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PROJECT TOPIC AND MATERIAL ON Studies on the Chemical Constituents of the Leaves and Seeds of Hyptis Spicigera Lam

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ABSTRACT

The extracts of the leaves of H.spicigera were screened for the presence of secondary
metabolites: alkaloids, glycosides, flavonoids, carbohydrates, tannins, sterols, terpenoids
and resins. The results of the phytochemical screening showed all the extracts with the
exception of hexane extract to contain the secondary metabolites analysed in high and
moderate amounts. Antimicrobial activities of the crude extracts of H.spicigera against a
broad spectrum of micro organisms namely: Staphylococcus aureus, Streptococcus
pyogenes, Bacillus subtilis, Corynebacterium ulcerans, Salmonella typii, Escherichia
coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Neisseria gonorrheae and
Candida albicans were carried out. All the extracts showed bactericidal activity against
the entire antibiotic resistant micro organisms tested with some degree of variations
against standard drug, penicillin. The methanolic extract of H.spicigera exhibited
significant bactericidal activity at low concentration of 2.5mg/ml while S.aureus,
N.gonnorhea and C.albicans were resistant at MBC of 5.0mg/ml. The insecticidal
properties of H.spicigera leaf extracts (hexane, ethyl acetate and methanol) tested against
Callosobruchus maculatus on cowpea was carried out. Azadirachtin was used as
standard check along with the extracts tested. Ethyl acetate extract showed the highest
percent mortality of 98% each at 72hrs, while 6% and 10% were recorded for the
emergence of the C.maculatus. Ethyl acetate extract and azadirachtin standard check
showed ovi-position deterrent at 4.16% and 2.78% respectively. Hexane extract showed
the least percent (2%) seed damage while the control gave the highest percent (88%) of
seed damage at 16 weeks. Hyptis spicigera seed oil was characterized using GC-MS and
UV-VIS spectrometry for its fatty acid profile, tocopherol and physicochemical
properties. The oil content was 21% while unsaturated fatty acids were linoleic acid
(71.85 %) and palmitic acid (16.06%) as predominant fatty acids. Tocopherol content
was 186.15mg/ml while Vitamin A was absent. The study showed potentials of Hyptis
spicigera seed oil to have high oxidative stability which could be suitable for food and
beverage as well as other industrial applications while the tocopherol content could
improve human health. Purification of the ethyl acetate fraction by High Performance
Liquid Chromatography (HPLC) yielded 3-buten-2-enol on the basis of GC-MS. The best
separation using the analytical High Performance Liquid Chromatography (HPLC) was
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achieved at 0.8ml/min. The hydrodistillation of the fresh leaves of H.spicigera gave a
colourless volatile oil with yield of 0.65%.The volatile oil gave forty compounds on the
basis of GC-MS with low composition of cineole (4.11%) and caryophyllene (2.61%)
while α-pinene (12.16%) β-pinene (9.47%) and α-phellandrene (10.19%) were
predominant compounds. Crude ethyl acetate extract of H.spicigera leaves was
fractionated through solvent separation followed by thin layer and column
chromatography. Two relatively pure constituents were obtained and characterized
through spectral studies. The structure elucidation of the constituents was established as
ursolic acid and its derivative (3β-hydroxy-urs-12-en-28-oic acid) and 3β-hydroxy-20-
isopropyl-urs-12-an-28-oic using spectroscopic techniques including 1D and 2D NMR
such as 1HNMR, 13CNMR, DEPT, HMQC, HMBC, COSY, NOESY as well as MS, and
IR. Toxicity studies on the dichloromethane and methanolic extracts of Hyptis spicigera
were carried out on mice intraperitoneally. The LD50 was calculated using the method of
Karber (LD50, 2534mg/kg) which showed the plant to be moderately toxic based on the
WHO toxicity rating. The biological activity of one of the new compounds (ursolic acid)
was determined against a trypanosome (T.brucei brucei).The compound was evaluated in
vitro for activity against Trypanosoma brucei brucei (Tbb) and was found to possess antitrypanosoma
activity in vitro in a dose dependent pattern at 0.4μg/ml in 3 minutes.

