IDENTIFICATION AND CHARACTERIZATION OF ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF SELECTED MEDICINAL PLANTS OF GALLYAT REGION, PAKISTAN
MUHAMMAD ISHAQUE 02-arid-270
Department of Botany Faculty of Sciences PirMehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan 2018
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IDENTIFICATION AND CHARACTERIZATION OF ANTIOXIDANT AND CYTOTOXIC ACTIVITIES OF SELECTED MEDICINAL PLANTS OF GALLYAT REGION, PAKISTAN
by
MUHAMMAD ISHAQUE (02-arid-270)
A thesis submitted in partial fulfillment of the requirements of the degree of
Doctor of Philosophy
in
Botany
Department of Botany Faculty of Sciences PirMehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan 2018
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THIS DISSERTATION IS
DEDICATED
TOMy Beloved Parents
For their endless love and support
and
To My Worthy Teachers
For their guidance, encouragement and
support
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CONTENTS
Page
List of Tables xiii
List of Figures xvi
List of Abbreviations xxiv
Acknowledgement xxvii
Abstract xxix
1 INTRODUCTION 1
1.1 BIOACTIVE NATURAL COMPOUNDS 1
1.2 ANTIOXIDANT AND CYTOTOXIC BIOACTIVE 3
COMPOUNDS
1.3 DETECTION OF ANTIOXIDANT AND CYTOTOXIC 3
BIOACTIVE COMPOUNDS
1.3.1 Brine Shrimp Lethality Test (BSLT) 5
1.3.2 Potato Disc Anti-tumor Assay 5
1.3.3 DPPH Free Radical Scavenging Bioassay 6
1.4 ISOLATION AND IDENTIFICATION OF BIOACTIVE 7
NATURAL PRODUCTS
1.4.1 Chromatography 8
1.4.1.1 Column chromatography 8
1.4.1.2 Thin layer chromatography (TLC) 9
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1.4.1.3 Preparative thin layer chromatography (PTLC) 9
1.4.1.4 Medium pressure liquid chromatography (MPLC) 11
1.4.1.5 High performance liquid chromatography(HPLC) 11
1.4.2 Spectroscopy 12
1.4.2.1 UV-Vis spectroscopy 12
1.4.2.2 Mass spectroscopy 13
1.4.2.3 Nuclear magnetic resonance spectroscopy (NMR) 13
1.5 STUDY AREA 13
1.6 SELECTION OF MEDICINAL PLANT SPECIES FOR 15
PRESENT STUDY
1.6.1 Dryopteris ramosa (Hope) C. Chr. 16
1.6.2 Quercus leucotricophora A. Camus ex Bahadur 16
1.6.3 Arisaema flavum (Forssk.) Schott 17
1.6.4 Bidens biternata(Lour.) Merr. &Sherff 18
1.6.5 Rosa brunonii Lindl 19
1.7 OBJECTIVES OF PRESENT STUDY 18
2 REVIEW OF LITERATURE 24
3 MATERIALS AND METHODS 33
3.1 SELECTION OF PLANT SPECIES 33
3.2 COLLECTION OF PLANT MATERIAL 33
3.3 PROCESSING, DRYING AND EXTRACTION 33
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3.4 BIOACTIVITIES OF CRUDE METHANOLIC 34
EXTRACTS
3.4.1 Antioxidant Activities 34
3.4.1.1 Preparation of DPPH solution 34
3.4.1.2 Preparation of extract samples 34
3.4.1.3 Bioassay (DPPH) 35
3.4.2 Cytotoxic Potential of Crude Methanolic Extracts 35
3.4.2.1 Hatching of brine shrimps 36
3.4.2.2 Bioassay (BSLT) 36
3.4.3 Antitumor Potential of Crude Extracts 37
3.4.3.1 Preparing Agrobacterium tumefaciens culture 37
3.4.3.2 Viability test of A. tumefaciens 38
3.4.3.3 Procedure of bioassay 38
3.5 FRACTIONATION OF CRUDE EXTRACTS 39
3.5.1 Bioactivities of Fractions 42
3.6 PRELIMINARY TESTS FOR PHYTOCHEMICALS 42
3.7 ESTIMATION OF PHENOLIC CONSTITUENTS OF 44
EXTRACTS
3.8 ESTIMATION OF TOTAL FLAVONOID 44
CONSTITUENTS
3.9 ISOLATION OF COMPOUNDS 45
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3.9.1 Column Chromatography 45
3.9.1.1 Normal phase 45
3.9.1.2 Reverse phase 46
3.9.1.3 Sephadex LH20 46
3.9.3 Thin Layer Chromatography (TLC) 46
3.9.3 High Performance Liquid Chromatography(HPLC) 47
3.9.4 Medium Pressure Liquid Chromatography(MPLC) 47
3.9.5 Preparative Thin Layer Chromatography (PTLC) 47
3.10 IDENTIFICATION of ISOLATED COMPOUND 48
3.10.1 Melting Point of Isolated Compounds 48
3.10.2 TLC Spraying Reagents 48
3.10.3 UV-Absorption Spectr0scopy 48
3.10.4 Mass Spectrometry 49
3.10.5 Fourier Transform Infrared Radiation (FT-IR) 49
Spectroscopy
3.10.6 Nuclear Magnetic Resonance (NMR) Spectroscopy 49
3.11 STATISTICAL ANALYSIS 50
4 RESULTS 51
4.1 BIOACTIVITIES OF CRUDE EXTRACTS 51
4.1.1 Antioxidant Potential of Crude Extracts 51
4.1.2 Cytotoxic Potential of Crude Extracts 52
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4.1.3 Potato Disc Anti-tumor Assay on Crude Methanol Extracts 56
of Selected Plants
4.2 FRACTIONATION AND BIOACTIVITIES OF 59
FRACTIONS
4.2.1 Antioxidant Potential of Fractions 59
4.2.2 Cytotoxic Potential of Fractions 59
4.2.3 Antitumor Potential of Fractions 60
4.3 PRELIMINARY PHYTOCHEMICALS ANALYSIS 60
4.3.1 Determination of Total Phenolic and Total Flavonoid 71
Contents
4.4 ISOLATION AND STRUCTURE ELUCIDATION OF 72
COMPOUNDS
4.4.1 Isolation of Pure Compound From Aqueous Fraction of D. 72
ramosa
4.4.1.1 Column chromatography of group 2 78
4.4.1.2 Isolation of compounds from group 3 80
4.4.2 Structural Elucidation of Isolated Compound From 83
Aqueous Fraction of D. Ramosa
4.4.2.1 Identification and structural elucidation of isolated 83
compound DAF-MI-01
4.4.2.2 Identification and structural elucidation of isolated 87
compound DAF-MI-02.1
4.4.2.3 Identification and structural elucidation of isolated 98
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compound DAF-MI-02.2
4.4.3 Isolation of Compounds From Ethyl Acetate Fraction of D. 119
ramosa
4.4.3.1 Identification of isolated compound (DEF-4MP1910) 120
4.4.4 Isolation of Pure Compound From Ethyl Acetate Fraction 134
of R. brunonii
4.4.4.1 Isolation of compounds from sub group 5 (Ethyl acetate 135
fraction of R. brunonii):
4.4.4.2 Isolation of Compounds From sub Group 4 (Ethyl acetate 135
fraction of R. brunonii).
4.4.4.3 Identification of Isolated Compound REF-5s5: 137
4.4.4.4 Identification of Isolated Compound REF-5s67 141
4.4.4.5 Identification of Isolated Compound REF-Mi-01-49 154
4.5 BIOACTIVITIES OF ISOLATED COMPOUNDS 173
4.5.1 Free Radical Scavenging Potential of Isolated Pure 173
Compounds.
4.5.2 Cytotoxic Potential of Isolated Compound Against Brine 174
Shrimps (BSLT).
5 DISCUSSION 181
5.1 BIOLOGICAL ASSAYS ON CRUDE METHANOL 181
EXTRACT (CME)
5.2 BIOACTIVITIES OF FRACTIONS 185
5.3 QUALITATIVE AND QUANTITATIVE ESTIMATION 187
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OF PHYTOCHEMICALS
5.4 IDENTIFICATION OF ISOLATED COMPOUNDS 189
FROM AQUEOUS FRACTION OF D. RAMOSA.
5.5 IDENTIFICATION OF ISOLATED COMPOUNDS 196
FROM ETHYL ACETATE FRACTION OF D. RAMOSA.
5.6 IDENTIFICATION OF ISOLATED COMPOUNDS 198
FROM ETHYL ACETATE FRACTION OF R. brunonii.
5.7 ANTIOXIDANT AND CYTOTOXIC PROPERTIES OF 206
ISOLATED COMPOUNDS
FUTURE RECOMMENDATIONS 208
SUMMARY 209
LITERATURE CITED 211
APPENDICES 240
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List of Tables
Table No. Page
1.1 Plants derived bioactive compounds and their uses in human 4
health care.
1.2 Taxonomic classification of selected plant species 21
3.1 Crude methanolic extract yield of selected plant species. 40
3.2 Amount of fractions obtained after solvent-solvent 40
fractionation of crude extracts.
4.1 Antioxidant potential of crude extracts of selected 53
plants in terms of IC50.
4.2a Mean percentage lethality of crude extracts against Brine 55
shrimp (BSLT).
4.2b Mean percentage lethality of Nicotine (standard) 55
against Brine shrimp (BSLT).
4.3 Determination of LD50 for crude extracts in BSLT. 57
4.4 Antitumor potential of crude extracts of selected plants. 61
4.5 Mean percentage scavenging and IC50 of fractions obtained 61
from crude extracts of D. ramosa, Q. leucotricophora and
R.brunonii
4.6 Mean percentage death (lethality) of different fractions of 64
selected plants against brine shrimps.
4.7 Calculation of LD50 of fractions of selected plants. 69
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4.8 Mean percentage tumor inhibition and IC50 of 69
fractionsobtained from CME of D. ramosa and R. brunonii.
4.9 Preliminary phytochemical analysis of crude extracts. 73
4.10 Qualitative phytochemical analysis of fractions obtained 74
from CME of D. ramosa, Q. leucotricophora and R.
brunonii
4.11 Total phenolic contents of crude extracts of selected plants. 75
4.12 Total flavonoid contents of crude extracts of selected plants. 76
4.13 Solvent mixture used as mobile phase in MPLC and number 79
of fractions obtained during purification of sub group 2a (D.
ramosa aqueous fraction)
4.14 NMR (1H &13C) chemical shift values and DEPT analysis 90
ofisolated compound DAF-MI-01.
4.15 NMR (1H &13C) chemical shift values and DEPT analysis 101
of isolated compound DAF-MI-02.1.
4.16 NMR (1H &13C) chemical shift values and DEPT 112
analysisofisolated compound DAF-MI-02.2.
4.17 Mobile phase used during column chromatography of ethyl 121
acetate fraction of D. ramosa.
4.18 NMR (1H &13C) chemical shift values and DEPT analysis 128
ofisolated compound DEF-MP1910.
4.19 Mobile phase used during column chromatography of ethyl 142
acetate fraction of R. brunonii.
4.20 NMR (1H &13C) chemical shift values and DEPT analysis 146
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ofisolated compound REF-5s5.
4.21 NMR (1H &13C) chemical shift values and DEPT analysis 157
ofisolated compound REF-5s67.
4.22 NMR (1H &13C) chemical shift values and DEPT analysis 166
ofisolated compound REF-MI-01-49.
4.23 Mean percentage scavenging potential and IC50 of isolated 176
pure compounds.
4.24 Mean percentage lethality and LD50of isolated compounds 178
inBrine Shrimp Lethality Test (BSLT).
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List of Figures
Page Figure No.
1.1 Mechanism of DPPH free radical scavenging bioassay 10
1.2 Instruments a) MPLC and b) HPLC instrument 10
1.3 Map of Pakistan highlighting study area (Galyat region) 22 and collection sites of medicinal plants for the present study. 1.4 Selected medicinal plant species of Gallyat region, 23
Pakistan
3.1 Fractionation scheme of crude methanolic extract 41
4.1 Dose dependent free radical scavenging potential of 53
crudemethanolic extracts of selected plants
4.2 Free radical scavenging potential of selected crude 54
extracts.
4.3 Mean percentage lethality of crude extracts against brine 54
shrimp
4.4 Viability of A. tumefaciens against crude extracts. 57
4.5 Mean percentage tumor inhibition of crude extracts of 58
selectedplants
4.6 Tumor inhibition of crude extracts of selected plants. 58
4.7 Mean percentage tumor inhibitions by fraction ofD. 68
ramosa and R. brunonii at various concentrations.
4.8 Gallic acid standard callibration curve 75
4.9 Quercetin standard callibration curve 76
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4.10 Correlation between antioxidant, cytotoxic and anti- 77
tumorpotential of CME of selected plants and total
phenolic (TPC)and flavonoid contents (TFC).
4.11 HPLC profile and UV-vis apex absorption spectra of 82
DAF-MI-02.
4.12 HPLC profile of DAF-MI-01 obtained from aqueous 88
fraction ofD. ramosa.
4.13 MS of isolated compound DAF-MI-01 showing 89
molecular ionat M+ 409m/z.
4.14 FT-IR spectroscopy of DAF-MI-01, isolated from 89
aqueousfraction of D. ramosa.
4.15 1HNMR plot of isolated compound DAF-MI-01 (from 91
aqueousfraction of D. ramosa.
4.16 13CNMR (DEPT) analysis of DAF-MI-01 showing 92
chemicalshift in ppm.
4.17 gs-HSQC spectra obtained from DAF-MI-01. 92
4.18 gs-COSY spectra obtained from DAF-MI-01 93
4.19 gs-HMBC spectra obtained from DAF-MI-01 93
4.20 Chemical structure of isolated compound DAF-MI-01 94
(Iriflophenone-3-C-β-D glucopyranoside.
4.21 Proposed fragmentation scheme of Iriflophenone-3-C-β- 94
D- glucopyranoside. Molecular ion is (M+H) 409 m/z.
4.22 HPLC profile of DAF-MI-02.1 obtained from aqueous 99
fractionof D. ramosa.
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4.23 FTIR spectroscopic analysis of isolated compound DAF- 100
MI-02.1.
4.24 Mass spectrum (HR-TOF) ESI in positive mode for DAF 100
-MI.02.1.
4.25 HNMR spectra of isolated compound (DAF-MI-02.1) 102
fromaqueous phase of D. ramosa.
4.26 13CNMR (DEPT) analysis of isolated compound (DAF- 103
MI-02.1) from aqueous phase of D. ramosa.
4.27 gs-HSQC of isolated compound (DAF-MI-02.1) from 104
aqueousphase of D. ramosa.
4.28 gs-HMBC of isolated compound (DAF-MI-02.1) from 104
aqueous phase of D. ramosa.
4.29 Structure of isolated compound (DAF-MI.02.1) from 105
aqueousphase of D. ramosa.
4.30 Proposed fragmentation scheme of Mangiferrin. 105
4.31 HPLC profile of DAF-MI-02.2 obtained from aqueous 110
fractionof D. ramosa.
4.32 Mass spectrum (HR-TOF) ESI in positive mode for DAF 111
MI-02.2.
4.33 FTIR spectroscopic analysis of isolated compound DAF- 111
MI-02.2.
4.34 HNMR spectra of isolated compound (DAF-MI-02.2) 113
fromaqueous phase of D. ramosa.
4.35 13CNMR (DEPT) analysis of isolated compound (DAF- 114
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MI- 02.2) from aqueous phase of D. ramosa.
4.36 gs-HSQC of isolated compound (DAF-MI-02.2) from 114
aqueousphase of D. ramosa.
4.37 gs-CoSY of isolated compound (DAF-MI-02.2) from 115
aqueousphase of D. ramosa.
4.38 gs-HMBC of isolated compound (DAF-MI-02.2) from 115
aqueous phase of D. ramosa.
4.39 Structure of isolated compound (DAF-MI-02.2) from 116
aqueousfraction of D. ramosa.
4.40 Schematic representation of compounds isolated from 117
aqueousfraction of D. ramosa.
4.41 Proposed fragmentation scheme of Iso-Mangiferrin. 118
4.42 HPLC profile of DEF-4MP-1910 obtained from ethyl 126
acetatefraction of D. ramosa.
4.43 Mass spectrum (HR-TOF) ESI in positive mode for 127
DEF-MP-1910.
4.44 FIR analysis of isolated compound DEF-MP-1910. 127
4.45 HNMR spectra of isolated compound (DEF-mi-4mp- 129
1910)from ethyl acetate fraction of D. ramosa.
4.46 13CNMR (DEPT) analysis of isolated compound (DEF- 130
mi-4mp-1910) from ethyl acetate fraction of D. ramosa.
4.47 gs-HSQC of isolated compound (DEF-mi-4mp-1910) 130
fromethyl acetate fraction of D. ramosa.
4.48 gs-HMBC spectra obtained from isolated compound 131
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(DEF-mi-4mp-1910) from ethyl acetate fraction of D.
ramosa
4.49 Structure of isolated compound (DEF-4MP-1910) from 131
ethylacetate fraction of D.ramosa.
4.50 Proposed fragmentation scheme of Kampferol- 3- O- β 132
glucopyranoside (Astragalin).
4.51 Schematic representation of isolation of compounds from 133
Ethylacetate fraction of D. ramosa.
4.52 Summary of compound isolated from ethyl acetate 143
fraction ofR. brunonii.
4.53 HPLC profile of REF-5s5 obtained from ethyl acetate 144
fractionofD. ramosa.
4.54 Mass spectrum (HR-TOF) ESI in positive mode for REF- 145
5s5.
4.55 FTIR analysis of 5s5. 145
4.56 HNMR spectra of isolated compound (REF-5s5) from 147
ethylacetate fraction of R. brunonii.
4.57 13CNMR (DEPT) analysis of isolated compound (REF- 148
5s5)from ethyl acetate fraction of R. brunonii.
4.58 gs-HSQC of isolated compound (REF-5s5) from ethyl 148
acetatefraction of R. brunonii.
4.59 gs-COSY of isolated compound (REF-5s5) from ethyl 149
acetatefraction of R. brunonii.
4.60 gs-HMBC spectra obtained from isolated compound 149
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(REF-5s5) from ethyl acetate fraction of R. brunonii.
4.61 Structure of isolated compound (REF-5s5) from ethyl 150
acetatefraction of R. brunonii.
4.62 Proposed fragmentation scheme of Quercetin-3-O- 150
rhamnos
4.63 HPLC Chromatogram of isolated compound REF-5s67 155
4.64 Mass spectrum (HR-TOF) ESI in positive mode for REF- 156
5s67.
4.65 FTIR analysis of REF-5s67. 156
4.66 1HNMR (400 MHz, dMeOD) of isolated compound 158
5s567.
4.67 13CNMR (DEPT) of REF-5s67. 159
4.68 gs-HSQC of isolated compound (REF-5s67) from ethyl 159
acetatefraction of R. brunonii.
4.69 gs-HMBC spectra obtained from isolated compound 160
(REF-5s67) from ethyl acetate fraction of R. brunonii.
4.70 Structure of isolated compound (REF-5s67) from ethyl 160
acetatefraction of R. brunonii.
4.71 HPLC profile of REF-Mi-01-49 obtained from ethyl 164
acetatefraction of R. brunonii.
4.72 MS spectra of isolated compound REF-Mi-49 from 165
EtoAcfraction of R. brunonii.
4.73 FTIR analysis of REF-Mi-01-49. 165
4.74 1HNMR (400 MHz, dMeOD) of isolated compound 168
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REF-Mi-01-49.
4.75 13CNMR (DEPT spectrum 135o) of isolated compound 169
REF-Mi-01-49.
4.76 gs-HSQC of isolated compound (REF-MI-01-49) from 169
ethylacetate fraction of R. brunonii.
4.77 gs-COSY of isolated compound (REF-MI-01-49) from 170
ethylacetate fraction of R. brunonii.
4.78 gs-HMBC spectra obtained from isolated compound 170
(REF-MI-01-49) from ethyl acetate fraction of R.
brunonii
4.79 Structure of isolated compound REF-MI-01-49 171
(Tiliroside)from ethyl acetate fraction of R. brunonii.
4.80 Proposed fragmentation scheme of Quercetin-3-O- 172
rhamnoside.
4.81 Best fit line regression equation (Calculation of IC50 for 177
isolated pure compounds).
4.82 Best fit regression line equation to calculate LD50 for 180
isolatedcompounds in Brine shrimp lethality test (BSLT).
5.1 HMBC correlation observed for Iriflophenone -3-C-β- D 204
glucopyrranoside (DAF-MI-01).
5.2 HMBC correlation observed for a)mangiferin (DAF-MI- 204
02.1), b)Isomangiferin (DAF-MI-02.2).
5.3 HMBC correlation observed for Astragalin(DEF-4mp- 205
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1910).
5.4 HMBC correlation observed for Quercetin-3-O- 205
rhamnoside
5.5 HMBC correlation observed for Tiliroside(REF-MI-01- 205
49)
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List of Abbreviations
1-D NMR One dimensional Nuclear magnetic Resonance
2-D NMR Two dimensional Nuclear magnetic Resonance
A. F Arisaemaflavum amu Atomic mass unit
AT Agrobacterium tumefaciens
BSLT Brine shrimp lethality test
CC Column chromatography
CME Crude methanolic extract
COSY Homonuclear Correlation Spectroscopy
D.R Dryopterisramosa
Da Dalton
DAF Dryopterisramosa aqueous Fraction
DCF Dryopterisramosa chloroform Fraction
DEF Dryopterisramosa Ethyl acetate Fraction
DEPT Distortion less Enhancement by Polarization Transfer
DMSO Dimethyl sulphoxide
DnHF Dryopterisramosa n- Hexan Fraction
DPPH 2,2-diphenyl-1-picrylhydrazyl
ESI-MS Electrospray Ionization-Mass Spectrometry
FTIR Fourier Transform Infrared Radiation
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GAE Gallic Acid Equivalent
GC-FID Gas Chromatography - Flame Ionization Detector
HMBC Heteronuclear Multiple Bond correlation
HPLC High Performance Liquid Chromatography
HPLC High performance liquid chromatography
HR-TOF High-Resolution Time-of-Flight
HSQC Heteronuclear Single Quantum Coherence
IC50 Inhibition Concentration 50%
IR- Infra-Red spectroscopy
LB Luria Bertani agar
LD50 Lethal Dose 50%
MeOH Methanol
MPLC Medium Pressure Liquid Chromatography
MS Mass spectroscopy
NIR Near infra-red absorption spectroscopy
NMR Nuclear magnetic resonance spectroscopy
PDA Potato disc antitumor ppm Part per million
PTLC Preparative Thin Layer Chromatography
Q. L Quercusleucotricophora
QE Quercetin Equivalent
R.B Rosa brunonii
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RAF Rosa brunoniiaqueous Fraction
RCF Rosabrunonii chloroform Fraction
REF Rosabrunonii Ethyl acetate Fraction
RnHF Rosa brunonii n- Hexan Fraction
RNS Reactive Nitrogen Species
ROS Reactive Oxygen Species
TFC Total Flavonoid Contents
TLC Thin layer chromatography
TLC Thin Layer Chromatography
TPC Total Phenolic Contents
UV-Vis Ultraviolet–visible spectroscopy
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ACKNOWLEDGEMENT
I want to regard all my admirations humble gratitude to Most Glorious and Merciful Sovereignty” ALLAH AZZAWAJALL” who blessed me with prospective ability to attain my task. All praises for him. I also pay my regards to who is an (ﷺ) ”the admirable and Peacemaker Holy Prophet “MUHAMMAD everlasting source of guidance and awareness for mankind and he enable us to recognize our creator and perceive the mysteries of his creations. Fortunately by the elegance of Almighty Allah, I had a provision of group of people who done their due diligence in the completion of this diligent inquiry, which was not less than an intimidating responsibility for me.
First and foremost I want to thank my worthy supervisor Dr. YaminBibi. It has been an honor to be her first Ph. D student. She has taught me, both consciously and unconsciously. I appreciate all her contributions of time, ideas, and encouragement to make my Ph.D. experience productive and stimulating. The joy and enthusiasm she has for her research was contagious and motivational for me, even during tough times in the Ph.D. pursuit. Without her guidance and constant feedback this Ph. D would not have been achievable.
I would like to express my sincere gratitude to members of my supervisory committee Prof. Dr. Muhammad Arshad and Prof. Dr. Syed MuhammadSaqlanNaqvi for their continuous support of my Ph.D study and research. I would also like to thanks all the faculty members of departments of Botany, PMAS arid Agriculture University Rawalpindi for their encouragement and support during my Ph. D. Some faculty members of the Institute have been very kind enough to extend their help at various phases of this research, whenever I approached them, and I do hereby acknowledge all of them.
I wish to express my deepest and whole hearted gratitude to Prof. Dr. Muhammad Gulfaraz and Prof. Dr. MuhammadSheraz Department of Biochemistry, PMAS AAUR for their support and providing lab facility at the very start of my Ph. D and their support continue for me till date. I am grateful to Dr.
xxvii
Tariq Masood(Director Islamabad Model College) and Prof. Aftab Tariq(IMCB- F-8/4) for taking serious efforts which enable me to avail study leave without which it would be very difficult for me to complete my Ph. D. I am also thankful to all the colleagues of IMCB F-7/3, Islamabad for their prayers and support.
I am also thankful To HEC Pakistan for providing me IRSIP scholarship. I am Thankful to Prof. Dr. Karin Vetschera University of Vienna, Austria for her cooperation and guidance during my stay in Austria under IRSIP program. Many thanks to Dr. John Schinerl and Stefan Mikulicicof chemo-diversity research group University of Vienna, Austria. I am also thankful to all the members of chemo-diversity research group University of Vienna, Austria especially toAndreas Berger, MariaTarazona-Montoya, NatthawadiWongthet, Markus Hofbauer, and Katharina Sandler for their support and company. I had never felt home sickness during my stay in Austria because of you guys. Thank you so much. Special thanks to Markus Bacher BOKU University Tullin, Austria for spectroscopic analysis.
I am thankful to Ejaz Ahmed, HumaMehreenSadaf,IqraRiaz, Kulsoom Zahra, Nadia Sardar, Muhammad Asad and ShehzadiTabasuumfortheir help. I am also thankful to my brothers (Muhammad Shahzad and Muhammad Obaid) for their support. I want to thanks my wife;she always supported me for all what I needed. I am also thankful to my little princess HaniaBatool, whenever I asked her not to come in my room, I am working. She always obeyed. In the last but not the least I would like to thanks my parents for their unconditional love and support for me. What I am today is only due to prayers of my parents. Above all, I owe it all to Almighty ALLAH for granting me the wisdom, health and strength to undertake this research task and enabling me to its completion.
Muhammad Ishaque
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ABSTRACT
Human have always relied on biological products and have continuously explored their potential to improve various aspects of humanlives.The research on natural product is one of the key of discovering bioactive natural substances. Since with little knowledge about the etiology of many human, animals and plants disorders, this become difficult to synthesized potentially active substance in the chemical laboratory for their treatment. The bioactive compounds obtained from natural sourcesprovideharmonizingattributes to synthetic compounds with respect to their composition, size, different functional groups, architectural and stereo chemical complexity. Importantly, these bioactive natural substances have potential to serve functions in specific biological system like scavenging of free radicals, inhibiting the tumor etc.Medicinal plants have been playing a vital role in combating various ailments including cancer. Many modern antitumor and anticancer drugs have plant-based origin. e.g.Podophyllotoxin (Podophyllumpeltatum), Vincristine
(Catharanthusroseus) etc.Study area i.e. Gallyat region has rich diversity of medicinal plants. Five ethnomedicinally important plant species
(Dryopterisramosa, Quercusleucotricophora, Bidensbiternata, Arisaemaflavum and Rosa brunonii) were selected from the study which were never been assessed for their antioxidant and cytotoxic potential. The selected plant species were collected from Gallyat region of Pakistan, identified and voucher specimens were deposited in herbarium of Quaid-i-Azam University Islamabad for future references.Crudemethanolic extract (CME) of each selected plant was prepared using maceration technique and each plant extract was evaluated for its antioxidant
xxix
and cytotoxic potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, Brine shrimp lethality (BSL) assay and Potato disc antitumor (PDA) assay respectively.The CME of D. ramosa showed maximum free radical scavenging potential (IC5088.67±0.73 µg/mL) followed by CME of Q. leucotricophora(IC5095.51±0.19µg/mL)and R. brunonii (IC50131.41±0.18 µg/mL).
CME of A. flavum and B. biternata showed minimum free radical scavenging potential. The crude extract of R. brunonii showed highest cytotoxicity against
Brine shrimps than all other crude extracts at all concentrations tested.The maximum antitumor potential was shown by crude extract of R. brunonii(IC50
655.65µg/mL) followed by D. ramosa (IC50 790.51µg.mL) and A. flavum (IC50
825.94 µg/mL).The CME of D. ramosa and R. brunonii were subjected to polarity based solvent-solvent fractionation. All fractions [n-hexan fraction (nHF),
Chloroform fraction (CF), Ethl acetate fraction (EF) and Aqueous fraction (AF)] so obtained were assessed for antioxidant and cytotoxic potential. The maximum antioxidant potential was showed by EF of D. ramosa (IC50 57.85±0.24µg/mL) followed by AF of same plant (IC50 108.98±0.28µg/mL). The EF of R. brunonii showed maximum cytotoxicity (LD50405.43±4.8µg/mL) followed by AF of D. ramosa (LD50830.95±2.0µg/mL). The maximum tumor inhibition in PDA was showed by EF of R. brunonii(753.68±0.48µg/mL) followed by AF of D. ramosa
(793.23±0.31µg/mL) and EF of D. ramosa (834.99±0.24µg/mL). In comparison with control, the vincristine a well-known antitumor pure compound showed IC50
232.34±0.58µg/mL. The EF and AF of D. ramosa and EF of R. brunonii were subjected to different chromatographic techniques (CC, HPLC, TLC, PTLC etc.) in order to isolate pure compounds. Iriflophenone-3-C-β-D- glucopyranoside,
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Mangiferrin, iso-mangiferrin and Astragalin were isolated from selected fractions
D. ramosawhile Quercetin-3-O-rhamnoside, Kampferol-3-O-β-glucopyranoside and Tilirosidewere isolated from ethyl acetate fraction of R. brunonii. The structure of isolated compounds were determine by UV-Vis spectroscopy, MS, FT-IR, 1-D
NMR (1H, 13C, DEPT) and 2-D NMR (COSY, HSQC, HMBC). All the isolated compounds showed high antioxidant potential except tiliroside (IC50923.67±
0.86µg/mL) while tiliroside showed excellent cytotoxic potential (LD5050.42±
0.88µg/mL). This is the first report of antioxidant and cytotoxic activities and isolation of compounds from D. ramosa and R. brunonii to the best of our knowledge. Further in-vivo and in-vitro testing of these isolated compounds is recommended. They may prove to be the potential future drugs.
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1
Chapter1
INTRODUCTION
1.1 BIOACTIVE NATURAL COMPOUNDS
The use of natural products to improve various aspects of human life is beyond any doubt. We have continuously explored natural resources such as plants for the welfare of human being. Plants have been used as flavoring agents, spices, perfumes, cosmetics, dyes and medicines since centuries. Some of the plant based natural products were identified as poison while some are used as insecticides and pesticides. The research on natural products is one of the key of discovering potential bioactive substances which have been used to treat different ailments of human and other animals. Since with little knowledge about the etiology of many human, animals and plants disorders, this become difficult to synthesized potentially active substance in the chemical laboratory for their treatment. On the other hand, the natural products have evolved in a biological system and perform variety of functions including scavenging of free radicals, protecting the cells from oxidative stress, pathogens and also protect the living organisms from environmental factors. In terms of stereo-chemical complexity, functional groups, size, composition and architectural properties they showed complete harmony with synthetic compounds (Colegate and Molyneux, 2008). One of the interesting fact that during the period 1981 to 2010 total 1073 organic molecules have been introduced as medicine worldwide out of which more than 50 percent were plants natural products (Newman and Crag, 2012).
In recent past one of the renowned chemical ecologist “Meinwald” made an
1
2
important statement “Chemists need to talk biologists, who could offer valuable guidance, Good field biologists are likely to notice interaction that might provide clues to interesting chemistry” (Rouhi, 2003). This statement becomes more pronounced when we consider biodiversity of the earth. For instance, about half of the predicted flowering plant species(there exist roughly 400,000 plant species in the world) have been described out of which only 10 percent were analyzed chemically but most of them in a preliminary manner. Similarly, less than 1 percent of spider‟s venom has been analyzed but spiders are represented on earth by 40,000 species approximately (Colegate and Molyneux, 2008). Screening of medicinal plants for bioactive phytochemicals, lead to the discovery of quite a few useful medicines. Some of them are listed in table 1.1.
The traditional medicines and ethno medicinal knowledge provide the evidence to the presence of bioactive substance and this triggered the scientific approach in the search of natural bioactive compounds. Some examples of traditional medicines and ethno medicinal knowledge which provided a leads to identification and isolation of natural bioactive compounds are; an antihypertensive agent “Forskolin” was obtained from Coleus forskohliiBriq. (Lamiaceae), the use of this plant was described in ancient Hindu Ayurevedic literature (Bhat et al.,
1977), The ginkolides-an unusual diterpenetrolactones containing a tertiary butyl group was isolated from ginko treeGinkgo biloba (Ginkgoaceae), it is reported in old Chinese medicinal book from 2800BC and was used in antiasthmatic and antitussive preparations (Nakanishi, 2005). In 1950s, a pharmacologist from
Russian reported that the villagers living at the foot of the Caucasian mountains used a plant Galanthusworonii(Amaryllidaceae) polio virus. European possessed
3
very little knowledge about the use of this genus as medicines. In 1951, an alkaloid
“Galanthamine” was isolated from this plant. This alkaloid not only decreases the decreases the progression of Alzheimer‟s disease but also present symptomatic treatment of this neurological disorder (Heinrich and Teoh, 2004). The anticancer commercial drugs like vincristine and vinblastine were obtained from ethno medicinally important plant species Catharanthusroseus. These ethnomedicinal informationattribute an anorexigenic affect to an infusionfrom the plants (Tyler,
1986).
1.2 ANTIOXIDANT AND CYTOTOXIC BIOACTIVE COMPOUNDS
Free radicals are generally produced in a cell as results of metabolism. The important classes of free radicals include ROS (reactive oxygen species) and RNS
(reactive nitrogen species). The substances that delay the oxidation process in the cell and terminate chain reactions initiated by free radicals are called antioxidants
(Halliwell, 1999). The role of these free radicals has been observed in several pathologies including cancer, atherosclerosis, diabetes, Alzheimer‟s disease and
Parkinson (Dhalla et al., 2000; Anderson, 2004; Hallliwell, 2006).Some of the plant based natural bioactive compounds are listed in table 1.1.
1.3 DETECTION OF ANTIOXIDANT AND CYTOTOXIC BIOACTIVE
COMPOUNDS
Bioassay testing is one of the first steps in the discovery of the bioactive compound from plants. A bioassay is a system (in vitro or in vivo) used to identify the potential biological efficacy of an extract or pure compound. A front line screening bioassay must be rapid, convenient, and inexpensive and be able to
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Table 1.1: Plants derived bioactive compounds and their uses in human health care.
