ADDIS ABABA UNIVERSITY

COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES

DEPARTMENT OF MICROBIAL, CELLULAR AND MOLECULAR BIOLOGY

Phytochemical Screening, Acute Toxicity and Anti-Rabies Activities of Extracts of Selected

Ethiopian Traditional Medicinal Plants

A Thesis Submitted to the School of Graduate Studies of Addis Ababa University, College of
Natural and Computational Sciences, Department of Microbial, Cellular and Molecular Biology
in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology
(Applied Microbiology)

By: Yeweynshet Tesera (GSR/9486/10)

Advisor: Asnake Desalegn (PhD)

Co-advisors: Abebe Mengesha

Ashenif Tadele

September, 2019

Addis Ababa, Ethiopia

Declaration
I hereby declare that this MSc thesis entitled “Phytochemical Screening, Acute Toxicity and
Anti-Rabies Activities of Extracts of Selected Ethiopian Traditional Medicinal Plants” is my
original work and has not been presented for a degree in any other University and all source of
materials used for the thesis have been duly acknowledged.

Yeweynshet Tesera Tewabe _____________________ ___________________

Name of the Designate Signature Date

ii
Acknowledgments
First and foremost, I would like to thank the Almighty God for giving me the opportunity, ability
and strength to undertake and complete this research study.

I would like to express my special appreciation and thanks to my advisor Dr. Asnake Desalegn
for his valuable comments that greatly improved this study. His support, encouragement and
credible ideas have been great contributors in the completion of the thesis. His careful editing
contributed enormously to the production of this thesis.

I owe my sincere thanks to my Co-advisors Mr. Ashenif Tadele and Mr. Abebe Mengesha who
made specific contributions to this work and helped out whenever needed.

My deepest gratitude is also expressed to Mr. Eyob Debebe at the Department of Traditional and
Modern Medicine, EPHI, for his technical help and support.

I would not forget to remember Mr. Anberbir Alemu and Mr. Birhanu Hurisa for their timely
support and guidance till the completion of the thesis.

I extend my thanks to Ethiopian Public Health Institute (EPHI) for providing necessary facilities
during this research work.

I would like to thank Addis Ababa University, Department of Microbial, Cellular and Molecular
Biology for financial support.

I would also like to thank Mr. Melaku Wondafrash, at the Department of Plant Biology and
Biodiversity management, Addis Ababa University for plant species identification.

Finally, I must express my gratitude to my parents for providing me with unfailing support and
continuous encouragement throughout the process of researching and writing this thesis.

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Contents
Declaration..................................................................................................................................... ii

Acknowledgments ........................................................................................................................ iii

List of Tables ............................................................................................................................... vii

List of Figures ............................................................................................................................. viii

List of Appendices ........................................................................................................................ ix

List of acronyms/abbreviations ................................................................................................... x

Abstract ........................................................................................................................................ xii

1. Introduction ........................................................................................................................... 1

1.1. Statement of the problem .............................................................................................. 3

1.2. Objectives ........................................................................................................................ 4

1.2.1. General objective .................................................................................................... 4

1.2.2. Specific objectives ................................................................................................... 4

1.3. Significance of the study ................................................................................................ 4

2. Literature Review .................................................................................................................. 5

2.1. Rabies Etiology and Epidemiology ............................................................................... 5

2.2. Global Distribution and Burden of Rabies .................................................................. 7

2.3. Rabies Transmission ...................................................................................................... 8

2.4. Pathogenesis and Incubation ......................................................................................... 9

2.5. Clinical Signs ................................................................................................................ 10

2.6. Diagnosis of Rabies ...................................................................................................... 10

2.7. Prevention and Control of Rabies............................................................................... 12

2.7.1. Prevention of Human Rabies ............................................................................... 13

2.7.2. Animal Rabies Control ......................................................................................... 14

2.8. Rabies Treatment ......................................................................................................... 14

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 2.9. Costs and Cost-Effectiveness of Control Measures ................................................... 15

2.10. Risks of Rabies vaccine ................................................................................................ 16

2.11. Traditional Medicinal Plants ...................................................................................... 17

2.11.1. Phytochemicals and Pharmacological Properties .......................................... 18

2.12. The Use of Traditional Medicinal Plants in Ethiopia ............................................... 19

2.13. Problems associated with traditional medicine ......................................................... 21

2.14. Description of selected medicinal plants used for treatment of rabies .................... 21

3. Materials and Methods ....................................................................................................... 25

3.1. Plant material collection and identification ............................................................... 25

3.2. Processing of the plants................................................................................................ 25

3.3. Extract preparation...................................................................................................... 26

3.4. Preliminary phytochemical screening ........................................................................ 27

3.4.1. Test for Alkaloids (Wagner’s test)....................................................................... 27

3.4.2. Test for Flavonoids ............................................................................................... 27

3.4.3. Test for Phenol (Ferric chloride test) .................................................................. 27

3.4.4. Test for Steroids (Libermann Burchard Test) ................................................... 27

3.4.5. Test for Saponins................................................................................................... 27

3.4.6. Test for Tannins (Ferric chloride test) ................................................................ 27

3.4.7. Test for Terpenoids ............................................................................................... 28

3.5. Cell culture and virus ................................................................................................... 28

3.6. In vivo acute toxicity and anti-rabies screening of crude extracts ........................... 29

3.6.1. Experimental animals and their management ................................................... 29

3.6.2. In vivo acute toxicity test ...................................................................................... 29

3.6.3. In vivo anti-rabies screening of crude extracts ................................................... 30

3.6.4. Determination of mean survival time.................................................................. 31

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 3.6.5. Fluorescent antibody test ..................................................................................... 31

3.7. Screening for in vitro cytotoxicity and anti-rabies activities of crude extracts....... 32

3.7.1. Cytotoxicity assay.................................................................................................. 32

3.7.2. In vitro anti-rabies assay ....................................................................................... 33

3.8. Ethical Clearance ......................................................................................................... 33

3.9. Data Analysis ................................................................................................................ 33

4. Results and Discussion ........................................................................................................ 34

4.1. Phytochemical study result of extracts ....................................................................... 34

4.2. The result of acute oral toxicity test ........................................................................... 36

4.3. Cytotoxicity determination of plant extracts in vero cell line .................................. 39

4.4. Determination of 50% end titer .................................................................................. 42

4.5. Determination of in vivo antirabies activity ............................................................... 42

4.6. The in vitro antirabies activity of plant extracts ........................................................ 44

5. Conclusions and Recommendations................................................................................... 47

5.1. Conclusions ................................................................................................................... 47

5.2. Recommendations ........................................................................................................ 47

6. References............................................................................................................................. 48

7. Appendices ........................................................................................................................... 59

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List of Tables
Table 1: The botanical identification of the plant specimens used in this study .......................... 25
Table 2: Different phytochemical components in the ethanol, 80% methanol and water extracts
of the leaves of Justicia schimperiana and Ricinus communis and the stem bark of Croton
macrostachyus plants .................................................................................................................... 35
Table 3: Mice treated with Justicia schimperiana extracts, and responses in acute toxicity testing
....................................................................................................................................................... 37
Table 4: Mice treated with Ricinus communis extracts, and responses in acute toxicity testing .. 38
Table 5: Mice treated with Croton macrostachyus extracts, and responses in acute toxicity testing
....................................................................................................................................................... 38
Table 6: Effect of the ethanol extracts of three plants on vero cells ............................................. 40
Table 7: Effect of the 80% methanol extracts of three plants on vero cells ................................. 40
Table 8: Effect of the water extracts of three plants on vero cells ................................................ 41
Table 9: Antirabies effect of plant extracts on mice survival time (n=6) at 3000mg/kg .............. 44

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List of Figures
Figure 1: Rabies-virus-shape-and-its-structures ............................................................................. 6
Figure 2: Photo of Justicia schimperiana plant ............................................................................ 22
Figure 3: Photo of Croton macrostachyus plant ........................................................................... 23
Figure 4: Photo of Ricinus communis plant .................................................................................. 24
Figure 5: A brief summary of extraction procedure ..................................................................... 26
Figure 6: LD50 values of 80% methanol extracts from the three selected plants……………….39

Figure 7: Microscopic photographs of vero cell lines treated with 80% methanolic extracts ...... 42
Figure 8: Negri bodies in mice brain tissue infected with rabies virus ......................................... 46

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List of Appendices
Appendix 1: Drying the plant samples…………………………………………………………..59

Appendix 2: Weighting and shaking samples at 130 rpm……………………………………….59

Appendix 3: Filtration and condensation process…………………….…………………………59

Appendix 4: Phytochemical screening……………………………………………….……….....60

Appendix 5: Animal (mice) management throughout experimentation period……………….…60

Appendix 6: Postmortem examination of mice brain……………………………………………60

Appendix 7: Titration of Rabies virus in vivo by intracerebral inoculation……………………..61

Appendix 8: Calculation of median lethal dose (LD50) of plant extracts in Swiss Albino
mice………………………………………………………………………………………………62

Appendix 9: Ethical clearance certificate………………………………………………………..64

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List of acronyms/abbreviations
AJADD American Journal of Advanced Drug Delivery

ARV Anti-Rabies Vaccine

ATRM African Traditional Medicines

CC50 Cytotoxicity Concentration 50%

CCEEVs Cell Culture and Embryonated Egg-Based RabiesVaccines

CDC Centers for Disease Control and Prevention

CNS Central Nervous System

DALYs Disability-Adjusted Life Years

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl Sulfoxide

DRIT Direct Rapid Immunohistochemical Test

ELISA Enzyme Linked Immune Sorbent Assay

EPHI Ethiopian Public Health Institute
FAT Fluorescent Antibody Test
FBS Fetal Bovine Serum

FCS Fetal Calf Serum

FITC Fluorescein Isothiocyanate
IACUC Institutional Animal Care and Use Committee
IC50 Inhibitory Concentration 50%

I/C Intra cranial

ITM Improved Traditional Medicine
LD50 Medium Lethal Dose
MEM Minimum Essential Medium
MOI Multiplicity of Infection

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MST Mean Survival Time
MTT 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide

NTV Nerve Tissue Vaccine

OECD Organization of Economic Corporation and Development
OIE World Organization for Animal Health
PBS Phosphate Buffer Saline
PEP Post Exposure Prophylaxis
PPE Personal Protective Equipment
PrEP Pre Exposure Prophylaxis

PV Pasteur Virus
RABV or RV Rabies Virus
RIG Rabies Immunoglobulin

RNP Ribo Nucleo Protein

Rpm Revolution Per Minute
RTCT Rapid Tissue Culture Infection Test

TCID50 Median Tissue Culture Infectious Dose

THP Traditional Health Practitioners
TMPs Traditional Medicinal Plants
UV Ultraviolet

VNA Virus-Neutralizing Antibodies

WHO World Health Organization

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Abstract
Despite the existence of safe and effective vaccines, rabies disease still causes an estimated
59,000 human deaths a year in the endemic areas in Asia and Africa. In most developing
countries people believe to cure rabies with different traditional and religious treatment rather
than seeking effective post exposure prophylaxis. The objective of this study was, therefore, to
investigate the phytochemical constituents, acute toxicity and antirabies activity of crude extracts
of the leaves of Justicia schimperiana and Ricinus communis and the stem bark of Croton
macrostachyus. Extraction was done by maceration technique. Standard procedures were used to
test the presence of various phytochemicals. For the determination of acute toxicity and
antirabies activities, Organization for Economic Corporation and Development (OECD)
Guideline No.423 was used. Different concentrations of extracts (0.4, 0.8, 1.6, 3.2, 6.4 and 12.8
mg/ml) were tested for their cytotoxic effect on vero cells through 3-(4, 5-Dimethylthiazol-2-yl)-
2, 5-Diphenyltetrazolium Bromide (MTT) assay. The mean cytotoxic concentration (CC50) was
estimated and the antirabies assay was carried out using the minimal cytotoxic concentration of
extracts. The phytochemical screening result has revealed the presence of alkaloids, flavonoids,
phenols, steroids, tannins and terpenoids in all plant extracts screened. The toxicity evaluation of
the extracts revealed that they are slightly toxic. The antirabies assay result showed that all plant
extracts had a moderate to good antirabies potential. The 80 % methanol extracts exhibited
higher antirabies activity compared to the other extracts under investigation. The present study
concluded that these medicinal plants have possessed different phytochemicals that helps in the
anti-rabies properties of the studied plants commonly used in Ethiopia. Further study on the
mechanism of those phytochemicals must be elucidated for the potential as antirabies agent.
Evaluation of those medicinal plants for their long-term toxicity would also be very important.

Keywords/phrases: Anti-rabies, Cytotoxicity, Pathogenicity, Phytochemicals, Traditional

medicinal plants

xii
 1. Introduction
Rabies is a fatal viral zoonosis, which causes encephalitis in warm-blooded animals and humans
(Zulu et al., 2009). It is caused by rabies virus, a neurotrophic virus which kills or causes
physiological disorders by infecting the neurons at the central nervous system (Vural et al.,
2016). All mammals are susceptible to rabies, but only a limited number of species also act as
reservoir hosts. They include members of the families Canidae (dogs, jackals, coyotes, wolves,
foxes and raccoons), Mustelidae (e.g., skunks), Viverridae (mongooses), and the order
Chiroptera (bats) (Balcha Chernet and Nejash Abdela, 2016). Having a large population of stray
dogs in a community is considered to be a risk factor for spreading of zoonotic diseases
such as rabies (Abraham et al., 2017). In developing countries, the majority of confirmed and
reported cases and over 90% of human exposures are from domestic dogs (Kaare et al., 2009). It
is prevalent throughout the world and endemic in many countries except in Islands like Australia
and Antarctica. It is most common in the world where stray dogs are presented in large numbers,
particularly in under developing countries (Neevel et al., 2018). Individuals from rural, remote
areas or developing countries are at high risk of dog bite which may be due to greater
unvaccinated stray dog (Chaudhary et al., 2018). In India, rabies affects mainly people of lower
socio-economic status and children between the ages of 5 and 15 years. Indian children often
play near stray dogs, which are many and roam freely, and are used to sharing their food with
them, which results in frequent bites (Kole et al., 2014). In Africa, the highest recorded human
death due to the disease for the year 1998 was reported from Ethiopia. The magnitude of the
problem is higher in big cities like Addis Ababa linked with the presence of large number of
uncontrolled dogs and the absence of regular vaccination of dogs (Tamiru Dabuma et al., 2017).
According to Richard Pankhurst, the first and only recorded of rabies case in Addis Ababa
occurred in August 1903.The main cause for an increase in the number of dog bites is its high
breeding rates. Sterilization is one of the methods for controlling the population of dogs
(Abraham et al., 2017).

