1.2.0 LITERATURE REVIEW 9
1.2.1.0 Spondias mombin
1.2.1.1 Taxonomical Classification of S. mombin 9
1.2.1.2 View of S. mombin 10
1.2.1.3 Origin, Botany and Morphology 11
1.2.1.4 Habitat 13
1.2.1.5 Cultivation details 13
1.2.1.6 Propagation 14
1.2.1.7 Edible Uses 14
1.2.1.8 Medicinal Uses 14
1.2.2 Phytochemical Composition 15
1.2.2.1 Alkaloids 15
1.2.2.2 Tannins 16
1.2.2.3 Saponins 17
1.2.2.4 Flavonoids 18
1.2.2.5 Steroids 19
1.2.2.6 Terpenoids 20
1.2.2.7 Glycosides 21
1.2.3 Antioxidants 22
1.2.4 Antioxidant assay 22
vii
1.2.4.1 Ferric ion Reducing Antioxidant Power (FRAP) 23
1.2.4.2
2-2, Diphenyl- 1- picrylhydrazyl (DPPH)
23
1.2.4.3
2-2’-Azino-di-(3-ethyl benzothiazoline sulphonate (ABTS)
24
1.2.5
Proximate Composition analysis
25
CHAPTER TWO
2.1.0 MATERIALS 25
2.1.1
Solvents and reagents
25
2.1.2
Glass wares and Apparatus
25
2.1.3
Equipment and Other Materials Used
26
2.1.4
Drugs
2.1.5 Animals
2.2.0
METHODS
27
2.2.1
2.2.2
Sample Collection and Preparation of
Stem Back of S. mombin
Extraction of Crude Powdered Sample
27
27
2.2.3
Phytochemical Screening of Stem bark of S. mombin
28
2.3
PROXIMATE ANALYSIS
29
2.3.1
Proximate analysis of stem bark of S. mombin
29
2.3.2
Moisture content
29
2.3.3
Total ash
29
MATERIALS AND METHODS
27
2.3.4 Acid-insoluble ash 29
2.3.5
Water-Soluble Ash
30
2.3.6
Determination of Crude Fat
30
2.3.7
Determination of Crude Fibre
30
2.4
ANTIOXIDANT ACTIVITY
31
2.4.1
Measurement of Free Radical Scavenging Activities
of Ethyl Acetate Extract of S. mombin
31
2.4.1.1 Preparation of sample extract solutions 31
2.4.1.2 Preparation of Ascorbic Acid Solution
2.4.1.3 Preparation of 0.0001mmol/dm3 of DPPH
31
(2, 2-diphenyl-1-picrylhydrazyl) 32
2.5 ANTIDEPRESSANT ACTIVITY 33
2.5.1 Preparation of Laboratory Animals 33
2.5.2 Forced Swim Test 35
2.5.3 Tail Suspension Test 35
CHAPTER THREE
3.0 RESULTS AND DISCUSSION 35
3.1
PHYTOCHEMICAL SCREENING
35
3.2 PROXIMATE ANALYSIS 37
3.3 ANTIOXIDANT ACTIVITIES 38
3.4 ANTIDEPRESSANT ACTIVITIES 41
3.5 FINDINGS 45
3.6 CONCLUSION
45
REFERENCES
47
APPENDICES
55
LIST OF TABLES
Table 2.1 Preparation of Blank, control, sample and standard 34
Table 3.1:
Phytochemical screening of stem bark of S. mombin
37
Table 3.2: Study of different parameters obtained from proximate analysis of powder of stem bark of S. mombin 39
Table 3.3: Percentage free radical scavenging ability of sample extract with
DPPH 42
Table 3.4: Percentage free radical scavenging ability of
ascorbic acid with DPPH 42
Table 3.5: Effect of ethyl acetate extract of stem bark of S. mombin as an antidepressant agent against Forced Swim Test in mice 44
Table 3.6: Effect of ethyl acetate extract of stem bark of S. mombin as an antidepressant agent against Tail Suspension Test in mice 45
Fig. 1.1
Morphine LIST OF FIGURES
16
Fig. 1.2
Quinine
16
Fig. 1.3
Gallic acid
17
Fig. 1.4
Saponin
18
Fig. 1.5
Catechin
19
Fig. 1.6
Steroids
20
Fig. 1.7
Limonene
21
Fig 1.8
Artemisinin
21
Fig. 1.9
Glycoside
21
Fig. l.10 O-phenanthroline Fe2+ 23
Fig. 1.11 DPPH 24
Fig. 1.12 ABTS 24
Fig. 3.1 % inhibition of DPPH against concentration of ethyl acetate extract of stem bark of S. mombin and ascorbic acid 42
Fig. 3.2: Effect of ethyl acetate extract of stem bark of S. mombin as an antidepressant agent against Forced Swim Test in mice 44
Fig. 3.3: Effect of ethyl acetate extract of stem bark of S. mombin as an antidepressant agent against Tail Suspension Test in mice45
Fig 3.4: Bar Chart of % Inhibition against Groups/Doses (mg/kg) for Forced Swim Test Fig 3.5: Bar Chart of % Inhibition against Groups/Doses (mg/kg) for Tail Suspension Test
LIST OF PLATES
Plate 1 S. mombin fruiting 11
Plate 2 Oval shaped fruits of S.mombin 11
Plate 3 S. mombin unripe fleshy fruits 11
LIST OF APPENDICES
APPENDIX I Proximate Analysis 58
APPENDIX II
APPENDIX III
Antioxidant Activities
Antidepressant Activities
69
67
ABSTRACT
S. mombin is a plant used by traditional medical practitioners in the treatment of many diseases, both physical and mental. All experiments in this study were carried out using standard methods. In view of the medicinal potential of the stem bark of S. mombin, this study was therefore undertaken to evaluate the phytochemical constituents, the proximate compositions, antioxidant and antidepressant activities of the ethyl acetate extract of stem back of S. mombin. Phytochemical screening of stem back extract of S. mombin revealed the presence of alkaloids, cardiac glycosides, steroids, phenols, tannins, saponins, terpenoids and flavonoids. The proximate analysis of the crude stem back yielded 9.33 0.40%, 1.40 0.33%, 2.00 0.17%, 17.40 0.20%, 6.50 0.48% for moisture content, total ash, acid insoluble ash, water soluble ash, crude fat and crude fibre respectively. Free radical scavenging ability ranging from 47.74 – 80.18% and
