is an effective tool for the extrapolation of chemical profile of plants for their
possible therapeutic implications. Medicinal plants have been known for centuries for
their numerous phytochemicals like tannins, glycosides, alkaloids, flavonoids,
polyphenols and many other. Nature is considered as an abundant pharmaceutical store
existing on this planet owing to their ability to produce various secondary metabolites
with a broad spectrum of bioactivities. Employing various phytochemical analysis
techniques many plant based chemicals have been characterized.
Chemotaxonomy of therapeutically important plants can be made with the help of
phytochemical analysis procedures. Phytochemicals are non-nutritive plant
constituents which play a prophylactic and defensive job against health complications
both in animals and plants. Thousands of chemicals proven to be active against pests
and diseases have been discovered (Fatima et al., 2015). In qualitative screening,
various phytoconstituents such as reducing sugars, steroids, alkaloids, flavonoids,
saponins, tannins, glycosides, anthraquinones, amino acids, triterpenoids etc. are tested
for their presence or absence. Quantitaive phytochemical analysis involve total content
determination of various phytoconstituents and include assays such as total alkaloid,
total phenolic content, total flavonoid, total saponin and total tannins contents
determination etc. In the current analysis, total phenolic and flavonoid content was
estimated by using colorimetric assays and various polyphenols were quantified using
RP-HPLC analysis against various external standards.
For the phytochemical evaluation of the leaf, stem and fruit parts of D. innoxia,
colorimetric assays were employed to determine the total phenolics and flavonoids
content whereas, specific polyphenols were quantified through RP-HPLC analysis.
The plant phenolics possessing antioxidant properties have an imperative role in
contesting oxidative stress, cytotoxicity and cellular death by quenching free radicals
or chelating trace elements and by this means fortifying the antioxidant defences
(Kumar et al., 2013). In most of the medicinal plants, phenolic and polyphenolic
compounds are the chief contributors to the antioxidant activity. The total phenolic
content was estimated using standard Folin-Ciocalteu (FC) reagent method (Fatima et
al., 2015b). The total phenols in different plant parts ranged from 29.91 ± 0.12 mg
GAE/g DW for the highly polar aqueous to 2.5 ± 0.12 mg GAE/g DW nonpolar n-
Ch.4: Discussion
104
hexane, which is in agreement with the results of previous studies that quantification of
phenolic compounds in plant extract is influenced by the chemical nature of the
extraction solvent (Kumaran et al., 2007). The greater extract yields observed in case
of aqueous extracts might be attributed to a higher solubility of proteins and
carbohydrates in water. These results affirm the effect of polarity on extraction
efficiency i.e. the extract yield decreased as the solvent polarity decreased which is in
agreement with the results of previous studies (Kolar et al., 2002). The antioxidant
mechanism of action of flavonoids is through scavenging or chelation (Jafri et al.,
2014). In search for anticancer remedies a number of reports from laboratories,
epidemiologic investigations and human clinical trials have demonstrated the role of
flavonoids in cancer chemoprevention and chemotherapy. (Tabassum et al., 2017). It is
also reported that flavonoids obtained from plants have the property of hampering
hydrolytic and oxidative enzymes and also have anti-inflammatory activity
(Atanassova et al., 2011). The aqueous fruit extract of D. innoxia unveiled highest total
flavonoid content as compared to other plant parts analysed. A positive correlation
(correlation coefficient; R2 = 0.9137 for leaf, 0.8026 for stem and 0.8999 for fruit) was
found to be present between the phenolic and flavonoid contents suggesting that the
antioxidant potential of phenols might be attributed to the presence of flavonoids (Jafri
et al., 2014). RP-HPLC based profiling was used for quantitative analysis of selected
plant phenolics and the chromatographic finger printing was done by comparing the
retention time and UV spectra of reference compounds with those of the test samples.
A significant amount of catechin, myricetin, quercetin, rutin and caffeic acid was
quantified in some of the analyzed extracts. Among the leaf extracts a substantial
amount of catechin and apigenin was present in the M and MC extracts. Significant
amount of catechin and apigenin were also quantified in ethanolic extract of fruit. The
presence of all these plant metabolites draw a parallel correlation of its potential with
their known bioactivities e.g. rutin is one of the phenolic compounds found in the
invasive plant species and contributes to its antibacterial and antioxidant properties,
caffeic acid outclassed the other antioxidants in alleviating aflatoxin synthesis by more
than 95% and apigenin induces autophagy in leukemia cells, which may support a
plant’s possible chemopreventive and anticancer role (Dai and Mumper, 2010).
4.3 Biological evaluation
4.3.1 Antioxidant potential
Ch.4: Discussion
105
Oxidation is a mandatory natural phenomenon in biological. The best antioxidant
activity in DPPH assay was demonstrated by the aqueous extract of leaf while, it was
lowest for n-hexane. Ethyl acetate-acetone extract of stem exhibited highest scavenging
activity followed by ethyl acetate and aqueous extracts. Among all the fruit extracts the
most potent radical scavenging potential was exhibited by the aqueous extract. The
relationship between total contents of polyphenols and the free radical scavenging
capacity as studied by many authors demonstrated that a linear correlation exists
between them (Oliveira et al., 2012). In the current analysis, a significant correlation
was also found between the radical scavenging capability and the total phenolic content
(leaf: R2 = 0.7094, stem: R2 = 0.7739, fruit: R2 = 0.8243). Therefore, it can be inferred
that the various polyphenols in D. innoxia extracts may be responsible for the radical
scavenging mediated antioxidant activity.
The total antioxidant capacity (TAC) of various leaf, stem and fruit extracts was
determined by phosphomolybdenum based colorimetric assay. The method relies on
the reduction of Mo (VI) to Mo (V) by the antioxidant mediators and the consequent
formation of a green coloured phosphate/Mo (V) complex with a maximal absorption
at 695 nm (Jafri et al., 2014). Maximum TAC was displayed by the aqueous extract of
the leaf, ethanol extract of the stem and ethanol-chloroform extract of the fruit. The
correlation between total phenolic content and antioxidant capacity was determined and
it was found to be linear with correlation coefficient, R2 of 0.7821 (leaf), 0.8544 (stem),
0.8702 (fruit) respectively. These results are in agreement with the previous studies
which showed that high phenolic content increases the antioxidant activity (Abdel-
Hameed, 2009, Brusotti et al., 2014). The redox property of a compound has a
significant role in neutralizing and absorbing free radicals which is manifested by the
transformation of Fe3+ to Fe2+ by extract/samples. Phenolic compounds have been
reported to serve as electron donors (Khan et al., 2015). This fact may elucidate current
interest in the employment of the reducing power assay to determine the antioxidant
capacity (reduction potential) of the solvent extracts of D. innoxia using ferric ion
reducing power assay. The assay relies on the conversion of ferric ion of Potassium
ferricyanide [K3Fe (CN)6] to ferrous ion (Fe3+ to Fe2+). The Potassium ferricyanide form
complex with antioxidant compound present in the extract in the acidic environment
causing the conversion of yellow colour of the test solution to green (Majid et al., 2015).
In D. innoxia crude extracts, the maximum extraction efficiency in terms of highest
Ch.4: Discussion
106
reducing power was achieved in the aqueous extract of leaf, ethyl acetate extract of
stem and methanol-ethyl acetate extract of fruit. In the present exploration a positive
correlation was also found to exist between the reducing power and antioxidant
potential of all the extracts with correlation coefficients, R2 = 0.8058, 0.8497, 0.80121
for leaf, stem and fruit, respectively.
4.3.2 Antimicrobial activities
The in vitro activity of D. innoxia extracts against L. tropica axenic promastigotes was
evaluated at different doses of plant extracts using standard MTT protocol. A number
of scientific studies have identified several types of flavonoids as antileishmanial
principles of plant extracts (Tasdemir et al., 2006, Fonseca-Silva et al., 2016, Fonseca-
Silva et al., 2015). Consequently, the HPLC based quantification of catechin, myricetin,
quercetin, rutin and caffeic acid in D. innoxia extracts reported in the present analysis
prompted to screen them for antileishmanial prospective. Among the three plant parts
analysed, leaf extracts demonstrated a remarkable antipromastigote activity in a
concentration dependent manner while the stem and fruit extracts displayed a low
inhibitory potential. Therefore, selection of appropriate plant part i.e. leaf was found to
be a critical factor in order to fully exploit the leishmanicidic potential of D. innoxia.
This is in line with scientific literature on D. stramonium that manifested a significant
antiprotozoal potential against extracellular promastigote and intracellular amastigote
forms of L. major (Nikmehr et al., 2014). A noteworthy in vitro antipromastigote
potential unveiled by the D. innoxia leaf extracts can be envisaged as an important
advancement in the search for novel antiprotozoan agents from natural sources and
isolation for the lead compounds was therefore, carried out.
Various microbes i.e. bacteria, fungi and algae have proven to be pathogenic in plants
and animals. Folk medicines have always shown promising results against these
transmittable diseases since antiquity. Haphazard usage of antimicrobials induces
resistance in infectious organisms regardless of advancement in medical knowledge
(Cowan, 1999a).
Spread of diseases throughout the world compels scientists to establish more advance
and systemized novel approaches to face such calamities. Folk knowledge about the
remedial properties of natural products including plants authenticates anti-infectious
properties. (Rios and Recio, 2005).
Ch.4: Discussion
107
In order to evaluate antimicrobial and antifungal screening of our test extracts, disc
diffusion method was followed. To determine MIC, both agar dilution and broth
dilution method was adopted.
Disc diffusion method is based on the development of zone of growth inhibition. Paper
discs impregnated with test extracts of particular concentration and volume is put on to
the seeded agar lawns (bacterial or fungal). Sample diffuse into its surroundings and
active samples form circular shaped zones of growth inhibition. Zone diameter is
criterion to determine antimicrobial property. It indicates vulnerability or susceptibility
of microorganisms which can be then statistically related with the corresponding
minimum inhibitory concentration (Andrews, 2001). Factors affecting zone size are;
tested specie and sample effectiveness. If a sample forms larger zone at lower
concentration then it is considered to be more effective and vice versa. It allows
simultaneous evaluation of large number of samples. Flexibility, easy accessibility, cost
effectiveness and quick results are advantages of this method. This method has its
limitations and is not considered suitable for quantification of antimicrobials because
constituents of less polar nature do not diffuse equally in agar plates therefore DMSO
is recommended to be used as an emulsifying agent (Lopes-Lutz et al., 2008).
The concentration of an antimicrobial agent at which growth of microbes is inhibited
under prearranged assay circumstances is called Minimum Inhibitory Concentration or
simply MIC. Broth and agar dilution methodologies were followed to determine MIC
of test extracts which already gave significant results in screening. Agar dilution
approach allows serially diluted test extracts of known concentration applied to paper
discs and then put them on agar plates having known concentration of microbial culture.
After incubation, presence of microbes on agar plates reveals microbial growth whereas
in broth dilution methodology, liquid growth medium inoculated with microbial strains
and having varying concentrations of test sample.
Among all the extracts 39% of leaf, 16% of stem and 29% of fruit extracts were found
to be active (zone ≥ 10 mm). Among all the bacterial strains tested, Mirocococcus
luteus was most susceptible with maximum inhibition by the n-hexane fruit extract.
Data indicated that extracts prepared from leaves possess better antibacterial activity
than those prepared from stem and fruit. Among all the leaf extracts a maximum zone
of growth inhibition was displayed by the ethyl acetate, ethanol and acetone extracts
against K. pneumonae, S. typhi, M. luteus and S. aureus respectively. Hydroxylated
Ch.4: Discussion
108
phenolic compounds such as caffeic acid and catechol from various plant extracts have
shown to be toxic to microorganisms (cowan, 1999b). In the present study, the
antimicrobial activity exhibited by various extracts might be due to these hydroxylated
phenols which are quantified through HPLC.
The plant’s antifungal potential was assessed against four strains of filamentous fungi.
The data indicate that acetone extract of leaf and stem while n-hexane extract of fruit
showed a prominent growth inhibition zone against A. niger respectively. Among all
the extracts, lowest minimum inhibitory concentration (MIC) of 12.5 µg/disc against
A. niger was presented by the n-hexane fruit extract. It has been reported that A.
niger produces potent mycotoxins on foodstuffs and is the most prevalent fungus
affecting corn (Quiroga et al., 2001). A moderate antifungal activity was shown by
almost all the extracts against Mucor sp. with an average diameter of growth inhibition
zone ranging between 7 and 14 mm. It was observed that mostly the antifungal activity
increased as the polarity decreased. Thus, the chloroform and n-hexane extracts showed
better antifungal activity than aqueous or ethanolic extracts. Well-known plant
secondary metabolites exhibiting antifungal activity (Quiroga et al., 2001). Therefore,
the antifungal activity might be attributed to the phenolic compounds such as
flavonoids.
