ABSTRACT
Peperomia pellucida (L.) Kunth is a plant of immense medicinal value. It is consumed as vege and also used in herbal medicine for the treatment of different ailments such as abdominal pain, abscesse, acne, boils and colic rhematic joint pain. This study investigates bioactive constituents of the leaves of P. pellucida. An ursane-type triterpenoid was isolated from the leaves of P. pellucida and was characterized using 1H, C-13, COSY, DEPT and HSQC NMR Spectroscopic techniques. The quantitative phytochemical screening of the extract revealed the presence of alkaloids (2.49 ± 0.02 %), phenols (0.05 ± 0.01 %), flavonoids (0.59 ± 0.01 %), saponins (0.64 ± 0.02 %) and tannins (0.08 ± 0.01 %). The results showed that the alkaloid content was the highest. Vitamins detected included thiamine (0.21 ± 0.03 mg/100g), niacin (0.57 ± 0.01 mg/100g), riboflavin (0.34 ± 0.01 mg/100g), ascorbic acid (8.74 ± 0.12 mg/100g) and β-carotene (2.33 ± 0.15 mg/100g), with ascorbic acid and thiamin having the highest and lowest concentrations respectively. Minerals detected included calcium (2.82 ± 0 mg/100g), magnesium (1.19 ± 0.06 mg/100g), potassium (0.63 ± 0.01 mg/100g), phosphorus (0.49 ± 0.01 mg/100g), sodium (0.03 ± 0.05mg/100g) and nitrogen (3.14 ± 0.03 mg/100g). Nitrogen had the highest concentration, while sodium was the lowest. The antioxidant activity of P. pellucida as determined by scavenging effect on 2,2-diphenyl-1-picryhydrazyl radical (DPPH) revealed that the fractions possessed antioxidant activity when compared with antioxidant standard ascorbic acid. Antimicrobial activity was investigated at varying concentration of 100mg/ml, 50mg/ml/, 40mg/ml, 20mg/ml and 10mg/ml against Escherichia coli, Bacillus cereus, Staphylococcus aereus and Salmonella typhi using the Agar diffusion method. The result showed that P. pellucida extract inhibited the growth of the test organisms, with the highest growth of inhibition Staphylococcus aureus and the lowest growth of inhibition against Bacillus cereus. The antimicrobial properties indicate the potential usefulness of this plant in the treatment of various pathogenic diseases which in future can be developed as a potential antimicrobial agent used in the treatment of infectious diseases. The results of this study revealed the presence of various pharmacologically active principles of medicinal importance including alkaloids, tannins, flavonoids, phenols and saponins. P. pellucida leave extracts are also good source of vitamins and minerals.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables x
List of Figures xi
List of Plate xii
Abstract xii
CHAPTER 1 INTRODUCTION
1.1 Background of Study 1
1.2 Statement of Problem 3
1.3 Aim and Objectives 3
1.4 Justification of Study 4
1.5 Scope of Study 4
1.6 Significance of Study 4
CHAPTER 2
LITERATURE REVIEW 5
2.1 Origin and Geographical Distribution of Peperomia pellucida 5
2.2 Description of the Plant 6
2.3 Piperaceae Family 7
2.4 Ethnomedicinal Uses of Peperomia pellucida 8
2.5 Pharmacological Activitiy of Peperomia pellucida 9
2.5.1 Antioxidant activity 9
2.5.2 Anti-microbial activity 10
2.5.3 Pharmacological activity 10
2.6 Economic Uses 11
2.7 Phytochemicals 11
2.7.1 Phenolic 12
2.7.2 Flavonoids 13
2.7.3 Tannins 15
2.7.4 Alkaloids 16
2.7.5 Terpenoids 17
2.7.6 Saponins 18
2.8 Spectroscopy
18
2.8.1 Nuclear magnetic resonance (NMR)
spectroscopy 19
2.8.2 One-dimensional nuclear magnetic resonance
(1d NMR) spectroscopy 20
2.8.3 Two-dimensional nuclear magnetic resonance
(2d NMR) spectroscopy 20
2.9 Chemical Constituents of Peperomia pellucida 20
2.10 Phytochemical Screening on Peperomia pellucida 23
CHAPTER 3: MATERIALS AND
METHODS 25
3.1 Materials for
Extraction 25
3.2 Sample Collection and Preparation 26
3.3 Solvent Extraction 26
3.4 Concentration of Peperomia Pellucida Using
Digital Heidolph
Rotary Evaporator. 26
3.5 Packing the Column 27
3.6 Thin
Layer Chromatography 28
3.6.1 Spotting, development and visualization 28
3.7 Quantitative Phytochemical Screening 29
3.7.1 Determination of alkaloids 29
3.7.2 Determination of flavonoids 30
3.7.3 Determination of tannins 30
3.7.4 Determination of saponins 31
3.7.5 Determination of phenols
32
3.8 Determination of Vitamin Content
32
3.8.1 Determination of ascorbic acid 32
3.8.2 Determination of niacin
33
3.8.3 Determination of riboflavin
34
