debre berhan university
TRANSCRIPT
ደብረ ብርሃን ዩኒቨርሲቲ የተፈጥሮና ኮምፒውቴሽናል ሣይንስ
ኮሌጅ ኬሚስትሪ ትምህርት ክፍል
Debre Berhan University
College of Natural and
Computational Sciences
Department of Chemistry
MSc Thesis
Comparative evaluation of the phytochemicals and antibactrial
activities crude extracts of the C.macrostachyus and S.incanum
plants
A Thesis submitted to the School of graduate studies, Debre Berhan
University in Partial Fulfillment of the Requirements for the Degree
of Master of Science in Organic Chemistry
BY
Shewaferaw Mengesha
ADVISOR
Abebe Tedla Mengstie, Ph. D.
Feb. 2021
Debre Berhan, Ethiopia
Approval Sheet
As a member of the board of examiners of Master of Science thesis defense examination, we
have read and evaluated the thesis work done by shewaferaw mengesha entitled “Comparative
Evaluation of the Phytochemicals and Antibactrial Activities crude Extract of
Croton Macrostachyus and Solanum Incanum plant parts”. We here by certify that the
thesis is accepted for fulfillment of the requirements for the award of the degree of Master of
Science (MSc) in chemistry from Department of Chemistry, College of Natural and
Computational Sciences, Debre Berhan University.
APPROVED BY BOARD OF EXAMINERS
1. _________________ ____/_____/_____
External Examiner Signature Date
2. _________________ ____/_____/_____
Internal Examiner Signature Date
3. _________________ ____/_____/_____
Chairperson Signature Date
4. Abebe Tedla (PhD) ________________ ____/_____/_____
Advisor Signature Date
Declaration
I, the undersigned, declare that this thesis is my original work and has been submitted in partial
fulfillment of the requirements for the degree of masters of Science in organic chemistry at Debre
Berhan University. All sources of materials used for this thesis have been duly acknowledged. This
paper has never been submitted to and/or presented in any other university, college or institution in
candidature of any other degree, diploma, or certificate.
Name: shewaferaw mengesha
Department: Chemistry
Signature………………………….
Date: …………………………..
ii
List of Abbreviations C. macrostachyus Croton macrostachyus
Con. H2SO4 Concentrated Sulfuric acid
Croton spp. Croton Species
DMSO Dimethyl sulfoxide
FeCl3 Iron chloride
H2SO4 Sulfuric acid
MHA Mueller-Hinton Agar
MHB Mueller-Hinton Broth
MDR Multi-Drug Resistant
TM Traditional Medicine
S. aureus Staphylococcus aureus
E.coli Escherichia coli
L. monocytogenes listeria monocytogenes
S.epidermidis Staphylococcus epidermidis
E.faecalis enterococcus faecalis
S. pyogenes streptococcus pyogenes
S. typhimurium salmonella typhimurium
S.sonnei shigella sonnei
P.aeruguinosa pseudomonas aeruguinosa
S.enteritidis salmonella enteritidis
C.albicans candida albicans
CM1 steam bark of croton macrostachyus
CM2 leaf of croton macrostachyus
CM3 root of croton macrostachyus
SI1 seed of solanum incanum
SI2 root of solanum incanum
SI3 leaf of solanum incanum
iii
Acknowledgments
First and foremost, I would like to thank almighty God for all giving me strength, peace and Well
being throughout this work. Then, I would like to thank to my mother, W/o gurdo werke, Next to
this, I would like to extend my gratitude and sincere thanks to my advisor Dr.Abebe Tedla for his
valuable guidance, continuing advice, moral and material supports for the accomplishment of this
work. In addition, I would like to thanks Dr. Minbale Gashu for giving continuing advice, material
supports and also extracting of my samples in Debreberehan University.
I would like to thanks AbuneMelketsadiq institution for material supports to achievable this sweet
result. I would like to thanks to my friends Temesgen Bires, Aregehagn gizaw and Abdu Mekonin
by sharing ideas for synthesis plant for this study and material supports. Last but not least, I would
like to thanks my parents and my family especialy for my wife ‘Meron Asefa’for giving me their
support and always encourage me emotionally and financially.
iv
Contents
Declaration .......................................................................................................................................... i
List of Abbreviations ......................................................................................................................... ii
Acknowledgments............................................................................................................................ iii
List of Tables .................................................................................................................................... vi
List of Schemes ................................................................................................................................ vii
List of figure .................................................................................................................................. viii
Abstract ............................................................................................................................................. ix
1. Introduction .................................................................................................................................... 1
1.1 Background of the study .................................................................................................................... 1
1.2 Statement of the Problem ................................................................................................................... 5
1.3 Significance of the Study ................................................................................................................... 5
1.4 Objective of the Study ........................................................................................................................ 6
1.4.1 General Objectives ............................................................................................................ 6
1.4.2 Specific Objectives ............................................................................................................ 6
2. Review of Literature’s ................................................................................................................... 7
2.1 The History of Medicinal Plant Use ................................................................................................. 7
2.2 Medicinal plants in Ethiopia.............................................................................................................. 7
2.3. The Genus Croton and solanum..................................................................................................... 10
2.3.1 The Genus Croton ............................................................................................................ 10
2.3.2 Medicinal Importance of genus Croton ........................................................................... 10
2.4 The Genus solanum .......................................................................................................................... 11
2.4.1 Medicinal Importance of genus Solanum ........................................................................ 12
2.5 chemical composition and Bioactive Compounds from genus Croton and Solanum .......... 13
2.5.1 Chemical composition and Bioactive Compounds from genus Croton .......................... 13
2.5.2 Chemical composition and Bioactive Compounds from genus Solanum ........................ 14
2.6 Botanical Description and Distribution of C.macrostachyus and S.incanum ........................... 14
2.6.1 Botanical Description and Distribution of C.macrostachyus .......................................... 14
2.6.2 Botanical Description and Distribution of S.incanum ..................................................... 16
2.7 Medicinal Value of C. Macrostachyus and S.Incanum ............................................................... 16
2.7.1 Croton Macrostachyus ..................................................................................................... 16
2.7.2 Medicinal properties of C. macrostachyus ...................................................................... 17
2.7.3 Solanum Incanum ............................................................................................................ 17
2.7.4 Medicinal properties of Solanum Incanum ...................................................................... 18
v
2.8 The Chemical Compositions and Biological Activities of C. Macrostachyus and S.Incanum
................................................................................................................................................................... 18
2.8.1 The Chemical Compositions and Biological Activities of C. Macrostachyus ................ 18
2.8.2 The chemical compositions and biological activities of S.incanum ................................ 22
2.10 Crude Extract from genus croton and solanum .......................................................................... 23
3. Materials and Methods ................................................................................................................. 25
3.1 Study Design and Period .................................................................................................................. 25
3.2 Collection of Plant Material ............................................................................................................ 27
3.3 Study area .......................................................................................................................................... 27
3.4 Apparatus, Instruments, Chemicals, Reagents and Media .......................................................... 27
3.4.1 Apparatus and Instruments .............................................................................................. 27
3.4.2 Chemicals, Reagents and Media ...................................................................................... 28
3.5 Preparation of Plant Material .......................................................................................................... 28
3.6 Extraction of plant material ............................................................................................................. 28
3.6.1 Extraction with ethanol .................................................................................................... 29
3.7. Phytochemical Screening Tests ..................................................................................................... 29
3.8 Antibacterial Test of the crude Extracts ........................................................................................ 30
3.8.1. Microorganisms Used, Preparation of Test Solution and Inoculum Preparation ........... 30
B. Preparation of Test Solution ................................................................................................ 31
3.8.3 Antibacterial Activity Test .............................................................................................. 31
4. Results and Discussion ................................................................................................................ 32
4.1. Phytochemical screening test ......................................................................................................... 32
4.2. Antibacterial activity test ................................................................................................................ 34
4.2.2 S. incanum ....................................................................................................................... 41
5. Conclusion and Recommendations .............................................................................................. 52
5.1 Conclusion ......................................................................................................................................... 52
5.2 Recommendations ............................................................................................................................. 53
6. Reference ..................................................................................................................................... 54
7. Appendix’s ................................................................................................................................... 68
vi
List of Tables Table 1: secondary metabolites from croton macrostachyus. .......................................................... 20 Table 2: secondary metabolites from Solanum incanum. ................................................................ 23 Table 3: Phytochemical constituents of ethanol extract of C.Macrostachyus ................................. 32
Table 4: Phytochemical constituents of ethanol extract of S.incanum ........................................... 33 Table 5: Phytochemical constituents of ethanol extract of C.macrosachyus and S.incanum ......... 34 Table 6: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the stem bark of Croton macrostachyus Against
11 microorganisms. .......................................................................................................................... 35 Table 7: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the leaf of Croton macrostachyus against 11
microorganisms. ............................................................................................................................... 37
Table 8: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the root of Croton macrostachyus against11
microorganisms. ............................................................................................................................... 39 Table 9: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the seed of Solanum incanum against 11
Microorganisms. .............................................................................................................................. 41 Table 10: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the root of Solanum incanum against 11
Microorganisms. .............................................................................................................................. 43
Table 11: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the leaf of Solanum incanum against 11
Microorganisms. .............................................................................................................................. 45 Table 12: Results from disc diffusion assay showing the evaluation of antibacterial
activity(Diameter of the Inhibition Zone, mm) the ethanolic crude extract the leaf of
C.macrostachyus and the ethanolic crude extract the root of S.incanum against Staphylococcus
aureus (Sa) (ATTC 25923) Bacteria. ............................................................................................... 51
vii
List Of Schemes
Scheme 1: Chemical stracture of secondary metabolite isolated from Croton spp.(1-4) ………...10
Scheme 2: Chemical stracture of secondary metabolite isolated from Solanum spps(5-8)……….12
Scheme 3: Some chemical stracture of bioactive compound isolated from Croton and solanum
spp (9-14)……………………………………………….....……………………...……………….13
Scheme 4: Chemical structure of some bioactive compound isolated from the
C.macrostachyus leaf, root and steam bark (15-32)…………………………….…………21
Scheme 5: Chemical structure of some bioactive compounds isolated from solanum incanum
Seed, leaf and root (33-37)……………………………………….………………………………...22
viii
List of figure
Figure 1: Photograph of Croton macrostachyus taken from welkitie abunemelketsediq primary
and secondary school, welkitie, Ethiopia in April 2020 ......................................................... …..16
Figure 2: Photograph of solanum incanum taken from welkitie abunemelketsediq primary and17
Secondary school, welkitie, Ethiopia in April 2020 ................................................................. …17
Figure 3: Flow chart of the study design ................................................................................ ….26
ix
Abstract Croton macrostachyus and solanum incanum are the most common traditional medicinal plants
used in Ethiopia. The parts of these plant Croton macrostachyus plant parts (stem bark, leaves and
roots) and solanum incanum plant part (seed, leaf and root) showed varieties of medicinal
properties; cure various human, animal diseases and ailments.C. macrostachyus have medicinal
value in treatment of wound, abdominal pain, toothache, malaria, rabies, gonorrhea, diarrhea,
hepatitis, jaundice, cancer, typhoidand gastrointestinal disorder and bioactive compounds which
presents including terpenoids, alkaloids, flavonoids, and cumarines.S. incanum has different
bioactive substances which have medicinal importance against skin diseases, treatment of
dandruff, chirt, wart and ringworm, foot rot, abdominal pains, fever, stomachaches, wounds, sore
throat, ear inflammation, liver disorders and treatment of cow driosis, dermatophilosis,
pasteurellosis, black leg, fasciolosis and snake bite and Bioactive compounds which present in S.
incanum are including tannins, flavonoid, phenol, cumarins and terpenoids. The Phytochemical
composition of C. macrostachyus of the leaf, root, stem bark and S.incanum of the seed, leaf and
root extracts were qualitatively assessed using standard procedures in these study. In the present
study, the crude extract from the Croton macrostachyus and solanum incanum were extracted by
maceration. Antibacterial activities of ethanol extracts Croton macrostachyus plant parts of leaf,
root, stem bark and solanum incanum plant parts of seed, root and leaf were screened against
listeria monocytogenes, Staphylococcus epidermidis, enterococcus faecalis,Staphylococcus
aureus, streptococcus pyogenes, salmonella typhimurium, shigella sonnei, Escherichia coli,
pseudomonas aeruguinosa, salmonella enteritidis and candida albicans.These were carried out by
the disc diffusion method on Mueller – Hinton agar (MHA).From those parts of the plants, the
Solanum incanum root ethanol extract showed more antibacterial activity than the other parts
against Staphylococcus aureus (Sa) bacteria with mean zone of inhibition 11.5 ± 0.70 mm.
indicating that Solanum incanum root ethanol extract may had variety of bioactive compounds than
others extracts. This result confirmed its ethnomedicinal use in the treatment of microbial
infections.
1
1. Introduction
1.1 Background of the study
Diseases are the major causes of death in the developing countries and accounts to 50% of
it.The extensive use of the antibiotics to control these diseases has led to the emergence of
multidrug resistance [1].
Nature has been a source of medicinal agents for thousands of years and an impressive number
of modern drugs have been isolated from natural sources; many of these isolations were based
on the uses of the agents in traditional medicine. This plant-based, traditional medicine system
continues to play an essential role in health care, with about 80% of the world’s inhabitants
relying mainly on traditional medicines for their primary health care [2].
Plants are invaluable and fundamental to almost all life on earth. They provide wide range of
uses to human beings such as medicine, food, shelter, clothing, fuel wood for cooking, timber
for construction, utensils, as well as fodder for cattle. They also recycle essential nutrients of
ecosystems, establishing soils and maintaining soil fertility in addition to protecting areas of
water catchments. Moreover, they keep ecological and climatic balance, facilitate, and control
rainfall through the process of evaporative transpiration. All our food comes from plants either
directly or indirectly. Plants have played a significant role in maintaining human health and
improving the quality of human life for thousands of years. The rural people live in the area
where plants are naturally growing have remarkable knowledge of the uses of plants growing
around them. Their livelihood depends on plant availability and their plant use knowledge,
which they gain from their previous generation.
According to World Health Organization, medicinal plants would be the best source to obtain
a variety of drugs. Therefore, such plants should be investigated to better understand their
properties, safety and efficacy [3].Plant products and their active constituents played an
important role in plant disease control by combating growth and development of pathogens
and including resistance in plants. Plant based natural constituents can be derived from any
part of the plant like bark, leaves, flowers, roots, fruits, seeds etc [4].As these plants and their
products are known to possess various secondary metabolites, Which showed significant
inhibitory effect against the growth of pathogens, therefore, the plant and their products should
be utilized to combat (fighting) the disease causing pathogens.Thus the wide spectra of
2
antimicrobial research are geared towards the discovery, development and antibacterial
activities. The demand for plant based medicines, health products, pharmaceuticals, food
supplement and cosmetics etc. are increasing in both developing and developed countries.
Since these natural products are non-toxic, have less side effects and easily available at
affordable prices they are recognize worldwide [5].
Plant crude extracts have been long known to have beneficial properties but have recently been
investigated for their use as antimicrobials activities. Antimicrobial activities are a chemical
that kill or inhibits that growth of microorganisms.Plant crude extracts are derived from
various types of plant material such as the flowers, seeds, bark, wood, buds, fruits, and roots.
These crude extracts are obtained through various methods such as maceration, hydro
distillation, steam distillation, soxhlate extraction, and solvent extraction [6].Compounds
found in crude extracts include phenols, polyphenols, terpenoids, flavonoids, flavones,
flavonols, tannins, quinones, coumarins, alkaloids, sapponins, lectins, and polypeptides
[7].According to [6], these compounds can be analyzed using techniques such as mass
spectrometry (GC-MS) and chromatography techniques.
The World Health Organization (WHO) reported that 80% of the emerging world’s population
relies on Traditional medicine for therapy. Traditional medicinal plants are widely used in
different part of the world for curing diseases. For instance, in China, about 30 to 50% of the
total medicinal consumptions were obtained from traditional herbal preparations. In Africa,
Ghana, Mali, Nigeria and Zambia, the first line of treatment for 60% of children; in Ethiopia
up to 80% of the population uses traditional medicine due to the cultural acceptability of
healers and local pharmacopeias, the relatively low cost of traditional medicine and difficult
access to modern health facilities [8]. Traditional medicine is an ancient medical practice,
which exists, in human societies before the application of modern science of health. It is the
sum total of knowledge, skills and practices on holistic healthcare, which is recognized and
accepted by the community for its role in the maintenance of health and the treatment of
diseases. Traditional medicine is based on indigenous theories, beliefs and experiences that are
handed down from generation to generation [9].Medicinal plants play a crucial role in the
search for alternative antimicrobial components. The literature indicates that medicinal plants
have secondary compounds that are great importance in human life in terms of acting as
antioxidants, anti-inflammatory and antimicrobial effects [10].
3
Research findings also support the idea that many plants are used in the treatment of various
diseases whose symptoms might involve microbial infection leading to the discovery of novel
bioactive compounds [11-13].In most African countries including Ethiopia, medicinal plants
have a major role to control microbial disease; like as bacteria, fungus, virus, cancer e.t.c.
The plants provide the basic nutrients needed for the growth of animals and humans like
proteins, carbohydrates, fats, vitamins and oils minerals. The phytochemicals are majorly
classified as primary and secondary metabolites. The primary metabolites are responsible for
the basic development of the plant which includes the sugars, amino acids, proteins, nucleic
acids, chlorophyll etc. Secondary metabolites are those which are needed for the survival of
the plants in a harsh environment. They forms the smell, color and taste of the plants and
secondary metabolites such as flavonoids, tannins, saponins, alkaloids, steroids, phytosterols
are found to have other commercial applications like they can be used as coloring agents, as
drugs as flavouring agents, insecticides, pesticides, anti-bacterial and antifungal products.
Moreover they can also be used to protect humans from many diseases like cancer, diabetes,
cardiovascular diseases, arthritis and aging etc.
Medicinal plants are used by almost 80% of the world’s population for their basic health care
because of their low cost and ease in availability. From the dawn of civilization, people have
developed a great interest in plant-based drugs and pharmaceutical products [14]. Some
medicinal plants have been used in the production of various drugs singly or in combination
and even as principal raw material for the production of other conventional medicines [15].
Medicinal plants are great role in care of primary health of humans and animals due to its
biological and medicinal activities, high safety margins and ability to overcome drug
resistance action of pathogens [16-17].They are an important source of traditional drugs,
modern medicines, folk medicines, nutraceuticals, pharmaceutical intermediates and entities
for synthetic drugs since plant extracts contain secondary metabolites like alkaloids,
flavonoids, saponins, tannins and glycosides which are used against different diseases and
relieve pain [18].they have been used to treat various health ailments for a long period of time
in different countries. Natural products have been playing dominant role in drug discovery
efforts for treatment of human and livestock diseases [19].With the upsurge(strong rise) in the
use of plants medicines, a through scientific investigation of these plants is imperative
(requiring) based on the need to validate their folklore use. Many studies have shown the
therapeutic activities of secondary metabolites obtained from medicinal plants [20-21].
4
In the last few decades, many bacterial organisms have continued to show increasing
resistance against current antimicrobial agents [3].Herbal drugs play an important role in health
care programs in world wide. The medicinal value of the herbs lies in their phytochemical compounds
which produce distinct physiological actions on the human body. The active components of herbal
remedies have the advantage of being combined with many other substances that appear to be inactive.
However, these complementary components give the plant as a whole a safety and efficiency much
superior to that of its isolated and pure active components [22].
