CYTOTOXIC AND PHYTOCHEMICAL ANALYSIS OF SELECTED MEDICINAL PLANTS FROM SALT RANGE, PAKISTAN
MUHAMMAD MAQSOOD
06-arid-672
Department of Botany Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2017 CYTOTOXIC AND PHYTOCHEMICAL ANALYSIS OF SELECTED MEDICINAL PLANTS FROM SALT RANGE, PAKISTAN
by
MUHAMMAD MAQSOOD (06-arid-672)
A thesis submitted in partial fulfillment of the requirement for the degree of
Doctor of Philosophy
in
Botany
Department of Botany Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2017
CONTENTS
Page
List of Tables ix
List of Figures xi
List of Abbreviations xvii
Acknowledgements xix
ABSTRACT 1
1 GENERAL INTRODUCTION 5
2 MEDICINAL PLANTS 9
2.1 INTRODUCTION 9
2.2 REVIEW OF LITERATURE 11
2.3 MATERIALS AND METHODS 13
2.3.1 Study Area 13
2.3.1.1 Geography 13
2.3.1.2 People 15
2.3.1.3 Farming 15
2.3.1.4 Culture 15
2.3.2 Collection of Ethnobotanical Information 17
2.3.3 Specimen Collection 18
2.3.4 Identification and Preservation of Specimens 18
2.3.5 Data Compilation 18
2.4 RESULTS 18
v
2.4.1 Inventory of Medicinal Plants 18
2.4.2 Fidelity Percentage 19
2.4.3 Disease Treated 19
2.4.4 Parts Used 19
2.4.5 Mode of Application 39
2.4.6 Selection of Plants for Cytotoxicity 39
2.5 DISCUSSION 39
2.6 CONCLUSION 44
3 CYTOTOXICITY 45
3.1 INTRODUCTION 45
3.2 REVIEW OF LITERATURE 52
3.3 MATERIALS AND METHODS 54
3.3.1 Chemicals 54
3.3.2 Collection of Plant Material 54
3.3.3 Preparation of Crude Extract 55
3.3.4 Column Chromatography Fractionation (CCF) 55
3.3.5 Preparation of Stock Solution of Crude Extract 57
3.3.6 Dilution of Stock Solution 57
3.3.7 Cell Culture 58
3.3.8 In vitro Cytotoxicity Tests 58
3.3.9 Presto Blue Cell Viability Assay 59
3.4 STATISTICAL ANALYSIS 59
vi
3.5 RESULTS 60
3.5.1 Apocynaceae 60
3.5.2 Asteraceae 61
3.5.3 Brassicaceae 66
3.5.4 Lamiaceae 71
3.5.5 Moringaceae 76
3.5.6 Solanaceae 86
3.5.7 Zygophyllaceae 93
3.5.8 Cytotoxicity of CCFs of P. brahuicus 98
3.6 DISCUSSION 105
3.7 CONCLUSION 113
4 PHYTOCHEMICAL SCREENING 115
4.1 INTRODUCTION 115
4.1.1 Phenolic Compounds 117
4.1.2 Flavonoids 120
4.3 MATERIALS AND METHODS 124
4.3.1 Plant Material 124
4.3.2 Preparation of Extract 124
4.3.3 Preparations of Standards 124
4.3.4 Conditions for RP-HPLC Analysis 126
4.3.5 Standards for Testing HPLC Column 126
4.3.6 LC-MS Analysis 128
vii
4.3.7 Identification of Compounds in CCFs 128
4.4 RESULTS 129
4.4.1 RP-HPLC Results 129
4.4.1.1 System suitability test 129
4.4.2 Phenolics and Flavonoids Results 131
4.4.2.1 Phenolic and flavonoids contents of CCFs of P. brahuicus 140
4.4.2.2 LC-MS results of CCFs of P. brahuicus 144
4.5 DISCUSSION 144
4.6 CONCLUSION 156
5 GENERAL DISCUSSION 159
SUMMARY 168
LITERATURE CITED 175
APPENDICES 195
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LIST OF TABLES
Table No. Page
2.1 List of medicinal plants along with local names, family, 20
method of preparation, disease treated, use reports and fidelity
percentage from the study area
2.2 Ailments treated by the use of native medicinal plants from the 36
Salt Range, Pakistan
2.3 Different plant parts used for the preparation of herbal recipes 41
3.1 Cancer incidence for 4 malignancies in worldwide, in the USA 46
and Pakistan. Summary of absolute number of cases and
ranking of the cancer within the respective sample area
3.2 Fraction weights and solvents used for the column 56
chromatography over silica gel separation of the methanolic
extract
3.3 IC50 values of Rhazya stricta (root) against the selected cell 65
lines
3.4 IC50 values of Artemisia scoparia (whole plant) against the 70
selected cell lines
3.5 IC50 values of Physorhynchus brahuicus (whole plant) against 75
the selected cell lines
3.6 IC50 values of Otostegia limbata (aerial parts) against the 80
selected cell lines
ix
3.7 IC50 value of Moringa oleifera (fruit and leaves) against the 85
selected cell lines
3.8 IC50 values of Withania coagulans (fruit and leave stalk) 91
against the selected cell lines
3.9 IC50 values of Fagonia indica (whole plant) against the 97
selected cell lines
3.10 IC50 values of CCFs against the selected cell lines 104
4.1 Certified reference standards of phenolics along with conc., 125
R.T and peak area
4.2 Comparison of calculated and standard values from regulatory 133
guidelines
4.3 Yield of phenolic acids (µgg-1) in various plant parts 142
4.4 Yield of phenolic acids (µgg-1) in various Column 143
Chromatography Fractions of P. brahuicus
4.5 Yield of flavonoids (µgg-1) in various plant parts 145
4.6 Yield of flavonoids (µgg-1) in various Column 146
Chromatography Fractions of P. brahuicus
x
LIST OF FIGURES
Fig. No. Page
2.1 Map of Salt Range, Punjab, Pakistan 14
2.2 Family Important Index of the flora of Salt Range, Pakistan 35
2.3 Mode of application of recipes employed by the people of Salt 42
Range, Pakistan
3.1 Cytotoxicity of Rhazya stricta (leaves and root) extracts against 62
HeLa cells
3.2 Cytotoxicity of Rhazya stricta (leaves and root) extracts against 62
MCF-7 cells
3.3 Cytotoxicity of Rhazya stricta (leaves and root) extracts against 63
RD cells
3.4 Cytotoxicity of Rhazya stricta (leaves and root) extracts against 63
INS-1 cells
3.5 Cytotoxicity of Rhazya stricta (leaves and root) extracts against 64
RG2 cells
3.6 Cytotoxicity of Artemisia scoparia (whole plant) extracts against 67
HeLa cells
3.7 Cytotoxicity of Artemisia scoparia (whole plant) extracts against 67
MCF-7 cells
3.8 Cytotoxicity of Artemisia scoparia (whole plant) extracts against 68
RD cells
xi
3.9 Cytotoxicity of Artemisia scoparia (whole plant) extracts against 68
RG2 cells
3.10 Cytotoxicity of Artemisia scoparia (whole plant) extracts against 69
INS-1 cells
3.11 Cytotoxicity of Physorhynchus brahuicus (leaves, root and 72
whole plant) extracts against HeLa cells
3.12 Cytotoxicity of Physorhynchus brahuicus (leaves, root and 72
whole plant) extracts against MCF-7 cells
3.13 Cytotoxicity of Physorhynchus brahuicus (leaves, root and 73
whole plant) extracts against RD cells
3.14 Cytotoxicity of Physorhynchus brahuicus (leaves, root and 73
whole plant) extracts against RG2 cells
3.15 Cytotoxicity of Physorhynchus brahuicus (leaves, root and 74
whole plant) extracts against INS-1 cells
3.16 Cytotoxicity of Otostegia limbata (aerial parts) extracts against 77
HeLa cells
3.17 Cytotoxicity of Otostegia limbata (aerial parts) extracts against 77
MCF-7 cells
3.18 Cytotoxicity of Otostegia limbata (aerial parts) extracts against 78
RD cells
3.19 Cytotoxicity of Otostegia limbata (aerial parts) extracts against 78
RG2 cells
xii
3.20 Cytotoxicity of Otostegia limbata (aerial parts) extracts against 79
INS-1 cells
3.21 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave 82
stalk) extracts against HeLa cells
3.22 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave 82
stalk) extracts against MCF-7 cells
3.23 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave 83
stalk) extracts against RD cells
3.24 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave 83
stalk) extracts against RG2 cells
3.25 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave 84
stalk) extracts against INS-1 cells
3.26 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and 88
root) extracts against HeLa cells
3.27 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and 88
root) extracts against MCF-7 cells
3.28 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and 89
root) extracts against RD cells
3.29 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and 89
root) extracts against RG2 cells
3.30 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and 90 root) extracts against INS-1 cells 3.31 Cytotoxicity of Fagonia indica (whole plant) extract against 94
xiii
HeLa cells
3.32 Cytotoxicity of Fagonia indica (whole plant) extract against 94
MCF-7 cells
3.33 Cytotoxicity of Fagonia indica (whole plant) extract against RD 95
cells
3.34 Cytotoxicity of Fagonia indica (whole plant) extract against RG2 95
cells
3.35 Cytotoxicity of Fagonia indica (whole plant) extract against 96
INS-1 cells
3.36 Column Chromatography Fractions of Physorhynchus brahuicus 99
against HeLa cells
3.37 Column Chromatography Fractions of Physorhynchus brahuicus 101
against MCF-7 Cells
3.38 Column Chromatography Fractions of Physorhynchus brahuicus 101
against RD cells
3.39 Column Chromatography Fractions of Physorhynchus brahuicus 102
against RG2 cells
3.40 Column Chromatography Fractions of Physorhynchus brahuicus 102
against INS-1 cells
4.1 Plant derived anticancer agents 118
4.2 Plant derived anticancer agents 119
4.3 The main classes of flavonoids: (A) Flavonoids (B) 122
xiv
Isoflavonoids (C) Neo-flavonoids
4.4 Subclasses of flavonoids: (A) flavonol, (B) flavone, (C) 123
dihydroflavonol, (D) flavan-3-ol, (E) flavanone, (F)
anthocyanidin, (G) chalcone, (H) dihydrochalcone and (I) aurone
4.5 Standard chromatogram of uracil (1), acetophenone(2),benzene(3) and 132
toluene (4) showing column performance
4.6 Chromatogram of vanillic acid (standard) 134
4.7 Chromatogram of Quercitin (standard) 134
4.8 Chromatogram of Myrecitin (standard) 135
4.9 Chromatogram of Gallic acid (standard) 135
4.10 Chromatogram of Ferulic acid (standard) 136
4.11 Chromatogram of p –Coumaric acid. (standard) 136
4.12 Chromatogram of p –Hydroxybenzoic acid (standard) 137
4.13 Chromatogram of Syringic acid (standard) 137
4.14 Chromatogram of Protocatechuic acid (standard) 138
4.15 LC-MS chromatogram of N,N-dipropyl propan-1-amine 147
4.16 Structure of N,N-dipropyl propan-1-amine 147
4.17 LC-MS chromatogram of 4-acetamidophenyl) 2-(diethylamino) 148
acetate
4.18 Structure of 4-acetamidophenyl) 2-(diethylamino) acetate 148
4.19 LC-MS chromatogram of 3-Methyl benzo furan-2-carboxylic 149
acid
xv
4.20 Structure of 3-Methyl benzo furan-2-carboxylic acid 149
4.21 LC-MS chromatogram of [2-(Diphenylphosphoryl) ethyl] 150
(methyl) oxo (phenyl) phosphine
4.22 Structure of [2-(Diphenylphosphoryl) ethyl] (methyl) 150
oxo(phenyl) phosphine
4.23 LC-MS chromatogram of 2-(3-Amino-1H-1, 2, 4-triazol-1-yl) 151
aceto-hydrazide
4.24 Structure of 2-(3-Amino-1H-1, 2, 4-triazol-1-yl) aceto-hydrazide 151
4.25 LC-MS chromatogram of 3-Methyl-N-(3-methylbutyl)-1- 152
butanamin
4.26 Structure of 3-Methyl-N-(3-methylbutyl)-1-butanamin 152
4.27 LC-MS chromatogram of 1, 3-Benzenedimethanol, α1-[[(1, 1- 153
dimethylethyl) amino] methyl]-4-hydroxy
4.28 Structure of 1, 3-Benzenedimethanol, α1-[[(1, 1-dimethylethyl) 153
amino] methyl]-4-hydroxy
4.29 LC-MS chromatogram of 3-Chloro-6-hydrazinopyridazine 154
4.30 Structure of 3-Chloro-6-hydrazinopyridazine 154
xvi
LIST OF ABBREVIATIONS
% age Percentage °C. Centigrade µg Microgram µL Microliter µM Micromolar ANOVA Analysis of Variance CCFs Column Chromatography Fractions CMEs Crude Methnolic Extracts Conc. Concentration dH2O Distilled water DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide EtAOH Ethyl Acetate EtOH Ethanol FA Ferulic acid FBS Fetal Bovine Serum FL Fidelity level g Gram GA Gallic acid GC Gas Chromatography HMP Herbal Medicinal products HPLC High Performance Liquid Chromatography Hrs Hours L Litre LC Liquid Chromatography LC-MS Liquid Chromatography Mass Spectrometery MEM Minimum Essential Medium MeOH Methanol
xvii mg Miligram mL Mililitre mM Milimolar MPs Medicinal Plants MTX Methotrexate mV Mili volts NMR Nuclear Magnetic Resonance PCA Proto Catechuic acid PCOA Para Coumaric Acid PHBA Para Hydroxybenzoic acid RP-HPLC Reverse Phase High Performance Liquid Chromatography RPMI Roswell Park Memorial Institute Medium RT Retention Time SA Syringic acid Spp Species SPSS Statistical Package for Social Sciences TFC Total flavonoid contents TLC Thin Layer Chromatography TPC Total phenolic contents VA Vanillic acid
xviii
ACKNOWLEDGEMENTS
For the completion of this work, I am thankful to Almighty Allah, whose Grace and Mercy blessed me with good health and enthusiasm. I offer my humblest thanks from my heart to Holy Prophet Hazrat Muhammad (Peace be Upon Him), who is forever a torch of guidance and knowledge for humanity as a whole. My deep appreciation goes to my dissertation advisor Dr. Rahmatullah Qureshi, Associate
Professor, Department of Botany Pir Mehr Ali Shah Arid Agriculture University
Rawalpindi, Pakistan for his constant help, guidance and countless hours of attention he devoted throughout the course of this work. His priceless guidance and suggestions enabled me to complete this project. I would like to place on record my great appreciation and indebted gratitude to Prof. Dr. Muhammad Arshad, Chairman,
Department of Botany, PMAS-AAU, Rawalpindi, for his supervision, cooperation and help to complete my thesis.
My deep appreciation goes to my dissertation advisor Prof. Dr. Masroor Ikram
(T.I), Chief Scientist, Director Academics, Pakistan Institute of Engineering and
Applied Sciences (PIEAS), Nilore, Islamabad for his continuous help, guidance and countless hours of attention he devoted throughout the course of this work. His priceless suggestion made this interesting and learning for me. Without his personal interest, I would not be able to complete this task. I am especially thankful and grateful to Prof. Junaid Ahmed Khan, Depty Chief Scientist, Mr. Muhammad Rafi Principal
Scientist and Dr. Ahmat Khurshid Senior Scientist Photo Medicine Research
Laboratory, PIEAS, Mrs. Farhat Waqar,Director (System & Services) PINSTECH, Dr.
xix
Sadaf Yaqoob CAFD,PINSTECH, Nilore, Prof. Dr. Muhammad Rafique Asi, Head
Plant Protection Division NIAB, Faisalabad, Dr. Mazhar Iqbal, Health Biotechnology
Division, NIBGE, Faisalabad for their co-supervision, helpful suggestions and encouragement to accomplish this task. I am especially thankful and grateful to. Dr.
Muhammad Sheeraz Ahmed and Dr. Bushra Jabeen, Department of Biochemistry, Pir
Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan for their co- supervision, helpful suggestion and encouragement to accomplish this task. I am also extremely grateful to my best friends Raja Tahir Mahmood, Dr. Muhammad Irfan, Dr.
Muhammad Inam and to my senior supervisor fellow Dr. Mubashrah Munir who has greatly helped me in my research work through discussion and helpful comments and guide me regarding write up. I feel great pleasure in offering thanks to my cooperative lab fellows Dr. Muhammad Ilyas, Mr. Waseem Ahmed, Mr. Muhammad Arshad and
Mr. Iqrar Habib, for their moral support and their efforts and prayers made it possible for me.
I am highly grateful to Higher Education Commission (HEC) Pakistan for
IRSIP .This program provide me an opportunity to learn and work in the World best research centre Princess Margaret Research Centre, UHN, University of Toronto,
Canada under the kind supervision of Prof. Dr. Lothar Lilge, Prof. Dr. Brian Wilson and all colleagues of Lilge Labs, especially Dr. Pavel and Dr. Fisher who has greatly helped me in my research work. Last but not the least, I am very much thankful to
Pakistan Science Foundation, Islamabad, who granted research project to my supervisor No. PSF/Res/P-PMAS-AAU/Bio (418) that enabled me in carrying out preliminary information about plant usage. Finally, I wish to express my heartfelt
xx gratitude to my Wife, Mother& Father in laws, Parents, Brothers and Sister in laws,
Brothers, Sisters and Friends for continuing support and constants prayers. I owe a lot of thanks to my dear for their extra patience and motivation and made this possible.
Muhammad Maqsood
xxi
ABSTRACT
Medicinal plants are providing raw material to herbal and pharmaceutical industry. These plants are endowed with variety of phytochemicals commonly known as secondary metabolites. A large number of secondary metabolites are being extracted and utilized against various disorders including cancer. Medicinal plants are fairly distributed in Salt Range, Pakistan and are traditionally used by local herbalists (Hakeems) to treat various noxious diseases including cancer. This is a malignant disease and is increasing at rapid pace. Cancer was responsible for over 8.8 million death in 2015, i.e. one out of six deaths globally. It is the second leading cause of death over the world and its incidence rate will increase by 70% over the next two decades. Keeping this into consideration, a study was carried out to record folk knowledge of medicinal plants, cytotoxic activity as well as phytochemical analysis of some selected plants from the studied area.
A total of 71 plants species belonging to 61 genera and 35 families were documented in using various allied complaints by the natives of the area. Based on fidelity level (FL %), some species such as Artemisia scoparia Waldst. & Kitam.,
Fagonia indica Burm. f., Moringa oleifera Lam., Otostegia limbata (Benth.)
Boiss., Rhazya stricta Decne., Physorrhynchus burhaicus Hook.f. and Withania coagulans (Stocks) Dunal were selected for screening cytotoxic activity against cancer cell lines viz., human breast cancer (MCF-7), human cervical cancer
(HeLa), human skin cancer (RD), rat pancreatic tumor (INS-1) and rat brain tumor
(RG2). The plant materials of the said species were dried and ground to make powder for extracts preparation by using methanol.
1 2
All plant extracts showed cytotoxic effects against the selected cell lines, however, the inhibitory activity was found specific to plant extracts versus cell lines. In the case of Artemisia scoparia, except HeLa cell, the whole plant extract showed 80% cytotoxicity against all cell lines. The whole plant extracts of Fagonia indica showed 80% cytotoxic effect on three cell lines such as MCF-7, INS-1 and
RG2. The fruit extract of Moringa oleifera had leading effects (> 80%) on all cell lines. Besides, leaves extract also inhibited 80% cells of MCF-7 and INS-1. The whole plant extract of Otostegia limbata showed maximum cytotoxic effect (80%) against all cell lines, except HeLa cell. From the Rhazya stricta, root extract showed highest cytotoxic effect (> 90%) for RG2 and HeLa cell lines and 80% for
MCF-7 and INS-1.
The whole plant and leaves extract of Physorhynchus brahuicus showed maximum cytotoxic effect (80%) on three cell lines viz., MCF-7, INS-1 and RG2 and root extract induced 80% RD cells. The leaves stalk and fruit extract of
Withania coagulans had highest cytotoxic effect on all cell lines in which MCF-7,
RG2, RD cells and HeLa cells were inhibited at 90%, while INS-1 was inhibited up to 80%. In order to examine the behavior of all these extracts, IC50 values were calculated against the selected cell lines. It was observed that the activity was very strong to moderate ranging from 0.99 to 23.15µgmL-1 after incubation of 48 hours.
The same trend was observed after 72 hours of application, however the magnitude of activity was very strong to strong that ranged from 0.86 to16.78 µgmL-1.
Physorhynchus brahuicus was subjected to bioactivity guided isolation of compounds through column chromatography (CC) which may be responsible for
3
activity. This species was selected due to higher ethnobotanical use (FL %), previous reported activities/biochemistry, preliminary cytotoxic screening and range of IC50 values. Six column chromatography (CC) fractions of Physorhynchus brahuicus (P. brahuicus) were subjected for cytotoxicity test against the five cell lines at the dose of 20µgmL-1 with Methotrexate (MTX) as comparator. All the fractions were found effective against the selected cell lines and the IC50 values were within the range of 0.38±0.08 to 12.86±0.10 µgmL-1. Except CCU, rest of fractions revealed very strong to strong inhibitory effects in terms of IC50 value.
Among them, CCO fraction was found highly effective which resulted IC50 ranging from 0.68±0.07 to 6.74±0.07 µgmL-1.
Out of 21 column fractions (CCFs), six fractions were selected based on their best performance. These were subjected for in depth evaluation of their IC50 values. According to the National Cancer Institute (NCI), USA, plant screening program, the plants extract and fractions can be considered active/putative if their
-1 IC50 value is less than 20 µgmL against cancer cell lines. The IC50 value was recorded between 0.38±0.08 to 12.86±0.10 µgmL-1 revealing very strong to strong inhibition against all the selected cell lines.
Bioassay guided isolation of compounds was carried out to identify the compound (s) responsible for the activities. The chemical structures were established with the aid of extensive LC-MS spectroscopic, mass-spectral analyses and published data. Eight compounds viz., N,N-dipropyl propan-1-amine, 4- acetamidophenyl) 2-(diethylamino) acetate, 3-Methyl benzo furan-2-carboxylic
4
acid, [2-(Diphenylphosphoryl) ethyl] (methyl) oxo(phenyl) phosphine, 2-(3-
Amino-1H-1, 2, 4-triazol-1-yl) acetohydrazide, 3-Methyl-N-(3-methylbutyl)-1- butanamin, 1, 3-Benzenedimethanol, α1-[[(1, 1-dimethylethyl) amino] methyl]-4- hydroxy and 3-Chloro-6-hydrazinopyridazine were identified from six column chromatographic fractions.
This study provided detailed scientific information about the cytotoxic activity and phytochemistry of P. brahuicus that would serve as benchmark towards anticancer drug development. Structural elucidation and in vivo activity is therefore suggested to test potential toxicity of the fractions that may be used in the cancer drug development programme.
5
Chapter 1
GENERAL INTRODUCTION
Plants are blessings of the nature for the human being.They have provided the basic needs of man in terms of food, fruit, vegetable, shelter, clothing, flavours, fragrances and spices as not the least, medicines. They have formed the basis of modern system of traditional medicine such as Ayurvedic, Unani, Chinese and others. These systems of medicine contained therapeutic drugs which are popularly used even today. Now a days, search for new therapeutic molecules is in progress.
For this purpose, the science of ethnobotany and ethnopharmacognosy are being used as a tool to guide and to lead the chemist towards different sources of compounds (Qureshi et al., 2016).
Plants are used as a source of natural drugs to cure various disorders worldwide. Traditionally used plants provide an important source for the development of novel drugs. In developed countries, there is increasing trend to search the use of medicinal plants rather than advanced medicine. Worldwide it has been recognized that medicinal plants are the best source on account of the presence of bioactive compounds in terms of their therapeutic potential. It has been estimated that about 80% of the world population use medicinal plants to treat diseases (Khan et al., 2012).
Plants have been used as medicament from the period of antiquity that formulated various healthcare systems, the most popular ones Ayurvedic, Unani
5
6
and Chinese systems. Many of herbal drugs are still in use that highlights the importance of such system of medicines. Even in developed world there is steady increase the demand of herbal drugs and natural products that shows recognition of such products. That appeals searching of new compounds of commercially importance (Koyama et al., 2006). Medicinal plants formulate the ground for searching new pharmaceutical and healthcare products. Using medicinal plants in disease prevention programme may be attributed to antioxidant properties of various phytoconstituents, normally found associated to a wide range of amphiphatic molecules, broadly termed as polyphenolic compounds. Efforts are done to explore the potential use of naturally occurring anti-inflammatory and antioxidant compounds (Hamamouchi, 2002; Bhakta et al., 2009).
All living organism contains metabolites and enzymes which prevent them from oxidative damage. Researchers have found a strong correlation between oxidative damage and the occurrence of diseases. A large number of plants and plants based products have been evaluated as potential chemo protective agents in the living cells. Natural products based on medicinal plants have played an important role in the treatment of cancer (Bhakta et al., 2009; Khan et al., 2012).
Medicinal plants are used for the cancer treatment that may contain anti- cancer compounds. The plant secondary metabolites in terms of phenolics and flavonoids are widely distributed and have been documented to have biological activities. In vitro and in vivo studies of antitumor and anti-allergic effects have proved their therapeutic potential (Koyama et al., 2006). Scientists are interested in
7
search of new chemotherapeutic agents from natural resource based or plant based.
The statistical data of the world shows that more than 50% natural product and their derivatives are in clinical use and angiosperm shared 25%. Interestingly, a dozen of potent drugs have been developed from higher plants during the last 40 years (Gurib-Fakim, 2006). Natural products derived from medicinal plants are known to play an important role in discovery of medicine. In fact synthetic medicines have some structural components that completely or principally derived from natural molecules (Hussain et al., 2011).
Cytotoxicity is a mechanism in which bioactive compounds cause toxicity in cells. This activity may be attributed to some toxic chemicals, toxic venom of various animals as well as immune cell. Medicinal plants are playing important role in the cancer therapy. Currently, more than 50% drugs have been isolated from medicinal plants are being used to check anticancer activity (Newman et al., 2003).
Purification and isolation of anticancer agents from medicinal plants are becoming an area of great interest. The main methods which are used for selection of medicinal plants including random screening and organized scientific approaches
(Pieters and Vlietnick, 2005).
Prior to 1983 and 1994 in the US, 92 anticancer drugs were commercially available and worldwide approved, among them 60% drugs are of natural origin
(Cragg and Newman, 2003). Certain anticancer drugs have been isolated from plants as a result of their traditional use in various indigenous communities and available in market. The most pronouncing such examples include vinblastine
8
(Cantharanthus roseus), etoposide, teniposide (Podophyllum spp.), pacilitaxel, docetaxel (Taxus spp.), topotecan, irinotecan HCl (Camptotheca acuminata), homoharringtonine (Cephalotaxus harringtonina), flavopiridol (Dysoxylum binectariferum) and combretastin (Combretum caffrum). These are originated from
Philippines, Eastern United States/Himalayas, Northwest United States, China,
India and South Africa respectively (Cragg and Newman, 2003).
Chemotherapy does not cure cancer patients due to susceptibility and toxic effects by the synthetic drugs as well as continuous use of such drugs may cause gradual resistance in the human body. Keeping into consideration, medicinal plants are screened for anticancer properties throughout the world. Therefore, present study is designed with the hypothesis that the study area may possess potential plants which may have bioactive compounds that can be employed in anticancer programme. No such study is previously reported from the study area. Followings are the objectives of work:
To screen medicinal plants of salt range for anticancer activities.
In vitro screening of selected plants extracts for anticancer activities.
