EVALUATION OF ANTIOXIDANT ACTIVITIES OF SOME NIGERIAN
MEDICINAL PLANTS BY THE USE OF 2, 2- DIPHENYL-1-
PICRYLHYDRAZYL (DPPH) FREE RADICAL.
BY
UCHE, FIDELIA IJEOMA
PG/M.PHARM/07/43544
DEPARTMENT OF PHARMACOGNOSY
FACULTY OF PHARMACEUTICAL SCIENCES
UNIVERSITY OF NIGERIA, NSUKKA.
FEBRUARY, 2010
1 Title page
EVALUATION OF ANTIOXIDANT ACTIVITIES OF SOME NIGERIAN
MEDICINAL PLANTS BY THE USE OF 2, 2- DIPHENYL-1-
PICRYLHYDRAZYL (DPPH) FREE RADICAL.
BY
UCHE, FIDELIA IJEOMA
PG/M.PHARM/07/43544
A THESIS SUBMITTED TO THE DEPARTMENT OF
PHARMACOGNOSY, FACULTY OF PHARMACEUTICAL SCIENCES,
UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILLMENT
OF THE REQUIREMENT FOR THE AWARD OF MASTER OF
PHARMACEUTICAL SCIENCES (M. PHARM) DEGREE IN
PHARMACOGNOSY.
SUPERVISOR: DR. CHRIS O. EZUGWU
DEPARTMENT OF PHARMACOGNOSY,
FACULTY OF PHARMACEUTICAL SCIENCES,
UNIVERSITY OF NIGERIA, NSUKKA.
2 FEBRUARY, 2010.
Certification
Uche, Fidelia Ijeoma is a postgraduate student in the Department of
Pharmacognosy, with registration number PG/M. Pharm/ 07/43544.
She has
satisfactorily completed the requirement for research work for the of
Master
of Pharmaceutical sciences in Pharmacognosy. The work embodied in this
project report is original and has not been submitted in part or full for
any other diploma or degree of this or any other University.
------
---
Supervisor Internal
Examiner/
Head of
Department.
3
DEDICATION
This is dedicated to the sweet memory of my beloved parents, late Chief
Felix N
and Laura Okorom.
4
ACKNOWLEDGEMENT
I am very grateful to almighty God for His endless mercy, grace, enduring love and
divine assistance in this study.
I am equally heavily indebted to my supervisor, Dr C.O. Ezugwu, who did
not relent in instruction and direction on this study.
My deep appreciation also goes to my beloved husband, Hon Uche Nwosu,
whose encouragement and financial support led to the successful
completion of this work.
I appreciate so much the sub-grant given to me for this study by Mac
Arthur Foundation through the University of Port Harcourt.
5 Moreover, the contributions of the entire staff and the Head, Department of
Pharmacognosy, are highly appreciated.
Finally, I very much appreciate the moral support given to me by
distinguished lecturers such as: Professors O.K. Udeala, S.I. Ofoefule and
F. C. Ohiri; Drs A.W. Obianime, E.C. Ibezim, O Okorie and C.E.C.
Ugwoke. I express my sincere gratitude to them all.
LIST OF TABLES
Pages
Table 1: Botanical names, Families and local/common names
of the plants under study…………………………………………………….6
Table 2: Results of the Phytochemical screening of the
6 plants under
study…………...... 62
Table 3: Results of the Phytochemical screening of the plants
under study………………………………………………………………….63
Table 4: Antioxidant activities and reducing potentials
of the plants under
study……………………………………………….….64
LIST OF FIGURES
7
P
a
g
e
s
Figure 1: The digital photograph of Kalanchoe pinnata in
its natural habitat……………………………………………………….8
Figure 2: The digital photograph of Aspilia africana in its
natural habitat……………………………………………………………11
Figure 3: The digital photograph of Mucuna pruriens flowers, fruits
and leaves……………………………………………………………………14
Figure 4: The digital photograph of Mucuna pruriens in its
8 natural habitat…………...... 15
Figure 5: The digital photograph of Emilia coccinea in its
natural habitat…………………………………………………………….18
Figure 6a and b: The digital photograph of Laportea ovalifolia in its
natural habitat…….
……………………………………………………….21
Figure 7: The digital photograph of Cucurbita moschata in its
natural habitat…………………………………………………………….25
Figure 8: The seeds of Cucurbita moschata……………………………………….26
9 Figure 9: The digital photograph of Celosia trigyna in its natural habitat……35
Figure 10a: The digital photograph of Asystasia gangetica in its
natural habitat……………...... 40
Figure 10b: The diagram showing different stages of development of
Asystasia gangetica……………………………………………………………41
Figure 10c: The digital photograph of Asystasia gangetica in its
natural habitat………………………………………………………………..42
Figure 11: Antioxidant activities of Nigerian medicinal plants…………………….66
Figure 12: Antioxidant activities of Nigerian
Vegetables……………………………67
10
Figure 13: Reducing potentials and antioxidant activities of the
plants under study…………………………………………….…………………68
Figure 14: IC50 inhibitory concentration and antioxidant activity………………….69
Figure 15: Relationship between antioxidant activity and
IC50……………………..70
Figures16a and 16b: Reducing potential and
IC50…………………………………..71
Figures 17a and 17b: Relationship between reducing potential and
Antioxidant activities of the plants…………………………………………………72
Figures 18a and b: Percentage antioxidant activities and
IC50…………………….73
Figure 19: Effects of concentration of A.G, C.M and C.I on
Antioxidant activities………………………………………………………….75
Figure 20: Effects of concentration on antioxidant activities
11 of L.O, E.C and
M.P…………………………………………………………..76
Figure 21: Effects of concentration on antioxidant activities of
A.A, K.P and ascorbic acid……………………………………………………77
ABSTRACT
The methanol extracts of the leaves of Kolanchoe pinnata, Aspilia africana,
Mucuna pruriens, Emilia coccinea, Laportea ovalifolia, Celosia trigyna,
Cucurbita moschata and Asystasia gangetica were evaluated for antioxidant activities by the use of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay.
The reducing potentials of these plants were also evaluated. The phytochemical screenings of the medicinal plants were equally carried out.
The percentage antioxidant activity values for the plants are: 31.0 ± 1.80%, 58.4
± 1.26%, 59.1 ± 1.60%, 60.0 ± 1.05%, 60.8 ± 1.28%, 62.4 ± 1.28%, 64.8 ±
2.10% and 75.7 ± 2.60% for A. gangetica, C. moschata, C. trigyna, L .ovalifolia,
E. coccinea, M. pruriens, A. africana and K. pinnata respectively. These values were dose – dependent and statistically significant at P < 0.05 (ANOVA).
The results indicated that K. pinnata has the highest antioxidant activity value
75.7 ± 2.60% while A. gangetica has the least value 31.0 ± 1.80%. The
12 percentage antioxidant activities of the plants were comparable to the standards used, the ascorbic acid and
- tocopherol which were found to be 86.7 ± 1.08% and 97.2 ± 1.06% respectively.
The reducing potentials of the plants were found to be proportionally correlated to the antioxidant activities of the plants. Phytochemical screenings revealed the presence of alkaloids, flavonoids, terpenoids, cardiac glycosides, steroids, saponins and tannins in the medicinal plants.
TABLE OF CONTENTS
Pages
Title page…………………………………………………………………………..ii
Certification……………………………………………………………………….i ii
Dedication………………………………………………………………………....i v
13 Acknowledgement……………………………………………..………………..… v
List of Tables……………………………………………………………..…….... vi
List of
Figures…………………………………………………………….……….vii
Abstract..………………………………………………………………….…….…. x
Table of contents………………………………………………………………….xii
CHAPTER ONE: INTORDUCTION
1. The Significance of
antioxidant…………………………………………….1
Types of antioxidants……………………………………………....………2
Activities of antioxidants………………………………………….……….4
Review of medicinal plants under study…………………...……….……...6
1.3.1. Pharmacognostic profile of the plants under
study……..……..….……..9
1.4. Justification of the
study…………………………………………………50
14 1.5. Objective of
study……………………….……………………………….51
CHAPTER TWO: Materials and Methods.
2.1. Materials/Chemicals used……………………………………………..……….52
2.2. Collection and drying……………………………………………....………….52
2.3. Preparation of extracts…………………………………………...... …………..53
2.4. Phytochemical screening………………………………………..……………..53
2.5. Antioxidant activity assay………………………………….…..…………...... 57
2.5.2 . Determination of reducing potentials……………………………………….58
2.6. Statistical analysis………………………………………….………………….59
15 CHAPTER THREE
3.1. Results
……………………………………………………………………..….60
3.2.
Discussion/Conclusion………………………………………………….……..78
Reference………………………………………………………………….……….
81
Appendix…………………………………………………………………………
…90
16 CHAPTER ONE
INTRODUCTION
1. The Significance of Antioxidant
Chemical compounds with unpaired radicals such as powerful oxidants and free radicals are capable, when present in the body, to damage lipids, proteins, and also DNA and consequently may bring about mutation (Ellinaim et al., 2003).
Free radicals play a prominent role in human health. Free radical reactions have been implicated in the etiology and pathogenesis of chronic diseases that are life limiting such as cancer, hypertension, cardiac infarction, arteriosclerosis, diabetes etc.
Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions
(Velioglu, et al., 1998). Antioxidant activity is a very important pharmacological property. Many of the pharmacological functions such as antimutagenicity, anticarcinogenicity, anti-aging etc originate from this property. (Cook and
Samman, 1996: Huang et al., 1992)
The most important free radicals in the body are the reactive oxygen and nitrogen species, such as super oxide, hydroxyl and nitric oxide radicals. They are
17 generated in the body as a consequence of cellular and metabolic activities. They also arise from exogenous sources (exposure to ionizing radiations, injury, oxidative drugs, pollutants, etc). Excessive production and leakages from their site of generation are damaging to cells and tissues due to their reactivity with other biologically functional compounds. The body maintains a balance in reactive oxygen and nitrogen species by various scavenging mechanisms. These include a number of antioxidant enzymes and antioxidant molecules. Oxidative stress occurs when there is an imbalance in free radicals and antioxidants in the body. This could lead to serious health problems.