Croton and Solanum species are among the most common traditional medicinal plants used in
Africa, Asia, and South America for treatment of diabetes [23], digestive problems [24],
Malaria [25-26], insomnia and head-ache [27], hemorrhoids and ulcers [28].The genus has
been reported to have a number of biological activities for instance anti-hypertensive, anti-
inflammatory, antimalarial, anti-viral [29-31] and angina, colic or indigestion, dandruff, fever,
general infection, headache, liver pain, painful menstruation, skin diseases, snake bites, sore
throat, stomach ache or abdominal pain, and wounds[32-33].Triterpenoids, either pentacyclic
or steroidal, volatile oils containing mono and sesquiterpenoids, shikimate-derived
compounds, phenolic and alkaloids, tannins, saponins, flavonoids compounds are among
secondary metabolites reported from the genus of Croton and solanum.
In guragae Zone, SNNP Regional state, south Ethiopia Croton macrostachyus and solanum
incanum have many traditional applications in human beings and livestock. The fresh leaves
of Croton macrostachyus attached to the wound which to form like water fluid and the flow of
fluid is abolition (destruction) and the juice of the leaves given to Cattle’s for the stomach
ache.And also people around this area the leaf of C. macrostachyus also used for the treatment
of the diseases in Amharic known as ‘chirt’ always displayed on the body of human beings.
The root of C.macrostachyus and S.incanum used to brush for teeth to kill bacteria, this
indicates due to the presence of phytochemicals. Solanum incanum is another medicinal plant
which used for treatment of disease when the human body part form wounds which carry fluid
water treated or covering the wound by fresh leaves and treatment of dandruff. The seed used
for tretement of abdominal pains and foot rot. The root which used for tretement of skin
disease and also used as teeth brush.
5
1.2 Statement of the Problem
The use of natural products for curing the symptoms of microbial and for manufacturing the
anti-microbial agents has been extensively discussed in academic resources. There are many
natural plants from which essential ingredients and components are extracted in order to cure
(treated) many of the diseases that are found today [34].In particular, the plant family of
Euphorbiaceae(Croton macrostachyus) and Solanaceae(solanum incanum) are known as
traditional medicinal plants and widely used to treat many infectious diseases in some
countries especially in Ethiopia. Many studies reported parts of this plants exhibit or show
different antibacterial activities and also there are many secondary metabolites isolated from
this plant parts or materials, but based on this result there is no more done on their comparison
and evaluation on antibactrial activities of these each plant parts. Due to these reason this
study was intended to deal with evaluation of antibacterial activity and comparison of
phytochemical analysis based on their experimental result data of the C.macrostachyus and
S.incanum plant parts of ethanol crude extracted.
1.3 Significance of the Study
Natural plant crude extract obtained from leaves, roots, flowers and fruits have wide
applications in pharmaceutical, foods, perfumery and cosmetics and in the scenting of soaps,
detergents, shampoos, lotions and antibacterial activities [35-36].C. Macrostachyus and S.
incanum species are an excellent source of crude extract. They grow abundantly in Ethiopia
and can be used as natural sources of essential oils for commercial applications. Therefore, the
results of this study can help people to aware about the plants to have the knowledge of the
antibacterial activity of the parts of the plant C. macrostachyus and S. incanum and to isolate
the compound exhibiting antibacterial activity and baseline information for further studies.
6
1.4 Objective of the Study
1.4.1 General Objectives
Comparative Evaluation of the Phytochemicals and Antibactrial Activities of Extract the
Croton Macrostachyus and Solanum Incanum plant based on experimental data.
1.4.2 Specific Objectives
To extract leaf, root and steam bark of C. macrostachyus and leaf, root and seed of
S.incanum using organic solvent (ethanol) by maceration method.
To carry out chemical test (phytochemical screening) of ethanol extract leaf, root and
steam bark of C. macrostachyus and leaf, root and seed of S.incanum in order to
determine the presence of secondary plant metabolites.
Compare the phytochemical screening among crude extracts of both medicinal plant
parts.
To test antibacterial activities of the crude ethanol extracts of the leaf, root and steam
bark of C. macrostachyus and leaf, root and seed of S.incanum plants against eleven
bacteria’s using disk diffusion method.
To evaluate the antibacterial activities among crude extracts of all medicinal plant
parts.
7
Review of Literature’s
2.1 The History of Medicinal Plant Use
Plants have been an integral part of life in many indigenous communities [37] and more so for
curative purposes. Before the advent of conventional medicine, man depended on plants for
their healing properties [38-41]. Phytomedicines derived from plants have shown great
promise in the treatment of intractable infectious diseases including opportunistic AIDS
infections [42]. The use of and search for drugs and dietary supplements derived from plants
have accelerated in recent years [7].
Ethnopharmacologists, botanists, microbiologists, and natural-products chemists are surveying
the earth for phytochemicals and "leads" which could be developed for treatment of infectious
diseases. Plants are a very important source of nutrients and a very important part in the
human diet. They provide us carbohydrates, protein, vitamins, cholesterol lowering
compounds, antioxidants and other important sources of biologically active substances. Many
nutritional values of plants have been discussed in the literature but there is very limited
research in the biologically active compounds that are present in them. These biologically
active compounds are called as phytochemicals. These phytochemicals are derived from every
part of the plant including roots, stem, leaves, flowers, fruits, seeds and sometimes used as
such in some cases they form the raw materials for a variety of other medicinally important
compounds.
The World Health Organization estimates that around 80% of the world population in
developing countries relies on traditional plant medicines for primary healthcare needs, of
which a major proportion corresponds to plant extracts or their active principles [43]. Most of
the currently available drugs for treatment of different human and animal diseases obtained
from natural products especially medicinal plants [44].Microorganisms are frequently a cause
of prevailing diseases, presenting a serious public health issues in a significant segment of the
population as showed by both private and official health care systems [45].
2.2 Medicinal plants in Ethiopia
Ethiopia is a country characterized by a wide range of climate and ecological condition,
Possesses enormous diversity of flora and fauna [46]. The country possesses a wide range
potentially useful medicine plants, more extensive indeed than available in many other parts of
the world [47].
8
Popular knowledge of plant used by human is based on thousands of years of experience by
trial and error people learn how to recognize and use plants, including those with a magic-
religious function. In Ethiopia, even though the traditional medicinal practitioners are the best
sources of information secret, only to be passed orally to their older son, at their oldest age
[48].Due to its long period of practice and existence, traditional medicine has become an
Integral part of the culture of Ethiopian people [49]. According to [50], there is a large
magnitude of use and interest in medicinal plant in Ethiopia due to acceptability and bio
medicinal benefits. In Ethiopia the long history use of medicinal plants is reflected in various
medico –religious manuscripts produced on parchments and believed to have originated
several centuries ago [51]. Medicinal text books written in Geez and Arabic in Ethiopia
between the 17 & 18 centuries employs that plants have been used as a source of traditional
medicine in Ethiopia health care system. In Ethiopia little emphasis has been given to
traditional medicinal studies over the past decades [52].
Traditional medicine is an ancient medical practice that is still widely used in prevention and
treatment of various health problems in Ethiopia. The use of traditional medicine is still
widespread in Ethiopia, and its acceptability, availability and popularity is no doubt as about
90% of the populations use it for their health care needs. Ethiopia is a Centre of diversity for a
number flora and fauna the sixth centers of biodiversity in the world. The country is endowed
with rich flora, having more than 6,500 species of vascular plants out of which an estimated
12% are endemic and about 887 species are used as medicinal plants. The majority (80%) of
Ethiopian community depends on traditional medicine for their health care, and more than
95% of traditional medicinal preparations made from plant origin. On the one hand, they have
limited access and economic capacity to modern health care services. On the other, they have
practical experiences and positive beliefs toward traditional medicine. It is also a home for
many languages, cultures and beliefs that have in turn contributed to the high diversity of
traditional knowledge and practice of the people, which, among others include the use of
medicinal plants.Herbal medicine in the simplest form are medicines or drugs made from
herbs or plants and can be said to process several synonyms all of which refer to plants as the
raw materials for medicine namely, phytomedicines, plant medicines, green medicines,
traditional medicine portions, traditional remedies plant drugs and forest health products
among others. There for it can be said that ethinobotanical studies are merely at the start in
Ethiopia there have been some attempts in investigating medicinal plants uses and there is as
9
yet, no in depth study on the relation between medicinal plants and indigenous knowledge on
sustainable management of such plant species.
In Ethiopia C.macrostachyus and S. incanum are the medicinal plants which have many
applications. A leaf extract (C.macrostachyus) is applied against itchy scalp. A decoction of
the leafy twigs mixed with Justicia schimperiana (Hochst. ex Nees) T.Anderson is taken to
treat jaundice and smallpox. The preparation is taken with pepper, butter and milk. An
infusion of the leafy branches and roots is used as a mouthwash to treat toothache. The leaves
or young shoots are eaten to treat fever and oedema and mashed leaves are applied to
haemorrhoids. A preparation of the seed is instilled into the ear to treat ear problems. The
seeds are poisonous and are used to make fish poison, while crushed seed and leaves in water
are taken to treat tapeworm infection; the seed is eaten to induce abortion and a fruit, bark or
root decoction or raw fruit is taken to treat venereal diseases. Bark maceration is drunk as an
abortifacient and uterotonic, to expel a retained placenta. These treatments are all considered
dangerous. The root or stem bark is chewed to treat toothache, but also rabies. Ripe crushed
fruits mixed with butter or honey and ground leaves are applied to skin diseases. The wood is
used in eastern and southern Africa to make tool handles, small stools, boxes, crates and
plywood, as flooring and building material and in carpentry. The wood is used as fuel that
burns even when green, but with a rather unpleasant spicy odour and much smoke; it is also
used to make charcoal. 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. It is also grown as a hedge plant and is suitable for intercropping.it
also an important medicinal plant especially in East Africa. It is widely used as a purgative
(stimulate) and vermifuge (medication), but also to treat venereal diseases. All parts, but
especially the seeds, are considered toxic and any medicine made from them should be used
with caution.
S. incanum is another bushy herbal plant, native to north and north eastern Africa including
Ethiopia. It found at forest edges and in bush land, grass land from sea level-up to 2500 m
altitude. Research investigation of S. incanum has been showed the medicinal value of these
plants which including in treatment of gastrointestinal infections, toothaches, dermatitis,
wound, malaria and dandruff. Despite the many medicinal uses, not much research has been
done concerning the chemical composition and pharmacology of the different plant parts and
more research is warranted [53].
10
About 80% of the Ethiopian population use traditional medicine due to the cultural
acceptability of healers, the relatively low cost of traditional medicine and the difficulty of
accessing modern health facilities [10]. The studies conducted on the traditional medicinal
plants in Ethiopia, especially medicinal plants are which known as C.macrostachyus and
S.incanum which used for mostly antimicrobial activities.
2.3. The Genus Croton and solanum
2.3.1 The Genus Croton
Croton is an extensive flowering plant genus in the’ spurge’ family, Euphorbiaceae. The
Common names for this genus are rushfoil and croton, but the latter also refers to Codiaeum
variegatum. The generic name comes from the Greek κροτον (kroton), which means "tick" and
Refers to the shape of the seeds of certain species. The genus croton is particularly rich in
secondary metabolites like alkaloids, Terpenoids, and flavonoids. The most common class of
compounds of croton is represented by diterpenoids. Apparently, clerodane is the widest
spread class of diterpenoids in Croton, which has been found in species from America (e. g. C.
cajucara), Africa (e. g. C. macrostachyus) and Asia (e. g. C. tiglium). The genus is also rich in
Constituents with biological activities, chiefly diterpenoids such as clerodane (1), trachylobane
(2), kaurane (3), pimarane (4) and labdane.
H
H
H1 2 43
Scheme 1: Some chemical structures of secondary metabolite isolated from Croton Spp.(1-4)
As most Euphorbiaceae, Croton species may contain latex, which is a characteristics of
medicinal properties. Several Croton species have a long role in the traditional use of
medicinal plants in Africa, Asia and South America. Popular uses include treatment of cancer,
constipation.
2.3.2 Medicinal Importance of genus Croton
Croton can be a tree, shrub or herbaceous plant which grows in tropical and warm regions.
The genus Croton belongs to the subfamily Crotonoideae of family Euphorbiaceae, one of the
largest families of plants, often characterized by being monoecious.
11
The predominant genera under the Euphorbiaceae family are Drypetes, Jatropha, Croton,
Euphorbia, Acalypha, Glochidion and Macaranga. A number of the Croton species are known
for their medicinal qualities especially in Africa, Asia and South America. Croton has been
found to possess secondary metabolites such as alkaloids, terpenoids, flavanoids and
compounds such as diterpenoids. Croton species are commonly used for the treatment of non-
communicable diseases such as diabetes, cancers and other ailments such as digestive
problems, dysentery, wounds, fevers, constipation, diarrhea, intestinal worms, malaria, pain
ulcers, and inflammation. The parts that are used for the treatments of the different kinds of
disease are the leaves, the roots, the stem barks, the fruit and the seeds [58].Some of the
Croton species are found in different regions; C.zambesicus ( Tannins, Saponins,
Anthraquinones , Alkaloids ) in Nigeria; C.macrostachyus (alkaloids, terpenoids , Saponins
,flavanoids and tanine in Kenya; C. tigliumproteins)[54] Pakistan; C. campestris (Tannin,
phlobaphenes, Flavones, Flavonols, Xanthones, Chalcones, Aurones, Flavononols, Catechins,
Flavonones, Alkaloids and Terpenes), C. zehntneri [55],C. cajucara [56],C. urucurana (Acetyl
aleuritic acid β-sitosterol-O-glucoside Sonderianin Steroids (stigmasterol, β-sitosterol,
campesterol) CatechinGallacatechin )[49]; And C. sonderianus (ent-Beyer-15-en-18-oic acid )
in Brazil.
2.4 The Genus solanum
Solanum can be an herb or soft wooded, shrub or herbaceous plant which abundant and
common as a weed, around houses, in overgrazed grassland and on roadsides. It is also found
at forest edges and in bush land and grassland, from sea-level up to 2500 m altitude. It is
considered an indicator for low-fertility soils.The genus solanum belongs to the subfamily of
Solonoaceae, one of the largest families of plants, often characterized by being monoecious.
The predominant genera under the Solonaceae family are it has been found plants of the
Solanaceae family with biological activity: Solanum americanum Mill., Solanum
bulbocastanum Dunal, Solanum corymbosum Jacq., Solanum elaeagnifolium Cav., Solanum
erianthum D., Solanum fructutecto Cav., Solanum heterodoxum Dunal, Solanum tridynamum
Dunal, Solanum lanceolatum Cav., Lycopersicon esculentum P. Mill., Solanum marginatum
L., Sol anum myriacanthum Dunal, Solanum nigrescens Mart and Gal, Solanum
pseudocapsicum L., Solanum rostratum Dunal, Solanum stoloniferum Schltdl.
12
H
H
O
HNH
H
H
H
Solasodine
N
O
H3C
H3CH3C
H
CH3
CHACOTRIOSE
a-chaconine
N
O
CH3
CH3
H3C H
CH3
SOLATRIOSE
a-solanine
O
HN
O
H3C
H3C
H
H
H3C
CH3
CHACOTRIOSE
a-solamargine5 6 7
8
Scheme 2: Some chemical structures of secondary metabolite isolated from solanum Spp. (5-
8)
2.4.1 Medicinal Importance of genus Solanum
Solanum species are the most potent plants against pathogenic microorganisms. Solanum
incanum (L) is one of the important traditional medicinal plant belongs to Solanacae family.
Antibacterial activity of Solanum incanum was studied [57]; and presences of analysis of
phytochemicals were also studied [58]. Other solanum species, Solanum torvum (leaf, stem
and roots) showed antibacterial and antifungal activity [59], and antibacterial activity of
Solanum surattense whole plant extract [60], and leaf extract [61] were studied. Analysis,
presence of phytochemicals and potent antibacterial activity of leaf, root and seed extracts
were studied in Solanum nigrum [62].
Solanum is the largest genus of the family Solanaceae and one of the largest among the
angiosperms with the potential for great food security in the developing world [63-64]. It is a
large and diverse genus of flowering plants, including two food crops of the highest economic
importance, the potato and the tomato. Solanum species show a wide range of growing habits,
such as annual and perennials, vines, subshrubs, shrubs and small trees. The genus was
established by Carl Linnaeus in 1753 [65]. Solanum is one of the ten most species-rich genera
of flowering plants and has approximately 1400 species that occur on all continents except
Antarctica in a wide variety of habitats from deserts to mountain slopes high above tree line
[66].
13
Three crops in particular have been bred and harvested for consumption by humans for
centuries, and are now cultivated on a global scale: Tomato: S. lycopersicum, Potato: S.
tuberosum, Eggplant: S. melongena. Majority of the members of this family are widely used in
folk medicine [67].
2.5 chemical composition and Bioactive Compounds from genus Croton and
Solanum
2.5.1 Chemical composition and Bioactive Compounds from genus Croton
Many parts of Croton macrostachyus exhibit different biological activities .This is the main
bases for the future investigation of this plant as it is sources of traditional medicines used to
cure many diseases. Several species of the genus Crotons are aromatic, indicating the presence
of volatile oil constituents [68]. Crude extracts are extracted from some member species of
genus Croton (e.g. C. cajucara and C. nepetaefolius) have various bioactivities including
antinociceptive, gastroprotective, antimicrobial, antiparasitic, cardiovascular, intestinal,
myorelaxant and antispasmodic [69-72]. These compounds isolated from genus Croton
include terpenes/terpenoids (monoterpenes, sesquiterpenes, diterpenes and triterpens),
alkaloids and flavonoids. For instance, lupeol, a triterpene, is one of a bioactive compound
isolated from the genus Croton. Other bioactive compounds such as crotin (a chalcone),
crotepoxide (a cyclohexane diepoxide), fatty acids and saponins are also reported from Croton
[73].
lupeol crotepoxide
H
H
H
HO
H
O
OO
O
O
O
O
O
methyl gallate
OH
HO
HO
O
O
sterols
HO
H H
H
H
O
OCH3 OH
O O
lichexanthoneMethyl 2,4-dihydroxy-3,6-dimethylbenzoate
O
O
HO
OH
9 10 11 12
13 14
Scheme 3: some chemical structures of bioactive compound isolated from Croton and
Solanum spp.(9-14)
14
2.5.2 Chemical composition and Bioactive Compounds from genus Solanum
The biological activities reported for genus solanums are secondary metabolites include
terpine, flavonoid, tannins, saponins, cyanates, oxalate, quinine and anthraquinones [74], as
well as steroid glycosides in the form of glycoalkaloids such as solanine and solasonine [75],
Which used for treatment for antimicrobial, anti-fungal, anti-cancer, antimalarial,
antispasmodic, antiviral, antiulcer, myorelaxant and cytotoxic. Antiseptic, laxative and anti-
inflammatory properties. The species of solanum leaves are rich in minerals such as K [76]
and Ca [77].
2.6 Botanical Description and Distribution of C.macrostachyus and S.incanum
Selection of candidate plant species for biological activity screening can be approached in
various ways. They include: a chemotaxonomic approach which is based on correlation
between plant taxonomy and the occurrence of specific chemical compounds, random
selection approach in which plants or plant parts are randomly selected and then subjected to
biological screening and ethnbotanical approach in which plants used in traditional medicine
are screened for biological activity [78].Several researchers have reported that the ethno
botanical approach is the best option [79]. The scientific discipline, ethno botany, is utilizing
the impressive array of knowledge assembled by indigenous peoples about the plant and
animal products they have used to maintain health. Plants also known to have special ability to
synthesize aromatic substances, most of which include alkaloids, quinones, flavones, tannins,
phenols or their oxygen-substituted derivative. Such a plant will have its parts including
leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control
or treatment of a disease condition and therefore contains chemical components that are
medically active [80].
