Toxicity of Abuleila (Detarium senegalense) Fruit Flour to Rats
By Hanan Mohamed Neimtalla Gibreil B.V.Sc. (2003), M.V.Sc. (2009) University of Khartoum
A Thesis Submitted to the University of Khartoum in Fulfillment of the RequirementsFor the Ph.D. Degree in Veterinary Science (Toxicology).
Supervisor: Professor Ahmed El Amin Mohammed
Department of Pharmacology and Toxicology Faculty of Veterinary Medicine University of Khartoum
May, 2015
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
لاي ذؼاٌٝ:
)فأنبتنا فيها حبا * وعنبا وقضبا * وزيتىنا
ونخال * وحذائق غلبا * وفاكهت وأبا * هتاعاً
لكن وألنعاهكن(
سىرة عبس االياث )27 - 32(
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
DEDICATION
I Would like to dedicate this research lovingly to: My kind Parents, who support me in everything. My Husband, who helped me to finished this research. My Sisters and brother, who encouraged me to reach this goal. My Son and Daughter, who were a great source of support, and Every person, who gave me a hint to exact my trail.
Hanan August , 2014
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
CONTENTS
Item Page List of Tables vii List of Figures ix List of Plates x List of Abbreviations xi Acknowledgements xii Abstract xiii Arabic abstract xvi INTRODUCTION 1 CHAPTER ONE LITERATURE REVIEW 3 1.1 Ethnotoxicology 3 1.2 Food trees 4 1.2.1 Adansonia digitata 4 Phytochemistry 4 Food uses 5 Medicinal uses 5 1.2.2 Ziziphus spina-christa 6 Phytochemistry 6 Food uses 6 Medicinal uses 7 1.2.3 Hyphaene thebaica 7 Food uses 8 Medicinal uses 8 1.2.4 Balanites aegyptiaca 8 Phytochemistry 9 Food uses 9 Medicinal uses 9 1.3 Fabales food trees 10 1.3.1 Family Fabaceae 10 Taxonomy and description 11 Uses 12 1.3.2 Caesalpinioideae 13 1.3.3 Detarieae 13 1.3.3.1 Tamarindus indica 13 Food uses 14 Medicinal uses 14 1.3.3.2 Detarium 14 1.3.3.2.1 Detarium macrocarpum 15 1.3.3.2.2 Detarium microcarpum 15 Phytochemistry 15
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Food uses 16 Medicinal uses 16 1.3.3.2.3 Detarium senegalense 17 Taxonomy 17 Distribution 17 Description 18 Nutrients composition 19 Proximate analysis 20 Phytochemical analysis 20 Nutritional uses 21 Medicinal uses 22 Toxicity 24 CHAPTER TWO MATERIALS AND METHODS 26 2.1 Materials and experimental designs 26 2.1.1 Toxicity of dietary Abuleila (D. senegalense) fruit flour to rats 26 2.1.1.1 Animals, housing and management 26 2.1.1.2 Test plant 26 2.1.1.2.1 Fruit flour preparation 27 2.1.1.2.2 Fruit flour proximate analysis 27 2.1.1.2.3 Microminerals 27 2.1.1.2.4 Preliminary phytochemical screening 28 2.1.1.3 Administration and dose rates 29 2.1.1.4 Data collected 29 2.1.2 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats 29 via s/c route 2.1.2.1 Animals, housing and management 29 2.1.2.2 Fruit flour extraction 29 2.1.2.3 Administration and dose rates 30 2.1.1.4 Data collected 30 2.1.3 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats 30 via i/p route 2.1.3.1 Animals, housing and management 30 2.1.3.2 Administration and dose rates 31 2.1.3.3 Data collected 31 2.2 Methods 31 2.2.1 Haematological methods 31 2.2.1.1 Red blood cells count (RBCs) 31 2.2.1.2 Packed cell volume (PCV) 32 2.2.1.3 Haemoglobin concentration (Hb) 32 2.2.1.4 Red blood cell haematostatistics 32 2.2.1.4.1 Mean corpuscular volume (MCV) 32 2.2.1.4.2 Mean corpuscular haemoglobin concentration (MCHC) 32 2.2.1.5 White blood cell count (WBCs) 33 2.2.1.6 Differential leukocyte count 33 2.2.2 Chemical methods 33 v
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
2.2.2.1 Serum metabolites 33 2.2.2.1.1 Glucose 33 2.2.2.1.2 Total protein 34 2.2.2.1.3 Albumin 35 2.2.2.1.4 Urea 35 2.2.2.2 Serum enzyme activities 36 2.2.2.2.1 Aspartate aminotransferase (Glutamicoxaloacetic transaminase, L, 36 Aspartate = 2 – OXO-glutarate aminotransferase E.C.2, 6,1.1,AST). 2.2.2.2.2 Alanine amino transferase, (Glutamic pyruvic transaminase, 36 L- aspartate,2-oxoglutamate ALT) 2.2.2.3 Test plant macrominerals 37 2.2.2.4 Test plant microminerals 37 2.2.3 Histopathological methods 39 2.3 Statistical analyses 39 CHAPTER THREE 40 RESULTS 3.1 Toxicity of dietary Abuleila (D. senegalense) fruit flour to rats 40 3.1.1 Clinical signs 40 3.1.2 Body weight changes 40 3.1.3 Haematological findings 41 3.1.4 Changes in serum constituents 42 3.1.5 Post mortem findings 43 3.1.6 Histopathological findings 43 3.2 Toxicity of Abuleila (D. senegalense) fruit flour water extract to 46 rats via subcutaneous route 3.2.1 Clinical sings 46 3.2.2 Body weight changes 46 3.2.3 Haematological findings 47 3.2.4 Changes in serum constituents 48 3.2.5 Post mortem findings 50 3.2.6 Histopathological findings 50 3.3 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats 51 via i/p route 3.3.1 Clinical sings 51 3.3.2 Body weight changes 51 3.3.3 Haematological findings 52 3.3.4 Changes in serum constituents 54 3.3.5 Post mortem findings 54 3.2.6 Histopathological findings 55 CHAPTER FOUR DISCUSSION 58 4.1 Conclusion 62 4.2 Suggestions for future work 62 REFERNCES 63 APPENDIX 80
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List of Tables
Table Page No. Title
1 Proximate percent crude chemical components (dry-matter basis) 27 of Detarium senegalense fruit flour.
Micromineral components (dry-matter basis) of Detarium 2 28 senegalense fruit flour.
3 Preliminary phytochemical screening of Detarium senegalense 28 fruit flour extract.
4 Average (mean ± s.d.) body weight changes of experimental rats 40 fed Detarium senegalense fruit flour for 6 weeks.
5 Average (mean ± s.d.) blood values of experimental rats fed 41 Detarium senegalense fruit flour for 6 weeks.
6 Average (mean ± s.d.) WBCs differential count of experimental 42 rats fed Detarium senegalense fruit flour for 6 weeks.
7 Average (mean ± s.d.) serum metabolites values of experimental 42 rats fed Detarium senegalense fruit flour for 6 weeks.
8 Average (mean ± s.d.) serum enzymes values of experimental rats 43 fed Detarium senegalense fruit flour for 6 week.
9 Average (mean ± s.d.) body weight changes of experimental rats 47 injected subcutaneously with Detarium senegalense fruit flour water extract for 6 weeks. 10 Average (mean ± s.d.) blood values of experimental rats injected 48 subcutaneously with D. senegalense fruit flour water extract for 6 weeks. 11 Average (mean ± s.d.) WBCs differential count values of 48 experimental rats injected subcutaneously Detarium Senegalense fruit flour water extract for 6 weeks. 12 Average (mean ± s.d.) serum metabolites values of experimental 49 rats injected subcutaneously Detarium senegalense fruit flour water extract for 6 weeks. 13 Average (mean ± s.d.) serum enzymes values of experimental rats 49 injected subcutaneously Detarium senegalense fruit flour water extract for 6 weeks.
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14 Average (mean ± s.d.) body weight changes of experimental rats 52 injected intraperitoneally with Detarium senegalense fruit flour water extract for 6 weeks. 15 Average (mean ± s.d.) blood values of experimental rats injected 53 intraperitoneally with D. senegalense fruit flour water extract for 6 weeks. 16 Average (mean ± s.d.) WBCs differential count values of 53 experimental rats injected intraperitoneally Detarium Senegalense fruit flour water extract for 6 weeks. 17 Average (mean ± s.d.) serum metabolites values of experimental 54 rats injected intraperitoneally Detarium senegalense fruit flour water extract for 6 weeks. 18 Average (mean ± s.d.) serum enzymes values of experimental rats 54 injected intrperitoneally Detarium senegalense fruit flour water extract for 6 weeks.
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
List of Figures
Figure Page No. Title 1 Distribution of test plant in Africa. 17
2 Detarium senegalense fruit. 19
Graphical representation of the concentrations of trace minerals using 3 38 XRF-Spectrometer system.
4 Average growth curves of treatment groups. 41
5 Average growth curves of treatment groups. 47
6 Average growth curves of treatment groups. 52
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List of Plates
Plate No. Title Page 1 The Detarium senegalense savanna type tree. 18
2 Detarium senegalense fruit. 18
3 Presser machine at 15 ton/cm2 38
4 The XRF Spectrometer system with liquid nitrogen. 38
5 Thickening of alveolar walls with slight interstitial proliferation of type 44 II cells. Peribronchial mononuclear cells infiltrate in the lung of rat in group C. H&E x100. 6 Thickening of alveolar walls with severe interstitial proliferation of type 44 II cells in the lung of rat in group D. H&E x100. 7 Thickening of alveolar walls with slight interstitial proliferation of type 45 II cells in the lung of rat in group D. H&E x100. 8 Peribronchial mononuclear cells infiltrate in the lung of rat in group D. 45 H&E x100. 9 Focal infiltration of mononuclear cells and neutrophils around blood 46 vessels in the lung of rat in group D. H&E x100. 10 Thickening of alveolar walls with slight interstitial proliferation of type 50 II cells. Peribronchial mononuclear cells infiltrate in the lung of rat in group B. H&E x100. 11 Thickening of alveolar walls with slight interstitial proliferation of type 51 II cells. Focal infiltration of mononuclear cells around blood vessels in the lung of rat in group D. H&E x100. 12 Thickening of alveolar walls with slight interstitial proliferation of type 55 II cells. Focal infiltration of mononuclear cells around blood vessels in the lung of rat in group C. 13 Dilatation of renal tubules, hemorrhage and coagulative necrosis in the 56 kidney of rat No.4, group D. H&E x100. 14 Congestion in the liver of rat No.3, group D. H&E x100. 56
15 Vesiculation of hepato cytes nuclei and hepatocytes necrosis in the liver 57 of rat No.3, group D. H&E x100. 16 Bridging necrosis in the liver of rat No.3, group D. H&E x100. 57
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
LIST OF ABBREVIATIONS
Abbrev- Full Abbrev- Full iation Wording iation Wording Atomic Absorption AAS mg Milligram Spectroscopy ALT Alanine aminotransferase ml Millliter AST Aspartate aminotransferase mm3 Cubic millimeter BCG Bromocresol green mmol Millimole Cal Calorie mol Mole cm Centimeter OD Optical density dl Deciliter PCV Packed cells volume EDTA Ethylene diaminetetraacetic acid PLT Platelets g Gram POD Peroxidaes Gas chromatography - Mass GC-MS ppm Parts per million Spectroscopy GOD Glucose oxidase RBCs Red blood cells Hb Haemoglobin r.p.m. Rotation per minute HIV Human Immunodeficiency Virus S/c Subcutaneous High performance liquid HPLC SD Standard deviation chromatography International Atomic Energy IAEA SE Standard error Agency Soluble non starch i/p Intraperitoneal s-NSP polysaccharide i.u. International unit U/L Units per liter Kcal. Kilocalorie UV Ultraviolet Kg Kilogram VIS Visual KJ Kilo joule WBCs White blood cells L Liter XRF X. Ray fluorescent m Meter > More than Mcal Megacalorie < Less than Mean corpuscular haemoglobin MCHC More than or equal concentration ≥ MCV Mean corpuscular volume ≤ Less than or equal
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ACKNOWLEDGMENTS
First, praise is to Allah, the Lord of the Worlds who permitted health and help to complete this work. My Lord is the hearer of prayers. I wish to express my hearty thankfulness and deep gratitude to my Supervisor Prof. Ahmed El Amin Mohammed, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, for his useful guidance, patience, encouragement and support throughout this work. Without his support this work would not have become possible. I am also grateful to Dr. Samia M.A. El Badawi, Head Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, for her personal interest and help and for allowing the use of Departmental facilities carrying out this work. I would like to express my thanks to Dr. Khairalla Mohamed Saied Khairalla, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, for his great assistance offered at all times needed. My thanks also are extended to Lecturer Ahmed Kamal, Department of Pathology, Faculty of veterinary Medicine, University of Khartoum, for his support during the histopathological work. My thanks also are offered to my truly great friends Afraa Tag Elsir ElAtta, Department of Microbiology and Rasha Yassin Fadlalmula, Department of Surgery for their considerably valuable assistance, availing their support in a number of ways. Many thanks are also expressed to my colleague Walliddeen Elsadig Elmagboul, Department of Physiology, Faculty of veterinary Medicine, University of Khartoum. Last but not least, my thanks are extended to the University of Khartoum for offering the chance to read for this degree and to all of those who supported me in any respect during the completion of this research.
Hanan August, 2014
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Toxicity of Abuleila (Detarium senegalense) Fruit Flour to Rats
Ph.D. in Veterinary Science (Toxicology)
Hanan Mohamed Niemtalla GibreilI
Abstract: The present study was carried out to investigate the toxic effect of Detarium senegalense fruit flour in rats. Three experiments were conducted using 60 rats; 20 in each experiment. In each experiment rats were divided into 4 groups (A, B, C, and D) of 5 rats each. Rats in all the experiments were fed on a basal ration of energy concentration 2.5 Mcal and 20% crude protein. In the first experiment; the rats were daily fed with D. senegalense fruit flour at 0%, 10%, 20% and 30% of the basal diet by dilution. In the second and third experiments, the rats were daily injected with D. senegalense fruit flour water extract subcutaneously at doses of 0, 50, 100 and 150 mg/kg body weight and intraperitoneally at doses of 0, 50, 100 and 150 mg/kg body weight respective to groups A, B, C and D. The experiments extended for 6 weeks. Clinical signs and mortalities were recorded daily. Body weights were recorded weekly. Blood and serum samples were collected fortnightly for haematology (RBCs, Hb, PCV, WBCs, differential count and Platelets), serum biochemical tests (glucose, total protein, albumin, globulin and urea) and enzymes (AST and ALT). Vital organ samples (lung, heart, liver, kidney, spleen and intestine) were collected for histopathological investigations at post mortem or in extremis. There were no changes in the general appearance or mortalities suffered in any dietary group. There were significant (P ≤0.05-0.01) increases in body weight gain in all test groups. In both parenteral routes, no changes were recorded in body weight gain but rats of groups C and D showed weakness and dullness, in addition group D in the intraperitoneal route showed beside weakness and dullness, recumbency with decreased appetite. In groups C and D, mortalities were 40% and 60%, respectively via subcutaneous route and 40% and 80%, respectively
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 intraperitoneally. WBCs values showed a significant (P ≤0.05) increases in group D on dietary route with an increase (P ≤0.001) in lymphocytes and a decrease (P ≤0.01) in monocytes and granulocytes of group B, whilst on both parenteral routes, WBCs and Platelets values showed a significant (P ≤0.05- 0.01) increases in all test groups with a significant increase (P ≤0.001) in monocytes of group C. RBCs values only increased significantly (P ≤0.05) in group B dosed intraperitoneally. In all groups in the dietary and parenteral routes, albumin concentration significantly (P ≤0.05-0.01) decreased. In dietary route, AST values significantly (P ≤0.05) increased in group B, whereas in the subcutaneous route showed significant (P ≤0.05-0.01) increases in all test groups with a significant (P ≤0.05) increase in ALT values in groups C and D. In the dietary route, all rat groups showed mild congestion with haemorrhagic foci in the liver and few scattered haemorrhagic foci in the lung. In the dietary route, rats of groups C and D showed alveolar walls thickening with slight to severe interstitial proliferation of type II cells. A peribronchial mononuclear cellular infiltrate was seen in group C. Mononuclear cells and neutrophils infiltrate peribronchially and around blood vessels were seen in group D. Parenteral dosing showed varying pulmonary pathies with few scattered haemorrhagic foci and focal infiltration of mononuclear cells around blood vessels. In both parenteral doses, livers were pale and mildly congested, whereas the lungs were pale and congested with focal infiltration of peribronchial mononuclear cells around blood vessels; there were different degrees of thickening of alveolar walls with slight to severe interstitial proliferation of type II cells. In intraperitoneal route, kidneys showed paleness with mild enlargement and dilatation of renal tubules, haemorrhage and coagulative necrosis. In conclution, dietary D. senegalense fruit flour was nutritious as food additive until 30% without observable clinical signs or mortalities. Parenteral D. senegalense fruit flour water extract (100 - 150 mg/kg body weight) was more toxic
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 intraperitoneally than subcutaneously to varying degrees of mortalities, pulmonary and hepatonephropathies.