TABLE OF CONTENTS

Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Abstract vii
Table of contents ix
CHAPTER ONE
1.0 Introduction 1
1.2 Aim 14
1.3 Statement of the problem 15
1.4 Significance of the study 16
1.5 Justification for the study 16
1.6 Statement of research hypothesis/research question 18
CHAPTER TWO
2.0 Literature Review 19
2.1 Botanical description of H. spicigera 19
2.3 Morphological description of H.spicigera plant 20
2.4 Uses of H.spicigera 20
2.5 Terpenoidal constituents 22
2.6 Biological and ethnopharmacological applications 39
2.8 Pesticidal properties 48
x
2.9 Other essential oils from plants 52
CHAPTER THREE
3.0 Materials and Methods 64
3.2.1 Instrumentation 64
3.2.3 Spectroscopy 65
3.2.4 Chromatography 65
3.3 Collection of the plant material 65
3.3.1 Extraction and isolation 65
3.3.2 Lipid extraction 66
3.3.3 Physicochemical analysis of H.spicigera seed oil 66
3.3.4 Preparation of the fatty acid methyl ester for GC-MS analysis 67
3.3.5 GC-MS analysis of derivatised oil 67
3.3.6 Extraction of the volatile oils and GC-MS analysis 67
3.4 Microorganisms and media 68
3.4.1 Antimicrobial studies 69
3.4.2 Minimum Bactericidal concentration (MBC) 69
3.5 Phytochemical screening of H.spicigera leaf extracts 70
3.10 Biological activity of sample ZL-14 77
3.10.2 Determination of LD50 78
3.11 Insecticidal evaluation of the plant extracts 79
3.12 Chromatographic Separations 80
3.12.1 Thin layer chromatography 80
3.12,2 Packing of column 80
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CHAPTER FOUR
4.0 Results and Discussion 81
4.1 Yields of extracts 81
4.2 Phytochemical screening results 83
4.3 Antimicrobial activities of H.spicigera 91
4.4 Physicochemical properties of seed oil 93
4.5 Fatty acid profile of H.spicigera seed oil 95
4.6 Insecticidal evaluation of extracts 97
4.7 Purification of ethylacetate fraction by HPLC 103
4.8 GC-MS analysis of the volatile oils of H.spicigera leaves 105
4.9 Chromatographic separation 109
4.10 Structural elucidation ZL-14 113
4.11 Structural elucidation EL-2 118
4.12 Toxicity evaluation of ethylacetate and methanolic extracts 121
4.13 Evaluation of the biological activity of ursolic acid derivative
against trypanososmes 122
CHAPTER FIVE
5.0. Summary, Conclusion and suggestion for further work 125
References 128
Appendices 154
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LISTS OF TABLES
1.0 Traditional plants with their uses 11
2.0 Diterpenes from Genus Hyptis 44
2.1 The chemical composition of essential oils from Hyptis suaveolens 61
4.0 Percentage extraction of the leaf part of H.spicigera 81
4.1 Phytochemical screening of the extracts of H.spicigera 81
4.2 Sensitivity tests of the extracts against some selected pathogenic
microorganisms 88
4.3 Zone of Inhibition of the extracts against the microorganisms 89
4.4 Minimum Inhibitory Concentration (MIC) 90
4.5 Physicochemical properties of H.spicigera seed oil 93
4.6 Fatty acid profile of the H.spicigera seed oil 94
4.7 Percent mortality and adult emergence of Callosobruchus maculates 97
4.8 Oviposition deterrent of extracts of H.spicigera on C.maculatus 98
4.9 Percent seed damage to cowpea 99
4.10 Purification of ethylacetate fraction by HPLC 103
4.11 Percentage composition of the essential oils of H.spicigera 105
4.12 Chromatographic separations using thin layer chromatography 109
4.13 Column chromatography of crude ethylacetate fractions 110
4.14 Column chromatography of fraction HF4 111
4.15 13CNMR and DEPT assignments of sample ZL-14 113
4.16 13CNMR and DEPT assignments of sample EL-2 118
4.17 Experimental results of toxicity tests 121
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APPENDICES
Lists of Figures:
Fig.1 A typical mature Hyptis spicigera plant 21
Fig.2 Regression data 155
Fig.3 Coefficient constants 156
Fig.4 1HNMR spectrum of sample ZL-14 157
Fig.5 13CNMR spectrum of sample ZL-14 158
Fig.6 DEPT spectrum of sample ZL-14 159
Fig.7 HMBC spectrum of sample ZL-14 160
Fig.8 HSQC spectrum of sample ZL-14 161
Fig.9 NOESY spectrum of sample ZL-14 162
Fig.10 COSY spectrum of sample ZL-14 163
Fig.11 FT-IR spectrum of sample ZL-14 164
Fig.12 MS spectrum of sample ZL-14 165
Fig.13 GC-MS of fatty acid profile 166
Fig.14 GC-MS profile of the volatile oils of H.spicigera 167
