ISOLATION, CHARACTERISATION AND ANALGESIC PROPERTIES OF CHLOROFORM EXTRACT FROM STACHYTARPHETA ANGUSTIFOLIA

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ABSTRACT

The plant Stachytarpheta angustifolia is a medicinal plant which comes from the genus Stachytarpheta of the family Verbenaceae. This study is aimed at isolating the plant extract, characterizing it and determining the phytochemical and analgesic properties of the plant. Extraction was done using chloroform and column chromatography was used for the elution of the crude extract using silica gel as the stationary phase. Solvents used as the mobile phase were petroleum ether, chloroform and methanol. These solvents had differing polarity; from non-polar to polar. A total of 26 fractions were collected after elution. Purity of the fractions were monitored using thin layer chromatography (TLC) on silica gel coated plates. Solvents used were petroleum ether, choroform and methanol. Quantitative and qualitative screening test were both carried out on the crude extract to determine the phytochemical properties of the plant. Analgesic analysis was carried out using acetic-acid writhing reflex method. After TLC, fraction 26 yeilded a single spot with the solvents, petroleum ether, chloroform and methanol in the ration 1:3:6 (Rf =0.55). The structure was elucidated using FTIR, 1HNMR, 13CNMR, DEPT-135 and C-H COSY and the structure had a molecular formula of C28H48O3. The compound was characterized as a triterpenoid. Phytochemical screening using the standard qualitative techniques revealed the presence of flavonoids, phenolics, tannins, steroids and terpenoids. The result showed very strong presence of Alkaloids, Flavonoids and terpenoids. The crude extract also showed good analgesic property. As a follow-up on the study, it is anticipated that the compound can further be explored to reveal its potential applications.







TABLE OF CONTENTS

 

Title Page                                                                                                        i

Declaration                                                                                                      ii

Certification                                                                                                    iii

Dedication                                                                                                      vi

Acknowledgements                                                                                        v

Table of Contents                                                                                           vi

List of Tables                                                                                                  xi

List of Figures                                                                                                 xii

List of Plates                                                                                                   xiii

Acronyms                                                                                                        xiv

Abstract                                                                                                          xvi

 

CHAPTER 1: INTRODUCTION                                                                          1

1.1       Background to the Study                                                                               1

1.2       Statement of the Research Problem                                                                4

1.3       Aim and Objectives of the Study                                                                   5

1.4       Justification of the Study                                                                               6

1.5       Scope and Limitation                                                                                      6

CHAPTER 2: REVIEW OF RELATED LITERATURE                                 7

2.1       Analgesics                                                                                                       7

2.1.1    Sources of Analgesic drugs                                                                            8

2.2       Starchytarpheta Species                                                                                  9

2.2.1    Stachytarpheta angustifolia                                                                             9

2.3       Phytochemical Studies of Stachytarpheta                                                      15

2.3.1    Alkaloids                                                                                                         17

2.3.2    Saponins                                                                                                          18

2.3.3    Phenols                                                                                                            19

2.3.4    Flavonoids                                                                                                      19

2.3.5    Tannin                                                                                                             21

2.4       Biological Activity of the Plant                                                                      22        2.4.1            Hypoglycemic activity                                                                                    22

2.4.2    Antioxidants                                                                                                   24

2.4.3    Anti-microbial                                                                                                 26

2.4.3.1 Antibacterials                                                                                                  28

2.5       Analgesic Effect of Stachytarpheta                                                                29

2.6.      Acetic acid induced writhing                                                                         30

2.7       Chromatography                                                                                             31

2.7.1    Principles of chromatography                                                                         32

2.7.2    Chromatography terms                                                                                   32

2.7.3    Types of chromatography                                                                               33

2.7.3.1 Column chromatography                                                                                34

2.7.3.2 Thin layer chromatography (TLC)                                                                  35

2.7.3.3 Paper chromatography                                                                                    37

2.8       Retention Factor                                                                                             37

2.9       Nuclear Magnetic Resonance (NMR) Spectroscopy                                      38

2.9.1    Principle of NMR                                                                                           41

2.10     Infra-Red Spectroscopy                                                                                  42

2.11     Mass Spectroscopy (MS)                                                                                43

2.12     Matrix Assisted Laser Desorption Ionization                                                 44

CHAPTER 3: MATERIALS AND METHODS                                                   45

3.1       Materials                                                                                                         45

3.1.1    Experimental animals                                                                          45

3.2       Methods                                                                                                          47

3.2.1    Summary of experimental procedures                                                47

3.2.2    Sample collection                                                                                            48

3.2.3    Sample processing                                                                                           48

3.2.4    Extraction                                                                                                       48

