CHAPTER ONE
INTRODUCTION
1.1 Background of The Study
Plants are considered as largely complicated chemicals factories which can turn the relatively
simple ingredients of air and water into so many compounds including liquids and oils (Haslam,
2016). Plants have been serving the animals’ kingdom as its source of energy (food, fuel) as well
as its means of shelter. In addition to the source of energy, plants have been synthesizing a large
variety of chemical substances. These substances in addition to basic metabolites include,
phenolic compounds, terpenes, steroids, alkaloids and other chemicals substances which as
known as “secondary metabolites” which have prominent effect on the animals systems and some
possess important therapeutic properties which can be and have been utilized in the treatment
and cure of human and other animals diseases for many centuries (Mabey, 2017). Secondary
metabolites differ from plants to plants. The plants which produce and accumulate constituents
have medicinal values are generally designated as “medicinal plants’ (Musa et al., 2011).
The Eucalyptus tree is a large, fast-growing evergreen that is native to Australia and Tasmania
so also Nigeria. The tree can grow to 375-480 feet (125-160 meters). Eucalyptus trees belong to
the myrtaceae family. Their name originates from the Greek word "eucalyptol" which means
"well covered". Eucalyptus trees thrive in environments that maintain average temperatures of
about 600C (Shaighal et al., 2012). Eucalyptus trees are well known for the medicinal properties
of the oil contained in their leaves. The oil was used in traditional aboriginal medicines to heal
wounds and fungal infections (Harborne, 2018). Teas made of Eucalyptus leaves were also used
to reduce fevers. Eucalyptus soon spread to other traditional medicine systems, including
Chinese, Indian and Greek and European. Eucalyptus oil is believed to possess a wide variety of
healing properties (Trease, 2012). It works very effectively as an antibiotic that is particularly
successful against some strains of bacteria. The oil also possesses anti-inflammatory properties.
It can help stimulate the flow of blood and works to ease muscle and joint pain. Eucalyptus oil
also acts as an antiseptic and works well in treating sore throats, mouth sores, gum disease and
gingivitis. The essential oil from the leaves is used as a disinfectant and in medicinal applications.
Although Eucalyptus oil has been used orally to treat some conditions, the oil is toxic when taken
by mouth and must be diluted (Sofowara, 2013).
1
Eucalyptus is used in many medicines to treat coughs and the common cold. It can be found in
many lozenges, cough syrups, rubs, and vapor baths throughout the United States and Europe.
Herbalists often recommend using fresh leaves in teas and gargles to soothe sore throats and treat
bronchitis and sinusitis. Ointments containing eucalyptus are also applied to the nose and chest
to relieve congestion (Mabey, 2017). The crude extract of the stem bark of Eucalyptus globulus
has been widely recognized for its medicinal properties and has been used in traditional medicine
for centuries. The therapeutic potential of Eucalyptus globulus stems from its rich chemical
composition, which includes various bioactive compounds such as essential oils, tannins,
flavonoids, terpenoids, and phenolic acids (Musa et al., 2011).
One of the primary medicinal uses of the crude extract of Eucalyptus globulus stem bark is its
effectiveness in treating respiratory conditions. The essential oil derived from this plant contains
a high concentration of cineole (also known as eucalyptol), which possesses expectorant and
mucolytic properties. These properties help to alleviate symptoms associated with respiratory
disorders such as coughs, colds, bronchitis, and sinusitis. Inhalation of eucalyptus oil can help to
clear congestion, reduce inflammation in the airways, and promote easier breathing (Shaighal et
al., 2012).
Phytochemical analysis of the crude extract of the stem bark of Eucalyptus globulus involves the
identification and quantification of various bioactive compounds present in the plant material.
This analysis provides valuable information about the chemical composition and potential
medicinal properties of the extract. The stem bark of Eucalyptus globulus is known to contain a
diverse range of phytochemicals, including terpenoids, phenolic compounds, flavonoids, tannins,
and alkaloids. These compounds contribute to the biological activities exhibited by Eucalyptus
globulus, such as antimicrobial, antioxidant, anti-inflammatory, and anticancer properties
(Harborne, 2018).
1.2 Statement of the Problem
Plants had been used for the healing of diseases ages ago before the use of recent clinical drugs.