4.3.3 Enzyme inhibition assays
4.3.3.1 α-amylase inhibitory activity
Post prandial hyperglycaemia culminating in type II diabetes ensues in the induction of
oxidative stress and formation of advanced glycation products, which are the promotors
of diabetic complications (Sudha et al., 2011). Pancreatic amylase inhibitors offers an
operative strategy to reduce the exaggerated spikes of post prandial hyperglycaemia
through control of starch hydrolysis. In general, all the samples demonstrated moderate
antidiabetic potential comparable to each other indicating that enzyme inhibition is not
effected either by plant part used or type of extraction solvent employed. Some of the
mechanisms through which herbal preparations exert their antidiabetic effect include;
simulation of insulin action, influencing the insulin secreting beta cells, or modification
of glucose utilization (Sangeetha and Vedasree, 2012). Inhibition of α-amylase
enzyme’s activity has also been reported for D. metel (Krishna Murthy et al., 2004) and
D. stramonium (Shobha et al., 2014). Several studies on α-amylase inhibitors isolated
from medicinal plants suggest that several potential inhibitors of this enzyme belong to
Ch.4: Discussion
109
flavonoid class of phytochemicals (Kyriakis et al., 2015, Najafian et al., 2010, Zhou et
al., 2009, Ma et al., 2012). Thus, we hypothesize that α-amylase inhibitory activity of
D. innoxia extracts might be due to the presence of flavonoids and phenolics (Fatima
et al., 2015a).
4.3.3.1 Protein kinase inhibition potential
In the recent years, there has been a significant surge for the development of inhibitors
of protein kinases from natural products especially plants. An advantage of the whole
cell Streptomycete assay is that it readily identifies cytotoxic activity of the compounds
being tested. This simple assay permits the identification of signal transduction
inhibitors for a variety of applications including anti-infective, antitumor agents and
several of the inhibitors of mycobacteria (Barbara et al., 2002).
Among all the extracts, a noteworthy protein kinase inhibition was manifested by the
ethyl acetate extract of both leaf and stem, while the most prominent hyphae formation
inhibition in case of fruit was exhibited by the ethanol extract. These findings suggest
that a moderately polar extraction solvent would be suitable for the extraction of
phytoconstituents of D. innoxia that may serve as a promising kinase inhibitory target
while the extremes of extraction solvent polarities exhibited little or no activity.
4.3.4 Cytotoxicity determination
The cytotoxicity evaluation provides a competent initial method to find antitumour,
antimcrobial, antimalarial and insecticidal activities. The larvae of A. salina (brine
shrimps) are a simple and suitable investigation for the initial screening of various
pharmacological activities such as cytotoxicity, antitumor and pesticidal activities
owing to the fact that most of the bioactive compounds are cidal to the shrimps at high
doses (Khan et al., 2015). It has also been proposed for pharmacological screening of
plant extracts and the resultant toxicity results can be correlated with their previously
documented ethnopharmacological role (Nguta et al., 2012). The cytotoxicity potential
of D. innoxia was tested against brine shrimp larvae to reveal its lethality profile. Out
of a total of 36 organic extracts screened for cytotoxic activity against brine shrimp
larvae, 25% of the leaf, 16% of the stem and 8.3% of the fruit extracts demonstrated
activity at or below 100 μg/ml and were categorized as highly toxic. The remaining
75% of the leaf, 84% of the stem and 91.7% of the fruit extracts exhibited LC50 values
≤ 250 μg/ml and were categorized as toxic. The positive control, doxorubicin
Ch.4: Discussion
110
demonstrated an LC50 value of 5.93 µg/ml. Among all the individual plant part extracts,
methanol-chloroform was found to be the most toxic exhibiting an LC50 of 85.94 μg/ml
for leaf and stem while 54.07 μg/ml for fruit; indicating that a moderately polar
extraction solvent would be the best choice for the isolation of such compounds rather
than a highly polar or nonpolar solvent. The degree of lethality was found to be directly
proportional to the concentration of the extract. In bioactivity evaluation of plant
extracts by brine shrimp lethality assay, an LC50 value of less than 1000 μg/ml is
considered to be cytotoxic. In the current study, 100% of all the screened organic
extracts demonstrated LC50 values < 1000 μg/ml, signifying the presence of cytotoxic
compounds responsible for the observed activity. These results recommended further
investigation of plant’s cytotoxic potential using in vitro anticancer cell lines.
The incidence of several cancers has increased exponentially with age from the fourth
to eighth decade of life. Over 6 million people decease due to cancer worldwide each
year, being the largest single cause of death in both men and women (Kumar et al.,
2013). One of the utmost clinical challenges is the therapeutic management of cancer
and nowadays phytotherapy is being extensively scouted to have integrated approach
for cancer cure (Khan et al., 2015). Keeping in view, the prodigious cytotoxic potential
as discovered through brine shrimp lethality assay; the plant extracts were further
screened for an in vitro cytotoxic activity using THP-1 and Hep G2 cell lines.
Standard MTT assay was employed to determine the cytotoxic potential of test extracts
against THP-1 cell line. Among all the leaf extracts, the chloroform extract was most
potent as it considerably inhibited the THP-1 cell line proliferation exhibiting 80.95 ±
1.77% cell mortality at 10 µg/ml concentration and an LC50 5.91 µg/ml. In the present
study, stem extracts did not display any cytotoxic potential against THP-1 cell line,
while in case of fruit, the most prominent cytotoxicity was demonstrated by the
chloroform and n-hexane extracts with an LC50 4.52 and 3.49 µg/ml, respectively which
is comparable to the standard drugs 5-florouracil and vincristine with IC50 of 5 µg/ml
and 8.1 µg/ml respectively. This is by far the first report (to the best of our knowledge)
highlighting the cytotoxic proficiency of D. innoxia fruit phytochemicals, which
according to all the previous studies have been reported in its leaf part only.
Hepatoma is amongst the two major forms of primary liver cancers (Machana et al.,
2012). It has been observed that the substantial cytotoxicity of D. innoxia against Hep
G2 cells as revealed in the current investigation resides in the leaf part only. Therefore,
Ch.4: Discussion
111
the antioxidant stature (Fatima et al., 2015a) and cytotoxicity of D. innoxia leaf extracts
suggest them to be worthy leads for the management of hepatocellular carcinoma. The
anticancer effects of D. innoxia have also been described previously. Methanol extract
of D. innoxia leaf part inhibits the proliferation of human colon adenocarcinoma (HCT
15) and human larynx cancer cell lines (Hep-2) through induction of apoptosis
(Arulvasu et al., 2010). In another study, a new withanolide, Dinoxin B isolated from
D. innoxia methanol extract of leaf part exhibited submicromolar IC50 values against
multiple human cancer cell lines (Vermillion et al., 2011).
Section-2: Preparative extraction, biological evaluation and isolation
4.4 Preparative extraction
The preliminary optimization studies on extraction efficiency of D. innoxia, in terms of
most efficacious plant part for bioactivity revealed that among the leaf, stem and fruit
parts, leaf and fruit are most proficient in terms of their bioactivity profile. Therefore,
these plant parts were selected to proceed for preparative extraction and isolation of
lead compounds. Keeping in view, the efficiency of ethyl acetate leaf extract of D.
innoxia as a potential source of phytochemicals inciting substantial antioxidant
capability, cytotoxic, kinase inhibitors and antibacterial compounds, it was selected as
one of the solvents for preparative extraction. Similarly, methanol extract of leaf was
substantially active against brine shrimps, THP-1 and Hep G2 cell proliferation.
Therefore, ethyl acetate and methanol (1:1) binary solvent combination was selected as
the extraction solvent system for preparative extraction of D. innoxia leaf part. In case
of fruit, nonpolar solvents unveiled prodigious cytotoxic, antimicrobial and
leishmanicidic potential; therefore, chloroform was selected for the preparative
extraction of fruit part. Maceration was used as the extraction technique for the
preparative extraction of crude leaf (DCL) and fruit (DCF) extracts.
4.5 Fractionation
For the fractionation of DCL and DCF, solid phase extraction (SPE) was used as the
partitioning technique. Extract loaded silica packed in glass column was eluted with
organic solvents of increasing polarity and the fractions thus obtained containing
compounds of different polarities were grouped together for further purification. A total
of 8 fractions i.e. DCL 1-8 was prepared from DCL whereas, 7 fractions (DCF 1-7)
were obtained from the fractionation of DCF.
Ch.4: Discussion
112
4.6 Biological evaluation of fractions
The preparative extracts DCL and DCF of D. innoxia leaf and fruit respectively, as well
as the fractions prepared thereafter, were evaluated phytochemically and biologically
using the assays as described in the section-1 of this chapter in order to identify fractions
for the hit to lead isolation and purification.
Antioxidant potential of the preparative extracts and fractions of D. innoxia leaf and
fruit parts was evaluated by %RSA, TAC and TRP assays. Among all the samples tested
for scavenging potential, highest free radical quenching potential was manifested by
the most polar DFL-8 and DFF-7 fractions of leaf and fruit respectively as compared to
their preparative crude extracts which shows that polar components are the major
contributors to the radical scavenging potential of DCF and DCL. It was also observed
that scavenging efficiency increased as the polarity of fractions increased. A positive
correlation was observed between the radical scavenging capability and the total
phenolic content (leaf fractions: R2 = 0.719, stem, fruit fractions: R2 = 0.84). Therefore,
it can be inferred that the various polyphenols in the test samples may be responsible
for the radical scavenging mediated antioxidant activity.
The phosphomolybdenum based total antioxidant capacity was highest in case of DFL-
7 and DFL-8 fractions of leaf whereas, in case of fruit fractions it was highest in DFF-
7 and DFF-6 fractions. The TAC of fractions decreased with a decrease in their polarity
which may be due to the lesser partitioning of antioxidant metabolites in nonpolar
solvents as compared to polar fractions. A positive correlation was observed between
TPC and TAC suggesting that antioxidant activity might be attributed to these
polyphenols.
In case of TRP, maximum reducing potential was manifested DFL-7 and DFL-8
fractions of leaf whereas, in case of fruit fractions it was highest in DFF-7 fraction. It
was also observed that the reducing power of leaf and fruit fractions decreased
exponentially with the polarity of fractions signifying that the reducing potential is
mostly due to the presence of polar reductones as compared to nonpolar ones. Overall,
the reductive potential of fruit and leaf part was comparable to each other for all the test
samples.
The antimicrobial spectrum of the preparative extracts as well as fractions was
evaluated by determining their antileishmanial, antibacterial and antifungal potential.
Ch.4: Discussion
113
Antileishmanial activity of various leaf and fruit test samples of D. innoxia was
determined using MTT assay. Among all the samples tested, the leaf part demonstrated
a conspicuous antileishmanial activity while none of the fruit samples exhibited greater
than 50% inhibition of growth of leishmanial promastigotes. Among the leaf samples,
maximum activity was manifested by the DFL-4 leaf fraction lagged by polar fractions
while the nonpolar fractions did not exhibit any leishmanicidal activity. The DFL-4
fraction manifested minimum IC50 value and was therefore, identified as the hit fraction
for the isolation of lead antileishmanial principles.
Disc diffusion assay was employed to determine the antibacterial spectrum of test
samples. Among the leaf samples, the most prominent antibacterial activity was
observed against B. subtilis as compared to other tested strains with maximum zone of
growth inhibition being formed around the preparative DCL extract loaded disc lagged
by DFL-5 and DFL-6 indicating the antibacterial proficiency of moderately polar
fractions as compared to nonpolar and polar fractions. Among the fruit samples, a
moderate antibacterial activity against K. pneumoniae was exhibited by most of the
samples with maximum ZOI being recorded around the DCF loaded disc.
The antifungal spectrum of the preparative extract and fractions of D. innoxia was
determined against four strains of filamentous fungi using standard disc diffusion assay.
The fractions exhibiting a growth inhibitory zone ≥ 10 mm in agar disc diffusion assay
were considered active. The crude DCL leaf extract exhibited a moderate inhibition
against only one strain i.e. A. flavus whereas, the crude DCF fraction of fruit did not
exhibit any antifungal activity. The data indicate that 37.50% of the leaf fractions were
active against A. flavus, 25% against A. niger whereas, none of the leaf fractions were
active against F. solani, A. fumigatus and Mucor sp. The maximum ZOI of 13 ± 0.65
mm around A. flavus was demonstrated by DFL-7. Among the fruit fractions a moderate
antifungal activity was observed only against A. flavus while there was little or no
activity against the other tested strains.
A moderate α-amylase inhibitory activity was recorded for both the leaf and fruit
samples indicating that the amylase inhibition potential was not affected by the plant
part used. Moreover, the amylase inhibitory activity did not significantly change with
the polarity of the fraction.
Ch.4: Discussion
114
Protein kinase inhibitory potential of the test samples was evaluated by monitoring their
capability to inhibit the hyphae formation in Streptomycetes 85E strain. Overall, the
leaf samples manifested better kinase inhibitory prospective as compared to the fruit
fractions with maximum bald phenotype being observed around the DCL preparative
extract lagged by DFL-6 and DFL-7 fractions. Among the fruit samples, only DFF-1,
DFF-3 and DFF-7 displayed a moderate protein kinase inhibition while the preparative
DCF crude extract exhibited no activity indicting that antagonistic interaction might be
responsible for the diminished activity.
Cytotoxic potential of the samples was evaluated using brine shrimp lethality assay and
in vitro cell viability assay against cell lines. Among all the leaf samples analysed,
minimum 50% inhibitory concentration was obtained in case of DFL-5, DFL-6 and
DFL-7. Presence of cytotoxic constituents in the DFL-5 fraction which was prepared
by eluting DCL with ethyl acetate shows that moderately polar phytochemicals might
be responsible for the activity. These results suggest that moderately polar fractions of
DCL such as DFL-4 and DFL-5 could be used as lead fractions for the isolation of
cytotoxic principles. Among all the fruit fractions, maximum lethality was observed
only in case of nonpolar fractions i.e. DFF-1 and DFF-2 as well as its preparative crude
extract DCF while none of the polar fractions exhibited any cytotoxicity against the
shrimp larvae. The absence of cytotoxic effects in fruit fractions indicate antagonistic
interactions among various phytochemicals. Therefore, both preparative extract and
fractions of leaf manifested better cytotoxicity as compared to the fruit part.
