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EVALUATION OF THE LOCAL ANAESTHETIC EFFECTS OF THE METHANOL LEAF EXTRACT OF STERCULIA TRAGACANTHA LINDL. (1830) IN WEST AFRICAN DWARF GOATS
BY
UDEGBUNAM, RITA IJEOMA D.V.M (Nig), M.V.SC (Ibadan) PG/Ph.D/06/42080
A THESIS SUBMITTED TO THE DEPARTMENT OF VETERINARY SURGERY, FACULTY OF VETERINARY MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA, FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN VETERINARY ANAESTHESIOLOGY
MAY, 2011
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CERTIFICATION
UDEGBUNAM, RITA IJEOMA, a post-graduate student in the Department of
Veterinary Surgery and with registration number PG/Ph.D/06/42080, has satisfactorily completed the requirements for the award of the degree of Doctor of Philosophy in
Veterinary Anaesthesiology. The work embodied in this thesis is original and has not been submitted in part or full for any diploma or degree of this or any other university.
…………………. ……………………. Prof. R.O.C. Kene Prof I.U. Asuzu Department of Veterinary Surgery, Department of Veterinary University of Nigeria. Physiology & Pharmacology (Supervisor) University of Nigeria (Supervisor)
…………………………….. Prof. E.O. Gyang External Examiner
…………………………… Dr. T.O. Nnaji (Acting Head of Department)
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DECLARATION
The studies presented in this thesis are original and were carried out by me under the supervision of Professors R.O.C Kene and I.U Asuzu. References made to the work of other investigators were duly acknowledged. No part of this thesis has been previously submitted elsewhere for a diploma or degree
……………………………………
Udegbunam, Rita Ijeoma
May, 2011
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DEDICATION
This thesis is dedicated in loving memory to Andrew Onebunne Nweke.
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ACKNOWLEDGEMENTS
I thank the Lord Almighty, the giver of all good gifts, knowledge and life for making this study possible. To Him be all Glory, honour and adoration forever.
I also say thank you to my Supervisors, Prof R.O.C Kene and Prof I.U Asuzu for their immeasurable contributions to this work. Without your guidance and assistance, this work would not have been. Remain blessed.
To by colleagues, in the Department of Veterinary Surgery, Drs. Eze, C.A; Nnaji, T.O;
Onuba, A.C and Offor, G.E, I am most honored to be part of the surgery family. Thank you for your support.
My thanks also go to the following: Prof J.O.A Okoye; Dr. E. Onuoha; Dr. R.C.
Ezeokonkwo; Dr. R.I. Obidike, Dr. M.C.O. Ezeibe; Dr (Mrs) U. Okoroafor; Dr (Mrs)
E.V. Ezenduka, Mrs. N. Nnaji; Mr. I.K. Ifedigbo, Mr I.I. Ogbudimkpa (late); Mr. A.
Ngene; Mr. C. Nwaehujor and Dr. C.C. Onah for their help and input into this work. May
God bless you all.
My outmost gratitude goes to my siblings Chike, Chidi, Ifeyinwa, Okey, Ifeanyi and
Chioma who supported me emotionally and financially. I lack the words to express my thanks. May the good Lord reward you all.
To my mum, Theresa Ekeamaka Nweke, your prayers, calls, visits and advice are appreciated. May your days be long because you deserve the best from us. Thank you so much.
My special thanks go to my husband, Dr. Sunday Ositadinma Udegbunam. I cannot thank you enough for your love, understanding and support. You were with me all the way. You are the best I can ask for. Somtochukwu, Chiagoziem and little Chiamaka, I say thank you to all of you for your understanding, love and support. May the Lord bless you all. 6
ABSTRACT
This study investigated the local anaesthetic effects of the methanol leaf extract of
Sterculia tragacantha (MEST) in WAD goats. The extract was prepared by cold maceration in 80% methanol to obtain a yield of 11.11%. The intraperitoneal (LD50) of the extract was found to be greater than 1600 mg/kg.
Four preliminary experiments were carried out in mice and guinea pigs to evaluate the anti-nociceptive and anti-inflammatory effects of the extract. The anti-nociceptive effects were evaluated using guinea pig wheal test and acetic acid-induced writhing test. The effects of the extract on acute and chronic inflammation were evaluated using carrageenan induced paw edema and cotton pellet induced granuloma tests respectively.
The preliminary screening of the extract for local anaesthetic activity in guinea pigs showed that injection of 10 mg/ml and 0.3 mg/ml solutions of the extract produced 100% and 86% analgesia respectively. Pretreatment of mice with the extract (300 and 600 mg/kg) significantly (p < 0.05) inhibited acetic acid induced pain and carrageenan- induced paw edema. Daily dosing of 300 and 600 mg/kg of the extract significantly
(p < 0.05) suppressed granuloma formation in mice.
The use of MEST (8 mg/kg) for infiltration anaesthesia prior to orchidectomy was also explored. The results of the study showed that the mean heart rate (HR) of MEST treated goats was significantly (p<0.05) lower than the mean HR obtained in group 1 (non anaesthetized orchidectomized) goats at 30 and 120 min of the study. The mean HR of the lignocaine (LIG) treated goats were significantly (p < 0.05) lower than that of the other groups throughout the post operative period. The mean respiratory rate (RR) of MEST group was significantly (p < 0.05) lower than RR of group 1 goats at 10, 30, 120 and 240 min. The LIG treated goats had significantly (p < 0.05) lower RR compared to groups 1 7 and 3 goats from 10 min of the study. The blood glucose of goats’ pretreated with MEST and LIG decreased at 30, 120 and 240 min while the glucose level of non anaesthetized orchidectomized goats increased at these time points post orchidectomy. The blood glucose values obtained in MEST and LIG groups were significantly (p < 0.05) lower than the glucose level of goats in group 1 at 30, 120 and 240 min post surgery. The mean pain scores obtained in the MEST and LIG pretreated orchidectomized goats were significantly (p < 0.05) lower than those obtained in non anaesthetized orchidectomized goats. No significant difference (p > 0.05) was observed between the pain scores of the
MEST pretreated and LIG pretreated orchidectomized groups. The degree of analgesia and distance of diffusion of the MEST and LIG after flank infiltration were not significantly (p > 0.05) different. LIG produced a significantly (p < 0.05) longer duration of anaesthesia when compared to MEST.
MEST was subjected to column and thin layer chromatography to separate its components. Seven fractions (F1-F7) were obtained at the end of chromatography and six fractions (F2-F7) were screened for local anaesthetic activity. F5 and F7 were more potent than LIG while F3, F5, F6 and F7 were more potent than MEST.
Preliminary phytochemical tests revealed the presence of carbohydrates, starch, glycosides, alkaloids, flavonoids, terpenes, tannins and saponins in the crude extract.
Fractions 5, 6 and 7 contained flavonoids, saponins and alkaloids.
It was concluded that the methanol extract of S. tragacantha possessed peripheral analgesic, local anaesthetic and anti-inflammatory properties. The extract was effective as a local anaesthetic for orchidectomy in WAD goats. It also showed potent anaesthetic activity on flank infiltration. The fractions obtained showed significant local anaesthetic activity. The observed local anaesthetic activity of the plant extract and its fractions may be due to the presence of alkaloids and saponins in the leaves of S. tragacantha. 8
TABLE OF CONTENTS
Title page ------i
Certification ------ii
Declaration ------iii
Dedication ------iv
Acknowledgments ------v
Abstract ------vi
Table of contents ------viii
List of tables ------xvi
List of figures ------xvii
List of abbreviations ------xviii
CHAPTER ONE
GENERAL INTRODUCTION ------1
1.1 Introduction ------2
1.2 Research objectives ------4
CHAPTER TWO
LITERATURE REVIEW ------5
2.1 Pain ------6
2.2 Physiology of Pain ------6
2.2.1 Transduction of pain ------6
2.2.2 Transmission of pain ------8
2.2.3 Modulation of pain ------9
2.2.4 Perception of pain ------10
2.3 Types of pain ------10 9
2.4 Local and systemic responses to noxious stimuli ------12
2.4.1 Local biochemical responses to pain ------12
2.4.2 Endocrine responses to pain ------13
2.4.3 Metabolic responses to pain ------14
2.4.4 Behavioral response to pain ------14
2.5 Analgesia ------16
2.6 Opioid analgesics ------17
2.6.1 Morphine sulfate ------18
2.6.2 Fentanyl citrate ------18
2.6.3 Buprenorphine hydrochloride ------19
2.6.4 Butorphanol tartrate ------19
2.6.5 Pentazocine ------19
2.7 Non steroidal anti-inflammatory drugs (NSAIDs) ------20
2.7.1 Inflammatory process ------20
2.7.2 Mechanism of action of NSAIDs ------21
2.7.3 Acetylsalicylic acid ------22
2.7.4 Phenylbutazone ------22
2.7.5 Flunixine meglumine ------22
2.7.6 Indomethacin ------23
2.7.7 Ketoprofen ------23
2.7.8 Carprofen ------23
2.7.9 Diclofenac ------24
2.8 Local anaesthetics ------24
2.8.1 General properties of local anaesthetics ------24
2.8.2 Mechanism of action of local anaesthetics ------25 10
2.8.3 Clinical pharmacology of local anaesthetics ------25
2.8.4 Side effects of local anaesthetics ------27
2.8.5 Procaine hydrochloride ------28
2.8.6 Lignocaine hydrochloride ------29
2.8.7 Mepivacaine hydrochloride ------29
2.8.8 Bupivacaine hydrochloride ------30
2.8.9 Ropivacaine hydrochloride ------30
2.9 Local anaesthetic techniques used in goats ------30
2.10 Medicinal plants with analgesic properties ------34
2.10.1 Plants used in traditional pharmacopeia for analgesia ------35
2.10.2 Plants with proven uses in pharmacopeia ------36
2.10.3 Phytochemical compounds identified in plants with analgesic activity-- 37
2.10.3.1 Alkaloids ------37
2.10.3.2 Volatile oils ------40
2.10.3.3 Glycosides ------40
2.10.4 Medicinal plants with proven local anaesthetic properties -- -- 40
2.10.4.1 Phytochemical compounds identified in plants with local anaesthetic
properties ------41
2.11 Studied medicinal plant ------42
2.12 Clinical assessment of pain ------44
2.12.1 Subjective assessment of pain ------45
2.12.2 Objective assessment of pain ------47
2.13 Evaluation of anti-inflammatory effects of drugs ------49
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CHAPTER THREE
EVALUATION OF THE ANALGESIC, ANTI-INFLAMMATORY AND TISSUE
EFFECTS OF THE METHANOL LEAF EXTRACT OF S. TRAGACANTA --
------52
3.1 Introduction ------53
3.2 Materials ------54
3.2.1 Instruments and equipments ------54
3.2.2 Reagents and solvents ------55
3.2.3 Glass wares ------55
3.2.4 Consumables ------55
3.2.5 Drugs ------56
3.3 Methods ------56
3.3.1 Plant collection and identification ------56
3.3.2 Extraction of plant materials ------56
3.3.3 Screening of the extract for local anaesthetic activity ------57
3.3.4 Determination of the solubility of MEST in distilled water and Tween 20 58
3.3.5 Determination of the pH of MEST ------58
3.3.6 Adjustment of the pH of MEST ------59
3.3.7 Acute toxicity test ------59
3.3.8 Screening of MEST for analgesic property ------59
3.3.9 Evaluation of the effect of MEST on acute inflammation -- -- 60
3.3.10 Evaluation of the effect of MEST on chronic inflammation -- -- 62
3.3.11 Evaluation of the tissue effect of MEST ------63
3.4 Results ------64 12
3.4.1 Calculation of plant yield ------64
3.4.2 Determination of the solubility of MEST ------64
3.4.3 Preliminary screening of MEST for local anaesthetic activity -- -- 65
3.4.4 Determination of the pH of MEST and pH adjustment ------65
3.4.5 Acute toxicity test ------65
3.4.6 Screening of MEST for analgesic activity ------65
3.4.7 Evaluation of the effect of MEST on acute inflammation -- -- 65
3.4.8 Evaluation of the effect of MEST on chronic inflammation -- -- 66
3.4.9 Evaluation of the tissue effect of MEST ------66
3.5 Discussion ------80
CHAPTER FOUR
EVALUATION OF THE EFFICACY OF MEST FOR LOCAL ANAESTHESIA IN
WAD GOATS ------84
4.1 Introduction ------85
4.2 Materials ------86
4.2.1 Instruments and equipments ------86
4.2.2 Reagents and solvents ------86
4.2.3 Glass wares ------87
4.2.4 Consumables ------87
4.2.5 Drugs ------87
4.3 Methods ------87
4.3.1 Evaluation of the anaesthetic efficacy of MEST for orchidectomy -- -- 87
4.3.1.1 Physiologic variables ------88
4.3.1.2 Blood glucose ------88 13
4.3.1.3 Pain estimation ------89
4.3.2 Evaluation of the efficacy of MEST for flank anaesthesia -- -- 90
4.4 Results ------91
4.4.1 Evaluation of the anaesthetic efficacy of MEST for orchidectomy -- 91
4.4.1.1 Physiologic changes ------91
4.4.1.2 Blood glucose ------91
4.4.1.3 Pain scores ------92
4.4.2 Evaluation of efficacy of MEST for flank anaesthesia ------92
4.5 Discussion ------98
CHAPTER FIVE
FRACTIONATION OF THE CRUDE EXTRACT OF S. TRAGACANTHA AND
IDENTIFICATION OF ITS ACTIVE FRACTION(S) ------101
5.1 Introduction ------102
5.2 Materials ------102
5.2.1 Instruments and equipments ------102
5.2.2 Reagents and solvents ------103
5.2.3 Glass wares ------103
5.2.4 Consumables ------103
5.2.5 Drugs ------104
5.3 Methods ------104
5.3.1 Column chromatography ------104
5.3.2 Thin layer chromatography ------105
5.3.3 Screening of the fractions of MEST for local anaesthetic activity -- 107 14
5.4 Results ------107
5.4.1 Fractionation of MEST ------107
5.4.2 Evaluation of the local anaesthetic effects of the fractions -- -- 108
5.4.3 Discussion ------116
CHAPTER SIX
PHYTOCHEMICAL ANALYSIS OF THE METHANOL EXTRACT AND
FRACTIONS OF S. TRAGACATHA ------117
6.1 Introduction ------118
6.2 Materials ------118
6.2.1 Instruments and equipments ------118
6.2.2 Reagents and solvents ------119
6.2.3 Glass wares ------120
6.2.4 Consumables ------120
6.3 Methods ------120
6.3 Phytochemical analysis of the crude MEST ------120
6.4 Phytochemical analysis of the MEST fractions ------123
6.5 Results ------124
6.5.1 Phytochemical analysis of the crude MEST ------124
6.5.2 Phytochemical analysis of the MEST fractions ------125
6.6 Discussion ------128
CHAPTER SEVEN
GENERAL DISCUSSION AND CONCLUSION ------130
7.1 Discussion ------131 15
7.2 Conclusion------133
REFERENCES ------134
APPENDICES ------161
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LIST OF TABLES
Table 1: Percentage local anaesthesia of lignocaine and MEST ------67
Table 2: pH of Lignocaine and MEST ------68
Table 3: Percentage local anaesthesia of MEST after pH adjustment -- -- 68
Table 4: Mean onset and number of contortions in the treatment and control
groups ------69
Table 5: Mean paw oedema in the treatment and control groups ------70
Table 6: Mean granuloma and transuda weights in the treatment and control
groups ------71
Table 7: Mean post operative pain scores of orchidectomized goats -- -- 93
Table 8: Duration of local anaesthesia of LIG and MEST ------93
Table 9: Degree of pain in the LIG and MEST goats ------94
Table 10: Distance of diffusion of LIG and MEST ------94
Table 11: Fractions obtained from MEST ------109
Table 12: Percentage anaesthesia of the fractions ------110
Table 13: Phytochemical tests result of MEST ------126
Table 14: Phytochemical tests result of MEST fractions ------127
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LIST OF FIGURES
Figure 1: Graph showing slopes of LIG and MEST ------72
Figure 2: Percentage inhibition of acetic acid induced contortions in mice -- 73
Figure 3: Percentage oedema inhibition in mice ------74
Figure 4: Percentage inhibition of granuloma in mice ------75
Figure 5: Skin section of distilled water treated mice on day 1 ------76
Figure 6: Skin section of MEST treated mice on day 1 ------77
Figure 7: Skin section of distilled water treated mice on day 5 ------78
Figure 8: Skin section of MEST treated mice on day 5 ------79
Figure 9: Heart rates (beats/min) of orchidectomized goats ------95
Figure 10: Respiratory rates (breaths/min) of orchidectomized goats -- -- 96
Figure 11: Blood glucose (mmol/l) of orchidectomized goats ------97
Figure 12: TLC plate showing the bands of the fractions ------111
Figure 13: Graph showing slopes of fractions 2 and 3 ------112
Figure 14: Graph showing slopes of fractions 4 and 5 ------113
Figure 15: Graph showing slopes of fractions 6 and 7 ------114
Figure 16: Graph showing slopes of LIG and MEST ------115
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LIST OF ABBREVIATIONS
ACVA: American college of Anesthesiology
IASP: International Association for the study of pain
NSAIDs: Non steriodal anti-inflammatory drugs
CNS: Central nervous system
HTMS: High threshold mechanoreceptors
MMT: Myelinated mechanothermal
NDHN: Nociceptive dorsal horn neurons
DNIC: Diffuse noxious inhibitory control
ADH: Antidiuretic hormone
ACTH: Adrenocorticotrophic hormone
K: Kappa
GIT: Gastrointestinal tract i.v: Intravenous i.m: Intramuscular i.p: Intraperitoneal
COX-1: Cyclo-oxygenase 1
COX-2: Cyclo-oxygenase 2
Na+: Sodium ion
K+: Potassium ion
%: Percentage
THP: Tetrahydropalmatine
ACC: Acetylaconitine
SDS: Simple descriptive scale 19
NRS: Numerical rating scale
VAS: Visual analogue scale
GCS: Glasgow coma scale
CHEOPS: Children’s Hospital of Ontario Pain Scale
A.A: Acetic acid
MEST: Methanol extract of Sterculia tragacantha
LIG: Lignocaine
INDO: Indomethacin
ANOVA: Analysis of variance
SPSS: Statistical package for the social sciences
DMRT: Duncan multiple range test
SE: Standard error of mean ml: Millilitre kg: Kilogramme g: Grammes
0C: Degree centigrade
DW: Distilled water
NaOH: Sodium hydroxide
LD50: Lethal dose 50
PMNS: Polymorphonuclear cells
IL-1: Interleukin-1
Hcl: Hydrochloric acid h: Hours min: Minutes p: Probability 20
UV: Ultraviolet
H2SO4: Sulphuric acid
TLC: Thin layer chromatography
Ppt: Precipitate
Solu: Solution
Mmol/l Millimoles per litre
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CHAPTER ONE
GENERAL INTRODUCTION
1.1 INTRODUCTION 22
Pain is as an unpleasant sensation associated with tissue injury (Merskey, 1979).
Pain signaling occurs through a distinct pathway that begins at the onset of noxious stimulus such as tissue trauma, surgical incision or heat (Busch et al., 2006). The presence of pain triggers off local biochemical, neuroendocrine, metabolic and behavioural changes (Bailey and Stanley, 1990; Nixon and Cummings, 1994; Martini et al., 2000). These immediate stress responses seen after an injury are important for the survival of the patient (ACVA, 2000). However, unrelieved pain leads to severe metabolic stress, functional derangement and maladaptive behaviours (ACVA, 2000).
These stress responses may induce severe suffering in the patient thus the need for post operative analgesia.
In modern medical practice, the aim of treating pain is to make it as tolerable as possible without eliminating it totally (Thurmon et al., 1996). Analgesia is achieved by interrupting the nociceptive processes: transduction, transmission, modulation and perception at one or more points between the peripheral nociceptors and the cerebral cortex (Thurmon et al., 1996). Transduction can be inhibited using local anaesthetics, non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids (Busch et al., 2006).
Local anaesthetics and alpha2- agonists inhibit impulse transmission while pain modulation can be interrupted by subarachnoid or epidural injection of local anaesthetics, opioids or alpha2 agonists (Busch et al., 2006). Pain perception can be inhibited by administration of local anaesthetics, opioids and alpha2-agonists (Kahn, 2005; Busch et al., 2006).
Despite the usefulness of the synthetic analgesics in pain management, their use is often associated with severe side effects in the patient. Opioids have the potential to cause hypoventilation and bradycardia (Dohoo and Dohoo, 1996a; Dohoo and Dohoo, 1996b).