Such medicinal plants are also recognized to have therapeutic properties or as precursors for the
synthesis of useful drugs (Sofowora, 2012). More than 50% of these synthetic drugs are
derivative of natural products. These natural products play a crucial role in drug development
(Jeyachandran et al., 2017). With the increasing use of chemicals, antibiotics many pathogens
2
have developed resistance against them; hence there is immense need to develop new anti-agent
with improved performance and wide applications.
1.3 Aim of the Study
The aim this study is to focus on the phytochemical profiling of crude stembark extract of
Eucalyptus globulus.
1.4 Objectives of the Study
1. To qualitatively determine the phytochemical composition of the crude stembark extract
of Eucalyptus globulus.
2. To quantitatively determine the phytochemical composition of crude stembark extract of
the Eucalyptus globulus.
1.5 Justification of the Study
The crude extract of the stem bark of Eucalyptus globulus has been widely recognized for its
medicinal properties and has been used in traditional medicine for centuries (Cowan, 2019). The
therapeutic potential of Eucalyptus globulus stems from its rich chemical composition, which
includes various bioactive compounds such as essential oils, tannins, flavonoids, terpenoids, and
phenolic acids (Hans-walter, 2015). One of the primary medicinal uses of the crude extract of
Eucalyptus globulus stem bark is its effectiveness in treating respiratory conditions. The essential
oil derived from this plant contains a high concentration of cineole (also known as eucalyptol),
which possesses expectorant and mucolytic properties. These properties help to alleviate
symptoms associated with respiratory disorders such as coughs, colds, bronchitis, and sinusitis.
Inhalation of eucalyptus oil can help to clear congestion, reduce inflammation in the airways, and
promote easier breathing (Okwu, 2011).
1.6 Scope of the Study
Scope of this study is to examine the phytochemical composition of crude extract of stem bark of
the Eucalyptus globulus in Jimeta metropolis, Adamawa State.
3
CHAPTER TWO
LITERATURE REVIEW
2.1 Medicinal Plant
Eucalyptus globulus is a flowering tree that belongs to myrtle family (Myrtaceae). It has been
used for thousands of years throughout human history. The genus eucalyptus contains more than
700 species and varieties and they have been successfully introduced worldwide. Eucalyptus is
native to Australia and Tasmania and also in Africa and tropical to southern temperate America
(Kaikini, 2011). Variability is prevalent in morphology, growth habit, flower colour, leaves,
stems and chemical composition. In case of Eucalyptus globulus, pollen competition favors
crosspollination over self-pollination. Controlled pollinations with self-pollen, cross-pollen and
a mixture of self-pollen and cross-pollen were conducted on three partially selfincompatible
trees. Paternity of individual seeds resulting from mixed pollination was determined by isozyme
analysis. No evidence for pollen competition was found. Instead, seed paternity reflected the
level of selfincompatibility of each trees as determined by separate selfpollinations and crosspollinations (Wilson et al., 2011).
Furthermore, number of seeds set per capsule following mixed pollination was significantly less
than that of following cross-pollination in the two least self-compatible trees. These results
suggest that both self-pollen and cross-pollen tubes reach ovules following mixed pollination and
that of a late-acting selfincompatibility mechanism operates to abort a certain proportion of selfpenetrated ovules (Gooding et al., 2013). The flowers of Eucalyptus globulus are mainly
pollinated by insects but birds and small mammals may also act as pollinating agents (Orwa et
al., 2009). Eucalyptus globulus is known by different names depending upon where you are in
the world and its common name is "Australian Fever Tree", "Tasmania Blue Gum", "Southern
Blue Gum" or "Blue Gum", "Blue Gum Tree" and "Stringy Bark". In Arabic language, it is known
as "ban" or "kafur". In Burmese language it is known by "pyilon-chantha". The trade name of
Eucalyptus globulus is "blue gum". In Amharic language it is called "nech bahir zaf". In English
language, it is commonly known as "turpentine gas", "Tasmanian blue gum eucalypt",
"Tasmanian blue gum", "southern blue gum", "fever tree", "blue gum eucalyptus" and "blue
gum". In Japanese language, it is called "yukari-no-ki". In Spanish language, it is known as
"eucalipto". In Swahili it is known as "mkaratusi" and in Tigrigna language it is called
"tsaedakelamitos" (Chen et al., 2015). Eucalyptus globulus is a complex species as consist of
4
four further subspecies which are Eucalyptus bicostata, Eucalyptus pseudoglobulus, Eucalyptus
globulus and Eucalyptus maidenii. The only one variety of Eucalyptus globulus is Eucalyptus
globulus var. compacta Labill-Dwarf blue gum (Mbuya et al., 2014). Eucalyptus oil has
numerous traditional uses especially in non-prescription pharmaceuticals but the market is small.
