chemical and molecular characterization of marigold ( tagetes...

Chemical and Molecular Characterization of

Marigold (Tagetes Species) Found in Pakistan

By

Irum Shahzadi

CIIT/SP05-R07-005/ATD

PhD Thesis

In

Environmental Sciences

COMSATS Institute of Information Technology Abbottabad - Pakistan

Fall, 2012

ii

COMSATS Institute of Information Technology

Chemical and Molecular Characterization of Marigold (Tagetes Species) Found in Pakistan

A Thesis Presented to

COMSATS Institute of Information Technology, Abbottabad

In partial fulfillment

of the requirements for the degree of

PhD

(Environmental Sciences)

By

Irum Shahzadi


Fall, 2012

iii


A Post Graduate Thesis submitted to the Department of Environmental Sciences

as partial fulfillment of the requirements for the award of Degree of PhD.

Name Registration Number

Irum Shahzadi CIIT/SP05-R07-005/ATD

Supervisor

Dr. Mohammad Maroof Shah Professor Department of Environmental Sciences COMSATS Institute of Information Technology (CIIT) Abbottabad

November, 2012

iv

Final Approval

This thesis titled


By

Irum Shahzadi


Has been approved

For the COMSATS Institute of Information Technology, Abbottabad

External Examiners: 1. ______________________________________________

Prof. Dr. Zahoor Ahmed Swati Director, Institute of Biotechnology and Genetic Engineering, KPK, Agricultural University, Peshawar

2. ______________________________________________

Dr. Uzaira Rafique Assoc. Prof. Department of Environmental Sciences Fatima Jinnah Women University, Rawalpindi

Supervisor: ______________________________________________ Prof. Dr. Mohammad Maroof Shah Department of Environmental Sciences, CIIT, Abbottabad

Head of Department: ______________________________________________ Prof. Dr. Arshid Pervez Department of Environmental Sciences, CIIT, Abbottabad

Dean, Faculty of Sciences: ______________________________________________ Prof. Dr. Arshad Saleem Bhatti, CIIT, Islamabad

v

Declaration

I, Irum Shahzadi, CIIT/SP05-R07-005/ATD hereby declare that I have produced the work presented in this thesis, during the scheduled period of study. I also declare that I have not taken any material from any source except referred to wherever due, and that the amount of plagiarism is within acceptable range. If a violation of rules on research has occurred in this thesis, I shall be liable to punishable action under the plagiarism rules of the HEC. Signature of the student: Date: ______________ ________________________

Irum Shahzadi CIIT/SP05-R07-005/ATD

vi

Certificate

It is certified that Miss Irum Shahzadi, CIIT/SP05-R07-005/ATD has carried out all the work related to this thesis under my supervision at the Department of Environmental Sciences, COMSATS Institute of Informational Technology, Abbottabad and the work fulfills the requirements for award of PhD degree. Date: ______________________

Supervisor: _________________________________ Prof. Dr. Mohammad Maroof Shah Department of Environmental Sciences CIIT, Abbottabad

Head of Department: ________________________________ Prof. Dr. Arshid Pervez Department of Environmental Sciences CIIT, Abbottabad

vii

DEDICATION

To

My most respected parents for their love, deep care and

encouragement that was a great strength to achieve this

remarkable objective of my life

viii

ACKNOWLEDGEMENTS

I am grateful to Almighty Allah (SWT) who helped me to complete this thesis and gave me courage and determination as well as true guidance in conducting my research work despite all difficulties. I need His help at every turn and every moment of my life.

First of all I am thankful to my supervisor Dr. Mohammad Maroof Shah, Professor, Department of Environmental Sciences, CIIT-Abbottabad for his excellent supervision, and guidance throughout my research and academic career. He always pushed me forward and encouraged to have firm belief in myself. I extend my gratitude to Professor Øyvind M. Andersen, Department of Chemistry, University of Bergen, Norway for his guidance in the identification and analyses of important chemical compounds in Tagetes. I acknowledge the Higher Education Commission of Pakistan (HEC) for providing me an opportunity to travel to the University of Bergen under International Research Support Initiative Program (IRSIP) where I completed a major objective of my study.

I express my gratitude to my supervisory committee including Dr. Raza Ahmad, Dr. Amjad Hassan, and Dr. Robina Farooq, for their help in my research work at CIIT Abbottabad. Particularly, I want to thank Dr. Raza Ahmad, for extending his expertise and auspicious efforts in carrying out molecular aspects of my research work and Dr. Amjad Hassan for assistance in thesis revisions. I also express my deepest thanks to Dr. Iftikhar A. Raja, Professor, Department of Environmental Sciences, for his help and continuous encouragement. Thanks are also extended to Dr. Arshid Pervez for support in research facilitation and to all faculty members of the Department of Environmental Sciences for long-lasting and unbendable support throughout the course of my studies and research work. Thanks to Dr. Manzoor Hussain, Professor and Taxonomist, Department of Botany, Post-Graduate College, Abbottabad, for species identification.

I also express my sincere thanks to lab staff especially Mr. Muhammad Amjad, Mr. Muhammad Jamshaid and Mr. Aurangzeb at CIIT, Abbottabad and the staff at the University of Bergen for their assistance in experiments. I am grateful to my friends for their support especially Maryam Mustafa, Ummara Waheed Khan and Maria Saddique who made my days joyous at and outside the department. I am highly obliged to my brothers Raja Yaser Ayaz, Raja Ihtsham, Raja Usman Akbar and sisters Noreen Ayaz and Aisha Ayaz who supported me throughout my life and academic career. My deepest gratitude goes to my parents who taught me the importance of having dreams, setting goals, achieving the targets and the art of hard work.

Irum Shahzadi

ix

ABSTRACT

Chemical and Molecular Characterization of Marigold (Tagetes

Species) Found in Pakistan Irum Shahzadi

Tagetes, a genus of flowering marigold is a medicinal plant grown over a wide range of climatic conditions. Molecular and chemical characterization of this important plant species may lead to identify novel compounds of high medicinal value. Major goal of the current research project was to characterize Tagetes species on chemical and molecular bases. The specific objectives of the study were to optimize extraction protocols for DNA and chemical compounds, molecular based genetic diversity of Tagetes plants, develop and characterize gene based markers, identify and characterize new/novel compounds present in the species. Fifteen genotypes of Tagetes including hybrids from different parts of Pakistan were collected and used. The plants of all Tagetes were grown in pots and field at CIIT Abbottabad for molecular analyses where as bulk amount of Tagetes minuta was collected and used for chemical analyses. The DNA from all genotypes was isolated using fresh, dried leaf tissues and seeds. Specific primers were developed for limonene synthase gene using sequence analyses and bioinformatic tools. Twenty-five random (RAPD) and 5 specific (degenerate-STS) primers were used for genetic diversity analysis. Chemical compounds were extracted using standard solvent extraction techniques and the crude fractions were tested for biological activities. The fractions were further purified and analyzed by chromatographic (TLC and HPLC) and spectroscopic (LCMS and NMR) techniques for compound identification and structural elucidation. The DNA isolated from fresh leaves and seeds of Tagetes was of good quantity and quality for PCR analyses. Significant variations were observed among all the genotypes of Tagetes species using PCR based markers. Twenty RAPD primers out of 25 and 3 degenerate-STS primers out of 5 revealed high polymorphism rate of 95.21% and 96.55% respectively. The phenograms generated using both marker systems grouped 15 Tagetes genotypes into 4 distinct groups, while T. minuta was placed genetically as most distantly related and unique species. PCR analysis with limonene synthase gene specific degenerate primers amplified candidate gene for limonene synthase in T. minuta. Bioactive extracts from flower parts of Tagetes exhibited antimalarial, phytotoxic, insecticidal and antibacterial activity. The HPLC profile revealed that butanol fraction contained good quality and quantity of flavonoids (17 flavonols) as compared to ethyl acetate (2 falvonols) and chloroform (mixture of other compounds). A total of 19 falvaonols were identified, of which 8 were structurally elucidated. Four new flavonols viz; 6-hydroxy quercetin 7-O-β-(6''-galloylglucopyranoside) (2), 6-hydroxy quercetin 7-O-β-(6''- caffeoylglucopyranoside) (9), 6-hydroxy kaempferol 7-O-β-glucopyranoside (5), 6-hydroxy kaempferol 7-O-β-(6''-galloylglucopyranoside) (7) were identified using butanol fraction. Four known flavonols, viz; 6-hydroxyquercetin 7-O-β-glucopyranoside (1), 6-methoxy quercetin 7-O-β-glucopyranoside (6), 6-methoxyquercetin (19), and 6-hydroxyquercetin (18) were also identified using butanol (1, 7) and ethyl acetate (18, 19) fractions. The combined molecular and biochemical information obtained will lead us to improve Tagetes for highly valuable compounds which may generate foreign exchange by exporting this important medicinal plant to other parts of the world.

x

TABLE OF CONTENTS

1 Introduction……………………………………………...................... 1

1.1 Genus Tagetes…...………………………………………………. 2

1.2 Tagetes species…………………………………………………… 2

1.2.1 Tagetes minuta L…………………………………………. 3

1.3 Molecular Characterization of Plant…………………................... 3

1.4 Chemical Characterization of Tagetes…………………………… 6

1.4.1 Essential Oils………………………………………............ 6

1.4.1.1 Terpenes...……………………………………..... 6

1.4.2 Flovonoids.………………………………………………... 7

1.4.2.1 Flavonols.………………………………………. 8

1.4.2.2 Structures of Flavonols.………………………… 8

1.4.2.3 Functions in Plant.……………………………… 12

1.4.2.4 Potential Health Effects………………………… 12

1.5 Uses of Tagetes Species.………………………………………..... 12

1.6 Objectives.………………………………………………………... 15

2 Materials and Methods..………………………………………………. 16

PART-I

Molecular Analysis..…………………………………………………….

17

2.1 Plant Material…………………..………………………………… 17

2.1.1 Plants Identification and Labeling..………………………. 17

2.1.2 Reagents, Chemicals and Buffer Solutions used in DNA

Analyses …………………………………………………..

18

2.2 DNA Isolation……………...…………………………………….. 19

2.2.1 DNA Extraction and Purification...…….…………………. 20

2.2.2 DNA Quantification……...……………………………….. 21

2.3 PCR Amplification and RAPD Analysis...……………………….. 21

2.3.1 STS Analysis..…………………………………………….. 22

xi

2.3.2 Data Analysis …………………………………………….. 23

2.4 Generation of Gene Specific Primers ……………………………. 25

2.4.1 Homology Search and Clustering………………………… 25

2.4.2 Degenerate Primers ………………………………………. 25

2.4.3 PCR and Nested-PCR Analysis .………………………….. 27

2.4.4 PCR Product Purification ………………………………… 27

2.4.5 DNA Sequencing and Data Analysis …………………….. 28

PART-II

Chemical Analysis..……………………………………………………..

29

2.5 Plant Material for Chemical Analysis …………………………… 29

2.6 Extraction and Fractionation …………………….......................... 29

2.7 Biological Assays.………………………………………………... 29

2.7.1 Antimalarial Bioassay .…………………………………… 30

2.7.2 Phytotoxicity Assay……………………………………..... 30

2.7.3 Insecticidal Activity Assay……………………………….. 31

2.7.4 Antibacterial Activity…………………………………...... 32

2.8 Isolation and Purification of Flavonoids ………………………… 33

2.8.1 Preparative HPLC ………………………….…………….. 33

2.9 Characterization and Structure Determination…………………… 34

2.9.1 Thin Layer Chromatography (TLC) ……………………… 34

2.9.2 Reversed Phase Analytical HPLC – DAD ……………….. 34

2.9.3 UV-Spectroscopy (UV)…………………………………… 35

2.9.4 NMR Spectroscopy……………………………………….. 36

2.9.4.1 1D 1H NMR ……………………………………. 36

2.9.4.2 1D 13C NMR …………………………………… 36

2.9.4.3 2D 1H 1H gradient selected, Double Quantum

Filter Correlation SpectroscopY (gs-DQF-

COSY)…………………………………………..

37

xii

2.9.4.4 2D 1H 1H TOtal Correlation SpectroscopY (2D

TOCSY)…………………………………………

37

2.9.4.5 2D 1H 1H Nuclear Overhauser and Exchange

Effect SpectroscopY(2D NOESY)………………

37

2.9.4.6 2D 1H 13C gradient- selected Heteronuclear

Single Quantum Coherence (gs-HSQC)…………

38

2.9.4.7 2D 1H 13C gradient-selected heteronuclear

Multiple bond Correlation (gs-HMBC)………….

38

2.9.5 LC-MS…………………………………………………….. 38

3 Results and Discussion……………………………………................... 39

PART- I

Molecular Analysis……………………………………………………..

40

3.1 Optimization of DNA Extraction……………………………….... 40

3.1.1 DNA Quantification……………………………………..... 44

3.1.2 DNA Amplification……………………………………...... 46

3.2 Analysis of Genetic Diversity in Tagetes……………………….... 49

3.3 Identification of Gene Specific Markers and their Relationship

With Biochemical Compounds……………………………………

68

PART- II

Chemical Analysis………………………………………………………

71

3.4 Biological Assays..……………………………………………..… 71

3.4.1 Antimalarial Activity………………..……………………. 71

3.4.2 Phytotoxicity .…………………………………………….. 72

3.4.3 Insecticidal Activity ……………………………………… 73

3.4.4 Antibacterial Activity .…………………………………..... 74

3.5 Isolation and Purification of Flavonoids (Flavonols)…………….. 76

3.5.1 NMR Elucidation of Flavonols...…………………………. 78

3.5.1.1 Structure Elucidation of Flovonols (1, 2, 9 and

xiii

18)……………………………………………….. 78

3.5.1.2 Structure Elucidation of Flavonols (5 and 7)……. 85

3.5.1.3 Structure elucidation of Flavonols (6 and 19)…... 85

3.6 Conclusions………………………………………………………. 87

4 References ……………………………………………………………... 89

Appendix A ……………………………………………………………. 104

Appendix B ……………………………………………………………. 116

Appendix C ……………………………………………………………. 129

xiv

LIST OF FIGURES

Fig. 1.1

Basic structure of flavonoid………………………………………. 7

Fig. 1.2 Structures of most common favonols occurring in nature ...……… 9

Fig. 1.3

Structures of monosaccharides found in flavonol structures……… 10

Fig. 1.4 Structures of aliphatic and aromatic acyl substitutions found in flavonols……………………………………………………………

11

Fig. 3.1 Genomic DNA of four Tagetes minuta from Haripur (a) before and (b) after treatment with RNaseA………………………………

41

Fig. 3.2 Genomic DNA of four Tagetes minuta from Abbottabad (a) before and (b) after treatment with RNaseA………………………………

41

Fig. 3.3 Genomic DNA of four Tagetes minuta from Manshera (a) before and (b) after treatment with RNaseA………………………………

42

Fig. 3.4 Genomic DNA of fourteen Tagetes genotypes (a) before and (b) after treatment with RNaseA……………………………………….

43

Fig. 3.5 Genomic DNA isolation from fresh leaf tissue of the three samples (lane 1, 2 and 3).……………………………………………………

45

Fig. 3.6 Genomic DNA isolation from dried seed powder of the three samples (lane 1, 2 and 3).…………………………………………..

46

Fig. 3.7 PCR amplification products of DNA isolated from fresh-leaf and seeds of 6 samples of T. minuta using random primer GL A-03…..

47


47


48

Fig. 3.10 PCR amplification products of fifteen Tagetes species with RAPD primer GL A-07. …....……………………………………...............

61

Fig. 3.11 PCR amplification of fifteen Tagetes species with primer GL A-09.………………………………………………………………….

61

Fig. 3.12 PCR amplification products of fifteen Tagetes species with primer GL A-13. .……………………………………................................

62

xv

Fig. 3.13 PCR amplification products of fifteen Tagetes species with primer GL A-16……………………………………....................................

62

Fig. 3.14 PCR amplification products of fifteen Tagetes species with primer GL A-18……………………………………....................................

63

Fig. 3.15 PCR amplification products of fifteen Tagetes species with STS primer LSTIII …………………..…………………………….........

63

Fig. 3.16 A dendrogram of fifteen (15) Tagetes genotypes generated using 20 RAPD primers and genetic distance estimates from UPGMA…

66

Fig. 3.17 A dendrogram of fifteen (15) Tagetes genotypes generated using 3 STS primers and genetic distance estimates from UPGMA……….

67

Fig. 3.18 Amino Acid Sequence of Several Limonene Synthases…………...

69

Fig. 3.19 Agarose gel analysis of PCR products using degenerate STS primers LSTI, LSTII, LSTIII, LSTIV and LSTV………………….

70

Fig. 3.20 Agarose gel analysis of PCR products using degenerate STS primers LSTI, LSTII, LSTIII, LSTIV and LSTV …………………

70

Fig. 3.21 Structures of the flavonols identified in the examined T. minuta …

77

Fig. 3.22 HPLC profiles showing the flavonols content of T. minuta extract (A) in butanol and (B) ethyl acetate……………………….………

78

Fig. 3.23 UV spectra of 2, 9 and 15 recorded on-line during HPLC analysis……………………………………………………………..

80

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LIST OF TABLES

Table 2.1

Sample code, flower color, collection site and putative species of Tagetes genotypes………………………………………………….

18

Table 2.2 Tissue types used for DNA extraction from 03 Tagetes species (T.

minuta, T. ecrecta and T. patula)…………………………………..

20

Table 2.3 Detailed information of RAPD and STS primers used in characterizing Tagetes genotypes…………………………………..

24

Table 2.4 Detail of primer sequences based on conserved DNA regions……. 26

Table 2.5 Composition of medium preparation for phytotoxic bioassay ……. 31

Table 3.1 Yield and quality of total DNA determined by spectrophotometer.…........................................................................

45

Table 3.2 Primer name, total bands, amplification products and homology of Tagetes genotypes (%) for the RAPD primers …………………….

50

Table 3.3 Primer, no of band fragments, band average and genetic distance of Tagetes genotypes for RAPD primers…………………………..

56

Table 3.4 Average genetic distances among 15 Tagetes genotype using RAPD primers…………………………………………...................

64

Table 3.5 Polymorphism parameters and homology of genotypes using STS primers……………………………………………………………...

64

Table 3.6 Primer, no of band fragments, band average and genetic distance of Tagetes genotypes using STS primers…………………………..

64

Table 3.7 Average genetic distances among 15 Tagetes genotype using STS primers...............................................................................................

65

Table 3.8 Anti-malarial activity of Tagetes minuta oil in the n-Hexane and Ether soluble fraction………………………………………………

72

Table 3.9 Phytotoxic bioassay of n-Hexane soluble fraction of T. minuta oil..