Until 1885, when Louis Pasteur and Emile Roux developed a vaccine, all human cases of rabies
were fatal as the case fatality rate almost 100% (Abebe Mengesha et al., 2016). Several modern
cell culture and embryonated egg-based rabies vaccines (CCEEVs) containing inactivated rabies
virus are available. The first licensed human rabies vaccine developed from cell culture was the
primary hamster kidney cell vaccine, which was created by cultivating viruses in primary
1
hamster kidney cells (Kissling, 1958). The disease is 100% preventable by either pre-exposure
prophylaxis (PrEP) or post-exposure prophylaxis (PEP) which together effectively prevent
approximately 372,000 deaths yearly. In resource-poor settings, however, these prophylaxes are
frequently not accessible, incomplete or delayed and consequently, almost 96% of all human
rabies cases occur in Africa and Asia despite the fact that rabies virus circulates worldwide
(Neevel et al., 2018). Specifically, prompt administration of vaccines in conjunction with rabies-
immunoglobulins and proper wound management after exposure prevent rabies even after high-
risk exposure (Nie et al., 2017). However, death is almost always inevitable in unimmunized
patients; only supportive measures are recommended after the onset of neurological signs and
symptoms. Older nerve tissue vaccines should no longer be used as they may induce severe
adverse reactions and are less effective than CCEEVs (Crowcroft and Thampi, 2016). The
vaccines recommended by WHO include those produced in Vero cells, available since the 1980s.
Unfortunately, the cell culture rabies vaccines are expensive and not readily available to
individuals living in developing countries where rabies is endemic in dogs. Sheep brain derived
Fermi type rabies vaccine is still being manufactured and utilized for the majority of exposed
patients in Ethiopia, even though this vaccine has been discouraged by the WHO (Birhanu
Hurisa et al., 2013). Hence, a continuing search for antirabies agents that is selectively virucidal,
accessible and cost effective remains crucial. Many traditional medicinal plants have been
reported to have strong antiviral activity and some of them have already been used to treat
animals and people who suffer from viral infections inhibiting the replication cycle of various
types of DNA or RNA viruses. Additionally, different secondary metabolites, including lignans,
tannins, saponins, flavonoids and phenolic acids exhibit promising antiviral activity. In the
search for such antiviral agents, the antirabies activity of medicinal plant extracts, including
South American plants, was evaluated (Müller et al., 2007).
According to a World Health Organization (WHO, 1993) report, at least 80 % of the populations
in most developing countries rely for their primary health care on traditional forms of health
care. Several countries of Africa have realized the need and importance to develop improved
traditional medicines (ITM) from native and endemic plants that are traditionally used at various
places for various ailments. Medicinal plants have been used in diagnosis, treatment or
prevention of various ailments since past decades in the name of traditional systems of medicine
such as Ayurvedic medicine, Chinese traditional medicine, Tibetan medicine, Unani medicine,

2
Japanese medicine and African traditional medicine (Chaudhary et al., 2018). Nearly 25% of
modern medicines are derived from plants first used traditionally (Petros Admasu et al., 2014).
In plants, the naturally occurring chemical compounds are phytochemicals. They give
organoleptic properties and color to the plant and they are beneficial to boost up immunolatory
responses and provide immunity against many diseases (Khalid et al., 2018). The phytochemical
analysis of the plants is very important commercially and has great interest in pharmaceutical
companies for the production of the new drugs for curing of various diseases (Wadood et al.,
2013).
Plant-derived substances have recently become of great interest owing to their versatile
applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of
medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical
intermediates and chemical entities for synthetic drugs (Tiwari et al., 2011). Phytolacca
dodecandra, Justicia schimperiana, Ricinuscommunis, Brucea antidysenterica, Croton
macrostachyus and Cucumis ficifolius were the most cited medicinal plant species utilized for the
management of rabies (Asfaw Meresa et al., 2017). Therefore, evaluations of the antirabies
activity of such medicinal plant extracts are a necessary and highly desirable task. The present
study had the objectives of assessing the phytochemical constituents, acute toxicity and
antirabies potential of extracts from the leaves of Justicia schimperiana and Ricinus communis as
well as the stem bark of Croton macrostachyus.
1.1. Statement of the problem
Rabies claims the lives of more than 24,000 people in Africa annually, but efforts to control the
disease are still not sufficient, particularly in sub-Saharan Africa such as Ethiopia. Prevention of
rabies in humans is complicated because those most commonly exposed to canine rabies lack the
resources necessary to treat or prevent exposure. As a result, individuals who are exposed to the
rabies virus often see traditional healers for the diagnosis and treatments of the disease.
Understanding the relationship among medicinal plants used in traditional medicine systems can
help identify plant materials with potential constituents applicable to modern medicine.
Knowledge of medicinal plants that has been passed on from generation to generation has been
proven to help millions in Ethiopia, but most of the traditional healers have poor knowledge on
the dosage, safety and antidote while prescribing remedies to their patients.

3
 1.2. Objectives
1.2.1. General objective

To determine the phytochemical constituents, acute toxicity and anti-rabies activities of the most
commonly used traditional medicinal plants used for the management of rabies in Ethiopia.
1.2.2. Specific objectives
To assess active ingredients within the ethanol, methanol and water extracts of selected
Ethiopian traditional medicinal plants
To evaluate the potential acute toxicity of the ethanol, methanol and water extracts of plants
collected from different parts of Ethiopia
To evaluate the in vivo and in vitro antirabies potential of ethanol, methanol and water extracts
of selected Ethiopian traditional medicinal plants
1.3. Significance of the study
Traditional medicine is used throughout the world as it is culturally acceptable, economically
affordable and effective against certain type of diseases as compared to modern medicines. The
findings of this study provide information on the phytochemical constituents, acute toxicity and
anti-rabies activities of the selected Ethiopian traditional medicinal plants.

4
 2. Literature Review
2.1. Rabies Etiology and Epidemiology
Rabies virion, a bullet shaped enveloped infectious particle (180 nm x 75 nm in size), having 12
Kb negative sense single-stranded RNA genome, belongs to the Lyssavirus genus of the
Rhabdoviridae family and Mononegavirale order (Singh et al., 2017). Rabies is a central nervous
system (CNS) disease that is almost invariably fatal. The causative agent is rabies virus (RV), a
negative-stranded RNA virus of the rhabdovirus family, which has a relatively simple, modular
genome organization and encodes five structural proteins: a RNA-dependent RNA polymerase
(L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M) and an external
surface glycoprotein (G) (Dietzschold et al., 2008). All the lyssaviruses share many biological
and physicochemical features as well as amino acid sequence characteristics that classify them
with other rhabdoviruses. These include the bullet shaped morphology helical nucleocapsid or
ribonucleoprotein core. The name Rhabdo comes from the Greek and identifies the characteristic
bullet or rod-shape of the viruses (Semayat Oyda and Bekele Megersa, 2017). Lyssaviruses are
usually confined to one major reservoir species in a given geographic area, although spillover to
other species is common. Identification of different virus variants by laboratory procedures such
as monoclonal antibody analysis or genetic sequencing has greatly enhanced understanding of
rabies epidemiology (Gemechu Regea, 2017). Rabies virus is very fragile outside of the animal
host, and is rapidly inactivated by drying or exposure to ultraviolet (UV) light (Tariku Jibat et al.,
2018). The ssRNA of rabies virus contains five monocistronic genes relate to five viral proteins
whose order is highly conserved (Singh et al., 2017). The negative-sense RNA genome is tightly
encapsulated by N, P, and L proteins to form a ribonucleoprotein complex that is responsible for
virus replication in the cytoplasm within infected cells. The RABV G protein is the only viral
protein exposed on the surface of the virus and is not only the major determinant of viral
pathogenicity, but also the major protective antigen responsible for inducing protective immunity
against rabies (Zhu and Guo, 2016).

5
Figure 1: Rabies-virus-shape-and-its-structures
Source: https dokuwiki.png. Accessed on 22 May, 2019

The RABV is not viable outside the host and can be inactivated by sunlight, heat and desiccation
(Singh et al., 2017). The virus affects virtually all mammals and infected species invariably die
from the disease once clinical signs are manifested and is a fatal viral zoonotic disease which
causes encephalitis in all warm blooded animals and humans (Semayat Oyda and Bekele
Megersa, 2017). Among the viral diseases, rabies is unique and it can affect a wide range of
victims including humans as well as a wide variety of wildlife species that act as reservoirs for
infection predominantly and it influences the population dynamics accordingly (Singh et al.,
2017). According to the Center for Disease Control (CDC), all mammal species have a risk of
becoming infected by the rabies virus. All mammals are susceptible to the virus, with reservoir
species most notably among carnivores of the family Canidae (Taylor and Nel, 2015). From an
epidemiologic perspective, the name of the mammalian species acting as the reservoir and vector
is used as an adjective to describe involvement in the infection process. For example, rabies
maintained by dog-to-dog transmission is termed canine rabies, whereas rabies in a dog as a
result of infection with a variant from a different reservoir mammal, e.g., skunk (or raccoon or
fox), would be referred to as skunk (or raccoon or fox, etc.) rabies in a dog (Gemechu Regea,

6
2017). Persons having frequent contact with wildlife, such as mammalogists, are at greater risk
than the general population for exposure to rabid animals (Krebs et al., 1995).

2.2. Global Distribution and Burden of Rabies

In the world, it has been estimated that about 59, 000 people die from rabies each year, of which
the highest death is in Africa and Asia due to the presence of endemic canine rabies and dogs
remain the major animal reservoirs in such areas (Wunner & Briggs, 2010). It is prevalent
throughout the world except in Islands (Singh, et al., 2017). The virus is a highly opportunistic
pathogen and canine rabies continues to spread to new areas, demonstrated by the last decade’s
epidemic across much of the People’s Republic of China and its emergence on previously rabies
free islands such as Flores and Bali (Taylor and Nel, 2015). The United States as with other
developed countries have seen a dramatic decrease in the number of human infections and deaths
due to the rabies virus. According to the Centers for Disease Control and Prevention (CDC) the
stark reduction in the number of rabies cases is attributable to the elimination of canine rabies
through vaccination, the vaccination of wildlife, education of the virus, and timely administration
of post exposure prophylaxis. Currently, in the U.S. only one to three cases of rabies are reported
annually (CDC, 2017).

Though rabies is prevalent throughout the globe, many countries and islands have got rabies-free
status due to strict quarantine or by virtue of their water locked geographical location. Among
the African countries Cape Verde, Congo, Libya Mauritius, Reunion and Seychelles are free
from rabies (Singh et al., 2017). Most of the human death cases occur in Asian and African
countries. Of these countries, Ethiopia is one of the worst affected, with domestic dogs being the
major sources of the infection to humans (Tariku Jibat et al., 2018). The first major outbreaks of
rabies in dog were reported in many parts of Ethiopia in 1884, especially in the former province
of Tigre, Begemder, Gojjam and Wollo. The reviewed rabies situation in Ethiopia revealed that
2172 cases of animal rabies had been confirmed in and around Addis Ababa during 1990-2000,
where dogs constituted 89.83 % with the incidence rate of 73.2 % (Semayat Oyda and Bekele
Megersa, 2017).

The global burden of canine rabies is not distributed equally, and it disproportionately affects
regions with limited resources, that are least capable of responding to the disease. It causes
economic loses directly or indirectly on local and national economy. There are two pathways

7
following human exposure: the individual either seeks medical treatment or is given PEP,
incurring direct and indirect costs, or he does not receive PEP, and either remains well or dies
from rabies, leading to further costs (Gemechu Regea, 2017). Rabies causes at least 24000 deaths
per year in Africa. The high death rates reported in poor rural communities and children. The
major cause of spread of rabies in this region is urbanization (Niloufer, 2003).

In Ethiopia, the national annual estimates from official reports indicate 12 exposure cases and 1.6
rabies deaths per 100,000 populations. Quantification of the health burden enhances the
understanding of its long term effects and of the comparative advantages of different levels of
treatment and prevention (Tariku Jibat et al., 2018). The annual reports of the EPHI (Ethiopian
Public Health Institute) indicated that a total of 488 human deaths had occurred from 1964 to
1975. During the period between 1996 and 2000, a total of 9593 post exposure, and a total of 153
fatal human rabies cases were recorded. The cases were originated from Addis Ababa and its
surroundings and other regions in the country (Semayat Oyda and Bekele Megersa, 2017).

