ABSTRACT
Phytochemical screening of the plant Pouteria alnifolia indicated the presence of tannins,
sterols, cardiac glycosides, flavonoids but no alkaloids. Two triterpenes have been isolated
from the stem bark and identified by GC-MS, and Carbon-13 NMR analyses as Betulin and
Lupeol. The extracts from the stem back were found to be antifungal: effective against
Aspergillus nigre, Trichophyton rubrum, and Candida albicans inhibiting their growths at
minimum concentrations of 10mg/ml, 20mg/ml and 5mg/ml respectively; and completely
killing Aspergillus nigre and Candida albicans at minimum fungicidal concentrations
of30mg/ml, 40mg/ml and 10mg/ml, respectively. Antibacterial screening of the methanol and
ethyl acetate extracts showed activity against Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Salmonella typhi, and Candida albicans, at 0.625mg/ml,
0.625mg/ml, 1.25mg/ml, 1.25mg/ml, and 1.25mg/ml respectively for the ethyl acetate
extracts but the methanol extract showed 1.25mg/ml for Staphylococcus aureus, Escherichia
coli, Pseudomonas aeruginosa, Salmonella typhi, and Candida albicans. Minimum
bactericidal concentrations of the extracts against Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Salmonella typhi, and Candida albicans were found to be
1.25mg/ml, 1.25mg/ml, 5mg/ml, 5mg/ml, and 5mg/ml respectively for ethyl acetate extract
and a 5mg/ml for the methanol extract in turn, with Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Salmonella typhi, and Candida albicans.The methanol extract was
found to be active against Trypanosoma brucei brucei, in an in vitro test, killing all the
parasites at a minimum concentration of 5mg/ml in 10 minutes as opposed to the standard
drug Diminaveto® 100mg/ml in 3 minutes. The above findings mark Pouteria alnifolia as an
important medicinal plant.
TABLE OF CONTENTS
DECLARATION ……………………………………………………………………………………………………………… ii
CERTIFICATION …………………………………………………………………………………………………………… iii
DEDICATION ………………………………………………………………………………………………………………… iv
ACKNOWLEDGEMENT ………………………………………………………………………………………………….. v
ABSTRACT …………………………………………………………………………………………………………………… vi
1.1 INTRODUCTION ………………………………………………………………………………………………….. 1
1.2 PHARMACOLOGICAL PRODUCTS IN PLANTS ………………………………………………………. 2
1.2.1 Terpenoids……………………………………………………………………………………………………………….. 2
1.2.2 Phenolic Compounds …………………………………………………………………………………………………. 4
1.2.3 Alkaloids …………………………………………………………………………………………………………………. 6
1.2.4 Tannins …………………………………………………………………………………………………………………… 8
1.2.5 Steroids …………………………………………………………………………………………………………………. 10
1.3 AIMS AND OBJECTIVES OF THE RESEARCH ………………………………………………………….. 12
1.3.1 Aims …………………………………………………………………………………………………………………….. 12
1.3.2 Objectives of the Research ………………………………………………………………………………………… 12
1.4 RESEARCH JUSTIFICATION ……………………………………………………………………………………. 13
1.5 SCOPE AND LIMITATION OF THIS RESEARCH ……………………………………………………….. 14
CHAPTER TWO ……………………………………………………………………………………………………………. 15
2.0 LITERATURE REVIEW ……………………………………………………………………………………………. 15
2.1 THE FAMILY SAPOTACEAE ……………………………………………………………………………………. 15
2.1.1 The Genus Pouteria …………………………………………………………………………………………………. 15
2.1.2 Chemical Composition of Pouteria Species …………………………………………………………………. 16
2.1.3 Biological Activity of Pouteria Species ………………………………………………………………………. 20
2.1.4 REVIEW OF POUTERIA ALNIFOLIA (BAKER) ROBERTY …………………………………………………. 25
2.2 BACTERIAL AGENTS ……………………………………………………………………………………………… 27
2.2.1 Staphylococcus “Gram – Positive” ………………………………………………………………………….. 27
2.2.2 Streptococcus “Gram – Positive” ………………………………………………………………………………. 28
2.2.3Klebsiella pneumonia “Gram-Negative” ………………………………………………………………………. 29
