ABSTRACT
Pausinystaliayohmibeis a member of the family Rubiaceae. It is a valuable medicinal tree, distributed in evergreen closed canopy forests in West Africa. It is traditionally used for treatment mostly on erectile dysfunction. This research was carried out to evaluate the immunomodulatory effect of (Pausinystaliayohmibe) stem bark methanolic extract. The yohimbe extract has been reported for its medicinal use for the treatment of impotence in men and for its anti-oxidants effects but have not be reported for any sign of immunomodulation or suppressive activity, which Is the basic aim for this research. This was achieved by assessments of the extract in the following parameters, using albino mice the phytochemical analysis which received the presence of alkaloids, flavonoids, tannnis . The acute toxicity effect of the extract which showed LD50 above 1000mg/kg and other signs of toxicity like dizziness and depression at dose 800mg/kg on a prolong administration . The animals were divided into four groups which received 100, 300 and 600 mg/kg , respectively while group four served as the control which revived a standard drug (levmisole) for the Delayed-type hypersensitivity response analysis while group four in the determination of humoral received distilled water .The extract significantly (p < 0.05) showed dose related stimulation of humoral immunity at 600mg/kg dose compared to the standard and also a significantly (p>0.05) anti-inflammatory activity at 300mg/kg compared with the control group. This result showed potential immunomodulatory effect because the immunomodulatory suppressed the antigenic response in system.
TABLE OF CONTENTS
Title Page pages
Certification i
Dedication ii
Acknowledgement iii
Abstract iv
Table Contents vi
List of Tables vii
List of Figures
CHAPTER ONE: INTRODUCTION
- Background of study 1
- Pausinystaliayohmibe 2
- Botancial information 2
- Traditional uses 3
- State of the problem 3
- Aim and objectives 3
CHAPTER TWO: LITERATURE REVIEW
2.1 pausinystaliayohmibe 4
2.2 General description of the plant 4
2.3 Botanical description 5
2.4Botanicalclassification 5
2.5 chemical formular 7
2.6 chemical composition 7
2.7 phytochemical constituens 8
2.7.1 Alkaloid 8
2 .7.2 Terpenoids 9
2.7.3 flavonids 9
2.7.4 saponins 10
2.8 Medicinal use 11
2.8.1 Pharmcology and toxicology 11
2.8.2 In virto data 11
2.9 Mechanism of immunomodulation 12
2.9.1 Benfits of immunomodulation 13
2.9.2 Drug used for immunomodulation 14
2.9.3 Immunosuppressants 15
2.9.4immunosutimulnts 15
2.9.4.1 Baciilluscalmette – Guren (BCG) 15
2.9.4.2 Levamisole 16
2.9.4.3 Thalidomide 16
2.9.4.4 Recombinant Cytokines 16
2.9.4.5Interfons 16
2.9.4.6 Interleukins 16
2.9.4.7 Isoprinosins 17
2.9.4.8 immunocynin 17
CHAPTER THRE : MATERIALS AND METHODS
3.1 Materials 18
3.1.1 Chemcials 18
3.1.2 Reagents 19
3.1.3 Preparation of reagents 19
3.1.4 Equipments 19
3.1.5 Biological materials 20
3.2 collection and identification of sample 20
3.2.1 Collection plants materials 20
3.2.2 collection of animals 20
3.2.3 Animal grouping 20
3.2.4 Preparation of extracts 21
3.2.5 Antigen 21
3.2.6 Preparation of sheep blood cell 22
3.2.7 Micro- organism 22
3.2.8. Handling of animals 22
3.2.8.1 Acute toxicity study 22
3.2.8.2Delay- type hypersensivitity response (DTHR) 22
3.2.8.3 Determination of humoral immune response (DIHR) 23
3.2.8.4 In vivo analysis 23
3.3 Preparation candida albicans 24
3.3.1 Evaluation of phagocytosis 24
3.4 Preparation of reagents 25
3.4.1 Preparation of drangedoff 25
3.4.2 Preparation of phosphate buffer saline 25
3.4.3 Preparation of normal saline 25
3.4.4 Preparation of Mc-Farland standard 25