2.3. Rabies Transmission

The lyssavirus infection is transmitted by all animals who are considered as warm-blooded,
while the lyssavirus can also grow up in cells of cold-blooded animals (Bano et al., 2017). The
disease is communicable during the period of salivary shedding of rabies virus. It is transmitted
when the virus is introduced into bite wounds, into open cuts in skin, or onto mucous membranes
from saliva or other potentially infectious material such as neural tissue (CDC,2011). On rare
occasions human rabies has been acquired by inhalation of airborne virus in laboratories working
with live rabies virus and in caves with millions of bats. The common mode of transmission of
rabies in man is by bite of a rabid animal or the contamination of scratch wounds by virus
infected saliva (Balcha Chernet and Nejash Abdela, 2016). Spread of the disease is often
seasonal, with high incidence in late summer and autumn because of large scale movement of
wild animals at the mating time and in pursuit of food. Respiratory and oral transmission can
also occur. The main determinant of transmission is the population density of non-immunized
susceptible key host species that are free roaming within an ecosystem (Semayat Oyda and
Bekele Megersa, 2017). It affects all warm blooded mammals and the virus shades in the saliva
of clinically ill animals and is transmitted through a bite. The main reservoir for humans is
known to be carnivores (Tariku Jibat et al., 2018). In some experimental models, virus was

8
found to immediately enter nerves at the site of inoculation and to appear in the CNS within a
very short time. In other systems, virus entered peripheral nerves after local replication in non-
nervous tissue. Transport to the CNS occurs by retrograde axoplasmic flow at an estimated rate
of 15 to 100 mm per day (Smith, 1996).

2.4. Pathogenesis and Incubation

Rabies virus entry occurs through wounds or direct contact with mucosal surfaces. The virus
cannot cross intact skin. The virus then either replicates in non-nervous tissues or directly enters
peripheral nerves and travels by retrograde axoplasmic flow to the central nervous system (CNS)
(WHO, 2004). The virus replicates in the bitten muscle (local viral proliferation in non-neural
tissue) and gains access (viral attachment) to motor endplates and motor axons to reach the
central nervous system (Semayat Oyda and Bekele Megersa, 2017). The virus is highly
neurotropic, and once it enters the body through a break in the skin or mucous membrane, it
migrates along the nerves from the site of infection to the brain where it causes fatal encephalitis.
From the brain, the virus travels back out to the organs of the body, eventually causing them to
shut down. The development of the virus is influenced by concentration of the virus, number of
bites and distance from head (Taylor and Nel, 2015).

The rabies virus (RABV) causes relatively slow but progressive disease without initial clinical
signs which turns fatal after onset of clinical signs. The virus at the injected site remains hidden
(eclipse) for variable time (a threshold must exceed to cause disease) (Singhet al., 2017). The
RAV G protein is the only viral protein exposed on the surface of the virus and the major
determinant of viral pathogenicity, and also the major protective antigen responsible for inducing
protective against it applied things (Tariku Jibat et al.,2018). The speed of virus uptake, the
ability of the virus to spread efficiently from cell-to-cell and the rate of virus replication are the
major factors that determine the pathogenicity of a RV. Neuroinvasiveness and neurotropism are
the main features that define the pathogenesis of rabies. Although RV pathogenicity is a
multigenic trait involving several elements of the RV genome, the RV glycoprotein plays a
major role in RV pathogenesis by controlling the rate of virus uptake and trans-synaptic virus
spread, and by regulating the rate of virus replication (Dietzscholdet al.,2008). The incubation
period is highly variable. In domestic animals, it is generally 3 to12 weeks, but can range from
several days to months, rarely exceeding 6 months (CDC,2011). In 98% of the cases, the

9
incubation period is under one year. The incubation period is only 12-21days when the bite site
is on the head and neck, while it is 25 days to two years when on the extremities. Generally, it
takes as short as 12 days to as long as two years for the development of clinical rabies
(Susilawathi et al., 2012).

2.5. Clinical Signs

Rabies infection in humans is triphasic, i.e., prodromal, acute neurologic, and coma proceeding
to death. The prodromal signs are mostly non-specific, including fever, headache, myalgia,
nausea, vomiting, and abnormal sensation around bite sites. During acute neurologic stage
patients show anxiety, agitation, dysphagia, hyper salivation, paralysis, and episodes of delirium
(Susilawathi et al., 2012). It is investigated that as the disease becomes advanced, the animal
shows strange behavior. The primary clinical signs are frequently non-specific and can comprise
anxiety, restiveness, anorexia or an improved appetite, nausea, diarrhea, a minor fever, dilation
of the pupils, hyperactivity to any stimuli in addition to extreme salivation. Symptoms are
produced after completion of the incubation period (Bano et al., 2017). Once clinical signs of
rabies appear, the disease is nearly always fatal, and treatment is typically supportive.
Symptoms of rabies are variable, but some distinctive signs including severe anxiety, fear of
water (hydrophobia), and fear of air (aerophobia) are recognizable in writings from millennia ago
(Taylor and Nel, 2015). It causes inflammation of the brain in humans and other mammals. In
Paralytic (dumb) phase, the gullet and masseter muscles turn into paralyzed; the animal might be
incapable of swallowing, and salivating abundantly. In addition to that, there might be facial
paralysis along with dropping of the lower jaw. Furthermore, this stage is also characterized by
dropping of foamy salivary secretion and paralysis of hind limbs eventually leading complete
paralysis followed by death (Bano et al., 2017). During the final stages of disease, the virus is
excreted in the saliva, and it is transmitted between hosts primarily through bites or scratches
from infected animals, but more rarely transmission can occur through viral contact with mucous
membranes (Taylor and Nel, 2015).

2.6. Diagnosis of Rabies

Rabies diagnostic tests were born with routine inoculation of rabies virus (RABV)-infected brain
or saliva samples to rabbits in 1880 followed by the identification of Negri bodies reported in
1903 (Mani & Madhusudana, 2013). Initial capture or confinement of a rabies-suspect animal

10
can be quite problematic. Rabid animals can be unpredictable, sometimes alternating from an
apparently weakened neurologic state to one of dangerous aggression (Hanlon and Nadin-Davis,
2013). The standard methods for rabies diagnosis in humans are virus isolation, antigen
detection, and viral genome detection (Susilawathi et al., 2012). The disease can only be
identified following the onset of the symptoms and it is not easy to diagnose via ante-mortem.
The virus is usually undetectable during the incubation period, and infections can also be
difficult to diagnose when the clinical signs first appear. The diagnosis of rabies virus is made by
taking some part of tissue from the brain of suspected animal. But mostly for confirmatory
diagnosis samples from the brain stem and cerebellum are taken (Bano et al., 2017). The clinical
signs of rabies are confused with other neurological sings caused by other neurotropic etiological
agents. The animals showing abnormal behavior should be kept in isolation where they cannot
bite others for 10 days (Singh et al.,2017).

One million to two million animal bites per year are treated by physicians in the United States.
Fortunately, only a small proportion of these bites involves a risk of rabies infection, and it is the
function of the rabies laboratory to rapidly and accurately identify rabies virus-infected animals
(Smith, 1996). Historically and in the research setting, RABV infection is identified by infecting
cells and detecting virus. This can be done either through the Mouse Inoculation Test (MIT) or
by inoculation of samples onto cultures of murine neuroblastoma or other cells (Rapid Tissue
Culture Infection Test - RTCT). Following intracerebral inoculation of mice aged 3-4 weeks;
MIT test results are available after an incubation period of up to 28 days. Some strains are
associated with a longer incubation period. In laboratories with cell culture facilities and
appropriate level of bio-containment, RTCT provides results within 24-48 hours, which is far
quicker than intracerebral inoculation (Mani and Madhusudana, 2013).

Diagnosis of rabies should not be assumed without laboratory confirmation as similar clinical
signs may be caused by a number of other viral or bacterial pathogens. Most commonly the
confirmation is made by detection of lyssavirus antigens in the mouse brain via direct fluorescent
antibody (dFA) test, although other methods for antigen or RNA detection can be implemented
as well (Kuzmin, 2015). Rabies diagnosis can be performed on fresh specimens from several
different tissue sources or on appropriate specimens stored at proper temperatures, preferably
refrigerated. Impressions (or smears) of tissue samples from brainstem, thalamus, cerebellum,

11
and the hippocampus (Ammon's horns) are recommended for increased sensitivity of the test
(WHO, 2005). The direct fluorescent antibody (dFA) testis rapid, sensitive, specific, easy to
perform, and relatively inexpensive (Smith, 1996) and it has served as the cornerstone of rabies
diagnosis for the past half century. The test is based upon microscopic examination under
ultraviolet light of impressions, smears or frozen sections of tissue after they have been treated
with anti-rabies serum or globulin conjugated with fluorescein isothiocyanate. The diagnostic
conjugate should be of high quality and the appropriate working dilution must be determined in
order to detect the different genotypes of lyssavirus (WHO, 2005).

The sensitivity of techniques for rabies diagnosis varies greatly according to the stage of the
disease, antibody status, intermittent nature of viral shedding and the training of the technical
staff. Molecular methods, although useful and extremely sensitive, may not always give positive
results for patients with rabies. This may be due to the intermittency of virus shedding, the
timing of sample collection, and the type of specimens collected (Hemachudha and
Wacharapluesadee, 2004). Laboratory diagnosis is critical to confirm the status of a suspect case,
in part, to justify prophylaxis in exposed persons or animals, to measure objectively the impact
of disease prevention programs, and to support certification of a country as free of disease
(Rupprecht et al., 2017). Current techniques for the definitive laboratory diagnosis of rabies
usually are nondiagnostic in the early days of infection, become useful only a week or more after
the onset of illness, and are of little benefit for the patient because no effective therapy is
available. However, the diagnosis of rabies should be confirmed as quickly as possible so the
number of persons exposed to the infection can be limited, and therapy for those exposed can be
initiated promptly (Wilkerson, 1995).

2.7. Prevention and Control of Rabies

Rabies is 100% fatal but it is also completely preventable with application of existing
vaccination technology (Garg et al., 2017). The majority of animal and human exposures to
rabies can be prevented by raising public awareness on rabies transmission routes, and avoiding
contact with wildlife (Balcha Chernet and Nejash Abdela, 2016). The earliest effective control
measures against rabies were based on movement restrictions and muzzling of dogs, and the
elimination of stray dogs. These methods were the backbone of the program that resulted in
canine rabies elimination from the United Kingdom in 1902 (Taylor and Nel, 2015). All rabies

12
vaccines registered for human and animal use must conform to established potency standards. A
minimum antigenic potency of 2.5 IU per dose is mandatory (Balcha Chernet and Nejash
Abdela, 2016).

2.7.1. Prevention of Human Rabies

Prevention of rabies in humans is complicated because those most commonly exposed to canine
rabies (e.g., children, the poor) also lack the resources necessary to treat or prevent exposure
(Gemechu Regea, 2017). Human rabies can be prevented by a) eliminating exposure to rabies
virus, b) providing appropriate rabies pre-exposure prophylaxis, and c) prompt local treatment of
bite wounds combined with appropriate rabies post exposure prophylaxis (Balcha Chernet and
Nejash Abdela, 2016). Currently, the disease is vaccine- preventable with pre- and post-
exposure prophylaxis (PrEP and PEP) (Shwiff et al., 2019). Pre-exposure vaccination may be
offered to high risk groups like laboratory staff handling the virus and infected material,
clinicians and persons attending human rabies cases, veterinarians, animal handlers and catchers,
wildlife wardens, quarantine officers and travelers from rabies free areas to rabies endemic areas
(Gemechu Regea, 2017). The first effective treatment for the rabies virus was a vaccine in 1885
by Louis Pasteur and Emile Roux. The vaccine was made from the nerve tissue of rabbits
infected with rabies (Petros Admasu et al., 2014). Rabies could potentially be eliminated from
the human population due to availability of efficacious vaccination tools which have been
validated across the world (Garg et al., 2017).

Modern rabies vaccines produced on cell cultures or embryonated eggs are both safe and
efficacious. Human rabies treatment is expensive and comprises a significant portion of the
overall economic impact of rabies. Treatment usually consists of a series of four to five
vaccinations over a span of several weeks, accompanied by a dose of rabies immunoglobulin
(RIG) (Shwiff et al., 2019). The essential components of rabies post exposure prophylaxis are
immediate thorough cleansing of all wounds with soap and water and the administration of anti-
rabies immune globulin and vaccine (Smith, 1996). Although there is an efficient vaccine, which
can also be administered after the exposure, once the clinical manifestation sets in death is
inevitable. The Milwaukee protocol for management of clinical rabies also has not produced
encouraging results. A major lacuna has been the non-availability of a successful specific anti-
viral. Ribavirin and interferon-α have proved to be disappointing agents for the therapy of rabies.

13
There is insufficient evidence to support the continued use of ketamine or amantadine for the
therapy of rabies (Sandeepan et al., 2017).