Phytochemical analysis of selected fractions having significant anticancer
activities
9
Chapter 2
MEDICINAL PLANTS
2.1 INTRODUCTION
Plants are fundamental component for the survival of human life on this globe. They supplied various ecosystem services such as food, shelter, clothing, and medicines. Based on miraculous medicinal properties, these are still used in our traditional healthcare systems of medicine like Ayurvedic, Unani, Chinese and various other ones. Based on their long historical use, toady plants are targeted in searching new biomolecules for developing new drugs. Ethnobotanical study is playing an important role for getting clues for the identification of potential plants which are being used by the communities that may lead to the identification of various compounds.
In the last 100 years, the science of ethnobotany has progressed and turned from mere documentation to a more practical one, which emphasized on conservation and sustainable use of plant resources. Reviews of ethnobotanical studies of many areas have been presented (Leporatti and Paresi, 1990; Padhye et al., 1992; Yang and Walter, 1992; Singh and Mheshwari, 1994). This field extracted valuable information about plant use throughout the world (Kumar et al.,
1980; Bhattarai, 1992; Girach et al., 1992; Motley, 1994 and Qureshi et al., 2001,
2002, 2011).
In Pakistan, ethnobotany is gradually going to be matured. Various studies
9
10
have been reported for different parts of the country (Hamayun et al., 2003;
Goodman and Ghafoor, 1992). However, still there are various pockets, localities in which the information about valuable plants is meager. In Pakistan, the major consumption of medicinal plants is in tibbi dawakhanas (medical centers of indigenous physicians known as Tabeeb/Hakeems). Unfortunately, very little attention has been paid to the important aspect of plants as Hakeems are only concerned with those parts of plants which fulfill their medicinal purpose. They have no concern to regard their botanical features or their distribution in the various ecological zones of the country i.e. Pakistan. No wonder these herbs are now being commercially exploited for the extraction of various ingredients.
Due to importance of the traditional practice of local flora by the traditional healers (Hakeems), this study was carried out to document important medicinal plants from the Salt Range, Pakistan with the view to undertake cytotoxic activity of selected medicinal plant based on their therapeutic potential in the form of ethnobotanical documentation. Some of the papers are written on the associated areas of this range showing qualitative attributes of ethnobotany of fewer plants.
For instance, Qureshi et al. (2007) carried ethnobotany of Mianwali District reporting 26 species used medicinally. Ahmad and Husain (2008) studied medicinal plants of Salt Range (Kallar Kahar) and documented 29 species. In both the studies only qualitative data were presented besides their uses other than jaundice and associated diseases. Furthermore, the present endeavor is based on quantitative appraisal of ethnobotany focusing on medicinal plants which are heavily used in treating complex of diseases. Based on previous experience of our research group
11
from the allied area i.e. Khushab (Qureshi et al., 2011), it was hypothesized that drought and salt stress may enhance secondary metabolites in various parts of plants and likewise salt range was selected for surveying and screening medicinal plants which are practically and particularly used by the local herbalists (Hakeem) to treat jaundice or complex nature of diseases such as edema, arthritis, asthma, pneumonia, etc.
2.2 REVIEW OF LITERATURE
The main objective of ethnobotanical knowledge is to record the indigenous knowledge about plants. A number of efforts have been made in this regard.
Reviews of ethnobotanical studies of other area have been presented (Leporatti and
Paresi, 1990; Padhye et al., 1992; Yang and Walter, 1992; Singh and Mheshwari,
1994). There are various studies reported from Baluchistan. Plant is commonly utilized by the people of Baluchistan (Shinwari and Malik, 1989). The inventory of the indigenous medicinal plants and herbs is also reported from Balochistan
(Goodman and Ghafoor, 1992). Preliminary ethnobotinical information is obtained by a team of scientists (Malik et al., 1990). Rasool (1998) reported 15 medicinally important plants from Saltanat of Oman. He described various parts of species which are used for the treatment of various diseases.
Ethnobotany is worldwide accepted as disciplines and a number of studies are reported from different corners of the globe. Kumar et al. (1980) carried out ethnobotanical research in Meghalaya. They reported medicinal uses of 54 plant species by local people in the area. Likewise South’s study (1993), Qureshi et al.
12
(2002) carried out ethnobotanical uses of Aloe barbadensis in Nara Desert and weeds of sugarcane crop in district Sukkur, Sindh (Pakistan) indicating 50 weed species in common use in the area under study.
Previously Kumar et al. (1980) carried out ethnobotanical research in
Meghalaya. They reported medicinal uses of 54 plant species by local people in the area. Another same type of study was reported by Bhattarai (1992). He described
60 recipes involving 58 plant species along with detail recording of administration and dosage. Girach et al. (1992) described 27 medicinal uses of Achyranthes aspera L. in India. Motley (1994) presented a comprehensive survey of past, present and future uses of sweet flag- Acorus calamus (Arecaceae).
South (1993) studied seven taxa of seaweed used in the diet of native
Fijians ethnobotanically. According to him the native Fijian population is organized through family and village groups in a cooperative manner.They consume them in a variety of recipes. Up to 36 metric tons of seaweed, valuing US $ 32,000 per annum are sold in Municipal Market of Fijian and other outlets. Another study was conducted from the tehsil Chakwal reporting 29 plants species belonging to 25 genera and 18 families used as medicinally by the inhabitants for curing certain type of diseases (Qureshi et. al., 2009).
During 1998-2001 the ethnobotanical survey of the remote area of the Nara
Desert they recorded six plant species of 4 genera of family Amaranthaceae which are highly used in medicine as well as ethnovetrinary purposes (Qureshi et. al.,
13
2009). An ethnobotanical survey was conducted in Nara desert during 1998-2001.
Local people depend on the plants to cure their certain disease such as asthma, cough, diabetes fever, flue, gas trouble gynecological diseases, male sexual diseases, ear diseases, tooth problem, piles, urinary diseases and miscellaneous uses. During the survey they recorded 63 medicinal plants species belonging to 50 genera and 29 families. The major plant family which has been used extensively as a source of medicine was Fabaceae 7 spp. followed by Boraginaceae 6 spp.,
Amaranthaceae 5 spp., and Cucurbitaceae 4 spp. (Qureshi et. al., 2010).
2.3. MATERIALS AND METHOD
2.3.1 Study Area
The Salt Range is named for rock salts deposits that extended in hilly range in the Punjab province of Pakistan. It lies from river Jhelum to the river Indus in north of the Punjab. There are great mines of Khewra, Khatha, Warchha and
Kalabagh that yield salt in large quantities. The area is situated between 32° 23′ –
33° N and 71° 30′ – 73° 30′ East on an area of 150 miles from east to west (Fig.
2.1).This area has significance since it lies between the Thal desert in the west and the Potohar plateau in the north east (Ahmad et al., 2007).
2.3.1.1 Geography
Geographically the area endowed with mountains, lush green harvesting lowland, lakes and river. The study area is rich in form of natural resources (salt & coal). Most people are associated with coal mining, salt mining, farming and agriculture Most of the area is hilly and there is no agriculture in the study area.
14
Figure. 2.1: Map of Salt Range, Punjab, Pakistan
15
Salt range, is basically situated between the districts of, Mianwali, Khushab,
Chakwal, and Jhelum.
2.3.1.2 People
Majority of people are serving in the armed forces and some other professions. The people are very hardworking and used agriculture to be the main profession. Consequently the people have migrated to large cities for jobs. As population is increasing with alarming rate per person square footage of land is decreasing. Majority of the population from this area goes into the armed forces.
Most of them have still a very hard life. They cultivate grains only. Mostly people are illiterate in the area and facing a problems regarding education, health and other basic necessities of life there.
2.3.1.3 Farming
The farmers of the area involved growing various agricultural corps like
Grams (Cicer arietinum) wheat (Triticum aestivum), Green Mong (Vigna radiata) mash (Vigna mongo), jawar (Sorgham bicolor) and bajra (Pennisetum typhoides) including makaee (Zea mays), Jao (Hordeum vulgare) peanut (Arachis hypogea) crops. In this way farmer is also playing a remarkable roll to full fill the local food requirement at large level.
2.3.1.4 Culture
The people of the area show intense feelings at all occasions whether it is the wedding ceremony or the funeral. They show their deep feelings with their own
16
relatives as well as with their community. The wedding ceremony usually takes place at masjid (mosques). People prefer the Nikkah in the masjid (mosque) instead of their home. They have firm belief that the Nikkah, which is recited in the mosque, bring harmony in the life of the couple. At the end of the nikkah, dried dates locally known as Chhuarah along with sweet items locally known as Laachi dana and Patasa are distributed among the participants.
Due to lack of education they waste lots of money at the event of Rasm-e- mehndi and Baraat. The close relative of groom arranged musical programme along with Lok Raqas. The bride is restricted in the room before three days of her
Rukhsati and ubton (locally known as chheeku) is applied on her face, arms and legs for whitening and softening of skin this Rasm (cutoms) is known as Medhhi.
Next day after noon, rasm-e-maaennyaan started from the Groom relatives. They distribute the Panjeery, a kind of sweet dish made up of wheat. After this, rasm-e- warry is started in which the parents of the groom show their wealth and distribute the money and clothes in their relatives and among the friends. They show their jewelry, clothes and all those things to their relatives which they are giving to their daughter in law.
The parents of the bride also display all material which they are gifting their daughter at this event. It contains clothes, jewelry, cooking utensils, trunks and all the essential items which are necessary for their life, but they waste lot of money on this custom which is known as Jahez. This custom has negative impact on the life of the rural areas community.
17
At the funeral occasion, the people lose patience especially women, just shouting/weeping over the dead body very loudly locally this is known as Waienr, a common practice in the rural areas. They serve their guest also at this event like wedding but this expense is met by the close relative till the end of Rasm-e-qul.
Musical programme, Kabbadi, Thobi ball, Walli ball, Cricket, Dog and Cock fight, horse and camel dance and bull race is highly appreciated and enjoyed by the people at the event of Eid festivals and specially organized at the event of Mela and
Urs.
2.3.2 Collection of Ethnobotanical Information
The indigenous knowledge of medicinal plants was gathered or extracted from inhabitants of Salt range, Pakistan by using semi-structured questionnaire
(Annexture-1) following the work of Qureshi et al. (2011). Regular field visits were arranged in order to collect information about the folk knowledge of plants. During field trips, the questionnaires were used on random basis to interview the local inhabitants, including older people, herbalists (Hakeems), midwives (Daai) and elderly known people who will be familiar with the traditional use of local plants.
In order to acquire medicinal information, 30 informant between ages of 40-80 were selected. The same was quantified through fidelity level (FL %) for each species. Following formula was employed for this purpose after Alexiades and
Sheldon (1996).
푁푃 퐹퐿 (%) = 푋100 푁
Where N= total number of informants that established its medicinal use in
different diseases and Np = number of informants which reported specific
18
use of medicinal plant for same disease.
2.3.3 Specimen Collection
The plant specimens were collected from different locations of the study area. All these specimens were dried and mounted on herbarium sheets after
(Qureshi et al., 2011).
2.3.4 Identification and Preservation of Specimens
The collected specimens were identified with the help of floristic literature
(Stewart 1972, Nasir and Ali 1971-2001, Nasir and Rafiq 1995, Qureshi, 2004) and deposited in the herbarium of Pir Mehr Ali Shah (PMAS) Arid Agriculture
University (AAU), Rawalpindi for record.
2.3.5 Data Compilation
By using excel spreadsheet the data were statistically analyzed and certain graphical presentations are made. All species were arranged alphabetically and presented in the form of thesis
2.4. RESULTS
2.4.1. Inventory of Medicinal Plants
The present study documented 71 plant species belonged to 61 genera and
35 families that served to the natives for fulfilling their primary healthcare needs.
The detailed inventory including botanical/vernacular names, parts used, mode of preparation, medicinal uses, number of use reports and fidelity are provided in
Table 2.1.Mimosaceae was found the most dominating family, which contributed 7
19
spp. (9.86%), followed by Solanaceae (8.45%), Brassicaceae, Euphorbiaceae,
Moraceae (5.63% each), Astraceae, Boraganaceae, Fabaceae, Zygophyllaceae
(4.23% each), while rest of the families had few numbers of species (Figure 2.2).
2.4.2 Fidelity Percentage
The ethnobotanical data reveals that Fagonia indica was highly utilized medicinal plant with 76.60% fidelity, followed by Otostegia limbta (73.3% FL),
Artemisia scoparia & Physorhynchus brahuicus (70% FL each), Moringa oleifera
& Withania coagulans (66.6% FL, each) and Rhazya stricta (63% FL). The rest of species had less than 60% fidelity determined by the natives (Table 2.1).
2.4.3 Disease Treated
Altogether, 56 different ailments/diseases were treated by using 71 plant species (Table 2.2). Boils was found the most prevalent disease in the area and most of the species (11 spp., 7.19%) were used to treat that complaint. It was followed by diabetes (10 spp., 6.54%), jaundice/hepatitis & skin allergy (9 spp.,
5.88% each), constipation & inflammation (7 spp., 4.58%, each), pimples and spermatorrhea (6 spp., 3.92%, each). The other diseases were treated by using species 1-6 (Table 2.2).
2.4.4 Parts Used
Native people and Hakeems of the study area consumed every part of the plant. However, a particular part of plant is used as per need of the user. Overall, 17 parts of medicinal plants were utilized by native communities for the preparation of
20
Table 2.1: List of medicinal plants along with local names, family, method of preparation, disease treated, use reports and fidelity
percentage from the study area.
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
1 Fagonia indica var. Dhaman Zygophyllaceae Whole plant Powder, Boils, skin 23 76.67
schweinfuthii Hadidi. decoction irritation, hepatitis
2 Otostegia limbta (Benth.) Chitta jand / Lamiaceae Leaves Gargle, Sore throat, boils 22 73.33
Boiss. Awani booti paste
3 Artemisia scoparia Jhau Asteraceae Whole plant Paste Urticaria, skin 21 70.00
Waldst & Kitam eruption
4 Physorhynchus Sanny pat Brassicaceae Whole plant Decoction Inflammation, skin 21 70.00
brahuicus Hook. f. warts
5 Moringa oleifera Lam. Sohanjna Moringaceae Leaves, fruits Powder, Joints pain, liver 20 66.67
potherb tonic
21
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
6 Withania coagulans Jangli paneer/ Solanaceae Leaves, root Juice, Skin allergy, boils, 20 66.67
(Stocks.) Dunal Akkri bark, fruits paste cooling agent
7 Rhazya stricta Dcne. Winraan Apocyanaceae Leaves, root Decoction, Skin irritation, 19 63.33
bark powder, diabetes, boils
viscous
extract
8 Aloe vera (L.) Burm.f. Kunwar Aloaceae Whole plant, Pulp Pimple, liver tonic, 17 56.67
gandal pulp jaundice
9 Ficus bengalensis L. Bohr Moraceae Juvenile Powder, Spermatorrhea, 17 56.67
leaves, decoction, impotency,
hanging roots, juice diarrhea,
buds, stem leucorrhea,
22
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
bark pregnancy, tonic,
premature
ejaculation
10 Ricinus communis L. Arind Euphorbiaceae Leaves, oil Roasted, Constipation, 17 56.67
oil inflammation,
painkiller
11 Schweinfurthia Sanny pat Plantaginaceae Whole plant Powder Antidiabetic, stop 17 56.67
papilionacea (L.) Boiss. bleeding from nose
12 Withania somnifera (L.) Aksan Solanaceae Roots, fruits Powder, Inflammation, 16 53.33
Dunal. juice, spermatorrhea,
decoction leucorrhea,
flatulence, tonic
23
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
13 Cicer arietinum L Chana Fabaceae Seed Potherb Diabetes 15 50.00
14 Tribulus terrestris L. Bakra Zygophyllaceae Fruits Powder Spermatorrhea, 15 50.00
kidney stone,
urinary tract
infection
15 Azadirachta indica (L.) Nim Meliaceae Leaves, Paste, Liver tonic, skin 14 46.67
A. Juss. flowers, seeds decoction, irritation, boils,
juice diabetes
16 Corchorus depressus L. Bophali Tiliaceae Whole plant Juice Appetizer 14 46.67
17 Solanum nigrum L. Katch match / Solanaceae Whole plant Juice Jaundice 14 46.67
Mako
18 Morus alba L. Toot Moraceae Leave, root, Decoction, Joints pain, liver 13 43.33
24
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
bark, fruit juice tonic
19 Morus nigra L. Shetoot Moraceae Leave, root, Decoction, Throat pain, 13 43.33
bark, fruit juice anthelmintic
20 Bombax malabaricum Simbal Bombacaceae Root Decoction Abdominal worm 11 36.67
DC.
21 Farsetia hamiltonii Latia Brassicaceae Whole plant Powder, Constipation, 11 36.67
Royale paste stomachache,
pimples
22 Lawsonia inermis Lam. Mehndi Lathyraceae Leave, Powder, Cooling agent, 11 36.67
branches paste, athlete foot
juice
23 Prosopis cineraria (L.) Jand Mimosaceae Gum Gum Semen production 11 36.67
25
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
Druce.
24 Solanum incanum L. Mahori Solanaceae Whole plant Powder Flatulence 11 36.67
25 Solanum surattense Kori wall / Solanaceae Leaves fruit Juice, Joint pain, asthma, 11 36.67
Burm.f. Peelay potherb, cough
chhiday/ powder
Mahori
26 Cynodon dactylon (L.) Khabal ghaa Poaceae Root, leaves Decoction, Fever, burning 9 30.00
Pers. paste sensation, pimple,
wound
27 Eruca sativa Miller. Jamaon Brassicaceae Leaves, Potherb, Earache, skin 9 30.00
flowers, oil itching, pimple,
seeds, oil antilice,
26
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
antimicrobial
28 Momordica balsamica L. Jangli Kareela Cucurbitaceae Fruit Potherb, Diabetes, liver 9 30.00
juice tonic, boils
29 Peganum hermala L. Hermal Zygophyllaceae Whole plant, Smoke, Appetizer, 9 30.00
seeds powder anthelmintic,
pimples, boils,
asthma
30 Prosopis juliflora Walaiti kikir Mimosaceae Leaves Paste Urinary problem 9 30.00
(Swartz) DC.
31 Tamarix aphylla (L.) Khaggal Tamaricaceae Leaves Smoke Measles, boils 9 30.00
Karst.
32 Ziziphus mauritiana Lam. Beri Rhamnaceae Leaves, fruit Poultice, Spine removing, 9 30.00
27
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
juice blood purifier,
diabetes, analgesic,
inflammation
33 Ziziphus nummularia Ghunger malla Rhamnaceae Leaves, fruit Poultice, Spine removing, 9 30.00
(Burm.f.) Wight & Arn. Juice blood purifier,
diabetes, analgesic,
inflammation
34 Acacia nilotica (L.) Del. Desi kikar Mimosaceae Bark Gargle, Pyorrhea, 8 26.67
subsp. indica (Benth.) douche, leucorrhea, eczema
Brenan paste
35 Acacia nilotica (L.) Desi kikar Mimosaceae Bark, gum, Powder, Spermatorrhea, 8 26.67
Delile. leaves,flowers paste skin irritation
28
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
and fruit
36 Carissa opaca Stafp ex Garanda Apocyanaceae Fruit Juice Jaundice, iron 8 26.67
Haines supplement
37 Cymbopogon jwarancusa Khavi Poaceae Whole plant, Smoke Measles, typhoid 8 26.67
(Jones) Schult. stem
38 Dalbergia sissoo Roxb. Tehli Fabaceae Stem Exude Athlete foot, 8 26.67
ringworm
39 Sisymbrium irio L. Jangli rai Brassicaceae Seeds Decoction Typhoid 8 26.67
40 Achyranthus aspera L. Puth kanda Mimosaceae Whole plant Decoction, Asthma, cough, 7 23.33
ash skin irritation
41 Albizzia lebbeck (L.) Bth. Shirin Mimosaceae Bark, seeds Powder, Headache, 7 23.33
decoction influenza, pyorrhea
29
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
42 Calotropis procera Akra Asclepiadaceae Leaves, Powder, Jaundice, 7 23.33
(Willd.) R. Br. flowers, fruit, Warm inflammation,
latex brown wound healing
43 Chenopodium album L. Bathu Chenopodiaceae Aerial parts Potherb Constipation 7 23.33
44 Chenopodium murale L. Bathu Chenopodiaceae Aerial parts Potherb Constipation 7 23.33
45 Ficus religiosa L. Peepal Moraceae Bark, leaves, Paste, Inflammation, 7 23.33
fruits powder, diarrhea, nausea,
juice, vomiting
decoction,
smoke
46 Glinus lotoides L. Baphara Molluginaceae Whole plant Powder Leucorrhea, 7 23.33
spermatorrhea
30
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
47 Heliotropium strigosum Gorakh paan Boraginaceae Whole plant Juice Cooling agent, 7 23.33
Willd. jaundice, liver tonic
48 Oxalis corniculata L. Khatti buti Oxalidaceae Leaves Powder Stomachache 7 23.33
49 Pluchea arguta Boiss. Jhau/Muther Asteraceae Leaves Smoke Insect repellent 7 23.33
50 Syzygium cumini (L.) Jammu Myrtaceae Bark, seeds Powder, Fever 7 23.33
Skeels. decoction
51 Capparis decidua Karri Capparaceae Twig, bark, Potherb, Pyorrhea, Athlete 6 20.00
(Forssk.) Edgew. fruit tooth foot
stick,
paste
52 Citrullus colocynthis (L.) Tumma/Kor Cucurbitaceae Roots, leaves, Pulp Gas trouble, 6 20.00
Schrad. tumma fruit, pulp, diabetes
31
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
seeds
53 Cuscuta reflexa Roxb. Peeli Cuscutaceae Whole plant Decoction Boils 6 20.00
wall/Akas bail
54 Datura fastuosa L. Dhatoora Solanaceae Leaves, seeds Paste, Asthma, cough, 6 20.00
decoction, swelling, antilice,
smoke insect bite
55 Melia azedirach L. Dharek/Bakain Meliaceae Leave, fruit Paste, Liver tonic, skin 6 20.00
decoction, irritation
juice
56 Alhagi maurorum Medic. Jawanha Fabaceae Leaves Paste Skin itching, boils, 5 16.67
eczema
57 Amaranthus viridis L. Cholai Amaranthaceae Foliage Potherb Constipation 5 16.67
32
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
58 Cleome brachycarpa Ganduli Capparaceae Whole plant Powder Stomachache 5 16.67
Vahl ex DC.
59 Cordia gharaf (Forssk.) Lasoora Boraginaceae Fruit Decoction Throat pain 5 16.67
Ehren. ex Asch.
60 Cordia myxa L Lasoora Boraginaceae Fruit Decoction Throat pain 5 16.67
61 Digera muricata Tandla Amaranthaceae Leaves Potherb Constipation 5 16.67
(L.)Mart.
62 Salvadora oleoides Wann Salvadoraceae Root bark Gargle, Pyorrhea, 5 16.67
Decne. seed oil ringworm
63 Carthamus oxycantha Pohli Asteraceae Seed Warm Hepatitis 4 13.33
M.B. brown
64 Convolvulus arvensis L. Lehli Convolvulaceae Whole plant Powder Appetizer 4 13.33
33
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
65 Euphorbia prostrata Ait. Dudhi Euphorbiaceae Whole plant Powder Diabetes 4 13.33
66 Euphorbia thymifolia L. Dudhi Euphorbiaceae Whole plant Powder Diabetes 4 13.33
67 Fumaria indica Shahtra papra Fumariaceae Whole plant Powder, Boils, pimples 4 13.33
(Hausskn.) H.N. Pugsley decoction
68 Acacia jacquemontii Kikri Mimosaceae Leaves Powder Urinary problem 3 10.00
Benth.
69 Asphodelus tenuifolius Bhokat Asphodelaceae Root, seeds Powder, Piles, constipation, 3 10.00
Cavan. paste, pulp jaundice,
ringworm, scorpion
bite
70 Boerhavia procumbens Itsit Nyctaginaceae Whole plant, Decoction, Liver tonic, 3 10.00
Banks ex Roxb. roots paste jaundice
34
Sr.# Plant species Local name Family Part(s) used Method Disease treated Use FL%
report
71 Chrozophora tinctoria Neeli Buti Euphorbiaceae Leaves Decoction Chest burning 3 10.00
(L.) Juss.
35
10
9
8
7
6
5 No. 4
3
2
1
0
Poaceae
Tiliaceae
Fabaceae Aloaceae
Moraceae
Meliaceae
Myrtaceae
Lamiaceae
Asteraceae
Solanaceae
Lathyraceae
Oxalidaceae
Cuscutaceae
Mimosaceae
Capparaceae Fumariaceae
Rhamnaceae
Brassicaceae
Moringaceae
Apocynaceae
Boraginaceae
Bombacaceae Tamaricaceae
Cucurbitaceae
Salvadoraceae
Nyctaginaceae
Euphorbiaceae Molluginaceae
Plantaginaceae
Asphodelaceae
Amaranthaceae Asclepiadaceae
Zygophyllaceae
Convolvulaceae Chenopodiaceae
Sp.# %age
Figure 2.2. Family Importance Index of the flora of Salt Range, Pakistan
36
Table 2.2: Ailments treated by the use of native medicinal plants from the Salt
Range, Pakistan
Sr.# Disease treated No. of species Percentage
1 Abdominal worm 1 0.65
2 Anthelmintic 2 1.31
3 Anti-lice 2 1.31
4 Antimicrobial 1 0.65
5 Appetizer 3 1.96
6 Asthma 4 2.61
7 Athlete foot 3 1.96
8 Blood purifier 2 1.31
9 Boils 11 7.19
10 Burning sensation 1 0.65
11 Chest burning 1 0.65
12 Constipation 7 4.58
13 Cooling agent 3 1.96
14 Cough 3 1.96
15 Diabetes 10 6.54
16 Diarrhea 2 1.31
17 Earache 1 0.65
18 Eczema 2 1.31
19 Fever 2 1.31
20 Flatulence 2 1.31
37
Sr.# Disease treated No. of species Percentage
21 Gas trouble 1 0.65
22 Headache 1 0.65
23 Impotency 1 0.65
24 Inflammation 7 4.58
25 Influenza 1 0.65
26 Insect repellent 1 0.65
27 Insect bite 1 0.65
28 Iron supplement 1 0.65
29 Jaundice/Hepatitis 9 5.88
30 Joints pain 3 1.96
31 Kidney stone 1 0.65
32 Leucorrhea 4 2.61
33 Measles 2 1.31
34 Nausea 1 0.65
35 Nose bleeding 1 0.65
36 Piles 1 0.65
37 Pimple 6 3.92
38 Pregnancy 1 0.65
39 Premature ejaculation 1 0.65
40 Pyorrhea 4 2.61
41 Ringworm 3 1.96
42 Scorpion bite 1 0.65
38
Sr.# Disease treated No. of species Percentage
43 Semen production 1 0.65
44 Skin allergy 9 5.88
45 Skin warts 1 0.65
46 Sore throat 4 2.61
47 Spermatorrhea 6 3.92
48 Spine removing 2 1.31
49 Stomachache 3 1.96
50 Swelling 1 0.65
51 Tonic 2 1.31
52 Typhoid 2 1.31
53 Urinary tract infection 3 1.96
54 Urticaria 1 0.65
55 Vomiting 1 0.65
56 Wound healing 2 1.31
Total 153 100.00
39
different kinds of recipes for the treatment of various ailments/diseases. Leaves were mostly used part for the preparation of herbal recipes (24.79%), followed by whole plant (18.18%), fruits (15.70%), seeds (9.9%), barks, root (6.61%, each), flower, stem (3.31%, each), root bark (2.48%) while rest of 8 parts were scarcely used (Table 2.3).
2.4.5 Mode of Application
Preparation of recipes is a traditional art that varies from region to region and culture to culture. Overall, 16 methods of application were used by the inhabitants of the area for treating common diseases. Powder formation (Phakki) is very commonly used mode of application and 21.24% (Fig. 2.3) of formulations discovered from the study area. It was followed by decoction (20.35%), juice
(15.93%), paste 13.27% and potherbs (8.85%), smoke (5.31%), while rest of ten formulations were within the range of 1-3% and scarcely used.
