Faculty o Biological Sciences - University Of Nigeria Nsukka RALEKE'S Somadina.pdf · Faculty o...
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Nwamarah UcheDigitally Signed by: Content manager’s
DN : CN = Weabmaster’s name
O= University of Nigeria, Nsukka
OU = Innovation Centre
Nwamarah Uche
Faculty of Biological Sciences
Department of Biochemistry
Paradoxical Effects of Methanol Extracts of
Ricinuscommunis Seeds on Smooth Muscle
Preparations
Chukwuka, Raleke Somadina
Reg. No PG/M.Sc/12/61667
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: Content manager’s Name
Weabmaster’s name
a, Nsukka
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Paradoxical Effects of Methanol Extracts of
Seeds on Smooth Muscle
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TITLE
Paradoxical Effects of Methanol Extracts of RicinuscommunisSeeds on Smooth
Muscle Preparations
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CERTIFICATION
Chukwuka, RalekeSomadina, a postgraduate student of the Department of Biochemistry with the
Reg. No PG/M.Sc/12/61667, has satisfactorily completed her requirements for research work, for
the degree of Master of Science (M.Sc) in Pharmacological Biochemistry. The work embodied in
this project is original and has not been submitted in part or full for any other diploma or degree
of this or any other university.
……………………. ………………………
PROF. OFC NWODO DR. PARKER. E. JOSHUA (Supervisor) (Supervisor)
………………………… ……………………………
PROF. OFC NWODO EXAMINER (Head of Department)
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DEDICATION
This work is dedicated to God Almighty for His immeasurable love, mercy and favour during
this programme and my life endeavours.
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ACKNOWLEDGEMENT
“The sword of ingratitude is greater than the sword of betrayal”. My immense gratitude goes to
my vibrant supervisors, Prof. OFC Nwodo and Dr. Parker. E. Joshua, for their tireless efforts,
encouragements, corrections and supports during this academic sojourn. The efforts of Dr.
Parker .E Joshua during the course of this work at various stages, cannot be underestimated. My
appreciation also goes to the ever promising lecturers of the Department of Biochemistry,
University of Nigeria,Nsukka most especiallyProf. F.C Chilaka, Prof. O. Njoku, Prof. I.N.E
Onwurah, Prof. L.U.S Ezeanyika, Prof. P.N Uzoegwu, Prof. Alumanah, Dr. B.C Nwanguma, Dr.
S.O Eze, Prof. H.A Onwubiko, Dr. C.O Enechi, Dr. C.S Ubani, Dr. ChiomaAnosike, Mr. P.A
Egbuna, Mr. Ozougwu, Mrs U.O Njoku and all the Graduate Assistants for their suggestions,
contributions and constructive criticisms at various stages of the work. I thank you all.My heart-
felt gratitude also goes to my exclusive Head of Department, Prof. OFC Nwodo, you are not only
an academic role model but also your careful scrutiny of my research work and quest for
academic excellence were the contributing factors in concluding this research work. I pray for
your long active life in Jesus Name Amen. I immensely thank the Chief technologist,
MrsM.Nwachukwu, the technicians of the Department and also the technicians of interest,
MrsJubilaEmelda and Mr. Okpe Aaron of the Department of Pharmacology and Therapeutics,
University Teaching Hospital, Enugu, for their efforts during the course of this work.
Moreover, my sincere acknowledgement also goes directly to my parents, Mr and Mrs P.C.A
Chukwuka, my sibilings and also Dr. and MrsIkennaIlechukwu for their inestimable financial
support, prayers and parental upbringing ever since I embraced the mother earth, the good Lord
will bless and beautify your life all with active long life in Jesus Name, Amen.
Lastly, I honestly appreciate the supports, advice and encouragements from my wonderful
friends and course mates in the persons of NdubuisiEbele, Tochi, Kingsley, Diogo,
AnokwuruGeraldine and Asiegbu Geraldine. I pray that the good Lord will flourish all our
efforts with resounding success in Jesus Name, Amen.
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ABSTRACT
The biphasic pharmacological activity of the aqueous methanol extract of Ricinuscommunisseeds
on smooth muscles was investigated in this research. The qualitative and quantitative
phytochemical analysis for bioactive compounds in the methanol extract of the fermented and
unfermented Ricinuscommunisseeds were carried out by the method of Trease and Evans and
Harborne. The median lethal doses of the two forms of extracts were determined by the method
described by Lorke.The qualitative phytochemical screening of the extracts showed relative
presence of alkaloids, flavonoids, hydrogen cyanides, steroids, soluble carbohydrates, tannins
and phenol in the fermented and unfermented extracts; while glycosides, saponins and reducing
sugars were not present in the fermented extract. The quantitative phytochemical screening of the
extracts showed the presence of high quantities of reducing sugars, 39.60±0.00mg/100g, soluble
carbohydrates, steroids and alkaloids for the unfermented methanol extracts of
Ricinuscommuniswhile the fermented extract revealed the presence of high quantities of tannins,
15.16±0.04mg/100g; flavonoids, 4.94±0.03mg/100g; and phenolics 12.62±0.04mg/100g. The
median lethal dose of the unfermented methanol seed extracts of Ricinuscommunisrecorded
nodeath at concentration of 5000mg/kg body weight while the fermented extract recorded death
at the same concentration.The smooth muscle effects of the extracts were determined on the
rabbit jejunum and pregnant rat uterus. A membrane depolarizing drug,acetycholinewas used to
initiate the normal rhythmic contraction and it contracted the rabbit jejunum at the concentration
of 1µg. Adrenaline, adrenergic receptor substance relaxed the jejunum at the concentration of
1µg. The unfermented extract relaxed the jejunum at different doses of 0.1, 0.2 and 0.4 ml at the
same concentration of 0.5µg/ml. The fermented extract also relaxed the jejunum at different
doses of 0.1, 0.2, 0.4 and 0.8 ml. Prazosin, an α- adrenergic antagonist, blocked the relaxing
effect of adrenaline at increasing doses of 0.4, 0.8 and 1.0 ml at a working concentration of 20
µg/ml. The extracts were also added in the bath with prazosin, it also blocked the relaxant effects
of the extracts at the doses of 0.2 and 0.4 ml. Indomethacin, a non-steroidal anti-inflammatory
drug, at a concentration of 20µg/ml, had no effect on the relaxant effect of adrenaline even at
higher dose of 1.0 ml. Indomethacin had no antagonistic effect on the extracts, at concentrations
of 0.5µg/ml. Oxytocin, a standard drug known for uterine contraction initiated a normal rhythmic
contraction on the uterus at a concentration of 10iu/ml and a dose of 0.1 ml. The unfermented
extract had no significant effect on the uterine tissue at a concentration of 0.5µg/ml dose of 0.1
ml while the fermented extract contracted the uterine tissue at the same concentration and dose.
Indomethacin, a prostaglandin synthesis blocker, also had no significant effect on the uterine
tissue against the standard drugs and the extracts at the concentrations of 20µg/ml and 0.5µg/ml
respectively. Ergotamine blocked the effects of the extracts at the concentration of 10µg/ml and a
dose of 1.0 ml. From these results, it can be concluded that the fermented methanol extracts of
Ricinuscommunisseeds can serve as an oxytocic agents for pregnant women during delayed
labour as claimed by the traditional birth attendants.
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TABLE OF CONTENTS
Title Page …………. ………. ………… ………….. …………………………………… i
Certification….. ……………….. ………………………………………………………… ii
Dedication………………………………………………………………………………… iii
Acknowledgement………………………………………………………………………… iv
Abstract………………………………………………………………………………… ..v
Table of Contents………………………………………………………………………… vi
List of Figures……………………………………………………..................................... ix
List of Tables……………………………………………………..………………………... x
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.0 Medicinal Plants…………………………………………………………………..1
1.1 Ricinuscommunis ………………………………………………………………………… 2
1.1.1 Morphology and classification of Ricinuscommunis seeds………………………… 2
1.1.2 Taxonomical classification of Ricinuscommunis seeds……………………………… 3
1.1.3 Pharmacological uses of Ricinuscommunis ……………………………………….. 6
1.2.0 Phytochemistry…………………………………………………………………. 7
1.2.1 Phytochemical constituents of plants…………………………………………… 8
1.2.1.1 Alkaloids……………………………………………………………………….. 9
1.2.1.2 Flavonoids……………………………………………………………………… 10
1.2.1.3 Glycosides……………………………………………………………………… 11
1.2.1.4 1.2.1.4 Tannins………………………………………………………………………… 12
1.2.1.5 1.2.1.5 Saponins……………………………………………………………………….. 13
1.2.1.6 1.2.1.6 Steroids………………………………………………………………………… 14
1.3.Depolarization………………………………………………………………….. 14
1.3.1 Hyperpolarization………….…………………………………………………… 14
1.3.2 Excitation-contraction coupling………………………………………………… 15
1.3.3 Action potential of cell membranes…….…………………………………………. 16
1.4.Muscles……………………..……………………………………………………. 17
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1.4.1 Muscle contraction and relaxation……………………………………………….. 19
1.4.2 Smooth muscles………….……………………………………………………… 20
1.4.2.1 Smooth muscle structure and organization……………………………………. 21
1.4.2.2 Smooth muscle contraction…………………………………………………….... 22
1.4.2.3 Smooth muscle relaxation……………………………………………………….24
1.5.0 Nervous system…….……………………………………………………………. 26
1.5.1 Autonomic nervous system……………………………………………………… 27
1.6.0 Uterus……………………….……………………………………………………. 29
1.6.1 Functions of the uterus……………………………………………………………30
1.7.0 Jejunum……………………………………………………………………………30
1.8.0 Aim and Objectives of the research………………………………………………32
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials……………………………………………………………………... 33
2.1.1 Plant material………………………………………………………………… 33
2.1.2 Animals………………………………………………………………………. 33
2.1.3 Drugs…………………………………………………………………………… 33
2.1.4 Equipment………………………………………………………………………. 33
2.1.5 Chemicals and reagents…………………………………………………………. 35
2.2 Methods…………………………………………………………………………. 36
2.2.1 Preparation of plant material…………………………………………………….. 36
2.2.2 Extraction of plant material………………………………………………………. 36
2.2.3 Experimental design …………………………………………………………….. 36
2.2.4 Preparation of reagents for phytochemical analysis…………………………... 37
2.2.5Drug dilutions………………………………………………………………….. 38
2.2.5.1 Composition of physiological salt solution (PSS)……………………………… 39
2.2.6 Qualitative phytochemical analysis of Ricinuscommunisseeds ……………… 40
2.2.6.1 Test for alkaloids………………………………………………………………. 40
2.2.6.2Test for glycosides………………………………………………………. ….. 40
2.2.6.3 Test for steroids…………………………………………………………... …... 40
2.2.6.4Test for flavonoids……………………………………………………………. 41
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2.2.6.5Test for saponin………………………………………………………………… 41
2.2.6.6 Test for tannins…………………………………………………………… 41
2.2.6.7 Test for reducing sugars…………………………………………………... 41
2.2.6.8 Test for carbohydrates………………………………………………………. 42
2.2.7Quantitative phytochemical analysis of Ricinuscommunisseeds…………….. 42
2.2.7.1 Alkaloid determination………………………………………………………… 42
2.2.7.2 Flavonoid determination……………………………………………………… 42
2.2.7.3 Glycoside determination…………………………………………………….. 42
2.2.7.4 Hydrogen cyanide determination…………………………………………… 43
2.2.7.5 Phenol determination………………………………………………………… 43
2.2.7.6 Saponin determination……………………………………………………… 43
2.2.7.7 Soluble carbohydrates determination…………………………………….. …. 43
2.2.7.8 Steroid determination………………………………………………………… 43
2.2.8Preparation of the methanol extract of unfermentedRicinuscommunisseeds for
the acute toxicity test………………………………………………………….. 44
2.2.8.1 Acute toxicity test of the methanol extract of unfermentedRicinuscommunis
seeds…………………………………………………............................................ 44
2.2.8.2 Preparation of the methanol extract of fermentedRicinuscommunisseeds for the
acute toxicity test………………………………………………………………… 44
2.2.8.3Acute toxicity test of the methanol extract of fermentedRicinuscommunisseeds .44
2.2.9Smooth muscle experiment………………………………………………………. 45
2.2.9.1Animal preparation………………………………………………………………. 45
2.2.9.2Determination of the effects of the extracts……………………………………….45
(a) On the Rabbit isolated jejunum……………………………………………….45
(b) On pregnant isolated uterus…………………………………………………...47
CHAPTER THREE: RESULTS
3.1.Percentage yield of the methanol extracts of fermented and
unfermentedRicinuscommunisseeds………………………………………………………
……... 49
3.2.Qualitative phytochemical screening of the methanol extracts of fermented and
unfermentedRicinuscommunisseeds…………………………………………. 51
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3.2.1 Quantitative phytochemical constituents of the methanol extracts of fermented and
unfermentedofRicinuscommunisseeds………………………………………… 53
3.3Median Lethal Dose (LD50) test of the methanol extract of unfermentedRicinus
communisseeds. ………………………………………………………………….. 55
3.3.1Median Lethal Dose (LD50) test of the methanol extract of fermentedRicinus
communisseeds…………………………………………………………………… 57
3.4.Effects of the extracts on the isolated rabbit jejunum ……………………………. 59
3.4.1 Effects of prazosin blockade of α- adrenoceptor…………………………………. 61
3.4.2 Effects of Indomethacin on extract induced relaxation…………………………… 63
3.4.3Effects of prazosin on the isolated rabbit jejunum ………………………………... 65
3.5Effects of the extracts on the isolated pregnant rat uterus……………………....... 67
3.5.1Effects of prostaglandin synthesis inhibition…..…………………………………. 69
CHAPTER FOUR: DISCUSSION
4.1Discussion.…………………………………………………………………………..... 71
4.2 Conclusion……………………………………………………………………………76
4.3 Suggestions for further studies ……………………………………………………... 76
REFERNCES………………………………………………………………………… 77
APPENDICES………………………………………………………………………..87
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LIST OF FIGURES
Fig. 1: Fruit of Ricinuscommunis………………………………………………………….…….4
Fig. 2: The whole plant of Ricinuscommunis……………………………………………….…...4
Fig. 3: The Seeds of Ricinuscommunis……………………………………………………….…5
Fig. 4: The Leaves of Ricinuscommunis………………………………………………………...6
Fig. 5: Basic structures of some pharmacologically important plant-derived alkaloids………..10
Fig. 6: Basic structures of some pharmacologically important plant-derived flavonoids………11
Fig. 7: Basic structures of some pharmacologically important plant-derived tannins…………..13
Fig. 8: The Three types of muscle………………………………………………………………15
Fig.9: Dense bodies and intermediate filaments which cause the muscle fibres to contract... 18
Fig. 10: Actin-myosin filaments………………………………………………………………....22
Fig. 11: Smooth muscle contraction…………………………………………………………..…2
Fig.12: Smooth muscle relaxation……………………………………………………………....22
Fig.13: Divisions of the nervous system…………………………………………………….…..27
Fig.14: Autonomic nervous system………………………………………………………….….28
Fig. 15: Anatomical characteristics and neurotransmitters of the somatic (Som), sympathetic
(Sym) and parasympathetic (Para) divisions of the PNS. Ach, acetylcholine; E,
epinephrine; NE, norepinephrine………………………………………………………29
Fig. 16: The structure of the digestive system showing the jejunum……………………………31
Fig. 17: Effects of the methanol extracts of Ricinuscommunisseeds on the isolated rabbit
jejunum…………………………………………………………………………………60
Fig. 18: Effects of the methanol extracts of Ricinuscommunisseeds on the isolated rabbit
jejunum…………………………………………………………………………………62
Fig. 19: Effects of the methanol extracts of Ricinuscommunisseeds on the isolated rabbit
jejunum………………………………………………………………………………...64
Fig. 20: Effects of the methanol extracts of Ricinuscommunisseeds on the isolated rabbit
jejunum………………………………………………………………………………...66
Fig. 21: Effects of the methanol extracts of Ricinuscommunisseeds on the isolated pregnant rat
uterus…………………………………………………………………………………...68
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LIST OF TABLES
Table 1: Table showing the differences between the three types of muscle……………………16
Table 2: Percentage yield of the methanol extracts of fermented and unfermented
Ricinuscommunisseeds…………………………………………………………………50
Table 3: Preliminary phytochemical screening of methanol extracts of fermented and unfermented
Ricinuscommunis seeds…………………............................................52
Table 4: Table showing the quantitative phytochemcial constituents of methanolextracts of
fermented and unfermented Ricinuscommunisseeds……………………………… 54
Table 5: Phase 1 and 11 of the median lethal dose (LD50) test of the methanol extracts of
unfermented Ricinuscommunisseeds……………………………………………...56
Table 6: Phase 1 and 11 of the median lethal dose (LD50) test of the methanolextracts of fermented
Ricinuscommunisseeds………………………………………………...58
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CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.0 Medicinal Plants
Plants serveas rich sources of organic compounds, many of which have been used for medicinal
purposes. Medicinal plants are the plants whose parts (leaves, seeds, stems, roots, fruits, foliage
etc), extracts, infusions, decoctions or powders are used in the treatment of different diseases of
humans, plants and animals (Jamil et al.,2007). In the last few decades, there has been an
exponential growth in the field of herbal medicine. It is getting popularized in developing and
developed countries, owing to its natural origin and lesser side effects. One of such medicinal
plant is Ricinus communis (Euphorbiaceae), which is commonly known as Castor. It is a small
tree which is found all over the India (Manpreet et al., 2012).There is a wide spectra of trees,
plants and shrubs whose seeds, roots, barks and leaves are used by humans throughout the globe
due to their nutritional or medicinal value (Abayomi, 1986). In the last few years, there has been
an exponential growth in the field of herbal medicine and these drugs are gaining popularity both
in the developing and developed countries because of their natural origin and less side effects
(Manisha et al., 2007).However, these complementary components give the plant as a whole, the
safety and efficiency much superior to that of its isolated and pure active components (Shariff,
2001). The World Health Organisation (WHO) report in 1993 showed that nearly 80 percent of
world population is dependent on the traditional system of medication, that is the use of plants
and their parts as medicine (Mathur et al.,2011).