3.3       Phytochemical Screening                                                                                48

3.3.1    Screening test                                                                                                  48

3.3.1.1 Alkaloids                                                                                                         49

3.3.1.2 Saponins                                                                                                          49

3.3.1.3 Tannins                                                                                                            49

3.3.1.4 Flavonoids                                                                                                      49

3.3.2    Quantitative test                                                                                             50

3.3.2.1 Tannin                                                                                                             50

3.3.2.2 Saponin                                                                                                           50

3.3.2.3 Alkaloids                                                                                                         51

3.3.2.4 Phenols                                                                                                            51

3.3.2.5 Flavonoids                                                                                                      52

3.4       Pharmacological Screening                                                                             53

3.4.1    Acute toxicity test                                                                                          53

3.4.2    Acetic acid-induced writhing in rat                                                                54

3.3.4    Statistical analysis                                                                                           54

3.5       Isolation                                                                                                          55

3.5.1    Spectra analysis                                                                                               56

 

CHAPTER 4: RESULTS AND DISCUSSION                                                    57

4.1       Phytochemical Screening                                                                                57

4.2       Thin Layer Chromatography                                                                           60

4.3       Spectral Analysis of Isolated Extract of S. angustifolia                                 62

4.3.1.   FT-IR analysis of the isolated chloroform extract of S. angustifolia              62

4.3.2    Proton-NMR of ST 26                                                                                    65

4.3.3    Carbon-Proton Cosy (HETCOR)                                                                   67

4.3.4    Carbon-13 (13 C) NMR                                                                                   69

4.3.5    Carbon-13(13 C) DEPT NMR                                                                         70

4.4       Analgesic Activity                                                                                          72

4.4.1    Acetic acid induced writhing reflex                                                   72

CHAPTER 5: CONCLUSION AND RECOMMENDATION                          74

5.1      Conclusion                                                                                                      74

5.2      Recommendations                                                                                          75

REFERENCES                                                                                                       76

 

 

 

 

LIST OF TABLES

2.1 Acetic acid-induced writhing in rats / mice                                                          31

3.1 Body weight, Food and water intake of Wister rats                                            45

3.2 Fractions of S. angustifolia extract by column chromatography                           56

4.1 Phytochemical qualitative screening of S. angustifolia                                         59

4.2 Phytochemical quantitative screening of S. angustifolia                                       59

4.3 IR absorption of S. angustifolia                                                                            64

4.1 S. angustifolia on Acetic induced Writhing Reflex in Albino Rats                      72

 

 

 


 

 

LIST OF FIGURES

2.1: Iridoid glycoside of S. angustifolia (Compound 1)                                           12

2.2: Iridoid Glycoside S. angustifolia (Compound 2)                                               13

2.3: Iridoid glycoside of S. angustifolia (Compound 3)                                           13

2.4: Lanostanoid triterpenoid glucoside of S. jamaicensis (Compound 4)               14

2.5: Steroidal Glycoside of S. jamaicensis (Compound 5)                                       14

2.6: Nnesodine B (Compound 6)                                                                              14

2.8: Saponin Structure                                                                                              18

2.9: Flavone structure                                                                                               20 2.10: Isoflavan structure                                                                                            20

2.11: Neoflavonoid structure                                                                                    20

2.12: Structures of some antioxidants                                                                      26

3.1: Flow chart of methodology                                                                               47

4.1: FT-IR Spectrum for the S. angustifolia extract                                                  63

4.2: Proton NMR (1H-NMR) for the S. angustifolia extract                                     65

4.3: Carbon-Proton COSY NMR for Stachytarpheta angustifolia extract               67

4.4: 13C NMR of Stachytarpheta angustifolia extract                                   69

4.5: 13C-DEPT (135) NMR of Stachytarpheta angustifolia extract                          70

4.6: Proposed structure from isolated compound of Stachytarpheta angustifolia.  71

 

 

 

LIST OF PLATES

2.1 Stachytarpheta angustifolia                                                                                 10

4.1 2D TLC showing a single spot for fraction ST26                                               62

 

 

  

 

 

ACRONYMS

%: Percentage.

3D: Three Dimension

4D: Four Dimension

ASA: Acetyl Salicylic Acid.

ADME: Absorption, distribution, metabolism and elimination

COX: Cyclooxygenase

COSY: correlation spectroscopy

DF: Dilution factor

Fig: Figure

FT-IR: Fourier Transform Infrared Spectroscopy

GC – MS: Gas Column Spectrophotometry.