72

Table 3.10 Phytotoxic bioassay of Ether soluble fraction of T. minuta oil……

73

Table 3.11 Insecticidal activity of T. minuta oil in the n-Hexane and Ether soluble fractions……………………………………………………

74

xvii

Table 3.12 Antibacterial activity of fraction extracts (Ether soluble, n-Hexane soluble, Chloroform soluble, Ethyl acetate soluble and Butanol soluble) and isolated compounds of T. minuta……………………..

75

Table 3.13a Relative amounts and on-line HPLC and high-resolution electrospray ionization mass spectral data recorded for flavonoids (1, 2, 4-9, 15-17) isolated from T. minuta (Butanol fraction)..

81

Table 3.13b Relative amounts and on-line HPLC and high-resolution electrospray ionization mass spectral data recorded for flavonoids (18-19) isolated from T. minuta (Ethyl acetate fraction)…………

82

Table 3.14 1H NMR spectral data for flavonoids (1, 2, 5-7, 9, 18, 19) isolated from T. minuta recorded in CD3OD at 25 °C……………………..

83

Table 3.15 13C NMR spectral data for flavonols (1, 2, 5-7, 9, 18, 19) isolated from T. minuta recorded in CD3OD at 25 °C……………………...

84

xviii

LIST OF APPENDICES

Fig. A-1 Tagetes patula and their hybrids (A), Tagetes minuta (B) ……….

105

Fig. A-2 View of Tagetes minuta in its natural habitat……………………...

105

Fig. A-3 Tagetes erecta and their hybrids species…………………………...

106

Fig. A-4 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-03. …...……………………………………………….

107

Fig. A-5 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-04. …………………………………………………....

107

Fig. A-6 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-05. …………………………………………................

108

Fig. A-7 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-06. …...………………………………….....................

108


109

Fig. A-9 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-12. …………………………………....……………....

109


110

Fig. A-11 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-15. …...……………………….……………................

110


111


111

Fig. A-14 PCR amplification products of fifteen Tagetes species with RAPD primer GLA-20. …..……………………………………………….

112

Fig. A-15 PCR amplification products of fifteen Tagetes species with RAPD primer GLB-01. …………………………………………................

112

Fig. A-16 PCR amplification products of fifteen Tagetes species with RAPD primer GLB-02. …...……………………………………………….

113

xix

Fig. A-17 PCR amplification products of fifteen Tagetes species with RAPD primer GLB-03. …...……………………………………………….

113

Fig. A-18 PCR amplification products of fifteen Tagetes species with RAPD primer GLB-04. ……………………………………………………

114

Fig. A-19 PCR amplification products of fifteen Tagetes species with STS primer LSTI. …….…………………………………………………

114

Fig. A-20 PCR amplification products of fifteen Tagetes species with STS primer LSTII. …..…………………………………….....................

115

Table B-1 Genetic distances among 15 Tagetes genotype using RAPD primer GLA03...............................................................................................

117


117


118


118


119


119


120

Table B-8 Genetic distances among 15 Tagetes genotype using RAPD primer GLA12……………………………………………………………...

120


121


121


122

xx


122


123


123


124


124

Table B-17 Genetic distances among 15 Tagetes genotype using RAPD primer GLB01……………………………………………………………...

125


125


126


126

Table B-21 Genetic distances among 15 Tagetes genotype using STS primer LSTI…..............................................................................................

127

Table B-22 Genetic distances among 15 Tagetes genotype using STS primer LSTII……………………………………………………………….

127

Table B-23 Genetic distances among 15 Tagetes genotype using STS primer LSTIII………………………………………………………………

128

xxi

LIST OF ABBREVIATIONS

α Alpha AFLP Amplified Fragment Length Polymorphism

β Beta CTAB Cetyltrimethylammonium Bromide 1D One Dimensional 2D Two Dimensional DMSO Dimethyl Sulfoxide g Gram

HPLC High Performance Liquid Chromatography λ Lambda

MS Mass Spectrometry mg Miligram

µg Microgram

µl Microliter

µm Micrometer mL Milli liter mM Milli molar M Molar ng Nano gram nm Nano meter nt Nucleotide

NMR Nuclear Magnetic Resonance PCR Polymerase Chain Reaction RAPD Random Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphism STS Sequence Tagged Site δ Sigma SDS Sodium Dodecyl Sulfate

TLC Thin Layer Chromatography TFA Trifluoroacetic Acid UV Ultra Violet

1

Chapter 1

Introduction

2

Medicinal plants are natural factories of medicines. Approximately 50% of

today’s prescribed drugs are derived either from a plant source or man made imitations of

plant compounds. The herbs have continuously been used to cure a number of illnesses

from ancient times. Today, research continues to prove that most of our present

medicines are the product of research on medicinal plants. Medicinal plants are under

investigations all over the world, yet a significant portion is still unexplored.

Asteraceae is the largest family of Angiosperm consisting of more than 1600

genera and 23000 species worldwide (Barkley et al., 2006; Panero and Funk, 2008) that

further distributed in 2 subfamilies (Asteroideae or Tubuliflorae and Cichorioideae or

Liguliflorae) and 17 tribes (Panero and Funk, 2002). Asteraceae is also considered as a

largest plant family in Pakistan with more than 650 identified species (Qaiser and Abid,

2002).

1.1. Genus Tagetes

Genus Tagetes (marigold) belongs to subfamily Asteroideae (or Tubuliflorae) of

family Asteraceae (Evans, 2002; Panero and Funk, 2002). Tageteae is a tribe of the plant

family Asteraceae (Loockerman, et al., 2003) consisting of 28 genera and approximately

216 species including genus Tagetes (Barkley et al., 2006). Tagetes contains cultivated

and wild marigolds having an offensive odor. Genus Tagetes is a flowering plant

represented by 56 species, of which 27 are annual and 29 are perennial species

throughout the world (Soule, 1993a; Soule, 1993b). Tagetes is indigenous to North and

South America (Soule, 1993a; Soule, 1993b) and have competetive nature and can grow

well in almost every type of soil. In Pakistan, three species of genus Tagetes namely: T.

erecta, T. minuta and T. patula are widely distributed throughout northern regions of the

country (Ghafoor, 2002).

1.2. Tagetes Species

The Tagetes (marigold) species grown all over the world, the plants of which are

stout and branched with height varying from 0.01-2.2 m. The leaves are segmented,

pinnate and fern like which are dark green in color and are strongly scented. Flowers are

3

varying in color from yellow and golden to orange, red and mahogany (Fig. A-1 and A-

3). Four annual species T. patula, T. lunulata, T. erecta and T. tenuifolia are commonly

cultivated throughout the world for ornamental purposes (Vasudevan et al., 1997). The

taller and large flowered T. erecta is known as African marigold while the smaller T.

patula is called French marigold. A number of hybrid varieties have been developed

from these two species. T. erecta, T. minuta, T. tenuifolia and T. patula are the most

common species while there are other species which are region specific.

1.2.1. Tagetes minuta L.

Tagetes minuta L. (Wild marigold) locally known as ‘Gul-e-Sad Barga’ is

an annual plant of the Asteraceae (Compositeae) family, probably originated from South

America and was later introduced to Central America, Europe, Australia and Eastern and

Southern Africa. The Tagetes species are grown widely all over the world for various

uses including ornamental, medicinal, cultural and therapeutic. This plant is growing

under a broad spectrum of climatic conditions starting from extreme temperate to tropical

regions of the world.

T. minuta is distinct being a very large plant with tiny pale yellow flowers

(Thomas, 1982). The plant grows to a height of 50-150 cm with a single and highly

branched stem at the top (Fig. A-2). It is generally grown in organic gardens due to its

useful properties on the soil, as roots give off chemicals which remove grasses (weeds)

and eelworms (Batish et al., 2007).

1.3. Molecular Characterization of the Plant

The information regarding molecular characterization of Tagetes species are

limited in general. The most reliable and powerful method to carry out molecular

characterization is through the DNA analyses, though it includes targeting living

organism at RNA and protein level. DNA analyses are an essential component for un-

derstanding organisms at molecular level. The quick, reliable and inexpensive protocols

for DNA isolation are always desirable at the initial stage of molecular analyses. DNA

extracted from Tagetes species is usually degraded or contaminated by essential oils,

4

polyphenols and tagetones and thus not useful in polymerase chain reaction (PCR) or

restriction products upon amplification or digestions (Hills and Van Staden, 2002). In

general, it is difficult to isolate high-quality DNA from medicinal plants because of the

presence of large quantities of secondary metabolites such as alkaloids, polyphenols,

polysaccharides and proteins. These compounds interfer by precipitating along with the

DNA, thus degrading its quality and reducing yield (Katterman and Shattuck, 1983;

Sarwat et al., 2006). An array of DNA isolation protocols have been optimized and were

used in various combinations to isolate quality DNA from plants for analyses (Dellaporta

et al., 1983; Doyle and Doyle, 1990; Suman et al., 1999; Shah et al, 2000; Warude et al.,

2003; Sarwat et al., 2006; Deshmukh et al., 2007). Liquid nitrogen has been extensively

used for DNA extraction from fresh leaf and or other tissues (Doyle and Doyle, 1990)

which is assumed to be a key factor of obtaining high quality DNA. However, it is not

always easily available or convenient to use especially in developing parts of the world.

Wide molecular biological techniques have developed highly informative and

useful DNA markers that are primarily used to determine the level of genetic

polymorphism followed by genetic fingerprinting, mapping and gene tagging. The

genetic based markers have been employed on phenotypic based experiments since

nineteenth century. These marker techniques such as Randomly Amplified

Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR), Amplified Fragment

Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLP)

are useful to phonological and morphological character analysis and genetic study.

These are also independent of environmental effects and can be used for the

identification of cultivars in their early stage of development (Agarwal et al., 2008).

Highly related individuals from different propagation sources can also be catagorized

by molecular markers thus facilitating the understanding of crop species (White and

Doebley, 1998; Metais et al., 2000; Lombard et al., 2000; Sun et al., 2001).

Random Amplified Polymorphic DNA (RAPD) markers are capable to

investigate the genetically distinct individuals, although not necessarily in a

reproducible way (Sreekumar and Renuka, 2006). The RAPD method is relatively

5

quick and easy to use and does not require any sequence information and can generate

large number of loci which can complement traditional morphological marker systems

(Fevzi, 2001). Generally short, single and arbitrary decamer of unlimited number of

oligonucleotide primer could be tested. Thus this method provides a valuable new

resource for phylogenic studies. The RAPD technique revealed the intra-specific

variation for screening the degree of inbreeding in animals and commercial plant

species. The polymorphic site is based on the presence and absence of same size of

RAPD fragment profile (Williams et al., 1990). RAPD molecular markers can be used

as good early selection techniques and their association with agronomic traits in crop

plants. RAPD technique has wide range of applications in molecular evolutionary

genetics, gene mapping and plant and animal breeding.

The usefulness of a particular category of molecular or biochemical markers is

limited by several attributes including level of polymorphism, environmental stability,

geographic distribution of chemotypes, the number of loci, molecular basis of the

polymorphism and the ease and cost of analysis (Grass et al., 2006). At DNA level

molecular markers reveal polymorphism and are considered a powerful tool to estimate

the genetic diversity for characterization of genotypes. Similarly, the parental selection

based on genetic diversity is better than pedigree method (Tinker et al., 1993).

Advances in molecular biology have developed new approaches and techniques

such as polymerase chain reaction (PCR) to facilitate the novel genomic DNA

sequencing and identification of target gene in genomic DNA. The known genomic

sequence of target DNA can be amplified by using specific oligonucleotide primers based

on those sequences. Degenerate primers can be used and designed from highly conserved

regions of amino acid sequence of target gene among several plants (Pytela et al., 1994;

Maruyama et al., 2002). This method is useful for identifying new members of a gene

family from different organisms where genomic information is not available (Lang and

Orqoqozo, 2011). PCR products can be sequenced directly in order to generate

information quickly and accurately (Slatko, 1996).

6

1.4. Chemical Characterization of Tagetes

Major compounds reported to be present in Tagetes are essential oil and

flavonoids. Majority of the studies regarding chemical composition of Tagetes focused

on the yield and distribution of essential oils (Singh et al., 1992; Chalchat et al., 1995;

Bansal et al., 1999; Gil et al., 2000). The main constituents of Tagetes oil are ocimene

(monoterpenes), limonene, dihydrotagetone and tagetenone (Kaul et al., 2005;

Upadhyaya et al., 2010).

1.4.1. Essential oils

Essential oils are secondary metabolites of plants. They are volatile in nature and

are complex mixtures of organic compounds. Essential oils are important to medicine,

pharmaceutical industry, agriculture, and biotechnology. Essential oils are composed of

different chemical groups of terpenic hydrocarbons and their oxidized derivatives.

Similarly terpenes are related with essential oils and are distributed throughout the plant

kingdom. These are also found in the species grown under unfavourable conditions.

1.4.1.1. Terpenes

Terpenes are natural products based on the five carbon framework arrangement

units. Terpenes represent a large group of natural compounds that do not contribute

much to flavour, fragrance or odour of the oil. Very often the hydrocarbon terpenes

represent a large percentage of the components of essential oils of plants and can be

found in a remarkable variety of closely related structures. Oils from thousands of plant

species have been extracted and are commercially available (Adams, 1995; Culp et al.,

1977). Oxygen-containing terpenic compounds (terpenoids) are also present in essential

oils. Terpenoids are the most abundant and widely distributed group of secondary

metabolites. The terpenoids structure is range in size from volatile oils of molecular

formula C10H16 to larger molecular structures such as rubber that contains about 4000

isoprene units (Martinez, et al., 1988). Terpenoids composition and components varies

within the species of Tagetes (Hethelyi, et al., 1986; 1987).

7

Essential oils contain monoterpenes. The general constituents of essential oils

and resins are monoterpenes (C10). Several hundred naturally occurring monoterpenes are

known in herbs and citrus fruits (Etherton et al., 2002). Limonene is cylic monoterpene.

Phytochemical investigations of Tagetes species indicate variation in the yield of

limonene (Kaul et al., 2005; Upadhyaya et al., 2010). The limonene and perillyl alcohol

are monoterpenes and have shown efficacy in both cancer prevention and therapy (Elson

and Qureshi, 1995; Gould, 1995; Crowell, 1999).

1.4.2. Flavonoids

The word flavonoid is derived from the Latin word flavus meaning yellow.

Flavonoids are polyphenolic compounds produced in plants, and more than 8150

different flavonoids have been reported (Andersen and Markham, 2006). The basic

flavonoid structure consists of a C15 skeleton as shown for flavone (Fig. 1.1), having two

benzene rings (A, B) joined together by a three-carbon link, with the latter forming a

pyrone ring (C ring). Flavonoids are classified on the basis of oxidation level and

substitution pattern of the C ring, while individual compounds within a class differ in the

substitution pattern on the A and B rings.

O

O

B

A C

2

3

4

10

5

6

7

8

9 1`

4`

5`

6`

3`

2`

Fig. 1.1: Basic structure of flavonoid

Flavonoids are reported to have potential antioxidant, antimicrobial, antiallergic,

antiviral, anti-inflammatory and possess vasodilating properties.

8

1.4.2.1. Flavonols

Flavonols are a family of polyphenols which constitute a particular class of flavonoids.

The important role of flavonols is ultra violet protection and copigmentation in fruits and

flowers of plant (Flint et al., 1985; Smith and Markham, 1998). The copigmentation of

flavonols and anthocynins also increase the red wine color (Baranac et al., 1997; Boulton,

2001). Flavones and flavonols and their derivatives are widely distributed in the

angiosperms, gymnosperms, mosses, liverworts and ferns. More than 1331 flavonol O-

glycosides have been reported in plants (Williams, 2006) and over 393 flavonols have

been identified with complete structure elucidation (Vetschera and Wollenweber, 2006).

The main flavonoids previously detected in the genus Tagetes belong to the

flavonols class. Patuletin, quercetin, quercetagetin and isorhamnetin and some of their

glycosides have previously been tentatively identified in T. minuta (Abdala and

Seeligmann, 1983; 1995).

1.4.2.2. Structure of Flavonols

Flavonols consist of an aglycone, and may contain sugar, acyl and sulfate groups.

The aglycone part possess two benzene rings joined by a linear three carbon chain (C2,

C3, C4), represented as the C6–C3–C6 system. The most important flavonols in the

genus Tagetes have been reported to be patuletin, quercetin, quercetagetin, kaempferol,

myricetin, isorhamnetin and some of their glycoside derivatives (Abdala and Seeligmann

1995; Parejo et al., 2004). These flavonol aglycones differ by the hydroxylation and

methoxylation pattern on their A and B-rings (Fig. 1.2).

9

Fig. 1.2: Structures of most common favonols occurring in nature

Highly methoxylated flavonols are found in the Rutaceae but a hexamethoxylated

flavonol is also isolated from Distemonanthus benthamianus and Fiscus altissima

(Malan, 1993; Sharaf et al., 2000). Similarly polymethoxylated derivatives are also

found in species of Asteraceae. Flavonols from the Asteraceae have a tendency towards

6-methoxylation rather than towards 8-methoxylation because of 5- and 7-hydroxylation

pattern (Halbwirth et al., 2004; Vetschera and Wollenweber, 2006).

O

O

OH

OH

OH

HO

HO

OH

O

O

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OH

OH

O

O

OH

OH

OH

HO

OCH3

O

O

OH

OH

OH

O

HO

OH

O

O

OH

OH

OH

HO

OH

Patuletin

Quercetagetin

Quercetin

Kaempferol

Myricetin Isorhamnetin

10

The sugar derivatives of flavonols are named as flavonol-O-glycosides. Various

types of sugar moieties are attached to the aglycone through O- or C-bindings. Flavonol-

glycosides are mono-, di-, tri- and tetra-glycosylated mainly at the 7- and 3-hydroxyl

positions, but can also be glycosylated at 2`, 4`, 6, 5, and 8. (Strack et al., 1989; Arisawa

et al., 1993; Fuchino et al., 1997; D’Agostino et al., 1997). In the genus Tagetes most of

the aglycones are mono-glycosylated at the 7- and 3-hydroxyl, and very few are di-

glycosylated (Parejo et al., 2004).

O

HO

HO

OH

OH

OH

O

HO

HO

OH

OH

OH

β-D-galactopyranose β-D-glucopyranose

O

HO

HO

OH

OH

O

OH

H3C

HO

HO

OH

α-L-arabinopyranose α-L-rhamnopyranose

Fig. 1.3: Structures of monosaccharides found in flavonol

The monosaccharides most commonly found in O-combination with flavonols are

glucose, galactose and rhamnose, and less frequently xylose, arabinose and glucuronic

acid, etc. (Fig.1.3) (Williams, 2006).