3.8.4 Determination of thiamin
35
3.8.5 Determination of b-carotene 35
3.9 Determination of Mineral Elements
36
3.9.1 Determination of potassium and sodium
37
3.9.2 Determination of magnesium and calcium 37
3.9.3 Determination of phosphorus
38
3.9.4 Determination of copper and zinc 39
3.10 Proximate Analysis of Peperomia pellucida 40
3.10.1 Determination of protein content
40
3.10.2 Determination of crude fibre content
40
3.10.3 Determination of lipid content
41
3.10.4 Determination of ash content 42
3.10.5 Determination of moisture content
42
3.10.6 Determination of carbohydrate content
43
3.11 Determination of Antioxidant Activity
43
3.12 Antibacteriall Assay of P. pellucida
Leaf Methanol Extract 44
3.12.1 Obtaining and confirming the test organisms 44
3.12.2 Antibacterial susceptibility test 44
3.12.3 Determination of minimum inhibitory
concentration (MIC) 45
CHAPTER
4: RESULTS AND DISCUSSIONS 46
4.1 Phytochemical Screening Result 46
4.2 Mineral Content Results 48
4.3 Vitamin Content Results 50
4.4 Proximate Composition Of P. pellucida Leaves 52
4.5 Column Chromatography Results 55
4.6 Thin Layer Chromatograph (TLC) Results 56
4.7 Spectral Analysis Result 57
4.7.1 1H-NMR spectra result 57
4.7.2 2-Dimensional NMR spectra interpretation 59
4.8 Antioxidant Activity of P .pellucida Leaves Extract 63
4.9 Antimicrobial Properties of P. pellucida Leave Extract 64
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 67
5.1 Conclusion 67
5.2 Recommendations 67
References
LIST OF TABLES
4.1: Phytochemical
compostition of the leaf of P. pellucida expressed
in percentage (%) 46
4.2: Mineral element composition of P.
pellucida leaves 48
4.3. Quantitative
determination of vitamin contents of P.
pellucida
leaves
50
4.4 Proximate composition of P. pellucida leaves 52
4.5: Fractionation
of chloroform extract of P .pellucida
leaves by
column
chromatograph 55
4.6 1H-NMR
chemical shift for fraction C20 58
4.7: 13C
(DEPT) chemical shift of fraction C20 61
4.8: Antioxidant determination of P. pellucida leaves 63
4.9: Zones
of growth inhibition at varying concentrations of
methanolic
extract of P. pellucida leaves against the test organisms 65
4.10: Minimum
inhibitory concentration of methanol leaves extract of
P.
pellucida 65
LIST OF FIGURES
4.1: 1H-NMR for
fraction C20 57
4.2: 13C (DEPT) NMR spectrum for
fraction C20 59
4.3: 1H-1H-COSY spectrum for fraction C20 60
4.4: 1H-13C
HSQC spectrum for fraction C20 60
LIST OF PLATE
2.1: Peperomia pellucida plant 5
CHAPTER ONE
INTRODUCTION
1.1
BACKGROUND OF
STUDY
The substances obtained from
plant have recently become of great interest due to their universal
applications. Medicinal plants are the richest bio-resource of drugs, they have
been discovered and used in traditional medicine practices since prehistory times.
These medicinal plants seem to be excellent remedies to cure all the ailments
of mankind. The importance of botanical, chemical and pharmacological
evaluation of plant-derived-agents used in the treatment of human ailments has
been increasingly recognized in the last decades (Gbile, 1986). Many drugs,
which are commonly used in modern day medicines, have been derived either
directly or indirectly from herbal sources. In spite of all the progress in
synthetic chemistry and biotechnology, plants are still a vital source of
medicinal preparations, both for prevention and cure. The World Health
Organization (WHO) estimates that
80% of the world’s population presently uses herbal medicines for
some aspects of primary healthcare (Srivastara et al., 1996).
‘Phyto’ is gotten from a Greek word which means plant. Plants are
made up of chemicals of various kinds. Plants have played an important role in
maintaining the human health and improving the quality of human life for
thousands of years (Winston, 1999). The chemical properties of plants have been
shown to be responsible for these nutritional and healing properties (Setchell
and Cassidy, 1999). More importantly, plants have been the main source of
medicine for man before the advancement of Science and Technology (Schmelzer
and Omino, 2003). Plants are the most vital sources of medicines that are used
in the treatment of various categories of human diseases. In Nigeria, various
indigenous plants are used both as food and as medicine. One of such plants is Peperomia pellucida also known as water
plant.
