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

s

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. 3: The seeds of Ricinus communis

(Sabina et al., 2009).

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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.