2.2.4 Salmonella “Gram – Negative” …………………………………………………………………………………… 29
2.3 FUNGI …………………………………………………………………………………………………………………….. 30
viii
2.4 TRYPANOSOMA BRUCEI BRUCEI ……………………………………………………………………………… 31
CHAPTER THREE …………………………………………………………………………………………………………. 34
MATERIALS AND METHODS ……………………………………………………………………………………….. 34
3.1CHEMICALS/REAGENTS …………………………………………………………………………………………. 34
3.1.2 Equipment ……………………………………………………………………………………………………………… 34
3.1.3 Plant Collection and extraction ………………………………………………………………………………….. 34
3.1.4. Preparation of stock solution for in vitro studies …………………………………………………………… 35
3.1.5 Phytochemical Screening ………………………………………………………………………………………….. 35
Molisch’s test………………………………………………………………………………………………………………. 35
Fehling’s test for free reducing sugars: ……………………………………………………………………………. 36
3.1.6Antimicrobial Screening ……………………………………………………………………………………………. 39
3.1.7 Cultivation and Standardization of Test Organisms ……………………………………………………….. 39
3.1.8 Preparation of Culture Media …………………………………………………………………………………….. 40
3.1.9 Antimicrobial Profile (susceptibility test) …………………………………………………………………….. 40
3.2.0 Determination of Minimum Inhibitory Concentration ……………………………………………………. 40
3.2.1 Determination of Minimum Bactericidal Concentration (MBC) ………………………………………. 41
3.2.2Trypanosoma brucei brucei ……………………………………………………………………………………….. 42
3.2.3 Determination of Parasitaemia …………………………………………………………………………………… 42
3.2.4 in vitro studies ………………………………………………………………………………………………………… 42
3.2.5 Identification and separation of the active principle ………………………………………………………. 43
3.2.5 Vacuum Liquid Chromatography ……………………………………………………………………………….. 44
3.2.6 Melting Point determination ……………………………………………………………………………………… 45
3.2.7 Spectroscopic Measurement………………………………………………………………………………………. 45
RESULTS ……………………………………………………………………………………………………………………… 46
4.1 EXTRACTION …………………………………………………………………………………………………………. 46
4.2. PHYTOCHEMICAL SCREENING……………………………………………………………………………… 47
4.2.1 Antimicrobial Screening …………………………………………………………………………………………… 47
4.2.2 Minimum Inhibitory Concentration (MIC) …………………………………………………………………… 49
4.2.4 Analysis of H1 ………………………………………………………………………………………………………… 53
4.2.5 GC-MS and 13C-NMR Spectroscopy of H1 …………………………………………………………………… 53
4.2.6 13C NMR spectrum of Isolated Compound …………………………………………………………………… 62
DISCUSSION ………………………………………………………………………………………………………………… 64
ix
5.1 PRELIMINARY PHYTOCHEMISTRY ………………………………………………………………………… 64
5.2 INVESTIGATION OF THE ETHYL ACETATE EXTRACT OF POUTERIA ALNIFOLIA …… 64
5.3 ANTIMICROBIAL PROFILE……………………………………………………………………………………… 67
RECOMMENDATIONS …………………………………………………………………………………………………. 68
REFERENCES ………………………………………………………………………………………………………………. 69
CHAPTER ONE
1.1 INTRODUCTION
With all the advancements made by mankind, man has continued to look at plants as
important sources of medication for his ailments and new drugs are still being discovered.
Hippocrates (460 – 377BC) was reported to have listed approximately 400 different plant
species for medicinal purposes (Sarker and Lutfun, 2007). Today, in China alone, 7295 plant
species are utilized as medicinal agents (Sarker and Lutfun, 2007).
To the organic chemist, plant constituents are merely a crude chemical brew that
needs to be simplified to classifiable active principles (Mills, 1994). A reductionist approach
is usually adopted for the following reasons: (i) the isolated principles provide a scientific
window into the medicinal usage of the herb, (ii) understanding the active constituents can
also provide answers to better isolation procedures and pharmacological behavior of a plant
(Bruneton, 1995).
