ABSTRACT
Evaluation of chemical composition of C. citratus and M. spicata, gave the
following proximate composition on dry weight basis: moisture; (1.67±0.29%)
and (2.67±0.29%), ash; (7.52±0.30%) and (24.4±0.35%), crude lipid;
(6.67±0.28%) and (6.17±0.27%), crude fibre; (24.67±0.29%) and (2.67±0.29%),
crude protein;(17.44±0.12%) and (25.32±0.13%), available carbohydrate
(42.03±0.32%) and (39.78±1.11%), energy value; (297.91) and (315.93)
respectively. The samples contain mineral elements, (mg/100g); Na; 15.83±0.38
and 13.17±0.38, K; 610±20.00 and 646±11.55, P; 0.32±0.01 and 0.33±0.01, Ca;
0.05±0.01 and 0.07±0.01, Mg; 6.41±0.01 and 6.94±0.02, Cu; 0.06±0.03 and
0.04±0.02, Fe; 0.08±0.01 and 0.37±0.03, Mn; 0.03±0.01 and 0.11±0.02, Zn;
0.15±0.02 and 0.32±0.01 respectively. Eight phytochemicals were qualitatively
tested out of which six were confirm present, with concentration of
alkaloid;(mg/100g) 3.20±0.60, and 7.60±0.20, tannins;(mg/100g) 4.73±0.08 and
8.52±0.40, phenolics;(mg/100g) 76.80±2.50 and 96.72±10.01,
flavonoid;(mg/100g) 74.59±05.97 and 94.33±6,65 proanthocyanidin;(mg/100g)
12.83±01.77 and 10.51±0.90 for C.citratus and M. spicata respectively. Both the
samples show antioxidant activities of 52% and 56% using Hydrogen peroxide
radical scavenging assay. The samples shows various components of essential
oil. The phytochemical and mineral content of both plants justify their medicinal
value. The result further indicated the potential of the two samples as sources of
therapeutic agents.
TABLE OF CONTENTS
Title page———————————————————————————- i
Certification—————————————————————————— ii
Acknowledgements———————————————————————–iii
Dedication———————————————————————————-v
Table of contents————————————————————————–vi
List of figures——————————————————————————-ix
List of Tables——————————————————————————x
Abstract————————————————————————————xi
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW—————————-1
1.1 Introduction—————————————————————————1
1.2 Literature Review——————————————————————-2
1.2.1 Botany of Mentha spicata——————————————————-2
1.2.2 Uses of Mentha Spicata———————————————————-2
1.2.3 Botany of Cymbopogon citratus———————————————–4
1.2.4 Uses and Applications of Cymbopogon Citratus————————-4
1.2.5 Alkaloid—————————————————————————-5
1.2.6 Classification of Alkaloids—————————————————–5
1.2.6.1 True Alkaloids——————————————————————6
1.2.6.2 Protoalkaloids——————————————————————6
1.2.6.3 Polyamine alkaloids———————————————————7
1.2.6.4 Peptide and cyclopeptide Alkaloids————————————-7
1.2.6.5 Pseudalkaloids————————————————————–7
1.2.7 Flavonoids——————————————————————–9
1.2.8 Saponins——————————————————————-10
1.2.8.1 Tannins——————————————————————–11
7
1.2.8.2 Hydrolysable Tannins——————————————————11
1.2.8.3 Condensed Tannins——————————————————-11
1.2.9 Essential Oils—————————————————————–14
1.3 Justification——————————————————————–14
1.4 Aim and Objectives————————————————————14
CHAPTER TWO
MATERIAL AND METHODS—————————————————15
2.1.1 Sample Collection and Treatment——————————————-15
2.1.2 Extraction—————————————————————————-15
2.1.3 Apparatus/Glassware used—————————————————-16
2.1.4 Reagents Used——————————————————————17
2.1.6 Preparation of Reagent ————————————————————19
2.2.1 Proximate Analysis—————————————————————–26
2.2.2 Phytochemical Screening——————————————————–30
2.2.3 Quantitative Phytochemical Analysis——————————————31
2.2.4 Antioxidant activity—————————————————————33
2.2.5 Mineral Analysis——————————————————————-34
2.2.6 Physico-Chemical Analysis—————————————————–36
2.2.7 Extraction and chemical Analysis of Essential oils———————-38
CHAPTER THREE
3.0. RESULTS AND DISCUSSION——————————————————-40
3.1. Results————————————————————————————–40
3.2 Discussion——————————————————————————–45
3.2.1 Proximate Analysis——————————————————————–45
