STUDENT NAME………….……….
ID NO
1) MAMUSH BEKELE..................... UGR/51937/13
2) AMANUEL WANA........................ UGR/52241/13
3) YACOB YOHANNIS....................... UGR/52144/13
Advisor: - Mr. MISGANA
April 2023
AMBO, ETHIOPIA
CONTENT
ABSTRACT:................................................................................................................................1
CHAPTER ONE...........................................................................................................................1
1. INTRODUCTION.............................................................................................................1
1.1 Background of the Study:................................................................................1
1.2 Statement of the Problem Recently......................................................................3
1.3. The objective of the study...................................................................................3
1.4 Significance of the study..............................................................................................4
2. LITERATURE REVIEW......................................................................................................5
2.1 Botanical descriptions of Eucalyptus camaldulensis............................................5
2.2 Traditional uses of Genus Eucalyptus camaldulensis...........................................6
2.3 Biological activities of plants Eucalyptus camaldulensis.....................................8
3. MATERIALS AND METHODS...........................................................................................9
3.1 Plant material.......................................................................................................9
3.2 Extraction procedure............................................................................................9
3.3 Phytochemical screening......................................................................................9
References..............................................................................................................................11
ABSTRACT:
Photochemical are active secondary plant metabolites responsible for most of the claimed
medicinal activities of plants. Eucalyptus camaldulensis is one of those plants that possess
these phytochemicals and claimed to possess medicinal activities on various ailments.
Qualitative screening revealed that tannins, saponins, glycosides, steroids and anthraquinones
were present in aqueous extract of all the parts of the plant, whereas alkaloids, flavonoids and
terpenoids were absent. On the other hand, tannins and steroids were present in the ethanolic
extract of all the parts of the plant, while saponins, alkaloids, flavonoids and terpenoids were
present only in some parts of the plant. However, glycosides and anthraquinone were absent
in all the ethanolic extracts. The quantitative screening revealed large amount of saponins in
both aqueous and ethanolic extracts across the various parts of the plant. Whereas small
amount of tannins, alkaloids and flavonoids were found only in the ethanolic extract of some
parts of the plant. The presence of these phytochemicals in Eucalyptus camaldulensis could
therefore justify the applications of the plant in management and curing of various ailments as
claimed traditionally.
Keywords: Eucalyptus camaldulensis, Phytochemical screening, Aqueous extract, Ethanolic extract.
CHAPTER ONE
1. INTRODUCTION
1.1 Background of the Study:
Eucalyptus camaldulensis is a plant belonging to the Myrtaceae family mostly found
in tropical, subtropical and temperate environments. For several years, attention has
been given to Eucalyptus species due to their medical characteristics. The
ethnomedicinal benefits of the plants have also been ascribed to several valuable
compounds such as flavonoids, phenols, sterols, terpenes, resins, saponin, cardiac
glycosides, tannin among others. Several studies have also reported the
antihyperglycemic, antibacterial activity, antioxidants, and antiinflammatory
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tendencies of leaves extracts of Eucalyptus species as well as antimicrobials activity.
Medicinal plants have been identified and used throughout human history.
Ethnobotany is recognized as an effective way to discover future medicines. Plants
have the ability to synthesize a wide variety of chemical compounds that are used to
perform important biological functions and to defend against attack from predators
such as insects, fungi and herbivorous mammals. At least 12,000 such compounds
have been isolated so far; a number estimated to be less than 10% of the total. In
2001, researchers identified 122 compounds used in modern medicine which were
derived from "ethnomedical" plant sources, 80% of these have had an ethnomedical
use identical or related to the current use of the active elements of the plant. Many of
the pharmaceuticals currently available to physicians have a long history of use as
herbal remedies including aspirin, digitalis, quinine, and opium. Chemical compounds
in plants mediate their effects on the human body through processes identical to those
already well understood for the chemical compounds in conventional drugs, thus
herbal medicines do not differ greatly from conventional drugs in terms of how they
work.
Eucalyptus is the most important genera in the botanical family Myrtaceae; it is
widely distributed in different regions around the world, with more than 800 species.
