ABSTRACT
Morphological and anatomical observation, cytological and molecular analysis, phytochemical, antimicrobial and anti-inflammatory potentials, toxicity, enzyme activity, and histopathological examination were carried out on the ‘epiflorous’ plant –Phyllobotyron spathulatum obtained from Cross River National Park, Akamkpa. Morphological characters were macroscopically observed and the following results recorded: Perennial, rarely branched erect shrub or tree up to 16ft high habit and terrestrial habitat. Cylindrical, hard, hairy brown stem. Lanceolate leaf about 65cm in length, serrate margin with small palm-like leaflet second outgrowth. ‘Epiflorous’ floral inflorescence on mature leaf midrib. Incomplete, white flower. Purple to yellow fruit nut, seed are five and white. Flowering and fruiting time is observed almost through all season. Venation is reticulate while rooting system is tapering with adventitious roots that support germination of new shoot. Observation of photomicrographs of the slides of the epidermal peel and anatomical sections showed the occurrence of hypostomatic and diacytic stomata with index of 28 and 20 in old and juvenile leaves respectively. Round epidermal circumference of stem adorned with trichome. Presence of starch sheath was observed just below the cortex of root and stem. Piths were less inconspicuous in stem and root. Chromosome number, determined through the squash method was observed to be 2n=22. Molecular extraction, purification, amplification and sequencing showed that the isolate of the plant sample have a band size of 650bp and BLAST result from NCBI data base got a pairwise identity (74.4) to Phyllobotryon spathulatum Müll.Arg. Phytochemical studies show that the root and leaf P. spathulatum contains these chemicals in appreciable quantity that has been recommended for various nutritional and pharmacological purposes. In vitro antimicrobial analysis using different solvent showed that the leaf of P. spathulatum possesses inhibitory activity through inhibition zone (IZ) against most of the tested pathogens at the various concentrations used. The minimum inhibitory concentration (MIC) study revealed an MIC value of up to 6.25mg/ml and the corresponding MBC/MFC. In vivo anti-inflammatory studies using animal model tests showed that the plant extract at 200mg/kg concentration inhibited carrageenan induced paw oedema by 81% when compared to 0% in water and 92% in the standardized drug after 3days. Acute toxicity testing of the leaf of the plant showed that the extract was well tolerated at a dose of 2000 mg/kg as the animals showed no sign of toxicity or death after 72 hours. Sub-acute study using the relative weight as well as liver and kidney enzyme profile showed that the extract of the leaf of P. spathulatum at various concentrations did not significantly alter viscerosomatic index of the rats. Activity in the analyzed liver enzymes; aspartate transaminase (AST), Alanine transaminase (ALT), alkaline phosphatase (ALP) and kidney enzymes; urea and creatinine were not affected as the extract concentration increased. Histopathological examination from the tissue slides did not show any changes in the features examined. Results obtained from this study can be employed in the identification, characterization, nutritional and pharmacological uses of this rarely known and underutilized plant.
TABLE OF CONTENTS
Title
page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table
of contents vi
List
of tables x
List
of figures xi
Abstract xii
CHAPTER
1: INTRODUCTION
1.1 Background of study 1
1.2 Statement
of the problem 5
1.3 Justification 5
1.4 Aim of the study 6
1.5 Objectives of the study 6
CHAPTER 2: LITERATURE
REVIEW
2.1
Morpho-anatomical reviews of the salicaceae family. 7
2.2
Nutrients and antinutrient content of Phyllobotryon.