Fig 15 HPLC spectrum of EAE at flow rate of 0.2ml/min. 168
Fig.16 HPLC spectrum of EAE at flow rate of 0.3ml/min. 169
Fig.17 HPLC spectrum of EAE at flow rate of 0.4ml/min. 170
Fig.18 HPLC spectrum of EAE at flow rate of 0.6ml/min. 171
Fig.19 HPLC spectrum of EAE at flow rate of 0.8ml/min. 172
Fig.20 HPLC spectrum of EAE at flow rate of 1.0ml/min. 173
Fig.21 GC spectrum of ethylacetate eluate from HPLC 174
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Fig.22 1HNMR spectrum of sample EL-2 175
Fig.23 13CNMR spectrum of sample EL-2 176
Fig.24 DEPT spectrum of sample EL-2 177
Fig.25 MS spectrum of sample EL-2 178
Fig.26 FT-IR spectrum of sample EL-2 179
Publications from this work 180
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Abbreviations
CDCl3 Deuterated chloroform
CD3OD Deuterated methanol
C5D5N Deuterated pyridine
(CD3)2CO Deuterated acetone
CHCl3 Chloroform
CH2Cl2 Dichloromethane
COSY Correlation Spectroscopy
d Doublet (NMR)
DEPT Distortionless enhancement by polarisation transfer
DMSO Dimethyl sulfoxide
DMSO-D6 Deuterated Dimethyl sulphoxide
EI Electronic impact
EtOAc Ethyl acetate
EtOH Ethanol
FT-IR Fourier transform Infrared
HIV Human immunodeficiency virus
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum coherence
HPLC High Performance Liquid Chromatography
m Multiplet (NMR)
Me Methyl
MeOH Methanol
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Min Minute
M pt. Melting point
MS Mass spectrometry
NMR Nuclear magnetic resonance
PE Petrol ether
ppm Parts per million
PTLC Preparative thin layer chromatography
Rf Retention factor
s Singlet (NMR)
t Triplet (NMR)
TLC Thin Layer Chromatography

CHAPTER ONE

INTRODUCTION
1.1 Background to the study
Natural products can either be of pre-biotic origin or originate from microbes,
plants, or animal sources (Nakanishi, 1999). As chemicals, natural products include such
classes of compounds as terpenoids, polyketides, amino acids, peptides, proteins,
carbohydrates, lipids, nucleic acids, ribonucleic acid (RNA), deoxyribonucleic acid
(DNA), and so forth. Natural products do not just occur by accident or products of
convenience of nature, but more than likely are a natural expression of the increase in
complexity of organisms (Jarvis, 2000). Interest in natural sources to provide treatments
for pain palliatives, or curatives for a variety of maladies or recreational use reaches back
to the earliest points of history. Nature has provided many things for humankind over the
years, including the tools for the first attempts at therapeutic intervention (Nakanishi,
1999). The Nei Ching is one of the earliest health science anthologies ever produced and
dates back to the thirtieth century B.C. (Nakanishi, 1999). Some of the first records on
the use of natural products in medicine were written in cuneiform in Mesopotamia on
clay tablets and date to approximately 2600 B.C. (Cragg and Newman, 2001; Nakanishi,
1999). Indeed, many of these agents continue to exist in one form or the other to this day
and are used for the treatment of inflammation, influenza, cough, and parasitic infestation
among others. Chinese herb guides document the use of herbaceous plants as far back in
time as 2000 B.C (Holt and Chandra, 2002).
For a variety of different reasons, the interest in natural products continues to this
very day (Barron and Vanscoy, 1993; Bhattaram et al., 2002; Chan, 1995; Holt and
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Chandra, 2002; Koh and Woo, 2000; Marriott, 2001). The first commercial pure natural
product introduced for therapeutic use is generally considered to be the narcotic
morphine, marketed by Merck in 1826 (Newman et al., 2000). The first semi-synthetic
pure drug based on a natural product, aspirin, was introduced by Bayer in 1899.
The World Health Organization estimates that approximately 80 percent of the
world’s population relies primarily on traditional medicines as sources for their primary
health care (Farnsworth et al, 1985). Over 100 chemical substances that are considered to
be important drugs that are either currently in use or have been widely used in one or
more countries in the world have been derived from a little under 100 different plants.