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سويت دقيق ثورة أبىليلى (Detarium senegalense) في الجرزاى
دكتىراة العلىم البيطريت )السوىم( حناى دمحم نعوت هللا جبريل
الوستخلص: أجز٠د ٘ذٖ اٌذراسح ؼٌّزفح اٌراث١ز اٌساَ ٌذل١ك ثّزج )أتٛ senegalense )ٍٝ١ٌ Detarium فٟ اٌجزساْ. اجز٠د ثالز ذجارب تاسرخذاَ ػذد 60 جزسا، فٝ وً ذجزتح ػشزْٚ جزسا ِمسّح ٍٝػ ارتغ ِجػّٛاخ )D ٚ C ،B ،A( فٝ وً ِجػّٛح خّسح جزساْ. ذّد ذغذ٠ح اٌجزساْ فٟ ج١ّغ اٌرجارب تغذاء لاػذٞ ترزو١ش ؽالح 2.5 ِماواٌٛرٚ ٞتزٚذ١ٓ خاَ 20٪ . فٟ اٌرجزتح األٌٟٚ اؼؽّد اٌجزساْ ١ِٛ٠اٍٟػ دل١ك ثّزج أتٛ ٍٝ١ٌ تؼّذي ٚ ٪20 ، ٪10 ٪0 30٪ ِٓ اٌغذاء اٌماػذٞ تاٌرخف١ف. دمٕد اٌجزساْ فٟ اٌرجزتر١ٓ اٌثا١ٔح ٚاٌثاٌثح ١ِٛ٠اً تاٌّسرخٍض اٌّائٟ ٌذل١ك ثّزج أتٛ ٍٝ١ٌ ذذد اٌجٍذ تجزػاخ ٚ 100 ٚ 50 ، 0 150 ٍِغُ / وغُ ِٓ ٚسْ اٌجسُ ٚداخً اٌظفاق تجزػاخ 0، 50، ٚ 100 150 ٍِغُ / وغُ ِٓ ٚسْ اٌجسُ ٍٟػ اٌرٛاٌٟ ِغ اٌّجػّٛاخ D ٚ C ،B ،A. اسرّزخ جاٌرجزب ٌىً اٌّجػّٛاخ سرح أسات١غ ٚ رطذخ اؼٌالِاخ اٌسز٠ز٠ح ٚإٌفٛق ١ِٛ٠ا ً. ٚسجٍد اٚساْ اٌجزساْ أسث١ػٛا ً. جؼّد ١ػٕاخ اٌذَ ٚاٌّظً ِزج وً أسث١ػٛٓ ٌٍفذٛطاخ اٌذ٠ِٛح )وزاخ اٌذَ اٌذّزاء، اّٛ١ٌٙغٍٛت١ٓ، دجُ اٌخال٠ا اٌّرزاص، اٌىز٠اخ اٌثؼ١اء ٚذفز٠ماذٙا ،اٌظفائخ اٌذ٠ِٛح( اٌفذٛطاخ جاٌثٛ١و١ّائٟ )اٌجٍٛوٛس، اٌثزٚذ١ٓ اٌىٍٟ، األٌث١ِٛ١ٓ، اٌجٍٛت١ٌٛ١ٓ ، اٛ١ٌر٠ا(، ٚاألٔش٠ّاخ )ٔالً األسثارذ١د ٚٔالً األ١ٔ٢ٓ(. جؼّد ١ػٕاخ ِٓ األجٙشج اٌذ٠ٛ١ح )اٌزئٗ ٚاٌمٍة ٚاٌىثذ ٚاٌىٚ ٍٟاٌطذاي ٚاالؼِاء ( تؼذ إٌفٛق أٚ اٌذتخ ػٕذ إٌشع إلجزاء اٌفذٛص اٌرشز٠ذ١ح اٌّز١ػح. ٌُ ذىٓ ٕ٘ان ذغ١١زاخ فٟ اٌّظٙز اؼٌاَ أٚإٌفٛق فٟ أٞ ِٓ ِجػّٛاخ اٌرجزتح اٌغذائ١ح. وأد ٕ٘ان س٠ادج ٠ٕٛؼِح ) P ≤0.05- 0.01( فٟ وسة اٌٛسْ فٟ وً اٌّجػّٛاخ. فٟ اٌّجػّٛر١ٓ اٌّذمٛٔر١ٓ ، ٌُ ذزطذ ذغ١زاخ فٟ س٠ادج اٌٛسْ ٌىٓ جزساْ اٌّجػّٛر١ٓ D ٚ C أظٙزذا اؼؼٌف ٚاٌخّٛي، اٌّجػّٛح D اٌّذمٛٔح داخً اٌظفاق تجأة اؼؼٌف ٚاٌخّٛي وأد ِسرٍم١ح ِغ لٍح فٟ اٌش١ٙح. واْ إٌفٛق فٟ ِجػّٛرٚ ٪40 ،D ٚ C ٟ ٍٝػ ٪60 اٌرٛاٌٟ تاٌذمٓ ذذد اٌجٍذ ٍٝػ ٪80 ٚ ٪40 ٚ اٌرٛاٌٟ تاٌذمٓ داخً اٌظفاق. أظٙزخ ل١ُ وز٠اخ اٌذَ اٌثؼ١اء س٠ادج ٠ٕٛؼِح ) P ≤0.05( فٟ ايِجػّٛح D اؼٌّاٌجح فٝ ا١ٍؼٌمح ِغ س٠ادج ) P 0.001≥( فٟ اٌخال٠ا ا١ٌٍّفا٠ٚح ٚأخفاع ) P ≤0.01( فٚ ٟد١ذاخ ِٚذثثح اٌخال٠ا فٟ اٌّجػّٛح B، ت١ّٕا أظٙزخ اٌّجّٛعذاْ اٌٍراْ اػط١را إٌثاخ تاٌشرق، س٠ادج ٠ٕٛؼِح )P ≤0.05-0.01( فٟ ل١ُ ػذد وز٠اخ اٌذَ اٌثؼ١اء اٌىٚ ٍٝاٌظفائخ اٌذ٠ِٛح فٟ وً ِجػّٛاخ اإلخرثار ِغ س٠ادج ٠ٕٛؼِح )P ≤0.001(
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فٟ خال٠ا وز٠اخ اٌذَ اٌثؼ١اء ٚد١ذج اٌخ١ٍح فٝ ايِجػّٛح C. سادخ ل١ُ ػذد وز٠اخ اٌذَ اٌذّزاء ٠ٕٛؼِاً )P ≤0.05( فمؾ فٟ اٌّجػّٛح B اٌّذمٛٔح داخً اٌظفاق. أخفغ ذزو١ش األٌث١ِٛ١ٓ ٠ٕٛؼِا ) P 0.01-0.05≥( فٟ وً اٌّجػّٛاخ اٌرٝ اػط١د إٌثاخ فٝ ا١ٍؼٌمح ٚاٌرٝ اػط١د إٌثاخ تاٌشرق سادخ ل١ُ ٔالً أِا٠ٓ األسثارذ١د فٟ اٌّجّٛعج اٌرٝ اػط١د إٌثاخ فٝ ا١ٍؼٌمح، ٠ٕٛؼِاً ) P ≤0.05( فٟ اٌّجػّٛح B ، فٟ د١ٓ أظٙزخ ل١ُ ٔالً أِا٠ٓ األسثارذ١د فٟ اٌّجػّٛح اؼٌّاٌجح ذذد اٌجٍذ س٠ادج ٠ٕٛؼِح )P ≤0.05-0.01( فٟ وً ِجػّٛاخ االخرثار ِغ س٠ادج ٠ٕٛؼِح) P ≤0.05( فٟ ل١ُ ٔالً أِا٠ٓ األال١ٔٓ فٟ ِجػّٛرD ٚ C ٝ. فٟ اٌّجّٛعج اٌرٝ اػط١د إٌثاخ فٝ ا١ٍؼٌمح، أظٙزخ ج١ّغ ِجػّٛاخ اٌجزساْ اٌرجز٠ث١ح ادرمأاخ خف١فح فٝ اٌىثذ ٚ تؤر ٔشف١ح ِغ ل١ًٍ ِٓ اٌثؤر إٌشف١ح اٌّرٕاثزج فٟ اٌزئح. فٟ اٌّجّٛعج اٌرٝ اػط١د إٌثاخ فٝ ا١ٍؼٌمح أظٙزخ اٌجزساْ ِٓ ِجػّٛرD ٚ C ٝ سّىا فٝ اٌجذراْ اٌسٕخ١ح ِغ أرشار خالٌٟ خف١ف إٌٝ داد ِٓ خال٠ا إٌٛع اٌثاٚ .ٟٔجذخ اٌخال٠ا ٚد١ذاخ اٌٜٕٛ ِذ١طح تاٌمظ١ثح ٚ ارذشاح خٍٞٛ فٟ اٌّجػّٛح ٚ .Cجذخ خال٠ا ٚد١ذاخ اٚ ٌٜٕٛاؼٌذالخ ِزذشذح ِٚذ١طح تاٌمظ١ثح ٚدٛي األ١ػٚح اٌذ٠ِٛح فٟ اٌّجػّٛح D . أظٙزخ اٌجزساْ فٝ اٌّجػّٛاخ اٌّذمٛٔح إِزا١ػاخ رئ٠ٛح ِرفاٚذح ِغ ل١ًٍ ِٓ اٌثؤر إٌشف١ح اٌّرٕاثزج ٚارذشاح خٍٞٛ ٌٍخال٠ا ٚد١ذج اٌٜٕٛ دٛي األ١ػٚح اٌذ٠ِٛح. وأد اٌىثذ فٟ اٌّجػّٛر١ٓ اٌٍر١ٓ اػط١را إٌثاخ تاٌشرق ، شادثح ِغ ادرماْ خف١ف. ت١ّٕا وأد اٌزئح شادثح ِٚذرمٕح ِغ ارذشاح ٟؼػِٛ ٌخال٠ا ٚد١ذاخ اٌٜٕٛ ِذ١طح تاٌمظ١ثح دٛي األ١ػٚح اٌذ٠ِٛح؛ ٕ٘ان درجاخ ِخرٍفح ِٓ سّه اٌجذراْ اٌسٕخ١ح ِغ أرشار خالٌٟ خف١ف إٌٝ داد ِٓ خال٠ا إٌٛع اٌثأٟ. أظٙز ا ٌذمٓ داخً اٌظفاق شذٛب اٌىٍٝ ِغ ذؼخُ خف١ف ٚذٛسغ األٔات١ة اٌى٠ٍٛح ٚٔشف ٚٔخز ِرخثز. خٍظد اٌذراسح اٌٝ اْ دل١ك ثّزج أتٛ ٍٝ١ٌ ِغذ٠اً وؼّاف ػٍفٝ درٝ 30٪ دْٚ ظٛٙر ػالِاخ سز٠ز٠ح ٚاػذح أٚ ٔفٛق. واْ اٌّسرخٍض اٌّائٟ ٌذل١ك ثّزج أتٛ ٍٝ١ٌ )100- 150 ٍِغ / وغ ِٓ ٚسْ اٌجسُ( أوثز س١ّح داخً اٌظفاق ِٕٗ ذذد اٌجٍذ تذرجاخ ِرفاٚذح ِٓ إٌفٛق ٚاإلِزا١ػح اٌزئ٠ٛح ٚاٌىثذو٠ٍٛح.
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INTRODUCTION
Plants are indispensable in the biosphere and probably it is not necessary to explain their importance. Surely, plant consciousness is ever considered from beneficial point of view and a wishful thinker prefers to assume that all plants are good. But, a reasonable person should be prepared to face the reality that many plants contain potent substances which can affect human and animals adversely. However, the existence of such hazardous substances in plants is a must which contributes to their survivability, provides resistance to diseases and insects and attack by birds and other predators (Morton, 1978). During the last two or three decades, there has been a revival of interest into research and utilization of medicinal plants, particularly the flora of the tropics (Soejarto, 1996). The revived interest in plant-derived drugs is attributed to the current widespread belief that “green medicine” is cheap, safe, more dependable and accessible than the costly synthetic drugs, many of which are associated with intolerable effects (Parek and Chanda, 2006 and Venkatesh and Krishnakumari, 2006). According to the World Health Organization, about 80% of the world‟s population depends wholly or partly on plant-derived pharmaceuticals (WHO, 1996) i.e. in most developing countries. There is a heavy dependence on herbal preparations for the treatment of human and animal diseases despite the availability of conventional pharmaceuticals (Nwabuisi, 2002). This is because the excessive cost of most conventional pharmaceuticals prevents utilization. Thus, there is need for toxicity evaluation of these plants to arrive at the safest use possible. Throughout much of tropical Africa the tree called Detar Detarium senegalense J. F. Gmelin is common and its rounded pods are well known. Fruits produced by the Detarium senegalense tree were first described as Detar in 1789, by De Juisieu in Senegal. Being discovered in Senegal, these trees still remain an important contributor to the country‟s local food system and economy 1
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(Cisse et al., 2010) it produce globular fruits (Adenkunle et al., 2011). This multipurpose plant, producing nutritious fruits (NRC, 2008), is also used for a number of medicinal purposes (Akah et al., 2012) beside its quality timber (Elkamali, 2011). Detarium senegalense has great potential for promoting food security, contributing to sustainable land care and rural development. In the Sudan the tree is found in the Ingassana and Nuba mountains. it‟s have an abundance of common names in languages across western Africa. In English: Sweet detar, Sweet dattock Detarium microcarpum, Tallow tree Detarium senegalense and in Arabic: Abu leita and Abu Leila (NRC, 2008 and NAS, 2008), in France Grand detar (Burkill, 1995). Detarium senegalense is mostly enjoyed in the West Africa still as traditional food plant (NRC, 2008). Most are eaten fresh, but some are dried in the sun and sold in the market. The hard shell and dry pulp give them an exceptional shelf life and the sweet and sour flavor appeals to all palates. In spite of the various uses of Detarium senegalense in food and as drug, their various phytoconstituents have not been fully documented. Thus, this study will investigate its phytochemical categories, proximate analysis and minerals constituents to assess the tree potential usefulness as food supplement or in pharmacognosy. Though used as safe non-conventional drought human food in most Sahil countries including Sudan, the toxicity of the Detar fruit flour was rarely reported. The fruit flour toxicity was not tested yet in country when similar savannah trees fruits B. aegyptiaca, Z. spinacrista, A. digtata and T. indica were studied before. This study intends to determine the extent to which Detarium senegalense fruit flour, originating from tree species inhabiting Sudan, has any toxic potential in rats, toxicity that variate according to the portal of entry, or differences, if any, in the distribution of the pathological lesions inflicted and put forward suggestions for future commencement on this line of research and point out practical implications foreseen. 2
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
CHAPTER ONE LITERATURE REVIEW
In this Chapter, ethnotoxicology, family Fabaceae, non-Fabale and Fabale food trees were reviewed with emphasis on test plant Detarium senegalense taxonomy, description, ecology and uses including medicinal. Profiles of phytochemical and proximate analyses and toxicity of the test plant were also reviewed.
1.1 Ethnotoxicology Ethnotoxicology, a branch of ethnobotany (Jain, 1987) deals with various toxic plants used as fish poison, arrow poison etc. (Schultes, 1970) but for a common scope, ethnotoxicology should be considered in much broader form (Morton, 1977). These poisonous plants contain powerful toxic ingredients phytochemicals which if introduced into the body of any animal system, may be of relatively smaller quantity, will affect deleteriously and may be fatal at times. These toxic substances injure the basic life principle i.e. the protoplasm and the harmful effects produced, may be immediate or accumulative, the later may appear after a period of time when the poison reaches up to a specific concentration due to repeated administration (Morton,1977 and Martin,1995). Aloe barbedensis, Datura stramonium, Euphorbia pulcherrima, Jatropha curcas, Lantana camara, Ricinus communis, etc., are some examples of toxic plants which have been used by ancient people from time immemorial. The poisonous plants around us are not only the flowering plants; rather some of the cryptogams like algae, fungi, mushrooms, lichens, ferns are also poisonous. The flowering plants from toxicological point of view can be divided into two main groups; plants poisonous to man and livestock and plants poisonous to insects and fishes. The first group acts toxic when taken in small
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 quantity as food and fodder for a longer time. The second group shows insecticidal or molluscicidal properties along with fish poisoning. A broad spectrum of plants was widely discussed from a forensic point of view (Morton, 1977).
1.2 Food trees Many trees were known to yield food as fruits having, as well as the mother tree, nutritive values as well as potential toxic properties. Most known in the Sudan are the Baobab (Adansonia digitata), Nabag (Ziziphus spinachrista), Doom palm (Hyphaene thebaica), Laloub (Balanites aegyptiaca) and the Fabales trees Tamrihindi (Tamarindus indica) and the Detarieae, Abuleila sweet Deter trees Detarium macrocarpum, D. microcarpum and D. senegalense
1.2.1 Adansonia digitata Adansonia digitata is the most widespread of the Adansonia gerera on the African continent, found in the hot, dry savannahs of sub-Saharan Africa. It belongs to the family Malvaceae (Bremer et al., 2003). The fruit pulp of the Adansonia digitata (Linn.), commonly known as Baobab, is an important human nutrition source in East, Central, and West Africa (Beckier, 1983 and Szolnoki, 1985).
Phytochemistry The dry baobab fruit pulp has a slightly tart, refreshing taste and is very nutritious, with particularly high values for carbohydrates, energy, calcium, potassium (very high), thiamine, nicotinic acid and very high vitamin C (Arnold et al., 1998). The fruit contains 50% more calcium than spinach, is high in antioxidants, and has three times the vitamin C (Sidibe and Williams, 2002). The nutritious seeds have high values for proteins, fats (oils), fibre and most minerals (Arnold et al., 1998). Besides, Baobab seeds have high levels of 4
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 lysine, thiamine, calcium and iron (Nnam and Obiakor 2003). Leaves are rich in vitamin C, sugars, potassium tartrate and calcium. Relative to fruits, leaves contain more essential amino acids, minerals and vitamin A.
Food uses The baobab is a traditional food plant in Africa. The dry pulp is either eaten fresh or dissolved in milk or water to make a refreshing drink. The powder is called Lalo in Mali and sold in many village markets in Western Africa. Oil extracted by pounding the seeds can be used for cooking but this is not widespread (Sidibe and Williams, 2002). Baobab leaves are sometimes used as forage for ruminants in dry season. The oil meal, which is a byproduct of oil extraction, can also be used as animal feed (Heuzé et al., 2013). The leaves can be eaten as relish, they are cooked fresh as a vegetable or dried and crushed for later use by local people. The sprout of a young tree can be eaten like asparagus. The root of very young trees is also reputed to be edible. The seeds are also edible and can also be roasted for use as a coffee substitute. Wild animals eat the fallen leaves and fresh leaves are good fodder for domestic animals. The fallen flowers are relished by wild animals and cattle alike. When the wood is chewed, it provides vital moisture to relieve thirst in animals at times of drought (Esterhuyse et al., 2001 and Germishuizen and Meyer, 2003).
Medicinal uses The fruit pulp traditionally used as an immune stimulant (El-Rawy et al,. 1997), anti-inflammatory, analgesic (Ramadan et al.,1994), pesticide (Tuani et al,. 1994), antipyretic, febrifuge, antimicrobial (Grand and Grand 1989, Hussain and Deeni,1991 and Locher et al., 1995) and astringent in the treatment of diarrhoea and dysentery (Ramadan et al., 1994). Also fruit pulp has hepatoprotective activity (Al-Qarawi et al., 2003). 5
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1.2.2 Ziziphus spina-christa Ziziphus spinachristi, the Christ's Thorn Jujube, Sidir, Nabak and Kurna is an evergreen tree native to northern and tropical Africa and southern and Western Asia (Zohary, 1972 and Yossef et al., 2011). It belongs to family Rhamnaceae (Abalaka et al., 2010). Sidir is a dense thorny tree of tropical Sudanese origin. It grows as a shrub or small tree, armed with short spines that are positioned in pairs along the branches, one of which is straight and the other curved or hooked, the fruit is round, 1-2 cm in diameter, brown yellowish when ripe. It contains a large stone in the center, which is surrounded by a dry and fleshy pulp (Saied et al., 2008 and Orwa et al., 2009).The ripe fruits are edible (Peter, 1992) and the flowers are an important source of honey in Eritrea and Yemen.
Phytochemistry The leaves contain various alkaloids, including ziziphine, jubanine and amphibine, alphaterpinol, linalol and diverse saponins. Flavonoids, triterpenoids, lipids, proteins, free sugar and mucilage are the main important compounds characterized in this plant (Adzu et al., 2003).
Food uses The fruits were found to have a very high energy value. Only dates, figs, raisins and a few other dried fruits have a higher value. The fruits contain 14.16% sugar and about 1.6% vitamin C (Orwa et al., 2009). The seeds were rich in protein and the leaves in calcium, iron and magnesium (Peter, 1992).The leaves provide valuable animal forage and fodder under open grazing conditions, but the nutritional value is apparently not high for most domestic livestock. The fruits are eaten by sheep and goats and the foliage by camels (Orwa et al., 2009). 6
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Medicinal uses In the Sahel region, the roots are used to treat headaches, while the spines or ashes of this species are applied to snake bites. Boiled leaves are applied to various surface wounds, and also have anthelmintic and antidiarrheal properties. In Egypt and the southern Sahara, a narcotic beverage is made from the fruits and which is considered to be a tranquilliser and sedative. In Morocco, the fruits are used as an emollient and astringent agent. It also is reputed to reduce abscesses and boils while acataplasm of young leaves is also used to reduce eye Inflammations (Orwa et al., 2009). The leaves are applied as poultices, helpful in liver troubles, asthma and fever (Michel, 2002). Z. spinachristi extract has also been reported to possess protective effect against aflatoxicosis (Abdel-Wahhab et al., 2007). The leaves were powdered and used as hair conditioner. From current studies, additional pharmaceutical applications of Z. spinachristi have revealed antifungal, antibacterial, antinociceptive, antioxidant, antidiabetic, antiplasmodial, antischistosomiasis, analgesic and anticonvulsant activities (Adamu et al., 2006; El-Kamali and Mahjoub, 2009; Adzu et al., 2001; 2011; Abalaka et al., 2011; Abdel-Zaher et al., 2005; El-Rigal et al., 2006; Adzu and Haruna, 2007 and Waggas and Al-Hasani, 2010).