It is true that without nature, it is impossible for human beings to survive. The food, clothes and
shelter are the three basic necessities of human beings and the most important is good health,
which is being provided by the plant kingdom. In traditional medicine, there are many natural
crude drugs for different health purposes, one of such plants is Ricinus communis (Jitendra and
Ashish, 2012).
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1.1 Ricinus communis
Castor bean, Ricinus communis is a species of flowering plant in the spurge family,
Euphorbiaceae (Momoh et al., 2012). It is an important drought-resistant shrub, also native to
the Ethiopian region of the tropical Africa and has become naturalized in tropical and temperate
regions throughout the world (Weiss, 2000). Ricinus communis (Euphorbiaceae family) is a soft
wooden small tree, grown throughout the tropics and warm temperate regions of the world
(Parkeh and Chanda, 2007). Its seed is the castor bean, which despite its name, is not a true bean.
Castor is indigenous to the South Eastern Mediterranean basin, Eastern Africa and India, but its
widespread throughout tropical regions and widely grown elsewhere as an ornamental plant
(Philips and Martyn, 1999). Ricinus communis is a small wooden tree which grows to about 6
meters in height and found in South Africa, India, Brazil and Russia (Singh et al., 2010). The
Euphorbiaceae is the fourth largest family of the angiosperms comprising over 300 genera and
about 7500 species and are distributed widely in tropical Africa (Gill, 1988). The Euphorbiaceae
plants are shrubs, trees, herbs or rarely lianas (Pandey, 2006). The family provides food and
varied medicinal properties used in ethnobotany (Etukudo, 2003).
The plant has many common names such as castor plant, castor oil plant, castor bean plant,
wonderboom, dhatura, eranda and palma Christi. Locally, the plant is known in Nigeria as
“Zurman” (Hausa), “Laraa” (Yoruba), “Ogili isi” (Igbo), “Kpamfinigulu” (Nupe), “Jongo” (Tiv)
and Era ogi (Bini) (Sani and Sule, 2007). The castor plant is considered by most authorities to be
native of the Tropical Africa and may have originated in Abyssinia, Ethiopia. The plant is a
native of India with about 17 species that have been grouped into two: as shrubs and trees that
produce large seeds or as annual herbs that produce smaller seeds (Weiss, 1971).
1.1.1 Morphology and classification of Ricinus communis seeds
It consists of several branches, each terminated by a spike. The mature spike is fifteen to 30cm
long and each spike bears 15 to 80 capsules (Oplinger et al.,1990). The leaves are alternate,
curved, cylindrical, purplish petioles, sub peltate, drooping, stipules large, ovate, yellowish,
united into a cap enclosing the buds, deciduous, blade 6-8 inches across, palmately cut for three
quarters of its depth into 7-11 lanceolate, acute, coarsely serrate segments, smooth blue green,
paler beneath, red and shinning when young (Manpreet et al., 2012).
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The flowers are monoecious and about 30-60cm long. The fruit is a three-celled thorny capsule.
The capsule of fruit covered with soft spins like processes and dehiscing into three 2-valved
cocci. The seeds are somewhat compressed, oval, 8-18mm long and 4-12mm broad. The testa is
very smooth, thin and brittle (Jitendra and Ashish, 2012). The male flowers are yellowish-green
with prominent creamy stamens and are carried in ovoid spikes up to 15 centimeters (5.9inches)
long; the female flowers, borne at the tips of the spikes, have prominent red stigmas. The fruit is
a spiny, greenish (to reddish-purple) capsule containing large, oval, shiny, bean-like, highly
poisonous seeds with variable brownish mottling. Castor seeds have a warty appendage called
the caruncle, which is a type of elaiosome. The caruncle promotes the dispersal of the seed by
ants (Myrrmecochony) (Brickell, 1996).
1.1.2 Taxonomical classification of Ricinus communis seeds
Castor bean plant (Ricinus communis) is a flowering plant that belongs to the Euphorbiaceae
family and is classified scientifically as
Kingdom Plantae
Phylum Magnoliophyta
Class Magnoliopsida
Order Malpighiales
Family Euphorbiaceae
Sub family Acalyphoideae
Tribe Acalypheae
Sub tribe Ricininae
Genus Ricinus
Species R. communisSource: (Jitendra and Ashish, 2012)
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Fig. 1: The fruit ofRicinus communis plant
(Jitendra and Ashish, 2012)
Fig.2: The whole plant of Ricinus communis
(Jitendra and Ashish, 2012)
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Fig. 4: The leaves of Ricinus communis
(Source; Rotblatt and Ziment, 2002).
1.1.3 Pharmacological Uses of Ricinus communis
Ricinus communis or castor plant has high traditional and medicinal value for maintaining
disease free healthy life. Traditionally, the plant is used as laxative, purgative, fertilizer and
fungicide. The plant also possess beneficial effects like anti-oxidant, antihistaminic,
antinociceptive, antiashmatic, antiulcer, immunomodulatory, antidiabetic, hepatoprotective, anti-
fertility, anti-inflammatory, antimicrobial, central nervous system stimulant, lipolytic, wound
healing, insecticidal, larvicidal and many other medicinal properties (Jitendra and Ashish, 2012).
All the parts of the plants are used medicinally (Obumselu et al., 2011). All these uses are due to
the presence of certain phytoconstituents in the plant. The major phytoconstituents in this plant
are rutin, gentistic acid, quercetin, gallic acid, kaempferol-3-o beta-d-rutinoside, kaempferol-3-0
beta-d-xylopyranoid, tannins, ricin A, B and C, ricinus agglutinin, indole-3-acetic acid and an
alkaloid ricinine (Manpreet et al., 2012). In the traditional system of medicines, Euphorbiaceae
plants are used to treat various microbial diseases such as diarrhoea, dysentery, skin infections
19
and gonorrhoea (Ajibesin et al., 2008). In the Indian system of medicine, the leaf, root and seed
oil of Ricinus communis have been used for the treatment of the inflammation and liver
disorders, hypoglycaemia and laxative (Kensa and Syhed, 2011). Ricinus communis of the family
Euphorbiaceae, is traditionally used by Traditional Birth Attendants (TBAs) in Machakos
district of Kenya to induce or augument labour, manage protracted labour, post partum
haemorrhage (Kaingu et al., 2012). It has also being the practice that, in the Middle Belt of
Nigeria, traditional healers administer to women three seeds of the variety minor as
contraceptives for a duration of 12 months (Okwusaba et al.,1997). Castor oil has many uses in
medicine and other applications. A water extract of the root bark showed analgesic activity in
rats; antihistamine and anti-inflammatory properties were found in ethanol extract of Ricinus
communis root bark (Lomash et al., 2010). It was also found out that the methanol extract of the
ether soluble fraction of Ricinus communis seed possesses anti-ovulatory activity and also
distorts the oestrous cycle of adult cyclic rats (Ogunranti, 1997).
1.2. Phytochemistry
Phytochemistry is the study of natural bioactive products found in plants that work with nutrients
and dietary fibre to protect against diseases (Doughari et al., 2009). “Phyto” is a Greek word that
means plant and phytochemicals are usually related to plant pigments. So fruits and vegetables
that have bright colours – yellow, orange, red, green, blue and purple, generally contain more
phytochemicals and more nutrients. Research suggests that phytochemicals, working together
with nutrients found in fruits, vegetables and nuts, may help slow the ageing process and reduce
the risk of many diseases including cancer, heart diseases, stroke, high blood pressure, cataracts,
osteoporosis and urinary tract infections (Gao et al., 2001).
Phytochemicals protect health. They can have complementary and overlapping mechanisms of
action in the body including antioxidant effects, modulation of detoxification enzymes,
stimulation of the immune system, modulation of hormone mechanisms and antibacterial and
antiviral effects (Conn, 1995). Medicinal plants are of great importance to the health of
individuals and communities. The medicinal value of these plants lies in some chemical
substances that produce definite physiological actions on the human body (Hill, 1952). Many
medicinal plants are used as spices and food plants. They are also sometimes added to foods
20
meant for pregnant and nursing mothers for medicinal purposes (Okwu, 2001). Medicinal herbs
are significant sources of synthetic and herbal drugs. Medicinal plants have active ingredients
which are responsible for most of the biological activities they exhibit (Fukumoto and Mazza,
2000).
1.2.1 Phytochemical constituents of plants
Phytochemicals are a heterogeneous group of chemical compounds with numerous biologically
active plant compounds that have potential disease inhibiting capabilities (Akinmoladun et al.,
2007). Phytochemicals (plant chemicals) are bioactive substances of plants that have been
associated with the protection of human health against chronic degenerative diseases (Fukumoto
and Mazza, 2000). The term ‘phytochemicals’ according to the American Cancer Society refers
to a wide variety of compounds produced by plants and can be found in fruits, vegetables, beans,
grains. They are chemical compounds formed during the plant normal metabolic processes.
These chemicals are often referred to as ‘secondary metabolites’ of which there are several
classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols,
tannins, terpenes and terpenoids (Harborne, 1998; Okwu, 2004). More than 900 different
phytochemicals have been found in plant foods and more probably will be discovered (Polk,
1996). These protective plant compounds are an emerging area of nutrition and health, with new
research reported every day. Some examples of phytochemicals in fruits and vegetables include –
carotenoids, β-carotene, lutein, lycopene, zeaxanthin, flavonoids, anthocyanin, limonene, indoles
and allium compounds. Phytochemicals are present in a variety of plants utilized as important
components of both human and animal diets. These include fruits, seeds, herbs and vegetables
(Okwu, 2005).
According to the World Health Organization, a medicinal plant is any plant which, in one or
more of its organs, contains substances that can be used for therapeutic purposes, or which are
precursors for chemo-pharmaceutical semi-synthesis. Such a plant will have its parts including
leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or
treatment of a disease condition and therefore contains chemical components that are medically
active. These non-nutrient plant chemical compounds or bioactive components are often referred
to as phytochemicals (‘phyto-‘ from Greek - phyto meaning ‘plant’) or phytoconstituents and are
21
responsible for protecting the plant against microbial infections or infestations by pests (Abo et
al., 1991; Liu et al., 2004; Nweze et al., 2004; Doughari et al., 2009).
1.2.1.1 Alkaloids
Alkaloids are natural products that contains heterocyclic nitrogen atoms; they are basic in
character. The name of alkaloids derives from the “alkaline” and it was used to describe any
nitrogen-containing base (Mueller-Harvey and McAllan, 1992). These are the largest group of
secondary chemical constituents; they are made largely of ammonia compounds comprising
basically of nitrogen bases synthesized from amino acid building blocks with various radicals
replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The
compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. In
fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1°, 2° or 3° amines,
contribute to the basicity of the alkaloid (Firn, 2010).
Alkaloids generally exert pharmacological activity particularly in mammals such as humans.
Many of our most commonly used drugs are alkaloids from natural sources and new alkaloidal
drugs are still being developed for clinical use (Roberts and Winks, 1998). Most alkaloids with
biological activity in humans affect the nervous system, particularly the action of neural
transmitters, example, acetylcholine, adrenaline, noradrenaline, gamma-aminobutyric acid
(GABA), dopamine and serotonin (Schmeller and Wink, 1998). They react with acidsto form
crystalline salts without the production of water (Firn, 2010). The majority of alkaloidsexist in
the solid state such as atropine, some as liquids containing carbon, hydrogen, and nitrogen.Most
alkaloids are readily soluble in alcohol. Though they are sparingly soluble in water,their salts of
are usually soluble. The solutions of alkaloids are intensely bitter. Thesenitrogenous compounds
function in the defence of plants against herbivores and pathogens,and are widely exploited as
pharmaceuticals, stimulants, narcotics, and poisons due to theirpotent biological activities
(Schmeller and Wink, 1998).
22
Fig. 5 Basic structures of some pharmacologically important plant derived alkaloids (Source;
Madziga et al., 2010).
1.2.1.2 Flavonoids
Flavonoids are an important group of polyphenols widely distributed among the plant flora.
Structurally, a flavonoid ismade of more than one benzene ring in its structure (a range of C15
aromatic compounds) and numerous reports support their use as antioxidants or free radical
scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans.
Flavonoids are also referred to as bioflavonoids. They are organic compounds that have no direct
involvement with the growth or development of plants, they are plant nutrients that when
consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the
human body. Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than
4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages
like tea, coffee and fruit drinks (Pridham, 1960).
Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids,
flavanones, flavonols and anthocyanidines (Nijveldt et al.,2001; Kuhnan, 1976). Flavones are
characterized by a planar structure because of a double bond in the central aromatic ring.
23
Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance
in onions, apples, broccoli and berries. Flavonones are mainly found in citrus fruit, an example is
narigin. Flavonoid is involved in scavenging of oxygen derived free radicals (Nijveldt et
al.,2001). It has been identified as a potent hypolipidemic agents in a number of studies (Harnafi
and Amrani, 2007; Narender et al.,2006). It has also been established that flavonoids from
medicinal plants possess a high antioxidant potential due to their hydroxyl groups and protect
more efficiently against free radical related diseases like arteriosclerosis (Vaya et al.,2003; Kris-
Etherton et al.,2002). Experimental studies showed that flavonoids enhance vaso-relaxant
process (Bernatova et al.,2002) and prevent platelet activity-related thrombosis (Wang et
al.,2002).
Fig. 6:Basic structures of some pharmacologically important plant derived flavonoids (Source;
Kar, 2007).
1.2.1.3 Glycosides
Glycosides in general, are defined as the condensation products of sugars (including
polysaccharides) with a host of different varieties of organic hydroxyl (occasionally thiol)
compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of
the carbohydrate must essentially take part in the condensation. Glycosides are colorless,
crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-
24
soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate
(glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007). Alcohol, glycerol or
phenol represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed
into its components with ferments or mineral acids. Glycosides are classified on the basis of type
of sugar component, chemical nature of aglycone or pharmacological action (Firn, 2010).