Spathulatum 11
2.3
Antimicrobial potentials of Phyllobotryon
spathulatum 13
2.4
Toxicity and anti-inflammatory studies of the salicaceae. 18
2.5
Effect of plant extract on enzyme activity, viscerosomatic index and
histopathology. 22
2.6
Chromosomal and molecular studies of salicaceae. 23
CHAPTER 3: MATERIALS AND
METHODS
3.1 Study
area 27
3.2 Morphological observation 28
3.3 Epidermal studies 29
3.4 Anatomical studies 29
3.5 Cytological analysis 29
3.6 Molecular study 32
3.6.1 Genomic
DNA extraction 32
3.6.2 Agarose gel electrophoresis 33
3.6.3 DNA
amplification 33
3.6.4 Data
analysis 34
3.7 Proximate study 34
3.7.1 Moisture content determination 34
3.7.2 Crude
protein determination 34
3.7.3 Crude
ash determination 35
3.7.4 Crude
fat determination 35
3.7.5 Crude
fibre determination 36
3.7.6 Carbohydrate
determination 37
3.8 Mineral
and vitamin study 37
3.9 Phytochemical
analysis 37
3.10 Quantitative
phytochemical analysis 37
3.10.1 Determination
of alkaloids 37
3.10.2 Determination
of phenols 38
3.10.3 Determination
of flavonoids 38
3.10.4 Determination
of tannins 38
3.11 Antimicrobial
activity 39
3.11.1 Extraction of plant material 39
3.11.2 Biochemical identification of the test
organisms 39
3.11.3
Isolation and identification of plant
pathogen 39
3.11.4 Screening of the extracts for antibacterial
activity 40
3.11.5 Determination
of MIC and MBC/MFC 41
3.12 Collection and
preparation of animal anti-inflammatory
studies 42
3.12.1 Anti-inflammatory
test 42
3.13 Acute
toxicity and lethal dose test (LD50) 43
3.14 Acute
toxicity testing 43
3.15
Sub-acute toxicity study 44
3.16
Relative organ weight estimation 46
3.17 Biochemical estimations 46
3.18 Histopathology of liver and kidney 46
CHAPTER
4: RESULTS AND DISCUSSION
4.1.1. Morphological
observation of Phyllobotryon spathulatum 47
4.1.2 Anatomical
observations of P. spathulatum 51
4.1.3 Leaf
anatomy of P. spathulatum 51
4.1.4 Stem anatomy of P.
spathulatum 54
4.1.5
Root anatomy of P. spathulatum 56
4.1.6 Chromosome study of P. spathulatum 57
4.1.7 Molecular study of P. spathulatum 58
4.1.8 Proximate analysis of P. spathulatum 61
4.1.9 Mineral contents of P. spathulatum 62
4.1.10 Vitamin content of P. spathulatum 63
4.1.11 Anti-nutrient
composition of P. spathulatum 64
4.1.12
Antimicrobial activity of different extracts of the leaf of P. spathulatum
(Inhibition zone). 65
4.1.13 MIC/MBC/MFC
(mg/ml) of chloroform extract of P.
spathulatum 66
4.1.14 MIC/MBC/MFC
(mg/ml) of ethanol extract of P.
spathulatum 67
4.1.15 MIC/MBC/MFC
(mg/ml) of petroleum ether extract of P. spathulatum 68
4.1.16 Anti-inflammatory effect of the leaves of p. spathulatum 69
4.1.17 Acute toxicity result of P. spathulatum 71
4.1.18 Subacute toxicity study of P. spathulatum 71
4.1.19 Effect of the leaf extract of P. spathulatum on enzyme activity 72
4.1.20 Sub-acute
histopathological examination. 72
4.2 Discussions. 75
CHAPTER 5: CONCLUSION
AND RECOMMENDATIONS
5.1
Conclusion. 89
5.2
Recommendations 91
References 93
Appendix 106
LIST OF TABLES
4.1 Morphological features of Phyllobotryon
spathulatum. 47
4.2
Proximate content of P. spathulatum. 61
4.3 Mineral contents of P. spathulatum. 62
4.4 Vitamin content of P. spathulatum. 63
4.5 Anti-nutrient composition of P. spathulatum. 64
4.6
Antimicrobial activity of extracts of the leaf of P. spathulatum
(Inhibition Zone). 65
4.7
MIC/MBC/MFC (mg/ml) of Chloroform leaf extract of P. spathulatum. 66
4.8
MIC/MBC/MFC (mg/ml) of Ethanol leaf extract of P. spathulatum. 67
4.9
MIC/MBC/MFC (mg/ml) of Petroleum Ether leaf extracts P. spathulatum. 68
4.10
Anti-inflammatory Effect of the leaves of P.
spathulatum. 70
4.11
Effects of P. spathulatum on
Viscerosomatic Index (Relative Organ Weight). 71
4.12
Effect of the Leaf extract of P.
spathulatum on enzyme activity 72
4.13
Effect of the leaf extracts of P.
spathulatum on kidney of the rats. 73
4.14 Effects of the leaf extracts
of P. spathulatum on liver of the rat. 74
LIST OF PLATES
2.1
Leaf of Phyllobotryum soyauxianum Baill 8
3.1
Map of Cross River
National Park Akamkpa 28
4.1a-4.1j Morphological
Features of Phyllobotryon spathulatum. 48
4.2a.-4.2e Anatomical Features of the leaf of P.
spathulatum. 52
4.3a-4.3b Stem Anatomy of P. spathulatum. 55
4.4a-4.4b Root Anatomy of P.
spathulatum. 56
4.5a-4.5b Chromosome observation of
P. spathulatum. 57
4.6a-4.6b Molecular
study of P. spathulatum. 58
4.7a-4.7h Histopathological Examination of Liver
and Kidney Tissues. 73
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
It
is a common knowledge that plants have had and currently have a crucial role in
the history of both living and non-living on earth. Plants provides for the
necessary oxygen needed for most organisms inhabiting the earth. Human depends
directly or indirectly on plants and its products, which play essential role in
nutrition and drugs. The
use of and search for medicine and nutritional supplements derived from plants
have increased over time. The chemical properties of most medicinal plants have
been used over the years to cure ailments. Historically, the Holy book of the
Christendom offers descriptions of about 30 plants with medicinal properties.