1.2.3 Hyphaene thebaica Hyphaene thebaica, with common names Doam palm and Gingerbread tree, is a type of palm tree with edible oval fruit. It is a native of the northern half of Africa. It common in Upper Egypt, originally native to the Nile valley, bearing an edible fruit which is glubose-quandrangular with a shinny orange- brown to deep chestnut skin (epicarp) (Burkill, 1997).
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Food uses The fruit is sold in herbalist shops, and is popular among children, gnawing its sweet yet sour hard fibrous flesh beneath the shiny hard crust. The rind (mesocarp) in some palm is unedible but very palatable in others, highly aromatic and sweet with a taste like ginger (Burkill, 1997).
Medicinal uses The fruit of the Doam palm has been used in folk medicine to treat hypertension. It was found that those receiving the supplement had lower systolic and diastolic pressures and lower total cholesterol, and the blood lipids and lipoproteins were changed in such a way as to decrease the risk of cardiovascular disease (El-Gendy et al., 2008). Doam palm fruit is rich in flavonoids (polyphenols), saponins and tannins (Dosumu et al., 2006). The chloroform extract of the fruits improve spermatic count of male rats at low concentration (Hetta and Yassin, 2006) but decrease it at high concentration (Hetta et al., 2005). Doam extracts are being used in the treatment of bilharziasis, haematuria, bleeding especially after child birth (Adaya et al., 1977) and also as hypolipidemic and hematinic suspension (Kamis et al., 2003). When eaten it serves as vermifuges and parasite expellant (Burkill, 1997). The tea of Doam is popular in Egypt and believed to be good for diabetes. It has been used by Egyptian people as a folk medicine for treatment of hypertension (Hetta et al., 2005).
1.2.4 Balanites aegyptiaca Balanites aegyptiaca is a species of tree known as Laloub or desert dates, classified either as a member of the Zygophyllaceae or the Balanitaceae families. The tree is native to much of Africa and parts of the Middle East (GRIN, 2008 and Daya and Vaghasiya, 2011). 8
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Phytochemistry The stem bark contains alkaloid, flavonoid, saponin, tanin and volatile oils (Sofowara, 1982). Fruit contains protein, lipid, carbohydrate, alkaloid, saponin, flavonoid, and organic acid (Daya and Vaghasiya, 2011). The seed contains 30-48% fixed (non-volatile) oil. Like the leaves, fruit pulp, bark and roots contains the sapogenins diosgenin and yamogenin. Diosgenin can be used to produce hormones such as those in combined oral contraceptive pills and corticoids (Ndoye, 2004). Saponins likewise occur in the roots, bark wood and fruit (Iwu and Maurice, 1993).
Food uses The yellow, single-seeded fruit is edible, but bitter. Many parts of the plant are used as famine foods in Africa; the leaves are eaten raw or cooked, the oily seed is boiled to make it less bitter and eaten mixed with sorghum, and the flowers can be eaten. Desert date fruit is mixed into porridge and eaten by nursing mothers, and the oil is consumed to improve lactation (Prashant et al., 2011). The tree is considered valuable in arid regions because it produces fruit even in dry times. The fruit can be fermented for alcoholic beverages (Wufem et al., 2007).
Medicinal uses It is traditionally used in treatment of jaundice, intestinal worm infection, wounds, malaria, syphilis, epilepsy, dysentery, constipation, diarrhea, hemorrhoid, stomach aches, asthma, and fever (Daya and Vaghasiya, 2011). Balanites aegyptiaca has many medicinal properties, anti inflammatory and antianalgasic activity (Kalpesh et al., 2008), anthelmintic activity (Gunasekhararan et al., 2006), antioxidant activity (Kamel et al., 1991), antidiabetic (Speroni et al., 2005), hepatoprotective activity (Majorie, 1999), 9
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 larvicidal activity (Chapagain and Wiesman, 2005) and antibacterial activity (Farid et al., 2002).
1.3 Fabales food trees 1.3.1 Family Fabaceae Legumes family Fabaceae have been recognized to be the 2nd most valuable plant source for human and animal nutrition (Vietmeyer, 1986). Fabaceae or Leguminosae (other names for Fabaceae are Leguminosae, Legume family, Legumes bean, Pea family, pulse family and Caesalpiniaceae) is a large and economically important family of flowering plants. Legumes include a large number of domesticated species harvested as crops for human and animal consumption as well as for oils, fiber, fuel, fertilizers, timber, medicinal, chemicals and horticultural varieties (R.B.G., 2005). In addition, the family includes several species studied as genetic and genomic model systems e.g., pea (Pisum sativum), barrel medic (Medicago truncatula) and trefoil (Lotus corniculatus). The name Fabaceae comes from the defunct genus Faba, now included into Vicia. Leguminosae is an older name still considered valid, and refers to the typical fruit of these plants, which are called legumes. Fabaceae is the third largest family of flowering plants, behind Orchidaceae (Orchids) and Asteraceae (Sunflower), with 730 genera and over 19,400 species, according to the Royal Botanical Gardens, and consisting of approximately 650 genera and 20000 species according to Doyle (1994). The largest genera are Astragalus with more than 2,000 species, Acacia with more than 900 species, and Indigofera with around 700 species. Other large genera include Crotalaria with 600 species and Mimosa with 500 species (Schrire et al., 2005 and R.B.G., 2005). The species of this family are found throughout the world, growing in many different environments and climates; deserts, savannas (African, neotropical and Australian savannas), seasonally dry tropical forests, rain forests 10
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(Atalantic and tropical rain forests) and temperate regions (Schrire et al., 2005 and Sprent, 2009). This preference for semi-arid to arid habitats is related to a nitrogen demanding metabolism. While many species have the ability to colonize barren and marginal lands because of their capacity to fix atmospheric nitrogen via a symbiotic association with root nodulating bacteria, this is just one of several ways in which legumes obtain high levels of nitrogen to meet the demands of their metabolism (McKey, 1994 and Sprent, 2001). Legumes play an important role in the terrestrial nitrogen cycle regardless of whether they from root nodules (Sprent, 2001). A number of legumes are important agricultural plants, including: Glycine max (soybean), Phaseolus (beans), Pisum sativum (pea), Cicer arietinum (chickpeas), Medicago sativa (alfalfa), Arachis hypogaea (peanut), Ceratonia siliqua (carob), and Glycyrrhiza glabra (licorice), which are among the best known members of Fabaceae. A number of species are also weedy pests in different parts of the world, including: Cytisus scoparius (broom) and Pueraria lobata (kudzu), and a number of Lupinus species (Schrire et al., 2005 and Sprent, 2009).
Taxonomy and description The Fabaceae or Leguminosae or Papilionaceae are placed in the order Fabales of the kingdom plantae according to most taxonomic systems. The Fabaceae comprise three subfamilies; Mimosoideae, 80 genera and 3,200 species, mostly tropical and warm temperate in Asia and America (Mimosa and Acacia), Caesalpinioideae 170 genera and 2,000 species, cosmopolitan (Caesalpinia, Senna, Bauhinia and Amherstia) and Papilionaceae (Faboideae), 470 genera and 14,000 species, cosmopolitan (Polhill and Raven, 1981 and Wojciechowski et al, 2006). These sub-families are sometimes recognised as three separate families: Papilionaceae, Caesalpiniaceae and Mimosaceae. The recognition of three subfamilies is based on miscellany botanic characteristics 11
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(R.B.G., 2005 and Wojciechowski et al, 2006). Moreover, there are a number of genera whose placement into either subfamily is not always agreed on e.g. Dimorphandra (Wojciechowski et al., 2004 and Wojciechowski et al, 2006). Fabaceae range inhabit from giant trees to small annual herbs, with the majority being herbaceous perennials (Rundel, 1989). Plants have indeterminate inflorescences, which are sometimes reduced to a single flower. The flowers have a short hypanthium and a single carpel with a short gynophore, and after fertilization produce fruits that are legumes.
Uses The history of legumes is tied in closely with that of human civilization, 6,000 BC, where they became a staple, essential for supplementing protein where there was not enough meat. Legume seeds and foliage have comparatively higher protein content than non-legume materials, due to the additional nitrogen that legumes receive through the nodules process (Sprent, 2009). Today legumes are an increasingly invaluable food source not just for humans, accounting for 27% of the world's primary crop production, but also for farm animals (Graham and Vance, 2003). Grain legumes alone contribute 33% of the dietary protein nitrogen needs of humans, while soybeans (Glycine max) and peanut (Arachis hypogeae) provide more than 35% of the world's processed vegetable oil and a rich source of dietary protein for the poultry and pork industries (Graham and Vance, 2003). Industrially, legumes have many uses in making biodegradable plastics, oils, dyes, and biodiesel fuel. Legumes are used traditionally in folk medicines, but also demonstrate importance in modern medicine. Isoflavones commonly found in legumes are thought to reduce the risk of cancer and lower cholesterol and soybean phytoestrogens are being studied for use in postmenopausal hormone replacement therapy (Graham and Vance,
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2003). Legumes also produce a hypoglycemic effect when eaten, making them a recommended food for diabetics (Gepts et al., 2005).
1.3.2 Caesalpinioideae Caesalpinioideae is a botanical name at the rank of subfamily, placed in the large family Fabaceae or Leguminosae. Its name is formed from the generic name Caesalpinia. The subfamily classified in four tribes, Caesalpinieae, Cassieae, Cercideae and Detarieae. The tribe Cercideae has sometimes been included in the subfamily Faboideae ( Martin et al., 2006).
1.3.3 Detarieae The tribe Detarieae is one of the subdivisions of the plant family Fabaceae. This tribe includes many tropical trees, some of which are used for timber or have ecological importance. The tribe consists of 81 genera, 53 of which are native to Africa. Pride of Burma (Amherstia nobilis) and tamarind (Tamarindus indica) are two of the most notable species in Detarieae (Watson and Dallwitz ,1993).
1.3.3.1 Tamarindus indica The Tamarind, Tamarindus indica, tamar hindi, or Indian date is a leguminous tree in the family Fabaceae indigenous to tropical Africa, particularly in Sudan. The genus Tamarindus is a monotypic taxon, having only a single species. The tamarind tree produces edible, pod-like fruit which are used extensively in cookeries around the world. Because of the tamarind's many uses, cultivation has spread around the world in tropical and subtropical zones (Penoeop, 1974 and Morton, 1987).
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Food uses The fruit pulp is edible. The hard green pulp of a young fruit is considered by many to be too sour. The ripened fruit is considered the more palatable, as it becomes sweeter and less sour (acidic) as it matures. A sour, chilled drink made from tamarind is served in Egypt. The tender leaves of T. indica are traditionally used with lentils in Southern India, replacing the tamarind fruit. A traditional food plant in Africa, tamarind has potential to improve nutrition, boost food security, foster rural development and support sustainable landcare (NRC, 2008*).
Medicinal uses Throughout Southeast Asia, fruit of the tamarind is used as a poultice applied to foreheads of fever sufferers (Doughari, 2006). The leaves are also used to treat throat infections, coughs, fever, intestinal worm infections, urinary problems and liver ailments. Leaves and pulp act as a cholagogue, laxative and anticongestant and exhibit anti-oxidant activity in the liver in addition to their blood sugar-reducing properties (El-Siddig et al.,2006).The leaf extracts were also shown to be antifungal and antimicrobial (Doughari, 2006 and Abubakar et al., 2010). Based on human study, tamarind intake may delay the progression of skeletal fluorosis by enhancing excretion of fluoride (Rupal and Narasimhacharya, 2012).
1.3.3.2 Detarium The family Fabaceae Detarium, is a genus that is represented by 8 species, however only three species of trees in west African forests, Detarium macrocarpum Harms, Detarium microcarpum Guillemin and Perrottet and Detarium senegalense J.F. Gmelin are of ethnomedicinal and pharmacological interest. These three species are very similar morphologically but appear to
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 differ in ecological distribution. The genus produces timber that may serve as a mahogany substitute. The fruit is edible ( Mabberley, 1987).
1.3.3.2.1 Detarium macrocarpum It is closely related to D. microcarpum as typical savanna species, but grows in humid forest (Kouyate and Van Damme, 2006). It differs from D. microcarpum trees which are drought tolerant and have the ability to grow on infertile sites as they are relatively insensitive to soil, altitude, heat, and humidity ( Elkamali, 2011).
1.3.3.2.2 Detarium microcarpum Detarium microcarpum, commonly known as sweet detar, sweet dattock or tallow tree, is an under-utilized tree legume that grows naturally in the drier regions of West and Central Africa. It is a small tree up to 10 m tall, with horizontal root system. It is typically a species of dry savanna (Leung et al., 1968) and differs from Detarium senegalense in its usually fewer, larger and more leathery leaflets, more compact inflorescences, sepals hairy outside and slightly smaller fruits. It is a multipurpose species, with a wide range of uses due to its medicinal properties, edible fruit eaten raw, cooked or made into flour with many uses of its own and hardwood used as fuel-wood (Keay et al.,1964 and Hopkins and Stanfield, 1966). The muco-adhesive properties of the defatted gum extracts have been reported also (Okorie and Chukwu, 2005).
Phytochemistry The fruit pulp has been found to have high proportions of carbohydrate (40-42.0%) and protein (29.1-30.9%) (Abdalbasit et al., 2009). The fruit is rich in vitamin C (3.2 mg), with 4.8 g protein and 64.5 g of sugar per 100 g (Abdalbasit et al., 2009). It was found to have the highest total phenolic, 15
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 flavonoid and antioxidant values among fourteen wild edible fruits from Burkina Faso (Abdalbasit et al., 2009). The seeds yield 7.5% oil with the predominant fatty acid being linoleic acid. The hulled seed flour contains per 100 g: 3.5–6.5 g water, 3 g crude fibre, 13–15 g crude fat, 13.5–27 g crude protein, 39 g carbohydrate, Ca 500 mg, Mg 500 mg, Fe 100 mg (Kouyaté and van Damme, 2006). The seed contain lipids, carbohydrates, proteins, crude fibre and the essential elements: Na, K, Mg, Ca, S, P and Fe (Abreu et al., 1998 and Abreu and Relva, 2002). Saponins, phytates and cyanides are reportedly present as anti-nutrients (Anhwange et al., 2004).
Food uses This species is highly appreciated by local peoples due to its variety of uses; it is said to be one of the most appreciated in the environments where it occurs naturally (Paulette, 2003). The fruit can be eaten raw or cooked, but for the most part, its pulp is transformed into flour (Kouyaté and Lamien, 2011). The seed flour is a traditional emulsifying, flavouring and thickening agent used to prepare cakes, bread, couscous, baby food and local beer (Kouyaté and van Damme ,2006). Its seed kernels are added to egusi soup, or are cooked and eaten as vegetables. The leaves are used as a condiment or vegetables, as are its flowers (Kouyaté and Lamien, 2011).
Medicinal uses Medicinal properties are in the roots, stems, bark, leaves and fruits to treat ailments including tuberculosis, meningitis and diarrhea (Abdalbasit et al., 2009). The species showed strong inhibitory effects on HIV-1 or HIV-2 infection in methanol extracts (Kouyaté and van Damme, 2006). Leaves and roots are also used to treat farm animals (Kouyaté, 2005). The plant and the 16
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 closely related species Detarium senegalense are used in the treatment of syphilis, dysentery, bronchititis, leprosy, sore throat, pneumonia, diarrhoea, malaria and meningitis (Daziel,1937; Abreu et al., 1998 and 1999; Okwu and Uchegbu 2008; Burkil, 1995 and Keay et al., 1989). Its antifungal and acetyl cholinesterase inhibitory activities have been reported (Cavin et al., 2006 and Adamu et al., 2006). The seed coat has antimicrobial and antifungal activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomona aeruginosa, Klebsiella pneumonia, Salmonella paratyphi and Candida albicans (Ebi and Afieroho, 2010). As one of the traditional medicinal plants, no toxic effects of D. microcarpum were reported in the literature (Nordeng et al., 2013).
1.3.3.2.3 Detarium senegalense Taxonomy Detarium senegalense J. F. Gmel. or Sweet detar is a species of plant in the Fabaceae family (Mabberley,1987; R.B.G.,2005; NRC,2008 and Adenkunle et al., 2011), subfamily: Caesalpinioideae and tribe: Detarieae. (Also placed in Leguminosae and Caesalpiniaceae (Mabberley, 1987 and R.B.G., 2005).
Distribution It grows naturally in the drier regions of West and Central Africa (Figure 1), Central African Republic; Zaire Cote D'Ivoire; Gambia; Ghana; Guinea; Guinea Bissau; Liberia; Nigeria; Senegal; Sierra Leone; Togo, from Senegal and Gambia east to Northeast Tropical
Africa and Sudan (Irvine, 1961 and GRIN, 2009) , Figure 1. Distribution of test plant in Africa where it inhabits the Ingassana and Nuba mountains. It is
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 more riparian and mostly occurs in dry forests (Kouyate and Van Damme, 2006).In Asia it grows in Indonesia and Singapore and Caribbean in Trinidad and Tobago (Elkamali, 2011).
Description It is found in two types, forestry type is tall to 40m and has reddish pods whose yellow pulp tends toward bitter and inedible. The savanna type is much smaller 5-10m with reddish brown scaly bark (Plate 1) with edible fruit, whose greenish pulp makes good eating. The latter trees are known as Sweet detar (Berhaut, 1967, NRC,2008 and NAS, 2008). The savanna form also these days usually classified as a separate species, Detarium microcarpum Guillemin and Perrottet (NAS,2008 and NRC, 2008). Detarium senegalense is a medium sized to fairly large tree up to 35–40 m tall (Adenkunle et al., 2011), bole is branchless up to 12–15 m, straight or irregular, cylindrical, up to 60–100 cm in diameter, without buttresses but sometimes swollen at base bark, surface finely fissured, becoming scaly, grayish to blackish, with large, round lenticels, inner bark thick, fibrous, red Plate 1. The Detarium senegalense brown, with some sticky gum, crown large, dark savanna type tree. green, with spreading branches. Savanna type leaves glaucous beneath, flowers are creamy white in dense inflorescences (Elkamali, 2011). The tree has large very leafy crown, leaves are bright green with common stalk 5-13 cm long (Keay et al., 1989). Detarium senegalense trees are propagated by stones which are often
Plate 2. Detarium senegalense fruit 18
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 distributed by elephants and chimpanzees who consume the fruits (Elkamali, 2011). Fruits are characterized by their dark green coating (Cisse et al., 2010). The fruits are about the size of an apricot (Plate 2). Fruits 4-6 cm diameter (Keay et al., 1989). Underneath the thin outer covering there is a quantity of green, farinaceous, edible pulp intermixed with stringy fibers that proceed from the inner and bony covering which encloses the single seed (Figure 2). There are two varieties one bitter, other sweet. The latter is the one sold in markets (Hedrick, 1919). It is noticeable that the fruit flour odor is very pleasant.