2.4.6 Selection of Plants for Cytotoxicity
The species having more than 60% fidelity percentage were selected for further activities. In this connection, seven plants namely Fagonia indica,
Otostegia limbta, Artemisia scoparia, Physorhynchus brahuicus, Moringa oleifera,
Withania coagulans and Rhazya stricta were selected (Table 2.1). The detailed activity is explained in succeeding chapters.
2.5 DISCUSSION
The present study documented 71 plant species belonged to 61 genera and
40
35 families that served to the natives for fulfilling their primary healthcare needs
(Table 2.1). According to plant families, Mimosaceae was found the most dominating family, which contributed 7 spp. (9.86%), followed by Solanaceae
(8.45%), Brassicaceae, Euphorbiaceae, Moraceae (5.63% each), Astraceae,
Boraganaceae, Fabaceae, Zygophyllaceae (4.23% each), while rest of the families had few numbers of species (Figure 2.2).
The results revealed that the inhabitants have good knowledge of local plants in order to fulfill their daily life requirements. Since the first step is naming to show attachment with the plants, therefore most of the species were culturally named that reflects their attachment with the native flora of the study area. The results of present study are in agreement of the work carried out by (Qureshi and
Bhatti 2009; Qureshi et al., 2011).The people of area faces high temperature in summer above 50º C during and below up to 4º C during winter. The area is scarce in term of water because of aridity and salinity which resultants skin boils, pimples and other types of skin allergies. Due to this reason people use large number of plants to cure boils disease in the area and most of the species (11 spp., 7.19%) were used to treat that complaint. It was followed by diabetes (10 spp., 6.54%), jaundice/hepatitis & skin allergy (9 spp., 5.88% each), constipation & inflammation
(7 spp., 4.58%, each), pimples and spermatorrhea (6 spp., 3.92%, each). The other diseases were treated by using species 1-6 (Table 2.2). This type of studies have been reported by various scientist such as Kumar et al., (2004), Qureshi et al.,(2011), Amujoyegbe et al., (2016); Sher et al., (2016).
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Table 2.3: Different plant parts used for the preparation of herbal recipes
Sr.# Plant part(s) used No. of species Percentage
1 Leaves 30 24.79
2 Whole plant 22 18.18
3 Fruits 19 15.70
4 Seeds 11 9.09
5 Bark 8 6.61
6 Root 8 6.61
7 Flowers 4 3.31
8 Stem 4 3.31
9 Root bark 3 2.48
10 Aerial parts 2 1.65
11 Gum 2 1.65
12 Oil 2 1.65
13 Pulp 2 1.65
14 Buds 1 0.83
15 Hanging roots 1 0.83
16 Latex 1 0.83
17 Stem bark 1 0.83
Total 121 100.00
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24 24 22 23
20
18 18 16
14 15 Species No. Species 12
10 10 8
6 6 4
2 3 3 3 2 1 1 1 1 1 1 0
Figure 2.3: Mode of application of recipes employed by the people of Salt Range,
Pakistan.
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From remote times, man used herbs as first source of medicine, which they were able to cater from their surrounding areas. This study reveals that natives of study area have good knowledge of plants from their settled areas to use as first source of medicines. The fundamental needs of human being were food shelter, clothing and medicines obtained from plant resources. Surveys on medicinal plants gathered valuable information about diverse uses of plant species from different areas of the world. In this regard, various studies have been reported from remote areas of the world (Bhatti et al., 2001; Qureshi, 2002; Abu-Dahab and Afifi, 2007;
Gilani et al., 2006; Qureshi and Bhatti, 2008; Qureshi et al., 2011; Amujoyegbe et al., 2016; Sher et al., 2016).
People of the study area and herbalists (Hakeems) used each and every part of plant to cure certain types of disease. They also used every opportunity for the utilization of natural resources (Bhatti et al., 1998). Results of present study revealed that leaves were mostly used for the preparation of herbal recipes
(24.79%), followed by whole plant (18.18%), fruits (15.70%), seeds (9.9%), barks, root (6.61%, each), flower, stem (3.31%, each), root bark (2.48%) while rest of 8 parts were scarcely used (Table 2.3). The current work is in endorsement of the study carried out by various scientific studies across the world (Amujoyegbe et al.,
2016; Sher et al., 2016).
Preparation of recipes is a traditional art and varies from region to region and culture to culture. In the area, powder formation (Phakki) is very commonly used mode of application and 24.21% (Fig. 2.3) of formulations discovered from
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the study area. It was followed by decoction (20.35%), juice (15.93%), paste
13.27% and potherbs (8.85%), smoke (5.31%), while rest of ten formulations were within the range of 1-3%. The finding of present study are in lines with various studies such as (Mradu et al., 2012; Qureshi et al., 2011; Amujoyegbe et al.,
2016).Various other studies reported similar type of results like Ahmad et al.
(2008), Qureshi and Bhatti (2008), Kumar et al. (2007); Sher et al., (2016).There are no organized shops for purchasing and selling of medicinal plants, therefore most people go to the field directly and collect fresh plant for their use. They also sells medicinal plants and plant based products in nearby market to earn money.
2.6 CONCLUSION
The present findings revealed that the plants of the area are providing most of the basic requirement for the survival of the traditional communities. Medicinal use of plants is a common practice by the natives. The present study is first report on the Salt range to record the indigenous knowledge of plants particularly recipes used for the treatment for various ailments. Some of the significant species having more than 60% fidelity percentage were required to authenticate their medicinal values. These species including Fagonia indica, Otostegia limbta, Artemisia scoparia, Physorhynchus brahuicus, Moringa oleifera, Withania coagulans and
Rhazya stricta were further explored for determining cytotoxicity. The detailed activity is explained in succeeding chapters.
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Chapter 3
CYTOTOXICITY
3.1 INTRODUCTION
Cancer caused over 8.8 million deaths worldwide in 2015. While in 1990 cancer was the 3rd leading cause of death worldwide it advanced to 2nd place in
2015. According to an estimate of the American Cancer Society (ACS) for 2015,
1,658,370 new cancer cases were diagnosed and 589,430 cancer deaths registered in the United States (US) alone. Its incidence rate will increase by 70% over the next two decades. Presently, one out of six death is due to cancer during 2015 globally (GBD, 2016).
For some cancers effective, albeit expensive, therapies are available, whereas no effective therapies are available for other. Breast cancer as example of the former has a high 5-year survival rate (89%) in the US when treated at stage
I/II, whereas brain and pancreatic cancer belong to the 2nd group of cancers with no effective therapy and poor prognosis expressed by a 5-year survival of only 35% and 7%, respectively. While cervical cancer can be treated the prognosis and 5-year survival 68% rate is still disappointing. Table 3.1 summarizes the incidence and death rates for the world, the US and Pakistan for these 5 cancers showing a clear survival difference between high and low income countries, and also that the worldwide statistics are dominated by high income countries either due to over- reporting of cancers in high income countries or conversely under -reporting of cancers in low income countries.
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46
Table 3.1. Cancer incidence for 5 malignancies in worldwide, in the USA and Pakistan. Summary of absolute number of cases and ranking
of the cancer within the respective sample area.
World USA Pakistan
Global Incidence Death 5 year Incidence Death 5 year Incidence Death 5 year ranking survival (%) survival [%] survival [%]
Breast 5 1,804,290 464,300 84 234,190 40,290 89 83,000 40,000 11.5 1st 5th 2nd 3rd 3 1st 2nd Cervix 8 527,624 265,672 68 12,900 4100 68 5,233 2,876 48 7th 10th 20th 18th 15th 16th Pancreas 9 350,361 330,000 6 48,960 40,560 7 575 552 < 1 14th 7th 10th 4th 24th 18th Brain 10 304,528 204,000 19.8 22,850 15,320 35 4253 3340 N/A 15th 12th 17th 12th 14th 13th Skin 23 120649 31390 61 80100 13340 89 196 124 N/A 17th 23rd 5th 16th 25th 26th Cancer Facts & Figure 2015,
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Among all cancers, breast cancer is ranked highest in incidence and prevalence in both high and lower income countries. Its incidence increases at an alarming rate throughout the World and ranks currently as second highest cause of cancer death in the world. According to reports of WHO, NCI and IARC, 1.8 million new cases and 464,300 deaths are reported annually worldwide. Every year about 2300 new cancer cases of breast cancer in men and 230,000 in women are reported by the U.S. In Pakistan breast cancer is the most frequent malignancy in women (Khan et al., 2012), and cervical cancer is the 2nd most common cancer for women age 15 to 44 years (Cancer facts & figure 2015). Survival rates in high income countries are significant better than in low income countries, attributable to late diagnosis and missing therapeutic options, predominantly due to excessive costs of chemotherapeutic drugs particular those related to personalized cancer medicine.
Therapeutic approaches for the eradication of the various cancer include next to standard of care comprised of surgery, radiation therapy and chemotherapy also alternative therapies such as laser based therapies, organ sparing and trans- orifice robotic surgery (TORS). While chemotherapy can be effective particular for early stage cancers, damage to healthy cells and organs cannot be voided (Ariyan,
1987).
Systemic therapies independent of the drug are often burdened with toxicity and morbidity. To minimize the unspecific toxicity and morbidity, personalized cancer therapy based on the genetic expression of oncogenes and lack of tumor
48
suppressors is actively being developed (Ariyan, 1987). So predominantly limited to high income countries. These pharmaceutical solutions will not necessarily provide adequate solutions for middle and low income countries or low social patients without universal access to healthcare, as these personalized cancer therapies are commonly associated with excessive costs. Hence, the present situation warrants to develop effective and less invasive procedures to target cancers (Cai et al., 2004).
Traditional medicine (TM) or Complementary and alternative medicine
(CAM) are generally population wide available, affordable and common practice in low income countries. For example a survey conducted by the WHO Roll Back
Malaria Program in 1998, noted that more than 60% of children with malaria are treated at home with herbal medicine (Cai et al., 2004). Overall, 80% of the world’s population has confidence in traditional medicine, and uses extracts for their primary healthcare needs (Sher et al., 2016; Qureshi et al., 2016). Mostly, the
TM/CAM therapies are supported by experimental or historical evidence pertaining safety and effectiveness. The increasing trend towards scientific studies investigating TM/CAM agents very frequently supports their therapeutic potential
(Ghani et al., 2011).
The main advantage of TM/CAM is due to its diversity, flexibility, availability and affordability in many parts of the world specifically in low and middle income countries. Compared with standard pharmaceutical drugs,
TM/CAM is low cost and requires relatively low level technology for its
49
implementation thus limiting the required capital investment needs. As a result,
CAM/TM therapies have the potential to improve health care delivery when embraced in low resource settings, however, the need to apply evidence based research remains to validate and/or improve the efficacy of these therapies (WHO,
2002).
Scientific studies of TM/CAM based therapies showed that they can be effective when use in AIDS, HIV patients. Based on their significant therapeutically potential, the United Nation (UN) AIDS control programme focuses on and advocates collaboration with TM practioners (Hakeems/Herbalist) in AIDS prevention and care in Sub-Saharan Africa (Gulcin et al., 2007; Qureshi et al.,
2011; UNAIDS., 2000).
It is estimated that worldwide more than 35,000 plant species are used in herbal medicine. In the previous five years, research has been conducted on more than 13,000 plants and an impressive body of evidence has been collected to demonstrate the considerable potential utility of medicinal plants in TM. In Asia medicinal plants are used widely as an alternative medicine for cancer related diseases and symptoms (Jaafar et al., 2012).
For one it is believed by the native population that plants have active naturally occurring compounds which can kill cancer cells (Jaafar et al., 2012).
However, limited studies have been reported on the tumor reducing efficacy of these medicinal plants (Balunas and Kinghorn, 2005). Nevertheless, there is a need to identify the components or active pharmaceutical ingredients (APIs) which are
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responsible for the therapeutic effects and the molecular pathway by which these compounds induce cancer cell death (Jaafar et al., 2012).
The World Health Organization (WHO) define medicinal plants as "a herbal preparation which are produced by subjecting plant materials to various processes including; extraction, fractionation, purification, concentration, among others, ultimately leading to herbal products for the immediate consumption” (WHO,
2008).
Medicinal plants can provide an alternate source of medication and a multitude has been used empirically for centuries for a range of indications including oncology (Fansworth and Soejarto, 1991; Doughari, 2006). These plants are rich in form of secondary metabolites, whereby the mechanism of action of these as a drug, is biological friendlier compared to most synthetic drugs. For example, flavonoids present a large group of plant secondary metabolites, used by the host for defensive purpose against photosynthetic stress, reactive oxygen species, wounds and herbivores. Flavonoids have been shown to play a significant role in inhibiting the growth of cancer cells, both in-vitro and in-vivo (Shoeb,
2006).
Ethnopharmacology and ethnobotany are playing a key role in searching new molecules with therapeutic potential and several drugs have been discovered so far based on these studies. Previous studies (Shoeb, 2006; Abu-Dahab and Afifi,
2007; Mothana et al., 2007; Kilani et al., 2008; Mesia et al., 2008; Shamim et al.,
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2009; Sufian et al., 2013; Maqsood et al., 2015) have reported on the in vitro cytotoxicity of plant extracts to examine anticancer activity in human tumor cell lines.
Oncologists are predominantly interested in active cancer treating compounds which induced cellular apoptosis, a property satisfied by the majority of secondary plants metabolites. Many studies reported that the mechanism of action of these anticancer drug is based on the induction of apoptosis (Doughari, 2006).
Recently, Hsu et al. (2016) reported on the effects of Ursolic acid (UA) derived from medicinal plant on A549 cells, a human lung cancer cell line, showing that exposure resulted in locking cells in G1 phase, which was linked to a decrease in cyclin D1, D2, E expression and activation of cdk2, 4 and 6, leading to the induction of p21 and WAF1, an accumulation of which might be through a p53 dependent manner. UA triggered apoptosis was demonstrated by DNA fragmentation.
Lin et al. (2014) reported effects of Eugenia jambolana L. on human leukemia HL-60 cells, which included induced apoptosis, a decreased cell population in G2/M phase and an increase in the G1 phase. Another study by Seo et al. (2001), reported on an extract of Artemesia asiatica which induced apoptosis in
HL-60 cells as determined by nuclear condensation, DNA fragmentation, release of mitochondrial cytochrome C into cytoplasm, proteolytic activation of caspases-9,-
3,-7 and cleavage of ADP-ribose.No studies have been reported to date on cytotoxicity from the study area in Pakistan. The selected plant species was
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collected during field survey in April, 2013 to October 2014 from the Salt range,
Punjab Pakistan at 32.2416º N, 72.0237ºE and 867m elevation above sea level. The present study is first report on the effects of the plant’s extracts cytotoxicity on
MCF-7 (Breast), HeLa (Cervix), RG2 (Brain), RD (Human rhabdomyosarcoma) and INS-1(pancreas) cancer cell lines following the guidelines and recommendation of the United States National Cancer Institute (NCI) plant screening program. According to the NCI, a plant extract is generally considered to have active cytotoxic effect if it inhibits the growth of 50% of the cells (IC50 value) at 20µgmL-1or less, following an incubation time of 48 to 72 hours (Geran et al.,
1972; Lee and Houghton, 2005; Sufian et al., 2013).
3.2 REVIEW OF LITERATURE
Since time immemorial, utilization of medicinal plants has been in practice by the practitioner/ herbalist (Hakeems) to treat range of ailments without knowing the active bio-compound which lead to the reduction or elimination of symptoms of the disease. Recent advancement in technologies became a turning point in health care where many medicinal plants which are gifted from the nature were being employed in research to synthesized variety of phytochemical compounds which were responsible for the various pharmacological properties of the plants. Few compounds which are plant derivative have been patented for the commercial use to treat illness.
Medicinal plants related to pharmacological properties like anticancer, antiviral, antidiabetics, antifungal, also the phytochemical constituents were
53
reviewed and documented. These reviews proved few related pharmacological properties of the plants where these plants are employed in the folk medicine.
Advancement in technologies and also research funding’s provide the suitable platform for the researchers to conduct extensive research on the medicinal plants.
Cytotoxic activities of 45 medicinal plants were determined through MRC-5 cell lines by Mesia et al. (2008). Results of these studies indicated comprehensive and dose dependent cytotoxic activity against the MRC-5 cell line. Mohanta et al.
(2007) characterized the methanolic extract of Yemeni medicinal plants against FL- cells lines cytotoxicity and found that activities of plant extracts are owing to the occurrence of some bioactive constituents in the extract.
The anticancerous effects of extract of Hemsleya amabilis was tested against different cell lines. Wei and his colleagues treated different types of cancer cells including U87, MDA-MB-231 and Jurkat cells with H. amabilis extract. This agent significantly inhibited tumor cell growth and colony formation at various concentrations. They also reported apoptotic assays with Annexin V staining at different concentrations (Shang et al., 2016).
Anti-inflammatory activity of 80% ethanolic extract of roots of Taraxicum officinale has been observed through carrageenan induced rat paw oedema test and before application of indomethacin showed 25% inhibition of oedema formation while showed 45% inhibition after the application of indomethacin. The methanolic extracts of flowers from T. officinale showed inhibition rate of 95% in
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Teradecanoylphorbol-13-acetate (TPA) induced ear edema test in mice. Extracts of
T. officinale leaves and roots exhibit slightly lower inhibition rates of 69 and 51% respectively in the same assay (Pieters and Vlietnick, 2005).
Anti-inflammatory activity of T. officinale has been investigating by aqueous methanolic extracts of root. The aqueous methanolic extracts of roots were portioned successively with hexane, ethyl acetate and butanol and observed significant inhibitory activity which was confirmed by the formation of leukotriene
B4 from human neutrophils. Butanol fraction resulted in highest 86% inhibition at
10 µgmL-1 and 32% at 3 µgmL-1 (Gulcin et al., 2007).
3.3 MATERIALS AND METHOD
3.3.1 Chemicals
Silica gel 60 (0,040-0,063) for column chromatograph (230-400 mesh
ASTM- Merck KGaA, Darmstadt, Germany), Ethyle acetate anhydrous, 99.8%
(EtOAc), Methanol HPLC grade, 99.9 % (MeOH) and n-Hexane 95% (n-hexane) all from Sigma-Aldrich, USA were used. Aluminium sheets pre-coated with silica gel 60 F254 (20 × 20 cm, 0.2 mm thick; E. Merck-Germany and DMSO was obtained by Fisher BioReagents® Fair Lawn, New Jersey.
3.3.2 Collection of Plant Material
Plant material was collected from different growing localities in the Salt
Range Punjab, Pakistan. One set of specimens was prepared and identified in the
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Taxonomy Lab., Department of Botany, Pir Mehr Ali Shah Arid Agriculture
University, Rawalpindi following the methodology of Qureshi et al. (2011).
3.3.3 Preparation of Crude Extract
The selected plant was washed, cut into pieces, dried in the shade and pulverized using a grinder. The fine plant powder was soaked in methanol for 5-10 days to extract solvable compounds with the remaining material removed by filter paper. The first crude methanolic extract (CME) was obtained by concentrating the filtrate under reduced pressure in a rotary evaporator and further dried in a vacuum oven at 40°C. The CME was stored at 4oC in dark until further use (Kaur and
Arora, 2009; Maqsood et al., 2015).
3.3.4 Column Chromatography Fractionation (CCF)
Chromatographic separations were carried out using aluminum sheets pre- coated with silica gel 60 F254 (20 × 20 cm, 0.2 mm thick; E. Merck) for thin layer chromatography (TLC) and silica gel (230–400 mesh) for column chromatography.
TLC plates were visualized under UV at 254 and 366 nm and by spraying with ceric sulphate reagent solution (by heating).
The methanolic extract (45 gm) was subjected to column chromatography over silica gel using n-hexane/EtOAc, EtOAc, EtOAc/MeOH and MeOH as eluent resulted into 21 fractions identified by letter (CCA-CCU) as listed in Table 3.2.
Fractions were combined according to similarity of Rf value on TLC according to the methodology as described earlier by Jabeen et al., 2013). In present study I am
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Table 3.2: Fraction weights and solvents used for the column chromatography over
silica gel separation of the methanolic extract.
Fraction Weight [g] EtOAc [%] n-hexane [%] MeOH [%]
A 1.91 5 95 0 B 2.05 10 90 0 C 0.21 20 80 0 D 0.34 20 80 0 E 0.62 20 80 0 F 1.41 30 70 0 G 0.51 30 70 0 H 0.55 30 70 0 I 0.89 40 60 0 J 2.31 50-70 50-30 0 K 1.13 80 20 0 L 4.57 80-90 20-10 0 M 1.78 100 0 0 N 5.46 98-95 0 2-5 O 1.85 10 0 90 P 0.32 20 0 80 Q 2.39 20 0 80 R 1.44 20 0 80 S 2.37 30 0 70 T 5.12 50 0 50 U 5.28 0 0 100
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focusing on the results of six CCFs which have shown significant results on the basis of preliminary screening.
3.3.5 Preparation of Stock Solution of Crude Extract
The dried plant extract of whole plant, leaves, root and six CCFs were weighted using a standard analytical balance (OHAUS®, model A560, S/N 2162-
USA) and dissolved in Dimethyl sulfoxide (DMSO Fisher Bio Reagents® Fair
Lawn, NJ) to a final stock solution concentration of 1 mgmL-1.
3.3.6 Dilution of Stock Solution
Dilutions of the stock solution were made in cell culture medium, utilizing, minimum essential medium (MEM) alpha for MCF-7 , Dulbecco’s Modified
Eagle’s Medium (DMEM) for RG2 and RD while Roswell Park Memorial Institute medium (RPMI-1640) for HeLa and INS-1 cells. All media containing phenol red and 2% fetal bovine serum (FBS), 1% L-glutamine (2mM), 1% sodium pyruvate
(1mM) and 1% antibiotics penicillin (5000UmL-1) and streptomycin (2500UmL-1)
(all from Gibco®, Invitrogen, CA, USA) as supplements. Logarithmic dilutions of the stock solution between 20 and 0.1 µgmL-1 to determine the extract fractions cytotoxicity (IC50 value). Dilutions were kept at 4 ºC until future use.
The INS-1 cells required additional supplements for their growth medium, comprised of 100 mL of Glutamax 1 (100X, 200 mM, Gibco), 100mL of MEM
Sodium Pyruvate (100 mM, Gibco) as energy soure during anabolic processes and
36 µL of β-mercaptoethanol (2.5mM, Sigma) as antioxidant reducing agent. The
58
pH of the solution was adjusted at 7.3 using 5N NaOH. The solution was filtered using a 0.2 µm pore size filter and stored at -20 ºC until future use. For INS-1 cell medium preparation 2 % concentration (by volume) of this was added to the RPMI-
1640 medium (Park et al., 2012).
3.3.7 Cell Culture
MCF-7 breast cancer cells were cultured in Minimum Essential Medium
(MEM) alpha, Human cervical cancer cells (HeLa) and Rat insulin secreting beta cell line (INS-1) in RPMI 1640 (1X) medium and Rat brain malignant glioma cells
(RG2) and human rhabdomyosarcoma (RD) were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) having phenol red supplemented with 10% fetal bovine serum (FBS),1% L-glutamine(2mM), 1% sodium pyruvate (1mM) and 1% antibiotics penicillin(5000U/mL) and streptomycin(2500U/mL) all were purchased
º from Gibco, Invitrogen, CA, USA. Cells were maintained at 37 C in 5% CO2 humidified incubator. Cells were passaged at 80% confluency level and media were completely exchanged every 2-3 days (Maqsood et al., 2015; Fong et al., 2015;
Kaspler et al., 2016).
3.3.8 In vitro Cytotoxicity Tests
For cytotoxicity tests, MCF-7, INS-1, RG2 and HeLa cells were seeded at a concentration of 10000 cells/well into 96 well cell polystyrene culture plates
(COSTAR ® 3596 Corning Incorporated, Invitrogen, CA, USA) for 24 - 48 hours prior to experiments, in 200 µL of culture media. Plates were maintained at 37 ºC in
5% CO2 humidified incubator. When plates reached 80% confluence, the medium
59
were replaced by 2% MEM medium containing different concentrations of a plant extract ranging from 0.1 µgmL-1 to 20 µgmL-1. Experiments were executed in triplicate. Plates were kept at 37 ºC for up to 72 hours in a humidified incubator with 5% CO2. After incubation, the cytotoxicity was evaluated by means of the
Presto Blue Cell Viability assay, see below.
3.3.9 Presto Blue Cell Viability Assay
Cell viability were determine using the Presto Blue metabolic assay (Presto
Blue TM, Invitrogen, Frederick, MD, USA). Presto Blue 10 µL along with 90 µL of
2% Fluorobrite TM Media supplemented with 2% fetal bovine serum (FBS), 1% L- glutamine (2 mM), 1% antibiotics penicillin (5000 UmL-1) and streptomycin (2500
UmL-1) (all Gibco, Invitrogen, CA, USA). The phenol red media was added to each well of 96 well plates for viability calculation. Plates were incubated at 37º C in an incubator for 60-90 min. Only metabolically active cells provide a color change from blue to purple. Fluorescence quantification was provided using a 96-well plate reader Spectra Max M5 (Molecular Devices, Sunnyvale, CA, USA) with λex = 560 nm and λem = at 600 nm via the bottom of the plate (Fong et al., 2015).The viability is defined as;
Viability [%] = Mean fluorescence of treated cells / Mean fluorescence of control cells ×100.
3.4 STATISTICAL ANALYSIS
The statistical package SPSS for Windows version 16.0 (SPSS Inc.,
Chicago, IL) was used to analyzed data by applying ANOVA and multiple comparisons were executed by Duncan’s multiple range tests (DMRT) at 95%
60
significant difference (p<0.05). For drawing graphs, standard error bar was applied based on three values and lettering is done to seek significant values at DMRT of p<0.05. There are statistically differences (p<0.05) of one sample over the other(s) if values carry different superscripts. To determine the IC50 value determination the logarithmic concentrations of the plant extract and calculated using Graph Pad
Prism Version 6.0(b), [Graphpad, La Jolla, CA, USA].
3.5 RESULTS
CMEs of Artemisia scoparia, Moringa oleifera, Physorhynchus brahuicus,
Fagonia indica, Withania coagulans, Rhazya stricta and Otostegia limbata were assessed for Cytotoxicity test and IC50 values against five selected cell lines viz.
HeLa, MCF-7, RD, RG2 and INS-1 by using Presto Blue cell viability assay.
Results are discussed families wise as follows:
3.5.1 Apocynaceae
3.5.1.1 Cytotoxicity of Rhazya stricta
Leaves and root CMEs of Rhazya stricta were tested at the concentration of
20 µgmL-1 against HeLa, MCF-7, RD, RG2 and INS-1. Chemo-drug (MTX), CCM and DMSO were also screened at the same concentration for comparison. No significant (p<0.05) effect was observed for DMSO and CCM on HeLa, MCF-7,
RD, RG2 and INS-1 cells even after 72 hours incubation. Both plant parts showed cytotoxic activity in which root was the leading one that showed highest cytotoxicity (> 90%) after 72 hours of incubation against the HeLa, MCF-7 and
RG2, which was up to six to seven time higher compared to chemo-drug (Fig. 3.1,
3.2 & 3.5). For INS-1, root extract inhibited up to 80% cell population after 72
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hours of incubation, followed by leaves extract. The root extract possessed up to two fold higher cytotoxicity, whereas, leaves extract was equally effective against the said cell line compared to the tested drug (Fig.3.4). RD cells were prone to tolerant against the plant extracts, therefore there was less cytotoxic effect and inhibited up to 50% growth of RD cells after incubation of 72 hours (Fig.3.3).