The diversity of flavonols is also related with the nature, number and linkage

position of acyl groups, including both aromatic and aliphatic acyl groups (Williams,

2006) (Fig. 1.4). These are linked to the hydroxyl groups of the sugar units. The

aliphatic acylation moieties include acetic, malonic, succinic, butyric, butanoic, lactic,

11

glutaric, tiglic, isovaleric and quinic acids. The aromatic acyl groups include coumaric,

gallic, cinnamic, caffeic, ferulic, benzoic and sinapic acids.

HO CH3

O

HO OH

OO

HO

O

OH

O

OH

O

OH

O

HO

O

OH

O

OH

Acetic acid Malonic acid Succinic acid Butyric acid Lactic acid Glutaric acid

OH

OO

OHH3CO

HO

O

OHHO

HO

HO

O

OHHO

OCH3

Cinnamic acid Sinapic acid Gallic acid p-hydroxybenzoic acid

O

OHHO

HO

O

OHH3CO

HO

O

OH

HO

p-coumaric acid Caffeic acid Ferulic acid

Fig. 1.4: Structures of aliphatic and aromatic acyl substitutions found in flavonols

The majority of the flavonoids reported to occur in genus Tagetes have been

characterized by chromatographic methods or by low-resolution LC-MS (Abdala and

Seeligmann 1995; Tereschuk et al., 2004; Parejo et al., 2004). One study dealt with

NMR characterization of flavonoid-O-glycoside isolated from T. maxima (Parejo et al.,

2005).

12

1.4.2.3. Functions in Plants

Flavonoids are involved in the plant reproduction, in which especially

anthocyanins make the most obvious contribution to flower color and other flavonoids

also assist in pigmentation. Various flavonols and flavones act as copigments with

anthocyanins leading to an intensification of flower color. Yellow color is produced by

flavonols following methylation, certain type of glycosylation, or certain A-ring

hydroxylation patterns (Forkmann, 1991). The main emphasis is on the flavonoids

function as antioxidant and as UV light protectant. There is increasing evidence that

flavonols and flavones accumulate in the epidermal cells in response to wounding,

pathogenic infection, nutrient deficiency, temperature changes, ozone (UV radiation) and

for protecting photosynthetic tissues (Koes et al., 1994; Shirley, 1996; Smith and

Markham, 1998; Simmonds, 2003).

1.4.2.4. Potential Health Effects

Flavonoids have attracted much interest during last fifteen years related to

potential beneficial health effects on humans. This is mainly because of their possible

antioxidant properties. They have potential therapeutic role in the resistance of tumour

diseases (Ickes, et al., 1973; Vasudevan et al., 1997; Kviecinski, et al., 2008; Mertens-

Talcott and Percival, 2005; Boots et al., 2008) and in the treatment of cancer (Kamei et

al., 1995; Andersen et al., 1997; Cecchini et al., 2005; Linda et al., 2006). Flavonoids

also have vital role in the prevention of neurological and cardiovascular diseases (Hertog

et al., 1993; Knekt et al., 2002; Ruiz et al., 2006; Lopez-Sanchez et al., 2007).

1.5. Uses of Tagetes Species

In tropics, especially in Brazil and adjoining regions it is grown for essential oil

production (Lawrence, 1985), which has numerous applications for the benefit of human

society. The oils are good insect repellents and are used in the treatment of certain

illnesses, such as smallpox, earache, and colds and to reduce fevers. In addition, it has

been recognized to possess hypotensive, spasmolytic, antimicrobial, antifungal and

nematicidal properties (Tereschuk et al., 1997; Mangena and Muyima, 1999). Tagetes

13

oil, which can be extracted with hexane (Wiese et al., 1992), is an established product of

flavor and a raw material for perfume production (Soule, 1993a).

The traditional use of insect repellent is widely spread among the communities

and cultures of East Africa, West Africa, China, northern Tanzania, Gambia and Kenya.

Like the other weeds T. minuta (55%) is also used in direct burning of plants which is

the most common application of plant (Seyoum et al., 2002). The ethnobotanical

survey of taditional use of medicinal plants also suggested the repellency of plants

against the malaria vector Anopheles gambiae (Seyoum et al., 2002). T. minuta has a

competitive nature towards light and space. It creates dense monotypic stands and

displaces other weeds and plants and is resistant to natural enemies. Due to the presence

of monotrepenes especially ocimenones it inhibit the germination of cohabitant species

and thus cause their delayed germination (Lopez et al., 2009). Now it is also cultivated

as crop for agrochemical and pharmaceutical products. It is used as natural herbicide to

manage the rice weeds (Tomova et al., 2005; Batish et al., 2007).

Almost any stored material, whether of plant or of animal origin, is prove to

attack by insects, especially common stored grain by Coleopetra species. Among the

products that are frequently damaged are grains and their derivatives. Stored grain pests

are a major problem in various countries. Similarly, in developing world weeds are a

major factor responcible for poor agriculture productivity. Synthetic weedicides

(herbicides) are often expensive, toxic and non specific. Weedicides from natural sources

having improved characteristic could, therefore, have promising future. In this regard

allelopathic plants can be used for managing the weeds (Singh et al., 2003). The search

for new weedicides/herbicides requires an assay, which can predict the general

phytotoxic effects of the tested samples. The specificity for certain weeds can be

determined at large stages. The Lemna minor hypotoxicity is a useful primary screen for

weedicides search. This bioassay has advantage to predict the growth stimulating effect

of the test sample.

14

The carotenoids are also important constituent of marigold flowers espacially

from T. patula and T. erecta (Vargas et al., 2000). The yellow carotenoid pigments are

used as source for food colouring and as a source of gum emulsification (Henken, 1992).

Lutein as carotenoids may help to reduce the risk of some age-related eye disorders such

as cataracts, photosensitivity disorders and cancer (Vargas and Lopez, 1996).

The medicinal plants have such type of chemical and biochemical compounds and

their derivatives which can be used as pharmaceutical medicines, perfumery products,

cosmetics, flavors and food colorant. In recent years, remarkable interest in plant based

drugs has developed and there is also a trend to adopt herbal medicines being safe for

humans (Giberti, 1983; Abbasi et al., 2010; Awodele et al., 2012). Moreover, regular

use of antibiotics has adverse effects and also develops low resistance in the host.

Wetlands and forests are important and main source of natural products and plant based

material. There are very few crops cultivated for the plant based drug material and

natural sources, thus there is a need to introduce the commercial cultivation of herbal

plants as crops into the cropping system. They will not only maintain the quality and

quantity of biochemical composition but will also meet the demands of industry. The

Tagetes minuta is wildly grown in Pakistan especially in northern hilly areas. Until now

the molecular and chemical characterization of Tagetes species in Pakistan has not been

performed comprehensively. It is, therefore, necessary and useful to study Tagetes

species found in Pakistan for their genetic and chemical analyses using advannced DNA

and biochemical techniques.

15

1.6. Objectives

Major goal of the current research was to characterize Tagetes species in terms of

plant’s DNA and chemical constituents, for the exploitation of its medicinal value in the

benefit of human society. The specific objectives were to:

1. Optimize extraction and purification protocols for DNA and chemical compounds

in the Tagetes

2. Investigate molecular marker based genetic diversity present in the Tagetes

3. Develop and characterize gene based specific markers controlling chemical

compounds in the Tagetes

4. Investigate biological activities of the crude and purified chemical compounds

isolated from the Tagetes

5. Isolate and characterize important chemical compounds such as flavonoids

present in the Tagetes

16

Chapter 2

Materials & Methods

17

Part-I

MOLECULAR ANALYSIS

2.1. Plant Material

A total of 27 sample plants (genotypes) of Tagetes species and their hybrids were

collected in the months of September and October (2007) at flowering stage from

different regions of Pakistan. The plant material was shipped as fresh and was preserved

in the lab after preliminary identification of the species.

2.1.1. Plant Identification and Labeling

The collected plants were identified by a qualified Taxonomist (Dr. Manzoor

Ahmed) of the Department of Botany, Post-Graduate College, Abbottabad, Pakistan.

Voucher specimens were deposited at the herbarium of the Botany Department of Post-

Graduate College, Abbottabad, Pakistan. The putative identification based on

morphologically distinct characteristics of plant especially of flower color and type

narrowed all 27 plants into 15 genotypes from 3 species (Table 2.1, Fig. A-2 and

A-3). Further genetic analyses were carried out on 15 genotypes of the 3 Tagetes

species, whereas chemical analyses were conducted only on Tagetes minuta.

18

Table 2.1: Sample code, flower color, collection site and putative species of Tagetes genotypes

Sample

code

Flower Color Collection site Species

T1 Light yellow flower (Sarai Saleh ) Haripur T. erecta

T2 Light orange flower (Sarai Saleh) Haripur T. erecta

T3 Dark yellow flower COMSATS Nursery Abbottabad T. erecta

T4 Dark orange flower COMSATS Nursery Abbottabad T. erecta

T5 Yellow flower (hybrid) (Sarai Saleh) Haripur T. patula

T6 Orange flower (hybrid) (Sarai Saleh) Haripur T. patula

T7 Dark orange to red (hybrid) COMSATS Nursery Abbottabad T. patula

T8 Yellow to red (hybrid) COMSATS Nursery Abbottabad T. patula

T9 Wild species *BZU, Multan T. patula

T10 Orange and yellow COMSATS Nursery Abbottabad T. patula

T11 Red flower COMSATS Nursery Abbottabad T. patula

T12 Yellow flower (hybrid) Kohat T. erecta

T13 Orange flower (hybrid) Mardan T. erecta

T14 Orange flower (hybrid) COMSATS Nursery Abbottabad T. erecta

T15 Pale yellow Composite sample from different localities of Abbottabad and Mansehra

T. minuta

* Baha-ud-din Zakariya University

2.1.2. Reagents, Chemicals and Buffer Solutions used in DNA Analyses

• Absolute Ethanol (Merck, Germany)

• 70% Ethanol (Merck, Germany)

• Absolute isopropanol (Sigma-Aldrich, Germany)

• EDTA (Merck, Germany)

• NaCl (Merck, Germany)

• CH3COONa (Merck, Germany)

• Chloroform (Merck, Germany)

• β-mercaptoethanol (Calbiochem, Merck, Germany)

19

• Tris-Cl (Merck, Germany)

• CTAB (Merck, Germany)

• SDS (Merck, Germany)

• Boric acid (Merck, Germany)

• Agarose gel (Fermentas Inc.)

• Ethidium bromide (10 mg/ml) (Merck, Germany)

• Enzyme: Taq DNA Polymerase (Fermentas Inc.), Rnase A (Fermentas Inc.)

• Buffer: Taq DNA Polymerase buffer (Fermentas Inc.)

• Nucleotides: dNTPs (G, A, T, C) (Fermentas Inc.)

• RAPD primers (Gene Link USA)

• 1.0 M Tris-Cl (pH 8.0, 9.5); 0.5 M EDTA (pH 8.0); 5.0 M NaCl; 3.0 M sodium

acetate (pH 5.2); CTAB (20%); chloroform:isoamyl alcohol (24:1, v/v); β-

mercaptoethanol

• CTAB extraction buffer: 0.1 M Tris-Cl (pH 9.5), 20 mM EDTA (pH 8.0), 1.4 M

NaCl, CTAB (2%, w/v) (mixture was autoclaved for 20 min at 121°C) and β-

mercaptoethanol (1%, v/v) (added to the buffer just before use)

• SDS extraction buffer: 0.1 M Tris-Cl (pH 8.0), 50 mM EDTA (pH 8.0), 0.5 M

NaCl, SDS (10%, w/v) (mixture was autoclaved for 20 min at 121°C)

• TE buffer: 10 mM Tris-Cl buffer (pH 8.0), 1 mM EDTA (pH 8.0)

• TBE 5X: 54 g Tris, 27.5 g boric acid, 20 mL 0.5 M EDTA

2.2. DNA Isolation

Fresh and dried leaves and seeds were used to isolate the DNA from Tagetes

species (Table 2.2) to compare the efficiency of each tissue type. Plants of each species

were grown at research farm of the COMSATS Institute of Information Technology in

Abbottabad using seeds collected from the plains and hilly areas of Hazara division, Paki-

stan. Leaf tissues were collected upon 6-8 leaved stage. The young leaf tissues were

taken to the laboratory in an icebox and were used directly to isolate DNA by crushing in

the CTAB extraction buffer. A portion of the leaves were subjected to sun-drying by

placing on clean mesh under sun in the farm. For shade drying the samples were brought

to the labs or store houses and were shade-dried by placing on the tables. Seeds of

20

Tagetes were collected from the farm grown plants and also from other parts and were

stored in plastic bags in the laboratory. Three major protocols, viz; chloroform:

isoamylalcohol (24:1, v/v), cetyltrimethylammonium bromide (CTAB) (Doyle and

Doyle, 1990; Warude et al., 2003; Sarwat et al., 2006), and sodium dodecyl sulfate (SDS)

(Aljanabi et al., 1999; Deshmukh et al., 2007) were used with modifications to isolate

and analyze DNA from the plant using different tissue types. A modified CTAB buffer

method (Kim and Hamada, 2005) was employed to post-extraction analyses of DNA

from seed and leaf tissues.

Table 2.2: Tissue types used for DNA extraction from 03 Tagetes species (T. minuta, T.

erecta and T. patula)

Tissue type Weight (g)

Seeds 0.1

Sun-dried leaf 0.1

Shade-dried leaf 0.1

Fresh leaf 0.2

2.2.1. DNA Extraction and Purification

Fresh leaf tissue (0.2 g) was ground in a 1.5mL Eppendorf tube with a micropestle

while seeds, sun-dried and shade-dried leaf samples (0.1 g) were ground to fine powder

with a pestle and mortar and transferred to a 1.5 mL eppendorf tube and 800 µL CTAB

extraction buffer was added. These eppendorf tubes were incubated at 65° C for 35-45

min, with inversion during incubation. An equal volume of chloroform: isoamyl alcohol

(24:1, v/v) was added and the tubes were inverted 8-10 times. The tubes were

centrifuged at 13,000 rpm for 15 min. The supernatant was placed in a new eppendorf

tube. If there was visible cellular debris, the steps of chloroform: isoamyl alcohol (24:1,

v/v) purifications were repeated until the removal of visible contaminants. An equal

volume of absolute ice-cold isopropanol was added. The tubes were centrifuged at

13,000 rpm for 10 min. The supernatant was discarded and the pellet was washed with

70% (v/v) ethanol. The pellet was air-dried for 1 h at room temperature and then

21

dissolved in 100 µL TE buffer. RNase A [2 µL (1 µg/µL)] was added and the tubes were

incubated at 37° C for 1 h.

For further purification, extraction with an equal volume of chloroform: isoamyl

alcohol (24:1, v/v) was performed. DNA was precipitated by adding 1/10th volume of

3M sodium acetate (pH 5.2) and 2.5 mL of ice-cold ethanol; the tube was inverted gently

and this mixture was maintained for 30 min at -20 ° C. It was then centrifuged at 13,000

rpm for 10 min. The supernatant was discarded and the pellet was washed with 70%

(v/v) ethanol. The pellet was dried and dissolved in 100 µL TE buffer. DNA

concentrations were measured by running aliquots on 0.8% agarose gel and by reading

absorbance at 260 nm and 280 nm (Kim and Hamada, 2005) using a UV-Vis

spectrophotometer (IRMECO GmbH, Model U2020, Geesthacht/Germany). The DNA

samples were stored at -20° C until further use.

2.2.2. DNA Quantification

DNA concentration was measured in a UV-Vis spectrophotometer (IRMECO

GmbH, Model U2020, Geesthacht/Germany), and purity of the DNA samples was

confirmed by absorbance (A260/A280) ratio (Kim and Hamada, 2005) to check DNA

quality and quantity for further PCR analysis. Total DNA obtained was run on 0.8%

agarose gel in 1X TAE or 1X TBE buffer containing ethidium bromide (0.01 mg/µl).

Initially same ratio of loading dye and DNA was used (2 µ L each). The samples were

loaded at 100 volt for suitable time (20 to 30 min). The gel was visualized under UV

light and the gels were photographed using the UVi-tec gel documentation system

(Cambridge, UK). The amount of DNA per µ L was judged from the visibility of the

band or ladder (λ DNA digested with restriction enzymes). When the bands for a sample

were not clear or showing total degradation the whole procedure was repeated for that

sample.

2.3. PCR Amplification and RAPD Analysis

PCR-based amplification of the purified DNA was carried out in a 20-µL reaction

mixture. The reaction mixture contained 25 ng template DNA, 0.125 U Taq DNA

22

polymerase, 1.6 mM dNTPs, 3.75 mM MgCl2, 1X Taq DNA polymerase buffer (Shah et

al., 2000), 2 mM primer and dilution up to the volume of 20 µ L was completed by

double distilled autoclaved water. Amplification of the DNA was done using a Perkin

Elmer 9700 Thermocycler (ABI, Foster City, CA, USA) with the following parameters:

initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 ° C for

30 s, primer annealing at 50 ° C for 1 min, and extension at 72 ° C for 1 min, with a final

extension at 72 ° C for 7 min. The reaction was stored at 4 °C, until it was loaded onto

the gel. Twenty five (25) decanucleotide primers were used for PCR amplification

products. Twenty (20) primers generated amplified of products by producing detectable

band patterns. Sequence information of twenty (20) RAPD primers randomly selected

for study is presented in Table 2.3. PCR products were fractionated on 0.8% agarose gel

using 1X TBE buffer containing 10 mg/mL ethidium bromide and were visualized under

UV light and the gels were photographed using the UVi-tec gel documentation and

analysis system (Cambridge, UK).

2.3.1. STS Analysis

Based on the highly conserved amino acid sequence among several plant

limonene synthases, the degenerate primers (15) were designed by reverse translating the

conserved regions using their corresponding genomic codons. The detailed decription is

given in section 2.4. Out of these fifteen (15) primers three (3) sequence-tagged-site

(STS) primers were used for amplification of products. These primers were 18-

nucleotide long for each forward and reverse fragment (Table 2.3). PCR reaction was

carried out in a 20-µL volume. The reaction mixture contained 25 ng to 30 ng template

DNA, 1x Taq DNA polymerase buffer with (NH4)2SO4, 0.8 µL of dNTPs (10 mM), 3 µL

of MgCl2 (25mM), 0.125-U Taq DNA polymerase, 4 µL of each STS forward and

reverse primer (Fermentas Inc.) and dilution up to the volume of 20 µ L was completed

by double distilled autoclaved water. PCR reactions were carried out in a Perkin Elmer

9700 thermocycler (ABI, Foster City, CA, USA) with the following parameters: initial

denaturation at 94 ° C for 5 min, followed by 30 cycles of denaturation at 94 ° C for 30

s, primer annealing at 54 ° C to 56 ° C for 1 min, and extension at 72 ° C for 1 min, with

a final extension at 72 ° C for 7 min (Shah et al., 2000; Maruyama et al., 2001). PCR

23

products were fractionated on 0.8% agarose gel using 1X TBE buffer containing 10

mg/mL ethidium bromide and were visualized under UV light and the gels were

photographed using the UVi-tec gel documentation and analysis system (Cambridge,

UK).