Approximately 75 percent of these substances were discovered as a direct result of
chemical studies focused on the isolation of active substances from plants used in
traditional medicine (Cragg and Newman, 2001).The number of medicinal herbs used in
China in 1979 has been estimated to be about 5267 (Nakanishi,1999).More current
statistics based on prescription data from 1993 in the United States show that over 50
percent of the most prescribed drugs had a natural product either as the drug or as the
starting point in the synthesis or design of the actual end chemical substance (Newman et
al,2000). Thirty-nine percent of the 520 new drugs approved during the period 1983
through 1994 were either natural products or their derivatives (Harvey, 2000). Of the 20
top-selling drugs on the market in the year 2000 that are not proteins, 7 of these were
either derived from natural products or developed from leads generated from natural
products. This select group of drugs generates over 20 billion U.S. dollars of revenue on
an annual basis (Grabley and Sattler, 2003; Harvey, 2000). Drug development over the
years has relied only on a small number of molecular prototypes to produce new
xix
medicines (Harvey, 2000). Indeed, only approximately 250 discrete chemical structure
prototypes have been used up to 1995, but most of these chemical platforms have been
derived from natural sources. While recombinant proteins and peptides are gaining
market share, low molecular- weight compounds still remain the predominant
pharmacologic choice for therapeutic intervention(Grabley and Sattler,2003).A small
sampling of the many available examples of the commercialization of modern drugs from
natural products along with their year of introduction, indication, and company are:
Orlistat, 1999, obesity, Roche; Miglitol, 1996, antidiabetic (Type II), Bayer; Topotecan,
1996, antineoplastic, SmithKline Beecham; Docetaxel, 1995, antineoplastic, Rhône-
Poulenc Rorer; Tacrolimus, 1993, immunosuppressant, Fujisawa; Paclitaxel, 1993,
antineoplastic, Bristol-Myers Squibb. The overwhelming concern today in the
pharmaceutical industry is to improve the ability to find new drugs and to accelerate the
speed with which new drugs are discovered and developed. This will only be successfully
accomplished if the procedures for drug target elucidation and lead compound
identification and optimization are themselves optimized. Analysis of the human genome
will provide access to a myriad number of potential targets that will need to be evaluated
(Grabley and Sattler, 2003). The process of high-throughput screening enables the testing
of increased numbers of targets and samples to the extent that approximately 100,000
assay points per day are able to be generated. However, the ability to accelerate the
identification of pertinent lead compounds will only be achieved with the implementation
of new ideas to generate varieties of structurally diverse test samples (Grabley and
Sattler, 2003; Harvey, 2000). Experience has persistently and repeatedly demonstrated
that nature has evolved over thousands of years a diverse chemical library of compounds
xx
that are not accessible by commonly recognized and frequently used synthetic
approaches. Natural products have revealed the ways to new therapeutic approaches,
contributed to the understanding of numerous biochemical pathways and have established
their worth as valuable tools in biological chemistry and molecular and cellular biology.
Just a few examples of some natural products that are currently being evaluated as
potential drugs are (natural product, source, target, indication, status): manoalide, marine
sponge, phospholipage- A2 Ca2+-release, anti- inflammatory, clinical trials; dolastatin 10,
sea hare,microtubules, antineoplastic, nonclinical; staurosporine, streptomyces, protein
kinase C, antineoplastic, clinical trials; epothilone, myxobacterium, microtubules,
antineoplastic, research; calanolide A, B, tree, DNA polymerase action on reverse
transcriptase, acquired immunodeficiency syndrome (AIDS), clinical trials; huperzine A,
moss, cholinesterase, Alzheimer’s disease, clinical trials(Grabley and Sattler,2003). The
costs of drug discovery and drug development continue to increase at astronomical rates,
yet despite these expenditures, there is a decrease in the number of new medicines
introduced into the world market. Despite the successes that have been achieved over the
years with natural products, the interest in natural products as a platform for drug
discovery has waxed and waned in popularity with various pharmaceutical companies.
Natural products today are most likely going to continue to exist and grow to become
even more valuable as sources of new drug leads. This is because the degree of chemical
diversity found in natural products is broader than that from any other source, and the
degree of novelty of molecular structure found in natural products is greater than that
determined from any other source (Cragg et al., 1997). The introduction of active agents
derived from natural sources into the anticancer weaponry has already significantly
xxi
changed the future of many individuals afflicted with cancer of many different types.