Drug/Chemical Action Plant Source Aescin Antiinflammatory Aesculus hippocastanum (Horse chestnut) Betulinic acid Anticancerous Betula alba (Comon birch) Cissampeline Skeletal muscle Cissampelos pareira relaxant (Velvet leaf) Colchicin Antitumor, Antigout Colchicum autumnale (Autumn crocus) Deslanoside Cardiotonic Digitalis lanata (Wooly foxglove) Etoposide Antitumor agent Podophyllum peltatum (Mayapple) Nordihydroguaiaretic Antioxidant Larrea divaricata acid (Creoste bush) Palmatine Antipyretic, detoxicant Coptis japonica (Chinese golden thread) Podophyllotoxin Antitumor, anticancer Podophyllum peltatum (Mayapple) Salicin Analgesic Salix alba (White willow) Silymarin Antihepatotoxic Silybum marianum (Milk thistle) Stevioside Sweetener Stevia rebaudiana(Stevia) Strychnine CNS stimulant Strychnos nux-vomica (Poison nut tree) Taxol Antitumor agent Taxus brevifolia (Pacific yew) Vincristine Antileukemic, Catharanthus roseus antitumor agent (Madagascar periwinkle)
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identify a broad spectrum of activities. The application of bioassay during isolation steps is calledbioassay guided isolation. Brine Shrimp lethality test (BSLT) is the simple and inexpensive bioassay which is used for screening of Cytotoxic compounds (Meyer, 1982). The potato disc anti-tumor assay is used for screening of anti-tumor substances from plants extracts (McLaughlinet al., 1998). The 2, 2- diphenyl-1-picrylhydrazyl (DPPH) is a free radical scavenging bioassay which is commonly used to asses antioxidant potential of an extract or substance (Takao et al., 1994). A brief introduction of these bioassays is given below.
1.3.1 Brine Shrimp Lethality Test (BSLT)
One of the important and widely used bioassay used for preliminary assessment of cytotoxicity is Brine shrimp lethality test. The recent studies revealed that this test has a positive strong correlation with toxicity analysis by pure cell culture like P388 and 9KB pure cell culture (Colegate and Molyneux,
2008).
For this test a crustacean Artemiasalina (Brine shrimp) is used. The cysts(eggs) are easily available and are hatched in marine (sea) water in 24-48 houro and give rise to large number of larvae (nauplii). The samples are tested at different concentrations in vials containing 5mL brine solution and 10-20 nauplii.
Survivors are counted after 24 hours and LD50 value is calculated at 95 percent
confidence. Minor adjustments have been made to suit particular case.
1.3.2 Potato Disc Anti-Tumor Assay
Potato disc anti-tumor assay is inexpensive, accurate and rapid bioassay to predict anti-tumor and cytotoxic potential of a sample with equivalent accuracy to
6
P388 (in vivo murine leukemia) pure cell culture. This assay is based on crown gall tumor inhibition on potato discs. Crown gall-a neoplastic plant disease caused by a gram negative bacterium “Agrobacterium tumefaciens”. This bacterium possess Ti- plasmid (tumor inducing plasmid) which is incorporated into plant chromosomal
DNA during infection. On activation (due to phenols produced by plants on injury)
Ti-plasmid, transformed normal, wounded cells of plants into autonomous tumor cells. The induce tumor is similar in DNA contents and histologicaly to human and animal cancers (McLaughlin et al., 1998).
A mandatory practice for this test is that the extracts or substances being tested do not show activity towards Agrobacterium tumefaciens. This can be asses by standard agar plate diffusion well method (Fadliet al., 1991). For potato disc anti-tumor assay, potatoes are surface sterilized and then cut into cylinders and make 2-3cm discs which are placed on plan ager (1.5 percent) in petri plates. Plant extract is dissolved in dimethyl sulfoxide (DMSO) and 5-10µL is spread over a disc and let the solvent to evaporate. The discs are then inoculated with 5-10µL of freshly prepared bacterial culture and plates are incubated at 27oC for 12 to 21 days. The assay measures the inhibition of tumors induced by the bacterium on potato discs. The solvent DMSO does not interfere with the bacterial activity or induce any tumors (Coker et al., 2003).
1.3.3 DPPHFree Radical Scavenging Bioassay
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) is an antioxidant assay which is easy, simple, rapid, accurate and economic bioassay. This free radical scavenging assay is used for the detection of polar as well as non-polar antioxidant
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substances. Also weak antioxidant compounds can be assessed by this method.
Another advantage of this bioassay is that several solvents like ethanol, aqueous acetone, methanol, aqueous alcohol and benzene etc. can be used without affecting the results (Yu, 2001; Parry et al., 2005).
As one of the few stable organic nitrogen radicals, the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical is used to analyze the antioxidant activity. The
DPPH possess a deep purple color and has a UV-vis absorption maximum at 517 nm (Huanget al., 2005). The test compounds (antioxidants) reduce DPPH radical to DPPH-H and the solution color fades(Figure 1.1). The reducing ability can be assessed by measuring the decrease of its absorbance. The percentage scavenging is calculating at various concentrations and antioxidant potential is expressed in terms of IC50.i.e. The necessary amount of antioxidant required to decrease the initial DPPH concentration by 50 percent and the time taken to reach the steady state to IC50 concentration (Antolovich, 2002).
1.4 ISOLATION AND IDENTIFICATION OF BIOACTIVE NATURAL
PRODUCTS
The recent advancements in isolation and identification techniques have provided tools for purification and structural elucidation of natural products.
Armed with these sophisticated tools, the natural product scientists (chemists, botanists, biochemists) have ventured into bio-assay guided isolation of metabolites and they are now turning their attention to the origin of bioactivity.
All isolation procedures depend mainly on some physical characteristics of the compounds. It is easier to separate compounds with large physical differences
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by simple techniques like solvent extraction while compounds with similar are closely related physical properties are isolated by combination of various techniques and usually one of their character is exploited. The separation and purification of plant constituents is mainly carried out by chromatographic techniques (Raaman, 2006).
1.4.1Chromatography
Chromatography is a technique used to separate molecules on the basis of their size, shape or charge (Heftmann, 1992). During chromatography, molecules in solvent/mixture move through a solid phase that acts as sieve material. As the molecules move through molecular sieve, they are separated. Chromatographic isolations are a result of interaction between analyte and the two phases (Miller,
2004). In general, there are five types of interaction: Adsorption, partition, ion- exchange, affinity and size exclusion. It is often noticed that the analyte of the interest may be isolated from the mixture of compounds that may be present in the plant‟s extract (Raaman, 2006).
1.4.1.1 Column chromatography
Chromatography is based on differential migration rates of components of a mixture as it moves through the solid phase. In column chromatography both liquid
(mobile) and solid (stationary) phases are placed in a glass column. The solid phase is neutral porous materials that allow molecules of different sizes to penetrate into the solid phase to different extent. Various stationary phases of different particle sizes and porosity are available. Silica is one of the most widely used stationary phase but several bonded silica phases (cyano, amino, hydroxyl,
9
nitro) are also used, although they are expensive. Poly acrylamide beads and
Sephadex LH20 are useful as inert packing in the chromatography of liable compounds (Colegate and Molyneux, 2008).
Dry and wet methods are using to load stationary phase in a column. In dry method, the dry adsorbent (silica gel) is filled in the column and then solvent
(liquid phase) is pour in the column. In wet method, a slurry of adsorbent is made with solvent and poured the slurry carefully (avoiding air bubble) in to the glass column. In normal phase chromatography, mobile phase is non-polar while stationary phase is polar while in reverse phase chromatography, the mobile phase is polar and stationary phase is non-polar.
1.4.1.2 Thin layer chromatography (TLC)
Thin layer chromatography was developed as a rapid method for the detection of bioactive compounds in a mixture. TLC is an adsorbent chromatography (Hahn- Deinstrop, 2000) in which molecules are separated based on specific interaction between adsorbent and a selected solvent/mixture of solvents. The fundamental measurement in chromatography is Rf. The Rf value is constant for a given compound under standard conditions.
Rf= Distance has traveled by a substance from the origin / Distance has traveled by a solvent from the origin.
1.4.1.3 Preparative thin layer chromatography (PTLC)
Preparative thin layer chromatography is most basic and most economical separation technique. The importance and usefulness of PTLC make it an essential tool in analytical chemistry laboratory. In PTLC the sample is load on PTLC plates
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Figure 1.1: Mechanism of DPPH free radical scavenging bioassay
a) b)
Figure 1.2:Instruments a) MPLC and b) HPLC instrument
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as uniform layer. When plat is developed in TLC chamber containing mobile phase, the component separated as continuous band rather than spots. Each band is then scratched from PTLC plates and dissolved in suitable solvent.
1.4.1.4 Medium pressure liquid chromatography (MPLC)
Medium pressure liquid chromatography was introduced in 1970s as and efficient technique for preparative separation of natural organic compounds.
MPLC provide separation of large quantities of organic compounds within less time as compared to open column chromatography. The MPLC apparatus is consist of a pump for continuous flow of solvent, a sample injector, pressure monitor gauge, a self-packed column (normal phase or reverse phase gel and is replaceable as required), a detector, a recorder, waste collector and a sample collector. The Figure 1.2a showing a MPLC setup at chemo diversity research center University of Vienna, Austria.
1.4.1.5Highperformance liquid chromatography (HPLC)
High Performance Liquid Chromatography is not only used to isolate the compound but also used for quantification and identification of compounds. It was introduced in 1970s and now it is greatly improved with respect to column packing and detectors. Advanced HPLC used a C-18 solid phase (non-polar) and a polar liquid phase mainly mixture of solvents (Hamilton and Sewell, 1982). High pressure up to 400bars is used to elute the analyte through the C-18 packed column and then they pass through a diode array detector (DAD). A detector
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(DAD)measures the absorption spectra of the eluted substance which help in identification molecule (Raaman, 2006).
1.4.2Spectroscopy
It is used to study qualitative or quantitative characters of a molecule by absorption, emission or scattering of electromagnetic radiation caused by a molecule. The interaction of radiations with matter can cause redirection of the radiations and or transition between energy level of the molecules. Spectroscopic techniques are widely used for identifications and structural elucidation of natural products. e.g. UV-Vis spectroscopy, Infra-Red spectroscopy (IR), Near infra-red absorption spectroscopy (NIR), Mass spectroscopy (MS), Nuclear magnetic resonance spectroscopy (NMR) etc. A brief introduction of some spectroscopic which have been used in present study are given below;
1.4.2.1 UV-Vis spectroscopy
When a beam of is passed through a sample. The beam of light is either reflected or attenuated. The measurement of reflection or attenuation of light is
UV-Vis spectroscopy. The UV-Vis spectra have applications mainly in quantifications but it provides limited information for identification. The concentration of a compound/ substance in a sample can be calculated by the absorbance of a sample at a given wavelength and applying Beer- Lambert law.
The absorption spectra of the compound is measured with the help of diode array detector (DAD) coupled with HPLC. The maximum absorption (ʎmax.)provides information about the chromophores and types of group may be attached with a molecule.
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1.4.2.2 Mass spectroscopy
This powerful technique enables us to identify a known compound from a mixture, determine the molecular formula and other properties of the isolated compounds. Mass spectrometry is a powerful analytical technique that is used to identify unknown compound, to quantify known compounds and to elucidate the structure and chemical properties of the molecules. MS relies on production of ions from a parent compound. The most powerful information that can be obtained from a MS spectrum is the molecular weight of the sample.
1.4.2.3Nuclear magnetic resonance spectroscopy(NMR)
Nuclear magnetic resonance spectroscopyhas been the single most important physical method for the determination of molecular structure for more than 40 years.NMR is a phenomenon on which occurs when nuclei of certain atoms are immersed in a static magnetic field and exposed to second oscillating magnetic field. There are numbers of important features of NMR which make it useful technique of industrial, commercial and research applications. Advancement in NMR spectroscopy enables us to perform a variety of NMR experiments and analysis including 1H-NMR, 13C-NMR, Distortion less Enhancement by
Polarization Transfer (DEPT), and 2-D NMR experiments like Homonuclear
Correlation Spectroscopy (COSY), Heteronuclear Single Quantum Coherence
(HSQC) and Heteronuclear Multiple Bond correlation (HMBC).
1.5 STUDY AREA
Gallyat area is located about 50-80 km north east of Islamabad, Pakistan. It is represented on both sides of border of Khyber Pakhtunkhwa (KPK) province and
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Punjab province. Gallyat hills are the part of great Himalayan Mountain. The name
“Gallyat” was derieved from Urdu language word “Galli” which mean apassageway between two mountains and both sides of the passageway has valleys. Many of the local area of the Gallyat region have the word gali as part of their name. e.g. Natiagali, Dungagali, etc. Murree, a famous hill station of Pakistan also located in Gallyat region. The Gallyat area is located at parameters 33-35°N latitude and 73-74°E longitude at 7000-9500 feet from sea level. The area is spread over 1011 Km2(250,00 acres) and it is one of the best representative of moist temperate forest (Irshad and Khan, 2012). The diversity in climate is found from pleasant warm to extreme cold type during different part of the year. This part of
Pakistan receives much rain fall during months of July, August and mid of
September as Monsoons. The area also receives snow during winter but melts quickly except on northern side of the study area (Above 6000 feet). December,
January and February are the coldest months of the year in the study area and usually snow fall occurred at this time of the year. Temperature usually starts increasing from end of February up to June which is the hottest month of the year.
In Pakistan, it is one of the rich areas in terms of species diversity (Waseem et al., 2005). The local inhabitants of the area are directly dependents on the forest for their timber, fuel, fodder and grazing requirements (Khan, 2003). In the British era, the administration had defined some area of forest (guzara forest) to fulfill above mentioned requirements of the local people. But since last few decades, the population of the area increased tremendously and it cause increasing pressure on guzara forest. As a result, these guzara forest quickly disappearing and are unable to fulfill timber, fuel, fodder and grazing requirements of the local population.
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Subsequently, the local community shifted their focused toward protected areas like Ayubia National Park (ANP) for fulfilling their requirements (Lodhi, 2007).
Due to these unsustainable practices, the soil, quality and quantity of water, wild life, medicinal plants and agriculture activities are adversely affected.This has also resulted in some social consequences due to inculcating abnormalities in wildlife behaviour (Irshad, 2010).
Gallyat is mostly hilly area of Pakistan and is rich with medicinal flora
(Khan, 2003). Various region of Gallyat are enriched with useful medicinal plants.
The local inhabitants are using these medicinal plants for combating their common ailments. They used these indigenous medicinal plants extensively. Due to extensive use of these medicinal plants of the area, some of them are over harvested, for example Skmmialaureola, Asparagus adscendense and
Taxusbaccata have become endangered due to their extensive and mismanaged use
(Ahmedetal., 2004).
1.6SELECTION OF MEDICINAL PLANT SPECIES FOR PRESENT
STUDY
Keeping in view the importance of bioactive natural compounds and medicinal plants diversity of the study area,five plant species of Galyat region of
Pakistan were selected based on two criteria.
a) Their ethnomedicinal importance mentioned in literature and
b) Those which were less explored or never explored for their antioxidant and
cytotoxic potential and phytochemical analysis.
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The selected plant species were Dryopteris ramosa, Quercus leucotricophora,
Bidens biternata, Arisaema flavum and Rosa brunonii.
A brief taxonomic classification (table 1.2) and description of each selected plant species is given below.
1.6.1Dryopteris ramosa (Hope) C. Chr.
Taxonomic description: Herbaceous plant, found in moist and shady places.
It has underground stem called rhizome having brownish hairs. Compound pinnate or bipinnate leaves present above the ground. Young leaves have brown hairs and shows circination. Brown sporangia on lower surface of the leaves and many in numbers. Spores period: December-March. It grows at moist, shady and relatively cold environmental conditions. In Pakistan it is commonly found in Kashmire,
Nrthern areas and Galyat region of Pakistan.Outside Pakistan, it is reported from
India and China also.
Ethnomedicinal uses: Young leaves are used to cure gastric ulcer and constipation (Abbasi etal., 2015, Amjad et al., 2015). The juice of the plant is given in stomach pain (Shafique and Habib, 2014).
1.6.2Quercus leucotricophora A. Camus ex Bahadur
Taxonomic description:Quercus leucotrichophora is an evergreen tree having acuminate, lancelet, serrate and leathery leaves. The upper surface of leaves is dark green while lower side is white or gray pubescent due to the presence of white hairs (hence the name leucotrichophora, meaning white hairs). Tip of the branches bears male inflorescence (catkin) while the female flowers are ting
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rounded and present at the base of leaves. The fruit is solitary acorn (orange-tan in colour). Flowering period: April – May, Fruiting period: August – October.Banj oak is the most common broadleaf tree in the mid-elevations of the Central
Himalayas in India. It is also found in Myanmar, Pakistan, Nepal, Thailand, and
Vietnam. Quercus leucotrichophora is best adapted to regions with a mild and moist climate. In the Himalayas, the banj oak is found at altitudes between 1500 and 2400 m above sea level. It thrives on loamy and clayey soils but not on sandy acidic soils. It does better on soils with a pH ranging from neutral to basic (alkaline soils). Young trees do well under shade while older trees require more sunlight.
The banj oak tree does better on moist soils.
Ethnomedicinal uses:In traditional medicines, gum is used against cold and considered as analgesic (Heuzé and Tran, 2016). The acorns are edible, and the seeds can be roasted to make a coffee substitute. A potential antitumor and pro- oxidant substance “gallic acid” has been obtained from galls that developed on leaves (Patni et. al., 2012).
1.6.3Arisaema flavum (Forssk.) Schott
Taxonomic description:A. flavum is 0.4 meters with sub-globosely tuber which is 1.5–2.5 centimeters in width. Leaves are green having green or yellowish petiole. The species' spathe is only 2.5–6 centimetres having ovoid, yellowish green spate tube. Dark purple throat, accumulated, slightly incurved, oblong-ovate limb (yellow or green in colour). Flower: bisexual with 1–2 centimetres longspadix. Female basal part has greenish ovaries while male part is slightly yellowish in colour. Two anthers are present with two loculed. Flowering period:
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June-July, Fruits: Berries with pale yellow three seeds in each berry. This species of Arisaema is found at relatively higher altitude than all other species of
Arisaema. It is found at Nathia Galli and Along side of Ayubia reserve forest. It is not found at lower sites of Galyat region like Murree, Patriata etc. This plant species is widespread across eastern Africa and southern Asia. It is native to Ethiopia, Somalia, the Pakistan, Afghanistan, Nepal, Assam, Himalayas, Tibet and Yunnan.
Ethnomedicinal uses:The plant is poisonous and used against snake‟s bites
(Gilani et al., 2006; Zubiullah et al., 2006).The root tuber is burn on coal fire and is used to treat dermal diseases, wart, edema (swelling of the body), and skin wound and to treat viral infection (Bhattarai et al., 2006).
1.6.4Bidens biternata (Lour.) Merr.&Sherff
Taxonomic description:A small erect herb commonly found in grass lands.
Leaves: pinnate or bipinnate, ovate, acuminate, serrate. Heads 18 - 20 mm long, oblong, in loose corym-bose cymes or solitary.Flowers: outer ligulated and whitish. Inner flowers bisexual; tubular corolla, 5-lobed, yellow, anthers obtuse at base.Achenes 16 mm long, linear, glabrous; pappus 3, retrorsely barbed, spinescent.B. biternata is one of the common species found in grass lands of
Galyat region but mostly low altitude regions of the study area and considered as weed.Widespread in tropical and subtropical Africa, Asia and Australia.
Ethnomedicinal uses:B. biternata is used against inflammation, bacterial infections, diabetes, malaria, leprosy, ulcers and diarrhea and digestive disorders
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(Zahara et al., 2015). Leaf juice of B. biternata is used for the treatment of Leprosy at initial stages, lactating mothers, cuts (Prajapati et al., 2003).
1.6.5Rosa brunoniiLindl
Taxonomic description:R. brononii is also known asHimalayan Musk Rose.
It has beautiful fragrance. It is a climber shrub with small curved prickles.
Compound leaves which are elliptic to oblong-lancelike and finely toothed. White flowers in clusters with five petals in each flower. Styles merge into a column,exserted, emerges out of the spreading numerous yellow stamens.Fruit purple-brown or dark red. Flowering period: April-May. It is found in
Afghanistan, Pakistan (Northern areas), Kashmir, India, Nepal, Sikkim, Bhutan to
S. W. China.
Ethnomedicinal uses:Fresh extract of flowers in water is considered useful in eye troubles, and perfumery. Poultice ofroots is useful against joint pains
(Ambasta, 1986). Decoction of flower is used to treat constipation (Amjad et al.,
2017). The fruits are used to cure skin diseases (Ishaque et al., 2017).
1.7 OBJECTIVES OF PRESENT STUDY
Keeping in mind the importance of natural product as bioactive compounds and the potential of natural products in drug discovery and also considering the biodiversity of medicinal plants of Gallyat area of Pakistan, the present study was aimed;
o To select medicinal plants from Gallayat region which were least
investigated for their antioxidant and cytotoxic potential.
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o To determine antioxidant and cytotoxic potential of crude extracts and
all the fractions by standard in-vitro bioassays. o To subject all the fractions and crude extracts for preliminary
phytochemical analysis including qualitative and quantitative analysis. o To select most potent fraction and to isolate some bioactive compounds
using chromatographic techniques. o To elucidate structure of isolated pure compound/s by spectroscopic
methods like UV-Vis spectroscopy, MS, NMR etc. o To determine antioxidant and cytotoxic potential of isolated compounds
by standard bioassays.
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Table 1.2: Taxonomic classifications of selected plant species
Plants Dryopteris Quercus leucotricophora Arisaema flavum Bidens biternata Rosa brunonii Taxa ramosa
Kingdom Plantae Plantae Plantae Plantae Plantae
Division Pterdophyta Tracheophyta Tracheophyta Tracheophyta Tracheophyta
Class Polypodiaceae Angiospermae Angiospermae Angiospermae Angiospermae
Order Polypodiales Fagales Alismatales Asterales Rosales
Family Dryopteridaceae Fagaceae Araceae Asteraceae Rosaceae
Genus Dryopteris Quercus Arisaema Bidens Rosa
Species Dryopteris Quercus leucotricophora A. Arisaema flavum Bidens biternata (Lour) Rosa brunonii ramosa (Hope) Camus ex Bhadur (Forssk) Schott. Merr. & Sherff. Lindl. C. Chr. Common Pakistan Wood Banj Oak Yellow Cobra Lilly Yellow Flowered Himalayam Musk Name Fern Blackjack, Spanish needles Rose
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Figure: 1.3 Map of Pakistan highlighting study area (Galyat region) and collection sites of medicinal plants for the present study.
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Dryopteris ramosa Quercus leucotricophora
Bidens biternata Arisaema flavum
Rosa brunonii
Figure 1.4: Selected medicinal plants species of Gallyat region of Pakistan
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Chapter 2
REVIEW OF LITERATURE
One of the oldest records about use of natural products were depicted on clay tablets in cuneiform from Mesopotamia (2600 B.C.). This literature mentioned the use of Cupressus sempervirens (Cypress) and Commiphora species (myrrh) to treat cough, cold and inflammation (Crag and Newman, 2005). Another ancient book “Ebers Papyrus” documented more than 700 medicinal plants based drugs used in Egypt about 2900 B.C. Some other examples of ancient use of medicinal plants and plant based drugs are; Chinese MateriaMedica(1100 B.C., 52 prescriptions), Shennong Herbal (~100 B.C., 365 drugs) and the Tang Herbal (659
A.D., 850 drugs). The Arabs in the 8th century with Avicenna revolutionized the world of medicines through work such as Canon Medicinae (Crag and Newman,
2005).
The use of medicinal plants in history laid down the basis of chemical, pharmacological and clinical studies of plants based drugs (Butler, 2004). One of the famous examples is synthesis of aspirin an anti-inflammatory substance which was derived from “salicin” a natural product found in bark of Salix alba(Marderosian and Beutler, 2002).The use of plant species as traditional medicines provides a real substitute in healthcare services for rural communities of the developing nations (Hayta et al., 2014). Various workers have reported indigenous knowledge of medicinal plants from different parts of Pakistan (Gilani and Rehman 2005; Ibrar et al., 2007; Ahmed and Hussain, 2008; Qureshi et al.,
2009; Mahmmood et al., 2011; Abbasi et al., 2013; Akhter et al., 2013; Murad et
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25
al.,2013; Mujtaba et al., 2014 and Saqibet al., 2014 etc.).Many investigators (Haq and Hussain, 1993; Shinwari and Khan, 1999; Ahmed et al., 2004; Qureshi et al.,
2009; Abbasi et al., 2013; Khan et al., 2013; Mujtaba etal., 2014. etc.) have reported the richness of medicinal flora of Gallyat region (lesser Himalaya and surroundings).
Shakooret al.(2013) proposed that Pteridophytes from District Shopian
(J&K) are rich source of plants secondary metabolites. In their preliminary phytochemical analysis study they reported the distribution of phytochemical within 34 species of pteridophytes. The pteridophytes of Shopian district are rich in flavonoids and phenolic contents (27and 26 species respectively) followed by glycosides (24species) terpenoids (23species), saponins (22species), and alkaloids(18species) and volatile oils (15species). Their study also indicated that
Dryopteris ramosa is a rich source of phenolics and flavonoid contents while alkaloids are absent in this specie.
Many researchers have reported the use of Dryopteris fragrans in traditional Chinese medicines. It was used to treat of skin diseases, like psoriasis, arthritis, rash, dermatitis and barbiers (Lee et al., 1999; Shen et al., 2012). Many phyto-constituents were isolated from D. fragrans which includes sesquiterpenes, phloroglucins, phenolic glycosides, itosterols and essential oils etc. Itoet al.(2000) has reported that more than ten isolated compounds from this plant species have anticancer activities. For example, aspidin BB and albicanol suppressed in vivo two-stage carcinogenesis on mouse skin. Zhaoetal. (2014) studies the cytotoxic properties of isolated compounds from ethyl acetate fraction of D. fragrans. They
26
reported total nine compounds out of which one novel and 8 known compounds.
Among the compounds two has showed strong cytotoxicity against A549 and
MCF7 cell lines while one compound is effective against MCF7 only.
Plants belonging to genus Dryopteris exhibited excellent antibiotic properties. For instance, D. crassirhizoma and D. filix-mas can be used against
Methicillin resistant S. aureus (Lee et al., 2009), whileD. cochleatashowed strong activity against variety of gram positive and negative bacteria and also against some species of pathogenic fungi (Banerjee and Sen, 1980). Alcoholic extract of
D. cochleatawas found to be containing anti-bacterial substances especially against
E. coli, Salmonella typhi etc. (Parihar et al., 2010).D. crassirhizomahas been patented in South Korea as an anti-tooth decay substance due to its high efficacy against Streptococcus. Acnes (pimple) are one of the major concerns especially among women. Propionibacteriumacnesare considered as major cause of acne
(Kim et al., 2006). Recent studies showed that extracts of plants belonging to
Dryopteris genus were showed strong antibiotic properties against
Propionibacteriumacnes(Lee and Shin, 2011).
Bidens biternata is an important medicinal plant which was used in Chinese medicines as well as in western herbalism for the treatment of various diseases like nausea, leprosy, fever, cough and asthma (Bhatt et al., 2012). In Chinese-English
Manual of Common-Used Herbs, under guizhencao, B. biternata along with
Bidens bipinnata and B. pilosa are listed as source materials in order to decapitate the heat for common cold of wind-heat type and prevention of influenza. It also clears away heat and toxic materials for sore throat, appendicitis, snake bite, and
27
centipede bite, diarrhea, dysentery and stomach ache. Bidens bipmnata, B. pilosa and B. biternata are called herba Bidens. The aqueous decoction of these Bidens species along with radix dichroae (Roots of Dichroa fibrifuga) is anti- inflammatory (Borten, 2015).
In the last few decades more than two hundered compounds were isolated from genus Bidens(Valdes, 2001). This genus is rich in chalcone glycosides; okanin is one of the most abundant chalcone (1,3- diphenyl-2-propen-1-one) found in genus Bidens (Kill et al., 2012). Recently, Surywanshi and Yadava (2015) has isolated several compounds from Bidensbiternata, one of them is identified as 5, 7,
8, 4' tetra hydroxy 3, 5' di-methoxy flavone- 7-O-α-Lrahmnopranosyl-4'-O-β-D- arabinopyranosyl(1→4)-Oβ-D-xylopyranoside.
Medicinal properties of members of genus Bidens are due to presence of large numbers of secondary metabolites such as polyacetylenic glycosides, aurons, auron glycosides, p-coumaric acidand its derivatives, flavonoids and flavonoid glycosides, sesquiterpenes, phenylpropanoid glucosides and diterpenes etc. (
Bairwaet al., 2010; Zulueta et al., 1995;Khemraj et al., 2010; Yutaka et al., 1991;
Carmelita et al., 1995).In last few decade, compound isolation of various species of genus Bidens is done i.e. about two hundred compounds have been isolated from
B. pilosa(Valdes, 2001).
Alzheimer‟s disease resulting in loss of memory is a neurologic disorder.
Controlling the activity of acetylcholinesterase (AChE) through acetylcholine esterase inhibitors is one of the ways to treat this disease. Nine extracts of B. biternata were tested for their acetylcholinesterase inhibitory potential by Ellman‟s
28
colorimetric method. All the extracts of B. biternata significantly inhibited acetylcholinesterase. Therefore, thisplant could be a better candidate for acetylcholinesterase inhibitors (Yang et al., 2006, Shahwar et al., 2011). Genus
Bidens has significant anti-malarial, anti-allergic, anti-ulcer, anti-diabetic, anti- cancer and antibacterial agent (Sandra et al., 2000; Parimalakrishnan et al., 2006;
Masako and Yoshiyuki, 2006; Maicon et al., 2008). Activities which are may be due to the presence of acetylene compounds. All species of genus Bidens having aliphatic acetylenes 6-14 each were also very active. However, different extracts of
B. biternata containing only 3 acetylenes showed only 38 percent growth inhibition of Plasmodium falciparum in vitro. Therefore, it considered to be inactive or have a borderline activity in vitro (Alonzo et al., 1999). Literatures suggest that essential oil from stems, leaves and flowers of genus Bidens have significant antioxidant activities (Yanget al.,2006 and Schlemmer et al., 2009).
In Northern Cameroon, leaves of Bidens pilosa were used as protectant tosaved store grains from decomposition (Goudoum et al., 2016). Goudoum et al.
(2016) determined the essential oil profile of Bidens pilosa by GC-FID and antioxidant potential by DPPH, Reducing power ad co-oxidation of β-carotene bioassays. They identified 27 compounds out of which form more than 97 percent of essential oil contents. Their results showed that the essential oil of B. pilosa leaves exhibits strong antioxidant potential that might be an added value for this essential oil preventing stored products from pest attacks (Goudoum et al.,
2016).Recently, a new potential allelochemical, has been isolated from ethanolic extract of the stems of B. biternata and the structure of the compound was characterized as 5, 7, 8, 4' tetra hydroxy 3, 5' di-methoxy flavone- 7-O-α-
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Lrahmnopranosyl-4'-O-β-D-arabinopyranosyl(1→4)-O- β-D-xylopyranoside
(C33H40O20). Some other unknown chemical compounds are also identified from this plant (Surywanshi and Yadava, 2015).
Free radical scavenging activity of crude extract, fractions, and compounds of B. biternata using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power assays showed that the butanolic and chloroform extract of B. biternata exhibited highest ferric reducing antioxidant power value in the range of
8.5 µmol/L/g of extract and in case of DPPH assay the hexane extract of B. biternata was the most active extract which showed a significant result with lowest
IC50 = (55 ± 3) µg/mL. However, butanol extract and chloroform extract exhibited very close results of percent inhibition with significantly different IC50 values
(Yang et al., 2006). Another study conducted by Swapna et al., (2014) revealed that this wild leafy plant possess high free radical scavenging properties (Swapna et al., 2014).
Cobra lilies belong to genus Arisaema representing more than 250 plant species worldwide especially in temperate to tropical climate (Bhagat et al., 2014).
In Indian and Chinese traditional medicines cobra lilies are being used as analgesic, antidote, anti-inflammatory and anti-arthritis (Choudhary et al., 2008;
Bibi et al., 2010 and Chunxiaet al., 2011) Essential oil obtained from cobra lilies
(A. jacquimontii) was grinded with its rhizome and this paste was applied on skin to cure skin diseases like pimples and blisters etc. (Khan et al., 2004). Many of the plants of genus Arisaema has been investigated for their bioactivities and quite a few pure bioactive compounds have been isolated from the plant species of genus
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Arisaema. For instance flavones C- glycicoside schaftoside and isoschaftoside which showed nematicidal activity were isolated from Arisaema erubescens(Du et al., 2011).
The Quercus genus belonging to the plant family fagaceae was found mainly in northern hemisphere and it has the status of national tree of United State of America (USA) ((Hogan, 2016).The china has more than 100 species of
Quercus (Michael, 2012). The fruit (nut) produced by members of genus Quercus are acorns which may contain one or two seeds. In traditional Chinese medicine, acorons are mainly used to treat diarrhea, evil sores, scrofula, etc. (Xin et al.,
2008). Recent studies showed that acorns are rich in antioxidant components like sterols and phenolic compounds (Cantoset al., 2003; Rakic et al., 2006 and
Tejerina et al., 2011). Acorns also contain anti-inflammatory agents liketriterpenes
(Deyrup et al.,2014).Huanget al.(2016) has isolated twelve compounds from acorn of Quercus species which includes eleven known compounds (paradrymoniside, oleanolic acid , arjunolic acid , arjunglucoside II , arjunglucoside I, arjunic acid ,
24-deoxysericoside, sericic acid, sericoside, 2α,3β,19α-trihydroxyolean-12-en-
24,28-dioic acid 28-β-D glucopyranoside ester ) while one new compound. On the basis of 1D and 2DNMR they identified the isolated compound as 2α,3β,19α- trihydroxy-24-oxo-olean-12-en-28-oic acid (Huang et al.,2016).
It was reported that Quercus species are rich in tannins and phenolic compounds. Many bioactive compounds were isolated from several species of this genus. For example compounds isolated from Quercus suber L. exhibited strong antioxidant potential (Fernandes et al., 2004). Researchers have also evaluated
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these isolated compounds against human cancerous cell lines including MCF-7,
Caco2 and HT-29. All the compounds showed inhibitory effects after 24 hours of continuous treatment in dose dependent manner. However the isolated pure compound Mongolicain-B showed maximum inhibition than all other tested compounds against all three cell lines (Fernandes et al., 2009).