2.7.2. Animal Rabies Control

The majority of human rabies deaths globally occur as a result of being bitten by dogs.
Adherence to a regular rabies vaccination schedule is critical to protect animals against
recognized and unrecognized rabies exposures (Gemechu Regea, 2017). Vaccination of pet
animals provides a barrier to transmission of rabies to humans. This has provided a major
mechanism for prevention by breaking the link between rabies cycles in wildlife and
transmission to domestic animals; the latter providing a ready means to pass the infection on to
humans (Krebs et al., 1995). According to the World Organization for Animal Health (OIE) and
the WHO recommendations, the critical percentage of dogs to be vaccinated to prevent rabies
cases should be at least 70% (WHO, 2005). Although rabies can be well controlled among
domesticated animals by different types of useful and widely available vaccines, canine rabies
continued to be a serious problem in Africa, including Ethiopia (Petros Admasu et al., 2014).
In Ethiopia, 94.01 percent of rabies cases are caused due to the bite of rabid dogs and the rest
cases incriminate domestic and wild animals (Tariku Jibat et al., 2018). Public education on the
risks of rabies transmission from wild animals is paramount to effective disease prevention. A
number of recently developed, highly-effective, thermostable, inactivated vaccines are available
for veterinary use. The vaccines may be used in young pups, but they must be boosted at three
months of age and again within the following year. Revaccination must be carried out every
three years thereafter. Cattle and sheep may be vaccinated annually or every two to three years,
depending on the vaccine manufacturer's instructions. Following an outbreak in domestic
livestock, vaccination of animals without visible bite wounds is strongly recommended (Balcha
Chernet and Abdela Nejash, 2016). Rabies vaccines may be administered under the supervision
of a licensed veterinarian to animals held in animal shelters before release. Within 28 days after
initial vaccination, a peak rabies virus antibody titer is expected, and the animal can be
considered immunized (Gemechu Regea, 2017).
2.8. Rabies Treatment
Rabies infection is always fatal unless prompt post exposure treatment is administered before
symptoms begin. Fortunately, rabies can be a vaccine-preventable disease, provided that post-
exposure prophylaxis (PEP) is given promptly and correctly. Protection against rabies correlates

14
with the presence of rabies-specific virus-neutralizing antibodies (VNAs). The recent advance of
modern cell cultivation techniques has made the production of high-quality rabies vaccines from
cell culture feasible (Zhu and Guo, 2016). Effective post exposure therapy includes wound
cleansing and active and passive immunization in a previously unimmunized individual. Active
immunization is achieved with five doses of a modern cell culture vaccine, including purified
chick embryo cell culture vaccine or human diploid cell vaccine (administered intramuscularly in
the deltoid muscle on days 0, 3, 7, 14, and 28). Passive immunization is performed with human
rabies immune globulin at a dosage of 20 IU/kg with local infiltration into and around the
wound(s); any remaining dose should be given intramuscularly into the gluteal area (Jackson,
2009). Just once rabies warning signs have appeared, the treatment is generally supportive. The
patients are sedated to manage their fear and pain (Bano etal., 2017).
Ribavirin and interferon-alpha are broad-spectrum antivirals that have significant in vitro activity
against rabies but in vivo experiments and treatment of several human patients with rabies
infection did not show any beneficial activity. Two other FDA approved drugs with some in vitro
anti-rabies activity are the N methyl-D-aspartate (NMDA) receptor antagonist ketamine and
amantadine (Jochmans and Neyts, 2017). Despite the wide availability of different types of
useful vaccines used for rabies treatment, a continuing search for new compounds having
antirabies agent remains crucial (Petros Admasu et al., 2014). Although live-attenuated virus-
based vaccines represent the most promising approach for rabies control and treatment, some
other novel modalities have also been investigated for their potential role in rabies treatment,
such as protein and peptide vaccine, nucleic acid-based vaccine, RNA interference (RNAi), and
RIG, coupled with BBB permeability enhancing agents such as monocyte chemotacticprotein-1
(MCP-1) (Zhu and Guo, 2016).
2.9. Costs and Cost-Effectiveness of Control Measures
The highest financial expenditure in any country is the cost of rabies post exposure prophylaxis.
The type of vaccine, vaccine regimen and route of administration as well as the type of
immunoglobulin used all significantly influence the cost of treatment (WHO, 2005). The total
cost of canine rabies to a country comprises a number of different aspects, including the direct
costs of vaccinating animals and people, the indirect costs (often borne by the patients and dog
owners) of lost salary and travel to seek vaccination, losses due to livestock that die of rabies,
surveillance costs, dog population management costs, and productivity losses. There is a strong

15
logical argument that mass dog vaccination is a more cost-effective long-term strategy than
relying on PEP provision alone (Taylor and Nel, 2015). Deaths due to rabies are responsible for
1.74million disability associated life years (DALYs) lost each year with 0.04 million DALYs are
lost through morbidity and mortality following side-effects of unsafe nerve-tissue vaccines
(Abebe Mengesha et al., 2016). About 563 million United States dollars are spent annually in the
world on measures to prevent rabies, yet in countries of south-eastern Asia the disease is still an
important public health problem (Kole et al., 2014). The need to pay for transport and expensive
post-exposure prophylaxis for rabies exposed family or community members can lead to the
unplanned sale of production animals and livelihoods assets, further impacting food and
economic security (Gemechu Regea, 2017). Veterinary services in Africa usually report very
limited budgets and often have to divert resources during outbreaks of other diseases. This is
clearly the most significant constraint to effective rabies control (Lembo et al., 2010). The cost
of upgrading nerve tissue based vaccine production facilities in order to produce cell culture
rabies vaccines is beyond the financial budget of most developing countries. Therefore, banning
the use of nerve tissue based vaccines alone will not solve the problem as this would remove
access to the post-exposure treatment (PET) available to many patients’ unable to afford modern
cell culture vaccines (Abebe Mengesha et al., 2016). Effective vaccination campaigns need to
reach a sufficient percentage of the population to eliminate disease and prevent future outbreaks,
which for rabies is predicted to be 70%, at a cost that is economically and logistically sustainable
(Kaare et al., 2012).
2.10. Risks of Rabies vaccine
Evidence suggests that rabies vaccine may have non-specific protective effects in animals and
children. Non-specific effects of vaccines are defined as those effects on the immune system of
the recipient that alter the risk of illness or death from conditions other than the specific
infectious disease the vaccine is designed to prevent (Knobel et al., 2017). The use of nerve
tissue vaccine from rabies virus–infected goat or sheep brain tissue has led to severe adverse
reactions and is currently being phased out in developing countries. Most reported adverse events
following immunization with rabies vaccine are local reactions such as pain at the injection site,
swelling, redness, and induration (Kang et al., 2018).

16
 2.11. Traditional Medicinal Plants
Traditional medicines have a very long history: it is the sum total of the practices based on the
theories, beliefs and experiences of different cultures and times, often inexplicable, used in the
maintenance of health, as like in the prevention, diagnosis, improvement and treatment of
illnesses (Firenzuoli and Gori, 2007). Traditional medicine is largely transmitted as oral
knowledge and around 4,000 species are used in ATRM which is predominantly (90%) plant
based (Payyappallimana, 2016). Medicinal plants have an unbelievable history in terms of
serving humanity in almost all continents of the world. Traditional healers have transferred that
incredible knowledge from generation to generation. Even modernity or cultural revolutions have
not altered the in-depth wisdom of this natural medical paradigm (Khan, 2014).
The World Health Organization (WHO) defines traditional medicinal plants as natural plant
materials which are used at least or in the absence industrial processing for the treatment of
diseases at a local or regional scale (Jamshidi-kia et al.,2018). The use of medicinally active plants
predates modern history. A large number of plants used in the traditional medicine have now become a
part of the modern world health care system because of their unique ability to synthesize a wide array of
compounds with diverse health-related benefits (Naithani et al.,2010). Since the ancient period plants
have been utilized by human beings as medicinal agents on the basis of ethnomedical
background (Divya et al., 2011). Plants that possess therapeutic properties or exert beneficial
pharmacological effects on the human body are generally designated as medicinal plants. The
therapeutic use of plants goes back to the Sumerian and the Akkadian civilization in about the
3rd millennium B. C (Kutama et al., 2018). Medicinal plants are used for discovering and
screening of the phytochemical constituents which are very helpful for the manufacturing of new
drugs (Wadood et al., 2013). Most developing countries, especially those in Asia, Africa, Latin
America and the Middle East, 70%–95% of their population rely on traditional medicines for
treatment of different diseases. The beneficial medicinal effects of plant materials typically result
from the combinations of secondary products present in the plant which used as sources of
medicines throughout history and continued to serve as the basis for many pharmaceuticals used
today (Petros Admasu et al., 2014).
More than a tenth of the plant species (over 50000 species) are used in pharmaceutical and
cosmetic products. However, the distribution of medicinal plants across the world is not uniform
and medicinal herbs are mainly collected from the wildlife population. In traditional methods,

17
plant materials are tested for pharmaceutical purposes. If any evidence of activity is observed,
the extract is fractioned, and the active compound is isolated and identified. Each step of
decomposition and isolation is usually guided by biological test, which is referred to as bioassay-
guided fractionation (Jamshidi-kia et al., 2018). Modern drug discovery which has its roots in
traditional medicine provides avenues to newer phytomolecules based therapies. Nowadays
major pharmaceutical industry are reducing their research focus and are indulging towards profit-
making venture by extracting and selling phyto-constituents (Kapoor et al., 2017). In Ethiopia,
medicinal plants contribute, to about 80% of the traditional medicines used in the country (the
others being animal and mineral origins). Most of these plants are obtained from local sources in
the wild by knowledgeable traditional practitioners (Worku Abebe, 2016).
2.11.1. Phytochemicals and Pharmacological Properties
Phytochemicals are chemical compounds formed during the plants normal metabolic processes.
These chemicals are referred to as secondary metabolites which comprises of several classes and
these includes; alkaloids, flavonoids, phenols, tannins, coumarins, glycosides, gums,
polysaccharides, terpenes, terpenoids (Kutama et al., 2018). There are chemical compounds with
complex structures and with more restricted distribution than primary metabolites (Zohra et al.,
2012). Plants and many of their secondary metabolites because of the healing properties have
been in traditional use throughout the world since ancient times. They provide us diverse
bioactive phytochemicals which play synergetic role in maintaining human health. Plants
produce a diverse array of more than 100,000 secondary metabolites and can be classified, on the
basis of composition and the pathway through which they are synthesized (Kapoor et al., 2017).
The medicinal plants are useful for healing as well as for curing of human diseases because of
the presence of phytochemicals. Phytochemicals are naturally occurring in the plants, leaves,
stem and roots that have defense mechanism and protect from various diseases (Wadood et al.,
2013). There has been increasing interest in the research on flavonoids from plant sources
because of their versatile health benefits reported in various epidemiological studies. Many
flavonoids are shown to have antioxidative activity, free radical scavenging capacity, coronary
heart disease prevention, hepatoprotective, anti-inflammatory, and anticancer activities, while
some flavonoids exhibit potential antiviral activities (Kumar and Pandey, 2013). The alkaloids,
substances extracted natural sources, show promising pharmacological activities, including
pharmacological activities for the treatment of neurodegenerative diseases such as Alzheimer's

18
disease, whose treatment is based on the use of various drugs (Chaves et al., 2016). Active
substances that have phenolic groups in their structure have great pharmacological potential
(Siqueira et al., 2012).
2.12. The Use of Traditional Medicinal Plants in Ethiopia
Traditional medicine plays an important role in the healthcare of the majority of the people in
developing countries, including Ethiopia, and medicinal plants serve as valuable sources of
natural therapeutic agents (Worku Abebe, 2016). In Ethiopia, about 800 species of plants are
used in the traditional health care system to treat nearly 300 mental and physical disorders
(Asmare Amuamuta et al., 2014). Traditional Ethiopian medicine is commonly used to treat a
variety of diseases including gastrointestinal disturbances, respiratory disorders, sexually
transmitted infections, tuberculosis, impotency, hemorrhoids, rabies, intestinal parasites, skin
problems, liver diseases, mental disorders, hypertension, diabetes, gynecological conditions
rheumatism, malaria and others (Kebede Deribe et al., 2006).
It is widely believed in Ethiopia that the skill of traditional health practitioners is 'given by God'
and knowledge on traditional medicines is passed orally from father to a favorite child, usually a
son or is acquired by some spiritual procedures. There is lack of accurate quantitative
information on rabies both in humans and animals and little is known about the awareness of the
people about the disease to apply effective control measures in Ethiopia (Semayat Oyda and
Bekele Megersa, 2017). The number of dog to human ratio is approximately assumed to be 1:6
and 1:8 in urban and rural areas, respectively. This indicates that rabies, which is maintained and
disseminated mostly by dogs, is a threat to public health in Ethiopia. People and traditional
rabies healers in Ethiopia claim that traditional plants can cure both animals and humans that are
exposed to rabies (Petros Admasu and Yalemtsehay Mekonnen, 2014). In rural Ethiopia,
individuals who are exposed to rabies often prefer to see traditional healers for the diagnosis and
treatment of the disease because of cultural background, lack of knowledge or limited
accessibility to medical treatment. These widespread traditional practices of handling rabies
cases might interfere with medical treatment seeking practice, resulting in an underreporting of
the actual number of rabies cases and its related health burden (Tariku Jibat et al., 2018).
According to Dabuma Tamiru et al., (2017), some of the interviewees believed that rabies is
caused by starvation; thirst and prolonged exposure to coldness and 30.6% of the respondents
didn’t knew the cause of rabies. A little more than the half (52.6%) believe that it is possible to

19
manage rabies by Holy water. Around 36.5% respondents accepted that there was specific
medical drug therapy in the Health Centre. Only 9.8% of the respondents were aware of the
presence of post exposure prophylaxis (PEP). Several traditional herbs have been formulated by
traditional healers to treat human and animal rabies and individuals who are exposed to rabies
virus often see traditional healers for the diagnosis and treatment of the disease (Petros Admasu
and Yalemtsehay Mekonnen, 2014). These widespread traditional practices of handling rabies
cases are believed to interfere with timely seeking of PEP. Rabies victims especially from rural
areas seek PEP treatment after exhausting the traditional medicinal intervention and usually after
a loss of life from family members (Tadesse Guadu et al., 2014).
Various traditional antirabies plants were reported in different indigenous people and parts of
Ethiopia which were used for the treatment of rabies in humans and animals. Among the reported
plants, Phytolacca dodecandra is widely used for the traditional treatment of rabies in both
humans and animals in Ethiopia (Petros Admasu et al., 2014). Phytolacca dodecandra, Justicia
schimperiana, Ricinus communis, Brucea antidysenterica, Croton macrostachyus,
Cucumisficifolius, Salix subserrata, Calpurnia aurea and Euphorbia abyssinica were the most
cited medicinal plant species utilized for the management of rabies by the Ethiopian traditional
health care system (Asfaw Meresa et al., 2017).
The folk drugs of rabies such as Cucumisficifolius, Datura stramonium, Dorsteniabarnimiana,
Dracaenasteudneri, Euphorbia abyssinica, Gnidiaglauca, Justicia schimperiana, Phytolacca
dodecandra, Salix subserrata, Silene macroselen, Vigna membrancea and Zehneriascabra were
one of the most widely used remedies in different parts of Ethiopia. Of these folk drugs, only
three (Salix subserrata, Silene macroselen and Phytolacca dodecandra) were evaluated in vivo to
see their efficacy against rabies virus (Petros Admasu and Yalemtsehay Mekonnen, 2014). About
twelve traditional antirabies plants in different indigenous people from different parts of Ethiopia
were reported by different investigators for the treatment of rabies in humans and animals (Petros
Admasu et al., 2014). However, efficacy of these plants against rabies were not evaluated with
modern pharmaceutical practices except findings of Asefa Deressa and his colleagues who
evaluated the efficacy of antirabies activities of crude extract of Salix subserrata and Silene
macroselen plants in mice which improved the survival period (Days) of experimental mice
compared to control group of mice.