2.3.2. Data Analysis

Only the visible band fragments were considered for data scoring. In all the

genetic analysis each single band fragment was considered as a single locus. In case of

RAPDs 15 Tagetes genotypes for PCR analysis were included. The amplified product

bands were scored in binary formula with the presence (1) and absence (0) in every

genotype for each primer. For assessing genetic diversity to prepare a dendogram,

Bivariate (1-0) data matrix was used on the basis of the number of shared amplification

products for estimating dissimilarity and similarity (Nei and Li, 1979). The distance

matrix and similarity matrix were obtained by computing binary data. These distance

matrices were used for cluster analysis. The dendrogram was generated by using

UPGMA (un-weighted pair-group method with arithmetic average) method of cluster

analysis of SAHN command on NTSYS-pc version 2.2 as follows:

GDxy=1 - 2Nxy

Nx + Ny

Where GDXY is genetic distance, Nxy is number of common bands (loci) in two

genotypes, Nx is number of loci in genotype 1, Ny is number of loci in genotype 2 (Nei

and Li, 1979).

24

Table 2.3: Detailed information of RAPD and STS primers used in characterizing Tagetes genotypes

Primer Sequence 5`-3` Size (nt) Melting

Temp (ºC)

Molecular

weight (amu)

GL A03 AGTCAGCCAC 10 29.5 2,997

GL A04 AATCGGGCTG 10 29.5 3,088

GL A05 AGGGGTCTTG 10 29.5 3,098

GL A06 CAAACGGGTG 10 29.5 3,004

GL A07 GGGTAACGCC 10 29.5 3,117

GL A09 GTGATCGCAG 10 29.5 3,053

GL A11 CAATCGCCGT 10 29.5 2,988

GL A12 TCGGCGATAG 10 29.5 3,068

GL A13 CAGCACCCAC 10 33.6 2,942

GL A14 TCTGTGCTGG 10 29.5 3,050

GL A15 TTCCGAACCC 10 29.5 2,984

GL A16 AGCCAGCGAA 10 29.5 3,046

GL A17 GACCGCTTGT 10 29.5 3,019

GL A18 AGGTGACCGT 10 29.5 3,068

GL A19 CAAACGTCGG 10 29.5 3,037

GL A20 GTTGCGATCC 10 29.5 3,019

GL B01 GTTTCGCTCC 10 29.5 2,970

GL B02 TGATCCCTGG 10 29.5 3,019

GL B03 CATCCCCCTG 10 33.6 2,924

GL B04 GGACTGGAGT 10 29.5 3,108

LSTI-F CAGCTTGAGTTGATCGAC 18 54.3 5,515

LSTI-R GCCATACACGTCGTAGAT 18 54.3 5,484

LSTII-F ATCTACGACGTGTATGGC 18 54.3 5,515

LSTII-R AGACTTTGGGACGTCACC 18 55.3 5,500

LSTIII-F CTGCAGCTGTATGAAGCT 18 54.3 5,515

LSTIII-R GCCATACACGTCGTAGAT 18 54.3 5,484

25

2.4. Generation of Gene Specific Primers

2.4.1. Homology Search and Clustering

The known genomic sequence of limonene synthase gene (accession numbers

AY055214, D49368, AF282875, L13459 and AB110637) were used for clustering. All

sequences producing significant alignments were downloaded from the NCBI Gen Bank

(http://www.ncbi.nlm.nih.gov/) for clustering. ClustalW sequence alignments program

(http://www.ebi.ac.uk/clustalw/) was used to obtain the conserved regions. Sequences

were scanned for short conserved amino acid regions with low permutations of possible

codons. Similarly sequences were also scanned directly for short conserved nucleotide

regions. Single primer contained up to three degenerate nucleotides. The resulting

groups were realigned with ClustalW to search conserved regions.

2.4.2. Degenerate Primers

Based on the highly conserved amino acid sequence among several plant

limonene synthases, the fifteen degenerate primers were designed and conserved regions

were reverse translated by using their corresponding genomic codons. The degenerate

primers were designed by reverse translating the conserved regions using their

corresponding genomic codons. Usually the degenerate primers were required to have a

certain length range and degeneracy. The designing criteria of primers were to have 18-

21 nt in length, 40-60% GC contents and Tm 50-65 ° C (optimum 60 ° C) (Rozen and

Skaletsky, 2000). The primer pairs having melting temperature difference within 4° C of

each other were selected to increase the consistency of results (Table 2.4). These

degenerate primer pairs were synthesized commercially from e-oligos Gene Link,

Hawthorne, USA. Detail of primer sequence, length, product size and degeneracy is

given in table 2.4.

26

Table 2.4: Detail of primer sequences based on conserved DNA regions

Primer name

Primer sequence (5′ to 3′) Length

Product

size (bp)

Degene

racy

GC

(%)

Tm

(ºC)

LSTI-F CAGCTTGAGTTGATCGAC 18 864 50 53.4

LSTI-R GCCATACACGTCGTAGAT 18

750

384 50 53.4

LSTII-F ATCTACGACGTGTATGGC 18 384 50 53.4

LSTII-R AGACTTTGGGACGTCACC 18

440

1536 56 55.4

LSTIII-F CTGCAGCTGTATGAAGCT 18 1152 50 53.4

LSTIII-R GCCATACACGTCGTAGAT 18

525

384 50 53.4

LSTIV-F ATCTACGACGTGTATGGC 18 384 50 53.4

LSTIV-R AGTTCCCAAGTCATCAGC 18

380

1536 50 53.4

LSTV-F TACATGCAGCTGTGCTTG 18 96 50 53.4

LSTV-R AGTTCCCAAGTCATCAGC 18

309

1536 50 53.4

LSTmI-F TACGATGTCTATGGTACC 18 44 49.8

LSTmI-R CCTCTTCTTCCGAACCAT 18

539

50 52.1

LSTmIII-F TTCAAGAACGAGGAGGGT 18 50 52.1

LSTmIII-R CATTCATCTTCTTCCACA 18

1063 39 47.5

LSTmIV-F TCCTCGCAACTCCCTACTATC 21 52 57.8

LSTmIV-R TGTTTTCGCCTTCCTTGAACA 21

475 43 53.9

LSTmVI-F GACGATCCGGAAACTACA 18 50 52.1

LSTmVI-R TACAGTTGCAACAATCCT 18

405 39 47.5

LSTmIX-F TTCAAGAACGAGGAGGGTGAG 21 52 57.8

LSTmIX-R CAACACTACTGGATTCATGTC 21

285 43 53.9

LSTmX-F TACGATGTCTATGGTACC 18 44 49.8

LSTmX-R CTCTTCTTCCGAAGCATT 18

522 44 49.8

27

2.4.3. PCR and Nested-PCR Analysis

Eleven degenerate primers were used for amplification of products with well-

marked fragments. These primers were 18 and 21-nucleotides in length for each of

forward and reverse primer sequence (Table 2.4). PCR reaction was carried out in a 20-

µL volume. The reaction mixture contained 25 ng to 30 ng template DNA, 1x Taq DNA

polymerase buffer with (NH4)2SO4, 0.125 U Taq DNA polymerase (Fermentas Inc), 0.8

µL of dNTPs (10 mM), 3 µL of MgCl2 (25mM), 4 µL of each STS forward and reverse

primer (e-oligos Gene Link, Hawthorne, USA) and dilution up to the volume of 20 µL by

double distilled autoclaved water. For nested-PCR all the ingredients of reaction mixture

were same except the template DNA, which was already PCR product. PCR reactions

were carried out in a Perkin Elmer 9700 Thermocycler (ABI, Foster City, CA, USA) with

the following parameters: initial denaturation at 94 ° C for 5 min, followed by 30 cycles

of denaturation at 94 ° C for 30 s, primer annealing at 54 ° C to 56 ° C for 1 min, and

extension at 72 ° C for 1 min, with a final extension at 72 ° C for 7 min (Maruyama et al.,

2001; Shah et al., 2000). PCR products were fractioned on 0.8% agarose gel using 1X

TBE buffer containing 10 mg/mL ethidium bromide and were visualized under UV light

and the gels were photographed using the UVi-tec gel documentation system.

2.4.4. PCR Product Purification

PCR products of degenerate primers were purified at specific size by using silica

beads DNA gel extraction kit (Thermo Fisher Scientific, USA). The procedure was same

as described in kit.

Agarose gel slice containing the DNA fragment was cut by sterile scalpel. Pre-

weighed 1.5 mL eppendorf tube was used to place for gel slice and again weighed to

obtain the weight of the gel slice. 3:1 (v: w) volume of binding buffer was added to the

gel slice. The gel mixture was incubated at 55 °C for five to seven min until the gel slice

was completely dissolved. During incubation the eppendorf tube was mixed by inversion

with intervals and 4.5 volumes of binding buffer and half volume of TBE conversion

buffer were added to a known volume of agarose gel. Then the silica powder suspension

(2 µL/µg) was added to the DNA/binding buffer mixture. The gel mixture was again

28

incubated for 5 min at 55 ° C for binding the DNA to silica matrix followed by vortexing

every few minutes to keep the silica powder in suspension. The mixture of DNA/silica

powder was spinned for 5 to 10 s to form a pellet. Supernatant was removed carefully

and discarded. An additional 300 µL of binding buffer was added in the pelleted silica

powder containing DNA to dissolve any residual agarose. The suspension was placed in

a water bath at 55 ° C for a few minutes to proceed for the next step. 500 µL of ice cold

washing buffer was added (diluted with ethanol) to wash the pellet. After resuspension

of pellet mixture was spun for 5 to 10 s and supernatant was discarded. The same

procedure was repeated for three times. The pellet was air-dried for 10-15 min until the

complete removal of residual ethanol. The pellet was resuspended in appropriate volume

of TE and again tubes were incubated for 5 min at 55 ° C. Tubes were spun again and

supernatant was removed while avoiding the pellet. The recovered supernatant

containing this specific sized PCR product was placed into a fresh tube and procedure

was repeated with another but with the same volume of TE.

2.4.5. DNA Sequencing and Data Analysis

The specific sized PCR products were shipped to Macrogen Inc. Seoul, Korea

for sequence analysis. The resulting sequence alignments were confirmed by the BLAST

program on the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi).

29

Part-II

CHEMICAL ANALYSIS

2.5. Plant Material for Chemical Analysis

Tagetes minuta L. was collected as whole plant in fresh form in the months of

September to October 2008 for the chemical analysis from Abbottabad, a district of

Khyber Pakhtunkhwa Province of Pakistan. These plants were separated into three

different parts and were air dried in the shadow. During shade drying the plants were

protected from fungus by proper aeration. The three different parts; A) flowers and fruit

(5.2 kg), B) roots (3.5 kg) and C) stem and leaves (10 kg) were thereafter crushed to fine

powder.

2.6. Extraction and Fractionation

The T. minuta parts (A–C) were separately extracted with methanol (5L) in three

intervals at room temperature for altogether 21 days. After extraction the extracts were

filtered, and methanol was removed by evaporation under reduced pressure at 30ºC to get

dark brownish masses: 356 g (A), 106.4 g (B) and 461 g (C).

The various dried extracts were suspended in dist. H2O and sequentially

partitioned with ether, hexane, chloroform, ethyl acetate and butanol yielding ether-

soluble: 178 g (A,B and C), n-hexane soluble: 105 g (A), 10.72 g (B), 146.42 g (C),

chloroform-soluble: 39.28 g (A), 3.80 g (B), 118.61 g (C), ethyl acetate-soluble: 21.36 g

(A), 1.33 g (B), 16.91 g (C), and n-butanol-soluble: 53.64 g (A), 17.88 g (B) fractions.

Twelve fractions with different polarity were thus obtained.

2.7. Biological Assays

Crude extracts of three different parts (A-C) and their fraction extracts

were analysed for following biological assays.

30

2.7.1. Antimalarial Bioassay

Plant extracts were analyzed for antiplasmodial activity by lactate dehydrogenase

method in vitro (human blood) (Makler et al., 1993). A drop of blood infected with

malarial parasite was taken and at least 500 erythrocytes were counted, making a note of

the number that contains parasites. The culture medium contained RPMI 1640 buffered

with 0.2% sodium bicarbonate, 25 mM HEPES (Gibco, Life Technologies) and 10%

human serum (Chan et al., 2004). The culture was diluted to final parasitemia of 2.0%

and grown at 37 ºC in the presence of 5% CO2 for 72 h. 100µ l aliquots were distributed

in to sterile 96 well microtiter plate and 10 µ l containing various concentrations of the

crude extracts (solublised in 0.5% Dimethyl sulfoxide (Merck) and culture was placed in

humidified CO2 (5%) incubator at 37 º C. Negative control was administered 10 µ l PBS

in place of the drug while positive control contained standard drug chloroquine

diphosphate. Thin blood films of the culture were prepared after 24, 36 and 72 h. The

slides were stained with Giemsa stains and the number of parasites per 1000 erythrocytes

was determined by microscopic examination. Total parasitemia was calculated and

plated as percentage control against concentration and IC50 was determined.

2.7.2. Phytotoxicity Assay

Phytotoxic bioassay was carried out for plant extracts of T. minuta against Lemna

minor (Tinny plant containing leaves with 1 - 8 mm long and 0.6 - 5 mm wide). The

medium was prepared containing various inorganic components in 1000 ml distilled

water and pH was adjusted approximately 5.5 - 6.0 by adding KOH pellets (Table 2.5).

The medium was autoclaved at 121º C for 15 minutes. 30 mg of each crude and fraction

extracts of T. minuta were dissolved in 1.5 ml organic solvent (methanol) serving as

stock solutions. Three sterilized flasks were inoculated with 1000 µ g, 100µ g, 10 µ g of

solutions from the stock solution for 500, 50, 5 ppm. The solvent was allowed to

evaporate overnight in sterilized conditions. 20 ml of medium was added to each flask

containing the plant extract under investigation. Ten plants of L. minor each containing

a rosette of three fronds were added to each flask. Other three flasks serving as control

were also supplemented with ten plants of L. minor in 20 ml of medium each. The flasks

31

were kept in the growth cabinet for seven days. Plants were observed during incubation.

Number of fronds per flasks were counted and recorded on the seven day (Atta-ur-

Rehman, 1991). Results were analyzed as growth regulation in percentage, calculated

with reference to the negative control.

Table 2.5: Composition of medium preparation for phytotoxic bioassay

Constituents mg/L

Potassium dihydrogen phosphate (KH2PO4) 680

Maganous chloride (MnCl2) 3.62

Magnesium sulphate (MgSO4 with 7H2O) 492

Potassium nitrate (KNO3) 1515

Boric acid (H3BO3) 2.86

Calcium nitrate tetrahydrate (Ca(NO3)2) 1180

Ferric chloride (FeCl3 with 6H2O) 5.40

Zinc sulphate (ZnSO4 with 7H2O) 0.22

Ethylene diamino tetraacetic acid (EDTA) 11.2

Sodium molybdate (Na2MoO4 with 2H2O) 0.12

Copper Sulphate (CuSO4 with 5H2O) 0.08

2.7.3. Insecticidal Activity Assay

Plant extracts of T. minuta were subjected to insecticidal bioassay to determine

the insecticidal activities of this plant extract and fractions. Three different insects

Tribolium castaneum (Red flour beetle), Rhyzopertha dominica (Lasser grain borer) and

Callosobruchus analis (Pulse beetle) were used to test insecticidal activity of the T.

minuta extracts. The insects were reared in the laboratory under controlled conditions (at

30 º C temperature and 50-60 % humidity), in 9.0 cm diameter plastic bottles containing

250 mg of sterile breeding media. The media was sterilized at 60 º C for one hour. The

uniform size and age of insects were used for bioassay test.

For fractions 100 mg each was taken and was dissolved in methanol. Insecticidal

studies were carried out by contact method using filter paper. The fractions of T. minuta

32

prepared by dissolving in 3-4 ml of methanol were used for the determination of solvent

effect using filter paper absorption method. One filter paper was absorbed by the

methanol only used to dissolve the samples to be used as check for determination of

solvent effect and by placing it overnight for evaporation. Next day 10 adults of the same

size and age were placed in Petri dishes containing samples. The controlled batch of

reference insecticide (Coopex) in the same quantity was used. All the insects were kept

for 24 h at 27 º C. Results were calculated as mortality (%) mean.

2.7.4. Antibactrial Activity Assay

Crude and fraction extracts of T. minuta were analyzed at 3 mg/ml in dimethyl

sulfoxide (DMSO) (Merck, Germany). Nutrient broth medium (Oxoid, England) was

used for bacterial growth to perform antibacterial assay. It composition was starch (1

g/L), peptone (5 g/L), sodium chloride (5 g/L) and agar (10 g/L). Nutrient agar media

(Britanialab) composition was plury peptone (5 g/L), sodium chloride (8 g/L), beef

extract (3 g/L) and agar (15 g/L). Both media were prepared according to manufacturer’s

instructions in distilled water and sterized by autoclave at 121 º C for 15 min.

The antibacterial activity was determined both in gram negative and gram positive

bacteria. Gram positive bacteria used in the experiment were Bacillus subtilis,

Staphylococcus aureus, Micrococcus luteus and gram negative bacteria were

Pseudomonas piket, Salmonella setubal.

To compare the turbidity of bacterial culture McFarland (0.5 BaSO4) solution was

used. The standard was prepared by adding 0.5 ml of 1 % solution (w/v) of anhydrous

barium chloride (BaCl2) to 99.5 ml of 1 % solution (v/v) of sulphuric acid (H2SO4).

Barium sulphate turbidity (4 to 6 ml) was taken in screw capped test tube and was used to

compare the turbidity of bacteria.

The agar well diffusion method (Carron et al., 1987; Naqvi et al., 1987) was used

to analyze the samples for antibacterial assay. The samples were prepared by dissolving

3 mg of crude samples of T. minuta in 1 ml of DMSO. The autoclaved nutrient agar

media was cooled at 45 º C and poured 25 mL in each petri plate in front of flame. These

33

Petri plates were incubated overnight at 37 º C in incubator to avoid contamination. The

wells were made by 6 mm sterile metallic borer at 24 mm distance from each other. Each

well was filled with 20 µ L of plant extract and labeled at the back of petri dishes.