79.28 – 88.29% was observed for ethyl acetate extract of stem back of S. mombin and ascorbic acid respectively. In the antidepressant study, the Forced Swim Test and Tail Suspension Test of the ethyl acetate extract of stem bark of S. mombin at (100 and
200mg/kg) showed a significant reduction in immobility time with 77.9% and 76.6% for Forced Swim Test and 65.7% and 91.5% for Tail Suspension Test. The result obtained proposed a sedative and antidepressant–like effect of ethyl acetate extract of stem bark of S. mombin, confirming the ethnomedicinal use of the stem bark of S. mombin for the management of depression.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1.0 INTRODUCTION
In recorded history, medicinal plants have been in use for the treatment of man and animal diseases (Osai, 1998 and Ibewike et al., 1997). A plant becomes a medicinal plant only when its biological activity has been ethnobotanically reported or scientifically established (Elujoba, 1997). In 1978, the World Health Organization (WHO) emphasized the importance of scientific research into herbal medicine. Many developing countries of the world look upon native medicinal plants as possible addition to the WHO’s list of ‘essential drugs’ once their values have been clinically proven. A simple first-principle biological technique in the evaluation of plant with known biological activity is to conduct a screening test (Elojoba, 1999). Various studies have been concerned worldwide in the identification and isolation of new therapeutic substances from diverse herbal plants to treat the existing and newly arousing diseases (Maria et al., 2016). S. mombin is one of such medicinal plant with a lot of potential, valuable, untapped resources of active drug for treating diseases (Ayoka, et al., 2008). Secondary metabolites are the classes of compounds which are known to display therapeutic activities against several sicknesses in man; they therefore explained the use of traditional therapeutic plants for the treatment of some diseases (Sabri et al., 2012). Unlike primary metabolites, secondary metabolites do not result in instant death, but rather in long-term weakening of the organism’s survivability, fertility, or aesthetics, or perhaps in no significant change at all (Wikipedia, 2018).
S. mombim linn is a small tree that grows up to 20m (60 ft) high and 1.5 m (5 ft.) in girth, moderately buttressed; stem bark is thick, corky, deeply fissured, slash pale pink, darkening rapidly, branchlets glabrous; leaves pinnate, leaflets 5-8 opposite pairs with a terminal leaflet. It belongs to the family Anarcardiaceae. Its flowers develop between January – May and fruits between July – September. The fruits have a sharp, somewhat acid taste and are edible. The matured fruit has a leathery skin and a thin
layer of pulp. The fruit pulp is either eaten fresh, or made into juice, concentrate, jellies, and sherbets. The fruit-juice is used as a febrifuge and diuretic. The roots are also used as febrifuge in Ivory Coast (Ayoka, 2006). The bark is used as a purgative and in local applications in the treatment of leprosy. The stem bark decoction is also used in the treatment of severe cough. It serves as an emetic, a remedy for diarrhea, dysentery, hemorrhoids and a treatment for gonorrhea and leucorrhoea (Ayoka, 2006). The decoction of the astringent stem bark is believed to expel calcifications from the bladder. The juice of crushed leaves and the powder of dried leaves are used on wounds and inflammations. The gum is employed as an expectorant and to expel tapeworms (Rodriguez, 2000). A decoction of the mashed leaves is used by the Ibos (Nigeria) for washing a swollen face. A leave infusion is a common cough remedy or used as a laxative for fever with constipation. A leave decoction is used in treatment of gonorrhea. The leaves are used in Ivory Coast for fresh wounds to prevent inflammation (Igbokwe,
2014).