Among all the leaf samples, DFL-8 most considerably inhibited the Hep G2 cell line
proliferation lagged by DCL and DFL-5. As the elution solvents of DFL-8 and DFL-5
were methanol and ethyl acetate, respectively while the extraction solvent system of
DCL was ethyl acetate: methanol; therefore, the greater cytotoxic potential of DFL-8
and DFL-5 might be due to better partitioning of cytotoxic phytoconstituents of DCL
in these fractions. Overall, the results suggest that polar fractions such as DFL-5 till
DFL-8 are the hit fractions for the isolation of cytotoxic principles.
The isolation and purification of lead compounds from the aforementioned fraction hits
one of the robust and cost effective techniques, liquid column chromatography was
used. The various sub fractions were combined on the basis of results of TLC
fingerprints. These combined fractions were subjected to various forms of normal phase
liquid column chromatography i.e. vacuum liquid chromatography and medium
Ch.4: Discussion
115
pressure liquid chromatography to get the pure compounds and gradient elution was
used. All the isolation and purification was performed keeping in view optimization of
process conditions.
Section-3: Biological evaluation and characterization of isolated compounds
The isolated compounds were subjected to antileishmanial, protein kinase inhibition,
cytotoxicity against cell lines and cancer chemopreventive assays in order to determine
their bioactivity profile. Total three compounds (CL-1, CL-3 and CF-5) were isolated
using normal phase vacuum and medium pressure column chromatography as the
isolation techniques. Compound CL-3 demonstrated noteworthy antileishmanial
activity and whereas, the other two compounds did not exhibit significant
antipromastigote activity. The compound CL-3 was isolated from the DFL-4
fraction of preparative crude extract of leaf that exhibited maximum activity against
L. tropica. Therefore, the highest antileishmanial activity observed in DFL-4
fraction might be due to CL-3. It was also observed that DFL-4 fraction was
prepared by SPE of the D. innoxia leaf part, which demonstrated highest
antipromastigote activity in MTT assay as compared to fruit and stem parts.
Therefore, these results endorse the role of bioassay guided extraction and
bioactivity profiling of fractions as an operative tool for the determination of hits
in lead compounds isolation. Among the isolated compounds, a phenomenal
cytotoxic potential against MCF-7, LU-1 and PC3 cancer cell lines was manifested
by CL-3. Therefore, the substantial cytotoxic activity of DFL-4 fraction as revealed
in brine shrimp lethality assay might be due to CL-3. The lethality of DFL-4 against
brine shrimps and the cytotoxicity of CL-3 against cancer cell lines reinforced the
previous findings that the preliminary brine shrimp assay manifests a good relationship
with cell line based cytotoxic potential determining assays (Nguta et al., 2012). In
protein kinase inhibition assay, maximum bald growth inhibition zone was formed
around the CL-1. Among the isolated compounds, CF-5 demonstrated remarkable
cancer chemopreventive activity through inhibition of NFĸB and NO production
whereas, none of the other isolated compounds exhibited significant cancer
chemopreventive activity. Crystallography and NMR spectroscopy characterized the
structure of CL-1, CL-3 and CF-5 as β-sitosterol, isowithametelin and (4a, 4b, 6b, 8b,
10b, 14a) 7, 10 dimethyl dinoroleanan-12 en-3-one (new terpenoid), respectively.
Phytosterols are naturally occurring lipophilic compounds with around 40 being
Ch.4: Discussion
116
reported so far. The chief phytosterols include stigmasterol, β-sitosterol, avenasteol and
campesterol. β-sitosterol, being the predominant phytosterol, occurs in some edible
plants, such as, fruits of Cucurbita moschata, seeds of Avena sativa, leaves of Spinacia
oleracea, and rhizomes of Daucus carota, and in some remedies used in traditional
Chinese medicines, such as fruits of Panax ginseng, and seed of Perilla frutescens,
Linum usitatissimum, and Platycladus orientalis. β-sitosterol manifests
antiproliferative action through mitochondrial and membrane death receptor pathway
and is reported to exert antiandrogen and antinflammatory activities on experimental
mice prostatic hyperplasia (Wu, 2013).
β-sitosterol has been reported previously as the phytoconstituent of various Datura
species however, isolation of β-sitosterol from D. innoxia in the current study has been
reported for the first time. Ramadan et al. (2007) characterized the crude n-hexane
extract of D. metel by using HPLC and reported a relatively high concentration of
phytosterols, in which the sterol indicators were β-sitosterol, sitostanol, stigmasterol,
D5-avenasterol, lanosterol, and sitostanol Gupta et al. (2012) reported that a bioactive
fraction from D. stramonium promotes human immune cells mediated cytotoxic effects
agianst MCF-7 breast cancer and A549 lung carcinoma. LC-MS analysis of this active
fraction showed sitosterol, stigma sterol, daturadiol and daturaolone as the chief
phytochemicals.
Isowithametelin is a withanolide that has been isolated in the present study. Its presence
as secondary metabolite in the leaf part of D. innoxia as well as antileishmanial potential
and cytotoxicity against MCF-7, LU-1 and PC-3 cancer cell lines have been reported
for the first time in the current study. Previously, withmetelin and isowithmetelin were
reported from D. metel leaves by Sinha et al. (1989). The withanolides are a class of
naturally abundant steroidal lactones that are reported to occur in various genera such
as Datura, Acnistus, Dunalia, Lycium, Jaborosa, Withania and Physalis, of the family
Solanaceae. A number of these compounds manifest an array of pharmacological
activities, such as antioxidant, immunosuppressive, anti-inflammatory and antitumor
properties.
Withanolides can prevent proliferation of tumour cell as well as angiogenesis and are
able to induce the phase II enzyme quinone reductase (Haq et al., 2012). Withanolides
have been reported as secondary metabolites in various species of Datura. Bioassay
directed fractionation of the methanol extract D. metel flowers led to the isolation of
ten withanolides, 1,10 seco withametelin B, withametelins I-P and 12â-hydroxy-1,10-
Ch.4: Discussion
117
seco-withametelin B (Pan et al., 2007). Another novel withanolide, designated as
withawrightolide and some other were reported from the aerial parts of D. wrightii. By
means of MTT viability assays, these withanolides presented antiproliferative potential
against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma
(MDA-1986), and normal foetal lung fibroblast (MRC-5) cells (Zhang et al., 2013). In
another study withanolide, Dinoxin B isolated from D. innoxia methanol leaf extract
exhibited submicromolar IC50 values against multiple human cancer cell lines
(Vermillion et al., 2011). Two withanolides named as withametelinol-A and
withametelinol-B were reported from the aerial parts of Datura innoxia (Siddiqui et al.,
1999). The CF-5 compound isolated from the fruit fraction of D. innoxia was found to
be a new terpenoid which was designated as (4a, 4b, 6b, 8b, 10b, 14a) 7, 10 dimethyl
dinoroleanan-12 en-3-one and demonstrated a substantial cancer chemopreventive
potential. Previously, two pentacyclic triterpenes, daturadiol and daturaolone, have
been isolated from Datura innoxia Mill. seeds (Maheshwari et al., 2012).
Terpenoids are hydrocarbons that occur naturally and are synthesized by a number of
animals and plants. They are categorised based on isoprene units as their basic building
unit. Wide-ranging useful roles of terpenoids have been studied extensively, some of
them include natural flavour additives for food or fragrances in perfumery and in
traditional and alternate medicines as aromatherapy. Most comprehensively studied
pharmacological activities of terpenes in prevention and neoplastic management.
Paclitaxel and docetaxel which are taxol derivative are amongst the extensively
employed medicines in cancer chemotherapy. Other important therapeutic uses of
terpenoids include antihyperglycemic, antifungal, antiviral, antimicrobial, antiparasitic,
anti-inflammatory, immunomodulatory, antioxidants and as skin permeation enhancer.
Since most of them exist in very low levels in nature, their extensive harvesting to
achieve adequate quantities utilizing metabolic engineering and synthetic biology
provides innovative methods to increase the production of terpenoids (Brahmkshatriya
and Brahmkshatriya, 2013).
Ch.4: Discussion
118
Research outcomes
• Antileishmanial and protein kinase inhibitory prospective of D. innoxia has
been described for the first time.
• The presence of β-sitosterol and isowithmetelin as phytoconstituents of D.
innoxia leaves has not been reported earlier.
• Determination of leishmanicidal and cytotoxic potential of isowithmetelin from
bioactive fraction of D. innoxia has not been reported yet.
• It is the first report on the isolation studies from D. innoxia fruit part and
purification of a new cancer chemopreventive terpenoid.
Conclusions
• Leaf and fruit parts of D. innoxia were found to be more bioactive as compared
to stem.
• Methanol and ethyl acetate (1:1) were most operative for preparative extraction
from D. innoxia leaf part whereas, chloroform was most suitable for the
extraction of bioactive phytoconstiuents from its fruit part.
• In case of leaf fractions, moderately polar and polar fractions were found to be
potential candidates for the isolation of antileishmanial compounds whereas,
polar fractions exhibited profound protein kinase inhibitory and cytotoxic
activities.
• Total three compounds (CL-1, CL-3 and CF-5) were isolated using normal
phase vacuum and medium pressure column chromatography as the isolation
technique.
• Compound CL-3 demonstrated noteworthy antileishmanial activity and
cytotoxic potential against MCF-7, LU-1 and PC3 cancer cell lines. In protein
kinase inhibition assay, maximum bald growth inhibition zone was formed
around the CL-1 loaded disc whereas, CF-5 demonstrated remarkable cancer
chemopreventive activity through inhibition of NFĸB and NO production.
• Crystallography and NMR spectroscopy characterized the structure of CL-1,
CL-3 and CF-5 as β-sitosterol, isowithametelin and (4a, 4b, 6b, 8b, 10b, 14a) 7,
10 dimethyl dinoroleanan-12 en-3-one (new terpenoid), respectively.
Ch.4: Discussion
119
Study limitations
Some of the limitations in the current analysis includes
• The Rf values of isolated compounds were very close to each and
sufficient purification could not be achieved by further
chromatographic resolution.
• Some of the isolated compounds were sufficiently pure, however their
quantities were not sufficient for biological evaluation and structure
elucidation and were therefore could not be reported in this study.
• Compounds that were isolated were not in quantities to proceed for
biological testing and structure elucidation.
• Isolated compounds were sufficiently pure but were not active in the
high through put screening assays employed in the current study.
Future prospects
• The isolated compounds could be a stupendous source for novel drug
molecules as anticancer drugs as well as to combat leishmaniasis.
• Preclinical studies could be performed for the comprehensive
determination of in vivo bioactivity profile and toxicity of lead
compounds.
• Quantification of the bioactive compounds in each plant part of D.
innoxia and the influence of collection time requires further studies.
Moreover, quantitative and qualitative analysis of variation of isolated
compounds with in different species of same genus (withanolides
bearing species) could be beneficial for the selection of most suitable
natural source.
• New bioactive lead compounds having anticancer potential can be
obtained from D. innoxia.
• Establishment of cell suspensions and hairy root cultures of D. innoxia.
Comparison of secondary metabolite produced in plants growing in
natural habitat and cell cultures could benefit to select the most
operative source for the enhanced quantification of bioactive
compounds on commercial scale.
References
120
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Appendix I
Appendix I
Table 1 Atomic coordinates ( x 10^4) and equivalent isotropic displacement
parameters (A^2 x 10^3) for CL-1. U(eq) is defined as one third of the trace of the
orthogonalized Uij tensor.
