biological and phytochemical studies of selected medicinal...
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BIOLOGICAL AND PHYTOCHEMICAL STUDIES
OF SELECTED MEDICINAL PLANTS FROM THE FAMILY
SCROPHULARIACEAE
By
Muhammad Riaz
2009-GCUF-393-114
Thesis submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
GC UNIVERSITY, FAISALABAD.
February 2013
DECLARATION
The work reported in this thesis was carried out by me under the supervision
of Dr. Nasir Rasool, Assistant Professor, Department of Chemistry, Government
College University, Faisalabad, Pakistan.
I hereby declare that the title of thesis “Biological and Phytochemical
Studies of Selected Medicinal Plants From the Family Scrophulariaceae” and
the contents of thesis are the product of my own research and no part has been
copied from any published source (except to references, equations and formulas
etc). I further declare that this work has not been submitted for award of any other
degree/diploma. The university may take action if the information provided is found
inaccurate at any stage.
Signature of the Student/Scholar
Name: Muhammad Riaz
Registration No: 2009-GCUF-393-114
CERTIFICATE BY SUPERVISORY COMMITTEE
We certify that the contents and form of thesis submitted by Muhammad Riaz,
Registration No. 2009-GCUF-393-114, has been found satisfactory and in
accordance with the prescribed format. We recommend it to be processed for the
evaluation by the External Examiner for the award of degree.
Signature of Supervisor ……………
Name: Dr. Nasir Rasool
Designation with Stamp: …………
Member of Supervisory Committee
Signature …………………………...
Name: Prof. Dr. Iftikhar Hussain Bukhari
Designation with Stamp……………
Member of Supervisory Committee
Signature ……………………………
Name: Dr. Ameer Fawad Zahoor
Designation with Stamp……………
Departmental Scrutiny Committee
1) Dr. Saira Shahzadi ____________________________
2) Dr. Tanveer Hussain Bokhari ____________________________
3) Dr. Matloob Ahmad ____________________________
4) Dr. Asim Mansha ____________________________
Chairman, Department of Chemistry
Signature with Stamp ………………
Dean/Academic Coordinator
Signature with Stamp ………………
CERTIFICATE BY THE SUPERVISORY COMMITTE
We certify that the contents and form of thesis submitted by Mr. Muhammad Riaz,
Registration No. 2009-GCUF-393-114 has been found satisfactory and in
accordance with the prescribed format. We recommend it to be processed for the
evaluation by the External Examiner for the award of degree.
Signature of Supervisor..…………………
Name: Dr. Nasir Rasool
Designation with Stamp………………….
Member of Supervisory Committee
Signature …………………………………
Name: Prof. Dr. Iftikhar Hussain Bukhari
Designation with Stamp…………………
Member of Supervisory Committee
Signature …………………………………
Name: Dr. Ameer Fawad Zahoor
Designation with Stamp…………………
Chairperson
Signature with Stamp……………………
Dean / Academic Coordinator
Signature with Stamp………………
Dedicated to My Parents and Wife
List of Abbreviations
Abbreviation Complete detail
13C-NMR Carbon nuclear magnetic resonance
1H-NMR Proton nuclear magnetic resonance
A.L. Anticharis linearis
A.M. Antirrhinum majus
Abs. Methanol Absolute methanol
CD Conjugated dienes
CT Conjugated trienes
DNA Deoxyribose nucleic acid
DPPH 1-1 diphenyl-2-picrylhydrazyl Radical
E.O. Essential oil
ESI-MS Electron spray ionization mass spectrometry
FFA Free fatty acids
FTC Ferric thiocyanate
GC-MS Gas chromatography mass spectrometery
HPLC High performance liquid chromatography
IC50 Inhibitory concentration fifty percent
L.F. Leucophyllum frutescens
M Molar solution
M.E. Absolute methanol extract
M.G. Mazus goodenifolious
MIC Minimum inhibitory concentration
N Nornal solution
NMR Nuclear magnetic resonance spectrometry
PBS Phosphate buffer saline
PV Peroxide value
R.E. Russelia equisetiformis
TAA Total antioxidant activity
TBA Thiobarbituric acid
TFC Total flavonoids content
TPC Total phenolics content
V.T. Verbascum thapsus
LIST OF CONTENTS
Acknowledgements i
Abstract iii
1 CHAPTER 1 INTRODUCTION 1-14
2 CHAPTER 2 REVIEW OF LITERATURE 15-23
2.1 Medicinal Plants 15
2.2 Essential Oils 16
2.3 Phytochemicals 17
2.4 Antioxidant 19
2.5 Antimicrobial Activity 21
2.6 Cytotoxicity 22
2.7 Stabilization of Lipid Containing Foods by Plant Sources 23
3 CHAPTER 3 MATERIALS AND METHODS 24-44
3.1 Chemicals and Reagents Used 24
3.2 Instruments Used 24
3.3 Preparation of Reagents 25
3.3.1 Sodium Carbonate Solution (10% W/V) 25
3.3.2 Ferrous Chloride Solution (20 mM) 25
3.3.3 Sodium Nitrate Solution (5%) 25
3.3.4 Aluminum Chloride (10%) 25
3.3.5 Sodium Phosphate Buffer 26
3.3.6 Sodium Hydroxide (1 M) 26
3.3.7 Ethanol (75%) 26
3.3.8 1,1 Diphenyl-2-picrylhydrazyl Radical (DPPH) 26
3.3.9 Wagner’s Reagent 26
3.3.10 Hager’s Reagent 26
3.3.11 Dragendroff Reagent 26
3.3.12 Mayer's Reagent 26
3.3.13 Aluminium Chloride Solution (1%) 27
3.3.14 Sodium Thiosulphate Solution (0.01 N) 27
3.3.15 Starch Solution 27
3.3.16 Peroxide Value Solution 27
3.3.17 Sodium Carbonate Solution (20%) 27
3.3.18 Oxalic Acid (0.1 N) 27
3.3.19 Potassium Iodide Solution (15%) 27
3.3.20 para-Anisidine Reagent 28
3.3.21 Sodium Thiosulfate (0.1 N) 28
3.3.22 Phenolphthalein Solution (1%) 28
3.3.23 Ceric Sulphate Reagent 28
3.3.24 Nutrient Agar 28
3.3.25 Sabouraud Dextrose Agar 28
3.3.26 Sterilization of Media for Antimicrobial Studies 28
3.3.27 Preparation of Petri Plates for Antimicrobial Activity 28
3.3.28 Laminar Air Flow Conditions 29
3.4 Standardization of Solutions 29
3.4.1 Standardization of Sodium Thiosulfate Solution (0.01 N) 29
3.4.2 Standardization of Sodium Hydroxide Solution (0.1 N) 29
3.5 General Procedure 29
3.5.1 Collection of Plant Material 29
3.5.2 Effect of Plant Sample Size on Extraction 30
3.5.3 Preparation of Extracts and Varoius Organic Solvent Fractions 30
3.6 Isolation of Essential Oils 30
3.6.1 GC-MS Analysis of Essential Oils 33
3.6.2 Identification of Compounds 33
3.7 Screening of Selected Plants 33
3.8 Phytochemical Analysis 33
3.8.1 Test for Alkaloids 33
3.8.2 Test for Tannins 34
3.8.3 Test for Steroids 34
3.8.4 Test for Saponins 34
3.8.5 Test for Terpenoids 34
3.8.6 Determination of Total Phenolic Contents (TPC) 34
3.8.7 Determination of Total Flavonoid Contents (TFC) 35
3.9 Evaluation of Antioxidant Activity 35
3.9.1 DPPH Free Radical Scavenging Assay 35
3.9.2 Percent Inhibition of Linoleic Acid Oxidation 36
3.9.3 Reducing Power 36
3.9.4 Bleachability of β-carotene in Linoleic Acid System 37
3.10 Antimicrobial Activity 37
3.11 Cytotoxicity Studies by Haemolytic Activity 39
3.12 Selection of Plant Which Showed Better Activity 40
3.13 Antioxidant Activity by DNA Protection Assay 40
3.14 Extraction of Phenolic Compounds from Russelia equisetiformis for LC-MS
1 Analysis 40
3.14.1 Analysis by Liquid Chromatography Mass Spectrometry (LC-MS) 41
3.15 Isolation of Phytochemicals 41
3.16 Antioxidant Activity by Stabilization of Sunflower Oil 43
3.17 Statistical Analysis 43
4 CHAPTER 4 RESULTS AND DISCUSSION 45-129
4.1 Effect of Sample Size on Extration of Plants 45
4.2 Percentage Yield of Plants Extracts and Fractions 47
4.3 Phytochemical Analysis 47
4.3.1 Qualitiative Analysis 47
4.3.2 Determination of Total Phenolic Contents (TPC) 49
4.3.3 Determination of Total Flavonoid Contents (TFC) 52
4.3.4 Percentage Yields of Essential Oils 52
4.3.5 GC-MS Assay of Essential Oils 52
4.4 Biological Studies 54
4.4.1 Evaluation of Antioxidant Avctivity 68
i. DPPH Free Radical Scavenging Assay 68
ii. Percentage Inhibition of Linoleic Acid Oxidation 69
iii. Reducing Power 74
4.4.2 Antioxidant Potential of Essential Oils by β-Carotene with Linoleic Acid
Assay 82
4.5 Antimicrobial Activity 82
4.6 Haemolytic Activity for Cytotoxicity Assay 104
4.7 DNA Protection Assay 106
4.8 Phenolic Compounds Analysed by LC-ESI-MS/MS 110
4.9 Isolated Compounds by Liquid Column Chromatography 116
4.9.1 Apigenin (76) 116
4.9.2 Ursolic acid (77) 117
4.9.3 Rutin (78) 119
4.9.4 Chlorogenic acid (79) 120
4.9.5 Ferulic acid (80) 121
4.10 Antioxidant Activity of Isolated Compounds Using Sunflower Oil as Oxidative
Substrate 122
4.10.1 Peroxide Value (PV) 122
4.10.2 Free Fatty Acids (FFA) 123
4.10.3 Conjugated Dienes (CD) and Conjugated Trienes (CT) 123
4.10.4 para Anisidine Value 124
Conclusion 130-131
REFERENCES 132-151
List of Publications 152
List of Tables
Table
No. Title
Page
No.
3.1 List of plants selected from the family Scrophulariaceae used in
present research work 31
4.1 Percentage yield of different sample sizes by selected plants using
absolute methanol solvent 46
4.2 Qualitative phytochemical analysis of selected plants extracts and
various organic fractions 50
4.3 Chemical compounds in essential oil of Anticharis linearis analysed by
GC-MS 55
4.4 Chemical compounds in essential oil of Leucophyllum frutescens
analysed by GC-MS 57
4.5 Chemical compounds in essential oil of Antirrhinum majus analysed
by GC-MS 59
4.6 Chemical compounds in essential oil of Verbascum thapsus analysed
by GC-MS 61
4.7 Chemical compounds in essential oil of Russelia equisetiformis
analysed by GC-MS 63
4.8 Chemical compounds in essential oil of Mazus goodenifolius analysed
by GC-MS 65
4.9
The screening of different concentrations on antibacterial activity by
inhibition zone (mm) by selected plants extracts and various organic
fractions against Staphylococcus aureus
85
4.10
The effect of different concentrations on antifungal activity by
inhibition zone (mm) by selected plants extracts and various organic
fractions against Rhizopus solani
86
4.11
The screening of different concentrations of selected plants essential
oils on antibacterial activity by inhibition zone (mm) against
Staphylococcus aureus
87
Table
No. Title
Page
No.
4.12
The effect of different concentrations of selected plants essential
oils against antifungal activity by inhibition zone (mm) against
Rhizopus solani
88
4.13 Antimicrobial activity in terms of inhibition zone (mm) by Russelia
equisetiformis extract and various organic fractions 91
4.14 The minimum inhibitory concentrations (MICs) in mg/mL by
Russelia equisetiformis extract and various organic fractions 92
4.15 Antimicrobial activity in terms of inhibition zone (mm) by
Leucophyllum frutescens extract and various organic fractions 93
4.16 The minimum inhibitory concentrations (MICs) in mg/mL by
Leucophyllum frutescens extract and various organic fractions 94
4.17 Antimicrobial activity in terms of inhibition zone (mm) by
Antirrhinum majus extract and various organic fractions 95
4.18 The minimum inhibitory concentrations (MICs) in mg/mL by
Antirrhinum majus extract and various organic fractions 96
4.19 Antimicrobial activity in terms of inhibition zone (mm) by
Anticharis linearis extract and various organic fractions 97
4.20 The minimum inhibitory concentrations (MICs) in mg/mL by
Anticharis linearis extract and various organic fractions 98
4.21 Antimicrobial activity in terms of inhibition zone (mm) by M.
goodenifolius extract and various organic fractions 99
4.22 Antimicrobial activity in terms of inhibition zone (mm) by
Verbascum thapsus extract and various organic fractions. 100
4.23 The minimum inhibitory concentrations (MICs) in mg/mL by
Verbascum thapsus extract and various organic fractions 101
4.24 Antimicrobial activity in terms of inhibition zone (mm) by selected
plants essential oils. 102
Table
No. Title
Page
No.
4.25 The minimum inhibitory concentrations (MICs) in mg/mL by selected
plants essential oils 103
4.26 Chemical compounds in Russelia equisetiformis extract analysed by
LC-ESI-MS/MS 112
List of Figures
Fig.
No. Title
Page
No.
1.1 Flavones (1) 3
1.2 Isoflavonoids (2) 3
1.3 Chalcone (3) 3
1.4 Shikimic acid (14) 4
1.5 Biosynthesis of catechin, a plant flavonoid (13) 5
1.6 Biosynthesis of plant phenolics 6
1.7 Biosynthesis of terpenes 7
1.8 Isoprene unit (40) showing head and tail 8
1.9 Linalool (41) 8
1.10 β-Pinene (42) 8
1.11 β-Caryophyllene (43) 9
1.12 β-Carotene (44) 9
2.1 General Structure of flavonoid (45) 18
3.1 Scheme for the extraction and fractionation of plant material 32
4.1 Percentage yield of selected plants extracts and various organic
fractions 48
Fig.
No. Title
Page
No.
4.2 Total phenolic contents in selected plants extracts and various
organic fractions 51
4.3 Total flavonoid contents in selected plants extracts and various
organic fractions 53
4.4 The GC-MS chromatogram of Anticharis linearis essential oil. 56
4.5 The GC-MS chromatogram of Leucophyllum frutescens essential
oil 58
4.6 The GC-MS chromatogram of Antirrnum majus essential oil 60
4.7 The GC-MS chromatogram of Verbascum thapsus essential oil. 62
4.8 The GC-MS chromatogram of Russelia equisetiformis essential
oil. 64
4.9 The GC-MS chromatogram of Mazus goodenifolius essential oil. 66
4.10 Structures of some major compounds identified by GC-MS
analysis 67
4.11 IC50 determined by DPPH assay of selected plants extracts and
various organic fractions 70
4.12 IC50 determined by DPPH assay of selected plants essential oils 71
4.13 % Inhibition of linoleic acid oxidation by selected plants extracts
and various organic fractions 72
4.14 % Inhibition of linoleic acid oxidation by selected plants essential
oils 73
4.15 Reducing power by Russelia equisetiformis extract and various
organic fractions 75
Fig.
No. Title
Page
No.
4.16 Reducing power by Antirrhinum majus extract and various organic
fractions 76
4.17 Reducing power by Mazus goodenifolius extract and various
organic fractions 77
4.18 Reducing power by Anticharis linearis extract and various organic
fractions 78
4.19 Reducing power by Verbascum thapsus extract and various
organic fractions 79
4.20 Reducing power by Leucophyllum frutescens extract and various
organic fractions 80
4.21 Reducing power of selected plants essential oils 81
4.22 Antioxidant activity of selected plants essential oils analysed by
bleaching of β-carotene-linoleic acid emulsion assay 83
4.23 Cytotoxicity by haemolytic activity of selected plants extract,
various organic fractions and essential oils 105
4.24 Electropherogram showing DNA protection effect by methanol
extract with H2O2 induced oxidative damage on pBR322 DNA. 107
4.25
DNA protection effect by Russelia equisetiformis extract, various
organic fractions and essential oils with H2O2 induced oxidative
damage on pBR322 DNA
108
4.26 The chromatogram of Russelia equisetiformis sample analyzed by
liquid chromatography 111
4.27 The structures of chemical compounds in Russelia equisetiformis
extract analysed by LC-ESI-MS/MS 113
4.28 A representative chromatogram of methyl protocatechuate (68) in
Russelia equisetiformis extract analysed by ESI-MS/MS 114
Fig.
No. Title
Page
No.
4.29 The fragmentation patteren of structures of methyl protocatechuate
(68) in Russelia equisetiformis extract analysed by ESI-MS/MS 114
4.30 A representative chromatogram of caffeic acid (72) in Russelia
equisetiformis extract analysed by ESI-MS/MS 115
4.31 The fragmentation patteren of structures of caffeic acid (72) in
Russelia equisetiformis extract analysed by ESI-MS/MS 115
4.32 Peroxide value (meq/kg) of sunflower oil stabilized with
compounds isolated from Russelia equisetiformis 125
4.33 Free fatty acid (percent oleic acid) of sunflower oil stabilized with
compounds isolated from Russelia equisetiformis 126
4.34 Conjugated dienes of sunflower oil treated with compounds
isolated from Russelia equisetiformis 127
4.35 Conjugated trienes of sunflower oil treated with compounds
isolated from Russelia equisetiformis 128
4.36 para-Anisidine value of sunflower oil treated with compounds
isolated from Russelia equisetiformis 129
1
1 CHAPTER 1 INTRODUCTION
INTRODUCTION
Plants have been known to be the richest source of natural antioxidants and antimicrobial
compounds (Farombi, 2003; Albayrak et al., 2010). In the view of the importance of the
plants as remedy for diseases, there is a need of proper evaluation of plants by biological
and phytochemical studies (FAO, 2004). In medicinal plants phenolic chemical
compounds are a large and diverse group of molecules, which include various families of
phytochemicals. The phenolics are the most abundant secondary metabolites such as
phenolic acids, condensed tannins, flavonoids and lignins. All these phytoconstituents are
involved in so many processes in plants and animals. Among other phytoconstituents the
phenolics and flavonoids are of most interest because of their various roles in plants and
their impact on human health (Harborne and Williams, 1992). In plants, phenolics and
flavonoids play an important function in flower, seed pigmentation, fertility, reproduction
and in different reactions to defend against abiotic stresses like Ulta Violet light or biotic
stresses such as pathogen attacks (Forkmann and Martens, 2001).
When generation of reactive oxygen species (ROS) overtakes, antioxidant defends the
cells. The free radicals start attacking cell lipids, proteins and carbohydrates and this leads
to various disorders in the body (Yu et al., 1992). Free radicals contribute to so many
diseases (Halliwell and Gutteridge, 1984). Both endogenous and exogenous antioxidants
(whether natural or synthetic) may be useful in the inhibition of free radical formation by
quenching (Maxwell, 1995). Therefore, the search for natural antioxidant has very much
increased in the present years. Since ages, plants have been traditionally used for curing
different diseases. Pakistan is an agricultural country and prosperous in pharmaceutically
and economically significant flora, so there is a need to evaluate the biological studies of
unexplored plants.
Many plants have potential to be used as natural remedy against diseases (Erdemgil et al.,
2007). The studies about antioxidant potential of phytochemical constituents found in
plants may also be necessary because of their medicinal properties (Zhu et al., 2004).
Antioxidants are the chemical constituents which can delay the oxidation of lipid
containing products. The antioxidant action of phytochemicals is mostly due to their
redox property, which takes part in a very important role for capturing free radicals and
scavenging of oxygen (Moosmann and Behl, 1999).
2
In present times, there is a rising attention in the defensive biochemical roles of
phytochemicals, especially flavonoids and phenolics related compounds to prevent
oxidative damage in the body caused by various ROS. There is a logical evidence that the
imbalance between reactive oxygen species and antioxidant defensive system may lead to
chemical modification of the biological macromolecules such as carbohydrates, proteins,
DNA and lipids (Halliwell and Gutteridge, 1984).
The demand to evaluate the sources of natural antioxidants is becoming stronger because
of the various uses of natural antioxidants in human. Although considerable work has
been reported on the dietary components of plants, yet somewhat little has been reported
on the cytotoxicity, antioxidant and antimicrobial potential of plants in Pakistan. There is
still need to explore natural sources for searching new plants having biologically active
compounds against infectious diseases. Phytochemicals from plant sources play a pivotal
role in human health (Sheikh et al., 2011). In fact, the production of free radicals during
metabolism and other activities beyond the antioxidant ability of a biological system leads
to oxidative stress (Astley, 2003). Foods or medicinal plants rich in natural antioxidants
such as phenolics are capable to decrease the risk of diseases. Today in food products the
use of synthetic antioxidants is mostly discouraged due to their adverse effects. On the
other hand, the use of natural antioxidants in foods is gaining a good deal of attention
because of their prospective health significances (Hussain et al., 2008). The plant exracts
with a good and smell pooled with a preservative action, have properties to evade lipid
deterioration and spoilage by microorganisms. The oxidation of food causes the
development of undesirable smell, creates toxicity and greatly affects the life of food
products (Farag et al., 1990). The use of natural sources as useful ingredients in food
items, cosmetics and drinks is gaining greater concern, because the synthetic additives
used as antioxidants are harmful (Reische et al., 1998). The food born diseases are main
problem in the developing countries, so there is an urgent need of safe preventives like
natural antioxidants (Sokmen et al., 2004).
The phytochemicals found in plants are organic biomolecules selected as naturally
occurring antibiotics (Seidil, 2000).
The phytochemicals such as flavonoids phenolics and terpenes are synthesized by various
pathways. The flavonoids are a class of plants secondary metabolites. The flavonoids
have two major classes such as flavones (1) and isoflavonoids (2) (McNaught and
3
Wilkinson, 1997). The flavones (Fig. 1.1) derived from 2-phenyl-1,4-benzopyrone
structure are apigenin, rutin, quercetin, kaempferol and myricetin etc.
O
O
Fig. 1.1: Flavones (1)
The isoflavonoids (Fig. 1.2) such as genistein is derived from 3-phenyl-1,4-benzopyrone
structure.
O
Fig. 1.2: Isoflavonoids (2)
Fig. 1.5 showed that flavonoids are produced by the phenylpropanoid metabolic pathway
in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA co enzyme
(Ververidis et al., 2007). This can be pooled with malonyl-CoA to synthesize the
backbone of flavonoids, called chalcones.The chalcones contain two phenyl rings.
Chalcone (3) is an aromatic ketone and an enone that yield the central core for various
important biological compounds (Fig. 1.3).
O
Fig. 1.3: Chalcone (3)
Conjugate ring-closure of chalcones results in flavonoids. The chemical pathway
continues through a chain of enzymatic reactions to synthesize catechin (13) (Fig. 1.5). In
this pathway, so many products are yielded, including the flavan-3-ols, flavonols, a host
of other various polyphenolics and proanthocyanidins.
4
The majority of the natural phenolics are derivative from metabolism of the shikimic acid
reaction pathway. The shikimic acid (14) (Fig. 1.4), more commonly known as its anionic
form shikimate, is a significant biochemical metabolite in plants.
Its name comes from the Japanese flower Illicium anisatum (shikimi), from which it was
first purified by isolation. The aromatic amino acid phenylalanine, yileded in the shikimic
acid biochemical pathway, is the ordinary originator of phenol containing amino acids
and phenolic chemical compounds (Fig. 1.6). The phenolics synthesized by shikimic acid
pathway are p-coumaric acid (26), caffeic acid (28), ferulic acid (29) and sinapic acid (30)
(Knaggs, 2003).
OHOH
OH
O OH
Fig. 1.4: Shikimic acid (14)
Cartenoids are terpenoids that are naturally occurring in chloroplasts and chromoplasts of
plants. The most common and concerned group are carotenoids due to their antioxidant
properties. The later was oxygenated carotene derivatives (Gorinstein et al., 2007).
Carotenoids act as antioxidant in the human body. Carotenoids such as α-carotene, β-
carotene and β-cypotoxantin have antioxidant properties (Armstrong and Hearst, 1996).
