i 

UNIVERSITY OF GHANA 

COLLEGE OF HEALTH SCIENCES 

SCHOOL OF PHARMACY 

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY 

 

ACUTE TOXICITY STUDIES ON APHRODISIACS OF HERBAL ORIGIN IN 

PHARMACIES WITHIN GREATER ACCRA METROPOLIS. 

 

DENNIS AMANKWAH 

(10803979) 

 

A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN 

PARTIAL 

FULFILLMENT OF THE AWARD OF A MASTER OF PHILOSOPHY DEGREE IN 

PHARMACOLOGY 

 

                                                       AUGUST, 2022 

 

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DECLARATION 

DECLARATION 

DECLARATION BY THE CANDIDATE 

I hereby declare that except for cited references duly acknowledged, this thesis is a true 

representation of my own work and has not been presented for a degree elsewhere. 

 

 

Dennis Amankwah                                             15th August, 2022                                          

(10803979)                                     Signature                                            Date 

 

 

 

DECLARATION BY THE SUPERVISORS 

We declare that we supervised the principal work and presentation of the thesis per 

guidelines on supervision of the thesis laid down by the University of Ghana. 

 

 

Prof. Mahama Duwiejua 

Principal Supervisor                                              Date: 15th August, 2022 

 

 

 

Prof. Patrick Amoateng 

Co-Supervisor                                                        Date: 15th August, 2022 

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 ii 

ABSTRACT 

Background: Erectile dysfunction (ED) is the inability of a man to attain and sustain adequate 

erection for satisfactory coitus.  Herbal medicines are widely promoted as remedies for ED.  

The safety profile of these products are largely unknown.  In this study, five products of herbal 

origin, sold as aphrodisiacs in pharmacies within the Greater Accra region, are screened for 

activity across three functional domains: autonomic, neuromuscular/motor, and CNS using the 

modified Irwin test.    

Method:  Groups of male Sprague-Dawley rats (150 - 200 g, n = 5) were used.  Each candidate 

herbal product was administered p.o. at three dose levels to different groups of rats.  

Chlorpromazine (30 mg/Kg, p. o) and caffeine (25 mg/Kg, p. o.) were used as positive controls.  

The vehicle-treated controls were given normal saline (1 ml/kg, p.o.).  Following drug 

administration, animals were monitored for changes in behaviours indicating alterations in 

autonomic, neuromuscular/motor, and CNS activity at 0-15 mins, 30 mins, 1 hr., 2 hr., 4 hr., 8 

hr., 24 hr. and 48 hr.  Hematological, biochemical, and histopathological analyses were 

performed on blood and isolated organs of the experimental animals 14 days’ post drug 

administration. 

Results:  No mortality was recorded after 48 h in all groups. Except for sedation, there were 

no clinical signs of interference with any system such as autonomic, CNS and motor activity 

for all the five herbal aphrodisiac products.  Products 1, 2 and 4 caused significant (p<0.05) 

increases in the weights of the liver, heart, and spleen at 1200, 150 and 400 mg/kg, p.o. 

respectively.  Products 1 and 2 caused elevated levels of only ALP at low doses while higher 

doses produced no change in enzyme levels.  From the biochemical indices, there was therefore 

no definite evidence of toxicity from the Products to the liver. Haematological parameters of 

animals were not adversely affected after 14 days of administration of the Products.  

Histopathological studies did not also provide any equivocal evidence of toxicity to any organ.   

Conclusion: The extracts produced no evidence of significant toxicity to major organs of the 

rats.  Transient behavioural alterations, lasting less than 48 hours, suggesting involvement of 

peripheral and central nervous systems were observed.  Caution should be exercised in the use 

of these Products while further tests are carried out to elucidate the safety profile of the 

commercial herbal aphrodisiacs. 

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DEDICATION 

This work is lovingly dedicated to my wife and children, who have been there for me at all 

times. 

 

 

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ACKNOWLEDGEMENT 

At last, the journey has come to an end after 2 years of intensive training and research in 

pharmacology and toxicology. Reflecting on this achievement, it is without doubt that I have 

come this far by grace and the inspiration of great individuals. I want to thank my uncle, Mr. 

Michael Appiah, for believing in me and investing in my education for many years. I am 

indebted to you eternally. I also want to thank my wife and children for their unrelenting 

support during these 2 years.  

I wish to also express my profound appreciation to my principal supervisor, Prof. Mahama 

Duwiejua, for his immense support provided from the start to the completion of this research 

work. To my co-supervisor, Dr. Patrick Amoateng, I appreciate your co-supervisory role you 

made towards the success of this research work. 

I am also thankful to Dr. Robert Kumoji, Consultant Pathologist at Korle-bu Teaching Hospital 

and Mr.  Daniel Potakey, Biomedical Scientist at Korle-bu Teaching Hospital for your selfless 

contributions made for the success of this research work. 

Finally, I acknowledge the technical inputs of the following people at Noguchi Memorial 

Institute for Medical Research, Department of Animal Experimentation. Dr. Samuel Adjei, Dr. 

Emmanuel Kodua, Mrs. Shirley Nyarko Adu-Poku, Mr. Bernard Addo, Mr. Samuel Anang, 

Mr. Richard Obeng, Mr. Daniel Amoah and Mr. Solomon Botchway of the Department of 

Animal Experimentation. God bless you all. 

 

 

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TABLE OF CONTENTS 

DECLARATION .......................................................................................................................................................... I 

ABSTRACT ................................................................................................................................................................. II 

DEDICATION ........................................................................................................................................................... III 

ACKNOWLEDGEMENT ........................................................................................................................................ IV 

TABLE OF CONTENTS............................................................................................................................................ V 

LIST OF TABLES ................................................................................................................................................... VII 

LIST OF FIGURES .................................................................................................................................................. IX 

APPENDIX................................................................................................................................................................ XII 

CHAPTER 1................................................................................................................................................................... 1 

BACKGROUND ....................................................................................................................................................... 1 
PROBLEM STATEMENT ....................................................................................................................................... 4 
JUSTIFICATION OF STUDY ................................................................................................................................. 5 
RESEARCH HYPOTHESIS .................................................................................................................................... 6 
AIM OF STUDY ....................................................................................................................................................... 6 
SPECIFIC OBJECTIVES ......................................................................................................................................... 6 

CHAPTER 2................................................................................................................................................................... 7 

LITERATURE REVIEW ........................................................................................................................................... 7 

INTRODUCTION ..................................................................................................................................................... 7 
HEPATOTOXICITY ................................................................................................................................................ 8 
RENAL TOXICITY ................................................................................................................................................ 10 
BRAIN TOXICTY .................................................................................................................................................. 10 
HEMATOLOGICAL STUDIES............................................................................................................................. 11 
MODIFIED IRWIN TEST ...................................................................................................................................... 12 

CHAPTER 3 VALIDATION .............................................................................................................................. 24 

INTRODUCTION ................................................................................................................................................... 24 
MATERIALS AND METHOD FOR THE VALIDATION .................................................................................. 25 
EXPERIMENT ........................................................................................................................................................ 26 
RESULTS OBTAINED FROM VALIDATION ................................................................................................... 27 
CONCLUSION ....................................................................................................................................................... 30 

CHAPTER 4................................................................................................................................................................. 31 

SCREENING OF PRODUCT .................................................................................................................................. 31 

ETHICAL STATEMENT ....................................................................................................................................... 31 
HERBAL PRODUCTS SELECTION .................................................................................................................... 31 
ACQUISITION AND ACCLIMATIZATION OF EXPERIMENTAL ANIMALS ............................................. 33 
SCREENING ........................................................................................................................................................... 34 
MEASUREMENT OF BODY WEIGHTS ............................................................................................................. 36 
BLOOD ANALYSIS............................................................................................................................................... 36 
HEMATHOLOGICAL ANALYSIS ...................................................................................................................... 37 
BIOCHEMICAL ANALYSIS ................................................................................................................................ 37 

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MEASUREMENT OF RELATIVE ORGAN WEIGHTS ..................................................................................... 37 
TISSUE PROCESSING AND HISTOLOGY ........................................................................................................ 37 
STATISTICS ........................................................................................................................................................... 38 

CHAPTER 5................................................................................................................................................................. 39 

RESULTS ................................................................................................................................................................ 39 
EFFECT OF PRODUCT 2 ON SPRAGUE-DAWLEY RATS ............................................................................. 42 
EFFECT OF PRODUCT 3 ON SPRAGUE-DAWLEY RATS ............................................................................. 45 
EFFECT OF PRODUCT 4 ON SPRAGUE-DAWLEY RATS ............................................................................. 48 
EFFECT OF PRODUCT 5 ON SPRAGUE-DAWLEY RATS ............................................................................. 51 
EFFECT OF PRODUCTS ON WEIGHT ............................................................................................................... 54 
RELATIVE ORGAN WEIGHT ............................................................................................................................. 55 
BIOCHEMISTRY ................................................................................................................................................... 60 
HEMATOLOGY ..................................................................................................................................................... 63 
HISTOPATHOLOGY ............................................................................................................................................. 71 

CHAPTER 6................................................................................................................................................................. 74 

DISCUSSION .............................................................................................................................................................. 74 

NEUROBEHAVIOURAL EVALUATION OF PRODUCTS .............................................................................. 75 
BIOCHEMISTRY ................................................................................................................................................... 75 
HEMATOLOGY ..................................................................................................................................................... 76 
WEIGHT DIFFERENCE AND RELATIVE ORGAN WEIGHT ......................................................................... 80 
HISTOPATHOLOGY ............................................................................................................................................. 81 

CHAPTER 7................................................................................................................................................................. 84 

CONCLUSION, LIMITATIONS AND RECOMMENDATIONS ...................................................................... 84 

CONCLUSION ....................................................................................................................................................... 84 
LIMITATONS ......................................................................................................................................................... 84 
RECOMMENDATION .......................................................................................................................................... 85 

REFERENCES ........................................................................................................................................................... 86 

APPENDIX.................................................................................................................................................................. 96 

 

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LIST OF TABLES 

Table 1: list of herbal aphrodisiacs and their respective ingredients. ..................................... 3 

Table 2: Domains and associated parameters in which animals were assessed post drug 

administration. ............................................................................................................ 26 
 

Table 3: list of herbal aphrodisiacs from survey .................................................................... 32 
 

Table 4: Comparison of weights of rats before and two weeks after treatment with test substances.  