Eucalyptus camaldulensis Dehnh, commonly known as the river red gum, is endemic
in Australia Leaves of E. camaldulensis are known to possess several biological and
pharmacological activities, including antioxidant and anti-dermatophytes. Literature
survey revealed the isolation and identification of some chemical ingredients from
different parts of E. camaldulensis including eucalyptanoic acid. The microbial
resistance to the existence antibiotics is well known, so it is very important to search
for alternative antimicrobial agents from natural source like plants or herbs to
overcome this challenge.
Eucalyptus camaldulensis, commonly known as the River Red Gum, is one of the
most widely distributed and ecologically significant tree species in Australia. Native
to the southern and eastern regions of the country, this large, evergreen tree has
played a crucial role in the continent's ecosystems, economy, and cultural heritage for
thousands of years.The River Red Gum is renowned for its ability to thrive in a wide
range of climatic conditions, from semi-arid to temperate regions. Its adaptability and
resilience have allowed it to colonize a diverse array of habitats, particularly along
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rivers, streams, and floodplains, where it plays a vital role in stabilizing riverbanks
and preventing soil erosion.Despite its ecological importance, the River Red Gum has
faced a range of threats in recent decades, including habitat loss and degradation,
overgrazing, and the impacts of climate change. These challenges have prompted
increased research and conservation efforts to better understand the biology, ecology,
and management of this iconic Australian tree species.The present study aims to
contribute to the growing body of knowledge on Eucalyptus camaldulensis by
investigating various aspects of its biology, ecology, and potential applications.
1.2 Statement of the Problem Recently
World Health organization (WHO) has issued on statement of great concern regarding
bacterial resistance which is becoming a global problem (Ajaily and Maihub , 2018).
In the past decade bacterial and viral infection and their resistance for many
antibiotics is becoming a problem. The increase of death rate is directly linked to the
bacteria and virus that have ability to resist and reduce the activity of the antibiotics.
So that for prevention of this serious medicinal problem, it is necessary to develop
new antimicrobial agents through extracting the bio active compounds from different
plants. Many phytochemical screening was performed on different parts of Eucalyptus
canaldulensis by using different solvent extract but till now their structures are not
reported, and society has no more information on curing abilities and biological
activities of stem barks of this plant. Therefore this study is mainly focuses on the
phytochemical investigation, anti-oxidant activity and antibacterial activities of stem
bark extracts of Eucalyptus canaldulensis.
1.3. The objective of the study
1.3.1. General Objective
The general objective of this study is to examine phytochemical constituents and
biological activities.study of stem bark extract of Eucalyptus canaldulensis. The
confirmatory qualitative phytochemical screening of plant extracts was performed to
identify the main classes of compounds (tannins, saponins, flavonoids, alkaloids,
phenols, glycosides, steroids, and terpenoids) present in the extracts following
standard protocols
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1.3.2. Specific objective of the study
To extract phytochemical
principles from the stem bark of
Eucalyptus canaldulensis using
maceration techniques.
To conduct selected qualitative tests
on the crude extract using standard
procedures.
To isolate compounds using by chemical techniques.
To purify the isolates and characterize using spectroscopic methods.
To conduct anti-bacterial and antioxidant activities of the crude extract.
1.4 Significance of the study
The extracts for phytochemical screening and antimicrobial activity were carried out
on dried fresh leaves, Eucalyptus globulus using water as solvent. The results of the
phytochemical screening showed that the plant parts contained saponins, tannins,
phenols and glycosides.It is important that medicinal plants be analysed to determine
their phytochemical constituents, which would be beneficial in understanding the
pharmacological importance and health risks. The areas of significance to humans of
Eucalyptus camaldulensis include agricultural, ecological, cultural, and recreational
significance. The speed of growth of the tree makes it a useful plantation timber.
Apiarists also use the tree's flowers for honey production. Maximum amount of
phytochemicals present in eucalyptus leaves were retained in methanolic extract. The
methanolic extract contained quinones, saponins, carbohydrates, tannins, phenols,
flavonoids & fat while in the acetonic extract quinones, flavonoids & fat was present.
Eucalyptus has become a wonderful symbol of strength, protection, and abundance.
As such, Australian Aboriginals practiced burning eucalyptus leaves to purify and
negate negative energy and saw eucalyptus as a sacred plant.