Sometimes, however, a straightforward natural product isolation route, irrespective of
bioactivity, is also applied, which results in the isolation of a number of natural compounds
(small compound library) suitable for undergoing any bioactivity screening(Sarker and
Lutfun, 2007).The most extensively used plant components can be classified into alkaloids,
tannins, steroids, phenolic compounds, terpenoids and carbohydrates(Hill, 1952).
2
Herbal medicine plays a very important role in the healthcare system of the
developing world especially those who do not have access to orthodox medicines, an
inclination supported by the (WORLD HEALTH ORGANISATION, 2002). Although about
40% of modern pharmaceuticals are derived from plants, none is used against viruses. In
contrast, traditional healers have long used phytomedicines to prevent or cure infectious
conditions. Scientists are interested in antimicrobial plant extracts because (i) of
dissatisfaction with antibiotics; (ii) of public inclination to take herbal medication; and (iii)
viral diseases have no viable cure yet. The earth’s resources are dwindling (Lewis, 1995)
leading to critical losses of structurally diverse and potentially useful phytochemicals (Borris,
1996). It is against this backdrop that the need arises to investigate plant resources for
medicinal uses.
1.2 PHARMACOLOGICAL PRODUCTS IN PLANTS
Alkaloids, tannins, steroids, phenolic compounds, terpenoids, proteins, waxes,
glycosides, carbohydrates etc. are responsible for physiological activity of plant products.
Some of the more important ones will be discussed piecemeal below.
1.2.1 Terpenoids
There are more than 23,000 known terpenoids. They are the largest class of natural products.
The terpenoids find wide ranging applications in industry and beyond. For example, the
antimalarial drug Artemisinin and the anticancer drug paclitaxel (Taxol®) are two of a few
terpenes with established medicinal applications (FAO, 1995).Artemisinin and its derivatives
are presently the most potent drugs against Plasmodium falciparum (White, 1997)
3
A
A B C
Figure 1.0 Conversion of Artemisinin to Dehydroartemisinin.
4
The group of functionalities namely peroxy, ketal, acetal, and lactone in the Artemisinin
molecule (A) can be selectively reduced to deoxyartemisinin. Deoxyartemisinin(C) lacks the
antimalarial properties of artemisinin indicating that the antimalarial character is conferred
by the peroxy moiety(Zeng B.-B. , Wu, Yu, Wu, Li, and Chen, 2000)(Zeng B.-B. , et al.,
2003).
1.2.2 Phenolic Compounds
This is a large group of structurally diverse naturally occurring compounds that possess at
least a phenolic moiety in their structures. For example, quercetin is a flavonoid that has four
phenolic hydroxyls at C-5, C-7, C-31 and C-41. The phenolic group of compounds
encompasses various structural types, for example: phenylpropanoids, coumarins, flavonoids
and isoflavonoids, lignans tannins etc. (Krishnamachari, Levine, and Pare, 2002)(Sarker and
Lutfun, 2007).
Quercetin possesses various degrees of antioxidant or free radical scavenging properties
(Sarker and Lutfun, 2007)
5
6
Figure 2.0 Quercetin
1.2.3 Alkaloids
Most alkaloids are obtained from plants. They are pharmacologically active, basic
compounds derived from amino acids. They may contain one or more heterocyclic nitrogen
atoms in the molecule (Cseke, Kirakosyan, Kaufman, Warber, Duke, and Brielmann,
2006)(Cordell, 1981). In fact, most nitrogen-containing secondary metabolites are usually
deemed alkaloids, but for amines or glucosamines may be grouped differently. The word
“alkaloid”originated from the Arabic, al-qali (an early form of soda ash) (Cordell, 1981).
Many alkaloids have a bitter taste, and a large number of them exhibit potent physiological
effects on mammals. For example, morphine shows narcotic effects; reserpine is an
antihypertensive agent; atropine is a smooth muscle relaxant; cocaine is a potent central
nervous system stimulant; and strychnine is a nerve stimulant.