Terpenes are very large class of most abundant natural hydrocarbons and commonly
synthesized in plants (Fig. 1.7) as constituents of essential oils. They are also major
components of resin. Most of the terpenes found in cyclic structures and their commercial
applications include their use as fragrance in perfumery, as constituent of flavours for
food (Paduch et al., 2007).
The fundamental building block of terpenes is the isoprene unit (2-methyl-1, 3-butadiene)
(40) linked in a head to tail and is represented by general structural formula (C5H8)n
where n is the number of linked isoprene units (Fig. 1.8). Terpenes are classified into
several classes depending on the number of isoprene units in the structure.
5
4-Hydroxycinnamoyl-CoA (4)
3 x Malonyl-CoAOOOO
CoAS
OH
O O
O
O
CoAS
OH
Chalcone synthesisClaisencondensation
O
OO
O
OHHChalcone synthesis
Enolization
OHOH
OH
OH
OH
OH
OH
michael-typenucleophic attack
Chalcone isomerase
O
OH
O
[O] Flavonoid 3'-hydroxylase
OH
OH O
OH
O
OH
OH
OH O
OH
O
OH
OHFlavonoid 3-hydroxylase
[O]
OH
OH O
OH
OH
OH
Dihydroflavanol4-reductase
NADPH
OH
OH
OH O
OH
OH
OH
Catechin
CoAS
O
OH
(5)
(6)
(7)
(8)
(9)
(10)(11) (12)
(13)
Fig. 1.5: Biosynthesis of catechin, a plant flavonoid (13)
6
Phosphoenol pyruvate (15) + Erythrose 4-phosphate (16)
Quinic acid (18)5-Dehydroquinic acid (17)
5-Dehydroshikimic acid (19)
Shikimic acid (14)
Shikimate 5-phosphate (20)
3-Enolpyruvylshikimate 5-phosphate (21)
Chorismate (22)
Arogenic Acid (23)
Tryosine (24) Phenylalanine (25)
p-Coumaric acid (26) Cinnamic acid (27)
Caffeic acid (28)
Ferulic acid (29)
Sinapic acid (30)
Fig. 1.6: Biosynthesis of plant phenolics
7
OH
OH
OP
O
OPP
Mevalonic acid (32)1-Deoxy-D-xylulose-5-phosphate (frompyruvic acid) (31)
Isopentenyl pyrophosphate (IPP) (33)
Isopentenyl pyrophosphate isomerase (34)
OPP
Dimethyl pyrophosphate (DMPP) (35)
Carvacrol (36) Cadinene (37) Abietic acid (38) Ganoderic acid (39)
MonoterpenesTriterpenesDiterpenesSesquiterpenes
OH
OHO
H
H
OH
OH
O
OH
O
O
H
OH
COOHHO
Fig. 1.7: Biosynthesis of terpenes
8
The isoprene rule, developed by Ruzicka which played key role in structure determination
(Ruzicka, 1953). Terpenes classification is based on number of isoprene units linked
together in a head to tail fashion. They are classified as monoterpenes, sesquiterpenes,
diterpenes, triterpenes, tetraterpenes and polyterpenes. The prefix represents the number
of isoprene unit present in a molecule (Bhutani, 2009). The monoterpenes consist of two
isoprene units and has molecular formula C10H16 (Figs. 1.9-1.10).
Terpens are volatile natural products found in plants as essential oils and are widely used
in perfumery industries for example linalool (41), menthol, α-pinene etc.
The turpentine contains two monoterpenes such as β-pinene (42). Camphor was extracted
from camphor tree (Cinnanomum camphora). It is used to protect cloths from moths. At
present, β-pinene is used as a raw material for the commercial synthesis of camphor.
Some of the other examples of monoterpenes include such as myrcene, limonene,
citronellal etc.
Head
Tail
Fig. 1.8: Isoprene unit (40) showing head and tail
OH
Fig. 1.9: Linalool (41)
Fig. 1.10: β-Pinene (42)
9
The sesquiterpens are also generally obtained from the essential oils but from higher
boiling point fractions. It contains three isoprene units and has molecular formula C15H24
(Fig. 1.11). For example β-Caryophyllene (43).
H H
Fig. 1.11: β-Caryophyllene (43)
The diterpenes are composed of four isoprene units having general molecular formula
C20H32 e.g. abietic acid (38). The triterpenes contain six isoprene units having molecular
formula C30H48 e.g. squalene.
The tetraterpenes are composed of eight isoprene units having molecular formula C40H56.
Some of the biologically active compounds such as β-carotene (44) are common
examples of tetraterpenes (Fig. 1.12).
Fig. 1.12: β-Carotene (44)
Ascorbic acid is naturally occurring organic compound and is the chief water soluble
antioxidant present in human plasma. It efficiently scavenges superoxide and other
reactive oxygen species (Gahler et al., 2003), its interaction with glutathione regulates the
intracellular redox state (Winkler et al., 1994). Due to the electron donating ability of
ascorbic acid, it acts as reducing agent for many reactive oxygen speceis. At the cellular
membrane it reduces tocopherol radical back to their active form and protects compounds
in the water soluble portions of cells and tissues.
10
The antioxidant properties of phenolics are primarily because of their redox property.
Condensed tannins are the most plentiful polyphenols, found in almost all families of
plants. Polyphenols can also be found in animals (Ferrazzano et al., 2011).
Tocopherol found in plants is a class of organic compounds consisting of various
methylated phenols. Tocopherols are grouped into two different families such as
tocotrienol and tocopherols. On the basis of methyl group position attached to chromane,
ring the tocopherols and tocotrienol are classified into α, β, γ and δ-tocols or trienols.
Tocotrienol have unsaturated side chain while tocopherols have saturated side chain.
They are synthesized by the plants (Syvaoja et al., 1985).
The synthetic antioxidants like butylated hydroxyanisol, butylated hydroxytoluene and
propyl gallate are used as food antioxidants, but these antioxidants have adverse effects
on health causing degenerative diseases and cancer (Iqbal et al., 2007).
An antimicrobial is a material that inhibits the growth of microorganisms, such as fungi
and bacteria. The disc diffusion assays are mostly used to analyze the antibacterial
potential of plant extracts and natural substances. These methods of analysis were based
on the use of holes or discs as reservoirs containing the solutions of natural substances to
be analyzed (Brantner et al., 1994).
Plant extracts has been used conventionally to treat a number of diseases such as those
caused by fungi and bacteria (Gao and Zhang, 2010). The literature and reports have been
available of plant extracts against disease causing micro organisms (Natarajan et al.,
2003). Many plant extracts, herbs and spices have antimicrobial activities against food
spoilage and pathogens (Bagamboula et al., 2003).
The biological activity is an expression explaining the adverse or beneficial effects of a
drug on living things. Phytochemicals are nutritional constituents that usually occur in
small quantities in food. The phytoconstituents are mostly studied to evaluate their effects
on living beings health. Many studies have shown protective effects of plant based diets
on cancer and cardiovascular disease. Many bioactive compounds have been discovered
which vary in functions and chemical structures. Much scientific research needs to be
conducted for the identification and evaluation of naturally occurring bioactive
compounds in plants. There are many plants which showed potential to be used as natural
source in traditional medicine (Erdemgil et al., 2007).
11
Food rich in phytochemicals such as flavonoids and phenolics are related to reduce risk of
many types of cancer have lead to the revival of attention in plants based diets (Choi et
al., 2007). The use of foods unhygienic with some microbes shows a health hazard to
human beings. The majority of the antibiotics existing today come from plant orgin.
Plants produce phytochemicals to defend themselves from attacks of microorganisms
(Sarker et al., 2007). The polyphenols are the most extensive spread phytochemicals in
plants. These polyphenol compounds have gained a greater attention as natural
antioxidants in terms of their capacity to act as efficient radical quenchers. This might be
believed due to having some redox properties (Zheng and Wang, 2001). Source of natural
antioxidant are plants phenolics that might be in plant parts such as seed, fruit, vegetable,
leaves, nuts, roots and barks (Pratt and Hudson, 1990).
The antioxidants obtained from plants are of greater importance in contrast to synthetic
ones, due to their natural origin. Phytochemicals play important role in preventing the
spoilage of food (Iqbal et al., 2007).
The plant extracts with a pleasing aroma pooled with a preservative action, have
properties to evade lipid deterioration and spoilage by microorganisms (Farag et al.,
1990). The essential oils are ahead in rising interest because of their comparatively safe
position, broad importance by users and some other multi purpose uses (Ormancey et al.,
2001). In current years, the herbal extracts and essential oils paved the path for the
scientific research due to the reason that they are naturally occurring as well as
biologically active (Bozin et al., 2006). The terms oxidant and reductant are used in
chemical reaction, whereas pro-oxidant and antioxidant have the same meaning in the
situation of a biological system. The antioxidant might be explained as a matter which is
present in very less amount as compared to those of an oxidizable substrate, extensively
prevents or delays a pro-oxidant initiated oxidation of the substrate. The pro-oxidant is a
toxic material that can cause oxidative damage to proteins, nucleic acids and lipids,
causing various diseases in human beings. On the other hand pro-oxidant is a similar
word used for the reactive oxygen species (ROS). The pro-oxidant initiates the oxidative
damage to substrate which is inhibited in the presence of an antioxidant. Thus the
percentage inhibition is determined and related to antioxidant potential of antioxidant
substance (Prior and Cao, 1999).
12
The cytotoxicity is the nature of substances being toxic for cells. The stability of the red
blood cells (RBCs) membrane is a excellent in vitro sign of the cytotoxicity studies of
substances administered (Riss and Moravec, 2004). The cells can bring to an end growing
and dividing cells (Baillie et al., 2009), that’s why there is need of cytotoxicity studies
plant sources.
The Scrophulariaceae is a family of flowering plants. The plants of family
Scrophulariaceae are mostly herbs or sometimes small shrubs rarely trees. This family
also includes medicinal plants (Ahmad, 1980). The plants are annual herbs with flowers
having bilateral or rarely with radial symmetry (Olmstead et al., 2001).
The Leucophyllum frutescens (Silvery) is a rounded shrub. It is an evergreen plant which
is four to eight feet tall, simple, alternate and pubescent (soft and furry to touch). The
flowers in leaf axis, lavender to purple, sporadic blooming season throughout the year.
Flowers are small but cover entire plant during main flush. The stems are silvery and are
very attractive. The flowers and leaves can be brewed into a pleasant herbal tea which is
said to be good as a bedtime drink, mildly sedative and for treating flu and colds
(Anonymous, 2012a). In earlier times the leaves of Leucophyllum frutescens have been
used for antihepatotoxicity (Balderas-Renteria et al., 2007).
Verbascum thapsus commonly called Gider tambakoo is a biennial plant. It grows in
differnt habitats Verbascum species (Scrophulariaceae), which are widespread plants in
Anatolia, are used as expectorant and mucolytic in folk medicine (Tatli and Akdemir,
2004). Verbascum L. is the major genus of the family Scrophulariaceae with about 2500
species world wide (Grieve, 1995). Various preparations of some species of this genus
have been used as expectorant and mucolytic, sedative, diuretic and constipate in
traditional medicine (Baytop, 1999). Traditionally, in Europe, Asia and Northern
America, several species of Verbascum have been reported as antiseptic and inflammative
(Grieve, 1995).
The Anticharis linearis (Benth.) Hochst. ex Aschers is commonly called Dhunnya. This
plant belongs to the family Scrophulariaceae. The plant has terminal (leaf) node. It is a
low land annual herb. Traditionally poultice of the whole plant is used in treating
swellings. Traditionally the pant also has been used as poultice, diuretic, in treating
jaundice and joints affections (Safi, 2006).
13
The Mazus goodenifolius (Hornem.) Pennel belongs to family Scrophulariaceae.
Traditionally plants related to this genus were studied and found that these has been used
as an aperient, emmenagogue, febrifuge and tonic, the juice of the plant Mazus pumilus is
used in the treatment of typhoid (Anonymous, 2012b). The Mazus goodenifolius is
recently sometimes related to the newly defined Phrymaceae sometimes has also cast
doubt on the inclusion of mazus genera in Phrymaceae. So the the Mazus goodenifolius
(Hornem.) Pennel belongs to family Scrophulariaceae.
Antirrhinum majus L. (Snapdragon) is a plant belong to Scrophulariaceae family and
traditionally has been used as a tonic, an aperient, an emmenagogue and an antipyretic.
Traditionally the leaves and flowers of Antirrhinum majus L. have been claimed to be
antinflammatory and are used to treat haemorrhoids. The plant is also used in the
treatment of scurvy, ulcer, liver disorders and as an antitumor (Harborne, 1963). The
Russelia equisetiformis (Firecracker) is an evergreen, perennial shrub with and attractive
look. When their cultivation requirements are met, firecracker plants produce their
distinguishing blossoms. Russelia equisetiformis is easily propagated from rooted
cuttings. It grows up to four feet with red flowers, and much reduced leaves. Russelia
equisetiformisis traditionally used as a medicinal plant and considered to have analgesic,
anti-inflammatory and membrane stabilizing ability (Awe et al., 2009). Some times the
Russelia equisetiformis and Antirrhinum majus L. is recently related to Plantaginaceae.
However, new phylogenetic research has indicated that Plantaginaceae in the strict sense
were nested within Scrophulariaceae (Anonymous, 2013).
The family Scrophulariaceae is very less published from Pakistan, an attempt has been
made to evaluate selected species of this family for biological and phytochemical studies.
As per our knowledge no literature has been available about the antimicrobial, antioxidant
and cytotoxicity studies of the selected plants, which has been represented in present
work by using plant extracts, fractions and essential oils.
The objectives of present research are as follows:
The selected plants extracts, fractions and essential oils were initially screened by in vitro
biological studies i.e. antioxidant, antimicrobial and cytotoxicity assays. The cytotoxicity
of selected plants were evaluated by in vitro haemolytic activity against the human red
blood cells (RBCs).
14
The Russelia equisetiformis showing better biological activity was put forward for the
evaluation of antioxidant activity by protection to DNA damaging assay. The Liquid
Chromtography-Mass Spectrometry (LC-MS) was carried out to analyze the extract of
Russelia equisetiformis. The isolation of Russelia equisetiformis fractions were also
carried out. The characterization of isolated phytochemicals using advanced spectroscopic
techniques such as 1H-NMR,
13C-NMR, IR, ESI-MS was also made. The isolated
compounds obtained were used to evaluate their antioxidant potential using sunflower oil
as oxidative substrate.
15
2 CHAPTER 2 REVIEW OF LITERATURE
REVIEW OF LITERATURE
2.1 Medicinal Plants
The medicinal plants are one of the major sources of novel pharmaceuticals.
Phytochemicals aid to maintain excellent health and fighting against diseases.
Phytochemicals now being described as functional constituents and neutraceuticals. The
health beneficial effects of the plant extracts has been credited to antioxidant,
antimicrobial, anticancer, antiulcerative and antidiabetic properties. At present the plant
extracts are still widely being used in ethnomedicines in the world (Mahesh and Satish,
2008).
There are so many plants which have been used as herbal medicine like Tinospora
cordifolia (Guduchi). The notable medicinal properties reported were antioxidant,
antiallergic, antidiabetic, antiperiodic, antimalarial, antispasmodic, antiarthritic,
antiinflammatory, antistress, antileprotic, immunomodulatory, hepatoprotective and
antineoplastic activities (Upadhyay et al., 2010).
Herbal plants have been identified to posses various phytochemicals. Natural products
and their derivatives contribute more than half of all clinically administered drugs (Koehn
and Carter, 2005). The natural product possessed a important position in drug discovery
for the treatment of cancer and infectious diseases (Fabricant and Farnsworth, 2001). The
P. sarmentosum has been one of the plants which possessed antimcirobial potential
(Chanwitheesuk et al., 2005). Antifungal and antibacterial potentential have been studied
in the plant M. arvensis (Imai et al., 2001).
Mathew and Abraham (2008) evaluated that medicinal plants showed antioxidant
potential of the methanol extract of Cinnamomum verum barks with reference to
antioxidant compounds like ascorbic acid, butylated hydroxyl anisole (BHA) and trolox.
By good feature of their hydrogen donating capacity, all of the tested compounds and
Cinnamomum verum exhibited reducing potential. They were found to be good in free
radical scavenging action particularly against 1,1-Diphenyl-2-picrylhydrazyl radical
(DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
radical cations. The hydroxyl (OH) and superoxide radicals (O-2
) were also found to be
scavenged. Cinnamomum verum also showed metal chelating action. The peroxidation
16
inhibiting potential of Cinnamomum verum using a linoleic acid emulsion system,
exhibted potent antioxidant potential.
Abbasi et al. (2012) evaluated the antioxidant action of medicinaly important palnt
Sauromatum venosum factory area Kotli, Azad Jammu and Kashmir. The effects of local
ethnopharmacological Sauromatum venosum were analyzed. The antimicrobial potential
of crude extracts of the fruit were examined against microorganisms using the method of
well diffusion. The antioxidant activity was analyzed by DPPH radical scavanging
activity. The results showed significant overall antioxidant activity, thereby reinforcing
the traditional medical practice.
Sarma and co workers described that the biological studies such as antioxidant, antiviral,
antifungal, antibacterial and cytotoxic studies of flavonoids have generated attention in
evaluation of plants containing flavonoid (Sarma et al., 2010). Biological evaluation of
plant products were carried out by cytotoxicity assay, antioxidant assay, DNA repair
assay and antibacterial assay (Bibi et al., 2011).
The monoterpenes are extremely hydrophobic compounds present in plant essential oils.
They show a broad spectrum of biological properties of vast significance in many diverse
areas such as antioxidant, antimicrobial and anticancer (Ozbek et al., 2008).
2.2 Essential Oils
Essential oils are extracted by the steam distillation of plants. The essential oils may be
defined as odiferous bodies of an oily nature, obtained almost exclusively from plant parts
such as flowers, barks, leaves, roots, seeds and fruits (Skocibusic et al., 2006).
Chemically, the essential oils were very complex and extremely changeable combination
of compounds that belong to two groups: aromatic compounds and terpenoids. The name
terpene was derived from the word turpentine (Guenther, 1985).
The essential oil considered beneficial in folk medicine to treat respiratory disorders,
allergies, diabetes, impotence, anxiety, stomach cramps, constipation, infertility,
headaches and vomiting. Furthermore, scientific data have proven that the anti oxidative
power of the volatile oils in plants makes it a healthy stimulant of the immune system
(Al-Maskria et al., 2011).
17
Essential oils from medicinal plants have been used for microbial resistance development
in food items. Essential oils have terpene hydrocarbons as well as their oxygenated
derivatives such as acids, alcohols, ketones, aldehydes and esters (Hanif et al., 2010).
The basis of varying degree of antimicrobial activity of test micro organisms may be due
to the intrinsic tolerance of microorganisms and the nature and combinations of phyto
compounds present in the essential oil (Baratta et al., 1998).
The essential oils contain complex mixture of numerous components. The major or trace
compounds might give rise to antimicrobial activity. However, some authors think that
essential oil activity should be considered as a whole (Knobloch et al., 1989). The
chemical components of essential oil exert their toxic effects, against studied
microorganisms through the disruption of fungal or bacterial microorganisms (Hanif et
al., 2010).
The essential oils were used in conventional medicine for their antiseptic potential. The
essential oil has been found in plants as phytochemicals and mainly represented by fatty
acid derivatives, terpenoids, amino acid derivatives and benzenoids (Derwich et al.,
2010). The essential oils and their chemical coonstituents have been known to show
biological properties (Iscan et al., 2002), antifungal (Sokovic and Vukojevic, 2009),
antibacterial (Eteghad et al., 2009) and antioxidant activities (Seun-Ah et al., 2010).
Essential oils have been used in ancient Rome, Egypt, Greece and throughout the world
as food flavours, perfumes, pharmaceuticals and deodorants (Baris et al., 2006). The
essential oils have been utilised centuries ago typically showed interesting biological
properties. The essential oils also assist to improve digestion, inhance the flow of
digestive fluids, stomach cramping and abolish gas (Uphof, 1968). In the light of
importance of essential oils, the plant which showed the better activity was also studied
for its essential oils composition and biological studies.
2.3 Phytochemicals
Earlier reports showed that plants contain a huge diversity of substances called
phytochemicals that possessed biological activities (Chanwitheesuk et al., 2005)
The natural phytochemicals present in plants have a world wide attention towards the use
as a medicine in recent years (Wang and Jiao, 2000). Flavonoids is a huge group of
naturally occurring plant phenolic compounds as well as flavonones, flavones, flavonols
18
and chalcones. Isoflavones, also known as natural drugs, possessed numerous
pharmacological properties and biological activities. The findings of the different
phytochemical assays showed that the plant Argemone mexicana to be rich in different
biologically active constituents which could serve as potential source of medicins (Sarma
et al., 2010).
Ow and Stupans (2003) studied that which components of T. sinensis were active as
antioxidant. It was found that the phenolic compounds such as gallic ester, gallic acid and
its catechin derivatives have rising consideration presently due to some interesting new
results for their antioxidant studies. So that antioxidant studies of T. sinensis has been
carried out.
Edeoga et al. (2005) evaluated that alkaloids, tannins, saponins, steroids, terpenoid and
flavonoids found in 10 medicinal plants belong to various families were analyzed. The
medicinal plants Emilia coccinea, Richardia bransitensis, Cleome nutidosperma,
Euphorbia heterophylla, Sida acuta, Physalis angulata, Stachytarpheta cayennensis
Scopania dulcis, Spigelia anthelmia and Tridax procumbens were investigated.
Antioxidant potential of phenolic contents has been studied and it was found that radicals
include to quinones to form stable radicals, permitting few quinones to be inhibitors in
free radical chain chemical reactions (Weng and Gordon, 2000). Therefore it has been
found that hydroxyl naphthoquinone moiety with obvious antioxidant activities which
might also be accountable for some of their pharmaceutical properties, like antibacterial
effects (Gao et al., 2000).
In general, antioxidant activity of flavonoids (45) depends on the structure and
substitution pattern of hydroxyl groups (Fig. 2.1).
A
O
OR4'
R5'
R3'
R3
R4R5
R6
OHB1
2
3
4
C
56
7
89
10
Fig. 2.1: General Structure of flavonoid (45)
19
The relationship between the chemical structure of flavonoids and their radical
scavenging properties were analyzed by (Bors et al., 2003).
2.4 Antioxidant
The antioxidants are the phytochemicals that decreased or inhibited the oxidation and
have the ability to scavange free radicals and thus were supposed to protect against heart
disease, cancer, arteriosclerosis and several other diseases. The majority used synthetic
antioxidants in diets were butylated hydroxytoluene (BHT), butylated hydroxyanisole
(BHA). But the probable toxicity of the synthetic antioxidants has been a area under
discussion of research from a lot of years. This lead to the attention in getting antioxidants
from plant sources (Bandyopadhyay et al., 2007). Plants may produce a broad diversity of
phenolic compounds (Loliger, 1991). Many investigations have showed that best part of
antioxidant activity has been from compounds such as flavones, flavonoids, anthocyanin,
catechin, isoflavones and other phenolics (Kahkonen et al., 1999). The different
antioxidant tests used to analyze the antioxidant activity of plants extracts have various
reaction mechanisms. It was advisable that multiple assays be adopted when screening
plant extracts for antioxidant activities (Kong et al., 2010).
Pszczola (2001) evaluated that a various antioxidant blends or mixtures may offer even
higher activity than any lone antioxidant. Herbal fortified dairy foodstuffs therefore serve
up as a better foundation of antioxidant. It has been observed that antioxidant activity of
the dairy manufactured goods and herbs were not depleted through oxidation reduction
chemical reactions upon mixing and storage of the foodstuffs.
Slavin (2003) evaluated the epidemiological and animal studies showed that the regular
use of plant foods, such as vegetables, whole grains diseases namely cardiovascular
diseases and also cancer associated with oxidative damage (Slavin, 2003).