3.2.2 Minerals Analysis———————————————————————46
3.2.3 phytochemical composition———————————————————49
3.2.4 Antioxidant activity——————————————————————-51
8
3.2.5 Physico-chemical properties of the fix oil————————————51
3.2.6 Essential oil composition————————————————————51
CHAPTER FOUR
4.0. CONCLUSION AND RECOMMENDATION————————————-53
4.1. Conclusion——————————————————————————–53
4.2. Recommendations———————————————————————-54
References——————————————————————————–55
Appendices
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Knowledge of the chemical constituents of plants is desirable, not only for the
discovery of therapeutic agents, but also because such information could be
valuable in disclosing new sources of economic and important materials as
tannins, oils, gums, and precursors for the synthesis of complex chemical
substances. Furthermore, the knowledge of the chemical constituents of plants is
valuable in discovering the actual value of folkloric remedies (Mojab et al., 2003).
Plants have a limitless ability to synthesize substances mainly secondary
metabolites, of which at least 12,000 are known, This figure is estimated to be
less than10% of the total (Mallikharjuna et al., 2007). The natural substances are
used by plants as defensive molecules against microorganisms, insects and
herbivores. However, some of these product are involved in determination of
plant odour (terpenoids), pigmentation (tannins and quinines), and flavour
(Capsacin) (Mallikharjuna et al., 2007). However, these defensive molecules
give plants their medicinal value which is exploited by human beings because of
their great importance in health care of individuals and communities. These
natural compounds formed the basis of modern drugs development today
(Edeoga et al., 2005).
13
1.2 LITERATURE REVIEW
1.2.1 Botany of Mentha Spicata
Spearmint (botanic name Mentha spicata) belongs to the mint family
(Lamiaceae) and is indigenous to most of the European regions as well as
places in southwest Asia. The species derives its name from the resemblance of
the tips of its leaves to pointed lances or spears. However, as spearmint has
been extensively cultivated since ancient times. Spearmint is a herb-like
rhizomatous (a flat, subversive steam that often sends out roots and shoots from
its nodes) plant that thrives best in damp soil conditions. This is a perennial herb
that grows up to a height of 30 to 100 cm. The spearmint plant is extremely
invasive in nature and was first noticed in the Great Lakes way back in 1843.
(Ditomaso and Healy, 2007).The stems of the spearmint plant may or may not
have hair-like bristles. The plant has dense undergrowth and an extensive fleshy
rhizome beneath the ground. The deep green leaves of spearmint grow up to
five to nine cm in length and 1.5 to 3.0 cm in width having finely dented edges.
The flowers of this species emerge on thin spikes and are found in white or pink
color. Usually the flowers are 2.5 to 3.0 mm in length as well as width (Ditomaso
and Healy, 2007;and Klinkenberg, 2010).
1.2.2 Uses of mentha spicata
Recent studies undertaken with spearmint have demonstrated that consumption
of tea prepared with the herb is effective in controlling moderate hirsutism or
excessive hairiness among women (Efloras, 2008).
14
Several researches have been conducted to find the antifungal actions of
spearmint.The essential oil obtained from spearmint has been found to possess
antifungal properties, albeit to a lesser extent compared to oregano (Konstantia
et al., 1998). In addition to being an antifungal, spearmint is also said to possess
exceptional antioxidant characteristics. Further researchers revealed that the
antioxidant properties of spearmint were equivalent to that of synthetic
antioxidant, butylated hydroxytoluene (BHT) (Konstantia et al., 1998).