1.2.1.4 Tannins
Tannins are polymerized phenols with defensive properties. Their name comes from their use in
tanning, rawhides to produce leather. In tanning, collagen proteins are bound together with
phenolic groups to increase the hide’s resistance to water, microbes and heat (Heldt and Heldt,
2005). Two categories of tannins that are of importance are the condensed and hydrolysable
tannins. The polymerization of flavonoid molecules produces condensed tannins, which are
commonly found in woody plants. Hydrolysable tannins are polymers, but they are a more
heterogeneous mixture of phenolic acids (especially gallic acid) and simple sugars. Though
widely distributed, their highest concentration is in the bark and galls of oaks (Heldt and Heldt,
2005). These are widely distributed in plant flora. They are phenolic compounds of high
molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem
and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things
into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of
phenolics or carboxylic group (Kar, 2007). They form complexes with proteins, carbohydrates,
gelatin and alkaloids.
Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins
and various other organic compounds including amino acids and alkaloids (Petridis, 2010). The
astringency from tanninsis what causes the dry and pucker feeling in the mouth following the
consumption of unripened fruit or red wine (Serafini et al.,1994). Many human physiological
activities, such as stimulation of phagocytic cells, host-mediated tumour activity, and a wide
range of anti-infective actions, have been assigned to tannins (Haslam, 1996). One of their
biological actions is to complex with proteins through nonspecific forces such as hydrogen
bonding and hydrophobic interactions, as well as by covalent bond formation (Haslam, 1996,
25
Stern et al.,1996). Thus, their mode of antimicrobial action may be related to their ability to
inactivate microbial adhesins, enzymes, cell envelope, transport proteins etc
Fig. 7 Basic structures of some pharmacologically important plant derived tannins (Source; Heldt
and Heldt, 2005).
1.2.1.5 Saponins
Saponins are glycosides of triterpenes and steroids which are characterized by bitter or astringent
taste, foaming properties (Okigbo et al., 2009), haemolytic effect on red blood cells and
cholesterol binding properties (Okwu, 2005). Saponins increase the permeability of intestinal
mucosa cells, inhibit active nutrient transport and facilitate the uptake of substances to which the
gut would normally be impermeable (Gee et al., 1997). It has also been shown to possess
beneficial effects such as cholesterol lowering properties and exhibits structure dependent
biological activity (Harborne, 1998).
Saponins, being both fat and water soluble, have surfactant and detergent activity. Thus they
would be expected to influence emulsification of fat-soluble substances in the gut, including the
formation of mixed micelles containing bile salts, fatty acids and fat-soluble vitamin (Okwu,
2005).
26
1.2.1.6 Steroids
Sterols are triterpenes which are based on the cyclopentane hydrophenanthrene ring system
(Harborne, 1998). Sterols were at one time considered to be animal substances (similar to sex
hormones, bile acids, etc) but in recent years, an increasing number of such compounds have
been detected in plant tissues. Sterols have essential functions in all eukaryotes. For example,
free sterols are integral components of the membrane lipid bilayer where they play an important
role in the regulation of membrane fluidity and permeability (Corey et al., 1993). While
cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants,
with sitosterol usually predominating. Sterols in plants are generally described as phytosterols
with three known types occurring in higher plants: sitosterol (formerly known as β-sitosterol),
stigmasterol and campesterol (Harborne, 1998). These common sterols occur both free and as
simple glucosides. Certain sterols are confined to lower plants; one of which is ergosterol, found
in yeast and many fungi. Others occur mainly in lower plants but also appear occasionally in
higher plants, e.g fucosterol, the main steroid of many brown algae and also detected in coconut
(Harborne, 1998).
1.3 Depolarization
Depolarization is a positive-going change in a cell's membrane potential, making it more
positive, or less negative, and thereby removing the polarity that arises from the accumulation of
negative charges on the inner membrane and positive charges on the outer membrane of the cell.
In neurons and some other cells, a large enough depolarization may result in an action potential.
Hyperpolarization is the opposite of depolarization, and inhibits the rise of an action potential
(Yellen, 2002).
1.3.1 Hyperpolarization
Hyperpolarization is a change in a cell'smembrane potential that makes it more negative. It is the
opposite of a depolarization. It inhibits action potentials by increasing the stimulus required to
move the membrane potential to the action potential threshold (Goldin, 2007).
Hyperpolarization is often caused by efflux of K+ (a cation) through K
+ channels, or influx of Cl
–
(an anion) through Cl– channels. On the other hand, influx of cations, e.g. Na
+ through
27
Na+channels or Ca
2+ through Ca
2+ channels, inhibits hyperpolarization (MacDonald and
Rorsman, 2006). If a cell has Na+ or Ca
2+ currents at rest, then inhibition of those currents will
also result in a hyperpolarization. Because hyperpolarization is a change in membrane voltage,
electrophysiologists measure it using current clamp techniques. In voltage clamp, the membrane
currents giving rise to hyperpolarization are either an increase in outward current or a decrease in
inward current (Yellen, 2002).
1.3.2 Excitation-contraction coupling
Excitation–contraction couplingis a term coined in 1952 to describe the physiological process of
converting an electrical stimulus to a mechanical response (Sandow, 1952). Excitation-
contraction coupling refers to the sequence of events by which an action potential in the plasma
membrane of a muscle fibre leads to cross-bridge activity by the mechanisms just described
(Widmaier et al., 2010). A smooth muscle is excited by external stimuli, which causes
contraction. It may contract spontaneously (via ionic channel dynamics) or as in the gut, special
pacemakers cells interstitial cells of Cajal produce rhythmic contractions. Also, contraction, as
well as relaxation, can be induced by a number of physiochemical agents (e.g., hormones, drugs,
neurotransmitters - particularly from the autonomic nervous system). Smooth muscle in various
regions of the vascular tree, the airway and lungs, kidneys and vagina is different in their
expression of ionic channels, hormone receptors, cell-signaling pathways, and other proteins that
determine function (Aguilar et al., 2010).
This process is fundamental to muscle physiology, whereby the electrical stimulus is usually an
action potential and the mechanical response is contraction. EC coupling can be dysregulated in
many diseases. Though E-C coupling has been known for over half a century, it is still an active
area of biomedical research. The general scheme is that an action potential arrives to depolarize
the cell membrane. By mechanisms specific to the muscle type, this depolarization results in an
increase in cytosolic calcium that is called a calcium transient. This increase in calcium activates
calcium-sensitive contractile proteins that then use ATP to cause cell shortening (Crespo, 1990).
It is important to note that contraction of smooth muscle need not require neural input that is, it
can function without an action potential. It does so by integrating a huge number of other stimuli
28
such as humoral/paracrine (e.g. Epinephrine, Angiotensin II, AVP, Endothelin), metabolic (e.g.
oxygen, carbon dioxide, adenosine, potassium ions, hydrogen ions), or physical stimuli (e.g.
stretch receptors, shear stress). This integrative character of smooth muscle allows it to function
in the tissues in which it exists, such as being the controller of local blood flow to tissues
undergoing metabolic changes. In these excitation-free contractions, then, there of course is no
excitation-contraction coupling (Fabiato, 1983).
Some stimuli for smooth muscle contraction, however, are neural. All neural input is autonomic
(involuntary). In these the mechanism of excitation-contraction coupling is as follows:
parasympathetic input uses the neurotransmitter acetylcholine. Acetylcholine receptors on
smooth muscle are of the muscarinic receptor type; as such they are metabotropic, or G-protein /
second messenger coupled. Sympathetic input uses different neurotransmitters; the primary one
is norepinephrine. All adrenergic receptors are also metabotropic. The exact effects on the
smooth muscle depend on the specific characteristics of the receptor activated—both
parasympathetic input and sympathetic input can be either excitatory (contractile) or inhibitory
(relaxing) (Cannell, 1994). The main mechanism for actual coupling involves varying the
calcium-sensitivity of specific cellular machinery. However it occurs, increased intracellular
calcium binds calmodulin, which activates myosin light chain kinase (MLCK). MLCK
phosphorylates the regulatory light chains of the myosin heads. Phosphorylated myosin heads
are able to cross bridge-cycle. Thus, the degree to and velocity of which a whole smooth muscle
contracts depends on the level of phosphorylation of myosin heads. Myosin light chain
phosphatase removes the phosphate groups from the myosin heads, thus ending cycling (and
leaving the muscle in latch-state) (Sandow, 1952).
1.3.3 Action potential of cell membranes
In physiology, an action potential is a short-lasting event in which the electrical membrane
potential of a cell rapidly rises and falls, following a consistent trajectory. Action potentials are
generated by special types of voltage-gated ion channels embedded in a cell's plasma membrane
(Barnett and Larkman, 2007). These channels are shut when the membrane potential is near the
resting potential of the cell, but they rapidly begin to open if the membrane potential increases to
a precisely defined threshold value. When the channels open (by detecting the depolarization in
29
transmembrane voltage (Barnett and Larkman, 2007). Action potentials occur in several types of
animal cells, called excitable cells, which include neurons, muscle cells, and endocrine cells, as
well as in some plant cells. In neurons, they play a central role in cell-to-cell communication
(Goldin, 2007). In other types of cells, their main function is to activate intracellular processes.
In muscle cells, for example, an action potential is the first step in the chain of events leading to
contraction. In beta cells of the pancreas, they provoke release of insulin (MacDonald and
Rorsman, 2006). Action potentials in neurons are also known as "nerve impulses" or "spikes",
and the temporal sequence of action potentials generated by a neuron is called its "spike train". In
animal cells, there are two primary types of action potentials, one type generated by voltage-
gated sodium channels, the other by voltage-gated calcium channels (Yellen, 2002). Sodium-
based action potentials usually last for under one millisecond, whereas calcium-based action
potentials may last for 100 milliseconds or longer. In some types of neurons, slow calcium spikes
provide the driving force for a long burst of rapidly emitted sodium spikes. In cardiac muscle
cells, on the other hand, an initial fast sodium spike provides a "primer" to provoke the rapid
onset of a calcium spike, which then produces muscle contraction (Doyle et al., 1998).
1.4 Muscles
The muscular systemis the biological system of humans that produces movement (Widmaier et
al., 2004). The muscular system, in vertebrates, is controlled through the nervous system,
although some muscles, like cardiac muscle, can be completely autonomous. Muscleis
contractile tissue and is derived from the mesodermal layer of embryonic germ cells. Its function
is to produce force and cause motion, either locomotion or movement within internal organs
(Widmaier, et al., 2004). The term ‘muscle’ refers to a number of muscle fibers bound together
by connective tissue. The relationship between a single muscle fiber and a muscle is analogous to
that between a single neuron and a nerve, which is composed of the axons of many neurons.
Muscles are usually linked to bones by bundles of collagen fibers known as tendons (Widmaier
et al., 2004). A muscle causing an action when it contracts is called an agonist, a muscle working
in opposition to the agonist moving structure in the opposite direction, is an antagonist. Most
muscle function as members of a functional group to accomplish specific movements (Seeley et
al., 2004). Muscles are categorized into smooth, cardiac and skeletal muscles. This
categorization is based on the structural and functional properties of these muscles (Ashlesha,
30
2011).Muscles generate force and movements used in the regulation of the internal environment,
and they also produce movements in the external environment. In humans, the ability to
communicate, whether by speech, writing or artistic expression also depends on muscle
contractions. Indeed, it is only by controlling the activity of muscles that the human mind
ultimately expresses itself (Widmaier, et al., 2004). There are three general types of muscle
tissues; Skeletal muscle responsible for movement, Cardiac muscle responsible for pumping
blood and Smooth muscle responsible for sustained contraction in the blood vessels,
gastrointestinal tract, uterus and other areas in the body (Gordon et al.,1966).
Fig. 8: The three types of muscle (Source; Widmaier, et al., 2004)
Table 1: Table showing the differences between the three types of
1.4.1 Muscle contraction and relaxation
The term contraction, as used in muscle physiology, does not necessarily mean “shortenin
simply refers to activation of the force
(Widmaier, et al., 2004). Following contraction, the mechanism that initiate force generation are
turned off, and tension declines, allowing relaxation
Muscle fibre generates tension through the action of actin and myosin cross
While under tension, the muscle may lengthen, shorten or remain the same. Although
‘contraction’ implies shortening, when referring to the muscular system, it means muscle fibres
generating tension with the help of motor neurons (the terms twitch tension, twitch force and
fibre contractions are also used
activated, however, each cross-bridge repeats its swiveling motion many times, resulting in large
displacements of the filaments. Thu
Table 1: Table showing the differences between the three types of muscles
Source: (Widmaier
.1 Muscle contraction and relaxation
The term contraction, as used in muscle physiology, does not necessarily mean “shortenin
simply refers to activation of the force-generating sites within muscle fibres-the cross
2004). Following contraction, the mechanism that initiate force generation are
turned off, and tension declines, allowing relaxation of the muscle fiber (Widmaier
generates tension through the action of actin and myosin cross
While under tension, the muscle may lengthen, shorten or remain the same. Although
tening, when referring to the muscular system, it means muscle fibres
generating tension with the help of motor neurons (the terms twitch tension, twitch force and
fibre contractions are also used) (Gordon et al.,1966). As long as a muscle fibre
bridge repeats its swiveling motion many times, resulting in large
displacements of the filaments. Thus, the ability of a muscle fibre to generate force and
31
Source: (Widmaier et al., 2004)
The term contraction, as used in muscle physiology, does not necessarily mean “shortening”. It
the cross-bridges
2004). Following contraction, the mechanism that initiate force generation are
of the muscle fiber (Widmaier et al., 2004).
generates tension through the action of actin and myosin cross-bridge cycling.
While under tension, the muscle may lengthen, shorten or remain the same. Although, the term
tening, when referring to the muscular system, it means muscle fibres
generating tension with the help of motor neurons (the terms twitch tension, twitch force and
). As long as a muscle fibre remains
bridge repeats its swiveling motion many times, resulting in large
to generate force and
32
movement depends on the interaction of the contractile proteins; actin and myosin (Widmaier et
al., 2004). The A-bands within each muscle fibre are composed of thick filaments and the I-
bands contain thin filaments. Movements of cross bridges that extend from the thick to the thin
filaments causes sliding of the filaments, and thus muscle tension and shortening. The activity of
the cross bridges is regulated by the availability of Ca2+
, which is increased by electrical
stimulation of the muscle fiber. Electrical stimulation produces contraction of the muscle through
the binding of Ca2+
to regulatory proteins within the thin filaments (Matsuoka et al., 1993).
1.4.2 Smooth muscles
Smooth muscles are involuntary, non-striated muscles, they are found within the walls of blood
vessels (termed vascular smooth muscles), small arteries, arterioles and veins. It is also found in
lymphatic vessels, the urinary bladder, uterus (termed uterine smooth muscle), male and female
reproductive tracts, gastrointestinal tract, arrector pili of skin, the ciliary muscle and iris of the
eyes (Aguilar et al.,2010). Smooth muscle is responsible for the contractility of hollow organs,
such as blood vessels, the gastrointestinal tract, the bladder, or the uterus. Its structure differs
greatly from that of skeletal muscle, although it can develop isometric force per cross-sectional
area that is equal to that of skeletal muscle. However, the speed of smooth muscle contraction is
only a small fraction of that of skeletal muscle (Dillon, 2004). Smooth muscle, like skeletal
muscle, uses cross-bridge movements between actin and myosin filaments to generate force, and
calcium ions to control cross-bridge activity. However, the organization of the contractile
filaments and the process of excitation-contraction are quite different in these two types of
muscle (Widmaieret al., 2004).
Smooth (visceral) muscles are arranged in circular layers in the walls of blood vessels and
bronchioles (small air passages in the lungs). Both circular and longitudinal smooth muscle
layers occur in the tubular digestive tract, the ureters (which transport urine), the ductus
deferentia (which transport sperm cells) and the uterine tubes (which transport ova). The
alternate contraction of circular and longitudinal smooth muscle layers in the intestine produces
peristaltic waves, which propel the contents of these tubes in one direction (search for the
reference). The properties of smooth muscle vary considerably in different organs and the link
between membrane events and contraction is less direct and less well understood than in other
kinds of muscle (Rang et al., 2007).