Amarowicz et al. (2003) studied the phenolic compounds in methanol, 80% of caryopses
and embryos of rye (Dankowskie zlote). In all extracts, the reducing power, scavenging
effect on DPPH radical and antioxidant activity in a β-carotene assay examined. The
highest TPC made from an extract of Caryopses amilo (7.93 mg/g extract). UV spectra of
all extracts were characterized by maxima originated from phenolic acids (320, 326 and
328 nm), and peak at shorter wavelengths (272 and 274 nm), attributed to other phenolic
compounds. All extracts showed a good antioxidant potential in a β-carotene linoleate
assay. This activity was similar to that reported before in leguminous seed extracts. The
20
activities of antioxidant extracts from caryopses of Dankowskie zlote and embryos of both
varieties were very similar, especially in the second part of the incubation period. The
extract embryos showed slightly weaker antioxidant activity.
Zhou and Yu (2004) studied that phytochemicals with antioxidant potential were
naturally found in food, are of great attention to the food industry and also to the
consumers because of their potential value, to prolong the life of food stuffs by protecting
them against oxidative decline and their possible useful effects on human and animal
health.
Souri et al. (2004) analyzed the antioxidant activity of methanol extracts of twenty six
frequently used vegetables in Iranian diet using linoleic acid oxidation method.
Antioxidant potential was also shown by some vegetable extracts including spirmint,
leek, savory, garden cress, radish leaf, chive (aerial part), dill and lettuce were compared
with those of quercetin and α-tocopherol.
Kulisic et al. (2006) investigated the antioxidant potential of water tea infusions made
from Origanum vulgare L., Thymus vulgaris L. and Thymus serpyllum L. Total
flavonoids, phenolic, catechin and anthocyanin contents were determined by
spectrophotometric assay. Origanum vulgare L. water infusion of tea showed highest
amount of total phenolics (12500 mg/L GAE) and flavonoids (9000 mg/L QAE).
Determination of polyphenolic compounds in aqueous tea infusions by High Performance
Liquid Chromatography assay exhibted a overriding presence of rosmarinic acid (in
mg/g) 123.11 in Origanum vulgare L., Thymus vulgaris L., 17.45 and 93.13 in the
Thymus serpyllum L.
Rocha-Guzman et al. (2007) analyzed the total phenolic contents (TPC) of 7 improved
common bean cultivars (Phaseolus vulgaris L.) namely, negro durango, egro altiplano
and negro sahuatoba. Methanol and acetone extracts from bean cotyledons were obtained
by successive extractions. Total phenolic contents (TPC) have been analysed using folin
ciocalteu method. They concluded that, in contrast, low correlation coefficients were
obtained for methanol extracts.
Jacob and Shenbagaraman (2011) studied the antioxidant potential of selected green leafy
vegetables. The focus of this rsearch was to determine the antioxidant potential of
selected green leafy vegetables such as Amaranthus tristis, Centella asiatica, Hibiscus
sabdariffa, Moringa oleifera, Sesbania grandiflora and Solanum trilobatum. The
21
antioxidant screening was done using aqueous and ethanol extracts by DPPH free radical
scavenging assay. This study showed strong antioxidant activity of Hibiscus sabdariffa,
Centella asiatica by using ethanolic extract and Moringa olifera by using aqueous
extract.
Sharma et al. (2011) evaluated the ethanolic and aqueous extracts from roots of Bauhinia
variegate for antioxidant activity. The antioxidant potential of both ethanolic and aqueous
extracts of roots of Bauhinia variegate were carried out by in lab experiment models such
as nitric oxide and superoxide assays. Both ethanolic and aqueous root extracts of
Bauhinia variegate produced significant free radical scavenging activity. The recent study
revealed that ethanolic and aqueous root extracts of Bauhinia variegate were a good
source of phytochemical with antioxidant potential. Phytoconstituents flavonoids, tannins,
steroids and saponins present in extracts might be responsible for antioxidant activity.
Reactive oxygen species and lipid peroxides were involved in numerous pathological
events, metabolic disorders, including cellular aging and inflammation (Victor et al.,
2006).
Lobo et al. (2010) investigated that extracts of various plants for evaluation of antioxidant
potential and it was found that some genus have been obsereved to show inhibitory effect
against ROS induced DNA damage. The plant extracts were rich in antioxidants such as
flavonoids, polyphenols and vitamin C. The compounds derived from plant and extracts
were economic with no or less toxicity.
2.5 Antimicrobial Activity
The plants have capability to synthesize phytochemicals, which provide as plant defence
mechanisms against microrganisms, herbivores and insects (Policegoudra et al., 2010).
Gupta described that plant origin antimicrobials substances were not linked with vast
pharmaceutical activity to cure so many diseases (Gupta, 2003). Medicinal plants
exhibted a rich source of antimicrobial substances. Although plant have been analysed for
antimicrobial properties yet so many plants have not been adequately evaluated (Mahesh
and Satish, 2008).
More study and screening of plants required because there has been an increase in the
incidence of new and re-emerging serious diseases (Singariya et al., 2012). The
antimicrobial potential of various extracts and fractions were analyzed by using disc
diffusion and MIC (Alzoreky and Nakahara, 2003). The plants extracts has been used for
22
the relief of coughs and as an expectorant and it possesses antibacterial, anti ulcer,
diuretic, antitumor, anti viral properties (Wang and Yu, 1998).
There were many microbial species but some of them caused serious diseases. The
phytochemicals such as flavonoid compounds showed antimicrobial potential. The
miroorganisms species were screened to evaluate the phytochemical of plant extracts.
(Smid and Gorris, 1999).
Bupesh et al. (2007) evaluated that antibacterial potenatial in the extracts of Mentha
piperita L. leaves against pathogenic bacteria like Serratia marcesens, Bacillus subtilis,
Pseudomonas aureus, Streptococcus aureus, and Pseudomonas aerogenosa. The water as
well as organic extracts of the leaves were exhibted to have potent antibacterial potential
against a range of pathogenic bacteria as revealed by agar well diffusion method.
2.6 Cytotoxicity
The cytotoxicity is used to determine the quality of substances being toxic to cells. The
stability of the red blood cells (RBCs) membrane was a good in vitro indicator for the
cytotoxicity studies of substances administered. When Treated cells with cytotoxic
chemical compounds can consequence in a diversity of cell fates. The cells may undergo
necrosis, in which they die rapidly as a result of cell lysis or lose membrane integrity
(Riss and Moravec, 2004).
The studies using plants extracts cytotoxic or cytoprotective potentials have been shown
from Bruguiera gymnorrhiza, Hygrophila auriculata (Uddin et al., 2009).
Aliyu et al. (2009) studied that Argemone mexicana (Papaveraceae) was an exotic weed
indigenous in South America but has widespread distribution in many tropical and sub
tropical countries including West Africa. Phytochemical screening and cytoxicity
evaluation was carried out on the leaves of this herb. Acute toxicity determination also
showed the plant to possess an LD50 value of 400 mg/kg body weight intraperitoneally in
mice with a weight of 18-25 g and averagely aged between 4-6 weeks. The findings of
different phytochemical assays showed that plant to be rich in biologically active
compounds which could serve as good source of drugs and in addition the plant was
slightly toxic.
23
The plants extracts, essential oils and volatile compounds may provide an significant
basis of new antimicrobial agents. However there was need to evaluate the cytoxicity of
the plants extracts and essential oils (Green et al., 2011).
2.7 Stabilization of Lipid Containing Foods by Plant Sources
The oxidative deterioration of food containing lipid was responsible for the flavours and
rancid odours during processing and storage, as a result declining the dietary quality and
safety of foods due to the formation of secondary products, potentially toxic cosntituents.
(Chanwitheesuk et al., 2005).
Lipids oxidation can also be bring about by other mechanisms such as photo-oxidation. In
present years confirmation has exhibted that UV induced oxidative damage occurs
through the formation of reactive oxygen species (Zhang et al., 1997). Plant based
antioxidants extracted from plant sources exhibited various degrees of efficiency when
used in various food uses like preservation of food (Yoo et al., 2008).
Suhaj (2008) investigated that antioxidant chemical constituents in food play significant
part as health defensive factors and stabilization of food. Antioxidants were also widely
used as additives in oils and fats and in food processing to prevent or delay foods
spoilage. The plant extracts were used for the stabilization of food. Plants spices and
herbs have been sources of many effective antioxidants. This review gave some
information about the most commonly used spices antioxidants, methods of their
preparation and their antioxidant properties. Recommended
Aslam et al. (2011) studied the antioxidant potential of the plant Carissa carandas roots
extracted with different polarity based solvents was assessed. The antioxidant activity was
evaluated by the measurement of total phenolic contents (TPC), total flavonoid contents
(TFC), reducing power, DPPH radical scavenging, IC50 and antioxidant activity in
linoleic acid system. The plant material contained the TPC (1.79-4.35 GAE mg/g of dry
extract), TFC (1.91-3.76 CE mg/g of dry extract), DPPH radical scavenging activity, IC50
(12.53-84.82%) and % inhibition of peroxidation in linoleic acid (41.0-89.21%) system
respectively. Furthermore the antioxidant effectiveness of extracts was assessed using
corn oil (CO) as the oxidation substrate. The oxidative alterations were evaluated by the
measurement of and peroxide values (PV), conjugated triene (CT), conjugated diene
(CD), free fatty acid (FFA) and p-anisidine value.
24
3 CHAPTER 3 MATERIALS AND METHODS
MATERIALS AND METHODS
The research work presented in this dissertation was conducted at Department of
Chemistry Governent College University, Faisalabad Pakistan.
3.1 Chemicals and Reagents Used
All the reagents and chemicals used such as linoleic acid (90%), gallic acid (99%),
catechin (99.9%), folin ciocalteu reagent, ascorbic acid, DPPH and homologous series of
C4-C28 n-alkanes were of analytical grade and purchased from Sigma Aldrich Chemical
Co. (St. Louis, MO, USA). The absolute methanol, sodium hydrogen phosphate buffer,
ammonium thiocyanate, ferrous chloride, butylated hydroxytoluene, potassium
ferricyanide, trichloroacetic acid, ferric chloride, absolute ethanol, hydrochloric acid,
sodium hydroxide, sodium nitrite, sodium carbonate, aluminum chloride, deionized water,
picric acid, n-hexane, chloroform, n-butanol, potassium iodide, iodine, ethyl acetate,
hydogen peroxide, tween-20, silica gel, aluminimum silca gel plates were purchased from
Merck Darmstadt, Germany. pBR322 DNA and 1 kb DNA ladder were purchased from
Fermentas Life Sciences Company Canada. All other reagents used were also of
analytical grade purchased from Merck Darmstadt, Germany.
3.2 Instruments Used
The instruments used for analysis during the study alongwith their company identification
detailed are as below.
1. GC-MS, GC 6850 network GC system equipped with a 7683B series auto injector
and 5973 inert mass selective detector (Agilent Technologies, Willmington, DE,
USA).
2. Gel documentation system, SYNGENE Model GENE GENIUS, Cambridge, United
Kingdom.
3. Gel electrophoresis cell, Bio-Rad model wide mini, Techview Singapore.
4. Grinder, Model CB 222, Cambridge United Kingdom.
5. Incubator, Sanyo, Germany.
6. IR spectrophotometer, JASCO-320-A, JASCO, Inc., Mary's Court, Easton, MD USA.
7. Laminar air flow, Dalton, Japan.
25
8. Magnetic stirrer, Gallen Kamp, England.
9. Microplate stirrer, New Jersey, USA.
10. NMR, AZ-300, Bruker spectrometers, Switzerland.
11. Orbitrary shaker, Gallen Kamp, England.
12. Oven, Memert GmbH, D-91126, Germany.
13. Rotary evaporator, Heidolph, model LABORATA 4000, Schwabach, Germany
14. Ultra low freezer, Sanyo, Germany.
15. UV detection lamp 254 and 365 nm, Fisher Scientific Ltd, Pandan Crescent, UE
Tech Park, Singapore.
16. UV/Visible spectrophotometer, Hitachi-UV-3200, Japan.
17. 96 micro titre well plate reader (spectrophotometer), BioTek, model µ-QuantTM
,
Winooski, VT, USA.
18. Autoclave , Omron, Japan.
19. Blender, Mamrelax, Fait Common, France.
20. Centrifuge, H-200 NR, Kokusan, Japan.
21. EI-MS, MAT 312 spectrometer, Scientific Instrument Services, Inc., Ringoes, NJ,
USA.
3.3 Preparation of Reagents
3.3.1 Sodium Carbonate Solution (10% W/V)
10 gram of sodium carbonate was dissolved in small quantity of distilled water in a 100
mL volumetric flask and volume was made up to the mark.
3.3.2 Ferrous Chloride Solution (20 mM)
The 0.254 g of ferrous chloride was dissolved in 100 mL of 3.5% HCl solution.
3.3.3 Sodium Nitrate Solution (5%)
5 gram of sodium nitrate was dissolved in some quantity of distilled water in 100 mL
volumetric flask. The volume was made up to the mark.
3.3.4 Aluminum Chloride (10%)
10 gram of aluminum chloride was dissolved in a 100 mL volumetric flask and volume
was made up to the mark with distilled water.
26
3.3.5 Sodium Phosphate Buffer
Sodium phosphate buffer (NaH2PO4/Na2HPO4) of pH = 7.0 was prepared by using
Henderson Hasselbalch equation:
pH = pKa + log [salt / acid]
3.3.6 Sodium Hydroxide (1 M)
4 gram of sodium hydroxide was dissolved in small quantity of distilled water in a 100
mL volumetric flask and volume was made up to the mark with distilled water.
3.3.7 Ethanol (75%)
75 mL of (99.9%) ethanol was taken in 100 mL volumetric flask and volume was made
up to the mark.
3.3.8 1,1 Diphenyl-2-picrylhydrazyl Radical (DPPH)
0.025 g of 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) was dissolved in small quantity
of absolute methanol in 1000 mL volumetric flask and volume was made up to the mark.
3.3.9 Wagner’s Reagent
Potassium iodide (2 mg) was dissolved in small quantity of distilled water and 3 mg of
iodine was added in 100 mL volumetric flask and volume was made up to the mark
distilled water.
3.3.10 Hager’s Reagent
1 gram picric acid was dissolved in in small quantity of distilled water in a 100 mL
volumetric flask and volume was made up to the mark.
3.3.11 Dragendroff Reagent
For the preparation of dragendroff reagent two stock solutions were prepared. Bismuth
nitrate (0.6 g) was dissolved in conc. hydrochloric acid (2 mL) and water (10 mL). The
second solution contained potassium iodide (6 g) in water (10 mL). The two solutions
were mixed together with concentrated hydrochloric acid (7 mL) and water (15 mL). The
mixture was diluted up to 400 mL with water (Harborne and Williams, 1992).
3.3.12 Mayer's Reagent
Mercuric chloride (1.36 g) was dissolved in 60 mL distilled water.
27
Potassium iodide (5 g) was dissolved in 20 mL distilled water.
The two solutions were mixed fully and volume was made up to 100 mL with distilled
water.
3.3.13 Aluminium Chloride Solution (1%)
One gram aluminium chloride was dissolved in small quantity of ethanol in a 100 mL
volumetric flask and volume was made up to the mark.
3.3.14 Sodium Thiosulphate Solution (0.01 N)
Sodium thiosulphate (2.48 g) were dissolved in a small quantity of distilled water in a
1000 mL volumetric flask and volume was made up to the mark. The solution was
standardized before use.
3.3.15 Starch Solution
Starch (1.0 g) was dissolved in distilled water to make a thin paste. Then this paste was
added in 100 mL of boiling water and thoroughly dissolved by continuously stirring for 1
minute.
3.3.16 Peroxide Value Solution
Peroxide Value solution was prepared by mixing chloroform and acetic acid (3:2 ratio
v/v).
3.3.17 Sodium Carbonate Solution (20%)
20 gram sodium carbonate dissolved in small quantity of distilled water in a 100 mL
volumetric flask and volume was made up to the mark.
3.3.18 Oxalic Acid (0.1 N)
6.3 g of oxalic acid in a small quantity of distilled water in a 1000 mL volumetric flask
and volume was made up to the mark.
3.3.19 Potassium Iodide Solution (15%)
Potassium iodide (15 g) was dissolved in small amount of distilled water in 100 mL
volumetric flask and volume was made up to the mark.
28
3.3.20 para-Anisidine Reagent
para-anisidine reagent was prepared by adding 2.5 g/L para-ansidine in glacial acetic
acid.
3.3.21 Sodium Thiosulfate (0.1 N)
Sodium thiosulfate (24.8 g) was dissolved in small quantity of distilled water in 1000 mL
volumetric flask and volume was made up to the mark.
3.3.22 Phenolphthalein Solution (1%)
Phenolphthalein (1 g) was dissolved in small quantity of ethanol in 100 mL volumetric
flask and volume was made up to the mark.
3.3.23 Ceric Sulphate Reagent
The ceric sulphate (0.1 g) and trichloroacetic acid (1 g) were dissolved in 4 mL distilled
water. The solution was boiled and conc. sulphuric acid was added drop wise till the
disappearance of turbidity.
3.3.24 Nutrient Agar
Nutrient agar (Oxoid) 28 g/L was dissolved in distilled water. Sterilized the medium by
autoclaving at 121ºC for 15 minutes.
3.3.25 Sabouraud Dextrose Agar
Sabouraud dextrose agar was dissolved in 3.3 g/L of distilled water in a 1 L conical flask,
stirred, boiled to dissolve and then autoclaved for 15 minutes at 121oC.
3.3.26 Sterilization of Media for Antimicrobial Studies
The conical flask containing media for antimicrobial studies was autoclaved. The lid of
autoclave was fixed, safety valve was adjusted to the required temperature (121oC) and
pressure (15 pound/sq. inch) then media was allowed to sterilize for 15 minutes. At the
end of this period, heater was turned off and autoclave was allowed to cool. Then, the
flask was removed.
3.3.27 Preparation of Petri Plates for Antimicrobial Activity
The agar media was added in glass petri plates 6 and 12 inches, approximately 15 mL and
25 mL respectively, cooled to 45-50oC was poured into pre sterilized 6 inches petri plates
29
respectively. The agar media formed a firm gel which on cooling gave a layer of 2-3 mm
thickness in each plate.
3.3.28 Laminar Air Flow Conditions
The aseptic conditions were maintained in laminar flow, which was cleaned with seventy
percent ethanol and irradiated with short wave UV light.
3.4 Standardization of Solutions
3.4.1 Standardization of Sodium Thiosulfate Solution (0.01 N)
Potassium dichromate (0.16 g) was taken in volumetric flask and dissolved it in 10 mL of
water. Added 1 mL of conc. hydrochloric acid and few drops of potassium iodide
solution. Mixed the solution and permitted to set for 5 min and then 100 mL of distilled
water was added. The contents of flask were titrated with 0.01 N sodium thiosulfate
(Na2S2O3) solutions shaking continuously until the yellow color faded. Then added 1-2
drops of starch as an indicator and continues the titration until the greenish blue color just
appeared.
3.4.2 Standardization of Sodium Hydroxide Solution (0.1 N)
10 mL of known concentration of oxalic acid was taken in conical flask with few drops of
phenolphthalein as an indicator. The contents of flask were titrated against sodium
hydroxide. Light pink color was noted as end point.
3.5 General Procedure
3.5.1 Collection of Plant Material
In present research work, the selected medicinal plants such as Leucophyllum frutescens,
Verbascum thapsus, Antirrhinum majus, Anticharis linearis, Mazus goodenifolius whole
plant and Russelia equisetiformis leaves, belonging to the family Scrophulariaceae were
collected from Pakistan (Table 3.1). The plants were further identified by the Taxonomist
Dr. Mansoor Hameed, Department of Botany, University of Agriculture, Faisalabad,
Pakistan. The voucher specimens (Table 3.1) were deposited in collection/herbarium
Department of Botany University of Agriculture, Faisalabad Pakistan.
30
3.5.2 Effect of Plant Sample Size on Extraction
To obtain maximum yield of extract the different mesh sizes of plant (25, 65 and 110 µm)
were optimized. The plants materials were washed with distilled water, shade dried and
ground. The 100 g of each was plant was soaked in absolute methanol at room
temperature for seven days. After soaking the plants extracts were filtered with filter
paper (Whatman No. 1) and extracting solevent (absolute methanol) was removed to
dryness using rotary evaporator (Heidolph, model LABORATA 4000, Schwabach,
Germany) at a temperature 45-50oC. The plant material was extracted thrice and extracts
were combined. The percentage yield of of absolute methanol extract (g/100 g of dry
plant) was calculated.
3.5.3 Preparation of Extracts and Varoius Organic Solvent Fractions
After the optimization of sample size it observed that 25 µm mesh size of plant provided
maximum yield. So the 25 µm mesh size of plants was used for further extraction. The
ground plant (5 Kg) 25 µm mesh size of each plant was soaked in absolute methanol at
room temperature. The extraction repeated thrice and extracts were combined. The
absolute methanol extract was further fractionated by using solvent extraction method
with different polarity based absolute solvents such as n-hexane, chloroform, ethyl acetate
and n-butanol (Fig. 3.1) as method described by (Tiwari et al., 2011; Zubair et al., 2011).
After fractionation, samples were concentrated to dryness using rotary evaporator at a
temperature 45-50oC. The samples were stored in a refrigerator at 4
oC, until used for
further analysis. The samples were stored at low temperature because storage at high
temperature may cause decomposition of phytochemicals and decline in biological
activity (Perez-Jimenez et al., 2008).
3.6 Isolation of Essential Oils
The dried and ground 500 g of selected plants were hydro-distilled for four hours using a
clevenger type apparatus as described by (Goren et al., 2003; Goze et al., 2009). The
percentage yield of essential oils was determined. The essential oils were collected and
dried over anhydrous sodium sulfate, filtered and stored at 4°C until analyzed.
31
Table 3.1: List of plants selected from the family Scrophulariaceae used in present research work
Names of selected plants Common name Abbreviation
used in thesis Place of collection
Voucher
specimen
number
Leucophyllum frutescens (Berland.) Silvery L.F. Mansehra Abbotabad Hills, Pakistan
4183
Russelia equisetiformis L. Fire cracker R.E. Botanical Garden University of
Agriculture Faisalabad, Pakistan 4203
Verbascum thapsus L. Gider tambakoo V.T. Mansehra Abbotabad Hills, Pakistan
4429
Antirrhinum majus L. Snapdragon A.M. Local areas from Faisalabad, Pakistan
4425
Anticharis linearis Hochst. ex
Aschers Dhunnya A.L.
Nara Desert Sindh, Pakistan
4204
Mazus goodenifolius (Hornem.)
Pennell Tiny Booty M.G.
Botanical Garden University of
Agriculture Faisalabad, Pakistan 4182
32
In absolute methanol exract added water for theseparatrion of dif ferent organic solvent layers
Added n-hexane
n-hexane fraction Residue
Added chloroform
Chloroform fraction Residue
Added ethyl acetate
Ethyl acetate f ractionResidue
Added n-butanol
n-butanol f raction Residue (water soluble)
Plant material
Extraction with absolute methanol
Fig. 3.1: Scheme for the extraction and fractionation of plant material
33
3.6.1 GC-MS Analysis of Essential Oils
The sample was analyzed using a GC 6850 network GC system equipped with a 7683B
series auto injector and 5973 inert mass selective detector (Agilent Technologies,
Willmington, DE, USA). Compounds were separated on an HP-5 MS capillary column with
a 5% phenyl polysiloxane stationary phase (30.0 m × 0.25 mm, film thickness 0.25 μm).
Oven temperature was programmed in a three step gradient: initial temp set at 45°C (held for
5 min), ramped till 150°C at 10°C/min, followed by a 5°C/min rise till 280°C and finally at
15°C/min to 325°C where it was held for 5 min. Helium gas flow rate was 1.1 mL/min
(pressure 60 KPa and linear velocity 38.2 cm/sec). Ions/fragments were monitored in
scanning mode through 40–550 m/z.
3.6.2 Identification of Compounds
The identification of the components was based on comparison of their retention index (RI),
relative to a standard alkane series (C9–C24). The compounds were further indentified and
authenticated using their MS data by comparison with those of the NIST 05 Mass Spectral
Library and published mass spectra (Adams, 2001). The quantitative data were obtained
electronically from the FID area percentage without the use of any correction factors.