Adverse effects common to NSAIDs include gastric and duodenal ulcers, renal failure 23 and haemorrhage caused by inhibition of prostaglandin synthesis (Grisneaux et al., 1999).
The local anaesthetics cause skeletal muscle toxicities and allergic reaction including hypersensitivity and anaphylaxis (Busch et al., 2006).
The use of crude herbs and plants for alleviating pain has a role in medical practice especially in Chinese and African traditional medicine (Subhuti, 2002). These ethno medicines are relied on by local West African dwellers for their primaty health care since the plant materials used in their preparation are cheap and readily available (Jodi et al., 2008).
There is currently a renewed search for safer analgesics and various researchers have demonstrated the inherent analgesic efficacy and safety of some medicinal plants used traditionally. Thus extracts from plants like Sigmatanthus trifoliatus, Culscasia scandens, Hyptis sauveolens, Lippia advensis, Olax viridis, Synedrella nodiflora,
Pseudocedrella kotschyii, Melanostoma malabathricum, Jatropha curcas and Ficus expasperate have been proved to have antinociceptive properties (Asuzu et al., 1998;
Makonnen et al., 2003; Okoli et al., 2006; Zakaria et al., 2006; Lima et al., 2006; Santos et al., 2007; Musa et al., 2007; Okoli et al., 2008; Woode et al., 2009b). Among plants shown to possess local anaesthetic properties are Corynanthe pachyceras, Picralima nitida, Mitragyna stipulosa, Pausinystalia johimbe, Cassia absus, Erythroxylum coca and
Voacanga africana (Oliver-Bever, 1986).
A wide range of medicinal plants used traditionally are yet to be screened for local anaesthetic activity. One of such plants is Sterculia tragacantha Lindl. (Family
Sterculiaceae) commonly known as “Uhobo” by Nsukka dwellers. It is a medium sized tree seen in the edges of lowland rain forests of Eastern Nigeria (Keay, 1989). The leaves, bark, shoots and seeds are used to prepare ethno medicines for the treatment of diarrhea, dysentery, helminthosis, pulmonary disorders, arthritis, rheumatism, syphilis, leprosy, 24 oedema, gout and whitlow (Iwu, 1993). Its aqueous leaf extract is used by a traditional bone setter for pain relief after closed fracture reduction (Oral communication). The methanol and aqueous leaf extracts of S. tragacantha have been reported to possess significant anti-ulcer, anti-cholinergic, antispasmodic and smooth muscle relaxant properties (Aguwa and Ukwe, 1997).
1.2 RESEARCH OBJECTIVES
1. To screen the methanol leaf extract of Sterculia tragacantha (MEST) for
analgesic and anti-inflammatory activities.
2. To investigate the tissue effect of subcutaneous injection of MEST.
3. To investigate the efficacy of MEST for local anaesthesia in West African
Dwarf goats.
4. To identify the active fraction(s) present in MEST.
5. To identify the phytochemical constituents present in MEST and its fraction(s).
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CHAPTER TWO
LITERATURE REVIEW
2.1 PAIN 26
The term pain is commonly used to describe any unpleasant sensory and emotional experience associated with actual or potential tissue damage (IASP, 1979).
Molony and Kent (1997) however, defined animal pain as an aversive sensory and emotional experience which indicates the awareness of the animal of damage or threat to the integrity of its tissues. Post-operative pain occurs due to tissue damage resulting from the pathological condition; the surgery used to correct the condition or may be a complication of both (Hosking and Welchew, 1985).
Pain is known to have a protective role by minimizing tissue damage (Molony and
Kent, 1997; ACVA, 2000). Acute pain frequently serves to change behaviours thus preventing further tissue damage. The metabolic and functional derangement characteristic of stress responses after an injury may be important for survival in an untreated patient (ACVA, 2000). Unrelieved, however, pain induces suffering which may lead to maladaptive physiological and behavioural responses (ACVA, 2000).
2.2 PHYSIOLOGY OF PAIN
Nociception involves four physiologic processes which include transduction, transmission, modulation and perception (Busch et al., 2006).
2.2.1Transduction of pain
Transduction starts when the free nerve endings (nociceptors) of the primary afferent neurons are exposed to noxious stimuli such as trauma or surgery (Wood, 2008;
Busch et al., 2006). These nociceptors are found in the skin, muscle, viscera, tendons, bones and subcutaneous tissues (Wood, 2008; Hanacek, 2010). Nociceptive nerve fibres include the A-delta fibers and the C fibers. A- delta fibres are large myelinated primary afferents which conduct impulses rapidly (Bullingham, 1985; Hill, 1986; Ferrell and
Koretz, 2010). These fibres are modality specific and carry high threshold mechanical or 27 thermal nociceptive information (Wood, 2008). Pain resulting from the stimulation of A- delta fibers is well localized, sharp, stinging, pricking or shooting (Wood, 2008;
Thamburaj, 2010). This type of pain is referred to as “first” or “fast” pain (Wood, 2008;
Thamburaj, 2010). Type C fibers are small unmyelinated primary afferents which are slow conducting (Wood, 2008; Thamburaj, 2010). These fibres are stimulated by mechanical, thermal and chemical agents (Hill, 1986; Bullingham, 1985). They are responsible for transmission of diffuse aching or burning sensations referred to as “slow” or “second pain” (Wood, 2008; Ferrell and Koretz, 2010).
In addition to noxious stimuli, nociceptors can be sensitized by chemical agents
(algogens) released after local injury (Wood, 2008; Thamburaj, 2010). The chemical irritants which commonly cause pain include potassium ion, hydrogen ion, bradykinin, histamine, prostaglandin, substance P and serotonin (Willis, 1983; Hughes and Lang,
1983; Boothe, 1984; Mckean, 1986; Dray, 1995). Bradykinin is released from plasma kininogens as a result of tissue damage (Boothe, 1984; Thamburaj, 2010). Bradykinin is a strong activator of polymodal nociceptors (Boothe, 1984; Mckean, 1986). It also stimulates histamine release from mast cells and prostaglandin synthesis by cell membrane (Mckean, 1986). Histamine is released from mast cells and promotes vasodilation, swelling and redness (Boothe, 1984; Mckean, 1986). Prostaglandin is released into inflamed tissues producing a long-lasting sensitization of pain receptors to mechanical and chemical stimulation (Boothe, 1984). Prostaglandin also sensitizes nociceptors to algesic substances such as bradykinin and histamine. They also decrease the pain threshold to both chemical and mechanical stimulation (Johnston, 1997).
Stimulation of peripheral nociceptors leads to depolarization and repolarization leading to an action potential and generation of pain impulse (Wood, 2008).
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2.2.2 Transmission of pain
Pain impulses from nociceptors are transmitted by small A-delta fibers and C fibers to the spinal cord (Hanacek, 2010). These primary afferents project to the spinal cord through the dorsal root. On entering the spinal cord, C fibers travel in the lateral side of the dorsal white matter while A-delta fibers travel in the medial side of the dorsal column. A-delta fibers conducting impulses generated from high threshold mechanoreceptors (HTMS) synapse at lamina I and II of the dorsal horn (Thurmon et al.,
1996). However, A-delta and C- fibers transmiting visceral nociception synapse on laminae I and V cells (Thamburaj, 2010). Thus, polymodal cutaneous /somatic and visceral nociceptive impulse converge on laminae I and V (Dubner and Bennett, 1983).
Cutaneous nociceptive afferents terminate in laminae I, II and V (Smith, 1984; Liss
1987). Visceral and muscle nociceptive afferents terminate in laminae I and V. Cells in substantia gelatinosa (lamina II) receive input from HTMS, myelinated mechanothermal
(MMT), A-delta heat nociceptors, C-polymodal receptors and low threshold mechanoreceptors.
There is a synaptic cleft between the terminal ends of the C fibers and A-delta fibers and the nociceptive dorsal horn neurons (NDHN). To enable the transmission of pain impulse across the synaptic cleft to the NDHN, excitatory neurotransmitters which bind to NDHN are released. These neurotransmitters include adenosine triphosphate, glutamate, bradykinin, substance P and nitrous oxide (Wood, 2008).
Pain impulses from the spinal cord travel in the anterolateral spinal quadrant to the thalamus through the spinothalamic tract (Hanacek, 2010; Thamburaj, 2010). The spinothalamic tract divides into the lateral and medial ascending pathways. The lateral pathways include the neospinothalamic, spinocervical and dorsal column postsynaptic tracts. The medial groups include the paleospinothalamic, propriospinal and spino 29 mesencephalic tracts (Thurmon et al., 1996). The neospinothalamic is the major ascending nociceptive pathway in humans and primates. It originates from the spinal cord and terminates in the lateral nucleus of the thalamus. It transmits nociceptive information leading to sensory discriminative aspects of pain (Price and Dubner, 1977). This tract rapidly transmits information about the onset and precise location of an injury, its quality
(sharp or aching), its intensity and duration (Price and Dubner 1977). The paleospinothalamic tract arises from WDR neurons and lies deep to the neospinothalamic tract. This pathway terminates in the reticular formation, the periaqueductal gray matter, hypothalamus and intralaminar thalamic nuclei. It transmits impulses leading to motivational-effective aspect of pain. It is also essential for species- specific behaviours directed towards gaining assistance in healing such as being fed, cared for or groomed (Dennis and Melzack, 1983)
The lateral and medial pathways synapse on neurons in the thalamus. Nerves from the thalamus then relay the pain signals to various areas of the brain where they are processed (Wood, 2008).
2.2.3 Modulation of pain
The process by which the nervous system modifies the nociceptive information to the spinal cord is called modulation (Thamburaj, 2010). It involves changing or inhibiting the transmission of pain impulse to the spinal cord (Wood, 2008). The multiple complex pathways involved in modulation are referred to as the descending modulatory pathways.
Activation of these pathways leads to either an increase in pain impulse transmission
(excitation) or a decrease in transmission (inhibition). Sensitization in the dorsal horn is reduced by the descending inhibitory control (Pud et al., 2009). This effect is termed diffuse noxious inhibitory control (DNIC) which leads to the abation of pain (McCracken et al., 2010). Pain inhibition also comes from the release of endogenous opiods such as 30 endorphins, dynorphins and enkephalins (Freudenrich, 2007). These endogenous opioids prevent the release of some excitatory neurotransmitters such as substance P therefore inhibiting the transmission of pain (Williams, 1986; Wood, 2008).
2.2.4. Perception of pain
Perception is the end result of the neuronal activity of pain transmission and leads to the conscious pain experience (Katz et al., 1992; Wood, 2008). Pain stimuli on getting to the brain stem and thalamus stimulate multiple cortical areas and responses are elicitated. The parts of the brain involved in the interpretation of pain signal include the reticular system, limbic system and somatosensory cortex. The reticular system is important in the integration of pain experience and behaviour. This system is responsible for the autonomic and motor response to pain and warns the individual to do something such as removing a hand when it touches a hot object (Wood, 2008). It also mediates the affective and motivational aspects of pain such as looking at and assessing the injury to the hand when it is removed from the hot object. The limbic system plays an important role in emotional and behavioural responses to pain (Wood, 2008). The Somatosensory cortex is involved in the perception and interpretation of pain sensations. It identifies the type, intensity and location of pain and relates the sensation to previous experiences, memory and cognitive activities (Hanacek, 2010). It also identifies the nature of the stimulus before it triggers a response (Wood, 2008).
2.3 TYPES OF PAIN
The International Association for the Study of Pain (IASP) recommends the description of pain based on its duration, severity and aetiology.
Pain is classified based on its duration as acute or chronic pain. Acute pain is the normal predicted physiologic response to chemical, thermal or mechanical stimulus (Kahn, 31
2005). It may be caused by tissue injury (Freudenrich, 2007). Acute pain stimulates reactive behaviour including evasive measures to avoid or eliminate the stimulus causing the pain (Sandford et al., 1986). This type of pain may be mild or severe (ACVA, 2000).
It can develop slowly or quickly lasting for a few minutes or days and generally improves within 3 days following an event such as surgery (Kahn, 2005). It goes away completely when the injury heals. Acute postoperative and traumatic pain accounts for much of the pain treated by veterinarians (ACVA, 2000). This type of pain responds favorably to analgesics (ACVA, 2000).
Chronic pain is the form of pain that persists after an injury has healed (ACVA,
2000). It occurs occasionally in the abscence of trauma due to chronic pathological processes like osteoathritis (Kahn, 2005). It usually lasts for days, months or even years
(Potthoff and Carithers, 1989). This type of pain is subjective and is not easily described.
Chronic pain is often more difficult to treat compared to acute pain and may require extensive diagnosis and multiple therapeutic approaches (ACVA, 2000). Humans with chronic pain often become depressed and loose weight (Atkinson et al., 1986).
Pain can be classified based on its cause as somatogenic pain, psychogenic pain, nociceptive pain and neuropathic pain (Keay et al., 2000). Nociceptive pain is initiated by the stimulation of somatic or visceral nociceptors. Activation of nociceptors in the skin or superficial tissues induces “superficial somatic” pain. This form of pain is sharp, well defined and localized. “Deep somatic” pain is however initiated by the stimulation of nociceptors in the ligaments, tendons, bones, blood vessels and fascia. This type of pain is dull, aching and diffuse (Keay et al., 2000).
Visceral pain is caused by activation of visceral nociceptors due to infiltration, compression, distension or inflammation (Payne, 1987). Common causes of visceral pain include pancreatic cancer and metastases in the abdomen. The resultant pain is poorly 32 localized, deep and associated with squeezing sensation that may seem to originate from a somatic tissue (Potthoff and Carithers, 1989).
Neuropathic pain is a persistent pain syndrome caused by inflammation or compression of nerve roots either by tumor or invertebral disc extension (Potthoff and
Carithers, 1989). This type of pain is severe and is usually described as vise-like with a burning or electric shock like sensation (Payne, 1987; Paice, 2003).
2.4 LOCAL AND SYSTEMIC RESPONSES TO NOXIOUS STIMULI
Tissue injury leads to local biochemical, metabolic, physiologic and neuroendocrine changes which are intended to be protective.
2.4.1 Local biochemical responses to pain
Local biochemical changes occur due to release of intracellular substances from damaged tissues into the extracellular fluid to induce local pain, tenderness and hyperalgesia (Thurmon et al., 1996). The intracellular substances releasesd include hydrogen ions, potassium ions, serotonin, histamine, prostaglandins and substance P.
(Thurmon et al., 1996). Activation of the peripheral nociceptors by prostaglandins results in transmission of impulse to dorsal horn (Nixon and cummings, 1994; Thurmon et al.,
1996). These transmitted impulses evoke somatomotor and sympathetic segmental autonomic nociceptive reflex responses. Ascending afferent impulses on reaching the brain stem initiate suprasegmental reflex responses and activate the descending modulating system. Segmental and suprasegmental reflexes lead to increased sympathetic tone, vasoconstriction, increased heart rate, increased metabolic rate and decreased gastrointestinal tone (Thurmon et al., 1996; Bonica, 1990).
33
2.4.2 Endocrine responses to pain
Surgery and post operative pain leads to an increased production of catecholamines, cortisol and growth hormones (Bailey and Child, 1987; Bailey and
Stanley, 1990). The most immediate endocrine response to injury is the stimulation of the sympathetic nervous system leading to the release of catecholamines (nor- epinephrine and epinephrine) from the adrenal medulla. Both hormones cause generalized and prolonged systemic response to injury (Breznock, 1980). Nor- epinephrine causes constriction of the vascular smooth muscles. Epinephrine on the other hand activates hepatic glycogenolysis leading to increase in available glucose. It also has a lipolytic effect facilitating the mobilization and utilization of fatty acids as energy sources
(Breznock, 1980). In an experiment carried out to compare the amount of pain induced by methods of branding in cattle, changes in the concentration of plasma epinephrine was used as an indicator of higher acute pain sensation (Lay et al., 1992a). Mean concentrations of plasma epinephrine were observed to be higher for hot branded calves 5 minutes post procedure compared to epinephrine concentration of sham-branded and freeze-branded calves (Lay et al., 1992a).
The endocrine reponse to surgery also leads to increase in catabolic hormones including cortisol (Ellis and Humphery, 1982). Plasma cortisol is an objective indicator of stress and pain in many species (Lay et al., 1992b; Schwartzkopf-Genswein et al., 1998;
Ley et al., 1994; Keita et al., 2010). For example, little change in plasma cortisol was observed in a group of lambs castrated using the rubber ring and burdizzor methods suggesting that the two methods produced minimal pain compared to the other castration methods tested (Kent et al., 1993). Also, post castration of calves, the highest cortisol response was seen in the surgically castration group compared to the burdizzor castrated calves (Robertson et al., 1994). In beef calves, abnormal posture and plasma cortisol were 34 recorded in the first three hours after castration (Molony et al., 1995). In pigs, surgical castration has been shown to induce an increase in plasma cortisol through the activation of the adrenal medulla (Prunier et al., 2006; Carroll et al., 2006; Llamas Moya et al.,
2006).
2.4.3 Metabolic responses to pain
Changes in the body’s sources of energy occur following injury. Cell metabolism requires glucose for oxidation. In a normal animal, glucose transport across many cell membranes is insulin dependent. With injury, the rate of removal of glucose from the plasma no longer varies as the square of glucose concentration but is independent of it
(Cutherbertson, 1976). Glucocorticoids, ACTH, growth hormones, glucagon and catecholamines all modify carbohydrate metabolism and induce hyperglycemia and carbohydrate intolerance (Allison et al., 1976). Blood glucose level thus rises transiently but returns to normal by the second day post trauma (Breznock, 1980). Also significant trauma often interferes with eating which compounds caloric starvation. To compensate for the caloric deficit, the body quickly mobilizes fat and protein and converts them to usable energy sources (Kinney, 1976; Davies and Liljedahl, 1976).
2.4.4 Behavioural responses to pain
Behavioural changes are often used to assess the degree of pain and discomfort in animals (Sammarco et al., 1996; Martini et al., 2000). Animals cannot describe pain sensations thus the veterinarian can only identify their patients’ pain through the knowledge of normal and abnormal behaviour (Morton and Griffin, 1985; Sandford et al.,
1986). Species specific and individual response to pain however varies, thus it is essential that the veterinarian evaluating an animal for pain must have a thorough knowledge of typical species specific and individual behaviours. The animal’s owner or handler may also be able to recognize subtle behaviours indicative of pain which may otherwise go 35 unnoticed (ACVA, 2000). The following are some examples of behaviours indicative of acute pain:
Change in personality or attitude: A normally docile animal may become
aggressive or an aggressive animal becomes quiet. Sheep in pain may become
lethargic and shows abnormal flock behaviour such as lagging behind or separates
itself from the flock (Dowd et al., 1998; Shafford et al., 2004).
Abnormal vocalization: Animals vocalize when a painful area is palpated or are
forced to move (Wright, 2002). Teeth grinding is often heard in rabbits, cattle,
sheep and goats experiencing pain (Church, 2000). However, vocalization appears
to be an insensitive and non specific behaviour indicative of pain and should not
be relied on as a sole criterion for determing whether an animal is in pain.
Change in posture or ambulation: Change in posture may be an attempt to lessen
pain by avoiding further stimulation of the injured tissue (Graham et al., 1997).
Pain may lead to subtle changes in behaviour or posture for example licking or
carrying of a painful appendage. Sheep may become lame on an operated limb
when walking (Shafford et al., 2004).
Changes in activity level: Increased frequency of restlessness is a widely accepted
response to pain (Wood et al., 1991). An animal may become restless and pace. It
may also repeatedly lie down, get up and lie down again. Some animals on the
contrast may be recumbent and lethargic or reluctant to move with guarding of the
painful area (Church, 2000). Sheep may however stand or become recumbent
showing little response when gently prodded (Shafford et al., 2004).
Changes in appetite: Decrease in food and water consumption may occur. Sheep
in pain have been shown to become lethargic with depressed appearance and
reduced or complete loss of appetite (Shafford et al., 2004). 36
Changes in facial expression: The eyes of sheep in pain may become dull, fixed
and staring or even closed (Shafford et al., 2004). The ears may also become
dropped (Shafford et al., 2004).
2.5 ANALGESIA
Analgesia strategies currently employed in pain management include preemptive analgesia, multimodal analgesia and post operative analgesia (Busch et al., 2006).
Preemptive analgesia refers to the administration of analgesics prior to exposing the patient to noxious stimuli (Sibanda et al., 2006; Tobias et al., 2006). Stimulation of peripheral nociceptors with surgical trauma can induce peripheral hypersensitivity and upregulation of central neuronal activity leading to prolonged and severe pain response with further manipulation of the site (Lascelles et al., 1994; Lamont, 2002). Once the neuronal pathways are sensitized, the physiological and behavioural pain responses persist even when the nerves are transected or blocked (Lascelles et al., 1994).