33
Smooth muscle is divided into two sub-groups; the single-unit (unitary) and multiunit smooth
muscle. Within single-unit smooth muscle tissues, the autonomic nervous system innervates a
single cell within a sheet or bundle and the action potential is propagated by gap junctions to
neighboring cells such that the whole bundle or sheet contracts as a syncytium (i.e., a
multinucleate mass of cytoplasm that is not separated into cells) (Matsuoka et al., 1993). Single-
unit smooth muscle is the most common type. The fibers of single-unit smooth muscle are
electrically coupled by gap junctions so that they become excited and contract as single unit.
Single-unit smooth muscle is sometimes called visceral smooth muscle because it is typical of
visceral organs, including the walls of the gastrointestinal tract, the reproductive and urinary
tracts and the smooth muscle of small blood vessels (Ramos-Franco, 2012). Multiunit smooth
muscle tissues innervate individual cells; as such, they allow for fine control and gradual
responses, much like motor unit recruitment in skeletal muscle (Matsuoka et al., 1993). Multi-
unit smooth muscles, have few, if any gap junctions. The cells must thus be stimulated
individually by nerve fibres. Multiunit smooth muscle is found in the walls of larger blood
vessels, the iris of the eyes, the airways of lungs and in the skin surrounding hair follicles. The
distinction between single-unit and multi-unit smooth muscle is an over-simplification, and
becomes difficult to separate in some tissues (Ramos-Franco, 2012). While skeletal muscle fibers
are multinucleate cells that are unable to divide once they have differentiated, smooth muscle
fibers have a single nucleus and have the capacity to divide throughout the life of an individual.
Smooth muscle cells can be stimulated to divide by a variety of paracrine agents, often in
response to tissue injury (Widmaieret al., 2010).
1.4.2.1 Smooth muscle structure and organization
Smooth muscle cells are spindle-shaped and are much smaller than skeletal muscle cells. They
are about 2-10 microns in diameter and about 50 to 400 microns long. Smooth muscle cells do
not have a transverse tubular system, they do have sarcoplasmic reticulum, but it is much more
poorly developed than in skeletal or cardiac muscle. The muscle cells do not extend the entire
length of the whole muscle (unlike skeletal muscle, but like cardiac muscle), typical smooth
muscle cells are arranged in sheets (Ramos-Franco, 2012).
The great diversity of the factors that can influence the contractile activity of smooth muscles
from various organs, has made it difficult to classify smooth muscle fibers. Two types of
34
filaments are present in the cytoplasm of smooth muscle fibers; thick-myosin containing
filaments and thin-actin containing filaments. The thin filaments are anchored either to the
plasma membrane or to cytoplasmic structures known as dense bodies, which are functionally
similar to the Z lines in skeletal muscle fibers (Widmaieret al., 2004).
1.4.2.2 Smooth muscle contraction
Smooth muscle cells (SMCs) are characterized by their phenotypic plasticity and diversity. The
activation mechanism that controls contraction, display a similar diversity, in that each smooth
muscle cell type has a signaling system that is uniquely adapted to control its particular function
(Berridge, 2008).
Fig. 9: The dense bodies and intermediate filaments which cause the muscle fibers to contract
(Source; Aguilar et al., 2010).
A substantial portion of the volume of the cytoplasm of smooth muscle cells are taken up by the
molecules- myosin and actin, which together have the capability to contract, and through a chain
of tensile structures, make the entire smooth muscle tissue contract with them (Matsuoka et
al.,1993).
Fig. 10: The Actin-myosin filaments (Source; Matsuoka
Changes in cytosolic calcium concentration control the contractile activity in smooth muscle
fibres, as in striated muscle. However, there are significant differences be
muscle in the way in which calcium activates cross
which stimulation leads to alteration in c
Intracellular Ca2+
plays a critical role in the contrac
observations using Ca2+
indicators revealed that the degree of contraction is not always
proportional to the Ca2+
concentration (Bradley and Morgan,
concentration of K+ evokes a membran
concentration. The force of contraction and the phosphorylation of MLC induced by agonist
stimulation are higher than those induced by a high concentration of K
Ca2+
. This phenomenon, in which a higher force is developed at an equal concentration of
intracellular Ca2+
is called Ca2+
-sensitization (Somlyo and Somlyo,
myosin filaments (Source; Matsuoka et al., 1993)
Changes in cytosolic calcium concentration control the contractile activity in smooth muscle
fibres, as in striated muscle. However, there are significant differences between the two types of
muscle in the way in which calcium activates cross-bridge cycling and in the mechanisms by
which stimulation leads to alteration in calcium concentration (Widmaier
plays a critical role in the contraction of smooth muscle. However, early
indicators revealed that the degree of contraction is not always
concentration (Bradley and Morgan, 1987). In smooth muscle, a high
evokes a membrane depolarization-dependent increment in the Ca
concentration. The force of contraction and the phosphorylation of MLC induced by agonist
stimulation are higher than those induced by a high concentration of K+ at an equal intracellular
on, in which a higher force is developed at an equal concentration of
sensitization (Somlyo and Somlyo, 1994).
35
Changes in cytosolic calcium concentration control the contractile activity in smooth muscle
tween the two types of
bridge cycling and in the mechanisms by
alcium concentration (Widmaieret al., 2004).
tion of smooth muscle. However, early
indicators revealed that the degree of contraction is not always
1987). In smooth muscle, a high
dependent increment in the Ca2+
concentration. The force of contraction and the phosphorylation of MLC induced by agonist
at an equal intracellular
on, in which a higher force is developed at an equal concentration of
36
Fig. 11: Smooth muscle contraction (Source; Webb, 2003).
1.4.2.3 Smooth muscle relaxation
Smooth muscle relaxation occurs either as a result of removal of the contractile stimulus or by
the direct action of a substance that stimulates inhibition of the contractile mechanism (e.g., atrial
natriuretic factor is a vasodilator). Regardless, the process of relaxation requires a decreased
intracellular Ca2+
concentration and increased MLC phosphatase activity (Webb, 2003). The
phosphorylation of the light chains by the myosin light-chain kinase is countered by a myosin
light-chain phosphatase, which dephosphorylates the MLC20 myosin light chains and thereby
inhibits contraction (Aguilar et al., 2010). The enzyme, myosin phosphatase is regulated by
cyclic nucleotides-cAMP and cGMP (Rang et al., 2007). To relax a contracted smooth muscle,
myosin must be dephosphorylated because dephosphorylated myosin is unable to bind to actin.
When cytosolic calcium rises, the rate of myosin phosphorylation by the activated kinase
exceeds the rate of dephosphorylation by the phosphatase and the amount of phosphorylated
myosin in the cell increases, producing a rise in tension, when the cytosolic calcium
37
concentration decreases, the rate of dephosphorylation exceeds the rate of phosphorylation and
the amount of phosphorylated myosin decreases, producing relaxation (Widmaieret al., 2004).
In general, the relaxation of smooth muscle is by cell-signaling pathways that increase the
myosin phosphatase activity, decrease the intracellular calcium levels, hyperpolarize the smooth
muscle, and/or regulate actin and myosin muscle can be mediated by the endothelium-derived
relaxing factor-nitric oxide, endothelial derived hyperpolarizing factor (either an endogenous
cannabinoid, cytochrome P450 metabolite, or hydrogen peroxide), or prostacyclin (PGI2)
(Aguilar et al., 2010). Removal of calcium from the cytosol to bring about relaxation is achieved
by the active transport of calcium back into the sarcoplasmic reticulum as well as out of the cell
across the plasma membrane. The rate of calcium removal in smooth muscle is much slower than
in skeletal muscle, with the result that a single twitch lasts several seconds in smooth muscle but
lasts only a fraction of a second in skeletal muscle (Widmaieret al., 2004).
Fig. 12: Smooth muscle relaxation (Source; Webb, 2003)
38
1.5 Nervous system
The nervous system is composed of the brain, spinal cord and nerves. The nervous system has
two divisions: the central nervous system (CNS) and the peripheral nervous system (PNS). The
brain and spinal cord make up the CNS, the nerves make up the peripheral nervous system. The
brain is divided into specific regions; each region is responsible for the performance of specific
functions within the body (Moini, 2009).
The nervous system is divided into two parts: the central nervous system (CNS) and the
peripheral nervous system (PNS). The CNS consists of the brain and spinal cord. The PNS
consists of all afferent(sensory) neurons, which carry nerve impulses into the CNS from sensory
end organs in peripheral tissues, and all efferent(motor) neurons, which carry nerve impulses
from the CNS to effector cells in peripheral tissues. The peripheral efferent system is further
divided into the somatic nervoussystemand the autonomic nervous system. The effector cells
innervated by the somatic nervous system are skeletal muscle cells. The autonomic nervous
system innervates three types of effector cells: (1) smooth muscle, (2) cardiac muscle, and (3)
exocrine glands (Craig and Stitzel, 2005).
39
Fig. 13: Divisions of the nervous system (Source; Moini, 2009).
1.5.1 Autonomic nervous system
The autonomic nervous system (ANS or visceral nervous system or involuntary nervous system)
is the part of the peripheral nervous system that acts as a control system, functioning largely
below the level of consciousness, and controls visceral functions (Schmitzet al., 1981). The ANS
affects heart rate, digestion, respiratory rate, salivation, perspiration, pupillary dilation,
micturition (urination), and sexual arousal. Most autonomous functions are involuntary but they
scan often work in conjunction with the somatic nervous system which gives voluntary control.
Everyday examples include breathing, swallowing, and sexual arousal, and in some cases
functions such as heart rate (Elliott, 1997). In general, ANS functions can be divided into
40
sensory (afferent) and motor (efferent) subsystems. Within both, there are inhibitory and
excitatorysynapses between neurons (Duttaroy et al., 2004). Relatively recently, a third
subsystem of neurons that have been named 'non-adrenergic and non-cholinergic' neurons
(because they use nitric oxide as a neurotransmitter) have been described and found to be
integral in autonomic function, in particular in the gut and the lungs (Kullmans et al.,2009).
The peripheral nervous system regulates both voluntary and involuntary functions in the human
body. The peripheral nervous system has two divisions, the somatic nervous system (SNS) and
the autonomic nervous system (ANS) (Moini, 2009). The SNS regulates voluntary or conscious
functions such as motor movement. The autonomic nervous system regulates all involuntary
functions such as secretion of hormones, contraction of the heart muscle, blood vessels and
bronchioles and the ability to move substances through the digestive tract. The ANS can further
be divided into the sympathetic and parasympathetic nervous systems (Craig and Stitzel, 2005).
Fig. 14:The autonomic nervous system (Moini, 2009).
41
Anatomical differences between the peripheral somatic and autonomic nervous systems have led
to their classification as separate divisions of the nervous system. The axon of a somatic motor
neuron leaves the CNS and travels without interruption to the innervated effector cell. In
contrast, two neurons are required to connect the CNS and a visceral effector cell of the
autonomic nervous system. The first neuron in this sequence is called the preganglionic neuron.
The second neuron, whose cell body is within the ganglion, travels to the visceral effector cell; it
is called the postganglionic neuron (Craig and Stitzel, 2005).
Fig. 15: Anatomical characteristics and neurotransmitters of the somatic (Som), sympathetic
(Sym) and parasympathetic (Para) divisions of the PNS. Ach, acetylcholine; E, epinephrine; NE,
norepinephrine (Source; Craig and Stitzel, 2005).
1.6 Uterus
The uterus is the central organ of reproduction. It is a thick, pear-shaped, muscular organ
approximately 7cm long and 4-5 cm wide at its widest point. It is divided functionally and
morphologically into three sections, namely the cervix, the isthmus and the main body of the
uterus (corpus uteri) (Symonds, 1998). The myometrium is the middle layer that makes up the
major proportion of the uterine body.
42
Myometrial smooth muscle is arranged in undefined layers and contractile forces can occur in
any direction enabling the uterus to assume virtually any shape. Through growth and stretch
during pregnancy, the myometrium provides the protective environment for the developing
foetus (Alberts et al.,1989). Then with the onset of labour, it contracts rhythmically to expel the
foetus and placenta. Smooth muscle fibres are composed of spindle-shaped cells, each with one
centrally located nucleus. Typically, they have a diameter of 2-10 µm and a length of several
hundred µm (Alberts et al., 1989). Smooth muscle cells are embedded in an extracellular matrix
composed principally of collagen fibres, which facilitate the transmission of contractile forces
generated by individual cells. They are organized into sheets of closely opposed fibres, oriented
at right angles to each other. These sheets form two distinct layers, the “longitudinal layer”,
which consists of a network of bundles of smooth muscle cells generally oriented in the long axis
of the organ, and in the “circular layer”, in which the fibres are arranged concentrically around
the longitudinal axis of the organ (Csapo, 1962). Contraction of the longitudinal layer causes the
organ to dilate and shorten, whereas contraction of the circular layer causes the organ to
elongate; thus alternating contraction and relaxation of these layers enables the uterus to expel its
contents at birth (Alberts et al.,1989).
1.6.1 Functions of the uterus
Functionally, the endometrium of the uterus is divided into two main zones: 1) the
stratumfunctionalis, which is built up and sloughed off mainly during menstruations, and 2) the
stratumbasalis, the epithelial and glandular elements that remain to supply replicative cells to
regeneratethe functionalis of the next cycle (Gunin et al., 2001). It is specialized for containing,
protecting, and nourishing ofthe nidating embryo from implantation to parturition. Physiological
changes in the uteruscorrelate with functional activity of the ovary (Pakarinen et al., 1998).
1.7 Jejunum
The jejunum is the middle section of the small intestine in most highervertebrates, including
mammals, reptiles, and birds. In fish, the divisions of the small intestine are not as clear and the
terms middle intestine or mid-gut may be used instead of jejunum. The jejunum lies between the
duodenum and the ileum (Guilaume, 2001). The change from the duodenum to the jejunum is
usually defined as the Duodenojejunal flexure and is attached, and thus "hung up", to the
43
ventricle (see stomach) by the ligament of Treitz. In adult humans, the small intestine is usually
between 5.5 and 6m long, 2.5m of which is the jejunum. The pH in the jejunum is usually
between 7 and 9 (neutral or slightly alkaline). If the jejunum is impacted by blunt force the
emesis reflex (vomiting) will be initiated (Van et al.,2011).
The jejunum and the ileum are suspended by mesentery which gives the bowel great mobility
within the abdomen. It also contains circular and longitudinal smooth muscle which helps to
move food along by a process known as peristalsis.
Fig. 16: The structure of the digestive system showing the jejunum (Guilaume et al.,2001)
The lumenal surface of the jejunum is covered in finger like projections of mucosa, called villi,
which increase the surface area of tissue available to absorb nutrients from ingested foodstuffs.
(Van et al.,2011). The transport of nutrients across epithelial cells through the jejunum and ileum
includes the passive transport of sugar fructose and the active transport of amino acids, small
peptides, vitamins, and most glucose. The villi in the jejunum are much longer than in the
duodenum or ileum. The jejunum contains very few Brunner's glands (found in the duodenum)
or Peyer's patches (found in the ileum). However, there are a few jejunal lymph nodes suspended
in its mesentery. The jejunum has many large circular folds in its submucosa called plicae
circulares which increase the surface area for nutrient absorption. The plicae circulares are the
best developed in the jejunum. These structures help protect the jejunum in the event that it is
punched or struck with blunt force, which can elicit a powerful emetic effect (Guilaume et al.,
2001).
44
1.8 Aim and Objectives of the research
This research work is aimed at investigating whether the aqueous methanol extract of Ricinus
communis seeds possesses the biphasic pharmacological activity on smooth muscles (non-
striated muscles).
The specific objectives of this research include;
• To determine both quantitatively and qualitatively the phytochemical composition of the
methanol fermented seed extracts of Castor bean plant (Ricinus communis).
• To determine both quantitatively and qualitatively the phytochemical compositions of the
methanol seed extracts of Castor bean plant (Ricinus communis)
• To determine the median lethal dose (acute toxicity) of the fermented seeds of Castor
bean plant.
• To determine the median lethal dose (acute toxicity) of the unfermented seeds of the
Castor bean plant.
• To determine the effect of the fermented and unfermented methanol extracts of Castor
bean plant (Ricinus communis) on the Isolated rabbit jejunum.
• To determine the effect of the fermented and unfermented methanol extracts of Castor
bean plant (Ricinus communis) on the pregnant rat uterus.