1.1. Types of Antioxidants
There are basically two categories of antioxidants: namely synthetic and natural.
Synthetic antioxidants are compounds with phenolic structures with various degrees of alkyl substitution. Natural antioxidants can be phenolic compounds
(tocopherols, flavonoids and phenolic acid), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids and amines) or carotenoids as well as ascorbic acid (Larson, 1988; Hudson, 1990; Hall and Cuppett, 1997). Synthetic antioxidants such as butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) have been used as antioxidants since the beginning of this century.Restrictions on the use of these compounds however are being imposed
18 because of their carcinogenicity (Braven 1975: Ito et al., 1983). Thus the interest in natural antioxidants has increased considerably (Loliger, 1991).
Many chemical studies have shown that the consumption of some medicinal plants, fruits and vegetables can exert positive effects upon human health and the aging process. Evidence points to these medicinal plants, fruits and vegetables as being rich in antioxidants. Phytochemicals such as vitamins, flavonoids, coumarins, phenols and lignin compounds act to prevent or reduce oxidative stress by scavenging free radicals.
Several researchers have investigated the antioxidant activities of flavonoid compounds and have attempted to define the structural characteristics of flavonoids that contribute to their activity (Nieto et al., 1993: Das and Pereira
1990: Foti et al., 1996). Phenolic acids such as caffeic and coumaric acids appear to be more active antioxidants than the hydroxyl derivatives of benzoic acid such as p- hydroxyl benzoic and vanillic acid (Dziedzic and Hudson, 1983: Larson,
1988). Burton and Ingold (1981) have shown that –tocopherol is one of the most active chain- breaking antioxidants. Carotenoids also have a protective function against oxidative damage and singlet oxygen is powerfully quenched by
-carotene (Foote et al., 1971). Free radical scavenging is one of the known
19 mechanisms whereby antioxidants inhibit lipid peroxidation (Bloknina et al.,
2003; Burns et al., 2000).
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay has been used extensively for screening antioxidants from fruit and vegetable juices or extracts (Sanchez- Moreno 2002). DPPH is a stable free radical which, on reaction with antioxidants, is reduced to DPPH-H. As a consequence, the absorbance is decreased and the original purple colour of DPPH is changed to yellow.
The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.
The scavenging reaction between DPPH and an antioxidant (A-H) can be represented as follows;
DPPH+ (A-H) = DPPH—H-(A)
1.2. Activities of Antioxidants
Many of the natural antioxidants, especially the flavoniods ,exhibit a wide range of beneficial pharmacological properties such as antibacterial, antiviral, anti- inflammatory, anti-allergic anti-ischemic, vasodilatory and anti-proliferative activity in cell and animal studies. (Cook and Samman, 1996). The antioxidant activities of several plant materials have been reported (Al-Saikhan et al., 1995;
20 Yen and Duh, 1995: Oomah and Muzza 1996: Wang et al., 1996: Cao et al., 1996
Amarowicz et al., 1996).
Presence of antioxidants has also been confirmed in soybeans (Facino et al.,
2000), garlic, red wine, green tea (Stajner et al., 1999) and in Tridax (Nia et al.,
2003).
21 1.3 LITERATURE REVIEW
1.3. Review of Medicinal plants to be investigated.
The antioxidants activities of the following medicinal plants will be investigated in this study. Table 1 lists the botanical names, Families and local or common names of the plants under study.
Table 1: The families, botanical and common names of the plants under
investigation.
Botanical names Family Common/Local name (s)
1. Aspilia africana Asteraceae English - Haemorrhage plant.
(Pers.) C.D Adams. Igbo - Oranjila.
Yoruba – yunyun.
Efik – odemedong.
Hausa – kalankuwa.
2. Kalanchoe pinnata Crassulaceae English-Restoration plant.
(Lam.) (Bryophyllum Igbo- oda opue pinnatum) Efik – afia-oyo
(Andr) Haw. Yoruba – abamoda.
3. Mucuna pruriens Fabaceae Igbo – Akugbara-uzo.
(Linn) English – velvet bean
22 4. Laportea ovalifolia Urticaceae Igbo - Akugbara.
(Schumach.) chew.
5. Celosia trigyna Amaranthaceae Igbo - Enune.
(Linn.)
6. Cucurbita moschata Cucurbitaceae Igbo - Ugbuguru.
(Duch.)
7. Emilia coccinea Asteraceae Igbo – Nti- ene.
(Sims) Yoruba - Odundunodo.
8. Asystasia gangetica Acanthaceae Igbo – Obi na epupe
(L.) T.Aderson.
These are widely distributed medicinal plants and vegetables in Nigeria. They have been consumed over hundreds of years in Nigeria and other African countries in health tonics or in remedies for the treatment of various ailments. In
Nigeria, many indigenous plants and vegetables are used in herbal medicine to cure diseases and heal injuries. These include: Kalanchoe pinnata, Aspillia africana, Mucuna pruriens, Emilia coccinea, Cucurbita moschata, Celosia trigyna, Asystasia gangetica and Laportea ovalifolia .
23
Fig.1: The digital photograph of Kalanchoe pinnata in its natural habitat. X400.
24 1.3.1. pharmacognostic profile of Kalanchoe pinnata (Lam.) Pers.
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Archichlamydeae
Order: Rosales
Family: Crassulaceae
Genus: Kalanchoe
Species: pinnata
Synonym: Bryophyllum pinnatum
K pinnata (formerly known as Bryophyllum pinnatum) is an erect, succulent, perennial shrub that grows about 1.5 cm height and is reproduced from seeds and also vegetatively from leaf bubble (Agoha, 1974). It is an introduced ornamental plant that is now growing as weed around plantation crops (Dalziel, 1955).
25 K. pinnata is used in ethnomedicine for the treatment of ear-ache, burns, abscesses, ulcers, insect bites, diarrhea and lithiasis (Chopra et al., 1956; Agoha,
1974; Ofokansi et al., 2005). In Southeastern Nigeria, this herb is used to facilitate the dropping of the placenta on daily basis (Dalziel, 1955). The plant leaf is mildly exposed to heat and the juice extracted and applied to the baby’s placenta on daily basis. The crushed leaves, as well as the extracted juice, are mixed with shear butter or palm oil and rubbed on abscesses or other swellings.
This is also applied on ulcers, burns and on the bodies of young children when they are ill (Agoha, 1974). Bryophyllin, potassium malate, ascorbic, malic and citric acids have been isolated from the leaves of B. pinnatum (McKenzie et al.,
1985; Siddigiuient et al., 1983; Singh, 1976; Ichikawa et al., 1986; Oliver, 1989).
The phytochemical composition of the leaves of K. pinnata were found to be
(expressed in mg/100g dry weight ): Alkaloids (1.48), Flavonoids (1.72), Phenols
(1.86), Tannins (0.51) (Okwu and Josiah 2006); Mineral composition was found to be (mg/100g dry weight): Magnesium (0.10), Calcium (0.32), Potassium
(0.04), Phosphorus (0.18), Sodium (0.02), Iron (1.85), Zinc (5.38) (Okwu and
Josiah, 2006 ). The Vitamin composition was found to be (mg/ 100g dry weight):
Ascorbic acid (44.03), Riboflavin (0.42), Thiamine (0.18), Niacin (0.02) (Okwu and Josiah, 2006).
26
Fig 2: The digital photograph of Aspilia africana in its natural habitat. X400.
27 1.3.2. Pharmacognostic profile of Aspilia africana (Pers.) C.D.Adams.
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Sympetalae
Order: Campanulales
Family: Asteraceae
Genus: Aspilia
Species: africana
A. africana is a perennial herb varying in height from 60 cm to about 1.5 m
depending on rainfall. It is a common weed of field crops in West Africa and
sometimes found in fallow land, especially the forest zones (Akobundu,
1987). The crushed leaves and flowers are used to stop bleeding and for
treating wounds and sores (Agoha, 1974). An infusion of the leaves is taken
by children and can also be mixed with clay as a medicine for stomach
28 troubles (Dalziel, 1955).It is also used in the treatment of rheumatic pains, scorpion stings and for removal of opacities and foreign bodies from the eyes
(Okoli, et al., 2007).It has antimicrobial potentials and accelerates wound healing (Okoli, et al., 2007).
The phytochemical composition of the leaves of A. africana were found to be
(expressed in mg/100g dry wieght : Alkaloids (1.24), Flavonoids (1.48),
Phenols (1.46), Tannins (0.04) (Okwu and Josiah 2006); Mineral composition were found to be (mg/100g dry weight): Magnesium (0.12), Calcium (1.04),
Potassium (0.03), Phosphorous (0.32), Sodium (0.02), Iron (3.78), Zinc (5.68)
(Okwu and Josiah, 2006 ). The Vitamin composition was found to be (mg/
100g dry weight): Ascorbic acid (26.42), Riboflavin (0.20), Thiamine (0.11),
Niacin (0.09) (Okwu and Josiah, 2006).
29
Fig. 3: The digital photograph of Mucuna pruriens’ leaves, fruits and flowers. x400.
30
Fig.4: The digital photograph of Mucuna pruriens in its natural habitat.
X400
31
1.3.3 Pharmacognostic profile of Mucuna pruriens Linn.
Kingdom: Plantae.
Sub kingdom :Tracheobionta, vascular Plants.
Division: Magnoliophyta (Angioserm).
Class: Magnoliopsida (Dicotyledon)
Sub class: Rosidae.
Order:Fabales
Family: Leguminoseae
Sub Family: Fabaceae
Genus: Mucuna
Species: pruriens
Growth and Distribution
Mucuna pruriens L (Leguminaceae) is an annual climbing legume that grows 3-
18m in height. It is indigenous to the tropical region especially Africa, India and
West Indies. It is distributed over most of the subcontinent and is found in bushes and hedges. It is a herbaceous twinge with long branches and opposite trifoliate lancelets leaves 15 to 30cm length. Leaflets broadly ovate, elliptic or rhomboid ovate, unequal at base.