At the end of week 2, weights of rats treated with Products 4 was not significantly 

different from their original weights. ......................................................................... 54 
 

Table 5: Relative weights of vital organs of Sprague Dawley male rats after 14 days of 

observation of Product 1 post administration. ........................................................... 55 
 

Table 6:  Relative weights of organs from rats 14 days after treatment with single dose of Product 

2. ................................................................................................................................. 56 
 

Table 7:  Relative weights of organs from rats 14 days’ post treatment with a single dose of 

Product 3. .................................................................................................................... 57 
 

Table 8: Relative weights of organs from rats 14 days after treatment with different doses of 

Product 4 administered ............................................................................................... 58 
 

Table 9: Relative weights of vital organs of Sprague Dawley rats 14 days’ post administration of 

3 different doses of Product 5 .................................................................................... 59 
 

Table 10Table : Influence of Product 1 on liver, kidney and cardiac enzymes of SD rats .. 60 
 

Table 11:  Influence of Product 2 on liver, kidney and cardiac enzymes of SD rats............ 61 
 

Table 12:  Influence of Product 3 on liver, kidney and cardiac enzymes of SD rats............ 61 
 

Table 13:  Influence of Product 4 on liver, kidney and cardiac enzymes of SD rats............ 62 

 
 

Table 14: Effect of Product 5 on organs from rats treated with the Product ........................ 63 
 

Table 15: Hematological parameters of rats treated with Product 1 at 12, 120 and 1200 mg/kg, 

p.o. .............................................................................................................................. 64 
 

Table 16:  Hematological parameters of rats treated with Product 2 at 15, 150 and 1500 mg/kg, 

p.o. .............................................................................................................................. 65 

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Table 17: Hematological parameters of rats treated with Product 3 at 16, 160 and 1600 mg/kg, 

p.o. .............................................................................................................................. 67 
 

Table 18: Hematological parameters of rats treated with Product 4 at 4, 140 and 400 mg/kg, p.o.

..................................................................................................................................... 68 
 

Table 19: Hematological parameters of rats treated with Product 5 at 0.3, 3 and 15 ml/kg, p.o.

..................................................................................................................................... 70 

 

 

 

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LIST OF FIGURES 

Figure 1. Image of Mr. Q herbal aphrodisiac ......................................................................... 20 

Figure 2. Image of Ajanta’s Stamina herbal aphrodisiac ....................................................... 21 
 

Figure 3. Image of Tinatett 230 Herbal Capsule .................................................................... 21 
 

Figure 4. Image of Original Tiger Herbal Capsule ................................................................ 22 
 

Figure 5. Image of Odyssey Herbal supplement.................................................................... 23 
 

Figure 6. Effect of Chlorpromazine, administered p.o. at 30 and 60 mg/Kg body weight, on male 

Sprague-Dawley rats.  Behavioral indices were determined according to the modified 

Irwin test. The results are an aggregate of observations over 48 hrs.  Raw data is presented 

in Appendix A. ........................................................................................................... 28 
 

Figure 7. Effects of caffeine (administered at 25 and 50 mg kg-1, p.o.) on male Sprague Dawley 

rats.  These results are aggregates of observations over 48 h.  The raw data indicating 

time-course effects is presented in Appendix C. ....................................................... 29 
 

Figure 8. Effect of Product 1 on neuromuscular and autonomic actions in Sprague-Dawley rats.  

Rats were administered 12 mg/kg, p.o., 120 mg/kg p.o. and 1.2 g/kg, p.o. and observed 

for the first 15 minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24 h and daily for 14 days.  

Results presented are number of rats exhibiting any parameter within the first 24 h.  The 

positive controls were Caffeine (red), Chlorpromazine (Blue) and vehicle (black) 

administered at 25 mg/kg, 30 mg/kg and 1 ml/kg; p.o. respectively. ....................... 39 
 

Figure 9. Effect of Product 1 on CNS activity in Sprague-Dawley rats.  Rats were administered 

12 mg/kg, p.o., 120 mg/kg p.o. and 1.2 g/kg, p.o. and observed for the first 15 minutes 

then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24 h and daily for 14 days.  Results presented are 

number of rats exhibiting any parameter within the first 24 h.  The positive controls were 

Caffeine (red), Chlorpromazine (Blue) and vehicle (black) administered at 25 mg/kg, 30 

mg/kg and 1 ml/kg; p.o. respectively......................................................................... 41 
 

Figure 10: Effect of Product 2 administered at 15, 150 and 1500 mg.kg, p.o., on the central 

nervous system of Sprague Dawley rats. ................................................................... 43 
 

Figure 11. Effect of Product 2 on autonomic and motor systems.  Rats (n = 5) were given p.o.; 

15, 150 and 1500 mg/kg doses of Product 2. ............................................................ 44 
 

Figure 12. Influence of Product 3 on neuromuscular and autonomic actions in Sprague-Dawley 

rats.  Rats were administered 16 mg/kg, p.o., 160 mg/kg p.o. and 1600 mg/kg, p.o. and 

observed for the first 15 minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24 h and daily for 14 

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days.  Results presented are number of rats exhibiting any parameter within the first 24 

h.  The positive controls were Caffeine (red), Chlorpromazine (Black) and vehicle (white) 

administered at 25 mg/kg, 30 mg/kg and 1 ml/kg; p.o. respectively. ....................... 45 
 

Figure 13. Influence of Product 3 on CNS activity in Sprague-Dawley rats.  Rats were 

administered 16 mg/kg, p.o., 160 mg/kg p.o. and 1.6 g/kg, p.o. and observed for the first 

15 minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24h and daily for 14 days.  Results presented 

are number of rats exhibiting any parameter within the first 24 h.  The positive controls 

were Caffeine (red), Chlorpromazine (Black) and vehicle (white) administered at 25 

mg/kg, 30 mg/kg and 1 ml/kg; p.o. respectively. ...................................................... 47 
 

Figure 14. Effect of Product 4 on sedation, excitation, stereotypy and motor systems.  Rats (n = 

5) were given p.o.; 4, 40 and 400 mg/kg doses of Product 4. The positive controls were 

Caffeine (red), Chlorpromazine (Black) and vehicle (white) administered at 25 mg/kg, 

30 mg/kg and 1 ml/kg; p.o. respectively. .................................................................. 49 
 

Figure 15.  Influence of Product 4 on neuromuscular and autonomic actions in Sprague-Dawley 

rats.  Rats were administered 4 mg/kg, p.o., 40 mg/kg p.o. and 400 mg/kg, p.o., and 

observed for the first 15 minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24h and daily for 14 

days.  Results presented are number of rats exhibiting any parameter within the first 24 

h.  The positive controls were Caffeine (red), Chlorpromazine (Black) and vehicle (white) 

administered at 25 mg/kg, 30 mg/kg and 1 ml/kg; p.o. respectively ........................ 50 
 

Figure 16. Influence of Product 5 on CNS activity in Sprague-Dawley rats.  Rats were 

administered 0.3ml/kg, p.o., 3ml/kg p.o. and 15ml/kg, p.o. and observed for the first 15 

minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24h and daily for 14 days.  Results presented 

are number of rats exhibiting any parameter within the first 24 h.  The positive controls 

were Caffeine (red), Chlorpromazine (Blue) and vehicle (yellow) administered at 25 

mg/kg, 30 mg/kg and 1 ml/kg; p.o. respectively. ...................................................... 52 
 

Figure 17 Effect of Product 5 on neuromuscular and autonomic systems.  Rats (n = 5) were given 

p.o.; 0.3, 3 and 15 ml/kg doses of Product 5. ............................................................ 53 
 

Figure 18.  Photomicrograph of sections of SD male rats in 14 days’ acute toxicity study. (a) 

healthy kidney (x10), (b) kidney presented with equivocal hyaline cast of the tubules 

(x10) evidential in low and medium dose of Product 1 administered SD rats and also in 

the medium dose of product 2 administered SD rats. ............................................... 72 
 

Figure 19. Photomicrograph of sections of SD male rats in 14 days’ acute toxicity study. (c) 

healthy liver (x20), (d) liver presented with mild focal steatosis (x20) observed in 

medium doses of Product 1 and Product 2 administered SD rats and (e) liver presented 

with cytoplasmic clamping (x20) as observed in low and medium doses of product 1 

administered SD rats. ................................................................................................. 73 
 

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Figure 20. Photomicrograph of sections of SD male rats in 14 days’ acute toxicity study. (f) 

healthy heart (x10) and (g) heart presented with equivocal focal acute inflammation of 

the heart (x4) as observed in both medium and high doses of Product 2 administered SD 

rats. .............................................................................................................................. 73 

 

 

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APPENDIX 

Appendix 1:  ETHICAL CLEARANCE CERTIFICATE .................................................... 96 

 

Appendix 2: EFFECT OF CHLORPROMAZINE ................................................................ 97 
 

Appendix 3: EFFECT OF CAFFEINE .................................................................................. 99 
 

Appendix 4:  EFFECT OF PRODUCT 1 (12MG/KG,120MG/KG AND 1200MG/KG) . 101 
 

Appendix 5:  EFFECT OF PRODUCT 2 (15MG/KG,150MG/KG AND 1500MG/KG) . 102 
 

Appendix 6:  EFFECT OF PRODUCT 3 (16MG/KG,160MG/KG AND 1600MG/KG) . 105 
 

Appendix 7:  EFFECT OF PRODUCT 4 (4.0MG/KG,40MG/KG AND 400MG/KG) .... 107 
 

Appendix 8: EFFECT OF PRODUCT 5 (0.3ml/kg, 3ml/kg and 15ml/kg) ........................ 108 

 

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 1 

Chapter 1  

BACKGROUND 

Erectile dysfunction (ED) is the inability of a man to attain and sustain adequate erection for 

satisfactory sexual coition (Kalsi and Muneer, 2013). Estimated data from US, Australia and UK 

studies revealed the same complete ED prevalence rate of about 5 % ,10 %, 15 % and 30-40 % 

among 40, 60, 70 and 80-year-olds men respectively (Saadat, 2015).  By 2025, ED is estimated 

to affect about 320 million adult males globally (Ahmed et al., 2011). Clearly, ED is regarded as 

a major health problem for the increasingly healthy ageing population (Chew, 2004). 

Erectile dysfunction is mostly caused by penile vascular disorders involving several 

pathophysiological mechanisms such as impaired arterial inflow, increased cavernosal smooth 

muscle contraction induced by chronic ischemia, impaired smooth-muscle cavernosal relaxation, 

veno-occlusive dysfunction and cavernosal fibrosis and chronic or episodic hypoxemia. Risk 

factors associated with vascular disorders are age, lack of exercise, hypertension, diabetes, 

abnormal lipid profile, smoking and obesity (Saadat, 2015).  Another known cause of ED is the 

loss of functional integrity of the endothelium of arterioles to relax known as endothelial 

dysfunction (Saenz de Tejada et al.,1989). Thus, any condition that damages the endothelial 

function can result in ED in general. Neurological, psychological (depression) and endocrine 

disorders such as hormonal deficiency, hyperprolactinemia, testosterone deficiency and raised 

sex hormone-binding globulin are other factors that can affect ED. 