2.6.1 Botanical Description and Distribution of C.macrostachyus
The name macrostachyus is given to it from the Greek words macro- (large) and –stachyus
(spike), referring to “a large spike” [81,58].The plant is crown rounded and opens with large
spreading branches and has large green leaves that turn orange before falling, with more or
less furry texture and slightly toothed margin. It has creamy to yellow-white flowers on
separate shoots but has green and grey fruits respectively at younger and maturation stages
[82].The genus Croton belongs to the family Euphorbiaceae (which commonly known as the
‘spurge’ family) [83]. It is known as Bisana’ (in Amharic) [84], and consists of approximately
1300 species of trees, shrubs and herbs distributed in tropical and subtropical regions of the
15
world. Croton macrostachyus which is called ‘rush foil’ or ‘broad-leaved croton is a
multipurpose, medium sized, drought-deciduous pioneer tree. It is a tall tree found in tropical
regions of Africa. Elsewhere in Africa, C. macrostachyus has been reported to occur in
Angola, Burundi, Cameroon, Central Africa, Ghana, Guinea, Ivory Coast, Kenya, Malawi,
Mozambique, Nigeria, Rwanda, Sudan, Tanzania, Uganda, Zaire, and Zambia [85].It is
commonly grows on forest edges along rivers, around lakes, woodlands, wooded grasslands,
in moist or dry evergreen forests and along roadsides [86]. It is native to Eritrea, Ethiopia,
Kenya, Nigeria, Tanzania and Uganda. In Ethiopia, C. macrostachyus occurs in regions
between 1300 and 2500 m with annual rainfall ranging between 200 and 2000 mm. The tree is
quite persistent, regenerating large numbers of coppices or shoots, even when it is repeatedly
lopped or degraded. Provided that environmental and soil conditions are favorable.
C.macrostachyus does establish well and can grow quite fast on reasonably good and well
drained soils, but prefers red or loam soils to verti soils. The latter soils are known for their
shrink-swell properties (during the dry and wet seasons, respectively), and for getting
waterlogged during the rainy season [83].it is a large tree with cylindrical trunk. The stem is
more or less pyramidal in shape with widespread branches. The stem is gray clear, smooth and
fissure with age. Leaves are almost as heart-shaped large that long, they have 10 to 15 cm of
length; they are flexible, green or brunette according to the season and present some prominent
ribs. Flowers are regrouped in inflorescence on stems of about 25 cm of long. They are visible
but their life span is very short. They are colour creamy and slightly fragrant yellow. Fruits are
regrouped along an axis.
Many parts of the Croton macrostachyus have medicinal value including boiled leaf decoction
is drunk or ashes taken orally as treatment for cough; juice from fresh leaves is applied on
wounds to hasten clotting. Roots are used as an anthlemintic for tape worm, for malaria,
venereal diseases, as antidiabetic, and the seeds are widely used as purgative, for constipation
and for stomach worms. Bark from stems and roots is boiled in water and newly born babies
are bathed in the mixture as a remedy for skin rash. The leaves of the tree are also used for
fodder and the tree is used for shade. The stem bark and the tips of the different branches of
the tree are used for the treatment of malaria and hepatitis in different parts of Ethiopia,
particularly in around welkite town in the south Regional State.
16
2.6.2 Botanical Description and Distribution of S.incanum
S. incanum have different names in different languages such as; Hiddii loonii (Afan Oromo),
Embouy (Amharic) and Sodom/bitter apple (English)) as indicated in Figure (B). The name S.
incanum is derived from Latin words, ‘Solamen’ meaning “relief” indicating the narcotic
effects of the plant and ‘incanum’ meaning “white”.it is commonly called the bitter apple or
African eggplant in English [87].in - Amharic is enbuaye. In Northern Nigeria among the
Hausa speaking people it is commonly called ‘’gauta‟, or ‘’gautan daaci‟, while among the
Bura/Babar speaking people of Southern Borno of North Eastern Nigeria, it is commonly
called ‘Tarku‟. It is an erect or spreading shrub up to 3m tall, occasionally a small tree [88]. It
is specie of nightshade native to Sub-Saharan Africa and the Middle East, Eastwards to India.
Solanum incanum is a controversial plant because it was given different descriptions by
different authors and these descriptions vary widely [89].
2.7 Medicinal Value of C. Macrostachyus and S.Incanum
2.7.1 Croton Macrostachyus
Croton macrostachyus belongs to the family Euphorbiaceae and it is commonly known as
broad-leaved Croton (English), and Bisana in the Amharic. C. macrostachyus (Figure 1) is a
deciduous tree which grows up to 3-25 m high, although more commonly it is 6-12 m. The
tree is crown rounded and open with large spreading branches commonly found in secondary
forests, around lakes, in moist or dry evergreen upland forests and woodlands. Croton
macrostachyus is widely distributed throughout tropical Africa and it is native to Ethiopia,
Eritrea, Kenya, Tanzania, Uganda, and Nigeria [86].
Figure 1: Photograph of Croton macrostachyus taken from welkitie abunemelketsediq
primary and secondary school, welkitie, Ethiopia in April 2020
17
2.7.2 Medicinal properties of C. macrostachyus Ethnobotanical and pharmacological studies revealed that various parts of C. macrostachyus
possess a wide range of activities [90-91, 86, 22]. These include antidiabetic, purgative and
anti-inflammatory, antibacterial and antifungal [22, 92-93] and antimalarial [22] activities. The
fruit extract showed promising antimalarial activity [94, 65]. Moreover, the methanol leaf
extract exhibited larvicidal activity against late third instar larvae of Anopiles arabiensis, a
predominant malaria vector in Ethiopia [84]. Stem bark decoction is used for bathing babies
against skin infections. Leaf decoction is employed against abdominal discomfort, sores,
dermatophilosis, ring worms, mange, scabie, wound and minor bleeding [91]. C.
macrostachyus also has therapeutic effect against fungal infections of skin (tinea versicolor)
(Quaqucha) by rubbing and covering with leaves at the affected area [95]. In view of the
above, the study aimed to evaluate the antibacterial activities of crude extracts C.
macrostachyus against the bacteria.
2.7.3 Solanum Incanum Solanum incanum belongs to the family solanceae and it is commonly known as broad-leaved
solanum (English), and enbuay in the Amharic. Solanum incanum (figure-2) is a herb or soft
wooded shrub up to 1.8 m in height with spines on the stem, stalks and calyces and with velvet
hairs on the leaves. Flowers pale to deep blue, mauve or purple. The leaves are alternate, egg-
shaped in outline with broad end at base (ovate) with slightly wavy margins (especially on
young leaves), with a grey-green upper surface and a green-white lower surface.
Figure 2: Photograph of solanum incanum taken from welkitie abunemelketsediq primary and
Secondary school, welkitie, Ethiopia in April 2020
18
2.7.4 Medicinal properties of Solanum Incanum S. incanum (L.) (Solanaceae) is used in the treatment of cough, cold and as expectorant [96].
The aqueous and methanol extracts of the leaves of S. incanum were studied for their
antibacterial effect against E. coli strains and results indicated that the extracts were
bacteriostatic at higher concentrations. The fruits extracts of S. incanum exhibited strong anti-
fungal activity against five opportunistic human fungal pathogens [94]. The plants selected
for the study are known for therapeutic uses as carminative, stomachic, antiseptic, laxative and
anti-inflammatory properties in the treatment of various ailments in traditional Indian
medicine. The leaf, root and seed decoction are gargled or drunk roots are chewed and sap
swallowed, leaf paste, root infusion and pounded seed are applied externally or rubbed in to
scarification, leaf sap is used for washing painful areas and ash of burnt plant is mixed with fat
and applied externally. The lack of scientific data regarding the presence of antibacterial
activity of these medicinal plants led us to investigate the antibacterial activity and
phytochemicals present in the organic extracts of these plants that may provide scientific
justification to the traditional uses in treating various ailments.
2.8 The Chemical Compositions and Biological Activities of C. Macrostachyus and
S.Incanum
Many parts of C.macrostachyus and S.incanum exhibit different biological activities .They
have the main bases for the future investigation of these plants as it is sources of traditional
medicines used to cure many diseases.
2.8.1 The Chemical Compositions and Biological Activities of C. Macrostachyus
Croton macrostachyus has widely utilized for the management of a large number of public
health and livestock problems in Ethiopian traditional medicines. Different parts of this plant
have been used as a remedy for malaria, abdominal pain, gonorrhea, wounds, ringworm
infestation, hemorrhoids, ascariasis, venereal diseases, cough and rheumatism [97-99].
C. macrostachyus have the activities of against diarrhea; traditional healers in Ethiopia use a
wide range of medicinal plants with antidiarrheal properties. An ethno botanical study in the
Shinasha, Agew-awi and Amhara, Chilga District, and Tigray region, for example, indicated
that the bark, seed, root and leaves of this plant are utilized for the management of rabies,
splenomegaly, ovine pasteurellosis, epilepsy, anti-termite, hyper-blurbia [100-101]. For
instance the fruit and decoction of the roots are used for the treatment of venereal diseases and
19
the seeds are used to induce abortion [102] and the slight larvicidal activity against Anopheles
arabiensis [103]. The bark of Croton macrostachyus is also used for the treatment of tapeworm
infection syphilis, and asthma in humans [104]. The seed is used by the local population of the
Bonga area of Southern Ethiopia for treatment of tapeworm infection in humans [105]. Roots
are used as a remedy for helminthiasis, malaria, venereal diseases and diabetic mellitus [106].
Whereas the leaves are used for the treatment of malaria, constipation, tetanus, epilepsy, skin
cancer and TB etc. in Ethiopia [107-108, 84]. The leaves and shoots of C. macrostachyus are
used to treat fever and oedema and also mashed leaves used for treatment of hemorrhoids and
for the treatment of various diseases such as malaria, hypertension, cancer, constipation,
diabetes, digestive problems, dysentery, external wounds, fever, leukemia, balsamic, narcotic,
rheumatism, leprosy, bronchitis, diarrhea, intestinal worms, psoriasis, urtcaria,
hypercholesterolemia, weight loss and ulcers [109]. Ethno pharmacological studies show that
hydro alcoholic extracts of C.macrostachyus leaves have promising activity against Neisseria
gonorrhoeae [110], Plasmodium berghei, Mycobacterium tuberculosis [111], analgesic and
Anti-inflammatory [112], anti-convulsant and sedative [113], anti-leishmanial activities [114]
and antidiarrhoeal activity. These showed that this plant exhibits many biological activities
that are used for the treatments of day to day diseases of human beings, livestock and the other
living things. These indicated that this plant is rich sources of biologically active secondary
metabolites [115].
Phytochemical screening of leaf extracts of Croton macrostachyus revealed the presence of
saponins, flavonoids, carbohydrate, free amino acids and vitamin C and proteins/peptides
were absent , by using both ethanol and water solvent extracts[116]. Also the general chemical
screening tests on the hydro-ethanolic root extract Croton macrostachyus confirm the presence
of alkaloids, phenolic compounds, tannins, terpenoids, saponins, phlobatannins and
Flavonoids [117].Recently the crude ethanol extract leaf of Croton macrostachyus revealed
that the presence of various metabolites such as terpenoids, flavonoids, saponins and alkaloids
whereas tannins and anthraquinones were not detected[118].Phytochemical constituents of the
leaf methanol extracts of this plant showed the presence of alkaloids, terpenoids, saponins,
flavonoids, phenol compounds and the absence of cardiac glycosides and anthraquinones.
Based on the solvent used and the parts of this plant used the chemical compositions obtained
from the plants are different [119]. The compounds isolated from leaf of croton
macrostachyus plant ; such triterpenes (lupeol (15), lupenone (16), betulinic acid (17), 28-O-
20
acetylbetulin (18),betulin (19), lupeol acetate (20), zeorin (21) phenolic compounds (benzoic
acid (22), methyl gallate (23), methyl 2,4-dihydroxy-3,6-dimethylbenzoate (24), lichexanthone
(25) and sterols (stigmasterol (26), β-sitosterol(27) and β-sitosterol palmitate(28) [120].
Chemical studies on the stem bark of the plant gave five known compounds namely; lupeol
(15) , floridolide A( 29), betulin (17), hardwickic acid (30) and 12-oxohardwickic acid
[121].The compounds isolated from roots of this plant are 3𝛽-acetoxy taraxer-14-en-28-oic
acid,trachyloban-19-oic acid ,trachyloban-18-oicacid,neoclerodan-5,10-en-19,6𝛽;20,12-
diolide,3𝛼,19-dihydroxytrachylobane(31) and 3𝛼,18,19-trihydroxytrachylobane(32) [122].
Table 1: secondary metabolites from croton macrostachyus.
Name of compound Plant part Stracture Reference
lupeol Stem bark , Leaf 15
120
lupenone
Leaf
16
betulinic acid 17
28-O-acetylbetulin 18
betulin Stem bark ,Leaf 19
lupeol acetate
Leaf
20
zeorin 21
benzoic acid 22
methyl gallate 23
methyl2,4-dihydroxy-3,6-
dimethylbenzoate
24
lichexanthone 25
stigmasterol 26
β-sitosterol 27
β-sitosterol palmitate 28
floridolide A
Steam bark
29
121 hardwickic acid 30
12-oxohardwickic acid
3𝛼,19-dihydroxytrachylobane Root 31
122 3𝛼,18,19-trihydroxytrachylobane 32
21
HO O
COOH
HO
lupeol lupenone betulinic acid
28-O-acetylbetulin
CH2OH
HO O
O
OH
OHO OH
betulin lupeol acetate
zeorin benzoic acid
HOH
OH
H
OH
15 16 17
18 1920
21 22
OH
OH
OH
O
HO
methylgallate
CH3
OH
H3C
HO
OH3CO
methyl 2,4-dihydroxy-3,6-dimethyl benzoate
O
CH3 O OH
O OCH3
lichexanthone sterols(stigmasterol)
23
24
2625
22
-sitosterol
-sitosterol palmitate
OO
CH2(CH2)13CH3
O
O
HO2C
H
H
Floridolide
O
HO2C
H
R
27
28
29
hardwickic acid
30
R''
RR'
31, R=CH2OH,R'=CH2OH,R''=OH32, R=CH3, R'=CH2OH, R''=OH
3a,19-dihydroxytrachylobane 3a,18,19-trihydroxytrachylobane
31 32
Scheme 4: chemical structure of some bioactive compound isolated from the C.macrostachyus
leaf, root and steam bark (15-32)
2.8.2 The chemical compositions and biological activities of S.incanum
The genus Solanum is rich in alkaloids which are widely distributed in all parts of the plants
[123]. Unlike some other members of the family Solanaceae mostly containing tropane
alkaloids, the genus Solanum is distinguished by its variety and productivity of alkaloids
joined to sugar moieties called steroidal glycoalkaloids [124-125].
23
Certain glycoalkaloids, such as solasonine and solamargine, stand out economically because
their chemical structures are very similar to steroidal hormones and therefore have been
proposed to be used as an important source for the production of medicines, such as
contraceptives and steroidal anti-inflammatory drugs [126]. These compounds bear the same
aglycone, solasodine, and differ from each other only in the nature of the involved trioses,
namely, solatriose for solasonine and chacotriose for solamargine. [127], reported the presence
of solamargine and solasonine in the leaves, stems and roots of S. incanum. It has been
reported that while solamargine and solasonine are in fruits of eggplant, solanine and
chaconine are found in the leaves of this plant [128]. Glycoalkaloids have also been studied
for their antidiabetic [129], antifungal [130], antiparasitic [131], and mostly for their
anticancer properties [132-133]. Solasonine and Solamargine showed selective cytotoxicity
against cancer cells in relation to normal cells [134].
Table 2: secondary metabolites from Solanum incanum.
Name of compound Plant part stracture Reference
Solasodine
leaf
Scheme 12
127
Chaconine
128
Solanine
Solamargine
Leaf, seed and root Solasonine
2.10 Crude Extract from genus croton and solanum
Crude extract and Essential oils are volatile aromatic compounds found in many plant organs
that give plants their wonderful scents and are related to various functions necessary for plant
survival playing a key role in defense against microorganism. They are complex mixtures,
constituted by terpenoid hydrocarbons, oxygenated terpenes and sesquiterpenes, alkaloid,
saponins, phenols. They originate from the plant secondary metabolism and are responsible for
their characteristic aroma. These crude and oils are often used for their flavor and their
therapeutic or odoriferous properties, in a wide selection of products such as foods, medicines,
and cosmetics. They are also improves or disguise the taste of unpleasant drugs. Crude extract
and Essential oils are frequently referred to as the “life force” of plants. these crude and oils
are volatile, highly concentrated substances extracted from flowers, leaves, stems, roots, seeds,
24
bark, and resin or fruit rinds and responsible for the biological activity. Crude extract and
Essential oils have a nice smell, and they are used in different industries, especially in
perfumes (fragancias and lotions), in foodstuff (like flavoring and preservatives) and in
pharmaceutical products (therapeutic action) [135].
The idea rised about medicinal plant, antibacterial activities and phytochemical analysis in the
review literature above by different scholar’s demonstrated with experiment in the
methodology part.
25
3.0 Materials and Methods
3.1 Study Design and Period
In vitro experimental design was adopted to conduct the study of antibacterial activity using
agar well diffusion method and phytochemical analysis of C.macrostachyus (leaf, root and
stem bark) and S.incanum (seed, leaf and root) crude extracts was carried out. Positive
(chloramphenicol) and negative (DMSO) controls were used to monitor the antimicrobial
activities of the extracts in all the assays.
The Raw plant product (C.macrostachyus (leaf, root and stem bark) and S.incanum (seed, leaf
and root)) was obtained from the south Regional State, Gurage Zone, welkite town, which is
258km from Addis Ababa in the month of April 2012. Specimens (sample) were identified and
confirmed by a botanist used selection for their authenticity. The plant specimens were
submitted to the herbarium of the chemistry department to extracting of concentrated crude
extrat, to test phytochemicals of secondary metaboilite and for microbiology lab room to
obtain the selected control drug (chloramphenicol) and bacteria (listeria monocytogenes
(ATCC19115), Staphylococcus epidermidis (ATCC12228),enterococcus
faecalis(ATCC29212), Staphylococcus aureus (ATTC 25923), streptococcus
pyogenes(ATCC19615), salmonella typhimurium(ATCC13311), shigella
sonnei(ATCC25931), Escherichia coli (ATTC 25922), pseudomonas
aeruguinosa(ATCC27853), salmonella enteritidis (ATCC13076) and candida albicans
clinical sample) in debreberhan university from June 2012 to July 2012 EC.
26
Figure 3: Flow chart of the study design
Preparation of ethanolic extract
Bacteria sampling and isolation
Lm SA Sh Ae
Se Sp Ec Ca
Ef Sal Pa
C.macrostachyus (leaf, root, stem bark)
S.incanum (leaf, seed, root)
Antibacterial activity (assey)
(Disc diffusion method)
Antibacterial
activity of plant
crude extract
Antibacterial activity
of antibiotics
Effect of plant
extract on bacterial
growth
Chloramphenicol
DMSO
Tannin phenol
Saponnin steroid
alkaloid Terpinoid
Flavonoid cumarin
Anthraquinone
Phythochemical activity test
Cleaned, shade dried, chopped & powdered
Use maceration techniques:
Selection of medicinal plant
27
3.2 Collection of Plant Material
The choice of plants for screening purposes may involve selecting plants with known
medicinal history, or randomly, or by basing on their taxonomic characteristics
[136].Alternatively, selection may be based on a common denominator such as family, genus
or species [32].The part of the plant for this study were collected from south Regional State,
Gurage Zone, welkite town, which is 258km from Addis Ababa in the month of April 2012.