32
Traditional and Pharmacological Uses. Mucuna pruriens is used traditionally for various purposes such as aphrodisiac,anthelmintic, tonic, ulcer, inflammation, general debility, fever, diuretic, dysentery, snake bite, sterility, gout, rheumatic disorder, muscular pain, gonorrhea (Sathiyanaranan and Arulmozhi, 2007).
Some of the biological activities of Mucuna pruriens include: Anti-Parkinson’ activity, hypoglycemic and hypercholesterolemia, Anti-tumour, Neuroprotetive,
Learning and memory enhancement, anti-diabetic, antivenom, antimicrobial, antiprotozoal, analgesic and anti-inflammatory activity. (Sathiyanaranan and
Arulmozhi, 2007). Mucuna pruriens is reported to have L-Dopa as a major constituent mainly in seeds. Alkaloidal constituents such as like mucunadine, mucunine, prurienidine, prurienine are reported from the seeds and many amino acids are reported from the plant (Sathiyanarayanan and Arulmozhi, 2007). Other phytochemicals reported to be present to in Mucuna pruriens are: Epoxy fatty acids, Lecithin, 1- methyl -3-carboxy-6,7-dihydroxy-1-,2,3,4- tetrahydroisoquinolone, 5-hydroxy tryptamine, 5-methoxy-n, n- dimethyltryptamine-n-oxide, 5-oxyindole- 3-alkylamine, 6-methoxyharman, alanine, arachidic acid, arginine, aspartic acid, behenic acid, choline, cystine, leucine, linoleic acid, nicotine, phenylalanine, phosphorus, praline, protein,
33 saponins, serine, stearic acid, tryptamine, tyrodine, valine and vernolic acid.(
Sathiyanarayanan and Arulmozhi, 2007).
Fig. 5: The digital photograph of Emilia coccinea in its natural habitat. X400
34
1.3.4. Pharmacognostic profile of Emilia coccinea (Sims) G. Don.
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Sympetalae
Order: Campanulales
Family: Asteraceae
Genus: Emilia
Species: coccinea
Common name: Nti ene (Igbo), Odundunodo (Yoruba).
35 Discription/Habitat
An annual herb, with weak stem of about 1m high with leaves clamping the stem and usually sessile. Emilia coccinea has orange inflorescence and grows in farmlands and forests.
Traditionally, it is used as remedy for eye and ear ailments, for fever, convulsion in children, ulcer, craw-craw, ringworm, (Agoha, 1981; Burkill, 1984; Edeogu et al., 2005), rashes, measles and other forms of inflammatory diseases. Leaves are used to cover sores. Decoction of leaves has mild laxative properties. The plant is used in the treatment of fibroid and stomach ulcer (Okujagu, et al., 2006;Agoha,
1981; Edeogu et al., 2005).
Chemical studies revealed the percentage of the crude to consist of : 0.92%
Alkaloids, 0.81% Phenol, 11.85% Tannins, 0.96% flavonoids, and 2.30%
Saponins ( Edeogu et al., 2005).
36
Fig.6a: The digital photograph of Laportea ovalifolia in its natural habitat.
X400
37
Fig.6b: The digital photograph of Laportea ovalifolia in its natural habitat.
X400
38
1.3.5. Pharmacognostic profile of Laportea ovalifolia (Schumach.) Chew.
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Archichlamydeae
Order: Urticales
Family: Urticaceae
Genus: Laportea
Species: ovalifolia.
Common name: akugbara (igbo).
39
Description
A robust- stemmed annual herb, a ruderal of waste places and riverine vegetation in Nigeria, and in central East- Africa. The leaves and stems bear stinging hairs
(Burkill, 1985). The plants yield a fibre which is used to make cordage and string in the Cameroon.
Traditional uses.
The aerial part of the plant is used as anti- diabetic agent in Cameroon.
40
Fig.7: The D igital photograph of Cucurbita moschata in its natural habitat. X400
41
Fig. 8: The seeds of Cucurbita moschata.
42 1.3.6. Pharmacognostic profile of Cucurbita moschata (Duch.)
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Archichlamydeae
Order: Cucurbitales
Family: Cucurbitaceae
Genus: Cucurbita
Species: moschata
Common name: ugboguru (igbo), Squash, Butter squash (English)
Habitat: Cultivated Beds.
43 Description/ Physical characteristics
Annual climber growing upto 0.6m x 5m at a fast rate. It is hardy to zone 10 and is frost tender. It is in flower from July to September and the seeds ripen from
August to October. The flowers are monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by insects. The plant is self-fertile.
The plant prefers light (sandy), medium (loamy) and heavy (clay) soils and requires well-drained soil. The plant prefers acid, neutral and basic (alkaline) soils. It can grow in semi-shade (light woodland) or no shade. It requires moist soil. The seeds are "about 2 cm long, broadly-ovate, flat, white or whitish, nearly smooth, with a shallow groove parallel to the edge; containing a short, conical radicle, and 2 flat cotyledons; inodorous; taste bland and oily.
Cucurbita is a genus in the gourd family Cucurbitaceae first cultivated in the
Americas and now used in many parts of the world. It includes species grown for their fruit and edible seeds (the squashes, pumpkins and marrows, and the chilacayote), as well as some species grown only as gourds. They have bicollateral vascular bundles. Many North and Central American species are visited by specialist pollinators in the apid group Eucerini, especially the genera
Peponapis and Xenoglossa, and these bees can be very important for fruit set.
44 Cucurbita pepo (common name: Pumpkin seed) is an annual plant, hispid and scabrous, with a procumbent stem and branching tendrils. Its leaves are large, cordate, palmately 5-lobed, or angled and denticulate. The flowers are yellow large, axillary, and the males long-pedunculate. Corolla campanulate; the petals united and coherent with the calyx. The calyx of the male flowers is 5-toothed; of the female the same, the upper part being deciduous after flowering; the stigmas are 3, thick, and 2-lobed; the pepo, or fruit, subligneous, very large, roundish, or oblong, smooth, yellow when ripe, furrowed and torulose, containing yellowish seeds, somewhat resembling those of the watermelon in form (Duke and Ayensu,
1985).
History.
The pumpkin flowers in July, and matures its fruit in September and October. It is a native of the Levant, and is extensively cultivated as a kitchen vegetable, and for cattle. The seeds of this plant are used in medicine, and have long been popular with the laity as a remedy for worms. The oil may be obtained from the pumpkin seeds by expression.
Chemical Composition.
Pumpkin seeds are composed of 25 per cent of husks and 75 per cent of kernels,
45 and contain upwards of 33 per cent of a reddish fixed oil, which consists of the glycerides of palmitic, myristic, and oleic acids. These also occur partly in the free state. The active (taenifuge) principle is a greenish-brown, acrid, bitter resin, not contained in the petroleum-benzin extract of the seeds, but in the extract obtained with ether. It is also soluble in alcohol and chloroform. Its dose, as a taenifuge, is 15 grains, in pill form. The fatty oil is soluble in absolute, but not in
95 per cent alcohol. Air-dried pumpkin seeds contain about 3.7 per cent of ash.
The juice of pumpkin pulp contains 1.6 per cent of dextrose and 0.9 per cent of cane sugar. The colouring matter of the pumpkin is due to carotene.
Action, Medical Uses, and Dosage.
Mucilaginous, taenicide, and diuretic, are of service in urinary infections, also in gastritis, enteritis, and febrile diseases. The infusion may be drunk freely. The expressed oil of the pumpkin seeds, in doses of 6 to 12 drops, several times a day, is said to be a most certain and efficient diuretic, giving quick relief in scalding of urine, spasmodic affections of the urinary passages, and has cured gonorrhea
(Duke and Ayensu, 1985).
Potential applications
Rheumatoid arthritis, elevated blood lipids and cholesterol, parasitic infestation, kidney/bladder disorders. Useful in maintaining skin health. The high tryptophan
46 content of the seeds may make the oil useful in cases of insomnia. A nutritious culinary oil.
ANTI-ARTHRITIC - Studies have shown that pumpkin seed oil is as potent as the drug indomethacin at relieving chronic rheumatoid arthritis. It is likely that this effect is due to the essential fatty acid profile, rich antioxidant content, and the synergistic effects of other minor components. Pumpkin seeds have been shown to have high levels of vitamin E, including all forms of the tocopherol family i.e. alpha, beta, delta, and gamma tocopherol, along with the tocotrienols.
PROSTATE FUNCTION - Pumpkin seed oil has been used in combination with saw palmetto in two double blind human studies to effectively reduce symptoms of Benign prostate Hyperplasia (BPH). Researchers have suggested that the zinc, free fatty acid, or plant sterol content of pumpkin seeds might account for their benefit in men with BPH. Studies have shown that pumpkin seed extracts can improve the function of the bladder and urethra, this might partially account for
BPH symptom relief (Burkill, 1985).
ANTI-PARASITIC - Cucurbitin is an amino acid that has shown anti-parasitic activity in vitro. Human studies conducted in China have shown pumpkin seeds to be helpful for people with acute schistosomiasis, a severe parasitic disease occurring primarily in Asia and Africa that is transmitted through snails.
Preliminary human research conducted in China and Russia has shown pumpkin
47 seeds can assist with resolving tapeworm infestations (Duke and Ayensu, 1985).
CHOLESTEROL LOWERING - Pumpkin seed oil (PSO) has been concurrently used with cholesterol lowering drugs and would appear to potentiate the overall lipid lowering effects. The positive effects on lowering low density lipoprotein
(LDL) levels and increasing high density lipoprotein (HDL) levels are most likely due to the antioxidant and essential fatty acid content of PSO. Side effects of the cholesterol drug were also reduced when PSO was administered. Similar positive results have been found in concomitant use of PSO with anti- hypertensive medication. The hypotensive action is due to the essential fatty acid and antioxidant capability of PSO (Agoha, 1974).
KIDNEY FUNCTION - Two studies in Thailand have demonstrated that eating pumpkin seeds as a snack can help prevent the most common type of kidney stone. Pumpkin seeds appear to both reduce levels of substances that promote stone formation in the urine and increase levels of compounds that inhibit stone formation. Some research has demonstrated that PSO could remarkably reduce bladder pressure, increase bladder compliance, and reduce urethral pressure
(Duke and Ayensu, 1985).