45
CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials
2.1.1 Plant material
The seeds of Ricinus communis were used for this study. The seeds were purchased from Eke
Amobi market, Otolo Nnewi in Anambra state and were identified by Mr. Alfred Ozioko of
Bioresource Development and Conservation Programme (BDCP) Research Centre, Nsukka. The
fermented seeds were purchased from Ogige Market, Nsukka LGA, Enugu State, Nigeria.
2.1.2 Animals
Two adult male rabbits and two female pregnant rats were used for the smooth muscle
experiment and 9 albino mice were used for the median lethal dose (LD50) study. The rabbit and
the female pregnant rat were obtained from the Animal House, Department of Pharmacology and
therapeutics, UNTH Enugu. The mice used for the toxicity study were obtained from the
Department of Vertenary medicine, UNN. The mice were fed with starter (Grand cereals and oil
mills Ltd, Jos, Nigeria) and water. The animals were handled in accordance with the rules
governing the use of laboratory animals as accepted internationally.
2.1.3 Drugs
The drugs used were purchased from pharmacy shops in front of UniversityTeaching Hospital,
Enugu and they are of clinical use. They include:Acetylcholine, Adrenaline, Prazosin,
Propranolol, Aminophylline, Oxytocin, Indomethacin and Ergotamine (Sigma Products, USA).
2.1.4 Equipment
The equipment used were obtained from the Department of Pharmacology and therapeutics,
University Teaching Hospital, Enugu and the Department of Biochemistry, University of
Nigeria, Nsukka and other scientific shops in Nsukka. They include the listed equipment and the
routine laboratory wares which were not listed;
46
Kymograph HAL, England
Weighing balance Metler HAS
Refrigerator Thermocool
Organ bathSRI, England
AeratorCAP, England
Writing leverHAL, England
Spectrophotometer Spectronic 20D
Aspirator bottlesPyrex, England
2.1.5 Chemicals and Reagents
The chemicals and reagents used were of analytical grade. The chemicals used in this study
include:
2,3- dinitrobenzoic acidBDH Analar, England
Absolute ethanolBDH, England
AcetoneSigma, London
Aluminium chlorideBDH Analar, England
Aqueous ethanolSigma, London
Ammonium chloride JHD, China
ChloroformSigma, London
Concentrated Hydrochloric acid BDH Analar, England
47
Ethyl acetate BDH, England
Fehling’s solution A&BBDH, England
Ferric chlorideMerck
Lead acetate BDH, England
MethanolSigma, London
Million’s reagentBDH, England
Picric acid Lab Tech Chemicals, London
Physiological solutions (Tyrode and De-jalons solutions)UNTH, Enugu
Sodium hydroxideMay & Baker, England
Concentrated sulphuric acidBDH Analar, England
α-napthtolSigma, London
2.2 Methods
2.2.1 Preparation of plant material
The seeds of Ricinus communis seeds were deshelled and manually separated from the shells.
2.2.2 Extraction of plant material
The unfermented seeds (949g) of Ricinus communis were deshelled and macerated in a mixture
of methanol and chloroform (1:2) for 24 hours. The extract was filtered using Whatmaan No. 1
filter paper and partitioned with 0.2 volume of distilled water to obtain two layers and separated
using a separating funnel. The upper aqueous methanol layer was concentrated using rotary
evaporator at a temperature of 40 to 60oC. The dry residue was used for the determination of
biological activity. The unfermented concentrated extract formed crystals or precipitates after
some days of concentration and their biological activities were also determined. The fermented
Ricinus communis seeds (329g) were treated similarly. The dry residue was used for the
determination of biological activity.
48
2.2.3 Experimental Design
Two (2) adult male albino rabbits and two (2) pregnant female albino rats were used for this
study. The rabbits were kept in a separate cage while the rats were also kept in a separate cage.
They were acclimatized for a period of four days, they had free access to animal feeds and water
ad libitum throughout the period of acclimatization. The rabbits were starved for 24 hours prior
to the smooth muscle experiment, while the female albino rats were not starved. After 24 hours,
they were sacrificed by cervical dislocation and the tissues of interest (jejunum and uterus) were
isolated and inserted in an organ bath. The organs were grouped according to the receptors as
follows:
Group 1: Determining the effects of the extracts (using rabbit jejunum) on;
(a) Muscarinic acetylcholine receptors
(b) Adrenoceptors (Akah et al., 2007).
Group 2: Determining the effects of the extracts (using pregnant rat uterus) on;
(a) Oxytocin receptors
(b) Prostaglandin synthesis
(c) Muscarinic receptors
(d) Adrenoceptors (Akah et al., 2007).
2.2.4 Preparation of reagents for phytochemical analysis
5% (w/v) Ferric chloride solution
A quantity, 5.0g of ferric chloride was dissolved in 100ml of distilled water.
Ammonium solution
Concentrated stock ammonium solution (187.5 ml) was diluted in 31.25 ml of distilled water and
then made up to 500 ml with distilled water.
45% (v/v) ethanol
A quantity, 45 ml of absolute ethanol was mixed with 55ml of distilled water.
49
Aluminium chloride solution
Aluminium chloride (0.5g) was dissolved in 100ml of distilled water.
Dilute sulphuric acid
A quantity, 10.9 ml of concentrated sulphuric acid was mixed with 5.0 ml of distilled water and
made up to 100ml.
Mayer’s reagent
A quantity, 13.5g of mercuric chloride was dissolved in 50 ml of distilled water. Also, 5.0g of
potassium iodide was dissolved in 20 ml of distilled water. The two solutions were mixed and
the volume made up to 100 ml with distilled water.
Dragendorff’s reagent
A quantity, 0.85g of bismuth carbonate was dissolved in 100 ml of glacial acetic acid and 40ml
of distilled water to give solution A. Another solution called solution B was prepared by
dissolving 8.0g of potassium iodide in 20 ml of distilled water. Both solutions were mixed to
give a stock solution.
Molisch reagent
A quantity, 1.0g of α-naphtol was dissolved in 100 ml of absolute ethanol.
1% (w/v) Picric acid
Picric acid (1.0g) was dissolved in 100 ml of distilled water.
2.2.5 Drug dilutions
Adrenaline
A concentration of 10µg/ml was prepared by adding 0.1ml of stock adrenaline into 9.9 ml of
distilled water.
50
Acetylcholine
A known quantity, 1mg/ml or 1000µg/ml of acetylcholine was prepared by weighing and
dissolving 10mg of acetylcholine in 10ml of distilled water. A concentration of 10µg/ml was
prepared by adding 0.1ml of the prepared 1mg/ml acetylcholine into 9.9ml of distilled water.
Aminophylline (25 mg/ml or 2500µg/ml)
Aminophylline (25µg/ml) was prepared by adding 0.1ml of 2500µg/ml into 9.9ml of distilled
water.
Ergotamine (0.5mg/ml-stock solution)
A measured quantity of 0.5mg, equivalent to 500µg of ergotamine was dissolved in 25ml of
distilled water to realize 500µg/ml or 20µg/ml of ergotamine.
Indomethacin (25mg capsule)
One capsule (25mg) was dissolved in 25ml of distilled water to realize 25mg/25ml or 1mg/ml or
1000µg/ml.
Prazosin
A known weight of 1mg of prazosin tablet was dissolved in 10ml of distilled water to realize
1000µg/10ml of prazosin. A concentration of 10µg/ml was prepared by the addition of 1mg/ml
of the initially prepared prazosin into 9.9ml of distilled water.
Propranolol (40mg tablet)
Propranolol tablet (40g) was dissolved in 40ml of distilled water to realize 40mg/ml or 1mg/ml
or 1000µg/ml of propranolol. A concentration of 10µg/ml was prepared by the addition of
1mg/ml of the initially prepared solution into 9.9ml of distilled water.
51
2.2.5.1 Composition of physiological salt solutions (PSS)
Tyrode solution De-Jalon’s solution
NaCl 60gm 90gm
KCl 10% solution 20ml 42ml
MgSO4. 7H2O 26ml _
NaH2PO4. 2H2O 5% Solution 13ml _
KH2PO4 10% Solution 10gm 5gm
Glucose 10gm 5gm
NaHCO3 10gm 5gm
CaCl2 1.8ml 2.7ml
Aerating Gas O2 or Air O2 + 5% CO2
Type of Preparation Intestine Uterine Muscle
2.2.6 Qualitative Phytochemical Analysis of Ricinus communis Seeds
The phytochemical analysis of the plant was carried out on both fermented and unfermented
methanol extracts according to the method of Harborne (1998) and Trease and Evans (1983) to
identify the active constituents of Ricinus communis.
2.2.6.1 Test for alkaloids
A quantity, 0.2g of the sample was boiled with 5ml of 1% aqueous HCl on a water bath for
45mins. The mixture was filtered and 1ml portion of the filtrate was distributed evenly in two
test tubes, with two drops of the following reagents.
(i) Drangendorff’s reagent: An orange-red precipitate indicates the presence of alkaloids.
(ii) Meyer’s reagent: A creamy-white precipitate indicates the presence of alkaloids
(Soforwora, 1993).
2.2.6.2 Test for glycosides
A quantity, 0.5g of the sample was mixed with 30ml of distilled water and heated in a water bath
for 5 minutes. The mixture was filtered and the filtrate used for the following test;
52
(i) A quantity, 0.2ml of Fehling’s solution A&B was added to 5ml of the filtrate until it
turned alkaline (tested with litmus paper) and heated on a water bath for 2 minutes. A
brick-red precipitate indicates the presence of glycosides.
2.2.6.3 Test for steroids
0.5g of the sample was mixed with 5ml of 1% lead acetate solution and 10ml of aqueous ethanol.
The mixture was placed on a boiling water bath for 2 minutes, it was allowed to cool and filtered.
The filtrate was extracted twice with 15ml of chloroform. 5ml of the chloroform layer was
evaporated . After the evaporation, 2, 3-dinitrobenzoic acid and 1ml of 1N NaOH were added. A
red colouration indicates the presence of steroids.
2.2.6.4 Test for flavonoids
A quantity, 0.5g of the sample was dissolved in ethanol, warmed and then filtered. 3 pieces of
magnesium chips were added to the filtrate, followed by few drops of concentrated HCl. A pink,
orange or red to purple colouration indicates the presence of flavonoids.
2.2.6.5 Test for saponin
One gramme (1g) of the sample was boiled with 5ml of distilled water for 5 minutes. The
mixture was filtered while still hot and the filtrate used for the following tests;
Frothing test: A quantity, 1ml of the filtrate was diluted with 3ml of distilled water. The mixture
was shaken vigorously for 5 minutes, frothing which persisted on warming was taken as
evidence for the presence of saponin.
2.2.6.6 Test for tannins
A known quantity, 0.5g of the sample was boiled with 5ml of 45% ethanol for 5 minutes. The
mixture was cooled and then filtered and the filtrate was treated with the following solutions
(i) Lead sub acetate solution: To 1ml of the filtrate was added 3 drops of lead sub acetate
solution. A gelatinous precipitate indicates the presence of tannins.
(ii) Bromine water: To 1ml of the filtrate was added 0.5ml of bromine water and then
observed for a pale brown precipitate.
53
(iii) Ferric chloride solution: A quantity, 2ml of the filtrate was diluted with distilled water
and then 2 drops of ferric chloride solution was added. A transient greenish to black
colour or blue black or blue-green precipitate indicates the presence of tannins.
2.2.6.7 Test for reducing sugars
A quantity, 0.5g of the sample was dissolved in 5ml of distilled water and filtered, the filtrate
was heated for 10 minutes with 5ml of equal volumes of Fehling’s solutions A&B and shaken
vigorously. A brick-red precipitate indicates the presence of reducing sugars.
2.2.6.8 Test for carbohydrates
A known weight, 0.5g of the sample was shaken vigorously with distilled water and filtered. To
the aqueous filtrate, few drops of Molisch reagent were added and vigorously shaken. Then, 1ml
of concentrated sulphuric acid was carefully added down the side of the test tube to form a layer
below the aqueous solution. A brown ring at the interface indicates the presence of
carbohydrates.
2.2.7 Quantitative phytochemical analysis of Ricinus communis seeds
2.2.7.1 Alkaloid determination
A measured weight, 1g of the sample was macerated in 20ml of ethanol and 20% sulphuric acid
(1:1). After the maceration, the solution was filtered and 1ml of the filtrate was collected using a
pipette, 5ml of 60% H2SO4 was added into the 1ml of the filtrate. After 5 minutes, 5ml of 0.5%
formaldehyde in 60% H2SO4 was added into the previous solution and mixed. The solution was
allowed to stand for 3 hours and the absorbance measured at 565nm.
2.2.7.2 Flavonoid determination
A quantity, 1g of the sample was macerated in 20ml of ethyl acetate for 5 minutes. After the
maceration, the solution was filtered, 5ml of the filtrate was collected using a pipette and added
to 5ml of dilute ammonia. The solution was shaken for 5 minutes, after which the upper layer
was collected and the absorbance measured at 490nm.
54
2.2.7.3 Glycoside determination
One gramme (1g) of the sample was macerated in 20ml of distilled water for 5 minutes, followed
by the addition of 2.5 ml of 15% lead acetate, the solution was filtered and 2.5 ml of chloroform
added to the solution and was shaken vigorously. The lower layer of the solution was collected
and evaporated to dryness. The residue was dissolved with 3ml of glacial acetic acid and 0.1 ml
of ferric chloride and 0.25 ml of concentrated H2SO4 were added and shaken vigorously. The
solution was put in the dark for 2hours and the absorbance measured at 530nm.
2.2.7.4 Hydrogen cyanide determination
A sample (1g),was macerated in 50 ml of distilled water and filtered. 1ml of the filtrate was
collected using a pipette and added into 4mls of alkaline picrate solution. The solution was
boiled for 5 minutes and cooled at room temperature. The absorbance was measured using a
spectrophotometer at the absorbance of 490nm.
2.2.7.5 Phenol determination
A quantity, 1g of the sample was macerated in 20ml of 80% ethanol and filtered. 5ml of the
filtrate was collected using a pipette and added to 0.5ml of Folinciocalteu’s reagent, the solution
was allowed to stand for 3 mins and 2ml of 20% Na2CO3 was added. The absorbance was
measured using a spectrophotometer at 650nm.
2.2.7.6 Saponin determination
A known weight, 1g of the sample was macerated in 10ml of petroleum ether and decanted into a
beaker and washed twice using 10ml of petroleum ether. The filtrate of the solution was
combined together and evaporated to dryness, the residue was dissolved in 6ml of ethanol and
2ml was collected using a pipette into a test tube while 2ml of chlomogen solution was added
and allowed to stand for 30 minutes. The absorbance was measured using a spectrophotometer at
550nm.
55
2.2.7.7 Soluble carbohydrates determination
The sample (1g) was macerated in 50ml of distilled water and filtered. 1ml of the filtrate was
collected using a pipette and added into 2ml of saturated picric acid. The absorbance was
measured using a spectrophotometer at 530nm.
2.2.7.8 Steroid determination
One gramme(1g) of the sample was macerated in 20ml of ethanol and filtered. 2ml of the filtrate
was collected using a pipette and added into 2ml of colour reagent and allowed to stand for 30
minutes. The absorbance was measured using a spectrophotometer at 550nm.
2.2.8 Preparation of the methanol extract of the unfermented Ricinus communis seeds for the
acute toxicity test
A quantity, 949 g of the cracked seeds of Ricinus communis was macerated in 1265 ml and 633
ml of chloroform and methanol respectively for 24hrs. The solution was filtered with Whatman
No.1 filter paper and separated, the supernatant (the methanol extract) was concentrated to a
semi-solid state using rotary evaporator at a temperature range of 40 to 600C. The methanol
extract was used for the study.
2.2.8.1 Acute toxicity test of the methanol extract of unfermented Ricinus communis seeds
The method of Lorke (1983) was used for the acute toxicity test of the methanol unfermented
extract of R.communis. Eighteen (18) albino mice were utilized in this study. The test involved
two phases. In phase one, the animals were grouped into three (3) groups of three mice each.
They were administered 10, 100 and 1000 mg/kg body weight of the extract respectively and in
the second phase, the animals were grouped into three (3) groups of three mice each and 1600,
2900 and 5000 mg/kg body weight of the extract were administered to the animals. The
administration of the extract was done orally.