20
 2.13. Problems associated with traditional medicine
Some plants used in traditional medicines, such as taenicides, are widely known to be toxic. For
example, blindness and changes in central nervous system function have repeatedly been found
in people who took over dosage of Hagenia abyssinica. Traditional healers may cause delays in
the treatment of communicable diseases such as TB if they fail to refer patients to modern health
services (Kebede Deribe et al., 2006). Though there is lack of published evidences, adverse fatal
side effects and cases of rabies deaths after traditionally treated with medicinal plants were the
most common problems reported by some health centers/institutes in Ethiopia including EPHI,
owning to the non-standardization of constituents, quality and efficacy of these traditionally used
antirabies herbal remedies (Petros Admasu and Yalemtsehay Mekonnen, 2014).
2.14. Description of selected medicinal plants used for treatment of rabies
Of the estimated 250,000 species of higher plants existing throughout the world, only a fraction
have been examined for pharmacological activities (Ali et al., 1996). According to Chaudhary et
al., (2018), the leaves of Justicia schimperiana, root and stem bark of Croton macrostachyus and
leaves and seed of Ricinus communis are mostly used to cure dog bite, used as ethno medicine by
different tribal society of the globe.

Justicia schimperiana (syn. Adhatoda schimperiana; Gendarussa schimperiana), belongs to the

genus Justicia of the family Acanthaceae. It is a shrub with much branched stems 2-3 m high,
with slightly unpleasant smell. Its ethnobotanical information indicated that the plant is used for
the treatment of various health problems such as scabies, fever, asthma, excessive pellagra and
constipation, gonorrhea, rabies and headache in Ethiopia (Jemal Abdela et al., 2014). It is known
by the common names in Amharic: Sensel and Simiza. Its stem is brittle i.e. it breaks easily. Its
leaves are simple and opposite, long oval to 13 x 4 cm, tip pointed, narrowed to a short stalk. Its
flowers are in conspicuous terminal heads on long stalks seen clearly above the leaves, each
small flower lies inside a green-yellow leafy bract 1.5 cm long, its edge clear and membranous,
flowers white or yellow white, tubular to 3 cm long, two-lipped with dark purple throat or lines
on the lip (Shemsu Umer et al., 2010). The plant has slightly unpleasant smell. The result of the
study by Tadesse Birhanu and Dereje Abera (2015) also indicated that the leaf and root of
Justicia schimperiana is used in the treatment of rabies and coccidiosis at Horro Guduru district,
western Ethiopia (Habtamu Abebe et al., 2014). In Ethiopia, Justicia Schimperiana is locally

21
utilized to heal ailments such as stomachache, burning, constipation, skin lesion, tooth ache and
Scabies (Asfaw Meresa et al., 2017).

Figure 2: Photo of Justicia schimperiana plant

Source: Yeweynshet Tesera. “Justicia schimperiana plant.” Menagesha, Ethiopia. 2019. JPEG
file.

Croton macrostachyus Hochst. ex Del. (Euphorbiaceae) another plant selected for this study is
one of the eight Croton species found in Ethiopia. The wide range medicinal uses of Croton
macrostachyus led scientists to isolate compounds from its different parts (Hadush Gebrehiwot et
al., 2018). It is widely distributed in Ethiopia and has been utilized as a remedy for malaria,
abdominal pain, gonorrhea, wounds, ringworm infestation, hemorrhoids, ascariasis, epilepsy,
rabies venereal diseases, cough, rheumatism, liver problem and other ailments in Ethiopian
traditional medicines (Asfaw Meresa et al., 2019). The leaves are large and green, turning to
orange before falling. It is also characterized by creamy to yellow-white colored flowers with
green (when young) to grey (at maturity) fruits (Abraham Fikru et al., 2016). It has a rounded
crown with slender trunk and massive spreading branches. It has a long taproot and numerous
side-roots, which makes it adapted to dry climates (Getu Alemayehu, 2018). It is widely used as
herbal medicine by the indigenous people of tropical Africa (Maroyi, 2017). It is regarded as a
multipurpose tree by subsistence farmers in Ethiopia, Kenya, and Tanzania and the species has

22
potential in playing an important role in the primary healthcare. The bark, fruits, leaves, roots,
and seeds of Croton macrostachyus are reported to possess diverse medicinal properties and it is
used as herbal medicine for at least 61 human and 20 animal diseases and ailments (Obey et al.,
2018). The plant Croton macrostachyus has been traditionally used to cure dog bite in Ethiopia
(Chaudhary et al., 2018).

Due to its drought hardiness and fast growth, Croton macrostachyus is considered useful for
afforestation of shifting sand dunes, degraded waste land, hill slopes, ravines and lateritic soils.
Throughout its distribution area a decoction, infusion or maceration of leaves, stem bark or root
bark is taken as a purgative and vermifuge (Pagadala et al., 2015). Croton macrostachyus has
compounds isolated from its root; fruit and bark and it has a medicinal value for treatment of
malaria, headache, skin rash, internal worms, abdominal pain, rabies, ringworm infestation,
hemorrhoids, ascarasis, and sexually transmitted diseases (Getu Alemayehu, 2018). It is
traditionally used for the treatment of wounds malaria, rabies, and gonorrhea, Tineaversi color,
diarrhea, hepatitis, jaundice, and scabies (Tigist Minyamer & Getnet Belay, 2018). It is used to
treat rabies, epilepsy, cough, skin disease, dysentery, lung complaints, plain full eyes, toothache
and others (Pagadala et al., 2015).

Figure 3: Photo of Croton macrostachyus plant

Source: Yeweynshet Tesera. “Croton macrostachyus plant.” Menagesha, Ethiopia. 2019. JPEG
file.

23
The other plant species used to treat many diseases and disorders traditionally is Ricinus
communis; Family: Euphorbiaceae popularly known as 'castor plant' and commonly known as
‘palm of Christ’, Gulo (Amharic). The plant is widespread throughout tropical regions as
ornamental plants (Jena and Gupta, 2012). The plant Ricinus communis is probably native to
Africa, and is cultivated in many tropical and subtropical areas of the world, commonly
appearing spontaneously (Kumar et al., 2015). The castor bean plant is a tropical perennial shrub
that originated in Africa, but is now cultivated in many tropical and subtropical regions around
the world. It can be self- and cross-pollinated and worldwide studies reveal low genetic diversity
among castor bean germplasm (Chan et al., 2010). The preliminary phytochemical study of
Ricinus communis showed the presence of steroids, saponins, alkaloids, flavonoids, and
glycosides. Its stem and leave extracts produce antioxidant activity due to the presence of
flavonoids in their extracts (Jena and Gupta, 2012).

Figure 4: Photo of Ricinus communis plant

Source: Yeweynshet Tesera. “Ricinus communis plant.” Menagesha, Ethiopia. 2019. JPEG file.

24
 3. Materials and Methods
3.1. Plant material collection and identification

The leaves of Justicia schimperiana and Ricinus communis and stem bark of Croton
macrostachyus were collected in January 2019 from Menagesha District, which is located 20km
from west of Addis Ababa, Ethiopia. The botanical identification and authentication was done by
Melaku Wondafrash, of the National Herbarium, Department of Plant Biology and Biodiversity
management, Addis Ababa University, Ethiopia, where voucher specimens were deposited
(Justicia schimperiana 001; Croton macrostachyus 002 and Ricinus communis 003). Plant
species used in the present investigation were given in Table 1.

Table 1: The botanical identification of the plant specimens used in this study

Scientific name Family Amharic name Location Part/s used Voucher

number
Justicia schimperiana Acanthaceae Sensel Menagesha Leaves 001
(Hochst. ex Nees)
Croton macrostachyus Euphorbiaceae Bisana Menagesha Stem bark 002
Del.
Ricinus communis L. Euphorbiaceae Gulo Menagesha Leaves 003

3.2. Processing of the plants

The collected leaves of Justicia schimperiana, Ricinus communis and the stem bark of Croton
macrostachyus were properly washed with tap water and separated from foreign material. For
evaporating the water content the washed plant samples were kept in a shaded area for two
weeks. The stem bark of Croton macrostachyus was cut into small pieces using a penknife and
was air-dried for additional one week. All the plant samples were then chopped, crushed and
powdered with electrical grinder and then the dried powdered samples were stored in
polyethylene bags at room temperature for further processes (Hussain et al., 2011).

25
 3.3. Extract preparation

The Plant samples were subjected to sequential solvent extraction using ethanol, 80% methanol
and water accordingly at room temperature and with agitation at 130 rpm on a rotary shaker and
the solvents were removed under reduced pressure to obtain the extracts. All solvents were of
analytical grade. The extracts of the tested medicinal plants were prepared according to the
procedures previously described by (Jemal Abdela, 2014). Briefly, the powder of each plant (200
g in 1600 ml) was separately extracted with ethanol by maceration for 72 h with frequent
agitation on a rotary shaker at 130 rpm and the resulting liquid was filtered (Whatman No. 1
filter paper). Accordingly the marc was re-macerated twice using the same volume of 80%
methanol for 72 h and finally with water for 72 h to exhaustively extract the plant material. After
exhaustive extraction, the filtered organic (ethanol and 80 % methanol) extracts were
concentrated under reduced pressure (Rota vapor at 72 rpm) at a temperature not exceeding 40°C
and the filtered water extracts were concentrated by lyophilization or freeze-drying. The
concentrated extracts were then dried in an oven at 40°C for about 48 h. Finally, the yields of the
extracts were stored at +4°C in airtight container throughout the study period until use.

Weighting Soaking

Drying Agitation

Concentratio Filtration
n

Figure 5: A brief summary of extraction procedure

26
 3.4. Preliminary phytochemical screening

Preliminary qualitative screening for phytochemicals of all the three plant species was carried
out according to (Sasidharan et al., 2011; Zohra et al., 2012; Wadood et al., 2013; Banu and
Cathrine, 2015; Khalid et al., 2018). The determination of metabolites was done by differential
coloring reaction and/or precipitation of the major families of chemical compounds contained in
plants.

3.4.1. Test for Alkaloids (Wagner’s test)

Alkaloids were identified by Wagner’s test; a few drops of Wagner’s Reagents (iodine solution
in potassium iodide) were added to a few ml of plant extract (Banu and Cathrine, 2015).

3.4.2. Test for Flavonoids

Flavonoids identification was carried out by alkaline reagent test; the plant extract was treated
with 2-3 drops of sodium hydroxide solution and observed the formation of acute yellow color.
To this mixture some drops of sulphuric acid were added (Khalid et al., 2018).

3.4.3. Test for Phenol (Ferric chloride test)

The extract (50 mg) was dissolved in 5 ml of distilled water. To this few drops of neutral 5%
ferric chloride solution were added (Banu and Cathrine, 2015).

3.4.4. Test for Steroids (Libermann Burchard Test)

To 1ml of extract, 1ml of chloroform, 2-3 ml of acetic anhydride and 1 to 2 drops of
concentrated sulphuric acid were added (Kumar et al., 2007 as cited in Sasidharan et al., 2011).

3.4.5. Test for Saponins

The saponins study was based on froth test; 50 mg extract was diluted with distilled water and
made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 minutes (Banu and
Cathrine, 2015).

3.4.6. Test for Tannins (Ferric chloride test)

Two ml of plant extract was added to 2 ml of water, a 1 to 2 drops of diluted ferric chloride
solution was added (Zohra et al., 2012).

27
 3.4.7. Test for Terpenoids
For the confirmation of terpenoids in the selected plants, 0.4 g of selected plant sample was
diluted with 5 ml of methanol, Then 2 ml chloroform and 3 ml sulphuric acid were added in
selected sample extract (Wadood et al., 2013).

3.5. Cell culture and virus

Vero cells obtained from Vaccine and Diagnostics production directorate, EPHI were cultured
using the T-75 culture flasks following the procedure described in (Ammerman et al., 2008).
Briefly, vero cells kept in liquid nitrogen were recovered from frozen stock in a 37°C water bath.
The cell suspension was transferred from the cryovial into a 15ml conical tube containing 10ml
of DMEM supplemented with FBS. The cells were pelleted by centrifugation at 200× g for 5
minutes at room temperature and the supernatant was removed and discarded. The cells were
resuspended in 5-10 ml DMEM supplemented with 10% FBS and transferred to tissue culture
flask with vented cap. Flasks were incubated in 37°C incubator with 5% CO2 for 2-3 days until a
monolayer was obtained. The growth medium from confluent monolayer of vero cells was
removed and cells were washed with 10 ml 1X DPBS. Five ml of 1X trypsin-EDTA was added
and the cells were incubated at 37°C for 2-3 minutes, until cells start to streak as they detached
from the flask. To inactivate the trypsin-EDTA, 5 ml DMEM with 10% FBS were added and
cells were washed down in media. The cell suspension was removed from flask and transferred
to a sterile 15 ml conical tube and centrifuged at 200 × g for 5 minutes at room temperature. The
cells were resuspended in 10 ml DMEM with 10% FBS after supernatant was removed and
discarded. The desired dilution of cells in a total of 15 ml DMEM with 10% FBS was prepared
and added to 75 cm2 cell culture flasks with vented caps. Flasks were incubated in 37°C
incubator with 5% CO2 until a monolayer was obtained. Finally, cells were collected within a
volume of 20-30 ml in cell culture medium with 10% heat-inactivated FBS. Cells were
maintained at 37 °C under a humidified 5 % CO2 atmosphere for bioassay.