During this time, there were heavy rains that are characteristic of this region, which promoted
more vegetative growth among most plants.
3.3 Study area
The studies included were in vitro studies carried out with a human bacterial pathogen. The
species under the genus Croton and Solanum were selected, Studies on the Croton
Macrostachyus and Solanum Incanum extract constituent and tested against common bacterial
infections by using standard drug test. The experimental study was done from June 14/10/2012
to July 8/11/2012 E.C. Extraction of crude extract by maceration methods was performed ,
phytochemical test was also done in Organic Chemistry lab room and In vitro antibacterial
activity test was conducted in microbiology lab room at Debreberehan University.
3.4 Apparatus, Instruments, Chemicals, Reagents and Media
3.4.1 Apparatus and Instruments The apparatus and instruments used in this study were : mortar, electronic Weighing balance,
beakers (different size), labler /marker/, sample stock holder, sponge, stand, plastic dropper,
conical flask, round bottom flasks (different size), measuring cylinder, test tubes, plastic
containers, Separating funnel, Rota vapor, autoclave, inoculating loop, incubator, filter paper,
(What man No.1 filter paper), paper disk, aluminum foil, sample vial, pipettes (different size),
Bunsen burner, spreader, hot plat, ruler, plaster, water bath, test tube holder, Petri-dishes,
laminar air flow hood, bottle brush, chamber (beaker), Spatula, elements for personal
biosecurity (gloves, gowns, goggles or protective eye wear, chemical/biological safety hood),
forceps, Photographic camera.
28
3.4.2 Chemicals, Reagents and Media The chemicals and reagents used in the study were: distilled water, organic solvents (ethanol),
appropriate media for bacteria (Muller hinter agar), test organisms, concentrated and dilute
hydrochloric acid, 10% ferric chloride solution(FeCl3), alkaline reagent sodium hydroxide,
concentrated and diluted sulfuric acid, ammonia solution, Mayer’s reagent, Chloroform, acetic
anhydride, DMSO, chloramphenicol, petroleum ether.
3.5 Preparation of Plant Material
The collected part of the plants was washed with distilled water thoroughly to free from Debris
and dried at room temperature in a shaded place (20-27 °C) for a period ranging from 2 to 4
weeks. The dried both plants were cut in to pieces via cutter, 500g of each sample was
measured and then kept in a sealed plastic bag at ambient temperature and protected from the
light until extraction. And on the later the prepared dried plant body parts (C.macrostachyus:
leaves, root, bark and S.incanum: leaves, root, seed were carried to the Organic Chemistry
laboratory at Debreberehan University for extracting the crude extract from the plant body
part.
3.6 Extraction of plant material
The extraction of plant materials was done as described by [137-138].The plant extract drugs
are new interest as antimicrobial agents in medicine which have safer biologically active
compounds with acceptable therapeutic index for development of novel drugs [139-140]. Plant
extracts have more active target sites against drug resistant pathogens [141]. it is the procedure
of the separation of medicinal active portions of plant from inactive component part of plants
which undergoes by using solvents diffuse into the solid plant material and solubilize
compounds with similar polarity [142].Plant extraction can be carried out in different steps
including collection of plant parts, drying, size reduction (grinding into pieces), extraction by
mixing with solvents, filtration, crude extracted. The quality and quantity of crude extraction
may be influenced by several factors including plant part, solvent, procedure and ration of
solvent to plant [143-144].
29
3.6.1 Extraction with ethanol
A. Maceration
This method is routinely employed in the labs where in a conical flask covered with aluminum
foil or parafilm is used to prevent evaporation of the solvent to avoid batch (collection) to
batch variations. The powdered sample is left to macerate for a known period after addition of
a suitable solvent. The maceration process is considered to be rather slow and sometimes
requires occasional or continuous shaking (or stirring) until the soluble compounds are
dissolved to ensures dispersal of saturated solution around the particle surface and bringing
fresh solvent to the surface of particle for further extraction. After maceration, the extract is
filtered through an appropriate filter or screen. In certain instances, the solid residues are
pressed and the occluded solutions are pooled with the extract before filtration. This method is
the best suitable method for the thermo labile compounds.Powdered plant materials 500 g each
were separately macerated in 500 mL of ethanol for 72 h with intermittent agitation. The
macerates were then filtered first by using gauze and Whatman No 1, filter paper [145-146).
3.7. Phytochemical Screening Tests
Following botanical identification of the selected plant crude extract (ethanol) were prepared
from the freshly collected material for the intended array of biological test systems. Portion of
the same extract that was subjected for phytochemical screening to determine the presence of
the following constituents: tannins, flavonoids, alkaloids, saponins, terpenes/terpenoids,
phenol, steriode, anthraquinones and cumarin by using the following procedures were carried
out according to the methods described next [147-153].
Test for Tannins: 2ml of various crude extract was mixed in 5 ml distilled water and
filtered. 1% aqueous Iron chloride (FeCl3) solution was added to the filtrate. Dark-green
solution indicates the presence of tannins.
Test for Flavonoids: 1 ml of crude extract was treated or mixed with a few drops of
concentration sulpheric acid. Which become or formation of orange color indicates the
presence of flavonoids.
Test for Alkaloids: A few milliliters of crude Extracts treated with the two drop of Mayer
reagent to form creamy or white precipitate indicates the presence of alkaloids.
30
Test for Saponins: To 2ml of each extract, 5 ml of distilled water (DI) was added in a test
tube. Then, the solution was shaken vigorously and observed for a stable persistent froth.
Formation of froth indicates the presence of Saponins.
Test for Terpenoids: 2ml of the crude extract was taken and 1ml of chloroform and 1.5 ml
of concentrated H2SO4 are added along the sides of the tube. The reddish brown color in the
interface is considered positive for the presence of terpenoids.
Test For Phenol: To 1ml of various crude extracts of sample mixed with 2ml of distilled
water (DI) followed by a few drops of 10% aqueous ferric chloride solution were added.
Formation of blue or green or blue black color indicated the presence of phenols.
Test for steroids: 1ml of crude extract was diluted with chloroform and followed by 1ml of
concentrated sulpheric acid added along the side of the test tube, appearance of two junction
/layer/ the reddish brown color interface form.
Test for Anthraquinones: A 3ml of crude extract treated with a few milliliter of
concentration sulpheric acid (H2SO4) and followed by 1ml of ammonia solution added to form
red, violet rose or pink confirms.
Cumarin: 2ml of crude extract treated with 3ml of 10% aqueous solution NaOH is added
after five minutes to form yellow color, which indicated the presence of phenols.
3.8 Antibacterial Test of the crude Extracts
3.8.1. Microorganisms Used, Preparation of Test Solution and Inoculum Preparation
A. Microorganisms Used
The antibacterial activity of the extracts was determined by the agar disc diffusion method
reported [154-158].A total of 11 microorganisms were used in this study. All are American
Type Culture Collection (ATCC) or reference strains: listeria monocytogenes (ATCC19115),
Staphylococcus epidermidis (ATCC12228),enterococcus faecalis(ATCC29212),
Staphylococcus aureus (ATTC 25923), streptococcus pyogenes(ATCC19615), salmonella
typhimurium(ATCC13311), shigella sonnei(ATCC25931), Escherichia coli (ATTC 25922),
pseudomonas aeruguinosa(ATCC27853), salmonella enteritidis (ATCC13076) and candida
albicans clinical sample.
31
B. Preparation of Test Solution
for each crude extract test solution was prepared to carry out their antibacterial activity
test.This were prepared by dissolving 150 mg of each of the crude extracts in 20 μl of dimethyl
sulfoxide(DMSO) to achieve final concentration of 150 and 250 mg/ml solution of test
sample.
C. Inoculum Preparation Inoculums of these bacteria were prepared in nutrient broth medium to obtain isolated
colonies. After incubation at 37Co
overnight, well-isolated colonies were selected with an
inoculating needle or loop and transferred in to a tube of sterile saline and vortex thoroughly.
Briefly, overnight bacterial cultures were diluted in the Mueller-Hinton broth (O.D. 600=0.08)
to obtain a bacterial suspension of 108 CFU/ ml. Petri plates containing 20 ml of Mueller-
Hinton agar media were inoculated with 200 μl of diluted cultures by the spread plate
technique and were allowed to dry in a sterile chamber. Seven filter paper discs (Whatman No.
4, 6 mm diameter) were placed on the inoculated agar surface. A 20 μl of the extracts (150
mg/ml, 250mg/ml) were loaded on to the filter paper discs and were allowed to dry
completely. Standard antibiotics 20 μl of Chloramphenicol as positive control and 20 μl of
DMSO negative control were placed as controls. Plates were incubated at 37Co for 24 h. The
antibacterial activity was assessed by measuring the inhibition zone. All the tests were
performed in doublate.
3.8.3 Antibacterial Activity Test
Antibacterial activity of ethanol extracts of C. macrostachyus (root, leaf, bark) and S. incanum
(root, leaf, seed) were evaluated by using the paper disc diffusion method against negative
bacteria.The paper discs which is prepared from the Whatmann filter paper Number 4 (6mm in
diameter) and sterilized in autoclave for placed individually with sterile forceps, and then
gently pressed down onto the agar. 20 μl of the concentrations (150 and 250mg/ml) of each
samples were pipetted to the discs in two replications. Antibiotic discs containing
Chloramphenicol were used as positive controls. Then the plate was inverted and incubated at
37°C for 24 hours. After incubation, the diameter of the zones of complete inhibition
(including the diameter of the disk) was measured and recorded in millimeters. The
measurement was made with a ruler on the undersurface of the plate without opening the lid
[159].By using experiment demonstration on the medicinal plant parts which is available for
antibacterial activities, extracting phytochemical screening and also the comparative
evaluation of beteween two plants parts discussed in the result and discussion part.
32
4.0 Results and Discussion
4.1. Phytochemical screening test
Phytochemical screening of the extracts of C.macrostachyus (leaves, bark, and root) and
S.incanum (leaf, seed and root) were presented as follows.
Table 3: Phytochemical constituents of ethanol extract of C.Macrostachyus
No Secondary
Metabolites
Crude extracts Plant parts
Stem bark Leaf Root
1 I. Tannins Ethanol extract - + -
2 Flavonoids >> + + +
3 Alkaloids >> + - +
4 Saponins >> - + +
5 Terpinoid >> + + +
6 Phenol >> - + +
7 steriod >> + - +
8 Anthraquinones >> - - -
9 cumarin >> + + +
+ Ve = Present - Ve = absent
The phytochemical screening of the ethanol extracts of these plant species revealed the
presence of flavonoid, cumarin and terpinoids are in all plant parts (stem bark, leaf and root),
the absences of phenol and saponin in stem barck but present in leaf and root.it also revealed
that the presence of steroid in stem bark and root but its absence in leaf. The absences of
alkaloids and steroid in leaf but present other plant parts. Tannins are absent in stem bark and
root but present in leaf of ethanol extracts of Croton macrostachyus. Saponin, phenol, steroid
are found the major components next to flavonoid, cumarin and terpinoids and tannin and
Anthraquinones rare in the plant.
33
Table 4: Phytochemical constituents of ethanol extract of S.incanum
No Secondary
Metabolites
Crude extracts Plant parts
Stem bark Leaf Root
1 J. Tannins Ethanol extract + + +
2 Flavonoids >> + + +
3 Alkaloids >> + - -
4 Saponins >> + + -
5 Terpinoid >> + + +
6 Phenol >> + + +
7 steriod >> - + +
8 Anthraquinones >> - - -
9 cumarin >> + + +
+ Ve = Present - Ve = absent
The phytochemical screening of the ethanol extracts of these plant species revealed the
presence of tannins, flavonoid, phenol, cumarin and terpinoids are in all plant parts (seed, leaf
and root), the absences of saponin in root but present in leaf and seed.it also revealed that the
presence of steroid in leaf and root but its absence in seed. Alkaloids are absent in leaf and
root but present in seed of ethanol extracts of S.incanum. Saponins, steroid are found the major
components next to tannins, flavonoid, phenol, cumarin and terpinoids and Anthraquinones
are rare in the plant.
34
Table 5: Phytochemical constituents of ethanol extract of C.macrosachyus and S.incanum
No P.chemicals reagents C.change C.macrostachyus S.incanum
S. bark Leaf Root seed leaf root
1 K. Tannins DI,FeCl3 dark green - + - + + +
2 Flavonoids con.H2SO4 orange + + + + + +
3 Alkaloids mayer
reagent
white ppt + - + + - -
4 Saponins froth test froth formation - + + + + -
5 Terpinoid salkowiski
test
redish brown + + + + + +
6 Phenol DI,FeCl3 green - + + + - +
7 steriod salkowiski
test
reddish brown + - + - + +
8 Anthraquinones H2SO4,NH3 Red - - - - - -
9 cumarin NaOH yellow + + + + + +
When compared the above present study of the two plant species (C.macrostachyus(steam
bark, leaf and root) and (S.incanum(seed, leaf and root)) which were that the ethanol crude
extracts phytochemical test(analysis) in both plant species revealed the presence of
flavoinoids, terpinoids and cumarins in all plant parts(steam bark, seed, leaf and root). But the
anthraquinones were absent in all plant parts.due to that of the ethanol crude extracted in both
plant parts not rich (contained) anthraquinone.
4.2. Antibacterial activity test
The results of antibacterial activity test for crude ethanol extract of plant parts were expressed
as mean value ± standard deviation (SD) of growth inhibition zone diameter obtained with
double trial shown in the following Table 4- table 9.The antibacterial efficacy of the two plant
part solvent extracts namely Croton macrostachyus (stem bark, root and leaf) and Solanum
incanum (seed, root and leaf) of the against the human pathogenic bacteria showed varied
level of mean zone of inhibition. The activity of the different extracts of all the screened plants
were compared with standard drugs chloramphinical.
35
4.2.1 C. macrostachyus
Results of bacterial test for crude ethanol extract of stem bark, ethanol extract of root and leaf
of croton macrostachyus were expressed as mean value ± standard Deviation (SD) of growth
inhibition zone diameters obtained with two trials as follows.
Table 6: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the stem bark of Croton macrostachyus
Against 11 microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Croton macrostachyus
\stem bark/
Chloramphenicol
/CD/
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.85± 0.21
7
250 6.9±0.141
7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 7.5±0.70
16
250 10.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 8.9±0.141 10
250 9.9±0.141 15
4 Staphylococcus aureus (Sa)
(ATTC 25923)
150 - 24
250 6.75±0.35 26
5 streptococcus pyogenes (Sp)
(ATCC19615)
150 7.9±0.141 -
250 8.4±0.84 -
6 salmonella typhimurium
(Sal)(ATCC13311)
150 6.85±0.21 9
250 9.75±0.35 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 16
250 - 27
8 Escherichia coli (Ec)
(ATTC 25922)
150 6.75±0.35 8
250 7±0 7
9 pseudomonas
aeruguinosa(Pa)
(ATCC27853)
150 6.8±0.28 16
250 7±0 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans (Ca) 150 6.95±0.07 7
250 7.05±0.07 7
-ve= Indicates no inhibition zone
36
As shown on the above Table 6, in the agar disc diffusion method of the 96% ethanol crude
extract of C. macrostachyus stem bark extract was subjected to the antibacterial activities and
a result were investigated as tabulated in the above table-6.chloramphinicol used as positive
control. The ethanol crude extract showed negative bacteria at the concentration of 250mg/ml
showed a better antibacterial activity against; listeria monocytogenes (ATCC19115),
Escherichia coli (ATTC 25922) and candida albicans with the mean zone of inhibition
6.9±0.141mm, 7±0mm, 7.05±0.07mm and the zone of inhibition of the control drug 7mm each
respectively; there zone of inhibitions are approchs when compared with positive controls. At
the concentration of 250mg/ml showed antibacterial activity against; Staphylococcus
epidermidis (ATCC12228), Enterococcus faecalis(ATCC29212), Staphylococcus aureus
(ATTC 25923), salmonella typhimurium (ATCC13311) , pseudomonas
aeruguinosa(ATCC27853) and salmonella enteritidis (ATCC13076) with the mean zone of
inhibition 10.5±0.70 mm(CD=17mm), 9.9±0.141mm(CD=15mm), 6.75±0.35mm(CD=26mm),
9.75±0.35mm(CD=12mm), 7±0mm(CD=12mm), 7±0mm(CD=18mm); there zone of inhibitions
are less when compared with positive controls respectively. Hence the extract is moderate
activity on those bacteria’s. At the concentration of 250mg/ml showed on the above table
streptococcus pyogenes (ATCC19615) bacteria was moderate antibacterial activities but there
is no zone of inhibition on control drug; this indicates the control drug is not active against
streptococcus pyogenes (ATCC19615) bacteria. At the concentration of 250mg/ml there is no
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 6.85 7.5 8.9 7.9 6.85 6.75 6.8 6.95
250mg/ml 6.9 10.5 9.9 6.75 8.4 9.75 7 7 7 7.05
6.85 7.5
8.9
7.9
6.85 6.75 6.8 6.95 6.9
10.5 9.9
6.75
8.4
9.75
7 7 7 7.05
0
2
4
6
8
10
12Z
on
e of
inh
ibit
ion
Bacteria(strains)
Ethanole crude extract of the stem bark 150mg/ml
250mg/ml
37
zone of inhibition on the gram positive bacteria shigella sonnei(ATCC25931) and the positive
control is Chloramphenicol showed 27mm zone of inhibitions; due to that the ethanol crude
stem bark extraction of croton macrostachyus is not effective on this bacteria.
When these crude extracted evaluated the C.macrostachyus stem bark showed higher value
mean zone of inhibition (10.5±0.70mm) and it has better antibacterial activities On
Staphylococcus epidermidis (Se) bacteria.