Spearmint is a widely used home-made herbal medication. Herbal
medicine practitioners have customarily used an infusion prepared with the
leaves of spearmint to treat medical conditions, such as headaches, fevers,
digestive problems as well as several other trivial maladies. The herb possesses
antiemetic (a substance that suppresses vomiting or nausea), carminative (a
medication that strengthens the digestive system), diuretic (a medication that
induces urine flow), and regenerative, tonic as well as stomachic properties
(Lawrence, 2006). The essential oil obtained from spearmint leaves possesses
antiseptic properties, but may prove to be noxious or fatal if taken in excessive
amounts. In traditional herbal medication, the stems as well as the essential oil
of spearmint are used for treating cancerous growths. In fact, a poultice made
with the spearmint leaves is believed to be an effective medication for tumors
(Abbaszadeh et al.,2009).
1.2.3 Botany of Cymbopogon Citratus
Cymbopogon citratus, (Lemon grass) formerly described as Andropogon citratus
by De Candolle and re-classified by Otto Stapf, belongs to Poaceae, a very large
plant family that comprises approximately 500 genus and 8,000 herb species,
15
generically named as grasses (Negrelle and Gomes, 2007). The genus
Cymbopogon includes around 30 grass species, (Tripplebrookfarm, 2003)The
name Cymbopogon is derived from the Greek words kymbe (boat) and pogon
(beard), referring to the flower spike arrangement (Plants, 2003). Citratus derives
from the ancient Latin, meaning lemon-scented leaves. The plant is a native herb
from india and is cultivated in other tropical and subtropical countries
(Figueirinha et al., 2008).
Cymbopogon citratus (Lemon grass) is an aromatic perennial tall
grass with rhizomes and densely tufted fibrous root. It has short underground
stems with ringed segments, coarse, green slightly leaves in dense clusters
(Carlin et al., 1986). Perennial herb, it grows forming dense clumps of up to 3 m
tall, with short rhizomes. Its leaves are erect, glabrous plane, more than 1m long,
5-15mm wide, whiter upper face and closed edge in the base,with rough margins
and membranaceous or arid ligules 4-5mm long. Erect inflorescences, usually in
pairs ofterminal spiciform racemes 30-60cm long. Sessile small spikes,
canaliculated ventral side, 4.5-5.0mm long, 0.8 -1.0mm wide, ciliated margins.
Equal or sub equal glumes. The inferior glume is lance-shaped, bicarinated, with
bilobulated apex and with acutely curved margins from the middle upwards. The
superior glume is lanceolated, 4.3-4.5mm long, usually 1- nervate. Sterile
lanceolated lemma, 3.5mm long, 2- nerved, ciliated. Lineal fertile lemma, 2.5mm
long, bifid, 1-nerved ciliated (Reitz, 1982 ; Negrelle and Gomes, 2007).
2.2.4 Uses and Applications of cymbopogon citratus
Cymbopogon.citratus is very used in the popular medicine in almost all the
continents and it comprises a wide range of indications by infusion prepared with
fresh or dry leaves. There is broad-range of uses of substances extracted from
16
the lemon grass, especially its essential oil (Negrelle and Gomes, 2007). Lemon
grass is also widely used in traditional medicine in Cuba and in many other
countries of the Caribbean region. Among its attributable popular properties are
those related to analgesic and anti-inflammatory actions (Ortiz et al., 2002). In
Trinidad and Tobago, it is used to combat diabetes (Mahabir & Gulliford, 1997).
In Suriname, lemon grass is used against coughing, cuts, asthma, bladder
disorders and as a diaphoretic and to relieve headaches. It is also used as an
insect repellent and a carminative (Lemon Grass, 2003).In Nigeria, lemon grass
have been found to confer important biological activities including antibacterial,
gastrointestinal infections and antimicrobial activities (Omotade, 2009 ; Ojo and
Anibijuwon,2010 ; Emmanuel et al.,2010).