Continued research into natural sources will continue to deliver newer and more
promising chemicals and chemical classes of anticancer agents with novel mechanisms of
action that will improve survival rates to even higher degrees. Human immunodeficiency
virus infection is a devastating, globally widespread disease that consumes significant
health-care dollars in the due course of management of patients (Jarvis, 2000). Most of
the currently useful anti-HIV agents are nucleosides and are limited in use due to severe
toxicity and emerging drug resistance. Natural products, with their broad chemical
structural diversity, provide an excellent opportunity to deliver significant therapeutic
advances in the treatment of HIV (Yang et al., 2001). Many natural products with novel
structures have been identified as having anti-HIV activities (Javis, 2000; Yang et al.,
2001). Betulinic acid, a triterpenoid isolated from Syzigium claviflorum, has been found
to contain anti- HIV activity in lymphocytes. The quassinoside glycoside isolated from
Allanthus altissima has been found to inhibit HIV replication. Artemisinin, isolated from
Artemisia anuua, is a sesquiterpene lactone that is of special interest because of its novel
structure, potent antimalarial activity, and activity against Pneumocystis carinii. A novel
phorbol ester isolated from Excoecaria agallocba has been reported to be a potent
inhibitor of HIV-1 reverse transcriptase. Indeed, most of the natural product chemicals
that are attracting interest in this area of research are secondary metabolites such as
terpenes, phenolics, peptides, alkaloids, and carbohydrates and are also inhibitors of HIV
reverse transcriptase. Other target opportunities in the life cycle of the human
immunodeficiency virus available for exploitation are: (1) attachment of virus to cell
surface, (2) penetration and fusion of the virus with the cell membrane, (3) reverse
xxii
transcription via reverse transcriptase, (4) integration into the host genome, (5) synthesis
of viral proteins including zinc fingers, and (6) processing of viral polypeptide with HIV
protease and assembly of viral proteins and DNA into a viral particle, maturation, and
extrusion of the mature virus (Yang et al., 2001). Infectious viral diseases remain a
worldwide problem. Viruses have been resistant to therapy or treatment longer than most
other forms of life because their nature is to depend on the cells that they infect for their
multiplication and survival (El-Sayed, 2000). Such a characteristic has made the
development of effective antiviral chemotherapeutic agents difficult. Today there are few
effective antivirals available for use. In order to confidently wage the war against viruses,
research efforts are now turning to the molecular diversity available from natural
products. For the period 1983 to 1994, seven out of 10 synthetic agents approved by the
Food and Drug Administration (FDA) for use as antivirals were based on a natural
product. Natural products are indeed viable sources and resources for drug discovery and
development (Artuso, 1997). Indeed, without natural products, medicine would be
lacking in therapeutic tools in several important clinical areas such as neurodegenerative
disease, cardiovascular disease, solid tumors treatment, and immune-inflammatory
disease (Banerji, 2000;Harvey, 2000; Nisbet and Moore, 1997).Furthermore, the
continual emergence of new natural product chemical structure skeletons, with interesting
biological activities along with the potential for chemical modification and synthesis
bode well for the utility of natural products. Finally, the uses of natural products need to
be by no means restricted to pharmaceuticals but can also be expanded to agrochemicals.
For example, the use of pyrethrins obtained from Chrysanthemum spp. as insecticides has
been very popular over the years and persists today. Research continues into the use of
xxiii
natural products as pesticides. While the success stories have not been as numerous or
spectacular for herbicides as they have been for drugs and pesticides, there have been
victories along the way and the future holds strong potential for this field also (Duke et
al., 2000; Majetich,1993).
In recent times, focus on plant research has increased all over the world and a
large body of evidence has accumulated to show immense potential of medicinal plants
used in various medical, pharmaceutical, cosmetic, agrochemical applications. Plants
have been the subject of human curiosity and use for thousands of years (Ram et al.,
2004).These plants have played important roles in many countries of the world for
centuries by providing food, shelter, clothing, agrochemicals, flavours and fragrances and
more importantly, medicines (Gurib-Fakim, 2006).Traditional people have relied on
medicinal plants to combat various ailments and many others, and this information has
been passed down from generation to generation (vonMaydell,1996). A large proportion
of these ailments occur due to the presence of microorganisms such as bacteria, fungi and
viruses that infect the body system. In some countries particularly the African continent,
medicinal plants are sold in open market places or prescribed by traditional herbal healers
in their homes (Fyhrquist et al., 2002) to help improve human and animal health. Plants
have indeed formed the basis of sophisticated traditional medicine systems glob will
continue to provide mankind with new remedies for all forms of ailments (Gurib-Fakim,
2006). Natural products and their derivatives represent more than 50% of all the drugs in
clinical use in the world. Higher plants contribute no less than 25% to the total (Gurib-
Fakim,2006).Natural products, particularly medicinal plants, will remain an important
source of new drugs, new leads and new chemical entities (NCEs)(Newman et
xxiv
al.,2000;Newman et al.,2003;Butler,2004). It has been indicated that in 2001 and 2002
approximately one-quarter of the best selling drugs worldwide were natural products, or
were derived from natural products (Butler, 2004). Bioactive natural products have
enormous economic importance as specialty chemicals. They can be used as drugs, lead
compounds, biological or pharmaceutical tools, feed stock products, excipients and
nutraceuticals (Pieters and Vlietinck, 2005). More than 80% of the population in
developing countries depends on plants for their medical needs (Farnsworth, 1988; Balick
et al., 1994). Nearly 88% of the global population turns to plant-derived medicine as their
first line of protection for maintaining health and combating diseases (Samy et al., 1999).