 A variety of plant, animal or mineral based supplements are known to possess potential sexual 

benefits, confirming older believes and inspiring newer hopes for male vitality and management 

of sexual dysfunctions. These sex enhancing substances are called Aphrodisiacs. Aphrodisiacs 

are either food or drugs sourced from plants, animals or minerals that arouse the sexual instinct, 

desire and performance (Kotta et al., 2013 and Malviya et al., 2011).  

In Africa, cultural values define masculinity based on sexual prowess and fertility placing a high 

premium on sex (Tabil, 2011). Thus, societal insensitivity and stigma associated with ED causes 

serious distress with noticeable effects on the self-esteem and the associations of men 

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experiencing it (Tabil, 2011). Therefore, aphrodisiacs are patronized for both recreational and 

therapeutic purposes. Ayurveda’s classify aphrodisiacs therapeutically as (i) drugs which boost 

or stimulate semen production for example. Asparagus racemosus, Microstylis wallichii, 

Mucuna pruriens, Roscoea procera and Polygonatum verticillatum. (ii) drugs which enhance the 

quality of semen and purifies it namely, Sesamum indicum, Myrica nagi, Anthocephalus 

cadamba, Vetiveria zizanioides, and Saussurea lappa. (iii) drugs which enhance ejaculatory 

functions namely, Cannabis sativa, Strychnos nux vomica Myristica fragrans, and Cassia 

occidentalis. (iv) drugs which prolong ejaculation and performance for example, Cannabis 

sativum, Asparagus racemosus, Mucuna pruriens, Anacyclus pyrethrum, Sida cordifolia, and 

Cinnamomum tamala. (v) drugs arousing desire to have sex, namely, Withania somnifera, 

Opium, Datura stramonium, Hibiscus abelmoschus, Anacyclus pyrethrum and Asparagus 

racemosus (Chauhan et al., 2014).  

Aphrodisiacs can also be classified based on their formulations or constituents, mechanism of 

action and origin. Classification based on formulation are firstly; psychophysiological stimulants 

involving visual, olfactory, tactile and aural stimulations that improve sexual drive, gratification 

and erection through hormonal alteration and increase blood flow in spongy tissues (Yidana et 

al., 2019). Secondly, ‘internal’ preparations that involves love portions, alcoholic drinks and food 

(Malviya et al., 2011).  

Classification based on the mechanism of action is in 3 groups: libido enhancers, effectiveness 

of erection and sexual delight. Generally, mechanism of action of aphrodisiac is through dilating 

blood vessels of corpus cavernoseum leading to the influx of blood into penile tissues hence 

erection (Mahima et al., 2017, Brunetti et al., 2020 and Kotta et al., 2013). 

Classification based on origin are natural or unnatural (synthetic). The natural aphrodisiacs are 

those from natural substances whether plant or animal based. Synthetic aphrodisiacs are 

manufactured to mimic the natural substance. Commonly found synthetic aphrodisiacs include 

Sildenafil (Viagra®), Vardenafil (Levitra®) and Tadalafil (Cialis®). 

Limitations with the use of these synthetic aphrodisiacs include limited efficacy, 

contraindications in certain disease conditions and unwanted side effects notably, dyspepsia, 

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 3 

headache, flushing and nasal congestion (Mathur, 2012 and Kotta et al., 2013 and Hammoud et 

al., 2017).  

Thus, plant-based remedies are and will keep being a common alternative for men with 

unimproved sexual life in spite of the increasing availability of effective synthetic medical 

treatment (Kotta et al., 2013). In Ghana, there are a number of these herbal aphrodisiacs, some 

of which are summarized as in table 1.0 

Table 1: list of herbal aphrodisiacs and their respective ingredients.  

PRODUCT INGREDIENTS 

Mr. Q Rehmanius root, Achyrmthes root, fruit of Puncturevine, 

Cnidium fruit, herb of Korean Epimedium, fruit of 

Magnoliavine, Songaria cynomorium herb, Red ginseng, 

Astragalus root, Rhodiola root, Aucklandia root. 

Tentex Royal Tribulus terrestis 100mg, Blepharis edulis 115mg, Crocus 

sativus 14 mg, Asteracantha longifolia 145mg,  Prunus 

amygdalus 126mg. 

New Kingdom Ginseng 

Power capsule 

Radix ginseng 100%, Ginko biloba, Daminana ,  

Givers P Capsules Penianthus zenkeri, Clausina anista 

Ziipman Herbal Capsules Paulina pinnata 

Vigomax Forte  Withania somnifera 500mg, Mucuna pruriens 200mg, Ricinus 

communis 150mg, Chlorophytum arundinaceum 100mg 

Pa-Kum Capsules Cissus popunea, Cyperus esculentus, Musa paradisiaca, 

Epimedium grandiflorum /Horney goat weed extract 

Extra Strength Adams 

Secret 

Epimedium sagitatum, Coleus froshkohlii, Maca extract, 

Cnidium monnieri, Catuaba, Xanthoparmelia scabrosa, 

Tongkat ali, Horny Goat Weed Extract, Damiana extract-50mg, 

L-Arginine-100mg, Vitamin B6-20mg , Vitamin B2- 20mg, 

Vitamin E- 50 iu , Vitamin B1-20mg , Saw Palmetto Extract -

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 4 

50mg, Rhodiola rosea 150mg, calcium sulphate, magnesium 

stearate.  

Today Man Capsule Penianthus zenkeri, Clausina anista 

Gidi Powa Herbal Capparis erythrocarpus, Khaya ivorensis, Clausina anista, 

Paulina pinnata 

 

Factors such as age, educational status, peer pressure, presence of sexual problems, continual 

advertisement and knowledge of unwanted effects of orthodox medicines are reported to 

influence the use of herbal aphrodisiacs (Manortey et al., 2018).  A study conducted in 

Ashaiman municipality on use of herbal aphrodisiac revealed 52.6 % of 352 men interviewed, 

reportedly used aphrodisiacs at some point in time with majority (68 %) between the ages of 

18 - 25 years. Approximately 50 % of aphrodisiac users had no sexual problems indicating 

recreational use only or just for the fun of it (Manortey et al., 2018).  

A similar study at Agbogbloshie, a suburb in Accra metropolis, by Atuobi-Bediako, 2019, 

revealed that out of 383 men interviewed, 42 % reportedly used aphrodisiacs at certain points 

in their lives; 82 % of the respondents were below 40 years.  Advertisement was a major 

determinant of the use of aphrodisiac.  

. 

 

PROBLEM STATEMENT 

Globally, male infertility is increasing with varying consequences (Mathur, 2012 and Kotta et 

al, 2013).  It is reported that about 10 million people living with erectile dysfunction globally 

do not opt for treatment (Lim, 2017).  Due to societal sensitivity and stigma, many sufferers of 

erectile dysfunction resort to herbal aphrodisiacs (Dike et al., 2020). It is expected that majority 

of people who patronize aphrodisiacs should be the elderly since age affects male sexual 

function and diminishes their libido (Chung, 2019).  To complicate issues, ED sufferers also 

have underlying conditions such as atherosclerosis, hypertension, diabetes mellitus, depression 

or other medications that are constantly been taken (Frimpong-Manso et al., 2016).  Also, many 

investigators have reported a high degree of use among the youth with normal sexual function.  

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 5 

Reasons adduced for usage of aphrodisiacs include recreational purposes, increasing erectile 

rigidity, maintaining an erection, improving sensation, prolong sexual intercourse, impress the 

other partner, satisfy ego, curiosity, increasing sex drive (Makwana et al., 2013 and Atuobi-

Bediako, 2019).  

Consumption of these products by especially the elderly exposes them to various risks like 

increased susceptibility to cerebrovascular accidents and priapism (Atuobi-Bediako, 2019) 

Little is known about the toxicity of herbal aphrodisiacs as there are no comprehensive 

precautionary labels on the products.  Additionally, there is general misconception that 

medicines of herbal origin are safe. It is therefore imperative that additional investigations on 

the potential and actual toxicity of herbal products be done to supplement Food and Drug 

Authority (FDA) pre-registration toxicity requirements.   

The Irwin test, a widely used model for quick and broad-spectrum screening 

compounds/products will be employed in this study to screen aphrodisiacs of herbal origin for 

safety.   

 

JUSTIFICATION OF STUDY 

Cultural values define masculinity based on sexual prowess and fertility placing a high 

premium on sex. Thus, societal insensitivity and stigma associated with ED causes serious 

distress with noticeable effects on the self-pride (Wattanathorn et al., 2019). Sufferers of ED 

and adventurous young men patronize herbal aphrodisiacs for both therapeutic and recreational 

purposes. Despite their therapeutic benefits, some components of the product are potentially 

harmful and pose health risks. Most herbal aphrodisiac are poly-herbals increasing the risk of 

possible interactions with either concomitant diseases or medicines. 

Vulnerable group for erectile dysfunction is mostly the aged (Gareri et al., 2014). The 

continuous use of aphrodisiacs exposes them to a higher risk of developing adverse effects due 

to: compromised organ functions, diminished ability to excrete drugs and multiple 

comorbidities (Nobili et al., 2011). This category of patients may be on other medications 

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which may also increase the risk of drug-drug interactions with any of their poly-herbal 

constituents (Gareri et al., 2014).  Additionally, Vardenafil, a synthetic medication used to treat 

erectile dysfunction has been reported to be a major adulterant in many herbal aphrodisiacs 

(Atuobi-Bediako, 2019).  Use of these products have been associated with heart attacks, kidney 

failures, cardiac arrest and even impotence (Ocloo, 2015).  

As part of the continuing efforts to ensure safety of herbal products, this study investigated the 

acute toxicity profiles of herbal aphrodisiacs. 

 

RESEARCH HYPOTHESIS 

Herbal aphrodisiacs could possess adverse effects. 

 

 

 

AIM OF STUDY 

To carry out acute toxicity screening of five herbal aphrodisiacs in pharmacies within the Accra 

metropolis. 

 

 

SPECIFIC OBJECTIVES 

• To determine the acute toxicity profiles of five herbal aphrodisiacs in Sprague 

Dawley rats using the modified Irwin test. 

• To perform histopathological studies on brain, livers, hearts, spleen and kidneys 

of the experimental animals. 

• To determine effects of the herbal products on hematological and biochemical 

parameters of the experimental animals. 

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Chapter 2  

LITERATURE REVIEW 

INTRODUCTION 

At the commencement of human existence, natural products have been used in various ways 

from generation to generations by man (Shakya, 2016). These natural products were used for 

prophylaxis and therapeutic purposes and came in the form of microorganisms, animals, 

marine organisms and plants (Yuan et al., 2016). 