H2SO4: Sulphuric acid.

HCl: Hydrochloric acid.

HETCOR: Heteronuclear Correlation

HMBC:Heteronuclear Multi-Bond Connectivity

HMQC:Heteronuclear Multi-Quantum Coherence

HOESY:HeteronuclearOverhauser Effect Spectroscopy

HPLC: High Pressure Liquid Chromatography

HSQC:Heteronuclear Single Quantum Coherence

IR: Infrared

KG: Kilogram.

L: Liter.

LD50: Median lethal dose.

MIC : Minimum inhibitory concentration

ML: Mililiter.

MHz: Mega Hertz

MM: Milimeter.

MRS: Magnetic Resonance Spectroscopy

NMR: Nuclear Magnetic Resonance

N/S: Normal saline.

NOE: Nuclear Overhauser Effect 

NOESY: Nuclear Overhauser Effect Spectroscopy

NSAIDs: Non-steroidal anti-inflammatory drugs.

Rf: Retention Factor.

TLC: Thin layer chromatography.

UV: Ultraviolet Light.

W/W: Weight per weight.

WHO: World Health Organization.

 

 

 




 

CHAPTER ONE

INTRODUCTION


1.1      Background to the Study

1.1.1 Plants with medicinal Properties

Medicinal plants with high therapeutic index are usually used for the treatment of many ailments (Chelaiah and Muniappan, 2006). This has led to an increased interest in their use rather than chemical drugs (Sasidharan et al., 2011). Research has shown significant progress in the use of medicinal plants for the treatment of various ailments such as diabetes, fever, hypertension, and veneral diseases. The total population of plant species in the world is estimated to be between 250, 000 and 500,000 and a large proportion has not been explored for medicinal purposes (Mehesh and Satish, 2008) and (Shinwari et al., 2013). Some of these claims by the traditional healers have been substantiated with scientific evidence. The active ingredients are usually extracted from the plant either traditionally or in the laboratary. The oldest written verification of medicinal plants usage for preparation of drugs was found on a Sumerian clay slab from Nagpur approximately 5000 years old (Sumboonnanonda and Lertsithichai, 2004). This Sumerian clay slab showed 12 recipes for drug preparation referring to over 250 various plants, some of the plants include poppy, henbane, and mandrake. Many of these recipes were also found in the ancient Chinese book on roots and grasses “Pen T’Sao,” written by Emperor Shen Nung circa 2500 BC. This book details 365 drugs from dried parts of medicinal plants, many of which are used even currently (Tesfahun et al., 2014).

While people in the past used medicinal plants primarily in simple pharmaceutical forms (infusion, decoctions and marcerations), the demand for compound drugs constantly increased with time. Plants have secondary metabolites which have been utilized by humans for making medicines (Daniel et al., 2012). According to World Health Organization (WHO), 80% of people in the world currently rely on herbs for their primary healthcare, to generate income and for livelihood improvement (Ozbilgin and Saltan, 2012). This is because herbs are considered to have a variety of secondary metabolites and are accessible, safer and affordable compared to the synthesized products which are regarded as having adverse side effects on humans and the environment (Gunjan et al., 2013). In addition, most of the secondary metabolites from plants provide protection against infections and are source of plant survival (Lauren and Peter, 2011).