Pumpkin Seeds Commercial Supplements
Chinese Raw Pumpkin Seeds - Pumpkin Seeds are a healthy snack that can be enjoyed all year long. This wonderful source of nutrients is naturally rich in
48 essential fatty acids, magnesium, iron, zinc, protein, and fibre (Duke and Ayensu,
1985).
Pumpkin Seed Oil - Pumpkin Seed Oil is nutritional oil rich in essential fatty acids. Most pumpkin seed oils are 100% natural and are screened for potency and purity (Duke and Ayensu, 1985).
Anti Parasite Formula - A regular natural detoxification program including Anti
Parasite Formula and a colon cleanser to promote proper elimination has been recommended by various naturopaths (Duke and Ayensu, 1985).
Pumpkin seed in cosmetics and combating fine lines
Pumpkin seed oil is a highly nourishing and lubricating oil, and is useful for all skin types. It is especially good if used to combat fine lines and superficial dryness and to prevent moisture loss (Duke and Ayensu, 1985).
African Cucurbita pepo L.: properties of seed and variability in fatty acid composition of seed oil
Pumpkin (Cucurbita pepo) seeds are used locally in Eritrea to treat tapeworm infestation. Seeds were found to be rich in oil ( 35%), protein (38%), - tocoferols (3 mg/100 g) and carbohydrate content ( 37%).
49
Traditional Uses Of Pumpkin Seed (Cucurbita Pepo)
Pumpkin seed is traditionally used to treat a wide variety of illnesses, and, through scientific investigation, most of the properties have been validated. It is used as an anthelmintic (to expel intestinal worms), taeniacide (killing tapeworms), as a diuretic, to treat bed-wetting in children and facilitate the passage of urine, while soothing an irritated bladder. It is also used to reduce the symptoms of an enlarged prostate, but does not help to reduce an enlarged prostate (Duke and Ayensu, 1985).
Anthelmintic; Galactogogue.
The seed is vermifuge. It is eaten fresh or roasted for the relief of abdominal cramps and distension due to intestinal worms (Duke and Ayensu, 1985). About
800 peeled seeds are said to make a safe and effective treatment for tape worm (
Duke and Ayensu, 1985). They are ground into a fine flour, then made into an emulsion with water and eat. It is then necessary to take a purge in order to expel the tapeworms or other parasites from the body.
The boiled root is galactogogue (Duke and Ayensu, 1985).
50
Fig.9: The digital photograph of Celosia trigyna in its natural habitat. X400
51 1.3.7. Pharmacognostic profile of Celosia trigyna (Linn.)
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Archichlamydeae
Order: Centrospermae
Family: Amaranthaceae
Genus: Celosia
Species: trigyna
Common name: enune (igbo).
Description/Physical characteristic: Celosia is grown throughout West Africa’s warmer and wetter sections. It is, for instance, Southern Nigeria’s most important leaf vegetable and is raised in myriad home gardens and farm plots, both for
52 family and the local market. Humidity and heavy rainfall fail to limit growth, so
Celosia is commonly cultivated during the wet season when other crops succumb to molds, mildews, and such like maladies. For maximum development the plants normally require at least moderate soil moisture. Although they survive dry periods, without irrigation, the level of leaf production is likely to be uneconomic in parched climes. The plant is well known in East Africa’s highlands under its
Swahili name, mfungu. Beyond Africa, throughout the world’s temperate regions, people enjoy this easy-to-grow short-lived (ornamental) annual during the summer months. Few, however, know that Celosia is a warm-weather spinach substitute. They plant it for show rather than soup. Celosia is also eaten in
India—although one report notes that it is eaten “in times of scarcity.” So maybe it lacks cachet as a food there as well.
USES: Generally, Celosia is used like leaf amaranth. Leaves, young stems and young inflorescences are eaten as potherbs. They soften up readily after being cooked in just minutes. The texture is soft; the flavour very mild and spinach- like. These boiled greens are often added to stews. They are also pepped up with such things as garlic, hot pepper, fresh lime, and red palm oil and eaten as a side dish.
53 Celosia is employed as forage for cattle. The foliage is, however, thought to accumulate oxalate.
Ornamental Uses: African families plant celosia as a vegetable not as an ornamental, but let a few plants grow to flowering to get seed. Its use as an ornamental is hardly known in Africa, but it could be. Elsewhere in the world, this is among the most popular choices for bedding and border plants, tall backgrounds, edging, and pot and container production. The blossoms also make ideal cut flowers. In addition, they are easy to dry, being merely hung upside down in a dark, dry place for several weeks. In this form they retain their form and colour and can be incorporated into dry bouquets and everlasting flower arrangements. One type, known as woolflower, is especially notable, producing elegant, chaffy flower spikes that glisten even when dry as dust.
Striga Suppression : The Celosia plant is believed to repress Striga, a parasitic weed that devastates sorghum, millet, and maize across Africa. This weed, which engenders both hunger and poverty, thrives where soils are infertile and crops ill- nourished, so it targets the poor most. Whether celosia can help farmers fight back is far from clear, but it is widely called “Striga chaser” owing to a reputation for sending the weed on its way. There is not complete confirmation of such
54 ability, but one study found that celosia stimulated Striga germination and lowered overall levels by 50% while increasing sorghum yields.
Medicinal Uses: Various medicinal benefits are widely claimed, including treatment for intestinal worms (particularly tapeworm), blood diseases, mouth sores, eye problems, chest complaints (seeds), and diarrhea (flowers). The leaves are employed as dressings for boils and sores, and the boiled vegetables are said to be slightly diuretic (Okujagu et al., 2006).
The leaves are believed to contain considerable protein and calcium as well as reasonable amounts of phosphorus and iron (which can be said for many dark- green leafy vegetables). They are also said to be good sources for vitamins A and
C. Other constituents found in Celosia include (measured per 100 g edible leaf portion): water 84 g, calories 44, protein 4.7 g, fat 0.7 g, carbohydrate 8 g, fibre
1.8 g, calcium 260 mg, phosphorus 43 mg, and iron 7.8 mg (Okwu and Josiah,
2006).
55
Fig.10a: Digital photograph of Asystasia gangetica x400
56
Fig.10b: The diagram showing different stages of developments of Asystasia gangetica. 1 = Flowering and fruiting; 2, flower in longitudinal section; 3, dehisced fruit.
57
Fig.10c: The digital photograph of Asystasia gangetica in its natural habitat.
X400
58
1.3.8. Pharmacognostic profile of Asystasia gangetica (L.) T. Anders.
Kingdom: Plantae, planta, Planter, Plants, Vegetal.
Sub Kingdom: Tracheophytes, Tracheobionta, Vascular Plants.
Division: Magnoliophyta. (Angiospermeae)
Class: Magnoliopsida: Dicote, Dicotyledon: Dicotyledoneae
Subclass: Sympetalae
Order: Tubiflorae
Family: Acanthacaea
Genus: Asystasia
Species: gangetica.
Common name: opina-epupe (igbo).
Origin and geographical distribution
Asystasia gangetica is native to tropical Africa, Arabia and tropical Asia, but has been introduced in many other tropical regions, where it is often naturalized.
59
Uses
Asystasia gangetica is locally used as a potherb and leafy vegetable, mainly in times of scarcity. In Kenya and Uganda it is locally a popular vegetable, mixed with beans and groundnut or sesame paste. It is also often prepared in a mix with other leafy vegetables. Asystasia gangetica is sometimes promoted as a cover plant in orchards because it checks erosion and prevents infestation by noxious weeds, and because it attracts bees to the orchard. Because of its ability to grow under shade and its high nutritive value, Asystasia gangetica is used as a forage for cattle, goats and sheep in Southeast Asia; it is either grazed or cut for stall feeding. Excessive consumption by sheep can result in bloat.
In Africa an infusion of the plant is used to ease pain during childbirth, and the sap is applied to sores, wounds and piles, and in embrocations to treat stiff neck and enlarged spleen in children. Powdered roots are considered analgesic and used in treating stomach-ache and snakebites. A leaf decoction is used as analgesic and to treat epilepsy and urethral discharge. In Nigeria the leaves are used to treat asthma. (Akah et al., 2003). In India the sap is applied to swellings.
It is also used as a vermifuge and to treat rheumatism. In the Moluccas
(Indonesia) the juice, together with lime and onion juice, is recommended for dry coughs with an irritated throat and discomfort in the chest. In the Philippines the
60 leaves and flowers are used as an intestinal astringent. In Tanzania, plants are pounded with water to make a wash against fleas for young animals. Asystasia gangetica is occasionally planted as an ornamental (Agoha, 1981).
Properties
The nutritional composition of Asystasia gangetica leaves per 100 g edible portion is: water 82.6 g, energy 234 kJ (56 kcal), protein 3.7 g, fat 1.2 g, carbohydrate 10.4 g, Calcium 226 mg, Phosphorus 30 mg, Iron 4.7 mg, carotene
6250 g, thiamin 0.19 mg, riboflavin 0.21 mg, niacin 1.0 mg, ascorbic acid 42 mg (Leung et al., 1972). Extracts of Asystasia gangetica have shown analgesic and anti-asthmatic properties in pharmacological tests (Akah et al., 2003).
Adulterations and Substitutes
Asystasia mysorensis (Roth) T.Anderson is used as a substitute for Asystasia gangetica.
Description
Perennial herb, with usually ascending, branched, quadrangular stem up to 2 m long, often rooting at the lower nodes. Leaves opposite, simple; stipules absent; petiole 0.5–6 cm long; blade ovate to lanceolate, 3–8(–13) cm × 1.5–4.5(–7) cm, base cuneate to cordate, apex acuminate or acute, margin entire, glabrous to sparsely pubescent, with 4–6 lateral veins at each side of the midrib, provided
61 with cystoliths. Inflorescence a terminal raceme up to 25 cm long, with flowers directed to one side. Flowers bisexual, slightly zygomorphic, 5-merous; pedicel up to 3 mm long; calyx with lanceolate lobes 4–10 mm long; corolla funnel- shaped, up to about 2.5(–4) cm long, usually white with purplish spots inside lower lobe, with rounded lobes. 1 cm wide, lower lobe slightly longer; stamens 4,
2 shorter and 2 longer; ovary superior, densely pubescent, 2-celled, style up to
1.5(–2) cm long, stigma with 2 short lobes. Fruit a club-shaped capsule 2–3 cm long, pubescent and glandular, usually 4-seeded. Seeds ovoid, flattened, 4–5 mm long, grey to brown, with crenate margins, tuberculate, supported by retinacula
(Agoha,1974).