Due to this strong dependence on plants as medicines, it is important to study their safety
and efficacy (Farnsworth, 1994). With the increasing acceptance of herbal medicines as
alternative form of health care delivery, the screening of medicinal plants for bioactive
compounds is imperative (Masoko et al., 2005; Cowan, 1999). It has been estimated that
one-quarter to one-half of all pharmaceuticals dispensed in the United States are from
higher plant origins. The use of plant extracts as well as other alternative forms of
medical treatments is currently enjoying great popularity. It is estimated that there are
over 250,000 higher plant species on this planet. Of these figure, only about 6% have
been screened for biological activity and a reported 15% have been evaluated
phytochemically (Ayensu and DeFilipps, 1978; Verpoorte, 2000).With increase now in
the popularity of medicinal plant research, these figures may have risen. An advantage of
natural bioactive molecule is that they have a milder side effect on the body in
comparison to chemically synthesized drugs (Badisa et al., 2003).
xxv
With considerable interest in the screening of plant and other natural product
extracts in modern drug discovery programs structurally novel chemotypes with potent
and selective biological activity may be obtained (Kinghorn and Balandrin, 1993; Gullo,
1994; Colegate and Molyneux, 1993; Cragg et al., 1997). A consideration of biological
activity in addition to the isolation and structure elucidation stages in a phytochemical
investigation may add a great deal to the overall scientific significance of the work.
Phytochemists may gain considerable information by using panels of simple bioassays
and/or more specialized in vitro bioassays to follow each step of a purification procedure
(Colegate and Molyneux, 1993).
Plant secondary metabolites also show promise for the cancer chemoprevention,
which has been defined as “the use of non-cytotoxic nutrients or pharmacological agents
to enhance physiological mechanisms that protect the organism against mutant clones of
malignant cells.” There has been considerable prior work on the cancer chemopreventive
effects of extracts and purified constituents of certain culinary herbs, fruits, spices, teas,
and vegetables, which have shown the ability to inhibit the development of cancer in
laboratory animal models (Ho et al.,1994).Clinical trials as cancer chemopreventive
agents under the auspices of the United States National Cancer Institute are planned for
plant products such as curcumin, ellagic acid, and phenethyl isothiocyanate (Kelloff et
al., 2000).
Figures show that about 25% of all prescriptions sold in the United States are
from natural products, while another 25% are for structural modifications of a natural
product (Farnsworth, 1977). Furthermore, Farnsworth (1977) claims that 119
characterized drugs are still obtained commercially from higher plants and that 74% were
xxvi
found from ethnobotanical information. Of the several hundred thousand plant species
around the globe, only a small proportion has been investigated both phytochemically
and pharmacologically. When one considers that a single plant may contain up to
thousands of constituents, the possibilities of making new discoveries become evident.
The crucial factor for the ultimate success of an investigation into bioactive plant
constituents is thus the selection of plant material.
In view of the large number of plant species potentially available for study, it is
essential to have efficient systems available for the rapid chemical and biological
screening of the plant extracts selected for investigation. Phytochemical studies on
medicinal plants have served the dual purpose of bringing up new therapeutic agents and
providing useful leads for chemotherapeutic studies directed towards the synthesis of
drugs modeled on the chemical structure of natural physiological products. The studies in
the correlation of chemical structure and physiological activity through functional
variation in active constituents of the plant materials in this case, can be promoted
further.
Today’s pharmaceutical companies rely heavily upon the exploration of botanicals
for providing “new leads”. Some traditional examples of natural product leads that have
worked their way into common pharmaceutical use (Table 1) are shown below.
xxvii
Table 1.0: Traditional plants with their uses
Name of Compound
d-tubocuraine
Quinine
Pilocarpine
Amphotericin
Cephalosporin
Reserpine
Cocaine
Morphine
Mitomycin
Mescaline
Botanical Source
Chondodendron
Cinchona
Pilocarpus jaborandi
Streptomyces nodosus
Cephalosporium
Rauwolfia serpentine
Erythroxylon coca
Papaver somniferum
Streptomyces caespitosus
Lophophora williamsii
Uses
muscle relaxant
Anti-malarial
Glaucoma
Anti-fungal
Antibiotic
Tranquilizer
Local anesthetic
Analgesic
Anti-cancer
Hallucinogen
xxviii
The exploration of plant extracts for effective therapeutic molecules is certainly
ongoing. In fact, industrial drug discovery process has historically relied heavily upon the
application of chemical methods as a means of screening plant/animal species for new
drugs. It is the job of the chemist to utilize certain techniques to isolate compounds that
may eventually lead to the discovery of the discrete components of a molecule
(pharmacophore) that are necessary for a molecule to elicit a biological action. In turn,
the pharmacophore can then be refined into a new molecule that can ultimately be
synthesized in the laboratory. The goal is to synthesize new molecules (that may, in some
cases, look radically different from the original compound) that retain the intrinsic
activity of the natural compound or even enhance its potency and efficacy. In the last few
decades there has been an increase in the demand for herbal drugs in various branches of
medicinal care and this has made it imperative for scientist to study the therapeutic claims
of reputed traditional medical practitioners. The pharmacopoeias of many countries have
recorded large number of drugs of plant origin. Interestingly, many African plants are
used in traditional medicine as antimicrobial agents and for treatment of other ailments,
but only a small proportion of such plants, plant parts and uses are documented. Also
very few of these uses have been subjected to scientific investigation and this necessitates
the screening of more plants species, since only a minute percentage of plants on earth
have been explored in research studies (Badisa et al., 2003).