3.7 Screening of Selected Plants
The initial screening of selected plants was made by using antioxidant and antimicrobial
assays.
3.8 Phytochemical Analysis
Phytochemical analysis was carried out by using the standard procedures to identify the
chemical constituents qualitatively in the plant using the method described by (Trease and
Evans, 1989; Edeoga et al., 2005) with some modifications.
3.8.1 Test for Alkaloids
The plant extracts and fractions (0.5 g) were boiled in a beaker containing 40 mL distilled
water and then 4 mL of conc. H2SO4 was added. The mixture was further boiled for 5
minutes before the mixture was filtered, cooled and added in four test tubes equally. These
parts were separately tested for alkaloids using Mayer’s Reagent, Hager’s reagent,
34
Dragendroff reagent and Wagener’s reagent. Any turbidity and precipitates formation with
these reagents confirmed the presence of alkaloids (Sofowora, 1993).
3.8.2 Test for Tannins
Each sample (1 g) was dissolved in distilled water (20 mL) for 5 min. the mixture was
filtered and then in 5 mL of filtrate solution, few drops of 1% FeCl3 solution was added,
occurrence of blue black or greenish coloration indicated the presence of tannins (Trease and
Evans, 1989).
3.8.3 Test for Steroids
Two mililiter of acetic anhydride were added to 0.5 g of sample with 2 mL sulphuric acid.
The colour changed from violet to blue or green in samples indicated the presence of steroids
(Edeoga et al., 2005).
3.8.4 Test for Saponins
Each sample (2 g) was boiled in 20 mL of distilled water in a water bath and filtered. 10 mL
of the filtrates was mixed with 5 mL of distilled water and shaken vigorously for a stable
persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then
formation of emulsion was observed (Sofowora, 1993; Edeoga et al., 2005).
3.8.5 Test for Terpenoids
Five mililiter of each sample was mixed in 2 mL of chloroform and concentrated H2SO4 (3
mL) was carefully added to form a layer. A reddish brown colouration at the inter face was
formed to show positive results for the presence of terpenoids.
3.8.6 Determination of Total Phenolic Contents (TPC)
Total phenolic contents (TPC) were determined using folin ciocalteau reagent (FCR) as
method described by (Chaovanalikit and Wrolstad, 2004) with some modification. This
method is based upon the reduction of folin ciocalteau reagent (FCR) by the phenolics
content present in the plant extracts or fractions. The 50 mg of the extracts and fractions were
dissolved with 0.5 mL folin ciocalteau phenol reagent and 7.5 mL of distilled water. The
mixture was vortexed and incubated in dark at room temperature for 10 minutes. After
incubation, added 1.5 mL of 20% Na2CO3 (w/v). The mixture was heated on water bath at
35
40°C for 20 minutes, cooled immediately in an ice bath and noted the absorbance at 755 nm.
The blank consisted of folin ciocalteau reagent (FCR), ethanol and sodium carbonate
solution. A linear dose response regression curve was generated in Microsoft Office Excel
2007 software using absorbance of gallic acid at different concentrations 20-200 µg/mL.
Amount of total phenolic contents were calculated using the gallic acid calibration curve. The
results of total phenolic contents were expressed as milligram of gallic acid equivalent
(GAE) per gram of dry extracts of plant. The experiments were performed in triplicate and
mean was taken.
3.8.7 Determination of Total Flavonoid Contents (TFC)
Spectrophotometric method described by (Dewanto et al., 2002) was used to determine the
total flavonoid contents with some modifications. The flavonoid contents were determined by
using catechin as a standard. The calibration curve using various concentrations of catechin
ranging from 10-200 µg/mL was used. The extract containing 10 mg/mL of dry matter was
dissolved in a flask then 5 mL of distilled water and 300 µL of 5% sodium nitrate was added.
After 5 minutes 300 µL of 10% aluminum chloride was added and after an other 5 minutes 2
mL of 1 M sodium hydroxide was added and mixed well. The absorbance was noted
immediately at 510 nm. The blank consists of all reagents except the extracts and fractions.
Total flavonoid contents were expressed as milligram catechin equivalent (CE) per g of dry
extracts and fractions.
3.9 Evaluation of Antioxidant Activity
Evaluation of antioxidant activity of extracts, various organic fraction and essential oils was
carred out using the following assays.
3.9.1 DPPH Free Radical Scavenging Assay
DPPH free radical scavenging assay of test samples was carried out using the method
described by (Iqbal et al., 2005; Rizwan et al., 2012) with some modification. The sample
solutions having concentration ranging from 0.1-1000 µg/mL made in absolute methanol
with dilution method. Then mixed the 0.025 mL sample solution, with 2.5 mL of DPPH at a
concentration of 0.025 g/L. After 30 minutes noted the absorbance at 517 nm. The DPPH
solution was highly sensitive to light so it was exposed to minimum possible light
36
(Parthasarathy et al., 2009). The IC50 values were calculated from the plot of concentration
against percentage scavenging. The percentage scavenging by DPPH assay was calculated
from the following equation. Three replicates were recorded for each sample.
Scavenging (%) = [(Ablank - Asample)/ Ablank] X100
Where Ablank was the absorbance of the control reaction containing all the reagents except the
test samples. Asample was the absorbance of the test samples.
3.9.2 Percent Inhibition of Linoleic Acid Oxidation
The antioxidant activities of extracts, fractions and essential oils were also carried out in
terms of measurement of percentage inhibition of per oxidation in the linoleic acid system
with some modifications as method described by (Iqbal et al., 2005; Rasool et al., 2011). 5
mg of each sample was added to 0.13 mL of linoleic acid, 10 mL of 0.2 M sodium phosphate
buffer (pH 7.0) and ethanol (10 mL). Then mixture was diluted with distilled water up to 25
mL. At 40ºC, the solution was incubated for 360 hours. The degree of ionization was
measured by peroxide value colorimetrically following the thiocyanate method of (Yen et al.,
2000). 0.2 mL of sample was added 10 mL of 75% ethanol, 0.2 mL of 30% aqueous solution
of ammonium thiocyanate and 0.2 mL of ferrous chloride solution (20 mM in 3.5% HCl)
were added and the mixture was stirred for 3 minutes. The absorption was noted at 500 nm.
A control was performed with linoleic acid but without extracts, fractions and essential oils.
Synthetic antioxidant butylated hydroxytoluene (BHT) at 200 ppm was used as a positive
control as World Health Organization (WHO) limits. In the sample, the maximum
peroxidation level was noted at 360 hours (15 days). The sample that contained no
antioxidant component was used as a blank. Percent inhibition of linoleic acid per oxidation
was determined by the following equation.
Inhibition (%) of linoleic oxidation = [100 - (Increase in absorbance of sample at 360 h /
Increase in absorbance of control at 360 h) X 100].
3.9.3 Reducing Power
The reducing power of the extracts, fractions and essential oils was determined according to
the procedure described by (Yen et al., 2000). The plants samples were prepared at
concentration of 2.5-10.0 mg/mL of respective solvent and were mixed with sodium
37
phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the
mixture was incubated at 50oC for 20 minutes. Then 5 mL of 10% trichloroacetic acid added,
centrifuged at 980 x g for 10 min at 5oC in refrigerated centrifuge. The upper layer of the
solution (5.0 mL) was diluted with 5.0 mL of distilled water. Then added 1 mL of 0.1% ferric
chloride and noted the absorbance at 700 nm. Increase in absorbance of the reaction mixture
on increasing concentration of samples indicated higher reducing power of samples analyzed
(Chen et al., 2007).
3.9.4 Bleachability of β-carotene in Linoleic Acid System
Antioxidant activity of essential oils was also analyzed by bleaching of β-carotene/linoleic
acid emulsion system as method described by (Kulisic et al., 2004). A stock solution of β-
carotene-linoleic acid mixture was prepared by dissolving 0.5 mg β-carotene, 25 µL linoleic
acid and 200 mg of tween 40 in 1.0 mL of chloroform (HPLC grade). In this assay Tween 40
was used as an emulsifier (Sacchetti et al., 2005). The chloroform was completely removed
under vacuum in rotary evaporator at 50oC. After this the 100 mililiter of distilled water
saturated with oxygen (30 min 100 mL/min) was added with higher shaking. The 250 µL of
this reaction mixture was added to test tubes containing 350 µL of the extract fractions and
essential oil prepared at 2 mg/mL concentrations and the absorbance at the zero time
measured at 490 nm against a blank, consisting of an emulsion without β-carotene. Then
emulsion was incubated for 50 hours at room temperature and the absorbance was recorded
at different time intervals. For the blank same procedure was applied.
3.10 Antimicrobial Activity
The selected bacterial stains such as Pasturella multocida, Escherichia coli, Bacillus subtilis
and Staphylococcus aureus were used to analyse the antimicrobial potential of plants
extracts, fractions and essential oils. The antimicrobial activity was also evaluated against
fungal strains like Aspergillus niger, Aspergillus flavus, Alternaria alternata and Rhizopus
solani by plant extracts, fractions and essential oils analyzed by disc diffusion method (CLSI,
2008).
The plants extracts, fractions and essential oils showed antimicrobial activity by disc
diffusion assay were put forwarded for minimum inhibitory concentration (MIC) as method
38
described by (NCCLS, 2002). The bacterial starins were cultured and maintained on nutrient
broth (Oxide) while the fungus strains were cultured and maintained on sabouraud dextrose
liquid medium (Oxide). The stock culture was maintained at 4ºC for further use. The
inoculum concetntation 1x108 colony forming unit (cfu/mL) of bacterial and fungal strains
1x107 spores/mL was used for the antimicrobial assay (Baker and Thornsberg, 1983).
For the disc diffusion assay nutrient agar (growth medium) was prepared, then autoclaved.
Agar media (15 mL) was transferred in 6 inches and 25 mL into the sterilized 12 inches per
petri dish. The wick paper sheet was used to prepare discs. The discs 6 mm size were
impregnated with the 100 µL tested samples. The ciprofloxacin and fungone were used as
standards for the bacterial and fungal strains respectively at the concentration of 1000
µg/mL. The respective solvent of extracts and fractions were used as negative control. The
discs were placed in the inoculated plates. After the incubation period, petri dishes were
incubated for 24 hours at 37ºC and for 48 hours at 28ºC against bacterial and fungal strains
respectively. The zones of inhibition around the discs were measured in millimeters (mm)
including the diameter of disc. For the minimum inhibitory concentration (MIC), different
concentrations of extracts, fractions and essential oils were made and added in 96 microtitre
wells separately. The dilution of samples for minimum inhibitory concentration (MIC) was
carried out for the test samples which showed better antimicrobial activity in the zone of
inhibition assay at less concentration and for the samples which showed less antimicrobial
activity their higher concentration was used. The minimum inhibitory concentration (MIC)
was the minimum amount of the extracts, fractions and essential oils in the broth medium
which inhibited the growth of tested microorganisms (NCCLS, 2002).
For the determination of MIC the 96 microtitre well plates were prepared under sterile
conditions. A volume of 50 μL of test material in 10% (v/v) DMSO or relevant solvent was
added into the each well. To each well 50 μL of nutrient broth was added. Different
concentrations of test samples were prepared using dilution formaula (C1V1=C2V2). In each
well 50 μL of test sample was added in descending order of concentration and tip was
discarded after single use. To each well 10 μL of resazurin indicator solution was added. The
resazurin solution was prepared by dissolving a 270 mg in 40 mL of sterile distilled water.
Finally, 10 μL of inoculum was added. Each 96 microtitre well plate with a set of controls; a
39
column with positive control, and a column with all solutions with the exception of the test
compound, also a column with all solutions with the exception of the bacterial solution
adding 10 μL of nutrient broth. The plates were prepared in triplicate and placed in an
incubator set at 37°C for 24 hours of bacterial and 28°C for 48 hours. The colour change was
observed and well number was noted. Any colour change from purple to pink or colourless
were recorded as positive. The lowest concentration at which colour change occurred was
taken as the MIC value (Sarker et al., 2007).
3.11 Cytotoxicity Studies by Haemolytic Activity
The cytotoxicity of the plants extracts, fractions and essential oils were analyzed by
haemolytic activity by the method described by (Powell et al., 2000; Sharma and Sharma,
2001; Aslam et al., 2011) with some modifications. The plant extracts at concentration of 1
mg/mL in 10% DMSO were prepared. Three mL of freshly obtained human blood was added
in heparinized tubes to avoid coagulation and smoothly mixed, poured into a sterile 15 mL
falcon tube and centrifuged for 5 min at 850 x g. The supernatant was poured off and RBCs
were washed three times with 5 mL of chilled (4°C) sterile isotonic phosphate buffer saline
(PBS) solution, adjusted to pH 7.4. The washed RBCs were suspended in the 20 mL chilled
PBS. Erythrocytes were counted on heamacytometer. The RBCs count was maintained to
7.068 x 108 cell per mL for each assay. The 20 µL of plant extract and fractions were taken in
2 mL eppendorfs, then added 180 µL diluted blood cell suspension. The samples were
incubated for 35 minutes at 37°C. Agitated it after 10 minutes, after incubation, then tubes
placed on ice for 5 minutes and centrifuged for 5 minutes at 1310 x g. After centrifugation
100 µL supernatant was taken from the tubes and diluted with 900 µL chilled PBS. All
eppendorfs were maintained on ice after dilution. After this 200 µL mixture from each
eppendorfs was added into 96 well plates. For each assay, 0.1% triton X-100 was taken as a
positive control and phosphate buffer saline (PBS) was taken for each assay as a negative
control. The absorbance was noted at 576 nm with a BioTek, μ CuantTM
instrument (BioTek,
Winooski, VT, USA). The % lysis of RBCs calculated by the following formula
% lysis of RBCs = (Absorbance of sample /Absorbance of triton X-100) ×100
40
3.12 Selection of Plant Which Showed Better Activity
Based on screening studies it was concluded that the Russelia equisetiformis showed better
antioxidant and antimicrobial studies. Therefore the Russelia equisetiformis selected for
further studies.
3.13 Antioxidant Activity by DNA Protection Assay
The antioxidant activity by DNA protection assay was carried out for the plant showing
better activity during initial screening so the Russelia equisetiformis was used for this assay
by the method described by (Kalpana et al., 2009) with some modifications. For this assay
pBR 322 DNA 0.5 µg/µL was diluted upto two folds (0.5 µg/3 µL using 50 mM sodium
phosphate buffer pH 7.4). The 3 µL of diluted pBR 322 DNA was treated with 5 µL test
sample. After this, 4 µL of 30% H2O2 was added in the presence and absence of different
concentrations of test samples. The volume was made upto 15 µL with sodium phosphate
buffer (pH 7.4). Initialy to optimize dose, different concentrations of absolute methanol
extract were used such as 10, 100 and 1000 µg/mL in the reaction mixture. The 1000 µg/mL
showed better protection to DNA damaging, it was proceeded for the organic fractions and
essential oil of Russelia equisetiformis to analysed for DNA protection assay. The relative
difference in migration between the native and oxidized DNA was then examined on 1%
agarose by horizontal DNA gel electrophoresis using a Bio-Rad wide mini system
(Techview, Singapore). The gels were documented by a Syngene model Gene Genius unit
(Syngene, Cambridge, UK).
3.14 Extraction of Phenolic Compounds from Russelia equisetiformis for
LC-MS Analysis
Phenolic compounds in Russelia equisetiformis leaves were extracted according to the
method reported by (Adom and Liu, 2002) with minor modifications. 1.0 g of whole plant
was mixed with 20 mL of absolute methanol for 10 min. After centrifugation at 2500 × g for
10 min, the supernatant was removed. The extraction was repeated thrice. Supernatants were
combined, evaporated at 45◦C to dryness using rotary evaporator. The extract was stored at
4◦C until use.
41
3.14.1 Analysis by Liquid Chromatography Mass Spectrometry (LC-MS)
The presence of phenolics in Russelia equisetiformis were analyzed by Liquid
Chromatography combined with Electon Spray Ionization Mass Spectrometry (LC-ESI-MS).
The plant extract was filtered through a 0.45 µm syringe filter before analysis. Separation of
phenolic compounds was performed on Surveyor Plus HPLC System equipped with
Surveyor auto (Thermo Scientific, San Jose, CA, USA). The pump equipped with a Luna RP
C-18 analytical column (4.6×150 mm, 3.0 µm particle size) (Phenomenex, USA). Solvent
elution consisted of LCMS grade methanol and acidified water (0.5% formic acid v/v) as the
mobile phases A and B, respectively. Solvent elution consisted of a gradient system run at a
flow rate of 0.3 mL/min. The gradient elution was programmed as follows: from 10% to 30%
A in 5 min; from 10% to 50% A in 20 min; and maintained this till the end of analysis. A 20
min re-equilibration time was used after each analysis. The column was maintained at 25°C
and the injection volume was 5.0 µL. The effluent from the HPLC column was directed to
the electron spray ionization mass spectrometer (LTQ XLTM
linear ion trap Thermo Scientific
River Oaks Parkway, USA). The mass spectrometer was equipped with an ESI ionization
source. Parameters for analysis were set using negative ion mode with spectra acquired over
a mass range from m/z 260 to 800. The optimum values of the ESI-MS parameters were:
spray voltage, +4.0 kV; sheath gas and auxiliary gas were 45 and 5 units/min. respectively;
capillary temperature 320◦C; capillary voltage, -20.0 V and tube lens -66.51 V. The accurate
mass spectra data of the molecular ions was processed through the Xcalibur Software
(Thermo Fisher Scientific Inc, Waltham, MA, USA).
3.15 Isolation of Phytochemicals
The Russelia equisetiformis showed better antioxidant and antimicrobial activity so it was put
forwarded for the isolation of phytochemical constituents. The ethyl acetate soluble fraction
(305 g) was subjected to flash column chromatography using silca gel (230-400 mesh size)
with increasing polarity of solvent (ethyl acetate:n-hexane). 105 fractions were obtained. The
thin layer chromatography (TLC) was applied to check the purity of compounds. The
fractions having spots on TLC plate at same Rf were combined to give three fraction (1 to 3).
The fraction 1 (3 g) was rechromatographed on flash column chromatography using silca gel
(230-400 mesh size) with ethyl acetate:n-hexane (15:85) and compound (76) 85 mg purified.
42
The fraction 2 (4.2 g) was rechromatographed on flash column chromatography using silca
gel (230-400 mesh size) with ethyl acetate:n-hexane (35:65) and compound (77) 72 mg was
purified.
The fraction 3 (5.6 g) was rechromatographed on flash column chromatography using silca
gel (230-400 mesh size) with ethyl acetate:n-hexane (45:55) and compound (78) 82 mg
purified.
Further n-butanol soluble fraction (156 g) was subjected to flash column chromatography
using silica gel (230-400 mesh size) with increasing polarity (methanol:chloroform as
solvent). The 46 fractions were obtained. The thin layer chromatography was applied to
check the purity of compounds. The fractions having spots on TLC plate at same Rf
(fractions 15-29) were combined to give one major fraction. The combined fraction was
rechromatographed on flash column chromatography using silica gel (230-400 mesh size)
with methanol:chloroform (32:78) and compound (79) (78 mg) purified.
The chloroform soluble fraction (198 g) of Russelia equisetiformis was subjected to flash
column chromatography using silca gel (230-400 mesh size) with increasing polarity
(methanol:chloroform as solvent). The 59 fractions were obtained. The thin layer
chromatographic (TLC) was applied to check the purity of compounds. The fractions having
sopts on TLC plate at same Rf (fractions 11-22) were combined to give one major fraction.
The fraction obtained was compound (80) (72 mg).
Thin layer chromatography (TLC) method was run on pre coated silica gel G-25- UV254
plates to check the purity of compounds. The spots on TLC plates were checked with UV
detection lamp at 254 nm and 365 nm before and after spraying ceric sulphate reagent.
The five compounds were finalized for NMR, IR, UV and ESI-MS analyasis. The 1H-NMR
analysis in CD3OD at 300 MHz on Bruker NMR spectrometer, where as 13
C-NMR
experiments were obtained on the same instrument at 75 MHz. Tetramethylsilane was used
as an internal standard. Electron ionization mass spectra (ESI-MS) of samples were recorded
with MAT 312 spectrometer. IR spectra were recorded on IR spectrophotometer, JASCO-
320-A, JASCO, Inc., Mary's Court, Easton, MD USA. Samples were prepared for IR
43
spectroscopy by incorporating the crystals into a KBr disc. The melting point was determined
by using Stuat SMP3 (SKS Science Products, West Watervliet, NY, USA).
3.16 Antioxidant Activity by Stabilization of Sunflower Oil
The Russelia equisetiformis samples showed better activity in initial screening so put forward
for the isolation of phytochemicals by column chromatography. The isolated compounds
were studied for antioxidant activity by stabilization of sunflower oil. The isolated
compounds from Russelia equisetiformis were separately added at a concentration of 300
ppm to prepare sunflower oil (100 mL) heated at 50oC for homogeneous distribution. The
sunflower oil without addition of test samples was used as a control for comparison of
antioxidant activity of isolated compounds. The treated and untreated sunflower oil samples
(100 mL), were transferred to brown glass bottles, stored in an electric oven at 60°C and were
analyzed after the interval of 10 days upto 70 days. The variation in oxidation of sunflower
oil was determined by the determination of free fatty acid (FFA), peroxide value (PV),
conjugated trienes (CT), conjugated dienes (CD) and para-anisidine values (PAV). The PV
and FFA of the stabilized and control sunflower oil samples were measured following the
American Oil Chemists Society (AOCS) official method Cd 8-53 and F 9a-44, respectively
(Fireston, 1997). The conjugated dienes and trienes were analyzed by following the IUPAC
Method II.D.23. The absorbance was noted for conjugated dienes (CD) and conjugated
trienes (CT) at 232 nm and 268 nm, respectively (Fireston, 1997). para-Anisidine value was
determined following the IUPAC method II.D.26 (IUPAC, 1987).
3.17 Statistical Analysis
All the experiments in present research work were conducted in thrice unless stated
otherwise and statistical analysis of the data was performed by analysis of variance, using
CoStat version 6.3 statistical software (CoHort Software, Monterey, CA, U.S.A.). For the
evaluation of probability value of difference P ≤ 0.05 was considered to denote a statistically
significance. The significant differences (P ≤ 0.05) between means were identified using
Least Significant Difference (LSD) procedures. The other calculations like percentage
inhibition, standard deviations were carried out using Microsoft Office Excel 2007. The
regression analysis also was carried out by Microsoft Office Excel 2007 to calculate the dose
44
respose effect for the determination of total phenolics and flavonoids contents in plants
extracts and fractions. All data were presented as average values ± standard deviation (S.D.)
(Zar, 1996; Steel et al., 1997).
45
4 CHAPTER 4 RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
The plants are being used throughout the history in conventional medicines. At present,
about two-thirds to three-quarters of the world population is dependent on plant based
medicins for the treatment of many diseases. Therefore there is an increasing interest to
study the phytochemical and biological properties of plants. Phytochemical contituents
found in plants have prominent pharmacological properties. In the present research work
biological and phytochemical studies of selected medicinal plants: Russelia
equisetiformis, Antirrhinum majus, Leucophyllum frutescens, Mazus goodenifolius,
Anticharis linearis and Verbascum thapsus belonging to the family Scrophulariaceae,
were carried out.
4.1 Effect of Sample Size on Extration of Plants
For the extraction of plants material, different sizes of sample, such as 25 µm, 65 µm and
110 µm, were screened. It was observed that the plant sample size (25 µm) provided
maximum percentage yield using absolute methanol as shown in Table 4.1. The
preparation of sample affects the extraction of phytochemicals greatly, therefore plants
materials were shade dried because by drying at high temperature causes a decline in the
biological properties and antioxidant activity due to the decomposition of phytochemicals
(Bravo et al., 1994). Larrauri and coworkers reported that drying the plant material above
60oC showed less antioxidant activity than when dried at lower temperature (Larrauri et
al., 1997). The decrease in particle size of plant material improved solvent penetration
(Mukhopadhyay et al., 2006). The plant material was grinded to get maximum percentage
yield. This was most probably because decrease in particle size breaks certain structures
releasing bound antioxidants and decreasing the distance for the solvent to reach the inner
surface easily. The percentage yield of plants extracted with absolute methanol was found
to be in the order: Russelia equisetiformis (23.5%) > Leucophyllum frutescens (19.25%)
> Mazus goodenifolius (17.21%) > Antirrhinum majus (16.20%) > Anticharis linearis
(14.89%) > Verbascum thapsus (9.40%) of plant’s dry weight (w/w). The phytochemicals
have been mostly extracted in methanol or mixture of water with methanol (Dalla-Valle
et al., 2007).