Table 7: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the leaf of Croton macrostachyus against 11
microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Croton macrostachyus
/leaf/
Chloramphenicol
1 listeria monocytogenes(Lm)
(ATCC19115)
150 - 7
250 7.5±0.7 7
2 Staphylococcus epidermidis (Se
)(ATCC12228)
150 7.5±0.7 16
250 10.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 9.5±0.70 11
250 9.5±0.70 16
4 Staphylococcus aureus(Sa)
(ATTC 25923)
150 - 25
250 9.5±0.70 26
5 streptococcus pyogenes(Sp)
(ATCC19615)
150 7.4±0.56 -
250 7.5±0.70 -
6 salmonella typhimurium(Sal)
(ATCC13311)
150 - 10
250 9.5±0.70 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 16
250 9.5±0.70 26
8 Escherichia coli(Ec)
(ATTC 25922)
150 6.5±0.70 7
250 7.5±0.70 7
9 pseudomonas aeruguinosa
(Pa)(ATCC27853)
150 9.5±0.70 17
250 9.5±0.70 20
10 salmonella enteritidis(Ae)
(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans(Ca) 150 6.5±0.70 6
250 7±0 6
-ve=Indicates no inhibition zone
CD=control drug
38
As shown on the above Table 7, in the agar disc diffusion method of the 96% ethanol extract
of C. macrostachyus leaf extract was subjected to the antibacterial activities and a result were
investigated as tabulated in the above table-7.chloramphinicol used as positive control.The
ethanol crude extracts showed negative bacteria; at the concentration of 250mg/ml showed a
better antibacterial activity against; listeria monocytogenes (ATCC19115), Escherichia coli
(ATTC 25922) and candida albicans with the mean zone of inhibition 7.5±0.7mm (CD=7),
7.5±0.70mm (CD=7) and 7±0mm (CD=6mm) there zone of inhibitions are approchs when
compared with positive controls each respectively. At the concentration of 250mg/ml showed
the antibacterial activity against; Staphylococcus epidermidis (ATCC12228), Staphylococcus
aureus (ATTC 25923), Enterococcus faecalis (ATCC29212), salmonella typhimurium
(ATCC13311), shigella sonnei (ATCC25931), pseudomonas aeruguinosa (ATCC27853) and
salmonella enteritidis (ATCC13076) with the mean zone of inhibition
10.5±0.70mm(CD=17mm), 9.5±0.70mm (CD=26mm), 9.5±0.70mm(CD=16mm),
9.5±0.70mm(CD=12mm), 9.5±0.70mm(CD=26mm), 9.5±0.70mm (CD=20mm) and
7±0mm(CD=18mm); there zone of inhibitions are less when compared with positive controls
respectively. Hence the extract is moderate activity on those bacteria’s. At the concentration of
250mg/ml there is no zone of inhibition on control drug. The negative bacteria streptococcus
pyogenes (ATCC19615); the mean zone of inhibition is 7.5±0.70mm. Due to this case, the
control drug is not effective on the negative bacteria.when these crude extracted evaluated the
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 7.5 9.5 7.4 6.5 9.5 6.5
250mg/ml 7.5 10.5 9.5 9.5 7.5 9.5 9.5 7.5 9.5 7 7
7.5
9.5
7.4
6.5
9.5
6.5
7.5
10.5
9.5 9.5
7.5
9.5 9.5
7.5
9.5
7 7
0
2
4
6
8
10
12
Zon
e o
f in
hib
itio
n
Bacteria(strains)
Ethanole crude extract of the leaf 150mg/ml
250mg/ml
39
C.macrostachyus leaf showed higher value mean zone of inhibition (10.5±0.70mm) and it has
better antibacterial activities On Staphylococcus epidermidis (Se) bacteria.
Table 8: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the root of Croton macrostachyus against11
microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
C. macrostachyus
(root)
Chloramphenicol
(CD)
1 listeria monocytogenes (Lm)
(ATCC19115)
150 6.9±0.14 7
250 7.5±0.70 7
2 Staphylococcus epidermidis
(Se) (ATCC12228)
150 8.5±0.70 16
250 10.5±0.70 17
3 Enterococcus faecalis
(Ef) (ATCC29212)
150 7.5±0.70 11
250 8.5±0.70 17
4 Staphylococcus aureus (Sa)
(ATTC 25923)
150 7.5±0.70 25
250 8±0 26
5 streptococcus pyogenes (Sp)
(ATCC19615)
150 6.8±0.28 -
250 7±0 -
6 salmonella typhimurium
(Sal) (ATCC13311)
150 7.5±0.70 10
250 7±0 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 16
250 7.5±0.70 26
8 Escherichia coli (Ec)
(ATTC 25922)
150 6.5±0.70 7
250 7.75±0.35 7
9 pseudomonas aeruguinosa
(Pa)(ATCC27853)
150 - 17
250 - 20
10 salmonella enteritidis(Ae)
(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans(Ca) 150 7.5±0.70 -
250 6.9±0.14 -
-ve= Indicates no inhibition zone,
40
The crude ethanol root of Croton macrostachyus extract was subjected to the antibacterial
activities and a result were investigated as tabulated in the above table-8.chloramphinicol used
as positive control.The ethanol crude extract showed negative bacteria; at the concentration of
250mg/ml showed a better antibacterial activity against; listeria monocytogenes
(ATCC19115), and Escherichia coli (ATTC 25922) with mean zone of inhibition of
7.5±0.70mm ,7.75±0.35mm and the positive control showed 7mm ; there zone of inhibitions
are approchs when compared with positive controls each respectively. At the concentration of
250mg/ml showed a moderate antibacterial activity against; Staphylococcus epidermidis
(ATCC12228), Enterococcus faecalis (ATCC29212), Staphylococcus aureus (ATTC 25923),
salmonella typhimurium (ATCC13311), shigella sonnei(ATCC25931), salmonella enteritidis
(ATCC13076) with mean zone of inhibition 10.5±0.70mm(CD=17mm),
8.5±0.70(CD=17mm), 8±0mm(CD=26mm), 7±0mm(CD=12mm), 7.5±0.70mm(CD=26mm)
and 7±0mm(CD=18mm) there zone of inhibitions are less when compared with positive
controls respectively. Hence the extract is moderate activity on those bacteria’s.but at the
concentration of 250mg/ml there is no zone of inhibition on the gram positive bacteria
pseudomonas aeruguinosa(ATCC27853) and the positive control is Chloramphenicol showed
20mm zone of inhibitions; due to that the ethanol crude root extraction of croton
macrostachyus is not effective on this bacteria.when these crude extracted evaluated the
C.macrostachyus root showed higher value mean zone of inhibition (10.5±0.70mm) and it has
better antibacterial activities On Staphylococcus epidermidis (Se) bacteria. So the crude
ethanol extract of stem bark, leaf and root were effective antibacterial activities on
Staphylococcus epidermidis (Se) bacteria.
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 6.9 8.5 7.5 7.5 6.8 7.5 6.5 7.5
250mg/ml 7.5 10.5 8.5 8 7 7 7.5 7.75 7 6.9
6.9 8.5
7.5 7.5 6.8 7.5 6.5
7.5 7.5
10.5 8.5 8
7 7 7.5 7.75 7 6.9
0
2
4
6
8
10
12Zo
ne
of
inh
ibit
ion
Bacteria(strains)
Ethanole crude extract of the root 150mg/ml250mg/ml
41
4.2.2 S. incanum
Results of bacterial test for crude ethanol extract of seed, root and leaf of Solanum incanum
were expressed as mean value ± standard Deviation (SD) of growth inhibition zone diameters
obtained with two trials as follows.
Table 9: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the seed of Solanum incanum against 11
Microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(seed)
Chloramphenicol
(CD)
1 listeria monocytogenes (Lm)
(ATCC19115)
150 6.5±0.70 7
250 6.5±0.70 7
2 Staphylococcus epidermidis (Se
)(ATCC12228)
150 6.5±0.70 15
250 7.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 6.5±0.70 10
250 7.5±0.70 16
4 Staphylococcus aureus
(Sa )(ATTC 25923)
150 - 24
250 9.5±0.70 26
5 streptococcus pyogenes
(Sp )(ATCC19615)
150 - -
250 6.9±0.14 -
6 salmonella typhimurium
(Sal )(ATCC13311)
150 - 9
250 7±0 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 15
250 - 27
8 Escherichia coli(Ec)
(ATTC 25922)
150 6.5±0.70 7
250 7±0 7
9 pseudomonas aeruguinosa
(Pa)(ATCC27853)
150 - 16
250 - 20
10 salmonella enteritidis (Ae)
(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans (Ca) 150 7±0 7
250 7±0 7
-ve= Indicates no inhibition zone
42
The crude ethanol seed of solanum incanum extract was subjected to the antibacterial activities
and a result were investigated as tabulated in table-9.chloramphinicol used as positive control.
The ethanol crude extracts showed negative bacteria; at the concentration of 250mg/ml
showed a better antibacterial activity against; listeria monocytogenes (ATCC19115),
Escherichia coli (ATTC 25922) and candida albicans with mean zone of inhibition of
6.5±0.70, 7±0, 7±0 mm and the positive control showed CD=7mm; there zone of inhibitions
are approchs when compared with positive controls each reaspectively .at the concentration of
250mg/ml showed a moderate antibacterial activity against; Staphylococcus epidermidis
(ATCC12228) , Enterococcus faecalis(ATCC29212), Staphylococcus aureus (ATTC 25923),
salmonella typhimurium (ATCC13311) and salmonella enteritidis (ATCC13076) with mean
zone of inhibition 7.5±0.70(CD=17), 7.5±0.70(CD=16), 9.5±0.70(CD=26), 7±0(CD=12) and
7±0(CD=18mm) ;there zone of inhibitions are less when compared with positive controls
respectively. Hence the extract is moderate activity on those bacteria’s. But there is no zone of
inhibition on the gram positive bacteria shigella sonnei and pseudomonas aeruguinosa and the
positive control is Chloramphenicol showed 27mm and 20mm zone of inhibitions.due to the
case of the ethanol crude root extract of solanum incanum is not effective on those
bacteria’s.when these crude extracted evaluated the S.incanum seed showed higher value mean
zone of inhibition (9.5±0.70mm) and it has better antibacterial activities On Staphylococcus
aureus(Sa )(ATTC 25923) bacteria.
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 6.5 6.5 6.5 6.5 7
250mg/ml 6.5 7.5 7.5 9.5 6.9 7 7 7 7
6.5 6.5 6.5 6.5 7
6.5 7.5 7.5
9.5
6.9 7 7 7 7
0
1
2
3
4
5
6
7
8
9
10
Zon
e of
inh
ibit
ion
Bacteria(strains)
Ethanole crude extract of the seed 150mg/ml
250mg/ml
43
Table 10: Results from disc diffusion assay showing the antibacterial activity (Diameter of the
Inhibition Zone, mm) of crude ethanol extract of the root of Solanum incanum against 11
Microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(root)
Chloramphenicol
1 listeria monocytogenes
(Lm )(ATCC19115)
150 6.9±0.14 7
250 7±0 7
2 Staphylococcus epidermidis
(Se) (ATCC12228)
150 - 15
250 7.5±0.70 17
3 Enterococcus faecalis
(Ef )(ATCC29212)
150 8.5±0.70 10
250 8.5±0.70 16
4 Staphylococcus aureus
(Sa )(ATTC 25923)
150 - 24
250 11.5±0.70 26
5 streptococcus pyogenes
(Sp )(ATCC19615)
150 7±0 -
250 7.5±0.70 -
6 salmonella typhimurium
(Sal )(ATCC13311)
150 6.5±0.70 9
250 8.5±0.70 12
7 shigella sonnei
(Sh )(ATCC25931)
150 - 15
250 - 27
8 Escherichia coli
(Ec )(ATTC 25922)
150 6.5±0.70 7
250 6.9±0.14 8
9 pseudomonas aeruguinosa
(Pa )(ATCC27853)
150 - 16
250 - 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 19
250 7±0 18
11 candida albicans (Ca) 150 7±0 6.5
250 7±0 7
-ve= Indicates no inhibition zone
44
The crude ethanol leaf of solanum incanum extract was subjected to the antibacterial activities
and a result was investigated as tabulated in table-10.chloramphinicol used as positive control.
The ethanol crude extract showed negative bacteria;at the concentration of 250mg/ml showed
a better antibacterial activity against; listeria monocytogenes (ATCC19115) , Escherichia coli
(ATTC 25922) and candida albicans with the mean zone of inhibition of 7±0(CD=7),
6.9±0.14 (CD=8) and 7±0(CD=7) mm ;there zone of inhibitions are approchs when compared
with positive controls each respectively.at the concentration of 250mg/ml showed a moderate
antibacterial activity against Staphylococcus epidermidis (ATCC12228), Enterococcus
faecalis(ATCC29212), Staphylococcus aureus (ATTC 25923), salmonella typhimurium
(ATCC13311) and salmonella enteritidis (ATCC13076) with the mean zone of inhibition
7.5±0.70(CD=17), 8.5±0.70(CD=16), 11.5±0.70(CD=26), 8.5±0.70(CD=12) and 7±0(CD=18)
; there zone of inhibitions are less when compared with positive controls respectively. Hence
the extract is moderate activity on those bacteria’s.but there is no zone of inhibition on the
gram positive bacteria shigella sonnei and pseudomonas aeruguinosa and the positive control
is Chloramphenicol showed 27mm and 20mm zone of inhibitions.due to the case of the
ethanol crude root extract of solanum incanum is not effective on those bacteria’s. when these
crude extracted evaluated the S.incanum root showed higher value mean zone of inhibition
(11.5±0.70mm) that of the S.incanum plant parts and it has better antibacterial activities On
Staphylococcus aureus(Sa )(ATTC 25923) bacteria.
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 6.9 8.5 7 6.5 6.5 7
250mg/ml 7 7.5 8.5 11.5 7.5 8.5 6.9 7 7
6.9 8.5
7 6.5 6.5 7 7 7.5 8.5
11.5
7.5 8.5
6.9 7 7
0
2
4
6
8
10
12
14Z
on
e of
inh
ibit
ion
Bacteria(strains)
Ethanole crude extract of the root 150mg/ml
250mg/ml
45
Table 11: Results from disc diffusion assay showing the antibacterial activity (Diameter of
the Inhibition Zone, mm) of crude ethanol extract of the leaf of Solanum incanum against 11
Microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(leaf)
Chloramphenicol
1 listeria monocytogenes
(Lm )(ATCC19115)
150 6.5±0.70 7
250 6.5±0.70 8
2 Staphylococcus epidermidis
(Se )(ATCC12228)
150 6.5±0.70 15
250 8.5±0.70 18
3 Enterococcus faecalis
(Ef )(ATCC29212)
150 9.5±0.70 11
250 9.5±0.70 16
4 Staphylococcus aureus
(Sa )(ATTC 25923)
150 - 24
250 - 26
5 streptococcus pyogenes
(Sp )(ATCC19615)
150 - -
250 6.5±0.70 -
6 salmonella typhimurium
(Sal )(ATCC13311)
150 8.5±0.70 9
250 9.5±0.70 12
7 shigella sonnei
(Sh )(ATCC25931)
150 - 15
250 - 27
8 Escherichia coli
(Ec )(ATTC 25922)
150 6.5±0.70 7
250 6.5±0.70 7
9 pseudomonas aeruguinosa
(Pa )(ATCC27853)
150 - 16
250 7.5±0.70 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 18
250 7.5±0.70 18
11 candida albicans (Ca) 150 7±0 7
250 7±0 8
-ve= Indicates no inhibition zone
CD-control drug
46
The crude ethanol leaf of solanum incanum extract was subjected to the antibacterial activities
and results were investigated as tabulated in table-11.chloramphinicol used as positive control.
The ethanol crude extract showed negative bacteria;at the concentration of 250mg/ml showed
a better antibacterial activity against;listeria monocytogenes (ATCC19115) , Escherichia coli
(ATTC 25922) and candida albicans with the mean zone of inhibition 6.5±0.70 (CD=8),
6.5±0.70(CD=7) and 7±0(CD=8) mm there zone of inhibitions are approchs when compared
with positive controls respectively.at the concentration of 250mg/ml were showed moderate
antibacterial activity against Staphylococcus epidermidis (ATCC12228) , Enterococcus
faecalis(ATCC29212), salmonella typhimurium (ATCC13311), pseudomonas
aeruguinosa(ATCC27853) and salmonella enteritidis (ATCC13076)with the mean zone of
inhibition 8.5±0.70 (CD=18),9.5±0.70(CD=16),9.5±0.70(CD=12), 7.5±0.70(CD=20),
7.5±0.70(CD=18mm) there zone of inhibitions are less when compared with positive controls
respectively. Hence the extract is moderate activity on those bacteria’s.but there is no zone of
inhibition on the gram positive bacteria; Staphylococcus aureus (ATTC 25923) and shigella
sonnei(ATCC25931) and the positive control is Chloramphenicol showed 26mm and 27mm
zone of inhibitions. Due to that reasons the ethanol crude leaf extract of solanum incanum is
not effectively on those bacteria’s. When these crude extracted evaluated the S.incanum leaf
showed higher value mean zone of inhibition (9.5±0.70mm) and they have better antibacterial
LM Se Ef Sa Sp Sal Sh Ec Pa Ae Ca
150mg/ml 6.5 6.5 9.5 8.5 6.5 7
250mg/ml 6.5 8.5 9.5 6.5 9.5 6.5 7.5 7.5 7
6.5 6.5
9.5
8.5
6.5 7
6.5
8.5
9.5
6.5
9.5
6.5
7.5 7.5 7
0
1
2
3
4
5
6
7
8
9
10
Zon
e of
inh
ibit
ion
Bacteria(strains)
Ethanole crude extract of the leaf 150mg/ml
250mg/ml
47
activities On salmonella typhimurium (Sal )(ATCC13311) and Enterococcus faecalis (Ef
)(ATCC29212) bacteria . So the crude ethanol extract of seed, root were effective antibacterial
activities on Staphylococcus aureus(Sa )(ATTC 25923) bacteria but the crude ethanol extract
of leaf were effective on salmonella typhimurium(Sal )(ATCC13311) and Enterococcus
faecalis (Ef )(ATCC29212) bacteria .
On the present study showed that, the ethanol extract of stem bark, leaf and root of
C.macrostachyus showed also antibacterial activities on both concentration (150 and
250mg/ml) agaist listeria monocytogenes (Lm )(ATCC19115), Staphylococcus epidermidis
(Se) (ATCC12228), Enterococcus faecalis(Ef )(ATCC29212), Staphylococcus aureus(Sa
)(ATTC 25923), streptococcus pyogenes (Sp )(ATCC19615), salmonella typhimurium (Sal )
(ATCC13311), shigella sonnei (Sh )(ATCC25931), Escherichia coli (Ec )(ATTC 25922),
pseudomonas aeruguinosa(Pa )(ATCC27853), salmonella enteritidis (Ae )(ATCC13076) and
candida albicans (Ca) with different mean zone of inhibitions. But have no active stem bark
crude extract on the shigella sonnei and the root crude extract on pseudomonas aeruguinose
bacteria’s.
In gram negative bacterias on listeria monocytogenes (Lm )(ATCC19115) the stem bark, leaf
and root crude extract showed 6.9±0.14mm,7.5±0.70mm and 7.5±0.70mm;for Staphylococcus
epidermidis (Se) (ATCC12228) the three crude extract showed that 10.5±0.70mm; for
Enterococcus faecalis(Ef )(ATCC29212) 9.9±0.141mm, 9.5±0.75mm and 8.5±0.70mm; for
Staphylococcus aureus(Sa )(ATTC 25923) 6.75±0.35mm, 9.5±0.70mm and 8±0mm; for
streptococcus pyogenes (Sp )(ATCC19615) 8.4±0.84mm,7.5±0.70mm and 7±0mm; for
salmonella typhimurium (Sal ) (ATCC13311) 9.75±0.35mm,7±0mm and 9.5±0.75mm;for
shigella sonnei (Sh )(ATCC25931) stem bark is not active, 9.5±0.70mm and 7.5±0.70mm;for
Escherichia coli (Ec )(ATTC 25922) 7±0mm, 7.5±0.70mm and 7.75±0.35mm; for
pseudomonas aeruguinosa(Pa )(ATCC27853) 7±0mm, 9.5±0.70mm and the root extract is not
active;for salmonella enteritidis (Ae )(ATCC13076) all extracts are 7±0mm; for candida
albicans (Ca) 7.05±0.07mm, 7±0mm and 6.9±0.14mm mean zone of inhibitions each
respectively. From the above three plant parts crude extracts data; the highest mean zone of
inhibitin on Staphylococcus epidermidis (Se) (ATCC12228) bacteria at all crude extracts. But
they have similar mean zone of inhibition (10.5±0.70mm) and can not evaluate the crude
extracts.To evaluet the crude extracted; showed the next bacteria mean zone of inhibition on
48
Enterococcus faecalis (Ef )(ATCC29212) the three extracts have different mean zone of
inhibition (stem bark=9.9± 0.14mm, leaf=9.5± 0.70mm and root=8.5± 0.75mm).