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2. LITERATURE REVIEW
2.1 Botanical descriptions of Eucalyptus camaldulensis
Eucalyptus is one of the world’s important and most widely planted genera belonging
to the family Myrtaceae This family includes 140 genera and about 3800 species
grown in tropical and subtropical parts of the world Eucalyptus is native to Australia,
and the genus contains about 600 species. A smaller number are also native to New
Guinea, Indonesia and the Phillipines. Eucalyptus is a fast-growing tree, and is
suitable for use in paper production. There has been extensive overseas forest
plantation of Eucalyptus trees.it have local such as names Amharic (key bahir zaf);
Arabic (ban,kafur); Burmese (pyilon-chantha);English (long beak eucalyptus,murray
red gum,red gum,river gum,river redgum,red river gum); French (eucalyptus
rouge,eucalyptus); German(Rotgummibaum,roter eukalyptus); Indonesian (ekaliptus);
Italian(eucalipto rostrato); Luganda (kalitunsi);
Eucalyptus camaldulensis commonly grows to 20 m tall, occasionally reaching 50 m,
with a trunk diameter of 1 (max. 2) m; in open formations has a short, thick bole and a
large, spreading crown; in plantations has aclear bole of 20 m with an erect, lightly
branched crown; bark smooth,white, grey, yellow-green, grey-green or pinkish grey,
shedding in strips orirregular flakes; rough bark occupies the 1st 1-2 m of the trunk.
Tree to 40 m tall. Bark smooth, mottled, shedding at intervals throughout the year to
showwhite, yellow and grey, becoming rough around the base.Juvenile leaves
petiolate, soon alternate, broadly lanceolate to lanceolate, to 11cm long,3cm wide,
dull green.Adult leaves petiolate, alternate, lanceolate, 10-20 cm long, 1-2 cm wide,
dull green. Veinsdense, generally with numerous, clear, yellow and green island oil
glands.Inflorescences unbranched, stalks to 2 cm long, 7 flowered. Buds on stalks,
hemispherical, with beaked cap to 1cm long, scar present.Fruit on stalks,
hemispherical to 0.6 cm long, 1cm diameter. Disc ascending, with 4 valves projecting
(Walsh & Entwisle 1996).
Leaves grey-blue, alternate, drooping, 8-22 cm long, 1-2 cm wide, often curved or
sickle shaped, tapering, short pointed at base.Inflorescence axillary, solitary, 7-11
flowered; flower buds white, globularrostrate or ovoid-conical;operculum
hemispherical, rostrate or conical, 4-6x 3-6 mm, obtuse.Fruit very small capsules at
the end of thin stalks, 5-8 mm, valves 4,containing minute seed The genus Eucalyptus
5
was described and named in 1788 by the French botanist l’Héritier. The flowers of the
various Eucalyptus species areprotected by an operculum, hence the generic name,
which is from theGreek words ‘eu’ (well) and ‘kalyptos’ (covered). The specific
epithet honours Count Camaldoni in whose garden E. camaldulensis was planted in
1803.
2.2 Traditional uses of Genus Eucalyptus camaldulensis
Used for centuries as a traditional Aboriginal herbal remedy, eucalyptus leaves and
their essential oils have found various applications in everyday life due to their
antiseptic, anti-inflammatory and antipyretic properties Ancient Aboriginal society in
Australia used E. camaldulensis plant in medicines to treat gastrointestinal symptoms
(including colic, diarrhea, and dysentery), respiratory disease (colds, coughs, asthma,
laryngalgia, laryngitis, pharyngitis, sore throat, trachalgia), arrest bleeding, open
wounds, and cuts, as well as its decoctions for the relief of spasms, aches, and pains in
muscles, but also pains in joints and even toothAs previously stated, E. camaldulensis
plant is also known as red gum eucalyptus, murray red gum, and river red gum,
because it produces red kino i.e. red gum, in significant amount. Thus besides
different plant parts Aborigines used its secondary products as folk remedies. They
made incisions in the tree trunks for obtaining the red kino and applied it directly to
abrasions and cuts. Except fresh kino, the dried, dehydrated kino was prepared and
used in the same way as fresh, but after softening in water
Another significant folk remedy is young leaves which were used for smoke bath,
where burning leaves smoke surrounds patient. The smoking medicine was used for
fevers, colds, flu and general sickness Being useful for treating various health
conditions, E. camaldulensis and its folk remedies then were transferred and
introduced to other parts of the world, such as Africa. In Sudan the red kino was used
for sore throat and diarrhea, while the smoke of burnt leaves was inhaled in case of
respiratory problems. In Senegal for stomach-ache decoctions from leaves were
prepared with sugar, while in Zimbabwe for cough, flu, and fever a decoction of E.