Today plant based products, essential oils, plant extracts, natural resins and their
preparations have a wide range of applications mainly in perfume and cosmetic industry,
in food technology, in aroma industry and in pharmaceutical industry. This large
spectrum of uses stimulated studies on natural products. The methods used in the analysis
xxix
of plants that started at the end of the 19th century, only allowed investigations on
crystalline constituents isolated from these extracts. Subsequent developments on vacuum
distillation techniques provided the possibility to determine the volatile components of
this extracts.Along with the developments in extraction techniques, the development of
chromatographic techniques primarily with planar chromatography (thin layer
chromatography (TLC)) and other novel analytical methods were introduced to the
benefit of scientists. Gas chromatography (GC) in the 1950’s had opened a new
dimension in the analysis of volatile compounds. In the meantime high performance
liquid chromatography (HPLC) was introduced for the fractionation and isolation of more
polar and non-volatile compounds.
The combination of gas chromatography and mass spectrometry (GC-MS) allows
the rapid identification of not only volatile components but also plant extracts, by
comparing their mass spectra with available libraries which build up with reference
substances recorded under the same experimental parameters. The same principle has
been applied in the last decade for liquid chromatography and mass spectrometry (LCMS)
for non-volatile plant constituents. Moreover, the invention of chiral stationary
phases for gas chromatography, mostly based on cyclodextrins, has facilitated the
identification of the enantiomeric composition of the isolated substances, especially in
essential oils. Simultaneously, the advances in spectroscopic methods such as mass
spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have increased
the speed of the identification and structure elucidation of natural products.
The present work aims at studying the chemical constituents of Hyptis spicigera
plant found in Northern Nigeria for better utility in industries such as pharmaceuticals,
xxx
agrochemicals, cosmetics, oleochemicals and as lead for the synthesis of biologically
active compounds.
1.2 Aim
The study aimed at achieving the following:
i. Phytochemical screening of the various extracts of leaves of Hyptis spicigera for
use as a bio-control agents in agriculture.
ii. Antimicrobial study of the extracts of the leaves of Hyptis spicigera on some
selected pathogens.
iii. Evaluation of the insecticidal properties of the various extracts on grain weevils
using cowpea.
iv. Isolation and purification of compounds from the extracts of Hyptis spicigera
plant.
v. To elucidate structures of the isolated compounds using Nuclear Magnetic
Resonance Spectroscopy (NMR), Mass Spectrometry (MS) and other
spectroscopic techniques.
vi. To identify active ingredient/s.
vii. To further conduct biological evaluation of the pure fractions.
The above aims are individually of special problem-solving relevance. Moreover,
they are mutually complimentary in helping to provide the much needed information on
the scientific data of the botanical extracts of the leaf part of Hyptis spicigera which may
find use as pharmaceuticals, agrochemicals, and cosmetics among others.
xxxi
1.3 Statement of the Problem
Plant resources have provided the basic needs of life such as food, fibre, fuel and
shelter and will continue to provide these needs and much more on a renewable basis.
Plants have also been a valuable source of flavours, fragrances, colourants, and
phytochemicals for industries and pharmaceuticals. The rising incidence of health related
problems in both developing and developed countries has prompted research in the
development of drugs from leads identified from traditional medicinal uses as an
alternative approach to manage new deadly diseases and those that have become resistant
to available drugs. There is also a resurgence of interest on plant- based medicines due to
the undue side effects of modern synthetic therapeutic agents and their inability to cure
diseases, many of them being for the treatment of symptoms. Herbal healthcare products
could also help in uplifting the quality of life of the ageing populations.