Currently somewhere between three and five thousand tonnes are traded each year on
international markets, with only two or three hundred tonnes being produced by Australia.
Eucalyptus oil based products have been used as a traditional non-ingestive treatment for coughs
and colds (Midgley et al., 2013).
2.2 Demography/Location
Eucalyptus globulus can be grown in variety of climatic conditions and environmental
modifications but the best known optimum conditions are evident to be found in countries having
warmer climate. Eucalyptus is preferably found in Albania, Tunisia, Argentina, Bangladesh,
Cambodia, Brunei, Eritrea, Greece, Ethiopia, Indonesia, Italy, Israel, Laos, Kenya, Malaysia,
Myanmar, Morocco, Namibia, Nigeria, Nepal, Pakistan, Spain, Philippines, Sudan, Uganda,
Tanzania, Thailand, Malta and United Kingdom (Moral et al., 2010). Nigeria is covered by
92,000,000 hectares that is equivalent to 227,336,951 acres of Eucalyptus globulus forest thereby
comprising three quarters of the whole area covered by native forests. Similarly, total area of
Eucalyptus globulus that is planted in India is supposed to be exceeding 2,500,000. The
"Tasmanian Blue Gum", "Southern Blue Gum" or "Blue Gum" are the other names for Eucalyptus
glabrous and is the most widely cultivated plant so far (Chingaipe, 2015). In the year 2006, it
comprised about 65 percent of all plantation hardwood in Australia with about 4,500 km planted
area. Eucalyptus globulus is the primary source for eucalyptus oil production all around the world.
During the last ten years, in the northwestern regions of Uruguay, Eucalyptus globulus was one of
the major cultivated crop (Iglesias, 2017)
That zone has a potential forested area of 1,000,000 hectares, approximately 29% of the national
territory dedicated to forestry among which approximately 800,000 hectares are currently forested
by monoculture of Eucalyptus globulus. In Brazil, there are around 7 million hectares planted area
that can produce upto 100 cubic metres per hectare per year.
2.3 Botanical Specifications
Eucalyptus glabrous is a broadleaf evergreen plant that can attain the maximum height of about
70 m as evident to found in Europe (Hardel et al., 2011). Although more than 700 different species
5
of this plant are found to exist but Eucalyptus glabrous is the most widespread among all other
species in East Bay (Paine, C. Hanlon. 2010). It is an aromatic plant that has straight trunk and
well-developed crown with tap root system exceeding the depth of 10 feet (Hall et al., (2010). The
most readily recognizable characteristics of eucalyptus species are the distinctive flowers and fruit
(capsules or gum nuts). Flowers have numerous fluffy stamens which may be white, cream, yellow,
pink or red in colour. In bud, the stamens are enclosed in a cap known as an operculum which is
composed of the fused sepals or petals or both. Thus flowers have no petals, but instead decorate
themselves with many showy stamens. As the stamens expand, operculum is forced off, splitting
away from the cup-like base of the flower; this is one of the features that unite the genus (Bhide et
al., 2014). The appearances of eucalyptus bark varies with the age of the plant, the manner of the
bark shed, the length of the bark fibers, the degree of furrowing, the thickness, the hardness and
the colour.