3.4.5 Preparation of diluted sodium hydroxide 25
3.4.6 Preparation of 1% lead acetate 25
3.5 Qualitative phytochemical analysis 25
3.5.1 Test for Alkaloids 25
3.5.2 Test for Flavonoids 26
3.5.3 Test for saponins 26
3.5.4 Test for Tanins 26
3.6 Statistical analysis 26
CHAPTER FOUR : RESULTS
4.1 Acute toxicity LD50 27
4.2 phytochemicals 28
4.3 Delay-type hypersensivity response (DTHR) 29
4.4 Determination of humoral immune response (DHIR) 32
4.5 Evaluation of Phagocytosis 35
CHAPTER FIVE:
5.1 Discussion 39
5.2 Conclusion 40
References 41
Appendices 45
LIST OF TABLES PAGES
Table 2.1 Classification of immunosuppressants 14
Table 3.1 Chemicals 18
Table 3.2 Reagents 19
Table 3.3 Equipments 19
Table 4.1 Phytochemical analysis 28
Table 4.2 Delay –type hypersensitivity response 29
Table 4.3 Determination humoral Immune response 32
Table 4.4 Phagocytic stimulation index 35
LIST OF FIGURES PAGES
Figure1 The whole tree of pausinsystaliayohmibe 6
Figure 2 The leaves of pausinsytaliayohmibe 6
Figure3 The stem bark of pausinsytaliayohmibe 6
Figure 4 The broken stem bark of pausinsytaliayohmibe 6
Figure 5 The grinded stem bark 6
Figure 6 Structure of Yohmibe 7
Figure 7 Structure of Alkaloid 8
Figure 8 Structure of Terpenoid 9
Figure 9 Structure of Flavonid 10
Figure 10 Structure of Saponin 10
Figure 11 Delay-type hypersensitivity 30
Figure 12 Percentage delayed-type 31
Figure 13 Primary titre on humoral immune 33
Figure 14 Secondary titre on humoral immune 34
Figure 15 Phagocytic index 36
Figure 16 Phagocytic effect 36
Figure 17 Phagocytic effect 38
CHAPTER ONE
1.1 BACKGROUND OF STUDY
Many plants have been screened for their medicinal properties; this includes yohimbine, which is an alkaloid chemically similar to reserpine. It is gotten from the bark of the yohimbe tree, it posses alpha-adrenergic blocking properties and is used as hydrochloride sympatholytic, mydriatic and for the treatment of impotence (Dorland’s medical dictionary 2007). Sexual dysfunction is a serious medical and social problem that occurs 10%- 52% in men and 25%-63% in women numerous central and peripheral neural circuits control sexual activity impairment one or more of these functional circuits may have a significant impact on personal , social and biological relationships. Although several aspects of sexual motivation and performance are known, complete picture of the various factors that control human sexual activity is still unknown and the available drugs and pleasant side effects and contraindications in certain disease conditions. A variety of botanical plants are known to have a potential effect on immunomodulatory (suppressive and stimulatory) functions thereby supporting older claims and offering newer hopes (Therakan and Manyam, 2005).
Presently, there is a growing interest in the use of various naturalplants parts and plants products as medicines and these folk medicines are being marked in almost all parts of Nigeria and the world at large.They range from herbal toothpaste to various drug supplements. Some of these herbal preparations may have some properties, which have contributed to their persistent use over the years,under scoring the need for validation of most them. It is said that only about 2% of all the plants on the earth have been subjected to pharmacological investigation. The rationalablefor utilization of medical plants has rested largely on long term clinical experience with little or no scientific data on their effect and safety (Zhu M, et.al 2002), with the upsurge in the use of herbal medicines through scientific investigation of these plants is imperative, based on the need to validate their folk usage (Sofowora E.A,1989).