The knowledge about these products being edible or medicinal developed as man went out 

searching for food and ended up taking poisonous substances that resulted in vomiting, 

stomach upsets, coma and death (Yuan et al., 2016). Such experiences brought distinguishing 

knowledge of what is food and what is not food. These natural products that were recognized 

and separated as medicines were called traditional medicines and their knowledge was then 

transferred from generation to generation in a more refined and acceptable way. Traditional 

medicine has therefore, been a kingpin in treating diseases since ages due to their inherent 

pharmacological effects like anti-infectives, anti-hyperlipidemia and anti-inflammatory etc. 

(Ezekwesili and Nwodo, 2013). World Health Organization (WHO) reports of more than 80 % 

of the population in the world rely on them for their basic healthcare needs currently 

(Vedhanarayanan et al., 2013). The dependency is more notable in developing countries. It has 

gained more acceptability due to its easy accessibility, affordability, personal believes and 

perception of harmlessness that has been linked to it (Amoah et al., 2014).  Despite the above 

advantages, imprecise dosage regimen, impure and sometimes unhygienic nature had been 

linked to preparations and usage over the years (WHO, 2004). 

With the onset of technology, traditional medicines usage and acceptability has become more 

acceptable among the elite with the knowledge that products have undergone an immense 

trans-formational treatment such as extraction, distillation, expression, fractionation, 

purification and concentration to become more effective and safer. Traditional medicines that 

basically use medicinal plants are called herbal drugs or medicine. Popularity of herbal 

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medicines as a natural or alternative way of treatment and prevention of various conditions has 

increased with time and has been widely accepted.  

Herbal medicines use various parts of the plant and their extracts for treatment and prevention 

of illnesses (Patrick-Iwuanyanwu and Nkpaa, 2015).  A typical example is herbal aphrodisiacs. 

Traditionally, these drugs are used for treating erectile dysfunction (ED) among the aged (Dike 

et al., 2020). They have been formulated into capsules, solutions, suspensions and tablets and 

packaged to not only be hygienically presentable but also appeal to all classes of people. Herbal 

aphrodisiacs are widely accepted and patronized by the young despite purposely being made 

for the aged (Manortey et al., 2018). 

The claims of these products been natural and therefore safe compared to the synthetic 

aphrodisiacs have made them popular products in Ghanaian pharmacies. But this claim of 

safety on human health should be continually be monitored since several reports on systemic 

toxicity and potential unwanted effects of several natural products have been made (Lüde, 

2016). There is a possibility for some natural products to possess ‘silent’ harmful effect that is 

not evidential within a short time after preparation (Patrick-Iwuanyanwu and Nkpaa, 2015). 

Natural products have different damaging effect on the different systems of an individual’s 

body especially central nervous system, respiratory system and cardiovascular system. The 

vulnerability of these systems in the body to these products are as a result of high 

vascularization of these organs making them easily accessible to natural products at high 

concentrations. In addition, when products reach some organs (heart, lungs, brain) notably 

known to regulate more sensitive bodily functions in high concentrations, they bring about 

significant changes that affect homeostasis (Gordan, 2015). Past generations have overlooked 

chronic and subtle forms of toxicity like hepatotoxicity, carcinogenicity and mutagenicity in 

relation to some herbal medicine’s toxicity. Some herbal medicines have been associated with 

serious liver toxicity (Patrick-Iwuanyanwu and Nkpaa, 2015).  

 

HEPATOTOXICITY 

The main target for toxic compounds is the liver due to its prior exposure to intestinally 

absorbed foreign substances before they enter into blood circulation (Alelign et al., 2020). The 

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liver detoxifies toxins even though these toxins may harm it (Ravikumar and Gnanadesigan, 

2012). Thus making liver function test for experimental animals very important. This test 

increases understanding of the toxic effects, which can be generalized for safety use in humans. 

Liver cells produce enzymes, these enzymes include alkaline phosphatase (ALP), alanine 

aminotransferase (ALT) and aspartate aminotransferase (AST). Alanine aminotransferase 

(ALT) produced in the hepatocytes is a very specific marker for liver damage compared to 

AST due to it lower concentrations in other tissues. It may go up with certain drugs. Aspartate 

aminotransferase (AST) which occurs in two isoenzymes namely mitochondria produced in 

hepatocytes and response to stress in membranes similar to alanine aminotransferase (ALT) 

and the cytosolic which is non-hepatically produced in the kidney tissue, heart muscle and 

skeletal muscle. ALT and AST high levels are used in conjunction for hepatocellular damage. 

ALP is produced bile cells lining canaliculi and bile ducts. Also produced in the intestine, 

kidney, WBC, placenta and the bone. Therefore, ALP levels may be raised even in the presence 

of healthy liver, with bone disease been the most common cause (Hall and Cash, 2012, Levick, 

2017). 

Test performed to ascertain the levels of these enzymes is called liver functioning test (LFT). 

The liver functioning test seeks for these enzymes (parameters) which are present in the serum 

at low concentrations under normal physiological conditions but becomes elevated in the serum 

when there are lesions (Agbodjento et al., 2020).  

Fairly specific liver enzymes are ALT and AST however, ALT or AST raised levels exceeding 

upper normal limit than ALP, indicates hepatocellular pattern i.e., drug or alcohol-induced 

hepatitis, metabolic liver disease, viral hepatitis and auto-immune hepatitis. In the same way, 

raised ALP levels more than the upper normal limit, than either AST or ALT indicates a 

cholestatic pattern suggesting a biliary pathology i.e., bile cholestatic drug-induced liver injury 

(new causative medication) in acute settings.  These liver enzymes abnormalities can be 

classified as mild if <5 times, moderate if 5-10 times or marked if >10 times the upper normal 

limit respectively (Levick, 2017). Many herbal medicines have been linked to hepatotoxicity 

and is from mild hepatitis to acute liver failure (Fatima and Nayeem, 2016).  

 

 

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RENAL TOXICITY 

Also, the kidney is a vital organ whose function can be analyzed to ascertain the toxic effect 

of a drug taken. i.e., functional analyses of the kidneys are another index of ascertaining toxic 

effects of plant extracts (Zainal et al., 2020). The integrity of the kidney is and not limited to 

excretion of toxins such as creatinine, urea and uric acid from the blood. Urea is a nitrogenous 

based compound formed by the liver and 85 % excreted by the kidneys and 15 % by the 

digestive tract.  Urea levels in serum can be attributed to either decrease renal clearance in the 

case of acute and chronic renal impairment or digestive tract disorders like upper gut bleeding, 

dehydration, high protein diets and catabolic states. Decrease in urea levels can be as a result 

of severe liver damage, severe hunger or low protein diet (Gounden et al., 2020). Hence urea 

levels help in renal function analyses to some extent but not a sure marker as creatinine. 

Creatinine is a more accurate indicator of the kidney glomerulus, produced constantly as a 

byproduct of muscle’s creatinine phosphate and excreted solely by the kidneys. High creatine 

levels show low blood cleansing function of the kidneys and vice versa making it more accurate 

indicator of renal function (Seriana et al., 2021). Damage to the kidney as a result of herbal 

medicines may be attributed to presence of heavy metals, adulterations and careless way of 

preparing them (Fatima and Nayeem, 2016). 

 

BRAIN TOXICTY 

Another vital organ whose functionality can help determine the toxic effect of a drug is the 

brain which is part of the central nervous system (CNS). Drugs that can penetrate the Central 

nervous system could have both pharmacological and detrimental effect on the brain. Toxicity 

to the brain in the addition to the spinal cord is the most dangerous since it controls other 

systems like endocrine, gastrointestinal, cardiovascular and respiratory which are sometimes 

exhibited as urine and stool retention, impotence and loss of activity (Fatima and Nayeem, 

2016). 

 

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HEMATOLOGICAL STUDIES 

To prevent toxic effects on vital organs, a higher predictive value like hematological 

parameters is employed to indicate potential health hazards on humans when results are 

translated from animal studies (Patrick-Iwuanyanwu and Nkpaa, 2015, Arsad et al., 2013 and 

Ghadirkhomi et al., 2016) or help determine the toxic effect in relation to dose and time 

response (Alelign et al., 2020).  

Some blood parameters like mean corpuscular hemoglobin concentration (MCHC) and the 

mean corpuscular volume (MCV) are used for diagnosis of anemia (Amna et al., 2013). MCV 

reflects red blood cell’s size; whilst mean corpuscular hemoglobin (MCH) and MCHC are used 

to define hemoglobin concentration mathematically (Mahmoud, 2013). Animal’s percentage 

of total red blood cells in whole blood are associated with hemoglobin (HB) and Parked cell 

volume (PCV) and are also anemic determinant (Alelign et al., 2020). Low levels of PCV, Hb, 

RBC and platelets suggest direct or indirect or both destruction of RBCs and low hemoglobin 

concentration. Oxidative damage is the indirect destruction of RBC.  Hb, RBC and PCV are 

associated with the total population of red blood cells whilst MCV and MCH are associated to 

individual RBCs. Production of RBCs is called erythropoiesis which is stimulated by a 

circulating hormone called erythropoietin (a glycoprotein). The higher the levels of this 

hormone the higher the production of RBC and vice versa which can lead to normochromic, 

normocytic anemia. The size of the RBC which is MCV also determines the category of anemia 

i.e.  MCVs below normal indicates RBCs smaller than normal and is termed as microcytic 

anemia whilst MCVs elevated than normally indicates RBC larger than normal and is described 

as macrocytic but normal size RBCs are termed normocytic (Patrick-Iwuanyanwu and Nkpaa, 

2015). 

In addition, platelet distribution width (PDW), platelet large cell ratio (P-LCR) and mean 

platelet volume (MPV), and are also needed for toxicity screening (Elsewefy et al., 2014). 

Clotting or bleeding abnormalities are indicative of platelets rise or fall respectively (Alelign  

et al., 2020). All the above information makes studies that evaluate pharmacological safety and 

toxicity of new natural products very important (Aydın et al., 2016). 

 In the development of new plant-based medicines, safety pharmacological studies are very 

important (Tornatore et al., 2019). This study aims at investigating the probable unwanted drug 

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effects on physiological functions, using doses that falls within and above therapeutic ranges 

(Bass et al., 2004).   

Safety Pharmacology predicts the safety of a new compound during the preclinical and clinical 

stages of its development using basic principles of pharmacology in a well regulatory-driven 

process.  

Different methods are employed in safety pharmacology, notable amongst them is the modified 

Irwin test. 