3.5.1.2 IC50 Values of Rhazya stricta
Based on initial screening results at the concentration of 20 µgmL-1, root extract showed maximum cytotoxic activity and therefore was selected to determine the IC50 values against the selected cell lines. Overall, root extract expressed cytotoxic effect on all the tested cell lines having IC50 values of 1.74 to
9.37 µgmL-1 after 48-72 hours incubation. There was very strong activity against
HeLa, MCF-7, INS-1 and RG2 (> 5 µgmL-1), while the same was strong (>10
µgmL-1) against RD (Table 3.3).
3.5.2 Asteraceae
3.5.2.1 Cytotoxicity of Artemisia scoparia
The CME of Artemisia scoparia (whole plant) was tested at the concentration of 20 µgmL-1 for cytotoxicity screening against HeLa, MCF-7, RD,
RG2 and INS-1. The chemo-drug (MTX), CCM and DMSO were also tested at the same concentration for comparison. There was no significant (p<0.05) effect observed for DMSO and CCM on HeLa, MCF-7, RD, RG2 and INS-1 cells even after 72 hours incubation. Maximum activity in terms of cytotoxicity (up to 90%) was recorded for RG2, followed by MCF-7 (80%) after 72 hours of incubation that is 2-3-fold higher than the chemo-drug (Figs.3.7 & 3.9). The same extract exhibited
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100 90 i h 80 g 70 60 d 50 e 40
Vibility(%) f 30 c 20 10 b a 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Leaves Root MTX
Figure 3.1 Cytotoxicity of Rhazya stricta (leaves and root) extracts against HeLa cells.
100 90 80 h 70 g 60 f f e 50 d
40 c Viability (%) Viability 30 b 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Leaves Root MTX
Figure 3.2 Cytotoxicity of Rhazya stricta (leaves and root) extracts against MCF-7 cells.
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100 90 80 f e 70 e e 60 d d c 50 b 40 a
Viability (%) Viability 30 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Leaves Root MTX
Figure 3.3 Cytotoxicity of Rhazya stricta (leaves and root) extracts against RD cells.
100 90 80 70 60 f f e 50 40
Viability(%) d 30 c b b b 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Leaves Root MTX
Figure 3.4 Cytotoxicity of Rhazya stricta (leaves and root) extracts against INS-1 cells.
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100 g 90 f 80 e 70 d 60 c 50 40 Viability(%) 30 b b 20 10 a a 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Leaves Root MTX
Figure 3.5 Cytotoxicity of Rhazya stricta (leaves and root) extracts against RG2
cells.
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Table 3.3: IC50 values of Rhazya stricta (root) against the selected cell lines
-1 Cell line Time period (hours) IC50 (µgmL )
48 3.72±0.03 MCF-7 72 1.94±0.02
48 2.23±0.02 INS-1 72 1.91±0.01
48 1.78±0.01 RG2 72 1.74±0.01
48 4.46±0.04 HeLa 72 2.43±0.02
48 9.37±0.09 RD 72 7.23±0.08
Activity range; ˃100: Not active:, Weak; 20–100: Moderate; 10–20: Strong; 5–
10: ˂ 5: Very strong (Wibowo et al., 2011).
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remarkable activity and inhibited up to 80% cell populations of Hela, RD and INS-
1 cell lines. The activity was at par with the standard drug (Fig. 3.6, 3.8 & 3.10).
3.5.2.2 IC50 values of Artemisia scoparia
In order to confirm the results of preliminary screening, the IC50 value was calculated against all the tested cell lines. The CME of A. scoparia had cytotoxic
-1 effect on all tested cell lines with IC50 values ranging from 6.57 to 21.15 µgmL after 48-72 hours incubation. Generally, the activity of A. scoparia remained strong for INS-1, RD and MCF-7 with the IC50 values of 6.57±0.05, 8.94±0.07 and
9.17±0.06 respectively after 72 hours incubation (Table 3.4). It was followed by moderate for RG2 (10.48±0.08) and HeLa (12.67±0.11) according to the criteria of
Wibowo et al. (2011).
3.5.3 Brassicaceae
3.5.3.1 Cytotoxicity of Physorhynchus brahuicus
Crude methanolic extracts (CMEs) of Physorhynchus brahuicus (leaves, root and whole plant) were tested at the concentration of 20 µgmL-1 for cytotoxicity against HeLa, MCF-7, RD, RG2 and INS-1. Besides, MTX, CCM and DMSO were also tested at the concentration of 20 µLmL-1 for comparison. No significant effect
(p<0.05) was observed for DMSO and CCM on HeLa, MCF-7, RD, RG2 and INS-
1 cells even after 72 hours incubation.
For RD cells, root extract was the most effective that showed the highest cytotoxicity (> 80%) after 72 hours of incubation. This activity was two-fold higher
67
100 90 e 80 70 d 60 c 50 bc
40 Viability (%) Viability 30 a a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs
A. scoparia MTX
Figure 3.6 Cytotoxicity of Artemisia scoparia (whole plant) extracts against HeLa cells.
100 90 80 e 70 d
60 c c 50 40 Viability(%) b 30 a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs
A. scoparia MTX
Figure 3.7 Cytotoxicity of Artemisia scoparia (whole plant) extracts against MCF-7 cells.
68
100 90 80 e 70 d 60 c 50 b 40 a
Viability(%) a 30 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs A. scoparia MTX
Figure 3.8 Cytotoxicity of Artemisia scoparia (whole plant) extracts against RD
cells.
100 90 80 f e 70 60 d 50 40
Viability (%) Viability c 30 b 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs A. scoparia MTX
Figure 3.9 Cytotoxicity of Artemisia scoparia (whole plant) extracts against RG2 cells.
69
100 90 80 e 70 d 60 c 50 40
Viability (%) Viability b 30 a a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs
A. scoparia MTX
Figure 3.10 Cytotoxicity of Artemisia scoparia (whole plant) extracts against INS-1 cells.
70
Table 3.4: IC50 values of Artemisia scoparia (whole plant) against the selected cell lines.
Cell line Time period (hours) -1 IC50 (µgmL )
48 14.15±0.09 MCF-7 72 9.17±0.06
48 10.15±0.07 INS-1 72 6.57±0.05
48 15.97±0.12 RG2 72 10.48±0.08
48 21.15±0.16 HeLa 72 12.67±0.11
48 11.15±0.09 RD 72 8.94±0.07
Activity range; ˃100: Not active: Weak; 20–100: Moderate; 10–20: Strong; 5–10:
˂ 5: Very strong (Wibowo et al., 2011).
71
than that of the tested synthetic drug MTX (Fig.3.13). In the case of RG2 cells, whole plant extract showed the highest cytotoxic activity (> 90%) after 72 hours of incubation that was up to three fold higher than the drug (Fig.3.14). The activity in terms of cytotoxicity against INS-1 cells revealed that whole plant extract, leaves and root possessed maximum cytotoxic effect up to two fold higher (> 80%) after
72 hours of incubation than that of the drug (Fig.3.15).
3.5.3.2 IC50 values of Physorhynchus brahuicus
The whole plant extract of P. brahuicus was subjected to determine the IC50 values against the selected cell lines. IC50 values were ranged from 2.44 to 12.15
µgmL-1 after 48-72 hours incubation indicating very strong to moderate activity
(Table 3.5). The extract showed very strong activity against RG2 (> 5 µgmL-1), strong against RD and INS-1 (>10 µgmL-1), strong to moderate against HeLa (< 5
µgmL-1 to > 20 µgmL-1), while in the case of MCF-7, the same was moderate (>20
µgmL-1).
All the tested plant parts showed cytotoxicity in which root extract was the most effective that showed the highest cytotoxicity (> 50%) after 72 hours of incubation against the HeLa cell line, followed by whole plant and leave (Fig.
3.11). The whole plant extract was the most effective that showed the highest cytotoxicity (> 80%) after 72 hours of incubation against the MCF-7 cell line and its cell inhibition effect was three fold higher than that of the tested drug MTX
(Fig. 3.12).
3.5.4. Lamiaeceae
72
100 k j 90 80 hi i 70 f g gh e 60 d c 50 b 40 Viability (%) Viability 30 a 20 10 0 24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs
Leaves Root Whole Plant MTX
Figure 3.11 Cytotoxicity of Physorhynchus brahuicus (leaves, root and whole
plant) extracts against HeLa cells.
100 90 80 j 70 i h 60 g f g 50 e 40 b d bc b
Viability (%) Viability 30 20 a 10 0 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Leaves Root Whole Plant MTX
Figure 3.12 Cytotoxicity of Physorhynchus brahuicus (leaves, root and whole
plant) extracts against MCF-7 cells.
73
100 h 90 80 g g 70 f f 60 e e 50 d 40 c c
Viability (%) Viability 30 b a 20 10 0 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Leaves Root Whole plant MTX
Figure 3.13 Cytotoxicity of Physorhynchus brahuicus (leaves, root and whole
plant) extracts against RD cells.
100 k j 90 i h 80 g f 70 60 e e 50 40
Viability (%) Viability d 30 c 20 b a 10 0 24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs24 hrs48 hrs72 hrs Leaves Root Whole Plant MTX
Figure 3.14 Cytotoxicity of Physorhynchus brahuicus (leaves, root and whole
plant) extracts against RG2 cells.
74
100 80 i g h 60 f e e d 40 c a a a b Viability(%) 20 0 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Leaves Root Whole Plant MTX
Figure 3.15 Cytotoxicity of Physorhynchus brahuicus (leaves, root and whole
plant) extracts against INS-1 cells.
75
Table 3.5: IC50 values of Physorhynchus brahuicus (whole plant) against the
selected cell lines.
-1 Cell line Time period (hours) IC50 (µgmL )
48 12.15±0.11 MCF-7 72 10.17±0.10
48 6.15±0.05 INS-1 72 5.20±0.04 48 4.77±0.05 RG2 72 2.44±0.02 48 10.15±0.10 HeLa 72 6.78±0.07 48 7.15±0.06 RD 72 6.62±0.05 Activity range; ˃100: Not active:, Weak; 20–100: Moderate; 10–20: Strong; 5–10:
˂ 5: Very strong (Wibowo et al., 2011).
76
3.5.4.1 Cytotoxicity of Otostegia limbata
The CME of Otostegia limbata (aerial parts) was screened at concentrationof 20 µgmL-1 for cytotoxicity against HeLa, MCF-7, RD, RG2 and
INS-1. Chemo-drug (MTX), CCM and DMSO were also tested at the concentration of 20 µLmL-1 for comparison. There was no significant (p<0.05) effect observed for DMSO and CCM on HeLa, MCF-7, RD, RG2 and INS-1 cells after 72 hours incubation Plant extract showed leading activity against MCF-7 and RD cell lines
(up to 80%) after 72 hours of incubation. Comparing with the chemo-drug the
CME exhibited three-fold higher activity against the former cell line and >1-fold against the later cell line (Figs. 3.17 &3.18). It was followed by Hela, RG2 and
INS-1 cells which were at par with drug (Figs. 3.16.3.19 & 3.20).
3.5.4.2 IC50 values of Otostegia limbata
The CME of O. limbata had cytotoxic effect on all tested cell lines having
-1 its IC50 values of 4.49 to 23.15 µgmL after 48-72 hours incubation, which are in the range of very strong to weak activity. There was very strong activity of the O. limbata CME for RG2 (4.49±0.05), strong for INS-1 (5.20±0.04) and MCF-7
(5.97±0.06), moderate for RD (14.69±0.15) and HeLa (16.78±0.17) after 72 hours incubation (Table 3.6).
3.5.5. Moringaceae
3.5.5.1 Cytotoxicity of Moringa oleifera
Crude methanolic extracts (CMEs) of Moringa oleifera bark, fruit, leaves and leave stalk were tested at the concentration of 20 µgmL-1 for cytotoxicity
77
100 90 80 70 e d 60 c 50 b 40
Viability (%) Viability 30 a a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Aerial parts MTX
Figure 3.16 Cytotoxicity of Otostegia limbata (aerial parts) extracts against HeLa cells.
100 90 80 f 70 e 60 d 50 c 40
b Viability (%) Viability 30 a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs
Aerial parts MTX
Figure 3.17 Cytotoxicity of Otostegia limbata (aerial parts) extracts against MCF-7
cells.
78
100 90 80 e 70 d d 60 c 50 40 b Viability (%) Viability 30 a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Aerial parts MTX
Figure 3.18 Cytotoxicity of Otostegia limbata (aerial parts) extracts against RD cells.
100 90 80 d 70 d c 60 b 50 40
Viability (%) Viability a 30 a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Aerial parts MTX
Figure 3.19 Cytotoxicity of Otostegia limbata (aerial parts) extracts against RG2 cells.
79
100 90 80 70 60 d d 50 c 40 b Viability (%) Viability 30 a a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Aerial parts MTX
Figure 3.20 Cytotoxicity of Otostegia limbata (aerial parts) extracts against INS-1 cells.
80
Table 3.6: IC50 values of Otostegia limbata (aerial parts) against the selected cell lines.
Cell line Time period (hours) IC50 (µgmL-1)
48 8.15±0.09 MCF-7 72 5.97±0.06
48 6.15±0.05 INS-1 72 5.20±0.04
48 6.77±0.07 RG2 72 4.49±0.05
48 23.15±0.19 HeLa 72 16.78±0.17
48 17.85±0.18 RD 72 14.69±0.15
Activity range; ˃100: Not active: Weak; 20–100: Moderate; 10–20: Strong; 5–10:
˂ 5: Very strong (Wibowo et al., 2011).
81
against HeLa, MCF-7, RD, INS-1 and RG2 cell lines. At the same concentration, standard drug (MTX), CCM and DMSO were also tested for comparison. All the tested plant parts showed remarkable activity and inhibited the cells of HeLa up to four times higher. Amongst the CMEs, fruit extract showed the highest cytotoxicity
(> 90%) after 24 hours leading up to 95% after 72 hours of incubation revealing almost four-fold higher activity than that of the tested drug. It was followed by leaves having 70% cytotoxicity (Fig. 3.21).
Against MCF-7, seed extract was the most effective that showed the highest cytotoxicity (95%) after 72 hours of incubation. The activity was almost ten-times higher than that of the tested drug (Fig. 3.22). It was followed by leaves (85%), leave stalk (70%) and bark (65%). Seed CMEs effectively controlled RD cells and inhibited the cell population up to two fold higher. The cytotoxicity was noted up to
85 % after 72 hours of incubation than that of the tested drug. It was followed by both bark and leaves with 50% cytotoxicity (Fig. 3.23).
The cytotoxicity of seed extract showed higher than the drug and the activity was four fold higher which reached up to 90 % after 72 hours of incubation against the RG2 (Fig. 3.24). It was followed by leaves, bark and leave stalk. In the case of INS-1 cells, the tested plant parts showed good activity and inhibited the cell population up to two fold higher.
Seed was the most effective part that showed the highest cytotoxicity (>
80%) after 72 hours of incubation against the INS-1 cell line revealing almost two
82
100 90 m 80 l 70 k j h i 60 g f 50 e d 40 c Viability(%) 30 b 20 10 a a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Fruit Bark Leaves Leaf stalk MTX
Figure 3.21. Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave stalk) extracts
against HeLa cells.
100 90 80 m l 70 k 60 j i 50 h 40 g f Viability (%) Viability 30 de ef d b 20 c b 10 a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Fruit Bark Leaves Leaf stalk MTX
Figure 3.22. Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave stalk) extracts
against MCF-7 cells.
.
83
100 90 i i 80 h 70 fg g ef efg e 60 d d 50 c 40 b
Viability(%) 30 a a 20 a 10 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Fruit Bark Leaves Leaf stalk MTX Figure 3.23. Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave stalk)
extracts against RD cells.
100 k j j 90 i 80 g h f f 70 60 e 50 d 40 Viability(%) c 30 b b 20 a 10 a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Fruit Bark Leaves Leaf stalk MTX
Figure 3.24 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave stalk)
extracts against RG2 cells.
84
100 90 l 80 70 k j 60 i h 50 g 40 f e f Viability(%) 30 d b d c 20 a a 10 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Fruit Bark Leaves Leaf stalk MTX
Figure 3.25 Cytotoxicity of Moringa oleifera (fruit, bark, leaves and leave stalk)
extracts against INS-1 cells.
85
Table 3.7: IC50 value of Moringa oleifera (fruit and leaves) against the selected cell
lines.
Cell Time period Fruit IC50 Leaves IC50 line (hours) (µgmL-1) (µgmL-1)
48 2.34±0.03 2.58±0.03 MCF-7 72 1.42±0.04 3.19±0.02
48 1.63±0.01 3.28±0.03 INS-1 72 0.89±0.01 2.44±0.02 48 8.61±0.07 4.13±0.04 RG2 72 2.58±0.05 0.86±0.06
48 4.24±0.03 1.79±0.02
HeLa 72 2.29±0.02 1.68±0.01
48 12.58±0.11 10.61±0.09
RD 72 10.73±0.09 6.88±0.07
Activity range; ˃100: Not active:, Weak; 20–100:
Moderate; 10–20: Strong; 5–10: ˂ 5: Very strong
(Wibowo et al., 2011).
86
fold higher than that of the tested drug (Fig. 3.25).It was followed by leaves, leave stalk and bark.
3.5.5.2 IC50 values of Moringa oleifera Since the leaves and seeds exhibited the highest toxicity (Fig. 3.21 To 3.25) against all the selected cell lines, therefore these extracts were subjected to calculate their
IC50 values. The IC50 value of seed extract were recorded between 0.89 to 12.58
µgmL-1 after incubation of 48 to 72 hours against all cell lines (Table 3.7). The
-1 lowest IC50 was recorded as 1.42 µgmL against MCF-7 after 72 hours, while the
-1 highest one was 12.58 µgmL against RD after 48 hours. The range of IC50 values in terms of its efficacy were very strong in the case of MCF-7, INS-1 and HeLa (<5
µgmL-1), strong to very strong in the case of RG2 (˂10 µgmL-1) and moderate against RD cells (10 to 20 µgmL-1) as per criteria of Wibowo et al., (2011).
The leaves extract of Moringa oleifera showed the IC50 values ranging from
0.86 to 10.61 µgmL-1 following the incubation period of 48 to 72 hours. The lowest
-1 IC50 value was recorded as 0.86 µgmL against RG2 after 72 hours and the highest
(10.61 µgmL-1) against RD after 48 hours (Table 3.7). Very strong activity in terms
-1 of IC50 was recorded in the case of MCF-7, INS-1, RG2 and HeLa (<5 µgmL ) and moderate against RD cells (10-20 µgmL-1).
3.5.6. Solanaceae
3.5.6.1 Cytotoxicity of Withania coagulans
The CMEs of root, leaves, leave stalk and fruit of Withania coagulans were tested at the concentration of 20 µgmL-1 against HeLa, MCF-7, RD, RG2 and INS-
87
1 for cytotoxicity purpose. Chemo-drug (MTX), CCM and DMSO were also tested at the same concentration for comparison. No significant (p<0.05) effect was observed for DMSO and CCM on HeLa, MCF-7, RD, RG2 and INS-1 cells even after 72 hours of incubation.
For HeLa cell, leave stalk extract was recorded as the most effective that showed the highest cytotoxicity (> 80%) after 24 hours leading up to 95% after 72 hours of incubation. This activity was almost four-fold higher than that of the tested drug (Fig. 3.26). It was followed by fruit which exhibited again the highest cytotoxicity (up to 90%) after 72 hours incubation time.
In the case of MCF-7, leave stalk was the most effective extract that killed more than 80% cells after 24 hours reaching up to 90% after 72 hours of incubation. The activity was almost nine-fold higher than that of the tested drug. It was followed by fruit which inhibited the growth of cells up to seven fold higher, leaves (five-fold higher) and root (up to two- fold) after 72 hours incubation time
(Fig.3.27).
The RD and RG2 cells were inhibited well through fruit and leave stalk extracts in which the cytotoxicity was recorded more than 80% after 24 hours that reached up to 90% after 72 hours of incubation. Such activity was almost two to five times higher than that of the tested drug. It was followed by leaves (>80) and root (>70) which inhibited the growth of cells from two- fold to four fold after 72 hours incubation time (Fig.3.28 & 3.29).
88
100 90 80 j i 70 h 60 g 50 f ef e ef
40 Viability(%) 30 d c 20 c b b 10 a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Fruit Root Leaves Leave stalk MTX
Figure 3.26 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and root)
extracts against HeLa cells.
100 90 80 m l 70 k 60 j 50 i 40 h g Viability(%) 30 f e 20 d bc cd b 10 a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Fruit Root Leaves Leave stalk MTX
Figure 3.27 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and root)
extracts against MCF-7 cells.
89
100 90 80 70 h g 60 f 50 e 40 d
Viability(%) 30 c c c 20 ab bc a a a a a 10 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Fruit Root Leaves Leave stalk MTX
Figure 3.28 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and root)
extracts against RD cells.
100 90 80 j j 70 i g gh f 60 e 50 40 d Viability(%) 30 20 c b ab 10 ab a ab a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs Fruit Root Leaves Leave stalk MTX
Figure 3.29 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and root)
extracts against RG2 cells.
90
100 90 80 70 60 j 50 i 40 h g Viability(%) 30 e f cd cd d c d 20 ab b a ab 10 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
Fruit Root Leaves Leave stalk MTX
Figure 3.30 Cytotoxicity of Withania coagulans (fruit, leaves, leave stalk and root) extracts against INS-1 cells.
91
Table 3.8: IC50 values of Withania coagulans (fruit and leave stalk) against the
selected cell lines.
Cell line Time period (Fruit) (Leave stalk) -1 -1 (hours) IC50 (µgmL ) IC50 (µgmL )
MCF-7 48 0.99±0.01 4.73±0.05
72 0.69±0.01 1.02±0.01
INS-1 48 2.84±0.03 4.56±0.04
72 1.95±0.02 2.97±0.03
RG2 48 6.69±0.06 2.55±0.03
72 1.97±0.02 1.68±0.02
HeLa 48 2.79±0.03 1.79±0.02
72 2.19±0.02 1.68±0.01
RD 48 5.16±0.04 1.74±0.02
72 2.98±0.03 0.96±0.01
Activity range; ˃100: Not active:, Weak; 20–100: Moderate; 10–20: Strong; 5–10:
˂ 5: Very strong (Wibowo et al., 2011).
92
As for as INS-1, all extracts of W. coagulans showed promising activity and inhibited the cell population (>90) revealing up to two-fold higher activity. Root, leaves, leave stalk and fruit extracts were the most effective that showed the highest cytotoxicity up to 90% after 72 hours of incubation. The activity was more than 1- fold higher than that of the tested drug (Fig.3.30).
3.5.6.2 IC50 values of Withania coagulans
Based on initial screening results, leave stalk and fruit extracts were
-1 selected to calculate their IC50 values at the concentration of 20 µgmL against all tested cell lines (Table 3.8). The leave stalk extract showed maximum cytotoxic
-1 effect on all tested cell lines with its IC50 values of 0.96 to 4.73 µgmL after 48-72 hours incubation revealing very strong activity for all the tested cell lines. Against
-1 HeLa cells, IC50 values were recorded between 1.68 to 1.79 µgmL after 48-72
-1 hours incubation. For MCF-7, IC50 values were 1.02 to 4.73 µgmL after 48-72 hours incubation. In the case of RD cells, the IC50 values were calculated as 0.96 to
-1 1.73 µgmL after 48-72 hours incubation. The IC50 values for RG2 cells were recorded between 1.68 to 2.55 µgmL-1 after 48-72 hours incubation, while in the case of INS-1 the same were between 2.97 to 4.56 µgmL-1 after 48-72 hours incubation.
The fruit extract had cytotoxic effect on all tested cell lines having its IC50 values of 0.69 to 6.69 µgmL-1 after 48-72 hours incubation, which are in the range of very strong to strong. Against HeLa cells the IC50 values were 2.19 to 2.79
-1 µgmL , which are in the range of very strong activity. For MCF-7, the IC50 values
93
-1 were calculated as 0.69 to 0.99 µgmL . In the case of RD cells, IC50 values were
-1 recorded as 2.98 to 5.16 µgmL . The IC50 values of RG2 cells were ranged between 1.97 to 6.69 µgmL-1; whereas, in the case of INS-1, the same were recorded as 1.95 to 2.84 µgmL-1 (Table 3.8).
3.5.7 Zygophyllaceae
3.5.7.1 Cytotoxicity of Fagonia indica
The CME of Fagonia indica (whole plant) was screened for cytotoxicity against HeLa, MCF-7, RD, RG2 and INS-1 at the concentration of 20 µgmL-1. At the same concentration, chemo-drug (MTX), CCM and DMSO were also tested for comparison. There was no significant (p<0.05) effect observed for DMSO and
CCM on HeLa, MCF-7, RD, RG2 and INS-1 cells even after 72 hours incubation.
The CME exhibited remarkable activity against the RG2, INS-1 and MCF-7 with approximately up to 90% cytotoxicity after 72 hours of incubation. The extract activity was 1-4 times higher than that of the tested drug (Figs. 3.32, 3.34 & 3.35).
In the case of RD and Hela cells, the whole plant extract also inhibited up to 80% cell population and the activity was equal to MTX (Fig.3.31 & 3.33).
3.5.7.2 IC50 values of Fagonia indica
Eight logarithmic concentrations of Fagonia indica (whole plant) were prepared to determine the IC50 values against HeLa, MCF-7, RD, RG2 and INS-1 cell line. CME of F. indica had cytotoxic effect on all the tested cell lines with IC50 values of 2.97 to 13.15 µgmL-1 after 48-72 hours incubation revealing the activity of very strong to moderate (Table 3.9). The CME of F. indica showed very strong
94
100 90 f 80 70 e 60 d 50 c 40
Viability (%) Viability 30 b a 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Whole plant MTX
Figure 3.31 Cytotoxicity of Fagonia indica (whole plant) extract against HeLa cells.
100 90 80 e 70 d 60 c c 50 40 30 b Viability (%) Viability 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Whole plant MTX
Figure 3.32 Cytotoxicity of Fagonia indica (whole plant) extract against MCF-7 cells.
95
100 90 f 80 70 e 60 d c 50 40 b a Viability (%) Viability 30 20 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Whole plant MTX
Figure 3.33 Cytotoxicity of Fagonia indica (whole plant) extract against RD cells.
100 90 80 f 70 e 60 d 50 40
Viability (%) Viability c 30 b 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs
Whole plant MTX
Figure 3.34 Cytotoxicity of Fagonia indica (whole plant) extract against RG2 cells.
96
100 90 80 70 60 d d 50 40
Viability (%) Viability c 30 b b 20 a 10 0 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Whole plant MTX
Figure 3.35 Cytotoxicity of Fagonia indica (whole plant) extract against INS-1 cells.
97
Table 3.9: IC50 values of Fagonia indica (whole plant) against the selected cell lines.
Cell line Time period (hours) IC (µgmL-1) 50
48 4.45±0.05 MCF-7 72 2.97±0.03 48 6.15±0.05 INS-1 72 5.20±0.04 48 5.79±0.06 RG2 72 3.46±0.04 48 11.15±0.10 HeLa 72 9.78±0.07 48 13.15±0.14 RD 72 10.62±0.11 Activity range; ˃100: Not active: Weak; 20–100: Moderate; 10–20:
Strong; 5–10: ˂ 5: Very strong (Wibowo et al., 2011).
98
activity in terms of IC50 values for MCF-7 (2.97±0.03) and RG2 (3.46±0.04) after
72 hours incubation. It was followed by strong for INS-1 (5.20±0.04) & HeLa
(9.78±0.07) and moderate for RD (10.62±0.11) µgmL-1.