Preemptive analgesic administration is thus aimed at preventing the establishment of spinal and central sensitization thus reducing severity of inflammation and pain (Katz et al., 1992; Tverskoy et al., 1994; Sibanda et al., 2006). It may also reduce an animal’s need for post operative analgesia (Woolf and Chong, 1993; Raffe, 1997; Pascoe, 2000;
Tobias et al., 2006). It also eliminates hyperalgesia and allodynia which are major component of post operative pain (Grineaux et al., 1999).
Analgesia strategies that include opioid analgesics, non steroidal anti inflammatory drugs and local analgesics have been widely used in veterinary patients
(Mathews, 2000; Pascoe, 2000; Budsberg, 2002; Muir, 2002; Wagner, 2002).
37
2.6 OPIOID ANALGESICS
Opioids have long been the drugs of choice for control of moderate and severe acute post operative pain (Grisneaux et al., 1999; Slingsby et al., 2006., Busch et al.,
2006). The term “opioid” is used to refer to all exogenous synthetic compounds that bind to specific opioid receptors (Thurmon et al., 1996; Kahn, 2005). These analgesics induce analgesia by binding to specific opioid receptors in both the central and peripheral nervous system (Busch et al., 2006). Opioids work at different locations inhibiting nociceptive signal transduction, modulation and perception (Busch et al., 2006). Based on the studies of Martin et al. (1976), the existence of three types of opioid receptors have been described viz the mu, kappa and delta opioid receptors. The mu and kappa opioid receptors are primarily responsible for producing analgesia (Busch et al., 2006). The effects of the morphine–like drugs appear to be mediated through the mu receptors. The enkephalin endogenous opioids and some opioid drugs selectively bind to the delta receptors. The delta receptor seems to mediate analgesia solely at the spinal level (Jiang et al., 1991; Mattia et al., 1991). The kappa (K) receptor is involved in both spinal and supraspinal antinociception (Millan, 1990). An opioid can interact with one or more types of opioid receptor. Drugs that bind to a receptor and cause expression of activity are called agonists while those that bind to receptors and block their activity are antagonists
(Busch et al., 2006). There are further sub classifications of opioids as pure agonists, pure antagonists, partial agonists and agonist antagonists (Jaramillo et al., 2006; Busch et al.,
2006). Pure agonists bind and stimulate all types of opioid receptors causing profound analgesia (Busch et al., 2006). Partial agonist opioids have affinity for specific opioid receptor where they show agonist activity but their maximal effect is less when compared with pure agonist (Roughan and Flecknell, 2002; Busch et al., 2006). Agonist antagonist opioids bind to more than one type of opioid receptor causing an effect on one type but 38 blocking effects at another receptor (Busch et al., 2006). Their analgesic activity is however low and they do not produce the same degree of analgesia as the pure agonist.
Commonly used opioid analgesics include the following:
2.6.1 Morphine sulfate
Morphine is an agonist opioid capable of producing very high degree of analgesia but of moderate duration (Busch et al., 2006). It relieves pain without blocking motor activity or conciouseness (Branson and Gross, 2001). Injection of 0.1 to 0.3mg/ kg morphine i.m will provide good analgesia in most species of animal (Hall et al., 2001b).
2.6.2 Fentanyl citrate
Fentanyl is a pure mu opioid agonist (Almeida et al., 2007). It is about 100 times more potent than morphine when given i.v but four times more potent when injecetd intrathecally (Yaksh et al., 1986; Palmer et al., 1998). It is more lipid soluble than morphine thus has a more rapid onset and shorter duration of action (Hug and Murphy,
1981). A single injection of fentanyl is used to produce short duration analgesia during surgery (Sano et al., 2006). To prolong its effect, injections are given repeatedly and this often leads to respiratory depression (Freye et al., 1991; Duke et al., 1994). To avoid this side effect, transdermal fentany patches (TDF) are used in veterinary patients for the alleviation of post operative pain (Schulthesis et al., 1995; Kyles et al., 1998., Robinson et al., 1999; Muriel et al., 2005; Egger et al., 2007). The use of these patches reduces the overall consumption of analgesics (Hofmeister and Egger, 2004). This analgesic delivery system is also non- invasive, ensures continuous delivery of the drug, allows long duration of analgesia, is easy to apply and is well tolerated by animals (Frank et al., 2000;
Lee et al., 2000; Gellasch et al., 2002)
39
2.6.3 Buprenorphine hydrochloride
This drug is a potent partial agonist opioid derived from thebaine (Roughan and
Flecknell, 2002; Dobbins et al., 2002; Stewart and Martin, 2003; Giordano, 2010). It partially binds to the mu receptor with great affinity (Busch et al., 2006). It is 20-30 times more potent than morphine (Martin, 1994). It thus produces analgesia of less intensity but of a longer duration lasting 8 to 12 hours (Busch et al., 2006). It is an effective opioid analgesic in both small and large animals (Roughan and Flecknell, 2002; Dobbins et al.,
2002; Stewart and Martin, 2003).
2.6.4 Butorphanol tartrate
This is a centrally acting agonist antagonist opioid used mostly in large animals
(Troncy et al., 1996; Skarda and Muir, 2003). It binds and activates kappa receptors to produce analgesia and sedation. It also binds to mu receptors antagonising these receptors
(Busch et al., 2006). It is 5 times more potent than morphine, 30 times more potent than pentazocine and 40 times more potent than meperidine (Vandam, 1980; Martin, 1994). It is indicated for the relief of moderate to severe pain (Martin, 1994). Doses of 0.1 to
0.5mg/kg given either i.m or s.c have been shown to produce effective analgesia (Garcia-
Pereira et al., 2007).
2.6.5 Pentazocine lactate
Pentazocine is an agonist antagonist with its primary agonist effect at the kappa receptors and weak antagonist effect at the mu receptors (Bailey and Stanley, 1994). Its analgesic potency is approximately one half to one fourth that of morphine. It is however about five times more potent than meperidine. Doses of 1 to 3mg/kg have been shown to give 3 hours of pain relief (Taylor and Houlton, 1984; Sawyer and Rech, 1987).
Pentazocine is used for control of colic pain in horses at a dose of 0.33mg/kg i.v followed
10-15 minutes later by a similar i.m dose. 40
2.7 NON- STEROIDAL ANTI-INFLAMMATORY DRUGS
Non- steroidal anti-inflammatory drugs (NSAIDs) are a group of drugs that are chemically unrelated but share certain therapeutic actions and side effects (Insel, 1990).
They are widely used to treat fever, inflammation and pain in most species (Busch et al.,
2006; Altaher et al., 2006). For maximum effect, they are administered preemptively to suppress inflammation (Busch et al., 2006). They can be combined with opioids to provide excellent multimodal analgesia (Busch et al., 2006). Aspirin is the prototype thus these compounds are often referred to as aspirin like drugs.
2.7.1 Inflammatory process
Inflammation is a fundamental protective response to noxious stimulation (Jones and Hamm, 1977). Its cardinal signs of heat, swelling, redness and pain reflect hyperemia, exudation and cellular infiltration are brought about by a coordinated action of substances released locally in response to trauma (Jones and Hamm, 1977; Snow, 1981).
The acute phase of inflammation is characterized by redness (hyperaemia) and heat which occurs due to vasodilation of cutaneous arterioles a response mediated by histamine, bradykinin and prostaglandins (Jones and Hamm, 1977., Snow, 1981). Heat has a pronounced effect on the inflammatory process since it leads to increased blood flow, enhanced supply of nutrients and oxygen, elimination of waste and noxious stimuli.
Swelling or edema is a result of exudation which results from increased permeability of the vascular bed especially the venules. The first phase of exudation is histamine mediated and lasts for a few minutes. Histamine along with heparin and serotonin are released at this time from mast cells, basophils and platelets. This phase is suppressed by histamine antagonists eg cathecholamines stored in cells away from the site of inflammation (Jones and Hamm, 1977). A second and more prolonged histamine independent phase ensues after a few hours and is induced by mediators such as kinins 41
(bradykinin, lysylbradykinin and methiony-lysyl-bradykinin), prostaglandins and
components of the complement system. Pain in this phase of inflammation is induced by
small amounts of prostaglandins released during inflammation which slowly sensitizes
the pain receptors to bradykinin, histamine and possibly to other prostaglandins and
thromboxanes (Snow, 1981).
The chronic phase of inflammation is however characterised by cellular
infiltration (monocyte) and fibroblast proliferation and exudation (Dunne, 1990).
Proliferation becomes widespread by proliferation of small vessels or granuloma
(Hosseinzadeh et al., 2000). This phase is also characterized by change in ground
substance, phagocytosis of necrotic cell, invasion of capillaries, fibroplasia and
regeneration (Jones and Hamm, 1977)
2.7.2 Mechanism of action of NSAIDs
NSAIDs interfer with pain perception peripherally by inhibiting pain input to the
peripheral nerve endings. This is achieved by inhibiting cyclo-oxygenase, the enzyme
responsible for the conversion of arachidonic acid to prostaglandin and thromboxane
precursor cyclic endoperoxidase (Martin, 1994; Lees et al., 2004; Keita et al., 2010).
Prostaglandins are known to cause hyperaemia, modulate inflammation and sensitise
nerve endings (Snow, 1981). Thromboxanes however function in aggregation of platelets
(Duncan et al., 2007).
Two distinct COX isoforms (COX 1 and 2) which are products of separate genes have been identified (Warner and Mitchell, 2004). COX 1 is expressed in most tissues leading to the production of prostaglandins (Duncan et al., 2007). COX-2 is expressed at sites of inflammation in response to inflammatory mediators such as interleukin-1 (Duncan et al., 2007). The therapeutic drug effects are mainly mediated via COX-2 inhibition while the unwanted side effects especially renal and gastro intestinal damage occur due to COX-1 42 inhibition (Lees et al., 2004). Aspirin like drugs such as ibuprofen, diclofenac and salicylates are COX-1 inhibitors. Newer NSAIDs called COX-2 selective inhibitors have been developed and inhibit only COX -2 with few incidences of gastrointestinal side effects
(Anon, 2002). COX-2 selective inhibitors include celecoxib, etoricoxib, lumiracoxib parecoxib, rofecoxib and valdecoxib (Krumholz et al., 1995; Julian et al., 1996).
Commonly used NSAIDs include
2.7.3 Acetylsalicylic acid (Aspirin)
This drug is one of the most commonly used NSAIDs in veterinary medicine (Hall
et al., 2001b). It acts as an analgesic peripherally (Hall et al., 2001b; Ferreira, 1979). A
direct effect on the CNS may also be involved in its analgesic effect (Insel, 1990). Aspirin
is primarily used for the relief of mild to moderate pain associated with inflammatory
joint diseases (Potthoff and Carithers, 1989; Martin, 1994). It may be given
preoperatively or after the first 48 to 72 hours post operatively when the most severe pain
has waned (Hall et al., 2001b). Aspirin and other NSAIDs have been combined with
opioids for the treatment of chronic pain.
2.7.4 Phenylbutazone
Phenylbutazone is widely used in horses to alleviate minor degress of pain due to
lameness (Boothe, 1984; Potthoff and Carithers, 1989; Hall et al., 2001b). Its analgesic
and antipyretic actions are similar to that of aspirin. However, its anti-inflammatory
property resembles those of the corticosteroids.
2.7.5 Flunixine meglumine
Flunixine is a potent NSAID with analgesic and antiflammatory properties
(Boothe, 1982). It is most commonly used for the treatment of endotoxic shock (Hardie et
al., 1983) and in the management of visceral pain (Hall et al., 2001b).
43
2.7.6 Indomethacin
Indomethacin has prominent anti-inflammatory and analgesic- antipyretic properties similar to those of salicylates (Insel, 1990). It is a potent inhibitor of cyclooxygenase. It also inhibits the motility of polymorphonuclear leucocytes (Insel,
1990). Its anti- inflammatory effect is evident in the treatment of rheumatoid and other arthritic conditions. In laboratory animals, 10 mg/kg indomethacin is used to relieve pain as well as acute inflammation (Choi and Hwang, 2004; Narendhirakaanan et al., 2007)
2.7.7 Ketoprofen
Ketoprofen is a propionic acid derivative (Grisneaux et al., 1999). It is approximately 15 times more potent than phenylbutazone and 30 times more potent than aspirin. It is a potent inhibitor of cyclooxygenase with some in-vitro inhibitory effect on lipoxygenase and bradykinin synthesis (Kantor, 1986). Therefore, it inhibits the synthesis and release of prostaglandin and to some extent synthesis of leukotrienes leading to a peripheral analgesic effect common to most NSAIDs (Vane, 1971). Ketoprofen also provides analgesic effect on the central level (Willer et al, 1989; De Beaurepaire et al.,
1990). Ketoprofen is a common NSAID antipyretic and analgesic used in horses and other equines. It is commonly used in dogs and cats following surgical procedures and in the treatment of musculoskeletal, joint and soft tissue pain.
2.7.8 Carprofen
Carprofen is the latest NSAID to be introduced into veterinary practice. It has been hypothesized to have a central action (Mckellar et al., 1990; Fox and Johnston,
1997). The drug is a weak inhibitor of cyclooxygenase and lipoxygenase. It is approved for preoperative use and appears to be safer than ketoprofen due to poor inhibition of
COX (Vane and Botting, 1996; Rickets et al., 1998). It is an effective long acting 44 analgesic (Nolan and Reid, 1993; Lascelles et al., 1994; Slingsby and Waterman-Pearson,
2001)
2.7.9 Diclofenac
This is a phenyl-acetic NSAID widely used in veterinary practice (Ramesh et al.,
2002). It is effective in the management of post-operative pain and lameness in horses
(Singh et al., 2001; Bertone et al., 2002). It has been used in the treatment of acute aseptic arthritis and myositis in cattle and buffaloes (Mahajan et al., 1994; Gupta et al., 2001).
Diclofenac has also been reported to reduce pain suffered by castrated calves (Graham et al., 1997; Molony et al., 1997).
2.8 LOCAL ANAESTHETICS
These drugs are used clinically to block pain sensation from specific areas of the body (Miller, 1998). The action of these drugs is reversible and no structural damage to nerve fibers or cells occurs (Ritchie and Greene, 1990; Mama and Steffey, 2001). The relative cost and minimal systemic absorption of local anaesthetic solution make them ideal alternatives or adjunts to opioids and NSAIDs (Wolfe et al., 2006).
2.8.1 General properties of local anaesthetics
Local anaesthetics have similar molecular configuration consisting of a lipophilic aromatic ring connected to a hydrophilic amine ring by a linkage chain (McLure and
Rubin, 2005). The linkage chain may be ester, amide, ketone or ether chain (Haddox and
Baumann, 1994; Strichartz and Berde, 1994; McLuren and Rubin, 2005). The nature of this bond determines certain of the pharmacological properties of these agents (Ritchie and Greene, 1990). Clinically important local anaesthetics are divided into two distinct chemical groups based on these intermediate chains as aminoesters or aminoamides. The aminoesters are anaesthetics with an ester link between the aromatic amide ends. 45
Procaine, chloroprocaine, tetracane and benzocaine belong to this group. The aminoamides have amide link between the aromatic and amine ends. Lignocaine, mepivacaine, bupivacaine, ropivacaine, etidocaine and prilocaine are in this class.
2.8.2 Mechanism of action of local anaesthetics
Local anaesthetics inhibit transduction and transmission of nerve impulse (Busch et al., 2006). They also modify the pain signals at the spinal cord (Busch et al., 2006).
They inhibit the generation and conduction of nerve impulses. Their main site of action is the cell membrane (Ritchie and Greene, 1990). They block the generation and transmission of nerve impulse by blocking Na+ in the neurons cell membrane (Busch et al., 2006). This slows the rate of depolarization of neuronal cell membrane and prevents the threshold potential from being reached (Busch et al., 2006). Nerve conduction eventually fails.
2.8.3 Clinical pharmacology of local anaesthetics
Local anaesthetics can produce temporary but complete anaesthesia of well defined body areas. The clinical important properties of these agents include the following: i. Anaesthetic potency
Lipid solubility plays a key role in the anaesthetic potency of local anaesthetics.
Smaller and more lipophilic molecules interact faster with sodium channel receptors (Hall et al., 2001a). The relative potencies of agents as determined in invivo preparation are highly dependent on intrinsic factors as well as anatomic and physicologic factors
(Strichartz et al., 1990). Water solubility (hydrophilicity) is also important for diffusion to the site of local anaesthetic action
46 ii. Onset of anaesthetic action
The onset of action of a local anaesthetic invitro has been shown to depend on the agent’s physicochemical properties. Invitro however, the onset of action of a local anaesthetic depends on the following:
A .Dose and concentration of the anaesthetic agent
The use of greater volume of the anaesthetic as well as solutions of high concentration increases the number of anaesthetic molecules in the nerve region (Hall et al., 2001a). These facilitate a more rapid onset of action and increase the duration of anaesthetic action. Injection of large volumes of these agents into the epidural or intrathecal space influences the spread of the agents. b. Carbonation and pH adjustment
In the isolated nerve preparation, addition of bicarbonate to the local anaesthetic solution lead to a more rapid onset of nerve blockade at a reduced anaesthetic concentration (Wong et al., 1993). The addition of bicarbonate is aimed at increasing the
PH of the solution. This leads to increase in the amount of the drug in uncharged base form thus leading to a faster diffusion of the anaesthetic through the cell membrane. This will lead to a faster onset of action. c. Use of hyaluronidase
Addition of hyaluronidaise is believed to enbance the diffusion of local anaesthetic to their site of action. However, there is increased risk of toxicity due to enhanced systemic absorption. iii. Duration of anaesthetic action
Invivo, the duration of action of a local anaesthetic is determined by 47
The anaesthetic’s action on the nerve and also by its action on local blood vessels.
At low concentration, local anaesthetics tend to cause vasoconstriction whereas
their clinical doses cause vasodilation.
The site of injection: The duration of action of a local anaesthetic varies inversely
with the absorption of the drug from the site of injection. Hence the shortest
duration of action is often seen after intrathecal administration and the longest
duration following peripheral nerve blocks.
Use of a vasoconstrictor: Addition of a vasconstricitor to a solution of a local
anaesthetic lowers the rate of diffusion thus delaying the rate of vascular
absorption of the agent. This effect in the long run leads to a prolongation of the
anaesthetic action
Pregnancy: Plasma cholinesterase activity is reduced during gestation and this
influences the duration of action of ester local anaesthetic. In pregnant patients,
the spread of epidurally injected local anaesthetic and the depth of anaesthesia are
also reported to be greater. A decrease in the size of epidural space due to
enlarged epidural vessels as well as higher progesterone level during pregnancy
have been incriminated as possible factors leading to these effects.
2.8.4 Side effects of local anaesthetics
Local anaesthetics interfere with function of all organs in which conduction and transmission of impulses occur with important side effect in the central nervous system and all forms of muscle (Covino, 1987; Garfield and Gugino, 1987; Gintant and Hoffman,
1987). They have been shown to depress contraction of the intact bowel and strips of isolated intestine (Zipf and Dittmann, 1971). They cause relaxation of vascular and bronchial smooth muscle (Covino, 1987). Spinal and epidural use as well as instillation of local anaesthetic into the peritoneal cavity leads to paralysis of sympathetic nervous 48
system with consequent increase in the tone of GIT musculature. These agents may also
increase the resting tone and decrease the contraction of isolated human uteri muscle.
The degree of tissue reaction caused by local anaesthetics has been assessed
experimentally. Direct injection of local anaesthetics into the muscle of rats has been
reported to cause intense inflammatory reaction leading to muscle necrosis (Benoit and
Belt, 1972). Basson and Carlson (1980) also reported skeletal damage after the use of
clinically recommended doses of these agents for local infiltration. According to De
Carvalho et al. (1976), Redd et al., 1990, Ribeiro et al. (2003) and Berto et al., (2011),
local anaesthetics with acidic pH (3.3-5.5) can cause local tissue irritation resulting in
inflammation after subcutaneous injection. Lignocaine has been shown to cause the least
inflammatory reaction while bupivacaine caused the most intense tissue reaction (Ribeiro
et al. 2003). Cassuto et al. (2006) reported that clinical doses of lidocaine and other local
anaesthetics studied showed anti-inflammatory activity inhibiting phagocytosis as well as
inflammatory mediators. Berto et al. (2011) opinned that local anaesthetics with pH (pH
5.5-7) close to physiologic pH do not cause tissue reaction
At clinical doses neuron toxicity is rarely seen following the use of these agents.