 

MODIFIED IRWIN TEST  

The Irwin test was developed originally to detect psychologically active compounds in mice 

(Irwin, 1968), hence use of the term ‘modified Irwin test’ when applied to rats.   The Irwin 

pharmacological test is used to evaluate both the therapeutic and toxicity effects of a new 

compound or substance. It is an observational test that studies behavior and physiological 

function of a new compound, from the first observable effective dose up to doses that induce 

clear behavioral toxicity. This is carefully carried out by trained observers and can be used in 

providing minimum dose lethal for a test substance. It gives initial estimate of a compound’s 

duration of action on different end point (Castagne´ et al., 2014, Jackson et al.,2019, Gauvin 

et al., 2016). 

The Irwin test is a powerful tool to detect potential issues of safety, including motor 

impairment, sedative problems and convulsive potentials. It is also useful in determining 

qualitative effects of compounds on physiological and behavioral functions thus a broad 

spectrum of activity a product can produce. Irwin test is preferred to other safety 

pharmacological studies like functional observational battery due to fewer animals required 

making it less costly and less time consuming (Mathiasen and Moses, 2018).  It gives us the 

initial signal that we can investigate with further specific tests to confirm these findings. 

Ahmed and Aslam, 2018 have used Irwin test to screen many aphrodisiacs of herbal base giving 

reproducible results. 

 

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Generally, experimental animals are used in modified Irwin test with the reason of close 

correlation to human toxicity.  When test substances are administered by the same route as 

intended, the systems of animals incorporate similar pharmacokinetic (absorption, distribution, 

metabolism) as well as taking into consideration, other physiological events that can influence 

toxicity.  

Potential toxicity is well predicted with cell-based assays but the added advantage with whole 

animal experiment is that it critically measures the toxicity of a test substance that manifest 

with increasing doses of the test substance gradually.  Nevertheless, there are some drawbacks 

with the use of whole animal experiment namely; high costs of the animals used, slight 

variations within species may affect the desired outcome to be observed and difficulty of 

arranging long term experiments if need be (Ifeoma and Oluwakanyinsola, 2013).  

Animal wellbeing is a major issue in safety and toxicity testing.  Pain and suffering of these 

animals in herbal research are of much concern to animal ethical committees. In other to 

minimize pain and suffering when using animal-based toxicity testing, it is important to reduce 

the number of experimental animals because increasing number of animals will correlate to 

inflicting pain on more animals. Also, the tests methods can be improved and where possible, 

replace animal tests with other alternatives that are valid like the human cells (Ifeoma and 

Oluwakanyinsola, 2013). These conditions must be considered before conducting animal 

experiment. 

Test animals used to perform Irwin test were initially mice but currently, rats are also being 

used as test subjects in what is termed as Modified Irwin test. The advantages of using mice 

are namely; more economical, both in terms of cost and the quantity of test substance needed 

for conducting the experiments and is the preferred animal for transgenic studies. Nevertheless, 

rats are considerably an acceptable option for studies of substances with probable effects on 

the CNS. Rats’ behavioral repertoire is considered as richer making it useful in acquiring other 

data in the species. It is also the desired species for chronic treatment, biochemistry, 

pharmacokinetics, toxicology and therefore often preferred for core CNS safety batteries 

(Mathiasen and Moses, 2018 and Yamada, 2015).   

The subject of exact relationship between ages of rats to that of human is still a matter of 

discussion and controversy.  Rats’ development is rapid during childhood, reach sexual and 

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social maturity at about six weeks old and five to six months later respectively. But in 

adulthood, every two and half years of human is approximately equivalent to every month of 

rat’s adult life. This has been confirmed by several experimental studies that has estimated 30 

days of human life to be equal to a day’s life of the animal (Andreollo et al., 2012).  

Based on the aim of the experiment, the test animals are sometimes pre-exposed to certain 

environmental conditions that will acclimatize them for some days prior to the experiment. 

Example is the exposure of the animals to dim light in the laboratory for a stipulated daily 

testing time for 6 days prior to an experiment that will be carried out in dim light (Owiredu et 

al., 2007) 

The route of administration in this test subjects are similar to that of humans as suggested by 

The ICH S7A guidelines in safety pharmacology. The per oral route used frequently in CNS 

core battery studies is not easy to predict in terms of similarity between rats and human 

absorption and metabolism of the new compound. Example is the poor absorption of some 

neuroleptics like haloperidol or sulpiride in rats after oral administration compared to that of 

humans with the same route of administration. More effective routes such as subcutaneous or 

intraperitoneal for safety pharmacological screening is therefore more justifiable (Castagne´ et 

al., 2011). Despite of the above advantages of these routes and the limitations of the oral route, 

the latter was used in this study because of the nature of the products where most were crude 

plant products, making them unsuitable for any parenteral route. 

Unlike traditional LD50 acute toxicity test that requires large numbers of test animals, the use 

of Irwin test in safety pharmacology generally limit the number of test subjects whilst ensuring 

that reliable results are obtained for making good regulatory decisions. 

The substance to be tested is usually evaluated at three or four different doses given 

immediately before the test. For safety pharmacological studies, the lowest dose is the closes 

therapeutically estimated dose from preclinical tests and the highest dose is 100 or 300 times 

this initial dose if possible, physically. The doses of both the test substance and control vehicle 

are given based on the calculated weight of the test animals. Up to 48h observations are usually 

performed post administratively thus continuously from 0 to 15 min post-dose, and separately 

at 15, 30, 1h, 2h, 4h, 24h and 48h post dose. Measurements at 48 post administration are for 

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agents with prolong action duration or for further safety evaluation can be added (Owiredu et 

al., 2007).  

 

Every safety pharmacology study aims at detecting risk. False negatives (type 2 errors) must 

decrease even if false positives (type 1 error) increases. To minimize type 2 errors, Irwin test 

incorporate controls without the test substance for comparisons (Castagne´ et al, 2011). 

Physiological saline or water for injection solutions are mostly the control vehicles used. In 

cases that involve the comparison of the test substance with a particular class of substance, a 

right reference substance can be added in the evaluation. 

Although it is essential to blind in most neurobehavioral testing and specifically during more 

subjective assays like the modified Irwin test, a non-blinded vehicle group is availably made 

to the experimenter, serving as a visual comparator for “normal” animal behavior subjected to 

the same testing conditions and at the same moment in time, in modified Irwin test (Lynch III 

et al., 2011). 

Specific items on modifications of behavior, neurotoxicity and physiological symptoms from 

Irwin test are recorded accordingly in a standardized observation grid (Porsolt, 2007). This  

grid is made up of  the following parameters: loss of grasping, altered activity, increase or 

decrease fear/startle response,  jumping, altered reactivity to touch, straub tail, aggression, fore-

paw treading, head twitches, convulsions, tremor, head movements, chewing, sniffing, 

scratching, catalepsy, akinesia, abnormal gait (rolling and  tip-toe), motor incoordination, loss 

of traction, writhing,  loss of righting reflex, analgesia, loss of corneal reflex, ptosis, 

exophthalmia, altered respiration, loss of balance, piloerection, salivation, defecation/diarrhea, 

pupil diameter, lacrimation ,altered rectal temperature and even death (Lynch III  et al., 2011). 

Some of these parameters can be broadly grouped, namely: excitation, sedation, stereotypy, 

autonomic, motor and others.  

 

Excitation 

Some parameters that fall under excitation are straub tail, increased reactivity, increase 

reactivity to touch, increase fear/startle, aggression, convulsions and tremors. 

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• The presence of straub tail phenomenon is noted when in the course of the 

experiment, the tail of the animal is consistently raised to 45° angle from bedding or 

standing upright or tail arched over the back of the animal in comparison to the 

normal which is flattened or elevated (very slightly) tail when walking.  

• Generally, animals move freely in their cages, the presence of increase reactivity to 

touch is noted in comparison to controls when the animals are slightly more, rapidly 

or very rapidly active with or without pauses. Rats in nature are typically passive 

when touched or handled. Increase reactivity to touch is the flight reactions when 

little finger pressure is exerted on the hind quarters or irritability when handling or 

vocalization and sometimes increased urination/defecation (non-formed or liquid 

stool or increased number of fecal boli presence) and jumping. 

• Fear/startle increase is noted and marked as presence when fingers are snapped above 

cage or a loud, sharp noise is made and immediately the animals respond by 

flinching, freezing, jumping slightly etc. compared with the control.  

• The animals can be aggressive compared with the control and this is observed by the 

biting response of the animals to a poke towards their head using a ball-point pen. 

Very similar to reactivity to touch (Roux et al., 2004 and Mathiasen and Moser, 2018) 

 

Sedation 

 Some parameters that fall under sedation are decreased fear/startle, decreased activity and 

decreased reactivity to touch. 

• Unlike excitation, and under the same condition of removing the cover of the cage 

and manipulation of animals, if the animals move more slowly, very slowly or no 

movement at all compared to the control then it is recorded as decreased activity.  

• If the fingers are snapped above cage or a loud, sharp noise is made and the animal 

does not respond by flinching, freezing, jumping slightly etc. compared with the 

control it will be noted as decreased fear/startle (Roux et al, 2004 and Mathiasen and 

Moser, 2018) 

 

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Stereotypy 

Stereotypic behavior is repeated movement that are abnormal to that of the control and some 

parameters that fall under this are head twitches, scratching, fore paw treading, chewing, 

sniffing and head movements. 

• Head twitches are noted with  flicking or sudden jerking of the head only whilst 

twitching in general is more than head flicks and often leads to convulsive behavior. 

•  Scratching of the animals takes place using fore-or-hind paws on any part of the 

body. It marked as present if it is observed in comparison with the control.  

• Presence of fore-paw treading is noted by the no displacement of the fore-paws 

despite repeated stamping around compared to the control.  

• It is normal for the rats to sniff around cages and in an open space but excessive 

sniffing compared to the control is marked as stereotypically present (Roux et al., 

2004 and Mathiasen and Moser, 2018). 

 

Motor 

Some parameters that fall under motor activity are abnormal gait (tip-toe), akinesia, abnormal 

gait (rolling), loss of grasping, catalepsy, loss of balance, loss of corneal reflex. 

 and righting reflex. 

Corneal reflex (eyeblink) is involuntary blinking of the eyelid in response to a nearby object 

or a ball-point pen drawn closer to the eyes of the rats. Failure to completely close or blink eye 

is termed as loss of corneal reflex  

• loss of righting reflex is the inability to resume optimal position when the animal is 

taking from its normal upright position. 

• Presence of abnormal gait is noted with tip-toe  or rolling movement of the animals in 

their cages compared to the control whilst loss of balance is noted when animals falls 

on its side during movement.  

• Motionless in the form of temporary paralysis is termed as akinesia whilst catalepsy is 

the animal being in the state of trance and as a result it is unable to respond to stimuli 

(Roux et al., 2004 and Mathiasen and Moser, 2018).  