3.5.8 Cytotoxicity of CCFs of Physorhynchus brahuicus
Six column chromatography fractions (CCFs) of Physorhynchus brahuicus were tested at the concentration of 20 µgmL-1 for cytotoxicity against HeLa, RD,
MCF7, RG2 and INS-1. Chemo-drug i.e. Methotrexate (MTX), cell culture medium
(CCM) and dimethyl sulfoxide (DMSO) were also tested at the concentration of 20
µLmL-1 for comparison. There was no significant (p<0.05) effect observed for
DMSO and CCM on HeLa, MCF-7, RD, RG2 and INS-1 cells even after 72 hours of incubation.
All the fractions showed remarkable activity and inhibited the cell population either equally or two fold higher. CCL was the most effective fraction that showed the highest cytotoxicity (> 80%) after 24 hours leading up to 90% even after 72 hours of incubation against the HeLa cell line revealing almost two-fold higher than that of the tested drug (Fig. 3.36). It was followed by CCN, CCO,
CCQ, CCT and CCU. Interestingly, three fractions viz., CCL, CCO, CCN exhibited the highest toxicity at 72 hours incubation time and showed two-fold higher cytotoxicity (up to 90%) than the standard drug (MTX).
Against MCF-7, all the CCFs showed remarkable activity and inhibited the cell growth either equally (after 24 hours of incubation time) or up to 5-fold higher
99
100 90 80 70 m l 60 k k j 50 i i i g h 40 Viability (%) Viability 30 f ef d de de 20 c b a a ab ab 10
0
48hrs 24hrs 24hrs 48hrs 72hrs 24hrs 72hrs 48hrs 72hrs 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs CCL CCN CCO CCQ CCT CCU MTX
Figure 3.36 Column Chromatography Fractions of Physorhynchus brahuicus
against HeLa cells.
100
than the standard drug (i.e. MTX). CCO, CCQ, CCT and CCU, respectively were the most effective fraction that showed the highest cytotoxicity (> 90%) at 72 hours of incubation, revealing almost six to eight-fold higher activity; while, CCN showed up to two fold effective than that of the tested drug (Fig.3.37).There was similar trend in activity compared to MCF-7, in which the toxicity was either at par with the drug or 4 times higher after 24 hours incubation time by the CCFs against the RD cells. Maximum cytotoxicity was observed in CCL and CCO that checked almost 95% cell population. It was followed by CCN, CCQ and CCU which inhibited up to 90% cell growth of RD cells, however, CCT showed 80% activity
(Fig. 3.38).
For RG2, all the tested CCFs showed highly significant cytotoxic activity more than that of MTX (Fig. 3.39). CCN and CCO were the most effective fractions that inhibited > 90% cells after72 hours of incubation, followed by CCQ,
CCT and CCL revealing up to three-fold higher than that of the tested drug
(Fig.3.39). The INS-1 cell line was significantly (p<0.05) inhibited by all the fractions, in which maximum cytotoxicity was shown by CCO, CCL, CCN and
CCQ having > 80% cytotoxicity after72 hours of incubation. The level of their activity was however either equal or 2-fold compared to the standard drug
(Fig.3.40). Eight logarithmic concentrations (i.e. 20, 10, 5, 2, 1, 0.5, 0.2 and 0.1
-1 µgmL ) of all CCFs were prepared to determine their IC50 values following the recommendations of the US National Cancer Institute (NCI) to screen the cytotoxic activity against HeLa, MCF-7, RD, RG2 and INS-1 cell lines. As a general rule, plant is considered to be cytotoxic if 20 µgmL-1 or less amount of its extract is
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100 90 m 80 l l 70 j j k i 60 h 50 40 g g
f f Viability (%) Viability 30 e d c d 20 b 10 a a a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
CCL CCN CCO CCQ CCT CCU MTX
Figure 3.37 Column Chromatography Fractions of Physorhynchus brahuicus
against MCF-7 Cells.
100 90 80 l 70 k j 60 h i 50 g 40 ef f d de Viability(%) 30 c c c c 20 b b b b b 10 a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrshrs CCL CCN CCO CCQ CCT CCU MTX
Figure 3.38 Column Chromatography Fractions of Physorhynchus brahuicus
against RD cells.
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100 90 80 l 70 60 k j 50 i 40 h g f g f g Viability (%) Viability 30 e e d cd c 20 b b b 10 a a a 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
CCL CCN CCO CCQ CCT CCU MTX
Figure 3.39 Column Chromatography Fractions of Physorhynchus brahuicus
against RG2 cells.
100 90 80 70 l k 60 i j h h 50 h 40 f g e e e Viability (%) Viability 30 d d c c c 20 b b a b 10 0 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs hrs
CCL CCN CCO CCQ CCT CCU MTX
Figure 3.40 Column Chromatography Fractions of Physorhynchus brahuicus
against INS-1 cells.
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enough to inhibit the growth of 50% cells (IC50) in 48-72 hours (Geran et al., 1972;
Lee and Houghton, 2005; Sufian et al., 2013).
3.5.8.1 IC50 values of CCFs of Physorhynchus brahuicus
All six CCFs showed cytotoxic effect on HeLa cell having IC50 values of
1.51 to 6.35 µgmL-1 after 48-72 hours incubation, which exhibited very strong to strong inhibition activity after Wibowo et al., (2011). Besides, there was increasing
-1 -1 trend in IC50 values for CCO (1.53 µgmL to 2.01 µgmL ), followed by CCN
(1.51 µgmL-1 to 2.19 µgmL-1), CCQ (2.21 µgmL-1 to 2.36 µgmL-1), CCT (3.58
µgmL-1 to 4.12 µgmL-1), CCL (1.62 µgmL-1 to 4.69 µgmL-1) and CCU (4.90 µgmL-
1 to 6.35 µgmL-1) after 72 hours of incubation time (Table 3.10).
The same trend has been observed for IC50 values by CCFs against the
MCF-7 cell line. There was very strong to strong activity that exhibited by the
-1 range of IC50 between 0.81 to 7.36 µgmL after incubation of 48-72 hours (Table
3.10). Similarly, there had been increasing trend in IC50 values in varying of time of incubation. The CCL possessed lowest IC50 after 72 and 48 hours, respectively (i.e.
0.81 µgmL-1 to 1.91 µgmL-1), followed by CCO (1.56 µgmL-1 to 2.32 µgmL-1),
CCQ (1.65 µgmL-1 to 2.38 µgmL-1), CCU (2.64 µgmL-1 to 3.45 µgmL-1), CCT
(3.45 µgmL-1 to 5.12 µgmL-1) and CCN (6.67 µgmL-1 to 7.38 µgmL-1).
For testing IC50 values against RD cell line, six CCFs have been evaluated
-1 for cytotoxic effect. The IC50 values were ranged in 4.94 to 12.86 µgmL following incubation period of 48-72 hours, reflecting very strong to moderate activity. The activity was increased with the increase in time of incubation period that reached
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-1 Table 3.10: IC50 values (µgmL ) of CCFs against the selected cell lines.
Time Fractions MCF-7 INS-1 RG2 HeLa RD (hours)
48 1.91±0.09 7.42±0.07 2.20±0.07 4.69±0.02 5.84±0.04 CCL 72 0.81±0.07 6.01±0.07 0.38±0.08 1.62±0.03 4.94±0.06 48 CCN 7.38±0.07 4.15±0.05 3.14±0.03 2.19±0.10 6.58±0.07 72 6.67±0.07 1.29±0.04 2.11±0.06 1.51±0.02 5.53±0.04 48 2.32±0.20 1.43±0.04 1.97±0.07 2.01±0.03 6.74±0.07 CCO 72 1.56±0.10 1.34±0.05 0.68±0.06 1.53±0.05 5.15±0.03 48 2.38±0.01 9.79±0.08 4.87±0.06 2.36±0.10 7.39±0.09 CCQ 72 1.65±0.01 4.24±0.07 3.61±0.05 2.21±0.08 5.04±0.05 48 5.12±0.30 9.93±0.09 3.16±0.05 4.12±0.07 8.66±0.07 CCT 72 3.45±0.28 5.43±0.07 1.69±0.05 3.58±0.06 7.39±0.09 48 3.45±0.11 4.88±0.06 1.81±0.01 6.35±0.08 12.86±0.10 CCU 72 2.64±0.06 1.28±0.05 0.22±0.02 4.90±0.08 10.17±0.08 Activity range; ˃100: Not active:, Weak; 20–100: Moderate; 10–20: Strong; 5–10:
˂ 5: Very strong (Wibowo et al., 2011).
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maximum after 72 hours of incubation as shown maximum by CCL (4.94 µgmL-1 to 5.84 µgmL-1), followed by CCN (5.53 µgmL-1 to 6.58 µgmL-1), CCO (5.15
µgmL-1 to 6.74 µgmL-1), CCQ (5.04 µgmL-1 to 7.39 µgmL-1), CCT (7.39 µgmL-1 to
8.66 µgmL-1) and CCU (10.17 µgmL-1 to 12.86 µgmL-1). The cytotoxicity in terms of IC50 values of CCFs was checked against RG2 cell line. Results showed that all
CCFs possessed cytotoxic effect on cells, having IC50 value ranging from 0.22 to
4.87 µgmL-1 following incubation period of 48-72 hours, expressing very strong activity. Maximum activity amongst CCFs expressed in CCU with the lowest IC50 values (i.e. 0.22 µgmL-1 after 72 hours and 1.81 µgmL-1 after 48 hours), followed by
CCO (0.68 µgmL-1 to 1.91 µgmL-1), CCL (0.38 µgmL-1 to 2.20 µgmL-1), CCN
(2.11 µgmL-1 to 3.14 µgmL-1), CCT (1.69 µgmL-1 to 3.16 µgmL-1) and CCQ (3.61
µgmL-1 to 4.87 µgmL-1).
In the case of INS-1 cell line, all CCFs showed cytotoxic effect on cells,
-1 with the IC50 values of 1.28 to 9.93 µgmL following incubation period of 48-72 hours, reflecting very strong to strong activity. The optimum activity with respect
-1 -1 to IC50 values was calculated in CCO (1.34 µgmL after 72 hours and 1.43 µgmL after 48 hours, respectively), followed by CCN (1.29 µgmL-1 to 4.15 µgmL-1), CCU
(1.28 µgmL-1 to 4.88 µgmL-1), CCL (6.01 µgmL-1 to 7.42 µgmL-1), CCQ (4.24
µgmL-1 to 9.79 µgmL-1) and CCT (5.43 µgmL-1 to 9.93 µgmL-1).
3.6 DISCUSSION
The use of medicinal plant as a therapeutics agent is very old in the history of human civilization. The early man discovered these green medicine through trial
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and error (Bhatti et al., 2001) and still acknowledged their potential even for treating common as well as complicated diseases/ailments. These plant resources are the basis for traditional medicine system which have been used in China, India and Pakistan for thousands of years (Bhatti et al., 2001: Qureshi et al., 2011).
Cancer being complex/noxious in nature is very difficult to cure. Scientists are targeting plant extracts to treat such complicated diseases hypothesizing their long history of utilization by the traditional communities (Qureshi et al., 2016).
According to Hartwell (1982) more than 3000 medicinal plants are being used in the treatment of cancer due to their anticancer properties. A remarkable number of anticancer drugs have been made from medicinal plants which are in the clinical use based on ethnobotanical as well as ethnopharmacological exploration (Cragg et al., 2003).
Cancer chemotherapy playing a vital role in curing malignancies and its main objective is to kill the cancer cells but not harms the healthy ones (Hoft et al.,
1999). Oncologists are predominantly interested in cancer treating compounds which induce cellular apoptosis, a property satisfied by the majority of secondary plants metabolites. Many studies reported that the mechanism of action of these anticancer drug is based on the induction of apoptosis (Ramasamy et al., 2013).
Keeping in view, present study was designed to explore medicinal plants of the Salt
Range, Pakistan for their potential use as well as their cytotoxicity which are ethnobotanically by the traditional herbal practitioner (THP) in treating such complications.
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The CME of M. oleifera seed showed the IC50 values ranging from 0.89 to
12.58 µgmL-1 after incubation of 48 to 72 hours against all cell lines (Table 3.7).
-1 The lowest IC50 was recorded as 1.42 µgmL against MCF-7 after 72 hours. The range of IC50 values in terms of its efficacy were very strong in the case of MCF-7,
INS-1 and HeLa (<5 µgmL-1), strong to very strong in the case of RG2 (˂10 µgmL-
1) and moderate against RD cells (10 to 20 µgmL-1) as per criteria of Wibowo et al.,
(2011). The cytotoxicity of same plant fractions/isolation of compounds was conducted against Raji cells by Guevara et al. (1999) and reported four compounds namely, 4- benzyl isothiocyanate, niazimicin, β -sitosterol-3-O-b-D- glucopyranoside and β- sitosterol. They reported higher IC50 values (i.e. 32.7, 35.3,
70.4 and 27.9 µgmL-1) compared to present study against Raji cells. Their calculated IC50 values falls in weak activity against the studied cell lines.
According to Lee and Houghton (2005), values higher than 20 µgmL-1 are not putative to be used as screening for drug development. However, results of present study fall in the range of putative against cancer.
CME of leaves of M. oleifera showed the IC50 values between 0.86 to 10.61
-1 µgmL following the incubation period of 48 to 72 hours. The lowest IC50 value was recorded as 0.86 µgmL-1 against RG2 after 72 hours. Very strong activity in terms of IC50 was recorded in the case of MCF-7, INS-1, RG2 and HeLa (<5
µgmL-1) and moderate against RD cells (10-20 µgmL-1). Another study on anticancer activity of methanolic extract of M. oleifera leaves against HepG2,
Caco-2 and MCF-7 cells was carried out by Charoensin (2014). He reported IC50 values of 120.37±2.55, 112.46±3.74 and 133.58±2.47 µgmL-1for HepG2, Caco-2
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and MCF-7, respectively. Due to higher than 20 µgmL-1 ranges (Lee and Houghton,
2005), the extract showed inactiveness and not putative against tested cell lines, while results of this study are vice versa from these ones. The similar case was that
-1 of Shang et al. (2016), who reported 170.1 µgmL IC50 against Hep-2 cell line from CME of Moringa oleifera leaves. Costa-Lotufo et al. (2005) carried out cytotoxic activity of M. oleifera root extract against HL-60, CEM, HCT-8 and B-16 cell lines and reported IC50 values against the studied cell lines as 60, 12.7, 113.8 and 28.8 µgmL-1, respectively. Among all tested cell lines, CEM was inhibited
-1 efficiently with the IC50 value of 12.7 µgmL revealing potentiality to be used as medicine according to Lee and Houghton (2005).
The crude aqueous and ethanolic extract of Artemisia annua stem and leaves were tested against HeLa and AGS cell lines by Penduka et al. (2014).
-1 According to them, the IC50 values for HeLa cells were recorded as <500 µgmL for aqueous leave extract, stem aqueous extract, stem ethanolic extract and 368.00
-1 ± 2.60 µgmL for leaves ethanolic extract. In the case of AGS cells, IC50 values for leave aqueous extract, stem aqueous extract, stem ethanolic extract was recorded <
500 µgmL-1 and leaves ethanolic extract 233.64±6.69 µgmL-1.
The study reported higher IC50 values for both the tested cell lines according to the criteria of Lee and Houghton (2005). The present study was carried out cytotoxic activity of the CME of A. scoparia against tested cell lines
-1 and reported 6.57 to 21.15 µgmL IC50 values after 48-72 hours of incubation.
Generally, the activity remained strong for INS-1, RD and MCF-7 with the IC50
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values of 6.57±0.05, 8.94±0.07 and 9.17±0.06, respectively after 72 hours incubation (Table 3.4). It was followed by moderate for RG2 (10.48±0.08) and
HeLa (12.67±0.11). The biological activity of CME of A. herba-alba was evaluated by Kilani et al. (2008) including anticancer activity against human bladder carcinoma (RT112), human laryngeal carcinoma (Hep2) and human myelogenous leukemia (K562). The study reported IC50 values of 81.59 ± 4.4, 59.05 ± 3.66 and
90.96 mgL-1 of the respective cell lines. Besides, they reported that CME had no effect on normal peripheral blood mononuclear cells and the biological activities were correlated with the phenolic contents of the extract. Comparing with the said study, the findings of present study in terms of IC50 values are vice versa (Table
3.4).
The bioassay guided isolation and purification of the CMEs of Withania coagulans of aerial parts has been reported by Ihsan-ul-haq et al. (2013) for understanding cancer chemo-preventive mechanisms. They isolated three new steroidal lactones namely, withacoagulin G, withacoagulin H and withacoagulin I.
The withacoagulin G and withacoagulin H were the most active compounds possessing the IC50 values of 3.1 to 1.9 μM inhibiting tumor necrosis factor-α
(TNF-α) and 1.60 to 12.4 μM, respectively. The present study first time reports leave stalk extract of W. coagulans showing maximum cytotoxic effect on all tested
-1 cell lines with the IC50 values of 0.96 to 4.73 µgmL after 48-72 hours incubation, revealing very strong activity for all the tested cell lines (Table 3.8). Against HeLa
-1, cells, IC50 values were recorded between 1.68 to 1.79 µgmL for MCF-7, the same were recorded as 1.02 to 4.73 µgmL-1 in the case of RD cells, 0.96 to 1.73 µgmL-
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1 -1 .The IC50 values for RG2 cells were recorded between 1.68 to 2.55 µgmL , while in the case of INS-1 the same were between 2.97 to 4.56 µgmL-1 (Table 3.8).
Interestingly, our findings are in agreement of Ihsan-ul-haq et al. (2013) who reported the IC50 more or less closer values. However, this study carried various plant parts (aerial parts) such as leaves stalk, fruit, etc. The fruit extract of
W. coagulans had cytotoxic effect on all tested cell lines having its IC50 values of
-1 0.69 to 6.69 µgmL after 48-72 hours incubation. Against HeLa cells the IC50 values were 2.19 to 2.79 µgmL-1, for MCF-7, the same were calculated as 0.69 to
-1 0.99 µgmL . In the case of RD cells, IC50 values were recorded as 2.98 to 5.16
-1 -1 µgmL . The IC50 values of RG2 cells were ranged between 1.97 to 6.69 µgmL ; whereas, in the case of INS-1, the same were recorded as 1.95 to 2.84 µgmL-1
(Table 3.8). Another study conducted by Esmaeili et al. (2014) reported higher IC50 values CMEs of W. coagulans than the present study. According to them, the IC50 values were > 50 µgmL-1 against tested five cell lines viz, HepG-2, MDBK, WEHI-
164 and A-549. The possible reason could be change of cell lines, except MCF-7 as well different assay (MTT).
The anticancer activity of CMEs of Otostegia michauxii and O. persica have been reported by Esmaeili et al. (2014) the against HepG-2, MCF-7, MDBK,
-1 WEHI-164 and A-549 cell lines. They reported >50 µgmL IC50 values for both plants the against the tested cell lines. The present investigation revealed very strong activity of the CME of O. limbata against RG2 (4.49±0.05), strong in the case of INS-1 (5.20±0.04) and MCF-7 (5.97±0.06), moderate for RD (14.69±0.15)
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and HeLa (16.78±0.17) after 72 hours incubation (Table 3.6).
CMEs of 27 medicinal plants from southern provinces of Iran were subjected for cytotoxicity against MCF-7, WEHI-164, HepG-2, MDBK and A-549 cell lines by means of MTT assay. Amongst these species, Calotropis procera and
Juniperus excelsa extracts were found more effective against the studied cell lines.
-1 The IC50 values of Calotropis procera were recorded as 4.96 µgmL for MCF-7,
21.42 µgmL-1 for MDBK, 4.84 µgmL-1 for A-549 and >50 µgmL-1 for HepG-2 and
WEHI-164 cell lines. While CMEs of Juniperus excelsa showed range of IC50
-1 -1 values as 7.52 µgmL for MCF-7, >50 µgmL for WEHI-164. The results of IC50 values of Calotropis procera against MCF-7 and A-549 cells support the findings of present study. The results of Juniperus excelsa against MCF-7 also agreement with the present study.
Wibowo et al., during 2011 isolated four compounds, malaysianol A, laevifonol, ampelopsin E and α-viniferin from the acetone extract of the stem bark of Dryobalanops aromatica and determine their anticancer activity against five cell lines namely HL-60, MCF-7, HepG-2, A-549 and WRL-68 by using MTT assay.
The range of IC50 values for compound malaysianol A was 39.6 (HL-60) ,25.1
(MCF-7) ,29.8 (HepG-2) ,59.8 (A-549) and 40.9 µgmL-1 (WRL-68), against laevifonol 37.6, 50.6, 33.8, not determined and 41.4 µgmL-1, for ampelopsin E ,
31.2, 14.3, 44.9, 60.7 and 50.1 µgmL-1 while for α-viniferin 2.7,15.7, 32.5, 49.7 and
41.5 µgmL-1 respectively. Results of compound ampelopsin E for MCF-7 and of α- viniferin for HL-60 and MCF-7 are agreement with the finding of present study after Sufian et al. (2013).
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A cytotoxic study of Muntingia calabura leaves CMEs was reported by
-1 Sufian et al. (2013), they have reported the IC50 values of 34.73 µgmL against
MCF-7, 30.90 µgmL-1 for HL-60 and 61.29 µgmL-1 against HCT cells. Seven
EtOAc fractions (F1-F7) resulted in higher cytotoxicity (lower IC50 value) following 48 to 72 hours incubation, against MCF-7, HL-60 and WRL 68 cell lines
-1 with the lowest IC50 value (3.98 µgmL ) reported against HL-60; however, the respective values for MCF-7 and WRL60 cells remained high at 30.80 µgmL-1 and
32.29 µgmL-1 respectively. The results of seven EtOAc fractions (F1-F7) with the
-1 lowest IC50 value of 3.98 µgmL against HL-60 are the in agreement after Lee and
Houghton (2005), Wibowo et al. (2011) and with the findings of present study.
Rhizomes CME of Curcuma zedoaria was subjected for anti-proliferative effect against MCF-7 cells by Hamdi et al. (2014) and they have reported IC50 value of 18.4 µgmL-1. These findings are agreement with present study after
Wibowo et al. (2011). Anticancer activity of CME of Peperomia pellucida (leaves) was reported by Liao et al. (2014), they determined cytotoxicity through MTT assay against MCF-7 cell line and the chemical compositions were screened and identified based on GC-MS technique. The CME possessed significant anticancer
-1 activities with the IC50 value of 10.4±0.06 μgmL . Phytol was the major compound found in the plant extract followed by 2- Naphthalenol, decahydro, Hexadecanoic acid, methyl ester and 9, 12-Octadecadienoic acid .This study indicated that CME of P. pellucid leaf possessed very high potential as medicinal drug especially against human breast cancer. These results support the findings of our study after
Wibowo et al. (2011).
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Nemati et al. (2013) reported on the antitumor effects of an Angelica sinensis extract both in vitro and in vivo against human (DBTRG-05) and rat (RG2) brain tumor cell lines, showing cell growth suppressed via cell cycle arrest and apoptosis. The plant extracts up regulated the expression of cdk inhibitors including p21 and decrease the phosphorylation of Rb proteins which causes the cell cycle arrest at G0/G1 phase for both. In vivo results showed that plant extract not only suppress growth of both human and rat brain tumor but could reduce their volumes.
3.7 CONCLUSION
Cancer is a group of an anxious diseases which leads to various complications. Current therapies have severe side effects, so the current shift to the herbal or natural preparation may be more effective as compare to synthetic ones.
Because herbal medicines are more effective, low cost, lesser side effects and having lower level of toxicities as compared to synthetics Kamalakannan et al.
(2016).Thus the ethnobotanical or native use of medicinal plants for the treatments of different diseases may be supported by present study. The management of cancer is still a challenge for oncologist, to cure it without any or lesser side effects.
Therefore, it is prudent/essential to look for alternative or better option in herbal or green medicine for cancer therapy as well.
Isolation, purification, identification and synthesis of the active bio compounds will definitely enhance the pharmacological properties. There is great need for the clinical trial or in-vivo studies for the authentications of biologically active compounds. Moreover, it is also necessary to study the effects and molecular
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mechanism/pathway of the compounds which are responsible for the cause of death cancer cells in higher animal models to ensure its potential and safety. All these advantages will prove the significance of natural product research for the treatment of cancer.
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Chapter 4
PHYTOCHEMICAL SCREENING
4.1 INTRODUCTION
Plants produce a vast diversity of secondary metabolites/phytochemicals that have potential use in pharmaceutical industry for new drug development purpose. Phytochemicals are naturally occurring bioactive plant compounds that act as a natural defense system for plant. They also provide color, aroma and flavor in plants. These includes terpenes and terpenoids, essential oils, alkaloids, lectins, polypeptides, phenolics and polyacetylenes.
Phenolics can be further divided into phenolic acids, flavonoids, quinones, tannins, coumarins and phenols. Flavonoids and flavones usually involve in the formation of complexes which in activates the enzymes of the pathogens.
Flavonoids are well known antioxidants and biologically active against liver toxins, tumors, viruses, allergies and inflammations (Penduka et al., 2014).
Medicinal plants formulate the ground for searching new pharmaceutical and healthcare products. Using medicinal plants in disease prevention programme may be attributed to antioxidant properties of various phytoconstituents, normally found associated to a wide range of amphipathic molecules, broadly termed as polyphenolic compounds. Efforts are done to explore the potential use of naturally occurring anti-inflammatory and antioxidant compounds (Hamamouchi, 2002).
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The NCI, US collected about 35,000 medicinal plant samples from 20 different countries and they found around 114,000 extracts putative against cancer
(Shoeb, 2006).Overall 92 anticancer drugs are commercially available prior to 1983 in the USand among globally approved anticancer drugs between 1983 and 1994,
60% are of natural origin (Cragg et al., 1996).
The strong evidence of plant based anticancer drug from the bark of Taxus brevifolia is the discovery of paclitaxel (Taxol®, 3) which is the success in natural product drug discovery. Native American tribe used various parts of T. brevifolia and other Taxus species mostly T. canadensis and T. baccata for the treatment of some non-cancerous cases also (Cragg and Newman, 2003) while in the Indian
Ayurvedic medicine T. baccata was reported to use for the treatment of cancer. The structure of paclitaxel was elucidated in 1971 and was clinically introduced to the
US market in the early 1990s and it has been found very significantly active against ovarian cancer, advanced breast cancer and lung cancer (Doughari, 2006).
Camptothecin (4) were isolated from the Camptotheca acuminate, a
Chinese ornamental tree to clinical trials by US National Cancer Institute(NCI) in the 1970s.Soon its clinical trial were dropped because of severe bladder toxicity
(Shoeb, 2006). Two semi synthetic derivatives of campothecin are Topotecan (5) and irinotecan (6) are used for the treatment of ovarian, lung cancers and colorectal cancers (Bertino, 1997).Another evidence of natural product based anticancer compound is podophyllotoxin (7) which is an isomers of epipodophyllotoxin which was isolated from the roots of P. peltatum and P. emodi. Two semi -synthetic
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derivatives of epipodophyllotoxin, etoposide (8) and teniposide (9) are used in the treatment of bronchial, testes cancers and lymphomas, (Cragg and Newman, 2003).
Homoharringtonine (10), is another plant based anticancer agent which is in clinical use and was isolated from the Chinese tree Cephalotaxus harringtonia var. drupacea (Sieb and Zucc.) (Itokawa et al., 2005). In France plant based anticancer drug elliptinium (11), which is a derivative of ellipticine is marketed for the treatment of breast cancer which was isolated from a Fijian medicinal plant
Bleekeria vitensis (Cragg and Newman, 2003).
Variety of bioactive compounds have been isolated from plant sources.
Most of them are in clinical, pre-clinical and under further investigation process.
Which is currently in phase I and II of clinical trials of different tumors like leukemia, lymphoma and solid tumors (Cheng-Hui, 2008). Continuous research on bioactive substances from medicinal plants can be a possible leads to a discovery and formulation of new potent anticancer compounds that may be helpful in alleviation the problem of anticancer resistance drugs for future use (Sultana and
Anwar, 2008).
