2. ii
DEDICATION
This research work is fore mostly dedicated to Almighty God, the beginning and the end, for
safely seeing me through my course of study. May all glory, honor and adoration be to his holy
name, Amen.
I would also like to dedicate this research work to my parents, Engr. and Mrs. Bimbo Makinde;
my brothers, Mr Olabiyi Makinde and Mr Olabode Makinde and the rest of my family. I am
indeed very grateful for their enormous support throughout the course of my study and even
during this research work. You are indeed the best family anybody can have. Thank you very
much.
3. iii
ACKNOWLEDGEMENT
I would like to use this opportunity to thank my supervisor, Dr. O. O. Babalola. I have not
known you for so long but with this little time that I have known you, you have imparted my life
positively.
Furthermore, I acknowledge my mentor, Dr. Tope Olomola. Thank you very much for your time,
thank you for your listening ears and thanks for all your words of encouragement.
I am also using this medium to appreciate all the Lecturers (Dr. F. K. Agboola, Dr. I. O.
Adewale, Dr. O. O. Babalola, Dr. E. M. Obuotor, Dr. R. E. Okonji, Dr. O. O. Osoniyi, Prof. O.
O. Oyedapo, Dr. (Mrs) B. A. Akinpelu, Dr. (Mrs) Akinwumi, Dr. (Mrs) A. Kuku) in
Biochemistry Department, this write up was put together as a result of the things you have taught
me years back. Thank you for your hard work.
I also want to thank Mr. J. O. Areola (Biochemistry Department) for assisting in the difficult
aspect of the assays done.
4. iv
LETTER OF CERTIFICATION
We hereby certify that the project work of Makinde Olatundun Yetunde with Matriculation
number BCH/2009/084 was personally supervised by us in partial fulfillment of the requirements
for the award of Bachelors’ degree in Biochemistry in the Department of Biochemistry, Obafemi
Awolowo University, Ile- Ife, Osun State, Nigeria.
………………………….. ………………………….
Dr. F. K. Agboola Dr. O. O. Babalola
H.O.D SUPERVISOR
BIOCHEMISTRY DEPARTMENT BIOCHEMISTRY DEPARTMENT
…………………………. ………………………….
Date Date
5. v
TABLE OF CONTENT
Dedication…….,………………………….…….………………………………………………...ii
Acknowledgement…………..……………………….…….……………………………………..iii
Letter of certification…………………………………………………………………………….iv
Abstract……………………………………………………………………………………………x
Chapter 1: Introduction………………..…...…..………..……………………………………...1
Chapter 2: Literature Review……………………..……...……………………………………..4
2.1 Review of Hedranthera barteri ……………………..……………………………..4
2.1.1 Botanical Identification………………………………..……………………...4
2.1.2 The family Apocynaceae……………………………………………………..5
2.1.3 Local uses, Ethno medical Importance…………………..…………………...7
2.1.4 Anti- depressant and Anxiolytic activity……...…………..………………….7
2.1.5 Antihistaminic activity…….……..…………………………………………..8
2.2 Phytochemical screening………….………………………………………………10
2.3 Toxicity Profile…………………….……………………………………………...10
2.4 Membrane Stabilizing Activity…………………………………………………...12
Chapter 3: Methods……………………………….……………………………………………14
3.1 Materials…………………………………………………………….…………….14
3.2 Reagent and Chemicals……………………………………………………….…...14
3.3 Apparatus…………………………………………………………………….……14
3.4 Procedures…………………………………………………………………….…...15
3.4.1 Plant Collection………………………….………………………………….15
3.4.2 Air drying and grinding………………………….………………………….15
3.4.3 Soaking and Solvent Extraction…………………….……………………....15
6. vi
3.4.4 Concentration and Fix drying…………………………….…………………16
3.4.5 Acute toxicity Testing…………………………………….………………...16
3.4.6 Acclamatization of Animals……………………………….………………..17
3.4.7 Administration of Extract………………………………….………………..17
3.4.8 Sacrifice and Harvest……………………………………….……………….18
3.5 Sub chronic Toxicity Testing……………………………………………………...19
3.5.1 Aspartate Amino Transferase (AST) Assay………………………………...20
3.5.2 Alanine Amino Transferase (ALT) Assay……….……………………...…..23
3.5.3 Gamma Glutamyl Transferase (GGT) Assay……………………………….26
3.5.4 Alkaline Phosphatase (ALP) Assay…………………………………………28
3.5.5 Bilirubin Assay……………………………………………………………...29
3.5.6 Total Protein Assay…………………………………………………………32
3.5.7 Albumin Assay………………………………………………………………34
3.5.8 Urea Assay…………………………………………………………………..36
3.5.9 Uric Acid Assay……………………………………………………………..39
3.5.10 Creatinine Assay………………………………………………………...…41
3.6 Membrane Stabilizing Activity……………………………………………………43
3.6.1 Principle……………………………………………………………………..43
3.6.2 Reagent and Chemicals……………………………………………………...43
3.6.3 Procedures…………………………………………………………………...44
3.6.4 Calculation…………………………………………..………………………47
3.6.5 Precautions, Limitations and Assumptions……………..……….………….47
3.7 Phytochemical Screening………………………………………………………...…48
3.7.1 Principle…………………..…………………………………………………48
7. vii
3.7.2 Reagent and Chemicals………………………………….………………….48
3.7.3 Procedure……………………………………………….…………………...48
3.7.4 Statement Of Observation………………………………..…………………50
Chapter 4: Results…………………………………….………………………………………..51
4.1 Graphs…………………………………………………………………………..…..51
4.1.1 AST Activity…...……………………………………………………….…....51