The one of the most prominent and widespread of Rosaceae family is its type genus “Rosa” which is represented by more than 100 species. It is found in
Europe, Asia and North America (Nilsson, 1997). Many researchers have studied the antioxidant potential of members of Rosa genus. Kumar et al.(2009) has studied the anti-oxidant potential of Rosa Brunonii, R. damascene and R. bourboniana.Their study exhibited that methanol extract of Rosa brunonii showed maximum free radical scavenging potential followed by crude extract of R. damascene and R. bourboniana.The chemical composition ofessential oil contents of R. brunonii were analyzed by Jangwan et al. (2007). The essential oil of this plant contain germacrene-D (9.05 percent), iso-eugenol (7.36 percent), heneicisane
(7.19 percent), 9-nonadecane (6.88 percent), geranial (6.27 percent), α-pinene
(5.24 percent), tricosane (5.07 percent), and β-caryophyllene (3.05 percent) as major constituents among the 65 identified compounds. The 20 percent dilution of essential oil was found to be most effective against the Xenthomonascomprestis
(Jangwan et al.,2007).Mavi etal. (2004) also reported high antioxidant properties of hips of R. pimpinellifolia. Another Rosa species R. canina also showed strong free radical scavenging potential and it was reported that this potential is mainly due to the presence of plant secondary metabolites chiefly of phenolics and
32
flavonoids classes (Serteser et al.,2008). The extract of R. canina also showed antibacterial properties (Serteser etal.,2008; Quave et al., 2008).
In order to compare the antioxidant and antiradical activity of wild Rosa phenolic extracts, two wild Rosa species (Rosa canina L. and Rosa pimpinellifolia
L.) were selected by Fattahi et al. (2012) from Takab, Oshnavieh and Qasemloo
Valley of Urmia, West Azerbaijan province, Iran, during 2011. The fruits of R. canina and R. pimpinellifolia were collected and then methanolic extracts were prepared from these fruits by Fattahiet al.(2012). The extracts' total phenolic and flavonoid contents and scavenging capacity for radicals‟ nitric oxide, hydrogen peroxide, and DPPH were analyzed. Fruit extracts, respectively, had a range of
176.48 ± 2.71 and 225.65 ± 2.50 mg gallic acid equivalents /100 g methanolic extract in total phenolic content, 0.41± 0.02 and 2.02 ± 0.03 mg quercetin/100 g methanolic extract in total flavonoid content, 22.41 ± 0.64 and 58.10 ± 0.72 percent in hydrogen peroxide, 79.16 ± 0.61 and 87.78 ± 0.1 percent in DPPH, and
76.93 ± 2.31 and 236.76 ± 16.04 percent in nitric oxide radical scavenging percentage. The findings suggested that radical scavenging capacities of R. canina and R. pimpinellifola extracts in different populations positively correlated with phenolic content (Fattahiet al., 2012). The reaction of Rosaextracts with biological molecules leads to cell and tissue injury by lipoperoxidation, proteolysis or DNA degradation (Weiss, 1989).
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Chapter 3 MATERIALS AND METHODS
3.1 SELECTION OF PLANT SPECIES
Five plant species of Galyat region of Pakistan were selected based on two criteria. a) Their ethnomedicinal importance mentioned in literature and b) Those which were less explored or never explored for their antioxidant and
cytotoxic potential.
The selected plant species wereDryopteris ramosa, Quercus leucotricophora, Bidens biternata, Arisaema flavum and Rosa brunonii.
3.2COLLECTION OF PLANT MATERIAL
The selected plants and their medicinally important parts (as mentioned in literature) were collected from study area during spring 2014 to spring 2015.
Leaves of D. ramosa and Q. leucotricophora, aerial parts of B. biternata, rhizome of A. flavum and fruits of R. brunonii were collected in polythene bags duly label with name, location and date of collection. Matured fully grown and disease free plants and their parts were selected for collection. The plant specimens were identified by using available literature and with the help of an expert taxonomist.
The voucher specimenswere deposited in the herbarium of Quaid-i-Azam
University Islamabad, Pakistan for future references.
3.3PROCESSING, DRYING AND EXTRACTION
All the unwanted and diseased parts were removed and plant materials were dried under shade. All the collected plants were grinded into powder form in
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34
grinding mill separately and stored in air tight container with labels. Powdered material (2 kg) of each plant was soaked in methyl alcohol (3000mL) and stored at room temperature for seven days with occasional shaking. It was then filtered using filter paper and glass funnel. This process was repeated with residue thrice and filtrates were combined and evaporated at 40oC under reduced pressure in rotary apparatus to obtained crude methanolic extract. The crude methanolic extract of each plant species was stored in air tight containers at 40C until further used. The quantity of the crude extract obtained after drying was given in table 3.1.
3.4BIOACTIVITIES OF CRUDE METHANOLIC EXTRACTS
3.4.1 AntioxidantActivities of Crude Extracts
Antioxidant potential of each crude extract of selected plant species was assessed by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) standard free radical scavenging bioassay as described by Kulisicetal. (2004) and Obeid etal. (2005) with some alterations.
3.4.1.1 Preparation of DPPH solution
DPPH solution (0.1mM) was prepared by dissolving 3.94mg of DPPH powder in 100mL of methanol in brown, dark glass vial as DPPH is sensitive to light.
3.4.1.2 Preparation of extract samples
200mg of each dry crude methanolic extract was dissolved in 50 mL of dimethyl sulfoxide (DMSO) to get 20mg/mL stock solution. From this stock
35
solution required concentrations (25, 50 100, 150 200 and 250µg/mL) were prepared. Ascorbic acid was used as positive control. Similar concentrations of ascorbic acid were prepared out of 5mg/mL stock solution of ascorbic acid. Pure
DMSO was used as negative control.
3.4.1.3 Bioassay (DPPH)
The reaction mixture contained 2800µL of DPPH solution (0.1mM) and
200µL of crude methanolic extract. The mixture was vortex and incubated at room temperature in dark for 30 minutes. After thirty minutes, the absorbance was measured at 517nm using UV-VIS-spectrophotometer. Reaction mixtures for positive and negative control were prepared by replacing crude methanolic extract with ascorbic acid and pure DMSO respectively.The control solution was prepared by mixing 0.2mL of methanol and 2.8mL of DPPH solution.The percentage free radical scavenging potential was calculated by using following equation;
Percentage scavenging potential = Ac-As/Ac x100
Where, Ac= Absorbance of control, As= Absorbance of sample
The IC50 was calculated by linear regression equation obtained by plotting concentrations against percentage scavenging activity.
3.4.2Cytotoxic Potential of Crude Methanolic Extracts
To determine the cytotoxic potential of crude methanolic extracts of selected plants Brine shrimp lethality test (BSLT)wasperformed as described by
McLaughlin and Rogers(1998) and Sasidharan etal.(2008) with
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somemodifications. The percentage lethality was calculated by comparing surviving larvae of the test and control tubes after 24 hours. By using regression line equation LD50 values were determined.
3.4.2.1Hatching of brine shrimps
Brine shrimp (Artemiasalina) eggs (JBL Artemiopur, Germany) were obtained from the market. The eggs were placed in well aerated artificial sea water in a two chambered container having one chambered covered while other chamber was open. Between the two chambers small openings were present. Artificial sea water was prepared by dissolving 38 grams of sea salt in one liter of distilled water. A pinch of yeast was also added to avoid starvation of hatched larvae. After
24 hours, the shrimp‟s larvae matured as nauplii and were ready to use. To attract the shrimp‟s larvae (nauplii) photactically a lamp was placed above the open side of the tank.
3.4.2.2 Bioassay (BSLT)
Brine shrimp lethality test (BSLT) was performed as described by
McLaughlin and Rogers (1998) and Sasidharanet al. (2008) with some modifications. Each crude methanolic extract (60mg/mL) was dissolved in 100 percent DMSO as stock solution. Less than 1.25 percent DMSO was used to prepare each dilution (v/v) to achieve the maximum tolerable concentration of working solution as described by Sahgalet al., 2013. From the stock solution, 10,
100, 300 and 600 µg/mL concentrations were prepared with artificial and well aerated sea water in glass vials with 5mL final volume. The largest concentration
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600 µg/mL has one percentof DMSO. Nicotine (C10H14N2) was used as positive control while well aerated artificial sea water was used as negative control. The phototropic nauplii (24-36 hours old) were collected with the help of pasture pipette and 20 nauplii were introduced in each concentration/ vial and incubated for 24 hours at room temperature. The toxicity was estimated by LD50 determined after 24 h incubation period (concentration of the solvent with 50 percent of the test animals killed after 24 hours of exposure). The nauplii were considered as dead if no movement was detected during the 10 seconds observation period.All the extracts and concentrations were tested in triplicate andthe mortality(percent death) of the brine shrimp was calculated using the formula:
Percentage of Death = (Total nauplii – Alive nauplii) × 100/Total nauplii
3.4.3AntitumorPotential of Crude Extracts
Antitumor potential of crude extracts of selected plants were evaluated by potato disc antitumor assay as described by Cocker et al., 2003 and Fatmaet al.,
2013 with some modifications.
3.4.3.1 PreparingAgrobacterium tumefaciens culture
Culture (LB) of Agrobacterium tumefaciens (AT10) strain was obtained from microbiology department, Quaid-i-Azam University Islamabad, Pakistan.
Luria Bertani (LB) agar media (2.5mg/100mL of distilled water) was autoclaved and wait until it cool down to 40-45oC, then add 20µL solution of ampicillin antibiotic(50mg/mL) in order to inhibits the growth of all other bacteria except A. tumefaciens. Now, LB culture of A. tumefacienswas added and incubated at 28oC
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for 48 hours with constant shaking. After 48 hours of incubation, suspension of A. tumefaciens on LB media was standardized to 107 CFU/mL with absorbance
0.956±0.031 at 600 nm.
3.4.3.2 Viability test of A. tumefaciens
Agar well diffusion method was followed to test the viability of A. tumefaciens. Nutrients agar plates were swabbed with LB culture of A. tumefaciens. Well 10mm diameter and 2-3cm apart were made with a sterile cork borer. Stock solutions (5mg/mL) of different plants crude extracts were prepared and about 50µL of crude extracts of different concentrations were added in wells.
The control experiment was without plant extracts. All the plates were incubated at
37oC for 24 hours. After that, the diameter of inhibition zone was measured and efficiency of extracts against A. tumefaciens was calculated. Antibiotic
(Cefotaxime) was used as control.
3.4.3.3 Procedure of bioassay
Red skinned potatoes were bought from the local market and their surface was sterilized with 1 percent mercuric chloride solution in water for 15minutes.
After sterilization, potatoes were rinsed with autoclaved distilled water and dry with sterilized towels. A cylindrical core was removed from potato by using sterilized cork borer (10mm diameter). A sterilized razor was used to cut the potato discs (0.5cm thick) out of these potato cylinders. These discs were placed in petri plates containing 15percent plan ager in water (autoclaved). Stock solution of each plants extract (5mg/mL) was prepared in DMSO. The concentrations (10, 100, 500
39
and 1000µg/mL) were prepared from this stock solution for each extract. Each concentration (10µL) and A. tumefaciens culture (10 µL) were mixed and poured at the top of potato disc. Vincristine (10, 100, 500 and 1000µg/mL concentrations) was used as positive control while DMSO was used as negative control. The plates were sealed with Parafilm and incubated at 28oC for 21 days. Lugol‟s solution was prepared and potato disc were stained after 21 days. Numbers of tumor were counted with the help of magnifying glass and dissecting microscope. Percentage tumor inhibition was calculated by using following formula;
Percentage tumor inhibition= 100 – [No. of tumor in sample / No. of tumor in control] x 100
While IC50 (50 percenttumor inhibition concentration) was calculated from regression line equation. Experiments were conducted in triplicates for each concentration of plant extracts and control.
3.5 FRACTIONATION OF CRUDE EXTRACTS
The most potent crude methanolic extracts of D. ramosa and R. brunonii(about 200 grams)were subjected to polarity based solvent-solvent fractionation as described by Bibi et al., 2003 (Figure 3.1). As a result n-
Hexanfraction (nHF), chloroform fraction (CF), ethyl acetate fraction (EF) and aqueous fractions (AF) were obtained. Each fraction was evaporated at 40oC under reduced pressure in rotary apparatus and stored in air tight containers at 40C until further used. The quantity of each fraction obtained after drying was given in table
3.2.
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Table 3.1: Crude methanolic extract yield of selected plant species
Crude Powder Plant species Plant’s family Part used extract used (g) yield (g) Dryopteris ramosa Dryopteridaceae Leaves 2000 535
Quercus Fagaceae Leaves 2000 583 leucotricophora
Aerial Bidens biternata Asteraceae 2000 340 parts
Arisaema flavum Araceae Rhizome 2000 318
Rosa brunonii Rosaceae Fruits 2000 440
Table 3.2: Amount of fractions obtained after solvent-solvent fractionation of crude extracts
CME (200 g) of selected plants species
Fractions D. ramosa Q. leucotricophora R. brunonii
(Grams) (Grams) (Grams) n-Hexan fraction 27 54 43
Chloroform fraction 23 47 55
Ethyl acetate fraction 59 42 61
Aqueous fraction 83 39 38
Impurities 8 18 3
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Figure 3.1: Fractionation scheme of crude methanolic extract
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3.5.1 Bioactivities of Fractions
Each fraction i.e. n-Hexan fraction (nHF), Chloroform fraction (CF), Ethyl acetate fraction (EF) and Aqueous fraction (AF) was subjected for the evaluation of its antioxidant, cytotoxic and anti-tumor potential.
3.6PRELIMINARY TESTS FOR PHYTOCHEMICALS
Various identification tests for categories of phyto-chemicals were conducted by using following standard procedures as cited by Yadav et al., 2014.
Dragendorff’s test: 20 mg of plant extract (solvent free) was stirred with 1 percenthydrochloric acid (4-5mL) and filter. Filtrate (2 mL) was mixed with 2mL of Dragendorff‟s reagent. A prominent yellow precipitation indicated the presence of alkaloid.
Dragendorff‟s reagent was prepared by mixing following solution “A” and
Solution “B” in 1:1 ratio.
Solution “A”: 1.7 grams of Basic Bismith nitrate and 20grams of tartaric acid was dissolved in 60mL of distilled water.
Solution “B”: 16 grams of Potassium iodide was dissolved in 40 mL of distilled water.
Mayer’s test: 20 mg of plant extract (solvent free) was stirred with 1 percenthydrochloric acid (4-5mL) and filter. Filtrate (2 mL) was mixed with 1 or 2 drops of Mayer‟s reagent. A creamy white precipitation indicated the presence of alkaloid.
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Mayer‟s reagent was prepared by mixing of following two solutions.
Mercuric chloride (1.358g) was dissolved in 60mL of distilled water and potassium iodide (5g) was dissolved in 10mL of distilled water. Both solutions were thoroughly mixed and final volume was 100mL in water.
Tests for sterols:Crudeextract (2 mg) was dissolved in 2mL of methanol and then added concentrated sulphuric acid (H2SO4). Formation of purple ring at upper surface indicated the presence of sterols.
Tests for Phenolics: The extract (10mg) was dissolved in distilled water (5mL) and to this; 3mL of 10 percent lead acetate was added. A bulky white precipitate indicates the presence of phenolic compounds.
Tests for Flavonoids: about 1 g of plant extract was shaken with petroleum ether to remove the fatty materials. The defated residue was dissolved in 80% ethanol (20mL) and filtered. The filtrate was used for the following tests; a) 3mL of filtrate was shaken with 1 percentaluminium chloride (3mL).
Formation of yellow colour indicates the presence of flavones, flavonol and
or chalcones. b) 3mL of filtrate + 4mL of 1 percentpotassium hydrooxide. A dark yellow
colour indicates the presence of flavonoids.
Tests for saponins:Plant extract (CME) about 500mg was dissolved in 2mL of distilled water in a test tube and boiled, then allow to cool at room temperature and shaken vigorously. Appearance of forth indicates the existence of saponins.
Test for Tannins:Plant extract (CME) about 500mg was dissolved in 20mL of
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distilled water in a test tube and boiled, then allow cooling at room temperature and then filtered. Few drops of Ferric chloride (0.1 percent) were added into the filtrate. Appearance of blue back or brownish green coloration indicates presence of tannins.
3.7ESTIMATION OF TOTAL PHENOLIC CONSTITUENTS (TPC)
For the purpose of estimation of total phenolic contents of the extracts, standard method was used as described by Singleton and Rossi (1965). For plotting a reference standard calibration curve, different dilutions (25, 50, 100, 150,200 and
250 µg/mL) of Gallic acid were used. The reaction mixture contained 500µL from each dilution of Gallic acid, 10x diluted 2.5mL of Follin-Ciocalteu reagent and 7 percent (w/v) 2.5mL Sodium carbonate. Gallic acid was replaced with 500µL of plant extract (1mg/mL) to obtained reaction mixture for the test sample. Each of this reaction tube was vortexes and incubated at 25-30oC for half an hour and then spectrophotometer analysis was carried out at ʎ760nm.
3.8 ESTIMATION OF TOTAL FLAVONOID CONSTITUENTS (TFC)
For the determination of Total flavonoid contents of each extract, the standard Aluminium chloride method was usedas suggested by Zhishenet al.
(1999) with slight modifications. For plotting a reference standard calibration curve, different dilutions (25, 50, 100, 150,200 and 250 µg/mL) of Queretine were used. The reaction mixture contained 500µL from each dilution of Quercetine, vortexes with 10 percent Aluminium chloride(100 µL), after one minute added 100
µL of Potassium acetate (1M) and vortexes and then after one minute added distilled water (2.8ml) and vortexes. Quercetine was replaced with 500µL of plant
45
extract (1mg/mL) to obtained reaction mixture for the test sample. Each of this reaction tube was incubated at room temperature for half an hour and then spectrophotometer analysis was carried out at ʎ415nm.
3.9 ISOLATION OF COMPOUNDS
Based on results the most potent fractions (ethyl acetate and aqueous fractions of D. ramosa and ethyl acetate fraction of R. brunonii) were selected for further study. For the isolation of pure compounds from selected fractions chromatographic techniques were used. Major isolations were carried out by using column chromatography (Normal and Reverse phase) by using slica gel of different partical sizes and Sephadex LH20. Purification of compounds was done by preparative TLC and Medium Pressure Liquid Chromatography (MPLC). High
Performance Liquid Chromatography (HPLC) was used as it required.
3.9.1 ColumnChromatography
3.9.1.1 Normal phase
Silica gel60 (Sigma-Aldrich) with pore size 0.02-0.05mm and 0.05-
0.07mm was used as required for column chromatography. Sufficient quantity of silica gel was added in petroleum ether to make slurry and then loaded to column avoiding air bubbles. Sample was adsorbed on small amount of dry silica gel and this dry powder of silica with sample was loaded at the top of column. One table spoon of dry sand was added at the top of sample to maintain uniformity of sample layer. Mobile phase was used in order of increasing polarity starting from
46
petroleum ether followed by chloroform, ethyl acetate and methanol in different ratios.
3.9.1.2 Reverse phase
For reverse phase column chromatography, reverse phase silica was used.
Sample was loaded in silica by using methanol and water 1:1 ratio. In reverse phase column chromatography, mobile phase was used in order of decreasing polarity started with water followed by methanol, ethyl acetate, chloroform and petroleum ether in different ratios. Fractions were collected and TLC plates were developed as in normal phase column chromatography.
3.9.1.3 Sephadex LH20
Sephadex LH20 was dissolved in methanol for overnight and then it was packed in glass column with continuous flow of methanol by gravity (without applying pressure). The sample was dissolved in methanol and was loaded at the top of Sephadex. When sample moved into the Sephadex then fill the top of the column with methanol (never let the Sephadex dry) and let the gravity to do the rest. Fractions were collected and subjected to TLC as described in normal phase column chromatography.
3.9.2 Thin Layer Chromatography (TLC)
Fractions so obtained (after CC) were subjected to thin layer chromatography (TLC) (Silica gel 60 coated plates F254 with florescent indicator).
TLC spotter was used to load the sample on TLC plates. An appropriate liquid
47
phase was adjusted and TLC plates were developed under UV light at 254nm.
Fractions with similar RF were grouped together and were again subjected to column chromatography to obtained further purity.
3.9.3 HighPerformance Liquid Chromatography (HPLC)
The fractions were also analysed by HPLC system: Series Agilent 1100 equipped with UV-DAD detector, Column; Hypersil BDS-C18, column size250 x
4.6 mm having 5 µm particle sizes. Solvent system used was MeOH in aqueous buffer (15 mM ortho-H3PO4 and 1.5 mM Bu4NOH) with a flow rate of 1 mL/min., sample injection volume: 10 µL and linear gradient starting from 20 percent
MeOH to 90 percent at 17 minutes to 100percent at 20 minutes kept for 8 minutes.
3.9.4 MediumPressure Liquid Chromatography(MPLC)
Further purification of selected groups was performed byMedium Pressure
Liquid Chromatography (MPLC). Detector: ISCO UA-6 UV / VIS detector;
Column: Lobar® glass column 440x37mm, filled with Merck silica gel
LiChroprep ™ SI 60 (particle size 40-63μm), reverse phase; Pump: Wobble Piston
Pump QD-1 SSY (1-10bar) Fluid Metering, Inc.Flow: 5-10mL / min at 2.0-3.0bar.
3.9.5Preparative Thin Layer Chromatography (PTLC)
In order to purify an impure sample, preparative thin layer chromatography
(PTLC) was used. The impure sample was dissolved in suitable solvent. Silica coated PTLC plates (20x20cm) were used. Sample was loaded on PTLC plates about 2.5cm above from bottom in a straight line like a band. This process was repeated 3-4 times. PTLC plates were allowed to dry. About 100-150mL selected
48
mobile phase was added in TLC chamber and covered with lid for 30 minutes.
TLC plates were developed in TLC chamber and visualized under UV light at
254nm. Clearly distant bands were marked by using lead pencil and physically separate that part from PTLC plates by scratching with spatula. This scratched silica from PTLC plates was washed 3-4 times with a suitable solvent (methanol).
Then it was filtered and allowed to evaporate on rotary apparatus under reduced pressure and then subjected to further analysis and identification by using spectroscopic techniques.
3.10 IDENTIFICATION OF ISOLATED COMPOUND
The isolated compounds were identified by using various techniques including chemical spraying reagents, UV-absorption spectrum analysis, Mass spectroscopy, NMR spectroscopy etc.
3.10.1 MeltingPoint of Isolated Compounds
Melting points of isolated compounds were measured on Gallen Kamp
(Sanyo) instrument.
3.10.2 TLCSpraying Reagents
TLC analyses were done on silica gel 60 F254 plates developed in the organic solvent. TLC spraying reagents used for identification of compounds were anisaldehyde spraying reagent, DPPH reagent (in methanol), Dragendorff‟s reagent and phosphomolybdic acid reagents.
3.10.3 UV-Absorption Spectroscopy
UV- absorption spectra of isolated compounds were recorded using HPLC
49
(as discussed earlier) with retention time and UV-absorption spectrum at 230nm and 254nm.
3.10.4MassSpectrometry
High-resolution time-of-flight (HR-TOF) mass spectrometer (maXis,
BrukerDaltonics) was used for mass analysis of isolated compounds. Direct infusion electrospray ionization (ESI) method (Both in positive and negative mode with mass accuracy ± 5 ppm and ± 10 ppm respectively) was used to record mass spectra between m/z 100-2500 of isolated compounds. Others specifications were:
Capillary voltage 4kV with a capillary current 30-50 nA, Nitrogen temperature
-1 180°C with a flow rate of 4.0 L min and the N2 nebulizer gas pressure at 0.3 bar.
3.10.5 Fourier Transform Infrared Radiation(FT-IR)Spectroscopy
FT-IR spectra of isolated compounds were obtained iD7 ATR Nicolet™ iS5 Spectrometer. The isolated compounds were dissolved in methanol and were scanned 400 to 4000cm-1 wave number.
3.10.6 NuclearMagnetic Resonance (NMR)Spectroscopy
All NMR spectra were recorded on a BrukerAvance II 400 (resonance frequencies 400.13 MHz for 1H and 100.63 MHz for 13C) equipped with a 5 mm observe broadband probe head (BBFO) with z–gradients at room temperature with standard Bruker pulse programs. The samples were dissolved in 0.6 mL of MeO-d4
(99.8 % D). Chemical shifts are given in ppm, referenced to residual solvent signals (3.31 ppm for 1H, 49.0 ppm for 13C). 1H NMR data were collected with 32k
50
complex data points and apodized with a Gaussian window function (lb = −0.3 Hz and gb = 0.3 Hz) prior to Fourier transformation. 13C-jmod spectra with WALTZ16
1H decoupling were acquired using 64k data points. Signal-to-noise enhancement was achieved by multiplication of the FID with an exponential window function
(lb = 1Hz). All two-dimensional experiments (COSY, HSQC, HMBC) were performed with 1k × 256 data points, while the number of transients and the sweep widths were optimized individually.Measurement temperature was 298 K ± 0.05
K. Residual CD3OD wasused as internal standard for 1 H (δH 3.34) and CD3OD for 13 C (δC 49.0) measurements.
3.11 STATISTICAL ANALYSIS
All the experiments were conducted in triplicates and results were shown in
± mean standard deviation. Regression line equations were used to calculate IC50 and LD50. A two way ANOVA was carried out by using SPSS 16.0 software in
CRD factorial design at significance (p) = 0.05. Tukey HSD was used for multiple comparisons between subject effects.
51
Chapter 4
RESULTS
Five ethno medicinally important plant species were selected from Gallyat region of Pakistan to identify their antioxidant and cytotoxic potential. The results of this study are divided into following sections;
4.1 Bioactivities of crude extracts
4.2 Bioactivities of selected Fractions
4.3 Preliminary phytochemical analysis
4.4 Isolation and structural elucidation of phytochemicals
4.5 Bioactivities of isolated compounds
4.1 BIOACTIVITIES OF CRUDE EXTRACTS
The dry powders of selected plants/parts were macerated with methyl alcohol to produce crude methanolic extract of each selected plant species. Crude methanolic extracts of Rosa brunonii, Arisaema flavum, Quercus leucotricophora,
Dryopteris ramosa and Bidens biternata were subjected to bioassays to determine their antioxidant, cytotoxic potential and anti-tumor potential.
4.1.1 AntioxidantPotential of Crude Extracts
Crude methanolic extract of Rosa brunonii, Arisaema flavum, Quercus leucotricophora, Dryopteris ramosa and Bidens biternata were evaluated for their
51
52
antioxidant potential using DPPH free radical scavenging assay. The results were expressed in terms of percentage inhibition and IC50.
All the crude extracts showed free radical scavenging potential in a dose dependent manner (Figure 4.1) but crude extract of D. ramosa showed maximum free radical scavenging potential followed by Q. leucotricophora and crude extract of R. brunonii.The antioxidant potential of all crude extracts of selected plants and standard ascorbic acid was expressed in terms of IC50. The IC50 was calculated using best fit regression equation. The lowest IC50 mean highest antioxidant potential. The crude extract of D. ramosa showed highest antioxidant potential among tested crude extracts (Table 4.1).
A two way ANOVA was conducted that examined whether our independent variables (CME and conc.) and their interaction (CME*Conc.) have a statistically significant effect on dependent variable (percent scavenging) or not. In sig. column of ANOVA table we see that there is significant effect of independent variable (F 25, 72 = 168.763, p< 0.05) on dependent variable at p= 0.05 level
(Appendix-1).
4.1.2 Cytotoxic Potential of Crude Extracts
Crude methanolic extract of Rosa brunonii, Arisaema flavum, Quercus leucotricophora, Dryopteris ramosa and Bidens biternata were assessed for their cytotoxic potential using Brine Shrimp Lethality test (BSLT). The results were expressed in terms of mean percentage lethality (Table 2) and LD50(Table 3).
There was a significance difference (Appendix-2) in lethality potential of all the crude extracts but the multiple comparisons test “Tuky HSD” has suggested
53
thatTable 4.1: Antioxidant potential (IC50) of crude extracts of selected plants.
Crude extracts 2 Regression equation R IC50 (µg/mL) B. biternanta y= 0.1482x + 8.6888 0.9972 278.76±0.23
D. ramosa y= 0.3167x + 21.919 0.9975 88.67±0.73
Q. leucotricophora y= 0.2926x +22.053 0.9232 95.51±0.19
A. flavum y= 0.1989x + 8.8738 0.9265 206.77±0.13
R. brunonii y= 0.2414x + 18.277 0.9978 131.41±0.18
Ascorbic acid y= 0.3415x + 35.758 0.9252 41.7±0.94
n=3 F (25, 72) = 168.76, p=0.00 (< 0.05)
120 25µg/mL 50µg/mL 100µg/mL 150µg/mL 200µg/mL 250µg/mL 100
80
60
40 Mean Percentage scavenging Percentage Mean
20
0 Ascorbic acid Bidens Dryopteris Quercus Arisaema Rosa brunonii biternata ramosa leucotricophora flavum
Figure 4.1: Dose dependent free radical scavenging potential of crudemethanolic extracts ofselected plants
54
Ascorbic acid Bidens biternata 110 Dryopteris ramosa Quercus leucotricophora Arisaema flavum Rosa brunonii 100 96.87 97.49 92.02 93.78 90 86.04 86.03 80 83.96 77.8 76.3 78.9 70 68.8 73.73 67.13 60 56.41 54.3 53.28 53.44 50 49.01 45.35 40 41.49 37.09 38.07 38.52 38.64
Mean % Scavenging % Mean 32.88 30 31.91 31.14 30.85 23.8823.74 24.68 23.17 20 21.48 17.16 10 11.5 5.9 0 25 50 100 150 200 250 Concentration ( µg/ml) Figure 4.2: Free radical scavenging potential of selected crude extracts
90 10 µg/mL 100 µg/mL 300 µg/mL 600 µg/mL 80
70
60 50
40 =0.00 (< 0.05) (< =0.00
30 p
Percentage lethality Percentage 20
10
0 F (12, 40)= 11.68, 40)= (12, 11.68, F
Crude extracts n=3, Figure 4.3: Mean percentage lethality of crude extracts against brine shrimp
55
Table 4.2a: Mean percentage lethality of crude extracts against Brine Shrimp (BSLT)
Crude . 10 µg/mL 100 µg/mL 300 µg/mL 600 µg/mL extracts
Conc After 24 hours After 24 hours After 24 hours After 24 hours
Mean % Mean % Mean % Mean %
lethality lethality lethality lethality
Total Live Dead % death Live Dead % death Live Dead % Death Live Dead % death D. ramosa 1 20 19 1 5 6.67 18 2 10 11.67 18 2 10 13.33 15 5 25 23.33 2 20 18 2 10 18 2 10 17 3 15 16 4 20
3 20 19 1 5 17 3 15 17 3 15 15 5 25 = 0.00 (< 0.00 = 0.05) Q. 1 20 19 1 5 10 18 2 10 13.33 15 5 25 18.33 15 5 25 35 p leucotricophora 2 20 17 3 15 17 3 15 16 4 20 13 7 35 3 20 18 2 10 17 3 15 16 4 20 11 9 45 A. flavum 1 20 17 3 15 11.6 14 6 30 28.33 10 10 50 51.67 8 12 60 63.33 2 20 18 2 10 15 5 25 10 10 50 7 13 65 3 20 18 2 10 14 6 30 9 11 55 7 13 65
R. brunonii 1 20 14 6 30 30 12 8 40 43.33 8 12 60 58.33 5 15 75 78.33 = 40) (12, 11.68, F
2 20 14 6 30 11 9 45 9 11 55 4 16 80
3 20 14 6 30 11 9 45 8 12 60 4 16 80 n=3, B. biturnata 1 20 15 5 25 26.67 12 8 40 36.67 11 9 45 43.33 8 12 60 58.33 2 20 14 6 30 13 7 35 11 9 45 9 11 55 3 20 15 5 25 13 7 35 12 8 40 8 12 60 Table 4.2b: Mean percentage lethality of Nicotine (standard) against Brine shrimp (BSLT) Concentrations 5µg/mL 10 µg/mL 20 µg/mL 40 µg/mL 80 µg/mL 100 µg/mL (Nicotin) Mean % lethality 11.67% 26.67% 36.67% 45% 66.67% 94.45%
56
there was a non-significance difference in percentage lethality against brine shrimp between CME of A. flavum and B. biternata.p> 0.05 (p=0.456), (Appendix-3).
The lethal dose 50 percent concentration (LD50) was calculated using regression line equation. The LD50 of crude extract of R. brunonii was
220.83±2.16µg/mL which is least and it proved to be most cytotoxic among tested crude extracts (Table 4.3).
4.1.3 Potato Disc Antitumor Assay on Crude Methanol Extracts of Selected
Plants
The crude methanolic extracts of all five selected plants species (Rosa brunonii, Arisaema flavum, Quercus leucotricophora, Dryopteris ramosa and
Bidens biternata) of Galyat region of Pakistan were subjected to evaluate their antitumor potential by potato disc antitumor bioassay. Prior to evaluation of anti- tumor properties of selected CME, the viability of Agrobacterium tumefaciens was assessed against all CME. Cefotaxime a known antibiotic was used as standard.
None of the CME was active against A. tumefaciens (Figure 4.4).
The anti-tumor potential of all the CME were expressed in terms of mean percentage tumor inhibition (Figure 4.5) and in terms of IC50 (Table 4.4). A well- known anti-tumor compound “Vincristine sulphate” was used as standard.
Inhibition concentration (IC50) was calculated by plotting mean percentage tumor inhibition versus various concentrations (µg/mL) and through linear regression equation. The lowest IC50 mean maximum antitumor potential of the extract.
57
Table 4.3: Determination of LD50 for crude extracts in BSLT
2 Crude extracts Regression equation R LD50 (µg/mL)
B. biternanta y= 0.0501x + 28.607 0.9708 427.01± 2.88
D. ramosa y= 0.0261x + 7.1711 0.9509 1640.95±2.88
Q. leucotricophora y= 0.0418x +8.6065 0.9705 990.28±5.19
A. flavum y= 0.0845x + 17.397 0.9056 385.83±2.88
R. brunonii y= 0.0785x + 32.665 0.9796 220.83±2.16
Nicotine(Standard) y = 0.7064x + 19.978 0.9749 50.50±4.81
n=3
Figure 4.4: Viability of A. tumefaciens against crude extracts.
58
10µg/mL 100µg/mL 500µg/mL 1000µg/mL 100 100 72.73 60.63 80 57.54 42.45 48.45 60
40
20 1000µg/mL 0 500µg/mL 100µg/mL
10µg/mL Mean percentage tumor inhibition tumor percentage Mean
Plant's crude extracts and control
Figure 4.5: Mean percentage tumor inhibition of crude extracts of selectedPlants
Figure 4.6:Tumor inhibition of crude extracts of selected plants.Potato discs were stain with Logo‟s solution. Unstained areas were representing the tumor induced by A.tumefaciens.a)Standardb)R. brunoniic)D. ramosad)A. flavume)B. biternataf)Q. leucotricophorag)Control (DMSO).
59
The maximum antitumor potential was shown by crude extract of R. brunonii (IC50 655.65µg.mL) followed by D. ramosa (IC50 790.51µg.mL) and A. flavum (IC50 825.94µg.mL). The lowest antitumor potential was found among crudeextracts of B. biternata(IC50 1010.73µg.mL) and Q. leucotricophora (IC50
1078.53µg.mL). The IC50 of control (vincristine) was 227.50µg/mL (Table 4.4).