All mature trees put on an annual layer of bark, which contributes to the increasing diameter of
the stems. Bark consist of long fibers and can be pulled off in long pieces, is hard rough and deeply
furrowed, bark is broken up into many distinct flakes, has short fibers, this has the bark coming
off in long thin pieces but still loosely attached in some places. The bark of tree is hard, rough and
deeply furrowed (Moral et al.,2010). It is soaked with dried sap exuded by the tree which gives it
a dark red or black coloration. The fruit looks like cone shaped woody capsules called "gum nuts",
distinctive for the genus and fruiting period is autumn and winter. The seed morphology of
Eucalyptus globulus is extremely variable, shape, size, colour and surface ornamentation are
strongly inherited traits and indicative of taxonomic groups. The primitive cotyledon shape is
reniform and this form of cotyledon occurs widely in the genus. The extreme modification is the
bisected cotyledon formed by emargination, resulting in a Y-shaped structure. A large number of
species have cotyledons shaped between these extremes and are usually described as bilobed,
although the distinction between the bilobed and the reniform is often blurred (Pacifici et al.,
2007). Eucalyptus globulus also grows in mild, warm and tropical climates having mean annual
temperature ranging from 3-22 to 21-40°C, but cannot live at temperatures lower than -5°C and
mean annual rainfall ranging from 250 to 2500 mm. Eucalyptus globulus are cultivated in
Mediterranean area and grow until 350 meters over the sea level. Usually the young plants are
planted in spring or at the end of summer. Eucalyptus globulus should be grown in climate with
high humidity otherwise suffers burning of leaf border. It can grow in wide range of soils and with
6
limited water supply (Dawoud et al., 2017). The soil type grows best on deep, silty or loamy soils
with a clay base and accessible water table. It is one of the species found to be most tolerated to
acid soils and soils optimum pH ranges from 5.5 to 6.5. In India, location was 10.2572°N latitude;
78.8861°E longitude; 216 ft above sea level with average temperatures ranging between 33.5°42.2°C and 1043.31 mm annual rainfall. The soil type of the study area is red soil (Sani et al.,
2014).
2.4. Chemistry
Eucalyptus globulus has a fresh mint like smell and a spicy, cooling taste and has various
concentrations of minerals. Eucalyptus is naturally occurring cellulose or protein, while synthetic
fibers are not found and identification of lipid constituents showed that this plant contains cutin
and soluble lipids. Eucalyptus essential oil is colorless and has a distinctive taste and odor and
typical volatility. Essential oil of eucalyptus is highly flammable and contains compounds that are
natural disinfectants and pest deterrents (Dawoud et al., 2017).
2.4.1 Chemical Composition
Essential oil of Eucalyptus glabrous is composed of mixtures of volatile organic compounds
including hydrocarbons, alcohols, aldehydes, ketones, acids, ethers and esters. Most of the
components are monoterpenes and sesquiterpenes in nature which consist of two or more isoprene
(C5H8) units. Essential oil has various concentrations of calcium, nitrogen, phosphorus, iron,
manganese, zinc, boron and copper. (Batish et al., 2008).
2.4.2 Phytochemistry
The essential oils obtained from the leaves, bare branches, flower buds and mature fruits of
Eucalyptus globulus contain large number of highly valuable chemical compounds. The leaf oils
were found to contain 1,8-cineole (4.10–50.30%) depending upon maturity and origin of their
collection site. Other major components of the leaf oils were α-pinene (0.05–17.85%), p-cymene
(trace-27.22%), cryptone (0.00–17.80%) and spathulenol (0.12–17.00%). In contrast, the essential
oil of fruit, bud and branch oils is known to contain α-thujene (0.00%, 11.95% and trace
respectively), 1,8-cineole (15.31%, 36.95% and 56.96% respectively) and aromadendrene
(23.33%, 16.57% and 8.24% respectively) (Kim et al., 2011).
7
Figure 1: Chemical structure of 1,8-cineole
2.6 Conventional Uses and Medicinal Applications
The uses of eucalyptus oils are very vast and wide ranging because there are so many species.
Traditionally, eucalyptus species have been used for supporting a healthy respiratory system and
to soothe the muscles after exercise. The Australian Aborigines used the leaves for soothing
physical and emotional discomfort. Unfortunately, with the broad uses and abundance of species
some confusions, are faced and even exploitation of the consumer takes place (Chingaipe, 2015).
This is similar to the problems often encountered with other popular essential oils such as
cinnamon essential oil and the Melaleuca species. Therefore, it is upto us as consumers and oil
users to have an understanding of the plant and the oil so we can use the oils safely and correctly.
2.6.1 General Uses
Uses of Eucalyptus globulus essential oil, distilled from Eucalyptus globulus tree, boast a long list
of traditional uses. Aboriginal Australians used Eucalyptus globulus to heal wounds, cure fungal
infections and as a fever reducer. Chinese, Greek, European and Ayurvedic medicine later adopted
Eucalyptus globulus as a disinfectant and expectorant (Kim et al., 2011). Present day medicinal
applications of Eucalyptus globulus oil may be seen in the majority of grocery stores and
pharmacies around the world including the oil’s use in vapor chest rubs, over-the-counter cough
and cold medications, sore throat sprays, topical pain relievers just to name a few (Bhide et al.,
2014).