4.1.1 Phenolic Compounds
Plant metabolites can be categories as secondary metabolites. Secondary metabolites often function, such as defensive compounds against herbivores, pathogens and stresses as well as playing their role in pigmentations (Bhakta and
Ganjewala, 2009). These are categorized as phenolics, alkaloid, saponin and terpenoids. Phenolic compounds are further distributed into three subgroups such as
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Figure 4.1. Plant derived anticancer agents
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Figure 4.2. Plant derived anticancer agents
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flavonoids, tannins and phenolic acids. (Bhakta and Ganjewala, 2009).
4.1.2 Flavonoids
These are polyphenolic compounds present in all plant. These have various effects on systems and structures of mammalian and protect biological membranes against ROS (Gulcin et al., 2007). The flavonoids have been highlighted by several studies because of their strong antioxidant effects (Sultana and Anwar, 2008). They have therapeutic effects on disease conditions which are caused by due to oxidative stress, such as atherosclerosis, aging cancer and diabetes (Madhuri and Pandey,
2009). Quercetin and rutin in Caenorhabditis elegans reduce the effects of thermal
ROS (Cragg and Newman, 2005).
Both these flavonoids are integral components of different vegetables and fruits and exhibit various health benefits which are attributed mostly due to free radical scavenging and antioxidant effects and exerts beneficial effects in various degenerative disorders (Noroozi et al., 1998; Middleton et al., 2000; Williams et al., 2004). According to Waterman and Mole (1994), flavonoids are based on a carbon backbone of C6‒C3‒C6 (Figure 4.4), which are categorized into three main classes: flavonoids, iso-flavonoids and neoflavonoids (Figure 4.3).
The class of flavonoids is further divided in several subclasses that are distinguished from each other by the chemical differences in their ring C (Figure
4.4; Iwashina, 2000). For example, different subclasses can have a double bond between C2–C3, a hydroxyl group at C3, or a carbonyl group at C4 (Figure 4.4).
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Flavonoids and phenolics are usually present in the form of complex mixtures in plants. For their analyses, a chromatographic step is often needed to separate individual compounds from each other. The most common separation techniques for flavonoids and phenolics compounds is HPLC (Marston and Hostettmann,
2006; Kalili and de Viliers, 2011).
In HPLC, a sample mixture in solution is eluted through a column with the help of eluent in the isocratic or gradient mode of elution. Different compounds in the sample mixture are retained with variable efficiencies inside the column, which causes their chromatographic separation. Mostly C-18 column is commonly used in the separation of flavonoids and phenolics (Marston and Hostettmann, 2006).
During LC column, the eluent flow is directed into a detector that detects flavonoids separated by the column by using several types of detectors are in combination with it. (Rijki et al., 2006). However, flavonoids can be derivatized with metalic cations to produce fluorescent complexes. Flavonoids are gradually oxidized in an electrochemical detector, as they move through an array of electrodes that are placed in the order of increasing potential (Brenes et al., 2000;
Guo et al., 1997). Ultraviolet– visible (UV-vis) and mass spectrometry (MS) is a common detection method of flavonoids in LC (Marston and Hostettmann, 2006).
If best accuracy is required than standards of phenolic compounds should be quantified with as the quantified compounds. The use of inappropriate standards causes errors, because these standards have different UV–vis absorption properties
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Figure 4.3. The main classes of flavonoids: (A) Flavonoids (B) Isoflavonoids (C)
Neoflavonoids (Waterman and Mole, 1994).
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Figure 4.4. Subclasses of flavonoids: (A) flavonol , (B) flavone , (C)
dihydroflavonol , (D) flavan-3-ol , (E) flavanone , (F) anthocyanidin ,
(G) chalcone , (H) dihydrochalcone and (I) aurone (Iwashina, 2000).
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than the quantified compounds (Guo et al., 1997; Brenes et al., 2000; Marston and
Hostettman, 2006).
4.3 MATERIALS AND METHODS
4.3.1. Plant Material
Out of 71 plant species, seven plants such as Artemisia scoparia, Fagonia indica, Moringa oleifera, Otostegia limbta, Physorhynchus brahuicus, Rhazya stricta and Withania coagulans were selected for cytotoxicity based on more than
60% fidelity percentage for further analysis (Table 2.1).
4.3.2. Preparation of Extract
The plant extracts were made from the selected species as mentioned in chapter 3 under subsection 3.3.3
4.3.3. Preparations of Standards
Identification of flavonoids standards (quercetin, myricetin) and phenolic acid standards (gallic acid, syringic acid, vanillic acid, 휌-hyrodxybenzoic acid, 휌- coumaric acid, ferulic acid and protocatechuic aicd) in plant extracts was made by comparing their retention times with standards (standards were purchased from
Sigma Chemicals Co., St Louis, MO, USA). Quantitative determination was carried by using calibration curves of the standards.
4.3.4. Conditions for RP-HPLC Analysis
The following conditions/parameters were used for the analysis of the sam-
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Table 4.1: Certified reference standards of phenolics along with conc., R.T and
peak area.
Sr. Name of standard Conc. R.T(min) Peak Inj.
No area (mV) mass (ng)
1 Gallic acid 20 µgmL-1 3.359 1743088 400
2 Protocatechuic acid 20 µgmL-1 5.256 1036161 400
3 p-Hydroxybenzoic 20 µgmL-1 8.598 270300 400
acid
4 Vanillic acid 20 µgmL-1 11.289 395127 400
5 Syringic acid 20 µgmL-1 12.399 2560395 400
6 p-Coumaric acid 20 µgmL-1 21.19 762981 400
7 Ferulic acid 20 µgmL-1 28.805 1299575 400
Key Conc. = Concentration, R.T= Retention Time, Inj.mass= Injected mass
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-ples. Column: Discovery ® HS C18-Supelco, cat. #.568523-U, Col.80634-06,
BL.5931. The length of column was 25 cm, internal diameter 4.6 mm and particle size of the stationary phase was 5 µm. Column Performance:
Performance/efficiency of column were tested according to the certificate of analysis of Discovery ® HS C18, 5 µm, cat. #. 568523-U.
4.3.5. Standards for Testing HPLC Column
Following standards were run through the column to check the efficacy of
C-18 column.
1. Uracil (7 µgmL-1 )
2. Acetophenone (7 µgmL-1)
3. Benzene (750 µgmL-1)
4. Toluene (750 µgmL-1)
Chromatographic conditions for Testing Column:
Test conditions:
Mobile phase: 30:70, Water: Methanol
Flow rate: 1.0 mLmin-1
Temperature: Ambient temperature
Injection: 10.0 µL
Wavelength: UV 254 nm detection
Chromatographic conditions for Phenolics Samples:
The following conditions were used for analysis of samples on HPLC.
HPLC System: LC-10, Shimadzu, Japan
System controller unit (SCL-10A), Shimadzu, Japan
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Solvent tray
Delivery pump
Test conditions:
Mobile phase: 88:10:2, Water: Acetonitrile: Acetic acid
Flow rate: 1.0 mLmin-1
Temperature: 30º C
Injection: 20.0 µL
Wavelength: UV 280 nm detection
Pressure: 145 Kgf/cm2
Acquisition Software: Class LC-10
Detector: UV-visible
Injection Loop = 20 μL (Rheodyn), USA
Chromatographic conditions for Flavonoids Samples:
Following conditions were used for the determination of flavonoids in herbal samples.
HPLC system = LC-10A
Column= C18 (250 x 4.6 mm, 5 µm), Supelco, USA
Column Temperature= 30o C (CTO-10A)
Detector = UV-Vis (SPD-10A), Shimadzu, Japan
Wavelength (λ) = 360 nm
Flow rate = 1 mLmin-1
Mobile Phase = 3% TFA (A), ACN: MeOH (80:20) (B), (50:50)
Injection volume = 20μL
Injection Loop = 20 μL (Rheodyn), USA
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Acquisition Software = LC-10
Reverse phase: If the stationary phase of column is non polar (C-18 bonded with silica, Octadecyl silyl that is polymer) and mobile phase is polar than it is called reverse phase.
Mobile phase: The mobile phase was acetonitrile 10%, water 88%, acetic acid 10% and isocratic (having constant conc.) mode.
Quantification of compounds was determined by using following formula.
푚푎푠푠(푛푔) 퐼푛푗푒푐푡푒푑 푃푒푎푘 푎푟푒푎 표푓 푠푎푚푝푙푒∗퐹푖푛푎푙 푣표푙푢푚푒 표푓 푠푎푚푝푙푒 Concentration of compound =( 푝푒푎푘 ) ∗ 푣표푙푢푚푒 표푓 푖푛푗푒푐푡푒푑 (µ퐿)∗ 푀푎푠푠 표푓 푠푎푚푝푙푒 (푔) 푎푟푒푎 표푓 푠푡푎푛푑푎푟푑
4.3.6 LC-MS Analysis
LC–MS of the CCFs of P. brahuicus was performed on a Micromass
(Waters, Manchester, UK). LC separation was attained by LC (Waters, Milford,
USA), consisting of 2695 Separation Module 2487 wavelength UV detector operated at 280 nm. Column, gradients and other conditions were same as described previously for RP-HPLC. Data was acquired by the Masslynx data system for both the MS and UV data.
4.3.7 Identification of Compounds in CCFs
Compounds were identified based on the following criteria.
4.3.7.1 LC-MS data of molecular mass
LC-MS chromatograms obtained were analyzed by Masslynx to get spectra
129
of various major peaks after LC-MS data can be a useful tool to identify compounds on the basis of molecular masses Spectra were recorded in negative and positive ion mode to estimate molecular masses of compounds..
4.3.7.2 Retention time of compounds
Retention times of components/compounds can also be used for the analysis of data by comparing it with already reported compounds data.
4.3.7.3 LC-MS data of fragmentation
Fragmentation data of a compound can give information about its structure and it can be compared to the data of already available or with known compounds.
4.4 RESULTS
4.4.1 RP-HPLC Results
4.4.1.1 System suitability test
According to the U.S. Pharmacopoeia 23-National Formulary 18 (USP), system suitability tests are an integral part of chromatographic methods (U.S.
Pharmacopoeia, 1995). These tests are used to verify that the resolution and reproducibility of a chromatographic system are adequate for an analysis. These tests are on the base of concept that the equipment, electronics, analytical operations and sample constitute an integral system that can be evaluated as a whole (Ishwana,
2000). By doing this test the following parameters can be determined to ensure the performance of the system before or during the analysis of the unknown samples.
i) Plate counts
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ii) Resolution iii) Retention factor k iv) Asymmetry
Unretained peak (t0) was received by injecting air or dichloromethane vapours
(2 L) and standard solution of organochlorine (lindane, heptachlor, - endosulfan, p,p’-DDE, p,p’-DDD and p,p’-DDT) was run on ECD and calculated the parameters (Table 4.2) to ensure the performance of the instrument by using the following formulas:
K= retention factor
tR= retention time of solute
Retention factor: t0= retention time of unretained peak t t t' K = R 0 R t’R= adjusted retention time t0 t0 of solute
Plate number:
t'R 2 Neff = 5.545 Wh Neff= number of effective plates
t’R= adjusted retention time of solute W = peak width at one-half peak height Resolution: h
tR1= adjusted retention time of solute 1
tR2 = adjusted retention time of solute 2 1.18tR2 tR1 Rs = W W h1 h2 Wh1= peak width at one-half peak height
Asymmetry: Wh2= peak width at one-half peak height
b As = a = half width of a peak at 0.1 height a b = half width of a peak at 0.1 height
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4.4.2 Phenolics and flavonoids results
Identification and quantification of polyphenols and flavonoids from medicinal plants was achieved by Reverse Phase High Performance Liquid
Chromatography (RP-HPLC). Altogether, 89 phenolic compounds were isolated from methanolic crude extract; while 26 phenolic compounds were quantified from six column chromatography (CC) fractions of P. brahuicus. With reference to flavonoids, overall 25 flavonols were identified from methanolic crude extract and
9 from CC fractions of P. brahuicus by using RP-HPLC method.
Among phenolics, p-hydroxy benzoic acid and gallic acid were found in all tested plant part (s) followed by protocatechuic acid, p-coumaric acid, vanillic acid, syringic acid and ferulic acid (Table 4.3).
Highest concentration of p-hydroxy benzoic acid was recorded in case of P. brahuicus 167.39 ± 2.13 µgg-1 followed by fruit extract of W. coagulans with the concentration of 157.46 ± 1.43 µgg-1 ,while all other tested parts having its concentration from 12.46 ± 0.8 µgg-1 to 143.69 ± 1.83 µgg-1 The other most common phenolic was gallic acid,which was present in all samples. The highest concentration of gallic acid was recorded in the leave stalk extract of W. coagulans
(83.62±0.7 µgg-1) followed by R. stricta root extract (78.02±1.3 µgg-1), A.scoparia whole plant extract (69.02±0.6 µgg-1), leaves extract of R. stricta (65.02±0.9 µgg-1) and rest of plant parts having its concentration from 17.62±0.7 µgg-1 to 52.82±0.6
µgg-1 (Table 4.3). The second most common phenolics were protocatechuic acid, p- coumaric acid and vanillic acid.
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1 3 4
2
Figure 4.5 Standard chromatogram of uracil (1), acetophenone (2), benzene (3) and toluene (4) showing column performance.
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Table 4.2 Comparison of calculated and standard values from regulatory guidelines
Parameter Calculated* Standard value
Retention factor 5.71-27.48 K’>2
Plate number 17835-6542 N>2000
Resolution 8.2-16.5 Rs>2
Asymmetry 2.5-2.86 0.7-1.8
*Values are mean of three injections
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Figure 4.6 Chromatogram of vanillic acid (standard).
Figure 4.7 Chromatogram of Quercitin (standard).
135
Figure 4.8 Chromatogram of Myrecitin (standard)
Figure 4.9 Chromatogram of Gallic acid (standard).
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Figure 4.10 Chromatogram of Ferulic acid (standard).
Figure 4.11 Chromatogram of p –Coumaric acid.
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Figure 4.12 Chromatogram of p –Hydroxybenzoic acid
Figure 4.13 Chromatogram of Syringic acid.
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Figure 4.14 Chromatogram of Protocatechuic acid
139
The second most common phenolics were protocatechuic acid, p-coumaric acid and vanillic acid. The p-coumaric acid was found abundantly in bark extract of
M. oleifera with the concentration of 212.46 ± 0.11 µgg-1 and it is the highest concentration amongst all quantified phenolic compounds. In case of other plant part (s) its concentration were recorded as 10.39 ± 0.6 to 131.64 ± 0.59 µgg-1.
The concentration of protocatechic acid was 39.09 ± 0.18 µgg-1 in fruit extract of W. coagulans, while for other tested part (s) its value were recorded between 9.39 ± 0.76 to 34.28 ± 0.53 µgg-1.The highest value for vanillic acid was recorded as 27.26 ± 0.11 µgg-1 against aerial parts extract of O. limbata while for others its range were observed as 1.26 ± 0.09 to 25.68 ± 0.17 µgg-1.
The quantity of syringic acid was recorded as 34.95 ± 0.17 µgg-1 for leave stalk extract of W. coagulans while for others its range was recorded from 2.89 ±
0.01 to 22.9 ± 0.49 µgg-1. For ferulic acid, its concentration was 43.02 ± 0.13 µgg-1 against fruit extract of W. coagulans followed by 19.92 ± 0.10 µgg-1 for root extract of said plant. In case of all other tested part (s) its value was 6.02 ± 0.01 to 19.02 ±
0.3 µgg-1.Overall the total phenolic contents (TPC) were recorded and the highest
TPC was recorded (421.62 ± 11.58 µgg-1) for bark of M. oleifera followed by
369.88 ± 6.46 µgg-1 against P. brahuicus, While for others the value of TPC was recorded 123.54 ± 6.30 to 364.93 ± 9.32 µgg-1 (Table 4.3).
Data for the flavonoids (myricetin and quercetin) contents of selected medicinal plant part(s)/organs also indicated that myricetin was the most common
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and abundant flavonoid present in tested plants (Table 4.5). The highest contents of myrecitin was quantified in leaves of W. coagulans with the concentration of
358.46 ± 2.91 µgg-1 followed by 321.98 ± 1.76 µgg-1 in the fruit extract of same plant. While for others, concentration of myrecitin were recorded from 63.92 ±
2.91 µgg-1 to 292.02 ± 1.10 µgg-1.
Quercitin is also an important flavonoid which is also quantified in the present study from the selected plant /part (s) and its highest concentration was recorded in 23.45 ± 0.99 µgg-1 followed by 13.96 ± 0.57 µgg-1 ,while for rest of plants it concentration was recorded between 3.27 ± 0.47 µgg-1 to 13.62 ± 0.96 µgg-
1 as shown in table 4.5.
Total flavonoid content (TFC) was calculated for all tested samples and highest TFC was recorded for root (394.34 ± 1.26 µgg-1) extract of Withania coagulans followed by leaves extract (358.46 ± 2.91 µgg-1) and fruit extract
(343.41 ± 1.96µgg-1) of the same plant. While for other plants, the concentration of
TFC were recorded from (75.26 ± 0.37µgg-1) to (281.52 ± 0.36 µgg-1) and their detailed results are shown (Table 4.5). Based on TFC values W. coagulans was ranked first amongst all tested plants.
4.4.2.1 Phenolic and flavonoids contents of CCFs of P. brahuicus
Six CCFs of P. brahuicus was determine for quantification of phenolic acids contents, amongst all the CCL fraction was the main fraction containing all phenolics except ferulic acid. The vanillic acid was detected in this fractions having
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the concentration of 7.26 ± 0.22 µgg-1 followed by syringic acid 3.42 ± 0.04 µgg-1in the same fraction (Table 4.4).In case of CCN fraction the highest concentration was recorded for vanillic acid 4.26 ± 0.13µgg-1 followed by Protocatechuic acid 3.09
±0.02 µgg-1.The CCO fraction having higher content of phenolic in the form of protocatechuic acid (2.68 ± 0.03 µgg-1).
The p-hydroxybenzoic acid was quantified in CCQ fraction with 2.38 ±
0.05 µgg-1, while for CCT fraction protocatechuic acid (2.09 ± 0.06 µgg-1) and for
CCU fraction the value of 2.85 ± 0.06 µgg-1 was recorded for gallic acid. Based on total phenolics contents (TPC), the CCL fraction was on top with 15.45 ± 0.06 followed by CCN fraction having 11.88 ± 0.03 values. The rest of fractions having values from 5.10 ± 0.01 to 6.66 ± 0.05 µgg-1 (Table 4.4). Flavonoids play key role regarding health and disease prevention due to this reason they have getting attention from the researchers. Amongst all flavonoids myrecitin and quercetin are abundantly occurring in medicinal plants.
Quantification of both flavonol was determine in CCFs and the highest value was recorded for myrecitin 206.07 ± 0.83 µgg-1 against fraction CCU followed by CCL fraction with the value of 197.62 ± 1.73µgg-1 (Table 4.6).The rest of fractions having the value of myrecitin 143.79 ± 0.81µgg-1 to 196.34 ± 0.78 µgg-
1. The highest concentration of quercetin was recorded against fraction CCL with the value of 3.85 ± 0.47 µgg-1 followed by CCU 3.62 ± 0.43 µgg-1. Total flavonoid content of CCU fraction was highest amongst all with the value of 209.69 ± 1.24
µgg-1, while its range for other fractions was recorded between 143.79 ± 0.81 to
201.47 ± 1.69 µgg-1 (Table 4.6).
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Table 4.3. Yield of phenolic acids (µgg-1) in various plant parts.
Plant Part(s) Gallic Protocatechuic p-Hydroxy Vanillic Syringic p-Coumaric Ferulic acid Total species acid acid benzoic acid acid acid acid phenolics R. stricta Leaves 65.02 ± 0.9 15.09 ± 0.6 12.46 ± 0.8 1.26 ± 0.09 N.D 19.79 ± 0.84 9.92 ± 0.30 123.54 ± 6.30
Root 78.02 ± 1.3 N.D 82.29 ± 1.7 N.D 11.29 ± 0.76 10.39 ± 0.67 11.79 ± 0.7 193.75 ± 8.02
A. scoparia Whole plant 69.02 ± 0.62 23.9 ± 0.28 112.16 ± 0.89 17.26 ± 0.09 21.97 ± 0.89 102.79 ± 0.81 19.02 ± 0.3 364.93 ± 9.32
P. brahuicus Whole plant 47.96± 0.58 13.98 ± 0.17 167.39 ± 2.13 N.D 2.89 ± 0.01 131.64 ± 0.59 6.02 ± 0.01 369.88 ± 6.46 O. limbata Aerial parts 37.02 ± 0.64 11.23 ± 0.38 102.43 ± 1.91 27.26 ± 0.11 15.29 ± 0.93 121.79 ± 0.10 N.D 315.02 ± 5.34
M. oleifera Bark 29.45 ± 0.65 13.78 ± 0.31 128.67 ± 1.62 17.26 ± 0.12 10.98 ± 0.94 212.46 ± 0.11 9.02 ± 0.62 421.62 ± 11.5
Fruit 52.82 ± 0.66 18.63 ± 0.45 143.69 ± 1.83 8.79 ± 0.04 13.29 ± 0.95 117.73 ± 0.18 13.02 ± 0.79 315.15 ± 10.6
Leaves 23.26 ± 0.67 34.28 ± 0.53 85.48 ± 0.94 25.68 ± 0.17 19.29 ± 0.96 N.D 11.84 ± 0.15 176.57 ± 5.37
Leave stalk 17.16 ± 0.68 21.78 ± 0.19 79.25 ± 0.65 16.24 ± 0.56 N.D 18.56 ± 0.14 N.D 135.83 ± 8.06
W. coagulans Root 28.09 ± 0.69 31.89 ± 0.35 116.46 ± 0.93 19.56 ± 0.15 17.29 ± 0.38 14.79 ± 0.09 19.92 ± 0.10 219.91 ± 4.89
Leaves 43.12 ± 0.85 9.39 ± 0.76 142.46 ± 1.47 13.27 ± 0.11 22.9 ± 0.49 29.49 ± 0.16 11.29 ± 0.08 228.8 ± 6.40
Leave stalk 83.62 ± 0.71 34.09 ± 0.09 123.46 ± 0.97 17.26 ± 0.18 34.95 ± 0.17 12.37 ± 0.17 N.D 222.13 ± 5.71 Fruit 35.28 ± 0.32 39.09 ± 0.18 157.46 ± 1.43 21.27 ± 0.19 14.26 ± 0.10 32.76 ± 0.21 43.02 ± 0.13 307.86 ± 9.42
F. indica Whole plant 27.06 ± 0.18 34.08 ± 0.39 83.49 ± 0.10 12.63 ± 0.22 11.56 ± 0.12 16.64 ± 0.13 7.02 ± 0.04 165.42 ± 4.86
ND; not detected Results are expressed in values of means ± SD, for analyzed in triplicate.
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Table 4.4. Yield of phenolic acids (µgg-1) in various Column Chromatography Fractions of P. brahuicus.
Fractions Gallic Protocatechuic p-hydroxybenzoic Vanillic Syringic p-coumaric Ferulic acid Total
acid acid acid acid acid acid phenolics
CCL 0.37 ± 0.01 0.89 ± 0.06 1.69 ± 0.02 7.26 ± 0.22 3.42 ± 0.04 1.79 ± 0.08 N.D 15.45 ± 0.06
CCN N.D 3.09 ± 0.02 1.46 ± 0.01 4.26 ± 0.13 N.D 2.79 ± 0.05 0.82 ± 0.01 11.88 ± 0.03
CCO N.D 2.68 ± 0.03 1.79 ± 0.06 N.D 0.63 ± 0.01 N.D N.D 5.10 ± 0.01
CCQ 1.02 ± 0.04 N.D 2.38 ± 0.05 0.42 ± 0.02 N.D 1.79 ± 0.24 1.02 ± 0.19 6.63 ± 0.08
CCT 1.62 ± 0.05 2.09 ± 0.06 N.D 0.89 ± 0.01 N.D N.D 1.87 ± 0.06 6.47 ± 0.04
CCU 2.85 ± 0.06 N.D N.D 1.47 ± 0.01 2.34 ± 0.09 N.D N.D 6.66 ± 0.05
ND; not detected Results are expressed in values of means ± SD, for analyzed in triplicate.
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4.4.2.2 LC-MS results of CCFs of P. brahuicus
Based on LC-MS altogether eight compounds were isolated and identified.
There molecular masses were confirmed from the available literature. These compounds are first time reported from column chromatographic fraction of P. brahuicus namely, N,N-dipropyl propan-1-amine (Fig. 4.15 & 4.16), 4- acetamidophenyl) 2-(diethylamino) acetate (Fig. 4.17 & 4.18), 3-Methyl benzo furan-2-carboxylic acid (Fig. 4.19 & 4.20), [2-(Diphenylphosphoryl) ethyl]
(methyl) oxo(phenyl) phosphine (Fig. 4.21 & 4.22), 2-(3-Amino-1H-1, 2, 4-triazol-
1-yl) acetohydrazide (Fig. 4.23 & 4.24), 3-Methyl-N-(3-methylbutyl)-1-butanamin
(Fig. 4.25 & 4.26), 1, 3-Benzenedimethanol, α1-[[(1, 1-dimethylethyl) amino] methyl]-4-hydroxy (Fig. 4.27 & 4.28) and 3-Chloro-6-hydrazinopyridazine (Fig.
4.29 & 4.30)
4.5 DISCUSSION
Plant polyphenolics have attained remarkable attention in the current era of
modern research because it act as a potential source of antioxidants and protect human beings from certain diseases like cardiovascular disease, tumors cancer, and infectious diseases etc. (Khan et al., 2012; Jahan et al., 2012).
Phenolics or phenolic acids are big class of compounds which are widely existing in plants. The antiradical activity or ROS is directly related to the number of hydroxyl groups on phenol ring. Antioxidant activity of phenolics increases with an increase in number of hydroxyl groups. Many researcher like (Karamac et al.,
2005; Gulçin,2006), have reported that important phenolics and also well reported their pharmacological actions and antioxidants potentials (Cai et al., 1997). HPLC based analysis of phenolics have been reported by many previous reports (Ozkan et
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Table 4.5. Yield of flavonoids (µgg-1) in various plant parts.
Plant species Part(s) Myrecitin Quercitin Total flavonoid
Leaves 78.02 ± 1.23 8.92 ± 0.93 86.94 ± 1.12 R. stricta Root 281.52 ± 0.36 N.D 281.52 ± 0.36
A. scoparia Whole plant 139.02 ± 1.3 3.92 ± 0.93 142.94 ± 1.19
P. brahuicus Whole plant 150.42 ± 1.46 13.96 ± 0.57 164.38 ± 0.73
O. limbata Aerial parts 134.56 ± 0.51 N.D 134.56 ± 0.51
Bark 63.92 ± 0.46 13.62 ± 0.96 77.54 ± 1.32
Fruit 87.47 ± 0.97 4.60 ± 0.16 92.09 ± 0.83
Leaves 116.07 ± 0.83 3.27 ± 0.47 119.69 ± 1.24
M. oleifera Leave stalk 139.02 ± 1.9 23.45 ± 0.99 162.47 ± 1.25
Root 292.02 ± 1.10 12.32 ± 0.100 394.34 ± 1.26
Leaves 358.46 ± 2.91 N.D 358.46 ± 2.91
Leave stalk 236.27 ± 1.85 14.57 ± 0.19 250.84 ± 1.28
W. coagulans Fruit 321.98 ± 1.76 21.43 ± 0.13 343.41 ± 1.96
F. indica Whole plant 69.02 ± 0.84 6.24 ± 0.104 75.26 ± 0.37
ND: not detected Results are expressed in values of means ± SD, for analyzed in triplicate.