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• When the animal is held gently by the tail and placed on a wire mesh with the held 

tail horizontally pulled slowly and drawn backwards (1 second) over the wire mesh 

and there is no resistance to pull despite being repeated three times, it is scored as loss 

of grasping. Some resistance will be provided by the control group. Loss of traction is 

measured by assessing the inability of the rats to pull themselves up onto a wire mesh 

frame made of stainless-steel placed in a horizontal position approximately 25 cm 

above a top of a table (Redfern et al., 2019).   

 

Autonomy 

Some parameters that fall under autonomy are ptosis, defecation/diarrhea, urination,  

 

piloerection, salivation and lacrimation. 

• Ptosis. Palpebral (eyelid) closure whilst exophthalmos is protrusion of the eyeball or 

eyeballs (bulging eyes).   

• Lacrimation is the visible dampness around eyes.  Lacrimal secretion appearing 

reddish is called chromodacyrrea. 

• The region around the  mouth of the rats are usually dry. If it has wet margins on 

entire submaxillary region or there is visible dampness around mouth then there is 

salivation. 

• Mostly, the furs of the animals do not stand on end, the presence of fur standing on 

end is termed as piloerection. This erection of fur can either be ruffled or puff-ball in 

appearance 

• It is usual for rats to defecate or urinate when handled but increased urination or 

defecation (number of fecal boli) is marked as increase urination and diarrhea 

respectively in comparison to the control (Roux et al., 2004 and Mathiasen and 

Moser, 2018). 

 

 

 

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Other measures 

• Analgesia/ Tail pinch response is inability of the animal to respond to pain (analgesia)  

when the tail is pinched at the base . It is scored as absence of analgesia or no tail 

pinch response if the animal shows some attention to pinch, bite, vocalize (make 

sounds). Unreactive animals (e.g., by vocalization or turning towards forceps) when 

pinched at the tip or base of the tail with forceps is noted as analgesic. 

• Digital thermometer is used to measure rectal temperature by positioning 2 cm above 

the anal sphincter. The temperatures taken could be the same (euthermia), lower 

(hypothermia) or higher (hyperthermia) than the normal.  Presence of hypothermia is 

recorded when baseline mean rectal temperatures are taken prior to the experiment for 

treated and the control groups and rats . Rats having mean temperatures decreased by 

1 to 3 oC during the actual work compared to the baseline temperatures is deemed as 

hypothermia (Roux et al., 2004 and Redfern et al., 2019) 

• Baseline mean rectal temperatures are taken prior to the experiment for treated and 

the control. Rat having mean temperatures increased by 1 to 3 o C during the actual 

work compared to the baseline temperatures is deemed as hyperthermia (Roux et al., 

2004). 

 

The above items in the grid are subjective to the experimenter depending on the type of 

research. Also, most endpoints are qualitative in nature, which requires training on the part of 

the experimenter to be able to distinguish ‘normal’ from ‘abnormal’ behaviors (Jackson et al., 

2019). After period of administration and observation, the animals are sacrificed and their 

organs are harvested and examined for organ damages during toxicological studies (Ossato et 

al., 2016).  

Compared to the automated system, the Irwin test is more labor intensive, performed in light 

phase and involves repetition of observations at short time intervals (Lynch III et al., 2011). 

Thus, experience observers or trained observers are needed in addition to the highly standardize 

procedure to reduce observational bias associated with qualitative studies and enhance it 

sensitivity and reproducibility (Castagne´ et al., 2014). 

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This Modified Irwin test was employed for safety screening of five herbal aphrodisiacs in 

community pharmacies within Greater Accra region. The profile for these five herbal 

aphrodisiacs sampled in pharmacies located in Greater Accra Metropolis were as follows: 

 

1. Mr. Q  

Mr. Q is a Chinese herbal product indicated for enhancing the body’s nature curing ability and 

promoting sexual coition. 

Each 10 ml bottle of Mr. Q contains 

Rehmanius root, Red ginseng, Achyrmthes 

root, Cnidium fruit, herb of Korean 

Epimedium, fruit of Puncturevine, fruit of 

Magnoliavine, Astragalus root, Rhodiola 

root, Songaria cynomorium herb and 

Aucklandia root.  One bottle is taken orally 

each time before sex or two bottles 

(maximum for a day) each time before 

sexual intercourse. 

 

                                                                         Figure 1. Image of Mr. Q herbal aphrodisiac 

 

2. Ajanta’s stamina capsule 

 Ajanta’s stamina capsule is an herbal formulation that ensures relaxation, anti-stress and action 

thereby ensuring a person of healthy and happy sex life. 

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One capsule contains Silajeet, Bang 

bhasma, Ext. of Jaiphal (Mysritca 

fragrans), Ext. of Ashwagandha (Withania 

somnifera), and Ext. of Kavach (Macuna 

pruriens). Two capsules are recommended 

to be taken before sexual intercourse.  It is 

produced by Ajanta Pharma Ltd. 

Figure 2. Image of Ajanta’s Stamina herbal aphrodisiac 

 

 

3. Tinatett 230 Herbal Capsule 

Tinatett 230 Herbal Capsule is indicated for enhancing energy and stamina for men.  

 

One capsule of Tinatett 230 

herbal capsule contains Roxb, 

Ginkgo biloba, Tribulus 

terrestris, Cayenne panex 

caltrop.   Two capsules are taken 

orally 30 minutes before sexual 

intercourse.  It is manufactured 

locally by Tinatett Herbal 

Manufacturing and Marketing 

Company Ltd. 

 

Figure 3. Image of Tinatett 230 Herbal Capsule 

 

 

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4. Original Tiger Herbal  

Original Tiger Herbal Formula distributed and marketed by Menzbek International Limited is 

indicated for premature ejaculation, sexual weakness, erectile dysfunction and improved sexual 

performance. 

 

One capsule contains fruit of Chinese 

Wolfberry, Kudzu root, fruit of 

Psoralea, Wild Jujube, Cinnamon lotus 

seed, Papaya, leaf of Epimedium, 

Gorgon fruit, root of Morinda 

officinalis, Mulberry fruit, Chinese 

yam, Fuling, rhizome of Polygonatum, 

and Walnut kernel. One capsule is 

taken one hour before sex. It is 

manufactured by Luoyang Kanghua 

Biological Preparations Company ltd. 

Figure 4. Image of Original Tiger Herbal Capsule 

 

 

 

 

 

 

 

 

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 23 

 

5. Odyssey herbal supplement  

Odyssey herbal supplement produced in Great Britain by alpha organics, is an herbal 

supplement indicated for male vitality.  

 

One tablet contains, Avena sativa, Gingko 

biloba, Tongkat ali, Chlorophylum, 

Borivilianum Withania somnifera, 

Capidium and Epimedium grandiflorum. 

Two tablets are taken orally two hours 

before engaging in sexual intercourse. 

Best taken on an empty stomach and with 

warm water. 

 

Figure 5. Image of Odyssey Herbal supplement. 

 

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 24 

Chapter 3  

VALIDATION 

INTRODUCTION 

The Irwin test as a systematic, comprehensive, qualitative observational assessment tool was 

introduced to assess the neurobehavioral effects of drugs on mice (Irwin, 1968). The model has 

since been modified for use in rats and is now used in safety pharmacological studies 

recommended by the International Conference on Harmonization (ICH) S7A guidelines for 

evaluating the effects of new chemical entities, to help protect clinical trials participants and 

patients from potential adverse effects ("International Council for Harmonization of Technical 

Requirements for Pharmaceuticals for Human Use (ICH) (2000),", Redfern and Wakefield, 

2006). 

Validation of the model is very essential in determining the probable pharmacological effects 

of drugs in rats.   

Once the model is validated, the certainty of any results obtained in the actual test will not be 

in doubt. Validation is mostly performed using two drugs that give opposing pharmacological 

effects which will aid in observing varying parameters.  Mostly, a drug that gives sedating and 

calming effect such as Diazepam or Chlorpromazine and a drug that gives a central nervous 

system (CNS) stimulating effect such as amphetamines and caffeine (Redfern et al., 2019).  In 

this validation, two established drugs, chlorpromazine a sedative and caffeine a stimulant will 

be used.  The drugs will serve as positive controls on which effects of the test agents can be 

compared. 

Chlorpromazine hydrochloride  is a dimethylamine derivative of phenothiazine.  It binds well 

with plasma proteins, with protein binding capacity of 90 – 99 %. Onset of action is 30 - 60 

minutes and half-life is 30 hours and duration of action is 4 - 6 hours.  It is mostly used as an 

antipsychotic drug and exerts its effect by antagonizing dopamine D2 receptors in the brain 

resulting in the inhibition of the release of hormones in the hypothalamic and hypophyseal. It  

possesses sedative, antiemetic, strong anti-adrenergic, weak peripheral anticholinergic, slight 

antihistaminic and anti-serotoninergic activity ( Mokrushin, 2017). 

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 25 

Generally, caffeine is a CNS stimulant which improves wakefulness, reduce fatigues and span 

attention. Varying consumption of caffeine has different effects on the body. Temporal 

increase in heart rate and blood pressure are induced by moderate consumption whilst adverse 

effects such as severe hypertension, vomiting, nausea, tachycardia, convulsions and even death 

can be induced by excessive consumption of caffeine (Schepici et al., 2020). 

Caffeine is absorbed orally in the gastrointestinal tract and metabolized by the liver cytochrome 

P450 enzymes into three metabolic products namely; theobromine, paraxanthine and 

theophylline. Caffeine and its metabolites cross all biological membranes and are found in all 

body fluids  but do not accumulate in tissues or organs and is excreted through urination 

(Arnaud, 2011 and Che et al., 2012). Caffeine is lipid soluble thus crossing the blood-brain 

barrier (BBB) influencing the CNS and inducing respiratory and skeletal stimulation (Monteiro 

et al., 2016).  

The mechanism of action of caffeine involves accumulation of intracellular cyclic adenosine 

monophosphate (cAMP) by the  inhibition of phosphodiesterase (PDE) resulting in 

vasodilation, increasing nutrient and oxygen availability in the brain and muscle (Sansone et 

al., 2017).  

In this experiment, caffeine and chlorpromazine were the drugs used to validate the model and 

served as the positive controls for all screening exercises with the herbal aphrodisiacs. 

 

 MATERIALS AND METHOD FOR THE VALIDATION 

 Acquisition and acclimatization of male adult Sprague Dawley rats  

Male Sprague Dawley rats (n =25, 100g - 200 g;) were purchased and housed at Noguchi 

Memorial Institute for Medical Research, University of Ghana, Accra. The animals were 

housed in groups of 5 in stainless steel cages (34×47×18 cm) with soft wood shavings as 

bedding, fed with standard feed and tap water ad libitum except during early periods of the test. 

All animals used were experimentally naïve and were handled in accordance with the Guide 

for the Care and Use of Laboratory Animals (Nih  et al., 2011). 