2.6.2 Pharmacological Uses
Eucalyptus globulus oil is used as an anti-septic and anti-spasmodic stimulant agent in bronchitis,
asthma and minor respiratory complaints (Hardel et al., 2011). By using externally, it has
increasing effects on blood flow and skin temperature. Therefore, it has been used in semi-solid
dosage forms for the treatment of cough, to promote scar formation in burns and injuries and as an
anti-rheumatic agent. It is used as an inhalant because 1,8-cineole is a well-known medicinal
8
component that causes a sensation of cold and this is accompanied with a facilitated respiration
(Moral et al., 2010). Thus it is often inhaled in asthma, pharyngitis and related conditions.
2.6.2.1 Anti-Microbial Activity
In comparison, crude Eucalyptus globulus oil seems to be more efficient against micro-organisms
grown in suspensions and biofilms compared with pure 1,8-cineole (Pacifici et al., 2007). The 1,8cineole was active against two grampositive bacteria while it was inactive against the gramnegative
bacteria Escherichia coli and Pseudomonas aeruginosa and also showed a positive effect against
Escherichia coli (Pacifici et al., 2007)
2.6.2.2 Anti-Fungal Activity
Eucalyptus globulus oil was found effective against twelve yeast-like fungi and filamentous fungi.
MICs values between 0.025 and 1% (V/V) were found (Dawoud et al., 2017). The plant’s oils for
anti-candida activity were tested against two different strains of Candida albicans. A concentration
of 0.05% (V/V) was enough to inhibit their growth completely, MIC values of 2-8 mg/ml. Antifungal effects of Eucalyptus globulus oil were also observed against five Fusarium species.
2.6.2.3 Anti-Viral Activity
The potential anti-viral effect of Eucalyptus globulus oil was determined against Herpes simplex
virus type I (HSV-1) (Sani et al., 2014). HSV-1 was incubated with various concentrations of
Eucalyptus globulus oil for one hour at room temperature. The IC50 could be given with 55µg/ml.
At maximum non-cytotoxic concentration (200 µg/ml = ~0.02%) plaque formation was
significantly reduced 3 days after cell infection by >96% after pre-incubation of HSV-1 and
essential oil compared with untreated control. Only moderate activity was seen when the essential
oil was added to host cells prior or after infection. Some scientists demonstrated that Eucalyptus
globulus oil (0.01%) reduced virus titers by 58-75% for HSV-1 and HSV-2 (Batish et al., 2008).
It could be shown that pre-treatment of virus with the essential oil showed best results while preincubation of the cells did not reduce virus production. The anti-viral activity of Eucalyptus
globulus essential oil on strains of adenovirus and mumps virus isolated from patients. In a
concentration of 0.25 µl/ml (0.025%), the essential oil showed a mild antiviral activity (~40%)
against mumps virus, but nor against adenovirus. The potential anti-viral effect of 1,8-cineole was
determined against Herpes simplex virus type I (HSV1). The IC50 could be given with 1200 µg/ml.
The potential antiviral effect of α-pinene was determined against Herpes simplex virus type I
(HSV-1) in-vitro. The IC50 could be given with 4.5µg/ml (Dawoud et al., 2017).
9
2.6.2.4 Anti-Inflammatory Activities
Anti-inflammatory effect of Eucalyptus globulus oil in the paw oedema test in rats after
subcutaneous injection in a dosage of 100 mg/kg (HED=16 mg/kg) (Sani et al., 2014). Eucalyptus
globulus oil to rats p.o. in a dosage of 12 mg/kg/day for 15 days (HED=1.9 mg/kg) to test whether
Eucalyptus globulus oil treatment could induce a recovery of peripheral blood mononuclear cells
activity after bone marrow suppression (by 5-fluorouracil on day 7). In the sets of experiment,
blood was collected on day 0, 7, 15 and 20. At day 15, an increase of circulating monocytes and
an increment in the phagocytic activity of granulocytes and monocytes were recorded for immunocompetent rats. In immuno-suppressed rats, a recovery of the percentage of circulating
granulocytes was observed as well as a nearly restored phagocytic activity of peripheral blood
granulocytes/monocytes (Batish et al., 2008). Eucalyptus globulus oil (~73 and 146 µg/ml)
increased the phagocytic activity of human monocyte derived macrophages after 24 h treatment,
while the release of immune-modulating cytokines.