1.2.5 ALKALOIDS
An alkaloid is, an amine that is produced by a plant. However, the term has been
extended to amines produced by animals and fungi as well (Hesse, 2002) The
name derives from the word alkaline; originally, the term was used to describe
any nitrogen-containing base. Alkaloids are usually derivatives of amino acids,
and many have a bitter taste. They are found as secondary metabolites in plants
(such as potatoes and tomatoes), animals (such as shellfish), and fungi. In many
cases, they can be purified from crude extracts by acid-base extraction
(Manfred, 2002 ; Fattorusso et al., 2008)
1.2.6 Classification of Alkaloids
Compared with most other classes of natural compounds, alkaloids are
characterized by a great structural diversity and there is no uniform classification
of alkaloids (Hesse, 2002). First classification methods have historically
combined alkaloids by the common natural source, e.g., a certain type of plants.
17
This classification was justified by the lack of knowledge about the chemical
structure of alkaloids and is now considered obsolete. More recent classifications
are based on similarity of the carbon skeleton (e.g., indole-, isoquinoline-, and
pyridine-like) or biogenetic precursor (ornithine, lysine, tyrosine, tryptophan,
etc.). However, they require compromises in borderline cases; for example,
nicotine contains a pyridine fragment from nicotinamide and pyrrolidine part from
ornithine and therefore can be assigned to both classes.(Dewick, 2002).
Alkaloids are often divided into the following major groups
1.2.6.1 True alkaloids, which contain nitrogen in the heterocycle and
originate from amino acids (Plemenkov, 2001). Their characteristic examples
are atropine 1, nicotine 2, and morphine 3. This group also includes some
alkaloids that besides nitrogen heterocycle contain terpene (e.g., evonine) or
peptide fragments (e.g. ergotamine). This group also includes piperidine
alkaloids coniine and coniceine although they do not originate from amino
acids (Dewick, 2002)
Figure:1.1 True alkaloids
1.2.6.2 Protoalkaloids, which contain nitrogen and also originate from amino
acids (Plemenkov,2001).Examples include mescaline, adrenaline and
ephedrine.
18
Figure: 1.2 Protoalkaloids
1.2.6.3 Polyamine alkaloids – derivatives of putrescine, spermidine, and
spermine.
Figure: 1.3 Polyamine alkaloids
1.2.6.4 Peptide and cyclopeptide alkaloids (Dimitris et al., 1997).The
cyclopeptide alkaloids are polyamide plant bases compound of amino acid
residue in common and highly modified forms. All so far known cyclopeptide
alkaloid with the exception of Lasiodine-4 are cyclic, containing 13-14 or 15
membered ring system. The 14 membered ring system containing alkaloid
represent the largest known group of the compounds (Shah and Pandey, 1985).
Figure;1.4 Peptide and cyclopeptide alkaloids
1.2.6.5 Pseudalkaloids – alkaloid-like compounds that do not originate from
amino acids (Aniszewski, 2007). This group includes, terpene-like and steroid19
like alkaloids (Plemenkov, 2001) as well as purine-like alkaloids such as caffeine
Figure 14, theobromine Figure 15, and theophylline Figure 16 (Aniszewski, 2007).
Some authors classify as pseudoalkaloids, compounds like ephedrine 6 and
cathinone.This is because they originate from the amino acid phenylalanine, but
acquire their nitrogen atom not from the amino acid but through transamination
(Aniszewski, 2007).
Figure:1.5 Pseudalkaloids
Properties of Alkaloids
Their molecular weight ranges from 100 to 900 daltons, Those without oxygen
atoms in their structure are usually liquid at ordinary temperature. Examples are
nicotine Figure 3, sparteine, and coniine. Those with oxygen atoms are
crystalline, such as berberine Most alkaloids are optically active, except for those
from the purine group (Manfred, 2002; fattorusso et al., 2008).
Application of Alkaloids
Various alkaloids have phamacological effects on humans and animals. Many
are poisonous, but some are used medicinally as analgesics (pain relievers) or
anesthetics, particularly morphine Figure 2 and codeine. Some, such as
vinblastine, are used to treat certain types of cancer (Manfred, 2002; Fattorusso
et al., 2008).