Solvent was used as negative control. Ampicillin (Sigma Aldrich) was used as standard

antibiotic drug, at the range of 50 µ g/mL. The incubation of sample plates was at 37 º C

in incubator for 24 h. Next day the results were noted in term of zone of inhibition from

one side to other side of circule and the well diameter was excluded.

2.8. Isolation and Purification of Flavonoids

The isolation, purification and structure identification of pure compounds

(flavonoids) is a time consuming process. Typical procedures for isolation and

characterization of flavonoids consist of several steps: i) extraction of plant material

followed by preliminary purification, ii) fractionation of the mixtures followed by the

isolation of pure pigments and finally iii) characterization and identification of pure

flavonoids (Strack and Wary, 1989; Marston and Hostettmann, 2006).

2.8.1. Preparative HPLC

This is a technique with high resolution. Preparative HPLC isolate the maximum

amount of a certain product at a desired purity in a minimum of time. The requirements

for a preparative HPLC system differ from analytical systems regarding sensitivity and

flow rates. The main difference however, is the fraction collection control for quality of

the product.

Various samples of the chloroform, ethyl acetate and butanol fractions were

examined by HPLC. Before injection to HPLC, samples were filtered by using 0.45 µ m

(Millipore membrane) filter.

The ethyl acetate fractions of A: seed flower, B: root part and similarly the

butanol fractions of A: seed flower, B: root part were subjected to preparative HPLC

using a Gilson 305/306 pump equipped with C18 reversed phase column (ODS-Hypersil

column (25 x 2.2 cm, 5 µ m)) coupled to a multidiode array detector (HP-1040 A)

(Supelco, Bellefonte, USA) for isolation of flavonoids 1-19. Compounds 18, 19 in the

34

ethyl acetate fraction, and compounds 1-17 in the butanol fraction were isolated by using

the solvents (A) H2O-TFA (0.5%) and (B) Acetonitrile-TFA (0.5%). The rate of flow

was 15 mL/min and aliquots of 500 µ L were injected. The initial profile of elution was

based on linear gradient from 10% to 100% of B for 40 min, after that with isocratic

elution of 100% B for 3 min and followed by final linear gradient from 100% to 10% B

for 5 min.

2.9. Characterization and Structure Determination

Chromatographic and spectroscopic techniques were used for characterization and

structure determination of purified compounds. TLC, HPLC and UV-Vis spectroscopy

may give a lot of characteristic information about the type of flavonoids, MS provides the

molecular mass of the compound, the application of powerful NMR instrument is usually

required for complete structure identification of compounds. Structure elucidation of

flavonoids comprises i) aglycone, ii) sugar units, and iii) acyl groups, as well as iv)

determination of linkage positions between the different sub-groups.

2.9.1. Thin Layer Chromatography (TLC)

TLC is considered to be one of the simplest of chromatographic techniques. TLC

facilitates short acquired time and is relatively inexpensive procedure. TLC silica plates

(0.1 mm) of cellulose F (Merck, Germany) was carried out on 0.1 mm cellulose F

(Merck, Germany) plates as a stationary phase with a mobile phase of polar solvent

(Formic acid: Hydrochloric acid: water; 25:24:51, v/v) system.

2.9.2. Reversed Phase Analytical HPLC - DAD

HPLC is the method of choice for accurate determination of both the composition

and concentration of flavonoids in a given sample (Andersen and Francis 2004). The

Agilent 1100 HPLC was operational with a HP 1050 diode-array detector and a 200 x 4.6

mm, 5 µ m ODS Hypersil column (Bellefonte, USA). The elution system was binary,

with (A) H2O-TFA (0.5%) and (B) Acetonitrile-TFA (0.5%) solvents. The rate of flow

was 1.0 mL/min. Samples of 20 µ L were injected with micro autosampler. The initial

profile of elution was based on linear gradient under conditions with 10% B and 90% of

35

A. Subsequently with 20% B for 18 min followed by linear gradient conditions for 18-26

min (to 23% B), 26-30 min (to 28% B), 30- 40 min (to 100% B), isocratic elution 40-43

min (100% B), and the final elution with linear gradient for 43-48 min (to 10% B).

During HPLC analysis online UV absorption spectrum was recorded under wavelength

range of 280-360 nm in steps of 2 nm.

The main chromatographical separation principle involved in reversed-phase

HPLC is the partition of solutes between mobile (polar) and stationary (non-polar) phase.

The overall polarity and stereochemistry of the flavonoids are the key factors for

separation (Strack and wray, 1989, 1994; Andersen and Francis, 2004). Elution of

flavonoids in reversed phase HPLC columns depends on the pattern of

hydroxylation/methoxylation of aglycone, the degree of glycosylation and acyl

subtitution as well as on the mobile phase composition and solvent gradient steepness.

The flavonoids glycosides eluted before aglycones with C18 phases and flavonoides

possessing more hydroxyl groups were eluted before the less substituted analogs.

Flavones C-glycosides generally elute with shorter retention times than the corresponding

O-glycosides. Flavanones elute before their corresponding flavones due to the

unsaturation effect between positions 2 and 3. The presences of aromatic or aliphatic

acylation increases retension times compared to the corresponding non-acylated

derivatives.

2.9.3. UV-Spectroscopy (UV)

Ultra Violet (UV) spectra of the compounds discussed in this work were obtained

online during various analytical HPLC analyses. Chromatograms can be recorded online

at different wavelengths by LC-UV with DAD (Diode Array Detection). Flavones and

flavonols can be found at 270 and 330 to 365 nm (Parejo et al., 2005), flavanones at 290

nm, isoflavones at 236 or 260 nm (Grayer and Veitch, 2006), chalcones at 340 to 360 nm,

dihydrochalcones at 280 nm (Veitch and Grayer, 2006),, anthocynins at 502 to 520 nm

(Andersen and Fossen, 2003; Andersen and Francis, 2004) and catechins at 210 or 280

nm (Merken and Beecher, 2000).

36

2.9.4. NMR Spectroscopy

Compensated attached proton test (CAPT), 2-D homonuclear correlation

experiment (1H-1H DQF-COSY), 2-D heteronuclear single quantum coherence (1H-13C

HSQC) and heteronuclear multiple bond correlation (1H-13C HMBC) were recorded on a

Bruker Biospin Ultrashield Plus AV-600 MHz instrument (Fallanden, Switzerland)

equipped with a cryogenic probe at 298 K at frequencies of 600.13 MHz and 150.90

MHz for 1H and 13C. The deuteriomethyl 13C signal and 1H residual signal of the

CD3OD solvent were used as secondary references (δ 49.0 and δ 3.40 from TMS,

respectively).

2.9.4.1. 1D 1H NMR

The 1D proton spectra of flavonoids provide quantitative information about

proton chemical shifts and their coupling constants (JHH) and give quantitative

information by integrated baseline-separated signals or selected spectral regions. The

information regarding the nature of aglycone, number and type of sugar unit and acyl

substituents can also be provided. The chemical shifts values also indicate linkage

positions between different sub-units of flavonoid.

2.9.4.2. 1D

13C NMR

Spin Echo Fourior Transform (SEFT) and compensated Attached Proton Test

(CAPT) was used along with different 2D techniques to obtain the accurate carbon

chemical shifts. The SEFT sequence suffers from the use of a 90º -excitation pulse,

which requires long repetition times. This feature has been significantly improved with

CAPT.

Due to low abundance (1.1%) and the lesser favorable magnetogyric ratio of 13C

compared to 1H, the 13C-spectra have lower signals to noise level than the corresponding

1H-spectra. In addition, with the 13C signal normally decrease in intensity because of the

JCH-couplings, which will split the signals into multiplets. But the last problem is

eliminated with proton decoupling where multiplets collapse into singlets. The signals

37

(C, CH, CH2, CH3) are differentiated with the aims of different delays in the pulse

program. The C and CH2 signals will be distinguished from the CH and CH3 signals by

having opposite phases.

2.9.4.3. 2D 1H-

1H gradient selected, Double Quantum Filter Correlation

Spectroscopy (gs - DQF- COSY)

This technique is used to assign the different proton signals based on the

couplings through bonds (J-coupling). COSY is 2D homo-nuclear technique where the

diagonal peaks represent the actual proton spectrum and the cross peaks show which

protons are J-coupled to each other.

2.9.4.4. 2D 1H-

1H Total Correlation Spectroscopy (2D TOCSY)

This is two dimensional homo nuclear NMR technique which has diagonal peaks,

and identical proton chemical shift axes as the COSY technique. The TOCSY is used to

find the proton shifts for all protons which belong to the same spin system, even if the

protons are not directly J-coupled. Thus, TOCSY is very useful for determination of

individual 1H shifts of the various sugar units linked to the flavonoid. This is particularly

relevant when the flavonoid contains more than one sugar unit.

2.9.4.5. 2D 1H-1H Nuclear Overhauser and Exchange Effect Spectroscopy (2D

NOESY)

This is 2D homo-nuclear technique which is based on coupling through space.

The method can provide information about the molecular geometry, confirmation and

linkage between flavonoid sun-units. Exchange cross peaks between analogous protons

of species that are in equilibrium with each other may be observed in NOESY spectra,

which will result in positive cross peaks. Across peak due to NOE correlation will be

negative (Santos et al., 1993; Jordheim et al., 2006).

38

2.9.4.6. 2D 1H-

13C gradient- selected Heteronuclear Single Quantum Coherence (gs-

HSQC)

The inverse-detected 2D-heteronuclear experiments correlate 1H and 13C

chemical shifts through single-bond heteronuclear couplings 1JCH. The HSQC spectrum

shows only protons that are directly attached to a carbon atom and vice versa.

2.9.4.7. 2D 1H-

13C gradient-selected Heteronuclear Multiple Bond Correlation (gs-

HMBC)

The HMBC correlates 1H and 13C chemical shifts through multiple-bond

heteronuclear couplings. The most important ones 2JCH and 3JCH, for which the strongest

cross peaks are observed. In addition, 1JCH correlations and some long distance

correlations may be observed. In the spectra recorded for the flavonoids the 1JCH large

doublet may be observed in the HMBC spectra because of incomplete suppression. In

the hetronuclear multiple-bond correlation spectra, most quaternary carbon resonances

may be assigned.

2.9.5. LC-MS

Mass Spectrometry in the present work was applied to measure the molecular

mass for verification of various structure determinations. The spectra of compounds 1-19

were obtained by high resolution LC electrospray mass spectrometry (ESI+/TOF). The

instrument JEOL AccuTOF JMS-T100LC in combination with an Agilent 1200 HPLC

system was used for recording of spectra. A Zorbax SB-C18 (50 mm x 2.1 mm (length x

i.d.), 1.8 µ m) column was used for combination, and separation of solvents A, H2O-TFA

(0.5%) (v/v) and B, Acetonitrile-TFA (0.5%) (v/v) were used for elution. The initial

solvent elution profile was based on linear gradient for 0 - 1.25 min with 10 to 22% B,

1.25 - 5 min with 22 to 30% B (linear gradient), followed by isocratic elution for 5 - 7

min with 30% B, 7 - 8 min with 30 to 40% B (linear gradient), 8 - 14 min 40% B

(isocratic) and finally linear gradient for 14 -15 min with 40 to 10% B. During this flow

rate was 0.4 mL/min.

39

Chapter 3

Results & Discussions

40

Part- I

MOLECULAR ANALYSIS

3.1. Optimization of DNA Extraction

The isolation of genomic DNA of T. minuta, T. patula, T. ertecta and their

hybrids was checked by means of agarose gel electrophoresis. Different tissue types

(fresh, sun-dried, shade-dried leaf tissues, and seeds) were used to isolate the DNA.

DNA obtained after application of DNA isolation protocol and RNase A treatment is

presented in Fig. 3.1, 3.2, 3.3 and 3.4. The quantity of the isolated DNA was optimum.

The DNA of T. minuta collected from four different areas of Haripur is presented

in Fig. 3.1. Seeds were used to isolate DNA from these genotypes of T minuta. The Fig.

3.1 (a) and (b) shows DNA before and after the treatment of RNase A respectively. The

bands of DNA are crispy and in good quality.

Fig. 3.2 shows the DNA of T. minuta taken from four different areas of

Abbottabad. Fig. 3.2 (a) and (b) shows DNA before and after the treatment of RNase A.

Fig. 3.3 shows the DNA of T. minuta taken from four different areas of Mansehra. The

sample TM12 shows excess amount of RNA in Fig. 3.3 (a) and similarly Fig. 3.1 (b)

shows DNA after the treatment of RNase A.

Fig. 3.4 shows the DNA of T. erecta T. patula and their hybrids collected from

the plains and hilly areas of Pakistan (Table 2.1). Seeds were used to isolate DNA from

fourteen genotypes of Tagetes species. Fig. 3.4 (a) and (b) shows DNA in good quantity

and before and after the treatment of RNase A.

41

(a) (b)

Fig. 3.1: Genomic DNA of four Tagetes minuta from Haripur (a) before and (b) after treatment with RNaseA TM1: Tagetes minuta collected from (Khanpur) Haripur.

TM2: Tagetes minuta collected from (Hattar) Haripur

TM3: Tagetes minuta collected from (Sarai Saleh) Haripur

TM4: Tagetes minuta collected from (Kotnagibullah) Haripur

(a) (b)

Fig. 3.2: Genomic DNA of four Tagetes minuta from Abbottabad (a) before and (b) after treatment with RNaseA

TM5: Tagetes minuta collected from (Havelian) Abbottabad

TM6: Tagetes minuta collected from (Habibullaha Colony) Abbottabad

TM7: Tagetes minuta collected from (Aspadar) Abbottabad

TM8: Tagetes minuta collected from (Qalanderabad) Abbottabad

42

(a) (b)

Fig. 3.3: Genomic DNA of four Tagetes minuta from Manshera (a) before and (b) after treatment with RNaseA

TM9: Tagetes minuta collected from (Daata) Mansehra

TM10: Tagetes minuta collected from (Chitta Bata) Mansehra

TM11: Tagetes minuta collected from (Rehar) Mansehra

TM12: Tagetes minuta collected from (Ghari Habibullah) Mansehra

(a)

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14

43

(b)

Fig. 3.4: Genomic DNA of fourteen Tagetes genotypes (a) before and (b) after treatment with RNaseA T1: Tagetes erecta collected from (Sarai Saleh) Haripur

T2: Tagete erecta collected from (Sarai Saleh) Haripur

T3: Tagetes erecta collected from COMSATS Nursery Abbottabad


T5: Tagetes patula collected from (Sarai Saleh) Haripur


T7: Tagetes patula collected from COMSATS Nursery Abbottabad


T9: Tagetes patula collected from (Baha-ud-din Zakariya) Multan



T12: Tagetes erecta collected from (Kohat) Peshawar

T13: Tagetes erecta collected from (Mardan) Peshawar


44

In the past various protocols and their modifications were developed and

employed to isolate quality DNA from different plant species (Murray and Thompson,

1980; Dellaporta et al., 1983; Saghai-Maroof et al., 1984; Rogers and Bendich, 1985;

Doyle and Doyle, 1990; Suman et al., 1999; Warude et al., 2003; Sarwat et al., 2006;

Deshmukh et al., 2007). Three of the reported protocols (isoamylalcohol, CTAB, and

SDS) were particularly used (with modifications) to isolate and analyze DNA from T.

minuta using different tissue types. Major aim was to optimize a protocol that may be

rapid and inexpensive with high quality and throughput. T. minuta is one of those

medicinal plant species that contain higher levels of secondary metabolites such as

polysaccharides, polyphenols, flavonoids, and essential oils, which may get co-

precipitated with DNA during its preparation, thus interfering with enzymatic analysis.

Removal of such compounds is key to obtain good-quality DNA. DNA isolated using

various extraction protocols was compared from preparation to PCR analysis in terms of

quantity and quality. DNA obtained was not of enough quantity and the quality was very

poor especially in case of dried (sun and shade) leaf tissues in all the tested protocols.

Preparations (including DNA) in the test tubes were highly viscous and dirty brown in

color which showed no or very faint bands (or smears of the bands) upon gel

electrophoresis while there were no amplification products after PCR analysis. However,

the results from the modified CTAB buffer method were encouraging and were far better

than the rest of the tested protocols especially in case of fresh leaf tissues without liquid

nitrogen and the seed samples. At that point, we stopped further testing for the protocols

except CTAB procedure. The modified CTAB buffer method of genomic DNA

extraction from different tissues of T. minuta was further tested and refined to compare

its efficiency in terms of quantity and quality of the DNA for various tissue types.

3.1.1. DNA Quantification

Successful DNA extraction was obtained from fresh, sun-dried, shade-dried leaf

tissues, and powdered (paste) seeds. DNA obtained from sun- and shade-dried leaves

was not of adequate quality for PCR analyses. DNA concentration was measured in a

spectrophotometer (UV/VIS) and an absorbance, i.e., A260/A280 ratio of 1.3, was obtained

45

that indicated high levels of contaminated proteins and polysaccharides (Dehestani and

Tabar, 2007).

Table 3.1: Yield and quality of total DNA determined by spectrophotometer

Absorbance Ratio Tissue type

OD 260/280

Yield (µg/0.1g)

Seed 1.84 ± 0.04a 83.9 ±10.6

Fresh leaf 1.89 90.6

Dry leaf 1.80 65.5

aValues are means ± SD (n = 6).

Total DNA isolated from fresh leaves and dried-seed powder of T. minuta was

checked by agarose gel electrophoresis. High-molecular weight DNA of larger quantities

and of good quality was obtained from fresh leaves without using liquid nitrogen and

dried seed samples (Fig. 3.5 and 3.6). The purity of the DNA samples was confirmed by

absorbance (A260/A280) ratio, which was 1.8 (Table 3.1). The modified protocol thus

yielded DNA of high purity, free from essential oils, polyphenols, flavonoides, and

polysaccharides from fresh leaf samples and seed tissues. However, an increase in the

amount of tissues used did not increase DNA yield from either types of plant tissues.

Fig. 3.5: Genomic DNA isolation from fresh leaf tissue of the three samples (lane 1, 2 and 3)

46

Fig. 3.6: Genomic DNA isolation from dried seed powder of the three samples (lane 1, 2 and 3).

3.1.2. DNA Amplification

The DNA obtained was suitable for enzymatic manipulations such as PCR and

showed high intensity amplification with arbitrary RAPD primers (Fig. 3.7, 3.8 and 3.9).

PCR amplification also indicated that the DNA was of good quality, free from interfering

compounds and it would be suitable for other DNA analyses such as restriction, Southern

transfer and hybridization when performing restriction fragment length polymorphism

(RFLP).