Nutrients composition Detarium senegalense fruit appears to be a very healthful product. Per 100 g, sweet detar fruit contains 27 mg calcium, 48 mg Figure 2. Detarium senegalense fruit phosphate, 0.14 mg thiamin and 0.05 mg riboflavin. The fruit pulp is rich in ascorbic acid 1000–2000 mg per 100 g (Elkamali,2011). Cisse et al. (2010) reported in fruit pulp also 2.8 mg iron, 0.6 mg niacin, and, most notably, about 1200 mg vitamin C i.e. very rich in vitamin C. In comparison to recommended daily vitamin and mineral requirements for an adult human, the fruit contains moderate amounts of thiamin and iron, an exceptional amount of vitamin C and lesser quantities of the other vitamins and minerals measured (Brown et al., 2011). The fruit seeds are composed of approximately 12% protein, and are rich in rare amino acids lysine and tryptophan, and thus the flour made from the seeds has an excellent amino acid composition (NRC, 2008). The mineral constituents of the seed and stem bark were Ca 1.44% - 1.80%, Mg 0.32% - 0.40%, K 0.50% - 0.85%, Na 0.53% - 0.40%, P 1.00% - 0.54%, Fe 7.11% - 6.97%, Mn 0.45% - 0.70% and Zn 5.40% - 6.15%. The plant 19
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 samples seed and stem bark were found to be rich in vitamins comprising riboflavin 0.62mg/100g – 0.60mg/100g, thiamin 0.14mg/100g – 0.27mg/100, niacin 2.06mg/100g – 8.11mg/100g and ascorbic acid 83.6mg/100 – 24.2mg/100g (Uchegbu and Okwu, 2012). Mineral content of the seed analysis revealed the concentrations of potassium 99.26, calcium 71.11, magnesium 77.83, sodium 55.26, iron 30.21, manganese 7.89, zinc 5.26 and copper 4.29 mg/g. These results show the nutritional value of the seeds of Detarium senegalense and justifies its use in the traditional treatment of skin diseases (Sowemimo et al., 2011).
Proximate analysis The proximate composition of the seed and stem bark revealed the presence of protein 20.5 - 9.60%, crude fiber 10.5 - 17.8%, fats/oil 55.6 - 3.56%, ash 5.00 - 5.50%, carbohydrates 8.40 - 63.54% and food energy 616.0 – 324.6 cal. / g (Uchegbu and Okwu, 2012). Detarium senegalense fruit appears to be a very healthful product. Per 100 g sweet detar fruit contains 67 % water, 1.9 % protein, 0.4 % fat, 29.6 % carbohydrates, 2.3 % fiber and 116 kcal (485 kJ) energy (Elkamali,2011) .Proximate analysis of seed revealed that seeds contain 24.43% carbohydrates, 7.23% protein, 31.16% fiber, 5.89% moisture and 1.93% ash (Sowemimo et al,. 2011).
Phytochemical analysis Phytochemical studies of the seeds and stem bark of Detarium senegalense Gmelin used as soup thickener and in herbal medicine in south Eastern Nigeria were studied and revealed the presence of alkaloids 0.37 - 0.72%, flavonoids 2.28 - 5.68%, tannins 0.47 - 0.79%, phenols 0.35 - 0.67% and saponins 1.85 - 4.60% (Uchegbu and Okwu, 2012).
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Nutritional uses Recent studies have shown that the Detarium seed contains a large amount of water-soluble, non-starch polysaccharide (s-NSP), which suggests it has important nutritional properties. The main monosaccharide residues of the extracted s-NSP were glucose, xylose, and galactose in ratios suggesting structural similarity to the xyloglucan group of cell wall storage polysaccharides similar to tamarind seed xyloglucan. The intrinsic viscosity was found to be 8.9 dl/g, indicating that the sample was of high molecular weight. Histochemical examination of Detarium seed using bright field and epifluorescence microscopy showed the presence of xyloglucan in highly thickened cell walls, which were particularly prominent at the cell junctions (Wang et al., 1996 and 1997). The greenish and sweet acidulous fruit pulp is edible, and can be eaten raw or cooked. It is also used to prepare sweetmeat or as an ingredient of ice cream. However, it may also be toxic, and caution is needed. The seed is oily and edible, and pounded seed is used as cattle feed. In Nigeria the seed flour is used traditionally as a emulsifying, flavouring and thickening agent in foods (Burkill, 1995) often used as a soup thickener (Adenkunl et al., 2011). The bark is added to palm wine to accelerate fermentation and to make it bitterer. Detarium senegalense J.F. Gmel. (ditax) fruits of which are characterized by an attractive green flesh with a high amount in ascorbic acid. It is generally consumed as a nectar in Senegal. The main pigments of ditax pulp were identified and quantified by HPLC-DAD. Pheophytin a (128 mg/kg), which represents 58% of the total pigments, followed by hydroxypheophytin a′ (33 mg/kg), chlorophyll b (24 mg/kg), and chlorophyll a (20 mg/kg), was the major pigment of ditax pulp. Lutein and β-carotene were present in lower amounts 4.6 and 3.6 mg/kg, respectively (Ndiaye et al., 2011). Analysis of the petroleum ether extract of the seeds with GC-MS produced ten constituents of which oleic and linoleic acids were the most prominent 30.8 and 44.1% respectively. The extract showed significant 21
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus kristinae, Streptococcus faecalis, Shigella flexneri, Klebsiella pneumonia and Serratia marcescens and antifungal activity against Aspergillus flavus, Aspergillus niger and Penicillium notatum (Sowemimo et al,. 2011), hence its use as a preservative of local palm wine in South Eastern Nigeria.
Medicinal uses There was a specific interest in the functionality of dietary polysaccharides starch and non-starch polysaccharides or NSP with respect to the bioavailability of nutrients and prevention and treatment of disease e.g. diabetes, coronary heart disease, and arthritis. Much of the work focuses on the behavior of water soluble NSP in the gastrointestinal tract and also the properties of supramolecular structures. Studies of the characterization of the structure and properties of new sources of NSP e.g. molecular weight and solution rheology from plant and microbial sources, we have recently shown that one of these, a xyloglucan extracted from a leguminous African plant food Detarium senegalense , has considerable promise in the treatment of diabetes and hyperlipidaemia. Its properties also indicate it has considerable commercial potential in the food, drugs and chemical industries (Wang et al., 1996 ; 1997 and Rayment et al., 2000). Detarium senegalense is an important medicinal plant that mostly overlaps with medicinal purposes of Detarium microcarpum Guill. and Perr. bark, roots, leaves and fruits (Elkamali, 2011). Several plant parts are used in traditional medicine. Bark is used to treat dropsy, swelling and odema. Bark decoctions or macerations are taken in case of heavy loss of blood, to treat digestive disorders, bronchitis, pneumonia, stomach ache, and to expel the placenta after childbirth ( Kaey et al., 1989; Adenkunle 22
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 et al., 2011 and Akah et al., 2012) Bark powder is applied to wounds, burns and skin complaints, and bark pulp is eaten against tuberculosis and as a tonic (Okwu and Uchegbu, 2008 and Elkamali, 2009 and 2011). The bark is used to alleviate pains such as headache, backpain, sore throat and painful menstruation (Keay et al., 1989 and Abrcu et al., 1999) and liquid from boiled bark used to cure indigestion (Keay et al., 1989). The bark is also used as arrow poison and soap substitute. Root decoctions are administered as anodyne and to treat intestinal complaints, marasmus, debility ( Elkamali, 2009 and 2011) , convulsions (Akah et al., 2012) and anaemia (Dalziel, 1995). Heated roots produce a sweet scent that is used as a perfume by women in Sudan, and as a mosquito repellent in Chad. Leaf and shoot decoctions are used to treat fever, trypanosomiasis, dysentery, anaemia, conjunctivitis,( Akah et al., 2012) arthritis, inflammations, fractures, boils and skin complaints( Elkamali, 2009 and 20 11).Fruit pulp is applied for the treatment of kidney pain, venereal diseases treatment, spinal tuberculosis, syphilis, cough, rheumatism and leprosy and in mixtures with other fruits as a stimulant (Burkill, 1995 and Neuwinger, 2000) and in the Sudan is used to treat abdominal pain. A detarium meal was observed to elicit significant reduction in the plasma glucose levels of the human subjects investigated (Onyechi et al., 1998). Seeds have been effective in controlling blood glucose levels in diabetic individuals (Cisse et al.,2010) also are taken as antidote against arrow poison and snake bites (Akah et al., 2012), also as emetic, insecticide, arachnicide and the smoke of burnt seeds as mosquito repellent .Bark and fruit are used to treat leprosy and pulmonary troubles. Bark and leaf generally heal skin and mucosa. The leaves are eaten as vegetable and are used traditionally as wash for itch, enema for dysentery and eye wash for conjunctivitis and skeletal structure. Root and fruit are used as painkillers (Burkill, 1995 and Neuwinger, 2000). Roots
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 and leaves use to stop diarrhoea in cattle. Decoctions of root and leaves use in paralysis, meningitis and difficult delivery ( Kaey et al., 1989). The gum from the plant is reported to have shown promising anti diabetic effect in experimental rats (Adikwu et al., 2004). Stem barks, seeds, leaves and root decoctions or infusions use to venereal diseases, urogenital infections, hemorrhoids, rheumatism, stomach ache, intestinal worms, diarrhea, malaria and leprosy (Abrcu et al., 1999 and Kaey et al., 1989). Leaves from the trees have demonstrated antiviral activity against a number of human and animal viruses and the bark has shown antibacterial activity against many pathogenic bacteria, justifying the medicinal properties of the plant (Elkamali, 2011). An anthocyanin alkaloid 2-methoxyamine 3,4,5,7-tetrahydroxyantho cynanidine has been isolated from the stem bark. Antibacterial studies showed that the isolated compound successfully inhibited Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumonia (Okwu and Uchegbu, 2008).All of the above results authenticate the use of Detarium senegalense in phytomedicine for the treatment of infections and disease prevention.
Toxicity Some trees produce toxic fruits and there is currently no method of differentiating these from trees that grow safe fruits (Cisse et al., 2010). Following a description of the habitat and morphological and physical characteristics, there seems to be existence of two different species or two different varieties of the edible/non edible characteristics of the detar fruit (Adam et al., 1991 and Berthelot et al., 2000), Though some Detarium senegalense trees produce toxic fruits, these are often identifiable by the presence of fruits remaining under the trees. If left behind, the trees are likely toxic as animals are normally very selective. A toxic, bitter compound has been 24
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 isolated from the fruit, as well as an acid tasting compound called detaric acid (Elkamali, 2011). The seeds contain a toxic substance which is removed by roasting or by prolonged soaking, vigorous washing and boiling (Aykroyd et al.,1982).
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
CHAPTER TWO MATERIALS AND METHODS
The study comprised three experiments on the toxicity of Abuleila (D. senegalense) fruit flour as dietary and as water extract injected via either s/c or i/p routes.
2.1 Materials and experimental designs 2.1.1 Toxicity of dietary Abuleila (D. senegalense) fruit flour to rats 2.1.1.1 Animals, housing and management Twenty, apparently healthy, adult male Albino rats weighing 60-120 gm were used in this experiment. Rats were purchased from Department of Pharmacology, Faculty of Pharmacy, University of Khartoum. Animals were transported to the premises of the Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Khartoum, Shambat, where they were housed in cages (each of dimensions 12x12x12cm. accommodating one dose-group) and maintained in a room under standard environmental conditions, controlled temperature (25±20C), relative humidity (60 %) with free access to water and formula rat basal feed (2.5 Mcal and 20% crude protein). Ventilation and drainage facilities were adequate. Rats were rested, kept for one week as an adaptation period prior to the start of the experiment, when rats were allotted randomly to four groups A, B, C and D in a 4x5 arrangement. Each group was accommodated separately and rats were identified by color tail marks. Group A was designated as the control group versus test groups B, C and D.
2.1.1.2 Test plant The test plant is a Fabaceae, Detarium senegalense J. F. Gmel., It was purchased from herbalists in El Obied Market, Northern Kordofan State, Sudan.
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
They were identified by the Medicinal and Aromatic Plants Research Institute, National Center for Research Khartoum, Sudan. 2.1.1.2.1 Fruit flour preparation The flour was obtained by decrusting the dried fruit. Flour was collected, cleaned and kept dry for further processing.
2.1.1.2.2 Fruit flour proximate analysis Proximate crude chemical values were determined for Detarium senegalense fruit flour (Table 1) according to AOAC (2008).
Table 1. Proximate percent crude chemical components (dry-matter basis) of Detarium senegalense fruit flour Component percentage Dry matter 94.45 Crude protein 08.45 Ether Extract 01.10 Crude fiber 09.78 Nitrogen-free extract 71.45 Na 0.15% Ca 0.30 % 03.68 Ash P 0.19% K 1.03%
2.1.1.2.3 Microminerals The microminerals in Detarium senegalense fruit flour were determined (Table 2) using the AAS (Atomic Absorption Spectroscopy) and the XRF analysis (X-Ray Fluorescent technique) (Ditrich and Cothern , 1971).
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Table 2. Micromineral components (dry-matter basis) of Detarium senegalense fruit flour
Micromineral Percentage Micromineral Percentage (mg%) (mg%) Br 0.296 Mg 0.325 Cd 0.0002 Mn 0.197 Co 0.004 Ni 0.417 Cr 0.002 Pb 0.037 Cu 0.174 Sr 0.044 Fe 5.170 Zn 0.203 Hg 0.041
2.1.1.2.4 Preliminary phytochemical screening General phytochemical screenings for the active constituents alkaloids, sterols, triterpenes, flavonoids, saponins, cumarins, tannins, anthraquenones and cyanogenics were carried out for Detarium senegalense extract (Table 3) using the methods described by Martinez and Valencia (1999), Sofowora (1993), Harborne (1984) and Wall et al. (1952) with few modifications (Appendix 1).
Table 3. Preliminary phytochemical screening of Detarium senegalense fruit flour extract
Test Result Observation Alkaloids + Turbidity Sterols - No observatiom Triterpenes ++ Pink-purple colour Flavonoids ++ Yellow colour Saponins +++ Foam Coumarins ++ UV florescence Tannins ++ Green-blue colour Anthraquenones - No observation Cyanogenic - No observation
Key: + Trace ++ Moderate +++ High - Negative
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
2.1.1.3 Administration and dose rates At the end of the adaptation period, experimental groups were dosed with Detarium senegalense fruit flour. Test flour was add daily to the basal meal at 10, 20 and 30 % rate by dilution to test groups B, C, and D respectively. Dosing was continued for six weeks.
2.1.1.4 Data collected Clinical signs and mortality rates were observed and recorded daily. Blood and serum samples were collected from the ocular vein before the start of the experimental dosing and thereafter fortnightly for hematological investigations and serum analysis. Post mortem or in extremis organ samples were collected and fixed in 10% formalin for further histopathological investigation.
2.1.2 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats via s/c route 2.1.2.1 Animals, housing and management Twenty, apparently healthy, adult male Albino rats weighing 60-120 gm were used in this experiment. Rats purchase, transport and housing conditions were the same as with rats of experiment 1. Formula rat basal feed and feeding were also similar to experiment 1. One week adaptation period was allowed prior to the start of the experiment. Experimental rats were allotted randomly to four groups A, B, C and D in a 4x5 arrangement. Each group was lodged separately and rats were identified by color tail marks. Group A was designated as the control group.
2.1.2.2 Fruit flour extraction Detarium senegalense fruit flour was dissolved in distilled water overnight at the room temperature. The solubility of Detarium senegalense fruit
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 flour was determined at 15%. The fruit flour extract was filtered, adjusted to a 2% solution and stored for further use.
2.1.2.3 Administration and dose rates At the start of dosing, experimental groups were dosed subcutaneously with Detarium senegalense fruit flour water extract daily at the rate of 0, 50, 100 and 150 mg/kg body weight to experimental groups A, B, C, and D respectively. Dosing was continued for six weeks.
2.1.1.4 Data collected Clinical signs and mortality rates were observed and recorded daily. Blood and serum samples collection and frequency, for hematological investigations and serum analysis were done as in experiment 1. Post mortem or in extremis organ samples were collected and fixed in 10% formalin for further histopathological investigation.
2.1.3 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats via i/p route 2.1.3.1 Animals, housing and management Twenty, apparently healthy, adult male Albino rats weighing 60-120 gm were used in this experiment. Rats avail and housing conditions were the same as with rats of experiment 1. Same formula rat basal feed and feeding were run as in experiment 1. One week adaptation period was allowed prior to the start of the experiment. Experimental rats were allotted randomly to four groups A, B, C and D in a 4x5 arrangement. Each group was kept separately and rats were identified by color tail marks. Group A was designated as the control group.
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
2.1.3.2 Administration and dose rates At the start of dosing, experimental groups were dosed intraperitoneally with Detarium senegalense fruit flour water extract daily at the rate of 0, 50, 100 and 150 mg/kg body weight to experimental groups A, B, C, and D respectively. Dosing was continued for six weeks.
2.1.3.3 Data collected Clinical signs and mortality rates were observed and recorded daily. Blood and serum collection and analysis, post mortem and in extremis organ samples treatment for histopathological investigation were done as in experiment 1.
2.2 Methods The blood samples were collected fortnightly from rat ocular vein, using 10 ml sterile plastic disposable syringes. Half volume of blood was transferred to a test tube containing ethylenediamine tetraacetate (EDTA) and used for hematological analyses (Kelly, 1984). The rest of blood was transferred into clean dry vials, allowed to clot overnight and serum was separated by centrifugation at 3000 r.p.m for 10 minutes and then stored at – 20°C until analyzed.
2.2.1 Haematological methods 2.2.1.1 Red blood cells count (RBCs) Red blood cells were counted with an improved Neubauer Haemocytometer (Hawksley and Sons Ltd., England). Hayem’s solution was used as a diluent (sodium chloride 1gm, sodium sulphate 0.5gm, mercuric chloride 0.5gm and made up to 200ml with distilled water).
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
2.2.1.2 Packed cell volume (PCV) Fresh blood samples were placed in the capillary haematocrit tube, centrifuged in a microhaematocrit centrifuge (Hawksley and Sons Ltd., England) for5 minutes and PCV percentage was read off on the microhaematocrit reader provided with the centrifuge.
2.2.1.3 Haemoglobin concentration (Hb) Haemoglobin concentration was determined by the cyanmethaemoglobin as described by Van Kampen and Zijlstra (1978) by using Drabkin,s solution (Potassium cyanide 0.2g, potassium ferricyanide 0.2g and sodium bicarbonate 1g in a liter of distilled water).Clean dry test tubes were prepared for sample and standard. To each tube 4 ml of reagent were added then 0.2 ml of blood sample. Hbstandard solution was added to the sample and standard test tubes,respectively. The tubes were allowed to stand for 15 min, and then the optical density (O.D) was read at 540 nm in a colorimeter. Haemoglobin concentration was expressed as follows: Hb concentration (g/dl) = (O.D samples/ O.D standard) x 15 (15g/dl is the concentration of the standard)
2.2.1.4 Red blood cell haematostatistics 2.2.1.4.1 Mean corpuscular volume (MCV) MCV was calculated from RBC number and PCV values as follows: MCV (m3) =10PCV /RBC in million/mm3
2.2.1.4.2 Mean corpuscular haemoglobin concentration (MCHC) MCHC was calculated from PCV and Hb values as follows: MCHC % =[100Hb (g/dl) ] /PCV %
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
2.2.1.5 White blood cell count (WBCs) White blood cells were counted with an improved Neubauer Haemocytometer (Hawksley and Sons Ltd., England) Turk’s fluid (1% glacial acetic acid, tinged with gentian violet) was used as diluent.