However it has been shown that high concentration of the local anaesthetic agents may
produce irreversible blockage of neuronal function (Strichartz and Berde, 1994).
Local anaesthetic agents used clinically include the following:
2.8.5 Procaine hydrochloride
Procaine is an ester local anaesthetic (Haddox and Baumann, 1994). It does not readily penetrate the mucous membrane thus it is not effective as a surface or topical anaesthetic. It is used for infiltration, nerve block and spinal anaesthesia (Haddox and
Baumann, 1994). For infiltration a concentration of 1% is used in small animals while 2% solutions are preferred in large animals. For nerve block, 0.5 to 2% solution is used. It 49 induces anaesthesia of a very brief duration because it is absorbed rapidly and destroyed quickly by plasma cholinesterase. To prolong its action vasoconstrictors are often added to solution of procaine to delay its absorption from the site of injection.
2.8.6 Lignocaine hydrochloride
This drug is the most widely used local anaesthetic in veterinary practice (Gray,
1986). Lignocaine is chemically N- diethylaminoacetyl 2,6-xylidine hydrochloride. It is an amide type local anaesthetic and is well tolerated (Haddox and Baumann, 1994).
Lignocaine HCl is very soluble in water and alcohol. It is presented as injections, ointments, jelly, topical solutions and topical aerosol. Solutions are often marketed in 0.5 to
2% concentration while some are sold as plain lignocaine solutions. Lignocaine solutions containing epinephrine are also available. Lignocaine has a more rapid onset of action, more intense and longer duration of action compared to procaine (Woolf and Chong, 1993;
Haddox and Baumann, 1994). Its spread through tissues is greater than that of procaine and injections made near nerve trunks penetrate effectively. Lignocaine has marked local anaesthetic activity when applied to the mucous membrane or the cornea. The drug is rapidly absorbed from tissues and mucous surfaces. To limit the penetration and toxicity of lignocaine, vasoconstrictors are added to solutions of lignocaine. This reduces the rate of its systemic absorption and increases the duration of action.
2.8.7 Mepivacaine
Mepivaciane is an amide type local anaesthetic. It has similar pharmacological effects as those of lignocine but is less toxic (Gray, 1986). Its action is more rapid in onset and more prolonged than that of lignocaine (Haddox and Baumann, 1994). It is marketed as
1%, 2%, 3% and 5% solution without vasoconstrictor. Its 2% solution often contains levonordefin as a vasoconstrictor to increase its effectiveness (Gray, 1986). Mepivacaine is used in infiltration and regional nerve block. It is the drug of choice in the diagnosis of 50 equine lameness because there is lower incidence of post injection edema after its use compared to lignocaine. Mepivacaine is however not indicated for obstetrical anesthesia due to its prolonged action which may affect the fetus.
2.8.8 Bupivacaine hydrochloride
This is also an amide local anaesthetic chemically similar to mepivacaine and
lignocaine (Gray, 1986). The drug is very stable and does not deteriorate or loose its
potency on boiling (with strong acid or alkali) or after autoclaving. Bupivacaine is
approximately four times more potent and more toxic than mepivacaine and lignocaine
and is well tolerated in goats (Gray, 1986; Haddox and Baumann, 1994). It has
particulary prolonged duration of action (Haddox and Baumann, 1994). It is available in
0.25, 0.5 and 0.75% solution with or without epinephrine (Haddox and Baumann, 1994).
It is commonly used for regional and epidural nerve blocks. It is the drug of choice for
obstetrical anesthesia since it does not cause significant sensory and motor blockage.
2.8.9 Ropivacaine hydrochloride
This is a new long acting amide local anaesthetic. The drug is the (S) enantiomer of a chain shortened homologue of bupivacaine. It is believed to have a wider safety margin than bupivacaine (Reiz et al., 1989). Its physicochemical properties are similar to those of bupivacaine. It is however less lipid soluble than bupivacaine (Rosenberg and Heinonen,
1983; Rosenberg et al., 1986). Its duration of action is similar to that of bupivacaine but it is less potent (Haddox and Baumann, 1994).
2.9 LOCAL ANAESTHETIC TECHNIQUES USED IN GOATS
For economic reasons, most abdominal surgeries in ruminants are performed
under local anaesthesia using infiltration, regional block and epidural block techniques
(Buback et al., 1996; Quandt and Rawlings, 1996; Sammarco et al., 1996; Winkler et al., 51
1997; Duke, 2000; Chevalier et al., 2004). Local anaesthetic techniques commonly used in goats include:
A. Infiltration anaesthesia
This technique involves the injection of small volumes of local anaesthetic solution into the skin, subcutaneous tissues or deeper structures (Ritchie and Greene,
1990; Haddox and Baumann, 1994; Hall et al., 2001a). By this method, the nerve fibres are affected at the actual site of injection. The drug also diffuses into surrounding tissue from the site of injection and anaesthetizes nerve fibres and endings. In ruminants the line block, inverted “L” or “L” block is commonly used for abdominal surgeries such as rumenotomy, cecotomy and ceasarean section (Skarda, 1986). Most minor surgeries of the digits, penis or teats can be performed under infiltration anaesthesia
Local anaesthetic solutions are also infiltrated into the testicles to allow castration.
This enables quick castration, reduces stress and provides post-operative analgesia (White et al., 1995; Nyborg et al., 2000; Haga and Ranheim, 2005).
The local anaesthetic solutions most frequently used in veterinaty practice are lignocaine, mepivacaine and bupivacaine (Gray, 1986). In large animals 2% solutions of lignociane are used while 0.125 to 0.250% bupivacaine is recommended (Hall et al.,
2001a). When used with epinephrine up to 4.5 mg/kg of lignocaine or 2.5 mg/kg bupivacaine may be used (Ritchie and Greene, 1990).
Infiltration anaesthesia produces good anaesthesia without interfering with the normal body function (Ritchie and Greene, 1990). It however, requires the use of relatively large volumes of drugs to anaesthetize small body areas predisposing the animal to systemic toxic reactions (Ritchie and Greene, 1990).
52
B. Peripheral nerve block/conduction block
This type of block is produced by injection of a local anaesthetic into the
immediate vicinity of a peripheral nerve /plexus (Hall et al, 2001a). This technique uses
small amounts of drugs to produce wide areas of anaesthesia (Ritchie and Greene, 1990).
Blockage of mixed peripheral nerves and nerve plexuses also leads to desensitization of
somatic motor nerves (Ritchie and Greene, 1990).
Paravertebral anaesthesia is a useful form of this technique and is often performed
to provide anaesthesia for caesarean section or laparotomy in goats or sheep (Gray, 1986).
Proximal paravertebral block involves the desensitization of T13, L1 and L2 paravertebral
nerves (Gray, 1986). The distal paravertebral block involves the desensitization of the
dorsal and ventral rami of the spinal nerves T13, L1 and L2 at the distal ends of L-1, L-2
and L-4 (Riebold et al., 1980). The use of paravertebral anaesthesia offers a major
advantage over field infiltration in that the abdominal wall as well as the peritoneum is
more likely to be uniformy desensitized.
C. Intravenous regional anaesthesia
This is a simple and safe technique used to produce anaesthesia for operations of the digit in cattle, small ruminants and pig (Weaver, 1972; Skarda, 1986). It involves the injection of large volumes of dilute local anaesthetic into a peripheral vein (Elmore, 1980;
Knight, 1980).
D. Caudal epidural block
Administration of a local anaesthetic epidurally is an established procedure for providing regional anaesthesia in many animal species (Keegan et al., 1995). Caudal block entails the injecting a local anaesthetic into the epidural space through the sacrococcygeal space (Gray, 1986). Caudal epidural anaesthesia is considered an extremely safe procedure
(Cruz et al.,1997). When it is properly performed, it can be used efficiently for obstetrical 53 and surgical intervenetions in the perineal region of large animals (Skarda, 1986; Marsico et al., 1999; Almeida et al., 2007). The extent of anaesthetic action is dependent on the spread of the drug and its diffusion to neural tissues from the site of injection. This technique is also used to provide long lasting post operative analgesia (Raffe, 1997; Pascoe,
2000; Smith and Kwang-An, 2001).
E. Lumbosacral epidural block
This block can be produced by the injection of local anaesthetic solution into the epidural space through the lumbosacral space (Hall et al., 2001c). This leads to complete analgesia and paralysis of the hindlimbs and abdomen to allow surgery (Trim, 1989).
The ideal local anaesthetic for epidural use should have a rapid onset of action, good analgesia and muscle relaxation (Howell et al., 1990). Lignocaine is the most frequently used although mepivacaine, bupivacaine and procaine are also used (Day and
Skarda, 1991). Bupivacaine has a long duration of action but its onset is slow and muscle relaxation is poor (Covino, 1986; Howell et al., 1990). With the exception of bupivacaine, the aforementioned agents provide relatively short duration of anaesthesia and blocks sensory, motor and sympathetic fibers (Day and Skarda, 1991). Thus other drugs like opioids and alpha2-adrenoceptor agonists are used epidurally (Valverde et al., 1990; Gross,
1993; Raffe and Tranquilli, 1993; Skarda and Muir, 1994; Gomez de Segura et al., 1998).
Opioids are administered in the epidural space in an attempt to induce long lasting analgesia and muscle relaxation (Bradley et al., 1980; Pascoe, 1992; McMurphy, 1993).
The systemic effect seen following the use of opioid epidurally is dependent on the lipophilic nature of the opioid used. In order to produce an effect after epidural injection, opioids must diffuse through the dura mater into the dorsal horn (Jones, 2001). The analgesia produced by epidural opioids is believed to be through the binding of these drugs to the opioid receptors in the dorsal horn of the spinal cord (Cousins and Mather, 1984; 54
Gustafsson, 1990). They are believed to prevent the release of substance P from the pre- synaptic sites. They thus abolish nociception without exerting any significant effect on motor function (Cousins and Mather, 1984). Opioids which have been used epidurally include morphine, pethidine, fentanyl, sufentanil, butorphanol and methadone (Bonath and
Saleh, 1985; Greene et al., 1990; Hosgood, 1990; Sawyer et al., 1991; Palmer et al., 1998;
Cohen et al., 1998; Lejus et al., 2000; Jones, 2001). Morphine was the first opioid used epidurally and is still the most useful opioid for epidural use due to its high potency and long duration of action of about 12 to 24 hours (Popilskis et al., 1993; Jones, 2001). The use of opioids however leads to respiratory depression, excessive sedation, vomiting and pruritis (Wood et al., 1994). The combination of local anaesthetics and opioids are thus used to minimize these effects (Kaneko et al., 1994; Christopherson et al., 1993).
Xylazine given epidurally produces selective inhibition of nociceptors. Alpha2 receptors are located in the dorsal horn neurons of the spinal cord where they inhibit the release of of nor-epinephrine and substance P thus decreasing neuronal activity and inhibiting rostral transmission of nociceptive impulse (Rang and Urban, 1995; Buerkle and
Yaksh, 1998; Prado et al., 1999). Epidural administration of xylazine has been proved to provide effective analgesia in sheep (Eisenach et al., 1986; Waterman et al., 1987; Eisenach and Grice, 1988). In cattle, xylazine given epidurally has been shown to produce perineal and flank analgesia (Caulkett et al., 1993; Rehage et al., 1994; Mosure et al., 1998; Gomez de Segura et al., 1998; Prado et al., 1999).
2.10 MEDICINAL PLANTS WITH ANALGESIC PROPERTIES
Treatment of pain is a primary function of all medical systems (Subhuti, 2002).
Throughout history, herbs have been used as analgesic substance to alleviate pain. One of 55
such plants was poppy from which opium was later isolated. The first anti-inflammatory
drug aspirin was also developed from a plant (willow)
The use of crude herbs for alleviating pain plays a major role in medical practice
in China and Africa. These herbs are primarily used in the treatment of chronic or recurrent
pain in patients. Also plants with anti-inflammatory properties are used traditionally for
pain relief since reduction of inflammation often brings secondary pain relief.
Plants in the families Capparaceae, Caesalipinoideae, Combretaceae, Tiliaceae,
Mimosoideae, Rutaceae, Sterculiaceae, Rutaceae, Polygalaceae, Rhamnaceae,
Verbenaceae, Bombacaceae, Annonaceae, Fabaceae, Euphorbiaceae, Maraceae,
Anacardiaceae, Araceae, Lamiaceae, Liliaceae and Olacaeceae are known to have analgesic
properties.
2.10.1 Plants used in traditional pharmacopoeia for analgesia
The following plants are used in traditional pharmacopoeia for analgesia.
Plant Family Part used Acacia ataxacantha DC. Mimosoideae Leaves
Acacia gerrardii Benth Mimosoideae Roots
Capparis tomentosa Lam. Capparaceae Roots
Clausena anisata (Willd) Rutaceae Roots Hoof.f.exbenth Delonix regia (Boj.) Raf Caesalpinioideae Leaves
Dialium guineense Willd Caesalpinioideae Bark
Securidaca longepedunculata Fres Polygalaceae Root
Vitex madiensis oliv. Verbenaceae Leaves
Ziziphus abyssinica Hochst.ex. A. Rhamnaceae Leaves Rich Ceiba petandra (L.) Gaertn Bombacaceae Flowers and fruits
Cola cardifolia(Cav.)R.Br. Sterculiaceae Root
Combretum collinum Fresen. Combretaceae Stems and Gums 56
Uvaria chamae P. Beauv Annonaceae Roots
Senna alata (L.) Roxb Caesalpinioideae Bark
Psoropsis Africana (Guill & Perr.) Mimosoideae Root, bark, Pod Taub Cordyla pinnata (Lepr.ex.A. Rich) Caesalpinioideae Bark
Erythrina senegalensis DC. Fabaceae Root
Croton zambesicus Mill. Arg Euphorbiaceae Leaves
Ficus asperifolia Miq. Maraceae Bark
Isoberlina tomentosa (Harms) Caesalpinioideae Root craib & stapf Lannea velutina A. Rich Anacardiaceae Root and bark
Lannea barteri (Oliv.) Engl Anacardiaceae Root Reference: Arbonnier (2004)
2.10.2 Plants with proven uses in pharmacopoeia
The following plants have been shown to possess analgesic properties.
Plants Family Part used Cadada farinose Forssk Capparaceae Leaves (Arbonnier, 2004) Erythrophyleum suaveolens Caesalpinioideae Root (Guill & Perr) Brenan (Arbonnier, 2004) Pericopsis laxiflora (Benth) Fabaceae Root Van Meeuwen(Arbonnier, 2004) Combretum glutinosum Combretaceae Leaves Perr.ex.DC. (Arbonnier, 2004) Combretum nigricans Combretaceae Bark and Lepr.ex.Guill & Perr Leaves (Arbonnier, 2004)
Ficus exasperate (Vahl) Moraceae Leaf (Woode et al., 2009a) Grewia lasiodiscus K.Scum Tiliaceae Root (Arbonnier, 2004) Culscasia scandens P. Beauv Araceae Leaves (Okoli et al., 2006) 57
Hyptis sauveolens Lamiaceae Leaves (Santos et al., 2007) Spilanthes acmella Compositae Shoot (Chakraborty et al., 2004) Sigmatanthus trifoliatus Rutaceae Roots Huber.ex Emmerich (Lima et al., 2006) Foeniculum vulgare Mill. Apiaceae Fruit (Choi and Hwang, 2004) (Umbelliferae) Ocimum suave Labiatae Leaves (Makonnen et al., 2003) Ocimum lamiifolium Labiatae Leaves (Makonnen et al., 2003)
Lippia adoensis Liliaceae Leaves, flowers (Makonnen et al., 2003) Ajuga remota Labiatae Leaves (Makonnen et al., 2003) Olax viridis Oliv (Asuzu et Olacaeceae Root bark al.,1998) Synedrella nodiflora (L.) Asteraceae Whole plant Gaertn ((Woode et al., 2009b)
Pseudocedrella kotschyii Meliaceae Leaves Harms (Musa et al., 2007) Melanostoma malabathricum Melanostomataceae Leaves (Zakaria et al., 2006) Jatropha curcas L. Euphorbiaceae Leaves (Okoli et al., 2008)
2.10.3 Phytochemical compounds identified in plants with analgesic activity (Anon,
2009a)
Four categories of plant phytoconstituents have been shown to have analgesic activity.
These categories are alkaloids, organic acids, volatile oils and glycosides.
2.10.3.1 Alkaloids
An alkaloid is a plant derived compound that is either toxic or physiologically active. Alkaloids contain nitrogen in a heterocyclic ring. The nitrogen generally makes these compounds basic and the compound exists in the plant as a salt. Many of the earliest isolated pure compounds with biological activity were alkaloids (Anon, 2009b) 58
The following subclasses of alkaloids have been identified
A. Tropane alkaloids
Many alkaloids in this class have useful medicinal properties. Tropane alkaloids compete with acetylcholine and block transmission of nerve signals. They may be used for pain relief and treatment of Parkinson’s disease. Hyoscyamus niger contains tropane alkaloids that have been used in treating pain due to gall bladder infection. Cocaine is a tropane alkaloid obtained from Erythroxylum coca. Cocaine is used for topical analgesia in ophthalmology. Datura stramonium (Jimson weed) which contains tropane alkaloids is known to have analgesic properties.
B. Isoquinoline alkaloids i. Morphine, codiene and other related compounds are isoquinoline alkaloids. They are the choice analgesic for the treatment of severe pain due to accidents, surgery and cancer. ii.Tetrahydropalmatine (THP) an isoquinoline alkaloid has been isolated from Corydalis ambigua, a Chinese herb used as a traditional analgesic. Some THP analogues have been isolated from species of Stephania. These analogues all share a naloxone resistant analgesic effect and had no affinity for opioid receptors (Xing-Zu, 1991). The effect of THP is thought to be due to blockage of dopamine. iii. Stepholidine is another isoquinoline alkaloid isolated from Stephania. This alkaloid induces sleep and analgesia. iv. Tetrandrine and cycleanine are analgesia alkaloids from Stephania species. They are also potent muscle relaxants. v. Higenamine is an isoqunoline alkaloid. It has beta adrenergic and analgesic activity.
C. Diterpene alkaloids
Two diterpene alkaloids 3 acetylaconitine (ACC) and aconitine were isolated from the root of Aconitum flavum, a plant used locally in China in the treatment of arthralagia. 59
The analgesic effect of ACC was studied by Tang et al. (1986) using the acetic acid induced writing test, hot plate test, formalin test and tail flick test. Their findings showed that the relative analgesic potency of ACC was 5.1 to 35.6 and 1250 to 3912 times that of morphine and aspirin respectively. Also the analgesic action of aconitine at 0.1mg/kg has been shown to be stronger than that of 6 mg/kg of morphine. Experiments to determine the mechanism of action of ACC showed that ACC was a non narcotic centrally acting analgesic.
D. Rauwolfia alkaloids
Rauwolfia is the source of reserpine an alkaloid which is currently used in modern drugs as an antihypertensive agent. This alkaloid was originally used for its sedative property. Uncaria contains several alkaloids notably rhynchophyllin. Uncaria is used in the treatment of headaches.
E. Organic alkaloids
Salicin an alkaloid was isolated from willow (Salix species). This alkaloid on hydrolysis yielded salicylic acid which is a useful analgesic, anti-inflammatory and antipyretie drug. Other organic alkaloids are i. Cinnamic acids such as methyl or ethyl cinnamate found in alpinia, liquiclamba, cinnamate and styrax which are employed in pain treatment. ii. Shikimic acid: This compound is similar to cinnamic acid and is contained in
illicumdunnianum. This compound has analgesic properties. iii. Achillea alpina contains organic acids that are analgesic, sedative and anti- inflammatory. iv. Ferulic acid.
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2.10.3.2 Volatile oils
Volatile oils of asarum which contain asarone, asatone and methyleugenol are sedative and analgesic. Plants like angelina have marked analgesic activity. The aromatic oils in these plants are thought to be responsible for their analgesic action. Murraja is an aromatic plant and is used to treat abdominal pain.
2.10.3.3 Glycosides
Glycosides are compounds that contain a sugar molecule. Most analgesic glycosides are terpene glycosides. Glycosides in Cynanchum species and Clematis henryi are strong analgesics. Sapanion glycosides (triterpenes) from bupleurum have analgesic and anti- inflammatory properties. Also monoterpene glycosides of Paeonia species such as paeoniflorin have anti-spasmodic, anti-inflammatory and analgesic properties
2.10.4 Medicinal plants with proven local anaesthetic properties
Some plants in the families Caesalpiniaceae, Euphorbiaceae, Rubiaceae,
Apocynaceae and Erythroxylaceae have been shown experimentally to have local anaesthetic properties.