 

 

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 26 

 

 Drugs 

Chlorpromazine hydrochloride was purchased from Ernest Chemist Limited, Ghana, and 

anhydrous caffeine was purchased from G.R. Lane Health Products Limited (Gloucester, UK). 

 

 EXPERIMENT  

The Irwin test was validated by randomly assigning the animals to five groups (n = 5) each.  

The sample size of n = 5 per treatment group was selected to have sufficient power to detect 

the effects reported on the modified Irwin test (Ewart et al., 2013). 

Solutions of chlorpromazine and caffeine were prepared by dissolving appropriate quantities 

in normal saline.  Rats in Group 1 were given saline 1 ml/Kg, p.o. as vehicle control, Groups 

2 and 3 was given chlorpromazine, 30 and 60 mg/Kg, p.o. respectively; Group 4 and 5 received 

caffeine, 25 and 50 mg/Kg, p.o. respectively.  Animals were monitored for effects of the drugs 

at the following times after drug administration: The first 0 -15 mins, then at 15 min, 30 min 1 

h, 2 h, 4 h, 8 h, 24 h and 48 h.  Specifically, animals were observed for changes in behaviours 

in three functional domains: motor/neuromuscular, autonomic and CNS activity (sedation, 

excitation, stereotypy) activities and death.  Table 2.0 represent parameters added in the 

observations.  

 

Table 2: Domains and associated parameters in which animals were assessed post drug 

administration. 

 Domain Parameters 

1 Autonomic • Lacrimation 

• Salivation 

• Diarrhoea/defecation 

• Urination 

• Piloerection 

• Pupil reflex 

2.  Neuromuscular/motor • Catalepsy 

• Akinesia 

• Abnormal gait 

• Loss of traction 

• Loss of grasping 

• Loss of righting reflex 

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 27 

• Loss of corneal 

reflex 

3.1 CNS: Sedation  • Decreased reactivity to touch 

• Decreased activity 

• Decreased fear/startle 

3.2 CNS: Excitation  • Convulsions 

• Tremors 

• Straub tail 

phenomenon. 

• Aggression  

 

• Increased fear/startle 

• Jumping 

• Increased activity 

3.3 CNS: Stereotypy  Fore-paw treading 

Head twitches 

Catalepsy 

sniffing 

Head movements 

Chewing 

Scratching 

 

 

Number of animals that exhibited a particular behavior was recorded and expressed as a 

percentage of total number of rats (n=5) in the cage.  

At the end of the assay, animals were anesthetized with sodium pentobarbitone 100 mg/kg 

intraperitoneally, killed by cervical dislocation and incinerated.  

 

 

 RESULTS OBTAINED FROM VALIDATION 

 

3.3.1.  Animal behavior post chlorpromazine administration. 

Figure 6 represents effects of chlorpromazine on functional domains in male Sprague-Dawley 

rats. The figure represents aggregates of behaviours observed over 48 hours at specified times.  

Appendix B contains the raw data collected at the following times t0: 0-15 mins, t1: 15 mins; 

t3: 30mins; t4: 1h; t5: 2 h; t6: 4 h; t7: 8 h; t8: 24 h; t9:48 h.   

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 28 

Behavior traits markedly affected following chlorpromazine administration were indicative of 

sedation.  Within 30 minutes’ post drug administration, all rats treated with both 30 and 60 

mg/kg, p.o. indicated marked decrease of fear, reactivity to touch as well as decreased 

activity/movement.  

There was also piloerection, an indication of autonomic involvement, after chlorpromazine 

administration at both dose levels.  No other behavioral parameter suggestive of autonomic 

involvement like lacrimation, defecation, salivation and ptosis was observed.   

 

Figure 6. Effect of Chlorpromazine, administered p.o. at 30 and 60 mg/Kg body weight, 

on male Sprague-Dawley rats.  Behavioral indices were determined according to the 

0 5 10 15

convulsions

tremors

Straub tail phenomenon

increased activity

aggression

increased fear

increased reactivity to touch

head movements

head twitches

chewing

sniffing

scratching

catalepsy

akinesia

abnormal gait

loss of traction

loss of righting reflex

loss of corneal reflex

decreased fear

deacreased activity

decreased reactivity to touch

analgesia

ptosis

defecation/diarrhoea

urination

piloerection

salivation

lacrimation

Number of Rats Exhibiting Behaviour

B
e
h

a
v
io

u
r

Saline 1 ml/Kg

Chlorpromazine 30 mg/kg

Chlorpromazine 60 mg/kg

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 29 

modified Irwin test. The results are an aggregate of observations over 48 hrs.  Raw data 

is presented in Appendix 2. 

 

 

Animal behaviour post caffeine administration. 

Figure 7 indicates the effects of caffeine administered at 25 and 50 mg/Kg, p.o. in male 

Sprague-Dawley rats.   

 

Figure 7. Effects of caffeine (administered at 25 and 50 mg kg-1, p.o.) on male Sprague 

Dawley rats.  These results are aggregates of observations over 48 h.  The raw data 

indicating time-course effects is presented in Appendix 3. 

 

0 5 10 15

convulsions

tremors

Straub tail phenomenon

increased activity

aggression

increased fear

increased reactivity to touch

head movements

head twitches

chewing

sniffing

scratching

catalepsy

akinesia

abnormal gait

loss of traction

loss of righting reflex

loss of corneal reflex

decreased fear

deacreased activity

decreased reactivity to touch

analgesia

ptosis

defecation/diarrhoea

urination

piloerection

salivation

lacrimation

Number of Rats Exhibiting Behaviour

B
e
h

a
v
io

u
r

Saline 1 ml/Kg

Caffeine 25 mg/Kg

Caffeine 50 mg/Kg/Kg

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 30 

There is concordance with effects of caffeine administered at 25 and 50 mg kg-1, p.o. in the 

rats.  Behaviours prominently indicating excitation were observed as: increased aggression, 

increased reactivity to touch, increased activity, increased fear/startle on snapping of 

observer’s fingers.  Though all animals exhibited these behaviours, rats treated with the higher 

dose exhibited the behaviours earlier. 

 

CONCLUSION 

In this validation experiment, dose- and time-related responses to chlorpromazine and caffeine 

were evaluated.  The model has been shown to be suitable for further screening of test 

substances for peripheral and central nervous system effects. 

The Irwin test is a validated approach for comprehensively observing and measuring the 

behavioral, neurological and autonomic nervous system responses of animals in pre-clinical 

pharmacological safety evaluation of test compounds.  It is an established fact that single 

behavioral units of measure are not only costly but also time-consuming and offer little 

relevance for predicting estimates or making inferences regarding the relative safety of drugs 

administered, for extrapolation to safety evaluation in humans (Irwin, 1968a, 1968b).  

 

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Chapter 4  

SCREENING OF PRODUCT 

ETHICAL STATEMENT 

The University of Ghana Institutional Animal Care and Use Committee (Protocol ID: UG-

IACUC 009/18-19) approved the experimental protocol and surgical procedures. National 

Institute of Health Guidelines for the care and use of laboratory animal procedures and 

techniques were adhered to in this study. The certification is presented as APPENDIX A. 

 

MATERIALS AND METHODS 

 HERBAL PRODUCTS SELECTION 

Most herbal aphrodisiacs in pharmacies located in the Greater Accra metropolis are taken on 

daily basis as supplements for male vitality.  Products selected for this study resulted from the 

following survey: 

Criteria for the selection of herbal aphrodisiacs for acute toxicity screening 

An online survey was made in December, 2020 for the selection of the five herbal aphrodisiacs 

in Community Pharmacies within Greater Accra Metropolis. 

 

Research Question 

Which herbal aphrodisiacs are of high demand in Community Pharmacies in the Greater Accra 

Metropolis? 

 

Research Participants 

Community Pharmacists in the Greater Accra Metropolis. 

 

Place of study 

Online Pharmacist’s Platforms 

 

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 32 

 

Results 

12 herbal aphrodisiacs were pooled from the platform.  

Below is the table with the listed herbal aphrodisiacs and their respective constituents from 

online survey. 

Table 3: list of herbal aphrodisiacs from survey  

PRODUCT INGREDIENTS 

Mr. Q  Rehmanius root, Achyrmthes root, fruit of Puncturevine, 

Cnidium fruit, herb of Korean Epimedium, fruit of 

Magnoliavine, Songaria cynomorium herb, Red ginseng, 

Astragalus root, Rhodiola root, Aucklandia root. 

New Kingdom Ginseng 

Power capsule 

Radix ginseng 100%, Ginko biloba, Daminana ,  

Original Tiger Herbal 

capsules 

fruit of Chinese Wolfberry, Kudzu root, fruit of Psoralea, Wild 

Jujube, Cinnamon lotus seed, Papaya, leaf of Epimedium, 

Gorgon fruit, root of Morinda officinalis, Mulberry fruit, 

Chinese yam, Fuling, rhizome of Polygonatum, and Walnut 

kernel. 

Givers P Capsules Penianthus zenkeri, Clausina anista 

Odyssey tablets Avena sativa, Gingko biloba, Tongkat ali, Chlorophylum, 

Borivilianum Withania somnifera, Capidium and Epimedium 

grandiflorum 

Ziipman Herbal Capsules Paulina pinnata 

Vigomax Forte  Withania somnifera 500mg, Mucuna pruriens 200mg, Ricinus 

communis 150mg, Chlorophytum arundinaceum 100mg 

Pa-Kum Capsules Cissus popunea, Cyperus esculentus, Musa paradisiaca, 

Epimedium grandiflorum /Horney goat weed extract 

Today Man Capsule Penianthus zenkeri, Clausina anista 

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 33 

Gidi Powa Herbal Capparis erythrocarpus, Khaya ivorensis, Clausina anista, 

Paulina pinnata 

Ajanta Stamina Silajeet, Bang bhasma, Ext. of Jaiphal (Mysritca fragrans), Ext. 

of Ashwagandha (Withania somnifera), and Ext. of Kavach 

(Macuna pruriens) 

Tinatett 230 Herbal 

capsule 

Roxb, Ginkgo biloba, Tribulus terrestris, Cayenne panex 

caltrop 

 

Selection of five herbal aphrodisiac products for screening was based on the following criteria 

1. Is it a registered product with the Food and Drugs Authority? 

2. Is it purely an herbal product or it contains other ingredients e.g., vitamins? 

3. What is the Manufacture’s dosage recommendation, thus is it a daily dose product or 

should be taken when needed? 

4. Have there been any issues in time past with the product e.g., adulteration 

5. Can the product be used as a one-time dose and desired effect obtained? 

Based on the above criteria, these products were sampled out for screening. 

Tinatett 230 herbal capsule 

Ajanta Stamina 

Odyssey tablets 

Mr. Q 

Original Tiger Herbal capsules 

These products were randomly coded as Product 1, 2, 3, 4, and 5 for this project. The identity of 

each product is known to only the supervisor of this project. 