2.2.1.6 Differential leukocyte count The percentage of neutrophils, monocytes, oesinophils, basophils and blood platelets were determined microscopically from a count of 100 leukocytes in thin Giemsa stained blood smear (Kelly, 1984).Giemsa stain was prepared by transferring 3.8g of Giemsa powder to a dry bottle, 250 ml of methanol were added to the stain and mixed well, and then 250 ml of glycerol were added and mixed well. For use, the prepared Giemsa stain was diluted 1:10 with distilled water. A fresh blood smear was prepared. The blood was spread using the spreader slide, then the smear was dried at room temperature and fixed by placing in absolute methyl alcohol for 5 minute. The smear was covered by stain for 15 minute, and then the smear was rinsed well in distilled water and allowed to dry. The blood film was examined using the oil immersion lens (x 100).
2.2.2 Chemical methods 2.2.2.1 Serum metabolites 2.2.2.1.1 Glucose Serum glucose values were determined using enzymatic kits (Plasmatic Labs. Products, Germany).
Test principle Glucose level was measured according to the method described by Paterson(1971):
Glucose + O2 + H2O Gluconate H2O 2H2O2 + phenol + 4 amino-antipyrine red chinonimine + H2O.
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Dilute enzyme reagent (GOD+ POD + 4-amio-antipyrine was mixed with buffer (phosphate buffer, pH 7.5+phenol). Non-haemolytic serum was mixed with the reagent solution and the mixture was incubated at 37°C for 15 minutes. The absorbance was read at wavelength 500 nm, against the reagent blank calculated as follows:
U/L= ( A sample / A standard) x standard concentration (where A = absorbance).
2.2.2.1.2 Total protein Total serum protein concentration was measured by the biuret method (Weichselbaum, 1946) using commercial kit (Arcomex, Arab Company for Medical Diagnostics,Amman, Jordan.)
Test principle The principle is based on the reaction of cupric ions in an alkaline solution, with peptide bonds resulting in the formation of a colored complex. The biuret reagent contains sodium hydroxide (0.I-N), Sodium potassium tartrate (16mmol/l), potassium iodide (15 mmol/l) and copper sulphate (6mmol/l). The serum sample was mixed with the reagent solution and the mixture was incubated for 15 minutes at 25°C. the absorbencies of sample (A-sample) and standard (A-standard) were read against the regent blank in a spectrophotometer (Scientific Technical Supplies Spectronic 501-Germany) at a wavelength of 546nm and the concentration (c) of serum total protein was calculated as follows: Conc. (g/dl) = (A- sample x 8) /A- standard (Where 8 denotes concentration of standard 8mg/dl)
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2.2.2.1.3 Albumin Serum albumin concentration was determined by a colorimetric method using a commercial kit (Plasmatec Labs. Products, Germany).
Test principle The method is based on using bromocresol green (BCG) at pH 4.2 Absorbencies were read at wavelength 628nm. Conc. (mg/dl) = ( A- sample x 5)/ A- standard (Where 5 denotes concentration of standard 5mg/dl)
2.2.2.1.4 Urea Urea concentration in serum measured using enzymatic colorimetric method described by Monica (1992). Test principle The principle of method that urea is hydrolyzed by urease to ammonia and carbon dioxide. The intensity of the color formed when ammonia react with alkaline hypochlorite and sodium salicylate is proportional to the concentration of urea in serum sample.
Urea + H2O 2NH 3 +CO2 The prepared serum and reagent were placed at room temperature . The color intensity was measured using spectrophotometer at wave length 600 nm and urea concentration was calculated as follows:
Urea (mg/dl) = Tested sample X 50 Standard sample Where50 was the standard concentration.
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2.2.2.2 Serum enzyme activities 2.2.2.2.1 Aspartate aminotransferase (Glutamicoxaloacetic transaminase, L, Aspartate = 2 – OXO-glutarate aminotransferase E.C.2, 6,1.1, AST). Serum aspartate aminotransferase activity was measured by the enzymatic reaction sequence employed by a commercial kit (Plasmatec Labs. Products, Germany).
Test principle L-aspartate + oxoglutarate AST L-Glutamate + Oxaloacetate.
AST activity was determined by measuring the rate of oxidation of NADH by the spectrophotometer (Spectronic 501, scientific Technical supply, Karl Kolb, Germany) included in the reagent to convert endogenous pyruvate in the sample to lactate during the lag phase prior to measurement. A unit per liter (U/L) of AST activity is the amount of enzyme which oxidizes one mole of NADH per minute. Serum AST activity was calculated as follows:
U/L = A 340/min.x 1768. (Where A 340= mean absorbance change at 340nm wavelength).
2.2.2.2.2 Alanine amino transferase, (Glutamic pyruvic transaminase, L- aspartate,2-oxoglutamate ALT) It is an enzymatic method ,which measures glutamic pyruvic transaminase in sera by monitoring the concentration of pyruvate hydrazone formed with 2- 4dinitrophenyl hydrazine.
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Test principle
Oxoglutarate + L-alanine ALT L-glutarate + pyruvate
The absorbance of samples were read against the reagent blank after 5 mintues at wavelength of 630 nm UV/VIS Spectrophotometer. The ALT was measured in U/L.
2.2.2.3 Test plant macrominerals Detarium senegalense fruit flour macrominerals concentrations for Ca, K, Na and P were determined by flame photometry as described by Varly (1967).
Protocol
Two grams of the sample were extremely dried in sand bath after HCl acidification. Cooled sample was then diluted to 200ml with distilled water as standard solutions (1:100) which was passed under controlled condition as a very fine spray in the air supply to a burner where the solution evaporates and the salt disassociates to give neutral atoms. Light of characteristic wavelength was emitted and passed through a specific fitter for macrominerals ion to a selenium cell and the amount of current produced was read on a galvanometer. Changes in the galvanometer reading for the sample against that of low standard concentration of NaCl (14 µmol/L) were recorded and macrominerals values were calculated in mEq/l as follows:
Macromineral concentration (mEq/l) = (T/S) × 140 Where T = test ; S = low standard.
2.2.2.4 Test plant microminerals Detarium senegalense fruit flour trace elements Br, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, Sr and Zn were determined in ppm using X- Ray Fluorescent (XRF) technique as described by Ditrich and Cothern ( 1971). 37
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Protocol Detarium senegalense fruit flour sample was first prepared into fine powder before pressed into pellet form (1g mass and 2.5 cm in diameter) using a 15 ton pressing machine (Plate 3). The pellets were then presented to the XRF-Spectrometer system (Plate 4) where each sample was measured for 2000 seconds. The spectra obtained as a result of X-ray excitation was Plate 3. Presser machine detected and displayed using CD-109 computer at 15 ton/cm2 program. The spectra were then analyzed and concentrations of the elements present in the samples were obtained using Axil XRF-Software available to the computer (Figure3). Plant standard (Hay standard obtained from the IAEA, Vienna) was used to ensure the reliability of the results.
Plate 4. The XRF Spectrometer system Figure 3. Graphical representation of the with liquid nitrogen concentrations of trace minerals using XRF- Spectrometer system
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2.2.3 Histopathological methods Tissue specimens collected from animals were liver, kidney, heart, lung, spleen and intestine after death or in extremes slaughter.Tissues were immediately fixed in 10% buffered formalin (sodium hydrogen 6.5 gm/L and sodium dihydrogen 4 gm/L) then embedded in paraffin wax, sectioned at 5µ and stained routinely with Haematoxylin and Eosin (H&E) using Drury and Wallington (1980) method.
2.3 Statistical analyses Mean values obtained in blood and serum parameters were statistically verified using the un-paired student t-test. All means were compared at the 5 or 1% probability level. (Mendehall, 1971).
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CHAPTER THREE RESULTS
3.1 Toxicity of dietary Abuleila (D. senegalense) fruit flour to rats 3.1.1 Clinical signs It was fairly obvious that there were no symptoms in all rat groups fed with D. senegalense fruit flour. It was also observed that there were no mortalities in all groups.
3.1.2 Body weight changes Table 4 shows the effects of various doses of D. senegalense fruit flour on body weight changes in rats. There were significant (p≤0.05) increases in final body weight values in all test groups. Weight gains showed significant (p≤0.05-0.01) increases in all test groups. Group B recorded the highest gain (88.20± 36.48). When final body weights were regressed on time (Figure 4) significant (p≤0.01) coefficients were experienced by all test groups compared to the control (p≤0.05).
Table 4. Average (mean ± s.d.) body weight changes of experimental rats fed Detarium senegalense fruit flour for 6 weeks.
Group Initial weight Final weight Weight gain ( Dosage) (gm/ head) (gm/head) (gm/head)
A ( Control) 91.00± 17.46 120.00± 22.96 29.00± 14.82 B ( 10%) 86.00±11.94 174.20*± 37.42 88.20**± 36.48 C (20%) 89.00±22.75 164.60*± 35.92 75.60*± 35.51 D (30%) 90.00±19.69 168.40*± 33.72 78.40**± 21.14 *Denotes mean value significant at (p≤ 0.05). ** Denotes mean values significantly different at (p≤ 0.01).
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Figure 4. Average growth curves of treatment groups
A y = 0.37x* + 97.31 (SE ± 1.53)
B y = 0.77x** + 86.23 (SE ± 2.16)
C y = 0.69x** + 85.41 (SE ± 2.48)
D y = 0.74x** + 87.87 (SE ± 2.27)
3.1.3 Haematological findings The haematological changes in rats fed with D. senegalense fruit flour are summarized in Table 5. All test groups values were similar (p>0.05) to the control except for WBCs value in group D which was significantly (p≤0.05) increased.
Table 5. Average (mean ± s.d.) blood values of experimental rats fed Detarium senegalense fruit flour for 6 weeks. Group RBCs WBCs Hb PCV MCHC MCV MCH PLT
( Dosage) (×106 mm) (×103 mm) g/dl % % % % (×103 mm) A 5.71± 5.72± 14.18± 41.60± 34.99± 77.51± 27.19± 443.22± ( Control) 1.34 1.71 2.03 4.76 1.51 24.89 9.18 130.51 B 5.68± 7.16± 14.11± 41.69± 33.13± 77.88± 25.31± 519.33± ( 10%) 1.29 3.41 1.53 4.90 3.04 23.4 6.03 165.17 C 5.92± 7.19± 13.05± 40.62± 32.82± 73.73± 23.59± 515.28± (20%) 1.15 2.84 1.34 4.12 3.43 22.96 8.13 194.82 D 5.74± 8.95*± 13.98± 41.02± 34.72± 76.59± 26.62± 553.78± (30%) 1.23 4.67 1.57 5.71 1.51 27.64 10.01 225.75 * Denotes mean values significantly different at (p≤ 0.05).
WBCs differential count in rats fed D. senegalense fruit flour is summarized in Table 6. There was a significant (p<0.001) increase in
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 lymphocytes values in group B with a significant (p<0.01) decrease in monocytes and granulocytes.
Table 6. Average (mean ± s.d.) WBCs differential count of experimental rats fed Detarium senegalense fruit flour for 6 weeks.
Group WBCs Lymphocytes % Monocytes % Gan.% ( Dosage) (×103 mm) A ( Control) 5.72± 1.71 68.78±3.38 15.18±1.14 15.38±2.63 B ( 10%) 7.16± 3.41 78.90***±5.97 11.68**±2.81 9.42**±3.43 C (20%) 7.19± 2.84 71.16±5.82 14.78±2.76 14.05±3.23 D (30%) 8.95*± 4.67 65.98±9.29 17.97±4.02 16.05±5.36 ** Denotes mean values significantly different at (p≤ 0.01). *** Denotes mean values significantly different at (p≤ 0.001).
3.1.4 Changes in serum constituents The effects of various doses of D. senegalense fruit flour feeding to rats on the concentrations of serum metabolites, are given in Table 7. All metabolites concentrations were not significantly (p>0.01) altered when compared to the control values. There was a significant (p≤0.05) decrease in albumin concentration in group B, as well as with groups C and D significance (p≤0.01).
Table 7. Average (mean ± s.d.) serum metabolites values of experimental rats Fed Detarium senegalense fruit flour for 6 weeks
Group Glucose Total protein Albumin Globulin Urea ( Dosage) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl)
A ( Control) 71.84±42.81 7.63±1.55 4.13 ±0.46 3.50±1.42 78.44±5.51
B ( 10%) 72.63±43.76 7.66±1.13 3.69* ±0.54 3.97±1.09 78.68±4.79
C (20%) 75.80±36.96 7.10±0.67 3.58**±0.40 3.53±0.60 76.87±6.18
D (30%) 71.77±38.04 7.18±0.46 3.60**±0.39 3.58±0.53 79.52±5.16 * Denotes mean values significantly different at (p≤ 0.05). ** Denotes mean values significantly different at (p≤ 0.01).
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The effects of various doses of D. senegalense fruit flour feeding to rats on the concentrations of serum enzymes ALT and AST are summarized in Table 8. Both enzymes concentrations were not significantly (p>0.01) changed when compared to the control values. There was a significant (p ≤0.05) increase in AST value of group B.
Table 8. Average (mean ± s.d.) serum enzymes values of experimental rats fed Detarium senegalense fruit flour for 6 week
Group AST ALT ( Dosage) I.U I.U
A ( Control) 17.16± 3.31 84.50±31.75 B ( 10%) 23.29*± 5.48 109.25±37.78 C (20%) 16.92± 11.14 98.38±35.94 D (30%) 17.44±10.13 78.94 ±38.55 * Denotes mean values significantly different at (p≤ 0.05).
3.1.5 Post mortem findings All experimental rat groups showed mild liver congestions with few scattered haemorrhagic foci in lung and liver. Only rat No. 4 of group B showed fragile and very pale liver.
3.1.6 Histopathological findings Rat of group C showed alveolar walls thickening with slight interstitial proliferation of type II cells. A peribronchial mononuclear cellular infilterate was also seen (Plate 5). Rat of group D showed thickening of the alveolar walls with slight to severe interstitial proliferation of type II cells (Plates 8 and 9). Mononuclear cells and neutrophils infiltrate peribroncially (Plate 5) and around blood vessels (Plate 11).
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Plate 5.Thickening of alveolar walls with slight interstitial proliferation of type II cells. Peribronchial mononuclear cells infiltrate in the lung of rat in group C. H&E x100.
Plate 6. Thickening of alveolar walls with severe interstitial proliferation of type II cells in the lung of rat in group D. H&E x100.
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Plate 7. Thickening of alveolar walls with slight interstitial proliferation of type II cells in the lung of rat in group D. H&E x100.
Plate 8. Peribronchial mononuclear cells infiltrate in the lung of rat in group D. H&E x100.
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Plate 9. Focal infiltration of mono- nuclear cells and neutrophils around blood vessels in the lung of rat in group D. H&E x100.
3.2 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats via subcutaneous route
3.2.1 Clinical sings Rats injected subcutaneously with the water extract of D. senegalense fruit flour showed weakness and dullness in both groups C (100mg/kg body weight) and D (150mg/kg body weight). The mortalities in groups C were 40% occurring between week 4-6 and 60% in groups D occurring between weeks 3-6.
3.2.2 Body weight changes Table 9 shows body weight changes of experimental rats injected subcutaneously with D. senegalense fruit flour water extract for 6 weeks. There were no significant (p>0.05) changes in initial weights, final weights or weight gain values in all groups compared to the control. Highest (p>0.05) final weight (133.00± 22.72) was that of group C and highest (p>0.05) gain (35.00± 35.36) points to group D. When final body weights were regressed on time (Figure 5) significant (p≤0.01) coefficients were experienced by all test groups compared to the control (p≤0.05).
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Table 9. Average (mean ± s.d.) body weight changes of experimental rats injected subcutaneously with Detarium senegalense fruit flour water extract for 6 weeks.
Group Initial weight Final weight Weight gain ( Dosage) (gm/ head) (gm/head) (gm/head)
A ( Control) 91.00± 17.46 113.40± 23.59 22.40± 14.57 B ( 50mg/kg) 85.00±14.58 118.20± 19.47 33.20± 18.21 C(100mg/kg) 97.00±21.39 133.00± 22.72 26.33± 11.15 D (150mg/kg) 84.00±18.51 127.50±03.54 35.00± 35.36
Figure 5. Average growth curves of treatment groups
A y = 0.37x* + 97.31 (SE ± 1.53)
B00 y = 0.68x** + 83.60 (SE ± 1.04)
C y = 0.44x** + 92.60 (SE ± 1.97)
D y = 0.68x** + 84.45 (SE ± 1.54)
3.2.3 Haematological findings The haematological changes in rats injected subcutaneously with D. senegalense fruit flour water extract are summarized in Table 10. All test groups values were similar (p≥0.05) to the control except for WBCs (p≤ 0.05- 0.01) and platelets values which showed significant (p≤ 0.01) increase in all test groups.
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Table 10. Average (mean ± s.d.) blood values of experimental rats injected subcutaneously with D. senegalense fruit flour water extract for 6 weeks.
Group RBCs WBCs Hb PCV MCHC MCV MCH PLT (×106 (×103 ( Dosage) g/dl % % % % (×103 mm mm) mm) )
A ( Control) 5.71 5.72 14.18 41.60 34.99 77.51 27.19 443.22 ±1.34 ±1.71 ±2.03 ±4.76 ±1.51 ±24.8 ±9.18 ±130.51 B 5.10 9.03* 14.48 44.00 33.43 93.26 31.84 760.00** ( 50mg/kg) ±1.79 ±4.29 ±0.85 ±4.18 ±1.93 ±33.41 ±10.52 ±112.25 C 4.87 10.57** 14.63 44.17 32.83 95.02 31.24 780.61** (100mg/kg) ±1.12 ±4.69 ±0.94 ±3.97 ±2.86 ±23.17 ±8.52 ±70.45 D 5.10 10.20** 14.09 41.76 34.19 87.64 29.01 849.50** (150mg/kg) ±1.21 ±4.61 ±2.4 ±5.75 ±8.02 ±30.03 ±10.68 ±330.22 * Denotes mean values significantly different at (p≤0.05). ** Denotes mean values significantly different at (p≤ 0.01).
WBCs differential count in rats injected subcutaneously with D.enegalense fruit flour water extract are summarized in Table 11. All test groups values were similar (p>0.05) to the control except for group C which showed a significant (p≤0.05-0.01) increase in monocytes and granulocytes values and a significant (p≤0.05) decrease in lymphocytes values.
Table 11. Average (mean ± s.d.) WBCs differential count values of experimental rats injected subcutaneously Detarium Senegalense fruit flour water extract for 6 weeks.
Group WBCs Lympho.% Mon.% Gan.% ( Dosage) (×103 mm) A ( Control) 5.72 ±1.71 68.78±03.38 15.18±1.14 15.38±02.63 B ( 50mg/kg) 9.03* ±4.29 62.57±10.59 16.66±03.89 20.78±.09.35
C(100mg/kg) 10.57** ±4.69 57.57*±11.39 18.96**±3.44 23.42*±10.07 D (150mg/kg) 10.20** ±4.61 62.03±15.55 16.65±02.44 21.67±05.12 * Denotes mean values significantly different at (p≤ 0.05). ** Denotes mean values significantly different at (p≤ 0.01).