Undoubtedly, frankincense and myrrh may have derived their status of great
worth as a result of their medicinal potentials. Recent report by Kochhar
(2016), revealed that about 7,000 species of plants are currently in use for
food, another 8,000 species of plants are used for industrial purposes such as
timber, pharmaceutical and aesthetic values. Plants contain a wide variety of
anti-nutrients, such as flavonoids, terpenoids, tannins and alkaloids which are
known to possess antimicrobial activities. Cowman (1999) classified these
phytochemicals into; Terpenoids, Phenolics, Essential oils (e.g capsaicin),
Lectins, Alkaloids, Polyacetylenes and Polypeptides. These compounds and their
derivatives are the major force driving ethno-medicine and mainstream
medicine.
Despite
the availability of numerous research works on the use of plants in the
following human areas; maintaining good health (Burkill, 1995; Moerman, 1996;
Schippers, 2000; Grubben and Denton, 2004): provision of raw materials needed
for commercial purposes especially in pharmaceutical industries (Gill, 1992;
Okwu and Okeke, 2003; Okwu, 2004; Constable and Ian, 2005; Osuagwu et al., 2007; Sofowara, 2008; Mali,
2010), Shackleton et al. (2009)
observed that a great number of plants and its products remain untapped. It is estimated
that there is about 250,000 to 500,000 species of plants on Earth of which a relatively
minute fraction (1 to 10%) of these percentage are used as foods by both man
and other animal species (Heinrich and Gibbons, 2001). It has been reported
earlier that possibly, more fraction of these estimated species are used for
medicinal purposes (Moerman, 1996) with more numbers still unknown and
underutilized (FAO, 1994; Demele and Abebe 2004).
Identification
and documentation has been a major challenge in the use of plants and its
products as antimicrobial agents for disease control. In addition to
physiochemical parameters, anatomical features of plant parts form in part a
source of taxonomic inference in different groups of angiospermic plants used
in ethno-medicine (Coopoosamy
and Naidoo, 2011). Microscopic observation enhances the identification of
medicinal plants and the isolation of distinct components of a mixture. The
presence of trichome in the leaves of Mucuna
species has been used in the identification of medicinal species (Edeoga et al., 2007). Crystals of calcium
oxalate found in most plant cortex is an anatomical feature used in identifying
plants with medicinal features (Idu et al.,
2009).
Overall
knowledge is a very important aspect in identification, preparation, safety and
efficacy of such plants especially those used as medicinal plants. Modi et al. (2010) stated that pharmacognosy
is a direct and reliable method, by which adequate knowledge of the natural drug
from plants can be assessed.
The
evolution of plants has led to an increase in the level of complexity of
structure and or morphology. As can be seen from the increase in structural and
morphological features from the earliest lower plants (algal mats, bryophytes,
lycopods), through higher lower plants -pteridophytes (ferns) to the complex lower
higher plants (gymnosperms) and true plants (angiosperms) of today. Alteration
in structure and or morphology associated with these categories of plants are
usually due to environmental factors influencing chromosomal and genetic
make-up of the proceeding plant genera to form a new species/variety different
from the parent plant (Leskinen and Alstroma-Rapaport 1999; De-Micco et al., 2014). Studies shows that aside
evolutionary change in chromosome number, other factors such as growth
conditions, stress like drought, salinity, ionizing irradiation, heavy metal
has been identified as possible factors that alters the structural, chemical
and genetic parameters in plant can cause new morphological changes (Edeoga et al., 2005; Osuagwu and Nwachukwu,
2007; Omosun et al., 2009; Esnault, et al., 2010; Temekorva, 2010; Al-Enezi
and Al-Khayri, 2012; Uwalaka and Osuagwu, 2015; Uwalaka et al., 2017).
The
complex and evolving nature and structure of plants has posed a challenge in
its identification, classification and use. Plants have been identified and
classified based on their morpho-anatomical features (Arroyo, 1986). However,
further studies on their genetic make-up have proved some of the previously morpho-anatomical
based classification wrong (Noer et al.,
2018). A major merit of using DNA data in classification is the high number of
possible characters usable for inferring relatedness. Another
benefit of DNA based studies is that changes within structural genes are
unlinked from alterations that could have occurred in the morphology (Nadler,
1995).
Misidentification
of plant is a serious challenge to man especially when used as food or drugs
(Serrano et al., 2010). For proper
identification and use of plants, it’s important to consider or study some or a
combination of the following; macroscopic and microscopic morpho-anatomical
features, molecular makeup, phytochemical and pharmaceutical constituents
before such plants can be used.