20
1.2.7 FLAVONOIDS
Flavonoids are water soluble polyphenolic molecules containing 15 carbon
atoms units. Flavonoids are structurally as two benzene rings which are joined
together with a sandwiched by a pyran unit.Based on this skeleton flavonoids
consist of 6 major subgroups: chalcone Figure 17, flavonone Figure 18, Flavonol
Figure 19,Anthocyanidins Figure 20,Aurones Figure 21,Anthocyanidin Figure 22
and Flavone Figure 23 (Aoki et al.,2000) flavanoids are also responsible for the
coloring of fruits, vegetables and herbs (Aoki et al., 2000).
Figure: 1.6 Flavonoids
Flavonoids are becoming very popular because they have many health
promoting effects. Such as anti-allergic, anti-cancer, antioxidant, antiinflammatory
and anti-viral properties (Harbone and Williams, 2000).The
21
contribution of flavonoids to the total antioxidant activity of components in food
can be very high because daily intake can vary between 50 to 500 mg (Harbone
and Williams, 2000).
1.2.8 SAPONINS
Saponins are glycosides with foaming characteristics. Saponins consist of a
polycyclic aglycones attached to one or more sugar side chains. The aglycone
part, which is also called sapogenin, is either steroid or a triterpene. The
foaming ability of saponins is caused by the combination of a hydrophobic (fatsoluble)
sapogenin and a hydrophilic (water-soluble) sugar part. Saponins have
a bitter taste. Some saponins are toxic and are known as sapotoxin.(Leslie et
al.,1999)
Figure: 1.7 SAPONINS
Saponins have many health benefits. Studies have illustrated the beneficial
effects on blood cholesterol levels, cancer, bone health and stimulation of the
immune system (Nassiri and Hosseinzadeh, 2008).
22
1.2.8.1 TANNINS
Tannins are distributed all over the plant kingdom.The are commonly found in leaves,
seeds, roots, and tissues of gymnosperms as well as angiosperms.They are mainly
located in the vacuoles or surface wall of the plants, where they do not interfere with
plants metabolism. They are also found in the heart wood of conifers and may play
role in inhibiting microbial activity, thus resulting in the natural durability of the wood.
Tannins are usually divided into hydrolysable and condensed tannins (Calvi et
al.,1995).
1.2.8.2 HYDROLYSABLE TANNINS
At the centre of hydrolysable tannins molecules, there is a carbohydrate
(usually D – glucose) the hydroxyl groups of the carbohydrate are partially or
completely esterified with phenolic groups such as gallic acid or ellagic acid.
Hydrolysable tannins are hydrolysed by weak acid or weak base to produce
carbohydrate and phenolioc acids.
1.2.8.3 CONDENSED TANNINS
Condensed tannins are polymers of 2 to 50 (or more) flavonoid units that are joined
by carbon-carbon bond which are not susceptible to cleavage by hydrolysis, while
hydrolysable tanninsand most condensed tannins are water soluble, some very large
condensed tannins are insoluble (Calvi et al., 1995). Condensed tannins give
precipited with bromine water and this test differenciate it from hydrolysable tannins
(Abubakar, 2001).
23
1.2.9 ESSENTIAL OILS
An essential oil is the volatile odoriferous oil extracted from aromatic vegetable
plant material by physical means. The physical methods used are steam
distillation, steam/water and water or expression (Quirin and Gerard,1999).
Essential oils are present in the plant within distinctive oil cells or secretory
glands either on the surface of the plant or within plant tissue. These secretory
glands, which are an important characteristic of many plant families, are
generally found to predominate in one particular part of the plant such as the
leaves, pericarp, wood, bark, roots, rhizomes or seeds (Stern et al.,2003).
Essential oils are primarily complex mixtures of mono (C10) and sesquiterpene
hydrocarbons (C15), and oxygenated compounds derived from these
hydrocarbons, which include alcohols, aldehydes, esters, ketones, phenols,
acids and oxides. (Bowles, 2003).
Figure:1.8 TANNINS
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Figure:1.9 Essential oils
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