Yohimbe bark extracts standardized to varying amounts of yohimbine are widely available in health food stores and through direct mail companies. Extracts are supplied as capsules tablets and liquids. Some of theseyohimbe preparations are sold in combination formula’s with other herbs. Yohimbine hydrochloride is a Food and Drug Administration (FDA) approved prescription drug for the treatment of impotence (BetJ.M, et.al, 1995).
Bet J.M and Coworkers (1995) also investigated yohimbe in commercialyohimbe products. Gas chromatography determinations were done on liquids and powders (from capsules and caplets). Virtually all the products tested did not specify on their labels that the products contained yohimbe bark extract concentration of yohimbine in the commercial products ranged from > 0.1 to 489pppm , compared with 7089ppm in authentic bark material of the 26 products examined , nine contained no quantifiable amount of yohimbe ; eight contained only trace amounts (0.1-1ppm). The authors suggest that the absence of alkaloids in the products indicated that the original extracts was aqueous (because the alkaloids are not particularly water soluble), the extract was extremely diluted in the final dosage form or no yohimbebark was used to make the product.
Yohimbe is available in research quantities at 98% purity from Aldrich chemical company. Yohimbinehydrochloride from Aldrich and Sigma are available at 99 and 80%purity, respectively (Aldrich chemical co., 199; sigma, 1999).
1.2PAUSINYSTALIA YOHIMBE
Pausinystaliayohimbe is also known as corynantheyohimbe. Another common name is Yocon, the Yorubas’ know it as Idagbon, while theHausas’ call itDankamaru.
1.3 BOTANICAL INFORMATION
It is an evergreen tree which grows to a height of 30m with a spread of 8m, the stem is erect and branching the less are oval, acuminate and about 10cm long. The seed are small winged silvers, almost paper thin. Pausinystaliayohimbe is a native of the rain forest of Nigeria, Cameroon and the Congo. It prefers rich soils in a protected part sun to shady position, and is drought and frost tender. The propagation of pausinystaliayohimbe is by seed or cutting. Seeds are sown in a free draining seed micorshagnummoss and will need temperature above 250C to germinate quickly. Seeds have a very short viability, which declines rapidly in dry and warm conditions (Shaman Australia Botanicals 1998).
1.4 TRADITIONAL USE
Yohmibe is the only natural medicinal aphrodisiac. It popularly used as an aphrodisiac in its native area and has been well documented and its unique effect was soon valued in many parts of the world, especially in Europe, Africa and Nigeria. Its modern times products have found a wide market ranging from medically treated impotence and self administered sexual enhancement “smart products” like “Cloud 9”™, “Viagra”, Barbecue for Suyaand other herbal ecstasy formulations.
1.5 STATEMENT OF PROBLEMS
Although yohimbe has been found to reduce blood sugar level in induced diabetic miceand impotency, but its administration has be seen to be an unsafe herbal practice because the percentage yohimbinein yohimbebark has not been documented, making it impossible to determine the exert percentage concentration presents in various yohimbe products used as folk medical and other health benefits of such as immunomodulatory potentials of this plant has not been fully investigated. Many countries especiallyin Nigerian,Yohimbe extract and its products are promoted and still been indiscriminately used.
1.6 AIM AND OBJECTIVES
The aim of this research work was to evaluate the immunomodulatory effects ofPausinystaliayohimbemethanolic extractin albino mice.
This aim was achieved through the following objectives;
- Quantitative and qualitative analyses of the phytochemical constituents of Pausinystaliayohimbe.
- Investigation ofthe toxicity effect (LD50) of the methanol extract.
- Investigation of the immunomodulatory potentials of Pausinystaliayohimbe methanol extract in mice via the following parameters;
- Evaluation of phagocytosis
- Determination of delayed-type hypersensivity response (DTHR).
- Determination of humoral immune response (IgG and IgM).
- In vivo leucocyte mobilization test.
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