In gram negative bacteria on Enterococcus faecalis (Ef )(ATCC29212) the steam bark, leaf
and root of croton macrostachyus plant parts exhibited different maen zone of inhibition(the
stem bark ethanol crude extracted showed 9.9± 0.14mm, the leaf ethanol crude extracted
showed 9.5± 0.70mm and the root ethanol crude extracted showed 8.5± 0.75mm).so that, the
stem bark ethanol crude extracte of the croton macrostachyus has better antibacterial activities
than the leaf ethanol crude extract of Croton macrostachyus and the root ethanol crude extract
of Croton macrostachyus has less antibacterial activities than the leaf ethanol crude etract of
Croton macrostachyus. But the leaf ethanol crude extract of Croton macrostachyus has
moderate antibacterial activities than root on the gram negative bacteria Enterococcus faecalis
(Ef )(ATCC29212) , when compared with their high concentration(250mg/ml). These due to
the three ethanol crude extract Croton macrostachyus plant parts have different content of
phytochemical compound.
In addition to the above; evaluation of the ethanol crude extracts of stem bark, leaf and root of
Croton macrostachyus showed also antibacterial activities on the leaft eight bacteria with
different mean zone of inhibition at 250mg/ml concentration.but the antibacterial activities are
less when compared from the above three bacteria Staphylococcus epidermidis (Se)
(ATCC12228), Enterococcus faecalis (Ef)(ATCC29212) and Staphylococcus aureus(Sal
)(ATTC 25923).From all of the ethanol extract (stem bark, root and leaf) of Croton
macrostachyus; the stem bark crude extract showed best antibacterial activities with the mean
zone of inhibition 9.9±0.141mm on Enterococcus faecalis(Ef)(ATCC29212) and
9.75±0.31mm on Staphylococcus aureus(Sal )(ATTC 25923);8.4±0.84mm on streptococcus
pyogenes (Sp )(ATCC19615);7±0mm on salmonella enteritidis (Ae )(ATCC13076), candida
albicans (Ca), pseudomonas aeruguinosa(Pa )(ATCC27853), Escherichia coli (Ec )(ATTC
25922);6.85±0.21mm on listeria monocytogenes (Lm )(ATCC19115);6.75±0.35mm on
Staphylococcus aureus(Sa )(ATTC 25923); 10.5±0.70mm on Staphylococcus epidermidis (Se)
(ATCC12228) and the left one bacteria(shigella sonnei (Sh )(ATCC25931)) has no mean zone
of inhibitions.this is might be due to the absence of
phytochemicals:tannins,terpinoids,sapponins,phenols,flavonoids,alkaloids, steroids,cumarins
and anthraquinones.The leaf crude extract showed moderate antibacterial activities next to the
49
stem bark crude extracts with mean zone of inhibitions 9.5±0.70mm and 9.5±0.75mm on
Enterococcus faecalis(Ef)(ATCC29212) and Staphylococcus aureus(Sa)(ATTC 25923)
respectively.
On the present study(on the other hand) showed that, the ethanol crude extract of seed, leaf
and root of S.incanum showed also antibacterial activities on both concentration (150 and
250mg/ml) agaist listeria monocytogenes (Lm )(ATCC19115), Staphylococcus epidermidis
(Se) (ATCC12228), Enterococcus faecalis(Ef )(ATCC29212), Staphylococcus aureus(Sa
)(ATTC 25923), streptococcus pyogenes (Sp )(ATCC19615), salmonella typhimurium (Sal )
(ATCC13311), shigella sonnei (Sh )(ATCC25931), Escherichia coli (Ec )(ATTC 25922),
pseudomonas aeruguinosa(Pa )(ATCC27853), salmonella enteritidis (Ae )(ATCC13076) and
candida albicans (Ca) with different mean zone of inhibitions. But seed and root ethanol crude
extract not active on the bacteria of the shigella sonnei and psuaudomonas aeruguinas and the
leaf ethanol crude extract not active on shigella sonnei and staphylococcus aureus bacteria’s.
In gram negative bacteria on Staphylococcus aureus(Sa)(ATTC 25923) the root extract
showed 11.5±0.70mm, the seed extract showed 9.5±0.70mm and the leaf extract showed no
mean zone of inhibition.So that, the root extract of the plant has better antibacterial activity
than the seed and leaf extract on the gram negative bacteria Staphylococcus aureus(Sa )(ATTC
25923) when compared with their high concentration (250mg/ml),this may be due to the
presence of difference chemical compositon of the plant parts.In gram negative bacteria
Staphylococcus aureus(Sa )(ATTC 25923) the seed extract and the leaf extract of solanum
incanum exhibited different mean zone of inhibition (seed extract 9.5±0.70mm and leaf extract
no mean zone of inhibition respectively).due to the ethanol crude extract seed has high
different content of phythochemical compounds. So, the ethanol crude extract seed of
S.incanum has moderate bacterial activity than tha ethanol crude extract leaf against
Staphylococcus aureus (Sa) (ATTC 25923), but less antibacterial activity than ethanol crue
extract root.In addition to the above evaluation of the ethanol crude extract of seed, root and
leaf of Solanum incanum showed also antibacterial activities on the left ten bacteria with
different mean zone of inhibition at 250mg/ml concentration, but the bacterial activities those
extracts are less when compered from the previous root, seed and leaf extract on the
Staphylococcus aureus (Sa) (ATTC 25923) bacteria.
50
From all the ethanol extract (root,seed and leaf) of Solanum incanum;the root crude extract
showed best antibacterial activities than the other two extracts with mean zone of inhibition
11.5±0.70mm on Staphylococcus aureus (Sa) (ATTC 25923); 8.5± 0.75mm on the salmonella
typhimurium (Sal ) (ATCC13311) and Enterococcus faecalis(Ef )(ATCC29212); 7.5± 0.70mm
on the Staphylococcus epidermidis (Se) (ATCC12228) and streptococcus pyogenes (Sp )
(ATCC19615); 7± 0mm on the listeria monocytogenes (Lm )(ATCC19115), salmonella
enteritidis (Ae )(ATCC13076) and candida albicans (Ca) ; 6.9± 0.14mm on the Escherichia
coli (Ec )(ATTC 25922) and the left two bacteria’s (shigella sonnei (Sh )(ATCC25931) and
pseudomonas aeruguinosa(Pa )(ATCC27853) have no mean zone of inhibition.
Evaluation of the antibacterial activity of ethanolic extracted of the studied plants were
determined initialy by the disc diffusion method against different bacterias. These bacterial
strains are showen on the above table gram negative species frequently encountering in
infectious disease.The result of the diameter each crude extracted inhibition zones were
showen in the above table-4 to table-11.All plant parts ethanolic crude extracts, exihibited
varying degree of antibacterial activity against all bacterial strain tested.The maceration
extracts of croton macrostachyus plant parts root, leaf, and stem bark were best antibacterial
activity on Staphylococcus epidermidis (Se) (ATCC12228) with zone of inhibition 10.5±
0.70mm for each greater than Solanum incanum plant parts seed, leaf and root antibacterial
activity on Staphylococcus epidermidis (Se) (ATCC12228) with zone of inhibition 7.5±
0.70mm, 8.5 ± 0.70mm and 7.5± 0.70mm respectively. But; in this study I focoused on
evaluated the antibacterial activity of ethanolic crude extracted Staphylococcus aureus (Sa)
(ATTC 25923) bacteria having a value of different concentration (150mg/ml and 250mg/ml)
listed in the table below.
51
Table 12: Results from disc diffusion assay showing the evaluation of antibacterial
activity(Diameter of the Inhibition Zone, mm) the ethanolic crude extract the leaf of
C.macrostachyus and the ethanolic crude extract the root of S.incanum against Staphylococcus
aureus (Sa) (ATTC 25923) Bacteria.
Microorganis
concentratio
C.macrostachyus S.incanum
Stem bark leaf root seed leaf root
Staphylococc
us
aureus(Sa)
150mg/ml - - 7.5± 0.70 - - -
250mg/ml 6.75± 0.35 9.5± 0.75 8 ± 0 9.5± 0.70 - 11.5±0.70
In gram negative bacteria Staphylococcus aureus (Sa) (ATTC 25923); the Croton
macrostachyus plant parts of the stem bark, leaf and root ethanolic crude extracted showed
mean zone of inhibitions 6.75± 0.35mm, 9.5± 0.75mm and 8 ± 0mm respectively and the
Solanum incanum plant parts of the seed ethanolic crude extracted showed mean zone of
inhibitions 9.5± 0.70mm, the root ethanolic crude extracted showed 11.5±0.70mm and the leaf
ethanolic crude extracted showed no zone of inhibition.Finally; when these ethanolic crude
extracted evaluated the Solanum incanum root showed higher value mean zone of inhibition
(11.5±0.70mm) that of the Croton macrostachyus plant parts and it is good antibacterial
activities when we shown on the figure as follows(reference- I2).
stem bark leaf root seed leaf root
150mg/ml 7.5
250mg/ml 6.75 9.5 8 9.5 11.5
7.5 6.75
9.5 8
9.5
11.5
0
2
4
6
8
10
12
14
zon
e o
f in
hib
itio
n
croton macrostachyus solanum incanum
plant species part
crude ethanol extract of C.macrostachyus and S.incanum on Staphylococcus
aureus (Sa) (ATTC 25923) bacteria
150mg/ml
250mg/ml
52
5. Summary and Recommendations
5.1 Summary
In this study, the nine phytochemical screenings tests of ethanolic crude solvent extracted
from each plant parts in C.macrostachyus revealed (shown) the presence of flavonoids,
alkaloids, terpenoids and cumarines and in S.incanum revealed (shown) the presence of
tannins,flavonoids, terpenoids, phenols and cumarins in the six plant parts respectivly.Finally,
to evaluate both plant specis (C.macrostachyus and S.incanum) were riched in flavonoid,
terpinoids and cumarins in the six tested plant parts respectively.The ethanolic crude extracted
of C.macrostachyus plant parts exhibited (displayed) best antibacterial activities against
Staphylococcus epidermidis (Se) bacteria. But the crude extracted from S.incanum plant parts
exhibited antibacterial activities against Staphylococcus aureus (Sa).So,in this study I focused
to evaluated the ethanol extracted of C.macrostachyus plant parts(stem bark,root and leaf) and
S.incanum plant parts(seed,root and leaf) antibacterial activities against Staphylococcus aureus
(Sa) bacteria.Finaly, when these ethanol crude extracts of plant parts evaluated the S.incanum
root crude extracted had better antibacterial activity with mean zone of inhibition 11.5
±0.70mm than the other C.macrostachyus and S.incanum plant parts.this may due to it
contains more or different bioactive compositions and use as a medicines.
The results of the present study provide evidence that medicinal plants continue to play an
important role in the health care system of these study sites.Knowledge and uses of herbal
medicine for the treatment of various ailments among both rural and urban people are still a
major part of their livelihood and culture.So, traditional knowledge (TK) of using and
preserving these plants is still being transferred from Generation to generation, but seems to be
aging. This shows that there is a problem in the transfer of knowledge from the elders to the
young generation. The problem is perhaps arises due to the introduction of modern education,
religious factors, spiritual and culture related problems.
53
5.2 Recommendations
Based on the findings of the study, the following recommendations were forwarded to the
concerned body.
This study extensively focused to the phytochemical screening and antibacterial testing was
conducted on a limited number of bacteria.so the same work should be carried out on a large
variety of bacteria and fungal strains so as to have a clear picture of the spectrum of the
antibacterial activity of the herbal drugs.Further work is needed to screen, to purfy, to know
and to characterize the pure chemical compouds of the plant that are responsible for
antibacterial properties and to increase the efficiency of these extracts.It is also recommended
to carried out in vivo screening test on the animal model to establish beneficial therapeutic
remedies to treat infections coused by most of antibiotic resistant organisms.It is
recommended to carry out antibacterial activity test on the other Croton and Solanum plant
species, which are not yet tested in the present work.Further studies should be taken with
different extraction solvent.
54
Reference
[1]. Westh H., Zinn C. S., Rosdahl V. T., 2004. An International multi cancer study of
Antimicrobial consumption and resistance in S. aureus isolates from 15 hospitals in
14 countries. Microbe Drug Resistance, 10: 169-176.
[2]. Owolabi MA, Coker HAB, Jaja SI (2007). Flavonoid metabolites in urine after oral
Administration of the aqueous extract of Persea Americana to rats. J. Nat. Med.
61: 2004.
[3]. Nascimento Gislene GF, Juliana Locatelli, Paulo C Freitas, Giuliana LSilva (2000).
Antibacterial activity of plant extracts and phytochemcals on antibiotic resistant
Bacteria. Braz. J. Microbiol. 31: 247-256.
[4]. Gordon MC, David JN (2001). Natural product during discovery in the next Millennium.
Pharm. Biol. 39: 8-17.
[5]. Kalia AN, (2005).Text book of Industrial Pharmacognosy Oscar publication, New Delhi,
India.
[6]. Burt, S. (2004). Essential oils: their antibacterial properties and potential Applications in
foods a review. International journal of food microbiology, 94(3),223-253.
[7]. Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology
reviews, 12(4), 564-582.
[8]. Ahmed YM, Muhammedawel K, Ashebir D (2016). Knowledge, Attitude and Practice
Of Community on Traditional Medicine in Jara Town, Bale Zone South East Ethiopia.
Science Journal of Public Health, 4(3):241-246.
[9]. Getahu A. some common medicinal and poisonous plants used in Ethiopia, folke
medicin in Addis abeba university, Addis abeba, Ethiopia; 1976.
[10]. Alamri SA, Moustafa MF. 2012. Antimicrobial properties of 3 medicinal plants From
Saudi Arabia against some clinical isolates of bacteria. Saudi Medical Journal, 33(3):
272-277.
[11]. Thankamma L. 2003. Hevea latex as wound healer and pain killer. Current Science,
84: 971-972.
[12]. Narendra N, Gaurav P, Lokesh D, Naveen KJ. 2009. Wound healing activity of Latex of
Calotropis gigantean. International Journal of Pharmaceutical Science, 1: 176-181.
55
[13]. Sequeira BJ, Vital MJ, Pohlit AM, Pararols IC, Caúper GS. 2009. Antibacterial And
antifungal activity of extracts and exudates of the Amazonian medicinal tree
Himatanthus articulates (Vahl) Woodson (common name: sucuba). Memórias do
Instituto Oswaldo Cruz, 104: 659-661.
[14]. Shahzadi, I., Hassan, A., Khan, U.W., and Shah, M.M. (2010). Evaluating Biological
Activities of the seed extracts from Tagetes minuta L. found in Northern Pakistan.
4(20), 2108-2112.
[15]. Tahir, L., and Khan, N. (2012). Antibacterial potential of crude leaf, fruit and
Flower extracts of Tagetes Minuta L. Journal of Public Health and Biological
Sciences, 1, 70-74.
[16]. Bekele D, Asfaw Z, Petros B, Tekie H. 2012.Ethnobotanical study of plants used for
Protection against insect bite and for the treatment of livestock health problems
In rural areas of Akaki District, Eastern Shewa, Ethiopia. Topclass Journal of Herbal
Medicine. 1(2): 40-52.
[17]. Bern MJ, Sturbaum CW, Karayalcin SS, Berschneider HM, Wachsman JT, Powell
DW. 1989.Immune system control of rat and rabbit colonic electrolyte transport. Role
of Prostaglandins and enteric nervous system. J Clin Invest. 83(6): 1810-1820.
[18]. Newman DJ, Cragg GM (2012). Natural products as sources of new drugs over the 30
years from 1981 to 2010. J. Nat. Prod. 75(3):311-335.
[19]. Sheng-Ji, P. 2001. Ethnobotanical Approaches of Traditional Medicine Studies: Some
Experiences from Asia. Pharm. Boil. 39, 74–79.
[20]. Hussain, M.S.; Fareed, S.; Ansari, S.; Rahman, M.A.; Ahmad, I.Z.; Saeed, M. 2012.
Current approaches toward production of secondary plant metabolites. J. Pharm.
Bioallied Sci. 4, 10–20.
[21]. Abate, G. B., T. Gebre Egziabher & M. Tadesse. 1976. A study on the medicinal
Plants of Ethiopia, Unpublished. Science Faculty, Addis Abeba University.
[22]. Exarchou V, Nenadis N, Tsimidou M, Gerothanassis IP, Troganis A, Boskou D. 2002.
Antioxidant activities and phenolic composition of extracts from greek oregano,Greek
sage summer savory. Journal of agriculture and food chemistry.50 (19):5294-99.
[23]. Yirga G, Teferi M, Kasaye M (2011). Survey of medicinal plants used to treat Human
Ailments in Hawzen district, Northern Ehiop. Inter. J. Biodivers. Conserv. 3:709-714.
56
[24]. Alshawsh MA, Mothana RA, Al-Shamahy HA, Alsllami SF, Lindequist U (2009).
Assessment of anti-malarial activity against Plasmodium falciparum and Phytochemical
screening of some Yemeni medicinal plants. Evid. Based Complement. Altern. Med.
6(4):453-6.
[25]. Mesfin A, Giday M, Anmut A, Teklehaymanot T (2012). Ethnobotanical study of
Antimalarial plants in Shinile District, Somalia Region, Ethiopia, and in vivo Evaluation
of selected ones against Plasmodium berghei. 139:221-227.
[26]. Bum EN, Ngah E, Ngo Mune RM, Ze Minkoulou DM, Talla E, Moto FCO,Ngoupaye
GT Taiwe GS, Rakotonirina A, Rakotonirina SV (2012). Decoctions of Bridelia
micrantha and Croton macrostachyus may have anticonvulsant and Sedative effects.
Epilepsy Behav. 24:319-323.
[27]. Antonio S, Maria L, Faria S, Giuseppina N (2007). Traditional uses, Chemistry and
pharmacology of croton species (Euphorbiaceae). J. Braz. Chem. Soc. 18:11-33.
[28]. Mbiantcha M, Nguelefack TB, Ndontsa BL, Tane P, Kamanyi A (2013). Preliminary
Assesement of Toxicity of Croton macroxtachyus stem bark extracts. Int. J. Pharm.
Chem. Biol. Sci. 3(1):113-132.
[29]. Habtamu G, Legesse A, Yinebeb T, Asrat H (2012). Isolation of Crotepoxide From
Berries of Croton macrostachyus and Evaluation of Its Anti-Leishmanial Activity.
1(4):15-24.]
[30]. Prozesky EA, Meyer JJ, Louw AI (2001). In vitro antiplasmodial activity and
Cytotoxicity of ethnobotanically selected South African plants. 76:239-244.
[31]. Y.-L. Lin, W.-Y. Wang, Y.-H.Kuo, C.-F.Chen, 2000. Nonsteroidal constituents from
Solanum incanum,” Journal of the Chinese Chemical Society, 47, 247–251.
[32]. L.-F. Liu, C.-H. Liang, L.-Y. Shiu, W.-L. Lin, C.-C. Lin, and K.-W. Kuo, 2004. “Action
of solamargine on human lung cancer cells - enhancement of the susceptibility of cancer
cells To TNFs,” FEBS Letters, 577, 67–74.