4.2 FRACTIONATION AND BIOACTIVITIES OF FRACTIONS
Three crude extracts i.e. D. ramosa, Q. leucotricophora and R. brunonii were selected for fractionation on the basis of antioxidant and cytotoxic potential of crude extracts. The selected crude extracts were subjected to polarity based solvent-solvent fractionation as mentioned in Figure 3.1.As a result each crude extract was fractionated as n-Hexane fraction (nHF), Chloroform fraction (CF), ethyl acetate fraction (EF) and Aqueous fraction (AF). The amount of each fraction was given in table 3.2. Each fraction was assessed for its antioxidant and cytotoxic potential.
4.2.1 Antioxidant Potential of Fractions
The antioxidant potential of fractions obtained from crude extracts of D. ramosa, Q. leucotricophora and R. brunonii were calculated by DPPH free radical scavenging assay. The results were expressed as mean percentage scavenging at various concentrations and in terms of IC50 (Table 4.5).
4.2.2 Cytotoxic Potential of Fractions
The results of BSLT of fractions obtained D. ramosa,Q. leucotricophora and R. brunonii were presented in table 4.6. The highest percentage lethality was
60
shown by ethyl acetate fraction of R. brunonii which was 33.33, 41.67, 58.33 and
91.67percent at 150, 300, 600 and 900µg/mL respectively (Table 4.6a). The LD50 was calculated for each fraction using regression line equation (Table 4.6b). The ethyl acetate fraction of R. brunonii showed maximum cytotoxicity
(LD50405.43z±4.8µg/mL) followed by aqueous fractions of D. ramosa
(LD50830.95±2.0µg/mL) and Q. leucotricophora (LD50 855.11±1.1µg/mL) (Table
4.7). The Ethyl acetate fractions of R. brunonii exhibited high cytotoxicity which indicated that cytotoxic substances were present in R. brunonii might be accumulated in ethyl acetate soluble fraction.
4.2.3 Antitumor Potential of Fractions Obtained From CME of D. ramosa and
R. brunonii
Antitumor potential of various fractions obtained from crude extracts of D. ramosaand R. brunonii were evaluated by using standard potato disc antitumor assay. The mean percentage tumor inhibition and IC50 value were calculated. The maximum tumor inhibition was showed by ethyl acetate fraction of R. brunonii(0,
20, 37.8 and 62.2 percents at 10, 100, 500 and 1000µg/mL concentration). The aqueous and ethyl acetate fractions of D. ramosa also showed significant tumor inhibition (Table 4.8). The control (vincristine) showed 2.2, 13.13 and100 percents tumor inhibition at 10, 100 and 500µg/mL respectively (Figure 4.6).
4.3 PRELIMINARY PHYTOCHEMICAL ANALYSIS
Crude extracts of all five plants and fractions of three plants were subjected to qualitative chemical tests to establish their chemical composition (Table 4.9).
These tests include; tests for alkaloids, sterols, Phenolics, flavonoids, saponins and
61
Table 4.4: Antitumor potential of crude extracts of selected plants
2 Crude extracts Regression equation R IC50 (µg/mL)
B. biternanta y = 0.0486x + 0.8787 0.9949 1010.73±1.07
D. ramosa y = 0.0605x + 2.1745 0.9876 790.51±1.16
Q. leucotricophora y = 0.0437x + 2.8683 0.9185 1078.53±0.74
A. flavum y = 0.059x + 1.2693 0.98 825.94±0.55
R. brunonii y = 0.0718x + 2.924 0.9892 655.65±1.45
Vincristine (Standard) y = 0.1884x + 7.1386 0.9812 227.5±1.76 n=3,
Table 4.5: Mean percentage scavenging and IC50 of fractions obtained fromcrude extracts of D. ramosa, Q. leucotricophora and R.brunonii.
2 Plants Fraction Conc. Mean % Regression equation R IC50 (µg/mL) scavenging (µg/mL) nHF 25 26.55±0.47 50 33.33±0.42
100 33.21±0.24 y=0.1356x ± 23.164
150 44.66±0.93 ± 0.26 ±
200 45.04±0.23
250 61.32±0.25
0.9081 198.81
CF 25 17.3±0.23
D. ramosa 50 19.97±0.16
100 21.63±0.27 y=0.0747x ± 15.358
150 26.08±0.69 ± 0.30 ± 200 31.04±0.07
250 34.08±0.81
0.9745 468.25
62
2 Plants Fraction Conc. Mean % Regression equation R IC50 (µg/mL) scavenging (µg/mL) EF 25 36.66±0.27 50 44.14±0.39 100 69.97±0.57 y= 0.2569x ± 35.199
150 76.72±0.71 ± 0.24 ± 200 91.35±0.45
250 91.48±0.02
0.9204 57.85 AF 25 26.21±0.65 50 36.77±0.32
100 58.69±0.75 y=0.2068x ± 27.557
150 59.28±0.73 ± 0.28 ± 200 69.97±0.16
250 74.68±0.61
0.9025 108.98 nHF 25 21.5±0.59 50 23.35±0.37
100 25.44±0.81 y= 0.0334x ± 21.425
150 26.55±0.39 ± 0.66 ± 200 28.24±0.61
250 29.35±0.19
0.9700 866.06 CF 25 21.51±0.77
50 24.17±0.29
100 26.46±0.21 y=0.1763x ± 14.899 ±0.5 150 44.4±0.37
200 50.51±0.67
0.9254 199.49 EF 25 17.68±0.38
Q. leucotricophora 50 22.64±0.02
100 33.08±0.17 y= 0.1507x ± 15.14
150 36.9±0.41 ± 0.33 ± 200 44.02±0.29
250 53.31±0.19
0.9860 232.40
AF 25 18.32±0.15 ±0. 50 19.08±0.13 y=0.0823x ± 16.326
100 26.97±0.50
0.9670 410.73 74
63
2 Plants Fraction Conc. Mean % Regression equation R IC50 (µg/mL) scavenging (µg/mL) 150 27.74±0.52 200 33.08±0.76 250 36.51±0.53 nHF 25 4.34±0.34 50 9.88±0.31
100 18.89±0.57 y= 0.1970x ± 5373
150 29.12±0.11 ± 0.31 ± 200 37.41±0.48
250 49.81±0.21
0.9975 251.08 CF 25 9.11±0.82 50 9.88±0.81
100 21.1±0.87 y= 0.220x ± 0.6153
150 32.23±0.89
± 0.81 ±
200 45.38±0.80
250 56.48±0.80
0.9906 224.48
EF 25 5.96±0.79 R. brunonii
50 12.11±1.21
100 25.11±0.98 y=0.2771x ± 1.6062 ±0.89 150 40.12±0.91
200 54.14±0.99
0.9990 186.24 AF 25 13.65±0.19 50 22.43±0.78
100 31.19±0.48 y=0.3526x ± 2.2909
150 51.66±0.64 ± 0.57 ± 200 77.45±0.39
250 90.63±0.71
0.9819 135.31 25 35.62±0.02
50 54.7 ± 0.12
100 80.41±0.11 y=0.3463x ± 35.616
150 92.24±0.10 0.10 ±
200 96.94±0.23
Ascorbic acid Ascorbic (Standard) 0.9023 41.59 nHF= n-Hexane fraction, CF= Chloroform fraction, EF= Ethyl acetate fraction, AF= Aqueous fraction, n=3.
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Table 4.6: Mean percentage death (lethality) of different fractions of selected plants against brine shrimps. .
Conc. 150µg/mL 300µg/mL 600µg/mL 900µg/mL
After 24 hrs. After 24 hrs. After 24 hrs. After 24 hrs.
(Larvae/ (Larvae/
Plants #
death
Fractions Total vial) Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % Mean death %
1 15 14 1 6.57 14 1 6.67 11 4 26.67 10 5 33.33
2 15 14 1 6.67 14 1 6.67 11 4 26.67 9 6 40
3 15 14 1 6.67 13 2 13.33 11 4 26.67 11 4 26.67
nHF 6.67 8.89 26.67 33.33
1 15 14 1 6.67 11 4 26.67 10 5 33.33 9 6 40
2 15 13 2 13.33 13 2 13.33 9 6 40 8 7 46.67
3 15 15 0 0 12 3 20 9 6 40 7 8 53.33
CF 6.67 20 37.78 46.67
1 15 14 1 6.67 12 3 20 11 4 26.67 10 5 33.33
D. ramosa
2 15 14 1 6.67 13 2 13.33 10 5 33.33 9 6 40
3 15 13 2 13.33 12 3 20 10 5 33.33 9 6 40
EF 8.89 17.78 31.11 37.78
1 16 13 3 18.75 10 5 33.33 9 6 40 8 7 46.67
2 15 12 3 20 10 5 33.33 8 7 46.67 7 8 53.33
3 15 12 3 20 10 5 33.33 9 6 40 7 8 53.33
AF 19.58 33.33 42.22 51.11
1 16 16 0 0 14 2 12.5 13 3 18.75 11 5 31.25
2 16 15 1 6.67 14 2 12.5 12 4 25 11 5 31.25
Q. Q.
phora 3 15 15 0 0 14 1 6.67 11 4 26.67 9 6 40
leucotrico
nHF 2.22 10.56 23.47 34.17
65
Conc. 150µg/mL 300µg/mL 600µg/mL 900µg/mL
After 24 hrs. After 24 hrs. After 24 hrs. After 24 hrs.
(Larvae/ (Larvae/
Plants #
death
Fractions Total vial) Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % Mean death % 1 15 14 1 6.6 13 2 13.33 11 4 26.67 9 6 40
2 15 14 1 6.67 13 2 13.33 11 4 26.67 9 9 40
3 15 15 0 0 12 3 20 11 4 26.67 8 7 46.67
CF 4.45 15.55 26.67 42.22
1 15 14 1 6.67 13 2 13.33 10 5 33.33 9 6 40
2 15 14 1 6.67 13 2 13.33 10 5 33.33 9 6 40
3 15 14 1 6.67 12 3 20 10 5 33.33 9 6 40
EF 6.67 15.55 33.33 40 1 16 13 3 18.75 12 4 25 10 6 37.5 8 8 50 15. 36.9 2 15 13 2 13.33 11 4 26.67 26.11 10 5 33.33 7 8 53.33 52.22
14 4
3 15 13 2 13.33 11 4 26.67 9 6 40 7 8 53.33 AF
1 15 14 1 6.67 11 4 26.67 11 4 26.67 10 5 33.33
2 15 14 1 6.67 13 2 13.33 11 4 26.67 9 6 40
3 15 13 2 13.33 12 3 20 11 4 26.67 9 6 40
nHF 8.89 20 26.67 37.78
1 15 14 1 6.67 12 3 20 11 4 26.67 10 5 33.33
R. brunonii
2 15 13 2 13.33 11 4 26.67 10 5 33.33 8 7 46.67
3 15 15 0 0 11 4 26.67 8 7 46.67 7 8 53.33
CF 6.67 24.45 35.56 44.44
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Conc. 150µg/mL 300µg/mL 600µg/mL 900µg/mL
After 24 hrs. After 24 hrs. After 24 hrs. After 24 hrs.
(Larvae/ (Larvae/
Plants #
death
Fractions Total vial) Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % death Mean death % Live Dead % Mean death %
91.67
1 12 8 4 33.33 7 5 41.67 5 7 58.33 1 11 33. 58.3 41.67 33 3
2 12 8 4 33.33 6 6 50 6 6 50 1 11 91.67
3 12 8 4 33.33 8 4 33.33 4 8 66.67 1 11 91.67
EF 91.67
1 12 11 1 8.33 10 2 16.67 10 2 16.67 9 3 25
2 12 10 2 16.67 9 3 25 9 3 25 8 4 33.33
3 12 11 1 8.33 9 3 25 8 4 33.33 6 6 50
AF 11.11 22.22 25 36.11 nHF= n-Hexane fraction, CF= Chloroform fraction, EF= Ethyl acetate fraction, AF= Aqueous fraction
67
Table.4.7: Calculation of LD50 of fractions of selected plants
2 Fractions Regression equation R LD50 (µg/mL)
nHF y= 0.0387x + 0.0422 0.957 1290.90±3.2
CF y= 0.0527x + 2.0731 0.9583 909.43±1.4 D. ramosa EF y= 0.0384x + 5.1612 0.9654 1167.68±0.0
AF y= 0.0391x + 17.501 0.9351 830.95±2.0
nHF y= 0.0422x - 2.9692 0.9933 1255.19±0.7
Q. CF y= 0.0482x - 1.2783 0.9887 1063.86±1.9
leucotricophora EF y= 0.0454x + 1.7319 0.9591 1063.17±1.9
AF y= 0.0473x + 9.5534 0.9882 855.11±1.1
nHF y= 0.0357x + 5.9514 0.96 1233.85±2.7
CF y= 0.0467x + 5.0168 0.9104 963.24±3.1 R. brunonii EF y= 0.0763x + 19.066 0.967 405.43±4.8
AF y= 0.0295x + 9.2263 0.9132 1382.16±3.8
Nicotine ( standard) y = 0.7064x + 19.978 0.9749 42.50±4.81
n=3, nHF= n-Hexane fraction, CF= Chloroform fraction, EF= Ethyl acetate fraction, AF= Aqueous fraction
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10µg/mL 100µg/mL 500µg/mL 1000µg/mL
100 90 80 70 60 50 40 30 20 10 0 1000µg/mL Mean percentage tumor inhibition percentage Mean 500µg/mL 100µg/mL 10µg/mL
Plants samples
Figure 4.7: Mean percentage tumor inhibitions by fraction of D. ramosa and R.
brunonii atvarious concentrations.
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Table 4.8: Mean percentage tumor inhibition and IC50 of fractions obtained from CME of D. ramosa and R. brunonii. Fractions Mean Percent tumor inhibition at concentrations Regression 2 (µg/mL) equation R IC50 (µg/mL) 10 100 500 1000
D. ramosa y = 0.0342x 0 13 24.47 37.8 0.9225 1311.97±0.48 n-HF + 5.1305
D. ramosa y = 0.0374x 0 11.13 24.47 40 0.9601 1234.53±0.50 CF + 3.8285
D. ramosa y = 0.0514x 0 17.8 37.8 55.53 0.9296 834.99±0.24 EF + 7.0812
D. ramosa y = 0.054x 0 17.8 40 57.8 0.9311 793.23±0.31 AF + 7.1657
R. brunonii y = 0.045x 0 20 31.13 51.13 0.9048 945.38±0.67 nHF + 7.4578
R. brunonii 0.0364x + 0 13.33 24.47 40 0.9372 1241.31±0.57 CF 4.8161
R. brunonii y = 0.0569x 0 20 37.8 62.2 0.9432 753.68±0.48 EF + 7.1155
R. brunonii y = 0.0461x 0 15.53 24.47 51.13 0.9454 993.21±0.47 AF + 4.2131
Vincristine y = 0.1913x 2.2 13.13 100 100 0.986 232.34±0.58 (Standard) + 5.5531
n=3 nHF= n-Hexan Fraction, CF= Chloroform Fraction, EF= Ethyl acetate Fraction and AF= Aqueous Fraction.
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tannins. In case of crude extract of D. ramosa, the results indicated the presence of
Phenolics, flavonoids and sterols while alkaloids, saponins and tannins were absent. The crude extracts of Q. leucotricophora and R. brunonii showed positive results for all the tests and both of these extracts indicated excellent results for phenolics and flavonoids. The crude extracts of A. flavum and B. biternata showed excellent positive results for alkaloids but results indicated that A. flavum extract is devoid of tannin while flavonoids are absent in case of B. biternata (Table 4.9).
The fractions obtained after solvent-solvent fractionation of CME of D. ramosa, Q. leucotricophora and R. brunonii were also subjected to determine the classes of plants secondary metabolites present in each fraction. The results were presented in the Table 4.10.
In case of D. ramosa, the n- hexan fraction showed negative test results for all phytochemicals except sterols. Simillar result was shown by chloroform fraction. The ethyl acetate fraction showed positive results for sterols, phenolics and flavonoids while negative results of saponins, tannins and alkaloids. The aqueous fraction showed presence of phenolics and flavonoids (Table 4.10).
In case of fractions obtained from Q. leucotrichophora, the n-Hexane fraction showed positive results for sterols, saponins and tannins. The chloroform fraction showed negative results for phenolics and flavonoids and positive for all other phytochemical tests. The ethyl acetate fraction showed positive tests for all phytochemicals while aqueous fraction showed positive results for phenolics, flavonoids and tannins only (Table 4.10).
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In case of R. brunonii, alkaloids were absent in n-hexane and aqueous fractions. The ethyl acetate and chloroform fraction showed positive results for alkaloids. Sterols were found in all fractions except aqueous fraction. The phenolics and flavonoids were shown positive results in ethyl acetate and aqueous fraction. Tannins were found only in aqueous fraction of R. brunonii (Table 4.10).
4.3.1 Determination of Total Phenolic and Total Flavonoid Contents
Three separate samples of Gallic acid were used with different concentrations and the mean value was used to calibrate standard Gallic acid calibration curve. The total phenolic contents of crude extracts and fractions were calculated by a linear regression equation (y= 0.0071x + 0.4332, R2= 0.9606) primed with a Gallic acid standard calibration curve (Figure 4.7), and expressed in terms of Gallic acid equivalent (GAE). Crude methanolic extract of D. ramosa had the highest amount of phenolic constituents122.64 ± 4.35µg/mg GAE followed by crude extract of Q. leucotricophora (74.06 ± 7.74µg/mg GAE) and R. brunonii
(66.73 ± 3.89µg/mg GAE). The crude extracts of A. flavum showed 19.3±6.29
µg/mg GAE, total phenolic contents which were least among all the crude extract tested (table 4.11).
The total flavonoid contents of crude extracts of Dryopteris ramosa,
Quercus leucotricophora, Arisaema flavum, Rosa brunonii and Bidens biternata were determined by standard method and using Querctin standard calibration curve
(Figure 4.8) and linear regression equation (y=0.0063x + 0.395, R2 = 0.9697) obtained from Quercetin standard calibration curve. The crude extract of D. ramosa showed highest (61.42 ± 17.89 µg/mg QE) flavonoid contents flowed by
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Q. leucotricophora (53.60 ± 1.43 µg/mg QE), R. brunonii (46.51 ± 0.96
µg/mgQE), B. biternata (38.73 ± 11.00 µg/mg QE) and leastflavonoid contents(4.55 ± 1.20 µg/mg QE) were found in crude extract of A. flavum (Table
4.12). In case of D. ramosa and B. biternata, the absorbance at 415nm was not significantly different among three samples of each crude extract with standard deviation 0.11 and 0.01 respectively. Figure 4.10 dipicted the correlation between bioactivities of CME of sleected plants and their TPC and TFC.
4.4 ISOLATION AND STRUCTURE ELUCIDATION OF COMPOUNDS
On the basis of antioxidant activities and cytotoxic potential of various fractions of crude extracts, following three fractions were selected for isolation of pure compounds.
i) Aqueous fraction of D. ramosa, ii) Ethyl acetate fraction of D. ramosa and iii) Ethyl acetate fraction of R. brunonii.
The fractions were subjected to column chromatography. The elutions were analyzed by TLC and HPLC. Finally the structure elucidation was done with the help of spectroscopic experiments like MS, 1HNMR, 13CNMR, COSY, gs-HSQC and gs-HMBC.
4.4.1 Isolation of Pure Compound fromAqueous Fraction of D. ramosa
Dryopteris ramosa aqueous fraction (2g) was subjected to column chromatography (Sephadex LH20, 75x3cm). HPLC grade methanol was used as
73
Table 4.9: Preliminary phytochemical analysis of crude extracts
Chemical test D. Q. R. A. B.
ramosa leucotricophora brunonii flavum biternata
Dragendorff‟s test - ++ + +++ +++
Mayer‟s test - ++ + +++ +++
Tests for sterols +++ +++ ++ + +
Tests for Phenolics +++ +++ +++ + +
Tests for Flavonoids +++ ++ ++ + -
Tests for saponins - ++ + +++ +
Test for Tannins - +++ ++ - ++
- Absent, + present + Good, ++ very good, +++ excellent
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Table 4.10: Qualitative phytochemical analysis of fractions obtained from CME of D. ramosa, Q. leucotricophora and R. brunonii.
Preliminary phytochemical tests Plants Fractions Dragendorff‟s test Mayer‟s test Sterols Phenolics Flavonoids Saponins Tannins n-Hexan fraction - - ++ - - - -
Chloroform fraction - - ++ - - - -
Ethyl acetate fraction - - + +++ ++ - - D. ramosa D. Aqueous fraction - - - ++ ++ - -
n-Hexan fraction - - + - - + +
Chloroform fraction + + ++ - - + +
Ethyl acetate fraction + + + ++ + + ++
Aqueous fraction - - - ++ ++ - + Q. leucotricophora Q.
n-Hexan fraction - - + - - - -
Chloroform fraction + + + - - + -
Ethyl acetate fraction + + + ++ ++ + - R. brunonii R. Aqueous fraction - - - ++ + - +
- Absent, + present + Good, ++ very good, +++ excellent
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2.5
2
1.5
y = 0.007x + 0.433 1 R² = 0.960
Absorbance 760nm at mean Absorbance 0.5
0 0 50 100 150 200 250 300 Concentrations µg/mL Fig. 4.8: Gallic acid standard callibration curve
Table 4.11: Total phenolic contents of crude extracts of selected plants
Absorbance at 760nm Total phenolic Sample Sample Sample Mean Crude extracts contents 1 2 3 Absorbance µg/mg GAE (Samples)
Dryopteris ramosa 1.268 1.326 1.281 1.292±0.03 122.64 ± 4.35
Bidens biternata 0.927 0.789 0.798 0.838±0.08 57.83 ± 11.03
Rosa brunonii 0.879 0.891 0.931 0.900±0.03 66.73 ± 3.89
Quercus leucotricophora 0.896 0.984 0.997 0.959±0.05 74.06 ± 7.74
Arisaema flavum 0.517 0.59 0.598 0.568±0.04 19.03 ± 6.29
n=3, GAE=Gallic acid equivalent
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2.5
2
1.5
y = 0.006x + 0.395 1 R² = 0.969
0.5 Absorbance nm 415 at mean Absorbance
0 0 50 100 150 200 250 300
Concentrations µg/mLl
Fig. 4.9: Quercetin standard callibration curve
Table 4.12: Totalflavonoid contents of crude extracts of selected plants
Absorbance at 415nm Total flavonoid Sample Sample Sample Mean Crude extracts contents 1 2 3 Absorbance µg/mg QE (Samples)
Dryopteris ramosa 0.666 0.789 0.891 0.782±0.11 61.42 ± 1.89
Bidens biternata 0.559 0.681 0.677 0.639±0.01 38.73 ± 1.00
Rosa brunonii 0.681 0.692 0.691 0.688±0.01 46.51 ± 0.96
Quercus leucotricophora 0.732 0.742 0.724 0.733±0.07 53.60 ± 1.43
Arisaema flavum 0.427 0.415 0.429 0.424±0.01 4.55 ± 1.20
n=3, QE= Quercetin equivalent
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1800 Antioxidant Potential (IC50)µg/mL
Cytotoxic potential (LD50) µg/mL 1600 Anti-tumor inhibition (IC50)µg/mL
1400 TPC (µg/mg GAE) TFC (µg/mg QE)
1200
1000 Values in µg in Values
800
600
400
200
0
Crude methanolic extracts
Figure 4.10: Correlation between antioxidant, cytotoxic and anti-tumor potential of
CME of selected plants and total phenolic(TPC) and total flavonoid
contents(TFC).
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mobile phase. A total 20 elution were collected in conical flasks each containing a volume of 50 mL. All the elution were dried under reduced pressure in rotaryapparatus and each of them is re-dissolved in methanol(2-5mL) and subjected to thin layer chromatography (TLC). The solvent system used was n-
Butanol: acetic acid: water (4:1:5, v/v/v). Fractions with similar TLC pattern were combined and as a result 4 groups were formed. i.e. Group-1 (Fractions 1-3), group-2 (Fractions 4-5), group-3 (Fractions 6-8), group-4 (Fractions 9-20). These four groups were subjected to High pressure liquid chromatography (HPLC). The groups 2 and 3 represented one and two major peaks along with other peaks
(impurities) in HPLCchromatogram. Both of these groups were rotary dried separately and weigh 139mg and 153mg respectively.
4.4.1.1Column chromatography of group 2
Group 2 was subjected to column chromatography (Sephadex LH20,
120x2cm) with HPLC grade methanol as mobile phase. A total 18 fractions were collected and each eluent was cut at 10mL volume. All the eluents were subjected to TLC and visualized under UV at 254 and 366nm. The solvent system n-Butanol: acetic acid: water (4:1:5, v/v/v) was used. On the basis of TLC pattern so obtained, the fractions were combined into two groups i.e. 2a (fractions 1-11) and 2b
(fractions 12-18). The HPLC profile showed that group 2a contain major peak (UV absorption spectra ʎmax.298nm and retention time 6.467 min.) along with other small peaks. The sub group 2a was dried and weighs 83.71mg.
Purification of sub-group 2a: The sub fraction 2a was subjected for purification through medium pressure liquid chromatography (MPLC) with the
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Table 4.13: Solvent mixture used as mobile phase in MPLC and number of
fractions obtained during purification of sub group 2a (D. ramosa
aqueous fraction)
Solvent mixture Fractioncollected Solvent mixture Fractioncollected
Water: Methanol Water:Methanol 1-5 36-40 (100:0) (80:20)
Water:Methanol Water:Methanol 6-10 41-45 (75:25) (97.5:2.5)
Water: Methanol Water:Methanol 11-15 46-50 (95:5) (70:30)
Water:Methanol Water:Methanol 16-20 51-55 (92.5:7.5) (70:30)
Water: Methanol Water:Methanol 21-25 56-60 (90:10) (0:100)
Water:Methanol Water:Methanol 26-30 61-65 (87.5:12.5) (0:100)
Water: Methanol 31-35 (85:15)
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solvent mixtures as indicated in table 4.13. A total 65 fractions were collected each fraction was cut at 20mL. All the fractions obtained from MPLC were dried and dissolved in methanol and then subjected to TLC. The fractions 29-42 showed similar pattern and single spot when observed under UV at 254nm. They were combined and subjected to HPLC analysis. HPLC chromatogram showed purity of the isolated compound (weighs 39.15mg) and finally spectroscopic data was taken.
It was a yellow coloured powder which has melting point 272 to 278oC. It was label as DAF-MI-01.
4.4.1.2 Isolation of compounds from group 3
The group 3 obtained from Sephadex column chromatography of aqueous phase of D. ramosa was subjected for purification to isolate compound by using
MPLC and PTLC.
4.4.1.2.1 Mediumpressure liquid chromatography (MPLC) of group 3
The group 3 obtained from Sephadex colomn chromatography of aqueous phase of D. ramosa was subjected (100mg) to reverse phase column chromatography by using MPLC system. Water: methanol (100:0, 97.5:2.5, 95:5,
92.5:7.5, 90:10. 87.5:12.5, 85:15, 80:20, 75:25, 70:30, 60:40, 40:60, 0:100 mL)solvent mixtures (100mL of each) were used and eluents were cut at 50mL. A total 26 eluent were collected, rotary dried and re-dissolved in HPLC grade methanol (2-3mL). Then TLC plates was developed with n-Butanol: acetic acid: water (4:1:5, v/v/v) solvent system. After drying, TLC plates were visualized under
UV lamp at 254 nm. The eluents (7-10) which showed similar pattern on TLC plates were combined and it was assigned as DAF-MI-02 and it weighs 63.72mg.
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Then the combined eluents (7-10 i.e. DAF-MI-02) was subjected for HPLC analysis. The HPLC chromatogram showed two peaks at Rt 8.520 and 8.729 minutes (Fig 4.11).The absorption spectrum of these two peaks also varies little at certain points indicating two compounds that might be the isomers of each other.
4.4.1.2.2 Preparative thin layer chromatography (PTLC) of DAF-MI-02
The sub-fraction DAF-MI-02 was subjected for purification through preparative TLC using n-Butanol: Acetic acid: water (4:1:5, v/v/v) solvent system.
Silica coated PTLC plats (20x20cm) was used. A line was drawn 2.5cm above from bottom and sample was loaded on entire length of the line by TLC spotter.
This process was repeated 3-4 times. Plate was left for some time to evaporate solvent. Then plate was observed under UV at 254 to confirm the complete and uniform adsorption of sample. About 100-150mL mobile phase was added in TLC chamber and covered with lid for 30 minutes. Then TLC plate was placed in TLC chamber and covered with lid. When mobile phase moved up to the top of the
PTLC plate then it was removed from the TLC chamber. Then PTLC plate was dried and visualized under UV light at 254nm.Two clear bands were seen under
UV-254nm. The lower band was designated as DAF-MI-02.1 while upper band was named as DAF-MI-02.2. Both bands were scratched from PTLC plates separately and washed with methanol three times and then filter and dried under reduced pressure. They weigh 23.5mg and 19.8mg respectively. Approximately 8 mg of each were sent for NMR and Mass spectrum analysis.
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Figure 4.11:HPLC profile and UV-vis apex absorption spectra of DAF-MI-02
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4.4.2Structural Elucidation of Isolated CompoundFrom Aqueous Fraction of
D. Ramosa
The chemical structure of three compounds (Labelled as DAF-MI-01,
DAF-MI-02.1 and DAF-MI-02.2) were determined by their physicalcharacteristics, chemical detection reagents and spectroscopic techniques.Summary of compound isolated from aqueous fraction of D. ramosa was presented in figure 4.40.
4.4.2.1 Identification andStructural elucidation of isolated compound DAF-
MI-01
4.4.2.1.1Physical characteristics of DAF-MI-01
Isolated compound (DAF-MI-01) was odourless, yellow coloured powder and was soluble in water and methanol. The melting point was observed 272-
2780C.
4.4.2.1.2 Detection reagents
TLC plates were developed with n-Butanol: acetic acid: water (4:1:5, v/v/v) solvent system. The TLC plates were dried and observed under UV at 230nm and
360nm. They showed a single spot with Rf 0.39. One of the developed TLC plates was sprayed with Dragendorff‟s reagent but there was no black or blue colouration indicating the absence of alkaloid nature of the molecule. Another TLC plates was sprayed with anisaldehyde and upon heating bright yellow spot appeared which indicated that isolated compound might belong to flavonoid class of plant secondary metabolite. Additionally, a third developed TLC plate was sprayed with one percent ethanolic solution of aluminum chloride (AlCl3). When observed under
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UV at 360nm it showed yellow inflorescence. This might be the additional evidence that the compound may be related to flavonoid class of plants secondary metabolites.
4.4.2.1.3UV-Vis Spectroscopy of DAF-MI-01
The HPLC chromatogram showed a peak at 6.467minutes (Figure 4.12a) and the UV-VIS absorption spectra (DAD at 230 nm) of isolated compound (DAF-
MI-01) exhibited two bands with ʎmax at 298 and 210nm for band I and II respectively (Figure 4.12b). This suggested that compound might have anaromatic ring system.
4.4.2.1.4 Mass spectroscopyod DAF-MI-01
ESI- HR-TOF mass spectra of isolated compound (DAF-MI-01) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 409m/z suggested the molecular weight of the isolated compound might be 408m/z (M-H).
4.4.2.1.5 Fourier Transfer- Infra-red (FTIR) spectroscopy of DAF-MI-01
The FTIR of isolated compound DAF-MI-01 was performed on iD7 ATR
Nicolet™ iS5 Spectrometer and isolated compound was scanned from 400-
4000cm-1 wavenumber (figure 4.14). It indicated the stretching of phenol-OH
(3675.57cm-1) and the stretching at wave number 1653.82 cm-1 indicated the presence of carbonyl group in the molecule.
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4.4.2.1.6 NMR spectroscopy of DAF-MI-01
1HNMR: The proton NMR (Figure 4.15a, 4.15b) of isolated compound was performed in Deuterated methanol. 1HNMR indicated the presence of 20 protons in the structure of DAF-MI-01 (Figure 4.15). The 1HNMR chemical shift value of 6.75, 6.78, 7.59, 7.61 ppm indicated the existence of aromatic ring system
(δ >6.5ppm) in the molecule (Fergoug and Bouhadda, 2014). The chemical shift towards low ppm suggested that molecule might have sugar moiety in the structure. The proton shift value and multiplicity is given in the table 4.14.
13CNMR: Deuterated methanol was used as solvent. The 13CNMR showed that isolated compound has 19 carbons and the shift value at 198.89ppm is the indication of carbonyl (C=O) group in the structure (Table 4.14). The Distortions enhancement by polarization transfer (DEPT) differentiate between the carbon atoms with CH2 or unsaturation or quaternary carbons (shown down wards) and
CH and CH3 carbons (shown upward directions) (Figure 4.16).
HSQC: The heteronuclear single quantum coherence or heteronuclear single quantum correlation (HSQC) spectroscopic experiment revealed the number of proton attached to a particular carbon. HSQC of isolated compound DAF-MI-01 is given in Figure 4.13. The HSQC of isolated compound DAF-MI-01 showed that
C-5 (δ= 104.668ppm) and C-8 (δ= 133.264ppm) are not directly bonded to hydrogen (proton). Carbon C-1/(δ= 76.61ppm), C-2/(δ= 73.34ppm) ,C-3/ (δ=
79.93ppm),C-4/ (δ= 71.46ppm) and C-5/ (δ= 82.55ppm) has one proton each while
C-6/ (δ= 62.48ppm) is directly bonded with two protons (Figure 4.17).
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Correlation spectroscopy/COSY (2-D NMR): This experiment determined the correlation between coupled protons. A point of entry into the COSY spectrum is one of the key to predict information from it accurately. Relation between coupling proton is determined by diagonal lines (correlation peaks and COSY spectrum) The H-COSY spectrum of isolated compound DAF-MI-01 suggested that H-1/(δ 4.88 ppm, d, j= 9.88)is coupled with proton of H-2/(δ 3.89, m).
Similarly H-13 (δ 7.61, s)was coupled with H-12(δ 6.78, s) and H-10 (δ 6.75, s)with H-9 (δ 7.59, s)(Figure4.18).
HMBC:HMBC is a two dimensional heteronuclear multiple bond correlation experiment developed to assist in the identification (correlation) of proton nuclei with carbon nuclei that are separated by more than one bond. The
HMBC plot of DAF-MI-01 was given in Figure 4.19.Three bonds coupling were observed between H-3 (δ5.94ppm) to C-1(δ107.17ppm) and C-
5(δ104.67ppm).Similarly 3-j-CH correlation was noticed between H-9 (δ 7.59ppm) to C-7 (δ 198.89ppm) and C-11 (δ 162.90ppm) and H-10 (δ 6.75ppm) to C-12 (δ
115.50ppm) & C-8 (δ 133.26ppm). Abundant 3-j-CH long range correlation signals were observed with cross peak H-1/ (δ 4.88ppm) to C-3/ (δ 79.93ppm), C-5/
(δ 82.55ppm), C-6 (δ 163.42ppm) and C-4 (δ 161.44ppm).
4.4.2.1.7 Structure and nomenclature of isolated compound DAF-MI-01
On the basis of evidences obtained from melting points of the isolated compound, chemical detection reagents spraying on TLC plates and UV-Vis absorption spectrum, we suggested that the molecule belong to the flavonoid class.
The mass spectrum analysis proposed molecular weight 408 m/z and on the basis
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1 13 of H-NMR and C-NMR, the molecular formula is C19H20O10. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in figure. 4.21. The HSQC and COSY experiments confirm the presence of glucose sugar moiety which is substituted at C-5 (δ 104.67ppm) as confirmed by HMBC.