________________________________________________________________
x y z U(eq)
________________________________________________________________
C(1) 9692(11) 4660(10) 120(1) 77(2)
C(2) 10796(10) 3476(9) 197(1) 72(2)
C(3) 10721(9) 3422(8) 403(1) 59(2)
C(4) 8832(8) 3353(7) 482(1) 52(2)
C(5) 7690(9) 4465(9) 384(1) 65(2)
C(6) 7815(10) 4479(10) 179(1) 75(2)
C(7) 8054(12) 1882(8) 440(1) 77(2)
C(8) 12115(9) 3438(8) 504(1) 69(3)
C(9) 12172(9) 3387(9) 703(1) 63(2)
C(10) 10412(7) 2920(6) 784(1) 47(2)
C(11) 8896(7) 3676(7) 688(1) 48(2)
C(12) 10352(8) 3182(7) 985(1) 51(2)
C(13) 8592(8) 2791(6) 1079(1) 52(2)
C(14) 7157(8) 3659(8) 989(1) 59(2)
C(15) 7112(8) 3435(9) 784(1) 62(2)
C(16) 8239(11) 1196(7) 1059(1) 70(2)
C(17) 11780(9) 2593(9) 1107(1) 69(2)
C(18) 11007(10) 2699(10) 1298(1) 79(2)
C(19) 9002(9) 3119(7) 1281(1) 58(2)
C(20) 7880(10) 2483(10) 1428(1) 76(2)
C(21) 5958(12) 2891(11) 1417(1) 90(3)
C(22) 8548(12) 2764(12) 1614(1) 92(3)
C(23) 8740(20) 1778(19) 1743(1) 159(6)
C(24) 9570(20) 2040(20) 1928(2) 164(7)
C(25) 8230(50) 1950(50) 2077(4) 240(30)
C(26) 6790(80) 3020(70) 2062(8) 440(60)
C(27) 7570(80) 490(60) 2041(6) 520(50)
C(28) 11190(30) 1160(20) 1953(3) 185(14)
C(30) 11840(40) 1410(30) 2148(4) 250(20)
C(31) 2439(10) 7768(9) 360(1) 66(2)
C(32) 1340(9) 8699(9) 481(1) 66(2)
C(33) 1499(8) 8303(7) 676(1) 54(2)
C(34) 3377(8) 8201(7) 750(1) 48(1)
C(35) 4444(8) 7289(8) 618(1) 56(2)
C(36) 4333(10) 7760(9) 422(1) 68(2)
C(37) 4189(10) 9678(8) 755(1) 63(2)
C(38) 63(8) 8061(8) 777(1) 57(2)
C(39) 83(8) 7688(7) 970(1) 57(2)
C(40) 1840(8) 7998(7) 1059(1) 50(2)
C(41) 3378(8) 7490(6) 938(1) 46(1)
C(42) 1961(8) 7367(7) 1245(1) 54(2)
C(43) 3698(9) 7624(7) 1343(1) 56(2)
C(44) 5168(9) 6987(9) 1227(1) 65(2)
C(45) 5177(9) 7583(9) 1038(1) 62(2)
C(46) 4084(11) 9200(9) 1374(1) 75(2)
C(47) 537(9) 7709(10) 1384(1) 77(2)
Appendix I
C(48) 1336(10) 7282(11) 1566(1) 86(3)
C(49) 3356(11) 6912(9) 1531(1) 70(2)
C(50) 4499(12) 7257(11) 1694(1) 90(3)
C(51) 6413(15) 6980(17) 1664(1) 133(5)
C(52) 3805(17) 6397(14) 1856(1) 117(4)
C(53) 3900(30) 7060(20) 2026(2) 179(7)
C(60A) 3000(200) 5610(160) 2450(16) 260(90)
C(61A) 5500(160) 6890(120) 2379(9) 360(70)
C(62A) 2790(110) 5130(130) 2187(9) 160(40)
C(64A) 1180(100) 5850(110) 2202(10) 90(40)
C(66A) -1930(170) 5250(130) 2420(15) 140(60)
C(68A) -70(90) 6290(60) 2098(8) 280(40)
C(69A) 4990(120) 5620(140) 2329(12) 360(70)
C(70A) 730(70) 5460(60) 2392(6) 290(40)
C(71A) 5540(150) 4610(130) 2201(15) 200(70)
C(62B) 29320(120) 20540(90) 21124(10) 280(50)
C(63B) 12700(30) 1620(30) 1814(3) 251(15)
C(64B) 1290(60) 6550(80) 2244(7) 80(30)
C(66B) 2500(110) 6140(70) 2201(5) 100(30)
C(67B) 3600(70) 5980(50) 2196(4) 80(20)
C(68B) 3220(80) 6820(80) 2189(6) 70(30)
C(69B) 790(100) 7430(110) 2254(9) 400(60)
C(70B) 6300(130) 2150(70) 2619(7) 480(60)
O(1) 9846(9) 4682(8) -72(1) 100(2)
O(2) 8399(11) 8766(9) 81(1) 127(3)
O(3) 2291(7) 8258(7) 174(1) 86(2)
___________________________________________________________
Table 2. Bond lengths [A] and angles [deg] for CL-1.
C(1)-O(1) 1.430(9)
C(1)-C(6) 1.495(11)
C(1)-C(2) 1.520(12)
C(1)-H(1) 0.9800
C(2)-C(3) 1.530(10)
C(2)-H(2A) 0.9700
C(2)-H(2B) 0.9700
C(3)-C(8) 1.292(10)
C(3)-C(4) 1.544(9)
C(4)-C(5) 1.550(10)
C(4)-C(7) 1.557(10)
C(4)-C(11) 1.564(9)
C(5)-C(6) 1.521(9)
C(5)-H(5A) 0.9700
C(5)-H(5B) 0.9700
C(6)-H(6A) 0.9700
Appendix I
C(6)-H(6B) 0.9700
C(7)-H(7A) 0.9600
C(7)-H(7B) 0.9600
C(7)-H(7C) 0.9600
C(8)-C(9) 1.480(10)
C(8)-H(8) 0.9300
C(9)-C(10) 1.524(9)
C(9)-H(9A) 0.9700
C(9)-H(9B) 0.9700
C(10)-C(12) 1.518(9)
C(10)-C(11) 1.528(8)
C(10)-H(10) 0.9800
C(11)-C(15) 1.540(9)
C(11)-H(11) 0.9800
C(12)-C(17) 1.513(9)
C(12)-C(13) 1.545(9)
C(12)-H(12) 0.9800
C(13)-C(14) 1.520(9)
C(13)-C(16) 1.558(9)
C(13)-C(19) 1.568(9)
C(14)-C(15) 1.535(9)
C(14)-H(14A) 0.9700
C(14)-H(14B) 0.9700
C(15)-H(15A) 0.9700
C(15)-H(15B) 0.9700
C(16)-C(62B)#1 1.14(9)
C(16)-H(16A) 0.9600
C(16)-H(16B) 0.9600
C(16)-H(16C) 0.9600
C(17)-C(18) 1.539(11)
C(17)-H(17A) 0.9700
C(17)-H(17B) 0.9700
Appendix I
C(18)-C(19) 1.573(10)
C(18)-H(18A) 0.9700
C(18)-H(18B) 0.9700
C(19)-C(20) 1.508(11)
C(19)-H(19) 0.9800
C(20)-C(22) 1.491(12)
C(20)-C(21) 1.507(12)
C(20)-H(20) 0.9800
C(21)-H(21A) 0.9600
C(21)-H(21B) 0.9600
C(21)-H(21C) 0.9600
C(22)-C(23) 1.355(16)
C(22)-H(22) 0.9300
C(23)-C(24) 1.526(18)
C(23)-H(23) 0.9300
C(24)-C(25) 1.50(2)
C(24)-C(28) 1.501(19)
C(25)-C(26) 1.50(3)
C(25)-C(27) 1.51(3)
C(28)-C(30) 1.54(2)
C(28)-C(63B) 1.60(3)
C(31)-O(3) 1.462(8)
C(31)-C(36) 1.503(10)
C(31)-C(32) 1.510(10)
C(31)-H(31) 0.9800
C(32)-C(33) 1.505(9)
C(32)-H(32A) 0.9700
C(32)-H(32B) 0.9700
C(33)-C(38) 1.335(9)
C(33)-C(34) 1.523(9)
C(34)-C(35) 1.538(9)
C(34)-C(37) 1.543(9)
Appendix I
C(34)-C(41) 1.553(9)
C(35)-C(36) 1.525(9)
C(35)-H(35A) 0.9700
C(35)-H(35B) 0.9700
C(36)-H(36A) 0.9700
C(36)-H(36B) 0.9700
C(37)-H(37A) 0.9600
C(37)-H(37B) 0.9600
C(37)-H(37C) 0.9600
C(38)-C(39) 1.476(9)
C(38)-H(38) 0.9300
C(39)-C(40) 1.515(9)
C(39)-H(39A) 0.9700
C(39)-H(39B) 0.9700
C(40)-C(42) 1.509(9)
C(40)-C(41) 1.548(9)
C(40)-H(40) 0.9800
C(41)-C(45) 1.550(9)
C(41)-H(41) 0.9800
C(42)-C(43) 1.519(10)
C(42)-C(47) 1.523(9)
C(42)-H(42) 0.9800
C(43)-C(44) 1.531(10)
C(43)-C(46) 1.555(10)
C(43)-C(49) 1.572(10)
C(44)-C(45) 1.516(9)
C(44)-H(44A) 0.9700
C(44)-H(44B) 0.9700
C(45)-H(45A) 0.9700
C(45)-H(45B) 0.9700
C(46)-H(46A) 0.9600
C(46)-H(46B) 0.9600
Appendix I
C(46)-H(46C) 0.9600
C(47)-C(48) 1.537(12)
C(47)-H(47A) 0.9700
C(47)-H(47B) 0.9700
C(48)-C(49) 1.589(12)
C(48)-H(48A) 0.9700
C(48)-H(48B) 0.9700
C(49)-C(50) 1.527(12)
C(49)-H(49) 0.9800
C(50)-C(51) 1.488(14)
C(50)-C(52) 1.545(14)
C(51)-H(51A) 0.9600
C(51)-H(51B) 0.9600
C(51)-H(51C) 0.9600
C(52)-C(53) 1.418(18)
C(52)-H(52) 0.9300
C(53)-C(68B) 1.33(5)
C(53)-C(67B) 1.64(4)
C(53)-C(66B) 1.90(7)
C(60A)-C(70B)#2 1.65(13)
C(60A)-C(70A) 1.78(14)
C(60A)-C(69A) 1.75(15)
C(60A)-C(66B) 1.95(13)
C(60A)-C(67B) 1.98(14)
C(60A)-C(62A) 2.01(13)
C(61A)-C(69A) 1.33(10)
C(61A)-C(70B)#2 1.39(8)
C(62A)-C(66B) 1.00(10)
C(62A)-C(67B) 1.03(10)
C(62A)-C(64A) 1.40(12)
C(62A)-C(68B) 1.65(14)
C(62A)-C(64B) 1.82(12)
Appendix I
C(64A)-C(64B) 0.75(7)
C(64A)-C(66B) 1.03(9)
C(64A)-C(68A) 1.29(8)
C(64A)-C(70A) 1.50(7)
C(64A)-C(69B) 1.59(16)
C(64A)-C(68B) 1.80(10)
C(64A)-C(67B) 1.83(8)
C(66A)-C(70A) 2.03(13)
C(68A)-C(64B) 1.52(9)
C(68A)-C(69B) 1.71(8)
C(69A)-C(71A) 1.42(14)
C(69A)-C(67B) 1.48(9)
C(69A)-C(70B)#2 1.80(15)
C(69A)-C(68B) 2.05(11)
C(70A)-C(64B) 1.57(7)
C(71A)-C(67B) 1.97(14)
C(64B)-C(69B) 0.92(12)
C(64B)-C(66B) 1.05(8)
C(64B)-C(68B) 1.54(9)
C(64B)-C(67B) 1.86(7)
C(66B)-C(67B) 0.85(6)
C(66B)-C(68B) 0.86(7)
C(66B)-C(69B) 1.83(13)
C(66B)-C(70B)#2 1.88(9)
C(67B)-C(68B) 0.85(6)
C(67B)-C(70B)#2 1.77(7)
C(68B)-C(70B)#2 1.51(6)
C(68B)-C(69B) 1.99(10)
O(1)-C(1)-C(6) 111.9(7)
O(1)-C(1)-C(2) 110.0(7)
C(6)-C(1)-C(2) 108.9(7)
Appendix I
O(1)-C(1)-H(1) 108.7
C(6)-C(1)-H(1) 108.7
C(2)-C(1)-H(1) 108.7
C(1)-C(2)-C(3) 112.4(6)
C(1)-C(2)-H(2A) 109.1
C(3)-C(2)-H(2A) 109.1
C(1)-C(2)-H(2B) 109.1
C(3)-C(2)-H(2B) 109.1
H(2A)-C(2)-H(2B) 107.8
C(8)-C(3)-C(2) 123.2(7)
C(8)-C(3)-C(4) 122.4(6)
C(2)-C(3)-C(4) 114.4(6)
C(3)-C(4)-C(5) 108.0(5)
C(3)-C(4)-C(7) 108.2(6)
C(5)-C(4)-C(7) 108.6(6)
C(3)-C(4)-C(11) 109.4(5)
C(5)-C(4)-C(11) 109.8(5)
C(7)-C(4)-C(11) 112.7(5)
C(6)-C(5)-C(4) 116.0(6)
C(6)-C(5)-H(5A) 108.3
C(4)-C(5)-H(5A) 108.3
C(6)-C(5)-H(5B) 108.3
C(4)-C(5)-H(5B) 108.3
H(5A)-C(5)-H(5B) 107.4
C(1)-C(6)-C(5) 110.8(6)
C(1)-C(6)-H(6A) 109.5
C(5)-C(6)-H(6A) 109.5
C(1)-C(6)-H(6B) 109.5
C(5)-C(6)-H(6B) 109.5
H(6A)-C(6)-H(6B) 108.1
C(4)-C(7)-H(7A) 109.5