Other botanical information
Asystasia comprises about 50 species, and is distributed in the tropics of the Old
World, with about 30 species in tropical Africa. Two subspecies can be distinguished within Asystasia gangetica. Subsp. micrantha (Nees) Ensermu, with corolla normally less than 2.5 cm long and style less than 1.5 cm long, is diploid (2n = 26) and distributed in tropical Africa, the Indian Ocean islands and
Arabia. Subsp. gangetica, with corolla normally more than 2.5 cm long and style more than 1.5 cm long, is tetraploid (2n = 52) and distributed in India, Sri Lanka,
South-East Asia and islands of the Pacific Ocean, and introduced in tropical
America. Both subspecies can be weedy, but subsp. micrantha is more serious as
62 it is more vigorous and tends to become decumbent, producing a dense carpet of rooting stems and foliage (Agoha, 1974).
Growth and development
The period from seedling emergence to seed dispersal can be as short as 8 weeks in open areas, but it can take 2 weeks longer in partially shaded areas. It takes one month from flower development to seed dispersal. The seeds are thrown as far as
6 m by an explosive opening mechanism of the fruits, triggered by hot afternoons
(Oliver,1989).
Asystasia gangetica is a shade-loving plant and optimum photosynthesis occurs between 30% and 50% full sunlight. With no weeding, its proportion in the undergrowth of a young oil palm plantation increased in a period of 2 years from
25% to 84%. It grows, though slowly, under a closed canopy of oil palm tree with less than 10%full sun light (Singh,1976).
Ecology
Asystasia gangetica is found along roadsides and river banks, in more or less waterlogged areas as well as well-drained cultivated areas, from sea-level up to
2500 m altitude. In areas with a dry season of 4 months or more it may not survive. It thrives on coastal alluvium, peat soils with 85% organic matter and pH
3.5–4.5, sandy loams and clay soils (Oliver,1989).
63 Management
Its agressiveness, high uptake of soil nutrients and ability to smother other species have characterized Asystasia gangetica as a weed in plantation management. However, the high palatability and digestibility of Asystasia gangetica make it attractive to grazing animals as plantation undergrowth (Akah etal.,2003).
Propagation and planting
Asystasia gangetica can be propagated by seed and stem cuttings with 1–3 nodes.
Single-node cuttings buried in soil produce flowers and fruits within 6 weeks
(Oliver,1989).
Diseases and pests
Asystasia gangetica is susceptible to the fungus Colletotrichum dematium, which causes necrosis, defoliation and stunted growth. In West Africa it was observed as a host plant for a mottle virus, transmitted by aphids (Larson, 1988).
Harvesting
Young tender leaves and shoots of Asystasia gangetica are collected as a vegetable. Frequent cutting for stall feeding induces early dieback because the stems have long internodes and growing points higher up the stems
(Miliauska et al., 2004).
64 Low grazing pressures or long intervals between grazing allow the plant to flower and set seed. It is usually consumed fresh by animals but it can be conserved as hay if properly dried.
Yield
For Asystasia gangetica grown under heavy shade (6–16% full sunlight) dry matter yields of 2–5 t/ha have been recorded, but under a more open canopy of
Leucaena leucocephala (Lamk) de Wit at 2 m × 1 m spacing, yields of 3.5–8 t/ha were obtained. Cattle production in the range of 110–135 kg/ha per year, equivalent to 270–310 g/head per day, can be achieved from native forages mixed with Asystasia gangetica grown under oil palm plantation (Akah et al.,
2003).
Handling and after harvest
Asystasia gangetica leaves can be dried, pounded, and the powder is stored.
Genetic resources.
There are no known germplasm collections of Asystasia gangetica and no breeding programmes. It is not at risk of genetic erosion. More attention to its different types may be desirable, focusing on vegetable use, forage use, medicinal properties, weedy characteristics and ornamental value (Akah et al., 2003).
65 Prospects
Asystasia gangetica may have potential and warrants research as a nutritious vegetable, as an auxiliary plant in agriculture and as a forage plant. It may be used as a substitute for legumes in the production of leaf meal.
Cucurbita moschata ,Celosia trigynea , Laportea ovalifolia, and Asystasia gangetica are edible green vegetables used in the Eastern parts of Nigeria. The fruit of Cucurbita moschata and aerial part of Laportea ovalifolia have some medicinal values that have not been scientifically proven and documented in
Nigeria.
1.4. Justification of the study
Many research works have been done on screening of some medicinal plants and vegetables for antioxidant activities (Mensor et al., 2001, Al-Saikhan, et al.,
1995, Amarowicz, et al., 1996, Kirby and Schmith, 1997; Sofidiya et al., 2006,
Nia et al., 2003, Stajner et al., 1999, Yen and Duh, 1995; Facino, et al., 2000,
Ellinaim, et al., 2003; Duan et al., 2007; Velioglu, et al., 1998, ).
However, no scientifically proven information is available on the antioxidant activity of K. pinnata, A. africana, L. ovalifolia, E. coccinea, A .gangetica,
C .trigyna and C. moschata .While insufficient or limited information is provided on the antioxidant potentials of M. pruriens.
66 Furthermore, the effect of the dose or concentration of the plants extracts on the antioxidant activity of these medicinal plants has not also been investigated.
Natural products still represent an important source of interesting leads for drug development. While the cost of orthodox medicine remain high and not easily affordable by the poor masses, the phytomedicines which are otherwise cheap and easily affordable, remain the first point of call for poor patients suffering the effects of free radical implicated diseases of oxidative origin.
Therefore, the search and discovery of new novel antioxidants will likely bring hope to many people afflicted with diseases originating from free radical activities or oxidative stress.
1.5. Objective of this study
This study aims to evaluate the antioxidant activities of eight Nigerian medicinal plants (Aspilia africana, Kalanchoe pinnata, Mucuna pruriens, Emilia coccinea,
Asystasia gangetica, Laportea ovalifolia, Celosia trigyna, and Cucurbita moschata). It will also investigate the reducing potentials of these plants.
The phytochemical screening of these medicinal plants will also be undertaken.
67 CHAPTER TWO
MATERIALS AND METHODS.
2.1. Materials/chemicals
All the chemicals used for the extraction, phytochemical screening, reducing potential and DPPH assay were of analytical grade; DPPH radical was a product of Sigma-Aldriech, U.S.A.
2.2. Collection and drying
The fresh leaves of the investigated plants were collected in May, 2008 from local gardens at the University of Port Harcourt and were authenticated by Edwin
Wosu, Department of Botany Herbarium, University of Port Harcourt. Voucher specimens are maintained at the Herbarium. The voucher specimen numbers are:
UPH 558; 559; 560; 561; 562; 563; 564 and 565 respectively for K. pinnata, A. africana, E. coccinea, C. moschata, L. ovalifolia, A. gangetica, C. trigyna and M. pruriens.
The leaves were cleaned of sand particles, air-dried for 10 days. They were pulverized to powder and stored in air-tight containers in the refrigerator for subsequent use.
These samples were brought out and allowed to assume room temperature prior to use for analysis.
68 2.3. Preparation of the Extracts:
Samples of the leaf powder of each plant (100g each) were macerated with
100ml of methanol for 72 hrs at room temperature.
Each extract was filtered (Whatman No. 1 filter paper) and the residue re- extracted with the same solvent. The extracts were combined and concentrated in a rotary evaporator under reduced pressure to give the methanol extract for phytochemical analysis and antioxidant- activity assay.
2.4. Phytochemical screening
Chemical tests were carried out on the methanolic extracts and on the leaf powder using standard procedures to identify the constituents (Trease and Evans,
1989; Harborne, 1973) by characteristic colour changes as described by
Sofowora, (1993); Odebedy and Sofowora, (1978) as follows:
Tannins
0.5g of dried leaf powdere plus 20ml water was boiled in a test tube and filtered.
Few drops of ferric chloride was added to the filtrate and observed .Brownish green or blue-black colouration indicates the presence of tannins.
69 Flavonoids
5ml of dilute ammonia solution was added to a portion of the aqueous filtrate of each plant extract followed by addition of concentrated sulphuric acid. Presence of flavonoids is indicated by formation of yellow colouration which disappears on standing.
Phlobatannins
Deposition of red precipitate when an aqueous extract of each plant powder is boiled with 1% aqueous hydrochloric acid is taken as evidence for the presence of phlobatannins.
Steroids
2 ml of acetic anhydride was added to 0.5g methanol extract of each sample plus
2 ml sulphuric acid .Presence of steroids is indicated by colour changes from violet to blue or green in some samples.
Terpeniods
5 ml of each extract was added to 2ml chloroform. The concentrated sulphuric acid (3 ml) was carefully added to form a layer. Formation of a reddish brown colouration at the interface shows a positive result for terpenoids
70
Saponins
2g of each leaf powder was boiled with 20ml of distilled water in a water bath and filtered. 10ml of the filtrate was mixed with 5ml of distilled water and shaken vigorously for stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaked vigorously. Formation of emulsion indicates presence of saponins.
Hemolytic test for Saponins
Sterile cork borer was used to make four wells on blood agar. Two of the wells are filled with different dilutions of the extract (5ml of extract prepared by shaking the powdered drug with normal saline and filtering).
The third well was filled with 1% w/v of white saponin in normal saline to serve as positive control. The last well was filled with normal saline to serve as negative control. The plates were incubated at room temperature for 6 hours.
Then the clear zones of hemolysis were observed and measured.
Alkaloids
2g of each leaf powder was moistened with a minimum quantity of dilute ammonia solution and allowed to stand for 10 minutes. The solution was shaken with 30ml chloroform for 5 minutes. The chloroform solution was mixed with
10ml dilute sulphuric acid. Dragendorff’s reagent (bismuth potassium iodide
71 solution) was added drop- wise to 5ml of the resulting mixture of chloroform solution. A reddish brown precipitate indicates presence of alkaloids.