On the basis of these evidences, we proposed that the isolated compound (DAF-
MI-01) from aqueous phase of D. ramosa was iriflophenone-3-C-β- D glucopyranoside and its chemical structure was given as below (Figure 4.20). This compound has four hydroxyl substituents distributed over two benzene rings that are linked through a carbonyl group. The IUPAC name of this compound is (2R,
3S, 4R, 6S)-2-(hydroxymethyl)-6-[2, 4,6-trihydroxy-3(4-hydroxybenzoyl) phenyl] oxane-3,4,5- Triol.
4.4.2.2 Identification and structural elucidation of isolated compound DAF-
MI-02.1
4.4.2.2.1 Physical characteristics of DAF-MI-02.1
The isolated compound (DAF-MI-02.1) was an odourless, orange yellowish colour powder with melting point 271oC. It was soluble in water and methanol easily.
4.4.2.2.2 Detection reagents
Three TLC plates were developed with Butanol: Ethyl acetate: Water
(5:1:4) v/v/v solvent system for DAF-MI-02.1. The Rf value was 0.49. First TLC plate was sprayed with dragendorff‟s reagent but there was no change in colour.
This mean the isolated compound DAF-MI-02.1 might not contain nitrogen in the
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c
b
a
Figure 4.12: HPLC profile of DAF-MI-01 obtained from aqueous fraction of D.
ramosa. a) Chromatogram, b) UV-Vis DAD Absorption spectra at
230nm and c) Purity of peak at 6.467 minutes.
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Figure 4.13: MS of isolated compound DAF-MI-01 showing molecular ion at M+
409m/z.
Figure 4.14: FT-IR spectroscopy of DAF-MI-01, isolated from aqueous fraction of D. ramosa
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Table 4.14: NMR (1H &13C) chemical shift values and DEPT analysis of isolated compound DAF-MI-01. Position 1H a 13C DEPT b (Carbon) δ(ppm) δ(ppm) 1 - 107.17 q C
2 - 161.07 q C
3 5.94 (1H, s) 94.61 CH
4 - 161.44 q C
5 - 104.67 q C
6 - 163.42 q C
7 - 198.89 q C
8 - 133.26 q C
9 7.59 (1H, s) 132.82 CH
10 6.75 (1H, s) 115.50 CH
11 - 162.90 q C
12 6.78 (1H,s) 115.50 CH
13 7.61 (1H, s) 132.82 CH
1/ 4.88 (1H, d, j= 9.88) 76.61 CH
2/ 3.89 (1H, m) 73.74 CH
3/ 3.44 (1H, m) 79.93 CH
4/ 3.48 (1H, m) 71.46 CH
5/ 3.35 (1H, m) 82.55 CH
/ 6 3.84 (2H, d, j=1.96) 62.48 CH2 aValues in parentheses are coupling constant (j) in Hz and Multiplicity.bq C= Quaternary carbon/ a carbon which is not directly bounded to „H‟ (determine by DEPT spectrum 135o).
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Figure 4.15a:1HNMR plot of isolated compound DAF-MI-01(from aqueous
fraction of D. ramosa.
Figure 4.15b:1HNMR plot of DAF-MI-01 showing integration values and frequencies in
joule mode.
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Figure 4.16:13CNMR (DEPT) analysis of DAF-MI-01 showing chemical shift in
ppm.
Figure 4.17:gs-HSQC spectra obtained from DAF-MI-01.
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Figure 4.18:gs-COSY spectra obtained from DAF-MI-01.
Figure 4.19:gs-HMBC spectra obtained from DAF-MI-01.
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Figure 4.20: Chemical structure of isolated compound DAF-MI-01 (Iriflophenone-
3-C-β-D glucopyranoside.
H+ OH H+ OH HO OH HO
HO O O HO OH OH O m/z = 392 O OH OH C19H19O9 OH
OH HO m/z = 409 OH M+H HO
C H O C6H11O5 7 5
H+ OH HO H+ HO OH OH O
HO OH OH m/z = 246 HO m/z = 288 C13H9O5 OH OH O C12H15O8
C6H11O5 C7H5O2 H+
HO OH
m/z = 126 C6H5O3 OH Figure4.21: Proposed fragmentation scheme of Iriflophenone-3-C-β-D glucopyranoside. Molecular ion is (M+H) 409 m/z.
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structure and it may not belong to alkaloid class of phytochemicals. The second plate was sprayed with ferric chloride (3%), after drying, when observed under
UV- long wavelength (365nm) it showed yellow florescence. This was the indication that it might belong to flavonoid or phenolic group of plants secondary metabolite. The third plate was sprayed with 1% ethanolic solution of Aluminum chloride and it also showed yellow florescence under longer UV-wavelength at
254nm. This mean the isolated compound DAF-MI-02.1 might belong to flavonoid or phenolic class of plants secondary metabolites.
4.4.2.2.3 UV-Vis spectroscopyof DAF-MI-02.1
The isolated compound was prepared at concentration of 50µg/mL in
HPLC grade methanol. Scanning of isolated compound was carried out at 200-
400nm wavelength. A single peak was observed at retention time 8.520 minutes
(Figure 4.22a) with peak purity (Figure 4.20c) and ʎmax240, 258, 318 and 366 nm was observed at 230 nm (Figure 4.22b).
4.4.2.2.4 Mass spectroscopy of DAF-MI-02.1
ESI- HR-TOF mass spectra of isolated compound (DAF-MI-02.1) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 423m/z suggested the molecular weight of the isolated compound might be 422m/z (M-H). The Other fragments observed were 351, 284 and 242 m/z
(Figure 4.24). The proposed fragmentation patern of DAF-MI-02.1 was given in figure 4.30 which correspondedto the observed signals in MS analysis of DAF-MI-
02.1.
96
4.4.2.2.5 Fouriertransfer- infra-red (FTIR) spectroscopy of DAF-MI-02.1
The FTIR of isolated compound DAF-MI-02.1 was presented in figure
4.23. It indicated the stretching of phenol-OH (3676.09cm-1) and also indicated the presence of carbonyl group in the molecule (1653.82 cm-1, C=O stretching).
4.4.2.2.6 NMR spectroscopy of DAF-MI-02.1
1HNMR: (d DMSO): The proton shift values and multiplicity are given in the table below (Table 4.15). The chemical shift value between 3.66ppm and
4.59ppm indicated the protons associated with glucose moiety. The integration showed the presence of eighteen protons in the structure (Figure 4.25). The δ=
13.77ppm suggested the hydroxyl group attached with carbon-1.
13CNMR: (solvent= d DMSO). The 13CNMR of isolated compound DAF-
MI-02.1 indicated presence of 19 carbon atoms in the structure (Figure 4.26). The
δ= 179.18 is the indication of carbonyl group (C=O). The chemical shift values of carbons are presented in the table. The Distortions enhancement by polarization transfer (DEPT) showed that 10 carbons are quaternary while one carbon has CH2.
It also showed that there are eight carbons in the structure which are directly bounded with a proton (H). The DEPT analysis of DAF-MI-02.1 showed that C-5
(δ= 102.72ppm) is a CH carbon while C-5a (δ=154.77ppm) is a quaternary carbon
(Table 4.15 and Figure 4.26).
HSQC: The heteronuclear single quantum coherence or heteronuclear single quantum correlation (HSQC) experiment of isolated compound DAF-MI-
02.1 provide information that C-6 /has two proton attached to it while Carbons C-
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1/,C-2/, C-3/, C-4/and C-5/ has one proton each. Similarly C-4 (δ=93.46ppm), C-5
(δ=102.72ppm) and C-8(δ= 107.94ppm) are bonded with one proton each. No signal was present in HSQC plot against C-1, C-1a, C-2, C-3, C-4a, C-5a, C-6, C-
7, C-8a and C-9 (Figure 4.27).This mean theses are quaternary carbons as also indicated by DEPT.
HMBC:The HMBC plot of isolated compound DAF-MI-02.1 showed long range bond correlation between various carbons present in skeleton of the isolated compound (Figure 4.28). A strong 3 bond coupling long-range correlation was observed between cross peaks from H-4 (δ 6.35ppm) to C-1a (δ 101.44ppm) and
C-2(δ 107.70ppm).
H-1/ (4.59ppm) showed strong correlation to C-3/ (δ 79.14ppm), C-5/(δ
81.68ppm), C-1 (δ 161.93ppm) and C-3(δ 163.96ppm). The C-4 (93.46ppm) has
/ week correlation with C-1 (73.26ppm). This mean C4 is not substituted with glucose. Similarly, the HMBC experiment also indicated that C3 (163.96ppm) is also not substituted as C-3 has a strong correlation with H-1/(δ 4.59ppm) .This strong correlation will only possible when both carbons are at one bond distance.
/ This clearly showed thatC-3 is not linked with C1/directly. However, H-2 (δ
4.05ppm) has a strong correlation with C-2 (δ 107.70ppm). This might be because
/ C-2 is bonded with C1/directly as cross peaks of H-2 showed three bond coupling correlation to C-2. Therefore we have strong evidences that glucose molecule may be attached at C-2 position (Figure 4.28).
4.4.2.2.7 Structure and nomenclature of isolated compound DAF- MI-02.1
The chemical reagent spraying analysis on TLC plates and UV-Vis
98
absorption spectra indicated that the molecule might have aromatic rings. The MS analysis showed a molecular ion (M+H) at 423m/z indicating molecular formula of
422amu. On the basis of HNMR, CNMR(DEPT) and MS analysis the molecular formula was determined as C19H18O11. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.30. The UV-Vis absorption spectra and HSQC suggested the presence of glucose moiety in the molecule. The HMBC experiments confirm the presence of sugar moiety which is substituted at C-2 of ring A. On the basis of these evidences we proposed following structure of isolated compound (DAF-MI-02.1) from aqueous fraction of
D. ramosa (Figure 4.29). It was identified as Mangiferrin and its IUPAC nomenclature is (1s)-1, 5- anhydro=1= (1, 3,6,7- tetra hydroxyl-9oxo-9H-xanthen-
2yl)- D-glucitol. It belongs to the xanthonoid sub-class of polyphenol. The four hydroxyl groups are attached with aromatic rings and it has a C- glycosylxanthone structure.
4.4.2.3 Identification and structural elucidation of isolated compound DAF- MI-02.2
4.4.2.3.1 Physical characteristics of DAF-MI-02.2
Isolated compound DAF-MI-02.2 was an odourless, orange yellowish colour powder with melting point 260oC. It was soluble in water and methanol easily.
4.4.2.3.2 Detection reagents
Three TLC plates were developed with Butanol: Ethyl acetate: Water
(5:1:4) v/v/v solvent system for DAF-Mi-02.2. The Rf value was 0.51. First TLC
99
b c a
Figure 4.22: HPLC profile of DAF-MI-02.1 obtained from aqueous fraction of D.
ramosa; a) Chromatogram b) UV-Vis DAD Absorption spectra at
230nm and c) Purity of peak at 8.520 minutes.
100
Figure 4.23: FTIR spectroscopic analysis of isolated compound DAF-MI-02.1
Figure 4.24: Mass spectrum (HR-TOF) ESI in positive mode for DAF-MI.02.1
101
Table 4.15: NMR (1H &13C) chemical shift values and DEPT analysis of isolated
compound DAF-MI-02.1.
Position 1Ha 13C DEPTb (Carbon) δ(chemical shift value)ppm δ(chemical shift value)ppm 1 13.77, (s, OH) 161.93 qC
1a - 101.44 qC
2 - 107.70 qC
3 - 163.96 qC
4 6.35. (s, 1H) 93.46 CH
4a - 156.38 qC
5 6.82, (s, 1H) 102.72 CH
5a - 154.77 qC
6 - 151.12 qC
7 - 144.06 qC
8 7.35, (s, 1H) 107.94 CH
8a - 111.55 qC
9 - 179.18 qC
1/ 4.59, (d, 1H , j= 9.95Hz) 73.26 CH
2/ 4.05, (d, 1H , j= 9.2Hz) 70.42 CH
3/ 3.14, (m, 1H) 79.14 CH
4/ 3.14, (m, 1H) 70.79 CH
5/ 3.14, (m, 1H) 81.68 CH
/ 6 3.66, (d, 2H, j= 11.8Hz) 61.65 CH2 aValues in parentheses are coupling constant (j) in Hz and Multiplicity.bq C= Quaternary carbon/ a carbon which is not directly bounded to „H‟ (determine by DEPT spectrum 135o).
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Figure 4.25a: HNMR spectra of isolated compound (DAF-MI-02.1) from aqueous phase of D. ramosa.
Figure 4.25b:1HNMR plot of DAF-MI-02.1 showing integration values and frequencies in joule mode.
103
Figure 4.26a:13CNMR (DEPT) analysis of isolated compound (DAF-MI-02.1) from aqueous phase of D. ramosa
Figure 4.26b: DEPT analysis of DAF.MI.02.1 isolated compound
104
Figure 4.27:gs-HSQC of isolated compound (DAF-MI-02.1) from aqueous phase of D. ramosa
Figure 4.28:gs-HMBC of isolated compound (DAF-MI-02.1) from aqueous phase of D. ramosa
105
Figure 4.29: Structure of isolated compound (DAF-MI.02.1) from aqueous phase of D. ramosa.
H+
H+ HO O OH
HO O OH
OH O m/z= 260 O C13H7O6 HO OH
OH OH O m/z = 406 C H O C19H17O10 6 11 5 HO OH C H O OH 6 11 5 H+ H+ HO O OH
HO O O HO OH
OH OH OH O m/z 423 M+H m/z = 244 HO OH O C13H7O5 OH CHO H+
C12H15O8 H+ HO O OH OH
O HO OH OH m/z = 396 m/z = 110 OH OH C H O C18H19O10 6 5 2 C H O HO 6 5 2 H+ OH C6H11O5 H+ HO O
HO O OH O HO
OH OH OH m/z= 234 m/z = 288 HO C H O OH C12H9O5 12 15 8 OH Figure 4.30:Proposed fragmentation scheme of Mangiferrin. Molecular ion is (M+H) 423 m/z.
106
plate was sprayed with dragendorff‟s reagent but there was no change in colour.
This mean the isolated compound DAF-MI-02.2 might not contain nitrogen in the structure and it may not belong to alkaloid class of phytochemicals. The second plate was sprayed with ferric chloride (3 percent), after drying, when observed under UV- long wavelength (365nm) it showed yellow florescence. This was the indication that it might belong to flavonoid or phenolic group of plants secondary metabolite. The third plate was sprayed with 1 percent ethanolic solution of
Aluminum chloride and it also showed yellow florescence under longer UV- wavelength at 254nm. This mean the isolated compound DAF-MI-02.2 might belong to flavonoid or phenolic class of plants secondary metabolites.
4.4.2.3.3UV-Vis Spectroscopy
The isolated compound was prepared at concentration of 50µg/mL in
HPLC grade methanol. Scanning of isolated compound was carried out at 200-
400nm wavelength. A single peak was observed at retention time 8.729 minutes
(Figure 4.31a) with peak purity (Figure 4.31c) and ʎmaxbetween 241, 255, 316 and
365nm (Figure 4.31b). Xanthones have absorption maximum between 230-245,
250-265, 305-330 and 340-400nm (Harborne, 1998).
4.4.2.3.4 Mass spectroscopy of DAF-MI-02.2
ESI- HR-TOF mass spectra of isolated compound (DAF-MI-02.2) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 423m/z suggested the molecular weight of the isolated compound might be 422m/z (M-H). The Other fragments observed were 406, 381, 357, 303,
245,218 and 94 m/z (Figure 4.32).
107
4.4.2.3.5 Fouriertransfer- infra-red (FTIR) spectroscopy of DAF-MI-02.2
The FTIR of isolated compound DAF-MI-02.2 was presented in figure
4.33. It indicated the stretching of phenol-OH (3676.09cm-1) and also indicated the presence of carbonyl group in the molecule (1653.82 cm-1, C=O stretching).
4.4.2.3.6 NMR spectroscopy of DAF-MI-02.2
1HNMR: (dDMSO): The proton shift values and multiplicity are given in the table 4.16. The δ= 13.35ppm suggested that C-1(161.73ppm) is substituted with hydroxyl group. Resonance singlet at H-5 ( δ6.75ppm), H-8 ( δ 7.34ppm) and
H-2 ( δ 6.21 ppm) representing a xanthone partial structure. The proton of the hydroxyl group at C-1 at δ 13.35 ppm was strongly down-field due to hydrogen bonding with the carbonyl C-9 (179.32ppm). The chemical shift value between
3.47ppm and 4.71ppm indicated the protons associated with glucose moiety. The integration showed the presence of eighteen protons in the structure (Figure 4.34).
13CNMR: (dDMSO); The13CNMR of isolated compound DAF-MI-02.2 indicated presence of 19 carbons atoms in the structure. The δ= 179.32ppm is the indication of carbonyl group (C=O). The chemical shift values of carbons are presented in the table 4.16. The Distortions enhancement by polarization transfer
(DEPT) analysis of DAF-MI-02.2 showed that C5 (δ= 102.72ppm) is a CH carbon while C-5a (δ=154.77ppm) is a quaternary carbon (Figure 4.35). The DEPT (135o) analysis of DAF-MI-02.2 indicated that C-4 (δ=104.9ppm) is a quaternary carbon instead of C-2 (δ=97.6ppm).
HSQC: The heteronuclear single quantum coherence or heteronuclear
108
single quantum correlation experiment (HSQC) of isolated compound DAF-MI-
02.2 provide information that C-6/(δ 61.65ppm) has two proton [3.72, (d, 2H, j=
12.6Hz)] attached to it while Carbons C-1/(δ 73.50ppm), C-2/(δ 71.15ppm),C-3/(δ
78.86ppm),C-4/(δ 70.79ppm) and C-5/ (δ 81.54ppm) has one proton each. Similarly
C-2 (δ=97.60ppm), C-5 (δ=102.72ppm) and C-8 (δ= 107.73ppm) are bonded with one proton at δ= 6.21, 6.79 and 7.34ppm respectively. No signal was present in
HSQC plot against C-1 (δ 161.73ppm), C-1a (δ 101.10ppm), C-4 (δ 104.86ppm),
C-3(δ 163.75ppm), C-4a (δ 156.34ppm), C-5a (δ 154.67ppm), C-6(δ 151.09ppm),
C-7(δ 144.06ppm), C-8a (δ 111.38ppm) and C-9 (δ 179.32ppm) (Figure 4.36).
Correlation spectroscopy/COSY (2-D NMR): The correlation peaks and H-
COSY spectrum of DAF-MI-02.2(at T= 340 K) suggested that H-1/(δ 4.27ppm) is
/ / coupled with cross peak of H-2 (δ 4.01ppm) while H-2 is associated with H-3/(δ
3.47ppm) .This indicated and confirmed the presence of glucose molecule in the structure of isolated compound. The cross peak of H-2 (δ 6.21ppm) do not coupled with any other peak in COSY spectra (Figure 4.37).
HMBC:The HMBC plot of isolated compound DAF-MI-02.2 showed long range bond correlation between various carbons present in skeleton of the isolated compound (Figure 4.36). A strong 3 bond coupling long-range correlation was observed between cross peaks from H-2(δ 6.21ppm) to C-1a (δ 101.10ppm) and C-
4(δ 104.90ppm). H-1/ (δ 4.27ppm) showed strong correlation to C-3/ (δ
78.86ppm), C-5/(δ 81.54ppm), C-4a (δ 156.34ppm) and C-3(δ 163.75ppm). This also indicated that C-3 is not substituted with glucose as C-3 (δ 163.75ppm) has a strong correlation with H-1/(δ4.27ppm).This strong correlation will only possible
109
when both carbons are at one bond distance. This clearly showed that C-3 (δ
163.75ppm) is not linked with C-1/(δ 73.50ppm)directly (Figure 4.38). However,
H-2/ (δ 4.01ppm) has a strong correlation with C-4 (δ 104.90ppm). This is because
/ C-2 9δ=97.60ppm) is bonded with C-1/(δ 73.15ppm) as cross peaks ofH-2 showed three bond coupling correlation to C-4 (δ 104.90ppm). Therefore we have strong evidences that glucose molecule may be attached at C-4 position.
4.4.2.3.7 Structure and nomenclature of isolated compound DAF-MI-02.2
The chemical reagent spraying analysis on TLC plates and UV-Vis absorption spectra indicated that the molecule might have aromatic rings. The MS analysis showed a molecular ion (M+H) at 423m/z indicating molecular formula of
422amu. On the basis of 1HNMR, 13CNMR (DEPT) and MS analysis the molecular formula was determined as C19H18O11. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.41.
The UV-Vis absorption spectra, HSQC and COSY suggested the presence of glucose moiety in the molecule. The HMBC experiments confirm the presence of sugar moiety which is substituted at C-2 of ring A. On the basis of these evidences we proposed following structure of isolated compound (DAF-MI-02.1) from aqueous fraction of D. ramosa (Figure 4.39). It was identified as Iso-mangiferin and the IUPAC name of this compound is “4- (β-D- glucopyranosyl)-1,3, 6, 7- tetrahydroxy-9H-xanthene-9-one”. It belongs to the xanthonoid sub-class of polyphenol.
110
c
a b
Figure 4.31: HPLC profile of DAF-MI-02.2 obtained from aqueous fraction of D.
ramosa; a) Chromatogram b) UV-Vis DAD Absorption spectra at
230nm and c) Purity of peak at 8.729 minutes.
111
Figure 4.32: Mass spectrum (HR-TOF) ESI in positive mode for DAF-MI.02.2
Figure 4.33: FTIR spectroscopic analysis of isolated compound DAF-MI-02.2
112
Table 4.16: NMR (1H &13C) chemical shift values and DEPT analysis of isolated compound DAF-MI-02.2. Position 1Ha 13C DEPTb (Carbon) δ(chemical shift value)ppm δ(chemical shift value)ppm 1 13.35, (s, OH) 161.73 qC
1a - 101.10 qC
2 6.21 (s, H) 97.60 CH
3 - 163.75 qC
4 - 104.86 qC
4a - 156.34 qC
5 6.79, (s, 1H) 102.72 CH
5a - 154.67 qC
6 - 151.09 qC
7 - 144.06 qC
8 7.34, (s, 1H) 107.73 CH
8a - 111.38 qC
9 - 179.32 qC
1/ 4.71, (2d, 1H , j= 6.81Hz, J= 73.50 CH
5.08Hz)
2/ 4.01, (d, 1H , j= 9.5Hz) 71.15 CH
3/ 3.47, (m, 1H) 78.86 CH
4/ 3.47, (m, 1H) 70.79 CH
5/ 3.47, (m, 1H) 81.54 CH
/ 6 3.72, (d, 2H, j= 12.6Hz) 61.65 CH2 aValues in parentheses are coupling constant (j) in Hz and Multiplicity (s= singlet, d=duplet, m= multiplet).
113
Figure 4.34a: HNMR spectra of isolated compound (DAF-MI-02.2) from aqueous phase of D. ramosa
Figure 4.34b:1HNMR plot of DAF-MI-02.2 showing integration values and frequencies in joule mode.
114
Figure 4.35:13CNMR (DEPT) analysis of isolated compound (DAF-MI-02.2) from aqueousphase of D. ramosa
Figure 4.36:gs-HSQC of isolated compound (DAF-MI-02.2) from aqueous phase of D. ramosa
115
Figure 4.37:gs-CoSY of isolated compound (DAF-MI-02.2) from aqueous phase ofD. ramosa
Figure 4.38:gs-HMBC of isolated compound (DAF-MI-02.2) from aqueous phase of D. ramosa
116
4' 6'
3' 5'
2'
1' 5 4 4a 5a 6 3
2 1a 9 8a 7 1 8
Isomangiferin
4- (β-D- glucopyranosyl)-1,3, 6, 7- tetrahydroxy-9H-xanthene-9-one
Figure 4.39: Structure of isolated compound (DAF-MI-02.2) from aqueous fraction
of D.ramosa
117
Figure 4.40: Schematic representation of compounds isolated from aqueous
fraction of D.ramosa
118
H+ OH H+ HO OH
HO HO OH O OH HO O m/z = 406 HO OH C19H17O10
O HO O HO
O OH OH m/z = 423 C6H11O5 M+H HO O OH H+
HO O CHO H+
OH OH OH HO O OH O m/z = 245 O C13H8O5 mz= 381 HO HO C18H20O9
H+ HO HO O OH
m/z = 126 C6H11O5 C6H5O3
OH C6H5OH H+ HO O H+
OH m/z= 218 C H O OH C6H5O3 OH 12 9 4 m/z = 94 C6H5O Figure 4.41:Proposed fragmentation scheme of Iso-Mangiferrin. Molecular ion is
(M+H) 423m/z.
119
4.4.3 Isolation of Compounds from Ethyl Acetate Fraction of D. ramosa
Ethyl acetate soluble fraction obtained from CME of D. ramosa was subjected to normal phase silica gel (Silica gel 60, 0.2-0.5 mm) column chromatography. The isocratic solvent mixtures were used (Table 4.17). A total twenty four elutions were collected in conical flasks each containing 50mL. All the elutions were dried under reduced pressure in rotary apparatus and each of them is re-dissolved in methanol(2-5mL) and subjected to thin layer chromatography
(TLC). The solvent system used was n-Butanol: Accetic acid: water (4:1:5, v/v/v).
Fractions with similar TLC pattern were combined and as a result 5 groups were formed. i.e. Group-1 (Fractions 1.1-3.1), group-2 (Fractions 3.2-4.2), group-3
(Fractions 5.1-5.2), group-4 (Fractions 6.1-8.2) and group-5 (fractions 9.1-10.4).
These five groups were subjected to High pressure liquid chromatography (HPLC).
Groups 2, 3 and 4 weigh 52.5, 20.2mg and 15.8mg respectively, while Groups 1 and 5yield less amount for further separation. Group 2, 3 and 5 were subjected to evaluate their antioxidant potential by spraying with DPPH on a developed TLC plate. The group 4 did not show antioxidant property while groups 2 and 3 showed clear bright spot indicating the presence of antioxidant substance/s. Therefore, groups 2 and 3 were selected for further purification.
4.4.3.1 Purification of group-2 (Obtained from ethyl acetate fraction ofD. ramosa
Group 2 (30mg) was subjected to column chromatography (Sephadex LH20,
120x2cm) with methanol as mobile phase. A total 21 fractions were collected and each eluent was cut at 10mL volume. All the eluents were subjected to TLC [The solvent system used was n-Butanol: Acetic acid: water (4:1:5, v/v/v)]and
120
visualized under UV at 254 and 366nm. On the basis of TLC pattern so obtained, the fractions were combined into two groups i.e. 2a (fractions 1-14) and 2b
(fractions 15-21). The HPLC profile showed that group 2a contains major peak
(UV absorption spectra ʎmax.(MeOH) 267, 350nm and retention time 12.487min.) and some impurities.It was dried and weighs 22.7mg while group 2b which weighs
6.9mg without any major peak in HPLC analysis.
The sub-fraction 2a was subjected for purification and removal of impurities by medium pressure liquid chromatography using Lobar® glass column
310x23mm, filled with Merck silica gel LiChroprep ™ SI 60 (particle size 40-
63μm). The solvent system used was ; Petroleum ether to ethyl acetate ( 70:30,
50:50, 30:70 and 00:100) followed by ethyl acetate to methanol ( 80:20, 60:40,
40:60, 20:80, 10:90 and 100 percent methanol). A total 23 fractions were collected.
All the fractions obtained from MPLC were dried and dissolved in methanol and then subjected to TLC with was n-Butanol: Acetic acid: water (4:1:5, v/v/v). The fractions 1, 9 and 10 showed similar pattern and single spot on TLC plate observed at 254 and 365nm. They were combined and subjected to HPLC analysis. HPLC chromatogram showed purity of the isolated compound (weighs 8.4mg) and finally spectroscopic data was taken and was labeled as DEF-4MP1910.
4.4.3.1 Identification of isolated compound (DEF-4MP1910)
4.4.3.1.1 Physical characteristics of DEF-4MP1910
It was a yellow amorphous powder, odourless, and is soluble in water and methanol. The melting point of isolated compound DEF-4MP1910 was determined as 175-1790C.
121
Table 4.17: Mobile phase used during column chromatography of ethyl acetate
fraction of D.ramosa.
S.No. Solvent mixture Ratio Volume Fraction collected used (50mL each (Solvent fraction) mixture)
1 Pet. Ether : Ethyl acetate 50:50 200mL 1.1, 1.2, 1.3, 1.4
2 Pet. Ether : Ethyl acetate 25:75 100mL 2.1, 2.2
3 Pet. Ether : Ethyl acetate 10:90 100mL 3.1, 3.2
4 Pet. Ether : Ethyl acetate 00:100 100mL 4.1, 4.2
5 Ethyl acetate : Methanol 90:10 100mL 5.1, 5.2
6 Ethyl acetate : Methanol 80:20 100mL 6.1, 6.2
7 Ethyl acetate : Methanol 60:40 100mL 7.1, 7.2
8 Ethyl acetate : Methanol 40:60 100mL 8.1, 8.2
9 Ethyl acetate : Methanol 20:80 100mL 9.1, 9.2
10 Ethyl acetate : Methanol 00:100 200mL 10.1, 10.2, 10.3, 10.4
.
122
4.4.3.1.2 Detection spraying reagents
Three TLC plates were developed with Butanol: Ethyl acetate: Water
(5:1:4) v/v/v solvent system for DEF-4MP1910. The Rf value was 0.59. First TLC plate was sprayed with dragendorff‟s reagent but there was no change in colour.
This mean the isolated compound DEF-MP1910 might not contain nitrogen in the structure and it may not belong to alkaloid class of phytochemicals. The second plate was sprayed with ferric chloride (3 percent), after drying, when observed under UV- long wavelength (365nm) it showed yellow florescence. This was the indication that it might belong to flavonoid or phenolic group of plants secondary metabolite. The third plate was sprayed with 1% ethanolic solution of Aluminum chloride and it also showed yellow florescence under longer UV-wavelength at
254nm. This mean the isolated compound DEF-4MP1910 might belong to flavonoid or phenolic class of plants secondary metabolites. Another developed
TLC plates was sprayed with anisaldehyde and on heating the spot turn yellow.
This indicated the presence of a flavonoid or related compound.
4.4.3.1.3 UV-Vis spectroscopyof DEF-4MP-1910
The isolated compound was prepared at concentration of 50µg/mL in
HPLC grade methanol. Scanning of isolated compound was carried out at 200-
400nm wavelength. A single peak was observed at retention time 12.487 minutes indicating the presence of a single compound (Figure 4.42a) with peak purity
(Figure 4.42c). The UV-Vis absorption spectra showed ʎmaxat 267nm and 350nm
(Figure 4.42b).
123
4.4.3.1.4 Mass spectroscopyof DEF-4MP-1910
ESI- HR-TOF mass spectra of isolated compound (DEF-4MP-1910) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 449m/z suggested the molecular weight of the isolated compound might be 448m/z (M-H). The Other major peaks were observed at 432, 356, 286, 254,
228, 193 and 110 m/z (Figure 4.41). The proposed fragmentation pattern of these peaks (m/z) for DEF-4MP-1910 was given in Figure 4.43.
4.4.3.1.5 Fourier transfer- infra-red (FTIR) spectroscopy of DEF-Mp-1910
The FTIR of isolated compound DEF-MP-1910 was presented in figure
4.44. It indicated the stretching of phenol-OH (3303.07cm-1) and also indicated the presence of carbonyl group in the molecule (1653.82 cm-1, C=O stretching) and
1448.48cm-1, Aromatic C=C bending.
4.4.3.1.6 NMR Spectroscopy of DEF-4MP-1910
1HNMR: (in deuterated Methanol): The proton shift value and multiplicity are given in the table below (Table 4.18). Some of the signal pattern was unclear due to overlapping. The proton signal appear at δ 5.25 (1H, d, j= 7.2Hz) with the other resonance in the 1H-NMR spectrum of DEF-4MP1910 was assigned to the anomeric proton of a β-glucose. The integration showed the presence of 20 protons
in the structure (Figure 4.45).
13 13 CNMR: (solvent= dCH3OH): The CNMR of isolated compound DEF-
4MP1910 indicated presence of 21 carbons atoms in the structure. The δ=
124
179.54ppm is the indication of carbonyl group (C=O). The chemical shift values of carbons are presented in the table 4.18. The Distortions enhancement by polarization transfer (DEPT) showed the carbon atoms with CH2 or unsaturation or quaternary carbons down wards while CH and CH3 carbons upward directions (Fig
4.46). The DEPT analysis of DEF-MP1910 showed that C-2 (δ 159.12ppm), C-
3(δ135.48ppm), C-4 (δ179.54ppm), C-4a (δ105.76ppm), C-5 (δ163.09ppm), C-7
(δ165.97ppm), C-8a (δ 158.53ppm), C-1/(δ 122.86ppm), C-2/ (δ132.27ppm), C-
3/(δ116.07ppm), C-5/ (δ116ppm) and C-6/ (δ131.89ppm) were quaternary carbons
// while C-6 (δ 62.66ppm) was CH2 carbon.
HSQC:The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment( HSQC) of isolated compound DEF-4MP-1910 provide information that Carbon- C-6//(δ 62.66ppm) has two proton attached to it while Carbons C-2/(δ 132.27ppm), C-3/(δ 116.07ppm), C-5/(δ 116.00ppm), C-6/(δ
131.89ppm), C-6(δ 99.89ppm)and C-8(δ 94.75ppm) has been bounded to a single proton(fig 4.47).
HMBC:HMBC is a two dimensional heteronuclear multiple bond correlation experiment developed to assist in the identification. The HMBC plot of isolated compound DEF-4MP-1910 showed long range bond correlation between various carbons and protons present in skeleton of the isolated compound. A strong
3 bond coupling long-range correlation was observed between cross peaks from H-
1// (δ 5.25ppm) to C-3 (δ 135.48ppm) and C-6//(δ 62.66ppm). This mean carbon C-
3 (δ 135.48ppm) is substituted with β-glucose (Figure 4.48). The HMBC data is in accordance with the literature published (Kuruzum-Uz 2013).
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4.4.3.1.7Structure and nomenclature of isolated compound DEF-4MP-1910
The chemical reagent spraying analysis on TLC plates and UV-Vis absorption spectra indicated that the molecule might have aromatic rings and it might belong to flavonoid class of plants secondary metabolites. The MS analysis showed a molecular ion (M+H) at 449m/z indicating molecular formula of
448amu. On the basis of HNMR, CNMR (DEPT) and MS analysis the molecular formula was determined as C21H20O11. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.50. The UV-Vis absorption spectra, HSQC and COSY indicated the presence of glucose moiety in the molecule. The HMBC experiments confirm the presence of sugar moiety which is substituted at C-3 of ring B. On the basis of these evidences we proposed following structure of isolated compound (DEF-4MP-1910) from ethyl acetate fraction of D. ramosa (Figure 4.49). It was identified as “Astragalin”. The other name of this compound is Kampferol- 3- O- β- glucopyranoside. The IUPAC name of this compound is “5,7 – dihydroxy – 2-(4-hydroxyphenyl) – 3-[3,4,5- trihydroxy – 6-(hydroxymethyl)tetrahydropyran – 2-yl]oxy-chromen – 4-one.