[33]. Newman, D.J. and Cragg, G.M., 2014. Marine-sourced anti-cancer and cancer pain
Control Agents in clinical and late preclinical development.Marine drugs, 12(1),
255-278.
[34]. Thibaud, H.K. (1954).Perfumary products from Citronella Oil, Perfum. Essent. Oil Rec.
45:186-191.
57
[35]. Gulati, B. and Sadgopal.C. (1972). Chemical Examination of oil of
Cymbopogonnardus.Jornal of Indian Oil Soap.37 (12):305-310.
[36]. Meresa, A., Gemechu, W., Basha, H., Fekadu, N., Teka, F., Ashebir, R., & Tadele, A.
(2017).Herbal Medicines for the Management of Diabetic Mellitus in Ethiopia and
Eretria including Their Phytochemical Constituents. American Journal of Advanced
Drug Delivery, 5(1).
[37]. Crevost, C., and A. Petalot. 1929. Catalogue des produits de I‟ indochine: Plantes
Medicinales. Bulletin Economique de I‟ indochine 37: 516-528.
[38]. Mayorga, R.P., and H. Espinoza. 1970. Coccidiomycosis in Mexico and Central
America. Mycopathologia ET mycologia applicata 41, 3-11.
[39]. Soleeki, (1975). Shanida IV, a Neanderthal flower burial in Northern Iraq. Science
190: 880-881.
[40]. Watt, J.M., and M.G. Breyer-Brandwijk. 1962. Medicinal and poisonous plants of
Southern and Eastern Africa, 2nd edn. E & S. Livingstone Ltd., Edinburgh, London.
[41]. Iwu, M.W., A.R. Duncan, and C.O. Okunji. 1999. New antimicrobials of plant Origin,
457–462.
[42]. Sampson, J. H.; Phillipson, J. D.; Bowery, N. G.; O’Neill, M. J.; Houston, J. G.; Lewis, J.
A.; 2000. Ethno botanical study Oftraditional plants in Gindebert district. 14, 24.
[43]. Mishra, B. B., and Tiwari, V.K. (2011). Natural products in drug discovery: Clinical
Evaluations and investigations opportunity challenge Scope natural Prod. MedChem 1‐62.
[44]. Nastro, A., Germano, M.A., D’Angelo, V., Marino A., Cannatelli M. A. Extraction
Methods and bioautography for evaluation of medicinal plant antimicrobial Activity.
Letters in Applied Microbiology. 30(5): 379-384.
[45]. Sidigia, I., C. Nyaigotti-Chacha, and M.P. Kanunah. 1990. Traditional medicine in
Africa. East African Educational Publishers, Nairobi.
[46]. Yirga, Gidey and Samuel, Zeraburk (2005). Ethno botanical study Oftraditional plants in
Gindebert district. Western Etiopia.62.
[47]. Janzen, J.M. 1981. the Need for a Taxonomy of Health in the Study of Mrican
Therapeutics. Social Science and Medicine. 12(2); 121-129.
[48]. Amenu, Endalew (2007). Use and manegment of medicinal plants by Indigenous people
People of Ejaji area (Chelya wereda) West shoa, Ethiopia. An ethno botanical approach.
MSc thesis AAU. Ethiopia.
58
[49]. Abebe, Dawit (2001).The role of medicinal plants in healthcare coverageOf Ethiopia.
The Possible benefits of integration. In; Conservation and sustainable use of medicinal
Plants in Ethiopia. Pp.6-21. (Medhne Zewdu and Abebe Demissieeds). Proceeding of the
National. Alemaehu Getu (2013). Plant biodiversity and ethno botany in Amaro and
Gelana wereda Southern Ethiopia with emphasis on .Medicinal and edible plants;13.
[50]. Kibebew Fassil (2001). Utilization and Conservation of Medicinal plants in Ethiopia. In
Proceeding of the workshop on Development Utilization of Herbal Remedies in
Ethiopia;EthiopianHealth and nutrition Institute. Addis Ababa. 46-52.
[51]. Hunde, Debela, Asfaw, Zemede, Kelbessa, Ensermu (2004). Use and management of
Ethno Veterinary medicinal plants used By indigenous people in “Boset ”. Welenchiti
area. Ethio. J.Biol.Sci.3 (2); 113-132.
[52]. Desta, B., 1995. Ethiopian traditional herbal drugs. Part 1: Studies on the Toxicity and
Therapeutic activity of local taenicidal medications. Journal of Ethnopharmacology
45(1): 27–33.
[53]. Kouki, A., Haataja. S., 2011. Identification of a novel streptococcal adhesin P
(SadP) protein recognizing galactosyl-α1-4-galactose-containing glycoconjugates:
Convergent evolution of bacterial pathogens to binding of the same host receptor. J
Biol Chem.; 286 (45):38854-64.
[54]. Rodrigues FF., 2009.Synergy effects of the antibiotics gentamicin and the essential
Oil of Croton zehntneri. Phytomedicine. 16(11):1052-5.
[55]. Alviano DS, Bizzo HR, Souto-Padron T, Rodrigues ML, Bolognese AM, Alviano
CS, Souza MM., 2005. Antimicrobial activity of Croton cajucara Benth linalool-
Rich essential oil on artificial biofilms and planktonic microorganisms. Oral
Microbiol Immunol.;20 (2):101-5.
[56]. John Britto S & Senthilkumar S, 2001. Antibacterial activity of Solanum incanum L. leaf
Extracts. Asian Journal of Microbiology Biotechnology Environmental Science, 3: 65-66.
[57]. Pavitra P.S. Janani V.S., Charumathi K.H., Indumathy R., Sirisha Potala and Rama S.
Verma (2012). Antibacterial activity of plants used in indian herbal medicine. 23-28.
[58]. Bari, M.A., Islam,1 W.,. Khan A.R. and Abul Mandal. (2010). Antibacterial and
Antifungal Activity of Solanum torvum (Solanaceae). International Journal of 1 (3): 386-
390.
59
[59]. Patil Suhas, Joshi Vijaya, Sutar Prasanna and Sambrekar Sudhir, (2009). Screening of
Whole plant extract of Solanum surattense for antibacterial activity. International
1(1):110-114.
[60]. Sheeba, E. (2010). Antibacterial activity of Solanum surattense Burm 6: (I). 1-4.
[61]. Sridhar T.M., Josthna P. and Naidu C.V. (2011). In Vitro Antibacterial Activity and
Phytochemical Analysis of Solanum nigrum (Linn.) – An Important Antiulcer Medicinal
Plant. Journal of Experimental Sciences, 2(8): 24-29.
[62]. Kochhar, S. L. (1981). Tropical Crops. A textbook of Economic Botany. New Delhi :
Macmillan Publication.
[63]. Nee, M. (1999).Synopsis of Solanum in the New World. In Nee M, Symon, D. E., Lester,
R. N. and Jessop, J. P. Solanaceae IV, Advances in Biology and Utilization. Royal
Botanic Gardens, Kew, 285-333.
[64]. Tsao, S. and Lo, H. (2006). Vegetables: Types and Biology. Handbook of Food Science,
Technology, and Engineering by Yiu H. Hui. CRC Press.
[65]. Frodin, D. (2004). History and Concepts of Big Plant Genera. Taxon 53: 753–776.
[66]. Tania, M. S., Silva, I., Jefferson, R. B., Batista, M. M., Agra, M. F. and Celso, A. (2007).
Brine Shrimp Biossay of some Specie of Solanum from North-Eastern Brazil. Brazilian
Journal of Pharmacognosy, 17(1): 35-38.
[67]. Lima, SGD, Citó, AMGL, Lopes, JAD, Neto, JMM, Chaves, MH and Silveira ER.
(2010). Fixed and volatile constituents of genus Croton plants: C. adenocalyx Baill –
Euphorbiaceae. Review of Latino America, 38(3): 133-144.
[68]. Magalhães, PJC, Criddle, DN and Tavares, RA. (1998). Effects of Croton nepetaefolius
on intestinal smooth muscle. Phytotherapy Research, 12: 172 – 177.
[69]. Hiruma-Lima, CA, Gracioso, JS, Rodriguez, JA, Haun, M, Nunes, DS and Brito,
ARMS. (2000). Gastroprotective effect of essential oil from Croton cajucara Benth.
(Euphorbiaceae). 69: 229 – 234.
[70]. Lahlou, S, Leal-Cardoso, JH and Magalhães, PJ. (2000). Essential oil of Croton
nepetaefolius decreases blood pressure through an action upon vascular smooth
muscle: studies in DOCA-salt hypertensive rats. Planta Medica, 66: 138 – 143.
[71]. Anthony, JP, Fyfe, L and Smith, H. (2005). Plant essential oils – a resource for
antiparasitic agents.Trends in Parasitology, 21: 462 – 468.
60
[72]. Schmelzer, GH and Gurib-Fakim, A. (2008). Plant Resources of Tropical Africa.
Medicinal plants I. Buckhuys Publishers, Wageningen, Netherlands. 203 – 206.
[73]. M. F. Asaolu, 2003 .“Chemical composition and phytochemical screening of the seeds
of Garcinia kola,” 46,145–147.
[74]. S. S. K. Alghamdi, 2013 “Topical depilatory and method of removing hair,” Patent
551,187.
[75]. R. Auta and I. Ali, 2011. “Nutritional and chemical value of Solanum incanum (L) (bitter
garden egg),” 1,96–107.
[76]. I. M. F. Abdalla, 2015. “Leaves value of Solanum incanum (L) at Khartoum, North
Sudan,” 4,25–28.
[77]. Liu WJH. 2011. Introduction to Traditional Herbal Medicines and their Study, in
Traditional Herbal Medicine Research Methods: Identification, Analysis, Bioassay,
And Pharmaceutical and Clinical Studies. John Wiley & Sons.
[78]. Chariandy CM,.1999.Screening of medicinal plants from Trinidad and Tobago For
antimicrobial and insecticidal properties. 64(3):265-70
[79]. Liu WJH. 2004. Introduction to Traditional Herbal Medicines and their Study, in
Traditional Herbal Medicine Research Methods: Identification, Analysis, Bioassay,
And Pharmaceutical and Clinical Studies. John Wiley & Sons.
[80]. Hedberg, I, Tadesse, M and Edwards, S. (1995). Flora of Ethiopia and Eritrea Addis
Ababa, Ethiopia ,2, 326-327.
[81]. TG. ,2014. Phytochemical and Pharmacological Properties of Crude Extracts and Pure
Compounds from Leaves of Vernonia Galamensis and Croton macrostachyus: Addis
Ababa University.
[82]. Negash L. 2010.A Selection of Ethiopia's Indigenous Trees: Biology, Uses and
Propagation Techniques. Addis Ababa University Press, Addis Ababa, Ethiopia.386.
[83]. Karunamoorthi K. and Ilango K. (2010).Larvicidal activity of Cymbopogon
citratus (DC) and Croton macrostachyus against Anopheles arabiensis Patton, a
Potent malaria vector. Eur Rev Med Pharmacol Sci; 14:57-62.
[84]. Friis I. (1992). Forests and forest trees of northeast tropical Africa. Their natural
Habitats and distribution patterns in Ethiopia, Djibouti and Somalia. Kew Bulletin,
Additional Series, XV. Her Majesty's Stationery Office, London, 396.
61
[85]. Giday M, Asfaw Z, Woldu Z. 2009. Medicinal plants of the Meinit ethnic group of
Ethiopia: an ethnobotanical study. 124(3):513-521.
[86]. GRIN (2006). Germplasm Resources Information Network. Solanum incanum L. United
States Department of Agriculture.
[87]. Matu E.N., (2008). Solanum incanum L. PROTA (Plant Resources of Tropical Africa);
Wageningen Netherlands. Edited by Schmelzer, G.H. & Gurib-Fakim.
[88]. Hutchinson, J., and Dalziel, J. M. (1963). Flora of West Tropical Africa.Second edn., F.
N. Hepper, London.
[89]. Giday, M., Teklehaymanot, T., Animut, A. and Mekonnen, Y., 2007. Medicinal plants of
the Shinasha, Agew-awi and Amhara peoples in northwest Ethiopia.110(3): 516-525.
[90]. Lulekal, E., Asfaw, Z., Kelbessa, E. and Van Damme, P., 2014. Ethnoveterinary plants of
Ankober District, North Shewa Zone, Amhara Region, Ethiopia. 10(1): 21.
[91]. Obey, J., von Wright, A., Orjala, J., Kauhanen, J. and Tikkanen-Kaukanen, C., 2016.
Antimicrobial activity of Croton macrostachyus stem bark extracts against several
human Pathogenic bacteria. J Pathog.
[92]. Wijesundara, S., Kannangara, B. and Abeywickrama, K., 2016. Antifungal Activity of
Croton aromaticus L. in vitro, Against Post-Harvest Fungal Pathogens Isolated from
Tropical Fruits. 11(2): 105 – 117.
[93]. Mohammed, T., Erko, B. and Giday, M., 2014. Evaluation of antimalarial activity of
leaves Of Acokanthera schimperi and Croton macrostachyus against Plasmodium berghei
in Swiss Albino mice. BMC Complement Altern Med, 14(1): 314.
[94]. Amuamuta, A., Mekonnen, Z. and Gebeyehu, E., 2015. Traditional therapeutic uses and
Phytochemical screening of some selected indigenous medicinal plants from Northwest
Ethiopia. Afri J Pharmacol Ther, 4(3): 80-85.
[95]. Thirugnanam S. Mooligai Maruthuvam. (Tamil) Trichy: Selvi Publishers; 2003. 33, 117,
131,139,147.
[96]. Kasa, M. (1991). Antimalarial activities of local medicinal plants. Traditional Medicine
Newsletter (Ethiopia), 2: 1 – 3.
[97]. Mesfin, F, Sebsebe, D and Tilahun, T. (2009). An ethnobotanical study of medicinal
Plants in Wonago Woreda, SNNPR, Ethiopia. Journal of Ethnobiology and
Ethnomedicine, 5: 28 – 34.
62
[98]. Asfaw M, Sileshi D, Ashenif T et al. 2017. Medicinal Plants Used for the Management
of Rabies in Ethiopia. Medicinal Chemistry. 7:795-806. 69
[99]. Teklay A, Abera B, Giday M. 2012. An ethnobotanical study of medicinal plants used in
Kilte Awulaelo District, Tigray Region of Ethiopia. 9(1):65.
[100]. Mekonnen LB. 2015.In vivo antimalarial activity of the crude root and fruit extracts of
Croton macrostachyus (Euphorbiaceae) against Plasmodium berghei in mice. 5(3):168-
173.
[101]. Amare G. , 1976. Some common medicinal and poisonous plants used in Ethiopian folk
Medicine. Addis Abeba, Ethiopia: Addis Abeba University.
[102]. Zarroug, I. M., Nugud, A. D., Bashir, A. K., & Mageed, A. A. (1988). Evaluation of
Sudanese plant extracts as mosquito larvicides. 26(2), 77-80.
[103]. Dessisa D, Medhin Z, Abebe D. 2001. A preliminary economic evaluation of medicinal
Plants in Ethiopia: trade, volume and price. Paper presented at: National Workshop on
Biodiversity Conservation and Sustainable use of Medicinal Plants In Ethiopia.
[104]. London, UK: Waterside Press, 2004. The Key Steps in Establishing Participatory Forest
Management a Field manual to guide practitioners in Ethiopia. V.1.
[105]. Zelalem Y., 2007.Phytochemical Investigation on the Stem Bark of Croton
Macrostachyus (Bisana): Chemistry, Addis Ababa University.
[106]. Behailu E. 2010. Ethnobotanical Study of Traditional Medicinal Plants of Goma
Wereda, Jima Zone of Oromia Region, Ethiopia: Biology, Addis Ababa University.
[107]. Reta R. 2018.Assessment of indigenous knowledge of medicinal plant practice and
mode Of service delivery in Hawassa city, southern Ethiopia. 7(9):517-535.
[108]. Giday, M, Teklehaymanot, T, Animut, A and Mekonnen, Y. (2007). Medicinal Plants
of the Shinasha, Agewawi and Amhara peoples in northwest Ethiopia.110: 516 – 525.
[109]. Tefera, M., Geyid, A., & Debella, A. (2012). In vitro anti-Neisseria gonorrhoeae
activity Of Albizia gummifera and Croton macrostachyus. Pharmacologyonline, 1, 75-
83.
[110]. Gemechu, A., Giday, M., Worku, A., & Ameni, G. (2013). In vitro Anti-mycobacterial
Activity of selected medicinal plants against Mycobacterium tuberculosis and
Mycobacterium bovis Strains. BMC Complementary and Alternative Medicine, 13(1),
291.
63
[111]. Kamanyi, A., Mbiantcha, M., Nguelefack, T. B., Ateufack, G., Watcho, P., Ndontsa, B.
L.,& Tane, P. (2009). Anti-nociceptive and anti-inflammatory activities of extracts from
the Stem bark of Croton macrostachyus (Euphorbiaceae) in mice and rats. 6(1).
[112]. Bum, E. N., Ngah, E., Mune, R. N., Minkoulou, D. Z., Talla, E., Moto, F. C. O. Moto,
G. T. Ngoupaye, G. S. Taiwe, A. Rakotonirina, and Rakotonirina, S. V. (2012).
Decoctions of Bridelia micrantha and Croton macrostachyus may have anticonvulsant
and sedative Effects. Epilepsy & Behavior, 24(3), 319-323.
[113]. Gelaw, H., Adane, L., Tariku, Y., & Hailu, A. (2012). Isolation Of crotepoxide from
berries Of Croton macrostachyus and evaluation of its anti-leishmanial activity.1(4),
2278- 4136.
[114]. Busa, Z., & Engidawork, E. (2014). Evaluation of in vivo anti-diarrheal activity of 80%
methanolic extracts of the Leaves of Croton macrostachyus Hochst.(Euphorbiaceae) in
Rats. Unpublished Master. Addis Ababa: Addis Ababa University Institutional
Repository
[115]. Amuamuta, A., Mekonnen, Z., & Gebeyehu, E. (2015). Traditional therapeutic uses and
Phytochemical screening of some selected indigenous medicinal plants from Northwest
Ethiopia., 4(3)
[116]. Meresa, A., Gemechu, W., Basha, H., Fekadu, N., Teka, F., Ashebir, R., & Tadele, A.
(2017). Herbal Medicines for the Management of Diabetic Mellitus in Ethiopia and
Eretria Including their Phytochemical Constituents.5(1).
[117]. Abebayehu, A., Mammo, F., & Kibret, B. (2016). Isolation and characterization of
terpene From leaves of Croton macrostachyus (Bissana). 10(19), 256-269.
[118]. Aylate, A. L. E. M. U., Agize, M. A. T. H. E. W. O. S., Ekero, D. E. S. T. A., Kiros, A.
S. H. E. N. A. F. I., Ayledo, G. E. W. A. D. O., & Gendiche, K. A. G. N. U. R. O.
(2017). In-Vitro and In-Vivo Antibacterial Activities of Croton macrostachyus Methanol
Extract Against E. coli and S. aureus. Advances in Animal and Veterinary Science, 5(3),
107-114.
[119]. Tala, M. F., Tan, N. H., Ndontsa, B. L., & Tane, P. (2013). Triterpenoids and phenolic
Compounds from Croton macrostachyus. Biochemical Systematics and Ecology, (51),
138-141.
64
[120]. Tene, M., Ndontsa, B. L., Tane, P., de Dieu Tamokou, J., & Kuiate, J. R. (2009).