Plants Family Part used Cassia absus Caesalpiniaceae Seeds
Jatropha podagrica Euphorbiaceae Stem
Erythrophylum suaveolens Caesalpinioideae Bark (Guill & Perr.) Brenan Mitragyna stipulosa Rubiaceae Root bark
Voacanga africana spapf Apocynaceae Stem, root bark
Picralima nitida Apocynaceae Stem, root bark
Corynanthe pachyceras schum Rubiaceae Bark
Crossopteryx febrifuga Rubiaceae Bark, leaves
61
Erythroxylum coca lam Erythroxylaceae Leaves
Pausinystalia johimbe Rubiaceae Bark
Reference: Oliver- Bever (1986)
2.10.4.1 Phytochemical compounds identified in plants with local anaesthetic
properties
The leaves, bark and seed of Erythroxylum coca plant contains the alkaloid cocaine. In India, the leaves contain 0.4-0.8% alkaloids largely cocaine
(methybenzoylecgonine) but also other pseudotropanol derivatives such as unnamylococaine, truxillines and tropacaine (benzylpseudotronanol) as well as some monocyclic N-methylpyrrolidine derivatives (Henry, 1949).
The seeds of Cassia absus L. contain fixed oils and a toxalbumin absin as well as two alkaloids Chaksine and isochaksine. The pharmacologic study of these two alkaloids by
Bukhari and Khan (1963) and Khan et al. (1963) showed that both alkaloids had local anaesthetic action on guinea pig skin when administered intradermally. Their action was however inferior to that of procaine which proved to be 3.6 times more active than chaksine and 1.7 times more active than isochaksine. This anaesthetic action was also confirmed in man (Oliver- Bever, 1986).
Tetramethylpyrazine has been isolated from the stem of Jatropha podagrica
(Odebiji, 1978). This compound showed antibacterial activity (Odebiji, 1978). It also blocked neuromuscular transmission and appeared to have spasmolytic activity on smooth muscles (Oyewole, 1980., Oyewole and Odebiji, 1980). Further studies by these authors confirmed blockage of adrenergic and cholinergic transmission by tetramethypyrazine.
Apart from its central actions, it was suggested from the result of this study that the 62 hypotensive effect in the experimental animals was likely contributed by or mediated via its local anaestheic (membrane stabilizing) activity (Oyewole, 1981).
The local anaesthetic effect of Erythrophyleum guineenses has been attributed to the presence of casssine while an indole alkaloid is the main constituent responsible for the local anaesthetic effect of Mitragyna spp (Oliver-Bever, 1986). Alkaloids of Picralima nitida namely akuammine, akuammidine and pseudo-akuammyine have been shown to possess local anaesthetic activities (Gabriella and Ameenah, 2008).
2.11 STUDIED MEDICINAL PLANT
Plant name: Sterculia tragacantha Lindl. was first identified by John Lindley in 1830.
This name (Sterculia tragacantha) is the accepted name for a species in the genus Sterculia with original publication details in Edwards Botanical Register. 16: t. 1353 (1830). The name was verified 07-11-1985 by Agricultural research services (ARS) systematic botanists.
English name: African tragacantha
Genus: Sterculia
Family: Malvaceae
Sub Family: Sterculiaceae.
Local names: Kukukin (Hausa); nyichi kuso (Nupe); alawefun (Yoruba); Oporipor
(Edo); apompir (Kwale); Oloko (Igbo); Udot (Ibibio); Uhobo (Okpatu).
Distribution: Widespread in tropical Africa.
Habitat: Open and dry parts of lowland rain forest, also forest outliners and transition woodlands.
Descriptions: It is a medium sized tree seen in rain forest zones. The tree grows up to 26 m high with up to 1.5 m girth sometimes buttresees. Bark grey-brown with longitudinal 63 fissures, slash pinkish brown, fibrous moist with a gummy sap. Branchlets densely covered with brown stellate hairs. Leaves 10 -30 cm long, 5 -15 cm broad ovate-elliptic or slightly obvate, rounded or slightly cordate at base, obtusely acuminate at apex; densely covered with small stellate hairs beneath; lateral nerves 7-9; stalk 1.5-7.5 cm long. Flowers
(October -June) reddish pink, in stalked inflorescence, crowded at first; florescence densely covered with brown stellate hairs, Calyx about 5 mm long, the lobes adhering together at apex. Fruits (September-May) composed of 4-5 boat shaped carpels, each carpel 5-7 cm long, bright red and finally brown when ripe, splitting along the top side to expose about 8 slate covered seeds, shortly and densely hairy outside, bristly inside, seeds about 18 mm long without arils.
Reference: Keay, 1989., Llamas, 2003.
Medicinal Uses: The bark, shoots and seeds are used to prepare traditional medicines for the treatment of nasopharyngeal affections, pulmonary disorders, arthritis, rheumatism, syphilis, leprosy, dropsy, oedema, gout, boils, whitlow, convulsion and epilepsy (Walt and
Breyer-Brabdwiju, 1962; Iwu, 1993). The leaves and bark are also used to make ethno medicines for the treatment of diarrhea, dysentery and helminthiasis. The leaves of this plant are squeezed in water by the traditional bone setter from whom the plant was collected and given to his patients for pain relief after closed fracture reduction.
Experiments: The methanol and aqeous extracts of S. tragacantha leaves have been reported by Aguwa and Ukwe (1997) to show significant anti ulcer activity in mice. The extracts also exhibited anticholinergic, antispasmodic and smooth muscle relaxant properties in isolated smooth muscle preparations
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STERCULIA TRAGACANTHA LEAVES
2.12 CLINICAL ASSESSMENT OF PAIN
Evaluation of pain in animals is often difficult because of the problem associated
with assessing the degree of pain (Carroll et al., 1998). It is well known that animals react
with behavioural and physiologic responses to painful stimuli; however, pain
management in veterinary patients is handicapped by lack of a validated method of
clinical assessment (Spinelli and Morish, 1987; Crane, 1987; Reid and Nolan, 1991;
Lascelles et al., 1994; Liles and Flecknell, 1994). Assessing pain is complicated because
observations of pain are subjective and developing a complete description of various 65 degree of pain is difficult. Thus pain assessment in animals involves multiple criteria to provide sufficient information on pain (Booker, 1996; Hansen et al., 1997). Any pain scale should consider the species, breed, cause of pain (trauma, surgery, pathology), body region affected (abdominal pain, musculoskeletal pain), type of pain (acute or chronic) and pain intensity (Kahn, 2005).
2.12.1 Subjective assessment of pain
Various pain scales have been developed but none have gained widespread acceptance in veterinary medicine (Firth and Haldane, 1999). These pain scoring systems are based on behavioural manifestation of pain (Morton and Griffiths, 1985; Pypendop and Verstegen, 1994). Some scoring systems, however, combine several independent variables such as physiological parameters (heart rate, respiratory rate and pulse rate), agitation, response to manipulation, vocalization etc to quantify the severity of pain
(Morton and Griffiths, 1985; Grisneaux et al., 1999 ; Gellasch et al., 2002 ; Lemke et al.,
2002).
The following pain scoring systems are often used for subjective pain assessment in animals.
Simple descriptive scale (SDS): This is the most basic pain scoring system (Firth
and Haldane, 1999). It assigns four or five degrees of severity of pain such as no
evidence of pain, mild, moderate, severe and very severe pain. The SDS is straight
forward and easy to use but it does not allow small changes in pain response to be
assessed (Bateman et al., 1994).
Numerical rating scale (NRS): This scale is produced by assigning numeric scores
to the categories of a simple descriptive scale or a similar scale. Scores assigned in
the NRS are usually whole numbers which implies that equal differences exist
between each of the categories but this is often not the case (Huskisson, 1974). A 66
NRS may include descriptive definition of each category of pain but may not
provide an improvement in usability or accuracy of the SDS (Firth and Haldane,
1999). It only facilitates tabulation or analysis of the results.
Visual analog scale (VAS): The scale is widely used in human medicine (Firth and
Haldane, 1999). It consists of a straight line usually 100 mm horizontal or vertical
line) in paper with a description of the limits of the scale written at each end of the
line (viz no pain, severe pain). The observer uses a pencil to mark a point on the line
to interpret the degree of pain. This scale is subject to a lot of observer variation but
is considered more sensitive than NRS and SDS since it does not use defined
categories. VAS is widely used to score pain in humans and can be administered by
nurses, medical staff or patients (Lascelles et al., 1994). It is however difficult to
apply in veterinary medicine since it requires pain interpretation by the observer.
Glasgow coma scale (GCS): This scale is based on behavioural responses. It is used
to assess and describe various states of impaired consciousness (Teasdale and
Jennet, 1974). The scale is multifactorial NRS that effectively assigns scores to
impaired consciousness using any clinical observations of behavioural responses.
The GCS assigns a value to various behaviours and a high score often denotes a
high degree of neurological impairment. In veterinary medicine, NRS based scales
similar to GCS are used to assess chronic pain (De Haan et al., 1994).
Composite pain scale: This is a scale for measuring acute post operative pain. It
includes both behavioural and physiologic variables. Most composite pain scoring
scale used to measure acute post operative pain in animals are modeled after the
children’s Hospital of Eastern Ontario pain scale developed for use in children
(McGrath et al., 1985). In this scale, pain associated behaviours are assigned scores
and these scores are summed to obtain a total pain score for the patient (McGrath et 67
al., 1985; Tyler et al., 1993). Unlike the NRS different behaviours are assigned the
same value. The GCS and CHEOPS have a potential to be applied in veterinary
patients since they clearly define observable behaviours. However behavioural and
physiologic variables tend to habituate with time thus the composite scale are not
useful for measuring pain several hours post surgery (McGrath and Unruh, 1999).
A major limitation of these pain rating scales is the difficulty associated with
scoring of animal behaviour in a relatively brief period (Church, 2000).
2.12.2 Objective assessment of pain
Objective assessment of pain can be achieved by measuring the level of the
following post surgery.
i. Plasma cortisol concentration (Grisneaux et al., 1999; Gellasch et al., 2002).
ii. Catecholamine concentration (Almeida et al., 2007).
iii. Serum glucose level (Lemke et al., 2002).
iv. Hemodynamic variables such as cardiac output, central venous pressure and
stroke volume (Skarda and Muir, 1994).
In laboratory animals experimental models used to assess the antinociceptive effect of drugs includes
i. Acetic acid induced writhing test (Koster et al., 1959)
This involves intraperitoneal injection of acetic acid to induce abdominal constrictions or stretching. The abdominal constriction response induced by acetic acid is a sensitive procedure to establish peripherally acting analgesics (Gene et al., 1998;
Chakraborthy et al., 2004). This response is thought to involve peritoneal receptors
(Bentley et al., 1983). Stimulation of these receptors causes acute inflammation leading to the release of arachidonic acid metabolites via cyclo-oxygenase and prostaglandin synthesis
(Franzotti et al., 2000). 68 ii. Formalin induced paw licking test
5% formalin is injected into the plantar surface of the paw. The time spent by the mice/rat in licking the injected paw (licking time) is recorded. The animals are observed for the first 5 min after injection (early phase) and for 10 min starting at the 20th minute post formalin injection (late phase). Subcutaneous injection of formalin produces a distinct biphasic nociception (Mehmet, 2002). The early phase starts immediately after formalin injection and lasts for 5 min. This phase marks the direct effect of formalin on nociceptors
(neurogenic non inflammatory pain). The late phase which begins 15-20 min post injection and continues for 60 min reflects the inflammatory pain (Olajide et al., 2000). iii. Hot plate test (Chakraborthy et al., 2004).
Each mouse is dropped on a heated hot plate (52 ± 10oC). The first trial familiarizes the animal to the test procedure while the second trial serves as the control reaction time. After the test and control drugs are administered, the animals are dropped again on the hot plate and reaction time is then remeasured. A cut off time of 40 seconds is usually selected to avoid tissue damage. This test is used to study central analgesic effect of drugs v. Tail flick test
In this method, the test and control analgesic are given to the mice. The tail flick latency is assessed by the analgesiometer. Current of 6Amp is then passed through a naked nicrome wire attached to the tail. The pain reaction time which measures the amount of pressure tolerated by each mouse before flicking the tail is determined by the analgesiometer post treatment. vi. Guinea pig wheal test (Shetty and Anika, 1982)
This is a twitch response test performed to assess local analgesic effect of a solution. It involves intradermal injection of the drug. The wheal formed is tested for 69 sensitivity every 5 min by pricking gently with a needle. Response to needle prick indicates no analgesia while insensitivity to pricking is an indication of local analgesia in the area.
2.13 EVALUATION OF ANTI- INFLAMMATORY EFFECTS OF DRUGS
Various methods are used to evaluate the effect of drugs and plant extracts on the development of acute inflammation. These methods include the following i. Carrageenan induced paw edema test (Winter et al., 1962).
This is a standard experimental model used to assess the effect of drugs on acute inflammation (Chakraborthy et al., 2004). Carrageenan is a phlogistic agent of choice used to induce inflammation since it is not known to be antigenic and is devoid of apparent systemic effects (Winter et al., 1962). Subplantar injection of this agent into the paw leads to extravasation and inflammation characterized by protein exudation (Gamache et al.,
1986; Szolesanyi et al., 1988). Edema induced by carrageenan is usually biphasic
(Chakraborthy et al., 2004). The first phase begins immediately after its injection and regresses in an hour. This phase is followed immediately by the second phase which peaks at 3 hours (Gamache et al., 1986; Garcia-Pastor et al., 1999). The early phase is characterized by hyperemia of the paw which is thought to be induced by histamine and serotonin release (Lalenti et al., 1992). The second phase is believed to be a result of the potentiating effect of prostaglandins on the release of bradykinins and neutrophil derived free radicals (Ferreria et al, 1974; Sumen et al., 2001). Hydrocortisones and some anti inflammatory drugs strongly inhibit the second phase of carrageenan edema. However some anti inflammatory drugs are effective against both phases. (Vinegar et al.,1969;
Kulkarni et al., 1986). ii. Croton oil ear edema test (Tubaro et al., 1985). 70
This method is used to evaluate the effect of drugs on topical acute inflammation.
It involves the application of croton oil on the inner ear of mice/rat to induce inflammation.
Ear plugs are obtained after croton oil application and compared with plugs taken from the control ear. Increase in ear plug weight reflects edema. This increase in ear plug weight has been suggested to be due to increased peroxidase activity. iii. Formaldehyde induced paw edema test.
This involves subplantar injection of 2.5% w/v formaldehyde. The paw volumes are determined before and after formaldehyde injection using a plethysmometer. The difference in paw volume is assumed to be the volume of edema. Several inflammatory mediators such as histamine, serotonin, prostaglandins, bradykinin and cytokines are incriminated to play roles in formalin induced paw edema (Kulkarni et al., 1986; Taylor et al., 2000). v. Histamine induced paw edema test.
This method involves the subcutaneous injection of 1% w/v histamine into the plantar surface of the paw. The edema formed is determined as in formaldehyde method.
The injection of histamine an inflammatory mediator and a potent vasodilator lead to increased vascular permeability and inflammation (Linardi et al., 2000; Cuman et al.,
2001).
Evaluation of the effect of drugs on chronic inflammation is carried out using any of the following experimental models i. Cotton pellet induced granuloma test (Niemegeers et al., 1975)
This method is widely used to determine the effect of drugs on transudative, exudative and proliferative components of inflammation (Mehmet, 2002). It involves subcutaneous implantation of sterile cotton pellets of known weights either in the axilla or dorsum of rats/mice. The test and control animals are treated with the test and control drugs 71 for a period of 7 days. The cotton pellets are then harvested and their wet and dry weights determined. This is followed by the determination of the weight of the granuloma and transudative fluid formed. ii. Formaldehyde induced arthritis test (Choi and Hwang, 2004).
Formaldehyde 2.5 % w/v is injected beneath the aponeurosis of the paw on the first and third days of the experiment to induce arthritis. This is followed by the administration of the test and control drugs for 7 days. The paw volumes before arthritis induction and after are determined using a plethysmometer.
72
CHAPTER THREE
EVALUATION OF THE ANALGESIC, ANTI-INFLAMMATORY AND TISSUE
EFFECTS OF THE METHANOL EXTRACT OF STERCULIA TRAGACANTHA
(MEST)
73
3.1 INTRODUCTION
Medicinal herbs and plants have been successfully used in modern health care in countries like China, India, Sri-Lanka, Nigeria, Cameroon and Ghana (Jagun et al., 1997).
In these countries, efforts are being made to blend the traditional medicine into orthodox practice (Jagun et al., 1997). In Cameroon, since 1989, Terminalia schimperiana and
Vernomia amygdalina are used in the treatment of helminthiasis in cattle (Jagun et al.,
1997). In Ghana an ethno medicine made from Mitragyna stipulosa used traditionally to cure guinea worm infestation have been adopted as first choice treatment against the disease (Sofowora, 1982).
In African traditional medicine, ethno medicines prepared from plant materials are used to treat a wide range of disease conditions including pain and inflammation. These ethno medicines are relied on by local West African dwellers for their primary health care since the plant materials used in their preparation are cheap and readily available (Jodi et al., 2008). Clinical experiments in rats and mice have shown that extracts from plants like Sigmatanthus trifoliatus, Culscasia scandens, Hyptis sauveolens, Lippia advensis,
Olax viridis, Synedrella nodiflora, Pseudocedrella kotschyii, Melanostoma malabathricum, Jatropha curcas and Ficus expasperate have antinociceptive properties
(Asuzu et al., 1998; Makonnen et al., 2003; Okoli et al., 2006; Zakaria et al., 2006; Lima et al., 2006; Santos et al., 2007; Musa et al., 2007; Okoli et al., 2008; Woode et al.,
2009b). Among plants shown to possess local analgesic properties are Corynanthe pachycera, Picralima nitida, Mitragyna stipulosa, Pausinystalia johimbe, Cassia absus,
Erythroxylum coca and Voacanga Africana. None of these plant extracts have been evaluated for analgesic property using any food animal.
A wide range of medicinal plants used traditionally including Sterculia tragacantha Lindl. (Family Sterculiaceae) are yet to be screened for analgesic property. 74
Sterculia tragacantha is a medium sized tree seen in the edges of lowland rain forests
(Keay, 1989). The tree grows to 80ft high with grey corky bark yielding a coloured gum.
The leaves, bark, shoots and seeds are used to prepare ethno medicines for the treatment inflammatory conditions such as arthritis and rheumatism (Walt and Breyer-Brabdwiju,
1962; Iwu, 1993). The leaves of this plant are squeezed in water by a traditional bone setter and given to his patients for pain relief after closed fracture reduction.
This experiment was carried out to screen the methanol leaf extract of Sterculia tragacantha for anti nociceptive (peripheral and local) and anti inflammatory activities.
The effect of subcutaneous injection of MEST was also evaluated.
3.2 MATERIALS
3.2.1 Instruments and equipments
Analytical weighing balance (Mettler, Switzerland)
Weighing balance (Ohaeus scale, New Jersey)
Electric oven (Gallenkamp, England)
Laboratory mill (Authur Willey, USA)
Venire caliper (ESALR)
Refrigerator
Test tube racks
Spatulas
Tally counter
Light microscope
Metal cages
Surgical scissors
Metre rule 75
Marker pen
Kidney dishes
Scapel blade holder
Surgical table
Artery forceps
Tissue forceps
3.2.2 Reagents and solvents
Tween 20 (Sigma Aldrich Co. USA)
70% alcohol
Methanol (Sigma Aldrich, Germany)
L- carrageenan(Fluka Biochem, Denmark)
Acetic acid (Sigma Aldrich, Germany)
Chloroform (Sigma Aldrich, Europe)
3.2.3 Glass wares
Beakers (50ml, 100ml, 500ml and 1000 ml)
Test tubes
Conical flask
Glass funnels
Glass rods
Measuring cylinder
Glass slides
Cover slips
3.2.4 Consumables
Whatman’s filter paper
Tuberclin syringe 76
Distilled water
5 ml syringe and needles
Cotton wool
Scapel blades
Razor blades
Gauze
3.2.5 Drugs
Indomethacin (Park Davis, Italy)
Lignocaine Hcl (Rotex medica, Germany)
Pentobarbitone Na (Kryon lab, Benrose)
3.3 METHODS
3.3.1 Plant collection and identification
Fresh leaves of Sterculia tragacantha were collected in September, 2007 from
Okpatu in Udi Local government area of Enugu state, Nigeria. They were authenticated by Mr. A.O. Ozioko, a taxonomist with the International Centre for Ethno medicine and
Drug Development, Nsukka. A voucher specimen (INTERCEED/819) was deposited in their herbarium.