The pulp of the fruit is sometimes eaten directly, especially when found in the forest, but it is too acidic to be considered attractive; It can also be boiled or dried. It is especially used for syrup, ice cream, drinks and jellies. Juices improve with keeping overnight as the mild astringency of the fresh fruit disappears. Fermented products are also good. The fruits are a good source of vitamins A and C; Vitamin C quantities vary between 34 and 54mg/g, and carotenoids are presumably present in reasonable concentrations. There is a great variation in fruit quality from region to region, some being sweet and pleasant and others quite disagreeable in flavour (Moronkola, 2003). Nworu et al., reported the several uses of the plant based on oral communication and not on any recorded scientific investigation, Infusion of its leaves has been used since long time without any report of collateral effects, due to its activities.
Proximate analysis helps to set up definition standard for dried crude drugs in order to avoid batch-to- batch dissimilarity and also to judge their quality (Backer and Hyne, 2012). Proximate analysis of
dehydrated crude drugs comprises determinations such as crude fibre, crude fat, Moisture content, total ash, acid-insoluble ash, water soluble ash.
Crude fibre contains cellulose, hemicellulose, lignin and other substances that are difficult to digest
(Yaovalak Suksong, 2013).
Crude fibre is a measure of the quantity of indigestible cellulose, pentosans, lignin, and other components of these types in foods. These components have little food value but provide the bulk necessary for proper peristaltic action in the intestinal tract.
Crude fat is more of a screening method because the test procedures do not identify a specific fat; instead, the method measures the materials that can be dissolved in some solvents. Crude fat is synonymous with “ether extract” and generally refers “free” lipids that can be extracted into less polar solvents such as petroleum ether or diethyl ether.
Ash is categorized as physiological ash which results from the plant tissue itself and non-physiological ash which is the remains after ignition of extraneous matter (example, sand and soil) (Khandelwe,
1998). The total ash comprises of carbonates, phosphates, silicates and silica (Backer and Hyne, 2012). Acid insoluble ash measures the occurrence of silica, mainly sand and siliceous earth (Anonymous,
1986). Water soluble ash is the difference in weight between the total ash and the residue gotten after boiling the ash in water (Backer and Hyne, 2012). Moisture content is the amount of moisture in the sample given as a percentage of the sample’s original weight. Typically, moisture content is determined via a thermogravimetric approach, i.e. by loss on drying, in which the sample is heated and the weight loss due to evaporation of moisture is recorded.
Oxidative process is one of the most important routes for producing free radicals in food, drugs and even in living systems (Halliwel, 1994). It has been established that oxidative stress is among the major causative factors of many chronic and degeneration diseases including ageing, diabetes milletus, cancer, ischemic heart disease, neurodegenerative diseases and others (Younge and Woodsick, 2001),
but a great number of aromatics, medicinal spice and other plants contain chemical compounds exhibiting antioxidant properties. Antioxidants are compounds that inhibit oxidation. Oxidation is a chemical reaction that can produce free radicals, thereby leading to chain reactions that may damage the cells of organisms. Antioxidants such as thiols and ascorbic acid (vitamin C) terminate these reactions (Wikipedia, 2019).
Depression is an effective disorder which results in a state of low mood and aversion to activities that can have a negative effect on a person’s thought, behavior, world view and physical wellbeing (American Psychiatric Association, 2013). Many psychiatric syndromes feature depressed mood as main symptom hence it has become a major global psychiatric problem (Pillemer, 2010). About 350 million people suffer from depression globally (WHO, 2012). People suffering from depression struggle with loss of energy and motivation, which influence their ability to be productive, making depression the leading cause of disability worldwide (Kessler, 2009). Treatment is varied but psychotherapy and pharmaceutical drug treatment are most commonly used. Pharmaceutical drug involves the use of antidepressant medications, which are based on neurotransmitters (naturally occurring endogenous neurochemicals) levels imbalance in the brain. Drug can influence mood/ behavior by altering neurotransmitters concentration in the brain hence the selection of antidepressant agent is based on the neurotransmitters that are thought to influence the symptoms of depression (Nutt,
2008). The Tricyclic Antidepressant (TAC), Selective Serotonin Reuptake Inhibitors (SSRI), Nor – Epinephrine Reuptake Inhibitors (NRI), “(DRI), and Serotonin-Nor-epinephrine Reuptake Inhibitors (SNRI) are the major classes of antidepressant used in pharmaceutical drug treatment (Millan, 2006). S. mombin is classified as an underutilized plant in Nigeria where it grows widely and as such, the associated health benefits are untapped. Based on the above evidences, the present study was designed to determine the antidepressant activity of the stem bark of the Nigerian grown S. mombin using the Forced Swim Test (FST) and Tail Suspension Test (TST).