3.2.4 Changes in serum constituents The effects of various doses of D. senegalense fruit flour water extract injected subcutaneously to rats on concentrations of serum metabolites, glucose, total protein, albumin, globulin and urea are given in Table 12. There were no 48
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 significant (p>0.05) changes in all concentrations of serum metabolites in all experimental groups.
Table 12. Average (mean ± s.d.) serum metabolites values of experimental rats injected subcutaneously Detarium senegalense fruit flour water extract for 6 weeks.
Group Glucose Total protein Albumin Globulin Urea ( Dosage) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) A ( Control) 71.84±42.81 7.63±1.55 4.13±0.46 3.50±1.42 78.44±5.51
B ( 50mg/kg) 62.15±36.74 8.01±1.13 3.80±0.31 4.20±1.19 80.71±4.48
C(100mg/kg) 59.86±38.31 7.77±1.13 3.96±0.38 3.78±0.96 78.18±4.67
D (150mg/kg) 48.45±41.81 7.86±0.64 3.81±0.45 4.05±0.48 80.70±3.09
The effects of various doses of D. senegalense fruit flour water extract injected subcutaneously to rats on concentrations of serum enzyme ALT and AST are summarized in Table 13. AST values showed a significant (p≤0.05- 0.01) increase in all test groups and also there were significant (p≤0.05) increase in ALT values in groups C and D. Group B value for ALT was higher (p>0.05) than the control group.
Table 13. Average ( mean ± s.d. ) serum enzymes values of experimental rats injected subcutaneously Detarium senegalense fruit flour water extract for 6 weeks .
Group AST ALT ( Dosage) I.U I.U A ( Control) 17.16 ±0 3.31 84.50 ±31.75 B ( 50mg/kg) 24.23*± 10.55 103.56±25.66 C(100mg/kg) 26.38*± 14.19 122.72*±52.31 D (150mg/kg) 29.20**±11.78 123.33*±43.44 * Denotes mean values significantly different at (p≤0.05). * * Denotes mean values significantly different at (p≤ 0.05).
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3.2.5 Post mortem findings All experimental rat groups showed mild liver congestions with few scattered haemorrhagic foci in the lungs.
3.2.6 Histopathological findings
All rat test groups showed thickening of alveolar walls with slight to severe interstitial proliferation of type II cells. Group B showed also peribronchial mononuclear cells infiltration in the lung (Plate 10). Group D showed also focal infiltration of mononuclear cells around blood vessels in the lung (Plate 11).
Plate 10. Thickening of alveolar walls with slight interstitial proliferation of type II cells. Peribronchial mononuclear cells infiltrate in the lung of rat in group B. H&E x100.
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Plate 11. Thickening of alveolar walls with slight interstitial proliferation of type II cells. Focal infiltration of mononuclear cells around blood vessels in the lung of rat in group D. H&E x100.
3.3 Toxicity of Abuleila (D. senegalense) fruit flour water extract to rats via i/p route
3.3.1 Clinical sings Rats injected intraperitoneally with water extract of D. senegalense fruit flour show weakness and dullness in group C and D which were given 100mg/kg body weight and 150mg/kg body weight respectively. Recumbency and decreased appetite were also noticed in rat Nos. 3-5 in group D. The mortalities were 40% (wks 3-6) and 80% (wks 2-6) in rats injected intraperitoneally 100mg/kg and 150 mg/kg body weight respectively.
3.3.2 Body weight changes Table 14 shows body weight changes of experimental rats injected intraperitoneally with D. senegalense fruit flour water extract for 6 weeks. There were no significant (p>0.05) changes in initial weights, final weights or weight gain values in all groups compared to the control. Highest (p>0.05) final weight (114.33± 31.07) and highest (p>0.05) gain (21.00± 12.53) were those of group
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C. When final body weights were regressed on time (Figure 6) significant (p≤0.01) coefficients were experienced by all test groups compared to the control (p≤0.05).
Table 14. Average (mean ± s.d.) body weight changes of experimental rats injected intraperitoneally Detarium senegalense fruit flour water extract for 6 weeks.
Group Initial weight Final weight Weight gain ( Dosage) (gm/ head) (gm/head) (gm/head)
A ( Control) 91.00± 17.46 113.40± 23.59 22.40± 14.57 B ( 50mg/kg) 84.00±19.81 102.20± 35.95 18.20±16.71 C(100mg/kg) 84.00±18.51 114.33± 31.07 21.00± 12.53 D (150mg/kg) 91.00± 17.46 100.00±0.00 10.00±0.00
Figure 6. Average growth curves of treatment groups
A y = 0.37x* + 97.31 (SE ± 1.53)
00 B y = 0.24x** + 86.22 (SE ± 2.05)
C y = 0.42x** + 89.27 (SE ± 1.79)
D y = 0.38x** + 93.17 (SE ± 2.09)
3.3.3 Haematological finding The haematological changes in rats injected intraperitoneally with D. senegalense fruit flour water extract are summarized in Table 15. All test groups values were similar (p>0.05) to the control except for RBCs values in
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 group B where they were significant (p<0.05) increase. Also WBCs values and platelet values recorded significant (p<0.05-0.01) increase in all test groups.
Table 15. Average (mean ± s.d.) blood values of experimental rats injected intraperitonially Detarium senegalense fruit flour water extract for 6 weeks.
Group RBCs WBCs Hb PCV MCHC MCV MCH PLT (×106 (×103 ( Dosage) g/dl % % % % (×103 mm mm) mm) ) A 5.66 5.72 14.18 40.62 34.89 75.56 26.53 443.22 ( Control) ±1.18 ±1.71 ±2.03 ±4.71 ±2.79 ±22.46 ±8.81 ±130.51 B 7.11* 9.59** 14.80 43.94 33.79 66.49 22.32 718.78** (50mg/kg) ±1.55 ±4.10 ±1.19 ±3.22 ±3.02 ±24.10 ±7.76 ±81.87 C 6.45 9.98** 14.41 41.27 35.17 68.25 23.61 772.33** (100mg/kg) ±1.31 ±4.24 ±1.86 ±6.32 ±3.33 ±26.31 ±7.59 ±126.97 D 5.76 10.94* 15.23 40.61 38.03 78.45 28.19 997.50** (150mg/kg) ±1.37 ±7.73 ±1.56 ±6.09 ±5.79 ±27.07 ±9.18 ±197.28 * Denotes mean values significantly different at (p< 0.05) ** Denotes mean values significantly different at (p< 0.01)
WBCs differential count in rats injected intraperitoneally with D.senegalense fruit flour water extract are summarized in Table 16. All test groups values were similar (p>0.05) to the control except for monocytes count in group C, where there were significant (p<0.05) increase.
Table 16. Average (mean ± s.d.) WBCs differential count values experimental of rats injected Iintraperitoneally Detarium senegalense fruit flour water extract for 6 weeks.
Group Lympho.% Mono.% Graulo.% ( Dosage) A ( Control) 69.98±02.49 15.02±1.13 14.84±2.07 B ( 50mg/kg) 65.83±10.63 15.59±4.43 14.17±4.88 C(100mg/kg) 62.22±09.49 18.27*±03.32 19.50±6.8 D (150mg/kg) - - - * Denotes mean values significantly different at (p< 0.05).
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3.3.4 Changes in serum constituents The effects of various doses of D. senegalense fruit flour injected intraperitoneally water extract to rats on concentrations of serum metabolites, glucose, total protein, albumin, globulin and urea are given in Table 17. All test groups values were similar (p>0.05) to the control except for group D when albumin concentration was significantly (p<0.05) decreased.
Table 17. Average (mean ± s.d.) serum metabolites values experimental of rats injected Iintraperitoneally Detarium senegalense fruit flour water extract for 6 weeks.
Group Glucose Total protein Albumin Globulin Urea ( Dosage) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) A ( Control) 71.84±42.81 7.61±1.55 4.13±0.46 3.50±1.42 78.44±5.51
B ( 50mg/kg) 54.08±33.74 8.40±0.75 4.11±0.50 4.29±0.51 80.78±4.50
C(100mg/kg) 51.37±40.69 8.00±0.80 3.98±0.38 4.03±0.57 79.50±2.15
D (150mg/kg) 53.36±61.25 7.16±1.49 3.4* 8±0.66 3.69±1.04 78.91±2.45 * Denotes mean values significantly different at (p< 0.05).
The effects of various doses of D. senegalense fruit flour water extract injected intraperitoneally to rats on concentrations of serum enzymes ALT and AST are summarized in Table 18. There were no significant (p<0.05) changes in AST and ALT values in all groups.
Table 18. Average ( mean ± s.d. ) serum enzymes values of experimental rats injected iintraperitoneally Detarium senegalense fruit flour water extract for 6 weeks .
Group AST ALT ( Dosage) I.U I.U A ( Control) 17.16 ± 3.31 84.50±31.75 B ( 50mg/kg) 18.88±11.16 119.88±52.38 C(100mg/kg) 19.99± 6.39 97.04±63.53 D (150mg/kg) 17.13 ±8.74 113.63±60.00
3.3.5 Post mortem findings Group B which was injected with 50mg/kg body weight water extract of D. senegalense fruit flour showed congested lung and liver and also there were
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 paleness in the lung and kidney. Rat members of Group C (injected with 100mg/kg body weight water extract of D. senegalense fruit flour) showed pale liver and lung with congestions in liver, spleen and lung. Group D, injected with 150mg/kg body weight water extract of D. senegalense fruit flour, showed pale lung, liver and kidneys, with mild enlargement in the kidneys .
3.3.6 Histopathological findings All rats groups showed different degrees of thickening of alveolar walls with interstitial proliferation of type II cells, similar to the section in group C (Plate 12). Rat No 1 also showed focal infiltration of mononuclear cells around blood vessels in the lung (Plate 12). Group D, rat No. 4 showed dilatation of renal tubules, hemorrhage and coagulative necrosis (Plate 13). Liver of rat No.3 showed congestion (Plate 14), vesiculation of hepatocytes nuclei, hepatocytes necrosis ( Plate 15) and bridging necrosis ( Plate 16)
Plate 12. Thickening of alveolar walls with slight interstitial proliferation of type II cells. Focal infiltration of mononuclear cells around blood vessels in the lung of rat in groupC.
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Plate 13. Dilatation of renal tubules, hemorrhage and coagulative necrosis. in the kidney of rat No.4, group D. H&E x100
Plate 14. Congestion in the liver of rat No.3, group D. H&E x100
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Plate 15. Vesiculation of hepato- cytes nuclei and hepatocytes necrosis in the liver of rat No.3, group D. H&E x100
Plate 16. Bridging necrosis in the liver of rat No.3, group D. H&E x100
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CHAPTER FOUR DISCUSSION
The food trees cited in this study were those growing in the Sudan and experienced by citizens as food, beverages or remedies. Nevertheless, side effects or toxicities were always noticed, though not scientifically studied. The leguminous Caesalpiniaceae test plant Detarium senegalense is a tree that produces globular fruits ( Adenkunle etal.,2011) commonly referred to - beside other names - as Detar. This multipurpose tree that, along with others producing nutritious fruits (NRC, 2008), is also used for a number of medicinal purposes (Akah et al., 2012). There are two different species or two different varieties of the edible/non edible characteristics of the Detar fruit (Adam et al., 1991 and Berthelot et al., 2000). There is no field differentiability between the toxic tree fruits whence the other (Cisse et al., 2010), minding D. senegalense toxicity proper was not studied. In this study the variety of test plant fruit was not earmarked as a dietary toxic variety. Hence no dietary toxicity assumptions were put forward, whereas for the parenteral routes, toxicity was assumed. Actually the present study has shown that, D. senegalense fruit flour introduced as dietary, subcutaneous and intraperitoneal water extracts has toxic manifestation in rats. For dietary D. senegalense fruit flour, there were no clinical signs or mortalities inflicted by all doses. Doses of 100 and 150 mg/kg body weight of D. senegalense fruit flour water extract subcutaneously and intraperitoneally induced clinical signs and mortalities, weakness and dullness. In addition, recumbency and decreased appetite were observed at the 150 mg/kg body weight dose of the test plant intraperitoneally. This means that the toxic effects of D. senegalense fruit flour water extract is severer intraperitoneally than subcutaneous. Evaluating the medium (group C) and high (group D) mortality
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Gibreil, Hanan M. N.,Ph.D. Thesis,2014 rates via the two parenteral routes, numerous phytochemical components therein the test plant can cause synergistic toxicities. There were significant increases in final body weight values and body weight gains in all dietary test groups. This marks D. senegalense as nutritious food or food additive of the energy yielding carbohydrate fraction of the chemical components (71.45% Nitrogen-free extract). There were no literature values cited for the nutrient composition of the D. senegalense fruit flour to compare with, but other nutrient composition values for the seed or bark were quoted (Elkamali, 2011). The D. senegalense fruit flour has many phytochemical components having considerable concentrations namely alkaloids, triterpens, flavonoids, saponins, coumarins and tannins. Whether these components have yielded individual or synergistic toxicities was only a symptomatic and/or histopathologic judgment, though some individual components has known toxicity symptoms and lesions (Wink, 1999). Blood as an index of physiological and pathological status in humans and animals is well documented (Schlam et al., 1975; Effraim et al., 1999 and Ogwumike, 2002). The results of the present study show no haematological changes in the RBCs counts but noticeable increases in WBCs and PLT values. White blood cells (leucocytes) defend the body from viruses, bacteria and parasites at such times, cell numbers will be raised .Neutrophils attack and destroy bacteria. Lymphocytes, the second most common white blood cell are divided into two types B lymphocytes make antibodies, while T lymphocytes destroy cells infected with viruses. Monocytes have several functions, including bacteria removal, and are active in inflammation and in repair of damaged tissues, with platelets count increases also in inflammation ( Blann ,2014) . Damage or proliferation of alveolar type II cells might involve secondary microbial infection. Such infection remained subclinical i.e. showing no clinical sings related to specific organ lesion in all test groups with alveolar type II cells 59
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 proliferation. The high WBCs and PLTs in the highest dietary group plus all parenteral test groups can be increased as a result of a direct microbial infection or affected specifically by coumarin the lung targeting phytochemical that increases platelets in rat (NTP, 1993 and Lake, 1999). Coumarin, though not cited in literature as a constituent of D. senegalense fruit flour, our test sample analysis has revealed moderate ammounts. Coumarin, a benzopyrone organic phytochemical compound (Bye et al., 1970), has marked differences in metabolism between rodents and other species including humans. Target organs for coumarin toxicity and carcinogenicity in the rat are primarily the liver and lung (Marles et al., 1987 and Lake, 1999). It appears that the 7-hydroxylation pathway of coumarin metabolism, the major pathway in most human subjects is a minor pathway in rats and mice. The major pathway of coumarin metabolism in the rat and mouse is the 3,4-epoxidation pathway resulting in the formation of toxic metabolites. Saponins are bioactive compounds produced mainly by plants, they foam up when they are shaken in water, similar to detergents. Chemically, they generally occur as glycosides of steroids or polycyclic triterpenes (Kensil, 1996). Both the stem barks and seeds of D. senegalense were found to contain high quantity of saponin (Uchegbu and Okwu, 2012). Also in the present study the fruit flour of D. senegalense contained high quantity of saponin, found as a major constituent. Saponins are known to make the bronchial secretion more liquid, reduce the congestion of the bronchi and ease coughing. This also may be the reason the plant fruit plup is used in herbal medicine for the treatment of coughing (Burkill, 1995). Some saponins reduce the feed intake and growth rate of non-ruminant animals while others are not very harmful (D.A.S., 2013). Saponins irritate mucous membranes of the mouth and the digestive tract, reduce feed intake, lower dietary protein quality, cause gastroenteritis and diarrhea, the reason why the above factors lead to decreased performance and growth rate (D.A.S., 2013). Besides, the aglycones in certain saponins increase the 60
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 erythrocytic permeability of membranes. In severe cases, the membranes burst and their hemoglobin escapes into the blood stream. In this study all of these sings of toxicity were not clear except for the decreased appetite in the 150mg/kg intraperitoneal dose which may be attributed to the effect of saponins. The liver is a vital organ it has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion. Serum albumin in all dietary test groups showed low values in spite of an ample good dietary protein source, which entails decreased liver protein synthesis. Decreased albumin was also noticed in group D which was given a high dose of D. senegalense fruit flour water extract intraperitoneally associated with damage of the liver and kidney. An obvious sign of hepatic injury is leakage of cellular enzymes into plasma. When the liver cell membrane is damaged, a variety of enzymes normally located in the cytosol are released into blood stream. The estimation of the AST and ALT in the serum is a useful quantitative marker for the extent and type of hepatocellular damage (Udem et al., 2009 and Kumar et al., 2004). AST is found in high constitutive levels in the heart and liver, whereas ALT is most active in the liver (Udem et al., 2009 and Maruo et al., 2003). The results of the present study showed increasing AST and ALT values, an obvious sign of hepatic injury that agrees with NTP (1993) report and Lake (1999) results. For the intraperitoneal route, early death in the higher dose allowed no time to effect significant elevations of AST and ALT values. Histopathological changes in the lung, liver and kidney revealed the likely toxic effects of coumarin in those organs. In addition of that, other toxic manifestations refered to may be due to the effect of other phytochemical components in synergistic toxicity like saponins which are found as major constituents. Hence the results of the present study demonstrated that D.senegalense fruit flour experience toxicity to varying extends route wise. Locally, the wide use of D.senegalense fruits for the traditional treatment of 61
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 various conditions might be life threatening since we do not know the concentrations or quantities being administered and the potential toxicity of the plant variety.
Conclusion Dietary D. senegalense fruit flour was considered nutritious as food additive up to 30% without observable clinical signs or mortalities. Parenteral D. senegalense fruit flour water extract (100 - 150 mg/kg body weight) was more toxic intraperitoneally than subcutaneously to varying degrees of mortalities, with pulmonary and hepatonephropathies. The absence of the dietary toxicity or enteropathies in this study leads to recommend D.senegalense as an alternative non-conventional dietary energy source in animal or human nutrition, likely in famines or other catastrophes.
Suggestions for future work Further studies should involve higher dose ranges and longer exposure time of D. senegalense fruit flour to different animal species to elucidate solid findings on the basic toxicity symptoms and lesions. Having an outstanding nutritional profile, studies should be initiated to reveal the dietary and dietary therapeutic values of D. senegalense fruit flour. Other medicinal deter tree synonym toxicities should be studied to cover meager toxicity reports in the literature. To study the toxicity of other plant parts of D. senegalense like the seed, bark and leaves extracts, both organic and inorganic. Study of medicinal uses of different extracts of D. senegalense fruit flour or its parts. 62
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REFERENCES
Abalaka, M.E.; Daniyan, S.Y.and Mann, A. (2010). Evaluation of the antimicrobial activities of two Ziziphus species (Ziziphus mauritiana L. and Ziziphus spina-christi L.) on some microbial pathogens. Afr. J. Pharm. Pharmacol.,4(4):135-139.
Abalaka, M.E.; Mann, A. and Adeyemo, S.O. (2011). Studies on in-vitro antioxidant and free radical scavenging potential and phytochemical screening of leaves of Ziziphus mauritiana L. and Ziziphus spina-christi L. compared with Ascorbic acid. J. Med. Gen. Geno., 3(2):28-34.