3.3.2 Extraction of the plant materials
Fresh leaves of S. tragacantha were air dried and later pulverized using a laboratory mill at the Crop Science Department, University of Nigeria, Nsukka. 1000 g of the plant materials were poured into a colourless glass bottle and allowed to soak in 80% methanol for 48 h. The extraction was by cold maceration at 370C with intermittent shaking every 2 h for the 48 h period. After 48 h, the content of the bottle was filtered 77 using whatman’s filter paper. The extract obtained was concentrated using a vacuum rotary evaporator at 400C. The dry extract was stored in a beaker at 4oC. The percentage yield was later determined as follows
% = Weight of extract x 100. Weight of plant material
3.3.3 Screening of methanol extract of Sterculia tragacantha (MEST) for local anaesthetic property (Shetty and Anika, 1982)
Experimental animals
Two female guinea pigs weighing 165 ± 0.4 g were used for this study.
Experimental protocols
Their lower backs were thoroughly shaved using a pair of scissors 24 h before the experiment. The back of each guinea pig was disinfected with 70% alcohol and divided into 4 equal parts with a marker. Solutions (10 mg/ml, 0.3 mg/ml and 0.03 mg/ml) of
MEST were prepared. Lignocaine (2%) solution was diluted with distilled water to obtain
0.1 mg/ml and 0.033 mg/ml solutions. This was followed by intradermal injection of 0.2 ml lignocaine and MEST as follows:
10 mg/ml solution of MEST was injected into the upper left quarter
0.3 mg/ml solution of MEST was injected into the lower right quarter.
0.1 mg/ml solution of lignocaine was injected into the upper right quarter.
0.033 mg/ml solution of lignocaine was injected into the lower left quarter.
In the second guinea pig (B) the intradermal injections were made as follows:
0.3 mg/ml solution of MEST was injected into the upper left quarter
0.033 mg/ml solution of MEST was injected into the lower right quarter.
0.1 mg/ml solution of Lignocaine was injected into the upper right quarter.
0.033 mg/ml solution of lignocaine was injected into the lower left quarter. 78
The sites of injection were outlined with a marker. The wheals formed were tested for sensitivity 5 min after the injection by pricking with a needle (six times lightly) and as control the skin far away from the site of injection was also pricked. The number of negative responses (failure to twitch) was recorded. The tests were repeated at 5 min interval for a period of 30 min after the injection. The total score for each wheal was added at the end of the experiment and expressed as the sum of negative responses out of
36 possible. The percentage anaesthesia was determined as the number of negative responses over the number of 36 possible responses multiplied by 100. Graphs of log concentration against percentage anaesthesia were plotted. The slopes of the lines for the two drugs were determined and compared.
3.3.4 Determination of the solubility of MEST in distilled water and Tween 20
Into two test tubes each containing 10 mg of MEST were added 1 ml of distilled water and Tween 20 respectively. The contents of each test tube were mixed and observed for complete solubility and presence of precipitate.
3.3.5 Determination of the pH of MEST
The pH of the following solutions were determined using a pH meter
A. Distilled water.
B. 2% Lignocaine.
C. 1% Lignocaine.
D. MEST (10 mg/ml) formed by dissolving 40 mg MEST with Tween 20
(0.4 ml) and distilled (3.6 ml).
E. MEST (10 mg/ml) formed by dissolving 40 mg MEST with distilled water (4 ml).
F. MEST (10 mg/ml) + 3 drops of 1% NaOH.
79
3.3.6 Adjustment of the pH of MEST
A 10 mg/ml solution of MEST was prepared. The pH of the solution was adjusted to
7.3 by adding 3 drops of 1% sodium hydroxide. The local anaesthetic effect of the final solution and that of the unadjusted MEST solution were tested in guinea pig using the wheal experiment as in experiment 3.8.5
3.3.7 Acute toxicity test
Experimental animals
Forty eight mice of both sexes of mean weight 23.4 ± 0.1 g were used for this experiment. They were housed in wire meshed cages 2ft by 1 ft. They were fed with pelleted finisher mash (Vital feeds®, Jos Nigeria) and water was provided ad libitium.
Experimental groups
The mice were divided into six groups of eight mice each.
Group 1: Mice were injected with 100 mg/kg MEST intraperitoneally (i.p).
Group 2: Mice were injected with 200 mg/kg MEST i.p.
Group 3: Mice were injected with 400 mg/kg MEST i.p.
Group 4: Mice were injected with 800 mg/kg MEST i.p.
Group 5: Mice were injected with 1600 mg/kg MEST i.p.
Group 6: Mice were injected with 1 ml/kg distilled water.
The mice were fed and allowed free access to water for 48 h. During the 48 h period, they were observed for signs of acute toxicity such as depression, convulsion, paralysis, salivation and death. Deaths within this period were recorded and the vital organs were examined grossly.
3.3.8 Screening of MEST for analgesic property
The acetic acid induced writing test (Koster et al., 1959) was used to evaluate the analgesic effect of MEST 80
Experimental animals
Forty mice were used for the experiment. They were of mean weight 21.3 ± 0.4 g
Experimental groups
The mice were divided into five groups of eight mice each
GP 1: Normal saline + 10 ml/kg of 0.7% acetic acid i.p. (negative control).
GP 2: 150 mg/kg extract i.p. + 10 ml/kg acetic acid (30 min later) i.p.
GP 3: 300 mg/kg extract i.p. + 10 ml/kg acetic acid (30 min later) i.p.
GP 4: 600 mg/kg extract i.p. + 10 ml/kg acetic acid (30 min later) i.p.
GP 5: 10 mg/kg indomethacin i.p. + 10 ml/kg acetic acid (30 min later) i.p.
Experimental protocol
The mice were pre-treated with 150 mg/kg, 300 mg/kg, 600 mg/kg MEST and 10 mg/kg indomethacin i.p. They were injected with 10 ml/kg acetic acid (A.A) 30 min post trea tment. The onset of contortions and number of contortions observed after A.A administration were recorded. The mean onset time and mean contortions were calculated per group. Percentage inhibition of contortion was calculated as shown below:
% inhibition = { Mean contortions in C – Mean contortions}x100 Mean contortions in C
C = Control group. T = Treatment groups.
Statistical analysis
The mean values obtained in the groups were compared using one way analysis of variance (ANOVA) in SPSS 12.0.1 software. Duncan multiple range test (DMRT) was used for Post Hoc test at p < 0.05.
3.3.9 Evaluation of the effect of MEST on acute inflammation
The effect of systemic administration of the extract on acute inflammation was assessed using the carrageenan induced paw edema test (Winter et al., 1962) 81
Experimental animals
Forty mice were used for the experiment. They were of mean weight 32.4 ± 0.1 g
Experimental groups
The mice were separated into five groups of eight mice as follows
GP 1: Normal saline + 0.05 ml carrageenan subplantar.
GP 2:150 mg/kg extract i.p 30 min before carrageenan injection.
GP 3: 300 mg/kg extract i.p 30 min before carrageenan injection.
GP 4: 600 mg/kg extract i.p 30 min before carrageenan injection.
GP 5: 10 mg/kg indomethacin i.p 30 min before carrageenan injection.
Experimental protocol
The thickness of the left hindpaw of each mouse was measured using a venire caliper before the experiment (Naved et al., 2005). Acute inflammation was induced by sub plantar injection of 0.02 ml of 1% carrageenan. The pad thicknesses were remeasured at 1, 2, 3, 4 and 5 h after carrageenan injection. Oedema thickness was calculated as difference between the original pad thickness measured at time zero and the pad thicknesses measured at the different time points after induction of edema. Mean inflammation was obtained by dividing the total edema thickness of each group by the total number of animals. Percentage edema inhibition at 1, 2, 3, 4 and 5 h were calculated as shown below:
% inhibition = { Mean oedema in control – Mean oedema in treatment gps.}x 100 Mean oedema in control
Statistical analysis
The mean edema volumes obtained in the groups were compared using one way
ANOVA in SPSS 11.0 software. DMRT was used for Post Hoc test at p < 0.05.
82
3.3.10: Evaluation of the effect of MEST on chronic inflammation
The effect of MEST on chronic inflammation was evaluated using the cotton pellet induced granuloma test (Niemegeers et al., 1975).
Experimental animals
Forty wistar mice of both sexes were used in this experiment. They were of mean weight
28.9 ± 0.6 g.
Experimental groups
The mice were grouped as follows
GP 1: 10 ml/kg normal saline i.p
GP 2: 150 mg/kg extract i.p
GP 3: 300 mg/kg extract i.p
GP 4: 600 mg/kg extract i.p
GP 5: 7 mg/kg indomethacin i.p
Experimental protocol
Formation of granulomatous tissues was induced by subcutaneous (s.c.) implantation of 50 mg (0.05 g) of sterile cotton pellets into the left and right axillae of mice under pentobarbitone (35 mg/kg) anaesthesia. Post cotton pellet implantation, the mice in groups 1 to 5 were treated daily with distilled water, 150 mg/kg MEST, 300 mg/kg MEST, 600 mg/kg MEST and 7 mg/kg indomethacin i.p. respectively for 7 days.
On day 8, the animals were euthanized using chloroform and the granulomatous tissues
(cotton pellets wrapped with granuloma) in the left and right axillae were carefully dissected out. The moist weights of the harvested granulomatous tissues were taken. They were subsequently dried in a hot air oven at 600C for 24 hr and reweighed to obtain their dry weights.
The following calculations were made at the end of the experiment 83 i. Weight of wet granuloma = Weight of freshly dissected granulomatous
tissue minus weight of implanted cotton pellet (0.05 g) ii. Weight of dry granuloma = Weight of dry granulomatous tissue minus
weight of implanted cotton pellet (0.05 g) iii. Weight of transudative fluid = Wet weight of granuloma minus dry
weight of granuloma. iv. Mean weight of dry granuloma/group = Sum of dry granuloma weight
divided by 16
Percentage inhibition of granuloma was calculated as shown below:
% inhibition = { Mean granulom wt. in C – Mean granuloma wt. in T}x 100 Mean granuloma wt. in C C = Control group. T = Treatment groups
Statistical analysis
The mean granuloma weights of the MEST treated groups and those of the controls were compared using ANOVA. DMRT was used for Post Hoc test at p < 0.05.
3.3.11 Evaluation of the tissue effect of MEST
Experimental animals
10 wistar mice weighing 28.4 ± 0.4 g were used.
Experimental groups
The mice were divided into two groups of 5 mice each as follows
GP 1: Distilled water s.c
GP 2: MEST (8 mg/kg, 10 mg/ml solution, s.c.)
84
Experimental protocol
10 mg/ml solution of MEST was prepared. The areas for injections were marked with an indelible ink. The animals in groups 1 and 2 were injected (s.c) with 0.02 ml of distilled water and MEST respectively. These areas were observed daily for signs of tissue reaction. On days 1 and 5 post injection, 2 mice were euthanized per group and skin sections taken for histology. Skin sections were fixed immediately in 10% formal saline.
These tissues were later processed and embedded in paraffin wax. Sections were cut and stained with haematoxylin and eosin and examined under the light microscope.
3.4 RESULTS
3.4.1 Calculation of plant yield
The following calculations were made to obtain the plant yield
Weight of original plant material = 1000 g
Weight of extract after drying = 111.15 g
Percentage yield (%) = 111.15 x 100 = 11.11% w/w.
1000
The methanol extract of S. tragacantha was dirty green in colour and sticky in consistency. The plant yield was calculated to be 11.11% w/w.
3.4.2 Determination of the solubility of the extract in distilled water and Tween 20.
The extract was observed to be completely soluble in Tween 20. The solution formed when dissolved in distilled water was not completely homogenous. Small extract particles were seen on the bottom of the test tube when the distilled water solution was allowed to stand for some minutes.
85
3.4.3. Preliminary screening of MEST for local anaesthetic property.
The result of the guinea pig wheal test presented in Table 1 showed that injection of 0.1 mg/ml solution and 0.03 mg/ml lignocaine produced 94.4% and 69.4% anaesthesia respectively. However, intradermal injection of 10 mg/ml and 0.3 mg/ml MEST produced
100% anaesthesia each while injection of 0.3 and 0.03 mg/ml MEST gave 100% and 86% anaesthesia respectively. When the graph of the log concentrations was plotted against percentage anaesthesia (Fig. 1), a slope of 48.1 was obtained for LIG (0.1 mg/ml against
0.03 mg/ml) while a slope of 14 was obtained for MEST (0.3 mg/ml against 0.03 mg/ml).
Comparison of both slopes showed that lignocaine was more active than MEST.
3.4.4 Determination of the pH of MEST and pH adjustment
The pH of MEST and lignocaine was in the acidic range as shown in Table 2. The pH of MEST dissolved in both Tween 20 and distilled water was in the acidic range. The result of the pH adjustment as presented in Table 3 showed that adjustment of the pH of
MEST from 4.6 to 7.3 decreased the anaesthetic potency of MEST by half (50%).
3.4.5 Acute toxicity test
No mortality or adverse reaction was detected in mice during the 48 h observation period following i.p injection of MEST up to a dose of 1600 mg/kg.
3.4.6 Screening of MEST for analgesic activity
The highest dose of the extract (600 mg/kg) and indomethacin significantly (p<
0.05) prolonged the onset of contortions following AA injection (Table 4). All test doses of the extract significantly (p<0.05) inhibited acetic acid induced pain (Table 4, Fig. 2).
Their effects were comparable to that of indomethacin.
3.4.7 Evaluation of the effect of MEST on acute inflammation
Subplantar injection of carrageenan into the hind paw of mice induced a progressive edema which reached its maximum thickness at 3 h (Table 5). The 86 administration of 150, 300 and 600 mg/kg MEST before carrageenan injection, significantly (p< 0.05) inhibited edema formation starting from one hour to four hours post carrageenan injection (Table 5). Indomethacin showed a similar anti inflammatory effect with the extracts only at 1 h. At 3, 4 and 5 h post edema induction, 300 mg/kg and
600 mg/kg MEST showed better anti inflammatory effect compared to indomethacin
(Fig.3).
3. 4.8 Evaluation of the effect of MEST on chronic inflammation
Daily injection of 300 and 600 mg/kg doses of MEST significantly (p<0.05) inhibited the formation of granuloma around the implanted cotton pellets (Table 6, Fig.
4). The weight of the transudative fluid was however significantly lower (p<0.05) in the indomethacin group (Table 6).
3.4.9 Evaluation of the tissue effect of MEST
Histological examination of the the skin sections did not reveal any sign of tissue reaction in the MEST treated group on days 1 and 5. However, neutrophilic infiltration of the epidermis was seen in the group given distilled water on day 1. By day 5 post distilled water injection, no tissue reation was observed in the distilled water treated mice
(Figs. 5-8).
87
Table 1: Percentage anaesthesia of lignocaine (LIG) and MEST
Drug Concentration % anaesthesia (mg/ml) LIG 0.1 94.4 0.033 69.4
MEST 10 100 0.3 100
MEST 0.3 100 0.03 86
88
Table 2: pH of solutions measured
Solutions pH
Distilled water (DW) 7.1
2% lignocaine 5.3
1% lignocaine 5.1
MEST + Tween 20 + DW 4.6
MEST + DW 4.5
MEST + 1% NaOH (3 drops) 7.3
Table 3: Percentage anaesthesia of MEST after pH adjustment
Drugs pH Conc.(mg/ml) % anesthesia LIG 5.1 10 94.4
MEST 4.6 10 100
MEST + NaOH 7.3 10 50
89
Table 4: Mean ± SE onset and number of contortions in treatment and control groups.
Treatments Dose Onset of No. of
mg/kg Contortions contortions
NS - 3.38 ±0.4a 183.5 ± 14.3a
INDO 10 12.0 ± 3.0b 31.8 ± 9.6b
MEST 150 5.5 ± 1.0a 43.0 ± 6.9b
MEST 300 5.4 ± 1.3a 25.1 ± 4.4b
MEST 600 8.0 ± 1.9ab 22.5 ± 6.4)b
NS- Normal saline; INDO- Indomethacin; MEST- Methanol Extract of S. tragacantha
90
Table 5: Mean ± SE paw edema (mm) in treatment and control groups.
Treat. Dose Time
(mg/kg) 1 h 2 h 3 h 4 h 5 h
NS - 0.06 ± 0.01a 0.07 ±0.00 a 0.08 ± 0.02 a 0.07 ± 0.01 a 0.06 ± 0.01 a
INDO 10 0.04 ± 0.01 b 0.07 ± 0.01 a 0.06 ± 0.01b 0.05 ± 0.00b 0.05 ± 0.00 a
MEST 150 0.02 ± 0.01b 0.04 ± 0.00 b 0.05 ± 0.01bc 0.05 ± 0.00b 0.05 ± 0.00 a
MEST 300 0.04 ± 0.00b 0.04 ± 0.00 b 0.04 ± 0.01dc 0.04 ± 0.00 bc 0.02 ± 0.01b
MEST 600 0.03 ± 0.00b 0.04 ± 0.00 b 0.04 ± 0.02d 0.03 ± 0.00 c 0.02 ± 0.01b
NS- Normal saline; INDO- Indomethacin; MEST- Methanol Extract of S. tragacantha
91
Table 6: Mean ± SE granuloma and transuda weight in treatment and control groups
Treatment Dose(mg/kg) Granuloma wt (g). Transuda wt.(g)
NS - 0.25 ± 0.02a 0.23 ± 0.03b
INDO 10 0.22 ± 0.02a 0.19 ± 0.01a
MEST 150 0.20 ± 0.02a 0.24 ± 0.01b
MEST 300 0.12 ± 0.02b 0.27 ± 0.02b
MEST 600 0.07 ± 0.01b 0.24 ± 0.02b
NS- Normal saline; INDO- Indomethacin; MEST- Methanol Extract of S. tragacantha
92
120
y = 14 100
y = 48.077
80
60 % Anaesthesia %
40
20
0 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 Log Concentration
LIG MEST Linear (LIG) Linear (MEST)
Figure 1: Graph showing slope of Lignocaine and MEST
93
90
88
86
84
82
80
78 % inhibition % of contortions
76
74
72
70 indo 150 mg/kg MEST 300 mg/kg MEST 600 mg/kg MEST Treatments
Figure 2: Percentage inhibition of contortions in treatment and control groups
94
70
60
50
40
30 % oedema % inhibition
20
10
0 1 h 2 h 3 h 4 h 5 h Time (hours)
Indo 150 MEST 300 MEST 600 MEST
Figure 3: Percentage oedema inhibition in treatment and control groups
95
80
70
60
50
40
30 % granuloma % inhibition
20
10
0 Indo 150 MEST 300 MEST 600 MEST Treatments
Figure 4: Percentage granuloma inhibition in treatment and control groups
96
H
D
N
Figure 5: Skin section of distilled water treated mouse on day 1 showing neutrophilic infiltration (N) of dermis (D) with normal hypodermis (H). H & E stain x 400
97
E
D
F
Figure 6: Skin section of MEST treated mouse on day 1 showing normal hair follicle (F) and dermis (D). H & E stain x 400
98
E
D
Figure 7: Skin section of distilled water treated mice on day 5 showing normal epidermis (E) and dermis (D). H & E x 200
99
E F
D
Figure 8: Skin section of MEST treated mouse on day 5 showing normal epidermis (E), dermis (D) and hair follicles (F). H & E x 100
100
3.5 DISCUSSION
The result of the solubility test showed that the crude MEST was moderately soluble in distilled water and completely soluble in Tween 20. This was an indication that the extract contained both lipophilic and hydrophilic components.
MEST at a high concentration produced anaesthesia which was superior to that of lignocaine. However, it was seen that the injection of a similar concentration of MEST as lignocaine produced anaesthesia that was lower than that of lignocaine. This might be attributed to the fact that MEST was not a pure drug and was still in a crude drug form. It can be concluded from this experiment that MEST possesses local anaesthetic property.
It was also noted that the pH of both 1% and 2% lignocaine were acidic in nature.
Adjustment of the PH of MEST from 4.6 to 7.3 in vitro reduced the local anaesthetic potency to half its initial strength. This result was not expected since pH adjustment is often done to increase the amount of uncharged basic form of the drug which is the physiologically active form (Anderson, 1983). This basic form is responsible for the diffusion through the cell membrane thus increasing the onset of action and potency of the drug. Thus, the reason for the reduction in the potency of the MEST after pH adjustment is not clear. However it appears that the additions of the base solution to the
MEST solution led to a chemical reaction which might have reduced the amount of free active compound (s) in the solution.