Recently
observed in Cross River National Park, Akamkpa is a plant –Phyllobotryon spathulatum,
with the unique feature of bearing flower in the leaf. This phenomenon has no known
physiological term in the field of botany. The best possible term to describe
the process is ‘epiflory’ –which implies growth on the leaf. The closest
phenomenon to this in the plant kingdom is cauliflory.
An
inquiry from the rangers and villagers shows that the plant has no local name.
Similarly, the plant has no known use within the locality. The plant was then
collected for identification and further investigation to unravel its potentials
in food, medicine, aesthetics as well as identification and probable cause of
this new development.
Placement
of plant in the Salicaceae family has varied
greatly. P. spathulatum was
hitherto placed in the obsolete flowering plant family of Flacourtiaceae
(Hutchinson and Dalziel, 1952), whose former members have been spread across
other families, especially to the Achariaceae and Salicaceae. This was done
using the molecular phylogeny-based classification, known as the APG IV system,
established by the Angiosperm Phylogeny Group. Wurdack and Charles
(2009), observed further division in the family of Salicaceae to include;
Salicaceae, Scyphostegiaceae, and Samydaceae. Akobundu and Agyakwa (1993);
Aigbokhan (2014) did not capture the plant in the publication
of plants within these region, probably due to the endemic and hidden habitat
of it. The moist tropical Nigeria environment facilitates the growth and
development of a wide variety of plant species that have been used in Nigerian
traditional medicine even before the introduction of conventional refined drugs
(Lifongo et al., 2014). Hence, the Nigerian flora is not only rich in
diversity but it is also mostly endemic to some plant species.
Most of the studies on Nigerian plants reported
expert identification of the selected plant(s), only few papers reported
deposition of plant in herbarium with the accompanying voucher number. Udegbunam
et al. (2015) stated that it is important that selected plant for study is
identified by an expert/plant taxonomist, as well should be deposited in a
reliable herbarium for future easy identification, use and reproducibility.
1.2 STATEMENT OF THE PROBLEM
Since
Cruz and Dierig (2014) reported that the number of unidentified plants is far
more than those identified and in use, there have been increasing research in
identifying plants with unique traits and the possible use of their products in
food, medicine and aesthetics. One of such plants currently identified is Phyllobotryon spathulatum. Found in the forest reserve of Akamkpa, Cross-River
State, Nigeria, this plant has a unique and non-existent feature in the plant
kingdom. The leaves bears flower in the upper surface –a non-existent feature
in plants. The conservator of the Park revealed that the plant has no known
established name as efforts have been made by visitors and locals to identify
the plant without any success. According to the villagers, this feature appears
recently developed by the plant. Overall the lack of scientific information on
the taxonomy, phytochemical yield and medicinal benefits of underutilized and
rarely known P. spathulatum has
contributed for the absence of its diversified use as food, medicine and
aesthetic source in Nigeria. Hence, it is classified as underutilized wild
plant.
1.3 JUSTIFICATION
The
numerous roles of plants in food, medicine and other industrial uses, calls for
the need to carry out physio-chemical, molecular and pharmacological studies on
this plant. Because it is wild plant, it may have different chemicals that are
secondary metabolites hence, they may be of anti-microbial importance. P. spathulatum has no known or published
anatomical as well as pharmaceutical studies. Considering the effects of the
soil and environmental factors on plants, studies on this plant will unravel
the potential use of the plant and its products in food, medicine and
aesthetics to mitigate the effect of malnutrition and lack of drugs in
developing countries of Africa where it grows. The unique feature on the leaf
of the plant calls for studies on it to unravel the possible cause of this
phenomenon. Cytological and molecular studies are needed to reveal possibly,
the genetic makeup of the plant –a major contribution to the existing data on
identified plants. Once it is studied, it is possible to announce the world to its
potentials for possible uses. Generally, this work will introduce P. spathulatum in class of plants with
known specific features and uses.
1.4 AIM OF THE STUDY
The
general aim is to determine the morpho-anatomical features, cytological and
molecular makeup, nutritional and anti-nutritional potentials, antimicrobial
and pharmacological activities of Phyllobotryon
spathulatum.
1.5 OBJECTIVES OF THE STUDY
The
objective of this research is to;
Ø Study
the morphological features of the root, stem, leaf and flower.
Ø Study
the anatomy of the root and leaf.
Ø Study
the chromosomal and genetic make-up of the plant.
Ø Study
the quantitative proximate, nutrient and antinutrient content of the root, stem
and leaf extract.
Ø Assess
the antimicrobial potentials of the leaf extract.
Ø Assess
the acute and sub-acute toxicity of the leaf on albino rats.
Ø Assess
the anti-inflammatory activity of the leaf extract.
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