C(4)-C(7)-H(7B) 109.5
Appendix I
H(7A)-C(7)-H(7B) 109.5
C(4)-C(7)-H(7C) 109.5
H(7A)-C(7)-H(7C) 109.5
H(7B)-C(7)-H(7C) 109.5
C(3)-C(8)-C(9) 127.0(6)
C(3)-C(8)-H(8) 116.5
C(9)-C(8)-H(8) 116.5
C(8)-C(9)-C(10) 112.0(6)
C(8)-C(9)-H(9A) 109.2
C(10)-C(9)-H(9A) 109.2
C(8)-C(9)-H(9B) 109.2
C(10)-C(9)-H(9B) 109.2
H(9A)-C(9)-H(9B) 107.9
C(12)-C(10)-C(9) 111.3(5)
C(12)-C(10)-C(11) 110.8(5)
C(9)-C(10)-C(11) 109.6(5)
C(12)-C(10)-H(10) 108.4
C(9)-C(10)-H(10) 108.4
C(11)-C(10)-H(10) 108.4
C(10)-C(11)-C(15) 111.9(5)
C(10)-C(11)-C(4) 112.5(5)
C(15)-C(11)-C(4) 113.2(5)
C(10)-C(11)-H(11) 106.2
C(15)-C(11)-H(11) 106.2
C(4)-C(11)-H(11) 106.2
C(17)-C(12)-C(10) 120.2(6)
C(17)-C(12)-C(13) 105.0(5)
C(10)-C(12)-C(13) 115.2(5)
C(17)-C(12)-H(12) 105.0
C(10)-C(12)-H(12) 105.0
C(13)-C(12)-H(12) 105.0
C(14)-C(13)-C(12) 106.6(5)
Appendix I
C(14)-C(13)-C(16) 111.9(6)
C(12)-C(13)-C(16) 110.0(6)
C(14)-C(13)-C(19) 116.9(5)
C(12)-C(13)-C(19) 102.1(5)
C(16)-C(13)-C(19) 108.7(6)
C(13)-C(14)-C(15) 111.9(5)
C(13)-C(14)-H(14A) 109.2
C(15)-C(14)-H(14A) 109.2
C(13)-C(14)-H(14B) 109.2
C(15)-C(14)-H(14B) 109.2
H(14A)-C(14)-H(14B) 107.9
C(14)-C(15)-C(11) 114.5(5)
C(14)-C(15)-H(15A) 108.6
C(11)-C(15)-H(15A) 108.6
C(14)-C(15)-H(15B) 108.6
C(11)-C(15)-H(15B) 108.6
H(15A)-C(15)-H(15B) 107.6
C(62B)#1-C(16)-C(13) 112(4)
C(62B)#1-C(16)-H(16A) 109.5
C(13)-C(16)-H(16A) 108.9
C(62B)#1-C(16)-H(16B) 109.5
C(13)-C(16)-H(16B) 107.2
H(16A)-C(16)-H(16B) 109.5
C(62B)#1-C(16)-H(16C) 109.5
C(13)-C(16)-H(16C) 3.0
H(16A)-C(16)-H(16C) 109.5
H(16B)-C(16)-H(16C) 109.5
C(12)-C(17)-C(18) 104.7(6)
C(12)-C(17)-H(17A) 110.8
C(18)-C(17)-H(17A) 110.8
C(12)-C(17)-H(17B) 110.8
C(18)-C(17)-H(17B) 110.8
Appendix I
H(17A)-C(17)-H(17B) 108.9
C(17)-C(18)-C(19) 108.1(6)
C(17)-C(18)-H(18A) 110.1
C(19)-C(18)-H(18A) 110.1
C(17)-C(18)-H(18B) 110.1
C(19)-C(18)-H(18B) 110.1
H(18A)-C(18)-H(18B) 108.4
C(20)-C(19)-C(13) 120.0(6)
C(20)-C(19)-C(18) 112.4(6)
C(13)-C(19)-C(18) 102.4(5)
C(20)-C(19)-H(19) 107.1
C(13)-C(19)-H(19) 107.1
C(18)-C(19)-H(19) 107.1
C(22)-C(20)-C(21) 109.4(7)
C(22)-C(20)-C(19) 113.8(8)
C(21)-C(20)-C(19) 113.4(7)
C(22)-C(20)-H(20) 106.6
C(21)-C(20)-H(20) 106.6
C(19)-C(20)-H(20) 106.6
C(20)-C(21)-H(21A) 109.5
C(20)-C(21)-H(21B) 109.5
H(21A)-C(21)-H(21B) 109.5
C(20)-C(21)-H(21C) 109.5
H(21A)-C(21)-H(21C) 109.5
H(21B)-C(21)-H(21C) 109.5
C(23)-C(22)-C(20) 124.4(11)
C(23)-C(22)-H(22) 117.8
C(20)-C(22)-H(22) 117.8
C(22)-C(23)-C(24) 124.3(15)
C(22)-C(23)-H(23) 117.8
C(24)-C(23)-H(23) 117.8
C(25)-C(24)-C(28) 115(2)
Appendix I
C(25)-C(24)-C(23) 112(2)
C(28)-C(24)-C(23) 110.7(15)
C(24)-C(25)-C(26) 113(4)
C(24)-C(25)-C(27) 99(3)
C(26)-C(25)-C(27) 112(4)
C(24)-C(28)-C(30) 107(2)
C(24)-C(28)-C(63B) 110.3(17)
C(30)-C(28)-C(63B) 109(2)
O(3)-C(31)-C(36) 111.2(6)
O(3)-C(31)-C(32) 109.0(6)
C(36)-C(31)-C(32) 110.3(6)
O(3)-C(31)-H(31) 108.8
C(36)-C(31)-H(31) 108.8
C(32)-C(31)-H(31) 108.8
C(33)-C(32)-C(31) 112.2(6)
C(33)-C(32)-H(32A) 109.2
C(31)-C(32)-H(32A) 109.2
C(33)-C(32)-H(32B) 109.2
C(31)-C(32)-H(32B) 109.2
H(32A)-C(32)-H(32B) 107.9
C(38)-C(33)-C(32) 121.0(6)
C(38)-C(33)-C(34) 123.3(6)
C(32)-C(33)-C(34) 115.7(5)
C(33)-C(34)-C(35) 107.4(5)
C(33)-C(34)-C(37) 108.7(5)
C(35)-C(34)-C(37) 109.2(5)
C(33)-C(34)-C(41) 110.4(5)
C(35)-C(34)-C(41) 108.7(5)
C(37)-C(34)-C(41) 112.2(5)
C(36)-C(35)-C(34) 114.1(6)
C(36)-C(35)-H(35A) 108.7
C(34)-C(35)-H(35A) 108.7
Appendix I
C(36)-C(35)-H(35B) 108.7
C(34)-C(35)-H(35B) 108.7
H(35A)-C(35)-H(35B) 107.6
C(31)-C(36)-C(35) 110.1(6)
C(31)-C(36)-H(36A) 109.6
C(35)-C(36)-H(36A) 109.6
C(31)-C(36)-H(36B) 109.6
C(35)-C(36)-H(36B) 109.6
H(36A)-C(36)-H(36B) 108.2
C(34)-C(37)-H(37A) 109.5
C(34)-C(37)-H(37B) 109.5
H(37A)-C(37)-H(37B) 109.5
C(34)-C(37)-H(37C) 109.5
H(37A)-C(37)-H(37C) 109.5
H(37B)-C(37)-H(37C) 109.5
C(33)-C(38)-C(39) 125.0(6)
C(33)-C(38)-H(38) 117.5
C(39)-C(38)-H(38) 117.5
C(38)-C(39)-C(40) 112.8(5)
C(38)-C(39)-H(39A) 109.0
C(40)-C(39)-H(39A) 109.0
C(38)-C(39)-H(39B) 109.0
C(40)-C(39)-H(39B) 109.0
H(39A)-C(39)-H(39B) 107.8
C(42)-C(40)-C(39) 112.1(5)
C(42)-C(40)-C(41) 111.1(5)
C(39)-C(40)-C(41) 110.0(5)
C(42)-C(40)-H(40) 107.8
C(39)-C(40)-H(40) 107.8
C(41)-C(40)-H(40) 107.8
C(40)-C(41)-C(45) 111.3(5)
C(40)-C(41)-C(34) 112.6(5)
Appendix I
C(45)-C(41)-C(34) 113.8(5)
C(40)-C(41)-H(41) 106.2
C(45)-C(41)-H(41) 106.2
C(34)-C(41)-H(41) 106.2
C(40)-C(42)-C(43) 115.0(5)
C(40)-C(42)-C(47) 119.2(6)
C(43)-C(42)-C(47) 104.8(5)
C(40)-C(42)-H(42) 105.6
C(43)-C(42)-H(42) 105.6
C(47)-C(42)-H(42) 105.6
C(42)-C(43)-C(44) 107.2(5)
C(42)-C(43)-C(46) 113.0(6)
C(44)-C(43)-C(46) 109.6(6)
C(42)-C(43)-C(49) 102.1(6)
C(44)-C(43)-C(49) 116.4(6)
C(46)-C(43)-C(49) 108.6(6)
C(45)-C(44)-C(43) 111.9(6)
C(45)-C(44)-H(44A) 109.2
C(43)-C(44)-H(44A) 109.2
C(45)-C(44)-H(44B) 109.2
C(43)-C(44)-H(44B) 109.2
H(44A)-C(44)-H(44B) 107.9
C(44)-C(45)-C(41) 114.6(5)
C(44)-C(45)-H(45A) 108.6
C(41)-C(45)-H(45A) 108.6
C(44)-C(45)-H(45B) 108.6
C(41)-C(45)-H(45B) 108.6
H(45A)-C(45)-H(45B) 107.6
C(43)-C(46)-H(46A) 109.5
C(43)-C(46)-H(46B) 109.5
H(46A)-C(46)-H(46B) 109.5
C(43)-C(46)-H(46C) 109.5
Appendix I
H(46A)-C(46)-H(46C) 109.5
H(46B)-C(46)-H(46C) 109.5
C(42)-C(47)-C(48) 105.0(6)
C(42)-C(47)-H(47A) 110.8
C(48)-C(47)-H(47A) 110.8
C(42)-C(47)-H(47B) 110.8
C(48)-C(47)-H(47B) 110.8
H(47A)-C(47)-H(47B) 108.8
C(47)-C(48)-C(49) 106.9(6)
C(47)-C(48)-H(48A) 110.3
C(49)-C(48)-H(48A) 110.3
C(47)-C(48)-H(48B) 110.3
C(49)-C(48)-H(48B) 110.3
H(48A)-C(48)-H(48B) 108.6
C(50)-C(49)-C(43) 121.2(7)
C(50)-C(49)-C(48) 111.3(6)
C(43)-C(49)-C(48) 102.1(6)
C(50)-C(49)-H(49) 107.2
C(43)-C(49)-H(49) 107.2
C(48)-C(49)-H(49) 107.2
C(51)-C(50)-C(49) 113.0(8)
C(51)-C(50)-C(52) 110.7(9)
C(49)-C(50)-C(52) 108.0(9)
C(50)-C(51)-H(51A) 109.5
C(50)-C(51)-H(51B) 109.5
H(51A)-C(51)-H(51B) 109.5
C(50)-C(51)-H(51C) 109.5
H(51A)-C(51)-H(51C) 109.5
H(51B)-C(51)-H(51C) 109.5
C(53)-C(52)-C(50) 115.7(12)
C(53)-C(52)-H(52) 122.2
C(50)-C(52)-H(52) 122.2
Appendix I
C(68B)-C(53)-C(52) 135(3)
C(68B)-C(53)-C(67B) 31(3)
C(52)-C(53)-C(67B) 113(2)
C(68B)-C(53)-C(66B) 23(3)
C(52)-C(53)-C(66B) 112(2)
C(67B)-C(53)-C(66B) 26.5(16)
C(70B)#2-C(60A)-C(70A) 108(9)
C(70B)#2-C(60A)-C(69A) 64(8)
C(70A)-C(60A)-C(69A) 135(8)
C(70B)#2-C(60A)-C(66B) 62(6)
C(70A)-C(60A)-C(66B) 67(6)
C(69A)-C(60A)-C(66B) 71(6)
C(70B)#2-C(60A)-C(67B) 58(5)
C(70A)-C(60A)-C(67B) 90(6)
C(69A)-C(60A)-C(67B) 46(5)
C(66B)-C(60A)-C(67B) 25(2)
C(70B)#2-C(60A)-C(62A) 86(7)
C(70A)-C(60A)-C(62A) 71(6)
C(69A)-C(60A)-C(62A) 65(6)
C(66B)-C(60A)-C(62A) 29(3)
C(67B)-C(60A)-C(62A) 30(3)
C(69A)-C(61A)-C(70B)#2 83(9)
C(66B)-C(62A)-C(67B) 49(6)
C(66B)-C(62A)-C(64A) 47(6)
C(67B)-C(62A)-C(64A) 97(9)
C(66B)-C(62A)-C(68B) 25(5)
C(67B)-C(62A)-C(68B) 26(4)
C(64A)-C(62A)-C(68B) 72(6)
C(66B)-C(62A)-C(64B) 27(5)
C(67B)-C(62A)-C(64B) 76(7)
C(64A)-C(62A)-C(64B) 22(4)
C(68B)-C(62A)-C(64B) 52(4)
Appendix I
C(66B)-C(62A)-C(60A) 72(7)
C(67B)-C(62A)-C(60A) 73(7)
C(64A)-C(62A)-C(60A) 83(7)
C(68B)-C(62A)-C(60A) 76(7)
C(64B)-C(62A)-C(60A) 70(6)
C(64B)-C(64A)-C(66B) 70(8)
C(64B)-C(64A)-C(68A) 92(9)
C(66B)-C(64A)-C(68A) 128(8)
C(64B)-C(64A)-C(62A) 112(10)
C(66B)-C(64A)-C(62A) 45(5)
C(68A)-C(64A)-C(62A) 139(8)
C(64B)-C(64A)-C(70A) 81(8)
C(66B)-C(64A)-C(70A) 107(7)
C(68A)-C(64A)-C(70A) 118(6)
C(62A)-C(64A)-C(70A) 99(5)
C(64B)-C(64A)-C(69B) 20(6)
C(66B)-C(64A)-C(69B) 86(8)
C(68A)-C(64A)-C(69B) 72(5)
C(62A)-C(64A)-C(69B) 130(8)
C(70A)-C(64A)-C(69B) 88(6)
C(64B)-C(64A)-C(68B) 58(7)
C(66B)-C(64A)-C(68B) 16(4)
C(68A)-C(64A)-C(68B) 115(6)
C(62A)-C(64A)-C(68B) 61(6)
C(70A)-C(64A)-C(68B) 112(6)
C(69B)-C(64A)-C(68B) 71(6)