Abdominal injection of acetic acid is often done when evaluating drugs or plant extracts for peripheral analgesic activity (Gene et al., 1998; Chakraborthy et al., 2004). It has been demonstrated that acetic acid irritates the peritoneal cavity leading to stimulation of local (C- polymodal) nociceptors located at the surface of the cells lining the cavity
(Deraedt et al., 1980; Bentley et al., 1983). Stimulation of these nociceptors leads to acute inflammation with subsequent release of inflammatory algogens such as prostaglandins 101
(PGE2 and PGE2α). The prostaglandins are known to slowly stimulate the pain receptors to bradykinin and histamine leading to non localized sharp aching inflammatory pain
(Johnston, 1997). It can thus be said that the plant extract has peripheral analgesic property. It probably reduced the pain response to acetic acid by its suppression of the release of inflammatory mediators like prostaglandin, bradykinin and histamine (Mehmet,
2002).
Acute inflammation often leads to exudation and release of chemical mediators which increase tissue permeability and cell migration (Jones and Hamm, 1977). The first phase of inflammation (exudation) is histamine mediated while the second phase is induced by algogens like prostaglandin, bradykinin and serotonin. To experimentally induce acute inflammation, carrageenan was injected subcutaneously into the plantar surface of the mice paw. The injection of this irritant reproduced the classical phases of acute inflammation in the paw of the animals. It has been suggested that the early hyperemia seen after carrageenan injection is due to the release of histamine and serotonin (Lalenti et al., 1992). The delayed phase of edema produced by carrageenan is believed to occur due to the release of bradykinin and neutrophil derived free radicals
(Ferreria et al., 1974; Sumen et al., 2001). It is also believed that macrophages in carrageenan insulted dermal tissues release interleukin- 1 (IL-1) to induce accumulation of polymorphonuclear (PMNs) cells into the inflamed area. The activated PMNs then produce lysosomal enzymes especially super oxides which destroy connective tissues leading to pain and swelling.
The extract was able to significantly reduce the edema in the paw of mice from the first hour, acting in both early and later phases of inflammation. This is an indication that it was able to inhibit various chemical mediators (histamine, prostaglandin, serotonin and bradykinin) involved in the early and late phases of acute inflammation (Kulkami et 102 al., 1986; Damas et al.,1986; Narendhirakannan et al., 2007; Woode et al., 2007; Woode et al., 2009a).
The extents of inhibition of edema by the MEST (300 mg/kg and 600 mg/kg) were more than the anti-inflammatory drug indomethacin. A similar report has been documented by Zakaria et al. (2006) following the investigation of the anti- inflammatory effect of Melastoma malabathricum chloroform leaf extract. These authors found out that the effect of the anti inflammatory drug aspirin (100 mg/kg) was lower than that of the extract throughout the experimental period. The reason for this finding was not postulated. However, it has been shown that plants contain several phytoconstituents which may exhibit complex interactions producing synergistic responses (Savelev et al.,
2003).
The use of a high dose of MEST was able to reduce the proliferative phase of chronic inflammation thus significantly reducing granuloma formation. This result is an indication that there was inhibition of cellular (granulocyte) migration and reduced accumulation of collagen and mucopolysaccarides on the implanted cotton pellets.
Numerous factors have been incriminated in the maintenance of chronic inflammatory response including prostaglandins, which are produced by polymorphonuclear cells
(Snow, 1981). Thus the effect of MEST on chronic inflammation can be related to its anti prostaglandin effect.
Inflammation is a protective response to noxious stimulation (Jones and Hamm,
1977). Most inflammatory process involves neutrophilic infiltration of damaged tissues
(Willard et al., 1994). The presence of neutrophils in the epidermis and dermis of distilled water treated mouse suggests that there was tissue reaction. This might have been a result of body response to needle puncture or the presence of distilled water. 103
There is considerable variation in research reports concerning the tissue effects of local anaesthetics. According to De Carvalho et al. (1976), Redd et al., 1990, Ribeiro et al. (2003) and Berto et al., (2011), local anaesthetics with acidic pH (3.3-5.5) can cause local tissue irritation resulting in inflammation after subcutaneous injection. Cassuto et al.
(2006) reported that clinical doses of lidocaine and other local anaesthetics studied showed anti-inflammatory activity inhibiting phagocytosis as well as inflammatory mediators. Berto et al. (2011) opined that local anaesthetics with pH (pH 5.5-7) close to physiologic pH do not cause tissue reaction. No tissue reaction was seen in mice injected with MEST (pH 4.6). The reason for this finding is not known. However, one may suggest that the absence of inflammation after MEST injection may be due to its anti inflammatory property.
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CHAPTER FOUR
EVALUATION OF THE EFFICACY OF MEST FOR LOCAL ANAESTHESIA IN
WEST AFRICAN DWARF (WAD) GOATS
105
4.1 INTRODUCTION
Goats are generally not stoic animals and have low pain threshold (Gray, 1986).
These animals thus tolerate few surgical procedures without the use of general or local anaesthesia (Gray, 1986). These anaesthetic techniques help to minimize pain, movement and vocalization during surgery (Taylor, 1991; Adetunji and Ogunyemi, 1998).
General anaesthesia in ruminants is associated with a lot of side effects such as passive regurgitation, ruminal tympany, increased salivation as well as cardiovascular depression (Taylor, 1991). Goats are thus often operated on under local anaesthetic techniques (Taylor, 1991).
Local anaesthetics are infiltrated intra testiculary to enable quick orchidectomy, reduce stress and provide anaesthesia (Haga and Ranheim, 2005). This also reduces the neuroendocrine and behavioural changes associated with orchidectomy (Molony and Kent,
1993; Molony and Kent, 1997; Mellor et al., 2000; Prunier et al., 2006; Carroll et al., 2006;
Keita et al., 2010).
Lignocaine is the most commonly used and tolerated local anaesthetic in goats and sheep (Gray, 1986; Taylor, 1991). Various studies have demonstrated the advantage of preorchidectomy intra testicular lignocaine injection (McGlone and Hellman, 1988;
VonWaldmann et al., 1994; Horn et al., 1999). No report exists on the use of a crude extract as a local anaesthetic for orchidectomy in goats.
This experiment was designed to evaluate the efficacy of MEST as a local anaesthetic for orchidectomy and flank anaesthesia in goats. To determine its anaesthetic efficacy for orchidectomy, the physiologic variables, blood glucose level and total pain score of the MEST pretreated orchidectomized goats were compared with those of lignocaine pre-treated orchidectomized goats and pain score of non anaesthezied orchidectomized goats. Also the duration of anaesthesia, degree of analgesia and distance 106 of diffusion obtained after the flank infiltration of MEST was compared with those obtained after lignocaine flank infiltration.
4.2 MATERIALS
4.2.1 Instruments and equipments
Weighing scale
Artery forceps
Ropes
Surgical scissors
Tissue forceps
Drapes
Towel clamps
Glucometer (AccucheckR)
Kidney dishes
Stop watch
Metre rule
Clinical thermometer
Stethoscope
Spatulas
Marker pen
Feeding bowls
Drinking troughs
4.2.2 Reagents and solvents
70% alcohol
Tween 20 107
4.2.3 Glass wares
Beakers
Glass rods
4.2.4 Consumables
Gauze
Needle and syringes
Cotton wool
Distilled water
Surgical gloves
Razor blades
Scapel blades
4.2.5 Drugs
Chlorhexidine
Lignocaine
4.3 METHODS
4.3.1 Evaluation of the anaesthetic efficacy of MEST for orchidectomy
Animals
Twenty male West Afican dwarf (WAD) goats of mean weight 5.7 ± 0.4 kg procured locally from Ibagwa market were used for this study. They were acclimatized for 2 weeks in the animal house of the Department of Veterinary Surgery, UNN. They were fed fresh grass (Cynodon aluefensis) supplemented with a concentrate (Bambara dusa) during the study period. Water was provided ad libitium. They were separated into four groups of five goats each.
108
Experimental groups
GP 1: Non anaesthesized orchidectomized
GP 2: MEST pretreated orchidectomized
GP 3: Lignocaine pretreated orchidectomized
GP 4: Non anaesthesized non orchidectomized
Experimental protocol
The scrotums of the goats in groups 1, 2 and 3 were shaved and disinfected using chlorhexidine. The areas around their scrotums were draped. Lignociane (LIG, 8 mg/kg,
1%) and MEST (8 mg/kg, 1%) were infiltrated into each testicle in groups 2 and 3 goats as described by (Hall and Clarke, 1991). The goats in the group 1 (control) did not receive any treatment before orchidectomy. Incisions were made through the median raphae of the scrotal sacs of the goats to allow removal of the testicles (Kumar, 2002).
4.3.1.1 Physiologic variables
The heart rates (HR) and respiratory rates (RR) of the goats in groups 1, 2 and 3 were measured before surgery (0 min), at 10 min (during surgery) and post surgery at 30,
120, 240, 480 and 720 min.
4.3.1.2 Blood glucose
The goats were fasted for 12 h before the experiment. The fasting blood glucose levels of goats in groups 1, 2, and 3 were determined before surgery (0 min) and post surgery at 30, 120, 240, 480 and 720 min. The fasting glucose levels of non anaesthesized non orchidectomized goats were also determined as in the orchidectomized groups.
Briefly, 0.5 ml blood was collected from the jugular vein of the goats. Drops of blood were touched and held at the edge of the test strips inserted into a glucometer (Accucheck advantage IIR). The blood glucose readings (mmol/l) on the glucometer were recorded. 109
The orchidectomized and non orchidectomized goats were feed after taking the 240 min glucose reading.
4.3.1.3 Pain estimation
A. Subjective pain estimation
1. Changes in physiologic parameters
The HR and RR of goats in groups 1, 2 and 3 were expressed as percentage change from their baseline readings (Gellasch et al., 2002 modified). The percentage increases above the baseline values were alloted scores as shown below:
< 10% increase above baseline = 1 (No pain).
11-20% increase above baseline = 2 (mild pain).
21-30% increase above baseline = 3 (Moderate pain).
> 30% increase above baseline = 4 (severe pain).
2. Behavioural changes
The changes in posture and appetite were scored as follows: a. Posture: Recumbent = 0; Standing = 1; Standing and hunched = 2 b. Appetite: Eating = 0; Inappetence =1
2. Objective pain estimation
The blood glucose readings of goats in groups 1, 2 and 3 were expressed as percentage change from their baseline readings. These percentages were alloted score as was done in the scoring of the physiologic parameters.
To obtain the mean pain scores of each group at each time point, the following calculations were made:
Mean pain scores = Total pain score 20 Total pain score = Subjective pain scores + Objective scores
110
Statistical analysis
The HR, RR, blood glucose and mean pain scores of the groups were compared using ANOVA. DMRT was used to separate variant means at p < 0.05.
4.3.2 Evaluation of the efficacy of MEST for flank anaesthesia.
Animals
Ten WAD goats of mean weight 6.5 ± 0.3 kg were used for this study. They were separated into two groups of five goats each
Experimental groups
Group 1: Lignocaine infiltrated
Group 2: MEST infiltrated
Experimental protocol
A marker was used to draw a line 8 cm long, 2 cm away from the thirteenth thoracic vertebrae on the left flank of each goat. 3 ml of 1% MEST and lignocaine were infiltrated as described by Hall et al. (2001c). The following were determined after the injections were made:
Onset of anaesthesia: This was calculated as the latency from the time of infiltration to loss of sensitivity to needle prick on the flank.
Duration of anaesthesia: This was calculated as the latency from the time of loss of sensitivity to needle prick on the flank to time of its return.
Degree of analgesia: The degree of analgesia obtained by the injection of the MEST and lignocaine were determined as described by Skarda and Muir (1994). 2.5 cm, 21G needles were inserted through the skin, subcutaneous (s.c) tissues (s.c) and muscle layers of the flank. Avoidance response to the needle insertion was scored as follows:
Twitching on skin prick = 1 (No analgesia). 111
No twitching of skin on skin prick = 2 (mild analgesia).
No response to insertion of needle into s.c tissues = 3 (moderate analgesia).
No response to insertion of needle to muscle layer = 4 (Deep analgesia).
Distance of diffusion: At 2, 7 and 12 min post infiltration, a meter rule was used to measure the distance (in cm) the drugs had diffused away from the lines of infiltration.
Statistical analysis
The duration of anaesthesia were compared between the two groups using K- independent sample T- test in SPSS 12.0.1 software. The degrees of analgesia obtained in the groups were compared using Mann Whitney U test.
4.4 RESULTS
4.4. 1 Evaluation of the anaesthetic efficacy of MEST for orchidectomy
4.4.1.1 Physiologic changes
The changes in HR and RR of goats are presented in Figures 9 and 10. As shown in Figure 9, the mean HR of the MEST treated goats was significantly (p<0.05) lower than the mean HR obtained in group 1 at 30 and 120 min of the study. The mean HR of the LIG treated goats was significantly (p < 0.05) lower than that of the other groups throughout the post operative period. The mean RR of MEST group was significantly (p
< 0.05) lower than RR of group 1 goats at 10, 30, 120 and 240 min. The LIG treated goats had significantly (p< 0.05) lower RR compared to groups 1 and 3 goats from 10 min of the study.
4.1.1.2 Blood glucose
The blood glucose of goats in groups 2, 3 and 4 decreased at 30, 120 and 240 min post orchidectomy. The glucose level of non anaesthesized orchidectomized goats increased post orchidectomy at 30, 120 and 240 min. The blood glucose values obtained 112 in MEST and LIG groups were significantly (p < 0.05) lower than the glucose level of goats in group 1 at 30, 120 and 240 min post surgery (Figure 11). The blood glucose level of goats in LIG, MEST and non orchidectomized group were not significantly (p > 0.05) different from 120 min of the study.
4.1.1.3 Pain scores
The mean post operative pain scores obtained in the treatments groups are shown in Table 7. The results of the experiment showed that at 30, 120 and 240 min, the pain scores of the goats infiltrated with LIG and MEST prior to orchidectomy were significantly (p< 0.05) lower than those obtained in group 1. The highest pain score was recorded for group 1 at 120 min post castration. Subsequent decrease in the pain score was recored thereafter. No significant difference (p> 0.05) was seen between the pain scores of the groups given LIG and MEST from 120 min.
4.4.2 Evaluation of efficacy of MEST for flank anaesthesia
The flank anaesthesia induced by lignocaine lasted for a significantly (p <
0.05) longer duration compared to the duration of anaesthesia in the MEST group (Table
8). At 2, 7 and 12 min, the degree of pain obtained in both groups were not significantly
(p> 0.05) different (Table 9). The distances of diffusion of MEST and LIG away from the lines of infiltration as shown in table 10 were not significantly different (p> 0.05).
113
Table 7: Mean post operative pain scores of orchidectomized goats
Time (min)
Treat. 30 120 240 480 720
Gp. 1 2.25±0.29a 2.55 ± 0.29a 2.45 ±0.25 a 2.25 ± 0.27a 1.65 ± 0.25 a
Gp. 2 1.35±0.12b 1.40 ± 0.80b 1.50 ± 0.20b 1.50 ± 0.18b 1.35 ± 0.21 b
Gp. 3 1.75±0.29c 1.10 ± 0.07b 1.20 ± 0.12b 1.55 ± 0.17 b 1.35 ± 0.20b
Group 1: Orchidectomy alone. Group 2: Lignocaine + Orchidectomy.different superscript in a column show significant difference (p< 0.05)
Table 8: Duration of anaesthesia of lignocaine and MEST
Treatments Duration of anaesthesia(min)
MEST 34.00 ± 11.60a
Lignocaine 70.00 ± 4.71b
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Table 9: Degree of pain in lignocaine and MEST groups
Treatments 2 min 7 min 12 min
MEST 4.00 ± 0.00 3.33 ± 0.67 2.67 ± 0.67
Lignocaine 4.00 ± 0.00 3.33 ± 0.67 3.33± 0.67
Table 10: Distance of diffusion (cm) of lignocaine and MEST
Treatments Distance of diffusion (cm)
2min 7 min 12 min
MEST 1.92 ± 0.14 3.37 ± 0.35 4.08 ± 0.39
Lignocaine 2.08 ± 0.26 3.85 ± 0.66 4.97± 0.78
115
250
200
150
100 Heart rates (beats/min) rates Heart
50
0 0 10 30 120 240 480 720 Time (min) Orch LIG MEST
Figure 9: Heart rates (beats/min) of goats orchidectomized goats
116
70
60
50
40
30 Resp. rates ( breaths/min) ( rates Resp.
20
10
0 0 10 30 120 240 480 720 Time (min) Orch LIG MEST
Figure 10: Respiratory rates of orchidectomized goats
117
7
6
5
4
3 Bloodglucose (mmol/l)
2
1
0 0 30 120 240 480 720 Time (min) orch. LIG MEST Non-orch
Figure 11: Blood glucose values (mmol/l) of orchidectomized goats.
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4.5 DISCUSSION
There is currently a lot of evidence showing that orchidectomy induces acute pain,
discomfort as well as physiological and behavioural changes (Molony and Kent, 1997;
Robertson et al., 1994; Hay et al., 2003; Prunier et al., 2006; Keita et al., 2010). High
frequency vocalization of long duration has been shown by piglets during orchidectomy
(White et al., 1995; Weary et al., 1998; Taylor and Weary, 2000; Marx et al., 2003). Also
the heart rate of these animals increased (White et al., 1995). Orchidectomized pigs were
also observed to be less active and showed pain related behaviours such as prostration and
trembling. They also suckled less (McGlone and Hellman, 1988; McGlone et al., 1993;
Hay et al., 2003). In lambs, all methods of orchidectomy led to changes in behaviour
which were indicative of considerable pain (Molony and Kent, 1993). Acute pain also
induced change in appetite, posture and ambulation (Chudler and Dong, 1983; Wright et
al., 1985; Morton and Griffin, 1985., Sandford et al., 1986). Pain and its associated
behavioural alteration induced by orchidectomy may persist for up to 5 days post surgery
(Hay et al., 2003).
The use of LIG prior to orchidectomy significantly reduced the pain response to
orchidectomy as shown by the nearly constant HR of goats in the group throughout the
study period. Previous studies have shown that animals felt less pain when
orchidectomized after lignocaine injection (White et al., 1995; Haga and Ranheim, 2005).
More intense physiologic changes were seen in the MEST group while marked
physiologic changes were noted in the non anaesthetized orchidectomized goats. The
physiologic changes observed in the non anaesthesized orchidectomized were similar to
that reported in a study done to evaluate the physiologic responses of pigs to surgical
castration (White et al., 1995). These researchers reported a consistent increase in the HR
and RR of pigs orchidectomized without the use of lignocaine. In all pain assessment 119 techniques used in animals, it is assumed that any change in a variable after a procedure is related to pain in the animal (Flecknell and Liles, 1991; Liles and Flecknell, 1993; Scott et al., 1994). According to these researchers, the administration of analgesics prior to the procedures prevented the occurrence of these changes.
Post operative pain leads to increased production of catecholamines (epinephrine and nor-epinephrine), glucocorticoids (eg. cortisol), growth hormones and ACTH (Bailey and Child, 1987; Bailey and Stanley, 1990). Epinephrine and glucagon activates glycogenolysis leading to increase in blood glucose (Breznock, 1980). Glucocorticoids,
ACTH and growth hormones also modify carbohydrate metabolism and induce hyperglycemia and carbohydrate intolerance (Allison et al., 1976). Blood glucose level thus increases in the immediate post trauma period but returns to normal by the second day (Breznock, 1980). In this study, the blood glucose level of LIG, MEST and non orchidectomized goats decreased at 30, 120 and 240 min of the study whereas the glucose level of the non anaesthetized orchidectomized goats increased at these time points. The result obtained in this study was similar to that of Lemke et al. (2002). These researchers investigated the effect of preoperative injection of ketoprofen on signs of post-operative pain in dogs undergoing ovariohysterectomy. They reported a decrease in the serum glucose of dogs between 4-20 h post surgeries. They concluded that the preoperative use of ketoprofen reduced signs of post operative pain in dogs. I therefore conclude that the use of MEST reduced post operative pain thus preventing post operative rise in blood glucose seen post trauma.
To estimate the amount of pain felt by the goats post orchidectomy, changes in blood glucose level, respiratory rates, heart rates, posture and appetite of the goats were scored post surgery. Comparison of the post operative pain scores obtained in the groups showed that the pain scores of LIG and MEST group were similar from 2 hours post 120 orchidectomy. This finding further supports my claim that administration of MEST before orchidectomy ameliorated the acute post operative pain induced by castration.
The degree of pain and distance of diffusion of LIG and MEST on the flank were similar throughout the period of assessment. However the duration of anaesthesia obtained in LIG group was longer compared to that obtained in MEST group. These results show that MEST exerted local anaesthetic effect on the nociceptors of the skin, subcutaneous tissues and muscles of the flank.