camaldulensis leaves were combined with Citrus limon (L.) Burm. f. fruits and
Psidium guajava L. leaves Further more, to prevent tooth decay and periodontitis in
Nigeria teeth cleaning sticks were made from tree and in traditional medicine for
healing wound infections, poultice of leaves containing eucalyptus oil have been used
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Nowdays, E. camaldulensis has been the subject of numerous studies, toconfirm
plant’s usefulness in traditional medicine for the treatment of various ailmentIts
essential oils are reported to be anesthetic, antiseptic andastringent In addition, a
decoction of the leaves is reported to be a remedy for sore throat and other bacterial
infections of the respiratory and urinary tracts Due to numerous contemporary data
regarding the antimicrobial activity of E. camaldulensis, this subject will be discussed
in special section/chapter.Except antimicrobial effects, E. camaldulensis plant extracts
(PEx) and essential oils (EOs) and its constituents possess numerous other beneficial
effects. One such effect is certainly gastrointestinal effect. In animal models, extracts
of the leaves of E. camaldulensis and E. torelliana R. Muell are reported to decrease
gastric acid production and thus appear useful for the treatment ofgastric ulcer.
The antioxidant effect represents one more useful E. camaldulensis characteristic. The
free radical scavenging activities of the essential oils is assessed by measuring their
scavenging abilities for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The
scavenging activity for the Eucalyptus camaldulensis was characterized as high
(81.9%) The results of E. camaldulensis EO antioxidant effect evaluation indicated
that the EO had high potent ferrous ions chelating and total antioxidant activities
comparing to ascorbic acid and BHT. Also, the E. camaldulensis var. brevirostris
leaves ethanol extract possessed antioxidant activity, where the prevailing
antioxidants in the extract were gallic and ellagic acid There are several more studies
dealing with antioxidant activity of E.camaldulensis but it is interesting to mention
here that E. camaldulensis flower EO inhibited melanogenesis through its antioxidant
properties and by down-regulating both mitogen-activated protein kinases (MAPK)
and protein kinase A (PKA) signaling pathways. This study indicated that the
essential oil has the potential to be developed into a skin care productThus, except the
medical application, E. camaldulensis extracts are also currently used in cosmetic
formulations, and leaf extracts have been approved as food additives . Also, essential
oils and their constituents have been used as flavoring agents in the formulation of
different pharmaceutical products, cosmetics, and food industry Except this, E.
camaldulensis PEx and EOs have the potential to be used as antibacterial and
antifungal agents in cosmetic and pharmaceutical products.
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2.3 Biological activities of plants Eucalyptus camaldulensis
2.3.1 Antioxidant activities
Antioxidants play an important role in foodpreservation by inhibiting oxidation
processesand contributing to the health promotionprovided by many dietary
supplements,nutraceuticals, and functional food ingredients
(Shahidi and Zhong, 2015). Furthermore, naturalantioxidants are in demand for
pharmaceuticalsproducts (Brewer, 2011). Therefore, in recentyears, considerable
attention has been detectedtowards the identification of plants withantioxidant
activity.