Other General Tests for Alkaloids
One percent aqueous solution of the extract was prepared in dilute H2SO4. 2mls of the solution prepared were put in three different test tubes. To the tests tube were respectively added few drops of Mayer’s reagent, Wagner’s reagent and
Hager’s reagent (picric acid solution). A blank test was carried out with aqueous
1% H2SO4. A positive result confirmed the presence of the alkaloids in the crude drugs.
General reactions /Tests for Glycosides
0.1g of the leaf powder was added to 20ml distilled water in a beaker and boiled gently for 3 minutes. It was filtered hot and cooled. 1ml of the filtrate was tested with Fehling’s solution and the result was noted.
The remainder of the filtrate was added to 5ml dilute H2SO4 and boiled gently for
5 minutes, then filtered. The filtrate was made neutral or slightly alkaline with sodium bicarbonate. Then Fehling’s solution was added and boiled. The result was noted.
72
Cardiac glycosides (Keller-Killani’s test)
5 ml of each plant extract was treated with 2ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1ml of concentrated sulphuric acid. A brown ring at the interface indicates the presence of cardiac glycosides.
2.5. ANTIOXIDANT ACTIVITY ASSAY
Chemicals:
• DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical.
• Ascorbic acid, -tocopherol and methanol
The ability of the extract to scavenge DPPH radical was determined according to
Mensor et al., (2001) with little modification:
2.5.1. DPPH assay for antioxidant activity:
(Mensor et al., 2001 method)
1.0ml of 0.3m M DPPH methanol solution was added to the solution of the extract or standard (250ug/ml, 2.5ml) and allowed to react at room temperature for 30 mins. The absorbance of the resulting mixture was measured at 518 nm with spectrophotometer and converted to percentage antioxidant activity (AA%).
Methanol (1.0ml plus extract solution (2.5ml) was used as a blank
73 1.0ml of 0.3mMDPPH plus methanol (2.5ml) was used as a negative control.
Solution of ascorbic acid served as positive control.
Antioxidant activity (AA) was calculated as percentage inhibition relative to control using the following equation (Al-Saikhan et al., 1995).
AA% =Rcontrol-Rsample/Rcontrol X 100
Where Rcontrol=absorbance of control
Rsample=absorbance with each sample.
AA%=percentage of antioxidant activity.
It should be noted that each extract (sample) at a particular dose or concentration was observed in triplicate so as to get the mean and the standard error of the mean (SED). It was the mean ± SEM that was used for analysis in this study.
2.5.2. Determination of Reducing Potential
Reducing potential was determined according to the method of Afolabi et al.,
(2007). The extract or standard (100µg/ml or 250µmg/ml respectively) was mixed with phosphate buffer and potassium ferricyanide. The mixture was incubated at 50oC for 20mins. Trichloroacetic acid (10%, 2.5ml) was added to the mixture. A portion of the resulting mixture was mixed with ferric chloride (FeCl3
; 0.1%, 0.5ml) and the absorbance measured at 700nm using a
Spectrophotometer.
Higher absorbance of the reaction mixture indicates higher reductive potential.
74
2.6. Statistical Analysis
Results were analyzed using one way analysis of variance (ANOVA). Data was expressed as Mean ± SEM and further subjected to Graph Pad prism 5 demo
(software) analyses, the differences between mean accepted as significant at P <
0.05 (ANOVA).
75 CHAPTER THREE
RESULTS AND DISCUSSION
3.1 RESULTS
The phytochemical screening of the medicinal plants investigated revealed the presence of alkaloids, favonoids, terpenoids, saponins, tannins, steroids and cardiac glycosides in the methanol extracts of the plants (tables 2 and 3).
From the DPPH assay carried out, the percentage antioxidant activities of the plants investigated were calculated to be 31.0 ± 1.80, 58.4 ± 1.26, 59.1 ± 1.80,
60.0 ±1.05,
60.8 ±1.20, 62.4 ±1.26, 64.8 ± 2.10 and 75.7 ± 2.60 respectively for
A. gangetica, C. moschata, C. trigyna, L. ovalifolia, E. coccinea, M. pruriens,
A.africana and K.pinnata (table 4, figures 11 and 12). While the standards used were found to have percentage antioxidant activities of 86.7± 1.08 and 97.2 ±
1.06 respectively for ascorbic acid and -tocopherol (table3, figures 11 and 12).
All these were statistically significant at P < 0.05 (ANOVA) (table 2, figures 11 and 12).
76 Also, the reducing potentials of the plants investigated were found to be proportional to the antioxidant activities of these plants (table3, figures 13, 17a and 17b).These are respectively 0.2 ± 0.01, 0.4 ± 0.05, 0.6 ± 0.04,0.8 ± 0.02, 1.0
± 0.07,1.2 ± 0.06±, 1.4 ± 0.03 and 1.6 ± 0.03 for A. gangetica, C. moschata, C. trigyna, L. ovalifolia, E. coccinea, M. pruriens, A.africana and K.pinnata
(table3, figure 13). Ascorbic acid and -tocopherol as standard used showed reducing potentials of 1.8 ± 0.02 and 2.0 ± 0.02 respectively (table3, figures 13,
17a and 17b).
Furthermore, figures 14 and 15 show the relationship between the antioxidant values of the plants investigated and their 50% inhibitory concentrations. While figures 16a and 16b show the relationship between the reducing potentials of these plants and their respective 50% inhibitory concentrations. More still, the figures 18a and 18b show the relationship between the 50% inhibitory concentrations of these plants and their respective antioxidant activities.
Finally, the results show that the antioxidant activities of the plants investigated are dose- dependent (figures 19, 20 and 21). Hence the 50% inhibitory concentration was used in the determination of the percentage antioxidant activities of the investigated plants.
77 Table 2: Phytochemical Screening
Phytochemicals K. A. M. E.
pinnata africana mucuna coccinea
Alkaloids + + + +
Flavonoids + + + +
Terpenoids + + + +
Saponins + + + +
Tannins + + + +
steroids + + + +
Resins - - - -
Cardiac + + + +
glycosides
+ = present
- = absent
78
Table 3: Phytochemical Screening
Phytochemicals L. C. C. A.
ovalifolia trigyna moschata gangetica
Alkaloids + - + -
Flavonoids + + + +
Terpenoids + + + +
Saponins + + + +
Tannins + + + + steroids + - + -
Resins - - - -
Cardiac + - + - glycosides
+ = present
- = absent
79 Table 4: The antioxidant activities and reducing potentials of the investigated medicinal plants.
Antioxidant Percentage Reducing
Activity IC50(µg/ml) Antioxidant Potential
(AOA) Activity (%AA) (RP)
Control/blank 0.560 2.5ml 0.0 0.0
(methanol)
A. gangastica 0.301 100 31.0 ±1.80* 0.2 ±0.01
C. moschata 0.234 150 58.4 ± 1.26* 0.4 ± 0.05
C.trigyna 0.266 120 59.1 ± 1.60* 0.6 ± 0.04
L. ovalifolia 0.221 100 60.0 ±1.05* 0.8 ± 0.02
E. coccinea 0.218 120 60.8 ± 1.20* 1.0 ± 0.07
M. pruriens 0.210 100 62.4 ± 1.26** 1.2 ± 0.06
A. africana 0.201 160 64.8 ± 2.10** 1.4 ± 0.03
K. pinnata 0.122 180 75.7 ± 2.60** 1.6 ± 0.03
Ascorbic acid 0.103 120 86.7 ± 1.08** 1.8 ± 0.02
-tocopherol 0.017 50 97.2 ± 1.06** 2.0 ± 0.02
80 **reperent significant at P < 0. 001; * significant at P< 0.01 (ANOVA);
AOA=Antioxidant activity, AA= Percentage antioxidant activity, RP= reducing potentials, IC50 = 50% inhibitory concentration. Experiments were carried out in triplicate and expressed as mean ± standard error of mean (SEM).
81 120
s 100 97.2 s 86.7 *** y t i
v 80 i 75.7 t c a Control
t ** *** n
a ** E.coccinea d
i 64.8 x 62.4 M.pruriens o 60.8 i t
n A.africana a 60
) K.pinnata % (
e Ascorbic acid g a t tocopherol n e c r e 40 P
20
0 0 Medicinal plants Figure 11: Antioxidant activities of Nigerian medicinal plants
S represents standard, ** P < 0.01, *** P < 0.001 significance
(ANOVA).
82 120
A. gangestica C.moschata s 100 C.trigyna L.ovalifolia s Ascorbic acid tocopherol
y control t i 80 v i t c a t n a d i x ** ** ** o i t n
a 60
) % ( e g a t n e c r e
P 40 *
20
0 Nigerian vegetables Figure 12: Percentage (%) antioxidant activity of Nigerian vegetables
S represents standard, * P < 0.05, ** P < 0.01 significance (ANOVA).