46
Table 4.1: Percentage yield of different sample sizes by selected plants using absolute methanol solvent
Sample size
(µm)
Leucophyllum
frutescens
(whole plant)
Mazus goodenifolius
(whole plant)
Antirrhinum majus
(whole plant)
Anticharis linearis
(whole plant)
Russelia equisetiformis
(leaves)
Verbascum thapsus
(whole plant)
25 19.25±0.21 17.21±0.12 16.20±0.15 14.89±0.13 23.50±0.21 9.40±0.08
65 17.22±0.16 15.02±0.10 14.22±0.11 12.04±0.09 20.32±0.18 7.45±0.06
110 15.42±0.12 11.16±0.09 12.42±0.13 10.36±0.07 18.23±0.14 6.01±0.04
The values were the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
47
So in the present research work for efficient extraction of phytochemicals from plant material
were extracted with absolute methanol as a solvent and further fractionated in different
polarity based solvents such as n-hexane, n-butanol, ethyl acetate and chloroform.
4.2 Percentage Yield of Plants Extracts and Fractions
The percentage yield (w/w) of plants extracts and fractions was found in the range of 0.56-
23.50 g/100g of dry plant. The maximum percentage yield was observed for absolute
methanol extract of Russelia equisetiformis while the minimum for n-hexane fraction of
Verbascum thapsus. The percentage yields of extracts and various organic fractions are
shown in Fig. 4.1. The methanol is a good solvent for the extraction of phytochemical
constituents from plant sources (Riaz et al., 2012c; Rizwan et al., 2012). Zhao and coworkers
also evaluated that solvent used also affected the extraction of plant sources (Zhao et al.,
2006).
4.3 Phytochemical Analysis
4.3.1 Qualitiative Analysis
The qualitative phytochemical analysis was used to determine the occurrence of chemical
constituents such as tannins, saponins, steroids, alkaloids and terpenoids. The findings are
shown in (Table 4.2). The analyzed phytochemicals constituents found to be present and
however some were also absent in certain plants extracts and fractions. It might be concluded
that the presence and absence of phytochemicals constituents was affected by the type of the
solvent used. It has been reported that plants extracts and various organic fractions showed
antioxidant and antimicrobial properties due to the presence of tannins, alkaloids, steroids,
saponins and terpenoids (Riaz et al., 2012a). Sofowora has also reported phytochemical
constituents of plants possess biological activities (Sofowora, 1993). The alkaloids prensent
in plants show biological properties such as antioxidant activity (Erol et al., 2010).
The phytochemicals are produced in many plant species and shows a wide range of
biological activities. The saponins have also been shown to exhibit antibacterial, antifungal
and antioxidant activities as a result it has been suggested that they to constitute part of some
plant defense systems.
48
Fig. 4.1: Percentage yield of selected plants extracts and various organic fractions
0
5
10
15
20
25
Absolute
methanol
n-Hexane Chloroform Ethyl
acetate
n-Butanol
% Y
ield
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
49
The saponins are common in plants used as herbs. The saponins are a class of
phytochemicals present in plant sources. The saponins present in abundance in different plant
species (Hostettmann and Marston, 1995). The saponins show biological properties like
antimicrobial activity (Kerem et al., 2005). The steroids are very important phytochemicals
found in plants, which also regulate the plants metabolism and show biological properties
like antioxidant (Mooradian, 1993). The terpenoids are found in palnts. The terpenoids have
great importance in traditional herbal remedies such as antimicrobial and so many
pharmaceutical functions (Michael, 2009).
4.3.2 Determination of Total Phenolic Contents (TPC)
The total phenolic contents were analyzed in plants extracts and various organic fractions.
The highest TPC were observed in absolute methanol extract of Russelia equisetiformis
(16.91 mg/g) and lowest TPC in n-hexane fraction of Verbascum thapsus (1.82 mg/g) gallic
acid equivalent (GAE) of dry plant material (Fig. 4.2).
The absolute methanol was found easier to penetrate the cellular membrane to extract the
intracellular ingredients like phenolics and flavonoids from the plant material (Sultana et al.,
2009). The absolute methanol has been reported as useful solvent to extract the phenolics and
flavonoids components (Siddhuraju and Becker, 2003) supporting the present findings. As
per earlier reports the role of phenolics compounds as a scavenger of free radicals have been
found (Komali et al., 1999). Significant differences (P ≤ 0.05) were found in the total
phenolic contents of the tested plants. The phenolics are vital component of plants. The
phenolics have been reported to eliminate the free radicals due to the presence of hydroxyl
group (Hatano et al., 1989). The phenolics compounds take part directly as an antioxidant in
the body (Duh et al., 1999). The phenolic compounds have an important role for the
stabilization of lipid and fat oxidation. The polyphenols also have been associated with
antioxidant activity (Cakir et al., 2003). Tanaka and coworkers described that phenolic
compounds might have the inhibitory effects on carcinogenesis and mutagenesis in human
beings when taken from a food such as vegetables and fruits (Tanaka et al., 1998). Further
the findings revealed that the organic solvents appreciably affected the phenolic contents of
the plants extratcs and fractions which agrees with the results from other plant sources
(Kahkonen et al., 2001).
50
Table 4.2: Qualitative phytochemical analysis of selected plants extracts and various organic
fractions
Plants Name of
phytochemical
Absolute
methanol
n-
Hexane
Chloroform Ethyl
acetate
n-
Butanol
R. equisetiformis
Alkaloids + + + + +
Steroids + + - + -
Tannins + - + + +
Terpenoids + + + + -
Saponins + - - + +
M. goodenifolius
Alkaloids + - + + +
Steroids + + - + -
Tannins + - + + +
Terpenoids + + + + +
Saponins + - - + -
A. linearis
Alkaloids + - + + +
Steroids + - + - -
Tannins + - - + +
Terpenoids + + + - -
Saponins + - - + +
L. frutescenes
Alkaloids + - + + +
Steroids + + - + -
Tannins + - + + +
Terpenoids + + + - -
Saponins + - - + +
V. thapsus
Alkaloids + - + + +
Steroids + + - + -
Tannins + - + + +
Terpenoids + + + - -
Saponins + - - + +
A. majus
Alkaloids + - + + +
Steroids + + - + -
Tannins + - + + +
Terpenoids + + + - -
Saponins + - - + +
Key: + = Presence; - = Absence of phytochemicals
51
Fig. 4.2: Total phenolic contents in selected plants extracts and various organic fractions
0
2
4
6
8
10
12
14
16
18
20
Absolute
methanol
n-Hexane Chloroform Ethyl
acetate
n-Butanol
TP
C (m
g/g
) G
AE
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
52
The antioxidant potential of plant extracts were often explained by the existance of phenolics
and flavonoid contents (Sheikh et al., 2011).
4.3.3 Determination of Total Flavonoid Contents (TFC)
The total flavonoid contents in plants extracts and various organic fractions were evaluated
and are shown in Fig. 4.3. The total flavonoid contents were determined in selected plants
extracts and various organic fractions as catechin equivalent. Significant differences (P ≤
0.05) were found in the total flavonoid contents of the selected medicinal plants (Fig. 4.3).
The absolute methanol extract of Russelia equisetiformis has the highest amount of TFC
compared to other fractions (Riaz et al., 2012a). The minimum total flavonoid contents were
found in Verbascum thapsus n-hexane fraction. Thus the properties of the extracting solvents
affected the antioxidant potential of the plants extracts and fractions (Sun et al., 2007).
Like phenolic contents there was also a contribution of flavonoid contents towards
antioxidant potential (Turkoglu et al., 2007). Flavonoids are plant secondary metabolites
(Prasain et al., 2004). This effect might be explained that the extracts and fractions have
phytochemicals which showed the antioxidant activity (Cakir et al., 2003).
4.3.4 Percentage Yields of Essential Oils
The percentage yields of selected plants essential oils were found to be in the range of 0.21 to
0.46%. The highest percentage yield was observed for Leucophyllum frutescens and
minimum was observed for Verbascum thapsus essential oil. The percentage yields of
selected medicinal plants were found to be in the order: Leucophyllum frutescens (0.46%) >
Russelia equisetiformis (0.42%) > Mazus goodenifolius (0.32%) > Anticharis linearis
(0.37%) > Verbascum thapsus (0.21%) (w/w of dry plant).
4.3.5 GC-MS Assay of Essential Oils
The chemical constituents identified by GC-MS technique of selected plants essential oils are
presented in Tables 4.3-4.8. The chromatograms of selected medicinal plants are shown in
Figs. 4.4-4.9. The major chemical constituents present in the Anticharis linearis essential oil:
β-ocimene (16.53%), α-guaiene (7.35%), hexadecanoic acid, methyl ester (7.24%), 1,8-
cineole (7.18%), β-phellandrene (6.17%), limonene (4.75%), carvacrol (4.22%).
53
Fig. 4.3: Total flavonoid contents in selected plants extracts and various organic fractions
0
2
4
6
8
10
12
14
16
Absolute
methanol
n-Hexane Chloroform Ethyl
acetate
n-Butanol
TF
C (
mg/g
) C
E
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
54
The major compounds determined in the Leucophyllum frutescens essential oil: germacrene
D (20.68%), octadecanoic acid methyl ester (15.21%), carvacrol (13.23%), β-caryophyllene
(6.87%), spathulenol (3.98%), α-muurolol (3.92%), valencene (2.81%), linalool (2.79%). The
major compounds determined in the Antirrhinum majus essential oil: germacrene D
(10.86%), stearic acid (6.61%), methyl tetradecanoate (6.26%), α-thugene (6.07%), α-
muurolol (5.61%), 2-undecanone (5.29%), octadecanoic acid methyl ester (5.28%), methyl
eugenol (5.19%). The major compounds determined in the Verbascum thapsus essential oil:
α-thugene (11.33%), camphene (8.26%), β-caryophyllene (7.87%), α-muurolol (5.19%),
valencene (4.96%), p-cymene (4.84%), germacrene D (4.55%). The major compounds
determined in the Russelia equisetiformis essential oil: hexadecanoic acid methyl ester
(11.04%), 11-methyltetracosane (8.44%), n-docosane (7.66%), α-pinene (7.26%), stearic acid
(7.02%), octadecanoic acid methyl ester (6.37%), ecosanoic acid methyl ester (6.16%),
octadecanoic acid ethyl ester (5.69%), geranic acid (5.60%), methyl tetradecanoate (5.27%),
α-pinene (4.60%). The major constituents in Mazus goodenifolius essential oil were: thymol
(15.16%), carveol (10.06%), linalool (9.61%), germacrene D (9.37%), 1,8-cineole (8.41%),
β-pinene (6.43%) and γ-terpinene (5.46%), respectively. Some other compounds found in
different concentrations were: β-ocimene (3.82%), carvacrol (2.73%), verbenone (2.67%), α-
fenchone (2.62%), limonene (2.13%), α-pinene (1.79%) and p-cymene (1.53%). The
structures of some major compounds are shown in Fig. 4.10. From the results it was observed
that antioxidant and antimicrobial activity of the Russelia equisetiformis essential oil was
greater than that of other medicinal plants used in present research work. Some studies have
shown that essential oils possess greater antioxidant activity due to the synergistic or additive
and some times antagonism effects of their components (Miraliakbari and Shahidi, 2008).
4.4 Biological Studies
For initial screening, the biological studies such as antioxidant potential and antimicrobial
activity of selected medicinal plants were carried out.
55
Table 4.3: Chemical compounds in essential oil of Anticharis linearis analysed by GC-MS
Retention Index (RI) Chemical compounds Area percentage (%)
933 α-thujene 1.19
940 α-pinene 1.83
951 α-fenchone 2.74
953 camphene 1.95
970 β-pinene 2.28
979 β-myrcene 4.99
1025 p-cymene 5.5
1028 limonene 4.75
1030 1,8-cineole 7.18
1033 β-phellandrene 6.17
1049 β-ocirmene 16.53
1058 γ-terpinene 0.91
1098 linalool 1.08
1138 carveol 1.5
1204 verbenone 1.03
1291 thymol 0.78
1293 2-undecanone 0.69
1297 carvacrol 4.22
1361 eugenol 1.10
1365 geranic acid 0.63
1392 β-elemene 0.69
1411 methyl eugenol 0.68
1411 α-copaene 1.28
1417 β-caryophyllene 0.78
1439 α-guaiene 7.35
1451 α-humulene 1.40
1483 germacrene D 0.53
1493 valencene 1.08
1513 γ-cadinene 0.62
1577 spathulenol 1.2
1647 α-muurolol 0.68
1654 α-cadinol 0.48
1719 methyl tetradecanoate 0.59
1962 hexadecanoic acid methyl ester 7.24
2000 eicosane t*
2124 octadecanoic acid methyl ester 0.67
2173 3-methylheneicosane 2.41
2194 octadecanoic acid ethyl ester 0.60
2288 n-docosane t
2527 docosanoic acid methyl ester 1.56
2934 hexacosanoic acid methyl ester 1.48
Mode of identification= Retention Index (RI) and comparison of mass spectra,*t= traces
56
Fig. 4.4: The GC-MS chromatogram of Anticharis linearis essential oil.
57
Table 4.4: Chemical compounds in essential oil of Leucophyllum frutescens analysed by
GC-MS
Retention Index (RI) Chemical compounds Area percentage (%)
940 α-pinene 0.84
953 camphene 2.53
970 β-pinene 1.20
979 β-myrcene 0.71
1025 p-cymene 1.44
1028 limonene 0.74
1030 1,8-cineole 1.67
1033 β-phellandrene 0.75
1098 linalool 2.79
1138 carveol 0.71
1204 verbenone 1.22
1291 thymol 3.41
1293 2-undecanone 0.71
1297 carvacrol 13.23
1392 β-elemene 2.54
1411 methyl eugenol 2.26
1411 α-copaene 1.88
1417 β-caryophyllene 6.87
1483 germacrene D 20.68
1493 valencene 2.81
1513 γ-cadinene 2.38
1577 spathulenol 3.98
1647 α-muurolol 3.92
1654 α-cadinol 1.50
1962 hexadecanoic acid methyl ester 1.78
2000 eicosane 2.04
2124 octadecanoic acid methyl ester 15.21
Mode of identification= Retention Index (RI) and comparison of mass spectra
58
Fig. 4.5: The GC-MS chromatogram of Leucophyllum frutescens essential oil
59
Table 4.5: Chemical compounds in essential oil of Antirrhinum majus analysed by
GC-MS
Retention Index (RI) Chemical compounds Area percentage (%)
933 α-thujene 6.07
940 α-pinene 1.26
953 camphene 1.78
970 β-pinene 1.64
979 β-myrcene 0.68
1025 p-cymene 4.43
1028 limonene 1.87
1030 1,8-cineole 1.57
1033 β-phellandrene 0.67
1049 β-ocirmene 2.80
1058 γ-terpinene 2.31
1098 linalool 0.65
1138 carveol 3.07
1204 verbenone 3.67
1291 thymol 1.03
1293 2-undecanone 5.29
1297 carvacrol 0.95
1361 eugenol 0.97
1392 β-elemene 0.61
1411 methyl eugenol 5.19
1411 α-copaene 3.38
1417 β-caryophyllene 0.86
1439 α-guaiene 1.07
1451 α-humulene 3.25
1483 germacrene D 10.86
1493 valencene 1.75
1513 γ-cadinene 0.61
1577 spathulenol 1.70
1647 α-muurolol 5.61
1654 α-cadinol 1.03
1719 methyl tetradecanoate 6.26
1962 hexadecanoic acid methyl ester 1.50
2000 eicosane 0.74
2124 octadecanoic acid methyl ester 5.28
2173 3-methylheneicosane 1.18
2194 octadecanoic acid ethyl ester 1.60
2200 stearic acid 6.61
Mode of identification= Retention Index (RI) and comparison of mass spectra
60
Fig. 4.6: The GC-MS chromatogram of Antirrnum majus essential oil
61
Table 4.6: Chemical compounds in essential oil of Verbascum thapsus analysed by GC-MS
Retention Index (RI) Chemical compounds Area percentage (%)
933 α-thujene 11.33
953 camphene 8.26
979 β-myrcene 3.89
1025 p-cymene 4.84
1028 limonene 2.13
1030 1,8-cineole 2.04
1033 β-phellandrene 3.80
1049 β-ocirmene 2.18
1058 γ-terpinene 1.99
1098 linalool 2.77
1138 carveol 2.46
1204 verbenone 2.94
1291 thymol 3.32
1361 eugenol 4.34
1392 β-elemene 4.28
1411 methyl eugenol 3.14
1411 α-copaene 3.06
1417 β-caryophyllene 7.87
1439 α-guaiene 2.11
1451 α-humulene 2.07
1483 germacrene D 4.55
1493 valencene 4.96
1513 γ-cadinene 4.34
1577 spathulenol 2.14
1647 α-muurolol 5.19
Mode of identification= Retention Index (RI) and comparison of mass spectra
62
Fig. 4.7: The GC-MS chromatogram of Verbascum thapsus essential oil.
63
Table 4.7: Chemical compounds in essential oil of Russelia equisetiformis analysed by
GC-MS
Retention Index (RI) Chemical compounds Area percentage (%)
940 α-pinene 7.26
953 camphene 0.15
970 β-pinene 4.60
1049 β-ocirmene 3.60
1058 γ-terpinene 3.25
1204 verbenone 3.27
1365 geranic acid 5.60
1411 methyl eugenol t*
1719 methyl tetradecanoate 5.27
1756 aristol-9-en-8-one 4.45
1962 hexadecanoic acid methyl ester 11.04
2000 eicosane 4.56
2124 octadecanoic acid methyl ester 6.37
2194 octadecanoic acid ethyl ester 5.69
2200 stearic acid 7.02
2288 n-docosane 7.66
2327 eicosanoic acid methyl ester 6.16
2435 11-methyltetracosane 8.44
2733 3-methylheptacosane t
2934 hexacosanoic acid methyl ester 3.79
Mode of identification= Retention Index (RI) and comparison of mass spectra,*t= traces
64
Fig. 4.8: The GC-MS chromatogram of Russelia equisetiformis essential oil.
65
Table 4.8: Chemical compounds in essential oil of Mazus goodenifolius analysed by GC-
MS
Retention Index (RI) Chemical compounds Area percentage (%)
933 α-thujene 0.33
940 α-pinene 1.79
951 α-fenchone 2.62
953 camphene 0.48
970 β-pinene 6.33
1025 p-cymene 1.53
1028 limonene 2.13
1030 1,8-cineole 8.41
1033 β-phellandrene 2.02
1049 β-ocirmene 3.82
1058 γ-terpinene 5.46
1098 linalool 9.61
1138 carveol 10.06
1204 verbenone 2.67
1291 thymol 15.16
1293 2-undecanone 1.58
1297 carvacrol 2.73
1365 geranic acid 1.97
1411 α-copaene 0.44
1417 β-caryophyllene 9.96
1439 α-guaiene 0.70
1451 α-humulene 0.73
1483 germacrene D 9.37
Mode of identification= Retention Index (RI) and comparison of mass spectra
66
Fig. 4.9: The GC-MS chromatogram of Mazus goodenifolius essential oil.
67
CH3
H3C CH3
HO
Thymol (53)
H2C CH3
CH3
OH
Carveol (52)
Germacrene D (56)
Linalool (50)
-Pinene (46) 1,8-Cineole (47)
-Terpinene (49)
-Caryophyllene (55)
CH3
H3C CH3
CH3
CH2
H3C
H3C
H
O
CH3H3C
H2C
CH3
CH3H3CCH3H3C
HCH2
CH3
H3C
H3C
CH3
-Pinen (58)
CH3H3C
OHCH3
O
Octadecenoic acid methyl ester (48)
O
O
Octadecenoic acid methyl ester (51)
O
C2H5
O
O
Hexadecanoic acid methyl ester (54)
Stearic acid (57)
OH
O
-Ocimene (59)
-Phellandrene (61) -Limonene (62)
-Phellandrene (64) Carvacrol (65)
para Cymene (63)
Geranic acid (60)
HO
O
Valencene (66)
Fig. 4.10: Structures of some major compounds identified by GC-MS analysis
68
4.4.1 Evaluation of Antioxidant Activity
i. DPPH Free Radical Scavenging Assay
The DPPH scavenging assay was used to determine the antioxidant activity of the plants
extracts, various organic fractions (Fig. 4.11) and essential oils (Fig. 4.12).
The IC50 of absolute methanol extract, various organic fractions and essential oil was
determined by DPPH radical assay. From the absolute methanol extracts, minimum IC50
value was found for Russelia equisetiformis (15.37 µg/mL). From the essential oils, IC50
observed for Russelia equisetiformis essential oil was 5.29 µg/mL (Fig. 4.12). The BHT used
a standard showed IC50 (9.96 µg/mL). Some studies have shown that plant essential oils
possess antioxidant activity and biological properties due to the synergistic or additive and
some times antagonism effects of their components (Miraliakbari and Shahidi, 2008).
The least value of IC50 represents the better antioxidant and greater value for less antioxidant
potential. The antioxidant potentail of methanol extracts was due to the occurrence of
phenolics, flavonoids and other phytochemicals (Lecumberri et al., 2007). The antioxidant
potential of essential oils is due to the presence of phytoconstituents. Derwich and coworkers
previously reported that 1,8-cineole, germacrene, limonene, pulegone, β-pinene and α-pinene
found in essential oils are all good antioxidants (Derwich et al., 2011a). Thymol also behaves
as an antioxidant (Undeger et al., 2009). As per earlier reports it was observed that presence
of chemical constituents such as linalool, β-pinene and α-pinene in essential oil also resulted
in antioxidant and antimicrobial properties (Hanif et al., 2011). The biological activities such
as antioxidant potential of essential oils phyto-components such as hexadecanoic acid ethyl
ester, unsaturated fatty acid, docosatetraenoic acid, hexadecanoic acid methyl ester and
octadecatrienoic acid (Kumar et al., 2010; Aslam et al., 2011). The essential oil showed
greater antioxidant activity as a whole rather than individual component, showing the
possible synergistic reaction of the essential oil compounds (Tepe et al., 2007).
So many studies have showed that essential oils hold pharmaceutical value for the treatment
of diseases (Carlson et al., 2002). The basic principle of the DPPH assay in color change of
DPPH solution from purple color to yellow when the radical was quenched by the
69
antioxidants (Karagozler et al., 2008). In DPPH assay the antioxidants interact with the stable
free radical, i.e. 1,1-diphenyl-2-picrylhydrazyl (deep violet colour) and change it to yellow.
The extent of discolouration indicated the scavenging properties of plants extracts, fractions
and essential oils (Jayaprakasha et al., 2007). As per earlier reports antioxidants donate
protons to DPPH free radicals, the absorption decreased with increase of concentration of test
sample. This dcrease in absorprtion was used to measure the extent of antioxidant to
scavange free radicals (Turkoglu et al., 2007).
The reduction in number of DPPH molecules can be attributed with the presence of hydroxyl
groups in the tested samples (Bhaskar and Balakrishnan, 2009). This was concluded that
antioxidant activities of tested extracts, various organic fractions and essential oils were due
to presence of phytoconstituents. The results showed that the plant extracts, various organic
fractions and essential oils have an antioxidant potential. Plant extracts may be used in food
as natural antioxidants to substitute synthetic antioxidants.
ii. Percentage Inhibition of Linoleic Acid Oxidation
The findings of percentage inhibitions of linoleic acid oxidation by plants extracts and
various organic fractions are shown in Fig. 4.13. The extracts, various organic fractions and
essential oils showed percentage inhibition ranged between 15.4 to 95.2%. From the extracts
the highest pecrcentage inhibition was also observed by absolute methanol extracts of
Russelia equisetiformis. The results of percentage inhibition of linoleic acid oxidation by
essential oils are shown in Fig. 4.14. The highest antioxidant activity in terms of % inhibition
was observed by essential oil of Russelia equisetiformis (95.2%), whereas BHT used as a
positive control showed 90.69% inhibition. However the synthetic antioxidants causes
carcinogenesis (Ito et al., 1986). Therefore, these results suggested that the plant extracts,
various organic fractions and essential oils can be used for the lowering of lipid oxidation
processes instead of synthetic antioxdants.