C(64B)-C(64A)-C(67B) 81(8)
C(66B)-C(64A)-C(67B) 12(4)
C(68A)-C(64A)-C(67B) 134(6)
C(62A)-C(64A)-C(67B) 34(5)
C(70A)-C(64A)-C(67B) 106(5)
C(69B)-C(64A)-C(67B) 97(6)
Appendix I
C(68B)-C(64A)-C(67B) 27(2)
C(64A)-C(68A)-C(64B) 30(4)
C(64A)-C(68A)-C(69B) 62(7)
C(64B)-C(68A)-C(69B) 32(4)
C(61A)-C(69A)-C(71A) 137(10)
C(61A)-C(69A)-C(67B) 100(9)
C(71A)-C(69A)-C(67B) 86(8)
C(61A)-C(69A)-C(70B)#2 50(6)
C(71A)-C(69A)-C(70B)#2 149(8)
C(67B)-C(69A)-C(70B)#2 64(5)
C(61A)-C(69A)-C(60A) 97(10)
C(71A)-C(69A)-C(60A) 126(10)
C(67B)-C(69A)-C(60A) 75(6)
C(70B)#2-C(69A)-C(60A) 55(6)
C(61A)-C(69A)-C(68B) 80(8)
C(71A)-C(69A)-C(68B) 104(7)
C(67B)-C(69A)-C(68B) 21(3)
C(70B)#2-C(69A)-C(68B) 46(3)
C(60A)-C(69A)-C(68B) 73(6)
C(64A)-C(70A)-C(64B) 28(3)
C(64A)-C(70A)-C(60A) 89(6)
C(64B)-C(70A)-C(60A) 82(6)
C(64A)-C(70A)-C(66A) 111(5)
C(64B)-C(70A)-C(66A) 114(5)
C(60A)-C(70A)-C(66A) 160(6)
C(69A)-C(71A)-C(67B) 49(5)
C(64A)-C(64B)-C(69B) 144(10)
C(64A)-C(64B)-C(66B) 68(7)
C(69B)-C(64B)-C(66B) 137(9)
C(64A)-C(64B)-C(68A) 58(7)
C(69B)-C(64B)-C(68A) 86(6)
C(66B)-C(64B)-C(68A) 108(6)
Appendix I
C(64A)-C(64B)-C(68B) 98(8)
C(69B)-C(64B)-C(68B) 105(8)
C(66B)-C(64B)-C(68B) 32(4)
C(68A)-C(64B)-C(68B) 119(4)
C(64A)-C(64B)-C(70A) 70(7)
C(69B)-C(64B)-C(70A) 116(7)
C(66B)-C(64B)-C(70A) 101(6)
C(68A)-C(64B)-C(70A) 102(5)
C(68B)-C(64B)-C(70A) 124(4)
C(64A)-C(64B)-C(62A) 45(7)
C(69B)-C(64B)-C(62A) 162(8)
C(66B)-C(64B)-C(62A) 26(5)
C(68A)-C(64B)-C(62A) 97(5)
C(68B)-C(64B)-C(62A) 58(5)
C(70A)-C(64B)-C(62A) 80(4)
C(64A)-C(64B)-C(67B) 76(7)
C(69B)-C(64B)-C(67B) 132(8)
C(66B)-C(64B)-C(67B) 9(4)
C(68A)-C(64B)-C(67B) 117(5)
C(68B)-C(64B)-C(67B) 27(2)
C(70A)-C(64B)-C(67B) 101(4)
C(62A)-C(64B)-C(67B) 32(3)
C(67B)-C(66B)-C(68B) 60(6)
C(67B)-C(66B)-C(62A) 67(6)
C(68B)-C(66B)-C(62A) 126(10)
C(67B)-C(66B)-C(64A) 154(9)
C(68B)-C(66B)-C(64A) 145(9)
C(62A)-C(66B)-C(64A) 87(9)
C(67B)-C(66B)-C(64B) 160(8)
C(68B)-C(66B)-C(64B) 107(8)
C(62A)-C(66B)-C(64B) 127(10)
C(64A)-C(66B)-C(64B) 42(5)
Appendix I
C(67B)-C(66B)-C(69B) 145(7)
C(68B)-C(66B)-C(69B) 88(6)
C(62A)-C(66B)-C(69B) 146(9)
C(64A)-C(66B)-C(69B) 60(7)
C(64B)-C(66B)-C(69B) 20(5)
C(67B)-C(66B)-C(70B)#2 69(6)
C(68B)-C(66B)-C(70B)#2 52(5)
C(62A)-C(66B)-C(70B)#2 118(7)
C(64A)-C(66B)-C(70B)#2 127(7)
C(64B)-C(66B)-C(70B)#2 90(6)
C(69B)-C(66B)-C(70B)#2 81(4)
C(67B)-C(66B)-C(53) 60(4)
C(68B)-C(66B)-C(53) 38(4)
C(62A)-C(66B)-C(53) 105(6)
C(64A)-C(66B)-C(53) 132(5)
C(64B)-C(66B)-C(53) 121(5)
C(69B)-C(66B)-C(53) 103(4)
C(70B)#2-C(66B)-C(53) 89(4)
C(67B)-C(66B)-C(60A) 79(6)
C(68B)-C(66B)-C(60A) 100(7)
C(62A)-C(66B)-C(60A) 79(7)
C(64A)-C(66B)-C(60A) 97(7)
C(64B)-C(66B)-C(60A) 89(6)
C(69B)-C(66B)-C(60A) 96(5)
C(70B)#2-C(66B)-C(60A) 51(4)
C(53)-C(66B)-C(60A) 131(6)
C(68B)-C(67B)-C(66B) 61(5)
C(68B)-C(67B)-C(62A) 123(8)
C(66B)-C(67B)-C(62A) 63(6)
C(68B)-C(67B)-C(69A) 120(8)
C(66B)-C(67B)-C(69A) 135(6)
C(62A)-C(67B)-C(69A) 106(7)
Appendix I
C(68B)-C(67B)-C(53) 54(4)
C(66B)-C(67B)-C(53) 94(5)
C(62A)-C(67B)-C(53) 122(5)
C(69A)-C(67B)-C(53) 124(5)
C(68B)-C(67B)-C(70B)#2 58(5)
C(66B)-C(67B)-C(70B)#2 84(6)
C(62A)-C(67B)-C(70B)#2 125(6)
C(69A)-C(67B)-C(70B)#2 67(6)
C(53)-C(67B)-C(70B)#2 101(3)
C(68B)-C(67B)-C(64A) 75(6)
C(66B)-C(67B)-C(64A) 14(5)
C(62A)-C(67B)-C(64A) 49(6)
C(69A)-C(67B)-C(64A) 132(5)
C(53)-C(67B)-C(64A) 102(4)
C(70B)#2-C(67B)-C(64A) 94(5)
C(68B)-C(67B)-C(64B) 55(5)
C(66B)-C(67B)-C(64B) 11(4)
C(62A)-C(67B)-C(64B) 72(6)
C(69A)-C(67B)-C(64B) 127(5)
C(53)-C(67B)-C(64B) 95(3)
C(70B)#2-C(67B)-C(64B) 73(4)
C(64A)-C(67B)-C(64B) 23(2)
C(68B)-C(67B)-C(71A) 152(7)
C(66B)-C(67B)-C(71A) 148(6)
C(62A)-C(67B)-C(71A) 85(7)
C(69A)-C(67B)-C(71A) 46(5)
C(53)-C(67B)-C(71A) 109(4)
C(70B)#2-C(67B)-C(71A) 112(5)
C(64A)-C(67B)-C(71A) 134(5)
C(64B)-C(67B)-C(71A) 153(4)
C(68B)-C(67B)-C(60A) 99(6)
C(66B)-C(67B)-C(60A) 76(6)
Appendix I
C(62A)-C(67B)-C(60A) 77(6)
C(69A)-C(67B)-C(60A) 59(5)
C(53)-C(67B)-C(60A) 151(5)
C(70B)#2-C(67B)-C(60A) 52(4)
C(64A)-C(67B)-C(60A) 75(5)
C(64B)-C(67B)-C(60A) 70(5)
C(71A)-C(67B)-C(60A) 92(6)
C(67B)-C(68B)-C(66B) 60(6)
C(67B)-C(68B)-C(53) 95(5)
C(66B)-C(68B)-C(53) 119(6)
C(67B)-C(68B)-C(70B)#2 93(6)
C(66B)-C(68B)-C(70B)#2 102(6)
C(53)-C(68B)-C(70B)#2 137(6)
C(67B)-C(68B)-C(64B) 99(7)
C(66B)-C(68B)-C(64B) 40(5)
C(53)-C(68B)-C(64B) 130(4)
C(70B)#2-C(68B)-C(64B) 90(5)
C(67B)-C(68B)-C(62A) 31(5)
C(66B)-C(68B)-C(62A) 29(5)
C(53)-C(68B)-C(62A) 104(4)
C(70B)#2-C(68B)-C(62A) 105(5)
C(64B)-C(68B)-C(62A) 70(5)
C(67B)-C(68B)-C(64A) 78(6)
C(66B)-C(68B)-C(64A) 19(5)
C(53)-C(68B)-C(64A) 118(4)
C(70B)#2-C(68B)-C(64A) 105(5)
C(64B)-C(68B)-C(64A) 24(3)
C(62A)-C(68B)-C(64A) 48(5)
C(67B)-C(68B)-C(69B) 125(7)
C(66B)-C(68B)-C(69B) 67(6)
C(53)-C(68B)-C(69B) 122(4)
C(70B)#2-C(68B)-C(69B) 86(5)
Appendix I
C(64B)-C(68B)-C(69B) 26(4)
C(62A)-C(68B)-C(69B) 96(5)
C(64A)-C(68B)-C(69B) 49(5)
C(67B)-C(68B)-C(69A) 39(6)
C(66B)-C(68B)-C(69A) 86(7)
C(53)-C(68B)-C(69A) 108(4)
C(70B)#2-C(68B)-C(69A) 59(5)
C(64B)-C(68B)-C(69A) 113(5)
C(62A)-C(68B)-C(69A) 65(5)
C(64A)-C(68B)-C(69A) 104(5)
C(69B)-C(68B)-C(69A) 130(4)
C(64B)-C(69B)-C(64A) 16(5)
C(64B)-C(69B)-C(68A) 62(7)
C(64A)-C(69B)-C(68A) 46(4)
C(64B)-C(69B)-C(66B) 23(5)
C(64A)-C(69B)-C(66B) 34(4)
C(68A)-C(69B)-C(66B) 72(5)
C(64B)-C(69B)-C(68B) 48(6)
C(64A)-C(69B)-C(68B) 59(5)
C(68A)-C(69B)-C(68B) 90(5)
C(66B)-C(69B)-C(68B) 26(3)
C(61A)#3-C(70B)-C(68B)#3 101(7)
C(61A)#3-C(70B)-C(60A)#3 99(9)
C(68B)#3-C(70B)-C(60A)#3 92(6)
C(61A)#3-C(70B)-C(69A)#3 47(5)
C(68B)#3-C(70B)-C(69A)#3 76(5)
C(60A)#3-C(70B)-C(69A)#3 61(6)
C(61A)#3-C(70B)-C(67B)#3 85(7)
C(68B)#3-C(70B)-C(67B)#3 29(3)
C(60A)#3-C(70B)-C(67B)#3 70(5)
C(69A)#3-C(70B)-C(67B)#3 49(4)
C(61A)#3-C(70B)-C(66B)#3 112(7)
Appendix I
C(68B)#3-C(70B)-C(66B)#3 26(3)
C(60A)#3-C(70B)-C(66B)#3 67(6)
C(69A)#3-C(70B)-C(66B)#3 72(5)
C(67B)#3-C(70B)-C(66B)#3 26.6(19)
_____________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 x-2,y-2,z-2 #2 -x+1,y+1/2,-z+1/2
#3 -x+1,y-1/2,-z+1/2
Appendix II
Appendix II
Table 1 Atomic coordinates ( x 10^4) and equivalent isotropic displacement
parameters (A^2 x 10^3) for CL-3. U(eq) is defined as one third of the trace of the
orthogonalize Uij tensor.
________________________________________________________________________________
x y z U(eq)
________________________________________________________________________________
O(1) 2318(7) 1848(5) 5803(2) 138(2)
O(2) 4009(6) 1470(4) 4842(2) 90(1)
O(3) 1559(4) 3165(4) 3337(2) 71(1)
O(4) 730(4) 2252(3) -844(2) 71(1)
C(28) 4117(8) 12(6) -1935(3) 88(2)
C(1) 546(12) 4045(8) 5047(4) 147(3)
C(2) 1895(8) 3434(7) 4742(3) 81(2)
C(3) 2737(10) 2219(8) 5176(4) 91(2)
C(4) 4647(7) 1842(5) 4089(3) 70(2)
C(5) 4569(7) 3415(5) 3993(3) 70(2)
C(6) 2599(8) 3879(5) 3971(3) 70(2)
C(7) 2355(8) 5421(5) 3791(3) 96(2)
C(8) 1566(7) 1689(6) 3402(3) 73(2)
C(9) 3480(7) 1068(5) 3446(3) 64(1)
C(10) 4408(6) 1156(5) 2671(2) 57(1)
C(11) 6358(6) 499(6) 2745(3) 76(2)
C(12) 6692(6) -80(5) 1937(3) 73(2)
C(13) 5070(5) 470(4) 1413(2) 46(1)
C(14) 3482(5) 440(4) 1935(2) 47(1)
C(15) 1908(5) 1237(4) 1502(2) 51(1)
C(16) 1451(5) 668(5) 680(2) 50(1)
C(17) 3080(5) 698(4) 182(2) 38(1)
C(18) 4696(5) -113(4) 598(2) 43(1)
C(19) 6384(5) 7(4) 148(3) 55(1)
C(20) 5977(7) -48(4) -708(3) 61(1)
C(21) 4334(6) 49(4) -1088(3) 53(1)
C(22) 2625(5) 206(4) -661(2) 42(1)
C(23) 1600(6) -1218(4) -697(3) 64(1)
C(24) 2895(6) -1078(4) 2118(3) 68(1)
C(25) 1388(6) 1243(5) -1131(3) 54(1)
C(26) 1053(8) 950(6) -1985(3) 87(2)
Appendix II
C(27) 2635(9) 470(6) -2349(3) 83(2)
Table 2 Bond lengths [Å] and angles [°] for CL-3.