47

Fig. 3.7: PCR amplification products of DNA isolated from fresh-leaf and seeds of 6 samples of T. minuta using random primer GL A-03. M: 100-bp DNA ladder; lanes 1, 2, and 3: fresh leaf; lanes 4, 5, and 6: seed tissue


48


DNA isolation is a primary and critical step for molecular analysis of any plant

species. This process becomes even more difficult when the plant species contain high

amounts of secondary metabolites and essential oils. These compounds, particularly in

medicinal plants, are considered to be as contaminants that cause DNA degradation

during preparation. T. minuta is one of such plant species and therefore the extraction of

genomic DNA from this plant is difficult. Polyvinylpyrrolidone (PVP), a compound

known to suppress polyphenolic oxidation, has been used frequently in CTAB extraction

protocols (Doyle and Doyle, 1990). The modified CTAB buffer containing PVP was also

employed to extract DNA from T. minuta using liquid nitrogen (Hills and Van Staden,

2002). However, it was reported elsewhere that this compound did not significantly

increase the yield or prevent contamination of the DNA (Schneerman et al., 2002). SDS-

based extraction buffer is being used to break open the cells and isolate DNA. But the

quality of DNA obtained is questioned due to precipitation of polysaccharides and

proteins. In addition, the SDS might not bind with the proteins in the purification step,

thus degrading the extracted DNA (Aljanabi et al., 1999; Deshmukh et al., 2007). Since

SDS and isoamylalcohol methods did not give significant results in either type of leaf

tissues or seeds that we tested in the case of T. minuta are therefore, hard to make any

49

conclusive comments on their efficacy or effectiveness. However, the use of PVP in

CTAB buffer did not improve the yield or quality rather we obtained significantly better

results without PVP use in our experiment of DNA extraction.

3.2. Analysis of Genetic Diversity in Tagetes

Following the protocol, the DNA concentrations obtained from plant seeds are

83.9± 10.6 µ g/0.1g and the absorbance ratio of A260/A280 was 1.84± 0.04 (Table 3.1).

The total DNA from seeds tissue was analysed on 0.8% agarose gels electrophoresis and

high molecular weight DNA was obtained.

Twenty RAPD primers were used to estimate the genetic dissimilarity in fifteen

Tagetes genotypes for the genetic polymorphism. These primers indicated 167 loci of

amplification products with total 757 detectable fragment bands scored against 15

genotypes of Tagetes found in Northern areas of Pakistan. Of 167 amplification

products 159 (95.21%) were polymorphic, while 8 (4.79%) were obtained monomorphic

(Table 3.2). Number of amplification products from 7 to 11, while 8.35 was average of

amplified product per RAPD primer. Average 53.18% homology was observed in 15

genotypes (Table 3.2).

50

Table 3.2: Primer name, total bands, amplification products and homology of Tagetes

genotypes (%) for the RAPD primers

Amplification products profile primers

Total loci Monomorphic Polymorphic

Homology of

genotypes (%)

GL A03 7 1 6 3.80

GL A04 8 0 8 62.85

GL A05 7 0 7 90.47

GL A06 9 0 9 89.52

GL A07 7 1 6 41.90

GL A09 10 1 9 5.71

GL A11 8 0 8 52.38

GL A12 7 0 7 54.28

GL A13 8 1 7 32.28

GL A14 9 0 9 78.09

GL A15 8 1 7 59.04

GL A16 7 1 6 37.14

GL A17 10 0 10 74.28

GL A18 8 0 8 35.23

GL A19 11 0 11 37.14

GL A20 7 1 6 80

GL B01 8 0 8 17.14

GL B02 9 0 9 68.57

GL B03 10 0 10 76.19

GL B04 9 1 8 67.61

Total mean 167 8 159 53.18

Different levels of genetic polymorphism was observed in all Tagetes genotypes

by using Genelink GLA03 primer. PCR amplification of this primer produced 57 band

fragments while band average per genotype was 3.8 (Table 3.3). The range of genetic

distance was observed from 0-100% in the genetic dissimilarity matrix. Only 5

combinations of Tagetes genotypes (for detail see Appendix Table B-1) showed

51

maximum genetic distance (100%). While 5 comparisons (T1-T2, T6-T7, T10-T11, T5-

T12 and T13-T15) showed 100% homology (GD = 0%) at DNA level when RAPAD

primer GLA03 was used.

Different levels of genetic polymorphism were also observed in all Tagetes

genotypes by using Genelink GLA04 primer. PCR amplification of this primer produced

29 band fragments while band average per genotype was 1.93. The range of genetic


combinations of Tagetes genotypes (for detail see Appendix Table B-2) showed

maximum genetic distance (100%). While 67 comparisons showed 100% homozygosis

at DNA level in respect to primer GLA04.

PCR amplification by using Genelink GLA05 primer in all Tagetes genotypes

produced 16 band fragments while band average per genotype was 1.06 (Table 3.3). The

range of genetic distance was observed from 0-100% in the genetic dissimilarity matrix.

Only 2 combinations (T3-T15, T6-T15) of Tagetes genotypes (for detail see Appendix

Table B-3) showed maximum genetic distance (100%). While 95 comparisons showed

100% homozygosis at DNA level while using Genelink primer GLA05.

Different level of genetic polymorphism was observed in all Tagetes genotypes


fragments while band average per genotype was 1.2. The range of genetic distance was

observed from 0-100% in the genetic dissimilarity matrix. Only 1 (T8-T15) comparison

of Tagetes genotypes showed maximum genetic distance (100%). While 97% genetic

distance was observed in 3 comparisons and 94 comparisons (for detail see Table B-4)

showed no polymorphism at molecular level using GLA06 primer.


by using Genelink GLA07 primer (Fig. 3.10). PCR amplification of this primer produced



52

comparisons of Tagetes genotypes (for detail see Table B-5) showed maximum genetic

distance (100%). While 44 comparisons showed no polymorphism at DNA level in

respect to primer GLA07.



54 band fragments while band average per genotype was 3.6 (Table 3.3). The range of

genetic distance was observed from 0-100% in the genetic dissimilarity matrix. 28


distance (100%). On the other hand 7 comparisons showed no polymorphism at DNA

level.

Similarly different level of genetic polymorphism was observed in all Tagetes



genetic distance was observed from 0-97% in the genetic dissimilarity matrix. Only one

(T8-T13) comparison of Tagetes genotypes (for detail see Appendix Table B-7) showed

maximum genetic distance (97%), while 55 comparisons showed 100% homozygosis at

DNA.

PCR amplification of GLA12 primer produced 39 band fragments while band

average per genotype was 2.6 (Table 3.3). The range of genetic distance was observed

from 0-100% in the genetic dissimilarity matrix. Eight (T3-T6, T3-T7, T3-T8, T3-T15,

T4-T6, T4-T7, T4-T8 and T4-T15) comparisons of Tagetes genotypes (for detail see

Appendix Table B-8) showed maximum genetic distance (100%). While 57 comparisons

showed 100% homozygosis at DNA level while using Genelink primer GLA12.




genetic distance was observed from 0-100% in the genetic dissimilarity matrix. Eight

53


distance (100%), while 34 comparisons showed 100% homozygosis at DNA level.




genetic distance was observed from 0-100% in the genetic dissimilarity matrix. Six


distance (100%), while 82 comparisons showed 100% homozygosis at DNA level.




observed from 0-100% in the genetic dissimilarity matrix. Six comparisons of Tagetes

genotypes (for detail see Table B-11) showed maximum genetic distance (100%). While

62 comparisons showed no polymorphism at DNA level, while using Genelink primer

GLA15.




genetic distance was observed from 0-80% in the genetic dissimilarity matrix. Four

comparisons (T1-T6, T1-T7, T1-T8 and T1-T12) of Tagetes genotypes (for detail see

Appendix Table B-12) showed maximum genetic distance (80%). While 39 comparisons

showed no polymorphism at DNA level, while using Genelink primer GLA16.




observed from 0-100% in the genetic dissimilarity matrix. Nine comparisons of Tagetes

genotypes (for detail see Table B-13) showed maximum genetic distance (100%). While

54

78 comparisons showed no polymorphism at DNA level, while using Genelink primer

GLA17.




distance was observed from 0-100% in the genetic dissimilarity matrix. Ten comparisons

of Tagetes genotypes (for detail see Table B-14) showed maximum genetic distance

(100%). While 37 comparisons showed no polymorphism at DNA level, while using

Genelink primer GLA18.




distance was observed from 0-100% in the genetic dissimilarity matrix. 21 comparisons



Genelink primer GLA19.




distance was observed from 0-100% in the genetic dissimilarity matrix. Three

combinations (T5-T6, T5-T15 and T9-T15) of Tagetes genotypes (for detail see Table B-

16) showed maximum genetic distance (100%). While 84 comparisons showed no

polymorphism at DNA level, while using Genelink primer GLA20.


by using Genelink GLB01 primer. PCR amplification of this primer produced 56 band


distance was observed from 0-100% in the genetic dissimilarity matrix. 17 combinations


55


Genelink primer GLB01.


genotypes by using Genelink GLB02 primer. PCR amplification of this primer produced


genetic distance was observed from 0-100% in the genetic dissimilarity matrix. 11

combinations of Tagetes genotypes (for detail see Table B-18) showed maximum genetic

distance (100%). While 72 comparisons showed no polymorphism at DNA level, while

using Genelink primer GLB02.

56

Table 3.3: Primer, no of band fragments, band average and genetic distance of Tagetes genotypes for RAPD primers


by using Genelink GLB03 primer. PCR amplification of this primer produced 27 band




Primer No of band

fragments

Band

average

GD (100%) GD (0%)

Homogeneity

GL A03 57 3.8 5 5

GL A04 29 1.93 8 67

GL A05 16 1.06 2 95

GL A06 18 1.2 1 94

GL A07 27 1.8 8 44

GL A09 54 3.6 28 7

GL A11 49 3.26 1 (97%) 55

GL A12 39 2.6 8 57

GL A13 64 4.26 8 34

GL A14 23 1.53 6 82

GL A15 41 2.73 6 62

GL A16 53 3.53 4 (80%) 39

GL A17 35 2.33 9 78

GL A18 40 2.66 10 37

GL A19 44 2.93 21 39

GL A20 22 1.46 3 84

GL B01 56 3.73 17 18

GL B02 32 2.13 11 72

GL B03 27 1.8 12 80

GL B04 41 2.73 1 (75%) 71

57


Genelink primer GLB03.


genotypes by using Genelink GLB03 primer. PCR amplification of this primer produced


genetic distance was observed from 0-75% in the genetic dissimilarity matrix. Only one

combination (T1-T3) of Tagetes genotypes (for detail see Table B-20) showed maximum

genetic distance (75%). While 71 comparisons showed no polymorphism at DNA level,

while using Genelink primer GLB04.

The average genetic distance in terms of genetic dissimilarity by using the set of

twenty RAPD primers in fifteen Tagetes genotypes is presented in Table 3.4. The range

of genetic distance was observed from 22-100% in the genetic dissimilarity matrix (Table

3.4). Maximum genetic distances (100%) were estimated in three comparisons (T3-T13,

T6-T14 and T7-T14) while the minimum genetic distance was observed for one

comparison T9-T10 (22%) closely followed by T10-T11 (23%), T13-T14 (25%) and T3-

T4 (26%). The remaining genotypes showed insignificant differences.

The dissimilarity coefficient matrix (Nei and Li, 1979) data of 20 RAPD primers

for 15 genotypes of Tagetes species was used to make a dendogram by UPGMA method

for cluster analysis (Fig. 3.16). In general the dendogram agreed with the average

dissimilarity matrix presented in Table 3.4. Fifteen genotypes were grouped into four

groups (A, B, C and D) in the phenogram which were putatively identified by

taxonomist. The group A comprised of five (T1, T2, T13, T14 and T12) genotypes of T.

erecta hybrids, in which T12 out-grouped as it a diverse genotype. T13 and T14 were

similar genotypes in this group. Group B comprised seven (T5, T9, T10, T11, T6, T8 and

T7) genotypes of T. patula hybrid. T9 and T10 genotypes were most similar, while T5

and T7 were out-grouped from the rest of this group. Group C comprised two (T3 and

T4) genotypes of Tagetes species. T15 genotype of T. minuta was the genetically most

distinct from the other genotypes and did not match with the any other genotype, which

58

indicated the high level of genetic diversity with other genotypes. On the other hand,

twelve T. minuta plants collected from different areas did not show any genetic diversity

because they presented 100% similarity with no polymorphic band.

Three STS primers were used for the genetic polymorphism and species selection

for essential oils. These primers indicated 29 loci of amplification products with total

139 detectable fragment bands scored against 15 genotypes of Tagetes found in northern

areas of Pakistan. Of 29 amplification products 28 (96.55%) were found to be

polymorphic and 1 (3.45%) was monomorphic (Table 3.5). The number of amplification

products from 7 to 12, while 9.67 was average of amplified products per STS primer.

Average 51.11% homology was observed in 15 genotypes (Table 3.5).


by using STS primer LSTI. PCR amplification of this primer produced 50 band





LSTI primer.


genotypes by using STS primer LSTII. PCR amplification of this primer produced 35

band fragments while band average per genotype was 2.33 (Table 3.6). The range of

genetic distance was observed from 0-66% in the genetic dissimilarity matrix. Three

combinations (T2-T3, T2-T4 and T2-T15) of Tagetes genotypes (for detail see Table B-

22) showed maximum genetic distance (66%). While 52 comparisons showed no

polymorphism at DNA level, while using LSTII primer.


genotypes by using STS primer LSTIII (Fig. 3.15). PCR amplification of this primer

produced 54 band fragments while band average per genotype was 3.6 (Table 3.6). The

59

range of genetic distance was observed from 0-100% in the genetic dissimilarity matrix.

20 combinations of Tagetes genotypes (for detail see Table B-23) showed maximum

genetic distance (100%). While 56 comparisons showed no polymorphism at DNA level,

while using LSTIII primer.

Genotype T1, T2, T13, and T14 showed no significant amplification products to

these STS primers that also proved in the Nei’s genetic diversity findings. UPGMA

dendrogram of STS primers illustrate the genetic distance between studies genotypes

(Fig. 3.17). The average genetic distance in terms of genetic dissimilarity by using the

set of three STS primers in fifteen Tagetes genotypes is presented in Table 3.7. The

range of genetic distance was observed from 9-100% in the genetic dissimilarity matrix

(Table 3.7). Maximum genetic distances (100%) were estimated in three comparisons

(T2-T15, T3-T7, T4-T7 and T4-T9) while the minimum genetic distance was observed

for one comparison T10-T11 (9%) closely followed by T3-T4 (10%) and T9-T10 (14%).

The remaining genotypes showed insignificant differences.

RAPD is a technique used widely for the analysis of genetic variability and

collection of germplasm identification as well as the identification of phylogenetic

relationship, cultivars, breeding programs and investigation of lines (Caetano-Anolles,

1996; Virk et al., 1995; Powell et al., 1996; Singh et al., 2009; Besse et al., 2004; Liu et

al., 2008; Przyborowski and Sulima, 2010). Tagetes is a genus of medicinal importance

(Soule, 1993b; Tereschuk, et al., 1997; Parejo, et al., 2005; Romagnoli, et al., 2005). So

many chemical compounds have been isolated from this genus but not enough data is

present on molecular analysis. Generally, molecular marker techniques have been

employed in the cereal crops such as wheat, maize and rice but less emphasis was laid on

such medicinal important species (Leach et al., 1992; Adhikari et al., 1999; Shah et al.,

2000; Jun et al., 2007; Selvi et al., 2003). It is highly significant to test the genetic

diversity and genetic polymorphism present in the Tagetes species to improve this

medicinal crop of commercial importance. Only one reference is reported on the genetic

diversity of Tagetes species in scientific literature. Darokar et al., (2000) found 90 bands

with 70% polymorphism in six accessions of T. patula. While in present study 757 bands

60

and 159 loci with 95.21% polymorphisim was observed in fifteen genotypes of Tagetes.

The increase number of polymorphic products also increases the effectiveness of

molecular markers, so that high number of primers should be used. The increased

number of polymorphic amplification products in present study proved the usefulness and

effectiveness of RAPD markers in determination of genetic diversity of Tagetes, while

STS markers in the current study were used for the first time for genetic polymorphism in

Tagetes genotypes.

One of the important aims of current study was to identify and measure the

genetic polymorphism in a set of Tagetes genotypes collected from the plain and hilly

areas of Pakistan. Significantly high percentage and degree of genetic diversity was

observed in the species of genus Tagetes for selection of species if they were similar or

dissimilar.

61

Fig. 3.10: PCR amplification products of fifteen Tagetes species with RAPD primer GL A-07. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

Fig. 3.11: PCR amplification of fifteen Tagetes species with primer GL A-09. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

62

Fig. 3.12: PCR amplification products of fifteen Tagetes species with primer GL A-13. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.


63


Fig. 3.15: PCR amplification products of fifteen Tagetes species with STS primer LSTIII. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

64

Table 3.4: Average genetic distances among 15 Tagetes genotype using RAPD primers

Genotype T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15

T1 - T2 0.37 - T3 0.67 0.42 - T4 0.76 0.56 0.26 - T5 0.94 0.58 0.84 0.77 - T6 0.81 0.50 0.73 0.82 0.39 - T7 0.64 0.43 0.78 0.76 0.50 0.33 - T8 0.58 0.38 0.67 0.68 0.44 0.29 0.36 - T9 0.89 0.61 0.89 0.83 0.32 0.41 0.58 0.31 - T10 0.67 0.44 0.67 0.68 0.40 0.38 0.46 0.27 0.22 - T11 0.66 0.46 0.70 0.63 0.36 0.42 0.38 0.36 0.29 0.23 - T12 0.66 0.58 0.84 0.98 0.67 0.53 0.45 0.65 0.80 0.86 0.76 - T13 0.66 0.53 1.00 0.98 0.74 0.58 0.64 0.58 0.56 0.49 0.62 0.54 - T14 0.46 0.62 0.83 0.80 0.85 1.00 1.00 0.62 0.60 0.62 0.75 0.78 0.25 - T15 0.69 0.53 0.61 0.78 0.73 0.70 0.75 0.69 0.70 0.51 0.69 0.83 0.69 0.82 -

Table 3.5: Polymorphism parameters and homology of genotypes using STS primers

Table 3.6: Primer, no of band fragments, band average and genetic distance of Tagetes genotypes using STS primers

Parameters Values

Amplification products 29 Polymorphic (%) 96.55 Monomorphic (%) 3.45 Average amplification product per primer 9.67 Homology of genotypes (%) 51.11

Primer No of band fragments Band average GD (100%) GD (0%) Homogeneity

LST I 50 3.33 12 53 LST II 35 2.33 3 (60%) 52 LST III 54 3.6 20 56

65

Table 3.7: Average genetic distances among 15 Tagetes genotype using STS primers



T1: Tagetes erecta collected from (Sarai Saleh) Haripur

T2: Tagetes erecta collected from (Sarai Saleh) Haripur







T9: Tagetes patula collected from (Baha-ud-din Zakariya) Multan



T12: Tagetes erecta collected from Kohat

T13: Tagetes erecta collected from Mardan


T15: Tagetes minuta (Composite sample from different localities of Abbottabad and Mansehra)

66

Fig. 3.16: A dendrogram of fifteen (15) Tagetes genotypes generated using 20 RAPD primers and genetic distance estimates from UPGMA

67

Fig. 3.17: A dendrogram of fifteen (15) Tagetes genotypes generated using 3 STS primers and genetic distance estimates from UPGMA

68

3.3. Identification of Gene Specific Markers and their Relationship with

Biochemical Compounds

T. minuta is reported to contain monoterpenes as essential oil component such as

d and l-limonene. Degenerate primers were designed for limonene synthase gene for T.

minuta by using ClustalW sequence alignments. The ClustalW sequence alignment

search showed conserved DNA and protein (amino acid) regions. The resulting

consensus sequence of amino acids at each branch was a potential primer. To assess the

performance of primers their maximum number was considered and selected from larger

multiple sequence alignments. The regions of the alignment with high conservation of

amino acids were used in designing of primers (Fig. 3.18). Several highly conserved

motifs were observed and used to design the degenerate primers of Limonen Synthase of

Tagetes (LST). Motifs QLELID, LQLYEA, IYDVYG, YMQLCF, ADDLGT and

GDVPKS consisting of 122, 217, 368, 404, 514 and 527 amino acid residues

respectively were used as forward and reverse primers with their specific product size.