7
1. 2. 3.
Figure 3.0 Some important alkaloids 1. Caffeine (a pseudo-alkaloid) 2. Quinine 3. Atropine
8
Alkaloids in plants serve as chemo-protective anti-herbivory agents or as growth regulators,
such as the well-known plant hormone, indole-3-acetic acid, (IAA) which is an indole
derivative synthesized from tryptophan (Buchanan, Gruissem, and Jones, 2000).
1.2.4 Tannins
Tannins are water-soluble oligomers, rich in phenolic groups, capable of binding or
precipitating water soluble proteins (Hagerman and Butler, 1989). The tannins, common to
vascular plants, exist primarily within woody tissues but can also be found in leaves, flowers,
or seeds. Plant tissues that are high in tannin content have a highly bitter taste and are
avoided by most feeders. Tannins fall into two groups: either condensed tannins or
hydrolysable tannins.
9
1.
2.
Figure 4.0 Structures of representative hydrolysable tannins (1) and condensed
proanthocyanidins (2)
10
Corilagin shows anti-human-immunodeficiency-virus (HIV) activity by inhibiting reverse
transcriptase(Notka, Meier, and Wagner, 2004)(Cseke, Kirakosyan, Kaufman, Warber, Duke,
and Brielmann, 2006).
1.2.5 Steroids
Steroids are chemical messengers. Some steroids are also hormones. All steroids are derived
from the acetate biosynthetic pathway. Hundreds of distinct steroids have been identified in
plants, animals and fungi, and most of them have interesting biological activity. They possess
a perhydrocyclopentaphenanthrene nucleus (Figure 1.4).
The four rings are lettered A, B, C and D, and the carbon atoms are numbered beginning in
the A ring as shown in figure 5.0. These fused rings can be trans fused or cis fused. In
steroids, the B, C and D rings are always trans fused. In most naturally occurring steroids,
rings A and B are also trans fused. Different steroids vary in the functional groups attached to
these rings.
11
Figure 5.0 Gonane
12
Phytosterols found in plants have many applications ranging from food additives, medicine
to cosmetics (Sarker and Lutfun, 2007).
1.3 AIMS AND OBJECTIVES OF THE RESEARCH
1.3.1 Aims
The aims of this project were to:
i. Authenticate or otherwise, the ethno medicinal claims on the vegetative parts of
Pouteria alnifolia.
ii. Isolate and characterize some compound(s) that may be responsible for the claimed
ethno medicinal values.
1.3.2 Objectives of the Research
1. Collection, proper botanical identification, drying and pulverizing of the vegetative
part of the plant.
2. Extraction of the powdered plant material using different solvents based on the
eluotropic series i.e. from non-polar pet-ether (60-80c) to methanol (polar).
3. Phytochemical screening for bio-active compounds using the crude extracts.
4. Antibacterial screening of the extracts.
13
5. Antifungal screening of the extracts.
6. Analytical separations involving several consecutive steps of chromatographic
techniques and purification.
7. Verification of the purity of the isolated compound(s).
8. Structural elucidation and characterization of the possible isolated compounds using
available spectroscopic techniques, 13CNMR, 1HNMR, IR and GC-MS etc.
1.4 RESEARCH JUSTIFICATION
Natural products present unparalleled chemical variety with structural complexity and
biological properties (Verdine, 1996). Natural products can model the design of synthetic
compounds (Breinbauer, Vetter, and Waldmann, 2002). Research on natural products has led
to the discovery of novel mechanisms of action for example, those of immune-suppressants
(Urizar, Liverman, Dodds, et al, 2002)(Zhang and Demain, 2005). Diseases like HIV/AIDS
still have no viable cure. The World Health Organization (World Health Organization, 2011)
estimates that 4 billion people, or 80 percent of the world’s population, use herbal medicine
for some aspect of primary health care.
It is against this background that Pouteria alnifolia, which is a plant used extensively as an
herbal remedy in some parts of Nigeria, West Africa, and throughout sub-Saharan Africa is
being investigated.
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