_____________________________________________________
O(1)-C(3) 1.199(6)
O(2)-C(3) 1.344(7)
O(2)-C(4) 1.459(5)
O(3)-C(8) 1.409(5)
O(3)-C(6) 1.447(5)
O(4)-C(25) 1.200(5)
C(28)-C(27) 1.323(7)
C(28)-C(21) 1.453(6)
C(28)-H(28) 0.9300
C(1)-C(2) 1.299(8)
C(1)-H(1A) 0.9300
C(1)-H(1B) 0.9300
C(2)-C(3) 1.483(8)
C(2)-C(6) 1.524(7)
C(4)-C(5) 1.506(7)
C(4)-C(9) 1.530(6)
C(4)-H(4) 0.9800
C(5)-C(6) 1.512(7)
C(5)-H(5A) 0.9700
C(5)-H(5B) 0.9700
C(6)-C(7) 1.507(7)
C(7)-H(7A) 0.9600
C(7)-H(7B) 0.9600
C(7)-H(7C) 0.9600
C(8)-C(9) 1.522(6)
C(8)-H(8A) 0.9700
C(8)-H(8B) 0.9700
C(9)-C(10) 1.550(6)
C(9)-H(9) 0.9800
C(10)-C(14) 1.544(5)
C(10)-C(11) 1.559(6)
C(10)-H(10) 0.9800
C(11)-C(12) 1.536(6)
C(11)-H(11A) 0.9700
Appendix II
C(11)-H(11B) 0.9700
C(12)-C(13) 1.524(5)
C(12)-H(12A) 0.9700
C(12)-H(12B) 0.9700
C(13)-C(18) 1.511(5)
C(13)-C(14) 1.532(5)
C(13)-H(13) 0.9800
C(14)-C(15) 1.524(5)
C(14)-C(24) 1.548(5)
C(15)-C(16) 1.525(5)
C(15)-H(15A) 0.9700
C(15)-H(15B) 0.9700
C(16)-C(17) 1.532(5)
C(16)-H(16A) 0.9700
C(16)-H(16B) 0.9700
C(17)-C(22) 1.533(5)
C(17)-C(18) 1.541(5)
C(17)-H(17) 0.9800
C(18)-C(19) 1.522(5)
C(18)-H(18) 0.9800
C(19)-C(20) 1.478(6)
C(19)-H(19A) 0.9700
C(19)-H(19B) 0.9700
C(20)-C(21) 1.327(6)
C(20)-H(120) 0.9300
C(21)-C(22) 1.516(5)
C(22)-C(25) 1.527(6)
C(22)-C(23) 1.549(5)
C(23)-H(23A) 0.9600
C(23)-H(23B) 0.9600
C(23)-H(23C) 0.9600
C(24)-H(24A) 0.9600
C(24)-H(24B) 0.9600
C(24)-H(24C) 0.9600
C(25)-C(26) 1.496(6)
C(26)-C(27) 1.441(7)
C(26)-H(26A) 0.9700
C(26)-H(26B) 0.9700
Appendix II
C(27)-H(27) 0.9300
C(3)-O(2)-C(4) 122.7(4)
C(8)-O(3)-C(6) 114.2(4)
C(27)-C(28)-C(21) 123.2(5)
C(27)-C(28)-H(28) 118.4
C(21)-C(28)-H(28) 118.4
C(2)-C(1)-H(1A) 120.0
C(2)-C(1)-H(1B) 120.0
H(1A)-C(1)-H(1B) 120.0
C(1)-C(2)-C(3) 116.4(6)
C(1)-C(2)-C(6) 123.7(6)
C(3)-C(2)-C(6) 119.8(5)
O(1)-C(3)-O(2) 118.1(6)
O(1)-C(3)-C(2) 123.7(7)
O(2)-C(3)-C(2) 118.2(5)
O(2)-C(4)-C(5) 109.1(4)
O(2)-C(4)-C(9) 108.8(4)
C(5)-C(4)-C(9) 112.7(4)
O(2)-C(4)-H(4) 108.7
C(5)-C(4)-H(4) 108.7
C(9)-C(4)-H(4) 108.7
C(4)-C(5)-C(6) 108.7(4)
C(4)-C(5)-H(5A) 110.0
C(6)-C(5)-H(5A) 110.0
C(4)-C(5)-H(5B) 110.0
C(6)-C(5)-H(5B) 110.0
H(5A)-C(5)-H(5B) 108.3
O(3)-C(6)-C(7) 104.8(4)
O(3)-C(6)-C(5) 109.0(4)
C(7)-C(6)-C(5) 112.7(5)
O(3)-C(6)-C(2) 109.2(4)
C(7)-C(6)-C(2) 113.9(5)
C(5)-C(6)-C(2) 107.1(4)
C(6)-C(7)-H(7A) 109.5
C(6)-C(7)-H(7B) 109.5
H(7A)-C(7)-H(7B) 109.5
C(6)-C(7)-H(7C) 109.5
Appendix II
H(7A)-C(7)-H(7C) 109.5
H(7B)-C(7)-H(7C) 109.5
O(3)-C(8)-C(9) 112.8(4)
O(3)-C(8)-H(8A) 109.0
C(9)-C(8)-H(8A) 109.0
O(3)-C(8)-H(8B) 109.0
C(9)-C(8)-H(8B) 109.0
H(8A)-C(8)-H(8B) 107.8
C(8)-C(9)-C(4) 107.9(4)
C(8)-C(9)-C(10) 114.2(4)
C(4)-C(9)-C(10) 109.6(4)
C(8)-C(9)-H(9) 108.3
C(4)-C(9)-H(9) 108.3
C(10)-C(9)-H(9) 108.3
C(14)-C(10)-C(9) 119.1(3)
C(14)-C(10)-C(11) 103.2(3)
C(9)-C(10)-C(11) 112.2(4)
C(14)-C(10)-H(10) 107.3
C(9)-C(10)-H(10) 107.3
C(11)-C(10)-H(10) 107.3
C(12)-C(11)-C(10) 106.7(4)
C(12)-C(11)-H(11A) 110.4
C(10)-C(11)-H(11A) 110.4
C(12)-C(11)-H(11B) 110.4
C(10)-C(11)-H(11B) 110.4
H(11A)-C(11)-H(11B) 108.6
C(13)-C(12)-C(11) 103.6(4)
C(13)-C(12)-H(12A) 111.0
C(11)-C(12)-H(12A) 111.0
C(13)-C(12)-H(12B) 111.0
C(11)-C(12)-H(12B) 111.0
H(12A)-C(12)-H(12B) 109.0
C(18)-C(13)-C(12) 119.5(4)
C(18)-C(13)-C(14) 116.7(3)
C(12)-C(13)-C(14) 104.1(4)
C(18)-C(13)-H(13) 105.0
C(12)-C(13)-H(13) 105.0
C(14)-C(13)-H(13) 105.0
Appendix II
C(15)-C(14)-C(13) 106.8(3)
C(15)-C(14)-C(10) 116.4(3)
C(13)-C(14)-C(10) 99.7(3)
C(15)-C(14)-C(24) 110.6(3)
C(13)-C(14)-C(24) 112.1(4)
C(10)-C(14)-C(24) 110.8(3)
C(14)-C(15)-C(16) 112.0(3)
C(14)-C(15)-H(15A) 109.2
C(16)-C(15)-H(15A) 109.2
C(14)-C(15)-H(15B) 109.2
C(16)-C(15)-H(15B) 109.2
H(15A)-C(15)-H(15B) 107.9
C(15)-C(16)-C(17) 113.0(3)
C(15)-C(16)-H(16A) 109.0
C(17)-C(16)-H(16A) 109.0
C(15)-C(16)-H(16B) 109.0
C(17)-C(16)-H(16B) 109.0
H(16A)-C(16)-H(16B) 107.8
C(16)-C(17)-C(22) 113.9(3)
C(16)-C(17)-C(18) 109.8(3)
C(22)-C(17)-C(18) 112.6(3)
C(16)-C(17)-H(17) 106.7
C(22)-C(17)-H(17) 106.7
C(18)-C(17)-H(17) 106.7
C(13)-C(18)-C(19) 110.7(3)
C(13)-C(18)-C(17) 108.6(3)
C(19)-C(18)-C(17) 110.8(3)
C(13)-C(18)-H(18) 108.9
C(19)-C(18)-H(18) 108.9
C(17)-C(18)-H(18) 108.9
C(20)-C(19)-C(18) 113.6(4)
C(20)-C(19)-H(19A) 108.8
C(18)-C(19)-H(19A) 108.8
C(20)-C(19)-H(19B) 108.8
C(18)-C(19)-H(19B) 108.8
H(19A)-C(19)-H(19B) 107.7
C(21)-C(20)-C(19) 125.9(4)
C(21)-C(20)-H(120) 117.0
Appendix II
C(19)-C(20)-H(120) 117.0
C(20)-C(21)-C(28) 120.7(5)
C(20)-C(21)-C(22) 121.7(4)
C(28)-C(21)-C(22) 117.7(4)
C(21)-C(22)-C(25) 106.7(3)
C(21)-C(22)-C(17) 111.5(3)
C(25)-C(22)-C(17) 112.1(3)
C(21)-C(22)-C(23) 108.3(3)
C(25)-C(22)-C(23) 106.2(3)
C(17)-C(22)-C(23) 111.8(3)
C(22)-C(23)-H(23A) 109.5
C(22)-C(23)-H(23B) 109.5
H(23A)-C(23)-H(23B) 109.5
C(22)-C(23)-H(23C) 109.5
H(23A)-C(23)-H(23C) 109.5
H(23B)-C(23)-H(23C) 109.5
C(14)-C(24)-H(24A) 109.5
C(14)-C(24)-H(24B) 109.5
H(24A)-C(24)-H(24B) 109.5
C(14)-C(24)-H(24C) 109.5
H(24A)-C(24)-H(24C) 109.5
H(24B)-C(24)-H(24C) 109.5
O(4)-C(25)-C(26) 121.0(4)
O(4)-C(25)-C(22) 122.8(4)
C(26)-C(25)-C(22) 116.2(4)
C(27)-C(26)-C(25) 114.5(4)
C(27)-C(26)-H(26A) 108.6
C(25)-C(26)-H(26A) 108.6
C(27)-C(26)-H(26B) 108.6
C(25)-C(26)-H(26B) 108.6
H(26A)-C(26)-H(26B) 107.6
C(28)-C(27)-C(26) 121.9(5)
C(28)-C(27)-H(27) 119.1
C(26)-C(27)-H(27) 119.1
_____________________________________________________________
Symmetry transformations used to generate equivalent atoms:
Appendix III
Appendix III
Table 1. Atomic coordinates ( x 10^4) and equivalent isotropic displacement
parameters (A^2 x 10^3) for CF-5. U(eq) is defined as one third of the trace of the
orthogonalized Uij tensor.
________________________________________________________________
x y z U(eq)
________________________________________________________________
O(1) 5560(4) -679(3) 7952(3) 103(2)
C(1) 4962(4) -304(3) 7404(3) 57(1)
C(2) 4165(4) -866(3) 6856(3) 63(1)
C(3) 4228(4) -599(2) 5914(2) 50(1)
C(4) 4046(3) 464(2) 5745(2) 36(1)
C(5) 4912(3) 1003(2) 6307(2) 38(1)
C(6) 4981(3) 763(3) 7290(2) 47(1)
C(7) 6102(4) 1140(3) 7660(3) 66(1)
C(8) 4012(4) 1147(4) 7856(3) 68(1)
C(9) 2794(3) 697(3) 5952(3) 55(1)
C(10) 4371(2) 649(2) 4782(2) 34(1)
C(11) 4533(2) 1689(2) 4493(2) 35(1)
C(12) 5230(3) 2238(2) 5173(2) 46(1)
C(13) 4942(3) 2064(2) 6115(2) 44(1)
C(14) 3900(3) 2508(2) 6361(2) 39(1)
C(15) 3386(3) 2195(3) 4383(2) 49(1)
C(16) 3594(3) 143(2) 4136(2) 44(1)
C(17) 3858(3) 391(2) 3222(2) 41(1)
C(18) 4553(2) 1067(2) 2952(2) 36(1)
C(19) 5201(3) 1675(2) 3599(2) 38(1)
C(20) 6395(3) 1233(3) 3702(3) 54(1)
C(21) 4755(3) 1199(2) 1989(2) 39(1)
C(22) 4790(3) 2244(2) 1732(2) 48(1)
C(23) 5637(4) 2755(3) 2303(3) 61(1)
C(24) 5362(4) 2692(3) 3267(3) 56(1)
C(25) 3618(4) 2686(3) 1853(3) 71(1)
Appendix III
C(26) 5802(3) 653(3) 1683(2) 51(1)
C(27) 6063(4) 737(3) 716(3) 58(1)
C(28) 6117(4) 1792(3) 491(3) 66(1)
C(29) 5085(4) 2330(3) 764(3) 64(1)
C(30) 7204(5) 284(5) 547(4) 94(2)
C(31) 5158(5) 228(3) 184(3) 74(1)
________________________________________________________________
Table 2 Bond lengths [A] and angles [deg] for CF-5.