2.2.8.2 Preparation of the methanol extract of fermented Ricinus communis seeds
Fermented castor bean plant (392 g) was purchased from Ogige market in Nsukka, Enugu State.
They were macerated in chloroform and methanolfor 24 hours. The solution was filtered with
Whatman no.1 filter paper and separated, the supernatant (the methanol extract) was
56
concentrated to a solid state using rotary evaporator at a temperature of 400c
to 600C. The
methanol extract was used for biological activity determination.
2.2.8.3 Acute toxicity test of the methanol extract of fermentedRicinus communis seeds
The method of Lorke (1983) was used for the acute toxicity test of the methanol fermented
extract of R.communis. Eighteen (18) albino mice were utilized in this study. The test involved
two phases. In phase one, the animals were grouped into three (3) groups of three mice each.
They were administered 10, 100 and 1000 mg/kg body weight of the extract respectively and in
the second phase, the animals were grouped into three (3) groups of three mice each and 1600,
2900 and 5000 mg/kg body weight of the extract were administered to the animals. The
administration of the extract was done orally.
2.2.9 Smooth muscle experiment
2.2.9.1 Animal preparation
Three (3) adult male rabbits with measured weightsof 2.8kg and three (3) pregnant female albino
rats weighing between 150 and 220g were obtained from the Animal house, Department of
Pharmacology and Therapeutics, University Teaching Hospital, Enugu were used for this study.
The animals were maintained on standard animal feeds and water ad libitum. The rabbits and
pregnant rats used for this study were sacrificed by cervical dislocation and the tissues of interest
(jejunum and uterus) were isolated and inserted in an organ bath for smooth muscle experiments.
2.2.9.2 Determination of the effects of the extracts
(a) On the isolated rabbit jejunum
This experiment was carried out using the method of Akah et al., (2007). The frontal writing
lever was balanced and plasticine was loaded. The physiological solutions (PSS) were prepared
and filled in the aspirator bottles. The physiological solution used for this tissue (jejunum) was
tyrode solution. The aspirator bottles were connected to the organ bath for the supply of
physiological solutions to the organ bath, the tap of the aspirator bottle was opened and enough
PSS was introduced into the tissue chamber of the organ bath in order to fill it up to a mark on
57
the wall of the tissue chamber. The drugs and the extracts were diluted and filled in the reagent
bottles.
The animals (rabbit) was sacrificed by cervical dislocation and the tissue of interest (the
jejunum) was isolated. Using a pair of forceps, the intestinal tissue (jejunum) was cut to a length
of 2cm and transferred into the plastic plate provided into which some PSS has been poured with
an aerator pump placed into the plate for the supply of oxygen to the tissue (if bare hands are to
be used instead of forceps, then they must be well washed and rinsed, and kept moist with PSS
whenever the tissue is to be touched). In the plastic plate with adequate aeration, one end of the
tissue was tied with thread and attached firmly to the hook of the aerator (Note: The thread
needle was carefully passed through the wall of the tissue by piercing from the lumen outward,
after which a firm knot was made on the tissue, and the needle end of the thread was then cut off
before attaching the tied tissue to the aerator). Having threaded the needle once more, the other
end of the tissue was tied and knotted firmly as before. With the aid of the aerator and the free
length of the thread, the tied tissue was picked up and transferred into the tissue chamber of the
organ bath. Care was taken to affix the aerator to its place on the organ bath, and to ensure that
the tissue was submerged in the PSS earlier introduced into the chamber and was well aerated.
The kymograph or recorder was set up such that the pointer or writing pen made adequate
contact with the smoked drum or recording paper, thus permitting the recording of a contraction
(shortening) or relaxation (lengthening) of the tissue. The kymograph was put on for about 30
seconds so as to obtain a baseline reading and then, with the aid of a 1ml syringe and needle.
0.1ml of 10µg of an agonist drug, acetylcholine, was administered into the organ bath. This was
to verify the viability of the tied tissue, as would be indicated by a definite contraction recorded
on the smoked drum/recording paper. The response of the tied tissue showed that it was
viable,the acetylcholine was washed off twice by running off the bath fluid and replaced with
fresh PSS from the aspirator bottle.
A known quantity, 0.1ml of 10µg of acetylcholine was added into the organ bath and allowed to
stay for 30 seconds and contraction occurred, after 30 seconds, it was washed off. 0.1ml 0f
adrenaline, was also added at a concentration of 10µg and allowed to stay in the bath for 30
seconds and washed off, the adrenaline relaxed the tissue. 0.1ml of the extract 1, the unfermented
methanol extract of Ricinus communis seeds was added in the bath and relaxation effect was
58
observed, the extract was washed off and the same doses and concentrations of the extract 2 and
3 were added differently, they both relaxed the tissues. Different doses and concentrations of the
extracts were added into the organ bath and washed off, relaxation also occurred. Prazosin, an α-
blocker or antagonist was added into the organ bath at different concentrations of 10and 20µg/ml
at doses of 0.1, 0.2, 0.4 and 1.0ml with different doses of adrenaline, both adrenaline and
prazosin were added in the bath inorder to determine the blocking effect of prazosin on
adrenaline, prazosin completely the effects of adrenaline and the extracts. Propranolol, a β-
blocker or antagonist was added into the organ bath with prazosin, at different doses and
concentrations of 20 and 10µg/ml at doses of 0.1 and 0.2ml. With prazosin and propranolol in
the organ bath, the extracts were added at concentrations of 10µg/ml at a dose of 0.1ml,
propranolol gave a weak blocking effect unlike prazosin that blocked the extracts completely.
Indomethacin, a NSAID drug was also added into the organ bath at different doses and
concentrations of 20µg/ml at 1.0ml, with indomethacin in the organ bath, different doses of the
extracts and adrenaline were added differently in the bath. Indomethacin had no blocking effect
on the extracts and adrenaline even at increasing doses and concentrations, indomethacin was
washed off and added again into the bath, the extracts and adrenaline were also added, a blocking
effect was not observed. Aminophylline, an adenosine receptor blocker was also added at a
concentration of 10µg/ml and a dose of 0.1ml, no blocking effect was also observed.
(b) On pregnant isolated uterus
Smooth muscle experiment on isolated pregnant rat uterus was carried out using the method of
Akah et al.,(2007). The frontal writing lever was balanced and plasticine was loaded. The
physiological solutions (PSS) were prepared and filled in the aspirator bottles. The physiological
solution used for this tissue (uterus) was De-jalon’s solution. The aspirator bottles were
connected to the organ bath for the supply of physiological solutions to the organ bath, the tap of
the aspirator bottle was opened and enough PSS was introduced into the tissue chamber of the
organ bath in order to fill it up to a mark on the wall of the tissue chamber. The drugs and the
extracts were diluted and filled in the reagent bottles.
The animal (rabbit) was sacrificed by cervical dislocation and the tissue of interest (the uterus)
was isolated. Using a pair of forceps, the piece intestinal tissue (uterus) cut to a length of about
59
2cm was transferred into the plastic plate provided into which some PSS has been poured with an
aerator pump placed into the plate for the supply of oxygen to the tissue (if bare hands are to be
used instead of forceps, then they must be well washed and rinsed, and kept moist with PSS
whenever the tissue is to be touched). In the plastic plate with adequate aeration, one end of the
tissue was tied with thread and attached firmly to the hook of the aerator (Note: The thread
needle was carefully passed through the wall of the tissue by piercing from the uterus outward,
after which a firm knot was made on the tissue, and the needle end of the thread was then cut off
before attaching the tied tissue to the aerator). Having threaded the needle once more, the other
end of the tissue was tied and knotted firmly as before. With the aid of the aerator and the free
length of the thread, the tied tissue was picked up and transferred into the tissue chamber of the
organ bath. Care was taken to affix the aerator to its place on the organ bath, and to ensure that
the tissue is submerged in the PSS earlier introduced into the chamber and is well aerated. The
kymograph or recorder was set up such that the pointer or writing pen made adequate contact
with the smoked drum or recording paper, thus permitted the recording of a contraction
(shortening) or relaxation (lengthening) of the tissue. The kymograph was put on for about 30
seconds so as to obtain a baseline reading and normal rhythmic contraction was also observed.
With the aid of a 1ml syringe and needle, 0.1ml of 10iu/ml of oxytocin was added into the organ
bath for 30 seconds and washed off, oxytocin exhibited a normal uterine contraction. The extract
1, the unfermented extract was added into the bath at a concentration of 0.5mg/ml at a dose of
0.1ml, allowed to stay for 30 seconds and washed off, the extract had no observable effect on the
tissue. The fermented extract was added into the organ bath at a concentration of 0.5mg/ml at
0.1ml, allowed to stay for 30 seconds and washed off, the extract contracted the tissue. The third
extract was also added and there was no observable effect.
Non-steroidal anti-inflammatory drug, indomethacin, was also added into the bath inorder to
inhibit the synthesis of prostaglandins, indomethacin and acetylcholine, were added together in
the bath at a concentration of 20µg/ml and washed off after 30 seconds, indomethacin had no
blocking effect on acetylcholine even at different concentrations. Indomethacin was also added
in the bath with the extracts at different doses and concentrations , the contractile effect of the
fermented extract was also observed this shows that indomethacin had no blocking effect on the
uterus and jejunum. Ergotamine, an α- adrenoceptor blocker was added into the organ bath at a
concentration of 20µg/ml and a dose of 1.0ml, with the ergotamine in the bath, adrenaline was
60
added at a concentration of 10µg/ml at a dose of 0.1ml, ergotamine blocked the effect of
adrenaline, the ergotamine and adrenaline were washed off after 60 seconds. With ergotamine in
the bath at a reduced concentration of 10µg/ml at a dose of 0.1ml, the three extracts were added
differently at a concentration of of 10µg/ml and doses of 0.1ml, the ergotamine blocked the
effects of the three extracts, especially the fermented extract that initially exhibited a contractile
effect.
61
CHAPTER THREE
RESULTS
3.1 Percentage Yield of the methanol extracts of fermented and unfermented Ricinus
communis seeds.
Table 1 shows that the unfermented Ricinus communisseeds, 949 gand the fermented Ricinus
communisseeds, 392 g, gave a percentage yield of 2.93 and 7.83 respectively. The high
percentage yield of the fermented extract after extraction might be as a result of high surface area
of the fermented seeds which allowed the passage of solvents into the pulp for proper extraction.
62
Table 2: Percentage yield of the methanol extracts of fermented and unfermentedR.
communisseeds
Unfermented extract (g) Fermented extract (g) Unfermented (%) Fermented (%)
949 392 2.93 7.83
63
3.2 Qualitative phytochemical screening of the methanol extracts of fermented and
unfermented Ricinus communis seeds.
Table 2 shows that both fermented and unfermented seeds ofRicinus communiscontain alkaloids,
flavonoids, steroids, hydrogen cyanide, soluble carbohydrates, phenol and tannin. Reducing
sugars, glycosides and saponins were highly, moderately and slightly detected respectively in the
unfermented seeds but they were not detected in the unfermented seeds of Ricinus communis.
Resins and terpenoids were not detected in both extracts.
64
Table 3: Preliminary phytochemical screening of methanol extracts of fermented and
unfermented Ricinus communis seeds
Phytochemicals Fermented extract Unfermented extract
Flavonoids ++ ++
Glycosides - ++
Hydrogen cyanides + +
Resin - -
Saponin - +
Steroid + ++
Soluble carbohydrates ++ ++
Tannin + + +
Reducing sugar - +++
Terpenoids - -
Phenol ++ +
Key: + slightly present
++ moderately present
+++ highly present
- Not detected
65
3.2.1 Quantitative phytochemical constituents of methanol extracts of fermentedand
unfermented Ricinus communis seeds
Table 3 shows that reducing sugars, saponins and glycosides were all detected in the
unfermented extract but were not detected in the fermented extract. The concentrations of
tannins, flavonoids, hydrogen cyanides and phenols increased in the fermented extract.
66
Table 4: Table showing the quantitative phytochemical constituents of the methanol
extracts of fermented and unfermented Ricinus communis seeds
Phytochemical constituents
(mg/100g)
Unfermented methanol
seed extract
Mean ± SD
Fermented methanol seed
extract
Mean ± SD
Reducing sugars 39.60 ± 0.00 ND
Soluble carbohydrates 3.25 ± 0.03 3.12 ± 0.05
Hydrogen cyanides 0.02 ± 0.00 0.04 ± 0.00
Steroids 4.58 ± 0.05 0.27 ± 0.04
Saponins 1.36 ± 0.04 ND
Tannins 5.74 ± 0.03 15.16 ± 0.04
Alkaloids 3.57 ± 0.04 2.74 ± 0.04
Flavonoids 3.63 ± 0.06 4.94 ± 0.03
Glycosides 2.56 ± 0.04 ND
Phenols 6.62 ± 0.04 12.62 ± 0.04
67
3.3The Median Lethal Dose (LD50) of the methanol extracts of unfermented of Ricinus
communis seeds
The median lethal dose of the unfermented seeds of Ricinus communis showed no casualty and
death at the dose of 5000 mg/kg body weight as shown in Table 4.
68
Table 5: Phases I and II of the median lethal dose (LD50) test of the methanol extract of
unfermented Ricinus communis seeds
Dosage mg/kg body weight Mortality
Phase I
Group 1 10 0/3
Group 2 100 0/3
Group 3 1000 0/3
Phase II
Group 1 1600 0/3
Group 2 2900 0/3
Group 3 5000 0/3
69
3.3.1 Median Lethal Dose (LD50) test of the methanol extract of fermentedRicinus
communis seeds
As shown in table 5, the median lethal dose of fermented seeds of Ricinus communis exhibited
no casualty and death at lower doses but at a high dose of 5000 mg/kg body weight, the animals
exhibited some behavioural differences and death of one of the mice.
70
Table 5: Phases I and II of the median lethal dose (LD50) test of the methanol extract of
fermented Ricinus communis seeds
Dosage mg/kg body weight Mortality
Phase I
Group 1 10 0/3
Group 2 100 0/3
Group 3 1000 0/3
Phase II
Group 1 1600 0/3
Group 2 2900 0/3
Group 3 5000 1/3
71
3.4 Effects of the extracts on the isolated rabbit jejunum
As shown in Fig. 17, acetylcholine induced contraction on the jejunum. Adrenaline and the
extracts relaxed the jejunum at the concentration of 10µg/ml. Prazosin blocked the relaxant
effect of adrenaline on the tissue at increasing doses and concentrations. Acetylcholine, a
muscarinic agent induced contraction of the rabbit isolated jejunum. Unlike acetylcholine,
adrenaline, the adrenoceptor active substance, at a concentration of 10µg/ml caused the tissue to
relax. In the presence of prazosin (10µg/ml), the relaxation produced by adrenaline was blocked.
Similarly, the extract induced relaxations were susceptible to prazosin blockade.
72
Fig. 17:Effects of the methanol extracts of Ricinus communis seeds on the isolated rabbit
jejunum
Key:
Ach- Acetylcholine
Adr- Adrenaline
Pra- Prazosin
Ext. 1- Methanol extracts of unfermentedR. communisseeds
Ext. 2- Methanol extracts of fermentedR. Communisseeds
Ext. 3- Crystals of methanol extracts of unfermented R. communis seeds
73
3.4.1 Effects of prazosin blockade of α- adrenoceptor
Fig. 18shows that the extracts relaxed the jejunum at different doses of 0.1, 0.2 and 0.4 ml.
Prazosin blocked the relaxant effects of adrenaline at different doses of 0.4 and 1.0 ml. The
blocking effect of prazosin against adrenaline was highly observed at the dose of 1.0 ml. It shows
further that adrenaline relaxed the tissue. When introduced into the bath 30 seconds before
adrenaline, prazosin an α- adrenoceptor blocker, abolished the relaxation. Similarly, each of the
extract induced relaxation was lost in the presence of prazosin.