The Vaccine and Diagnostics Production Directorate, Ethiopian public health Institute, provided
rabies virus Pasteur virus strain (RV PV) used in the present study. The virus (PV) was
propagated in vero cells as previously described by (Webster and Clow, 1937) and titer of
infectious virus was obtained by the limit-dilution method and expressed as 50% tissue culture
infections dose per ml (TCID50/ml). Briefly, serial 10-fold dilutions (10-1 to 10-7) of virus in

28
serum-free MEM were added into the confluent monolayer in 96-well tissue culture plate and
were incubated for 72 h at 37°C. After incubation, the medium were decanted and the cells were
fixed by adding cold acetone (50 µl per well) and kept at 4°C for 30 minutes. After discarding
the acetone, cells were stained by direct polyclonal florescent-labeled antibody for 30 minutes
and washed three times with 0.01 M phosphate buffer saline (PBS), air dried, and visualized
under an inverted fluorescence microscope. The titer was calculated by using Spearman-Karber
method and expressed as TCID50. The virus titration was also performed in mice through
intracerebral inoculation using serial ten-fold dilutions at a volume of 0.03 ml. Six mice were
inoculated per virus dilution, and the end point was death. The statistical method of Spearman-
Karber was used and the mortality at each dilution was calculated to determine the 50% end
point titer (Ramakrishnan, 2016).

3.6. In vivo acute toxicity and anti-rabies screening of crude extracts

3.6.1. Experimental animals and their management

Female Swiss albino mice aged 3-4 weeks and with weight of 15-25g obtained from the
laboratory animal unit of Ethiopian Public Health Institute (EPHI) were used for the studies. The
mice were maintained at a temperature of 220 C ± 2°C and with a 12 h light/12 h dark cycle and
were given clean pellet diet and water ad libitum. As described by (OECD, 2001), all
experimental mice were treated under similar feeding management and left under controlled
condition for three days to acclimatize before conducting the experimental procedure and each
mouse were used only for one experiment. The handling of mice was done following
internationally accepted ethical principles.

3.6.2. In vivo acute toxicity test

The extracts from the leaves of Justicia schimperiana and Ricinus communis and stem bark of
Croton macrostachyus plants, proposed for their antirabies activity, were assessed for their
toxicity in non-infected female Swiss albino mice according to the standard guideline of
Organization for Economic Cooperation and Development (OECD). In the acute toxicity study, a
single administration of the extracts at doses of 1000, 2000, 3000, 4000 and 5000 mg/kg
respectively, was given orally. A total of 276 Swiss albino mice were used and the extract was
administered to mice by using oral gavage in a volume of 20 ml/kg body weight. All mice were
randomly divided into control and treatment groups; six mice per cage. They were fasted for 4 h

29
prior to dosing and 2 h after the administration of the extract. Following the period of fasting, the
mice were weighed and the extracts were administered. The mice in the treatment groups (A, B
and C) received 0.5 ml of the ethanol, methanol and water extracts of each plant respectively, at
doses of 1000, 2000, 3000, 4000 and 5000 mg/kg. And the mice in the control group received 0.5
ml of respective vehicle of each extract (4% Polysorbate-80) (OECD, 2001).

The mice were observed individually after dosing at least once during the first 30 minutes, and
daily thereafter, for a total of 14 days (Assefa Deressa et al., 2010). For determination of LD50 of
extracts, an exploratory assay with each tested mouse strain was performed in order to exactly
determine the dose range to be used with accuracy. The LD50 value was expressed as micrograms
of extract per mouse body weight. All observations were systematically recorded, with individual
records being maintained for each mouse. Weight changes were calculated and recorded and at
the end of the test surviving animals were weighed by a sensitive digital weighing balance and
then humanely killed by cervical dislocation (OECD, 2001).

3.6.3. In vivo anti-rabies screening of crude extracts

Mice were randomly assigned into three treatment groups and two controls, six mice per cage,
and each mouse was used only for one experiment. The extracts were diluted before
administration to the mice using a 4% Polysorbate-80 solvent and sterile distilled water. Except
group I, all groups of mice were inoculated with PV virus strain. As described by Assefa Deressa
et al., (2010) after virus inoculation, the mice were allowed to stay in their respective cages for
about 1 hour so as to make them calm. Then, the diluted extracts were orally administered to the
mice in the treatment groups by using an intra-gastric needle based on the animal’s body weight
in 1ml vehicle via gauge. Briefly, Group I was negative control and received water as a placebo.
Group II was inoculated intracerebrally (30 μl) with 10 LD50 RV PV. Group III, IV and V were
inoculated intracerebrally (30 μl) with the challenge dose of 10 LD50 RV PV followed by the oral
administration (after 1 h) of the ethanol, methanol and water extracts (20 mg/ml) respectively, at
dose of 3000 mg/kg. Dosage was setup based on the acute toxicity assay result i.e. the highest
dose which showed the lowest toxicity would be selected. Volume administered was calculated
based on individual mouse body weight and 0.5 ml was the maximum volume administered.
Extracts were administered via the oral route using gavage and all the groups of mice were
observed for mortality up to 28 days.

30
 3.6.4. Determination of mean survival time
Anti-rabies activities of the selected medicinal plants were evaluated on mice survival period
compared to negative control group. Mortality rates as a result of rabies virus challenge were
determined by clinical signs and direct fluorescent antibody test (Petros Admasu et al., 2014).
The mice were monitored daily for the sign of paralysis and mortality for about 28 days after
inoculation of the virus. Death was recorded for each mouse in the treatment and control groups
throughout the follow up period on the mouse history cards. Confirmatory diagnosis of rabies
through direct FAT was conducted by opening of the skulls and collection of the brain of mice
was done according to the procedure specified by Dean and Albelseth, 1973. Briefly, a midline
incision was made on the dorsal surface of the head using scalpel and blade. The brain sample
consists of cerebellum, hippocampus and brain stem and any available brain tissues were taken
and an impression smears were made for direct FAT. Standardized protocol for the direct FAT
was carried out in accordance with the procedures described by Kissling, 1975. The number of
days each mouse survived was recorded for the mice in each group and mean survival time were
calculated using the formula (Nafiu et al.,2013).

3.6.5. Fluorescent antibody test

Fluorescent antibody test was done to confirm the presence of the virus. Brain impressions were
made upon microscope slides, which were fixed by acetone and incubated with fluorescein
isothiocyanate (FITC)-labeled antibodies to rabies virus. The stained impressions were viewed
using fluorescence microscopy (Mayes and Rupprecht, 2015). Briefly, the mice infected with
PV-RV were observed closely for 2 hours after treatment with different extracts. After that, the
mice were checked daily for 28 days to register clinical signs. The mice with rabies related signs
were killed humanely by placing them into the airtight plastic container. The skull was grasped
‘by forceps in the orbits, and the cranium was cut away by curved scissors or scalpel to expose
the brain. The brain was removed from the skull; a section of the brain just anterior to the
cerebellum was homogenized gently and very thin impression smears were made on slides. The
smears were allowed to air dry for 30 minutes and then fixed in acetone at -200c for about 1h and
air dried for about 5-10 minutes. A sufficient quantity of clarified conjugate (FITC) were added

31
on each smear and incubated at 37oc for 30 minutes. The smears were soaked in PBS, rinsed with
distilled water and air dried. Finally, the slides were examined under a fluorescence microscope.
When labeled antibody was incubated with rabies suspect brain tissue, it would bind to rabies
antigen (Hosseini and Asgary, 2015).

3.7. Screening for in vitro cytotoxicity and anti-rabies activities of crude extracts

3.7.1. Cytotoxicity assay

Evaluation of the cytotoxicity of ethanol, methanol and aqueous extracts towards the vero cells
was performed using MTT [3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide]
assay. Briefly, vero cells were seeded in a 96-well flat-bottom microtiter plate at a density of 2 ×
105 cells/ml and allowed to adhere for 24 h at 37°C in a CO2 incubator. After incubation, culture
medium was replaced with a fresh medium and different concentrations (0.4, 0.8, 1.6, 3.2, 6.4
and 12.8 mg/ml) of extracts were serially diluted in DMEM and added to each culture wells in
triplicate. To the control cells only 100 μl of the culture medium was added. The plates were
incubated for 3 days at 37°C with 5% CO2. Subsequently, the medium was removed and the cells
washed by phosphate buffer saline (PBS) followed by the addition of 100 μl of DMEM and 50μl
of MTT working solution (5mg/ml in phosphate buffer solution) to each well. The plates were
then incubated for 3h. After incubation, MTT was aspirated, and the formed formazan crystals
were solubilized by adding 50μl of DMSO per well, followed by gentle shaking for 15 min
(Fayyad et al., 2014). In this regard, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assay uses a water-soluble yellow tetrazolium salt (MTT), which is reduced to
an insoluble purple formazan by viable cells. The absorbance of dissolved formazan, quantified
spectrophotometrically, correlates with the number of intact viable cells. Finally, the intensity of
the dissolved formazan crystals (purple color) was quantified using the ELISA plate reader at
490 nm (Sandeepan et al., 2017). Percentage of viable cells was obtained by dividing the mean
absorbance at 490 nm of treated cells (for each concentration of extract) to the mean absorbance
of its control cells (Soltanian et al.,2017).

Cell viability (%) = Treated cells Mean OD/Control OD x 100%

The percentage of cytotoxicity is calculated as [(A-B) / A] x 100 where, A and B are the OD490
of untreated and of treated cells, respectively.

32
 3.7.2. In vitro anti-rabies assay
The in vitro antirabies activity of medicinal plant extracts (Sensel, Bisana and Gulo) were
evaluated by FAT assay. Vero cell lines were trypsinized and 2 × 105 cells/ml were seeded in a
96-well tissue culture micro plate and incubated at 37°C for 72 h. After the incubation period,
50μl of rabies virus suspension were added to confluent cell monolayers in a 96-well plate and
allowed to stand for 1h to enable virus adsorption. Thereafter, different concentrations (2mg/ml,
4mg/ml and 8mg/ml) of each extract based on cytotoxicity test result were added in triplicate
into all the wells with the exception of the negative control wells that contained only vero cells
and the virus control that contained an equal virus concentration but lacked the plant extract. The
plates were incubated at 37 °C in 5% CO2 humidified incubator for 72 h. The medium were
decanted and the cells were fixed by adding cold acetone (50µl per well) and kept at 4°C for 30
minutes. After discarding the acetone, cells were stained by direct polyclonal florescent-labeled
antibody for 30 minutes and then washed (3 times) with 0.01 M PBS, air dried, and visualized
under an inverted fluorescence microscope. Reading was qualitative, every well that shows
specific fluorescence was considered to be positive.

3.8. Ethical Clearance

Ethical approval for the study was sought from the college of Natural and Computational Science
Institutional Review Board (CNS IRB), Addis Ababa University. The animals were handled
according to the international guidelines for the care and use of laboratory animals (Clark et al.,
1997).

3.9. Data Analysis

Data were entered into an excel spreadsheet and then transferred to a statistical package for
social sciences (SPSS version 20). Student’s t test was used to compare means of treatment and
control groups and to evaluate the significance of observed differences between groups of mice
in the mean survival time (Days). Paired t test statistic was applied for body weight change of mice
in acute toxicity determination. The 50% cytotoxic (CC50), 50% inhibitory (IC50) concentrations
and selectivity index (SI) were calculated from concentration-effect curves after linear regression
analysis.

33
 4. Results and Discussion
4.1. Phytochemical study result of extracts

The property of extracting solvents significantly affects the phytochemical content, the higher
the polarity, the better the solubility of compounds. In the present study the result of qualitative
phytochemical analysis of all extracts from the three plants using different standard methods
showed positive results for alkaloids, but negative for saponins. The study also revealed the
presence of terpenoids in all the extracts except the ethanol extract from leaf of Justicia
schimperiana. One of the possible causes of negative results for saponins in all plant extracts
could be ecological factor. The type, content, and proportion of phytochemicals may vary
depending on ecological factors in areas where the plants are grown (Liu et al., 2015). In this
study, the ethanol, 80% methanol and water showed differential extraction of some of the
compounds within the same organ of the plant. For example, the methanol extracted most of the
bioactive compounds such as, alkaloids, flavonoids, phenols, steroids, terpenoids and tannins
while the ethanol extracted only alkaloids, steroids and tannins and the water extracted alkaloids,
flavonoids and terpenoids in the leaf of Justicia schimperiana. The variations in each solvent
extract may be due to degrading enzymes that may be active or denatured in any of the three
extractants. For example, the enzyme polyphenol oxidase, which degrade polyphenols in water
extracts, whereas in methanol and ethanol they are inactive (Tiwari et al., 2011).