2.6.2.5 Analgesic/Anti-Nociceptive Activity
Eucalyptus globulus oil induced analgesic effects. Analgesic effect was demonstrated by i.p.
injection at doses of 10 or 100 mg/kg (rats, positive control: morphine; HED=1.6 and 16 mg/kg)
and by subcutaneous injection at doses of 0.1, 10 and 100 mg/kg (acetic acid induced writhing
mice; HED=0.16, 1.6 and 16 mg/kg) (Batish et al., 2008). The effect of 1,8-cineole (oral
administration) in mice on chemical (acetic acid and formalin) nociception (Kim et al., 2011). In
the formalin test, a dosage of 400 mg/kg (HED=32.5 mg/kg) inhibited significantly the paw licking
response while a dosage of 200 mg/kg (HED=16.2 mg/kg) inhibited only the second phase (Batish
et al., 2008). The incidence of abdominal constriction response was found to be significantly less
even in the lowest dose of 100 mg/kg (HED=8.1 mg/kg). Anti-nociceptive effects of 1,8-cineole
was examined in rats and mice tail-flick, hot plate, (Pacifici et al., 2007) A dosage of 0.3 mg 1,8cineole/kg in rats (i.p.) provoked a significant effect on reaction time to nociceptive effects in rats,
while changes in reaction in mice, could not be seen, (Moral et al., 2010). The β-pinene in-vivo
studies: anti-nociceptive effects of βpinene were examined in rats and mice (tail-flick, hot plate).
The β-pinene provoked a supra spinal anti-nociceptive action in rats only (0.3 mg/kg, i) (Sofowara,
2013).
2.6.2.6 Anti-Oxidant Activities
10
Anti-oxidant properties of essential oils are well known and in order to prove the ability of
essential oils to reduce reactive oxygen species (ROS) production even confirmed an anti-oxidant
effect of eucalyptus oil (1 µg/ml) cultured and stimulated alveolar macrophages from patients with
chronic obstructive pulmonary disease (COPD) (Dawoud et al., 2017). But the exact mechanism
on how essential oils exert this function on inflammatory cells is still unknown. Whether this effect
correlates with clinical measurable benefits for the patients has also to be studied. 6.2.7 AntiDiabetic and Repellent Effects Anti-diabetic effects and repellent Eucalyptus globulus oil (Kim et
al., 2011).
Terpenoids are one of the major classes of phytochemicals found in Eucalyptus globulus. They
are responsible for the characteristic aroma of the plant and have been extensively studied for
their therapeutic potential. The main terpenoids identified in the stem bark extract include
eucalyptol (1,8-cineole), α-pinene, β-pinene, limonene, and camphor. Eucalyptol is particularly
abundant and has been reported to possess antimicrobial, anti-inflammatory, and bronchodilatory
activities (Sofowara, 2013).
Phenolic compounds are another important group of phytochemicals present in the stem bark
extract. These compounds exhibit strong antioxidant properties and contribute to the overall
medicinal value of Eucalyptus globulus. Some of the phenolic compounds identified in the
extract include gallic acid, ellagic acid, caffeic acid, and quercetin. These compounds have been
shown
to
possess
anticancer,
anti-inflammatory,
and
neuroprotective
activities.
Flavonoids are a class of polyphenolic compounds that are widely distributed in plants and have
diverse biological activities. Several flavonoids have been identified in the stem bark extract of
Eucalyptus globulus, including rutin, kaempferol, and quercetin. These compounds have been
reported to exhibit antioxidant, anti-inflammatory, and antimicrobial properties(Trease, 2012).
Tannins are polyphenolic compounds that are known for their astringent properties. They have
been identified in the stem bark extract of Eucalyptus globulus and contribute to its medicinal
value. Tannins have been shown to possess antimicrobial, antioxidant, and anticancer activities.
Alkaloids are nitrogen-containing compounds that often exhibit pharmacological activities.