4.4.3.2 Compound isolated from group 3
(Obtained from ethyl acetate fraction of D. ramosa after column chromatography)
Group 3 was obtained by combining fraction 5.1 and 5.2 after column chromatography of ethyl acetate fraction of D. ramosa. A TLC plate was developed with Butanol: Ethyl acetate: Water (5:1:4) v/v/v solvent system and group three showed a single spot. The Rf was0.39. The HPLC chromatogram of
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Figure 4.42: HPLC profile of DEF-4MP-1910 obtained from ethyl acetate fraction
of D. ramosa; a) Chromatogram b) UV-Vis DAD Absorption spectra
at 230nm and c) Purity of peak at 12.487 minutes.
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Figure 4.43: Mass spectrum (HR-TOF) ESI in positive mode for DEF-MP-1910
Figure 4.44: FIR analysis of isolated compound DEF-MP-1910.
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Table 4.18: NMR (1H &13C) chemical shift values and DEPT analysis of isolated
compound DEF-MP1910.
Position (Carbon) 1H a 13C DEPT b δ(chemical shift value) δ(chemical shift value) ppm ppm 2 - 159.12 qC 3 - 135.48 qC 4 - 179.54 qC 4a - 105.76 qC 5 - 163.09 qC 6 6.21 (d, 1H, J= 2.05Hz) 99.89 CH 7 - 165.97 qC 8 6.41(d, 1H, J= 1.98Hz) 94.75 CH 8a - 158.53 qC 1/ - 122.86 qC 2/ 8.06 (d, 2H,J= 8.91Hz ) 132.27 CH 3/ 6.88 (d, 2H,J= 9.0 Hz ) 116.07 CH 4/ - 161.56 qC 5/ 6.88 (d, 2H,J= 9.0 Hz ) 116.00 CH 6/ 8.06 (d, 2H,J= 8.91Hz ) 131.89 CH 1// 5.25 (d, 1H,J= 7.20Hz ) 104.36 CH 2// ¶ 75.74 CH 3// ¶ 78.13 CH 4// ¶ 71.38 CH 5// ¶ 78.41 CH // 6 3.52 (dd, 1H, J= 12.0/5.4) 62.66 CH2 3.69 (dd, 1H, J= 12.0/2.0)
aValues in parentheses are coupling constant (j) in Hz and Multiplicity.bq C=
Quaternary carbon/ a carbon which is not directly bounded to „H‟ (determine by
DEPT spectrum 135o).¶ Unclear signal due to overlapping
129
a
b
c
Figure 4.45: a) HNMR spectra of isolated compound (DEF-mi-4mp-1910) from
ethyl acetate fraction of D. ramosa, b) and c) HNMR with joule
mode showing integration.
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Figure 4.46:13CNMR (DEPT) analysis of isolated compound (DEF-mi-4mp-1910)
from ethyl acetate fraction of D. ramosa
Figure 4.47:gs-HSQC of isolated compound (DEF-mi-4mp-1910) from ethyl
acetate fraction of D. ramosa.
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Figure 4.48:gs-HMBC spectra obtained from isolated compound (DEF-mi-4mp- 1910) from ethyl acetate fraction of D. ramosa
Figure 4.49: Structure of isolated compound (DEF-4MP-1910) from ethyl acetate
fraction of D.ramosa.
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H+ H+ O HO O HO HO O O O C6H5 m/z = 356OH HO OH OH O C H O 15 15 10 OH OH O OH O HO H+ OH m/z = 432 HO HO O C21H19O10 OH
O H+ OH C6H11O6 OH O O m/z = 449 HO M+H HO HO O OH m/z = 254 C6H11O5 OH C15H9O4 H+ OH O
HO O H+ m/z = 286 C15H9O6 CHO O
OH O HO O
C6H5O m/z = 228 C14H11O3 OH H+
HO O C8H6O H+ HO O m/z = 110 OH O m/z = 193 C9H4O5 C6H5O2 OH Figure 4.50:Proposed fragmentation scheme of Kampferol- 3- O- β-
glucopyranoside(Astragalin). Molecular ion is (M+H) 449 m/z.
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Figure 4.51: Schematic representation of isolation of compounds from Ethyl
acetate fractionofD. ramosa.
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group three showed a single peak at 6.467 minutes and UV-VIS absorption maxima at ʎmax at 298 and 210 nm for band I and II respectively. It was odourless, yellow coloured powder and was soluble in water and methanol. The melting point was 272-2780C.
It was sent for mass analysis and NMR analysis. The data so obtained was identical with the information received during analysis of DAF-MI-01 compound
(from aqueous fraction of D. ramosa). The mass spectrum analysis proposed molecular weight 408 m/z and on the basis of HNMR and CNMR, the molecular formula is C19H20O10. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4. 21. The HSQC and COSY experiments confirm the presence of glucose sugar moiety which is substituted at
C-5 (δ 104.67ppm) as confirmed by HMBC. On the basis of these evidences, we proposed that the isolated compound (DAF-MI-01) from aqueous phase of D. ramosa was iriflophenone-3-C-β- D glucopyranoside and its chemical structure was given in Figure 4. 19. The IUPAC name of this compound is (2R, 3S, 4R, 6S)-
2-(hydroxymethyl)-6-[2, 4,6-trihydroxy-3(4-hydroxybenzoyl) phenyl] oxane-3,4,5-
Triol. The figure 4.51 showed a Schematic representation of isolation of compounds from Ethyl acetate fraction of D. ramosa. i.e Kampherol-3-O-β- glucopyranoside and iriflophenone-3-C-β-D glucopyranoside.
4.4.4 Isolation of Pure Compound from Ethyl Acetate Fraction of R. brunonii
Ethyl acetate soluble phase of R. brunonii (2 g) was subjected to column chromatography (Silica gel 60, 0.5-0.6 mm, normal phase, 75 x 3 cm). Non-polar to polar solvent mixtures were used (Table 4.19). A total 12 solvent mixtures
135
(100mL of each) were used and total 26 sub-fractions were collected. Each eluent was cut at 50 mL. All the eluents were evaporated in rotary apparatus and re- dissolved in Methanol (2-5 mL) and subjected to thin layer chromatography (TLC) using Toluene: Dioxane: Water (19:5:1) v/v/v and n-Butanol: Acetic acid: water
(4:1:5 v/v/v) solvent systems. Fractions with similar TLC pattern were combined and as a result six groups were formed. i.e. group1 (fraction 1.1), group2 (fractions
1.1 - 1.2), group-3 (fractions 1.3-3.2), group-4 (fractions 4.1-4-2), group-5
(fractions 5.1-5-2) and group-6 (fractions 6.1-12.2). The TLC plates were also sprayed with DPPH to identify the antioxidant potential of these groups. The group
5 weighs 481mg while group 4 weighs 402mg. The groups 4 and 5 were selected for isolation studies. A schematic diagram presenting isolation of compounds form ethyl acetate fraction of R. brunonii was shown in figure 4.52.
4.4.4.1 Isolation of compounds from sub group 5 (Ethyl acetate fraction of R.
brunonii)
The sub group 5 (that was obtained from silica gel column chromatography of ethyl acetate soluble fraction of R. brunonii) was subjected to Sephadex column
LH20 (120x2 cm) with methanol as mobile phase. Each eluent was cut at 20mL. A total 12 eluents were collected. All the eluents were dried and re-dissolved in methanol. A TLC plate was developed with n-Butanol: Acetic acid: water (4:1:5, v/v/v) solvent systems. After drying, it was observed under UV-at 254nm. The fraction 5s5 showed a single spot with Rf 0.62 while fractions 5s6 and 5s7 also showed a single spot at Rf 0.59. Both fractions 5s6and 5s7 were combined as 5s67 and weighs 78.4mg. The fraction 5s5 weighs 114.1mg. Both fractions i.e 5s5 and
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5s67 were subjected to HPLC analysis. The HPLC profile of sub fraction 5s5 showed a single peak at retention time 12.540 minutes while 5s67 showed a peak at 12.487 minutes.
4.4.4.2 Isolation of compounds from sub group 4 (Ethyl acetate fraction of R.
brunonii)
The sub group 4 (that was obtained from silica gel column chromatography of ethyl acetate soluble fraction of R. brunonii) was subjected to Sephadex column
LH20 (75x3 cm) with pure methanol as mobile phase. A total 22 (4s1-4s22) eluents
(Figure 4.52) were collected each of 30mL volume. All the eluents were dried and re-dissolved in methanol and subjected to TLC analysis. Based on TLC profile, the eluents 4s13 to 4s18 were combined and subjected to medium pressure liquid chromatography (MPLC) system. The solvent mixtures with increasing polarity
(100 mL of each) were used (Petroleum ether: Ethyl acetate 80:20, 60:40, 40:60,
20:80 and 100 percent ethyl acetate and then Ethyl acetate : Methanol 80:20,
60:40, 40:60, 20:80 and 100 percent methanol). The eluents were cut at different volumes based on absorption spectra showed by ISCO UV-6 UV/VIS detector associated with MPLC system. All the eluents were rotary dried and re-dissolved in methanol and subjected to TLC (silica gel 60, F254 TLC plates) using n-
Butanol: Acetic acid: water (4:1:5, v/v/v) solvent systems. The eluents 4m4 to 4m9 showed similar TLC pattern. All these six eluents (4m4-4m9) were subjected to
HPLC analysis. The HPLC chromatogram presented a single peak at 14.310 minutes (retention time). These eluents were combined as REF-MI-49 and it weighs 89.8 mg.
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4.4.4.3Identification of isolated compound REF-5s5
4.4.4.3.1 Physical characteristics of REF-5s5
It was a yellow coloured crystal, odourless, and was soluble in water and methanol. The melting point was 176-1790C.
4.4.4.3.2 Sprayingreagents
Three TLC plates were developed with Butanol: Ethyl acetate: Water
(5:1:4) v/v/v solvent system for REF-5s5. The Rf value was 0.61. First TLC plate was sprayed with dragendorff‟s reagent but there was no change in colour. This mean the isolated compound REF-5s5 might not contain nitrogen in the structure and it may not belong to alkaloid class of phytochemicals. The second plate was sprayed with ferric chloride (3 percent), after drying, when observed under UV- long wavelength (365nm) it showed yellow florescence. This was the indication that it might belong to flavonoid or phenolic group of plants secondary metabolite.
The third plate was sprayed with 1 percent ethanolic solution of Aluminum chloride and it also showed yellow florescence under longer UV-wavelength at
254nm. This mean the isolated compound REF-5s5 might belong to flavonoid or phenolic class of plants secondary metabolites. Another developed TLC plates was sprayed with anisaldehyde and on heating the spot turn yellow. This indicated the presence of a flavonoid or related compound.
4.4.4.3.3 UV-Vis spectroscopyof REF-5s5
The HPLC analysis of isolated compound(50µg/mL) was carried out at
200-400nm wavelength. The HPLC chromatogram showed a single peak at 12.540
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minutes (Figure 4.53a) with peak purity was given in fig 4.53b. The UV-VIS absorption spectra of isolated compound (REF-5s5) exhibited ʎmax (in methanol)
355, 265 and 257 nm (Figure 4.53c).
4.4.4.3.4 Mass spectroscopy of REF-5s5
ESI- HR-TOF mass spectra of isolated compound (REF-5s5) were recorded in positive ionization mode. The mass spectrum showed a molecular ion (M+H) at
449m/z suggested the molecular weight of the isolated compound might be 448m/z
. The Other major peaks were observed at 434, 303, 286, 270, 178, 164, and 94m/z
(Figure 4.54). The proposed fragmentation pattern of these peaks (m/z) for REF-
5s5 was given in Figure4.62.
4.4.4.3.5 Fouriertransfer- infra-red (FTIR) spectroscopy of REF-5s5
The FTIR of isolated compound REF-5s5 was presented in figure 4.55. It indicated the stretching of phenol-OH (3308.54cm-1) and also indicated the presence of carbonyl group in the molecule (1684.72cm-1, C=O stretching) and
1448.48cm-1-1418.99 cm-1, Aromatic C=C bending.
4.4.4.3.6 NMR spectroscopy of RE- 5s5
1HNMR: (in deuterated Methanol): The proton shift value and multiplicity are given in the table 4.20. The proton signal appear at δ 5.35 ppm (1H, s) with the other resonance in the 1H-NMR spectrum of REF-5s5 was assigned to the anomeric proton of a sugar moiety. The integration showed that the molecule has twenty protons (Figure4.56).
13 13 CNMR: (solvent= dCH3OH): The CNMR of isolated compound REF-
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5s5 indicated presence of 21 carbon atoms in the structure (Figure 4.57). The δ=
179.67ppm is the indication of carbonyl group (C=O). The chemical shift values of carbons are presented in the table 4.20. The Distortions enhancement by polarization transfer (DEPT) showed five aromatic methines (C-6 δ99.81; C-8
δ94.71; C-2/ δ 116.96; C-5/ δ 116.07; C-6/ δ122.86ppm), five oxygenated methines
(C-1// δ103.55; C-2// δ71.38; C-3// δ72.15; C-4// δ73.28; C-5// δ71.91ppm), methyl
(C-6// δ17.64ppm) and nine signals for quaternary aromatic carbons (C-2 δ159.31;
C-3 δ 136.25; C-4a δ 105.93; C-5 δ163.22; C-7 δ165.85; C-8a δ158.53; C-1/
δ123.0; C-3/ δ146.42; C-4/ δ149.79ppm) and one carbonyl carbon ( C-4 δ179.67 ppm).
HSQC:The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment (HSQC) of isolated compound REF-5s5 differentiated between C/CH2 carbons and CH/CH3 carbons. The HSQC plot is given in the figure 4.58. It clearly indicated that carbons (C-2 δ159.31; C-3 δ
136.25; C-4a δ 105.93; C-5 δ163.22; C-7 δ165.85; C-8a δ158.53; C-1/ δ123.0; C-
3/δ146.42; C-4/ δ149.79ppm) are not bounded to any proton and hence they are quaternary carbons Similarly, carbon C-4 δ179.67ppm) is a carbonyl carbon
(Figure 4.56).
Correlation spectroscopy/COSY (2-D NMR): The COSY spectrum of REF-
5s5 (Figure 4.59) suggested that proton H-2// (δ 4.22ppm) was coupled with H-1//
(δ 5.35ppm) and H-3// (δ 3.75ppm) while cross peak of H-1// (δ 5.35ppm) was only coupled with H-2// (δ 4.22ppm). Similarly, The cross peak of H-6// (δ 0.95ppm) was coupled with H-5// (δ 3.48ppm).
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HMBC:The heteronuclear multiple bond correlation (HMBC) experiment of isolated compound REF-5s5 showed long range bond correlation between various carbons and protons present in skeleton of the isolated compound (Figure
4.60). The HMBC correlation of H-2/ (δ 7.34ppm) to C-2, C-1/, C-3/ and C-4/ while H-5/ (δ6.90ppm) to C-1/, H-5/to C-3/ suggested the 3/, 4/ dihydroxy substitution of aromatic ring attached with C-2. Similarly, the position of rhamnose sugar moiety was also confirmed in HMBC spectra by observing of peaks between
H-1// (δ 5.35ppm) and C-3 (136.25ppm).
4.4.4.3.7 Structure and nomenclature of isolated compound REF-5s5:
The chemical reagent spraying analysis on TLC plates and UV-Vis absorption spectra indicated that the molecule might have aromatic rings and it might belong to flavonoid class of plants secondary metabolites. The MS analysis showed a molecular ion (M+H) at 4449m/z indicating molecular formula of
448amu. The H-NMR spectra indicated the ABX spin coupling system between H-
2/, H-6/ and H-5/ while AB spin coupling system was observed between H-6 and
H-8.
The anomeric position at δH 5.35ppm indicating α configuration. The
DEPT experiment revealed 10 quaternary and 11 primary/ tertiary carbons. The
HMBC experiment confirmed the position of rhamnose at position C-3. On the basis of HNMR, CNMR (DEPT) and MS analysis the molecular formula was determined as C21H20O11. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.62. The UV-Vis absorption spectra, HSQC and COSY suggested the presence of rhamnose moiety in the
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molecule. The HMBC experiments confirm the presence of rhamnose sugar moiety which is substituted at C-3 of ring B. On the basis of these evidences we proposed following structure of isolated compound (REF-5s5) from ethyl acetate fraction of
R. brunonii (Figure 4.61). It was identified as “Quercetin-3-O-rhamnoside”. 2-
(3,4-dihydroxyphenyl)-5,7 – dihydroxy – 3-(3,4,5 – trihydroxy – 6 – methyl- tetrahydropyran-2-yl)oxy – chromen – 4-one.
4.4.4.4Identification of isolated compound REF-5s67:
4.4.4.4.1 Physical characteristics of REF-5s67
Isolated compound REF-5s67 was a yellow amorphous powder, odourless, and is soluble in water and methanol. The melting point was 175-1790C.
4.4.4.4.2 Detectionspraying reagents
Three TLC plates were developed with Butanol: Ethyl acetate: Water (5:1:4) v/v/v solvent system for REF-5s67. The Rf value was 0.59. First TLC plate was sprayed with dragendorff‟s reagent but there was no change in colour. This mean the isolated compound REF-5s67 might not contain nitrogen in the structure and it may not belong to alkaloid class of phytochemicals. The second plate was sprayed with ferric chloride (3 percent), after drying, when observed under UV- long wavelength (365nm) it showed yellow florescence. This was the indication that it might belong to flavonoid or phenolic group of plants secondary metabolite. The third plate was sprayed with 1 percent ethanolic solution of Aluminum chloride and it also showed yellow florescence under longer UV-wavelength at 254nm.
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Table 4.19: Mobile phase used during column chromatography of ethyl acetate
fraction of R.brunonii.
S.No. Solvent mixtures Ratio Volume used Fraction collected
(Solvent (50mL each mixture) fraction)
1 Pet. Ether : Ethyl acetate 50:50 200mL 1.1, 1.2, 1.3, 1.4
2 Pet. Ether : Ethyl acetate 25:75 100mL 2.1, 2.2
3 Pet. Ether : Ethyl acetate 10:90 100mL 3.1, 3.2
4 Pet. Ether : Ethyl acetate 00:100 100mL 4.1, 4.2
5 Ethyl acetate : Methanol 90:10 100mL 5.1, 5.2
6 Ethyl acetate : Methanol 80:20 100mL 6.1, 6.2
7 Ethyl acetate : Methanol 60:40 100mL 7.1, 7.2
8 Ethyl acetate : Methanol 40:60 100mL 8.1, 8.2
9 Ethyl acetate : Methanol 20:80 100mL 9.1, 9.2
10 Ethyl acetate : Methanol 00:100 100mL 10.1, 10.2
11 Methanol: Water 90:10 100mL 11.1, 11.2
12 Methanol: Water 80:20 100mL 12.1, 12.2
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Figure 4.52: Summary of compound isolated from ethyl acetate fraction of
R. brunonii.
144
a b c
Figure 4.53: HPLC profile of REF-5s5 obtained from ethyl acetate fraction of D.
ramosa; a) Chromatogram b) UV-Vis DAD Absorption spectra at
230nm and c) Purity of peak at 12.487 minutes.
145
Figure 4.54: Mass spectrum (HR-TOF) ESI in positive mode for REF-5s5
Figure 4.55: FTIR analysis of isolated compound Ref-5s5.
146
Table 4.20: NMR (1H &13C) chemical shift values and DEPT analysis of isolated
compound REF-5s5.
Position (Carbon) 1H a 13C DEPT b δ(chemical shift value) δ(chemical shift value) ppm ppm 2 - 159.31 qC 3 - 136.25 qC 4 - 179.67 qC 4a - 105.93 qC 5 - 163.22 qC 6 6.21 (d, 1H, J= 2.06Hz) 99.81 CH 7 - 165.85 qC 8 6.37(d, 1H, J= 2.05Hz) 94.71 CH 8a - 158.53 qC 1/ - 123.00 qC 2/ 7.34 (d, 1H,J= 2.08Hz ) 116.96 CH 3/ - 146.42 qC 4/ - 149.79 qC 5/ 6.90 (d, 1H,J= 8.36Hz ) 116.07 CH 6/ 7.29(d, 1H,J= 8.30Hz ) 123.00 CH 1// 5.35 (d, 1H, J= 1.2 ) 103.55 CH 2// 4.22 (m) 71.38 CH 3// 3.75 (m) 72.15 CH 4// 3.18 (m) 73.28 CH 5// 3.48(m) 71.91 CH // 6 0.95 (d, 3H, J= 6.01Hz) 17.64 CH3
aValues in parentheses are coupling constant (j) in Hz and Multiplicity.bq C=
Quaternary carbon/ a carbon which is not directly bounded to „H‟ (determine by
DEPT spectrum 135o).
147
a
b
c
Figure 4.56: a) HNMR spectra of isolated compound (REF-5s5) from ethyl acetate fraction of R. brunonii,” b” and “c” HNMR with joule mode showing integration.
148
Figure 4.57:13CNMR (DEPT) analysis of isolated compound (REF-5s5) from ethyl acetate fraction of R. brunonii
Figure 4.58:gs-HSQC of isolated compound (REF-5s5) from ethyl acetate fraction
of R. brunonii
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Figure 4.59:gs-COSY of isolated compound (REF-5s5) from ethyl acetate fraction
of R. brunonii
Figure 4.60:gs-HMBC spectra obtained from isolated compound (REF-5s5) from
ethyl acetatefraction of R. brunonii
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Figure 4.61: Structure of isolated compound (REF-5s5) from ethyl acetate fraction of R. brunonii.
H+ HO H+ O OH C H O O 15 9 6 CH3
OH O HO O CH O 3 m/z = 449 HO HO OH M+H O m/z = 164 C6H11O5 HO HO OH C6H11O5 CH3 H+ H+ OH HO OH O OH
OH O HO O m/z = 286 O m/z = 434 C15H9O6 O C5H9O5 C20H17O11 HO HO HO OH O H+ OH
HO O OH C6H5O2 H+
m/z = 178 C6H5OH C9H5O4 OH O HO O OH H+ OH m/z = 270 C15H9O5
HO O m/z = 94 C9H5O4 C6H5O
Figure 4.62:Proposed fragmentation scheme of Quercetin-3-O-rhamnoside.
Molecular ion is (M+H) 449 m/z.
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This mean the isolated compound REF-5s67 might belong to flavonoid or phenolic class of plants secondary metabolites. Another developed TLC plates was sprayed with anisaldehyde and on heating the spot turn yellow. This indicated the presence of a flavonoid or related compound.
4.4.4.4.3 UV-Vis spectroscopyof REF-5s67
The isolated compound was prepared at concentration of 50µg/mL in
HPLC grade methanol. Scanning of isolated compound was carried out at 200-
400nm wavelength. A single peak was observed at retention time 12.487 minutes
(Figure 4.63a) with peak purity and ʎmaxat 267nm and 350nm (Figur 4.63b).The
UV-Vis DAD absorption spectra and retention time was simillar with the isolated compound DEF-4MP-1910 (Astragalin) from D. ramosa.
4.4.4.4.4 Mass spectroscopy of REF-5s67
ESI- HR-TOF mass spectra of isolated compound (REF-5s67) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 449m/z suggested the molecular weight of the isolated compound might be 448m/z (M-H). The Other major peaks were observed at 432, 356, 286, 254,
228, 193 and 110 m/z (Figure 4.64). These peaks (Figure 4.64) and their fragmentation patterns were similar with isolated compound DEF-MP-1910 from ethyl acetate fraction of D. ramosa (Figure 4.50).
4.4.4.4.5 Fouriertransfer- infra-red (FTIR) spectroscopy of REF-5s67
The Fourier transfer- infra-red spectroscopy (FTIR) analysis of isolated compound REF-5s67 was presented in figure 4.65. It indicated the stretching of
152
phenol-OH (3303.07cm-1) and also indicated the presence of carbonyl group in the molecule (1653.82 cm-1, C=O stretching) and bending at wave number 1448.48cm-
1 (a typical C=C bending) indicating the presence of aromatic ring system in the molecule.
4.4.4.4.6 NMR spectroscopy of REF-5s67
1HNMR: (in deuterated Methanol): The proton shift value and multiplicity are given in the table below (Table 4.21). Some of the signal pattern was unclear due to overlapping. The proton signal appear at δ 5.25 (1H, d, j= 7.2Hz) with the other resonance in the 1H-NMR spectrum of DEF-4MP1910 was assigned to the anomeric proton of a β-glucose. The integration showed the presence of 20 protons in the structure (Figure 4.66).
13 13 CNMR: (solvent= dCH3OH): The CNMR of isolated compound REF-
5s67 indicated presence of 21 carbon atoms in the structure. The δ= 179.54ppm is the indication of carbonyl group (C=O). The chemical shift values of carbons are presented in the table 4.21. The Distortions enhancement by polarization transfer
(DEPT) showed the carbon atoms with CH2 or unsaturation or quaternary carbons down wards while CH and CH3 carbons upward directions (Fig 4.67). The DEPT analysis of DEF-MP1910 showed that C-2 (δ 159.12ppm), C-3 (δ135.48ppm), C-4
(δ179.54ppm) C-4a (δ105.76ppm), C-5 (δ163.09ppm), C-7 (δ165.97ppm), C-8a (δ
158.53ppm), C-1/(δ 122.86ppm), C-2/ (δ132.27ppm), C-3/(δ116.07ppm), C-5/
(δ116ppm) and C-6/ (δ131.89ppm) showed signals in downwards direction and hence they were quaternary carbons while C-6// (δ 62.66ppm) also showed signal in downwards direction and hence was CH2 carbon.
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HSQC: The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment( HSQC) of isolated compound REF-5s67 provide information that Carbon- C-6//(δ 62.66ppm) has two proton attached to it while Carbons C-2/(δ 132.27ppm), C-3/(δ 116.07ppm), C-5/(δ 116.00ppm), C-6/(δ
131.89ppm), C-6(δ 99.89ppm)and C-8 (δ 94.75ppm) has been bounded to a single proton(fig 4.68).
HMBC:The HMBC plot of isolated compound REF-5s67 showed long range bond correlation between various carbons and protons present in skeleton of the isolated compound. A strong 3 bond coupling long-range correlation was observed between cross peaks from H-1// (δ 5.25ppm) to C-3 (δ 135.48ppm) and
C-6//(δ 62.66ppm). This mean carbon C-3 (δ 135.48ppm) is substituted with β- glucose (Figure 4.69).
4.4.4.4.7Structure and nomenclature of isolated compound REF-5s67
The chemical reagent spraying analysis on TLC plates and UV-Vis absorption spectra indicated that the molecule might have aromatic rings and it might belong to flavonoid class of plants secondary metabolites. The MS analysis showed a molecular ion (M+H) at 4449m/z indicating molecular formula of
448amu. On the basis of HNMR, CNMR (DEPT) and MS analysis the molecular formula was determined as C19H18O11. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.50. The UV-Vis absorption spectra, HSQC and COSY suggested the presence of glucose moiety in the molecule. The HMBC experiments confirm the presence of sugar moiety which is substituted at C-3 of ring B. On the basis of these evidences we proposed
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following structure of isolated compound (REF-5s67) from ethyl acetate fraction of R. brunonii (Figure 4.70). It was identified as “Astragalin”. The other name of this compound is Kampferol- 3- O- β- glucopyranoside. The IUPAC name of this compound is “5,7 – dihydroxy – 2-(4-hydroxyphenyl) – 3-[3,4,5 – trihydroxy – 6-
(hydroxymethyl)tetrahydropyran – 2 – yl]oxy – chromen – 4-one.
4.4.4.5 Identification of isolated compound REF-Mi-01-49
4.4.4.5.1 Detectionspraying reagents
Three TLC plates were developed with isolated compound using n-Butanol:
Acetic acid: water (4:1:5, v/v/v) solvent system. The one TLC plate was sprayed with dragendorff‟s reagent but there was no black or blue coloration. This mean the isolated compound is not belonging to alkaloid class of plant secondary metabolites. The second TLC plate was sprayed with anisaldehyde and upon heating bright yellow spot appeared which indicated that isolated compound might belong to phenolic or flavonoid or related class of plants secondary metabolites.
The third plate was sprayed with 1 percent ethanolic solution of Aluminum chloride and it also showed yellow florescence under longer UV-wavelength. This also added evidences that the isolated compound REF-Mi-01-49 belong to phenolic or flavonoid or related class of plants secondary metabolites
4.4.4.5.2 UV-Vis spectroscopyof REF-Mi-01-49
The HPLC chromatogram showed a single peak at 14.310 minutes and the
UV-VIS absorption spectra of isolated compound (REF-Mi-01-49) exhibited two bands with ʎmax at 260 and 315nm for band II and I respectively (Figure 4.71).
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a
b
Figure 4.63: a) HPLC Chromatogram of isolated compound REF-5s67 b) UV-Vis absorption spectra of isolated compound REF-5s67.
156
Figure 4.64: Mass spectrum (HR-TOF) ESI in positive mode for REF-5s67
Figure 4.65: FTIR analysis of isolated compound REF-5s67
157
Table 4.21: NMR (1H and13C) chemical shift values and DEPT analysis of isolated
compound REF-5s67
Position (Carbon) 1H a 13C DEPT b δ(chemical shift value) δ(chemical shift value) ppm ppm 2 - 159.12 qC 3 - 135.48 qC 4 - 179.54 qC 4a - 105.76 qC 5 - 163.09 qC 6 6.21 (d, 1H, J= 2.05Hz) 99.89 CH 7 - 165.97 qC 8 6.41(d, 1H, J= 1.98Hz) 94.75 CH 8a - 158.53 qC 1/ - 122.86 qC 2/ 8.06 (d, 2H,J= 8.91Hz ) 132.27 CH 3/ 6.88 (d, 2H,J= 1.62Hz ) 116.07 CH 4/ - 161.56 qC 5/ 6.88 (d, 2H,J= 1.62Hz ) 116.00 CH 6/ 8.06 (d, 2H,J= 8.91Hz ) 131.89 CH 1// 5.25 (d, 1H,J= 7.20Hz ) 104.36 CH 2// ¶ 75.74 CH 3// ¶ 78.13 CH 4// ¶ 71.38 CH 5// ¶ 78.41 CH // 6 3.52 (dd, 1H, J= 12.0/5.4) 62.66 CH2 3.69 (dd, 1H, J= 12.0/2.0)
aValues in parentheses are coupling constant (j) in Hz and Multiplicity.bq C=
Quaternary carbon/ a carbon which is not directly bounded to „H‟ (determine by
DEPT spectrum 135o).¶ Unclear signal due to overlapping
158
a
b
c
Figure 4.66:1HNMR (400 MHz, dMeOD) of isolated compound 5s567 (Kampferol-3-O-β-glucipyranoside). a) Normal mode, b)&c) magnified view showing integration values.
159
Figure 4.67:13CNMR (DEPT) of isolated compound REF-5s67.
Figure 4.68:gs-HSQC of isolated compound (REF-5s67) from ethyl acetate
fraction of R. brunonii.
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Figure 4.69:gs-HMBC spectra obtained from isolated compound (REF-5s67) from
ethylacetate fraction of R. brunonii.
Figure 4.70: Structure of isolated compound (REF-5s67) from ethyl acetate
fraction of R. brunonii.
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This suggested that flavonoid is acelyted as it showed 315nm absorption spectra for band I. This is the main property of p-coumaroyl glycosylated flavonols (Negri et al., 2013; Shahat et al., 2005; Guerreroet al., 2009).
4.4.4.5.3Massspectroscopyof REF-Mi-49
ESI- HR-TOF mass spectra of isolated compound (REF-Mi-49) were recorded in positive ionization mode. The mass spectrum showed a molecular ion
(M+H) at 595m/z suggested the molecular weight of the isolated compound might be 594 m/z (M-H). The Other major peaks were observed at 578, 448, 326, 270,
233 and 94 m/z (Figure 4.72).
4.4.4.5.4 Fouriertransfer- infra-red (FTIR) Spectroscopy of REF-Mi-01-49
The FTIR of isolated compound REF-Mi-01-49 was presented in figure
4.73. It indicated the stretching of phenol-OH (3568.01cm-1) and also indicated the presence of carbonyl group in the molecule (1653.82 cm-1, C=O stretching) and
1560.09cm-1, Aromatic C=C bending while 2358.73 cm-1alkyl C=C stretching.
4.4.4.5.5NMRspectroscopy of RE-Mi-01-49
1HNMR: (in deuterated Methanol): The proton shift value and multiplicity are given in the table below 4.22. The proton signal appear at δ 5.24 (1H, d, j= 7.4
Hz) with the other resonance in the 1H-NMR spectrum of REF-MI-01-49 was assigned to the anomeric proton of a sugar moiety (Figure 4.74).
13 13 CNMR: (solvent= dCH3OH): The CNMR of isolated compound REF-
Mi-01-49 indicated presence of 30 carbon atoms in the structure (Figure 4.75). The
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δ= 180.08ppm and 169.37ppm indicated the presence of carbonyl groups (C=O).
The chemical shift values of carbons are presented in the table 4.22. The
Distortions enhancement by polarization transfer (DEPT) showed Twelve
Aromatic methines (C-6 δ 100.6; C-8 δ 95.4; C-2/ δ 132.8; C-3/ δ 116.6; C-5/ δ
116.6; C-6/ δ132.8; C-2/// δ 131.8; C-3/// δ 117.4; C-5/// δ 117.4; C-6/// δ 131.8; C-7///
δ 147.1; C-8/// δ 115.4ppm), five oxygenated methines ( C-1// δ 104.6; C-2// δ
76.43; C-3// δ 76.32; C-4// δ 72.34; C-5// δ 78.6 ppm), One oxygenated methane (C-
6// δ 64.88), ten quaternary aromatic carbons (C-2 δ 159.97; C-3 δ 135.78; C4a δ
106.21;C-5 δ 159.06; C-7 δ 166.62; C-8a δ 163.60; C-1/ δ 123.36; C4/ δ 162.13; C-
1/// δ 127.72; C-4/// δ 161.79 ppm) and two carbonyl carbons ( C-4 δ 180.06; C-9/// δ
169.37ppm).
HSQC:The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment (gs- HSQC) of isolated compound REF-Mi-01-49 differentiated between C/CH2 carbons and CH/CH3 carbons. The gs-HSQC plot for REF-Mi-01-49 is given in figure (Figure 4.76). and is clearly indicated that C-6
δ 100.6; C-8 δ 95.4; C-2/ δ 132.8; C-3/ δ 116.6; C-5/ δ 116.6; C-6/ δ132.8; C-2/// δ
131.8; C-3/// δ 117.4; C-5/// δ 117.4; C-6/// δ 131.8; C-7/// δ 147.1; C-8/// δ 115.4ppm were directly attached with a single proton while C-6// δ 64.88ppm was bounded with two protons.
Correlation spectroscopy/COSY (2-D NMR): The correlation peaks and gs-
H-COSY spectrum of REF-Mi-0149 suggested that proton H-2/// (δ 7.31ppm) was coupled with H-3/// (δ 6.80ppm) and H-6// (δ 4.29 and 4.18ppm) are coupled protons (Figure 4.77).