The antioxidant potential of essential oils was due to the occurrence of phytoconstituents.
The level of % inhibition in oxidation offered by the absolute methanolic extract was due to
the occurence of higher concentration of total flavonoid contents (TFC), total phenolic
contents (TPC) and other phytoconstituents.
70
Fig. 4.11: IC50 determined by DPPH assay of selected plants extracts and various organic
fractions
The BHT used as reference standard showed IC50 9.96 µg/mL
0
20
40
60
80
100
120
140
Absolute
methanol
n-Hexane Chloroform Ethyl
acetate
n-Butanol
IC5
0 (µ
g/m
L)
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
71
Fig. 4.12: IC50 determined by DPPH assay of selected plants essential oils
0
5
10
15
20
IC5
0 (µ
g/m
L)
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
72
Fig. 4.13: % inhibition of linoleic acid oxidation by selected plants extracts and various
organic fractions
0
10
20
30
40
50
60
70
80
90
100
Absolute
methanol
Chloroform Ethyl
acetate
n-Butanol n-Hexane
% I
nhib
itio
n
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
73
Fig. 4.14: % Inhibition of linoleic acid oxidation by selected plants essential oils
82
84
86
88
90
92
94
96
98
100
% I
nhib
itio
n
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
74
Vardar-Unlu and coworkers described that the biological properties of plants such
antioxidant and antimicrobial activity based on synergistic and antagonism effects of their
components (Vardar-Unlu et al., 2003).
The antoxidant activity of selected plant’s essential oils is credited to the occurrence of
chemical constituents as analyzed by GC-MS in present research work.
iii. Reducing Power
The absolute methanol extracts of selected plants, various organic fractions and essential oils
showed considerable reducing power as shown in (Figures 4.10-4.15). The increase in
antioxidant activity when the concentration of analyzed samples increased was observed by
noting the absorbance spectrophotometrically. An increase in reducing power was observed
as concentration of samples increased. The highest out of the selected plants was observed in
the essential oils of Russelia equisetiformis and lowest for Verbascum thapsus. The highest
reducing power was observed by absolute methanol extract of Russelia equisetiformis among
analyzed samples. The reducing power of extracts, various organic fractions and essential
oils is in agreement with our findings that the selected plants extracts which have higher
values of TPC, TFC and other phytochemicals analysed during qualtitative analysis.
Signifcant differences (P ≤ 0.05) were found in various concentrations used for the reducing
power analysis. The reducing power of phytochemical constituents could be associated with
the antioxidant potential (Siddhuraju and Becker, 2003). The highet reducing power of
essential oils was attributed due to the phytoconstituents as analyzed by GC-MS. The
reducing power of plant’s extracts, various organic fractions and essential oils was probably
due to hydrogen donating capacity (Shimada et al., 1992), and was usually linked with the
occurrence of phytoconstituents (Duh, 1998). Therefore reducing power assessment could be
taken as an significant assay for the evaluation of antioxidant potential of plant sources. The
reducing power of plant’s sources were often used to study the capability of antioxidant to
donate electron to inhibit free radicals (Yildirim et al., 2000).
75
Fig. 4.15: Reducing power by Russelia equisetiformis extract and various organic fractions
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.5 5 7.5 10
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
76
Fig. 4.16: Reducing power by Antirrhinum majus extract and various organic fractions
0
0.5
1
1.5
2
2.5 5 7.5 10
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
77
Fig. 4.17: Reducing power by Mazus goodenifolius extract and various organic fractions
0
0.5
1
1.5
2
0 5 10 15 20 25
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
78
Fig. 4.18: Reducing power by Anticharis linearis extract and various organic fractions
0
0.5
1
1.5
2
5 10 15 20
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
79
Fig. 4.19: Reducing power by Verbascum thapsus extract and various organic fractions
0
0.5
1
1.5
2
0 5 10 15 20 25
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
80
Fig. 4.20: Reducing power by Leucophyllum frutescens extract and various organic fractions
0
0.5
1
1.5
2
0 5 10 15 20 25
Abso
rban
ce
Concentration (mg/mL)
Absolute methanol
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
81
Fig. 4.21: Reducing power of selected plants essential oils
0
0.5
1
1.5
2
2.5
2.5 5 7.5 10
Abso
rban
ce
Concentration (mg/mL)
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
82
Various studies have shown that there was a direct association between antioxidative
potential and reducing power of phytochemical constituents. In this reseatch work, the
capability of plants extracts and different organic fractions to reduce from Fe+3
to Fe+2
was
determined. For the reducing power assay, the existence of reductants (antioxidant) in
analyzed samples consequences in the reduction of Fe+3
ion Fe+3
/ferricyanide complex to
ferrous form (Bougatef et al., 2009). Zhu and coworkers reported the reducing power of
plant’s extracts enhanced on increasing concentration of test samples (Zhu et al., 2006). The
findings of present research work suggested that the the plants sources can be used as a good
source of pharmaceutical applications and natural antioxidants.
4.4.2 Antioxidant Potential of Essential Oils by β-Carotene with Linoleic Acid Assay
The antioxidant potential of selected medicinal plants essential oils by bleaching of β-
carotene with linoleic acid system was studied and shown in Fig. 4.22. The decline in
absorbance of β-carotene shown a lower rate of oxidation of linoleic acid and higher
antioxidant potential in the presence of essential oils. Essential oils of R. equisetiformis
exibted good antioxidant potential than other plants essential oil when compared with control
(without addition of antioxidant). The antioxidant potential of essential oils have formed the
basis of so many uses such as pharmaceuticals, natural therapies, preservation of food and
medicine (Bozin et al., 2006). The antioxidant potential of essential oils have probably
imparted due to the presence of terpenoids and some other phytoconstituents. The chemical
components like terpenoids had a significant biological activities (Ruberto and Baratta,
2000). Furthermore, some researchers showed that some essential oils rich in phytochemicals
such as terpenoids, fatty acid/methyl esters also have antioxidant potentials (El-Massry et al.,
2002) previously reported as potent radical scavengers (Bozin et al., 2006). During the
review of literature it was also found that the biological studies such as antioxidant potential
of some phyto-components such as hexadecanoic acid ethyl ester, unsaturated fatty acid,
docosatetraenoic acid, hexadecanoic acid methyl ester, α-pinene, β-pinene and
octadecatrienoic acid (Kumar et al., 2010; Aslam et al., 2011; Riaz et al., 2012b).
4.5 Antimicrobial Activity
In order to evaluate the antimicrobial activity initially different concentrations of sample to
be analysed were optimized by disc diffusion assay using methanol extract and essential oils
83
Fig. 4.22: Antioxidant activity of selected plants essential oils analysed by bleaching of β-
carotene-linoleic acid emulsion assay
0
0.2
0.4
0.6
0.8
1
1.2
5 10 20 30 40 50
Abso
rban
ce
Time (Hours)
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
Control
84
at different concentrations, against the bacterial strain (Staphylococcus aureus) (Table 4.9)
and fungal strain (Rhizopus solani) (Table 4.10). It was found that maximum zone of
inhibition (mm) obtained at different concentrations for selected plants extracts. The further
analyses were carried at the optimized concentrations of respective plant. From the results it
was found that the maximum zone of inhibition against the bacterial strain (Staphylococcus
aureus) and fungal strain (Rhizopus solani) by absolute methanol extract was found at
different concentration of the plants such as: Russelia equisetiformis (3 mg/mL),
Leucophyllum frutescens (3 mg/mL), Mazus goodenifolius (20 mg/mL), Antirrhinum majus
(4 mg/mL), Anticharis linearis (4 mg/mL), Verbascum thapsus (5 mg/mL). It was observed
that further incease in concentration the zone of inhibition linearized. For the selected plants
essential oils the maximum zone of inhibition (mm) was observed at 2 mg/mL and linearized
after 2 mg/mL against the bacterial and fungal strains (Tables 4.11-4.12). The antimicrobial
activity using disc diffusion assay by selected plants against pathogenic microorganisms like
bacterial strains such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus and
Pasturella multocida were used. The antimicrobial activity was also evaluated against fungal
strains such as Aspergillus niger, Rhizopus solani, Aspergillus flavus and Alternaria
alternata was analyzed. The results of zone of inhibition (mm) and MIC for antibacterial and
antifungal activity are presented in Tables 4.13-4.25. The plants extracts and fractions
exhibited antimicrobial activity. After the antimicrobial screening assay extracts, various
organic fractions and essential oils which showed positive results by zone of inhibition (mm)
were selected for the evaluation of minimum inhibitory concentrations (MICs).
The tables 4.13, 4.15, 4.17, 4.19, 4.21, 4.22 and 4.25 showed that the zone of inhibition (mm)
by selected plants absolute methanol extract were as: Russelia equisetiformis (37.7 mm
against Staphylococcus aureus) > Leucophyllum frutescens (34.2 mm against Staphylococcus
aureus) > Mazus goodenifolius (31.7 mm against Staphylococcus aureus) > Antirrhinum
majus (30.8 mm against Staphylococcus aureus) > Anticharis linearis (31.3 mm against B.
Subtilis > Verbascum thapsus (28.5 mm against E. coli).
85
Table 4.9: The screening of different concentrations on antibacterial activity by inhibition zone (mm) by selected plants extracts and various
organic fractions against Staphylococcus aureus
Concentration
(mg/mL)
R.E.
(M.E.)ǂ
L.F.
(M.E.)
M.G.
(M.E.)
A.M.
(M.E.)
A.L.
(M.E.)
V.T.
(M.E.)
1 18.35± .12 *N.A. N.A. 14.4±0.12 N.A. N.A.
2 25.5±0.19 18.5±0.14 N.A. 17.3±0.16 13.4±0.12 N.A.
3 35.4±0.23 34.2±0.26 N.A. 20.7±0.19 19.2±0.17 12.6±0.10
4 35.4±0.23 34.2±0.26 N.A. 33.6±0.28 29.2±0.26 13.4±0.12
5 †N.T.
(after this
the inhibition
zone linearized)
N.T.
14.2±0.13 33.6±0.28 29.2±0.26 27.2±0.23
10 16.7±0.14
N.T.
N.T.
27.2±0.23
20 31.7±0.29 N.T.
25 31.7±0.29
The values were the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
*N.A. = No activity; †N.T.
= Not tested as the concentration was optimized above this concentration;
ǂ (M.E.)
= Absolute methanol extract
R.E. (Russelia equisetiformis), L.F. (Leucophyllum frutescens), M.G. (Mazus goodenifolius), A.M. (Antirrhinum majus),
A.L. (Anticharis linearis), V.T. (Verbascum thapsus)
86
Table 4.10: The effect of different concentrations on antifungal activity by inhibition zone (mm) by selected plants extracts and various organic
fractions against Rhizopus solani
Concentration
(mg/mL)
R.E. (M.E.)ǂ L.F. (M.E.) M.G. (M.E.) A.M. (M.E.) A.L. (M.E.) V.T. (M.E.)
1 15.33± .12 *N.A. N.A. 11.0±0.09 N.A. N.A.
2 25.7±0.17 16.2±0.14 N.A. 17.3±0.12 11.3±0.09 N.A.
3 31.5±0.29 32.6±0.24 N.A. 20.7±0.18 15.8±0.12 10.7±0.08
4 31.5±0.29 32.6±0.24 N.A. 25.4±0.21 28.7±0.24 11.9±0.10
5 †N.T.
(after this the
inhibition zone
linearized)
N.T.
14.2±0.11 31.1±0.27 28.7±0.24 25.6±0.21
10 16.7±0.14 31.1±0.27
N.T.
25.6±0.21
20 23.1±0.19 N.T.
N.T.
25 23.1±0.19
The values were the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
*N.A. = No activity; †N.T.
= Not tested as the concentration was optimized above this concentration;
ǂ (M.E.)
= Absolute methanol extract
R.E. (Russelia equisetiformis), L.F. (Leucophyllum frutescens), M.G. (Mazus goodenifolius), A.M. (Antirrhinum majus),
A.L. (Anticharis linearis), V.T. (Verbascum thapsus)
87
Table 4.11: The screening of different concentrations of selected plants essential oils on antibacterial activity by inhibition zone (mm) against
Staphylococcus aureus
Concentration
(mg/mL) R. equisetiformis A. majus V. thapsus L. frutescens A. linearis M. goodenifolius
1 19.2 ±0.28 16.1±0.18 13.1±0.14 17.7±0.15 14.5±0.13 18.1±0.16
2 39.7±0.34 33.5±0.30 28.3±0.30 37.5±0.31 31.5±0.30 35.2±0.19
3 39.8±0.32 33.7±0.31 28.4±0.29 37.7±0.32 31.6±0.29 35.4±0.21
The values were the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
88
Table 4.12: The effect of different concentrations of selected plants essential oils against antifungal activity by inhibition zone (mm) against
Rhizopus solani
Concentation
(mg/mL) R. equisetiformis A. majus V. thapsus L. frutescens A. linearis M. goodenifolius
1 18.8±0.11 15.3±0.08 12.2±0.11 16.8±0.17 14.2±0.20 13.1±0.31
2 35.1±0.24 31.4±0.29 26.9±0.24 33.4±0.31 30.6±0.20 29.7±0.21
3 35.1±0.24 31.5±0.28 27.1±0.25 33.6±0.32 30.7±0.21 29.9±0.22
The values were the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
89
The highest antimicrobial activity was observed by the essential oils of Russelia
equisetiformis from the selected plants and lowest for Verbascum thapsus.
From the extracts and various organic fractions the absolute methanol extracts of each plant
showed comparatively better antimicrobial activity. The maximum zone of inhibition by
seleted plants essential oils as: Russelia equisetiformis (39.4 mm against Staphylococcus
aureus > Leucophyllum frutescens (37.5 mm against Staphylococcus aureus) > Mazus
goodenifolius (35.2 mm against Staphylococcus aureus) > Antirrhinum majus (33.5 mm
against Staphylococcus aureus) > Anticharis linearis (33.1 mm against E. coli > Verbascum
thapsus (29.6 mm against E. coli). From the activity results it observed that antioxidant and
antimicrobial activity of the selected plants essential oils was greater than that of absolute
methanol extracts and various organic fractions. Some research works have also shown that
plant essential oils can have greater antimicrobial potential due to the synergistic or additive
effects of their components (Miraliakbari and Shahidi, 2008). From the results it was
observed that maximum zone of inhibition (mm) showed by Russelia equisetiformis essential
oil from the selected plants against bacterial strains (Table 4.24) such as Staphylococcus
aureus (39.7 mm), Bacillus subtilis (37.2 mm), Pasturella multocida (34.6 mm), Escherichia
coli (30.6 mm). The maximum zone of inhibition (mm) showed by Russelia equisetiformis
essential oil against fungal strains was: Rhizopus solani (35.1 mm), Aspergillus niger (34.7
mm), Aspergillus flavus (31.5 mm), Alternaria alternata (28.9 mm). From antimicrobial
results table 4.24 showed that over all least activity in zone of inhibition was observed by
Verbascum thapsus essential oil against the bacterial strains such as: Staphylococcus aureus
(29.3 mm), Escherichia coli (28.6 mm), Pasturella multocida (No activity), Bacillus subtilis
(27.9 mm), against the fungal strains: Rhizopus solani (26.9 mm), Alternaria alternata (24.8
mm), Aspergillus niger (No activity), Aspergillus flavus (21.2 mm). The absolute methanol
extract of Russelia equisetiformis also showed activity against the fungal strains maximum
zone of inhibition was observed against Rhizopus solani (34.2 mm) and lowest for Alternaria
alternata (27.2 mm).
The minimum inhibitory concentration (MIC) results showed that the essential oils showed
better activity at lower concentration than absolute methanol extracts and various organic
fractions (Tables 4.14, 4.16, 4.18, 4.20, 4.23 and 4.25). The least minimum inhibitory
90
concentration (MIC) showed that the less concentration was required to inhibit the selected
bacterial and fungul strains. The higher MIC represents that the tested samples have less
activity and greater concentration was required to inhibit the selected bacterial and fungul
strains.
The results showed that the MIC of selected plants essential oils (E.O.) and absolute
methanol extract (M.E.) were: Russelia equisetiformis (E.O. = 1.05 mg/mL, M.E.= 2.05
mg/mL against Staphylococcus aureus ˂ Leucophyllum frutescens (E.O. = 1.10 mg/mL,
M.E. 2.15 mg/mL against Staphylococcus aureus ˂ Antirrhinum majus (E.O. = 1.30 mg/mL,
M.E. = 3.05 mg/mL against Staphylococcus aureus) ˂ Anticharis linearis (E.O. = 1.45
mg/mL against E. coli; M.E. = 3.10 mg/mL against B. subtilis ˂ Verbascum thapsus (E.O.=
1.55 mg/mL, M.E. 4.45 mg/mL against E. coli).
The better antimicrobial potential of the essential oil was attributed to the presence of some
major components thymol, carveol, linalool, germacrene D, 1,8-cineole, β-pinene, γ-
terpinene, β-ocimene, carvacrol, limonene, α-pinene and p-cymene. Essential oils containing
these major compounds have been reported to show antimicrobial properties (Hanif et al.,
2011). Antibacterial material can easily obliterate the cell wall of bacteria, cytoplasmic
membrane and result in a seepage of cytoplasm or its coagulation (Kalemba and Kunicka,
2003). The synergistic or antagonistic activity between some components may affect the
observed antimicrobial potential of the essential oil (Giweli et al., 2012), which exerts its
toxic effects against microorganisms through the disruption of bacterial and fungal
membrane integrity. The presence of hexadeconoic acid, stearic acid and methyl esters also
showed antimicrobial activities (Riaz et al., 2012b).
The ciprofloxacin and fungone were used as positive control for fungal and bacterial strains
respectively. Some of the secondary metabolites estimated during the phytochemical assay in
plants in this study have steroids, tannins, saponins, alkaloids and terpenoids. These
compounds have variously been reported to showed antimicrobial activity (Field and
Lettinga, 1992).
91
Table 4.13: Antimicrobial activity in terms of inhibition zone (mm) by Russelia equisetiformis extract and various organic fractions
Extract and various
organic fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute methanol 35.4±0.25 31.3±0.27 37.6±0.31 33.2±0.24 30.8±0.29 32.4±0.21 34.2±0.27 27.2±0.21
n-Butanol 24.0±0.18 20.3±0.18 N.A.* 22.9±0.19 18.8±0.14 24.7±0.22 N.A. 16.3±0.26
Chloroform 27.2±0.17 24.8±0.15 28.4±0.24 25.9±0.21 21.2±0.19 N.A. 28.7±0.17 14.5±0.12
Ethyl acetate 33.8±0.14 28.5±0.21 31.6±0.25 29.7±0.24 28.2±0.22 30.1±0.27 32.5±0.21 25.1±0.22
n-Hexane 19.0±0.12 16.4±0.14 20.0±0.17 18.4±0.12 N.A. 17.3±0.14 19.7±0.16 N.A.
†Standard 29.2±0.15 27.2±0.21 30.1±0.16 28.3±0.15 22.5±0.18 28.1±0.26 29.7±0.21 24.6±0.23
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
†Ciprofloxacin and fungone were used as reference standards for bacterial and fungal strains, respectively.
92
Table 4.14: The minimum inhibitory concentrations (mg/mL) by Russelia equisetiformis extract and various organic fractions
* N.A. = No activity
Extract and
various
organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
2.07 2.20 2.05 2.12 2.22 2.18 2.10 2.30
n-Butanol 2.72 2.80 N.A.* 2.75 2.85 2.82 N.A. 2.90
Chloroform 2.52 2.57 2.45 2.55 2.62 2.57 2.45 2.67
Ethyl acetate 2.40 2.50 2.32 2.42 2.42 2.40 2.37 2.50
n-Hexane 2.82 2.90 2.80 2.85 N.A. 2.92 2.87 N.A.
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
93
Table 4.15: Antimicrobial activity in terms of inhibition zone (mm) by Leucophyllum frutescens extract and various organic fractions
Extract, various
organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
31.1±0.21 30.3±0.21 34.2±0.30 32.2±0.21 25.7±0.19 30.2±0.24 32.6±0.22 27.2±0.24
n-Butanol 23.1±0.20 22.2±0.13 N.A.* 25.7±0.15 13.2±0.11 N.A. 25.4±0.13 20.2±0.17
Chloroform 20.1±0.14 18.3±0.14 28.4±0.24 22.0±0.14 14.3±0.24 22.9±0.25 N.A. 16.9±0.19
Ethyl acetate 27.1±0.21 27.8±0.19 30.7±0.22 30.2±0.19 23.1±0.27 29.3±0.21 31.1±0.20 26.2±0.21
n-Hexane 17.1±0.11 15.1±0.13 19.0±0.14 20.0±0.18 N.A. 16.3±0.17 17.9±0.14 N.A.
Standard 28.30±0.15 27.20± 0.21 30.10±0.16 29.20±0.15 22.50±0.18 24.60 ±0.23 29.70±0.21 28.10±0.26
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
94
Table 4.16: The minimum inhibitory concentrations (MICs) in mg/mL by Leucophyllum frutescens extract and various organic fractions
Extract and
various
organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
2.30 2.35 2.15 2.27 2.30 2.45 2.20 2.35
n-Butanol 2.75 2.85 N.A.* 2.70 2.80 2.60 2.55 2.75
Chloroform 2.85 2.90 2.55 2.62 2.90 2.85 N.A. 2.90
Ethyl acetate 2.60 2.70 2.40 2.50 2.64 2.50 2.40 2.55
n-Hexane 2.95 3.00 2.85 2.90 N.A. 3.00 2.90 N.A.
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
* N.A. = No activity
95
Table 4.17: Antimicrobial activity in terms of inhibition zone (mm) by Antirrhinum majus extract and various organic fractions
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol 30.5±0.21 29.4±0.32 31.8±0.32 31.4±0.34 25.3±0.19 27.2 ±0.21 31.1±0.32 30.3±0.23
n-Butanol N.A.* 18.10±0.17 17.2±0.21 28.2±0.27 18.7±0.26 22.6 ±0.27 16.2±0.11 23.1±0.21
Chloroform 28.4±0.28 20.2 ±0.19 N.A. N.A. 20.7±0.05 N.A. 22.1±0.13 N.A.
Ethyl acetate 29.2±0.24 28.2±0.21 31.4±0.23 30.3±0.29 24.1±0.21 25.3 ±0.12 30.2±0.29 29.4±0.26
n-Hexane 16.1±0.21 N.A. N.A. 18.0±0.15 N.A. 14.3±0.17 15.9±0.14 N.A.
Standard 28.3±0.15 27.2± 0.21 30.1±0.16 29.2±0.15 22.5±0.18 24.6 ±0.23 29.7±0.21 28.1±0.26
* N.A. = No activity.
The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
96
Table 4.18: The minimum inhibitory concentrations (MICs) in mg/mL by Antirrhinum majus extract and various organic fractions
Extract and
various
organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
3.20 3.25 3.05 3.10 3.57 3.50 3.30 3.40
n-Butanol N.A.* 3.70 3.65 3.85 3.70 3.65 3.75 3.60
Chloroform 3.70 3.92 N.A. N.A. 3.85 N.A. 3.70 N.A.
Ethyl acetate 3.65 3.77 3.45 3.55 3.67 3.55 3.45 3.50
n-Hexane 3.00 N.A. N.A. 3.95 N.A. 4.00 3.90 N.A.