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Table 4.6. Yield of flavonoids (µgg-1) in various Column Chromatography
Fractions of P. brahuicus
Fractions Myrecitin Quercitin Total flavonoid
CCL 197.62 ± 1.73 3.85 ± 0.47 201.47 ± 1.69
CCN 154.87 ± 1.46 N.D 154.87 ± 1.46
CCO 143.79 ± 0.81 N.D 143.79 ± 0.81
CCQ 196.34 ± 0.78 1.68 ± 0.06 198.02 ± 1.32
CCT 187.47 ± 0.97 N.D 187.47 ± 0.97
CCU 206.07 ± 0.83 3.62 ± 0.43 209.69 ± 1.24
ND; not detected Results are expressed in values of means ± SD, for analyzed in triplicate.
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Figure 4.15. LC-MS chromatogram of N, N-dipropyl propan-1-amine identified
from CCL
Figure.4.16. Structure of N,N-dipropyl propan-1-amine.
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Figure.4.17. LC-MS chromatogram of 4-acetamidophenyl) 2-(diethylamino)
acetate.
Figure.4.18. Structure of 4-acetamidophenyl) 2-(diethylamino) acetate.
149
Figure.4.19. LC-MS chromatogram of 3-Methyl benzo furan-2-carboxylic acid.
Figure.4.20. Structure of 3-Methyl benzo furan-2-carboxylic acid.
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Figure.4.21. LC-MS chromatogram of [2-(Diphenylphosphoryl) ethyl] (methyl)
oxo (phenyl) phosphine.
Figure.4.22. Structure of [2-(Diphenylphosphoryl) ethyl] (methyl) oxo (phenyl)
phosphine.
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Figure.4.23. LC-MS chromatogram of 2-(3-Amino-1H-1, 2, 4-triazol-1-yl)
acetohydrazide.
Figure.4.24. Structure of 2-(3-Amino-1H-1, 2, 4-triazol-1-yl) acetohydrazide
152
Figure.4.25. LC-MS chromatogram of 3-Methyl-N-(3-methylbutyl)-1-butanamin.
Figure.4.26. Structure of 3-Methyl-N-(3-methylbutyl)-1-butanamin.
153
Figure.4.27. LC-MS chromatogram of 1, 3-Benzenedimethanol, α1-[[(1, 1-
dimethylethyl) amino] methyl]-4-hydroxy.
Figure.4.28. Structure of 1, 3-Benzenedimethanol, α1-[[(1, 1-dimethylethyl) amino]
methyl]-4-hydroxy.
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Figure 4.29. LC-MS chromatogram of 3-Chloro-6-hydrazinopyridazine.
Figure.4.30. Structure of 3-Chloro-6-hydrazinopyridazine
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al., 2006; Oslzewskai, 2007; Amber et al., 2012; Jahan et al., 2012). Quercetin and myricetin are potent free radical scavengers (Middleton, 2000; Levites, 2001; Silva et al., 2002; Dukic et al., 2008). Considerable variations have been observed in phenolic compounds of all tested medicinal plants. The highest contents of myrecitin was quantified in leaves of W. coagulans with the concentration of
358.46 ± 2.91 µgg-1 followed by 321.98 ± 1.76 µgg-1 in the fruit extract of same plant. While for others, concentration of myrecitin were recorded from 63.92 ±
2.91 µgg-1 to 292.02 ± 1.10 µgg-1. The quantity of syringic acid was recorded as
34.95 ± 0.17 µgg-1 for leave stalk extract of W. coagulans while for others its range was recorded from 2.89 ± 0.01 to 22.9 ± 0.49 µgg-1. For ferulic acid, its concentration was 43.02 ± 0.13 µgg-1 against fruit extract of W. coagulans followed by 19.92 ± 0.10 µgg-1 for root extract of said plant.
Higher concentration of phenolics and flavonoids content of W. coagulans, and CCL, CCN and CCU, the CCFs of P. brahuicus are responsible for its excellent cytotoxic potential (Table 4.6). In literature antioxidant potential of different species has been reported by various researchers like, Ozturk et al.,
(2007).
Findings of present study are in line with previous reports of (Nagulendran et al., 2007; Jahan et al., 2011, Jahan et al., 2012) Myricetin was predominate flavonol in Euphorbia tirucalli. Phenolic acids including p-coumaric acid, gallic acid chlorogenic acids and catechin are identified by HPLC as major antioxidant constituents. In some previous reports plants of family Euphorbiaceae such as
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Euphorbia hirta (Sharma et al., 2007) and Euphorbia tirucalli (Jyothi et al., 2008) showed high phenolic contents and antioxidant activity.
Eight compounds viz., N,N-dipropyl propan-1-amine, 4-acetamidophenyl)
2-(diethylamino) acetate, 3-Methyl benzo furan-2-carboxylic acid, [2-
(Diphenylphosphoryl) ethyl] (methyl) oxo(phenyl) phosphine, 2-(3-Amino-1H-1, 2,
4-triazol-1-yl) acetohydrazide, 3-Methyl-N-(3-methylbutyl)-1-butanamin, 1, 3-
Benzenedimethanol, α1-[[(1, 1-dimethylethyl) amino] methyl]-4-hydroxy and 3-
Chloro-6-hydrazinopyridazine are isolated from P. brahuicus. These compounds belong to the classes of Alkaloids and flavonoids. Their chemical structures are closely related to the alkaloid class i.e. Vinca alkaloids obtained from Catharanthus roseus and flavones isolated from Dysoxylum binectariferum. Presently, both are in clinical use (Yokoshima et al., 2002).
4.6 CONCLUSION
The highest contents of myrecitin was quantified in root, leaves, leave stalk and fruit of W. coagulans followed by root of R. stricta. While from CCFs of P. brahuicus flavonoid content of CCU fraction was highest amongst all and regarding phenolics content CCL fraction was the main fraction containing all phenolics except ferulic acid.
Plant polyphenolics have attained remarkable attention in the current era of modern research because it act as a potential source of antioxidants and protect human beings from certain diseases like cardiovascular disease, tumors cancer, and
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infectious diseases etc. (Khan et al., 2012; Jahan et al., 2012). Phenolics are larger class of compounds which are widely existing in medicinal plants. The antiradical activity or ROS is directly related to the number of hydroxyl groups on phenol ring.
Antioxidant activity of phenolics increases with an increase in number of hydroxyl groups. Various study have reported that phenolics and flavonoids have strong potential against ROS (Jahan et al., 2012). Many researcher like (Balasobashini et al., 2004 ;Karamac et al., 2005,; Gulçin, 2006; Khan et al., 2012; Jahan et al.,
2012),have reported that important phenolic acids namely, caffeic acid ferulic acid
,gallic acid and chlorogenic acid are also well reported for their strong pharmacological actions and antioxidants potentials (Cai et al., 1997).
HPLC based analysis of phenolics have been reported by many pervious reports (Ozkan et al., 2006; Oslzewskai, 2007; Amber et al., 2012; Jahan et al.,
2012). HPLC analysis showed high phenolics and flavonoid contents in all medicinal plants but varying in concentration. Quercetin and myricetin are very important free radical scavengers (Bouchet et al., 1998; Middleton, 2000; Levites,
2001; Silva et al., 2002; Dukic et al., 2008).Considerable variations have been observed in phenolic compounds of all tested medicinal plants. The highest contents of myrecitin was quantified in leaves of W. coagulans with the concentration of 358.46 ± 2.91 µgg-1 followed by 321.98 ± 1.76 µgg-1 in the fruit extract of same plant. While for others, concentration of myrecitin were recorded from 63.92 ± 2.91 µgg-1 to 292.02 ± 1.10 µgg-1. The quantity of syringic acid was recorded as 34.95 ± 0.17 µgg-1 for leave stalk extract of W. coagulans while for others its range was recorded from 2.89 ± 0.01 to 22.9 ± 0.49 µgg-1. For ferulic
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acid, its concentration was 43.02 ± 0.13 µgg-1 against fruit extract of W. coagulans followed by 19.92 ± 0.10 µgg-1 for root extract of said plant
Higher concentration of polyphenolics and flavonols content are responsible for the excellent cytotoxic potential of W. coagulans. These results are in agreements with earlier reports which confirm the antioxidant activity of rhizomes of C. rotundus (Nagulendran et al., 2007; Jahan et al., 2011) and may be the reason of its strong cardioprotective potential (Jahan et al., 2012). The extract of C. rotundus showed significantly (p<0.05) fewer amount of total phenolics than the methanolic extract of rhizome. Euphorbia tirucalli has good quantity of antioxidant polyphenolics and demonstrated strong action towards free radicals. Myricetin was predominate flavonol in E. tirucalli. During the process of photosynthesis variety of secondary metabolites are synthesized as earlier reported by various scientist like, (Jahan et al., 2012).
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Chapter 5
GENERAL DISCUSSION
Medicinal plants are providing raw material to herbal and pharmaceutical industry. These plants are endowed with variety of phytochemicals commonly known as secondary metabolites. A large number of secondary metabolites are being extracted and utilized against various disorders including cancer. Medicinal plants are fairly distributed in Salt Range, Pakistan and are traditionally used by local herbalists (Hakeems) to treat various noxious diseases including cancer. This is a malignant disease and is increasing at rapid pace in Asian countries. Keeping this into consideration, a study was carried out to record folk knowledge of medicinal plants, cytotoxic activity as well as phytochemical analysis of some selected plants from the studied area.
The present study documented 71 plant species belonged to 61 genera and
35 families that served to the natives for fulfilling their primary healthcare needs
(Table 2.1).According to plant families, Mimosaceae was found the most dominating family, which contributed 7 spp. (9.86%), followed by Solanaceae
(8.45%), Brassicaceae, Euphorbiaceae, Moraceae (5.63% each), Astraceae,
Boraganaceae, Fabaceae, Zygophyllaceae (4.23% each), while rest of the families had few numbers of species (Figure 2.2). The results revealed that the inhabitants have good knowledge of local plants in order to fulfill their daily life requirements.
The people of study area faces high temperature in summer above 50º C
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during summer and below up to 4º C during winter The area is scarce in term of water because of aridity and salinity which resultants skin boils, pimples and other types of skin allergies. Due to this reason people use large number of plants to cure boils disease in the area and most of the species (11 spp., 7.19%) were used to treat that complaint. It was followed by diabetes (10 spp., 6.54%), jaundice/hepatitis & skin allergy (9 spp., 5.88% each), constipation & inflammation (7 spp., 4.58%, each), pimples and spermatorrhea (6 spp., 3.92%, each). The other diseases were treated by using species 1-6 (Table 2.2).This type of studies have been reported by various scientist such as Kumar et al., (2004), Qureshi et al. (2011), Amujoyegbe et al. (2016), Sher et al. (2016).
From remote times, man used herbs as first source of medicine, which they were able to cater from their surrounding areas. This study reveals that natives of study area have good knowledge of plants from their settled areas to use as first source of medicines. The fundamental needs of human being were food shelter, clothing and medicines obtained from plant resources. Surveys on medicinal plants studies gathered valuable information about diverse uses of plant species from different areas of the world. In this regard, various studies have been reported from remote areas of the world (Bhatti et al., 2001; Qureshi, 2002; Gilani et al., 2006;
Qureshi and Bhatti, 2008; Qureshi et al., 2011; Amujoyegbe et al., 2016; Sher et al., 2016).
People of the study area and herbalists (Hakeems) used each and every part of plant to cure certain types of disease. They also used every opportunity for the
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utilization of natural resources (Bhatti et al., 1998).Results of present study revealed that leaves were mostly used for the preparation of herbal recipes
(24.79%), followed by whole plant (18.18%), fruits (15.70%), seeds (9.9%), barks, root (6.61%, each), flower, stem (3.31%, each), root bark (2.48%) while rest of 8 parts were scarcely used (Table 2.3). The current work is in endorsement of the study carried out by various scientific studies across the world (Amujoyegbe et al.,
2016; Sher et al., 2016).
Preparation of recipes is a traditional art and varies from region to region and culture to culture. In the area, powder formation (Phakki) is very commonly used mode of application and 21.24% (Fig. 2.3) of formulations discovered from the study area. It was followed by decoction (20.35%), juice (15.93%), paste
13.27% and potherbs (8.85%), smoke (5.31%), while rest of ten formulations were within the range of 1-3%. The finding of present study are in lines with various studies such as (Saikia et al., 2006; Qureshi et al., 2011; Amujoyegbe et al.,
2016).Various other studies reported similar type of results like Ahmad et al.
(2008), Qureshi & Bhatti (2008), Mohan et al. (2008), Kumar et al. (2009), Sher et al. (2016).There are no organized shops for purchasing and selling of medicinal plants, therefore most people go to the field directly and collect fresh plant for their use. They also sells medicinal plants and plant based products in nearby market to earn money.
The use of medicinal plant as a therapeutics agent is very old in the history of human civilization. The early man discovered these green medicine through trial
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and error (Bhatti et al., 2001) and still acknowledged their potential even for treating common as well as complicated diseases/ailments. These plant resources are the basis for traditional medicine system which have been used in China, India and Pakistan for thousands of years (Kumar et al. 2009; Sher et al., 2016).
Cancer being complex/noxious in nature is very difficult to cure. Scientists are targeting plant extracts to treat such complicated diseases hypothesizing their long history of utilization by the traditional communities (Qureshi et al., 2016).
According to Hartwell (1982) more than 3000 medicinal plants are being used in the treatment of cancer due to their anticancer properties. A remarkable number of anticancer drugs have been made from medicinal plants which are in the clinical use based on ethnobotanical as well as ethnopharmacological exploration (Cragg et al., 2001).
Cancer chemotherapy playing a vital role in curing malignancies and its main objective is to kill the cancer cells but not harms the healthy ones (Halliwell et al., 1988). Oncologists are predominantly interested in cancer treating compounds which induce cellular apoptosis, a property satisfied by the majority of secondary plants metabolites. Many studies reported that the mechanism of action of these anticancer drug is based on the induction of apoptosis (Ramasamy et al.,
2012). Keeping in view, present study was designed to explore medicinal plants of the Salt Range, Pakistan for their potential use as well their cytotoxicity which are ethnobotanically by the traditional herbal practitioner (THP) in treating such complications.
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The CME of M. oleifera seed showed the IC50 values ranging from 0.89 to
12.58 µgmL-1 after incubation of 48 to 72 hours against all cell lines (Table 3.7).
-1 The lowest IC50 was recorded as 1.42 µgmL against MCF-7 after 72 hours. The range of IC50 values in terms of its efficacy were very strong in the case of MCF-7,
INS-1 and HeLa (<5 µgmL-1), strong to very strong in the case of RG2 (˂10 µgmL-
1) and moderate against RD cells (10 to 20 µgmL-1) as per criteria of Wibowo et al.
(2011). The cytotoxicity of same plant fractions/isolation of compounds was conducted against Raji cells by Guevara et al. (1999) and reported four compounds namely, 4- benzyl isothiocyanate, niazimicin, β -sitosterol-3-O-b-D- glucopyranoside and β- sitosterol. They reported higher IC50 values (i.e. 32.7, 35.3,
70.4 and 27.9 µgmL-1) compared to the present study against Raji cells. Their calculated IC50 values fall in weak activity against the studied cell lines. According to Lee and Houghton (2005), values higher than 20 µgmL-1 are not putative to be used as screening for drug development. However, results of this study are within the range of putative against the tested cancer cell lines.
CME leaves extract of M. oleifera showed the IC50 values between 0.86 to
-1 10.61 µgmL following the incubation period of 48 to 72 hours. The lowest IC50 value was recorded as 0.86 µgmL-1 against RG2 after 72. Very strong activity in terms of IC50 was recorded in the case of MCF-7, INS-1, RG2 and HeLa (<5
µgmL-1) and moderate against RD cells (10 to 20 µgmL-1). Another study on anticancer activity of methanolic extract of M. oleifera leaves against HepG2,
Caco-2 and MCF-7 cells was carried out by Charoensin (2014). He reported IC50 values of 120.37±2.55, 112.46±3.74 and 133.58±2.47 µgmL-1for HepG2, Caco-2
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and MCF-7, respectively. Due to higher than 20 µgmL-1 ranges (Lee and Houghton,
2005), the extract showed inactiveness and not putative against tested cell lines, while results of this study are vice versa from these ones. Costa-Lotufo et al.
(2005) carried out cytotoxic activity of M. oleifera root extract against HL-60,
CEM, HCT-8 and B-16 cell lines and reported IC50 values against the studied cell lines as 60, 12.7, 113.8 and 28.8 µgmL-1, respectively. Among all tested cell lines,
-1 CEM was inhibited efficiently with the IC50 value of 12.7 µgmL revealing potentiality to be used as medicine according to Lee and Houghton (2005).
The crude aqueous and ethanolic extract of Artemisia annua stem and leaves were tested against HeLa and AGS cell lines with very good results. The
-1 IC50 values for HeLa cells were recorded as <500 µgmL for aqueous leave extract, stem aqueous extract, stem ethanolic extract and 368.00±2.60 µgmL-1 for leave ethanolic extract (Inayat et al., 2010). In the case of AGS cells, IC50 values for leave aqueous extract, stem aqueous extract, stem ethanolic extract was recorded
<500 µgmL-1 and leaves ethanolic extract 233.64±6.69 µgmL-1. The study reported higher IC50 values for both the tested cell lines according to the criteria of Lee and
Houghton (2005).
The present study was carried out cytotoxic activity of the CME of A.
-1 scoparia against tested cell lines and reported 6.57 to 21.15 µgmL IC50 values after 48-72 hours incubation. Generally, the activity remained strong for INS-1, RD and MCF-7 with the IC50 values of 6.57±0.05, 8.94±0.07 and 9.17±0.06, respectively after 72 hours incubation (Table 3.4). It was followed by moderate for
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RG2 (10.48±0.08) and HeLa (12.67±0.11). The biological activity of CME of A. herba-alba was evaluated by Khlifi et al. (2013) including anticancer activity against human bladder carcinoma (RT112), human laryngeal carcinoma (Hep2) and human myelogenous leukemia (K562). The study reported IC50 values of 81.59 ± 4.4, 59.05 ± 3.66 and 90.96 mgL-1 of the respective cell lines. Besides, they reported that CME had no effect on normal peripheral blood mononuclear cells and the biological activities were correlated with the phenolic contents of the extract. Comparing with the said study, the findings of present study in terms of
IC50 values are vice versa (Table 3.4).
The bioassay guided isolation and purification of the CMEs of Withania coagulans of aerial parts has been reported by Ihsan-ul-haq et al. (2013) for understanding cancer chemo-preventive mechanisms. They isolated three new steroidal lactones namely, withacoagulin G, withacoagulin H and withacoagulin I.
The withacoagulin G and withacoagulin H were the most active compounds possessing the IC50 values of 3.1 to 1.9 μM inhibiting tumor necrosis factor-α
(TNF-α) and 1.60 to 12.4 μM, respectively.
The present study first time reports leave stalk extract of W. coagulans showing maximum cytotoxic effect on all tested cell lines with the IC50 values of
0.96 to 4.73 µgmL-1 after 48-72 hours incubation, revealing very strong activity for all the tested cell lines (Table 3.8). Against HeLa cells, IC50 values were recorded between 1.68 to 1.79 µgmL-1, for MCF-7, the same were recorded as 1.02 to 4.73
-1 -1 µgmL and in the case of RD cells, 0.96 to 1.73 µgmL .The IC50 values for RG2
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cells were recorded between 1.68 to 2.55 µgmL-1, while in the case of INS-1 the same were between 2.97 to 4.56 µgmL-1 (Table 3.8).
Interestingly, our findings are in agreement of Ihsan-ul-haq et al. (2013) who reported the IC50 more or less closer values. However, this study carried various plant parts of the aerial parts such as leaves stalk, fruit, etc. The fruit extract of W. coagulans had cytotoxic effect on all tested cell lines having its IC50 values
-1 of 0.69 to 6.69 µgmL after 48-72 hours incubation. Against HeLa cells the IC50 values were 2.19 to 2.79 µgmL-1, for MCF-7, the same were calculated as 0.69 to
-1 0.99 µgmL . In the case of RD cells, IC50 values were recorded as 2.98 to 5.16
-1 -1 µgmL . The IC50 values of RG2 cells were ranged between 1.97 to 6.69 µgmL ; whereas, in the case of INS-1, the same were recorded as 1.95 to 2.84 µgmL-1
(Table 3.8). Another study conducted by Esmaeili et al. (2014) reported higher IC50 values CMEs of W. coagulans than the present study. According to them, the IC50 values were > 50 µgmL-1 against tested five cell lines viz, HepG-2, MDBK, WEHI-
164 and A-549. The possible researn could be change of cell lines, except MCF-7 as well different assay (MTT).
The anticancer activity of CMEs of Otostegia michauxii and O. persica have been reported by Esmaeili et al. (2014) the against HepG-2, MCF-7, MDBK,
-1 WEHI-164 and A-549 cell lines. They reported >50 µgmL IC50 values for both plants the against the tested cell lines. The present investigation revealed very strong activity of the CME of O. limbata against RG2 (4.49±0.05), strong in the case of INS-1 (5.20±0.04) and MCF-7 (5.97±0.06), moderate for RD (14.69±0.15)
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and HeLa (16.78±0.17) after 72 hours incubation (Table 3.6). CMEs of 27 medicinal plants from southern provinces of Iran were subjected for cytotoxicity against MCF-7, WEHI-164, HepG-2, MDBK and A-549 cell lines by means of
MTT assay. Amongst these species, Calotropis procera and Juniperus excelsa extracts were found more effective against the studied cell lines. The IC50 values of
Calotropis procera were recorded as 4.96 µgmL-1 for MCF-7, 21.42 µgmL-1 for
MDBK, 4.84 µgmL-1 for A-549 and >50 µgmL-1 for HepG-2 and WEHI-164 cell lines. While CMEs of Juniperus excelsa showed range of IC50 values as 7.52
-1 -1 µgmL for MCF-7, >50 µgmL for WEHI-164. The results of IC50 values of
Calotropis procera against MCF-7 and A-549 cells support the findings of present study. The results of Juniperus excelsa against MCF-7 also agreement with the present study.
Some of the studies reported higher IC50 values than the present study for various crude methanolic extracts of various species. For instance, Sufian et al.
-1 -1 (2013) reported IC50 values of 34.73 µgmL against MCF-7, 30.90 µgmL for HL-
-1 60 and 61.29 µgmL against HCT cells. Lin et al. (2014) reported IC50 value of
Curcuma zedoaria (rhizome) CME of 18.4 µgmL-1 against MCF-7 cells.
Anticancer activity of CME of Peperomia pellucida (leaves) was reported by Liao et al. (2014) against MCF-7 cell line. The CME possessed significant anticancer
-1 activities with the IC50 value of 10.4±0.06 μgmL . The later study supports the findings of our study.
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SUMMARY
Medicinal plants are providing raw material to herbal and pharmaceutical industry. These plants are rich in chemical substances in the form of secondary metabolites. A large number of secondary metabolites are being extracted and utilized against various disorders including cancer. Medicinal plants are fairly distributed in Salt Range, Pakistan and are traditionally used by local herbalists
(Hakeems) to treat various noxious diseases including cancer. This is a malignant disease and is increasing at rapid pace in Asian countries. Keeping this into consideration, the present study was undertaken to document medicinal uses of native plants, cytotoxic activity as well as phytochemical analysis of some selected plants from the studied area. Altogether, 56 different ailments/diseases were treated by using 71 plant species. Boils was found the most prevalent disease in the study area and most of the species (11 spp., 7.19%) were used to treat that complaint. It was followed by diabetes (10 spp., 6.54%), jaundice/hepatitis & skin allergy (9 spp., 5.88% each), constipation & inflammation (7 spp., 4.58%, each), pimples and spermatorrhea (6 spp., 3.92%, each).
Overall, 17 parts of medicinal plants were utilized by native communities for the preparation of different kinds of recipes for the treatment of various ailments/diseases. Leaves were mostly used part for the preparation of herbal recipes (24.79%), followed by whole plant (18.18%), fruits (15.70%), seeds
(9.9%), barks, root (6.61%, each), flower, stem (3.31%, each), root bark (2.48%) while rest of 8 parts were scarcely used. Overall, 16 methods of application were used by the inhabitants of the area for treating common diseases in which, powder
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form (Phakki) is very commonly used mode of application and 21.24% of formulations discovered from the study area. It was followed by decoction
(20.35%), juice (15.93%), paste 13.27% and potherbs (8.85%), smoke (5.31%).
Based on more than 60% fidelity level (FL%), seven species such as
Artemisia scoparia Waldst. & Kitam., Fagonia indica Burm. f., Moringa oleifera
Lam., Otostegia limbata (Benth.) Boiss., Rhazya stricta Decne., Physorrhynchus burhaicus Hook.f. and Withania coagulans (Stocks) Dunal were selected for screening cytotoxic activity against cancer cell lines viz., human breast cancer
(MCF7), human cervical cancer (HeLa), human skin cancer (RD), rat pancreatic tumor (INS-1) and rat brain tumor (RG2). The plant materials of the said species were dried and ground to make powder for extracts preparation by using methanol.
All plant extracts showed cytotoxic effects against the selected cell lines, however, the inhibitory activity was found specific to plant extracts versus cell lines. In the case of Artemisia scoparia, except HeLa cell, the whole plant extract showed 80% cytotoxicity against all cell lines. The whole plant extracts of Fagonia indica showed 80% cytotoxic effect on three cell lines such as MCF-7, INS-1 and
RG2. The fruit extract of Moringa oleifera had leading effects (> 80%) on all cell lines. Besides, leaves extract also inhibited 80% cells of MCF-7 and INS-1. The whole plant extract of Otostegia limbata showed maximum cytotoxic effect (80%) against all cell lines, except HeLa cell. From the Rhazya stricta, root extract showed highest cytotoxic effect (> 90%) for RG2 and HeLa cell lines and 80% for
MCF-7 and INS-1. The whole plant and leaves extract of Physorhynchus brahuicus
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showed maximum cytotoxic effect (80%) on three cell lines viz., MCF-7, INS-1 and RG2 and root extract induced 80% RD cells. The leaves stalk and fruit extract of Withania coagulans had highest cytotoxic effect on all cell lines in which MCF-
7, RG-2, RD cells and HeLa cells were inhibited at 90%, while INS-1 was inhibited up to 80%. In order to examine the behavior of all these extracts, IC50 values were calculated against the selected cell lines. It was observed that the activity was very strong to moderate ranging from 0.99 to 23.15 µgmL-1 after incubation of 48 hours.
The same trend was observed after 72 hours of application, however the magnitude of activity was very strong to strong that ranged from 0.86 to16.78 µgmL-1.
Interestingly, the results revealed that the cytotoxicity was directly proportional to the incubation period.
The most active fractions were subjected to HPLC for identifying phytochemicals. Among phenolics, p-hydroxy benzoic acid and gallic acid were found in all tested plant part (s) followed by protocatechuic acid, p-coumaric acid, vanillic acid, syringic acid and ferulic acid. Highest concentration of p-hydroxy benzoic acid was recorded in case of P. brahuicus 167.39 ± 2.13 µgg-1 followed by fruit extract of W. coagulans with the concentration of 157.46 ± 1.43 µgg-1 ,while all other tested parts having its concentration from 12.46 ± 0.8 µgg-1 to 143.69 ±
1.83 µgg-1 The other most common phenolic was gallic acid, which was present in all samples. The highest concentration of gallic acid was recorded in the leave stalk extract of W. coagulans (83.62±0.7 µgg-1) followed by R. stricta root extract
(78.02±1.3 µgg-1), A. scoparia whole plant extract (69.02±0.6 µgg-1), leaves extract
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of R. stricta (65.02±0.9 µgg-1) and rest of plant parts having its concentration from
17.62±0.7 µgg-1 to 52.82±0.6 µgg-1.