83
2.5
2 s l A.gangetica a i t 1.5 C.moschata n C.trigynea e t L. ovalifolia o
p E. coccinea
g e M.pruriens n
i A.africana c K. pinnata u d 1 Ascorbic acid e &-tocopherol R
0.5
0 31.0 58.4 59.1 60.8 60.0 62.4 64.8 75.7 86.7 97.2 (%) Percentage anti-oxidant activity (%) Figure 13: Reducing potentials and anti-oxidant activities of Nigerian plants
84
300
250 n o i t a r t n 200 e c n o c
y 150 r o t i b i h 100 n i
% 0 5 50
0 0 10 20 30 40 50 60 Antioxidant activity (x 0.01)
Figure14: IC50 inhibitory concentration and antioxidant activity
Green,purple, yellow, blue red and darkblue colours are respectively K.pinnata, A. Africana
,M.pruriens, E.coccinea, L.ovalifoli, C.trigyna,and C.moschata,
85
0.6
0.5 ) A O A
( 0.4 y t i v i t c a 0.3 t n a d i x o i
t 0.2 n A 0.1
0 0 50 100 150 200 250 300 50% inhibitory concentrations (ug/ml) of the plants
Figure15: Antioxidant activity and 50% inhibitory concentrations ( IC50) the plants
86 2.5 s t 2 n a l p
e h t
f
o 1.5
s l a i c n e t
o 1 p
g n i c u d e 0.5 R
0 0 50 100 150 200 250 300 50% inhibitory concentration (ug/ml)
Figure 16a: The reducing potential and 50% inhibitory concentration
Series1
300 Series2 Series3 Series4 250 Series5 ) l m / Series6 g u ( Series7 n 200 o i t
a Series8 r t n
e Series9 c
n 150
o Series10 c
y r o t i b i 100 h n i
% 0 5 50
0 0 10 20 30 40 50 60 Reducing potential (x0.1)
Figure 16b: The 50% inhibitory concentration and reducing potential of the plants
Series 1,2,3,4.5,6,7,8,9 and 10 are respectively tocopherol, ascorbic acid, K.pinnata, A. africana,M.pruriens, E.coccinea,
L.ovalifoli, C.trigyna, C.moschata,and A.gangeastica
87 2.5 s t n
a 2 l p
e h t
f
o 1.5
l a i t n e t o
p 1
g n i c u d
e 0.5 R
0 0 20 40 60 80 100 120 Percentage antioxidant activity Figure17a: The relationship between reducing potential and antioxidant activity
100 Series1 90 Series2 80 Series3 ) %
( Series4
70 y t i Series5 v i 60 t c Series6 a
t 50 n Series7 a d i 40
x Series8 o t n 30 Series9 A 20 Series10 10 0 0 50 100 150 Reducing potential (x0.01) Figure 17b: Antioxidant activity and reducing potentials of the plants
Series 1, 2, 3 ,4, 5, 6, 7, 8, 9 and 10 are respectively tocopherol, ascorbic acid, K.pinnata, A. africana,
M.pruriens, E.coccinea, L.ovalifoli, C.trigyna, C.moschata,and A.gangeastica
88
Tocopherol Ascorbic acid 200 K.pinnata A.africana M.pruriens 180 75.7 E.coccinea L.ovalifolia C.trigyna 160 64.8 C.Moschata A. gangeestica 58 ) l m / 140 g u ( n o i t 120 596.01.8 86.7 a r t n e c n 100 31 6062.4 o c y r o t i 80 b i h n i
% 60 0 5 97.2 40
20
0 0 20 40 60 80 100 120 Antioxidant activity (%) Figure 18a: The percentage antioxidant activity and 50% inhibitory concentration of the plants
89
300 Series1 Series2 Series3 250 Series4 Series5 Series6 n o
i Series7 t 200 a r
t Series8 n e
c Series9 n o
c Series10
y 150 r e t i b i h n i
% 100 0 5
50
0 0 50 100 150 Antioxidant activity (%) Figure 18b: The 50% inhibitory concentration and % antioxidant activity
Series 1,2,3,4.5,6,7,8,9 and 10 are respectively tocopherol, ascorbic acid, K.pinnata, A. africana,M.pruriens, E.coccinea,
L.ovalifoli, C.trigyna, C.moschata,and A.gangeastica.
90 80 Series2 Series4 70 Series6
60 ) % ( y t i 50 v i t c a t
n 40 a d i x o i
t 30 n A
20
10
0 0 100 200 300 400 Inhibitory concentration (ug/ml) Figure 19: The effects of concentration of A.G, C.M and C.T on Antioxidant activity
Series 2, 4 and 6 represent A.gangestica (A.g), C .trigyna (C.t) and C. moschata (C.m)respectively.
91
80
70 )
% 60 ( y t i v i 50 t c a
t Series2
n 40 a d Series4 i x 30 o i t Series6 n
A 20 10 0 0 50 100 150 200 250 300 Concentration (ug/ml) Figure 20: The effects of concentration on antioxidant activity of L.o, E.c and M.p
Series 2, 4 and 6 represent L.ovalifolia (L.o), E. coccinea (E.c) and M. pruriens respectively.
92
100 90 80 Series2 ) Series4 % ( 70 y
t Series6 i v i
t 60 c a t
n 50 a d i x 40 o i t n 30 A 20 10 0 0 100 200 300 400 Concentration (ug/ml) Figure 21: The effects of concentration on antioxidant activityof A.a, K.p and ascorbic acid
Series 2, 4 and 6 represent Aspilia africana (A.a), Kalanchoe pinnata (K.p) and Ascorbic acid respectively.
93 3.2 DISCUSSION/CONCLUSSION
Tables 2 and 3 show the phytochemicals, detected in the methanolic extracts of the leaves of K. pinnata, A. africana, M. pruriens and E.coccinea. The extracts tested positive for alkaloids, flavonoids, terpeniods, saponins and cardiac glycosides. These compounds detected have been documented to possess medicinal properties and potent therapeutic effects (Afolabi et al., 2007; Edeoga et al., 2005; Okwu and Josiah, 2006; Liu, 1991;) These are consistent with the previous works of Larson, 1988; Hudson, 1990; Hall and Cuppett, 1997.
The results of the DPPH scavenging assay are shown in the table 4, Figures
11and 12. The percentage antioxidant activity of K. pinnata was found to be the highest at 75.7 ± 2.60%. This is very comparable to the antioxidant activities of
- tocopherol and ascorbic acids which were used as standards and obtained as
97.2 ± 1.06% and 86.7 ± 1.08% respectively. The percentage antioxidant activity of K.pinnata was found to be statistically significant at P = 0.001 (ANOVA). The high percentage antioxidant activity value of K. pinnata could be attributed to its high content of Flavonoids, Phenols and ascorbic acid which have been evaluated to be 1.72, 1.86 and 44.03mg/100g dry weight respectively (Okwu and Josiah
2006).This is equally consistent with the works of Nieto et al., (1993); Das and
Pereira, (1990); and Foti et al., (1993).
94 The least percentage antioxidant activity was obtained with A. gangestica at 31.0
± 1.80%. This is also comparable to the percentage antioxidant activities of the - tocopherol and ascorbic acid used as standards (97.2 ± 1.06 and 86.7 ± 1.08 respectively) This is statistically significant at P < 0.05 (ANOVA).
The percentage antioxidant activity of A. africana was found to be 64.8 ± 2.10%.
This value is comparable to the standards used (97.2 ± 1.06% and 86.7 ± 1.08%).
The value is also statistically significance at P < 0.001 (ANOVA). The high percentage antioxidant activity value of A. africana can be attributed to its high content of flavonoids, ascorbic acid and phenols which have been evaluated to be
1.48, 26.42 and 1.46.mg/100g dry weight respectively (Okwu and Josiah 2006).
This is also consistent with the previous works of Larson, 1988; Hudson, 1990;
Hull and Cuppett, 1997.
The percentage antioxidant activities of M. pruriens, and E. coccinea were 62.4 ±
1.26% and 60.8 ± 1.20% respectively. These were significant at P < 0.01
(ANOVA). The values were comparable to the standards used. These are consistent with the works of Sathiyanaranan and Arulmozhi, ( 2007);
Edeoga et al., 2005; Miliuaska et al., 2004.
The percentage antioxidant activities of C. moschata, C.trigyna and L.ovalifolia were found to be 58.4 ± 1.26%, 59.1 ± 1.60% and 60.0 ± 1.05 respectively. These
95 were statistically significant at P < 0.01 (ANOVA). These results are consistent with the works of Foote et al., 1971; Miliauska et al., 2004.
The reducing potentials of the plants were found to have a direct linear relationship with the percentage antioxidant activity (Figure13). This is consistent with the work of Duan et al., 2007.
Finally, the antioxidant activities of the plants investigated were found to be dose
– dependent (figures 19, 20 and 21). Hence 50% inhibitory concentrations (IC50) of the plants were used.
These plants can be good potential sources for new drug development.
The findings from this study have revealed the potentials of these plants as antioxidants. This could be exploited in drug development in the search of powerful antioxidants which are urgently needed to challenge free radicals in biological systems. It will consequently help to prevent the body from free radicals originating ailments. However, further study needs to be done to isolate and characterize the active principles in these plants.
96
REFERENCE
Afolabi C; Akinmoladum, E.O; Ibukun, I.A; Dan-Ologe (2007).
Phytochemical constituents and antioxidant properties of extracts from the
leaves of Chromolaena odorata. Scientific Research and Essay 2 (6)
:191-194
Agoha, R.C (1974). Medicinal Plants of Nigeria. Offset Drakerij.Faculfcitder
Wiskunde in Naturwetenschappen, the Netherlands pp. 33, 41.
Agoha, R.C (1981). Medicinal Plants of Nigeria. Offset Drakerij.Faculfcitder
Wiskunde in Naturwetenschappen, the Netherlands pp. 22-159.
Akah, P.A., Ezike, A.C., Nwafor, S.V., Okoli, C.O. & Enwerem, N.M., 2003.
Evaluation of the anti-asthmatic property of Asystasia gangetica leaf extracts.
Journal of Ethnopharmacology 89(1): 25–36.
Akobundu, I.O (1987). Weed Science in the Tropics; Principles and Practices,
John Wiley and Sons, Chichester, U.K. pp.522.
AL –Saikhan, M.S: Howard, L.R: Millar, J.C., Jr. (1995). Antioxidant activity and total phenotics in different genotypes of potato (Solanum tuberosm) J. Food Sci/;
60 (2): 341 – 343
97 Amarowicz,R: Wanasundara, U.N; Karamac, M: Sakidi, F.(1996). Antioxidant activity of ethanolic extract of mustard seed. Nahrung , 40 (5): 261 – 263.
Bloknina, O.; Virolainen, E., Fagerstedt,K.W. (2003).Antioxidants, oxidative damage and oxygen deprivation stress: a Review. Ann. Bot. 91: 179-194.
Braven, A.L.(1975) Toxicology and Biochemistry of butylated hydroxy anisole and butylated hydroxy toluene. J. An old chain Sco , 52: 59 -63
Burkill, (1985). The useful Plants of West Tropical Africa .5:2-30
Burns, J; Gardner, P.T; Mcphail , D.B; O’nell, J; Crawford, S; Morocroft,
I; L ister, C; Mathew, D; Maclean, M.R; Lean, M.E.J; Duthie, G.G; Crozier, A.