2.3.2 Antimicrobial activities
The antimicrobial activity of theEucalyptus camaldulensis essential oils has been
studied by severalresearchers and discussed in the text; theessential oils exhibit
toxicity against a widerange of microbes, including bacteria, fungi,yeast but the
bioactivity against virus has not investigated
2..4 Antifungal activities
An antifungal activity of the different part(stems, adult leaves, fruits, and immature
flowers) of E. oleosa essential oils were testedagainst three pathogenic fungi
Aspergillusochraceus, Mucor ramamnianus and Fusariumculmorum and demonstrated
that immatureflowers and stems had strongest antifungalactivity with minimal
inhibition concentrationvalue between 2.79-3.88 mg/ml (Srinivasan et
al., 2001). Additionally, these findings wereconsistent with another study performed
byKouki et al. (2023) who reported that E. oleosaEOs exhibited a
significantantifungal activityagainst five Fusarium ssp.Generally, E. oleosa essential
oils showedvariable antimicrobial activity against thedifferent test organisms. This
variability could berelated to several factors such as chemicalcomposition of essential
oils and geographiclocation of the plant material, also the sensitivityof the bacterial
strains and its nature (Sabo andKnezevic, 2019)
2.3.5 Antiviral activities
8
The antimicrobial activity of plant extract with different solvent was determined by
the well diffusion method on Mueller Hinton agar medium . Briefly, 15 ml of sterile
medium (at 45°C) was uniformly mixed 100 μL of inoculum (corresponding to 10
CFU/ml) of different strains and then poured in Petri dishes. After solidification of
medium, wells (6 mm diameter) were made in each Petri‐dish, to which 30 μL of the
different extracts were added. At the center of Petri dish, antibiotics disc of
Chloramphenol (30 μg/mL) placed and used for the control. The plates were
incubated at 36±2 °C for 24 h and the diameter of the resulting zone of inhibition, if
any, was measured.
3. MATERIALS AND METHODS
3.1 Plant material
The leaves of Eucalyptus camaldulensis were collected from the area of Ambo
Universitry.The leaves were air-dried for 15 days, milled into powder and sieved with
1 mm mesh. The various powders obtained were stored in glass bottles and then kept
in a refrigerator at 4 °C. until the extraction period.
3.2 Extraction procedure
The extraction procedure was cold maceration method as used by Perry et al. (2007) .
One kilogram of each leaves powder plant were macerated in 3 L of hexane for 3 days
in the glass jar (5 L) and the maceration was turned twice a day. Then the maceration
were filtrated through Whatman No.1 filter paper to obtain hexane filtrate and residue
for each plant. Then, the same procedure was reproduce respectively with acetone and
methanol to give acetone and methanol extracts. The dry plant extracts were stored at
-4°C until use for phytochemical screenings and bioassays.
3.3 Phytochemical screening
The phytochemical composition of different extracts was evaluated according to the
methods described by Harborne with slight modifications.
9
3.3.1 Test for identification of phenolic compounds
3.3.1.1 Ferric chloride test: To 0.2 mL of extract solution was added a drop of the
3% ferric chloride solution. The appearance of a darkish blue or green coloration
more or less dark characterizes the presence of phenolic derivatives.
3.3.2 Test for identification of Flavonoids
3.3.2.1 Shinoda test: Magnesium chips and concentrated hydrochloric acid were
added drop wise to the test solution. Reddish-pink colour with effervescence indicates
the presence of flavonoid.
3.3.3 Test for identification of Tannins
Five grams of the ground powder was extracted with 10 mL ammonical chloroform
and 5 mL chloroform. The mixture was filtered and the filtrate was shaken with 10
drops of 0.5 M sulphuric acid. Creamish white precipitate was observed for the
presence of tannins.
3.3.4 Test for identification of coumarins
In test tube containing 2 mL of extract, 3 ml of NaOH (10%) were added. After
shaking the solution, the appearance of a yellow colour indicates the presence of
coumarins.
3.3.5 Test for identification of alkaloids
3.3.5.1 Dragendorff’s test: To 1 mL of each of the sample solution taken in a test
tube few drops of Dragendorff’s reagent (potassium bismuth iodide solution) was
added. A reddish brown precipitate was observed indicating the presence of alkaloids.
3.3.5.2 Meyer’s test: To 1ml of each of the sample solution few drops of Meyer’s
reagent (potassium mercuric chloride solution) was added. A creamish white
precipitate was formed indicating the presence of alkaloids.
3.3.6 Test for identification of Terpenoids
Five milliliter of the methanol extract was mixed with 2 mL of chloroform and 2mL
of concentrated sulphuric acid. The layer interface was observed for reddish brown
coloration which indicates the presence of Terpenoids.
3.3.7 Test for identification of Steroids
In a test tube containing 0.5 mL of extract solution, 2 mL of acetic anhydride was
added and along the sides of the tube 2 mL of sulfuric acid were also added. The
appearance of the purple or green-blue colour indicates the presence of steroids.
10
3.3.8 Test for identification of saponins
Persistent foam, resulting from shaking the extract in distilled water for 10-15 min
indicated the presence of saponins.
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