Antimicrobial diterpenoids and triterpenoids from the stem bark of Croton
macrostachys. 3(3).
[121]. Kapingu, M. C., Guillaume, D., Mbwambo, Z. H., Moshi, M. J., Uliso, F. C., &
Mahunnah,R. L. (2000). Diterpenoids from the roots of Croton macrostachys.
Phytochemistry, 54(8), 767-770.
[122]. Cronquist, A. (1981). An Integrated System of Classification of Flowering Plants.
Colombia university press, New York. 1262.
[123]. Harrison, D. M. (1990). Steroidal Alkaloids, Natural Product Reports, 7(2): 139–147.
[124].Blankemeyer, J. T., McWilliams, M. L., Rayburn, J. R., Weissenberg, M., and
Friedman,M.(1998). Developmental Toxicology of Solamargine and Solasonine
Glycoalkaloids in Frog Embryos. Food and Chemical Toxicology, 36(5): 383–389.
[125]. Goswami, A., Kotoky, R., Rastogi, R. C., and Ghosh, A. C. (2003). A One-pot Efficient
Process for 16-dehydropregnenolone Acetate. Organic Process Research and
Development, 7(3): 306–308.
[126]. Sana, S., Al Sinani, E., A. Eltayeb, Y. T., Kamal, Masood S., Khan and Ahmad, S.
(2015).Variations in the Cytotoxic Glycoalkaloids Solamargine and Solasonine in
Different Parts Of the Solanum incanum Plant during its Growth and Development in
Oman. 4-8.
[127]. Chen, Z. and Miller, R. (2001). Steroidal Alkaloids in Solanaceous Vegetable Crops.
Horticultural Review, 25: 171-196.
[128]. Yoshikawa, M., Nakamura, S., Ozaki, K., Kumahara, A., Morikawa, T. and Matsuda,
H.(2007). “Structures of Steroidal Alkaloid Oligoglycosides, Robeneosides A and B,
and Antidiabetogenic Constituents from the Brazilian Medicinal Plant Solanum
lycocarpu 70(2): 210–214.
[129]. Fewell, A. M., Roddick, J. G. and Weissenberg, M. (1994). Interactions between the
Glycoalkaloids Solasonine and Solamarrgine in Relation to Inhibition of Fungal
Growth. Phytochemistry, 37(4): 1007–1011.
[130]. Hall, C. A., Hobby, T., and Cipollini, M. (2006). Efficacy and Mechanisms of α-
Solasonine- and α-solamargine-induced Cytolysis on two Strains of Trypanosoma
cruzi. 32(11): 2405–2416.
65
[131]. Cham, B. E. (2007). Solasodine Rhamnosyl Glycosides Specifically Bind Cancer Cell
Receptors and Induce Apoptosis and Necrosis. Treatment for Skin Cancer and Hope for
Internal Cancers, Research in Biological Sciences, 2(4): 503–514.
[132]. Kuo, K. W., Hsu, S. H., Li, Y. P. (2000). Anticancer Activity Evaluation of theSolanum
Glycoalkaloid Solamargine: Triggering Apoptosis in Human Hepatoma Cells.
Biochemical Pharmacology, 60(12): 1865–1873.
[133]. Cham, B. E. and Daunter, B. (1990). Solasodine Glycosides. Selective Cytotoxicity for
Cancer Cells and Inhibition of Cytotoxicity by Rhamnose in mice with Sarcoma 180.
Cancer Letters, 55(3): 221–225.
[134]. Barbosa, A. G., Oliveira, C. D., Lacerda-Neto, L. J., Vidal, C. S., Saraiva, R. D. A., da
Costa, J. G., Coutinho H., Galvao H. & de Menezes, I. R. (2017). Evaluation of
chemical Composition and antiedematogenic activity of the essential oil of Hyptis
martiusii Benth. 24(2), 355-361.
[135]. Taniguchi, M., and I. Kubo. 1993. Ethnobotanical drug discovery based on
medicinemen‟s trials in the African savannah: screening of East African plants for
antimicrobial activity II. Journal of Natural Products, 56(9): 1539-1546.
[136]. Ikan, R. 1969. Natural products: A laboratory guide, Jerusalem: Israel: Israel University
Press. International Archives of Applied Immunology, 94: 262-265.
[137]. Harborne, J.B. 1984. Phytochemical methods. A guide to modern techniques of plant
Analysis, 2nd edition, London: Chapman and Hall, 2: 89.
[138]. VanBelkum A, Melles DC, Nouwen J, et al. 2009. Co-evolutionary aspects of human
colonisation And infection by Staphylococcus aureus. Infect Genet Evol. 9: 32-47.
[139]. Verma S, Singh S. 2008. Current and future status of herbal medicines. Vet World.
1(11): 347.
[140]. Wadood A, Ghufran M, Jamal S, et al. 2013. Phytochemical analysis of medicinal
plants Occurring in local area of mardan. Biochem Anal Biochem. 2: 1-4.
[141]. Wakjira K. 2007.Seed germination physiology and nursery establishment of Croton
macrostachyus Hocht. Ex Del. [master’s thesis]. Addis Ababa, Ethiopia: Addis Ababa
University.
[142]. Wakjira K, Negash L. 2013.Germination responses of croton macrostachyus
(Euphorbiaceae) to various physic chemical pre-treatment conditions. 87: 76-83.
66
[143]. Gossman W, Wasey A, Salen P.; 2018. Escherichia Coli (E Coli 0157 H7). FL, USA:
Stat Pearls Publishing.
[144]. Nduagu, C., Ekefan, E. and Nwankiti, A., 2008. Effect of some crude plant extracts on
Growth of Colletotrichum capsici (Synd) Butler & Bisby, causal agent of pepper
Anthracnose. 6(2): 184-190.
[145]. Jones WP, Kinghorn AD. 2005. Extraction of plant secondary metabolites.In: Sarker
SD,Latif Z, Gray AI, editors. Natural products isolation. 2nd ed. New Jersey: Humana
Press, 323–51.
[146]. Singh J. 2008. Maceration, percolation and infusion techniques for the extraction of
medicinal and aromatic plants. In: Handa SS, Khanuja SPS, Longo G, Rakesh DD,
editors. Extraction technologies for medicinal and aromatic plants. Italy: International.
Centre for Science and High Technology, 67–82.]
[147]. Evans’ W.C. 1996. Trease and Evans Pharmacognosy, 14th edition, WB Sauders
Company Ltd; London. 224–228, 29 –309, 542–575.
[148]. Harbone J.B.,1998. Phytochemical Methods: A Guide toModern Techniques of Plant
Analysis. Edn 3, Chapman & Hall, London, UK, 60-66.
[149]. Kebede, T., Kibret, F., Fikre, M., & Milkyas, E. (2015). Phytochemical Screening and
Characterization of Olean-18-ene Type Triterpeniod from the Roots of Lantana camara.
Science, , 4(1), 91-94.
[150]. Brian KR, Turner TD (1975). Practical Evaluation of Phytochemicals. Wright
Scentechnical, Bristol, UK. 57-59.
[151]. Vishnoi NR (1979). Advanced practical chemistry. Ghaziabad-India: Yikas Publication
House, PVT Ltd. 447-449.
[152]. Markham KR (1982). Techniques of Flavonoids Identification. Academic Press: New
York, USA.1-113.
[153]. Silva LG, Lee IS, Aflinnghorn DA (1998). Special problem with extraction of plant in
Natural product isolation. Human Press Inc. 999, Review drive, suite 208, Totowa, New
Jersey, USA 072512. 343- 364.
[154]. Sofowora A (2008). Medicinal plants and traditional medicine in Africa.3rd Edition,
Spectrum Books limited, Ibadan, Nigeria. 289.
[155]. Kokate CK. 2001. Practical Pharmacognosy (4th ed).Vallabh Prakashan, New Delhi,
India 107- 111.
67
[156]. Rios JL, Recio MC, Villar A. 1988. Screening methods for natural products with
antimicrobial Activity: A review of the literature. 23:127-49.
[157]. Abo KA, Ogunleye VO, Ashidi JS. 1999.Antimicrobial potential of Spondias mombin,
Croton zambesicus and Zygotritonia crocea. Phytother Res.; 13(6):494-7.
[158]. Shalid M, Tayyab M, Naz F, Jamil A, Ashraf M, Gilani AH. 2008. Activity-guided
isolation of a Novel protein from Croton tiglium with antifungal and antibacterial
activities. Phytother Res.; 22(12):1646-9.
[159]. Salem, M. Z. M., Aly, H., Gohar, Y., & El-Sayed, A. W. (2013). Biological activity of
Extracts from Morus Alba L., Albizzia lebbeck (L.) Benth. And Casuarina glauca
Sieber Against the growth of some pathogenic bacteria. 2(1), 9-22.
68
6. Appendix’s Appendex 1.1: instruments and apparatus (photo by: shewaferaw)
Mortar electronic Weighing balance beker conical flask
Round bottom flask measuring cylinder test tube rota vapor
Fennul autoclave incubator Bunsen burner
Pitridish bottle brush water bath spatula
69
Appendix 1.2: Photography of the solanum incanum materials (taken by shewaferaw me
ngesha)
S.incanum leaf (A) S.incanum seed (B) S.incanum root (C)
Appendix 1.3: Photography of the croton macrostachyus materials (taken by shewaferaw me
ngesha)
C.macrostachyus leaf (A) C.macrostachyus root (B) C.macrostachyus stem bark(C)
Appendix 1.4: S.incanum and C.macrostachyus plant parts(powdered form) (photo by:
shewaferaw)
Seed of enbuay (SI1) (A) root of Enbuay (SI2) (B) leaf of enbuay (SI3) (C)
70
Root of bisana (CM1) (D) leaf of bisana(CM2) (E) stem bark of bisana (CM3)(F)
Appendix 1.5: S.incanum and C.macrostachyus plant parts(material) maceration (photo by:
shewaferaw)
Appendix 1.6: S.incanum and C.macrostachyus plant parts(material) filtrate (photo by: shewaferaw)
Seed of enbuay filtrate (SI1) (A) root of Enbuay filtrate(SI2) (B) leaf of enbuay filtrate (SI3) (C)
Root of bisana filtrate(CM1) (D) leaf of bisana filtrate(CM2) (E) stem bark of bisana filtrate
(CM3)(F)
71
Appendix 1.7: Phytochemical constituents of ethanol extract of C.macrosachyus and
S.incanum(comparation)
No P.chemicals reagents C.change C.macrostachyus S.incanum
S. bark Leaf Root seed leaf root
1 L. Tannins DI,FeCl3 dark green - + - + + +
2 Flavonoids con.H2SO4 orange + + + + + +
3 Alkaloids mayer
reagent
white ppt + - + + - -
4 Saponins froth test froth formation - + + + + -
5 Terpinoid salkowiski
test
redish brown + + + + + +
6 Phenol DI,FeCl3 green - + + + - +
7 steriod salkowiski
test
reddish brown + - + - + +
8 Anthraquinones H2SO4,NH3 Red - - - - - -
9 cumarin NaOH yellow + + + + + +
72
Appendix 1.8: Results from disc diffusion assay showing the antibacterial activity (Diameter
Of the Inhibition Zone, in mm) of crude ethanol extract of Croton
macrostachyus (S.bark, leaf and root) plant parts against 11 microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Croton macrostachyus
\stem bark/
Chloramphenicol
/CD/
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.85± 0.21
7
250 6.9±0.141
7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 7.5±0.70
16
250 10.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 8.9±0.141 10
250 9.9±0.141 15
4 Staphylococcus aureus (Sa)
(ATTC 25923)
150 - 24
250 6.75±0.35 26
5 streptococcus pyogenes (Sp)
(ATCC19615)
150 7.9±0.141 -
250 8.4±0.84 -
6 salmonella typhimurium
(Sal)(ATCC13311)
150 6.85±0.21 9
250 9.75±0.35 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 16
250 - 27
8 Escherichia coli (Ec)
(ATTC 25922)
150 6.75±0.35 8
250 7±0 7
9 pseudomonas
aeruguinosa(Pa)
(ATCC27853)
150 6.8±0.28 16
250 7±0 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans (Ca) 150 6.95±0.07 7
250 7.05±0.07 7
73
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Croton macrostachyus
/leaf/
Chloramphenicol
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 - 7
250 7.5±0.7 7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 7.5±0.7 16
250 10.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 9.5±0.70 11
250 9.5±0.70 16
4 Staphylococcus aureus (Sa)
(ATTC 25923)
150 - 25
250 9.5±0.70 26
5 streptococcus pyogenes (Sp)
(ATCC19615)
150 7.4±0.56 -
250 7.5±0.70 -
6 salmonella typhimurium
(Sal)(ATCC13311)
150 - 10
250 9.5±0.70 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 16
250 9.5±0.70 26
8 Escherichia coli (Ec)
(ATTC 25922)
150 6.5±0.70 7
250 7.5±0.70 7
9 pseudomonas aeruguinosa(Pa)
(ATCC27853)
150 9.5±0.70 17
250 9.5±0.70 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans (Ca) 150 6.5±0.70 6
250 7±0 6
74
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
C. macrostachyus
(root)
Chloramphenicol
(CD)
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.9±0.14 7
250 7.5±0.70 7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 8.5±0.70 16
250 10.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212) 150 7.5±0.70 11
250 8.5±0.70 17
4 Staphylococcus aureus (Sa)
(ATTC 25923) 150 7.5±0.70 25
250 8±0 26
5 streptococcus pyogenes (Sp)
(ATCC19615) 150 6.8±0.28 -
250 7±0 -
6 salmonella typhimurium
(Sal)(ATCC13311) 150 7.5±0.70 10
250 7±0 12
7 shigella sonnei(Sh)
(ATCC25931) 150 - 16
250 7.5±0.70 26
8 Escherichia coli (Ec)
(ATTC 25922) 150 6.5±0.70 7
250 7.75±0.35 7
9 pseudomonas aeruguinosa(Pa)
(ATCC27853) 150 - 17
250 - 20
10 salmonella enteritidis
(Ae )(ATCC13076) 150 - 18
250 7±0 18
11 candida albicans (Ca) 150 7.5±0.70 -
250 6.9±0.14 -
75
Appendix 1.9: Results from disc diffusion assay showing the antibacterial activity (Diameter
Of the Inhibition Zone, in mm) of crude ethanol extract of Solanum incanum
(Seed, leaf and root) plant parts against 11 microorganisms.
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(seed)
Chloramphenicol
(CD)
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.5±0.70 7
250 6.5±0.70 7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 6.5±0.70 15
250 7.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212)
150 6.5±0.70 10
250 7.5±0.70 16
4 Staphylococcus aureus (Sa)
(ATTC 25923)
150 - 24
250 9.5±0.70 26
5 streptococcus pyogenes (Sp)
(ATCC19615)
150 - -
250 6.9±0.14 -
6 salmonella typhimurium
(Sal)(ATCC13311)
150 - 9
250 7±0 12
7 shigella sonnei(Sh)
(ATCC25931)
150 - 15
250 - 27
8 Escherichia coli (Ec)
(ATTC 25922)
150 6.5±0.70 7
250 7±0 7
9 pseudomonas aeruguinosa(Pa)
(ATCC27853)
150 - 16
250 - 20
10 salmonella enteritidis
(Ae )(ATCC13076)
150 - 18
250 7±0 18
11 candida albicans (Ca) 150 7±0 7
250 7±0 7
76
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(root)
Chloramphenicol
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.9±0.14 7
250 7±0 7
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 - 15
250 7.5±0.70 17
3 Enterococcus faecalis(Ef)
(ATCC29212) 150 8.5±0.70 10
250 8.5±0.70 16
4 Staphylococcus aureus (Sa)
(ATTC 25923) 150 - 24
250 11.5±0.70 26
5 streptococcus pyogenes (Sp)
(ATCC19615) 150 7±0 -
250 7.5±0.70 -
6 salmonella typhimurium
(Sal)(ATCC13311) 150 6.5±0.70 9
250 8.5±0.70 12
7 shigella sonnei(Sh)
(ATCC25931) 150 - 15
250 - 27
8 Escherichia coli (Ec)
(ATTC 25922) 150 6.5±0.70 7
250 6.9±0.14 8
9 pseudomonas aeruguinosa(Pa)
(ATCC27853) 150 - 16
250 - 20
10 salmonella enteritidis
(Ae )(ATCC13076) 150 - 19
250 7±0 18
11 candida albicans (Ca) 150 7±0 6.5
250 7±0 7
77
No Name of Microorganisms Concentration
(mg/ml)
Zone of inhibition(mm)
Solanum incanum
(leaf)
Chloramphenicol
1 listeria monocytogenes
(Lm)
(ATCC19115)
150 6.5±0.70 7
250 6.5±0.70 8
2 Staphylococcus epidermidis
(Se)
(ATCC12228)
150 6.5±0.70 15
250 8.5±0.70 18
3 Enterococcus faecalis(Ef)
(ATCC29212) 150 9.5±0.70 11
250 9.5±0.70 16
4 Staphylococcus aureus (Sa)
(ATTC 25923) 150 - 24
250 - 26
5 streptococcus pyogenes (Sp)
(ATCC19615) 150 - -
250 6.5±0.70 -
6 salmonella typhimurium
(Sal)(ATCC13311) 150 8.5±0.70 9
250 9.5±0.70 12
7 shigella sonnei(Sh)
(ATCC25931) 150 - 15
250 - 27
8 Escherichia coli (Ec)
(ATTC 25922) 150 6.5±0.70 7
250 6.5±0.70 7
9 pseudomonas aeruguinosa(Pa)
(ATCC27853) 150 - 16
250 7.5±0.70 20
10 salmonella enteritidis
(Ae )(ATCC13076) 150 - 18
250 7.5±0.70 18
11 candida albicans (Ca) 150 7±0 7
250 7±0 8
78
Appendix 1.10: Results from disc diffusion assay showing the evaluation of antibacterial
activity (Diameter of the Inhibition Zone, mm) the ethanolic crude extract the leaf of
C.macrostachyus and the ethanolic crude extract the root of S.incanum against Staphylococcus
aureus (Sa) (ATTC 25923) Bacteria.
Microorganism
concentration
C.macrostachyus S.incanum
Stem
bark
leaf root seed leaf root
Staphylococcus
aureus(Sa) (ATTC
25923)
150mg/ml - - 7.5±
0.70
- - -
250mg/ml 6.75±
0.35
9.5±
0.75
8 ± 0 9.5±
0.70
- 11.5±0.70
Appendix’s 1.11: Some Petri dishes showing the activity of parts of C.macrostachyus and
S.incanum on eleven bacteria’s
S.incanum and C.macrostachyus S.incanum and C.macrostachyus plant
parts Ethanol Plant parts Ethanol crude crude extract on Staphylococcus
Extract on Enterococcus faecalis aureus
S.incanum and C.macrostachyus plant parts S.incanum and C.macrostachyus plant
parts Ethanol crude extract on Ethanol crude extract on Staphylococcus
listeria monocytogenes epidermidis
79
S.incanum and C.macrostachyus S.incanum and C.macrostachyus plant parts
Plant parts Ethanol crude extract Ethanol crude extract on Escherichia coli
on salmonella typhimurium
S.incanum and C.macrostachyus plant parts S.incanum and C.macrostachyus plant parts
Ethanol crude pseudomonas aeruguinosa Ethanol crude extract on salmonella enteritidis
S.incanum and C.macrostachyus plant parts S.incanum and C.macrostachyus plant parts
Ethanol crude extract on candida albicans Ethanol crude extract on streptococcus pyogenes