_____________________________________________________________
O(1)-C(1) 1.230(5)
C(1)-C(2) 1.504(6)
C(1)-C(6) 1.527(5)
C(2)-C(3) 1.516(5)
C(2)-H(2A) 0.9700
C(2)-H(2B) 0.9700
C(3)-C(4) 1.548(4)
C(3)-H(3A) 0.9700
C(3)-H(3B) 0.9700
C(4)-C(5) 1.552(4)
C(4)-C(9) 1.557(4)
C(4)-C(10) 1.569(4)
C(5)-C(13) 1.537(4)
C(5)-C(6) 1.569(5)
C(5)-H(5) 0.9800
C(6)-C(7) 1.547(5)
C(6)-C(8) 1.548(6)
C(7)-H(7A) 0.9600
C(7)-H(7B) 0.9600
C(7)-H(7C) 0.9600
C(8)-H(8A) 0.9600
C(8)-H(8B) 0.9600
C(8)-H(8C) 0.9600
Appendix III
C(9)-H(9A) 0.9600
C(9)-H(9B) 0.9600
C(9)-H(9C) 0.9600
C(10)-C(16) 1.542(4)
C(10)-C(11) 1.556(4)
C(10)-H(10) 0.9800
C(11)-C(15) 1.550(4)
C(11)-C(12) 1.553(4)
C(11)-C(19) 1.601(4)
C(12)-C(13) 1.525(5)
C(12)-H(12A) 0.9700
C(12)-H(12B) 0.9700
C(13)-C(14) 1.440(5)
C(13)-H(13) 0.9800
C(14)-H(14A) 0.9600
C(14)-H(14B) 0.9600
C(14)-H(14C) 0.9600
C(15)-H(15A) 0.9600
C(15)-H(15B) 0.9600
C(15)-H(15C) 0.9600
C(16)-C(17) 1.497(5)
C(16)-H(16A) 0.9700
C(16)-H(16B) 0.9700
C(17)-C(18) 1.334(4)
C(17)-H(17) 0.9300
C(18)-C(21) 1.528(5)
C(18)-C(19) 1.533(4)
C(19)-C(24) 1.547(4)
C(19)-C(20) 1.558(5)
C(20)-H(20A) 0.9600
C(20)-H(20B) 0.9600
C(20)-H(20C) 0.9600
Appendix III
C(21)-C(22) 1.539(4)
C(21)-C(26) 1.541(5)
C(21)-H(21) 0.9800
C(22)-C(23) 1.526(6)
C(22)-C(25) 1.538(6)
C(22)-C(29) 1.551(5)
C(23)-C(24) 1.537(6)
C(23)-H(23A) 0.9700
C(23)-H(23B) 0.9700
C(24)-H(24A) 0.9700
C(24)-H(24B) 0.9700
C(25)-H(25A) 0.9600
C(25)-H(25B) 0.9600
C(25)-H(25C) 0.9600
C(26)-C(27) 1.540(5)
C(26)-H(26A) 0.9700
C(26)-H(26B) 0.9700
C(27)-C(30) 1.522(7)
C(27)-C(31) 1.538(7)
C(27)-C(28) 1.541(6)
C(28)-C(29) 1.506(7)
C(28)-H(28A) 0.9700
C(28)-H(28B) 0.9700
C(29)-H(29A) 0.9700
C(29)-H(29B) 0.9700
C(30)-H(30A) 0.9600
C(30)-H(30B) 0.9600
C(30)-H(30C) 0.9600
C(31)-H(31A) 0.9600
C(31)-H(31B) 0.9600
C(31)-H(31C) 0.9600
O(1)-C(1)-C(2) 121.7(4)
Appendix III
O(1)-C(1)-C(6) 120.2(4)
C(2)-C(1)-C(6) 118.1(3)
C(3)-C(2)-C(1) 112.5(3)
C(3)-C(2)-H(2A) 109.1
C(1)-C(2)-H(2A) 109.1
C(3)-C(2)-H(2B) 109.1
C(1)-C(2)-H(2B) 109.1
H(2A)-C(2)-H(2B) 107.8
C(2)-C(3)-C(4) 113.7(3)
C(2)-C(3)-H(3A) 108.8
C(4)-C(3)-H(3A) 108.8
C(2)-C(3)-H(3B) 108.8
C(4)-C(3)-H(3B) 108.8
H(3A)-C(3)-H(3B) 107.7
C(3)-C(4)-C(5) 107.1(3)
C(3)-C(4)-C(9) 107.8(3)
C(5)-C(4)-C(9) 114.3(3)
C(3)-C(4)-C(10) 107.0(2)
C(5)-C(4)-C(10) 107.0(2)
C(9)-C(4)-C(10) 113.3(3)
C(13)-C(5)-C(4) 113.0(3)
C(13)-C(5)-C(6) 113.7(3)
C(4)-C(5)-C(6) 118.4(3)
C(13)-C(5)-H(5) 103.1
C(4)-C(5)-H(5) 103.1
C(6)-C(5)-H(5) 103.1
C(1)-C(6)-C(7) 108.3(3)
C(1)-C(6)-C(8) 105.8(3)
C(7)-C(6)-C(8) 107.8(3)
C(1)-C(6)-C(5) 109.2(3)
C(7)-C(6)-C(5) 109.4(3)
C(8)-C(6)-C(5) 116.1(3)
Appendix III
C(6)-C(7)-H(7A) 109.5
C(6)-C(7)-H(7B) 109.5
H(7A)-C(7)-H(7B) 109.5
C(6)-C(7)-H(7C) 109.5
H(7A)-C(7)-H(7C) 109.5
H(7B)-C(7)-H(7C) 109.5
C(6)-C(8)-H(8A) 109.5
C(6)-C(8)-H(8B) 109.5
H(8A)-C(8)-H(8B) 109.5
C(6)-C(8)-H(8C) 109.5
H(8A)-C(8)-H(8C) 109.5
H(8B)-C(8)-H(8C) 109.5
C(4)-C(9)-H(9A) 109.5
C(4)-C(9)-H(9B) 109.5
H(9A)-C(9)-H(9B) 109.5
C(4)-C(9)-H(9C) 109.5
H(9A)-C(9)-H(9C) 109.5
H(9B)-C(9)-H(9C) 109.5
C(16)-C(10)-C(11) 109.2(3)
C(16)-C(10)-C(4) 113.4(2)
C(11)-C(10)-C(4) 117.7(2)
C(16)-C(10)-H(10) 105.1
C(11)-C(10)-H(10) 105.1
C(4)-C(10)-H(10) 105.1
C(15)-C(11)-C(12) 108.1(3)
C(15)-C(11)-C(10) 111.3(2)
C(12)-C(11)-C(10) 110.3(3)
C(15)-C(11)-C(19) 110.2(3)
C(12)-C(11)-C(19) 109.5(2)
C(10)-C(11)-C(19) 107.5(2)
C(13)-C(12)-C(11) 117.0(3)
C(13)-C(12)-H(12A) 108.1
Appendix III
C(11)-C(12)-H(12A) 108.1
C(13)-C(12)-H(12B) 108.1
C(11)-C(12)-H(12B) 108.0
H(12A)-C(12)-H(12B) 107.3
C(14)-C(13)-C(12) 112.1(3)
C(14)-C(13)-C(5) 111.0(3)
C(12)-C(13)-C(5) 110.5(3)
C(14)-C(13)-H(13) 107.7
C(12)-C(13)-H(13) 107.6
C(5)-C(13)-H(13) 107.7
C(13)-C(14)-H(14A) 109.5
C(13)-C(14)-H(14B) 109.5
H(14A)-C(14)-H(14B) 109.5
C(13)-C(14)-H(14C) 109.5
H(14A)-C(14)-H(14C) 109.5
H(14B)-C(14)-H(14C) 109.5
C(11)-C(15)-H(15A) 109.5
C(11)-C(15)-H(15B) 109.5
H(15A)-C(15)-H(15B) 109.5
C(11)-C(15)-H(15C) 109.5
H(15A)-C(15)-H(15C) 109.5
H(15B)-C(15)-H(15C) 109.5
C(17)-C(16)-C(10) 112.6(3)
C(17)-C(16)-H(16A) 109.1
C(10)-C(16)-H(16A) 109.1
C(17)-C(16)-H(16B) 109.1
C(10)-C(16)-H(16B) 109.1
H(16A)-C(16)-H(16B) 107.8
C(18)-C(17)-C(16) 126.7(3)
C(18)-C(17)-H(17) 116.6
C(16)-C(17)-H(17) 116.6
C(17)-C(18)-C(21) 119.6(3)
Appendix III
C(17)-C(18)-C(19) 120.6(3)
C(21)-C(18)-C(19) 119.7(3)
C(18)-C(19)-C(24) 111.6(3)
C(18)-C(19)-C(20) 107.3(3)
C(24)-C(19)-C(20) 107.3(3)
C(18)-C(19)-C(11) 109.2(2)
C(24)-C(19)-C(11) 109.9(3)
C(20)-C(19)-C(11) 111.5(3)
C(19)-C(20)-H(20A) 109.5
C(19)-C(20)-H(20B) 109.5
H(20A)-C(20)-H(20B) 109.5
C(19)-C(20)-H(20C) 109.5
H(20A)-C(20)-H(20C) 109.5
H(20B)-C(20)-H(20C) 109.5
C(18)-C(21)-C(22) 112.2(3)
C(18)-C(21)-C(26) 111.6(3)
C(22)-C(21)-C(26) 112.5(3)
C(18)-C(21)-H(21) 106.7
C(22)-C(21)-H(21) 106.7
C(26)-C(21)-H(21) 106.7
C(23)-C(22)-C(21) 109.0(3)
C(23)-C(22)-C(25) 109.3(3)
C(21)-C(22)-C(25) 109.7(3)
C(23)-C(22)-C(29) 112.3(3)
C(21)-C(22)-C(29) 109.6(3)
C(25)-C(22)-C(29) 106.9(3)
C(24)-C(23)-C(22) 113.6(3)
C(24)-C(23)-H(23A) 108.8
C(22)-C(23)-H(23A) 108.8
C(24)-C(23)-H(23B) 108.8
C(22)-C(23)-H(23B) 108.8
H(23A)-C(23)-H(23B) 107.7
Appendix III
C(23)-C(24)-C(19) 114.0(3)
C(23)-C(24)-H(24A) 108.7
C(19)-C(24)-H(24A) 108.7
C(23)-C(24)-H(24B) 108.7
C(19)-C(24)-H(24B) 108.7
H(24A)-C(24)-H(24B) 107.6
C(22)-C(25)-H(25A) 109.5
C(22)-C(25)-H(25B) 109.5
H(25A)-C(25)-H(25B) 109.5
C(22)-C(25)-H(25C) 109.5
H(25A)-C(25)-H(25C) 109.5
H(25B)-C(25)-H(25C) 109.5
C(21)-C(26)-C(27) 115.2(3)
C(21)-C(26)-H(26A) 108.5
C(27)-C(26)-H(26A) 108.5
C(21)-C(26)-H(26B) 108.5
C(27)-C(26)-H(26B) 108.5
H(26A)-C(26)-H(26B) 107.5
C(30)-C(27)-C(31) 109.3(4)
C(30)-C(27)-C(28) 109.6(4)
C(31)-C(27)-C(28) 111.4(4)
C(30)-C(27)-C(26) 108.3(4)
C(31)-C(27)-C(26) 110.4(3)
C(28)-C(27)-C(26) 107.8(3)
C(29)-C(28)-C(27) 113.4(4)
C(29)-C(28)-H(28A) 108.9
C(27)-C(28)-H(28A) 108.9
C(29)-C(28)-H(28B) 108.9
C(27)-C(28)-H(28B) 108.9
H(28A)-C(28)-H(28B) 107.7
C(28)-C(29)-C(22) 114.7(4)
C(28)-C(29)-H(29A) 108.6
Appendix III
C(22)-C(29)-H(29A) 108.6
C(28)-C(29)-H(29B) 108.6
C(22)-C(29)-H(29B) 108.6
H(29A)-C(29)-H(29B) 107.6
C(27)-C(30)-H(30A) 109.5
C(27)-C(30)-H(30B) 109.5
H(30A)-C(30)-H(30B) 109.5
C(27)-C(30)-H(30C) 109.5
H(30A)-C(30)-H(30C) 109.5
H(30B)-C(30)-H(30C) 109.5
C(27)-C(31)-H(31A) 109.5
C(27)-C(31)-H(31B) 109.5
H(31A)-C(31)-H(31B) 109.5
C(27)-C(31)-H(31C) 109.5
H(31A)-C(31)-H(31C) 109.5
H(31B)-C(31)-H(31C) 109.5
_____________________________________________________________
Symmetry transformations used to generate equivalent atoms:
Appendix III