The PCR with degenerate primers designed with reference to the conserved amino

acid sequences among several plant terpene synthases (Rozen and Skaletsky, 2000;

Bertomeu, et al., 2008) and following sequence analysis, by using the DNA isolated from

the fresh leaf tissue of T. minuta as a template produced clear bands. The degenerate

primer LSTI showed the product size of 750-bp (Fig. 3.20) at annealing temperature of

54° C while showed no results at 56° C of annealing temperature. Similarly LSTII

showed the clear band pattern at product size of 440-bp, LSTIII primer has clear band

pattern at 525-bp product size and LSTV primer showed clear band pattern at product

size of 309-bp with annealing temperature of 56° C (Fig. 3.19). While the LSTIV primer

did not show any band at annealing temperatures ranging from 52°C, 54° C, 56° C and

even at 58° C. The primer dimmers appeared at the lower ends of all degenerate primers.

69

Fig. 3.18: Amino Acid Sequence of Several Limonene Synthases AY055214, AF282875, L13459 are Limonene synthase from Agastache rugosa, Schizonepeta, tenuifolia, Mentha spicata and D49368 limonene cyclase from Perilla

frutescens respectively. Stars indicate highly conserved amino acid residues and arrows show forward and reverse primers.

70

These bands of interest were recovered at their specific sizes and were sequenced. The resulting nucleotide sequences were BLAST that showed variation in sequence producing significant alignments.

Fig. 3.19: Agarose gel analysis of PCR of T. minuta products using degenerate STS primers LSTI, LSTII, LSTIII, LSTIV and LSTV. M, 100-bp DNA ladder. The degenerate primer has annealing temperature of 56°C.

Fig. 3.20: Agarose gel analysis of PCR products of T. minuta using degenerate primers LSTI, LSTII, LSTIII, LSTIV, LSTV. M, 100-bp DNA ladder. The degenerate primer has annealing temperature of 54°C.

71

Part- II

CHEMICAL ANALYSIS

3.4. Biological Assays

Crude extracts of three different plant parts; A) seed flower, B) roots, C)

stem and leaves and their fraction extracts were analysed for biological assays. Crude

extract of part-A showed good results of biological assays. Therefore, fraction extracts of

part-A were further analysed for different biological assays.

3.4.1. Antimalarial Activity

The anti-malarial results of T. minuta are presented in Table 3.8. The n-hexane

fraction showed better anti-malarial activity at 2.78 µ g/ml than did ether fraction at

25µ g/ml which was inactive against Plasmodium falciparum 3D7 strain (Table 3.8). The

chloroquine diphosphate was used as standard drug with 0.025 µ g/ml. The ratio of n-

hexane fraction and chloroquine diphosphate showed that it was moderately active.

Biological activities are commonly applied to analyze the phytochemicals of medicinal

plants. Like most other developing countries of the world the practice of herbal treatment

is well established in Pakistan. Few studies have been made to investigate phytotoxic

and insecticidal activities but no report on anti-malarial activity of T. minuta extracts was

seen. In the present study malaricidal, phytotoxic and insecticidal activities of T. minuta

seeds extracts were evaluated.

In the previous study it was found that the direct burning and thermal expulsion of

plant as mosquito-repellent was the most common method of application for T. minuta

(54.8 and 56.0%) (Seyoum et al., 2002). The results of present study proved the

importance and usefulness of T. minuta extract in determination of anti-malarial activity

against Plasmodium falciparum 3D7 strain.

72

Table 3.8: Anti-malarial activity of Tagetes minuta oil in the n-Hexane and Ether soluble fraction

Test Samples lC50(µg/ml)

Test organism

Standard Drug lC50(µg/ml) n-Hexane fraction Ether fraction

Plasmodium

falciparum

3D7

Chloroquine

diphosphate

0.025

2.78

>25

3.4.2. Phytotoxicity

Phytotoxic effect of T. minuta oil on Lemna minor at different concentrations

(1000, 100, 10 µ g/ml) was significantly lower in n-hexane soluble and ether soluble

fractions (Table 3.9 and 3.10). Paraquat was used as standard drug and incubation

condition was 28± 1 º C. It was observed that n-hexane fraction extract showed high

activity as compared to ether soluble extract. The n-hexane fraction showed 11.11,

16.66, 22.22% growth inhibition at 10, 100, 1000 µ g/ml, respectively (Table 3.9). As

the concentration of sample fraction increased the inhibition effect also increased

proportionately. The ether fraction extract showed low inhibition activity 11.11% at

1000 µ g/ml (Table 3.10).

Table 3.9: Phytotoxic Bioassay of n-Hexane soluble fraction of T. minuta oil

No. of Fronds Name of plant

Conc. of

compound

(µg/mL) Sample Control

%Growth

Regulation

Conc. of

Std. Drug

(µg/mL)

1000 14 22.22

100 15 16.66

Lemna minor

10 16

18

11.11

0.015

Std. Drug Paraquat

73

Table 3.10: Phytotoxic Bioassay of Ether soluble fraction of T. minuta oil

No. of Fronds Name of plant

Conc. of

compound

(µg/mL) Sample Control

%Growth

Regulation

Conc. of

Std. Drug

(µg/mL)

1000 16 11.11

100 17 5.5

Lemna minor

10 18

18

0

0.015

Std. Drug Paraquat

It has been found that secondary metabolites including terpenes especially

monotrepenes like tagetones and ocimenones are abundant in essential oil of T. minuta.

These secondary metabolites of plant inhibit the germination of cohabitant species and

thus cause their delayed germination (Zygadlo et al., 1993; Simon et al., 2002; Lopez et

al., 2009). Further more, phytotoxicity of the plant inhibit or reduced the growth of

weeds rather then causing any negative effect on growth of crop (Batish et al., 2007).

3.4.3. Insecticidal Activity

The insecticidal activity of crude and fraction extracts of the T. minuta were also

tested against three species of common grain pests namely Tribolium castaneum (Red

flour beetle), Rhyzopertha dominica (Lasser grain borer), Callosobruchus analis (Pulse

beetle) (Table 3.11). In treated samples 70% mortality was considered as significant and

active effect of the extract. The n-hexane fraction proved highly active and caused 70-

80% growth inhibition of C. analis and R. dominica, respectively. While the same

hexane fraction presented nearly 20% inhibitions against T. castaneum proving virtually

ineffective.

Ether soluble fraction was significantly effective against R. dominica with 70%

mortility, and showed non-significant mortility of 40% against T. castaneum and was

inactive against C. analis.

74

Table 3.11: Insecticidal activity of T. minuta oil in the n-Hexane and Ether soluble fractions

Mortality (%)

Insect used n-Hexane

soluble

fraction

Ether

soluble

fraction

Control Coopex

Tribolium castaneum 20 40 0 100

Rhyzopertha dominica 70 70 0 100

Callosobruchus analis 80 0 0 100

Previous studies on T. minuta extract showed weak repellant effect against C.

maculatus (Boeke, 2004). Krishna et al., (2005) found that essential oil of T. minuta

was also toxic against beetle species like S. oryzae (Linnaeus) and C. maculatus

(Fabricius). The composition of essential oil varies with plant parts. A fine powder of

leaves of T. minuta were moderately toxic against A. obtectus and Z. subfasciatus as

compared to whole leaves for the management of stored beans (Paul et al., 2009).

3.4.4. Antibacterial Activity

The antibacterial activity of crude and fraction extracts (Ether, n-Hexane,

Chloroform, Ethyl acetate and butanol) of the T. minuta were tested against gram

positive (M. leteus, S. aureus, B. subtilis) and gram negative (P. piket, E. coli, S.

setubal) bacteria (Table 3.12). All the samples showed significant antibacterial activity

while these samples were inactive against E. coli and S. setubal. The crude fractions

were also highly significant against B. subtilis and S. aureus as gram positive bacteia

and as well also againt P. piket (gram negative). This study indicated that fraction

extracts and isolated flavonols of T. minuta have a broad spectrum of antibacterial

activity.

75

Table 3.12: Antibacterial activity of fraction extracts (Ether soluble, n-Hexane soluble, Chloroform soluble, Ethyl acetate soluble and Butanol soluble) and isolated compounds of T. minuta

Zone of Inhibition of sample (mm) Samples

M. luteus S. aureus B. subtilis P. piket

Crude extract -- 6 9 5

Ether fration -- -- -- --

Hexane fraction -- 5 -- 4

Chloroform fraction 4 3 8 2

Ethly acetate fraction 3 3 21 6

Butanol fraction -- 1 8 5

Control (DMSO) 0 0 0 0

Standard (Ampicillin) 14 16 8 8

-- shows no activity

The results of the present study provided convincing evidence that bioactive

extracts of wild T. minuta that grows as weed is potentially valuable plant containing

economically important compounds effective against human disease and insect pests.

These results showed that there is a need to evaluate this plant for further investigation of

bactericidal and insecticidal compounds. These results also justified the traditional use of

T. minuta as insecticide. The study paved the way for further exploration of the

compounds in the plant that still are mystery.

76

3.5. Isolation and Purification of Flavonoids (flavonols)

HPLC/DAD analysis of the chloroform, ethyl acetate and butanol fractions of T.

minuta were detected in the UV spectral region. The HPLC profiles revealed that the

butanol fractions contained relative high quantities of flavonols as compared to the ethyl

acetate fractions, while the chloroform fractions contained other compounds than

flavonoids, and these latter fractions were not used for further spectroscopic analysis.

The HPLC profiles of the butanol fractions (A: seed flower, B: root part) showed

seventeen flavonols, while the ethyl acetate fractions (A: seed flower, B: root part)

showed two flavonols as major compounds (Fig. 3.22). The absorbtion maxima in the

UV spectra of respective compounds obtained online during HPLC analysis in the 320–

360 nm region indicated flavonol 7-O-glycosides based on quercetagetin, 6-hydroxy

kaempferol quercetin and patuletin aglycones.

The flavonols were separated by preparative HPLC. The spectroscopic analysis

of 1, 2, 4-9, 15-19 by UV, high resolution LC/MS (Table 3.13a and 3.13b), and 1D and

2D NMR (Table 3.14 and 3.15) established that 2, 4, 5, 7, 9 and 15-17 (Fig. 3.21) have

not been reported before in T. minuta. This is the first complete identification of

compound 7, which has previously been tentatively identified in T. maxima (Parejo et

al., 2004). Compounds 3, 10-14 were not separated and analysed by spectroscopic

analysis because of having very low concnration. Compounds 1, 6, 8, 18 and 19 have

previously been reported to occur in T. minuta (Abdala and Seeligmann, 1995), however,

this is the first NMR characterization of 1, 6, 18 and 19 isolated from this plant.

77

Fig. 3.21: Structures of the flavonols identified in the examined T. minuta 1: 6OH Qc 7-glc, 4, 5: 6OH Kf 7-glc, 6, 8: 6MeO Qc 7-glc, 2: 6OH Qc 7Gao Glc, 7: 6OH Kf 7Gao Glc, 9: 6OH Qc 7Caf Glc, 15: 6OH Qc 7Cum Glc, 16: 6MeO Qc 7Caf Glc, 17: 6MeO Qc 7Caf Glc with a OH on aglycone. Qc = quercetin; Kf = kaempferol; Glc = glucoside; Gao = galloyl; Caf = caffeoyl; Cum = coumaroyl

78

Fig. 3.22: HPLC profiles showing the flavonols content of T. minuta extract (A) in butanol and (B) ethyl acetate. The HPLC chromatograms were recorded at 320 ± 20 nm.

3.5.1. NMR Elucidation of Flavonols

3.5.1.1. Structure Elucidation of Flavonols (1, 2, 9 and 18)

The 1D 1H NMR spectrum of 1 and 18 confirmed a 3H ABX system at 7.76 ppm

(1: dd 2.0 Hz, 8.0 Hz; H-6'), 7.72 ppm (18: dd 3.6 Hz, 7.2 Hz; H-6'), 7.86 ppm (1: d 2.0

79

Hz; H-2'), 7.82 ppm (18: d 3.6 Hz; H-2'), 6.98 ppm (1: d 8.4 Hz; H-5') and 6.97 ppm (18:

d 7.8 Hz; H-5'), and an unresolved singlet at 7.03 ppm (1: H-8) and 6.58 ppm (18: H-8).

In the HMBC spectrum of 1 the aromatic proton at 7.03 ppm showed correlations to the

aromatic carbons at 106.2 ppm (C-10), 130.3 ppm (C-6), 150.0 ppm (C-9), 152.5 ppm

(C-7), as well as to the carbonyl function at 177.1 ppm (C-4), revealing the aglycone to

be 6-hydroxyquercetin. The molecular mass at m/z 319.0435 of 1 and 319.0433 of 18 in

their ESI high-resolution MS spectra were in accordance with 6-hydroxyquercetin

(C15H10O8 + H+; calc: 319.0454).

1D 1H NMR spectrum of the sugar region of 1 presented the existence of one

sugar unit by the single anomeric proton signal at 5.14 ppm (J = 7.4 Hz). In the COSY

spectrum of this proton the observed crosspeak at 5.14/3.71 and the following crosspeaks

were in accordance with 7 sugar protons, which indicated that this sugar unit was a

hexosyl. The corresponding crosspeaks in the HSQC spectrum allowed the assignment

of H-2'', H-3'', H-4'', H-5'', H-6A'' and H-6B'', and the coupling constants and chemical

shifts values of 1 were in agreement with β-glucopyranosyl. Similarly in the HMBC

spectrum a cross peak at δ 5.14/152.5 (H-1''/C-7) varified the connection point of the

sugar unit to be in the 7-position of the aglycone. The molecular mass at m/z 481.0963 in

the ESI mass spectrum of 1 was in agreement with 6-hydroxyquercetin 7-O-β-

glucopyranoside (C21H20O13 + H+; calc: 418.0982).

The 1H NMR spectrum of compound 2 shared many resemblance with the

corresponding resonances of 1 (Table 3.7). The chemical shift values of H-6A'' (4.78

ppm), H-6B'' (4.55 ppm), H-5'' (3.94 ppm) and C-6'' (64.2 ppm) of the sugar, indicated

the presence of acylation at the 6''-hydroxyl. The two-proton singlet at 7.13 ppm of H-2'''

and H-6''' in the 1D 1H NMR spectrum of 2 was in accordance with a galloyl unit. The

crosspeaks at δ 4.78/168.3 (H-6A''/C=O galloyl) and δ 4.55/168.3 (H-6B''/C=O gallyol)

confirmed the linkage between the 7-glucosyl and the galloyl moiety to be at the 6''-

hydroxyl. Fig. 3.23 shows the UV spectra of the three compounds, 2 with UVmax at 365

nm, 9 with UVmax at 359 nm and 15 with UVmax at 339 nm respectively. The molecular

mass at m/z 633.1066 (C28H24O17 + H+; calc: 633.1092) of 2 was similar with 6-

80

hydroxyquercetin 7-O-β-(6''-galloylglucopyranoside) as obtained in the high-resolution

mass spectrum.

The 1H and 13C resonances of the aglycone and monosaccharide of 9 were

assigned by a combination of 1D 1H NMR, DQF-COSY, HSQC and HMBC experiments

(Tables 3.7 and 3.8) to be 6-hydroxyquercetin 7-O-β-glucopyranoside. The HMBC

spectra presented the crosspeak at δ 5.18/151.7 (H-1''/C-7) confirmed the aglycone and

sugar unit linkage to be at the 7-hydroxyl. The doublets at δ 6.83 (1.5 Hz; H-2'''), δ 6.62

(8.1Hz; H-5'''), δ 7.53 (15.8 Hz; H-α) and δ 6.27 (15.8 Hz; H-β), and the double doublet

at δ 6.66 (dd, 7.8Hz, 1.8Hz; H-6''') in the 1D 1H NMR spectrum of 9 where in accordance

with a caffeoyl unit. The HMBC spectra showed crosspeaks at δ 4.74/168.9 (H-6A''/C=O

caffeoyl) and δ 4.42/168.9 (H-6B''/C=O caffeoyl) confirmed that an acyl group was

linked to be at the 6''-hydroxyl, and the molecular mass at m/z 643.1272 (C30H26O16 + H+;

calc: 643.1299) of 9 was similar to 6-hydroxyquercetin 7-O-β-(6''-

caffeoylglucopyranoside) as obtainedin the high resolution MS spectra.