Abdalbasit, A. M.; Mohamed Elwathig, S.; Mirghani, A. B. A. and Siddig, I. A. (2009). Detarium microcarpum Guill. and Perr. fruit proximate chemical analysis and sensory characteristics of concentrated juice and jam. African Journal of Biotechnology 8 (17): 4217-4221.
Abdel-Wahhab, M.A.; Omara, E.A.; Abdel-Galil, M.M.; Hassan, N.S.; Nada, S.A.; Saeed, A.and ElSayed, M.M. (2007). Ziziphus spina-christi extract protects against Aflatoxin B1-intitiated hepatic carcinogenicity. Afr. J. Trad. CAM., 4(3): 248-256.
Abdel-Zaher, A.O.; Salim, S.Y.; Assaf, M.H.and Abdel-Hady, R.H. (2005). Antidiabetic activity and toxicity of Zizyphus spina-christi leaves. J. Ethnopharmacol., 101(1-3):129-138.
Abreu, P.M.; Martins, E.S.; Kaysar, O.; Bindseil, K.U.; Siems, K.; Seemann, A. and Frevert, J. (1999). Antimicrobial, antitumor and antileishmania screeming of medicinal plants from Guinea Bissau. Phytomedicine, 6 (3):187-195.
Abreu, P. and Relva, A. (2002). Carbohydrates from Detarium microcarpum bark extract. Carbohydr. Res., 337: 1663-1666.
Abreu, P.M.; Rosa, V.S.; Araujo, E.M.; Canda, A.B.; Kayser, O.; Bindseil, K.U.; Siems, K. and Seemann, A. (1998). Phytochemical analysis and
63
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 antimicrobial evaluation of Detarium microcarpum bark extracts. Pharmaceutical and Pharmacological Letters 8(3): 107–109.
Abubakar, M.G.; Yerima, M.B.; Zahriya, A.G.and Ukwuani, A.N. (2010) Acute toxicity and antifungal studies of ethanolic leaves, stem and pulp extract of Tamarindus indica. Res J Pharm Biol Chem Sci., 1(4):104–111.
Adam, F.; Adam, J. C. and Hasselot, N.(1991). Poisoning by ingestion of ditakh (Senegal). Med. Trop., 51(4): 455-458.
Adamu, H.M.; Abayeh, O.J.; Ibok, N.U.and Kafu, S.E. (2006). Antifungal activity of extracts of some Cassia, Detarium and Ziziphus species against dermatophytes. Nat. Prod. Radiance 5(5):357-360.
Adaya, A.L.; Bitrus, H.; Fanjaji, H.; Eaton, M. and Gambo, D. (1977). Hidden harvest project in research series; 3: 14-27, 47-53.
Adenkunle, A.; Afolayan, A.; Okoh, B.; Omotosho, T.; Pendota, C. and Sowemimo, A. (2011). Chemical composition, antimicrobial activity, proximate analysis and mineral content of the seed of Detarium senegalense JF Gmelin. African Journal of Biotechnology, 10(48): 9875-9879.
Adikwu, M.U.; Yoshikawa, Y. and Takada, K. (2004). Pharmacodynamic- Pharmacokinetic profiles of metformin hydrochloride from amucoadhesive formulation of a polysaccharide with antidiabetic property in streptozotocin- induced diabetic rat models. Biomat., 25:3041-3048.
Adzu, B. and Haruna, A.K. (2007). Studied on the use of Zizyphus spina- christi against pain in rats and mice. Afr. J. Biotechnol., 6(11):1317-1324.
Adzu, B.; Amos, S.; Amizan, M.B.and Gamaniel, K. (2003). Evaluation of the antidiarrhoeal effects of Zizyphus spina-christi stem bark in rats. Acta. Trop.,87(2): 245-50.
Adzu, B.; Amos, S.; Wambebe, C. and Gamaniel, K. (2001) . Antinociceptive activity of Zizyphus spina-christi root bark extract. Fitoterapia.,72(4): 344-350.
64
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Adzu, B.; Haruna, A.K.; Ilyas, M.; Pateh, U.U.;Tarfa, F.D.; Chindo, B.A. and Gamaniel, K.S. (2011). Structural characterization of ZS – 2A: An antiplasmodial compound isolated from Zizyphus spina-christi root bark. J. Pharm. Nut. Sci., 1:48-53.
Akah, P.; Nworu, C.; Mbaoji, F.; Nwabunike, I., and Onyeto, C. (2012). Genus Detarium: Ethnomedicinal, phytochemical and pharmacological profile. Phytopharmacology, 3(2): 367-375.
Al-Qarawi, A. A. ; Al-Damegh, M.A. and El-Mougy, S.A. (2003). Hepatoprotective Influence of Adansonia digitata Pulp. Journal of Herbs, Spices & Medicinal Plants, Vol. 10 (3): 1-6.
Anhwange, B.A.; Ajibola, V.O.and Oniye, S. (2004). Chemical studies of the seeds of Moringa oleifera (Lam) and Detarium microcarpum (Guill and Sperr). J. Biol. Sci.,4(6): 711-715.
AOAC (2008). Official Methods of Analysis. Association of Official Analytical Chemists. 12th ed., Washington, USA.
Arnold, T.H.; Wells, M.J. and Wehmeyer, A.S. (1998). Khoisan food plants: taxa with potential for future economic exploitation. In: Plants for Arid Lands, Wickens, G.E.; Goodin, J.R.and Field, D.V. (eds.), Allen and Unwin, London, UK, 69-86.
Aykroyd,W.R.; Doughty, J. and Walker, A.(1982). Legumes in human nutrition. FAO Food and Nutritrition Paper. No. 20.
Beckier, B. (1983). The contribution of wild plants to human nutrition in the Ferlo (Northern Senegal). Agroforestry-Systems 1(3):257-267.
Berhaut, J.(1967). Flore du Senegal. Les légumineuses vivrières, Le Technicien d'Agriculture Tropicale, éd. and Taxonomy of Culicine larvae, 2d Edition, London: Printed by Order of the Trustees, 355 p.
65
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Berthelot, P. G.; N'Diaye, M.; Diatta, B.; Guerry, B.; Angel, G. and Saissy, J. M.(2000). Acute intoxication after ditakh fruit ingestion. Care. Med., 26(10): 1587-1587.
Blann, A. (2014) .Routine blood tests 4: functions and diseases of red and white blood cells. Nursing Times, 110(8):16-18.
Bremer, B.; Bremer, K.; Chase, M.W.; Reveal, J.L.; Soltis, D.E.; Soltis, P.S.;Stevens, P.F.; Anderberg, A.A; Fay, M.F.; Goldblatt, P.; Judd, W.S.; Kallersjo, M.; Karehed, J.; KRON, K.A.; Lundberg, J.; Nickrent, D.L.; Olmstead, R.G.; Oxelman, B.; Pires, J.C.; Rodman, J.E.; Rudall, P.J.; Savolainen, V.; Sytsma, K.J.; Van der Bank, M.; Wurdack, K.; Xiang, J.Q.Y.and Zmarzty, S. (2003). An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants. APG II. Botanical Journal of the Linnean Society, 141, 399-436.
Brown, J.; Isaacs, J.; Krinke, U.; Lechtenberg, E.; Murtaugh, M.; Sharbaugh, C.; Splett, P.; Stang, J. and Wooldrige, N. (2011). Detarium senegalense. Nutrition Through the Life Cycle (4th Ed.). Belmont, CA: Wadsworth.
Burkill, H.M. (1995). The Useful Plants of West Tropical Africa, Detareium Senegalense. Royal Bot. Gardens, Kew, 3: 102-105.
Burkill, H.M. (1997). The useful plants of West Tropical Africa, 2nd (Edn), Royal Botanical garden, Kew, 4:371-373.
Bye, A. and H. K. King. (1970). The biosynthesis of 4-hydroxycoumarin and dicoumarol by Aspergillus fumigatus Fresenius. Biochemical Journal, 117: 237- 745. Cavin, A.L.; Hay, A.E.; Marston, A.; Stoeckli-Evans, H.; Scopelliti, R.; Diallo ,D. and Hostettman, K. (2006). Bioactive Diterpenes from the Fruits of Detarium microcarpum. J. Nat. Prod., 69(5): 768-773.
Chapagain, B. and Wiesman, Z. (2005). Larvicidal effects of aqueous extract of Balanites aegyptiaca (desert date) against the larvae of Culex pipiens mosquitoes. African J. of Biotechnol., 4 (11): 1351-1354. 66
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Cisse, M.; Dieme, O.; Diop, N.; Dornier, M.; Ndiaye, A. and Sock, O. (2010). Detarium senegalense J. F. Gmel., principales caractéristiques et utilisations au Sénégal. Fruits, 65(5): 293-306.
D.A.S. (2013).Plants Poisonous to Livestock, Saponin. Department of Animal Science. Cornell university web servers, USA.
Dalziel, J.M. (1995).The useful plants of west tropical Africa. Gown Agents for Overseas Colonies, London.
Daya, L. C. and H. U. Vaghasiya. (2011). A review on Balanites aegyptiaca Del (desert date): phytochemical constituents, traditional uses, and pharmacological activity. Pharmacogn Rev. ,5(9): 55–62.
Daziel, J.M. (1937). The useful plants of west tropical Africa. Crown Agents, London.
Ditrich, T.and Cothern, R. (1971). Analysis of trace metal particulate in atmospheric samples using X-ray fluoresence. J. Air Boll. Control Assoc., 12:716.
Dosumu, O.O.; Nwosu, F.O.and Nwogu, C.D. (2006). Antimicrobial studies and phytochemical screening of extracts of Hyphaene thebaica (Linn.) Mart fruits. Intl. J. Trop. Med., 1(4): 186-189.
Doughari, J. H. (2006). Antimicrobial Activity of Tamarindus indica. Tropical Journal of Pharmaceutical Research 5 (2): 597–603.
Doyle, J.J. (1994). Phylogeny of the legume family: an approach to understanding the origins of nodulation. Ann Rev Ecol Syst., 25:325-349.
Drury, R.A. and Wallington, E.A. (1980). Carleton‟s Histological Techniques. 5th ed., Oxford, New York, Toronto.
Ebi, G.C. and Afieroho, O.E. (2010). Phytochemical and antimicrobial studies on Detarium microcarpum Guill and Sperr (Caesalpinioceae) seeds coat. African Journal of Biotechnology 10(3): 457-462.
67
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Effraim, K. D.; Salami, H. A. and Nwafor, P. A. (1999). The effect of aqueous seed extract of Trigonella foenum graecum (Fenugreek) on hematological parameters in Albino rats. African Journal of Biomedical Research, 2:47-51.
El-Gendy, A.; El-Mileegy, A.; Ghyaty, E.; Malek, H.and El-Hamid Mousa, A. (2008). The Beneficial Dietary Hypotensive and Hypolipidaemic Effects of Hyphaene Thebaica ( Doum ) . Internet Journal of Alternative Medicine 7 (1).
Elkamali, H.H. (2009). Ethnopharmacology of Medicinal Plants used in North Kordofan (Western Sudan). Ethnobotanical Leaflets 13: 89-97.
Elkamali, H.H. (2011). Detarium senegalense J.F.Gmel. Prota., 7(2): PROTA, Wageningen, Netherlands.
Elkamali, H.H. and Mahjoub, S.A. (2009). Antibacterial activity of Francoeuria crispa, Pulicaria undulata, Ziziphus spina-christi and Cucurbita pepo against seven standard pathogenic bacteria. Ethnobot. Leaflets 13:722-733.
El-Rawy, M.; Eman, S.A.M.; Gergis, S.B., and ElMougy, S.A.. (1997). The immunostimulant effect of Adansonia digitata on the immune response of chicken vaccinated with avian cholera vaccine. Journ. Egypt. Veterinary Med. Asso., 57:959-970.
El-Rigal, N.S.; Aly, S.A.; Rizk, M. and Said, A. (2006). Use of Ailanthus altissima and Ziziphus spina-christi extracts as folk medicine for treatment of some hepatic disorders in Schistosoma mansoni infected mice. Trends. Med. Res., 1(2):100-112.
El-Siddig, K.; Gunasena, H.P.M.; Prasada, B.A.; Pushpkumara, D.K.N.G.; Ramana, K.V.R. and Vijayanand, P. (2006) Tamarind: Tamarindus indica L. Southampton: Southampton Centre for Underutilized Crops,UK.
Esterhuyse, N.; Von Breitenbach, J. and Sohnge, H. ( 2001). Remarkable trees of South Africa. Briza publications, Pretoria.
68
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Farid, H.A.; Haslinger, O.; Kunat, C.; Wegener and Harnburger, M. (2002). New glycosides from Balanites aegyptiaca . Helvetica Chimica. Acta., 88(4):1019-1026.
Gepts, P. W. D.; Beavis, E. C. ;Brummer, R. C.; Shoemaker, H. T.; Stalker,N. F. ;Weeden and N. D. Young.(2005). Legumes as a model plant family.Genomics for food and feed report of the cross-legume advances through genomics conference. Plant Physiology, 137: 1228–1235.
Germishuizen,G. and Meyer, N.L.( 2003). Plants of southern Africa: an annotated checklist. Strelitzia 14.
Graham, P. H. and C. P. Vance. (2003). Legumes: importance and constraints to greater use. Plant Physiol., 131: 872 – 877.
Grand, Le A. and Le-Grand, A. (1989). Anti-infectious phytotherapies of the tree-savanna, Senegal (West Africa) III: a summary of the phytochemical substances and the antimicrobial activity of 43 species. Journ. Ethnopharmacology 25:315-338.
GRIN. (2008). Balanites aegyptiaca (L.) Del., .Germplasm Resources Information Network. United States Department of Agriculture.
GRIN. (2009). Detarium Senegalense J.F.Gemel. Germplasm Resources Information Network, United States Department of Agriculture.http://www.ars- grin.gov/cgi-bin/npgs/html/taxon.
Gunasekhararan, R.A. Divyakant and Senthilkumar, K.L. (2006). Anathematic activity of bark of Neolarnarchia cadarba Roxb. Indian J. of Natural product, 22(1): 11-13.
Harborne, J. B. (1984). Phytochemical Methods. 2nd edition. Chapman and Hall. London , 288 pp.
Hedrick,U.P. (1919). Sturtevant,s Notes On Edible Plants of the World. Report of the New York, Agricaltural Experiment station. http://Food Oregonstate. Edu.
69
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Hetta, M.H. and Yassin, N.Z. (2006). Comparative studies on hypocholesterolemic effect of different fractions of Hyphaene thebaica (Doum) in experimental animals. Pharmazie., 61(3):230-232.
Hetta, M.H.; Yassin, N.Z.and El Shaer, M.A. (2005). Effect of Hyphaene thebaica on the spermatogenesis of male rats. Egypt. Med. J. N.R.C., 4(3): 35- 39.
Heuzé, V.; Tran, G.; Bastianelli, D.and Archimède, H. (2013). African baobab (Adansonia digitata) . Feedipedia.org. A programme by INRA, CIRAD, AFZ and FAO.
Hopkins, B. and Stanfield, D.P. (1966). Savannah Trees of Nigeria. University Press, Ibadan. pp. 6-7, 12.
Hussain, H.S.N. and Y.Y. Deeni. (1991). Plants in Kano ethnomedicine; screening for antimicrobial activity and alkaloids. Internat. Journ. Pharmacognosy 29:51-56.
Irvine, F.R. (1961). Woody plants of Ghana with Special Reference to their Uses. Oxford Univ. Press London 868 pp.
Iwu and Maurice M. (1993). Handbook of African medicinal plants. Boca Raton u.a.: CRC Press. p.129.
Jain, S.K. (1987). Ethnobotany, its scope and various subdisciplines. In: A Manual of Ethnobotany. Jain, S.K. (Ed.) pp.1-11. Scientific Publisher, Jodhpur .
Kalpesh, G.R.K.; Newma,M.L.; Kori, C.S.; Sharma and Singh, V. (2008).Anti inflammatory and anti analgestic activities of Balanites aegyptiaca in experimental animals. Intl. J. Green pharmacy., 214-217.
Kamel, M.S.;Ohtan, K.; Kurokawa, T;Assaf, M.H;El-shanawany, M.A.; Ali, A.A; Kasai,R.; Ishibashi, S and Tanaka, O. (1991). Studies on Balanites aegyptiaca fruits and antidiabetic Egyptian folk medicine . Chem.Pharm. Bul., (Tokyo) 39 (5) 1229- 1233.
70
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Kamis, A.B.; Modu, S.; Zanna, H.and Oniyangi, T.A. (2003). Preliminary biochemical and haematological effects of aqueous suspension of pulp of Hyphaene thebaica (L) Mart in rats. Biokemistri., 13-17.
Keay, R.W.J.; Onochie, C.F.A. and Stanfield, D.P. (1964). Nigeria trees. National press Ltd. Federal Department of Forest Research, Ibadan, 2: 42-50, 227-228.
Keay, R.W.J.; Phil, D. and Biol, F.T. (1989). Trees of Nigeria. Oxford University press, New York. pp 204-207.
Kelly, W.R. (1984). The blood and blood forming organs. In:Veterinary Chemical Diagnosis. Pp 312- 337, Bailiere Tindall, London.
Kensil, C.R.(1996). Saponins as vaccine adjuvants. Crit Rev Ther Drug Carrier Syst., 13(1–2):1–55.
Kouyaté, A.M. (2005). Ethnobotanique Detarium microcarpum Guill. and Perr. In Mali, in: Domestication des espèces agroforestières au Sahel: Situation actuelle et perspectives ICRAF Working Paper No. 5. World Agroforestry Centre.
Kouyaté, A.M. and Lamien, N. (2011). Detarium microcarpum, sweet detar. Conservation and Sustainable Use of Genetic Resources of Priority Food Tree Species in sub-Saharan Africa. Bioversity International 4 : 1-8.
Kouyate, A.M. and van Damme, P. (2006). Medicinal plants/Plantes médicinales: Detarium microcarpum Guill. and Perr. Prota 11, no. 1 .Accessed November 24, 2012.
Kumar, G.; Sharmila Banu, G.; Vanitha Pappa, P.; Sundararajan, M. and Rajasekara Pandian, M. (2004). Hepatoprotective activity of Trianthema portula castrum rats L. against paracetamol and thioacetamide intoxication in albino. J. Ethnopharmacol., 92: 37-40.
Lake,B.G. (1999). Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment. Food Chem.Toxicol., 37(4):423-453.
71
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Leung,W.T. ; Busson, F.and Jardin, C. (1968). Food consumption table for use in Africa. FAO, Rome Italy p. 306.
Locher, C.P.; M.T. Burch; H.F. Mower; J. Berestecky, H. ;Davis, B.; Van- Poel, A. ;Lasure, D.A.; Vanden-Berghe, and A.J. Vlietinck. (1995). Antimicrobial activity and anticomplement activity of extracts obtained from selected Hawaiian medicinal plants. Journ. Ethnopharmacology 1:23-32.
Mabberley, D. J. (1987). Detarium. The Plant Book: A Portable Dictionary of the Higher Plants. Cambridge: Cambridge University Press. ISBN 0-521- 34060-8.
Majorie, M.C. (1999). Plant product at antimicrobial agents. Clin. Microbiol. Rev., 12(4):564-582.