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HMBC:The heteronuclear multiple bond correlation (gs-HMBC) experiment of isolated compound REF-Mi-01-49 showed long range bond correlation between various carbons and protons present in skeleton of the isolated compound. The gs-HMBC plot of REF-Mi-01-49 is given in Figure 4.79and itconfirmed that B-ring of flavonoid was substituted with glucose moiety at C-3 as
H-1// (δ 5.24ppm) showed a strong correlation with C-3 (δ 135.78ppm). Thegs-
HMBC also indicated that glucose moiety is attached with p-Coumaric acid at C-6// as H-6// protons showed a strong correlation with C-9/// (δ169.37ppm).
4.4.4.5.6 Structure and nomenclature of isolated Compound REF-Mi-01-49
The chemical reagent spraying analysis on TLC plates and UV-Vis absorption spectra indicated that the molecule might have aromatic rings and it might belong to flavonoid class of plants secondary metabolites. The MS analysis showed a molecular ion (M+H) at 595m/z indicating molecular formula of 594 amu. On the basis of HNMR, CNMR (DEPT) and MS analysis the molecular formula was determined as C30H26O13. The proposed fragmentation pattern which corresponded to the MS- ESI (+) peaks (m/z) is given in Figure 4.80.
The information gathered through various spectroscopic techniques (as discussed above) the isolated compound REF-Mi-01-49 was identified as
Tiliroside (Figure 4.79) and the IUPAC name is [(2R,3S,4S,5R,6S) – 6-[5,7 – dihydroxy – 2-(4-hydroxyphenyl) – 4 – oxochromen – 3 – yl]oxy-3,4,5 – trihydroxyoxan – 2-yl]methyl (E) – 3 - (4 - hydroxyphenyl) prop – 2 - enoate.
164
Figure 4.71: HPLC profile of REF-Mi-01-49 obtained from ethyl acetate fraction
of R. brunonii; a) Chromatogram b) UV-Vis DAD Absorption
spectra at 230nm and c) Purity of peak at 14.309 minutes.
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Figure 4.72:MS spectra of isolated compound REF-Mi-49 from EtoAc fraction of
R. brunonii
Figure 473: FTIR analysis of isolated compound REF-Mi-01-49
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Table4.22: NMR (1H &13C) chemical shift values and DEPT analysis of isolated
compound REF-MI-01-49.
Position 1H a 13C DEPT b (Carbon) δ(chemical shift value) δ(chemical shift value)
2 - 159.97 qC
3 - 135.78 qC
4 - 180.06 qC (C=O)
4a - 106.21 qC
5 - 159.06 qC
6 6.14,( m) 100.63 CH
7 - 166.62 qC
8 8.32,(m) 95.43 CH
8a - 163.60 qC
1/ - 123.36 qC
2/ 7.99,(d, j= 8.80Hz) 132.79 CH
3/ 6.82, (d, j= 8.96) 116.65 CH
4/ - 162.13 qC
5/ 6.82, (d, j= 8.96) 116.65 CH
6/ 7.99, (d, j= 8.80Hz) 132.79 CH
1// 5.24, (d, j= 7.4Hz) 104.55 CH
2// 3.42, (m,) ¶ 76.43 CH
3// 3.84,( m,) ¶ 76.32 CH
4// 3.49,( m,) ¶ 72.34 CH
5// 3.90, (m) 78.63 CH
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Position 1H a 13C DEPT b (Carbon) δ(chemical shift value) δ(chemical shift value)
// 6 4.29, (dd, j= 2.08, 11.76 Hz) 64.88 CH2
4.18, (dd, j= 6.52, 11.92Hz )
1/// - 127.72 qC
2/// 7.31, (d, j= 8.5Hz) 131.77 CH
3/// 6.80, (d, j= 8.96Hz) 117.40 CH
4/// - 161.79 qC
5/// 6.80, (d, j= 8.96Hz) 117.40 CH
6/// 7.31, (d, j= 8.5Hz) 131.77 CH
7/// 7.40, ( d, j= 15.92Hz) 147.14 CH
8/// 6.07, ( d, j= 15.88Hz) 115.36 CH
9/// - 169.37 qC (C=O)
aValues in parentheses are coupling constant (j) in Hz and Multiplicity. ¶ Signals overlapped. bq C= Quaternary carbon/ a carbon which is not directly bounded to
„H‟ (determine by DEPT spectrum 135o).
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a
b
c
Figure 4.74:1HNMR (400 MHz, dMeOD) of isolated compound REF-Mi-01-49
(Tiliroside). a) Normal mode, b)&c) magnified view showing
integration values.
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13 Figure 4.75: CNMR (DEPT spectrum 135o) of isolated compound REF-Mi-01-49.
Figure 4.76:gs-HSQC of isolated compound (REF-MI-01-49) from ethyl acetate
fraction of R. brunonii.
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Figure 4.77:gs-COSY of isolated compound (REF-MI-01-49) from ethyl acetate
fraction of R. brunonii.
Figure 4.78:gs-HMBC spectra obtained from isolated compound (REF-MI-01-49)
from ethylacetate fraction of R. brunonii.
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Figure 4.79: Structure of isolated compound REF-MI-01-49 (Tiliroside) from ethyl
acetatefraction of R. brunonii.
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H+ H+
OH OH HO O O HO O (E)
(Z) O O OH O OH OH m/z= 448 HO OH O m/z= 270 C21H19O11 C15H9O5
O H+
C15H17O8 C9H7O2 HO O HO OH H+ HO O C15H9O5 HO O O m/z= 326 O O (E) (E) (E) C15H17O8
O O OH O OH OH OH HO m/z= 595 OH OH M+H C9H12O7 H+ H+ C6H5O HO
O O H+ (E) OH HO HO O O OH O HO O O O m/z= 94
(E) C6H5O HO O HO OH OH m/z= 578 m/z= 233 O C30H25O12 C9H12O7
Figure 4.80:Proposed fragmentation scheme of Quercetin-3-O-rhamnoside.
Molecular ion is (M+H) 595m/z.
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4.5BIOACTIVITIES OF ISOLATED COMPOUNDS
All the six isolated compounds (Iriflophenone 3-C-β-D glucopyranoside,
Mangiferrin, Isomangiferrin, Astragalin, Quercetin-3-O-rhamnoside and
Tiliroside) were subjected for the evaluation of their antioxidant and cytotoxic potential by DPPH free radical bioassay and Brine Shrimp Lethality Test (Table
4.24).
4.5.1FreeRadical Scavenging Potential of Isolated Pure Compounds
All the isolated pure compounds from D. ramosa and R. brunonii were subjected to DPPH free radical scavenging assay.Table 4.23 showing the free radical scavenging potential of isolated compounds at various concentrations. The
IC50 was calculated by using best fit line regression equation (fig 4.81).
The minimum free radical scavenging potential was shown by tiliroside i.e.
38.8, 19.87, 16.07, 13.21 and 12.84 percent at 625, 312.5, 156.25, 78.12 and
39.06µg/mL respectively. All other compounds showed significant free radical scavenging potential in DPPH free radical scavenging assay. More than 50 percent free radical scavenging was observed at 78.12µg/mL concentration or above in case of all the compounds except Quercetin-3-O-rhamnoside (44.24%) and tiliroside(13.21%).
In terms of IC50, except tiliroside(923.63µg/mL) all compounds under investigation (iriflophenone – 3 – C – β-D glucopyranoside, Mangiferrin, isomangiferrin, astragalin and Quercetin-3-Orhamnoside) showed significant antioxidant potential. i.e. low IC50(70.81µg/mL, 60.34µg/mL, 55.98µg/mL,
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50.91µg/mL and 79.04µg/mL respectively). Ascorbic acid was used as standard.
The IC50 of ascorbic acid was calculated 57.97µg/mL. It is important to notice that
IC50 of astragalin (50.91µg/mL) and isomangiferrin (55.98µg/mL) were less than ascorbic acid (57.97µg/mL). This mean the antioxidant potential of these two compounds is better than standard ascorbic acid. The Mangiferrin also showed
IC50very close to standard (Table 4.23).
4.5.2 Cytotoxic Potential of Isolated Compound Against Brine Shrimps
The isolated pure compounds i.e. Iriflophenone 3-C-β-D glucopyranoside,
Mangiferrin, Isomangiferrin, Astragalin, Quercetin-3-O-rhamnoside and Tiliroside were test against brine shrimp nauplii for the assessment of their cytotoxicity. The results were expressed in terms of mean percent lethality and LD50(Table 4.24).The
LD50 of individual compound was calculated by plotting concentration versus mean percent lethality and from best fit line (Linear regression as shown in figure
4.82).
The maximum cytotoxicity was exhibited by tiliroside at all concentration i.e. 33.33, 53.33, 73.33 and 96.67 percent at 25, 50, 100 and 250µg/mL respectively. In all the experiments, the tiliroside showed 100 percent lethality to brine shrimp nauplii at concentration 275µg/mL or above. The minimum mean percent lethality was found in case of Mangiferrin (25 percent) at 500µg/mL followed by Astragalin (31.67percent), isomangiferrin(33.33 percent) and iriflophenone 3-C-β-D glucopyranoside (56.67 percent). At concentrations
25µg/mL mangiferrn, isomangiferrin, and Quercetin-3-O-rhamnoside did not cause death of any nauplii and hence showed zero lethality.Queretin-3-O-rhamnoside
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showed 0% lethality at 25µg/mL but 8.33, 13.33, 45 and 68.33 percent lethality at
50, 100 and 250µg/mL respectively.
In terms of LD50, the lowest LD50 was found in case of tiliroside
(50.42±0.88µg/mL) followed by Queretin-3-O-rhamnoside (345.22 ± 0.92 µg/mL) and astragalin (391.72±0.09µg/mL).Iriflophenone 3-C-β-D glucopyranoside showed moderate cytotoxic potential against brine shrimp nuplii. However, other isolated compounds showed higher LD50 and it represented their less cytotoxic potential.
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Table 4.23:Mean percentage scavenging potential and IC50 of isolated pure
compounds
Mean percentage scavenging
-
-
3
-
-
D
-
β
n
-
de
roside
C
-
3
rahmnoside
Tili
Astragalin (standard)
-
Mangiferrin
Quercetin
Ascorbicacid
Isomangiferri
glucopyranosi
Iriflophenone O
Conc. Conc. (µg/mL) 625 89.13 91.34 90.14 93.45 90.56 38.8 96.28
312.5 86.81 89.23 87.29 91.27 89.83 19.87 94.88
156.25 64.8 71.92 74.11 83.08 73.43 16.07 81.03
78.12 53.57 53.29 57.71 64.46 44.24 13.21 58.3
39.06 25.94 41.78 46.77 37.45 22.76 12.84 25.4
19.53 19.6 17.16 19.7 24.85 11.67 11.06 16.05
9.77 12.53 13.51 10.51 11.48 3.66 10.64 10.78
4.88 10.46 8.29 5.32 7.65 1.64 10.22 8.7
50
0.93
IC
70.81 60.34 79.04 57.97
55.98 50.91
±0.18 ±0.39 ±0.92 ±0.84 ±1.23 ±
± 0.21 ±
923.63 (µg/mL)
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Figure 4.81: Best fit line regression equation (Calculation of IC50 for isolated pure
compounds).
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Table 4.24: Mean percentage lethality and LD50of isolated compounds in Brine Shrimp Lethality Test (BSLT)
25 µg/mL 50 µg/mL 100 µg/mL 250 µg/mL 500 µg/mL
. Conc
Pure isolated After 24 hours After 24 hours After 24 hours After 24 hours
compounds Mean % Mean % Mean % Mean % Mean %
%
Live Live Live Live Live
Dead Dead Dead Dead
Dead lethality lethality lethality lethality lethality
Total
Death
% % death % death % death % death
Iriflophenone-3- 1 20 19 1 5 18 2 10 16 4 20 13 7 35 10 10 50 C-β-D 2 20 19 1 5 5 18 2 10 8.33 17 3 15 18.33 12 8 40 40 8 12 60 56.67 glucopyranoside 3 20 19 1 5 19 1 5 16 4 20 11 9 45 8 12 60 1 20 20 0 0 20 0 0 19 1 5 18 2 10 16 4 20 Mangiferrin 2 20 20 0 0 0 20 0 0 o 19 1 5 5 17 3 15 11.67 15 5 25 25 3 20 20 0 0 20 0 0 19 1 5 18 2 10 14 6 30 1 20 20 0 0 20 0 0 19 1 5 18 2 10 13 7 35 Isomangiferrin 2 20 20 0 0 0 20 0 0 0 18 2 10 6.67 18 2 10 10 14 6 30 33.33 3 20 20 0 0 20 0 0 19 1 5 18 2 10 13 7 35 1 20 18 2 10 18 2 10 18 2 10 15 5 25 12 8 40 Astragalin 2 20 19 1 5 6.67 18 2 10 10 17 3 15 11.67 15 5 25 23.33 14 6 30 31.67 3 20 19 1 5 18 2 10 18 2 10 16 4 20 15 5 25 1 20 15 5 25 18 2 10 17 3 15 11 9 45 6 14 70 Quercetin-3-O- 2 20 14 6 30 0 18 2 10 8.33 18 2 10 13.33 11 9 45 45 7 13 65 68.33 rhamnoside 3 20 15 5 25 19 1 5 17 3 15 11 9 45 6 14 70 1 1 20 13 7 35 10 50 6 14 70 1 19 95 0 20 100 0 1 10 Tiliroside 2 20 13 7 35 33.33 9 55 53.33 5 15 75 73.33 0 20 96.67 0 20 100 100 1 0 1 3 20 14 6 30 9 55 5 14 75 1 19 95 0 20 100 1
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Table 4.24: Mean percentage lethality and LD50 of isolated compounds in Brine Shrimp Lethality Test (BSLT) Iriflophenone-3-C-β-D Mangiferrin Isomangiferrin Astragalin Quercetin-3-O- Tiliroside Standard LD 50 glucopyranoside rhamnoside (Nicotine) (µg/mL) 406.67±0.35 969.77±0.67 768.92±0.81 391.72±0.09 345.22±0.59 50.42±0.88 46.76±4.81
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Figure 4.82:Best fit regression line equation to calculate LD50 for isolated
compounds in Brine shrimp lethality test (BSLT).
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Chapter 5
DISCUSSION
Medicinal plants have played a pivotal role in the healthcare of human for millennia. Medicinal plant species have been used worldwide as a vital source in the discovery of new drugs. (Ahn, 2017). In recent past, since 1981 to 2010 a total
1073 small organic molecules have introduced worldwide as drugs, out of which more than 60 percent belongs to natural products (Newman and cragg, 2012). The scientific evaluation of medicinal plants follows logical pathways. Usually plants are selected for scientific evaluation by following the ethnomedicinal lead and traditional uses of medicinal plants species in a particular geographical area. In the present investigation, the selected plants species (Bidens biternata,Arisaema flavum, Quercus leucotricophora, Dryopteris ramosa and Rosa brunonii) has been using by the inhabitants of Gallyat region of Pakistan for their primary health care
(Prajapati et al., 2003, Gilani et al., 2006, Patni et al., 2012, Abbasi et al., 2015
Amjad et.al., 2017) . The selected plants were never been evaluated for their antioxidant and cytotoxic potential and isolation of bioactive phytochemicals. This is the first report regarding antioxidant, cytotoxic potential and isolation of bioactive phytochemicals from these selected plant species of Gallyat region of
Pakistan to the best of our knowledge.
5.1 BIOLOGICAL ASSAYS ON CRUDE METHANOL EXTRACT
The antioxidant properties of crude extracts were evaluated by DPPH free radical scavenging in-vitro bioassay. Crude extract of D. ramosa showed percentage inhibition 23.74±0.82 percent at concentration of 25µg/mL which is
181
182
highest among all tested crude extract at this concentration. However, at concentrations 50µg/mL the crude extract of Q. leucotricophora (38.07±0.06 percent) showed more free radical percentage inhibition than D. ramosa
(30.85±1.64percent). At all other concentrations tested (100 µg/mL, 150 µg/mL,
200 µg/mL and 250 µg/mL), the crude extract of D. ramosa showed better free radical scavenging potential than all other crude extracts (Figure 4.2). The lowest free radical scavenging was exhibited by crude extract of B. biternata and A. flavum. At highest concentration 250 µg/mL, the percentage inhibition was
45.35±0.9 percent and 56.41±0.4 percent for crude extract of B. biternata and A. flavum respectively. In contrast, at same concentration (250 µg/mL), crude extracts of D. ramosa, Q. leucotricophora and R. brunonii showed 93.78±0.2, 86.03±0.2 and 78.9±0.7 percent free radical inhibition respectively. These results showed a dose dependent free radical scavenging among all the crude extrats tested. This mean increasing the concentration of crude extract, increases the free radical scavenging potential of the extracts. So, we have used mean percentage scavenging to standardized the results and to calculate IC50 to determine the antioxidant potential of crude extracts of selected plants. On the other hand, present finding suggested that B. biternata and A. flavum might have poor concentration of antioxidants as compared to Q. leucotricophora, D. ramosa and R. brunonii. The standard (ascorbic acid) showed free radical scavenging 37.09, 54.3, 77.8, 92.02,
96.87 and 97.49 percent at 25, 50, 100, 150, 200 and 250µg/mL respectively. A two way ANOVA was conducted that examined whether our independent variables
(CME and conc.) and their interaction (CME*Conc.) have a statistically significant effect on dependent variable (%scavenging) or not. In sig. column of ANOVA
183
table we see that there is significant effect of independent variable (F 25, 72 =
168.763, p< 0.05) on dependent variable at p= 0.05 level (Table 4.2). Similar finding was reported by several scientists working in this field. For instance,
Soareetal., 2012 studied the antioxidant potential of Romaian ferns (Athyriumfilix- femina, Dryopteris affinisand Dryopteris filix-mas). Sarwar et al., 2015, discussed the antioxidant properties of Quercus incana collected from Abbottabad, Pakistan.
Garniet al., 2017, reported the antioxidant potential of Madeni rose (Rosa damascene). Yang et al.,2006, reported the free radical scavenging potential of
Bidens pilosa. Baba and Malik (2014) studied the antioxidant properties of
Arisaemajacquemontii. In terms of IC50, the lowest IC50 mean maximum antioxidant potential of the extract. The crude methanol extract of D. ramosa showed maximum antioxidant potential (IC50 =88.67±0.73µg/mL) followed by Q. leucotricophora (IC50= 95.51±0.1973µg/mL) and R. brunonii (IC50
=131.41±0.1873µg/mL).
The crude extract of R. brunonii showed highest cytotoxicity against Brine shrimps than all other crude extracts at all concentrations tested. All the concentrations were tested in triplicate and mean percentage lethality was calculated (Table 4.3). The mean percentage lethality of brine shrimp naupli caused by crude extract of R. brunonii was 30, 43.33, 58.33 and 78.33 percent at
10, 100, 300 and 600µg/mL respectively. The least cytotoxicity exhibited by crude extract of D. ramosa (6.33, 11.67, 13.33 and 23.33 percent respectively) at same concentrations (Figure 4.3). Nicotine a known cytotoxic compound was used as reference standard (Table 4.4). LD50 of crude extract of R. brunonii was
220.83±2.16µg/mL which is least among all the crude extract tested. The standard
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showed LD50 = 42.50±4.81µg/mL. Statistically, there is significance difference between different crude extracts and their cytotoxicity toward Brine shrimps at p=0.05.In recent published literature, Khan et al., 2017 reported that R. brunonii has showed maximum cytotoxicity among all the extracts studied. Krishnaraju et al. (2005) has assessed cytotoxicity of 120 medicinal plants by using Brine Shrimp
Lethality test. Many researchers used BSLT to determine cytotoxicity of extracts because BSLT is simple reliable and convenient method (Krishnaraju et al., 2005).
Potato disc antitumor assay was used to confirm antitumor (cytotoxic) potential of medicinal plants (Atalay, 2001). The viability test for Agrobacterium tumefaciens against all the selected crude extracts showed no inhibition zone indicated that all the crude extracts were not active against A. tumefaciens.
However, a previous study (Galsky et al., 1980) showed that CME of Quercus dilatata was active against A. tumefaciens. The known antibiotic cefotaxime showed inhibition zone which suggested anti A. tumefaciens activity (Figure
4.4).Usually, more than 20 percent tumor inhibition was considered as significant tumor inhibition for crude plant extracts (Ferrigni et al., 1982). Our results suggested that tumor percentage inhibition increases by increasing the concentration of crude extract.At concentration 10µg/mL, all the crude extracts showed zero percent tumor inhibition while the control (Vincristine) showed 3 percent tumor inhibition. The crude extract of R. brunonii showed maximum tumor inhibition (12.09, 42.45and 72.73 percent) among all the crude extracts at all concentrations (100, 500 and 1000µg/mL) tested. In comparison the control showed 33.36 percent and 100 percent tumor inhibition at concentration 100 and
500µg/mL respectively. At concentration of 100µg/mL, the crude extract of R.
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brunonii showed 12.09 percent and D. ramosa showed 9.09 percent tumor inhibition while A. flavum, Q. leucotricophora and B. biternata showed 6.09 percent tumor inhibition. At 500µg/mL,R. brunonii inhibits 42.45 percent tumors followed by D. ramosa (36.36 percent) and A. flavum (36.36 percent) while Q. leucotricophora and B. biternata inhibits 33.36 percent and 27.27 percent tumors respectively. At highest concentration (1000µg/mL), the mean percent tumor inhibition were 72.73, 60.63,57.54, 48.45 and 42.45 percent among crude extracts of R. brunonii, D. ramosa, A. flavum, Q. leucotricophora and B. biternata respectively (Figure 4.4). Hussain et al., 2007 discussed similar observation by reporting the Cytotoxic and Antitumor Potential of Fagoniacretica L.In terms of
IC50, minimum IC50 was showed by crude extract of R. brunonii (IC50=
655.65±1.45µg/mL) followed by D. ramosa(IC50= 790.51±1.16µg/mL). This suggested the presence of antitumor and cytotoxic phytochemicals in these extracts. While crude extract of Q. leucotricophora (IC50= 1078.53±0.74 µg/mL) showed minimum antitumor potential. In a similar study, Q. dilatatashowed tumor inhibition (Jamil et al., 2012) but the crude extract of Q. dilatataalso showed antibacterial activity and it was believed that Q. dilatatashowed anti-tumor activity due to its antibacterial activity(Galsky et al., 1980).
5.2 BIOACTIVITIES OF FRACTIONS
On the basis of bioactivities of CME of all selected plants species, three plants species i.e. D. ramosa, Q. leucotricophora and R. brunonii were selected for further study. Among all the fractions obtained from selected CME of plants, the ethyl acetate fraction of D. ramosa, aqueous fraction of D. ramosa and Ethyl
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acetate fraction of R. brunonii showed maximum free radical scavenging potential.
The Ethyl acetate soluble fraction obtained from D. ramosa showed maximum scavenging abilities than all other fractions tested. Then aqueous fraction of the same plant (D. ramosa) exhibited second best scavenging potential followed by ethyl acetate fraction of R. brunonii while the least scavenging activity was shown by n-Hexane soluble fraction of Q. leucotricophora followed by chloroform fraction of D. ramosa(table 4.10). The lowest IC50 (57.85±0.24µg/mL and
108.98±0.28µg/mL) were calculated for ethyl acetate and water soluble fractions of D. ramosa. While IC50for ethyl acetate fraction of R. brunonii was calculated as
IC50 186.24±0.8928µg/mL.The standard (ascorbic acid) a known antioxidant compound showed IC50 41.59±0.10 µg/mL. Similar observations were also reported by Andrensek et al., 2004 and Karimiet al., 2015 working on Quercus species.
The aqueous fractions of D. ramosa and Q. leucotricophora also showed some cytotoxic effects on brine shrimps but their percentage lethality were too less than ethyl acetate fraction of R. brunonii. For instance the percentage lethality caused by ethyl acetate fraction of R. brunoniiat 900 µg/mL was 91.67 percent while at same concentration aqueous fractions of D. ramosa and Q. leucotricophora showed 51.11 percent and 52.22 percent lethality respectively
(Table 4.6). The LD50 calculated for ethyl acetate fraction of R. brunonii was
LD50405.43±4.8µg/mL while LD50830.95±2.0µg/mL was calculated for aqueous fraction of D. ramosa. The significant lethality of various fractions is an indicative of presence of potent cytotoxic components which warrants further investigation
(Krishnaraju et al., 2005).
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Potato disc antitumor assay (PDA) is a valuable tool which indicates the presence of antitumor components in an extract. PDA was a sensitive, bench-top antitumor bioassay for chemicals and extracts that interfere with the mitosis during cell division irrespective of their mechanism of inhibition (Coker et al.,
2003).Results indicated the inhibition of tumor by plant‟s extracts and their fractions were increases by increasing the concentration of the extracts. All the experiments were done in triplicate and mean tumor inhibition were calculated with standard deviation. The IC50 values were calculated for each extract and fractions to standardize the results at any given set of concentrationstested. In present study, the ethyl acetate fraction of R. brunonii while aqueous and ethyl acetate fractions of D. ramosa also showed significant tumor inhibition (Table
4.15). In terms of IC50, the lowest IC50 was shown by ethyl acetate fraction of R. brunonii (753.68±0.48µg/mL) followed by aqueous fraction of D. ramosa
(793.23±0.31µg/mL) and ethyl acetate fraction of D. ramosa (834.99±0.24µg/mL).
In comparison with control, the vincristine a well-known antitumor pure compound showed IC50 232.34±0.58µg/mL. Antitumor potential of different plants extracts and their fractions were evaluated by many researchers working in this field by using Agrobacterium tumefaciens. (Islam et al.,2010; Kanwal et al.,2011 and
Turkeret al., 2012).
5.3 QUALITATIVE AND QUANTITATIVE ESTIMATION OF
PHYTOCHEMICALS
Preliminary tests for phytochemicals indicated that CME of D. ramosa is rich in phenolic andflavonoid groups of plants metabolites while CME of D.
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ramosalack alkaloid group ofsecondary metabolites. Similar results were also presented by Shakooret al., 2013. CME of Q.leucotricophora showed presence of alkaloids, steroids and flavonoids but in contrast to anotherspecies of the same genus Q. infectoria which showed negative results for these Phytochemicals (
Roshni and Ramesh 2013). Another study indicated the presence of steroids and flavonoids inQ. Robr(Din and Rauf, 2012). This suggested that different species of the same genus may have different chemical constituents. The CME of R. brunonii also showed presence ofplants secondary metabolites like alkaloids, phenolics, flavonoids and steroids. CME of R.damascena also showed similar results (Tatke et al., 2015). CME of A. flavum showed positiveresults of alkaloids, steroids, flavonoids and phenolic compounds. These results are inaccordance with previous study on the same plant species (Kunwar et al., 2010, Kant et al., 2016).The fractions obtained from CME of D. ramosa, R. brunonii and Q. leucotricophora showed thatmajority of flavonoids and phenolics secondary metabolites were concentrated in ethyl acetate fraction and aqueous fraction (Table 4.17). These results are in accordance withthe previous studies (Roshni and Ramesh 2013;
Aliyu et al., 2014) on preliminary screening ofphyto-chemicals.
The total phenolic contents (TPC) were determined by a linear regression equation (y= 0.0071x+ 0.4332, R2= 0.9606) primed with a Gallic acid standard calibration curve (Figure 4.8), andexpressed in values of Gallic acid equivalent
(GAE). The CME of D. ramosa showed maximumTPC (122.64 ± 4.35 µg/mg
GAE) followed by CME of Q. leucotricophora(74.06 ± 7.74 µg/mgGAE) and R. brunonii(66.73 ± 3.89 µg/mg GAE) as shown in table 4.13. The TPC of CME
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interms of GAE were reported by many scientists like Fatma et al., 2013; Karimi and Moradi 2015; Garniet al., 2017 etc. The total flavonoid contents (TFC) were calculated determined bystandard method and using Querctin standard calibration curve (Figure 4.9) and linear regressionequation (y=0.0063x + 0.395, R2 = 0.9697)
(Figure 4.9). The TFC were expressed in terms of Quercetin equivalent (QE). The
CME of D.ramosa showed maximum TFC (61.42 ± 17.89µg/mg QE) followed by
CME of Q.leucotricophora (53.60 ± 1.43µg/mg QE) and R. brunonii (46.51 ±
0.96µg/mg QE) asshown in table 4.14. The quantitative estimation of TFC of crude extracts in terms of QE was reported in literature (Kashani et al., 2011; Ali et al.,
2012).
5.4 IDENTIFICATION OF ISOLATED COMPOUNDS FROM QUEOUS
FRACTION OF D. ramosa
A total three compounds were isolated from aqueous fraction of Dryopteris ramosa(Figure 4.40) by using chromatographic techniques. The compound-1 isolated from aqueous fraction of Dryopteris ramosa(DAF-MI-01) was a yellow orange powder with melting point 272-2780C. When loaded on TLC plates, it gave a yellow spot and inflorescence under UV light at 254-360nm upon sprayed with anisaldehyde and aluminum chloride followed by heating. This indicated that compound may have belonged to phenolic class of secondary metabolites. The identification of phenolic compounds by spraying reagents has been reported in literature (Esmaeili, 2011).
In HPLC analysis the DAF-MI-01 was eluted with methanol in aqueous buffer (15 mM ortho-H3PO4 and 1.5 mM Bu4NOH) with RT. 6.467 minutes
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indicated the hydrophilic nature of molecule. The UV-Vis spectroscopy with DAD at 230nm showed a ʎmax 298nm indicated the presence of unsaturation in the molecule. Similar data (ʎmax 294nm) was recorded by Beer et al. (2012) from the aqueous fraction of Cyclopiasubternata (Honeybush). The mass analysis showed that the molecular mass of isolated compound DAF-MI-01 was 408 (molecular ion
409m/z M+H). The major ions fragments in ESI positive mode were given in figure 4.10. A peak at 392m/z (M+H-17) corresponds with the hydroxyl group in the molecule. Another major peak was found at 288m/z (M+H-121) showed the presence of C-glycoside structure of the molecule. Additionally the fragment peak at 246m/z represented the loss (M+H-163) which corresponds to C-glycoside
(Murakami et al.,1986; Beer et al., 2012).
The 1H-NMR of the isolated compound (DAF-MI-01) indicates the presence of a signal at δ 4.88ppm (j=9.8Hz) which is suggested to be due to anomeric proton of sugar moiety (Bock and Tho¨gersen, 1982). They concluded that 1H spectra of carbohydrates do contain some well-resolved signals of anomeric protons (δ 4.4– δ5.5ppm). The signal present at δ 3.31-3.35ppm represented the solvent used (deuterated methanol) (Fulmer et al., 2010). The 1HNMR chemical shift value of 6.75, 6.78, 7.59, 7.61 ppm indicated the existence of aromatic ring system (δ >6.5ppm) in the molecule (Fergoug and Bouhadda, 2014). In the 1H-
NMR spectrum, two sets of doublets at δH7.60 and 6.79ppm revealed the presence of a para-substituted symmetric phenolic ring derivative with two equivalent pairs of ortho-coupled protons, and the aromatic proton of the other phenolic ring displayed a singlet at δH 5.94ppm (Figure 4.14, Table 4.21). Similar observation was also presented by Jinetal. (2009). The 13C-NMR indicates the presence of 19
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carbons and chemical shift down field δ= 198.89ppm suggested the presence of carbonyl group (C=O) in the structure. The chemical shift signals of anomeric carbon (δ76.61ppm) indicated the presence of C- glycoside (Xu et al., 2012). In the
DEPT spectrum, six carbon signals from the sugar moiety resonated at δ C 76.61
(CH), 73.74 (CH), 79.93 (CH), 71.46 (CH), 82.55 (CH) and 62.48 (CH2). All the information described above supported that compound DAF-MI-01 was a mono- glycoside derivative of iriflophenone.
The ge-HSQC (Heteronuclear Single Quantum Coherence) of DAF-MI-01 was shown in figure 4.16.The spectrum was readily analyzed with the horizontal and vertical coordinates of each peak defining the chemical shift of a proton and its directly attached carbon respectively. The ge-HSQC of DAF-MI-01 showed a signal which correspond to H-resonance of δ6.75ppm and δC115.50 ppm. This showed that carbon (δC 115.50 ppm) was bonded directly to one proton
(δ6.75ppm). Similarly, Hδ5.94 ppm corresponded to δC 96.61ppm. But two H- resonance signals were present at Hδ3.84 and Hδ3.82ppm which correspond to carbon at δC62.48ppm. This indicated that carbon at δ62.61ppm was directly attached to two protons.The data is in accordance with the literature (Jin et al.,
2009). The spectra obtained as a result of COSY experiment represented the signals of proton (1H) along both X and Y axis (Keeler 1990).COSY spectra show two types of peaks. Diagonal peaks and cross peaks. The Diagonal peaks present along the diagonal of the plot and are much more similar to the peaks in 1H NMR.
On the other hands, the cross peaks are present off the diagonal and are representing coupling between pair nuclei like multiplicity and splitting pattern in
1-D-1HNMR (Keeler 1990). The H-COSY spectrum of isolated compound DAF-
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MI-01 suggested that H-1/ (δ 4.88 ppm, d, j= 9.88) is coupled with proton of H-2/
(δ 3.89, m). Similarly H-13 (δ 7.61, s) was coupled with H-12(δ 6.78, s) and H-10
(δ 6.75, s) with H-9 (δ 7.59, s) (Figure 4.13). Similar cross peak relationship was found in literature (Jin et al., 2009).
Heteronuclear multiple-bond correlation spectroscopy (HMBC) is a 2D-
NMR which is used for the elucidation of molecular structure, function and dynamics. This long range correlation experiment enables us to predict neighboring carbons which are at 2-3 bond distance. HMBC has wide range of applications (Vasaviet al., 2011).The HMBC plot of DAF-MI-01 was given in
Figure 4.18.Three bonds coupling were observed between H-3 (δ5.94ppm) to C-
1(δ107.17ppm) and C-5(δ104.67ppm). Similarly 3-j-CH correlation was noticed between H-9 (δ 7.59ppm) to C-7 (δ 198.89ppm) & C-11 (δ 162.90ppm) and H-10
(δ 6.75ppm) to C-12 (δ 115.50ppm) & C-8 (δ 133.26ppm). Abundant 3-j-CH long range correlation signals were observed with cross peak H-1/ (δ 4.88ppm) to C-3/
(δ 79.93ppm), C-5/ (δ 82.55ppm), C-6 (δ 163.42ppm) and C-4 (δ 161.44ppm).
This strong correlation of H-1/ (δ 4.88ppm) with distant (one bond distance) carbons suggested that molecule is substituted with glucose moiety at C-5 (δ
104.67ppm).Based on the UV-Vis spectral data and MS fragmentation patterns,
1 H-NMR, DEPT analysis, the proposed molecular formula is C19H20O10and molecular weight is 408amu. This compound (DAF-MI-01) was identified as iriflophenone-3-C-β-glucopyranoside, which has previously been isolated and described by Kokotkiewicz etal., (2012).The DEPT, COSY, HMQC and HMB
NMR spectra show complete correlation between the protons and carbons. The structure of DAF-MI-01 was elucidated to be identical to that of iriflophenone-3-
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C-β- D glucopyrranoside, and our data for this compound agree with those described in the literature (Murakami et al., 1986). DAF-MI-01 has also been found in Hypodematiumfaurieiand Hypodematiumcrenatum (Murakami etal.,
1986) and in the leaves of Aqualaria sinensis L. (Fing et al., 2011) and Mangifera indica(Zhang etal., 2011). This is the first report of the isolation of benzophenoneglucosides from Genus Dryopteris to be the best of our knowledge.