 

 ACQUISITION AND ACCLIMATIZATION OF EXPERIMENTAL ANIMALS  

Male Sprague Dawley rats (n =100, 100g - 200 g;) were purchased and housed at Noguchi 

Memorial Institute for Medical Research, University of Ghana, Accra. The room temperature 

was 28oC + 2, humidity 60%.  Lighting was maintained at 12 h light, 12 h dark. The animals 

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 34 

were housed in groups of 5 in stainless steel cages (34×47×18 cm) with soft wood shavings as 

bedding.  The animals allowed unrestricted access to standard feed and tap water except during 

early periods of the test. All animals used were experimentally naïve and were handled in 

accordance with the Guide for the Care and Use of Laboratory Animals (Nih  et al., 2011). 

 

SCREENING 

The five herbal aphrodisiac products were screened using a combination of criteria under the 

modified Irwin test and the Organization for Economic Cooperation and Development (OECD) 

chemical tests guideline no. 420.   

Each of the five products were screened as follows: 

 

PRODUCT 1 

The recommended dose of two capsules of product 1 was emptied, weighed and recorded as 

0.81g.  Based on 70 kg man, this weight gave a working standard dose of 12 mg/kg. The content 

was transferred into a mortar and with an aid of a pestle it was grounded into a smooth powder.  

A solution of 1.2 mg/ml was prepared with normal saline and administered as low dose to the 

first set of rats (n=5) in relation to their respective weights. The medium and high dosed sets of 

rats (n=5) received 12 mg/ml and 1200 mg/ml respectively.  Test substance (herbal products) 

were administered orally in a single dose with a gavage. Rats in the saline control group were 

administered a single oral dose of normal saline (1ml/kg, p.o.)   

 

PRODUCT 2 

Two capsules of product 2 were emptied, weighed and recorded as 1.04g (equivalent to the 

manufactures’ recommended daily dose for 70 kg man).  This weight gave a working standard 

dose of 15 mg/kg. The content was transferred into a mortar and with an aid of a pestle it was 

grounded into a smooth powder.  A solution of 1.5 mg/ml was prepared with normal saline and 

administered as low dose to the first set of rats (n=5) in relation to their respective weights. The 

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 35 

medium and high dosed sets of rats (n=5) received 15mg/ml and 1500mg/ml respectively. Test 

substance (herbal products) were administered orally in a single dose with a gavage. Rats in the 

saline control group were administered a single oral dose of normal saline (1ml/kg, p.o.)   

PRODUCT 3 

Two caplets of product 4 were emptied, weighed and recorded as 1.1g (equivalent to the 

manufactures’ recommended daily dose of 70kg man). This weight gave a working standard dose 

of 16 mg/kg. The content was transferred into a mortar and with an aid of a pestle it was grounded 

into a smooth powder.  A solution of 1.60mg/ml was prepared with normal saline and 

administered as low dose to the first set of rats (n=5) in relation to their respective weights. The 

medium and high dosed sets of rats (n=5) received 16mg/ml and 1600mg/ml respectively. Test 

substance (herbal products) were administered orally in a single dose with a gavage. Rats in the 

saline control group were administered a single oral dose of normal saline (1ml/kg, p.o.)   

 

PRODUCT 4 

Two capsules of product 4 were emptied, weighed and recorded as 0.28g (equivalent to the 

manufactures’ recommended daily dose of 70kg man). This weight gave a working standard dose 

of 4.0mg/kg.  The content was transferred into a mortar and with the aid of a pestle it was 

grounded into a smooth powder.  A solution of 0.4 mg/ml was prepared with normal saline and 

administered as low dose to the first set of rats (n=5) in relation to their respective weights. The 

medium and high dosed sets of rats (n=5) received 4.0 mg/ml and 400mg/ml respectively. Test 

substance (herbal products) were administered orally in a single dose using a gavage. Rats in the 

saline control group were administered a single oral dose of normal saline (1ml/kg, p.o.)   

 

PRODUCT 5 

 Two ampoules of product 5 was emptied into a measuring cylinder giving a volume of 20ml 

(recommended daily dose of 70kg man). This volume gave a working standard dose of 0.3ml/kg.  

0.3ml/kg was administered as low dose to the first set of rats (n=5) in relation to their respective 

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 36 

weights. The medium and high dosed sets of rats (n=5) received 3ml/kg and 15ml/kg 

respectively. Test substance (herbal products) were administered orally in a single dose using a 

gavage. Rats in the saline control group were administered a single oral dose of normal saline 

(1ml/kg, p.o.) 

 

Prior to dosing of any of the above herbal products, the animals were allowed free access to water 

but feed was withheld overnight.  Following the fasting period, the animals were weighed and 

the test substances administered.  After the test substances were administered, food was furtherly 

withheld for 2 hours.   

Each Animal was observed for effects of the test substances at the following times after test 

substance administration: 0-15 mins, 15 min, 30 min 1 h, 2 h, 4 h, 8 h, 24h and daily for 14 days. 

Specifically, animals were observed for changes in behaviours at three functional domains: 

neuromuscular/motor; autonomic and CNS.  Death in any group was also recorded. The body 

weights of the animals were taken on days 0 and 14.  

The study was qualitative using behavioral changes as end-points.  Number of animals exhibiting 

a particular behavior were recorded and expressed as a percentage of total number (5) in the cage 

in  comparison to that of the control animals. 

 

MEASUREMENT OF BODY WEIGHTS 

The animals were weighed on the first day and  the 14th  day  of the experiment. Difference in 

body weights was determined and compared with the control using a two-tailed paired 

students t-test. 

 
 BLOOD ANALYSIS 

On day 14, the animals were anesthetized with sodium pentobarbitone (100 mg/kg 

intraperitoneally). Blood (10 ml) was collected once through cardiac puncture and placed in 

bottles containing ethylene diamine tetra-acetic acid (EDTA), an anticoagulant and serum 

separating tubes for hematological and biochemical analysis respectively. 

 

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 37 

HEMATHOLOGICAL ANALYSIS 

 Automated hematology analyzer (Mytaic 22, Germany) was used in performing the 

hematological analysis. Analyzed parameters were erythrocyte (RBC), platelet count (PLT), total 

leukocyte (WBC), differential leukocyte, mean corpuscular hemoglobin concentration (MCHC), 

mean corpuscular hemoglobin (MCH), mean platelet volume (MPV), hemoglobin (Hgb), mean 

corpuscular volume (MCV), hematocrit (HCT), platelet distribution width (PDW) and red 

distribution width (RDW). 

 

 BIOCHEMICAL ANALYSIS 

Serum separating tubes containing blood were centrifuged at 3000 rpm at 4 ◦C for 10 min to 

obtain the serum and transferred into Cryo valves. The serum was then stored at -20 ◦C for 

biochemical analysis. Analyzed parameters were aspartate aminotransferase (AST), alanine 

aminotransferase (ALT), alkaline phosphatase (ALP) indicative of liver function, creatinine (Cr) 

and Urea indicative of renal function and creatinine kinase (CK-MB) indicative of brain function 

using URIT 880 Semi-automatic Chemistry Analyzer. 

 

MEASUREMENT OF RELATIVE ORGAN WEIGHTS 

After euthanisation, the brains, livers, hearts, spleen and kidneys, were excised and quickly 

weighed and compared to the body weights to determine the Relative organ weight of each 

animal. This was mathematically expressed as follows: Relative organ weight (%) = (Organ 

weight / final body weight) x 100. All carcasses were disposed of by incineration. 

 

 

TISSUE PROCESSING AND HISTOLOGY 

These organs of interest for each animal were maintained in 10 % (v/v) neutral buffered formalin 

for a week. It was afterwards washed with water. They were trimmed into 3mm thick slices using 

a scalpel, the slices obtained from the organs under investigation were dehydrated using 

increasing concentrations of alcohol (70 %, 80 %, 90 % and 100 %). The tissues were cleared 

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 38 

with xylene to remove the alcohol to enable infiltration with paraffin wax. The tissues were 

embedded in molten paraffin wax, slice sections of 4 µm thickness were made from the paraffin 

embedded tissues using a rotary microtone and scooped with glass slides after being floated on 

water of 46 oC of temperature. The frosted sides of the tissue slides were labelled with codes and 

oven dried at a temperature between 40 o C - 50 o C for 3 hours followed by deparaffinization in 

xylene and subsequent hydration with descending series of alcohol (100 %, 95 % and 80 %). 

These tissues were thereafter stained with hematoxylin and eosin dyes, dried and mounted on a 

light microscope for histopathological examination. Scoring used were 0-none of the above 

changes, 1- equivocal (uncertain), 2-slight and 3-severe. 

 

STATISTICS 

The results were analyzed statistically using GraphPad Prism Version 5.0 for Windows 

(GraphPad Software, San Diego, CA, USA). Data from the study was expressed as mean±SEM 

(n = 5). Comparison of test groups with that of the vehicle-control was made using a 

nonparametric paired sample t-test and a one-way ANOVA followed by Dunnett’s multiple 

comparison test. P < 0.05 was considered statistically significant. 

 

 

 

 

 

 

 

 

 

 

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 39 

Chapter 5  

RESULTS 

  

 

Figure 8 indicates the effects of Product 1 administered at 12 mg/kg, p.o., 120 mg/kg p.o. 

and 1.2 g/kg, p.o. on behaviours under the modified Irwin’s test to monitor for the 

involvement of autonomic and neuromuscular involvement.  There was no indication of 

autonomic or neuromuscular activation by the Product at the dose levels administered 

even though two rats in the group treated with 1200 mg/kg had watery stools within 24 

hours (Figure 8, Appendix 4). 

 

Figure 8. Effect of Product 1 on neuromuscular and autonomic actions in Sprague-

Dawley rats.  Rats were administered 12 mg/kg, p.o., 120 mg/kg p.o. and 1.2 g/kg, p.o. 

and observed for the first 15 minutes then at 30 mins, 1 h, 2 h, 4 h, 8 h, 24 h and daily 

for 14 days.  Results presented are number of rats exhibiting any parameter within the 

Lacrim
atio

n

Salivatio
n

Defaecatio
n

Urin
atio

n

Pilo
erectio

n

Pupil r
efle

x
0

2

4

6
A.  AUTONOMIC

Parameter

Nu
mb

er 
of 

rat
s e

xh
ibi

tin
g P

ara
me

ter

vehicle 1 mL/kg

Caffeine (25 mg/kg)

Chlorpromazine (30 mg/kg)

Product 1 (12 mg/kg) 

Product 1 (120 mg/kg) 

Product 1 (1200 mg/kg) 

Catalepsy

Akinesia

Abnorm
al g

ait

loss of tr