Pain however is a common ailment suffered by many people, therefore the need to find drugs that can relief pain. Many scholars have reported the use of plants in relieving pain (Fabricant and Farnsworth, 2001; Kennedy, 2004; Scheid, 2007; Khare, 2007). Researchers have identified a number of plants whose extracts have shown analgesic activity (Ezeja et al., 2011; Panda et al., 2009; Okwu and Iroabuchi 2009; Kondangala et al., 2011; Rahul et al., 2011) and this has especially been prompted by the fact that many synthesized analgesic drugs that relieve pain such as morphine are known to be associated with adverse effects like ulceration, gastrointestinal bleeding, addictive potential, respiratory distress, drowsiness and nausea (Modi et al., 2012). Ibuprofen, another pain killer can increase the risk of heart failure (Forman et al., 2005). Analgesic drugs act in various ways on the peripheral and central nervous systems. There are many analgesic and synthetic drugs like Paracetamol, non steroidal anti-inflamatory drugs (NSAIDs), Ibuprofen, diclofenac whose use is limited due to their significant adverse side effect on the stomach, kidney and other body tissues. This has led to the need to find natural sources to relieve pain. Many phytochemicals from herbal plants have been found to provide analgesic solutions which are safe and broadly effective with fewer side effects (Sasidharan et al., 2011; Ali et al., 2014). Plants which are rich sources of analgesics include: Aloevera barbedensis, Andrographis paniculata, Elettaria cardamomum, Punica granatum, Eugenia caryophyllus, Curcuma alismatifolia, Phoenix sylvestris, Stachys schtscheglee, Cissus quadrangularis, menthol, Bunts longifolia, Buxus sempervirens, Burns sempervirens, Fumaira vaillantii, Rumex crispus, Urtica dioica and Morinda Citrifolia, Stachytarpheta jamaicensis (Okwu et al., 2010), Pimenta officinali (Priya et al., 2012), Andrographis paniculata, Argemone mexicana, Pimenta racemosa, Monarda didyma, Citrus bergamia, Boswellia thurifera which have been used as herbal solutions to relieve pain (Dana, 2010; Remedy, 2015). Providers of medications from medicinal plants do not always consider fully the harmful side effects of their prescriptions (Huntsman Cancer Institute, 2013). Although opioid drugs are normally much safer than synthetic drugs which is the reason some medical professionals prefer to prescribe more of opioid drugs. However, opoid drugs still have some side effects which may incluse; cognitive impairment, respiratory depression, gastrointestinal e.g. constipation, nausea, vomiting and gastrointestinal bleeding. Rosiglitazone which is used to treat diabetes is known to increase the risk of heart failure (Martinez, 2006).

Stachytarpheta angustifolia extract has also been usedfor a number of ailments as an anti-diabetic, anti-asthmatic, arbotifacient, sedative, antihypertensive, and antifever (Yakubu et al, 2005). Iridoid glycosides have also been extracted from Stachytarpheta angustifolia. They are a large group of naturally occurring Monoterpenoid with a glucose moiety attached at C-1 in the pyran ring (Song et al., 2006). They occupy an important position in the field of natural product chemistry and biology, as they provide a structural link between terpenoids and indole alkaloids and as well display a broad spectrum of biological activities (Sticher, 1977). Iridoids are useful phytochemicals in a number of folk medicinal plants and many of them poses significant biological and pharmacological activities; some of them are chemo taxonomically useful as markers of genus in various plant families (Sticher, 1977).

The main hindrance for herbal therapies nevertheless is the blending of native knowledge with modern medical practices with little or no scientific data available regarding the safety and efficacy of the herbal drugs (Ali et al., 2014).


1.2 Statement of the Research problem

Pain is an accompaniment of many medical conditions and pain control is among the important therapeutic priorities (Ezeja et al., 2011). It is mostly a warning signal that causes discomfort which leads to many adverse effects (Raquibul et al., 2010). Since most of the analgesic drugs available have serious side effects, it is necessary to explore biodiversity and heighten the search for new analgesic drugs with minimal side effects. This study is set to investigate the analgesic property of the chroloform extract of Stachytarpheta angustifolia.

 

1.3 Aim and objectives of the Study

The concern of this study is to extract, characterize and isolate the active components in Stachytarpheta angustifolia plant using chloroform and determine the analgesic properties of the plant. The aim of this study is achieved by:

1.  identification and collection of samples of Stachytarpheta angustifolia and drying under room temperature.

2. merceration of the pulverized sample using chloroform to obtain the crude extract for further analysis

3. conducting phytochemical screening on the chloroform crude extract to determine the secondary metabolites in the plant extract.

4. conducting an acetic acid induced screening on the crude chloroform extract to determine the analgesic properties of the plant.

5. conducting statiscal analysis to analyze results gotten from phytochemical screening and acetic acid-induced writhing in rats.

6. separation of the crude extract into fractions by using column chromatography

7. isolation of the pure fraction using Thin Layer Chromatography

8. carrying out spectroscopic analysis to propose a structure for the isolated pure fraction

1.4 Justification

The use of compound drugs for relieving pain has led to the development of more challenging health problems which is of great concern. This may include ulcer, heart palpitation, hypertension etc. This brings researchers to seek alternative ways of relieving pain with lesser or milder side effects. This research is therefore targeted at finding the analgesic effect of the plant extract of Stachytarpheta angustifolia in comparison with compound drug aspirin.


1.5 Scope and Limitation

The study focuses on the isolation, characterization, phytochemical studies as well as analgesic activity of Stachytarpheta angustifolia. The analgesic activity of Stachytarpheta angustifolia was compared with the synthetic drug Aspirin. The study was limited by the non-availability of Nuclear Magnetic Resonance instruments in Nigeria which necessitated the quest to obtain spectral analysis from sophisticated laboratories abroad.

 

 

 

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