Marles, R.J.; Compadre, C.M. and Farnsworth, N.R. (1987). Coumarin in vanilla extracts: Its detection and significance. Econ. Botany, 41(1):41-47.
Martin, G.J. (1995). Ethnobotany: A Methods Manual. Chapman and Hall, London.
Martinez, A. and Valencia,G. (2003) Marcha fitoquimica. In Manual de prácticas de Farmacognosia y Fitoquímica: 1999. 1. st edition. Medellin: Universidad de Antioquia; Phytochemical screening methods 59-65.
Maruo, V.M; Bernardi, M.M. and Spinosa, H.S. (2003). Toxicological evaluation of long term consumption of Salanumly cocarpum St. Hill fruits in male and female adult rats. Phytomedicine, 10:48-52.
McKey, D. (1994). Legumes and nitrogen: the evolutionary ecology of a nitrogen-demanding lifestyle. In Advances in Legume Systematics, part 5, the nitrogen factor (J. I. Sprent and D. McKey, eds.) pp 211-228. Royal Botanic Gardens, Kew, UK.
72
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Mendehall, W. S. (1971). Introduction of Probability Statistics. 3rd. ed., Wadswarch Publishing Co., Belmont, California, USA.
Michel, A. (2002). Tree, Shrub and Liana of West African Zone. Margraf Publishers GMBH, Paris p. 440.
Monica, C. (1992). Medical Laboratory Manual for Tropical Countries. 2 nd ed., pp 465-472, Butterworth-Heinemann Ltd, UK.
Morton, J.F. (1977). Poisonous and injurious higher plants and fungi. In: Forensic Medicine a Study in Trauma and Environmental Hazards, Chapter 71, Vol. III, C.G. Tedeschi, W.G.
Morton, J.F. (1978). Adverse reactions to plants in Florida. The Jour. of Florida Medical Association, 65:159-170.
Morton, J. F. (1987). Fruits of Warm Climates. Inc., 115-121.
NAS. (2008). Fruits. Lost Crops of Africa: Vol.III,Washington. National Academy of Science, National Acadmies Press, USA.
Ndiaye, N.D.; Mayer,C.D.; Cisse, M. and Dornier ,M. (2011). Identification and Thermal Degradation Kinetics of Chlorophyll Pigments and Ascorbic Acid from Ditax Nectar (Detarium senegalense J.F. Gmel). J. Agric. Food Chem., 59: (22) 12018–12027.
Ndoye, M. (2004). Reproductive biology in Balanites aegyptiaca (L.) Del., a semi-arid forest tree. African Journal of Biotechnology. 3:1 40-46.
Neuwinger, H.D. (2000). African traditional medicine. A dictionary of plant use and applications. Medpharm Scientific, Stuttgart, Germany. 589 pp.
Nnam, N.M. and Obiakor, P.N. (2003). Effect of fermentation on the nutrient and antinutrient composition of baobab (Adansonia digitata) seeds and rice (Oryza sativa) grains. Ecology of Food and Nutrition, 42: 265-277.
Nordeng, H.;Al-Zayadi,W.; Diallo, D.; Ballo,N. and Paulsen, B.S.(2013). Traditional medicine practitioners knowledge and views on treatment of
73
Gibreil, Hanan M. N.,Ph.D. Thesis,2014 pregnant women in three regions of Mali. Journal of Ethnobiology and Ethnomedicine, 9:67.
NRC. (2008). Sweet detar. Lost Crops of Africa:Vol.III, FruitsWashington. National Research Council, National Acadmies Press, 330-337, Washington.
NRC .(2008*). Tamarind. Lost Crops of Africa: Volume III: Fruits. National Research Council, Washington.
NTP. (1993). NTP Toxicology and Carcinogenesis Studies of Coumarin (CAS No. 91-64-5) in F344/N Rats and B6C3F1 Mice (Gavage Studies). National Toxicology Program. Toxicol. Program .Tech .Rep. Ser. ,422:1-340.
Nwabuisi, C. (2002). Prophylactic effect of multi herbal extract „Agbo-Iba‟ on malaria induced in mice .East African Medical Journal, 79(7): 343 – 346.
Ogwumike, O. O. (2002). Hematopoietic effect of aqueous extract of the leaf sheath of Sorghum bicolor in albino rats. African Journal of Biomedical Research, 5: 69-71.
Okorie, O. and Chukwu, A. (2005). Studies on Defatted Detarium microcarpum Gum, DDG I: Granule and Tablet Properties in: book of Abstracts of the Nigerian Association of Academic Pharmacists. Annual National Scientific conference, Abuja, pp. 27-29.
Okwu, D.E. and Uchegbu, R. (2008). Isolation, characterization and antibacterial activity screening of ethoxyamine tetrahydroxy-anthocyanidines from Detarium senegalense Gmelin stem bark. African Journal of Pure and Applied Chemistry, 3(1): 1–5.
Onyechi, U.A.; Judd, P.A.and Ellis, P.R. (1998). African plant foods rich in non starch polysaccharides reduce postprandial blood glucose and insulin concentrations in healthy human subject. Br. J. Nutr., 80:419-428.
Orwa, C.; Mutua, A.; Kindt, R.; Jamnadass, R. and Simons, A. (2009) Ziziphus spina-christi. Agroforestree Database: A tree reference and selection guide, World Agroforestry Centre. Available at: http:/ /www.worldagroforestry. org/treedb2/AFTPDFS/Zizyphus_spina-christi.pdf. 74
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Parek, J. and Chanda ,S. (2006). In-vitro Antimicrobial Activities of Extracts of Launaea procumbens Roxb. (Labiateae), Vitis vinifera L. (Vitaceae) and Cyperus rotundus L. (Cyperaceae). African Journal of Biomedical Research, 9: 89 – 93.
Paterson, Y. (1971). A new screening test for serum creatinine phosphokinase. Clin. Chem. Acta. , 34:373.
Paulette T. (2003).Use of woody plants by locals in Mare aux Hippopotames Biosphere Reserve in western Burkina Faso. Biodiversity and Conservation 12 (6):1211.
Penoeop, W. (1974). Tamarind. Manual of Tropical and Subtropical Fruits, 432-436 Hafner Press.
Peter, S. (1992). Nutritional study on Ziziphus spina Christi. Eden Foundation, Sweden.
Polhill, R. M. and P. H. Raven .(1981). Legume (Fabaceae) Fruits and Seeds. Advances in legume systematics, parts 1 and 2. Royal Botanic Gardens, Kew, UK.
Prashant, K.D.; Mahesh, Y.; Akhil, B.;Soni, M.L.;Ajeet, S. and Anupam, Kr.S. (2011). Balanites aegyptiaca (L.) Del., a semi –arid forest tree. Acad. J. plant Scien., 4(1):12-18.
Ramadan, A.; F.M. Harraz and S.A. ElMougy. (1994). Anti-inflammatory, anlalgesic and antipyretic effects of the fruit pulp of Adansonia digitata. Fitoterapia 65:418-422.
Rayment, P.; Ross-Murphy, S. B. and Ellis, P. R. (2000). Effect of size and shape of particulate inclusions on the rheology of galactomannan solutions. Carbohydrate Polymers, 43: 1-9.
R.B.G. (2005). Legumes of the World., G. B. Lewis, B. Schrire, B. Mackinde and M. Lock (Edits.). Royal Botanic Gardens, Richmond, U.K.
75
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Rundel, P. W. (1989). Ecological success in relation to plant form and function in the woody legumes. In C.H. Stirton and J.L. Zarucchi (eds.). Advances in legume biology, Monographs in Systematic Botany from the Missouri Botanical Gardens 29: 377-398.
Rupal A. Vasant and A. V. R. L. Narasimhacharya. (2012). Ameliorative effect of tamarind leaf on fluoride-induced metabolic alterations. Environ Health Prev. Med. ,17(6): 484–493.
Saied, A.S.; Gebauer, H.; Hammer, K. and Buerkert, A. (2008) Ziziphus spina-christi (L.) Willd: a multipurpose fruit tree. Genetic Resources and Crop Evolution, 55: 929-937.
Schlam ,O. W.; Jain, N. C. and Caroll, E. J. (1975).Veterinary Hematology, 3rd ed. Lea and Tebiger Publishers, Philadelphia, U.S.A., pp. 207-209 .
Schrire,B.D.;Lewis,G.B. and Lavin,M. (2005). Biogeography of the leguminasae. In Lewis,G; Schrire,G. and Mackinder,B. Legumes of the world. England, Royal Botanic Gardens, pp 21-54.
Schultes,R.E.(1970). Several ethnotoxicological notes from the Columbia Amazon. Bot. Mus. Leaflets, Harvard Univ., 22: 345-352.
Sidibe, M.and Williams, J.T. (2002). Baobab – Adansonia digitata. Fruits for the future 4, International Centre for Underutilised Crops, Southampton, UK,P 96.
Soejarto, D. D. (1996). Biodiversity prospecting and benefit-sharing perspective from the field. Journal of Ethnopharmacology ,51: 1 – 5.
Sofowora, A.F. (1993). Medicinal Plants and Traditional Medicines in Africa. Chichester John, Willey and Sons New York 256.
Sofowora, A.F. (1982). Medicinal plant and traditional medicine in Africa part 2. Pitnan Press Ltd, London 128-146.
76
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Sowemimo, A.A.; Pendota,C.;Okoh, B.; Omotosho ,T.; Idika, N.; Adekunle, A.A. and Afolayan, A.J. (2011). Chemical composition, antimicrobial activity, proximate analysis and mineral content of the seed of Detarium Senegalense J.F. African Journal of Biotechnology , 10(48): 9875-9879.
Speroni, E.R., Cervellati, G.; Innocenti, S.; Costa,M.C.; Guerra, S.; Dall, A. and Govoni, P. (2005). Anti inflammatory , antinociceptive and antoxident activities of Balanites aegyptiaca (L.) Delile . J. Ethnopharmacol, 98: 117-125.
Sprent, J. I. (2001). Nodulation in legumes. Royal Botanic Gardens, Kew, UK.
Sprent, J. I. (2009). Some legumes for the future. Legume NodulationA Global Perspective.pp97.
Sprent, J. I. (2009).Global distribution of legumes. Legume NodulationA Global Perspective.pp.21-54.
Szolnoki, T.W. (1985). Food and Fruit Trees of the Gambia. Published in conjunction with the Bundesforschungsanstalt fur Forst-und Holzwirtschaft, Stiftung Walderhaltung in Afrika, Hamburg. 132 p.
Tuani, G.K.; J.R. Cobbinah and P.K. Agbodaze. (1994). Bioactivity of phytochemical studies on extractives from some Ghanaian plants. Ghana Journ. Forestry,1:44-48.
Uchegbu,R.I. and Okwu, D.(2012). An Evaluation of the Pytochemical and Nutrient Composition of the Seed and Stem Bark of Detarium senegalense Gmelin. Journal of Natural Sciences Research., 2:(5) 107- 111.
Udem, S.C.; Obidoa, O. and Asuzu, I.U. (2009). Acute and chronic toxicity studies of Erythrina Senegalensis DC stem bark extract in mice. Comparative Clinical Pathology,19: 275-282. Van Kampen, E.J. and Zijlstra, W.G. (1978). Recommendations for reference method for haemoglobinometry in human blood (ICSH standard EP 6/2: 1977) and specifications for international haemiglobincyanide reference preparation (ICSH standard EP 6/3: 1977). J Clin. Pathol. , 31(2): 139–143.
77
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Varley, H. (1967). The determination of Na and K by flame photo. In: Pract. Clin. Biochem., 4th ed., Heineman W. Med. Books Ltd, Int. Book Inc., New York.
Venkatesh Babu, K. C. and Krishnakumari, S. (2006). Cardiospermum halicacabum suppresses the production of TNF-alpha and Nitric oxide by Human Peripheral Blood Mononuclear cells. African Journal of Biomedical Research, 9: 95 – 99.
Vietmeyer, N.D. (1986). Lesser-known plants of potential use in agriculture and forestry. Science., 232:1379–1384.
W.H.O. (1996). W.H.O.Guidelines for the Assessment of herbal medicines.World Health Organization. W.H.O. Expert Committee on specification for pharmaceutical preparations. Technical Report series No. 863. Geneva.
Waggas, A.M. and Al-Hasani, R.H. (2010). Neurophysiological study on possible protective and therapeutic effects of Sidr (Zizyphus spina-christi L.) leaf extract in male albino rats treated with pentylenetetrazol. Saudi J. Biol. Sci., 5(3):1-6.
Wall, M. E.; Eddy, C. R.; McClenna, M. L.; and Klump, M. E. (1952). Detection and estimation of steroid and sapogenins in plant tissue. Analytical Chemistry, 24:1337-1342.
Wang, Q.; Ellis, P.R.; Ross-Murphy, S.B. and Burchard, W. (1997) Solution characteristics of the xyloglucan extracted from Detarium senegelense Gmelin. Carbohydrate Polymers., 31: 115-124.
Wang, Q.; Ellis, P.R.; Ross-Murphy, S.M. and Reid, J.S.G. (1996). A new polysaccharide from a traditional Nigerian plant food: Detarium senegalense Gmelin. Carbohydr. Res., 284:(2) 229-239.
78
Gibreil, Hanan M. N.,Ph.D. Thesis,2014
Watson, L. and Dallwitz, M.J. (1993) The Genera of Leguminosae- Caesalpinioideae and Swartzieae. DELTA database.
Weichselbaum, T.E. (1946). An accurate and rapid method for the determination of proteins in small amount of blood serum and plasma. Amer. J. Clin. Pathol., 16:40.
Wink, M. (1999). Introduction: biochemistry, role and biotechnology of secondary products. In Biochemistry of Secondary Product Metabolism. ed Wink (CRC Press, Boca Raton, FL), pp 1–16.
Wojciechowski, M. F.; Lavin, M. and Sanderson, M. J. (2004). A phylogeny of legumes (Leguminosae) based on analysis of the plastid mat K gene resolves many well supported subclades within the family. American Journal of Botany, 91:(11) 1846-1864.
Wojciechowski, M.F.; Mahan, J. and Jones, B. (2006). Fabaceae. legumes. Version 14. The Tree of Life webproject. The Tree of Life Web Project, http://tolweb.org/.
Wufem, B.M.; Adamu, H.A.; Cham, Y.A. and Kela, S.L. (2007). Preliminary studies on the antivenin potential and phytochemical analysis of Balanites aegyptiaca (Lin.) Delile on albino rats. Natural Product Radiance, 6(1): 18-21.
Yossef, H.E.; Khedr, A.A.and Mahran, M.Z. (2011). Hepatoprotective activity and antioxidant effects of El Nabka (Zizyphus spina-christi) fruits on rats hepatotoxicity induced by carbon tetrachloride. Nat. Sci., 9(2):1-7.
Zohary, M. (1972). Flora Palaestina. II. Jerusalem: The Israel Academy of Science and Humanities, pp. 307–308 cited in Amots Dafni, Shay Levy, and Efraim Lev, The ethnobotany of Christ's Thorn Jujube (Ziziphus spina-christi) in Israel.
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APPENDIX
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Appendix I Preliminary Phytochemical Screenings
Preparation of the extract: - 25 g of the plant powder was soaked in 100 ml hot distilled water four about 4 hours. The cooled solution was filtered and stored till used for the screening for the presence of the secondary metabolites compounds.
Test for Unsaturated Sterols and Triterpenses: - 10 ml of the solution was evaporated to dryness on a water path and the cooled residue was washed several times with petroleum ether to remove most of the coloring materials. The residue was then dissolved in 20 ml chloroform. The chloroform solution was Pale purple colour in dehydrated over sodium sulphate anhydrous. 5 ml portion of the the lower phase, chloroform solution was mixed with 0.5ml of acetic anhydride which was a result followed by 2drops of conc. Sulphuric acid. The gradual appearance for the presence of of green, blue pink to purple color was taken as an evidence of the tritepens. presence of sterols (green to blue) and or triterpenses (pink to purple) in the sample.
Test for Alkaloids: - 10 ml of the solution was evaporated to dryness on a water bath 5 ml of 2N Hcl was added and stirred while heating for 10 minutes, cooled filtered and divided into tow test tubes. To one test tube few drops of Mayer’s reagent was added while to the other tube few drops of Valser’s reagent was added. A slight turbidity or heavy precipitate in either Formation of Appearance of of the tow test tubes was tanked as turbidity, which yellow colour, presumptive evidence for the presence of was result for the which was result for alkaloids. presence of the presence of alkaloids. flavonoids. Tests for Flavonoids: - 17.5 ml of the solution was evaporated to dry ness on water bath, cooled and the residue was defatted by several extractions with petroleum ether and the defatted residue was dissolved in 30 ml of 80% ethanol and filtered.
The filtrate was used for following tests: - A/ to 3 ml of the filtrate in a test tube 1ml of 1% aluminum chloride solution was in methanol was added. Formation of a yellow color indicated the presence of Flavonoids. Flavones or and chalcone.
B/ to 3ml of the filtrate in a test tube 1ml of 1% potassium hydroxide solution was added. A dark yellow color indicated the presence of Flavonoids compounds (flavones or flavonenes) chalcone and or flavonols.
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C/ to 2 ml of the filtrate 0.5ml of magnesium turnings were added. Producing of defiant color color to pink or red was taken as presumptive evidence that flavonenes were present in the plant sample.
Tests for Tannins: - For this test 10 ml of the solution was evaporated to dryness on water bath. The residue was washed several times with n-hexane and filtered. The insoluble residue was stirred with 10 ml of hot saline solution. The mixture was cooled, filtered and the volume of the filtrate was adjusted to 10ml with more saline solution. 5ml of this solution was treated with few drops of gelatin salt reagent. Formation of immediately precipitate was taken as evidence for the presence of tannin in the plant sample. To another portion of this solution, few Appearance of blue- drops of ferric chloride test reagent were added. The formation of greenish colour, blue, black or green was taken as an evidence for the presence of which was result for tannins. the presence of tannins.
Test for Saponins: - 1 g of the original dried powder plant material was placed in a clean test tube.10 ml of distilled water was added and the tube was stoppered and vigorously shaken for about 30 seconds. The tube was then allowed to stand and observed for the formation of foam which was taken as evidence for presence of Saponins.
Test for cyanogenic glycoside: - 1 g of the powdered plant sample were placed in erlenmeyer flask and Formation of foam, sufficient water was added to moisten the sample, followed by 1ml of which was result for chloroform (to enhance every activity). A piece of freshly prepared the presence of sodium picrate paper was carefully inserted between a split crock saponins. which was used to stopper the flask, a change in color of the sodium picrate paper from yellow to various shades of red was taken as an indication of the presence of cyanogenic glycoside.
Test for Anthraquinone glycoside: - 1 g of the powdered plant sample were boiled with 10ml of 0.5N KOH containing 1ml of 3% hydrogen peroxide solution. The mixture was extracted by shaking with 10ml of benzene. 5ml of the benzene solution was shaken with 3ml of 10% ammonium hydroxide solution and the two layers were allowed to separate. The presence of anthraquinones was indicated if the alkaline layer was found to have assumed pink or red color.
Test for Coumarins: - Fluoresces under One g of the original powdered plant sample boiled with 20ml UV254, which took distilled water in test tube with filter paper attached to it to be as positive result for saturated with the vapor after a spot of 0.5N KoH put on it. Then the the presence of filter paper was inspected under UV light, the presence of coumrins Coumarins. was indicated if the spot have found to be adsorbed the UV. light. 2