74
Fig.18: Effects of prazosin blockade of α- adrenoceptor on the isolated rabbit jejunum
Key:
Extract 1: Methanol extract of unfermented Ricinus communis seeds
Extract 2: Methanol extract of fermented Ricinus communis seeds
Extract 3: Crystals of methanol extract of methanol extract of unfermented Ricinus communis
seeds
Pra: Prazosin
Adr: Adrenaline
75
3.4.2 Effects of Indomethacin on extract induced relaxation
Indomethacin, a non-steroidal anti-inflammatory drug had no effect on the extracts and
adrenaline at a concentration of 20 µg/ml, this means that the extracts do not enhance
prostaglandin synthesis. Propranolol exhibited a weak blocking effect against the extracts as
shown in fig. 19. The effect of propranolol on the isolated rabbit jejunum in fig. 19 shows that all
the extracts relaxed the rabbit jejunum, the β- adrenoceptor antagonist, propranolol reduced the
amplitude of the relaxation.
76
Fig.19: Effects of Indomethacin on extract induced relaxation
Key:
Pra: Prazosin
Adr: Adrenaline
Indo: Indomethacin
Extract 1: Methanol unfermented extract of Ricinus communis seeds
Extract 2: Methanol fermented extract of Ricinus communis seeds
Extract 3: Crystals of methanol unfermented extract of Ricinus communis seeds
77
3.4.3 Effects of prazosin on the isolated rabbit jejunum
Fig. 20 reveals that the α- adrenoceptor blocker, prazosin reduced the relaxation of the rabbit gut
induced by the extract. In the presence of combined α- and β- blockade by prazosin and
propranolol at a concentration of 10µg/ml, the relaxation effect of adrenaline was not abolished
but rather reduced.
Effect of aminophylline on extract activity
As the relaxation of the tissue to the extract was not abolished, the effect of adenosine blockade
by aminophylline was investigated. The drug did not affect the residual activity of the extract.
78
Fig. 20: Effects of the methanol extracts ofRicinus communis seeds on isolated rabbit jejunum
Key:
Pra: Prazosin
Adr: Adrenaline
Amino: Aminophylline
Extract 1: Methanol extract of unfermented Ricinus communis seeds
Extract 2: Methanol extract of fermented Ricinus communis seeds
Extract 3: Crystals of methanol extract of unfermented Ricinus communis seeds
79
3.5:Effects of the extracts on the isolated pregnant rat uterus
Oxytocin, a potent uterotonic drug, contracted the gravid uterus at different doses as revealed in
Fig. 20. Extract 1, the unfermented extract had no effect on the tissue at the concentration of 0.5
µg/ml and a dose of 0.1 ml . On the other hand, extract 2, the fermented extract at the same
concentration and dose, contracted the tissue.
80
Fig.21: Effects of the methanol extracts of Ricinus communis seeds on the isolated pregnant
rat uterus
Key:
Oxy: Oxytocin
Adr: Adrenaline
Indo: Indomethacin
Ach: Acetylcholine
Ext. 1: Methanol extract of unfermented Ricinus communis seeds
Ext. 2: Methanol extract of fermented Ricinus communis seeds
Ext. 3: Crystals of methanol extract of unfermented Ricinus communis seeds
81
3.5.2 Effects of prostaglandin synthesis inhibition
Fig. 22 shows that indomethacin, a prostaglandin synthesis inhibitor had no effect on the extracts
at different doses and concentrations, this showed that the extracts do not enhance prostaglandin
synthesis.
Effect of ergotamine on the extracts
Ergotamine, an α-adrenoceptor blocker fully blocked the contractile effects of the extracts and
the oxytocin at different doses and concentrations
82
.Fig. 22: Effects of the methanol extract of fermented and unfermented Ricinus communis
seeds on pregnant rat uterus
Key:
Ach: Acetylcholine
Ind: Indomethacin
Oxy: Oxytocin
Erg: Ergotamine
Adr: Adrenaline
Extract 1: Methanol extract of unfermented Ricinus communis seeds
Extract 2: Methanol extract of fermented Ricinus communis seeds
Extract 3: Crystals of methanol extract of unfermented Ricinus communis seeds
83
CHAPTER FOUR
DISCUSSION
The preliminary phytochemical screening showed that the unfermented methanol extract of
Ricinus communis seeds contained alkaloids, flavonoids, tannins, glycosides, steroids, soluble
carbohydrates and phenols, as also reported by Monisha et al., (2013) and Kensa and Syhed,
(2011) while the fermented methanol extract contained alkaloids, flavonoids, hydrogen
cyanides, steroids, soluble carbohydrates, tannin and phenol. From the results, some of the
phytochemicals such as glycosides, saponins and reducing sugars were present in the
unfermented methanol extract but were not detected in the fermented extract. The reducing sugar
which was detected in the unfermented methanol extract of Ricinus communis seeds was not
detected in the fermented extract. This could be as a result of fermentation process which
resulted to the breakdown of the reducing sugar to alcohols (phenols), this breakdown led to the
non-detection of the reducing sugars in the fermented extract and also an increase in the quantity
of the phenolic content of the fermented methanol extract of Ricinus communis seeds from
6.62±0.04 mg/100g to 12.62 ± 0.04 mg/100g in the quantitative analysis. The quantitative
phytochemical analysis showed the variations in the phytochemical content of the unfermented
and fermented methanol extracts respectively, alkaloids (3.57 ± 0.04, 2.74 ± 0.04), flavonoids
(3.63 ± 0.06, 4.94 ± 0.03), tannins (5.74 ± 0.03, 15.16 ± 0.04), soluble carbohydrates (3.25 ±
0.03, 3.12 ± 0.05), hydrogen cyanides (0.02 ± 0.00, 0.04 ± 0.00), steroids (4.58 ± 0.05, 0.27 ±
0.04) and phenols (6.62 ± 0.04, 12.62 ± 0.04). The flavonoids, saponins and alkaloids are said to
have medicinal properties in animal (Living stone et al., 1997). The high increase in the tannin,
flavonoid and phenolic content of the fermented methanol extract suggested the increase in its
contractile effect on the isolated smooth muscle tissues, this is because they affect the calcium
availability of cells and calcium enhances the smooth muscle contraction (Polya et al., 1995).
The acute toxicity or median lethal dose (LD50) of the unfermented methanol extract of Ricinus
communis indicated that the seed extract is not toxic. The result showed that no casualty was
recorded at a dose as high as 5000mg/kg body weight, this result also ascertains that the organic
solvents used for the extraction did not extract the toxic glycoprotein, known as ricin. The acute
toxicity or median lethal dose (LD50) of the fermented methanol extract of Ricinus communis
indicated that the extract is not toxic at lower concentrations but toxic at a high dose
84
of5000mg/kg body weight, the result showed that death was recorded at high dose of 5000mg/kg
body weight. Fermentation increases the quantity of organic acids in the fermented foods,
organic acids such as lactic acid, citric acid, tartaric acids etc, this was suspected to be the cause
of the death that was recorded at high concentrations of 5000 mg/kg body weight.
Isolated organ bath assays are the classical pharmacological screening tool for isometric
recordings to assess concentration-response relationships in contractile tissues (Fry, 2004). The
uterus is the central organ of reproduction. It is a thick, pear shaped, muscular organ
approximately, 7cm long and 4-5 cm wide at its widest point. It is divided functionally and
morphologically into three sections, namely the cervix, isthmus and the main body of the uterus
(Symonds and Symonds, 1998). The use of herbal medicine is to alleviate problems associated
with gynaecological conditions of menstruation and menopause, to support health during
pregnancy and to facilitate childbirth is common amongst many traditional cultures (Gruber and
O’Brien, 2010). Some traditionally used medicines, such as raspberry leaves (Rubus idaeus.l),
castor oil (Ricinus communis) and cotton bark root (Gossypium hirsutum) are again receiving
attention from midwives for application during pregnancy and labour (Bayles, 2007).
Oxytocin, a potent uterotonic nonapeptide hormone, known to act both directly and indirectly to
stimulate uterine smooth muscle contraction, is widely used for the induction of labour. It
circulates as a free peptide in the blood stream and, as with all hypothalamic hormones, is
released discontinuously in a pulsatile fashion (Chard, 1989). From the results obtained in this
study in fig. 21, it was found out that oxytocin at a concentration of 0.1u/ml at a dose of 0.1ml,
contracted the uterine tissue. Oxytocin contracts the uterus through prostaglandin synthesis.
Prostaglandins are members of the eicosanoid family of proteins. They are lipid mediators
produced by the uterus, foetal membranes and the placenta and are capable of modulating uterine
contractions and have been used in pregnancy for a variety of treatments (Mitchell, et al., 1995).
The contractile effect of prostaglandin is based on their ability to mobilize calcium and inhibit
adenylyl cyclase activity. Unlike oxytocin, the extract 1 and the unfermented methanol extract of
Ricinus communis seeds, had no significant effect on the uterus at the concentration of 0.5mg/ml
and a dose of 0.1ml, this shows that the extract has no α- receptor activity. The results obtained
from this study also indicated that the fermented methanol extract of Ricinus communis
contracted the uterine tissue, this contractile effect was thought to be as a result of the high
85
content of tannins (15.16 ± 0.04). From the previous studies, tannins as one of the phytochemical
constituents of R. communis have been reported to affect calcium availability for the contraction
of uterine smooth muscles (Polya et al., 1995). An increase in free intracellular calcium can
result from either increased flux of calcium into the cell through calcium channels or by release
of calcium from internal stores (e.g sarcoplasmic reticulum; SR) (Klabunde, 2007).
Contraction of smooth muscle occurs when there is an unequal distribution of ions in the semi
permeable membrane of cell membrane, giving rise to membrane potential. Any event that
causes the positive ions to flow into the cell, is known as depolarization. Depolarization gives
rise to action potential, thereby leading to the influx of Ca2+
ions from the sarcoplasmic reticulum
(SR) into the cell membrane. The free calcium binds to a special calcium binding protein called
“Calmodulin”. Calcium-calmodulin activates myosin light chain kinase (MLCK), an enzyme that
is capable of phosphorylating myosin light chains (MLC) in the presence of ATP. In
pharmacology, contraction of the smooth muscle gives rise to the increase in the peristaltic
movement, known as diarrhoea, in the uterus, contraction of the uterus by the extract 2,
fermented methanol extract of Ricinus communis seeds depicts its tendency to cause abortion in
early pregnancies, when consumed frequently. It can also be recommended during cases of
delayed labour, for the quick expulsion of foetus from the womb.
Acetylcholine, a cholinergic neurotransmitter and a membrane depolarizing drug, binds to the
muscarinic receptors to induce contraction. From this research study, it contracted the uterus at a
concentration of 10µg/ml and a dose of 0.1ml. Indomethacin is a non-steroidal anti-inflammatory
analgesic used in the treatment of disorders such as rheumatoid arthritis, ankylosing spondylitis
and osteoarthritis (Norton, 1997). Indomethacin, β-adrenergic antagonist, inhibits the actions of
prostaglandins during uterine contractions. Indomethacin (NSAID), blocks the synthesis of
prostaglandins, from the results obtained, indomethacin was not able to abolish the contractile
effect of oxytocin at increasing doses and concentrations. Indomethacin blocked the effect of the
unfermented methanol extract of Ricinus communis at a concentration of 20µg/ml and a dose of
0.1ml. Indomethacin had no blocking effect on the contractile effect of the extract 2, the
fermented methanol extract of Ricinus communis at a dose of 0.1ml of indomethacin and
0.5mg/ml and 0.1ml of the extract. It had no effect on the third extract, though the extract 3, the
crystals or precipitates, initially had no observable effect on the tissue.
86
Ergotamine, is an α- adrenoceptor blocker or antagonist. The effect of ergotamine was also
carried out on the pregnant rat uterus at different concentrations and doses. Ergotamine fully
blocked the extracts at different concentrations and doses, all other drugs had no blocking effect
on the extracts except ergotamine which is an �- blocker, this confirmed the adrenoceptor
activity of Ricinus communis extract and substantiating the dangers of taking it in excess, which
might increase the risk of blood pressure and aggravate cardiovascular diseases.
The small intestine is used in the pharmacological experiments, because it is long, slender and
can be cut into smaller pieces. Thus, the intestine is unusual in that both α- and β- receptor types
mediate a similar biological response, that is inhibitory. The β-receptors are located on smooth
muscle fibres, whilst theα- receptors are located presynaptically on the ganglion cells of the
myentric plexus (Peddireddy, 2010).Acetylcholine, a cholinergic neurotransmitter also
contracted the uterine tissue at a concentration of 10µg/ml at a dose of 0.1ml. Acetylcholine is a
depolarizing drug that induces contraction through muscarinic receptors and intracellular
messengers.Smooth muscle relaxation occurs either as a result of removal of the contractile
stimulus or by the direct action of a substance that stimulates inhibition of the contractile
mechanism (e.g., atrial natriuretic factor is a vasodilator). Adrenaline, an adrenergic
neurotransmitter, relaxed the jejunum at a concentration of 10µg/ml at 0.1ml, adrenaline relaxes
the jejunum through theα- and β- receptors of the sympathetic adrenergic neurons.The α-
receptors of the sympathetic adrenergic neurons has the α- adrenoceptor activity which causes
peripheral resistance that leads to high blood pressure of the heart and increase in blood volume,
also the β- adrenoceptor activity leads to the increase in the contraction, force and rate of the
heart which causes high blood pressure. The β- adrenoceptor activity also stimulates rennin or
angitensinoginase (an enzyme) which catalyses the conversion of angiotensinogen to
angiotensin1, these metabolic processes causes peripheral resistance of the heart.From the results
also obtained from this study, like adrenaline, at increasing concentrations of the fermented and
unfermented methanol extracts, relaxation occurred. The crystal form of the unfermented
methanol extract (extract 3) had no significant effect on the tissue.
Prazosin is a sympatholytic drug used to treat high blood pressure and anxiety. It is an alpha-
adrenergic blocker or antagonist that is specific for the alpha-1 receptors. It acts by inhibiting the
post synaptic alpha (1) adrenoceptors on vascular smooth muscle. These receptors are found on
87
vascular smooth muscle, where they are responsible for the vasoconstrictive action of
norepinephrine. Prazosin, an α- adrenergic blocker or antagonist of adrenaline, antagonizes or
blocks the contractile effect of the intestinal tissues. From this study, it was observed that
prazosin actually blocked the relaxant effect of adrenaline. At higher concentrations and doses,
prazosin showed its antagonizing effect. The doses of adrenaline (0.1ml, 0.2ml, 0.4ml, 1.0ml)
were not changed rather that of prazosin were continuously increased.
Propranolol, a β- adrenergic blocker or antagonist, abolished the residual effect of adrenaline, at
different doses and concentrations, therefore exhibiting a weak effect on the β- adrenoceptor
activity.These receptors are either activated or inhibited by some drugs used for this study and
also the fermented and unfermented methanol extracts of Ricinus communis. From the results
obtained in this study, the unfermented methanol extract of Ricinus communis had no effect (no
contraction) on the uterine tissue at a concentration of 10µg/ml and dose of 0.1ml.Indomethacin
is a non-steroidal anti-inflammatory analgesic used in the treatment of disorders such as
rheumatoid arthritis, ankylosing spondylitis and osteoarthritis (Norton, 1997). Indomethacin, β-
adrenergic antagonist, inhibits the actions of prostaglandins during uterine contractions.
Prostaglandins have a significant stimulatory effect on established labour, indomethacin acts by
inhibiting the activity of cyclo-oxygenase enzyme necessary for the synthesis of prostaglandins,
prostacyclins and thromboxanes (Norton, 1997). From the study, indomethacin exhibited its
normal inhibitory effect on the uterine tissue at a concentration of 10µg/ml at different doses.
Indomethacin had no effect on the jejunum at a concentration of 20µg/ml and a dose of 0.1ml,
meaning it’s not a prostaglandin synthesis blocker unlike in Abrus seed extract where the
relaxation was being blocked by indomethacin. With an increasing dose-dependent manner of
indomethacin in the organ bath, the relaxant effect of adrenaline was still observed showing that
the antagonizing effect of indomethacin was not observed. Likewise indomethacin,
aminophylline, an adenosine receptor blocker or antagonist also had no observable effect on the
extracts even at increasing concentrations and doses, this means that the extracts do not possess
an adenosine-like activity.