Although present in smaller quantities compared to other phytochemicals, alkaloids have also
been detected in the stem bark extract of Eucalyptus globulus. Some of the alkaloids identified
include eucalyptine, globuline, and macrocarpine. These alkaloids have been reported to possess
antimalarial, antifungal, and analgesic properties (Haslam, 2016)
11
CHAPTER THREE
MATERIALS METHOD
3.1 Plant collection and Identification
Stem bark of Eucalyptus globulus will be collected from Jimeta modern Market North Local
Government Area of Adamawa State. The stem bark will be identified and authenticated by,
Lecturers of Adamawa State Polytechnic, Yola. The plant will be deposited in the laboratory,
Department of Science and Laboratory Technology, Adamawa State Polytechnic, Yola.
Chemical and reagents
Methanol, Chloroform and Ethyl acetate, n-Hexane, Butylated hydroxytoluene (BHT), FRAP
(ferric reducing antioxidant power) assay. All other chemicals and reagents will be of Anarlar.
Extract Preparation
Stem bark powder (500 g) will be macerated with 70% ethanol (v/v) in a glass jar for 2 days at
room temperature. The extract will be filtered, concentrated to dryness in Oven under reduced
temperature (Evans, 2009).
3.2 Qualitative phytochemical analysis
Alkaloid: To 2 mL of the extract, 2 mL of 10% HCl was added, followed by 2 mL of Mayer’s
reagent. The formation of an orange precipitate indicated a positive result.
Saponin: To 2 mL of the extract, 2 mL of distilled water was added. The mixture was agitated in
a test tube for 5 minutes.
The appearance of a layer of foam indicated a positive result.
Tannin: To 2 mL of the extract, five drops of 0.1% ferric chloride were added. The formation of
a brownish-green or blueblack coloration indicated a positive result.
Steroid: To 2 mL of the extract, 10 mL of chloroform was added, and then 10 mL of concentrated
sulphuric acid was added by the side of the test tube. The formation of a reddish upper layer and
yellow sulphuric acid layer with green fluorescence indicated a positive result.
Glycoside: To 2 mL of acetic acid, 2 mL of the extract was added. The mixture was cooled in a
cold-water bath, and 2 mL of concentrated H2SO4 was added. Color development from blue to
bluish-green indicated the presence of glycosides.
Terpenoid: To 2 mL of the extract, 2 mL of chloroform, and 1 mL of concentrated sulphuric acid
were carefully added to form a layer. A transparent upper and lower layer with reddish-brown
interphase indicated a positive result.
12
Flavonoid: To 2 mL of the extract, 10% sodium hydroxide was added. A yellow color was
formed, which turned colorless upon the addition of 2 mL of dilute hydrochloric acid, indicating
a positive result.
3.2.1 Determination of total alkaloids content
Total alkaloids were determined by the gravimetric method as previously described12. Briefly, 0.5
g of the extract was weighed into a conical flask containing 10 mL of 10% ammonium hydroxide
to convert alkaloidal salts into the free base; the mixture was stirred and allowed to stand for 4
hours before filtering. The filtrate was evaporated to one-quarter of its original volume on a water
bath, and concentrated ammonium hydroxide solution was added dropwise to the mixture to
precipitate the alkaloids. The precipitate was filtered using a weighed filter paper and washed with
10% ammonium hydroxide solution. The precipitate was dried with the filter paper in an oven at
60°C for 30 minutes and then reweighed and calculated thus Equation 1.
% 𝑇𝑜𝑡𝑎𝑙 𝑎𝑙𝑘𝑎𝑙𝑜𝑖𝑑𝑠 = 𝑤𝑒𝑖𝑔
ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 × 100
[1]
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3.2.2 Determination of total saponins content
Total saponins were determined according to the previous method13. Briefly, 0.5 g extract was
introduced into a conical flask, and 10 mL of 20% aqueous ethanol was added. The sample was
heated over a water bath for one hour with continuous stirring at about 55°C. The concentrate was
transferred into a 250 mL separator funnel, and 5 mL of diethyl ether was added and shaken
vigorously. The aqueous layer was recovered, and the ether layer was discarded. About 10 mL of
n-butanol was added, followed by 2 mL of 5% aqueous NaCl. The remaining solution was heated
over a water bath. After evaporation, the sample was dried in the oven to a constant weight and
calculated thus Equation 2.