The 80% methanol extract of the leaf of Justicia schimperiana showed the presence of alkaloids,
flavonoids, phenols, steroids; tannins and terpenoids, however, saponins were absent. From
previous studies it was confirmed that flavonoids and alkaloids (Mukherjee et al., 2017);
polyphenols, tannins, terpenoids and steroids (Limenew Abate and Tadesse Mengistu, 2018);
phenols, tannins, flavonoids, saponins, terpenoids, glycosides, and anthraquinones (Belay
Mekonnen et al., 2018); saponins, alkaloids and flavonoids (Jemal Abdela et al., 2014) were
present in the methanolic extract of Justicia schimperiana.
From the three plants studied, only Croton macrostachyus showed flavonoids in its three
extracts. Flavonoids have been reported to possess a wide variety of biological activities among
which are antimicrobial, anti-inflammatory, anti-allergic, antiviral, anticancer as well as
antidiarrheal properties (Middleton et al., 2000). The test of alkaloids, flavonoids, and terpenoids
gave positive results in the three extracts of the stem bark of Croton macrostachyus. The

34
Libermann Burchard test failed to show the presence of steroids in the ethanolic extract, but gave
a positive result with the methanolic and water extracts. Steroids are involved in diverse
physiological functions ranging from regulation of metabolism, behavior, and fertility to
inflammation and immune control (Libert and Dejager, 2014). Previously, it has been
documented that the leaves fruits and stems from Croton macrostachyus contains broad spectrum
of phytochemicals including alkaloids, aminoacids, saponins, flavonoids, steroids, triterpenoids
and tannins (Letha et al., 2016).

Presence of alkaloids, phenols, steroid, tannins and terpenoids were recorded in the three extracts
of Ricinus communis while flavonoids were recorded only in the methanolic extract.
Phytochemicals such as, tannins, saponins, terpenoids, flavonoids and alkaloids were detected in
methanol extract of the leaves of Ricinus communis (Suurbaar et al., 2017). Pandhure (2015)
identified alkaloids, flavonoids, steroid, terpenoids, cardiac glycosides and saponins in the leaves
and stem of Ricinus communis. Phytoconstituents detected in plant samples are shown in (Table
2).
Table 2: Different phytochemical components in the ethanol, 80% methanol and water extracts
of the leaves of Justicia schimperiana and Ricinus communis and the stem bark of Croton
macrostachyus plants

Plant name Solvent Secondary metabolites

Alkaloids Flavonoid Phenols Steroids Saponins Tannins Terpenoids
s
Justicia Ethanol + _ _ + _ + _
schimperiana Methanol + + + + _ + +
Water + + _ _ _ _ +
Croton Ethanol + + + _ _ + +
macrostachyus Methanol + + + + _ + +
Water + + _ + _ _ +
Ricinus Ethanol + _ + + _ + +
communis Methanol + + + + _ + +
Water + _ + + _ + +
+, the secondary metabolite was present; _, the secondary metabolite was absent.

35
 4.2. The result of acute oral toxicity test

The acute toxicity results showed no evidence of toxicity of the ethanol, 80 % methanol and
water extracts of Justicia schimperiana (leaf), Croton macrostachyus (stem bark) and Ricinus
communis (leaf) in mice administered at 1000 mg/kg, 2000 mg/kg and 3000 mg/kg. No
abnormalities were recorded at three doses with regard to food consumption, water intake and
body weight of the mice. This shows that the plant extracts could be well tolerated up to the dose
of 3000 mg/kg body weight of Swiss albino mice. It was also noticed that the oral LD50 of
Justicia schimperiana leaves aqueous extract (Andualem Tesfaye et al., 2016) and 80%
methanolic extract (Belay Mekonnen et al., 2018) was greater than 2000 mg/kg as there was no
mortality or remarkable signs of toxicity noted in the 14-day observation of the mice which were
used for the test. From previous studies it was confirmed that the aqueous extract of Croton
macrostachyus stem bark did not provoke death until the dose 16 g/kg during 7 days of
observation; whereas the organic extract caused general behavior, adverse effects and mortality
(Mbiantcha et al., 2013). However, it the present study, at higher doses, i.e. 4000 mg/kg and
5000 mg/kg, mice showed common signs of toxicity like low locomotion, weakness and erection
of hairs including death in the course of acute study. As a result, the LD50 of the extracts could
be greater than 3000mg/kg body weight. It suggested that the extracts may not be completely
safe at a dose higher than 3000 mg/kg. According to the Hodge and Sterner, (1943) toxicity
scale, the ethanol, methanol and aqueous extracts of Justicia schimperiana (leaf), Croton
macrostachyus (stem bark) and Ricinus communis (leaf) were placed in category IV (500 mg/kg-
5000 mg/kg, p.o.), and hence classified as slightly toxic.

The percentage of mice that died at each dose was transformed to probits using Finney’s method
(Appendix 7). According to Finney’s method the log dose at probit 5.0 (Log LD50) for the
ethanol, 80 % methanol and water extracts of Justicia schimperiana were found to be 3.60, 3.54
and 3.65 and hence, LD50 was calculated by taking antilog of the Log LD50 values of each extract
and found to be 4000mg/kg, 3500mg/kg and 4500mg/kg body weight respectively (Table 3). The
acute toxicity study in LD50 determination showed that 80 % methanol leaf extract of Justicia
schimperiana is more toxic than the ethanol and water leaf extracts of the plant. This might be
due to active ingredients responsible for toxic effects, which were more abundant in methanolic
extract of the plant leaf than in ethanolic and water extracts.

36
Table 3: Mice treated with Justicia schimperiana extracts, and responses in acute toxicity testing
Group Dose (mg/kg) Log Dose Death n (%) Probits
A1 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 3 (50%) 5.00
5000 3.70 5 (83%) 5.95
B1 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 6 (100%) 7.24
C1 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 0 (0%) 2.76
5000 3.70 4 (67%) 5.44
Where, A1= group of mice treated with ethanol extracts of Justicia schimperiana, B1= mice
treated with 80 % methanol extracts of Justicia schimperiana and C1= mice treated with water
extracts of Justicia schimperiana.

Similarly, the lethal dose (LD50) of the ethanol, 80% methanol and water extracts of the leaves of
Ricinus communis was also greater than 3000 mg/kg body weight of mice. The Log LD50 for the
ethanol and water extracts of Ricinus communis was found to be 3.54 but, its methanolic extract
was found to be 3.60. Thus, the LD50 values were found to be 3500 mg/kg for the ethanol and
water extracts and 4000 mg/kg for the methanol extract of the plant which were calculated by
taking antilog of 3.54 and 3.60 (Table 4). Therefore, the ethanol and water leaf extract of Ricinus
communisis more toxic than the methanol extract of the plant. At doses of 4000 mg/kg and 5000
mg/kg, there were significant differences (p=0.008) in changes in calculated body weights of test
animals compared to the control after acute administration of Ricinus communis leaf extracts.
The body weight gain after treated with 4000 mg/kg and 5000 mg/kg extract in treatment group was
significantly (p<0.05) increased compared with control group. The increase in body weight of the
treated animals with leaf extracts of Ricinus communis at 4000 mg/kg and 5000 mg/kg might be
due to the fact that Ricinus communis leaf extracts contain some phytochemical compounds with
nutritional factors, which increase the appetite, especially in high doses.

37
Table 4: Mice treated with Ricinus communis extracts, and responses in acute toxicity testing
Group Dose (mg/kg) Log Dose Death n (%) Probits
A3 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 5(83%) 5.95
B3 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 3 (50%) 5.00
5000 3.70 5 (83%) 5.95
C3 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 6 (100%) 7.24
Where, A3=group of mice treated with ethanol extracts of Ricinus communis, B3= mice treated
with 80 % methanol extracts of Ricinus communis and C3= mice treated with water extracts of
Ricinus communis.

The lethal dose (LD50) of the ethanol, 80 % methanol and water extracts of the stem bark of
Croton macrostachyus were also greater than 3000 mg/kg body weight of mice. The Log LD50
for the ethanol and 80% methanol extracts of Croton macrostachyus was found to be 3.54. Thus,
LD50 was calculated by taking antilog of 3.54 and found to be 3500 mg/kg body weight.
Whereas, the Log LD50 for the water extract was found to be 3.59 hence, the LD50 value was
3900 mg/kg (Table 5).

Table 5: Mice treated with Croton macrostachyus extracts, and responses in acute toxicity testing
Group Dose (mg/kg) Log Dose Death n (%) Probits
A2 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 5 (83%) 5.95
B2 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 5 (83%) 5.95
C2 1000, 2000, 3000 3, 3.30, 3.48 0 (0%) 2.76
4000 3.60 4 (67%) 5.44
5000 3.70 4 (67%) 5.44

38
Where, A2= group of mice treated with ethanol extracts of Croton macrostachyus, B2= mice
treated with 80% methanol extracts of Croton macrostachyus and C2= mice treated with water
extracts of Croton macrostachyus. A graph was plotted between probit vs. Log dose and LD50
values of extracts were confirmed by the graph which was the dose at probit 5.0, i.e. 50%
mortality.

Figure 6: LD50 values of 80% methanol extracts from the three selected plants

4.3. Cytotoxicity determination of plant extracts in vero cell line

Evaluation on vero cell lines by using MTT assay showed that the 50% cytotoxic concentration
(CC50) values of the ethanol extracts from three plants; Justicia schimperiana, Croton
macrostachyus and Ricinus communis were found to be above 12.8 mg/ml (Table 6).

39
Table 6: Effect of the ethanol extracts of three plants on vero cells

Concentration (mg/ml) Absorbance 490nm % cell viability % cytotoxicity

JS CM RC JS CM RC JS CM RC
A 12.8 0.28 0.27 0.25 62.2 60 55.5 37.8 40 44.5
B 6.4 0.29 0.28 0.33 64.4 62.2 73.3 35.6 37.8 26.7
C 3.2 0.31 0.34 0.35 68.9 75.5 77.8 31.1 24.5 22.2
D 1.6 0.31 0.37 0.36 68.9 82.2 80 31.1 17.8 20
E 0.8 0.32 0.39 0.42 71.1 86.7 93.3 28.9 13.3 6.7
F 0.4 0.36 0.42 0.44 80 93.3 97.8 20 6.7 2.2
G Control cells 0.45 0.45 0.45 100 100 100 0 0 0
Where, JS= Justicia schimperiana; CM= Croton macrostachyus; RC= Ricinus communis

The 50% cytotoxic concentration (CC50) value of the methanol extracts from Justicia
schimperiana and Croton macrostachyus were found to be 9.6mg/ml and 8mg/ml whereas,
Ricinus communis showed above 12.8 mg/ml (Table 7). In contradiction to the results from the
present study (CC50>12 mg/ml), Mukherjee et al., 2017 observed the median cytotoxicity (CC50)
of methanol extract of Ricinus communis in BHK_21 cells at 9.79 mg/ml.
Table 7: Effect of the 80% methanol extracts of three plants on vero cells

Concentration (mg/ml) Absorbance 490nm % cell viability % cytotoxicity

JS CM RC JS CM RC JS CM RC
A 12.8 0.21 0.22 0.24 46.7 49 53.3 53.3 51 46.7
B 6.4 0.23 0.21 0.25 51 46.7 55.5 49 44.5 44.5
C 3.2 0.24 0.23 0.28 53.3 51 62.2 46.7 49 37.8
D 1.6 0.26 0.25 0.30 57.8 55.5 66.7 42.2 44.5 33.3
E 0.8 0.31 0.28 0.31 68.9 62.2 68.9 31.1 37.8 31.1
F 0.4 0.34 0.36 0.33 75.5 80 73.3 24.5 20 26.7
G Control cells 0.45 0.45 0.45 100 100 100 0 0 0
JS= Justicia schimperiana; CM= Croton macrostachyus; RC= Ricinus communis

40
The 50% cytotoxic concentration (CC50) values of the aqueous extracts from three plants were
also found to be above 12.8mg/ml (Table 8).

Table 8: Effect of the water extracts of three plants on vero cells

Concentration (mg/ml) Absorbance 490nm % cell viability % cytotoxicity

JS CM RC JS CM RC JS CM RC
A 12.8 0.29 0.31 0.34 64.4 68.9 75.5 35.6 31.1 24.5
B 6.4 0.33 0.35 0.35 73.3 77.8 77.8 26.7 22.2 22.2
C 3.2 0.32 0.28 0.35 71.1 62.2 77.8 28.9 37.8 22.2
D 1.6 0.36 0.39 0.38 80 86.7 84.4 20 13.3 15.6
E 0.8 0.27 0.41 0.42 60 91.1 93.3 40 8.9 6.7
F 0.4 0.40 0.43 0.43 88.9 95.5 95.5 11.1 4.5 4.5
G Control cells 0.45 0.45 0.45 100 100 100 0 0 0
JS= Justicia schimperiana; CM= Croton macrostachyus; RC= Ricinus communis

Generally, the percentage viability was found to be increasing with decreasing concentration of
test extracts. The MTT assay results revealed that all extracts tested were non-cytotoxic and
exhibited CC50 values above the cut-off point which is 30µg/ml. An extracts can be considered
as non-cytotoxic if the CC50 is higher than 30µg/ml (Nondo et al., 2015). The MTT assay is
based on the reduction of MTT into formazan crystals by living cells. The reducing power is
mainly provided by NAD(P)H which is derived from dehydrogenase activity in mitochondria,
endoplasmic reticulum and plasma membrane (Stockert et al., 2012).

41
Figure 7: Microscopic photographs of vero cell lines treated with 80% methanolic extracts (6.4 mg/ml
left and 12.8mg/ml right) of the leaf of Justicia schimperiana.

4.4. Determination of 50% end titer

Titration of rabies virus (PV) was performed in vero cell line as well as in Swiss Albino mice
and the titer of 104 TCID50/ml and 105.6 LD50/0.1ml of RV PV respectively, were obtained.

4.5. Determination of in vivo antirabies activity

Group of mice infected with rabies virus but not treated with any of plant extracts showed 0%
survival rate and a mean survival period of 9.5days. However, oral treatment of mice infected
with rabies virus with ethanol, methanol and water extracts from the leaves of Justicia
schimperiana and Ricinus communis as well as the stem bark of Croton macrostachyus at a dose
of 3000 mg/kg significantly (p<0.05) increased the mean survival time compared to those of
negative control group (Table 9). Relatively higher mean survival time (22.16 days) was
obtained when methanol extract of Ricinus communis was administered to mice at a dose of 3000
mg/kg. This indicates that the methanolic extract from the leaf of Ricinus communis has some
more antirabies activity than others. The different extracts from the leaf of Justicia schimperiana
(p=0.018), stem bark of Croton macrostachyus (p=0.037) and leaf of Ricinus communis
(p=0.031) at 3000 mg/kg dose level, significantly (p<0.05) increased the survival period of mice.