Smooth muscle relaxation occurs either as a result of removal of the contractile stimulus or by
the direct action of a substance that stimulates inhibition of the contractile mechanism (e.g., atrial
natriuretic factor is a vasodilator). From the results obtained, adrenaline, an adrenergic
88
neurotransmitter relaxed the intestinal tissue at a concentration of 10µg/ml at a dose of 0.1ml.
Adrenaline, an adrenergic neurotransmitter, relaxes the intestinal tissues, probably due to the
presence of the α- and β- receptors on this smooth muscle tissue, this was confirmed in this
particular study when adrenaline at different concentrations relaxed the smooth muscle of the
jejunum.
4.2 Conclusion
From the present study, due to the fact that the jejunum contractile activity was sensitive to
prazosin, it was an α- adrenoceptor effect. Insensitivity of the uterotonic activity to indomethacin
revealed that the extract had no effect on both oxytocin receptor and prostaglandin synthesis.
However, the abolition of this contraction by ergotamine in both the jejunum and uterus showed
that α- adrenoceptor activity was evident. Also, the contraction of the uterus by the fermented
extract suggests its use during delayed labour by pregnant women and also the relaxation of the
jejunum by the same extract also suggests its use in diarrhoeal conditions and can boost
constipation.
4.3 Suggestions for further studies
The paradoxical effects of methanol extract of the seeds of Ricinus communis on smooth muscle
preparations was investigated in this study and it is therefore suggested that further studies be
done on
• The effect of the methanol extracts of the fermented and unfermented Ricinus communis seeds
on rat prostate
• The use of different solvents in the extraction of Ricinus communis seeds and its subsequent
effect on the smooth muscle tissues (the jejunum, uterus, ileum and prostate). This will reveal
the solvent that may have a significant paradoxical effect on those tissues.
• The use of purified forms of the extracts of the fermented and unfermented Ricinus communis
seeds on smooth muscle preparations.
• Identifying the actual phytochemical constituents responsible for the paradoxical effects of the
fermented and unfermented methanol extracts of Ricinus communis seeds.
89
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Descriptives
Fermented Methanol Extract of Ricinuscommunis Seeds
Phytochemicals N Mean Std. Deviation Std. Error 95% Confidence Interval for
Mean
Minimum Maxim
um
Lower Bound Upper Bound
Flavonoids 3 4.940000 .0026458 .0015275 4.933428 4.946572 4.9380 4.9430
Hydrogen Cyanide 3 .039533 .0002517 .0001453 .038908 .040158 .0393 .0398
Tannin 3 15.155667 .0041633 .0024037 15.145324 15.166009 15.1510 15.159
0
Soluble
Carbohydrate 3 3.123667 .0045092 .0026034 3.112465 3.134868 3.1190 3.1280
Alkaloids 3 2.743000 .0036056 .0020817 2.734043 2.751957 2.7400 2.7470
Phenol 3 12.625667 .0015275 .0008819 12.621872 12.629461 12.6240 12.627
0
Steroids 3 .274000 .0036056 .0020817 .265043 .282957 .2710 .2780
Total 21 5.557362 5.6745937 1.2382979 2.974318 8.140406 .0393 15.159
0
ANOVA
Fermented Methanol Extract of Ricinuscommunis Seeds
Sum of Squares df Mean Square F Sig.
Between Groups 644.020 6 107.337 10283637.095 .000
Within Groups .000 14 .000
Total 644.020 20
Multiple Comparisons
Dependent Variable: Fermented Methanol Extract of Ricinus communis Seeds
LSD
(I) Phytochemicals (J) Phytochemicals Mean Difference
(I-J)
Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Flavonoids
Hydrogen Cyanide 4.9004667* .0026379 .000 4.894809 4.906124
Tannin -10.2156667* .0026379 .000 -10.221324 -10.210009
Soluble Carbohydrate 1.8163333* .0026379 .000 1.810676 1.821991
100
Alkaloids 2.1970000* .0026379 .000 2.191342 2.202658
Phenol -7.6856667* .0026379 .000 -7.691324 -7.680009
Steroids 4.6660000* .0026379 .000 4.660342 4.671658
Hydrogen Cyanide
Flavonoids -4.9004667* .0026379 .000 -4.906124 -4.894809
Tannin -15.1161333* .0026379 .000 -15.121791 -15.110476
Soluble Carbohydrate -3.0841333* .0026379 .000 -3.089791 -3.078476
Alkaloids -2.7034667* .0026379 .000 -2.709124 -2.697809
Phenol -12.5861333* .0026379 .000 -12.591791 -12.580476
Steroids -.2344667* .0026379 .000 -.240124 -.228809
Tannin
Flavonoids 10.2156667* .0026379 .000 10.210009 10.221324
Hydrogen Cyanide 15.1161333* .0026379 .000 15.110476 15.121791
Soluble Carbohydrate 12.0320000* .0026379 .000 12.026342 12.037658
Alkaloids 12.4126667* .0026379 .000 12.407009 12.418324
Phenol 2.5300000* .0026379 .000 2.524342 2.535658
Steroids 14.8816667* .0026379 .000 14.876009 14.887324
Soluble Carbohydrate
Flavonoids -1.8163333* .0026379 .000 -1.821991 -1.810676
Hydrogen Cyanide 3.0841333* .0026379 .000 3.078476 3.089791
Tannin -12.0320000* .0026379 .000 -12.037658 -12.026342
Alkaloids .3806667* .0026379 .000 .375009 .386324
Phenol -9.5020000* .0026379 .000 -9.507658 -9.496342
Steroids 2.8496667* .0026379 .000 2.844009 2.855324
Alkaloids
Flavonoids -2.1970000* .0026379 .000 -2.202658 -2.191342
Hydrogen Cyanide 2.7034667* .0026379 .000 2.697809 2.709124
Tannin -12.4126667* .0026379 .000 -12.418324 -12.407009
Soluble Carbohydrate -.3806667* .0026379 .000 -.386324 -.375009
Phenol -9.8826667* .0026379 .000 -9.888324 -9.877009
Steroids 2.4690000* .0026379 .000 2.463342 2.474658
Phenol
Flavonoids 7.6856667* .0026379 .000 7.680009 7.691324
Hydrogen Cyanide 12.5861333* .0026379 .000 12.580476 12.591791
Tannin -2.5300000* .0026379 .000 -2.535658 -2.524342
Soluble Carbohydrate 9.5020000* .0026379 .000 9.496342 9.507658
Alkaloids 9.8826667* .0026379 .000 9.877009 9.888324
Steroids 12.3516667* .0026379 .000 12.346009 12.357324
Steroids
Flavonoids -4.6660000* .0026379 .000 -4.671658 -4.660342
Hydrogen Cyanide .2344667* .0026379 .000 .228809 .240124
Tannin -14.8816667* .0026379 .000 -14.887324 -14.876009
Soluble Carbohydrate -2.8496667* .0026379 .000 -2.855324 -2.844009
Alkaloids -2.4690000* .0026379 .000 -2.474658 -2.463342
Phenol -12.3516667* .0026379 .000 -12.357324 -12.346009
101
*. The mean difference is significant at the 0.05 level.
Descriptives
Unfermented Methanol Extract of Ricinuscommunis Seeds
Phytochemicals N Mean Std.
Deviation
Std. Error 95% Confidence Interval for
Mean
Minimum Maximu
m
Lower Bound Upper Bound
Reducing Sugar 3 39.565333 .0005774 .0003333 39.563899 39.566768 39.5650 39.5660
Soluble
Carbohydrate 3 3.254333 .0025166 .0014530 3.248082 3.260585 3.2520 3.2570
Hydrogen Cyanide 3 .021567 .0002517 .0001453 .020942 .022192 .0213 .0218
Steroids 3 4.584333 .0047258 .0027285 4.572594 4.596073 4.5790 4.5880
Saponin 3 1.355000 .0040000 .0023094 1.345063 1.364937 1.3510 1.3590
Tannin 3 5.735667 .0025166 .0014530 5.729415 5.741918 5.7330 5.7380
Alkaloids 3 3.566333 .0037859 .0021858 3.556929 3.575738 3.5620 3.5690
Flavonoids 3 3.630667 .0060277 .0034801 3.615693 3.645640 3.6250 3.6370
Glycoside 3 2.549667 .0258134 .0149034 2.485543 2.613791 2.5200 2.5670
Phenol 3 6.503667 .2023619 .1168337 6.000972 7.006361 6.2700 6.6210
Total 30 7.076657 11.1681173 2.0390099 2.906413 11.246900 .0213 39.5660
ANOVA
Unfermented Methanol Extract of Ricinuscommunis Seeds
Sum of Squares df Mean Square F Sig.
Between Groups 3616.995 9 401.888 96332.832 .000
Within Groups .083 20 .004
Total 3617.078 29
Multiple Comparisons
Unfermented Methanol Extract of Ricinuscommunis Seeds
LSD
(I) Phytochemicals (J) Phytochemicals Mean Difference
(I-J)
Std. Error Sig. 95% Confidence Interval
Lower Bound Upper
Bound
Reducing Sugar Soluble Carbohydrate 36.3110000* .0527375 .000 36.200991 36.421009
102
Hydrogen Cyanide 39.5437667* .0527375 .000 39.433758 39.653775
Steroids 34.9810000* .0527375 .000 34.870991 35.091009
Saponin 38.2103333* .0527375 .000 38.100325 38.320342
Tannin 33.8296667* .0527375 .000 33.719658 33.939675
Alkaloids 35.9990000* .0527375 .000 35.888991 36.109009
Flavonoids 35.9346667* .0527375 .000 35.824658 36.044675
Glycoside 37.0156667* .0527375 .000 36.905658 37.125675
Phenol 33.0616667* .0527375 .000 32.951658 33.171675
Soluble Carbohydrate
Reducing Sugar -36.3110000* .0527375 .000 -36.421009 -36.200991
Hydrogen Cyanide 3.2327667* .0527375 .000 3.122758 3.342775
Steroids -1.3300000* .0527375 .000 -1.440009 -1.219991
Saponin 1.8993333* .0527375 .000 1.789325 2.009342
Tannin -2.4813333* .0527375 .000 -2.591342 -2.371325
Alkaloids -.3120000* .0527375 .000 -.422009 -.201991
Flavonoids -.3763333* .0527375 .000 -.486342 -.266325
Glycoside .7046667* .0527375 .000 .594658 .814675
Phenol -3.2493333* .0527375 .000 -3.359342 -3.139325
Hydrogen Cyanide
Reducing Sugar -39.5437667* .0527375 .000 -39.653775 -39.433758
Soluble Carbohydrate -3.2327667* .0527375 .000 -3.342775 -3.122758
Steroids -4.5627667* .0527375 .000 -4.672775 -4.452758
Saponin -1.3334333* .0527375 .000 -1.443442 -1.223425
Tannin -5.7141000* .0527375 .000 -5.824109 -5.604091
Alkaloids -3.5447667* .0527375 .000 -3.654775 -3.434758
Flavonoids -3.6091000* .0527375 .000 -3.719109 -3.499091
Glycoside -2.5281000* .0527375 .000 -2.638109 -2.418091
Phenol -6.4821000* .0527375 .000 -6.592109 -6.372091
Steroids
Reducing Sugar -34.9810000* .0527375 .000 -35.091009 -34.870991
Soluble Carbohydrate 1.3300000* .0527375 .000 1.219991 1.440009
Hydrogen Cyanide 4.5627667* .0527375 .000 4.452758 4.672775
Saponin 3.2293333* .0527375 .000 3.119325 3.339342
Tannin -1.1513333* .0527375 .000 -1.261342 -1.041325
Alkaloids 1.0180000* .0527375 .000 .907991 1.128009
Flavonoids .9536667* .0527375 .000 .843658 1.063675
Glycoside 2.0346667* .0527375 .000 1.924658 2.144675
Phenol -1.9193333* .0527375 .000 -2.029342 -1.809325
Saponin
Reducing Sugar -38.2103333* .0527375 .000 -38.320342 -38.100325
Soluble Carbohydrate -1.8993333* .0527375 .000 -2.009342 -1.789325
Hydrogen Cyanide 1.3334333* .0527375 .000 1.223425 1.443442
Steroids -3.2293333* .0527375 .000 -3.339342 -3.119325
103
Tannin -4.3806667* .0527375 .000 -4.490675 -4.270658
Alkaloids -2.2113333* .0527375 .000 -2.321342 -2.101325
Flavonoids -2.2756667* .0527375 .000 -2.385675 -2.165658
Glycoside -1.1946667* .0527375 .000 -1.304675 -1.084658
Phenol -5.1486667* .0527375 .000 -5.258675 -5.038658
Tannin
Reducing Sugar -33.8296667* .0527375 .000 -33.939675 -33.719658
Soluble Carbohydrate 2.4813333* .0527375 .000 2.371325 2.591342
Hydrogen Cyanide 5.7141000* .0527375 .000 5.604091 5.824109
Steroids 1.1513333* .0527375 .000 1.041325 1.261342
Saponin 4.3806667* .0527375 .000 4.270658 4.490675
Alkaloids 2.1693333* .0527375 .000 2.059325 2.279342
Flavonoids 2.1050000* .0527375 .000 1.994991 2.215009
Glycoside 3.1860000* .0527375 .000 3.075991 3.296009
Phenol -.7680000* .0527375 .000 -.878009 -.657991
Alkaloids
Reducing Sugar -35.9990000* .0527375 .000 -36.109009 -35.888991
Soluble Carbohydrate .3120000* .0527375 .000 .201991 .422009
Hydrogen Cyanide 3.5447667* .0527375 .000 3.434758 3.654775
Steroids -1.0180000* .0527375 .000 -1.128009 -.907991
Saponin 2.2113333* .0527375 .000 2.101325 2.321342
Tannin -2.1693333* .0527375 .000 -2.279342 -2.059325
Flavonoids -.0643333 .0527375 .237 -.174342 .045675
Glycoside 1.0166667* .0527375 .000 .906658 1.126675
Phenol -2.9373333* .0527375 .000 -3.047342 -2.827325
Flavonoids
Reducing Sugar -35.9346667* .0527375 .000 -36.044675 -35.824658
Soluble Carbohydrate .3763333* .0527375 .000 .266325 .486342
Hydrogen Cyanide 3.6091000* .0527375 .000 3.499091 3.719109
Steroids -.9536667* .0527375 .000 -1.063675 -.843658
Saponin 2.2756667* .0527375 .000 2.165658 2.385675
Tannin -2.1050000* .0527375 .000 -2.215009 -1.994991
Alkaloids .0643333 .0527375 .237 -.045675 .174342
Glycoside 1.0810000* .0527375 .000 .970991 1.191009
Phenol -2.8730000* .0527375 .000 -2.983009 -2.762991
Glycoside
Reducing Sugar -37.0156667* .0527375 .000 -37.125675 -36.905658
Soluble Carbohydrate -.7046667* .0527375 .000 -.814675 -.594658
Hydrogen Cyanide 2.5281000* .0527375 .000 2.418091 2.638109
Steroids -2.0346667* .0527375 .000 -2.144675 -1.924658
Saponin 1.1946667* .0527375 .000 1.084658 1.304675
Tannin -3.1860000* .0527375 .000 -3.296009 -3.075991
Alkaloids -1.0166667* .0527375 .000 -1.126675 -.906658
Flavonoids -1.0810000* .0527375 .000 -1.191009 -.970991
104
Phenol -3.9540000* .0527375 .000 -4.064009 -3.843991
Phenol
Reducing Sugar -33.0616667* .0527375 .000 -33.171675 -32.951658
Soluble Carbohydrate 3.2493333* .0527375 .000 3.139325 3.359342
Hydrogen Cyanide 6.4821000* .0527375 .000 6.372091 6.592109
Steroids 1.9193333* .0527375 .000 1.809325 2.029342
Saponin 5.1486667* .0527375 .000 5.038658 5.258675
Tannin .7680000* .0527375 .000 .657991 .878009
Alkaloids 2.9373333* .0527375 .000 2.827325 3.047342
Flavonoids 2.8730000* .0527375 .000 2.762991 2.983009
Glycoside 3.9540000* .0527375 .000 3.843991 4.064009
*. The mean difference is significant at the 0.05 level.