by HUMAIRA FATIMA CHUGHTAI

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


In partial fulfillment of the requirements for degree of

Doctor of Philosophy

in

Pharmacy (Pharmacognosy)


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


2.2.8 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




2.2.9.4 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


3.2.1 Total phenolic content 50

3.2.2 Total flavonoid content 51

3.2.3 RP-HPLC analysis 51



3.3.1.1 %RSA 60

3.3.1.2 TAC 60

3.3.1.3 TRP 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




3.3.4 Assays for the determination of cytotoxic potential 63


3.3.4.2 Cytotoxicity against cell lines 71

3.4 Summary 71


3.5 Fraction yield 72



3.6.1.1 %RSA 72

3.6.1.2 TAC 73

3.6.1.3 TRP 74



3.6.2.2 Antibacterial activity 77

3.6.2.3 Antifungal activity 77

3.6.3 Enzyme inhibition assay 77



3.6.4 Cytotoxicity assays 78


3.6.4.2 Cytotoxicity against Hep G2 cell line 78

3.7 Summary 83



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



4.3.1 Antioxidant potential 105

4.3.2 Antimicrobial activities 106


4.3.3.1 α-amylase inhibitory activity 108

4.3.3.1 Protein kinase inhibition potential 109

4.3.4 Cytotoxicity determination 109


4.4 Preparative extraction 111

4.5 Fractionation 112

4.6 Biological evaluation of fractions 112


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


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


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


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


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


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).


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).


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


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.


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)


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).


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)


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).


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).


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


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


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.


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)


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).


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)


(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


(10 ml each)

Crystals in fractions 29-34 to yield CL-3

(43 g)


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)


(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)


(10 ml each)

Crystals in vials 44-51 were washed with n-hexane to give CF-

5 (125 mg)


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.


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).


The protein kinase inhibition assay was performed using standard protocol and is

attached as annexure A (Fatima et al., 2015).


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).


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


3.2.1 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

57

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

Ch.3: Results

58

Figure 3.2(b) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia stem


thrice.

Ch.3: Results

59

Figure 3.2(c) TPC, TFC, TAC, TRP and %RSA determination in D. innoxia fruit


thrice.

Ch.3: Results

60



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.


3.3.2.1 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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62

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.

Ch.3: Results

63



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.


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


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

64

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.

Ch.3: Results

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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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66

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

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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)


%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

-- -- --

Ch.3: Results

71

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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72

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



3.6.1.1 %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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73

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


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).



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


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


Ch.3: Results

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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


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).


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


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.


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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84

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


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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93

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).

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Figure 3.15 13C (100 MHz, in CDCl3) spectrum of CL-3

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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.

Ch.3: Results

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

Ch.4: Discussion

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).


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).


4.3.1 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.


4.3.3.1 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

_____________________________________________________________


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

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Appendix III

Documents

by HUMAIRA FATIMA CHUGHTAI