% 𝑇𝑜𝑡𝑎𝑙 𝑠𝑎𝑝𝑜𝑛𝑖𝑛𝑠 = 𝑤𝑒𝑖𝑔
ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 × 100
[2]
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3.2.3 Determination of total glycosides content
Total glycosides were determined as described previously14. Briefly, 0.5 g of the extract was
weighed into a 100 mL volumetric flask with 10 mL of 70% of ethanol in it. It was boiled for 2
minutes in the water bath, filtered, and the filtrate was diluted with 20 mL of distilled water.
Afterward, 2 mL of 10% lead acetate was added to this volumetric flask to precipitate the
13
chlorophyll, tannins, and alkaloids. It was then filtered with the filtrate transferred to a separating
funnel with 10 mL of chloroform. The funnel was rotated repeatedly. Two layers were formed,
and the lower organic layer was collected (chloroform), dried, and weighed. The percentage of
total glycosides contents was determined thus Equation 4.
ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 × 100
% 𝑇𝑜𝑡𝑎𝑙 𝑔𝑙𝑦𝑐𝑜𝑠𝑖𝑑𝑒𝑠 = 𝑤𝑒𝑖𝑔
[4]
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3.2.4 Determination of total terpenoids
Total terpenoids were determined by the gravimetric method described previously11. Briefly, 0.5
g of the sample was taken and soaked in 10 mL of ethanol for 24 hours. The extract, after filtration,
was extracted with 10 mL of petroleum ether using a separating funnel. The ether extract was
separated in pre-weighed crucibles and waited for its complete drying. Ether was evaporated, and
the yield (%) of total terpenoids contents was measured thus Equation 5.
% 𝑇𝑜𝑡𝑎𝑙 𝑡𝑒𝑟𝑝𝑒𝑛𝑜𝑖𝑑𝑠 = 𝑤𝑒
𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 × 100
[5]
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3.2.5 Determination of total flavonoids content
Total flavonoids were determined according to the method described previously12. About 0.5 g of
the extract was mixed with 10 mL of 80% aqueous methanol. The whole solution was filtered
through the Whatman filter paper. The filtrate was transferred to a pre-weighed crucible and
evaporated into dryness over a water bath, and weighed thus Equation 6.
% 𝑇𝑜𝑡𝑎𝑙 𝑓𝑙𝑎𝑣𝑜𝑛𝑜𝑖𝑑𝑠 = 𝑤𝑒𝑖𝑔
ℎ𝑡 𝑜𝑓 𝑟𝑒𝑠𝑖𝑑𝑢𝑒 × 100
[6]
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
3.2.6 Determination of antioxidant activity
Evaluation of the DPPH radical scavenging method was adopted, as reported previously15. The
free radical scavenging activity of the extract was measured by DPPH. Here 0.1 mM solution of
DPPH in methanol was prepared and added to different concentrations of the extract (20, 40, 60,
80, and 100 µg/mL) prepared in methanol. The mixture was shaken vigorously and allowed to
stand at room temperature for 30 minutes. The absorbance was then measured at 517 nm using a
spectrophotometer, with ascorbic acid as standard. The procedure was done in triplicate. The
lower absorbance of the reaction mixture indicated higher free radical activity. The half-maximal
inhibition concentration (IC50) value was determined. The percentage DPPH scavenging effect
was calculated by using the following Equation 7.
14
% 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑒𝑑 = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 × 100
[7]
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
3.3 Gas chromatography-mass spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis GC-MS analysis will be carried out
in a combined 7890A gas chromatograph system (Agilent 19091-433HP, USA) and mass
spectrophotometer, fitted with a HP-5 MS fused silica column (5% phenyl methyl siloxane 30.0
m × 250 μm, film thickness 0.25 μm), interfaced with 5675C Inert MSD with Triple-Axis detector.
Helium gas will be used as carrier gas and will be adjusted to column velocity flow of 1.0 ml/min.
Other GC-MS conditions are ion-source temperature, 250 °C; interface temperature, 300 °C;
pressure, 16.2 psi; out time, 1.8 mm; and 1 μl injector in split mode with split ratio 1:50 with
injection temperature of 300 °C.
3.4 Statistical Analysis
Data will be expressed as mean ± Standard error of mean (SEM). Data will be statistically
evaluated using statistical package for the social science (SPSS) version 22 software.
15
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