The World Health Organisation has initiated global efforts to urge governments to
take steps to upgrade the traditional medical systems and treatments of their respective
countries through validation of their quality, safety and efficacy. Guidelines have been
developed by WHO on standardisation, quality control and analysis of herbal medicinal
products. These initiatives have sparked off considerable interest in the international
health-related scientific community to re-evaluate traditional therapies based
predominantly on the use of medicinal plants. Presently, research on the untapped plant
resources and leads from traditional uses that would hopefully identify useful bioactive
compounds/extracts with therapeutic relevance is being extensively carried out by
scientists in developed and developing countries. Additionally the role of plant- based
products as dietary/ health supplements, nutraceuticals, cosmecuticals and personal care
xxxii
products are being investigated and many countries have recognized the need for such
products. It is clear from the high number of ongoing and completed studies in this
research area that plant- derived pharmaceuticals and healthcare products can
meaningfully contribute towards the management of the biochemical and physiological
functions of a wide range of disorders. However in many cases, the issue of mechanism
of action and pharmacodynamics is a major concern and it is necessary to conduct
research extensively focused on molecular level activities and clinical trials that will
contribute to our understanding of the efficacy and safety of many extracts with
therapeutic potential.
1.4 Significance of the study
The significance of the study lies in the need to authenticate claims by traditional
herbal healers and farmers on the efficacy of the plant in treating ailments and as pest
repellents in protecting grains from damage. This will also lead to the generation of
scientific data on the biological properties of the plant which is in abundance in the
Northern part of Nigeria. The scientific data generated will help in the development of
soft natural bio-control agents that can replace chemical insecticides imported into the
country. The use of bio-control agents will reduce pesticide load in the environment and
conserve foreign exchange from the importation of synthetic pesticides.
1.5 Justification for the study
Medicinal plants constitute an important source of bioactive molecules (Sharma,
2002).Plants synthesize secondary metabolites as defense mechanisms to protect them
xxxiii
against microbial infections. The presence of these compounds provide an invaluable
resource that has been used to find new drug molecules (Gurib-Fakim,2006).Compounds
such as muscarine, physostigmine, yohimbine, forskolin, colchicines and phorbol esters,
all obtained from plants, are important tools used in pharmacological, physiological and
biochemical studies (Williamson et al.,1996).It is estimated that 60% of anti-tumor and
anti-infectious drugs already on the market or under clinical trials are of natural origin
(Yue-Zhong,1998).Many of these chemical compounds cannot be easily synthesized but
can be obtained from wild or cultured plants (Rates,2001).Recently, there has been
growing interest in alternative therapies and the therapeutic use of natural products,
especially those derived from plants (Goldfrank et al.,1982;Vulto and Smet,1988;Mentz
and Schenkel,1989).The interest in drugs of plant origin is due to several reasons, for
example, conventional medicine can be ineffective or may result in side effects, can be
very costly and unaffordable especially by the poor population in the African continent
and lot of other problems. A large percentage of the world population does not have
access to to conventional pharmaceutical products (Rates, 2001).
The test plant Hyptis spicigera is commonly found in the Northern part of Nigeria.
The choice of this plant for the present study is based principally on the result of a survey
(Abubakar and Abdulrahman, 1998) carried out on some plants in Kaduna State, Nigeria,
for pesticidal properties of which Hyptis spicigera showed a remarkable repellency
against some weevils that attack grains. Also, claims by local farmers and traditional
herbal healers on the medicinal properties of this plant with excellent results have further
added interest on the need to study the biological properties of this plant.
Insect pests form a major component of adverse environmental factors militating
xxxiv
against sufficient food production in Nigeria. They reduce crop yields in the field and
equally destroy stored food. Rural farmers suffer especially from the destructive tendency
of these insect pests as they cannot afford the high cost of pesticides available in the
market. These pests are vectors of many human diseases and need to be eradicated.
Nigeria currently depends heavily on the importation of synthetic insecticides. There is
no single indigenous company as far as we know that is involved in the manufacture of
bio-insecticides in Nigeria. Synthetic insecticides are not environmentally friendly and
their massive importation affects our foreign exchange earnings.
Several varieties of plants have been reported to possess insecticidal properties
(Pushpalatha and MuthuKrishna, 1995). The active compounds responsible for the
insecticidal property in some of these plants have also been isolated and characterized
and the mechanisms of action elucidated (Chauret et al., 1976; Dwuma Badu et al., 1976;
Madrigal et al., 1979; Gbewonyo and Candy, 1992). There is little or no detailed
scientific report or study on the secondary metabolites and the biological properties of
Hyptis spicigera. (NAPRELERT, 2005). This study is aimed at providing some scientific
data on the secondary metabolites and the biological properties of the plant Hyptis
spicigera.
1.6 Statement of research hypothesis/research question.
All the various extracts of the leaf part of the plant exhibit biological activity
against some microorganisms. The claim by the local users of this plant will be
established. Scientific methods will be used to isolate some of the bio-active compounds
of the leaf part for structural elucidation.

 

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