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
* N.A. = No activity
97
Table 4.19: Antimicrobial activity in terms of inhibition zone (mm) by Anticharis linearis extract and various organic fractions
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
31.3±0.32 28.70±0.22 29.2 ±0.28 30.20±0.30 26.30±0.29 25.20 ±0.21 28.7±0.26 29.75±0.27
n-Butanol N.A.* 20.26 ±0.19 N.A. N.A. 19.70±0.25 N.A. 21.15±0.23 N.A.
Chloroform N.A. 17.15 ±0.12 N.A. 26.20±0.14 N.A. 21.80 ±0.27 N.A. N.A.
Ethyl acetate 28.90±0.24 N.A. 30.40±0.29 N.A. 23.90±0.21 N.A. 31.20±0.39 29.30±0.26
n-Hexane 17.1±0.13 N.A. N.A. 16.0±0.14 N.A. 13.8±0.17 14.3±0.18 N.A.
Standard 28.3±0.15 27.2± 0.21 30.1±0.16 29.2±0.15 22.5±0.18 24.6±0.23 29.7±0.21 28.1±0.26
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
98
Table 4.20: The minimum inhibitory concentrations (MICs) in mg/mL by Anticharis linearis extract and various organic fractions
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute methanol 3.10 3.45 3.35 3.20 3.50 3.55 3.45 3.35
n-Butanol N.A.* 3.80 N.A. 3.70 N.A. 3.80 N.A. N.A.
Chloroform N.A. 3.90 N.A. N.A. 3.85 N.A. 3.75 N.A.
Ethyl acetate 3.45 N.A. 3.55 N.A. 3.75 N.A. 3.60 3.65
n-Hexane 3.95 N.A. N.A. 3.90 N.A. 4.00 3.95 N.A.
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
* N.A. = No activity
99
Table 4.21: Antimicrobial activity in terms of inhibition zone (mm) by Mazus goodenifolius extract and various organic fractions
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute methanol 25.4±0.21 28.1±0.21 31.7±0.12 25.1±0.21 19.0±0.13 28.6±0.21 30.1±0.23 23.1±0.21
n-Butanol 17.2±0.16 15.2±0.17 10.6±0.08 16.3±0.21 14.8±0.11 14.6±0.14 N.A. 15.6±0.14
Chloroform 15.7±0.08 19.3±0.14 14.1±0.12 19.2±0.18 17.2±0.13 N.A. 22.3±0.17 12.7±0.07
Ethyl acetate 23.3±0.17 26.2±0.19 23.1±0.29 20.1±0.15 18.6±0.14 20.2±0.21 15.2±0.16 20.3±0.22
n-Hexane 11.2±0.14 12.2±0.10 N.A.* 15.2±0.13 N.A. 13.2±0.12 32.4±0.31 11.4±0.17
Standard 29.2±0.15 27.2±0.21 30.1±0.16 28.3±0.15 22.5±0.18 28.1±0.26 29.7±0.21 24.6±0.23
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
100
Table 4.22: Antimicrobial activity in terms of inhibition zone (mm) by Verbascum thapsus extract and various organic fractions.
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol
22.3±0.31 24.4±0.28 27.2 ±0.19 28.5±0.22 20.1±0.29 19.8 ±0.21 25.6±0.22 23.4±0.27
n-Butanol N.A.* 18.1±0.12 N.A. N.A. 13.2±0.21 N.A. 15.2±0.14 N.A.
Chloroform 15.4 ±0.11 N.A. N.A. N.A. 9.7±0.20 N.A. N.A. N.A.
Ethyl acetate 18.9±0.24 N.A. N.A. 20.9±0.23 15.6±0.19 N.A. 22.2±0.27 N.A.
n-Hexane N.A. N.A. N.A. 15.0±0.12 N.A. 10.8±0.12 12.2±0.14 N.A.
†Standard 28.3±0.15 27.2± 0.21 30.1±0.16 29.2±0.15 22.5±0.18 24.6±0.23 29.7±0.21 28.1±0.26
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
101
Table 4.23: The minimum inhibitory concentrations (MICs) in mg/mL by Verbascum thapsus extract and various organic fractions
Extract and
various organic
fractions
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
Absolute
methanol 4.60 4.50 4.65 4.45 4.45 4.60 4.65 4.55
n-Butanol 4.80 N.A. N.A. N.A. 4.75 N.A. N.A. N.A.
Chloroform N.A.* 4.85 N.A. N.A. 4.80 N.A. 4.85 N.A.
Ethyl acetate 4.70 N.A. N.A. 4.75 4.70 N.A. 4.75 N.A.
n-Hexane N.A. N.A. N.A. 4.97 N.A. 4.90 5.00 N.A.
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
* N.A. = No activity
102
Table 4.24: Antimicrobial activity in terms of inhibition zone (mm) by selected plant essential oils.
Essential oils
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
R. equisetiformis 37.2±0.31 34.6±0.27 39.7±0.34 30.6±0.34 31.5±0.27 34.7±0.31 35.1±0.24 28.9±0.18
A. majus 30.9±0.29 31.6±0.32 33.5±0.30 33.1±0.31 25.2±0.17 29.9±0.28 31.4±0.29 28.9±0.23
V. thapsus 27.9±0.24 N.A.* 28.3±0.30 29.6±0.37 21.2±0.17 N.A. 26.9±0.24 24.8±0.19
L. frutescens 29.9±0.31 32.6±0.36 37.5±0.31 34.2±0.37 26.4±0.23 32.4±0.32 33.4±0.31 29.4±0.29
A. linearis 31.9±0.29 N.A. 31.5±0.30 33.1±0.31 24.32±0.27 N.A. 30.6±0.20 28.7±0.21
M. goodenifolius 32.2±0.26 32.2±0.26 35.2±0.19 30.2±0.27 29.2±0.27 31.8±0.30 29.7±0.21 30.4±0.24
†Standard 29.2±0.15 27.2±0.21 30.1±0.16 28.3±0.15 22.5±0.18 28.1±0.26 29.7±0.21 24.6±0.23
* N.A. = No activity. The values are the average of triplicate samples (n = 3) ± S.D., (P ≤ 0.05).
†Ciprofloxacin and fungone were used as reference standards for bacterial and fungal strains, respectively
103
Table 4.25: The minimum inhibitory concentrations (MICs) in mg/mL) by selected plants essential oils
Essential oils
Bacterial strains Fungal strains
B. subtilis P. multocida S. aureus E. coli A. flavus A. niger R. solani A. alternata
R.
equisetiformis
1.07 1.22 1.05 1.15 1.35 1.32 1.20 1.25
A. majus 1.40 1.50 1.30 1.35 1.68 1.55 1.35 1.60
V. thapsus 1.60 N.A.* 1.70 1.55 1.80 N.A. 1.60 1.75
L. frutescens 1.35 1.45 1.10 1.20 1.65 1.50 1.30 1.55
A. linearis 1.55 N.A. 1.50 1.45 1.70 N.A. 1.55 1.65
Standard 0.062 0.125 0.025 0.075 0.100 0.050 0.015 0.062
* N.A. = No activity
104
The plants extracts, various organic fractions and essential oils were found to have significant
antimicrobial activity and therefore can be used as a natural antimicrobial agent for the
treatment of several infectious diseases caused by the studied microorganism, which have
developed resistance to antibiotics (Derwich et al., 2011b). The Pasquale described that the
medical properties are not essentially limited to a single compound present in plant. But
various phytocostituents combinely showed antimicrobial activity (Pasquale, 1984). The
tannins have been observed to form complexes with proline rich protein resulting in the
inhibition of protein synthesis (Shimada, 2006). The tannins have been known to interact
with proteins to provide the typical tannin effect which are significant for the treatment of
ulcerated tissues. Tannins were also toxic to bacteria, fungi and viruses and inhibit their
growth (Scalbert, 1991). Alkaloids are also the largest group of secondary metabolites in
plants have good effects against diseases and this has lead to the production of powerful
antimicrobial effects (Kam and Liew, 2002). It has been revealed that the saponins have
antimicrobial properties (Killeen et al., 1998). Flavonoids, another constituent found in
plants extracts and various organic fractions showed a broad range of biological prpoerties
like antimicrobial activity and antioxidant potential (Hodek et al., 2002). Anthraquinones
possessed anti inflammatory and bactericidal effects (Feroz et al., 1993).
The earlier reports showed that growth of microorganism are inhibited very effectively by
plants extracts due to presence of phenolics and flavonoids (Mori et al., 1987). The present
study showed the value of plants material used in present research work may be of significant
interest for the development of plant based herbal medicine.
4.6 Haemolytic Activity for Cytotoxicity Assay
To evaluate the cytotoxicity of the selected medicinal plants extracts, various organic
fractions and essential oils, haemolytic activity method was used against human red blood
cells (RBCs). The triton X-100 was used as a positive control and phosphate buffer saline as
a negative control for haemolytic activity. The results were shown in Fig. 4.23. The highest
percentage lysis of RBCs was observed in Anticharis linearis absolute methanol extract
(7.25%) and lowest for mazus goodenifolius essential oil (0.96%). The mechanical stability
of the red bllod cells membrane was a good sign of the effect of various in vitro studies by
different compounds.
105
Fig. 4.23: Cytotoxicity by haemolytic activity of selected plants extract, various organic
fractions and essential oils
0
1
2
3
4
5
6
7
8
Absolute
methanol
n-Hexane Chloroform Ethyl
acetate
n-Butanol Essential
oil
L
ysi
s of
RB
Cs
(%)
R. equisetiformis
A. majus
V. thapsus
L. frutescens
A. linearis
M. goodenifolius
106
The percentage lysis of human red blood cells was below 10.0% for all samples, so it can be
expected that the extract and various organic fractions have a minor cytotoxity (Sharma and
Sharma, 2001). Uddin and coworker worked on cytotoxicity, described that minor cytotoxic
properties of the extracts is of great importance for their conventional use in the dealing of
different disorder other than cancer (Uddin et al., 2009). As per our findings the selected
plants used in present research work showed minor cytotoxicity. The plants extracts or
fractions haivinig minor Cytotoxicity might be used as a herbal medicine (Aslam et al.,
2011).
4.7 DNA Protection Assay
The results of antioxidant and antimicrobial studies of selected plants showed that Russelia
equisetiformis has comparatively better activity, so the Russelia equisetiformis was selected
for futher studies.
The antioxidant activity by DNA protection assay was carried out for the plant showing
better activity during initial screening so the absolute methanol extract Russelia
equisetiformis leaves, various organic solvent fractions and essential oil were used for this
study. Initially the different concentration (10, 100 and 1,000 µg/mL of absolute methanol
extract were applied to optimize the concentrations of samples to be used.
The results in (Fig. 4.24) showed the changes in the extent of protection of plasmid pBR322
DNA by H2O2 induced damage when treated with different concentrations of R.
equisetiformis methanol extract to evaluate the antioxidant activity. It was found that the
protection occurred with the inhance in concentration of extract. In the lane (1) plasmid
pBR322 DNA present without any treatment that might be due to dominant super coiled
form. The lane (2) showed the 1 Kb DNA ladder. The lane (3) contain pBR322 DNA that
was exposed to 4 µL H2O2 that caused damage in plasmid pBR322 DNA, in this lane the
DNA and H2O2 as a result damaging of DNA strand might occurred that converted the super
coiled form of pBR322 DNA into open linear form and remained behind than the untreated
DNA can be seen from Fig. 4.24. In the lane (4 to 6) 5 µL (10, 100 and 1000 µg/mL)
respectively of R. equisetiformis methanol extract added in pBR322 DNA to observe their
protective effect. The R. equisetiformis methanol extract protected H2O2 induced strand
breaks in plasmid pBR322 DNA in a concentration dependent manner.
107
Fig. 4.24: Electropherogram showing DNA protection effect by methanol extract with H2O2
induced oxidative damage on pBR322 DNA.
Lane 1 = Plasmid pBR322 DNA without treatment (Super coiled); Lane 2 = Plasmid pBR322 DNA treated with
H2O2 (Open circular); Lane 3 = 1 Kb DNA ladder; Lane 4 = Plasmid pBR322 DNA treated with methanol
extract (10 µg/mL) + H2O2; Lane 5 = Plasmid pBR322 DNA treated with methanol extract (100 µg/mL) + H2O2;
Lane 6 = Plasmid pBR322 DNA treated with methanol extract (1000 µg/mL) + H2O2
6 5 4 3 2 1
108
Fig. 4.25: DNA protection effect by Russelia equisetiformis extract, various organic fractions
and essential oils with H2O2 induced oxidative damage on pBR322 DNA
(Lane 1 = Plasmid pBR322 DNA without treatment (Super coiled); Lane 2 = Plasmid pBR322 DNA treated
with essential oil + H2O2), Lane 3 = Plasmid pBR322 DNA; treated with H2O2 (open circular or damaged); Lane
4 = Plasmid pBR322 DNA treated with absolute methanol extract + H2O2; Lane 5 = Plasmid pBR322 DNA
treated with n-butanol fraction + H2O2; Lane 6 = 1 Kb DNA ladder; Lane 7 = Plasmid pBR322 DNA treated
with chloroform fraction + H2O2; Lane 8 = Plasmid pBR322 DNA treated with ethyl acetate fraction + H2O2;
Lane 9 = Plasmid pBR322 DNA treated with n-hexane fraction + H2O2).
1 2 3 4 5 6 7 8 9
109
Fig. 4.24 showed that the R. equisetiformis methanol extract at 1000 µg/mL protected the
DNA, it may have not allowed to damage the DNA and remained protected to super coiled
form (protected) when compared with other bands. The band at 1000 µg/mL absolute
methanol extract is equal to the untreated DNA (lane 1). The protective effect was observed
by absolute methanol extract, which may be due to higher amount of total phenolics,
flavonoid contents and their antioxidant potential. The result was comparable with the other
antioxidant assays carried out in present work. Thus the R. equisetiformis absolute
methanolic extract was a valuable antioxidant that protected plasmid pBR322 DNA from free
radical induced oxidative damage. Apart from lipid oxidation, reactive oxygen species may
cause damage to the cellular genetic material.
The protective effect of R. equisetiformis absolute methanol extract on DNA can also be
described by its ability to scavenge reactive oxygen species due to its property as an
antioxidant. By comparing with other results there was an explanation for the protective
effect, which was the direct interaction of phytochemicals with DNA (Yoshikawa et al.,
2006).
Further to evaluate the protective effect on plasmid pBR322 DNA by H2O2 induced damage
exerted by absolute methanol extract, various organic fractions and essential oil at a
concentration of 1,000 µg/mL of Russelia equisetiformis was used (Fig. 4.25). In the first
lane plasmid pBR322 DNA constitute the control (without treatment) and it may be in super
coiled form. When the plasmid was exposed to H2O2, the DNA damage was evident; may be
with the conversion of the super coiled form of pBR322 DNA into open linear form (third
lane) leaving behind from the untreated DNA (first lane) (evident from Fig. 4.25). When
compared with other lanes, the protective effects of the essential oil (second lane) showed the
band almost equal to the pure DNA band while the n-hexane fraction (nine lane) showed less
protective effect on DNA the band of DNA was near to the damaged DNA (third lane treated
with H2O2). Similarly the other fractions showed protective effect on DNA (seven to nine
lane) but the protection was less than the essential oil and absolute methanol extract and
higher than the n-hexane fraction when compared with the other lanes (evident from Fig.
4.25). So it is concluded that the highest effective DNA protection showed by absolute
methanol extract, the lowest by the n-hexane fraction. The plant essential oil at 1,000 µg/mL
110
protected the DNA, perhaps by scavenging the oxidation products that damage the DNA and
this did not allowed the H2O2 to open the coiled DNA so it remained in protected form. The
protective effect by samples might be due to the higher concentration of phenolics,
flavonoids and the antioxidant activity that scavenges the free radicals and oxidation
products. The protective effect observed by absolute methanol extract was probably linked to
its antioxidant activity, as the protective effects of plant extract on DNA can also be
explained by its ability to scavenge reactive oxygen species (ROS) (Yoshikawa et al., 2006).
The hydroxyl radicals have very short life span than any other free radicals and these radicals
attack the DNA double strands (double helical) and break down into single strand (open
circular). The antioxidants play important role in inhibiting the DNA damage by quenching
of free radicals. The phytochemicals such as phenolics have a role in scavenging the free
radicals and thus reduce the DNA damage. The studies also showed that phenolics and
flavonoids from T. cordifolia leaves from different organic solvents posses a major DNA
protection activity due to presence of phytochemicals (Premanath and Lakshmidevi, 2010).
4.8 Phenolic Compounds Analysed by LC-ESI-MS/MS
The liquid chromtography-mass spectrometry analysis (Fig. 4.26) of Russelia equisetiformis
sample showed the presence of phytochemicals such as chlorogenic acid (67), methyl
protocatechuate (68), p-coumaric acid (69), 4-hydroxybenzoic acid (70), gallic acid (71),
caffeic acid (72), caftaric acid (74), syringic acid (75) and catechin (76) (Table 4.26). The
identification of compounds was done after the elution from liquid chromatography (LC)
coupled with ESI-MS/MS. The structures of compounds identified are shown in Fig. 4.27.
The identication of compounds was made by comparison of retention time with the known
standards analysed with same solvent system and conditions. The ESI-MS/MS spectra of
analysed samples were also compared with the standards and data in the literature.
The presence of methyl protocatechuate (68) was also confirmed by comparing the retention
time with standards and ESI-MS/MS pattern in chromatogram (Fig. 4.28). The presence of
peak in negative mode at m/z = 167.2 corresponds to the molecular formula C8H8O4.
111
Fig. 4.26: The chromatogram of Russelia equisetiformis sample analyzed by liquid
chromatography
112
Table 4.26: Chemical compounds in Russelia equisetiformis extract analysed by LC-ESI-MS/MS
Retention Time
(min)
Major MS/MS
m/z (intensity) Molar mass Name of identified Compound
2.14 353.1 (45), 191.1(42), 179(62), 173(100) 354.31 Chlorogenic acid (67)
3.10 167.1 (30), (152.1 (100), 108.0 (11) 168.12 Methyl protocatechuate (68)
3.66 163.05 (45), 119(100) 164.16 p-Coumaric acid (69)
4.60 137.25 (20), 121(60), 93(90) 138.12 4-Hydroxybenzoic acid (70)
4.70 169.15 (40), 125(100) 170.12 Gallic acid (71)
10.40 179.3 (100) 161.1 (10), 135.1 (27) 180.16 Caffeic acid (72)
11.80 311.15 (25), 178 (45), 148 (90) 312.22 Caftaric acid (73)
16.20 197.10 (35), 179.10 (60), 135.10 (100) 198.17 Syringic acid (74)
19.81 289.15 (35), 271.2 (15), 245.1 (45) 290.27 Catechin (75)
113
HO
OH
O
Catechin (75)
O
O
OH
OHOH
O
HO
O
OH
Caftaric acid (73)
OH
O
HO
HO
Caffeic acid (72)
OHO
HO
Gallic acid (71)
OH
OH
OH
OH
OH
O
H3C
OH
O CH3
O
HO
Syringic acid (74)
COOCH3
OH
HO
Methyl protocatechuate (68)
OHO
HO CO2H
O
OHOH
OH
Chlorogenic acid (67)
COOH
p-Coumaric acid (69)
HO
HO
OH
p-Hydroxybenzoic acid (70)
O
Fig 4.27: The structures of chemical compounds in Russelia equisetiformis extract analysed
by LC-ESI-MS/MS
114
Fig 4.28: A representative chromatogram of methyl protocatechuate (68) in Russelia
equisetiformis extract analysed by ESI-MS/MS
COOCH3
O
HO
COO-
O
HO
m/z = 167.2
m/z = 152.1m/z = 108.0
m/z = 168
COOCH3
OH
HO
Methyl protocatechuate (68)
O
HO
-COOCH3
-49
-CH3-15
Fig 4.29: The fragmentation patteren of structures of methyl protocatechuate (68) in Russelia
equisetiformis extract analysed by ESI-MS/MS
115
Fig 4.30: A representative chromatogram of caffeic acid (72) in Russelia equisetiformis
extract analysed by ESI-MS/MS
O
OH
OH
O
m/z = 179.3
OH
OH
OH
O -H2O
-CO2
m/z = 180.01
m/z = 161.1
m/z = 135.1
Caffeic acid (72)
Fig 4.31: The fragmentation patteren of structures of caffeic acid (72) in Russelia
equisetiformis extract analysed by ESI-MS/MS
116
The fragments ions peaks at m/z = 152.1 due to loss of –15 (–CH3) and at m/z = 108.0 due to
loss of –COOCH3 (–49) confirmed the methyl protocatechuate (68) compound (Fig. 4.29).
The caffeic acid (72) has a molecular ion fragment peak in negative mode at m/z =179.3
which corresponds to the molecular formula of caffeic acid C9H8O4 as evident from
chromatogram (Fig. 4.30). The further fragment ion peak at 135.1 was due to loss of –44 [–
CO2]. The other fragment ion at m/z = 161.1 due to loss of –18 [–H2O]. These fragments
confirmed the caffeic acid (72) (Fig. 4.31). The use of mass spectrometry coupled to liquid
chromatography is ideal for the assay of phenolics found in plants. The advantage of Liquid
Chromatography MS/MS is the separation and structural elucidation of compounds can be
obtained in a continuous manner (Kallenbach et al., 2009). Ionization by using electrospray
method is one of the extensively used for LC-ESI-MS/MS studies (Johnson, 2005). In the
ESI negative mode, analysis of small molecules containing free carboxyl groups, yields
mainly the ion [M-H]-, relating to their carboxylate anion (Nishikaze and Takayama, 2007).
The identification of phenolic compounds in Russelia equisetiformis extract was based on
chromatograms obtained by HPLC and EIS-MS/MS and comparison with literature data
(Dinelli et al., 2009).
4.9 Isolated Compounds by Liquid Column Chromatography
The Russelia equisetiformis plant showed better antioxidant and antimicrobial activity due to
this it was put forward for isolation of bioactive constituents. The chemical structure
confirmation of isolated compounds was accomplished by analyzing the samples with
advanced spectroscopic techniques such as ESI-MS, IR, 1H-NMR,
13C-NMR and UV. It was
observed that the isolated compounds were known but first time reported in the Russelia
equisetiformis.
4.9.1 Apigenin (76)
OOH
HO O
OH
1
2
3456
78
9
10
1'2'
3'4'
5'6'
117
1H-NMR (300 MHz), CD3OD, chemical shift δ in ppm, coupling constant J in Hz:
6.75 (1H, s, H-2), 6.20 (1H, d, J=2.1, H-8), 6.12 (1H, d, J=2.0, H-6), 7.66 (2H, d, J=8.6, H-
2', H-6'), 6.85 (2H, d, J=8.6, H-3', H-5'), 10.16 (1H, s, OH-4'), 11.96 (1H, s, O-5H'), 10.65
(1H, s, OH-7'.
13C-NMR (75 MHz), CD3OD, chemical shift δ in ppm: 164.1 (C-2),104.1 (C-3), 182.1 (C-4),
160.3 (C-5), 96.8 (C-6), 163.8 (C-7), 94.2 (C-8), 162.6 (C-9),104.2 (C-10), 122.1 (C-1'),
128.7 (C-2'), 116.5 (C-3'), 159.2 (C-4'), 116.5 (C5'), 128.7 (C-6').
ESI-MS m/z (intensity): 269 (100), 158 (45).
IR (KBr), cm-1
: 1172, 1616, 3144, 3298.
The fraction was yellow crystalline solid with melting point = 347-349 °C. The flavone
structure was supported by the UV spectrum due to absorption at λmax = 267 nm and 335 nm
of methanolic solution of apigenin (76). The mass spectrum in negative mode showed a
stronger molecular ion peak at m/z = 269 which leads to molecular formula C15H10O5 and the
fragmnet ion peak at m/z = 158. The IR spectra showed that there are no intensive bands for
glycoside bond in the 1100-1000 cm-1
. The intensive band at 1616 cm-1
is most probably the
result of C=O group from the central heterocyclic ring, while the C-O vibration occured at
approximately 1172 cm-1
. In the spectrum a band found, at approximatly 3144 cm-1
, that was
most probably the result OH of phenol groups. The 1
H-NMR and 13
C-NMR data for apigenin
(76) were in good agreement with that of apigenin (Breitmaier and Voelter, 1989).