(2000). Antioxidant activity, vasodilation capacity and phenolic content of red wines.J.Agric. Food Chem. 48: 220-230.
Burton, G. W ; Ingold, K. U. (1981). Antioxidation of biological molecules 1. The antioxidant activity of Vitamin E and related chain-breaking phenolic antioxidants in vitro.J.Am Chem. Soc 103: 6472-6477
Cao, G: Sofic E ; Prior R.L (1996). Antioxidant capacity of tea and common vegetable. J Agric Food Chem.. 44: 3426 – 3431.
98 Chopra R. N, Nayar SL, Chopra I.C (1956). Glossary of Indian Medicinal Plants 1.
Council of Scientific and Industrial Res, New Delhi pp 330.
Cook N.C and Samman, S. (1996). Flavonoids chemistry: metabolism, cardiopoctive effects, and diatary sources. Nutr Biochem. ,7 : 66 -67.
Dalziel, JM (1955). The useful Plants of West Tropical Africa. Grown Agents for
Oversea Governments and Administrations, London. pp. 28, 53, 415.
Das, N P.and Pereira, T.A. (1990) . Effects of flavonoids on terminal autoxidation of palm oil: structure- activity relationship J. Agric food chem. 44: 497-501.
Duan, X; Wu, G; Jiang, Y. (2007). Evaluation of the antioxidant properties of
Litchi fruit phenolics in relation to pericarp browning prevention. Molecules, 12 :
759-771.
Duke. J. A. and Ayensu. E. S (1985). Medicinal Plants of China Reference
Publications, Inc. ISBN 0-917256-20-4 Details of over 1,200 medicinal plants of
China and brief details of their uses.
Dziedzic, S.Z: Hudson, G.J.F.(1983). Polyhydrochalcones and flavonones as antioxidants for edible oils. Food Chem. 12: 205 – 212.
99 Edeogu, H.O; Okwu, D .E; Mbaebie B.O (2005) .Phytochemical constituents of some Nigerian medicinal plants. African Journal of Biotehnology 4 (7): 685-688).
Ellinaim, W.M: Krucyyanalci Z and Kaapfak J. (2003). Investigation of the free radical scavenging activity of Cunkgo Kuoba L. Leaves. Filloton tapla 74 : (1-6).
Facino, M.R: Carinin, N: Aldini, G; Piccine, M Morazzoni, p: Bombardelli E
(2000). Antioxdant profile of a soy standardized extract in book abstract. 2000 years of national products research 680.
Foote, C.S: Denny R.W: Weaver, L: Chang .Y. Peter, N.Y(1971) Oxygen , Ann.
Acad Sci. . 139 -145.
Foti M: Plattelli M: Barattas M.T. Ruberto, G. (1996) Flavonoids coumarins and annamic acids as antioxidant in a micellar system structure – activity relationship
J. Agric Food Chem. . 44: 497-501.
Hall, C A and Cuppett. S.L.(1997). Structure activities of natural antioxidants in antioxdant methodology in vivo and in vitro concepts Aruma, O.L, cuppett S.L.,
Eds : Aocs Press: Champsign il : pp. 2 – 29.
Harborne, J .B (1973). Phytochemical methods. Chapman and Hall Ltd . London pp.49-188.
100 Huang, M.T: HO, C.T: Lee, C.Y (1992). Phenolic compounds in food and their effects on Health II. Antioxidants and cancer prevention, AC s symposium series
507: American Chemical Society Washington, DC.
Hudson, B.J.F, (1990). Ed. Food Antioxidant; Elsevier Applied Science London.
Ichikawa M; Ogura M; Ujuman T (1986). Antiallergic flavour Glycoside from
Bryophyllum pinnatum. Kokai, Tokyo, Kayo J. 84 : 280-282.
Ito N: Fukushima, S: Hasegawa A: Shibato M: Pgiso T. (1983). Carcinogenicity of butylated hydroxyl anisole in F344 rats. J. Natt Cancer Inst . 70 : 343 – 347.
Kirby, A.J. and Schmith, R.J (1997) The antioxidant activity of Chinese herbs for eczema and macebo herbs – I . J Ethnomacol, 56 : 103 – 108.
Larson, R.A (1988). The antiotidants of higher plants. Phytochermistry 27 (4): 969
– 978.
Leung, W.-T.W., Butrum, R.R. & Chang, F.H.( 1972). Food composition table for use in East Asia. Department of Health, Education and Welfare, Bethesda, United
States. pp.334
Liu, X.S; Jiang,Y.M.; Chen, F.; Zhang, D.L; Li, Y.B. (1991). The relationship between the browning in the pericarp of Litchi ( Litchi chinensis Sonn) fruit and
101 poly phenol oxidase, peroxidase, phenolics and their compartmentation.Acta Bot.
Austro sin. 7: 95-98.
Loliger, J. (1991). The use of antioxidants in foods. In free Radicals in food
Additive. Arouma, O.I, Halluell, B, Eds: Taylor and Francis London : pp. 121 –
150.
Mensor L.I; Menezes, FS: Leitao, G G; Reis A S; Dossantos , T; Coube, C S;
Leitao, SG (2001). Screening of Brazilian plant extract for antioxidant activity by the use of DPPH free radical method Phytother Res. 15 :127 – 130.
Mckenzie, RA; Franke, FO; Duster, PJ (1985). Flavonoids and Glycosides of
Bryophyllum pinnatum Antivet J. 64: 10-15.
Miliauska, G.P.R, Venskuloris, P R and T.A Beek (2004). Screening of radical scavenging activity of some Medicinal and aromatic plant extracts. Food Chem.,
85: 231-237.
Nia, R: Paper, D.H; Easien, E. E; Oladimeji, O.H : Iyadi, K.C and Franz G (2003).
Investigations into in vitro radical scavenging and in vivo anti inflamniatory potential of Tridax procumbens. Nig. J physiological sciences 18 : (1-2), 39-43.
102 Nieto, S: Garrido, A: Sanhueza J: Loyola, L: Morales, G: geighton; F. Valenzuda
(1993). A Flavonoids as stabilizers of fish oil. Alternative to synthetic antioxidant
J.Ann. Oil Chem. Soc. 70: 773 -778.
Odebedy O and Sofowora (1978). Phytochemical screening of Nigerian medicinal plants, Lloydia 41: 41-234.
Ofokansi, KC; Esimone, CO; Anele, C.K (2005). Evaluation of the in vitro combined anti bacterial effects of the leaf extracts of Bryophyllum pinnatum.(Fam:
Crassulaceae) and Ocimum gratissium (Fam: Labiatea).
Plant Prod. Res. J. 9: 23-27.
Okoli, C.O; Akah, P.A; Okoli, A.S (2007). Potentials of leaves of Aspilia africana
(Fam: Compositae) in wound care: An experimental evaluation BMC.
Comparative and Alternative medicine 7 : 24
Okujagu, T.F; Etatuvie S. O; Eze I; Jimoh B; Nweke C; Mbaoji C; (2006).
Medicinal Plants of Nigeria; South-West Nigeria. In collaboration with Lagos
State Traditional Medicine Board and Botany Department, University of Lagos. 1:
20.
Okwu, D.E; Josiah, C (2006).Evaluation of the chemical composition of two
Nigerian Medicinal plants. African Journal of Biotechnolgy, 5 (4) : 357-361.
103 Oliver, A.S (1989). Plant Taxonomy and Biosystematics, 2nd ed.University Press
Cambridge 125-152.
Oomoh, B.D: Mazza, G.(1996) Flavonoid and antioxidative activities in burckwheat. J. Agric. Food Chem. 44 (7): 1746 – 1750.
Sanchez-Moreno, C (2002). Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci. Technol. Int. 8 : 121-137.
Sathiyanarayanan, L; Arulmozhi, S (2007). Mucuna pruriens Linn . A
Comprehensive Review. Pharmacognosy Reviews 1 ( 1): 157-162.
Siddigiuient, S.M; Bina, S. F; Sultana, N (1983). Triterpenoids and phenanthrenes triterpenoids from leaves of Bryophyllum pinnatum. Res. Inst, Chem, University of
Karachi, Pakistan 88: 45-51.
Singh, R.C.P (1976). Chemical analysis on Valendoe fluoribunda medicinal plants of India Vol 1. Indian Council of medical Research, New Delhi 149-150.
Sofidiya, M.O; Odukoya, A.O; Familoni, O.B; Inya- Agha, S.I (2006). Free radical scavenging activity of Some Nigerian medicinal plant extracts. Parkistan
Journal of Biological Sciences 1438 – 1441.
104 Sofowora, A (1993). Medicinal Plants and Traditional Medicine in Africa.
Spetrum books.
Stajner D. De Mairno M.M and Conadow B.J. (1999). Antixtidant and scavenger activities of cultivate and wild allium species Flto terapla 74: (1-60.
Trease, G.E; Evans W.C (1989). Trease and Evans Pharmacognosy. 13th edition:
Ballere tindal London
Velioglu, Y.S. ;Mazza, G; Gao, L; Oomah, B.D (1998). Antioxidant Activity and
Total Phenolics in Selected Fruits, Vegetables, and Grain Products.J. Agric. Food
Chem. 46: 4113-4117.
Wang H: Cao G.H Prior, R.L. (1996). Total antioxidant capacity of fruits .J Agric
Food 44 : 248 -251.
Yen, G.C; Duh P.D (1995). Antioxidant activity of methanolic extracts of peanut hulls from various cultivars J. Am old Chem. Soc. 72 (9): 1065 – 1067.
105 Appendix
Percentage Antioxidant activity (AA%) = Rcontrol-Rsample/Rcontrol x100.
Where Rcontrol = Absorbance of control, Rsample = Absorbance of sample
0.561-0.103/0.561x100 = 86.76%
0.561-0.122x100/0.561 = 75.7%
0.561 – 0.201x100/0.561 = 64.8%
0.561 – 0.210 x 100/0.561 = 62.4%
0.561-0.221x100/0.561= 60.8%
0.561 – 0.226 x 100/0.561 = 59.1%
0.561 – 0.234 x100/ 0.561 = 58.3%.
106