Fig. 3.23: UV spectra of 2, 9 and 15 recorded on-line during HPLC analysis

8

1

Ta

ble

3.1

3a

: R

elat

ive

amo

unts

an

d o

n-l

ine

HP

LC

an

d h

igh-r

eso

luti

on

ele

ctro

spra

y i

oniz

atio

n m

ass

spec

tral

dat

a re

cord

ed f

or

flav

ono

ids

(1,

2,

4-9

, 1

5-1

7)

iso

late

d f

rom

T.

min

uta

(B

uta

nol

frac

tio

n)

Co

mp

ou

nd

R

ela

tiv

e a

mo

un

ts

(%)

UV

ma

x

(nm

)

t R (

min

) [F

+H

]+ m

/z

ob

serv

ed

[F+

H]+

m/z

calc

ula

ted

[M+

H]+

m/z

ob

serv

ed

[M+

H]+

m/z

calc

ula

ted

Mo

lecu

lar

form

ula

1

68.0

7

359

1

4.9

0

319

.04

35

3

19

.04

54

4

81

.09

63

4

81

.09

82

C

21H

20O

13

+

2

30.1

8

365

1

6.2

7

319

.04

45

3

19

.04

54

6

33

.10

66

6

33

.10

92

C

28H

24O

17

+

4

6.6

5

369

1

9.1

8

303

.04

93

3

03

.05

05

4

65

.10

15

4

65

.10

33

C

21H

20O

12

+

5

24.0

6

345

1

9.9

8

303

.04

80

3

03

.05

05

4

65

.10

09

4

65

.10

33

C

21H

20O

12

+

6

33.8

0

369

2

0.4

9

333

.05

97

3

33

.06

10

4

95

.11

37

4

95

.11

39

C

22H

22O

13

+

7

19.3

4

349

2

1.3

8

303

.04

85

3

03

.05

05

6

17

.11

37

6

17

.11

43

C

28H

24O

16

+

8

40.0

7

343

2

2.6

5

333

.05

95

3

33

.06

10

4

95

.11

40

4

95

.11

39

C

22H

22O

13

+

9

29.3

6

359

2

3.1

0

319

.04

43

3

19

.04

54

6

43

.12

72

6

43

.12

99

C

30H

26O

16

+

15

14.5

0

339

2

7.6

9

319

.04

58

3

19

.04

54

6

27

.13

48

6

27

.13

50

C

30H

26O

15

+

16

37.4

2

337

2

8.3

2

333

.06

13

3

33

.06

10

6

57

.14

50

6

57

.14

56

C

31H

28O

16

+

17

10.1

9

320

2

9.4

0

673

. 13

87

673

.14

05

C

31H

28O

17

+

8

2

Ta

ble

3.1

3b

: R

elat

ive

amo

unts

an

d o

n-l

ine

HP

LC

and

hig

h-r

eso

luti

on

ele

ctro

spra

y i

on

izat

ion

mas

s sp

ectr

al d

ata

reco

rded

fo

r fl

avo

no

ids

(18

-19

) is

ola

ted f

rom

T.

min

uta

(E

thyl

acet

ate

frac

tio

n)

Co

mp

ou

nd

R

ela

tiv

e a

mo

un

ts

(%)

UV

ma

x

(nm

)

t R (

min

) [F

+H

]+ m

/z

ob

serv

ed

[F+

H]+

m/z

calc

ula

ted

[M+

H]+

m/z

ob

serv

ed

[M+

H]+

m/z

calc

ula

ted

Mo

lecu

lar

form

ula

18

89.5

4

359

2

5.7

8

319

.04

33

3

19

.04

09

C

15H

10O

8+

19

72.3

6

371

3

5.4

2

333

.06

11

3

33

.05

66

C

16H

12O

8+

8

3

Ta

ble

3.1

4:

1H

NM

R s

pec

tral

dat

a fo

r fl

avo

noid

s (1

, 2

, 5

-7,

9,

18,

19)

iso

late

d f

rom

T.

min

uta

rec

ord

ed i

n C

D3O

D a

t 2

5 °

C

1

2

5

6

7

9

1

8

19

Agly

con

e

6

3.9

8 s

(O

CH

3)*

3.9

7 s

(O

CH

3)

8

7.0

3 s

6

.97 s

B

7.0

3 s

6

.91 s

6

.96 s

6

.92 s

6

.58 s

6

.58

s

2'

7.8

6 d

2.0

7

.85 d

2.0

8

.21 d

8.8

7

.82 d

2.0

7

.9 d

8.8

7

.82 d

5.2

7

.82 d

3.6

7

.82

d 1

.9

3'

6.9

9 d

8.9

6.9

2 d

8.9

5'

6.9

8 d

8.4

6

.97 d

8.4

B

6.9

9 d

8.9

6

.90 d

8.6

6

.92 d

8.9

6

.92 d

5.0

6

.97 d

7.8

6

.97

d 8

.2

6'

7.7

6

dd

2.0

, 8

.0

7.7

5 d

d 2

.0,

8.5

8

.21 d

8.8

7

.52 d

d 2

.0,

8.5

7

.9 d

8.8

7

.70 d

d 3

.3,

7.5

7

.72 d

d 3

.6,

7.2

7

.72

dd 1

.9,

8.0

7

-O-β

-glu

cop

yran

osy

l

1''

5.1

4 d

7.4

5

.20 d

7.4

5

.14 d

7.5

5

.19 d

7.6

5.1

8 d

7.4

2

'' 3

.71 m

C

3.7

1 d

d 7

.7,

9.4

3

.68 m

B,C

3

.65 m

B,C

3.7

2 m

C

3''

3.6

5 m

C

3.6

7 m

B

3.6

1 m

B,C

3

.60 m

B,C

3.6

6 m

C

4''

3.5

3 m

3

.68 m

B,C

3

.509

t 9

.3B

3

.510

t 9

.3B

3.5

5 t

9.3

5''

3.6

6 m

B,C

3

.94 m

3

.66 m

B,C

3

.66 m

B,C

3.9

6 m

C

6(A

)''

4.0

3

dd

2.0

, 1

2.0

B

4.7

8 d

d 1

.9,

12.0

4

.04 d

d 2

.0,

12.0

B

4.0

4 d

d 2

.0,

12.0

B

4

.74 d

d 1

.9,

11.9

6(B

)''

3.9

dd 6

.2,

12.2

B

4.5

5 d

d 5

.0,

12.0

3

.81 d

d 6

.2,

12.2

B

3.8

1 d

d 6

.2,

12.2

B

4

.42 d

d 1

2.0

, 7

.2

6''-

O-a

cyl

6

''-O

- g

all

oyl

6

''-O

- g

all

oyl

6

''-O

-caff

eoyl

1

'''

2'''

7.1

3 s

B

7.1

5 s

6

.83 d

1.5

3

'''

4'''

5

'''

6

.62 t

8.1

6

'''

7

.13 s

B

7.1

5 s

6

.66 d

d 7

.8,

1.8

Α

7

.53 d

15

.8

Β

6

.27 d

15

.8

BO

ver

lapp

ed b

y o

ther

sig

nal

. CF

rom

CO

SY

. s

(sin

gle

t),

d (

do

uble

t), d

d (

do

ub

le d

oub

let)

and

m (

mult

iple

t)

84

Table 3.15: 13C NMR spectral data for flavonols (1, 2, 5-7, 9, 18, 19) isolated from T.

minuta recorded in CD3OD at 25 °C

$These values are ranked from lowest to highest shift-values from HSQC. Exact shift-values were then determined from CAPT.

1 2 5 6 7 9 18 19

Aglycone 2 146.3 147.0B 149.0 149.3B 148.8B 148.8 148.8 3 137.1 137.4 137.1 137.1 137.1 136.9 137 4 177.1 177.6 177.5 177.5 177.5 177.6 177.3 177.5 5 147.0 153.15 146.8 147.0 147.0 146.8 147.5 6 130.3 133.3 130.9 130.9 130.7 129.7 132.1 6 (OCH3) 61.5 61.0 7 152.5 157.6 152.7 152.7 151.7 154.6 158.2 8 95.4 95.4 95.4 95.1 94.9 94.0 94.7 9 150.0 152.9 150.4 150.4 146.5 151.1 153.7 10 106.2 106.67 106.67 106.74 106.8 104.8 105.0 1' 123.9 123.9 123.7 124.1 123.9 124.4 124.1 2' 116.2 116.18$ 130.9 116.4 133 116.2 116.0 116.0 3' 145.9 146.2 116.30$ 146.0 146.1 146.2 146.1 4' 148.5 148.8 160.7 149.0 149.3B 148.7 148.7 5' 116.1 116.23$ 116.30$ 116.25$ 122 116.2 116.2 116.2 6' 121.8 121.9 130.9 121.7 133 121.9 121.7 121.7 Glycosyl 1'' 102.8 102.0 102.6 102.4 102.1 2'' 74.4 74.6 74.7 74.8 74.5 3'' 78 77.2 77.5 77.9 77.4 4'' 71.1 71.3 71.3 71.3 72.0 5'' 77.9 75.8 78.5 78.5 75.7 6'' 62.7 64.2 62.50 62.55 64.6 6''-O-acyl 6''-O-

galloyl 6''-O-

galloyl 6''-O-

caffeoyl

1''' 121.3 127.2 2''' 110.1 110.1 115.6 3''' 146.4 146.4 4''' 139.8 149.2

5''' 146.4 116.4

6''' 110.1 110.1 122.2 Α 147.5

Β 114.4 C=O 168.3 168.9

85

3.5.1.2. Structure Elucidation of Flavonols (5 and 7)

The UV spectra of 5 and 7 showed UVmax at 345 nm and 349 nm, respectively.

The 1H NMR spectrum of 5 presented a singlet at 7.03 ppm (H-8) and a 4H ABXY

system of two doublets at 8.21 ppm (d, 8.8 Hz; H-2'' and H-6'') and 6.99 ppm (d 8.9

Hz; H-3'' and H-5'') in accordance with the flavonol, 6-hydroxykaempferol. The

signals recorded on the basis of 1D 1H NMR, 1H-1H COSY, 1H-13C HSQC 1H-1H

TOCSY and 1H-13C HMBC spectra, the chemical shifts values (1H and 13C) of 5 were

in agreement 6-hydroxykaempferol linked to one β-glucopyranose unit. The

crosspeak at 5.14/152.7 ppm (H-1''/C-7) in HMBC spectra were in agreement with

linkage between the aglycone and the sugar unit to be at the 7-hydroxyl. The

molecular mass at m/z 465.1009 in the ESI-MS spectrum of 5 was in accordance with

6-hydroxykaempferol 7-O-β-glucopyranoside (C21H20O12 + H+; calc: 465.1033).

The chemical shift values in 1D 1H NMR spectrum of 7 were similar to those of

5. It showed a singlet at 6.96 ppm (H-8) and a 4H ABXY system of two doublets at

7.9 ppm (d, 8.8 Hz; H-2'' and H-6'') and 6.92 ppm (d 8.9 Hz; H-3'' and H-5'') in

accordance with the flavonol, 6-hydroxykaempferol (Table 3.14). The ESI-MS spectra

showed the fragment ion due to cleavage of the glycosidic bond, which suggested the

presence of 6-hydroxykaempferol (m/z 303.0485). Some of the NMR signals are

missing or were very weak, but the singlet at 7.15 ppm of H-2''' and H-6''' in the 1D 1H

NMR spectrum of 7 was in accordance with a galloyl unit. The molecular mass at m/z

617.1137 in the high-resolution ESI-MS spectrum were in accordance with 6-

hydroxykaempferol 7-O-β-(6''-galloylglucopyranoside) (C28H24O16 + H+; calc:

617.1143).

3.5.1.3. Structure elucidation of Flavonols (6 and 19)

The 1D 1H NMR spectra of 6 and 19 revealed the presence of 6-

methoxyquercetin aglycone. Each of their 1H NMR spectra showed four aromatic

proton resonances, a singlet at 6.91 ppm (6: H-8), 6.58 ppm (19: H-8) 7.52 ppm (6: dd

2.0 Hz, 8.5 Hz; H-6'), 7.72 ppm (19: dd 1.9 Hz, 8.0 Hz; H-6'), 7.82 ppm (6: d 2.0 Hz;

H-2'), 7.82 ppm (19: d 1.9 Hz; H-2'), 6.90 ppm (6: d 8.6 Hz; H-5') and 6.97 ppm (19: d

86

8.2 Hz; H-5') along with a methoxy group at 3.98 ppm (6) and 3.97 ppm (19). The

molecular mass at m/z 333.0597 of 6 and 333.0611 of 19 in the ESI high-resolution MS

spectra were in accordance with 6-methoxyquercetin (C16H12O8 + H+; calc: 333.0610).

1H and 13C resonances of the monosaccharide of 6 were assigned by a combination of

1D 1H, DQF-COSY, TOCSY and experiments, in accordance with β-glucopyranosyl.

The molecular mass at m/z 495.1137 in the ESI-MS spectrum of 6 was in accordance

with 6-methoxy quercetin 7-O-β-glucopyranoside (C22H22O13 + H+; calc: 495.1139).

In the scientific literature, only a few papers dealing with compounds isolated

from Tagetes species have addressed full structure elucidation of individual flavonoids

including NMR assignments. Particularly in T. minuta no reference has been found

with respect to NMR investigations of flavonoids. In T. minuta Abdala and

Seeligmann (1995) and Tereschuk et al., (2004) have reported the occurrence of the

flavonols quercetagetin, patuletin, isorhamentin and quercetin as aglycones with

glycosides linkages without acylations. In this study we have also identified 6-

hydroxy- and 6-methoxyquercetin glycosides with acylation in T. minuta. 6-

hydroxykaempferol as found in 5 (6-hydroxykaempferol 7-O-β-glucopyranoside) and 7

(6-hydroxykaempferol 7-O-β-(6'' galloylglucopyranoside)) has previously not been

identified in T. minuta. The compounds 2 (6-hydroxyquercetin 7-O-β-(6''-

galloylglucopyranoside)) and 9 (6-hydroxyquercetin 7-O-β-(6''-

caffeoylglucopyranoside)) have previously in nature been identified only in extracts of

Tagetes maxima (Parejo et al., 2005), and this is the first complete identification of

compound 7, which has previously been tentatively identified in T. maxima (Parejo et

al., 2004).

87

3.6. Conclusions

• Modified Cetyltrimethylammonium Bromide (CTAB) extraction protocol was

found to be highly suitable for extracting high-molecular weight DNA from T.

minuta and other species including T. erecta, T. patula and their hybrids con-

taining large amounts of secondary metabolites and essential oils.

• PCR-based primers such as RAPD and STS may successfully be employed to

estimate genetic diversity in different Tagetes genotypes which may further be

used for the species assortment of parents for breeding and mapping purposes.

• Gene based specific primers were successfully developed as DNA markers

(STS) to distinguish genotypes for limonene synthase gene in Tagetes.

• Tagetes extracts showed promising results against pathogenic bacteria, malaria

and insect pests.

• Findings of the bioassays showed that Tagetes minuta contains valuable

compounds which can be effective against other human diseases and pests.

• Of the three solvents used Butanol may be used as an effective solvent for

extracting new flavonols in T. minuta.

• Seven (07) new flavonols were identified via spectroscopic analyses among

which the structure of 04 was elucidated where as 03 are yet to be characterized

which may turned to be the noval flavonols.

• The structure of additional 4 known flavonols was also elucidated via NMR

which was putatively reported earlier by LC/MS.

88

Future Prospects

• More molecular studies may be carried out to explore in-depth diversity and

structure of Tagetes genome including cytogenetics and chromosome mapping.

• The identified biochemical compounds (flavonols) and the gene responsible for

these can further be characterized with more specific DNA and biochemical

markers in medicinal plants other than Tagetes.

• Efficacy analyses of the identified compounds (flavonols) for therapeutic uses

may further be expanded.

• Identification and complete profiling of additional compounds useful in

pharmaceutics and agriculture may be targeted.

89

Chapter 4

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APPENDEX-A

105

Fig. A-1: (A) Tagetes patula and their hybrids, (B) Tagetes minuta

Fig. A-2: A view of Tagetes minuta in its natural habitat

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Fig. A-3: Tagetes erecta and their hybrids species

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M T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15

Fig. A-4: PCR amplification products of fifteen Tagetes species with RAPD primer GL A-03. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.


Fig. A-5: PCR amplification products of fifteen Tagetes species with RAPD primer GLA-04. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

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109




Fig. A-9 PCR amplification products of fifteen Tagetes species with RAPD primer GL A-12. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

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111





112




Fig. A-15: PCR amplification products of fifteen Tagetes species with RAPD primer GL B-01. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

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114



Fig. A-19: PCR amplification products of fifteen Tagetes species with STS primer LSTI. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

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Fig. A-20: PCR amplification products of fifteen Tagetes species with STS primer LSTII. Lanes 1 M: 100-bp DNA ladder; lanes 2-16 contains T1 to T15.

116

APPENDEX-B

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Table B-1: Genetic distances among 15 Tagetes genotype using RAPD primer GLA03


T1 - T2 0.00 - T3 0.22 0.22 - T4 0.40 0.40 0.11 T5 0.46 0.46 0.46 0.35 - T6 0.66 0.66 0.26 0.14 0.20 - T7 0.66 0.66 0.26 0.14 0.20 0.00 - T8 0.09 0.09 0.31 0.49 0.55 0.75 0.75 - T9 0.40 0.40 0.40 0.69 0.35 0.55 0.55 0.20 - T10 0.31 0.31 0.31 0.20 0.55 0.35 0.35 0.18 0.49 - T11 0.31 0.31 0.31 0.20 0.55 0.35 0.35 0.18 0.49 0.00 - T12 0.46 0.46 0.46 0.35 0.00 0.20 0.20 0.55 0.35 0.55 0.55 - T13 0.66 0.66 0.66 0.55 0.90 0.41 0.41 0.75 1.24 0.35 0.35 0.90 - T14 0.46 0.46 1.15 1.00 0.69 0.90 0.90 0.55 1.04 0.55 0.55 0.69 0.20 - T15 0.66 0.66 0.66 0.55 0.90 0.41 0.41 0.75 1.24 0.35 0.35 0.90 0.00 0.20 -




118







119







120







121







122







123







124







125

Table B-17: Genetic distances among 15 Tagetes genotype using RAPD primer GLB01






126







127

Table B-21: Genetic distances among 15 Tagetes genotype using STS primer LSTI



Table B-22: Genetic distances among 15 Tagetes genotype using STS primer LSTII.



128

Table B-23: Genetic distances among 15 Tagetes genotype using STS primer LSTIII



129

APPENDEX-C

130

List of Publications

I. Shahzadi, R. Ahmed, A. Hassan and M.M. Shah. (2010). Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes

minuta) for polymerase chain reaction analysis. Genet. Mol. Res. 9: 386-393. I. Shahzadi, A. Hassan, U. W. Khan and M. M. Shah. (2010). Evaluating biological activities of the seed extracts from Tagetes minuta L. found in Northern Pakistan. JMPR Vol. 4: 2108-2112. I. Shahzadi, R. Ahmad, M. M. Shah. (2012). Genetic diversity analysis of Tagetes species using PCR based molecular markers. Scientia Horticulturae [Submitted] I. Shahzadi, O. M. Andersen, I. Skaar, M. Jordheim, M. M. Shah. (2012). Flavonoid compounds from Tagetes minuta characterized by high-resolution LC-MS. Phytochemistry [Submitted]

chemical and molecular characterization of marigold ( tagetes...

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