Phytochemicals are chemical compounds formed during the plants
normal metabolic processes (Okigbo et al,
2009). They are defined as natural chemical compounds found in plants which
possess biological activities beneficial to human health. Phyotochemicals are
chemicals manufactured by plants via secondary or primary metabolism. They
primarily have biological effects in plant and act in plant development and
defense against competitors, pathogens and predators. Phyotochemicals are
considered as research compounds rather than essential nutrients because
evidence of their possible health effects has not been recognized yet. They are
found in fruits, vegetables, whole grains, nuts, seeds, fungi, herbs and
spices. Broccoli, cabbage, carrot, onions, garlic, whole wheat bread, tomatoes,
grapes, cherries, strawberries, raspberries, beans, legumes and soy foods are
common sources. Phytochemicals accumulate in different parts of the plants such
as the roots, stems, leaves, flowers, fruits or seeds.
In modern medicine, plants are used as sources of direct healing
agents, as models for new synthetic compounds, and as a systematic marker for
discovery of new compounds. They serve as a raw material base for the
elaboration of more complex semi synthetic chemical compounds (Akerele,
1992). According to Heldt (2005), most
of these phytochemicals are produced through biosynthesis in the metabolic
pathways. The primary metabolites are of major importance to plants (Trease and
Evans, 1989). The secondary metabolites are of medicinal value to man and these
can equally be obtained from various anatomical structures of plants (Fahn,
1974). Literature of many researchers proves that plant contains alkaloids,
saponins, tannins and cardenolides (Khan et
al., 2010), flavonoids, essential oils and carotol (Khan et al., 2002).
Peperomia pellucida (L.) Kunth is an annual, shallow rooted herb that belongs to the
family piperacea
(Ghani, 1998). It is commonly known as shiny bush. It is found in Nigeria,
Sierra Leone and Ghana.
Other places where it is found are Southern America, Asian
countries, China, Brazil and DR Congo (Ghani et al., 1998; Bayma et al.,
2000; Dos Santos et al., 2001 and
Arrigona-Black et al., 2004). It
grows in clumps and thrives in loose humid soils, and about 15 to 45cm and is
characterized by succulent stems and shiny heart shaped, fleshy leaves and tiny
dot like seed attached to several fruiting spikes (Dos Santos et al., 2001).
1.2
STATEMENT OF
PROBLEM
The leaves of Peperomia
pellucida are widely used as food and flavouring agent in various parts of
the world, and also used in traditional medicine to cure various ailments like
acne, fever, boil, haemorrhages etc. The phytoconstituents of this plant have
not been fully documented, it is therefore necessary to delve into the
isolation and characterization of the bioactive constituents available in the
leaves.
1.3
AIMS AND
OBJECTIVES
This research work is aimed
at the isolation and NMR characterization of Ursane-type triterpenoid and the
bioprotective investigation of secondary metabolites from the leaves of P. pellucida. This aim is hoped to be
achieved through the following specific objectives:
1. Carrying out soxhlet extraction on the leaves of P. pellucida
2. Isolation of secondary metabolites using column and thin layer
chromatography techniques.
3. Characterization and identification of the isolated secondary
metabolites using 1H, C-13, Heteronuclear Single Quantum Coherence
(HSQC), Distionless Enhancement Polarization by
Transfer
(DEPT), Correlation Spectroscopy (COSY) NMR spectral techniques.
4. Determination of the phytochemicals, vitamins, minerals and
proximate composition of P. pellucida
5. Determination of the antimicrobial and antioxidant activities of the
secondary metabolites from the leaves of
P. pellucida
1.4 JUSTIFICATION OF THE STUDY
An alternative source of
antimicrobial medications can be found in Peperomia
pellucida. However, unless its bioactivity is recognized and its bioactive
principles are discovered and characterized, it cannot be developed and
standardized. Investigating P. pellucida
is therefore necessary to learn more about its chemical makeup, characteristics,
and effectiveness.
This study will provide a
database for the active compounds that were isolated, the phytochemicals,
minerals and vitamins found in the leaves, as well as the antimicrobial
properties of P. pellucida leaves.
1.5 SCOPE OF STUDY
The study covers a wider area of scientific research such as plant
collection and identification, extraction, phytochemical screening, vitamin and
mineral element determination, proximate analysis, partitioning followed by
chromatography and spectral analysis. The study also covers the antimicrobial
and antioxidant determination of P.
pellucida
1.6
SIGNIFICANCE OF
STUDY
The study on the leaves of Peperomia
pellucida is important from both literature and experimental analysis. The
research is built on previous work done so far on the leaves of the plant for
the literature aspect, and this gives the insight on the ethnomedicinal uses of
the plant. On the experimental aspect, the findings may be of importance to
researchers, pharmaceutical industries and for traditional herbalists for
effective cure of various ailments. The findings will also help researchers by
providing the basic empirical information to do further work on the plant.
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