4.1.2 ALT Activity…………………………………………………………………54
4.1.3 GGT Activity……………………………….……………..…………….…...57
4.1.4 ALP Activity……………………………………………...…………….…....60
4.1.5 Total Protein Activity……………………………………...………….……..63
4.1.6 Albumin Activity……………………………………………...…….……….66
4.1.7 Globulin Activity………………………………………………...….…….....69
4.1.8 Total Bilirubin Activity………………………………………………………72
4.1.9 Direct Bilirubin Activity……………………………………….…...………..75
4.1.10 Urea Activity……………………………………………….………...……..78
4.1.11 Creatinine Activity…………………………………….……………...….....81
4.1.12 Uric Acid Activity……………………………….……………………...…..84
4.2 Phytochemical Screening...…..……………………………………………………..87
4.3 Membrane Stabilizing Activity..……………………………………………………88
4.3.1 Membrane Stabilizing Activity of Ibuprofen (Standard)……………………88
4.3.2 Membrane Stabilizing Activity of the herbal mixture………..…………….88
Chapter 5: Discussion/ Interpretation of results………………….…………………..…..…..90
Conclusion……………………………………………………………………………………….97
Recommendation………………………………………………………………………………...98
8. viii
References……………………………………………………………………………………….99
Appendix………………………………………………………………………………………..104
Table 1.1 AST Activity………………………………………………………….……….…....104
Table 1.2 ALT Activity………………………………………………………………….…….104
Table 1.3 GGT Activity………………………………………………………….....................105
Table 1.4 ALP Activity………………………………………………......................................105
Table 1.5 Total Protein Concentration..……………………………………………………....106
Table 1.6 Albumin Concentration..…………………………………………………………...106
Table 1.7 Globulin Concentration…..……………………………….......................................107
Table 1.8 Total Bilirubin Concentration………..…………………………………………….107
Table 1.9 Direct Bilirubin Concentration……..……………………………………………....108
Table 1.10 Urea Concentration……………..…………………………………………………108
Table 1.11 Creatinine Concentration..…………………………………...................................109
Table 1.12 Uric Acid Concentration...………………………………………………………...109
Table 2.1 Row statistics of AST Concentration………………………………………………110
Table 2.2 Row statistics of ALT Concentration………………………………………………110
Table 2.3 Row statistics of ALP Concentration………………………………………………110
Table 2.4 Row statistics of GGT Concentration………………………………………………110
Table 2.5 Row statistics of Total protein Concentration……………………………………...110
Table 2.6 Row statistics of Albumin Concentration…………………………………………..110
Table 2.7 Row statistics of Globulin Concentration…………………………………………..111
Table 2.8 Row statistics of Total bilirubin Concentration…………………………………….111
Table 2.9 Row statistics of Direct bilirubin Concentration…………………………………...111
Table 2.10 Row statistics of Uric acid Concentration………………..……………………….111
9. ix
Table 2.11 Row statistics of Creatinine Concentration…………………..…………………...111
Table 2.12 Row statistics of Urea Concentration………………………..……………………111
Table 3: Showing P values of one way anova of in vivo toxicological studies……………….112
Table 4: Showing Standard deviation in each group for all assays done………………….…..113
10. x
ABSTRACT
Statistics has shown that 80% of the worlds’ population relies on traditional concoctions and
local herbs for the treatment of one ailment or another. However, science has found out that
majority of these herbs (leaves, barks and roots) most times contain harmful phytochemicals.
This research work was carried out to investigate the harmful effects of the ethanolic extracts of
Hedranthera barteri in conjunction with seven other plant constituent of a popular herbal
mixture on the blood, liver and Kidney.
The sub-chronic toxicity testing includes in vivo assays of AST (Aspartate aminotransferase),
ALT (Alanine aminotransferase), GGT (Gamma Glutamyl Transferase), ALP (Alkaline
Phosphatase) activities, Determination of Total Protein, Albumin, Globulin, Direct and Total
Bilirubin, Uric Acid, Urea and Creatinine Concentrations using standard procedures. The acute
toxicity of the Ethanolic joint plant extract was determined using the method of Lorke, 1983.
Membrane Stability tests were carried out in addition to a complete phytochemical screening of
both Hedranthera barteri and the extract of the Herbal mixture under study.
The result of the research shows that the herbal mixture can stabilize cell membranes up to 66.34
± 4.31 (%). The phytochemical screening of the ethanolic extract of Hedranthera barteri shows
the presence of phytochemicals such as: alkaloids, flavonoids, tannins and saponins. Moreover,
the phytochemical screening of the ethanolic joint plant extracts revealed the presence of
phytochemicals such as: alkaloids, tannins, saponins, flavonoids and steroids. The trado medical
product was found not to be acutely toxic or sub- chronically toxic upon prolonged
administration and increased concentration.
From the findings of this investigation, we can deduce that the herbal mixture can function as a
fairly good nerve tonic and the popular herbal mixture is not toxic.