42
The presence of some antirabies activity of the extracts may be due to the presence of multiple
classes of phytochemicals such as, alkaloids, flavonoids, phenols, steroids, tannins and
terpenoids. From the literature survey it was found that flavonoids have wide range of biological
properties such as anti-inflammatory, antibacterial, antiviral, anti-allergic, cytotoxic antitumor
properties. Alkaloids and phenolic compounds along with hypoglycemic, antidiabetic properties
also exhibit anti-inflammatory, antimicrobial and antioxidant effects (Bansode et al., 2015).
Terpenoids have been reported for its anti-inflammatory, anti-viral, anti-malarial, inhibition of
cholesterol synthesis and anti-bacterial properties (Wadood et al., 2013). Hence, the presence of
pharmacologically useful substances in the leaves of Justicia schimperiana and Ricinus
communis as well as stem bark of Croton macrostachyus confirm the claims and application of
such part of the plants in local treatment of ailments like rabies.

For the confirmation of antirabies properties of the plant extracts, FAT were done by taking only
one sample from each parameter per group of mice. One mouse from the negative control group
died within four days of inoculation with PV but, didn’t show any antigen against rabies virus
which indicate the death was due to accidental rather than the effect of the virus. Death from
samples taken from those at moribund state was due to the action of rabies virus PV strain
because all the samples showed a viral antigen but, most of the samples from survivors indicate
negative for antigen detection.

43
Table 9: Antirabies effect of plant extracts on mice survival time (n=6) at 3000mg/kg

Group Treatment Route of Survival FAT results

administration time/days Death Moribund Survivors/sac
(mean±SD ) within four mice rificed mice
days
I Distilled water Os 24±2.529 ND NM _
II PV IC 9.5±0.438 _ + NS
IIIA JS ethanol extract IC, Os 13.8±0.327 ND + +
IVA JS methanol extract IC, Os 14.3±0.413 ND + _
VA JS water extract IC, Os 13.3±0.413 ND + _
IIIB CM ethanol extract IC, Os 18±1.132 ND + +
IVB CM methanol extract IC, Os 21.3±0.413 ND + _
VB CM water extract IC, Os 21±1.78 ND + _
IIIC RC ethanol extract IC, Os 17.5±1.497 ND + _
IVC RC methanol extract IC, Os 22.16±2.287 ND + _
VC RC water extract IC, Os 20.16±1.468 ND + _
Where, I= Placebo administrated 1ml of dH2O; II=Negative control; JS=Justicia schimperiana;
CM= Croton macrostachyus; RC=Ricinus communis; IC= Intracerebral; Os= Oral; ND= No
death; NM= No moribund mice; NS= No survivor; - =No antigen detected and + = Antigen
detected

4.6. The in vitro antirabies activity of plant extracts

The antirabies activity for the leaves of Justicia schimperiana and Ricinus communis and the
stem bark of Croton macrostachyus against PV strain was summarized in (Table 10). Although,
differences were observed between antirabies activities of the extracts, each of the extracts tested
in the present study displayed antirabies activity against PV strain. These differences could be
due to variations in the type and nature of solvents (extractants), which can determine quantity
and diversity of phytochemicals to be extracted. Plant extracts from organic solvents (ethanol
and methanol) gave more antirabies activity compared to water extract. It can be attributed to the
presence of higher amounts of polyphenols as compared to aqueous extracts (Tiwari et al.,
2011).

44
Methanol extracts from all plant samples at a concentration of 8mg/ml showed a 100% RV-PV
inhibition. The half maximal inhibitory concentration (IC50) values ranged from 3mg/ml to
6mg/ml with the selectivity indices (SI) of each tested material above 2.13. The aqueous extracts
of all tested samples and the ethanol extract of Justicia schimperiana showed relatively the
highest IC50 values. The results exhibit that smaller the IC50 value higher the antirabies activity.
Ethanol extracts of Croton macrostachyus stem bark and Ricinus communis leaf as well as
methanol extract of Ricinus communis shows highest SI value against RV-PV with the value of
>4.27. The selectivity indices (SI = CC50/IC50) of Ricinus communis (leaf) methanol extract
(SI>4.27) was found to be higher than the methanol extracts of Justicia schimperiana (SI=3.2)
and Croton macrostachyus (SI=2.7). SI values less than one considered as weak, greater than one
moderate, and greater than three considered as good antiviral activities (Bagla et al., 2012).
The IC50 value and the selective index (SI) of ethanol, methanol and aqueous extracts of Justicia
schimperiana (leaf) were calculated and found to be 6mg/ml (SI>2.13), 3mg/ml (SI=3.2), and
6mg/ml (SI>2.13) respectively. The methanol extract exhibited smaller IC50 value when
compared to the other extracts. Hence, the methanol extracts of Justicia schimperiana leaf
contain antirabies active compounds with good activity whereas; its ethanolic and aqueous
extracts had shown a moderate antirabies activity against PV strain.
Croton macrostachyus stem bark showed 50% inhibition of RV-PV at 3mg/ml(ethanol and
methanol extracts) and 6mg/ml(aqueous extracts).The ethanol extract of Croton macrostachyus
stem bark contain antirabies active compounds with good activity (SI>4.27) whereas, the
methanol (SI=2.7) and aqueous (SI>2.13) extracts had shown a moderate antirabies activity
against PV strain.

The IC50 values of ethanol, methanol and aqueous extracts of Ricinus communis (leaf) was found
to be 3 mg/ml (SI> 4.27), 3mg/ml (SI> 4.27) and 6 mg/ml (SI>2.13) respectively. This indicates
that methanol and ethanol extracts had a good antirabies activity against PV strain while,
aqueous extract with moderate activity. However, Mukherjee et al., (2017), reported the
methanol extract of Ricinus communis exhibited the most effective activity against rabies with an
IC50 value of 19.70 µg/ml (SI=496.95), which is many times less than the value determined in
the present study (IC50=3 mg/ml and SI> 4.27). This may be due to the antirabies effect of the
extract depends on the type of cell line, the virus strain and the methodology used to evaluate
antirabies activity of the extracts.

45
Table 10: Cytotoxicity (CC50), inhibition concentration (IC50) and selectivity index (SI) of plant
extracts

Plant name Solvent CC50 IC50 SI

Justicia schimperiana Ethanol >12.8 mg/ml 6 mg/ml > 2.13
Methanol 9.6 mg/ml 3 mg/ml 3.2
Aqueous >12.8 mg/ml 6 mg/ml > 2.13
Croton macrostachyus Ethanol >12.8 mg/ml 3 mg/ml > 4.27
Methanol 8 mg/ml 3 mg/ml 2.7
Aqueous >12.8 mg/ml 6 mg/ml > 2.13
Ricinus communis Ethanol >12.8 mg/ml 3 mg/ml > 4.27
Methanol >12.8 mg/ml 3 mg/ml > 4.27
Aqueous >12.8 mg/ml 6 mg/ml > 2.13
SI: Selectivity index, CC50 value was divided by the IC50 value

The figure below shows the Green bodies (Negri bodies) in brain tissue observed by using
fluorescence microscope confirming the rabies infection.

Figure 8: Negri bodies in mice brain tissue infected with rabies virus

46
 5. Conclusions and Recommendations
5.1. Conclusions

The phytochemical analysis showed that the ethanol, methanol and water extracts of the leaves
of Justicia schimperiana and Ricinus communis as well as the stem bark Croton macrostachyus
contains a mixture of phytochemicals as alkaloids, flavonoids, phenols, steroids, tannins and
terpenoids but lack saponins. All the extracts are slightly toxic in animal model but non cytotoxic
in vero cell lines. All the Plant extracts had a moderate to good antirabies activity against PV
strain. The methanol plant extracts gave more antirabies activity compared to ethanol and water
extracts in mice.
5.2. Recommendations
❖ Further studies are necessary for isolation, identification and characterization of
biologically active substances that are more affordable, effective and safer alternative to
Imovax Rabies, Rab Avert rabies vaccine and others.
❖ Toxicity tests should be examined in major vital organs by histopathological examination
to setup the starting dose for antirabies activity.

❖ The antirabies activity of plant extracts was conducted by direct fluorescent antibody test
(DFA) method. However, according to the CDC, the DFA “can only be interpreted by
laboratory workers with special skills, extensive training, and a specific type of
microscope. Autolysed tissue samples can reduce the sensitivity of this test and often are
unsuitable for confirming the presence of rabies antigen. This makes the test hard to use
and access in resource-poor areas. RT-PCR is a suitable alternative for FAT in rabies
diagnosis as the cost incurred per diagnostic sample is lesser than that of FAT (Babu et
al., 2012). Therefore, it is recommended to carry out antirabies tests by PCR-based tests
as it is simpler and more precise than direct fluorescent antibody testing, and could be
considered in place of the current standard test, the DFA.

47
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 7. Appendices
Appendix 1: Drying the plant samples

Appendix 2: Weighting and shaking samples at 130 rpm

Appendix 3: Filtration and condensation process

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Appendix 4: phytochemical screening

Appendix 5: Animal (mice) management throughout experimentation period

Appendix 6: Postmortem examination of mice brain

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Appendix 7: Titration of Rabies virus in vivo by intracerebral inoculation

Microbiological safety cabinet, sterile pipettes (10ml and 1ml), a tray with ice flasks, rabies virus
suspension (PV strain, stored at –80°C), mice (Swiss albino) 3-4 weeks old, cages for housing
mice, one ml syringe and needle for I/C inoculation, test tubes, sterile PBS pH 7.4 and sterile
bovine (Calf) serum, were used in the in vivo rabies virus titration test.

Procedure:

Working in microbiological safety cabinet, the diluting fluid which is PBS containing 2%
serum was prepared and 9ml in test tubes labeled 10-1 to 10-7 were dispensed and the test
tubes in rack were kept immersed in plenty of ice. To make 10 fold (log) dilutions of the
virus material, 1ml of virus in 9ml of diluent was diluted to get the initial dilution i.e.
10-1. Subsequently 1ml of previous virus dilution was transferred to next dilution by
using at each step a fresh pipette, to achieve serial tenfold dilutions. 0.03 ml of each
virus dilution was inoculated intracerebrally into mice, starting from the highest dilution
(in this case 10-7). At least 6 mice per dilution were used and transferred these into cages
appropriately labeled. The mice were observed for 14 days. Any death occurring within first 5
days should be considered non-specific (due to stress/bacterial infection etc). Observe for
specific signs and symptoms of rabies. Note down total number of specific deaths in each
dilution and calculate the virus titre by using Spearman-Karber formula.

Table 1: Calculation of virus titre in mice using the Spearman Karber method

Dose Mice

Inoculated Died Survived Cumulative Cumulative Cumulative Cumulative

dead alive mortality ratio % mortality
10-1 6 6 0 31 0 31/31 100
10-2 6 6 0 25 0 25/25 100
10-3 6 6 0 19 0 19/19 100
10-4 6 6 0 13 0 13/13 100
10-5 6 4 2 7 2 7/9 78
-6
10 6 2 4 3 6 3/9 33
10-7 6 1 5 1 11 1/12 8
The LD50 lies between the LD78 (10-5) and the LD33(10-6)

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Spearman-Karber method

Log10 50% end point dilution = - (x0 - d/2 + d ∑ ri/ni)

X0 = log10 of the reciprocal of the highest dilution (lowest concentration) at which all animals
are positive;

d = log10 of the dilution factor;

ni = number of animals used in each individual dilution (after discounting accidental deaths);

ri= number of positive animals (out of ni).

Summation is started at dilution x0.

To determine the LD50, first calculate the proportion distance (PD):

X0 = 4; d = 1; log10 of 50% endpoint dilution = - [4 - ½ + 1 (13/6)] = -5.6;

50% end point dilution = 10-5.6; the titre of the virus = 105.6 LD50/0.1ml

Appendix 8: Calculation of median lethal dose (LD50) of plant extracts in Swiss Albino mice

Median LD50 is usually an initial screening step in the assessment and evaluation of the toxic
characteristic of a substance. It is the amount (dose) of an extract, which produce death in 50%
of the population of test animals to which it is administered by any of the methods such as oral,
dermal inhalation or intravenous. Determination of this test examines the relationship between
dose and the most extreme response-death. The more potent or toxic the chemical (extract),
lower the LD50 and the smaller the dose needed to cause death. LD50 figures are frequently used
as a general indicator of a substance’s acute toxicity (Chandra et al., 2014). The percentage of
mice that died at each dose was transformed to probit using Finney’s method (Table 2).

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Table 2: Transformation of percentage mortalities to probits

% 0 1 2 3 4 5 6 7 8 9

0 - 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66

10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12

20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45

30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72

40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97

50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23

60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50

70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81

80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23

90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33

Source: (Yadav and Trivedi,2016)

According to the methods of Finney and Miller-Tainter, Probits values for mortality of 0% and
100% at n=6 was found to be 2.76 and 7.24 respectively.

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Appendix 9: Ethical clearance certificate

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Advisors´ Approval Sheet

This is to certify that the thesis entitled “Phytochemical Screening, Acute Toxicity and Anti-
Rabies Activities of Extracts of Selected Ethiopian Traditional Medicinal Plants” submitted in
partial fulfillment of the requirements for the Degree of Master of Science in Applied
Microbiology and has been carried out by Yeweynshet Tesera Tewabe, under my supervision.
Therefore, I recommend that the student has fulfilled the requirements and hence hereby can
submit the thesis proposal to the Department.

Asnake Desalegn (PhD) ___________________ ___________________

Name of Advisor Signature Date

65