The isolation of benzophenones might indicate the presence of xanthones in the plant (Kaya et al.,2011).
Compound-2 (DAF-MI-02.1) and Compound-3 (DAF-MI-02.2) were isolated from group 3, aqueous fraction of Dryopteris ramosa. The Rf for DAF-
MI-02.1 was 0.49 while for DAF-MI-02.2 was 0.51. Both were easily soluble in water, methanol and DMSO. The melting point of DAF-MI-02.1 was determined as 270-2740C (Finnegan, 1968) while DAF-MI-02.2 melt at 259-2620C. When applied on TLC for spraying reagent analysis, both showed yellow florescence under UV light at 254 after spraying with ferric chloride followed by heating
(Bhuvaneswari, 2013). The UV-Vis spectroscopic analysis of DAF-MI-02.1 showed ʎmax at 258, 318 and 366nm with DAD at 230nm and RT 8.520 minutes.
The DAF-MI-02.2 had a RT 8.729 minutes and ʎmax at 255, 316 and 365nm. These information indicated that both DAF-MI-02.1 and DAF-MI-02.2 might be the isomers of each other and they might be benzophenone or its derivative (Beelders et al., 2014.). The xanthones, the sub-class of phenolic compound and derivatives of benzophenone have ʎmax between 230-245, 250-265, 305-330 and 340-400nm
(Harborne, 1998).
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The mass analysis showed that the molecular mass of isolated compounds
DAF-MI-02.1 and DAF-MI-02.2 was same as 422 (molecular ion 423m/z M+H).
The major ions fragments in ESI positive mode for DAF-MI-02.1 was given in figure 4.24 while figure 4.32 showing the fragment ion peaks of ESI MS for DAF-
MI-02.2. A peak at 406m/z (M+H-17) corresponds with the hydroxyl group in the molecule. The fragment peat at 260m/z represented the loss (M+H-163) which corresponds to C-glycoside in the structure (Murakami et al., 1986: Beer et al.,
2012).
In xanthones, the 1H-NMR signals predominantly appear between 0-14 ppm depending upon the substitutions (Negi et al., 2013). The signlets δ13.77ppm
(DAF-MI-02.1) and 13.35ppm (DAF-MI-02.2) suggested that aromatic ring of xanthone structure was substituted with hydroxyl proton at C-1 while singlet δH
7.35(DAF-MI-02.1) and singlet δH7.34ppm (DAF-MI-02.2) indicated that aromatic ring was not substituted at C-8 with hydroxyl group (Kaldas et al., 1974).
1H-NMR spectrum of DAF-MI-02.1 showed a complex 7-spin system between 3.0 and 5.0 ppm. Analysis of this second order system revealed coupling constants typical of a glucose moiety (Silva and Pinto, 2005). 1H-NMR spectrum of DAF-
MI-02.2 in d-MeOD also showed three singlets very close to the spectrum of DAF-
MI-02.1 and complex 7-spin system between 3.0 and 5.0 ppm. Talamond et al.
(2008) reported broad signals for DAF-MI-02.2 between 3.0 to 5.0ppm in d6-
DMSO that did not allow the calculation of coupling constants (Talamond etal.,
2008).
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13C-NMR spectra for DAF-MI-02.1 and DAF-MI-02.2 showed that both have nineteen carbons in their structures. A signal at δC 61.65ppm which represent the carbon of primary alcohol in glucose i.e C-6/. The chemical shift for carbonyl was 179.18ppm (DAF-MI-02.1) and 179.32ppm (DAF-MI-02.1) were indicating that either C-1 or C-8 was substituted with hydroxyl group (Purev et al., 2002).
The chemical shift signals of anomeric carbon (δC 73.26 for DAF-MI-02.1 and δC
73.50ppm for DAE-MI-02.2) indicated the presence of C- glycoside (Xu et al.,2012). The Distortions enhancement by polarization transfer (DEPT) analysis of DAF-MI-02.2 showed that C-4 (δ=104.9ppm) is a quaternary carbon instead of
C-2 (δ=97.6ppm). This might be due to sugar moiety substitution at C-4. While the
DEPT analysis of DAF-MI-02.1 showed that C-2 (δC 107.70ppm) is a quaternary carbon instead of C-4(δC 156.38ppm). This might be due to the presence of C- glycoside substitution at C-2(δC 107.70ppm) in DAF-MI-02.1. The HSQC spectra of both DAF-MI-02.1(Figure 4.23) and DAF-MI-02.2(Figure 4.31) confirmed the fact that C-2 is a quaternary carbon in DAF-MI-02.1 while C-4 in case of DAF-
MI-02.2. The HMBC plot of DAF-MI-02.1(Figure 4.28) showed that it was substituted with C-glycoside at C-2 and based on these observations (ESI-MS, H-
NMR, C-NMR, DEPT, HSQC&HMBC) DAF-MI-02.1 was identified as mangiferin (Fujita and Inoue, 1982; Catalano et al., 1996; Talamond et al., 2008).
The HMBC plot of DAF-MI-02.2 (Figure 4.38) showed that DAF-MI-02.2 has glucose moiety attached at C-4 as C-glycoside. Based on ESI-MS, H-NMR, C-
NMR, DEPT, HSQC &HMBC data and value published for the 1,3,6,7- tetrahydroxyxanthone, the DAF-MI-02.2 was identified as isomangiferin.
Mangiferin and isomangiferin are C-glucosyl-xanthones. Mangiferin was
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isolated from Mangiferaindica L. (Anacardiaceae) and named euxanthogen in
1908 by Wiechowski(Gorter, 1922). It has also been successively called alpizarin, aphloïol, chimonin, hedysarid and shamimin, depending on the author and on the plant analyzed. Its structure, 2 – C – β – D – glucopyranosyl-1,3,6,7 - tetrahydroxyxanthone, was only established in 1957 by Iseda(Iseda.
1957).Isomangiferin, isolated later from Anemarrhenaasphodeloides Bunge
(Asparagaceae), was identified as 4-C-β-D-glucopyranosyl-1,3,6,7- tetrahydroxanthone (Aritomi and Kawasaki, 1970).The great dispersal of mangiferin-containing families and species in the plant kingdom suggests that mangiferin could be a marker of specific adaptation to environmental modifications. Nevertheless, studying bearded Iris species, Williams etal. (1997) considered that O- and C-glucosyl-xanthones could be used as phylogenetic and evolutionary markers at the sub-family and/or genus level.
5.5 IDENTIFICATION OF ISOLATED COMPOUNDS FROM ETHYL
ACETATE FRACTION OF D. ramosa
Two compounds were isolated from ethyl acetate fraction of Dryopteris ramosa after silica gel and Sephadex LH20 column chromatography followed by purification through MPLC using isocratic solvent mixture (non-polar to polar)
Figure 4.51. The compound obtained from group 3 (after CC of ethyl acetate fraction of D. ramosa) showed a single spot on TLC with Rf 0.39. It was yellow orange powder having melting point 272-2780C. The HPLC chromatogram showed a single peak at 6.467 minutes and UV-Vis absorption spectrum ʎmax 210 and
298nm. The ESI-MS revealed that this compound has molecular weight of 408m/z.
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The H-NMR showed 20 protons and C-NMR showed 19 carbons (Keeler 1990; Jin et al 2009). All the spectroscopic data obtained was similar with the data obtained for DAF-MI-01(isolated from aqueous fraction of D. ramosa). So, the compound isolated from group 3 was identified as iriflophenone-3-C-β-glucopyranoside
(Kokotkiewicz etal., 2012).
The compound DEF-4MP-1910 was isolated from ethyl acetate fraction of
D. ramosa by CC (Figure 4.51). It was a yellow amorphous powder with melting point 175-1790C (Hiraoka and Hasegawa. 1975). HPLC analysis ofDEF-4MP-
1910 showed aRt 12.487 minutes with ʎmax266, 294 and 350nm (Figure 4.37). The
ʎmax350nm suggested that compound was 3-O substituted (Francescato et al.,
2013). The shoulder on the UV spectra can also indicate substitution on aglycones:
Kaempferol (266, 294sh and 349nm for 3 glycoside while 266, 318sh and 349nm for 3,7diglycosides) (Romani et al., 2006). On TLC, when detection chemical spraying reagents were applied, the DEF-4MP-1910 showed characteristic spot of flavonoid structure (Prabhu et al., 2011). ESI-MS revealed molecular weight 448da
(M+H=449). The peak at 286 indicated the loss of glucose residue (M+H-163).
The peak at 286(M+H)+ was a characteristic peak of kaempferol ((Francescato et al., 2013; Ablajan et al., 2006). This also confirms that kaempferolhas 3-O- glycosylation (Ablajan et al., 2006; Luet al., 2010).
The 1H-NMR spectra of DEF-4MP-1910 [ δH6.21 (d, 1H, j= 2.05Hz, H-6),
δH6.41(d, 1H, j= 1.98Hz, H-8), δH8.06 (d, 2H, j= 8.91Hz, H-2/, H-6/), δH6.88 (d,
2H,j= 9.0 Hz, H-3/, H-5/ )] indicated that DEF-4MP-1910 had kaempferol as aglycon(Furusawa et al., 2005).The 13C-NMR and DEPT analysis of DEF-4MP-
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1910 was in agreement with the published literature (Lee et al., 2011; Wei et al.,
2011). The HSQC and HMBC spectra confirmed the 3-O-glycosylation of kaempferol (Figure 4.47 and 4.48). On the basis of these evidences the isolated compound DEF-4MP-1910 was identified as Kaempferol-3-O-β-glucopyranoside
(Astragalin).
Astragalin (Kaempferol 3-O-glucoside) was very first time reported from genus Dryopteris in present study. Although, previously astragalin was reported in fern family Dryopteridaceae e. g. Kaempferol 3-O-glucoside was isolated from the fronds of Cyrtomiumfalcatum, C.Presl(Iwashina et al., 2006). Astragalin was also found in fern family Schizaeaceae (Iwashina and Matsumoto, 2013). Same compound was also isolated from fern family Thelypteridaceae
(Phegopterishexagonoptera) by Adam(1999).
5.6 IDENTIFICATION OF ISOLATED COMPOUNDS FROM ETHYL
ACETATE FRACTION OF R. brunonii
Three compounds were isolated from ethyl acetate fraction of Rosa brunonii by chromatographic procedure; silica gel60 (0.5-0.6mm) followed by LH20 and MPLC (Figure 4.52). The isolated compounds were labeled as REF-5s5
(compound-1), REF-5s67 (compound-2) and REF-MI-01-49(compound-3).
All the three isolated compounds from ethyl acetate fraction of R. brunonii had showed yellow inflorescence on TLC under UV long wavelength (365nm) when sprayed with 3 percent Ferric chloride. The same results were observed under UV 254nm when TLCs were treated with 1 percent ethanolic solution of aluminium chloride (AlCl3). This mean the isolated compounds might have related
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to phenolics or flavonoid class of plants secondary metabolites. The use of TLC spraying reagents for the detection of phenolic and flavonoid contents are routinely used (Wardas et al., 2000; Pyaka et al., 2002). The isolated compound-1(REF-5s5) from R. brunonii showed a single peak in HPLC chromatogram at 12.540 minutes
(Figure 4.53). The UV-Vis spectroscopy of REF-5s5 showed ʎmax 257, 265 and
355nm when scan with DAD between 200-400nm. The ʎmax 350nm suggested that compound was 3-O substituted (Francescato et al., 2013). The mass spectrum showed a molecular ion (M+H) at 449m/z suggested the molecular weight of the isolated compound REF-5s5 might be 448da. The Other major peaks were observed at 434m/z (M+H-OH), 286m/z (M+H-sugar moiety) and 94 m/z (may be a phenol residue C6H5OH) (Plazonic et al.,2009; Ablajan et al., 2006). The proposed fragmentation pattern of these peaks (m/z) for REF-5s5 was given in
Figure 4.62.
The 1H-NMR spectra of REF-5s5 showed an ABX coupling spin system at
[δH7.34 (d, 2H,j = 2.08Hz, H-2/), δH 6.90 (d, 1H,j= 8.36Hz, H-5/) and δH7.29(d,
1H,j = 8.30Hz, H-6/). Two protons at δH6.21 (d, 1H, j = 2.06Hz, H-6) and
δH6.37(d, 1H, j = 2.05Hz, H-8) showed AB spin coupling system (Rice et al.,
1996).The δH 5.35 (d, j = 1.2Hz) represented the anomeric proton of sugar moiety.
The δH 0.95ppm (d, 3H, j= 6.01Hz) indicating the presence of methyl group. It was assigned to H-6//. This indicated the presence of rhamnose sugar in the molecule (Yaya et al., 2012). 13C-NMR of isolated compound REF-5s5 has showed 21 signals which corresponds to 21 carbons. The δ= 179.67ppm is the indication of carbonyl group (C=O) which was assigned to C-4. The DEPT analysis showed five aromatic methines (C-6 δ99.81; C-8 δ94.71; C-2/ δ 116.96;
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C-5/ δ 116.07; C-6/ δ122.86ppm), five oxygenated methines (C-1// δ103.55; C-2//
δ71.38; C-3// δ72.15; C-4// δ73.28; C-5// δ71.91ppm), methyl (C-6// δ17.64ppm) and nine signals for quaternary aromatic carbons (C-2 δ159.31; C-3 δ 136.25; C-4a
δ 105.93; C-5 δ163.22; C-7 δ165.85; C-8a δ158.53; C-1/ δ123.0; C-3/ δ146.42; C-
4/ δ149.79ppm) and one carbonyl carbon ( C-4 δ179.67) (Yaya etal., 2012). The
HSQC confirmed the above informations obtained from 13C-NMR. The correlation peaks and H-COSY spectrum of REF-5s5 suggested that proton H-2// (δ 4.22ppm) was coupled with H1// (δ 5.35ppm) and H-3// (δ 3.75ppm) while cross peak of H1//
(δ 5.35ppm) was only coupled with H-2// (δ 4.22ppm). Similarly, The cross peak of H-6// (δ 0.95ppm) was coupled with H-5// (δ 3.48ppm). The position of rhamnose was confirmed in HMBC spectrum of REF-5s5 by observing the resonance signals (peaks) between δH 5.35ppm (H-1//) and δC 136.25ppm (C-3)
(Yaya et al., 2012).
On the basis of TLC analysis and UV-Vis absorption spectra it was concluded that isolated compound REF-5s5 might have flavonoid/phenolic structure. The ESI-MS revealed the molecular weight 448 dalton. On the basis of
HNMR, CNMR (DEPT) and MS analysis the molecular formula was determined as C21H20O11. The HSQC, COSY and HMBC confirmed the presence of rhamnose sugar associated with aglycone. The HMBC indicated the 3-O position of rhamnose. On the basis of these evidences the isolated compound REF-5s5 was identified as Quercetin-3-O-rhamnoside. This compound was previously also isolated from Bauhinia longifolia (Bong.) (Santos et al., 2014), Mango-fruits
(Berardini et al., 2005), Pepper- fruits (Materska and Perucka, 2005), Cranberry
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and Lingonberry (Zeng and Wang 2003). This is the first report of isolation of
Quercetin-3-O-rhamnoside from Rosa brunonii.
Compound 2 (REF-5s67) isolated from ethyl acetate fraction of Rosa brunonii was yellowish powder with melting point ranges 171-1790C. When sprayed with aluminum chloride and anisaldehyde, a characteristic yellowish spot appear on TLC after heating. This might be the indication of flavonoid or related compound (Prabhu et al., 2011). The UV-Vis spectroscopy showed a typical ʎmax of shoulder at 294nm indicating that it might be substituted with 3-O glycoside
(Romani et al., 2006). The MS-analysis showed that REF-5s67 have molecular weight 448da. A positive ion mode of ESI_MS showed a characteristic kampferolaglycon peak at 286m/z (Francescato et al., 2013). The 1H-NMR spectra of REF-5s67 [ δH 6.21 (d, 1H, j = 2.05Hz, H-6), δH 6.41(d, 1H, j = 1.98Hz, H-8),
δH8.06 (d, 2H, j = 8.91Hz, H-2/, H-6/), δH 6.88 (d, 2H, j = 9.0 Hz, H-3/, H-5/ )] indicated that REF-5s67 had kaempferol as aglycon (Furusawa etal., 2005). The
13C-NMR and DEPT analysis of REF-5s67 was in agreement with the published literature (Lee et al., 2011; Wei et al., 2011). The HSQC and HMBC spectra confirmed the 3-O-glycosylation of kaempferol. On the basis of these evidences the isolated compound DEF-4MP-1910 was identified as Kaempferol-3-O-β- glucopyranoside (Astragalin). It was the same compound which we have also isolate in the present study from ethyl acetate fraction of Dryopteris ramosa.
Compound-3 (REF-MI-01-49) was isolated from ethyl acetate fraction of
Rosa brunonii (Figure 4.52). It was light yellow in coloured having melting point
256-2610C. It showed a yellow spot on TLC after spraying with 10 percent
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Sulphuric acid followed by heating (Seo et al., 2016). UV-VIS absorption spectra of isolated compound (REF-Mi-01-49) exhibited two bands with ʎmax at 260 and
315nm for band II and I respectively (Figure 4.57). This suggested that flavonoid is acelyted as it showed 315nm absorption spectra for band I. This is the main property of p-coumaroyl glycosylated flavonols(Negri et al., 2013;Shahat et al.,
2005;Guerreroetal., 2009).
The ESI-MS analysis of REF-MI-01-49 has showed a molecular ion at
595m/z (M+H) indicating 594da molecular weight. The major peak in ESI-MS positive mode; [578m/z (M+H-OH), 448m/z (M+H-p-coum.), 270m/z (M+H-p- coum.-glu.)]. The molecular formula of REF-MI-01-49 was determined as
C30H26O13 based on Molecular ion peak and major fragment peaks (Seo et al.,
2016). The 1H-NMR spectra of REF-MI-01-49 [ δH 6.14 (d, 1H, j = 2.05Hz, H-6),
δH 8.32(d, 1H , H-8), δH7.99 (d, 2H, j = 8.80Hz, H2/, H6/), δH 6.82 (d, 2H, j =
8.96 Hz, H-3/, H-5/ )] indicated that REF-MI-01-49 had kaempferol as aglycon
(Furusawa et al., 2005). The 1H and 13C-NMR showed that REF-MI-01-49 has similar structure as Astragalin with additional signals. These additional signals were representing the phenylpropanoid moiety. 1H-NMR of REF-MI-01-49 showed signals [δH 7.31 (2H, d, j = 8.5 Hz, H-2''', H-6''') and 6.80 (2H, d, j = 8.96
Hz, H-3''', H-5''')] representing the methines Para substitution of benzene ring. The signals [δH 7.40 (1H, d, j = 15.92 Hz, H-7''') and 6.07 (1H, d, j = 15.88 Hz, H-8''') representing olefin proton due to double bonds in trans position (Sanchez et.al.,
2014). The additional (other than astragalin) signals in 13C-NMR of REF-MI-01-
49 showed δC 169.37ppm (C-9///) indicated the carbonyl group; δC 161.79ppm (C-
4///) indicated the oxygenated methines; δC 127.72ppm (C-1///) quaternary carbon
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of aromatic ring and δC [ 131.77ppm (C-2///, C-6///), 117.40ppm (C-3///, C-5///),
147.14ppm (C-7///) and 115.36ppm (C-8///)] were representing the olefins methines carbons. On the basis of these observation it was proposed that additional phenylpropanoid moiety was a p-coumaroyl group (Seo et al., 2016).The esterification shift of the oxygenated methylene proton signals (δH 4.29, H-6//a; δH
4.18, H-6//b) indicated that the p-coumaroyl group was linked to the OH-6// of the glucopyranose moiety. It was confirmed by gs-HMBC plot of REF-MI-01-49 by correlating the δC 169.37ppm(C-9///) signals with δH 4.29, H-6//a and δH 4.18, H-
6//b signals. Finally, the compound-3 REF-MI-01-49 was identified as tiliroside, a kaempferol-3-O-β-D-(6''-O-coumaroyl)-glucopyranoside.
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Figure 5.1: HMBC correlation observed for iriflophenone-3-C-β- D
glucopyrranoside (DAF-MI-01).
a
b
Figure 5.2: HMBC correlation observed for a)Mangiferin (DAF-MI-02.1),
b)Isomangiferin (DAF-MI-02.2).
205
Figure 5.3: HMBC correlation observed for Astragalin(DEF-4mp-1910).
Figure 5.4: HMBC correlation observed for Quercetin-3-O-rhamnoside(REF-5s5).
Figure 5.5: HMBC correlation observed for Tiliroside(REF-MI-01-49).
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This is the first report of isolation of this compound from Rosa brunonii.
Although tiliroside has been isolated from many plants
Lasiopetalummacrophyllum(Timmers and Urban, 2012), Tiliacordata(Neigri et al.,
2013), Heliocarpusterebinthinaceus (Sanchez et al., 2014) and many others.
5.7ANTIOXIDANT AND CYTOTOXIC PROPERTIES OF ISOLATED
COMPOUNDS
All the isolated compounds has showed antioxidant potential but in comparison with the standard known antioxidant compound ascorbic acid (IC50=
57.97±0.49µg/mL), the astragalin (IC50= 50.91±0.61µg/mL) showed maximum
(better than ascorbic acid) antioxidant potential. This supports our initial finding about antioxidant potential of ethyl acetate fraction of D. ramosa which had showed maximum potential among all the fractions. Isomangiferrin also exhibited better antioxidant potential than ascorbic acid. In a study, Choi et al. (2013) has induced oxidative stress artificially to human RBCs for hemolysis and they treated the cells with three compounds they had isolated from leaf extract of mulberry
(Morus alba). Their study showed that astragalin had greatest protective effect against oxidative stress. A study on agar wood plant (Aquilaria sinensis) lead to the isolation of iriflophenone-3-C-β-D glucopyranoside and it is found to be effective in lowering the blood glucose level (Pranakhon et al., 2015). Mangiferrin and isomangiferrin has proved to be strong antioxidants. Researcher has found that mangiferrin is not only has antioxidant potential but also it is analgesic and has hepatoprotective abilities (Dar et al., 2005; Sellamuthu et al., 2013). A recently published study demonstrated that Quercetin-3-O-rhamnoside is also found in
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Rhus chinensis and it has strong antioxidant activity. The study also proposed pancreatic lipase inhibitory potential of quercetin-3-O-rhamnoside (Zhang et al.,
2017).
The cytotoxicity of all isolated compounds against BSLT was assessed.
Tiliroside showed maximum cytotoxic potential against Brine shrimp‟s nauplii with LD50 50.42±0.88µg/mL in comparison with Nicotine (46.76±4.81µg/mL).
Among the others isolated compounds Quercetin 3-O rhamnoside (LD50
345.22±0.59µg/mL) followed by iriflophenone-3-C-β-D glucopyranoside (LD50
406.67±0.35µg/mL). Tiliroside not only showed cytotoxic potential against brine shrimp larvae but several studies also confirmed the cytotoxic potential of tiliroside on various cell lines (Dimas et al., 2000; Souza et al., 2002;Savietto, Jóiceet al.,
2013; Da‟i et al., 2016).
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FUTURE RECOMENDATIONS
We have suggested following recommendations based on preset research work. o These isolated compounds should be studied against different pure culture cell
lines to identify their potential invivo and also to understand their mode of
action as antioxidant and cytotoxic compounds. o These plants should be further investigated for the isolation and identifications
of more bioactive components. o An online liberaray of bioactive phytochemical should be developed specifying
the bioactive phytoconstituents from medicinal plants of Pakistan. It should
include spectroscopic data for individual compound. o We proposed that Higher Education Commission (HEC) Pakistan should
encourage pharmaceutical companies to work in close collaboration with
universities and researchers working on medicinal aspects of natural products. o This work has shown the true potential of bioactive compounds in a range of
key species of Pakistan. A detail screening of these medicinal plants may be
carried out in future to identify potential newer bioactive compounds. o DNA sequencing mand maping for these medicinal plants and their bioactive
compounds may be carried out in future not only to find biosynthetic pathways
of these and more bioactive compounds from these plant species but to identify
the phylogenetic lineage and adoptability of these species in various
environmental conditions.
208
209
SUMMARY
Plants are blessing for human beings who not only provide food and basic needs of life but also a miraculous and endless source of bioactive constituents.
These natural compounds are active against many human diseases. Use of plants to cure human ailments is as old as human history. The ethnomedicinal flora of
Gallyat region of Pakistan has rich potential for the source of antioxidant and
Cytotoxic natural bioactive compounds. In the present study we have selected five medicinal plants of Gallyat region, Pakistan which were never been explored for their bioactive phytoconstitiuents. These species included Dryopteris ramosa,
Quercus leucotricophora, Bidens biternata, Arisaema flavum and Rosa brunonii.
Crude methanole extracts (CME) of all the selected plants were subjected to DPPH free radical scavenging biassay, Brine shrimp lethality test (BSLT) and potato disc antitumor assay. Strong free radical scavenging and antitumor potential was showed by CME of D. ramosawhile same extracts showed minimum lethality towards brine shrimp nauplii in BSLT in-vitro bioassay. CME of R. brunonii has showed maximum cytotoxicity against brine shrimp. It also exhibited significant free radical scavenging potential and strong antitumor abilities. Q. leucotricophora also showed strong antioxidant potential. Other two plants showed comparatively less Cytotoxic and antioxidant potential in our study. So we have selected D. ramosa, Q. leucotricophora and R. brunonii for further studies.
CME of selected D. ramosa, Q. leucotricophora and R. brunonii were subjected to solvent-solvent fractionation and each CME was portioned in four fractions i.e. n-Hexan (nHF), chloroform (CF), ethyl acetate (EF) and aqueous(AF)
209
210
fraction. All the fractions were subjected to in-vitro bioassay to assess their antioxidant and cytotoxic potential. On the basis of results of these experiments we have selected ethylacetate and aqueous fraction of D. ramosa and ethyl acetate fraction of R. brunonii for further study and isolation of bioactive secondary metabolites.
By using chromatographic techniques we have isolated total of seven bioactive pure compounds. Their identification and structural elucidation was carried out by using spectroscopic techniques in cluding MS, UV-Vis.
Spectroscopy, 1HNMR, 13CNMR, DEPT, CoSY, HSQC and HMBC. Four compounds were isolated from D.ramosa. These include irriflophenone-3-C-β-D glucopyranoside, Mangiferrin, Isomangiferrin and Astragalin. While three compounds were isolated from R. brunonii that includ Quercetin-3-O-rhamnoside,
Astragalin and Tiliroside. This is the first report of isolation of Phytochemicals and their structural elucidation from these plant species to the best of our knowledge.
These isolated pure compounds showed invitro strong antioxidant/ free radical scavenging potential. The isolated compound tiliroside exhibited maximum cytotoxic potential as compared to all other isolated compounds in the present study. These plants species have considerably higher amount of these phytochemicals. We have isolated considerably higher yield of these pure compounds as compared to previous studies from other plant species. Theresults of bioactivities of CME, fractions and more importantly isolated compounds have underline the importance of these ethnomedicinally important plants species of the
Gallyat region, Pakistan.
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APPENDICES
Appendix 1
ANOVA Table for Free Radical Scavenging Potential of CME of Selected Plants. Dependent Variable:: free radical percentage scavenging Source SS df MS F Sig. Corrected Model 76269.678a 35 2179.134 2.2343 .0001 Intercept 289155.200 1 289155.200 2.9645 .0001 CME 29616.456 5 5923.291 6.0723 .0001 Concentrations 42537.454 5 8507.491 8.7213 .0001 CME * 4115.767 25 164.631 168.763 .0001 Concentrations
Error 70.237 72 .976 Total 365495.114 108 Corrected Total 76339.915 107 a. R Squared = .999 (Adjusted R Squared = .999)
Appendix 2
ANOVA Table, mean percent lethality of CME of selected plants against brine Shrimp. Dependent Variable:Percent lethality Source SS df MS F Sig. Corrected Model 24107.917a 19 1268.838 95.163 .0001 Intercept 66333.750 1 66333.750 4.975E3 .0001 CME 12247.500 4 3061.875 229.641 .0001 Concentrations 9991.250 3 3330.417 249.781 .0001 CME * 1869.167 12 155.764 11.682 .0001 Concentrations Error 533.333 40 13.333 Total 90975.000 60 Corrected Total 24641.250 59 a. R Squared = .978 (Adjusted R Squared = .968)
Treatments in each appendix are different concentrations of plant extracts. SS= Sum of Square, df= Degree of Freedom and MS= Mean Square.
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Appendix 3
Tukey HSD multiple comparison of cytotoxicity between all crude extractsMultiple Comparisons Percent lethality (Tukey HSD)
(I) Plant (J) Plant Mean Std. Error Sig. 95% Confidence Interval crude crude Difference (I- Lower Bound Upper Bound extract extract J) D.R Q.L -6.2500* 1.49071 .001 -10.5076 -1.9924 A.F -25.0000* 1.49071 .000 -29.2576 -20.7424 R.B -38.7500* 1.49071 .000 -43.0076 -34.4924
B.B -27.5000* 1.49071 .000 -31.7576 -23.2424 Q.L D.R 6.2500* 1.49071 .001 1.9924 10.5076 A.F -18.7500* 1.49071 .000 -23.0076 -14.4924 R.B -32.5000* 1.49071 .000 -36.7576 -28.2424 B.B -21.2500* 1.49071 .000 -25.5076 -16.9924
A.F D.R 25.0000* 1.49071 .000 20.7424 29.2576 Q.L 18.7500* 1.49071 .000 14.4924 23.0076 R.B -13.7500* 1.49071 .000 -18.0076 -9.4924 B.B -2.5000 1.49071 .459 -6.7576 1.7576 R.B D.R 38.7500* 1.49071 .000 34.4924 43.0076
Q.L 32.5000* 1.49071 .000 28.2424 36.7576 A.F 13.7500* 1.49071 .000 9.4924 18.0076 B.B 11.2500* 1.49071 .000 6.9924 15.5076 B.B D.R 27.5000* 1.49071 .000 23.2424 31.7576 Q.L 21.2500* 1.49071 .000 16.9924 25.5076
A.F 2.5000 1.49071 .459 -1.7576 6.7576 R.B -11.2500* 1.49071 .000 -15.5076 -6.9924
Based on observed means.The error term is Mean Square(Error) = 13.333.*. The mean difference is significant at the .05 level. (DR= D. ramosa, Q.L= Q. leucotricophora, R.B= R. brunonii, A.F= A. flavum and B.B= B. biternanta).
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Appendix 4 Anova Table: Antitumor potential of CME of selected plants Dependent Variable: percent tumor inhibition Source SS df MS F Sig. Corrected 65481.714a 24 2728.405 1.265E4 .0001 Model Intercept 63576.013 1 63576.013 2.949E5 .0001 Conc 46135.780 3 15378.593 7.132E4 .0001 CME 14674.337 6 2445.723 1.134E4 .0001 Conc * CME 5474.171 15 364.945 1.693E3 .0001 Error 10.134 47 .216 Total 132794.149 72 Corrected Total 65491.847 71 a. R Squared = 1.000 (Adjusted R Squared = 1.000)
Appendix 5
ANOVA Table for free radical scavenging abilities of fractions of selected plants. Dependent Variable:Mean percentage free radical scavenging Source SS df MS F Sig. Corrected Model 141076.937a 88 1603.147 6.485E3 .0001 Intercept 457517.611 1 457517.611 1.851E6 .0001 Fractions 54974.334 14 3926.738 1.588E4 .0001 Concentration 67870.013 5 13574.003 5.491E4 .0001 Fractions * 17452.164 69 252.930 1.023E3 .0001 Concentration Error 44.005 178 .247 Total 595101.592 267 Corrected Total 141120.942 266 a. R Squared = 1.000 (Adjusted R Squared = 1.000) Treatments in each appendix are different concentrations of plant extracts. SS= Sum of Square, df= Degree of Freedom and MS= Mean Square.
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Appendix 6
Tukey HSD multiple comparisons for Free radical scavenging of different fractions of selected plant species. Multiple Comparisons: Mean percentage free radical scavengingTukey HSD
(I) Mean 95% Confidence Interval Fraction Difference Lower s (J) Fractions (I-J) Std. Error Sig. Bound Upper Bound DR nHF DRCF 15.6689* .16574 .0001 15.0988 16.2390 DREF -27.7039* .16574 .0001 -28.2740 -27.1338 DRAF 13.7306* .16574 .0001 13.1605 14.3006 DRCME -22.1339* .16574 .0001 -22.7040 -21.5638 DRCF DREF -43.3728* .16574 .0001 -43.9429 -42.8027 DRAF -1.9383* .16574 .0001 -2.5084 -1.3682 DRCME -37.8028* .16574 .0001 -38.3729 -37.2327 DREF DRAF 41.4344* .16574 .0001 40.8644 42.0045 DRCME 5.5700* .16574 .0001 4.9999 6.1401 DRAF DRCME -35.8644* .16574 .0001 -36.4345 -35.2944 QLnHF QLCF -7.6717* .17383 .0001 -8.2696 -7.0737 QLEF -8.8667* .16574 .0001 -9.4368 -8.2966 QLAF -28.5283* .16574 .0001 -29.0984 -27.9582 QLCME -34.1133* .16574 .0001 -34.6834 -33.5432 QLCF QLEF -1.1950* .17383 .0001 -1.7929 -.5971 QLAF -20.8567* .17383 .0001 -21.4546 -20.2587 QLCME -26.4417* .17383 .0001 -27.0396 -25.8437 QLEF QLAF -19.6617* .16574 .0001 -20.2318 -19.0916 QLCME -25.2467* .16574 .0001 -25.8168 -24.6766 QLAF QLCME -5.5850* .16574 .0001 -6.1551 -5.0149 RBnHF RBCF -4.1150* .16574 .0001 -4.6851 -3.5449 RBEF -9.3511* .16574 .0001 -9.9212 -8.7810 RBAF -22.9261* .16574 .0001 -23.4962 -22.3560 RBCME -24.5506* .16574 .0001 -25.1206 -23.9805 RBCF RBEF -5.2361* .16574 .0001 -5.8062 -4.6660 RBAF -18.8111* .16574 .0001 -19.3812 -18.2410 RBCME -20.4356* .16574 .0001 -21.0056 -19.8655 RBEF RBAF -13.5750* .16574 .0001 -14.1451 -13.0049 RBCME -15.1994* .16574 .0001 -15.7695 -14.6294 RBAF RBCME -1.6244* .16574 .0001 -2.1945 -1.0544 Significance difference at 0.05 levels. The error term is Mean Square (Error) = .247 DR= D.ramosa, QL= Q.leucotricophora, RB= R. brunonii, CME= Crude Methanol Extract, nHF= n-Hexan Fraction, CF= Chloroform Fraction, EF= Ethyl Acetate Fraction and AF= Aqueous Fraction.
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