The second most common phenolics were protocatechuic acid, p-coumaric acid and vanillic acid. The p-coumaric acid was found abundantly in bark extract of
M. oleifera with the concentration of 212.46 ± 0.11 µgg-1 and it is the highest concentration amongst all quantified phenolic compounds. In case of other plant part (s) its concentration were recorded as 10.39 ± 0.6 to 131.64 ± 0.59 µgg-1. The concentration of protocatechic acid was 39.09 ± 0.18 µgg-1 in fruit extract of W. coagulans, while for other tested part (s) its value were recorded between 9.39 ±
0.76 to 34.28 ± 0.53 µgg-1.The highest value for vanillic acid was recorded as 27.26
± 0.11 µgg-1 against aerial parts extract of O. limbata while for others its range were observed as 1.26 ± 0.09 to 25.68 ± 0.17 µgg-1.
The quantity of syringic acid was recorded as 34.95 ± 0.17 µgg-1 for leave stalk extract of W. coagulans while for others its range was recorded from 2.89 ±
0.01 to 22.9 ± 0.49 µgg-1. For ferulic acid, its concentration was 43.02 ± 0.13 µgg-1 against fruit extract of W. coagulans followed by 19.92 ± 0.10 µgg-1 for root extract of said plant. In case of all other tested part (s) its value was 6.02 ± 0.01 to 19.02 ±
0.3 µgg-1.Overall the total phenolic contents (TPC) were recorded and the highest
TPC was recorded (421.62 ± 11.58 µgg-1) for bark of M. oleifera followed by
369.88 ± 6.46 µgg-1 against P. brahuicus, While for others the value of TPC was recorded 123.54 ± 6.30 to 364.93 ± 9.32 µgg-1.
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Data for the flavonoids (myricetin and quercetin) contents of selected medicinal plant part(s)/organs also indicated that myricetin was the most common and abundant flavonoid present in tested plants. The highest contents of myrecitin was quantified in leaves of W. coagulans with the concentration of 358.46 ± 2.91
µgg-1 followed by 321.98 ± 1.76 µgg-1 in the fruit extract of same plant. While for others, concentration of myrecitin were recorded from 63.92 ± 2.91 µgg-1 to 292.02
± 1.10 µgg-1.Quercitin is also an important flavonoid which is also quantified in the present study from the selected plant /part (s) and its highest concentration was recorded in 23.45 ± 0.99 µgg-1 followed by 13.96 ± 0.57 µgg-1; while for rest of plants it concentration was recorded between 3.27 ± 0.47 to 13.62 ± 0.96 µgg-1.
Total flavonoid content (TFC) was calculated for all tested samples and highest TFC was recorded for root (394.34 ± 1.26 µgg-1) extract of Withania coagulans followed by leaves extract (358.46 ± 2.91 µgg-1) and fruit extract
(343.41 ± 1.96µgg-1) of the same plant. While for other plants, the concentration of
TFC were recorded from (75.26 ± 0.37µgg-1) to (281.52 ± 0.36 µgg-1) and their detailed results are shown. Based on TFC values W. coagulans was ranked first amongst all tested plants.
Identification and quantification of polyphenols and flavonoids from medicinal plants was achieved by Reverse Phase High Performance Liquid
Chromatography (RP-HPLC). Altogether, 89 phenolic compounds were isolated from methanolic crude extract; while 26 phenolic compounds were quantified from six column chromatography (CC) fractions of P. brahuicus. With reference to
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flavonoids, overall 25 flavonols were identified from methanolic crude extract and
9 from CC fractions of P. brahuicus by using RP-HPLC method.
Physorhynchus brahuicus, an endemic species of Irano-Turanian element, was subjected to bioactivity guided isolation of compounds through column chromatography (CC) which may be responsible for activity. This species was selected due to higher ethnobotanical use (FL%), previous reported activities/biochemistry, preliminary cytotoxic screening and range of IC50 values, since there is no previous work reported worldwide on its cytotoxic activity as well as phytochemistry. Six column chromatography (CC) fractions of Physorhynchus brahuicus were subjected for cytotoxicity test against the five cell lines at the dose of 20µgmL-1 with Methotrexate (MTX) as comparator. All the fractions were found effective against the selected cell lines and the IC50 values were within the range of
0.38±0.08 to 12.86±0.10 µgmL-1. Except CCU, rest of fractions revealed very strong to strong inhibitory effects in terms of IC50 value. Among them, CCO fraction was found highly effective which resulted IC50 ranging from 0.68±0.07 to
6.74±0.07µgmL-1.
Plant extracts showing significant activities were subjected to column chromatography fractionation, based on solvent-solvent extraction and four different fractions were prepared i.e. n-hexane, n-hexane/EtOAc, EtOAc,
EtOAc/MeOH and MeOH as eluent. Those fractions which showed significant activities were characterized for active constituents by physical techniques like
HPLC and LC-MS. Out of 21 column fractions (CCFs), six fractions were selected
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based on their best performance. These were subjected for in depth evaluation of their IC50 values. According to NCI, USA, plant screening program, the plants extract and fractions can be considered active/putative if their IC50 value is less
-1 than 20 µgmL against cancer cell lines. The IC50 value was recorded between
0.38±0.08 to 12.86±0.10 µgmL-1 revealing very strong to strong inhibition against all the selected cell lines.
The chemical structures were established with the aid of extensive LC-MS spectroscopic, mass-spectral analyses and published data. Eight compounds viz.,
N,N-dipropyl propan-1-amine, 4-acetamidophenyl) 2-(diethylamino) acetate, 3-
Methyl benzo furan-2-carboxylic acid, [2-(Diphenylphosphoryl) ethyl] (methyl) oxo(phenyl) phosphine, 2-(3-Amino-1H-1, 2, 4-triazol-1-yl) acetohydrazide, 3-
Methyl-N-(3-methylbutyl)-1-butanamin, 1, 3-Benzenedimethanol, α1-[[(1, 1- dimethylethyl) amino] methyl]-4-hydroxy and 3-Chloro-6-hydrazinopyridazine were identified from six column chromatographic fractions.
This study provided detailed scientific information about the cytotoxic activity and phytochemistry of P. brahuicus that would serve as benchmark towards anticancer drug development. Structural elucidation and in vivo activity is therefore suggested to test potential toxicity of the fractions that may be used in the cancer drug development programme.
175
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(ANNEXURE-1)
PROFORMA FOR THE INTERVIEW FROM THE INHABITANTS REGARDING USES OF PLANT SPECIES
Date…………
Name/ Address of Respondent: ______ ______ ______ ______
Enumerator: ______
Botanical Name: Vernacular Name: Language: Locality:
______USES A-Medicine:
Part of plant used: ______ Method of preparation: ______
Type of Ailment: ______
B-Veterinary:
Disease: ______ Part of plant used: ______ Method of preparation: ______
C-Fodder: All parts of Plants: [ ] Leaves: [ ] Fruits: [ ] Seeds: [ ] Availability of forage: [ ] D-Fuel: [ ] E-Any other usage/Cultural Beliefs:
195
Pak. J. Bot., 49(1): 353-359, 2017.
PRELIMINARY PHYTOCHEMICAL SCREENING, ANTIFUNGAL AND CYTOTOXIC ACTIVITIES OF LEAVES EXTRACT OF MORINGA OLEIFERA Lam. FROM SALT RANGE, PAKISTAN
MUHAMMAD MAQSOOD1, RAHMATULLAH QURESHI1*, MUHAMMAD ARSHAD1, 2 3 MUHAMMAD SHEERAZ AHMED AND MASROOR IKRAM
1Department of Botany, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan. 2Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan, 3Department of Physics and Applied Mathematics, Pakistan Institute of Engineering and Applied Sciences, Nilore, Islamabad, Pakistan *Corresponding author’s email: [email protected], [email protected]
Abstract
Moringa oleifera Lam. is a miraculous plant that endowed with variety of medicinal properties and traditionally used as herbal drug as well as nutraceutical agent. There is sporadic information on phytochemical and antifungal activity of various solvents based leaves extracts. Therefore, the present study was designed to explore In vitro antifungal activity of M. oleifera leaves against Aspergillus fumigatus, A. niger and Candida albicans at four different concentrations (50-300 mg/ml) by ager well diffusion method. Leaves of this plant were collected from the Thal Desert, Pakistan, dried under shade, powdered and kept in air tight sterilized bottles. Polarity based solvent extraction of powdered samples were carried out with different solvents. The ethanolic and methanolic extracts were found the most effective against all selected fungal strains. The maximum zone of inhibition was recorded in the case of methanolic leaves extract (16 mm) against A. niger at a concentration of 300 mg/ml, which was at par to the standard antibiotic. Methanolic extract showed the highest MIC value (70 mg/ml) against A. niger. There was very strong activity in terms of IC50 against MCF-7, INS-1, RG2 and HeLa (<5 µgmL-1). The quantitative phytochemical analysis revealed that leaves possessed high amount of flavonoids (21.76±0.68), followed tannins (14.3±0.26), saponins (12.56±0.51) and alkaloids (2.4±0.85). This piece of research would be used as benchmark to carry-out further detailed study ranging from isolation, characterization, pharmacological diagnosis and clinical trials prior to launching marketable drug.
Key words: Moringa oleifera, Medicinal properties, Phytochemicals, Antifungal activity, Leaves extracts.
Introduction activity (Shukla et al., 1981), ethanolic extract have shown activity against a number of pathogenic fungi Moringa oleifera Lam. locally known as Suhanjana, (Chuang et al., 2007), whereas leaf methanolic extract has belongs to family Moringaceae (David et al., 2015), a strong depressant action on central nervous system (Pal mostly cultivated in the Sindh province and irrigated et al., 1996). The seeds of M. oleifera possess strong anti- plains as well as arid areas of the Pakistan (Iqbal & microbial and coagulative properties (Eilert et al., 1981). Bhanger, 2006; Qaiser, 1973). This species is distributed Since most of the work is reported from various parts of in India, Pakistan, Bangladesh and Afghanistan (Fahey, the world, however no detailed study is reported from 2005). M. oleifera is a shrub or small tree of 2.5-10 m in Pakistan, therefore present work was planned to screen height (Vlahof et al., 2002). This is a dynamic plant with phytochemicals, antifungal and cytotoxic activities of rich medicinal properties. The entire plants have protein, leaves extracts of M. oleifera. vitamins, mineral and carbohydrates, seeds have oil content, while the coagulant of seeds is used for the Materials and Methods treatment of the wastewater (Foidl et al., 2001). Various parts of M. oleifera are used as vegetables Collection and identification of plant material: The and for treatment of variety of human ailments in the folk collection of leaves of M. oleifera were carried out from medicine (Sastri, 1962; Nadkarni & Nadkharni, 1976). In Thal Desert, Pakistan and identified through floristic Ayurvedic system of medicine, its root, root bark, leaves, material (Qureshi, 2012). The voucher specimen (2056) flowers and unripe pods are reported as antipyretic, was deposited in the Herbarium of Department of Botany, abortificient, galactogogue and anthelmintic. The bark is Pir Mehr Ali Shah Arid Agriculture University Rawalpindi. used as an emmenagogue in Siddha and Unani system of medicine and root decoction is used as gargle, Preparation of plant sample: The collected leaves were abortifacient, rubefacient, counter-irritant in rheumatic washed thoroughly with water, chopped, air dried under shade cases (Chopra, 1938). Several studies reported that it has at room temperature, oven dried and ground to powder with some pharmacological activities including antibacterial electric grinder and stored in airtight jars for further process. (Eilert et al., 1981; Dayrit et al., 1990, Elumalai et al., 2015), anti-inflammatory (Ezeamuzie et al., 1996), Preparation of plant extract: For the preparation of hepatoprotective (Pari & Kumar, 2002), anti-hypertensive extracts the powdered samples were extracted in seven (Faizi et al., 1995) and anti-tumor (Murakami et al., polarity based solvents i.e. n-hexane, chloroform, acetone, 1998), antispasmodic, diuretic, antioxidant (Kumbhare et ethyl acetate, ethanol, methanol and water. Initially, al., 2012) and antifungal activities (Caceres et al., 1992). samples were extracted in n-hexane (1:10) and shaken for Leaves have been used for wound healing, as 24 hours and centrifuged at 10,000 rpm for 15 min. The diuretic, antiulcer and anti-inflammatory (Caceres et al., supernatant was decanted into labeled containers and 1992; Udupa et al., 1994; Pal et al., 1995; Jaiswal et al., residue was extracted in next solvent, which was slightly 2009). The leaves aqueous extract possess antifertility higher in polarity than n-hexane. The evaporation of the all 354 MUHAMMAD MAQSOOD ET AL., extracts were carried out under reduced pressure at 40C were taken in triplicates. and stored at 4C in refrigerator. For the antifungal assay Minimum inhibitory concentration (MIC): The the dried extracts were dissolved in Dimethylsulfoxide minimum inhibitory concentration (MIC) of leaves (DMSO) for antifungal assay. extracts was carried out by the agar well diffusion method as described by Thongson et al., 2004. The lowest Phytochemicals screening concentration of extract caused complete inhibition of fungal growth was taken as MIC. a. Qualitative identification of phytochemicals: The Leaves extracts of all solvents were subjected to qualitative Cytotoxicity activity: Dilutions of the stock solution determination of flavonoids (Trease & Evans, 1996; were made in cell culture medium, utilizing, minimum Kalimuthu et al., 2010), saponins (Tyler, 1994; Hussain et essential medium (MEM) alpha for MCF-7 , Dulbecco’s al., 2011), alkaloids (Tyler, 1994; Hussain et al., 2011), Modified Eagle’s Medium (DMEM) for RG2 and RD phenols (Roopashree et al., 2008) and tannins (Roopasheer while Roswell Park Memorial Institute medium (RPMI- et al., 2008). 1640) for HeLa and INS-1 cells. All media containing phenol red and 2% fetal bovine serum (FBS), 1% L- b. Quantitative determination of phytochemicals: glutamine (2mM), 1% sodium pyruvate (1mM) and 1% Quantitative determination of major phytochemicals of antibiotics penicillin (5000UmL-1) and streptomycin leaves extracts of M. oleifera was carried out as described (2500UmL-1) (all from Gibco®, Invitrogen, CA, USA) as by Obodoni & Ochuko (2001), Hussain et al. (2011) and supplements. Logarithmic dilutions of the stock solutions Sutharsingh et al. (2011), Tyler (1994). were between kept 20 and 0.1 µgmL-1 to determine the extract fractions of cytotoxicity (IC50 value). Dilutions Determination of antifungal activity: Different solvent were kept at 4ºC until future use. based leaves extract of M. oleifera were assessed for its antifungal activity against fungal pathogens viz., A. niger, A. Statistical analysis: All the results were expressed as fumigatus and C. albicans by ager well method. Their mean ± SD (standard deviations). Statistical analysis was clinical isolates were obtained from Pakistan Institute of performed using MSTATC and MS-Excel 2007. The Medical Sciences (PIMS), Islamabad and used in this study. significant difference was considered at α values ≤ 0.05.
Preparation of inoculum: Sabouraud Dextrose Agar Results and Discussion (SDA) was used to maintained stock of fungi. A loopful of cells from the stock cultures was transferred to test Phytochemical screening tubes of sterile Sabouraud Dextrose Broth (SDB) to prepare 106 colony forming units (CFU/ml) and tubes Qualitative analysis of leaves extracts for were placed at 25C for 24 hours in shaker incubator. phytochemicals: Qualitative phytochemicals analysis of Agar Well Diffusion method: The antifungal activity was Moringa leaves showed the presence of all tested tested by agar well diffusion method (Nejad et al., 2010). phytochemicals and the results are summarized in Table SDA media was prepared, autoclaved for 15 minutes at 1. Ethanol and methanol were found the most effective 121C, cooled and poured in laminar flow cabinet in petri solvents that isolated all phytochemicals. Various studies plates. After allowing the media to solidify at room discovered that ethanol extract has capability to isolate temperature, one hundred microlitres of inoculums (106 maximum number of compounds (Tijjan et al., 2009; CFU/ml; 0.5 McFarland) of each tested fungus evenly spread Bennett et al., 2003). Interestingly Bukar et al. (2010) onto separate agar plates using a sterile glass spreader isolated saponins and flavonoids from ethanol extract but (Fenner et al., 2005; Thongson et al., 2004). The agar plates failed to separate tannins and alkaloids from it. The were dried and punch wells were formed by sterile borer of 6 absence of later phytochemicals was also reported by mm in diameter. The wells were filled with 100 µl of plant Kasolo et al. (2010) from ethanol extract. extract. The extract were allowed to diffuse into agar media The water as solvent in the present endeavour isolated for 2 hours and incubated at 27C for 72 hours. Dimethyl alkaloids, phenols and tannin, but unable to detect flavonoids sulfoxide (DMSO) was used as a negative control and and saponins which contradicts with the study of Kwaghe &
Fluconazol was used as a positive control. Ambali (2009) and Kasolo et al. (2010). Quantitatively, phenols and tannins were present in very higher amount in Measurement of zone of inhibition: The antifungal ethanol, methanol and water (Table 1). Tannins are a group activity was measured as zone of inhibition in diameter of polymeric phenolic substances capable of killing (mm) after 72 hours of incubation at 27C. All readings microorganisms (Cowan, 1999; Hausteen, 2005).
Table 1. Qualitative phytochemicals analysis of leaves extracts. Sr. No Solvent Flavonoids Saponins Alkaloids Phenol Tannins 1. n-Hexane ─ + ─ ─ ─ 2. Chloroform + + ─ ─ ─ 3. Acetone + ─ ─ + ─ 4. Ethyl acetate + ─ ─ ++ + 5. Ethanol ++ ++ + +++ +++ 6. Methanol +++ ++ + +++ +++ 7. Water ─ ─ ++ +++ +++ Key: Absent = ─, Trace = +, moderately present = ++, highly present = +++ PRELIMINARY PHYTOCHEMICAL SCREENING AND ANTIFUNGAL ACTIVITY OF LEAVES EXTRACT 355
The ethyl acetate showed the presence to flavonoids, acetone (Table 3). It was found that leaves extract phenols and tannins; whereas acetone leaves extract showed maximum antifungal activity against A. niger isolated phenols and flavonoids which were present in (16 mm), followed by A. fumigatus (13.83 mm) and C. low amount (Table 1). There was a little difference found albicans (13 mm). Similar findings are reported by in ethyl acetate and acetone that the former extract Kekuda et al. (2010), who observed that steam contained tannins in addition to flavonoids and phenols distillation of leaves showed more inhibition against A. which is in agreement with those of Kwaghe & Ambali niger (46.51%). Therefore, A. niger was the most (2009). In the case of chloroform, flavonoids and susceptible fungus among all the tested fungi. saponins were extracted that corresponds with the finding The all concentrations of ethanol and methanol of Kwaghe & Ambali (2009), however, present study based M. oleifera leaves extracts were found the most failed to isolate further compounds as mentioned by effective against all selected fungal strains except 50 previous authors. Interestingly, Bukar et al. (2010) found mg/ml. The maximum zone of inhibition (16±2 mm) alkaloids, saponins and tannins from chloroform leaves was recorded in the case of methanol leaves extract extract but unable to detect flavonoids which contradicts against A. niger at concentration of 300 mg/ml, which with our findings. The n-hexane was found least effective was almost equivalent to standard antibiotic (Table 3; solvent and solely isolated saponins. The present study Fig. 5). Therefore, this study suggests that methanolic revealed that both phenols and tannins were richly found, leaves extracts of screened plant would be helpful in whereas, saponins in moderate amount in methanol and treating diseases caused by A. niger. At the same ethanol extract (Table 1). concentration A. fumigatus and C. albicans showed inhibition of 13.83±1.25 mm (Fig. 1) and 13±1 mm Quantitative phytochemicals determination of leaves: respectively. Chuang et al. (2007) reported that ethanol The data provided in Table 2 shows that phytochemicals extracts showed In vitro antifungal activity against composition of leaves were found in the order of Trichophyton rubrum, T. mentagrophytes, flavonoids (21.76±0.68%)> tannins (14.3±0.26%)> Epidermophyton Xoccosum, and Microsporum canis. saponins (12.56±0.51%)> and alkaloids (2.4±0.85%). The results of present study confirmed that ethanolic Flavonoids were found in higher proportion (21.76%) and methanolic Moringa leaves extract possess broad than rest of chemicals and are reported to be of high spectrum antifungal activity. value in deriving a wide range of biological activities It has been observed that inhibition of A. niger and (Hodek et al., 2002). Similarly, Munazir et al. (2015) C. albicans was directly proportional with reported higher amount of flavonoids from L. pyrtotechnica. Thus high antifungal activity determined concentrations. It has been found 15.3±1.52 mm (Fig. by this study reveals that the activity may be attributed 5), 13.41±2.30 mm, 11.17±1.44 mm inhibition zones due to presence of high proportion of flavonoids and (Table 3) were produced by ethanol extract against A. tannin content. Krishnaiah et al. (2009) investigated niger at 300 mg/ml, 200 mg/ml and 100 mg/ml leaves of medicinal plants for five major groups of respectively. In the case of C. albicans 12.66±1.60 mm, phytochemicals and reported tannins (9.2%), saponins 11.33±2.08 mm and 8.5±1.32 mm inhibition zones (2.3%), flavonoids (0.51%), alkaloids (0.36%) and recorded at 300 mg/ml, 200 mg/ml and 100 mg/ml phenols (0.08%). The present investigation discovered respectively. In the case of A. fumigatus, there was no significantly higher proportion compared with former change found in inhibition zone when concentrations study. There are multiple factors which govern in were kept 200 (Fig. 2) to 300 mg/ml (Fig. 3) and results proportion within chemical composition and were same i.e. 13 mm (Table 3; Fig. 4). concentration in plants. These might be due to The present study reveals that with increase of environmental, edaphic and nature of plant growth stage, concentration of extract the antifungal activity also etc. Mustafa et al. (2016) carried phytochemical increased which is in agreement with findings of screening of some plants from Cholistan desert. Wanchaitanawong et al. (2005), that used various
concentration of extract of selected plant on the Table 2. Quantitative estimation of phytoconstituents inhibition of A. niger, A. oryzae and penicillium spp. (%) in M. oleifera leaves. The aqueous extracts of Moringa in this investigation Flavonoids Saponins Alkaloids Tannins exhibited moderate antifungal activity and zones of 21.76 ± 0.68 12.56 ± 0.51 2.4 ± 0.85 14.3 ± 0.26 inhibition were in the order of 12.3 ± 0.57 mm > 12 ± 1 mm against A. fumigatus and A. niger respectively at Values are expressed as means ± S.D after triplicate analysis 300 mg/ml. However, no activity recorded in the case
Antifungal activity of leaves extracts: Antifungal of C. albicans that corresponds to the findings of activity of different leaves extracts (n-hexane, Caceres et al. (1991) (Table 3; Fig. 3). Rahman et al. chloroform, acetone, ethyl acetate, ethanol, methanol (2009) reported that M. oleifera fresh leaves juice and water) of M. oleifera was tested against A. niger, A. showed stronger antibacterial activity and higher zone fumigatus and C. albicans (Table 3). The extracts of inhibitions (15-25 mm) than present findings. The showed significant differences in their efficacy. The strong antibacterial activity of the same solvent base activity of different leaves extract was in the order of extract may be attributed due to difference in methanol > ethanol > ethyl acetate > water > and prokaryotic cell composition. 356 MUHAMMAD MAQSOOD ET AL.,
Table 3. Antifungal activity of leaf extracts of Moringa oleifera. Extract conc. Fungal strains diameter of zone of inhibition in mm Solvents mg/ml A. fumigatus A. niger C. albicans n- hexane 300 _ _ _ 200 _ _ _ 100 _ _ _ 50 _ _ _ Chloroform 300 _ _ _ 200 _ _ _ 100 _ _ _ 50 _ _ _ Acetone 300 10.16 ± 1.25 11.6 ± 0.76 _ 200 8.75 ± 0.66 10.08 ± 0.87 _ 100 _ 8 ± 1.5 _ 50 _ _ _ Ethyl acetate 300 11.16 ± 1.52 13.91 ± 1.37 11 ± 1 200 10 ± 1.73 12 ± 2 10 ± 2 100 8.75 ± 1.29 9.5 ± 0.86 _ 50 _ _ Ethanol 300 13±2 15.3 ± 1.52 12.66 ± 1.60 200 13±1 13.41 ± 2.30 11.33 ± 2.08 100 10.8±1.25 11.17 ± 1.44 8.5 ± 1.32 50 _ _ _ Methanol 300 13.83 ± 1.25 16 ± 2 13 ± 1 200 12.75 ± 1.29 14.8 ± 1.89 12.08 ± 1.58 100 11.16 ± 0.57 12 ± 1 9.5 ± 0.86 50 _ _ _ Water 300 12.3 ± 0.57 12 ± 1 _ 200 10 ± 2 10.5 ± 1.5 _ 100 _ 9.66 ± 0.76 _ 50 _ _ _ Fluconazole 10 µg/ml 15.1 ± 0.86 16.5 ± 0.1 17.2 ± 0.62
Fig. 1. Antifungal activity of ethyl acetate and methanol extract Fig. 2. Antifungal activity of ethyl acetate and water extract against A. fumigatus at 300 mg/ml. against A. fumigatus at 200mg/ml. PRELIMINARY PHYTOCHEMICAL SCREENING AND ANTIFUNGAL ACTIVITY OF LEAVES EXTRACT 357
The ethyl acetate extract was found effective against all tested strains (Table 3). A. niger was highly susceptible to extract and inhibited at 13.91±1.37 mm, followed by A. fumigatus (11.16±1.52 mm) (Fig. 1) and C. albicans (11±1 mm) at the concentration of 300 mg/ml (Table 3). The acetone leaves extract reasonably inhibited A. niger (11.16±0.76 mm), followed by A. fumigatus (10.16±1.25 mm) at the concentration of 300 mg/ml, while C. albicans was found resistant to extract and not inhibited at any concentration (Fig. 3). The result of ethyl acetate extracts authenticates superiority on acetone extracts in terms of antifungal activity that may be due to the isolation of tannins, as confirmed by the phytochemical analysis. Banso & Adeyemo (2007) stated that the tannins from the medicinal plants possess remarkable toxicity against bacteria and fungi and may have pharmacological importance. The tannins from S. adstringens have strong ability to inhibit the growth of C. Fig. 3. Antifungal activity of water and acetone leaves extracts albicans (Luiz et al., 2015). The Zn nanoparticles against C. albicans. produced from of M. oleifera possess antimicrobial potential because of amino acids, alkaloids, flavonoids and phenolics of the leaf extracts which further validate a the result of present study (Elumalai et al., 2015).
Cytotoxic activity: The crude methanolic extract (CME) of Moringa oleifera showed IC50 values that was ranged from 0.86 t 10.61 µgmL-1 following the incubation period of 48 to 72 hours. The lowest IC50 value was recorded as 0.86 µgmL-1, revealing a very strong activity against RG2 after 72 hours (Table 4). Very strong activity in terms of IC50 was recorded in the case of MCF-7, INS-1, RG2 and HeLa (<5 µgmL-1) and moderate against RD cells (10-20 µgmL-1) according to the criteria of Wibowo et al. (2011).
Table 4. IC50 value of Moringa oleifera (fruit and leaves) against the selected cell lines
Time period Leaves IC50 Cell line b (hours) (µgmL-1) 48 2.58 ± 0.03 MCF-7 72 3.19 ± 0.02 48 3.28 ± 0.03 INS-1 72 2.44 ± 0.02 48 4.13 ± 0.04 RG2 72 0.86 ± 0.06 48 1.79 ± 0.02 HeLa 72 1.68 ± 0.01 48 10.61 ± 0.09 RD 72 6.88 ± 0.07
Legend: ˃100: not active; 20–100: weak; 10–20: moderate; 5– Fig. 4. Comparison of antifungal activity of ethanol leaf extract 10: Strong; ˂ 5: very strong (Wibowo et al., 2011) against A. fumigatus at 300 (a) and 200 mg/ml (b). 358 MUHAMMAD MAQSOOD ET AL.,
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(Received for publication 6 December 2015)