4.9.2 Ursolic acid (77)
Ursolic acid (77) was isolated as colorless needles, from the ethyl acetate soluble fraction of
Russelia equisetiformis.
118
1
3
5 7
9
11
13
15
17
21
30
24
2526
27
28
29
23
2
4
810
6
12
16
18
20
HOCH3
CH3 COH
O
H3C
H3C
H3C
CH3
CH3
1H-NMR (300 MHz), CD3OD, chemical shift δ in ppm, coupling constant J in Hz: 5.25 (m,
1H, H-12), 3.12 (m, 1H, H-3), 2.33 (d, 1H, J=11.1 Hz, H-18), 2.01-1.13 (m, H-22), 1.04 (s,
3H, H-23), 0.99 (s, 3H, H-27), 0.93 (s, 3H, H-26), 0.90 (s, 3H, H-24), 0.89 (d, 3H, H-29),
0.84 (d, 3H, H-30), 0.81 (s, 3H, H-25).
13C-NMR (75 MHz), CD3OD, chemical shift δ in ppm: 179.8 (C-28), 138.9 (C-13), 125.7 (C-
12). 78.9 (C-3), 55.9 (C-5), 54.1 (C-18), 48.3 (C-17), 47.8 (C-9), 42.5 (C-14), 39.6 (C-8),
39.4 (C-20), 39.1 (C-19), 38.9 (C-4), 38.6 (C-1), 38.1 (C- 10), 37.0 (C-22), 34.1 (C-7), 30.9
(C-21), 29.1 (C-15), 28.9 (C-2), 27.6 (C-23), 26.0 (C-24); 25.4 (C-16), 25.2 (C-11), 24.8 (C-
27), 20.5 (C-30), 19.2 (C-6), 17.3 (C-29), 17.2 (C-26), 16.1 (C-25).
ESI-MS m/z (intensity): 456.3 (100), 411.3 (35), 248.1 (25), 219 (28.7), 207 (27), 203.1
(42.6), 189 (10.1), 133 (32.2), 119 (11.3), 69 (13.3).
IR (KBr); cm-1
: 3750, 3405, 1692, 1540 1272, 1092, 996.
The fraction was slightly greenish powder with melting point = 284-285 °C. The molecular
formula C30H48O3 was established through ESI-MS showing molecular ion peak at m/z 456.3
(calculated for C30H48O3, 456.360). Further diagnostic peaks at m/z 411.3 representing the
loss of COOH group. Another prominent peak at m/z 248.1 represented Retro Diels-Alder
fragmentation, characteristic of ursane type triterpenes with COOH group at C-17
(Budzikiewicz et al., 1963). The peak, at m/z 203.1 was attributed to the loss of COOH from
the fragment at m/z 248.1. The structure was supported by the UV spectrum due to absorption
at λmax = 470 nm, 443 nm and 422 nm of methanolic solution of ursolic acid (77). The 1H-
NMR and 13
C-NMR data was in agreement with ursolic acid.
119
The IR spectrum showed absorptions at 3405 cm-1
(hydroxyl group) and 1692 cm-l for
carbonyl group. Comparison of the 13
C-NMR signals with the literature values especially
those for C-12, C-13, C-18, C-19, C-20, C-23, C-24, C-25, C-29 and C-30 indicated that the
compound was ursolic acid. This conclusion was further supported by the 1H-NMR spectrum
where a doublet at 2.20 ppm with J = 11.3 Hz indicated that H-18 and H-19 are trans to one
another.
4.9.3 Rutin (78)
O
OH
HO O
O
A B
C12
3
456
78
9
10
1'
2'
3'4'
5'6'
OO
HOHH
OH
HO
HOHOH
H
OHCH3
HHO
OH
OH
1"
2"3"
4"
5"6"
1"'
2"'3'"
4"'
5"'
1H-NMR (300 MHz), CD3OD, chemical shift δ in ppm, coupling constant J in Hz: 9.61 (1H,
s, OH-4'); 9.61 (1H, s, OH-3'); 11.98 (1H, s, OH-5'); 10.84 (1H, s, OH-7); 6.24 (1H, d, J=2,
H-6); 6.42 (1H, d, J=2, H-8); 7.53 (1H, d, J=2.1, H-2'); 6.88 (1H, d J=9, H-5'); 7.54 (1H, dd,
J=9,2.1, H-6'); 5.65 (1H, d, J=7.4, H-1''); 5.08 (1H, d, J=1.9, H-1'''); 1.15 (3H, d, J=6.1, Me
C-5''').
13C-NMR (75 MHz), CD3OD, chemical shift δ in ppm: 150.9 (C-2), 135.2 (C-3), 177.6 (C-
4), 160.1 (C-5), 98.5 (C-6), 165.3 (C-7), 93.2 (C-8), 159.7 (C-9), 103.2 (C-10), 121.3 (C-1'),
117.2(C-2'), 144.4 (C-3'), 148.2 (C-4'), 116.6 (C-5'), 121.3 (C-6'), 101.8 (C-1''), 74.7 (C-2''),
76.9 (C-3''), 71.8 (C-4''), 77.1 (C-5''), 68.1 (C-6''), 103.1 (C-1''), 71.2 (C-2''), 72.0 (C-3''), 72.1
(C-4'''), 69.3 (C-5'''), 17.9 (C-6''').
ESI-MS, m/z (intensity): 610.1 (25), 609.2 (30), 301 (90).
IR (KBr); cm-1
: 3430, 1656 and 1296.
The fraction was crystalline solid with melting point = 241-242 °C. The structure was
supported by the UV spectrum due to absorption at λmax = 205 nm, 255 nm and 355
120
methanolic solution of rutin (78). The ESI-MS mass spectrum showed a stronger molecular
ion peak at 610.1 and the fragmnet ion peaks at m/z 609.2 and 301 which correspond to
molecular formula C27H30O16. The 1H-NMR and
13C-NMR data was in agreement with rutin.
The presence of 1H-NMR signals at chemicals shift (ppm): 6.24 (d, H-6); 6.42 (d, H-8); 7.53
(d, H-2'); 6.88 (d, H-5') and 7.54 (dd, H-6') was indication of rutin (3). The presence of signal
at 5.65 (d, H-1'') showed the occurrence of glucose moiety. The occurrence of signals at 5.08
(d, H-1''') and 1.15 (3H, d, Me, C-5''') indicated the rhamnose moiety.
The presence of 13
C-NMR signals at chemicals shift (ppm): 98.5 (C-6), 93.2 (C-8), 117.2 (C-
2'), 116.6 (C-5'), 121.3 (C-6') confirmed rutin (3). Further the presence of signals at 101.8 (C-
1''), 68.1 (C-6'') for glucose moiety and 103.1 (C-1'''), 17.9 (C-5''') represents the rhamnose
moiety. From the above data the structure of rutin (3) was confirmed. The IR spectra showed
the occurrence of peaks at 3,430 (OH), 1,656 (C=O) and 1,296 cm-1 (C-O-C) supported the
structure of rutin.
4.9.4 Chlorogenic acid (79)
1 2
34
6
7
1'
3' 4'
5'
6'
8'9'
COOHHO
HO
OH
O
O
OH
OH
7'
1H-NMR (300 MHz), CD3OD, chemical shift δ in ppm, coupling constant J in Hz: 12. 3 (1H,
d, J=16 Hz, H-7'), 9.42 (1H, s, OH-4'), 9.38 (1H, s, OH-3'), 7.43 (1H, d, J=16 Hz, H-7'), 7.13
(1H, s, H-2'), 6.93 (1H, dd, J=8.0, 2.0 Hz, H-6'), 6.81 (1H, d, J=8.0 Hz, H-5'), 6.15 (1H, d,
J=16.0 Hz, H-8'), 5.06 (1H, ddd, J=10.0, 6.0 Hz, H-3), 3.92 (1H, s, H-5), 3.42 (1H, s, H-4),
2.03 - 1.77 (4H, m, H-2 and H-6).
13C-NMR (75 MHz), CD3OD, chemical shift δ in ppm: 176.4 (C-7 for (COOH)), at 164.9 (C-
9' for OCOCH=CH-), 146.9 (C-4'), 145.8 (C-7'), 145.1 (C-3'), 125.7 (C-1'), 121.6 (C- 6'),
116.9 (C-5'), 114.8 (C-8'), 114.5 (C-2'), 74.5 (C-1), 71.2 (C-4), 70.2 (C-5), 69.0 (C-3), 36.9
(C-6) and 36.8 (C-2).
IR (KBr); cm-1
: 3421, 2929, 1697, 1635, 1456, 1398, 1278, 1182 and 812.
121
ESI-MS m/z (intensity): 354.3 (25), 353.1 (10), 191.1 (70), 179.4 (32).
The fraction was slightly yellow amorphous powder with melting point = 208-209 °C. The
ESI-MS data showed a stronger molecular ion peak at 354.3 which corresponds to molecular
formula C16H18O9. The further fragmnet ion peaks at 353.1, 191.1 and 179.4 confirmed the
presence of chlorogenic acid (79). The structure was also supported by the UV spectrum due
to absorption at λmax = 325 nm of methanolic solution of chlorogenic acid (4). The 1H-NMR,
and 13
C-NMR data was in agreement with chlorogenic acid.
The signals for protons like 9.42 (1H, s, H-4'), 9.38 (1H, s, OH-3'), 7.43 (1H, d, H-7'), 7.13
(1H, s, H-2'), 6.93 (1H, dd, H-6'), 6.81 (1H, d, H-5'), 5.06 (1H, ddd, H-3), 3.92 (1H, s, H-5),
2.03 - 1.77 (4H, m, H-2 and H-6). Indicated the chlorogenic acid (4), The 13
C-NMR spectra
showed two carbonyl carbons at 176.4 (C-7) and 164.9 (C-9’). Further there were 8 signals at
146.9 (C-4'), 145.8 (C-7'), 145.1 (C-3'), 125.7 (C-1'), 121.6 (C- 6'), 116.9 (C-5'), 114.8 (C-8'),
114.5 (C-2') ppm described the presence of six aromatic and two olefenic carbons. The 6
signals of carbons from the cyclohexane ring appeared at 74.5 (C-1), 71.2 (C-4), 70.2 (C-5),
69.0 (C-3), 36.9 (C-6) and 36.8 (C-2).
4.9.5 Ferulic acid (80)
1
23
4
5
6
1'
2'3'
4'
HO
H3COOH
O
1H-NMR (300 MHz), CD3OD, chemical shift δ in ppm, coupling constant J in Hz: 11.98
(broad s, OH-3'), 9.80 (broad s, OH-4'), 7.45 (1H, d, J=14 Hz, H-1'), 7.12 (1H, dd J=6 Hz and
2 Hz, H-5), 7.06 (1H, d, J=1 Hz, H-3), (1H, dd, J=8 and 2 Hz, H-5), 7.08 (1H, d, J=2 Hz, H-
3), 6.32 (1H, d, J=14 Hz and 2 Hz, H-2'), 6.91 (1H, d, J=8 Hz, H-6) and 3.96 (3H, s, H-4').
13C-NMR (75 MHz), CD3OD, chemical shift δ in ppm: 149.8 (C-1), 134.3 (C-2), 113.4 (C-
3), 121.3 (C-4), 110.5 (C-5), 119.05 (C-6), 145.7 (C-1'), 115.7 (C-2'), 172.1(C-3') and 55.2
(C-4').
ESI-MS m/z (intensity): = 194.18 (20), 193.2 (45), 178 (21), 149 (55).
122
IR (KBr), cm-1
= 3440, 1680, 1600, 1505, 1270 and 940.
The fraction was crystalline powder with melting point = 170-172°C. The mass spectrum
showed a stronger molecular ion peak at m/z 194.18 which corresponds to molecular formula
C10H10O4. The fragmnet ion peaks at 193.23, 178 and 149 confirmed the ferulic acid (80). The
structure was also supported by the UV spectrum due to absorption at λmax = 278 nm and 321
nm by methanolic solution of ferulic acid (80).
The IR spectrum with the peaks at 3440 cm-1
(carboxylic acid O-H stretching), 1680 cm-1
(carboxylic acid C=O stretching), 1270 cm-1
(carboxylic acid C-O stretching) and 1505;
1600 cm-1
(aromatic C=C) confirms the skeleton of ferulic acid (80).
1H-NMR spectrum displayed the characteristic signal for a methoxy group showed singlet at
chemical shift 3.96. The chemical compound spectrum also exhibted three aromatic protons
at chemical shift 6.91 (H-6), 7.12 (H-5) and 7.06 (H-3), characteristics for the aromatic part
of isolated compound (80). The presence of further two proton doublets with J= 14 Hz at
chemical shift 7.45, 6.32 and indicated in the presence of H-1' and H-2' in the side chain of
compopund respectively. The 13
C-NMR spectrum indicated the occurrence of 10 signals (4
aliphatic chain carbons and 6 aromatic carbons) in agreement with the proposed structure of
ferulic acid. Due to the phenolic nucleus and extended conjugated side chain, ferulic acid is
an antioxidant (Graf, 1992).
4.10 Antioxidant Activity of Isolated Compounds Using Sunflower Oil as
Oxidative Substrate
The isolated compounds were put forward for the antioxidant studies by using sunflower oil
as oxidative substrate. Yen and coworker shaowed that the phenolic compounds were linked
with antioxidant potential and play a significant function in stabilizing lipid peroxidation
(Yen et al., 1993).
4.10.1 Peroxide Value (PV)
Fig. 4.32 showed the peroxide values of sunflower oil untreated (control) and treated with
isolated compounds (76-80) of Russelia equisetiformis. At the end of the storage period of 70
days, the control had the highest level of PV (relative to the initial value), indicating a higher
extent of primary oxidation. The decresase in peroxide values after 70 days of storage was
123
found to be in the order: control > ferulic acid (80) > chlorogenic acid (79) > apigenin (76) >
rutin (78) > ursolic acid (77). The lesser is the peroxide values after the storage period of 70
days, greater is the antioxidant activity. So the samples of sunflower oil stabilized with
isolated ursolic acid (77) showed the maximum antioxidant activity. The variations in
peroxide value of stabilized sunflower oil were found to be significant (P ≤ 0.05) for the
incubation period and PV values among the isolated compounds. The peroxide value is a
good parameter used to analyze the degree of primary oxidation products in the prersnce of
antioxidants (Mcginely, 1991).
4.10.2 Free Fatty Acids (FFA)
The free fatty acid formation increased in the sunflower oil with an increase in storage period
(Fig. 4.33). The control sample of the sunflower oil (without antioxidant) showed the higher
FFA value, while the oil sample with ursolic acid (77) exhibited the lowest FFA values
(Figure 4.31). The variations in free fatty acids constituents of stabilized sunflower oil were
found to be significant (P ≤ 0.05) for incubation period and FFA values among the isolated
compounds (76-80) analysed. The free fatty acids (FFA) were considered as a significant
measure of deterioration of food products. The free fatty acids were formed by the hydrolysis
of triglycerides and may increase by reaction of edible oil with oxygen (Frega et al., 1999).
4.10.3 Conjugated Dienes (CD) and Conjugated Trienes (CT)
Among the tested samples, ursolic acid (77), showed lower values of CD and CT reflecting
its better antioxidant activity for stabilization of sunflower oil against oxidation. However
among the tested samples, ferulic acid (80), showed higher values of CD and CT reflecting
its lower antioxidant activity for stabilization of sunflower oil against oxidation (Fig. 4.34
and 4.35 respectively). The results were found to be statistically significant (P ≤ 0.05). The
estimation of conjugated dienes (CD) and conjugated trienes (CT) are valuable indications
for the early stages of peroxidation in the study of edible oils (Dekkers et al., 1996).
After 70 days, the untreated sample of sunflower oil (control) showed higher levels of these
oxidation products. The sample treated with isolated compounds (76-80) showed the lowest
increase in conjugated dienes and trienes values. During the period of the experiment, the
contents of CD and CT in oils increased in control sample without isolated compounds;
124
however these levels were considerably lower for samples treated with Russelia
equisetiformis isolated compounds. The control exhibited higher levels of these oxidation
products predicting that it had undergone extensive oxidative deterioration.
4.10.4 para Anisidine Value
The results of para-anisidine assay, which typically described the formation of aldehydic
secondary oxidation products in the edible oils. The control sunflower oil showed the
maximum increase in para-anisidine value indicating a higher rate for the formation of
secondary oxidation products. The smallest increase in para-anisidine value of the oils was
observed with ursolic acid (77), while the maximum increase was with ferulic acid (80) (Fig.
4.36). The smaller the para-anisidine values during the storage period, better is the
antioxidant activity oils (Mcginely, 1991; Anwar et al., 2006). There was a significant
difference (P ≤ 0.05) among the para-anisidine values PV values among the isolated
compounds (76-80) analysed.
125
Fig. 4.32: Peroxide value (meq/kg) of sunflower oil stabilized with compounds isolated from
Russelia equisetiformis
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
PV
(m
eq/K
g)
Incubation period (days)
Control
Apigenin (76)
Ursolic acid (77)
Rutin (78)
Chlorogenic acid (79)
Ferulic acid (80)
126
Fig. 4.33: Free fatty acid (percent oleic acid) of sunflower oil stabilized with compounds
isolated from Russelia equisetiformis
0.0
0.2
0.4
0.6
0.8
1.0
0 10 20 30 40 50 60 70
FFA
(%
)
Incubation period (days)
Control
Apigenin (76)
Ursolic acid (77)
Rutin (78)
Chlorogenic acid (79)
Ferulic acid (80)
127
Fig. 4.34: Conjugated dienes of sunflower oil treated with compounds isolated from Russelia
equisetiformis
0
2
4
6
8
10
12
14
16
18
0 10 20 30 40 50 60 70
Conju
gat
ed d
ienes
Incubation period (days)
Control
Apigenin (76)
Ursolic acid (77)
Rutin (78)
Chlorogenic acid (79)
Ferulic acid (80)
128
Fig. 4.35: Conjugated trienes of sunflower oil treated with compounds isolated from Russelia
equisetiformis
0
2
4
6
8
10
12
14
16
0 10 20 30 40 50 60 70
Conju
gat
ed t
rien
es
Incubation period (days)
Control
Apigenin (76)
Ursolic acid (77)
Rutin (78)
Chlorogenic acid (79)
Ferulic acid (80)
129
Fig. 4.36: para-Anisidine value of sunflower oil treated with compounds isolated from
Russelia equisetiformis
0
2
4
6
8
10
12
0 10 20 30 40 50 60 70
para
ansi
din
e val
ue
Incubation period (days)
Control
Apigenin (76)
Ursolic acid (77)
Rutin (78)
Chlorogenic acid (79)
Ferulic acid (80)
130
Conclusion
From the results it is conlcuded that the research work was carried out in a biotargeted assay.
After the collection of selected plants the percentage yields of plants extracts and fractions
was studied. The phytochemical analysis of plants extracts and fractions showed the presence
of alkaloids, tannins, steroids, saponins and terpenoids. The plants extracts, fractions and
essential oils showed considerable antioxidant activity when analyzed by DPPH free radical
scavenging assay, reducing power and % inhibition by linoliec acid oxidation. The
antioxidant activity of essential oils was better than that of extracts and fractions of selected
plants studied.
The essential oils of these plants were extracted by clavenger type apparatus and further
analyzed by GC-MS. The percentage yield of essential oils from selected plants in this study
was found in the range of 0.21 to 0.46%. The major chemical compounds found in selected
plants essential oils were indentified. From the results it was observed that antioxidant and
antimicrobial activities of the Russelia equisetiformis essential oil was greater than those of
other plants used in present research work. The plants extracts, fractions and essential oils
were assayed for cytotoxicity study by haemolytic activity against human blood erythrocytes
(RBCs). From the results, it was observed that the selected plants have a minor cytotoxity as
compared to the positive control. The highest percentage lysis of RBCs was observed in
Anticharis linearis absolute methanol extract (7.25%) and lowest for mazus goodenifolius
essential oil (0.96%).
The results of antimicrobial activity using disc diffusion assay by selected plants extracts,
fractions and essential oils showed considerable potential against pathogenic microorganisms
like bacterial strains such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus and
Pasturella multocida. The findings of antimicrobial activity also showed that the selected
plants possess considerable potential against fungal strains such as Aspergillus niger,
Aspergillus flavus, Alternaria alternata and Rhizopus solani.
From the initial screening of plants by antioxidant and antimicrobial studies it was observed
that Russelia equisetiformis leaves showed better activities. So Russelia equisetiformis was
put forward for further studies like protection to DNA damaging and isolation of
phytoconstituents by column chromatography. The protective effect Russelia equisetiformis
131
extract, fractions and essential oil by H2O2 induced oxidative damage in plasmid pBR322
DNA was evaluated and found that it protected the DNA due to its antioxidant activity.
Hydroxyl radicals are mainly involved in DNA damage and hence leading to abnormalities in
DNA functions. The quenching of these free radicals by antioxidants might be a way to
protect DNA.
The liquid chromtography-mass spectrometry analysis of Russelia equisetiformis sample
showed the presence of phytochemicals such as chlorogenic acid (67), methyl
protocatechuate (68), p-coumaric acid (69), 4-hydroxybenzoic acid (70), gallic acid (71),
caffeic acid (72), caftaric acid (73), syringic acid (74) and catechin (75).
The Russelia equisetiformis leaves showed better antioxidant and antimicrobial activity due
to this it was put forward for isolation of bioactive constituents. The isolation of
phytochemicals in ethyl acetate, n-butanol and chloroform soluble fractions showed the
presence of compounds such as apigenin (76), ursolic acid (77), rutin (78), cholorogenic acid
(79) and ferulic acid (80). The confirmation of the chemical structure was accomplished by
analyzing the samples with advanced spectroscopic techniques such as 1H-NMR,
13C-NMR,
IR and ESI-MS. It was observed that the isolated compounds were known but first time
reported in the Russelia equisetiformis. The antioxidant acitivity of the isolated compounds
by using the sunflower oil as oxidative substrate was carried out and considerable antioxidant
potential was found in the isolated compounds.
132
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List of Publications
Journal Articles from PhD Research Work
1. Muhammad, Riaz, Nasir Rasool, Iftikhar Hussain Bukhari, Muhammad Shahid,
Muhammad Zubair, Komal Rizwan and Umer Rashid. 2012. In vitro Antimicrobial,
Antioxidant, Cytotoxicity and GC-MS Analysis of Mazus goodenifolius. Molecules, 17:
14275–14287.
2. Muhammad Riaz, Nasir Rasool, Iftikhar Hussain Bukhari, Komal Rizwan, Muhammad
Zubair, Fatima Javed, Atif Ali Altaf and Hafiz Muhammad Abdul Qayyum. 2013.
Antioxidant, Antimicrobial Activity and GC-MS Analysis of Russelia equsetiformis
Essential Oils. Oxidation Communications, 36:1: 272–282.
3. Muhammad Riaz, Nasir Rasool, Iftikhar Hussain Bukhari, Muhammad Shahid, Ameer
Fawad Zahoor, Mazhar Amjad Gilani, and Muhammad Zubair. 2012. Antioxidant,
Antimicrobial and Cytotoxicity Studies of Russelia equisetiformis. African Journal of
Microbiology Research, 6(27): 5700–5707.
4. Muhammad Riaz, Nasir Rasool, Shahid Rasool, Umer Rashid, Iftikhar Hussain
Bukhari, Muhammad Zubair, Mnaza Noreen, and Mazhar Abbas. 2013. “The Chemical
Analysis, Cytotoxicity and Antimicrobial Studies by Snapdragon a Medicinal Plant.”
Asian Journal of Chemistry 25:10: 5479-5482.
5. Muhammad Riaz, N. Rasool, I.H. Bukhari, M. Zubair, M. Shahid, T.H. Bokhari, Y.
Gull, K. Rizwan, M. Iqbal, and M. Zia-Ul-Haq. 2013. “Antioxidant Studies Using
Sunflower Oil as Oxidative Substrate and DNA Protective Assay by Antirrhinum
majus.” Oxidation Communications, 36:2: 542–552.