The shorter duration of flank anaesthesia obtained in the MEST group may be given two interpretations. One is that it can be said that LIG may be more lipophilic in nature compared to MEST. Local anaesthetics which are more lipophilic are more potent and have a more prolonged duration of action compared to less lipophilic drugs. This is because association of the drug at the lipohilic sites enhances the partitioning of the drug to its site of action and decreases the rate of metabolism by plasma esterases and hepatic enzymes (Courtney and Strichartz, 1987). The shorter duration of anaesthesia in the
MEST group may also mean that LIG produced a longer duration of action since it was a pure compound and contained more active anaesthetic compounds as against the MEST which was still in a crude form.
121
CHAPTER FIVE
FRACTIONATION OF THE CRUDE EXTRACT OF S. TRAGACANTHA AND
IDENTIFICATION OF ITS ACTIVE FRACTIONS
122
5.1 INTRODUCTION
The use of crude extracts of medicinal plants is preffered by many researchers to their purified compounds or fractions (Ajali and Okoye, 2009). These researchers are of the opinion that purification may lead to loss of bioactivity (Ajali and Okoye, 2009).
However, Ajali and Okoye (2009) while screening Olax viridis root bark for antimicrobial activity demonstrated that the fractions had better antimicrobial activity than the crude extract. This finding according to them may be due to the purification of the extract and subsequent removal of some inert compounds that may interact antagonistically with the active compound (Savelev et al., 2003; Ajali and Okoye, 2009).
Techniques used to purify crude extracts include column chromatography, thin layer chromatography, partition chromatography and paper chromatography (Trease and
Evans, 1984). Column and thin layer chromatography enables the separation of the components of a mixture (Trease and Evans, 1984).
The crude MEST has been shown in experiments 1 and 3 to possess local analgesic property. The aim of this experiment is to separate the crude MEST into its components and identify the fraction(s) with local anaesthetic activity.
5.2 MATERIALS
5.2.1 Instruments and equipments
Tripod stand
TLC chamber
UV lamp
Atomizer
Hot plate
Weighing scale 123
5.2.2 Reagents and solvents
Silica Gel 70-30 mesh, 60A (Sigma Aldrich, Germany)
Hexane (Sigma Aldrich, Germany)
Chloroform (Sigma Aldrich, Europe)
Ethylacetate (Sigma Aldrich, Brazil)
Methanol (Sigma Aldrich, Germany)
Vanillin (AnalarR, England)
Sulphuric acid
Tween 20
5.2.3 Glass wares
Glass column
Test tubes
Beakers
Glass funnel
Glass rod
Flat bottom flask
5.2.4 Consumables
Glass wool
Distilled water
Needle and syringes
Nose mask
Precoated SIL G/UV254 thin layer chromatography plates with aluminium base
(polygramR, Germany)
Gloves
Micro pipette tips 124
Filter paper
5.2.5 Drugs
Lignocaine
6.3 METHODS
Experimental protocols
5.3.1 Column chromatography
The following steps were taken in running the column chromatography.
The glass column was set up on a tripod stand.
Silica gel (70-230 mesh, 60A) was mixed with hexane and the slurry was poured
into the column up to the 500ml mark
The column was allowed to settle for 24 h.
MEST was mixed with silica gel at a ratio of 1:3 (10 g crude MEST To 30 g
silica)
The mixture was dried over a hot plate and allowed to cool properly.
The hexane in the column was drained up to the level of silica gel before the dry
mixture of MEST and silica were introduced on the column.
More hexane was immediately poured over the mixture through the sides of the
glass column.
The separation of MEST was done using the following solvents at the ratios stated
below:
Hexane 100 (200 ml)
Hexane: chloroform….. 80:20 (320 + 80 ml)
Hexane: chloroform: ethyl acetate….30:60:10 (90+180+30 ml)
Chloroform: ethyl acetate….80:20 (400 + 100 ml) 125
Ethyl acetate: methanol ……60:40 (180 +120 ml)
Ethyl acetate: methanol ……40:60 (80 + 120 ml)
Ethyl acetate: methanol ……20:80 (40 +160 ml)
Methanol …………………...100 (400 ml)
The column was allowed to run at the rate of 8 drops/15 secs.
10 ml aliquots were collected into test tubes.
At the end of the separation, the test tubes were left open to allow evaporation
and concentration of their contents before thin layer chromatography was done.
5.3.2 Thin layer chromatography.
The following solvent systems were used in the preliminary test to determine the solvent system that produced the best separation of the eluates on pre coated silica gel
GF254 aluminium TLC plates.
Hexane: chloroform: methanol (1:1:2).
Hexane: chlororform: methanol (2:1:1).
Chloroform: methanol (0.5:2).
Chloroform: methanol (1:2).
Chloroform: ethylacetate: methanol (1:2:1).
Ethylacetate: methanol (2:2).
Ethylacetate: methanol: hexane (1:2:1).
Ethylacetate: methanol (2:1).
15 ml of the eluting solvents were prepared in a TLC tank and the tank was saturated using a white filter paper. One end of the TLC chromatography plate (precoated silica gel
60 F254 plate) was marked at 1.2 cm using a pencil. Micro pipette tips were used to collect small quantities of the eluates which were then spotted on the line marked on the TLC plate. The spots were allowed to dry and the TLC plate was inserted into the TLC tank to 126 allow separation of the spots. The solvent system was allowed to ascend up the plate until the solvent front was about two-third of the plate before the plate was removed from the tank and allowed to dry. The plate was viewed under UV lamp to identify flourescent bands. Positions of the separated compounds were marked and the paper sprayed with a mixture of vanillin and sulphuric acid. The TLC plate was dried in a hot air oven at 1000C and re examined. Test tubes whose eluates showed similar bands and spots on separation were pulled together and were then dried in the hot air oven at 40 0C.
After drying the fractions, the following solvents were prepared and used in the trial separation of all six fractions as well as the crude extract.
Chloroform: ethylacetate: methanol (1:2:1).
Chloroform: ethylacetate: methanol (3:2:1).
Chloroform: ethylacetate: methanol (1:2:2).
Chloroform: ethylacetate: methanol (2:1:2).
The fractions and crude exract were streaked on small (4cm x 4cm) TLC plates and developed in a TLC tank. On drying, the plates were viewed under the UV lamp at
365nm. They where later sprayed with vanillin sulphuric acid. The best solvent system for the final separation of the fractions was identified. A large (19cm x 7cm) TLC plate was cut and the fractions and crude extract streaked on it. The TLC plate was inserted into the TLC tank to allow separation of the fractions and crude extract. The solvent system was allowed to ascend up the plate until the solvent front was about two-third of the plate before the plate was removed from the tank and allowed to dry. The plate was viewed under UV lamp at 365nm to identify flourescent bands, the positions of the separated bands and similar bands. Positions of the bands were marked and the plate sprayed with a mixture of vanillin and sulphuric acid. The TLC plate was dried in a hot 127
0 air oven at 100 C and re examined. The Rf values of the identified bands were calculated with the following formula:
Rf = Distance travelled by the band from the starting point Distance travelled by the solvent from the starting point
5.3.3 Screening of fractions for local anaesthetic activity
Animals
Four guinea pigs were used for the experiment. They were of mean weight 125 ± 0.4 g.
Experimental protocol
0.1 mg/ml and 0.033 mg/ml solutions of lignocaine were prepared using distilled water.
Also 0.15 mg/ml and 0.015 mg/ml solution of the fractions and crude MEST were prepared with Tween 20 in distilled water. 2 ml of these solutions were injected intradermally. The test procedure was done as described in experiment 2.3.3.
5.4 RESULTS
5.4.1 Fractionation of the crude MEST and identification of the active fractions
After the preliminary thin layer chromatography, the eluates were pooled into seven fractions as shown in Table 11. The following solvents mixtures gave the best separation of the components of the test tubes in the preliminary TLC:
Test tubes 0-50: hexane: chloroform: methanol (1:1:2)
Test tubes 51-59: hexane: chlorofom: methanol (1:1:2)
Test tubes 96-116: hexane: chloroform: methanol (2:1:2)
Test tubes 117-162: chlorofom: methanol (0.5:2) 128
In the second thin layer chromatography, the best separation of all six fractions as
well as the crude extract, was achieved using chloroform: ethylacetate: methanol (1:2:1)
solvent mixture. On viewing the paper under the UV lamp at 365nm F5 was showed a
characteristic red colour. Five bands (a, b, c, d and e) were identified in the crude extract
and F5 as shown in Table 11. The bands identified in other fractions are listed in Table 11
and shown in figure 9. The Rf values of the bands are also shown in Table 11.
5.4.2 Evaluation of the local anaesthetic effects of the fractions
The result of the guinea pig wheal experiment as presented in Table 12 showed
that the different fractions of MEST produced varying degrees of anaesthesia. When the
graph of the log concentrations was plotted against the percentage anaesthesia (Fig. 13-
16), the fractions 2, 3, 4, 5, 6 and 7 had slope of 16, 30.8, 8.3, 100, 36 and 83.3
respectively. LIG and the crude MEST had slopes of 69.5 and 27.1 respectively.
Comparison of the slopes showed that F5 was the most active fraction. Two MEST
fractions (F5 and F7) were more potent than LIG. These two fractions shared similar
bands (b and a) which were not seen in the other fractions. Also four MEST fractions (F3,
F5, F6 and F7) were more potent than the crude MEST.
129
Table 11: Fractions obtained from the crude MEST
Test tubes Fractions Yield (g) Band (Rf) F1 0.01 - 0-50 F2 0.34 e (0.948) 51-95 96-116 F3 0.08 d (0.897) e (0.948) 117-124 F4 0.08 b (0.707) c (0.828) d (0.897) e (0.948) 125-127 F5 0.42 a (0.534) b (0.707) c (0.828) d (0.897) e (0.948) 128-147 F6 2.14 c (0.828)
148-162 F7 0.75 a (0.534)
11.1 a (0.534) MEST b (0.707) c (0.828) d (0.897) e (0.948)
130
Table 12: Percentage local anaesthesia of the fractions Drug Concentration(mg/ml) % anaesthesia Slope
LIG 0.1 47.22 LIG 0.015 25 69.47
F2 0.15 97.22 F2 0.015 80.5 16.00
F3 0.15 77.77 F3 0.015 47.0 30.80
F4 0.15 58.33 F4 0.015 50 8.30
F5 0.15 100 F5 0.015 100 100.00
F6 0.15 100 F6 0.015 63.89 36.10
F7 0.15 100 83.34 F7 0.015 16.66
MEST 0.15 77.77 27.09 MEST 0.015 8.33
131
Figure 12: TLC plate showing bands of fractions
132
120
100
y = 16.7
80
y = 30.8
60 % Anaesthesia %
40
20
0 -2 -1.8 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0
Log concentration
F2 F3 Linear (F2) Linear (F3)
Fig 13: Graph showing slope of fractions 2 and 3
133
120
y = 100 100
80
60
y = 8.3 % Anaesthesia %
40
20
0 -2 -1.8 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0
Log Concentration
F5 F4 Linear (F5) Linear (F4)
Fig 14: Graph showing slope of fractions 4 and 5 134
120
100
y = 36.11
80
y = 83.34
60 % Anaesthesia %
40
20
0 -2 -1.8 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 Log Concentration
F6 F7 Linear (F6) Linear (F7)
Fig 15: Graph showing slope of fractions 6 and 7
135
90
80 y = 69.47
70
60
50 y = 27.098
% Anaesthesia % 40
30
20
10
0 -2 -1.8 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 Log Concentration
LIG MEST Linear (LIG) Linear (MEST)
Fig 16: Graph showing slope of LIG and MEST
136
5.5 DISCUSSION
The results of this experiment showed that the various fractions obtained from
MEST possessed various degrees of local anaesthetic activity. It was seen that fractions
F5 and F7 were the most potent having shown superior anaesthetic effect compared to the standard drug lignocaine.
Proper separation of the components of F5 and F7 during thin layer chromatography was only possible in solvent systems containing a mixture of chloroform
(non polar solvent) and methanol (polar solvent). This finding suggests the presence of both lipophilic and hydrophilic compounds in these two fractions which might have been responsible for their profound local analgesic effect. The lipophilic end of a local anaesthetic facilitates easy association with lipid membrane while its hydrophilic end increases its potency and duration of action (Mama and Steffey, 2001).
It has been shown that plants contain several phytoconstituents which may exhibit complex interactions producing synergistic or antagonistic responses (Savelev et al.,
2003). Thus it was not surprising that F3, F5, F6 and F7 were more potent than the crude
MEST showing that these fractions may contain only the phytochemical compounds responsible for local anaesthetic effect of the plant.
137
CHAPTER SIX
PHYTOCHEMICAL ANALYSIS OF THE METHANOL EXTRACT AND
FRACTIONS OF S. TRAGACANTHA
138
6.1 INTRODUCTION
Plants have been shown to contain chemicals which are responsible for their biologic activity (Ahmadiani et al., 2000; Ayinde et al, 2007; Ijeh and Uweni, 2007).
These phytochemicals include flavonoids, alkaloids, tannins, saponins, glycosides and terpenes.
Flavonoids show wide pharmacological activities and are present in many edible plants and beverages (Havesteen, 1983). A varierty of in vitro and in vivo experiments have shown that flavonoids isolated from medicinal plants possessed anti inflammatory, anti allergic, anti viral and anti oxidant activities (Middleton, 1998; Manthey, 2000;
Rajnarayana et al., 2001; Kim et al., 2004; Musa et al., 2007).
Many of the earliest isolated pure compounds with medicinal activity were alkaloids (Anon, 2009b). Tropane, isoquinoline and diterpene alkaloids have been shown to have analgesic activity (Odebiji, 1978; Tang et al., 1986; Xing-Zu, 1991). The local analgesic effect of Cassia absus was attributed to the presence of two alkaloids-chaksine and isochacksine (Bukhari and Khan, 1963; Khan, 1963). Also the local analgesic effect of Erythrophyleum guineenses was attributed to the presence of cassine while an indole alkaloid is the main constituent of Mitragyna spp responsible for its local analgesic property (Oliver-Bever, 1986).
The aim of this experiment is to identify the phytochemical compounds present in
MEST and its fractions.
6.2 MATERIALS
6.2.1 Instruments and equipments
Hot plate
Funnels 139
Water bath
Spatula
Weighing scale
6.2.2 Reagents and solvents
Molisch reagent
1% NaOH
Tween 20
0.5M Hcl
20% picric acid
95% ethanol
10% ferric acid
10% lead acetate
Conc. H2SO4
2% iodine solution
1% aluminium chloride
Ammonia solution
Fehlings I solution
Fehlings II solution
95% Chloroform
95% ethylacetate
25% H2SO4
Olive oil
Mayer’s reagent (Potassium mercuric iodide solution)
Dragendorff’s reagent (Potassium bismuth iodide solution)
Wagner’s reagent (Iodine and potassium iodide solution) 140
Conc. Acetic anhydride
6.2.3 Glass wares
Beakers
Test tubes
Glass rod
Petri dishes
6.2.4 Consumables
Filter paper
Needle and syringes
6.3 METHODS
6.31 Phytochemical analysis of the crude MEST
The crude MEST was subjceted to phytochemical analysis as described by Trease and
Evans (1984).
Experimental protocols
2 g of the crude MEST was weighed and mixed with 20 ml of distilled water to form a
100 mg/ml solution. The solution was filtered to obtain a clear filtrate. The filtrate obtained was used for the alkaloid, flavonoid, tannin, saponin and polyuronides tests as shown below. Distilled water was used as the control solution
Test for alkaloids
Mayer’s test: 2 ml of the filtrate and control solution were pipetted into two separate test tubes. To the test tubes were added 3 drops of Mayer’s reagent. The solutions were mixed and allowed to stand for 5 min and then observed for presence of precipitate and colour change. 141
Wagners test: 2 ml of the filtrate and control solutions were pipetted into two separate test tubes. To the test tubes were added 3 drops of Wagner’s reagent. The solutions were mixed and allowed to stand for 5 min and then observed for presence of precipitate and colour change.
Dragendorrf’s test: 2 ml of the filtrate and control solutions were pipetted into two separate test tubes. To the test tubes were added 3 drops of dragendorff’s reagent. The solutions were mixed and allowed to stand for 5 min and then observed for presence of precipitate and colour change.
Test for flavonoids i. 2 ml of the filtrate and control solutions were pipetted into two separate test tubes. To
the test tubes were added 3 drops of NaOH. The mixtures were allowed to stand for 2
min and then observed for presence of precipitate and colour change. ii. 2 ml of the filtrate and control solutions were pipetted into two separate test tubes. To
the test tubes were added 3 drops of NaOH and 3 drops of 0.5N Hcl. The mixtures
were observed for presence of precipitate and colour change.
Test for tannins i. 2 ml of the filtrate and control solution were pipetted into two separate test tubes. To
the test tubes were added 3 drops of 10% ferric chloride. The mixtures were observed
for presence of precipitate and colour change. ii. 2 ml of the filtrate and control solution were pipetted into two separate test tubes. To
the test tubes were added 3 drops of 10% lead acetate. The mixtures were observed
for presence of precipitate and colour change.
142
Test for polyuronoids
5 ml of ethanol and control solutions were pipetted into separate test tubes. 1 ml (100 mg/ml) of filtrate was added dropwise into the test tubes. The mixtures were observed for presence of precipitate and colour change
Test for saponins
Emulsifying test: 2 ml of the filtrate and control solution were pipetted into two separate test tubes. To the test tubes were added 3 drops olive oil and the mixture shaken vigorously. The Mixtures were observed for presence of brown emulsion.
Frothing test: 1 ml of the filtrate and control solution were pipetted into two separate test tubes. To the test tubes were added 4 ml distilled water. The mixture was shaken vigorously and then observed for presence of frothing.
Test for terpenes
0.1 g of the crude MEST was dissolved in 10 ml concentrated chloroform. The solution was filtered and used for this test. i. To 1 ml of filtrate and control solutions in separate test tubes were added 1 ml acetic anhydride. The solutions were mixed thoroughly with a glass rod. The test tubes were then placed in a slanting positions and 1 ml H2SO4 was added by the side of each test tube into the mixture. The junction of the two liquid layers was observed for presence of colour change.
Test for arthroquinone
0.1 g of the crude MEST was dissolved in 10 ml concentrated chloroform. The solution was filtered and used for this test. i. To 5 ml of filtrate and control solution in separate test tubes was added 5 ml ammonia solution. The mixtures were shaken vigorously. The mixtures were observed for presence of precipitate and colour change. 143
Test for carbohydrates
0.5 g of the crude MEST was mixed with 20 ml distilled water. The mixture was boiled for 3 min in a water bath and filtered. The filtrate was used for the following tests i. Test for reducing sugar: To 2 ml of filtrate and control solutions in separate test tubes
were added 3 drops of Molisch reagent. The mixtures were observed for presence of
precipitate and colour change. ii. Test for glycoside: To 2 ml of filtrate and control solutions in separate test tubes were
added 2 ml of Fehlings I and Fehlings II solutions. The solutions were mixed
thoroughly and boiled in a water bath for 2 min. The mixture was observed for
presence of precipitate and colour change. iii. Test for starch: To 2 ml of filtrate and control solutions in separate test tubes were
added 3 drops of 2% iodine solution. The solutions were mixed thoroughly and boiled
in a water bath for 2 min.The mixtures were observed for presence of precipitate and
colour change.
6.4 Phytochemical analysis of MEST fractions
1 mg/ml solutions of F5, F6 and F7 were prepared by solubulizing 0.02 g of the fractions in 0.2 ml Tween 20 followed by the addition of 1.8 ml distilled water. The control solution was prepared by mixing 0.6 ml tween 20 with 5.4 ml distilled water. The following tests were subsequently performed:
Test for alkaloids
Dragendorff’s test: 0.2 ml of F5, F6, F7 and control solutions was pipetted into 4 test
tubes. 1 drop of dragendorrf’s reagent was added to each test tube. The mixtures were
observed for colour change and presence of precipitate. 144
Mayer’s test: 0.2 ml of F5, F6, F7 and control solutions was pipetted into 4 test tubes. 1
drop of Mayer’s reagent was added to each test tube. The mixtures were observed for
colour change and presence of precipitate.
Test for flavonoids
0.2 ml of F5, F6, F7 and control solutions was pipetted into 4 test tubes. To each test tube was added 1 drop of NaOH. The mixtures were observed for colour change and presence of precipitate.
Test for tannins
0.2 ml of F5, F6, F7 and control solutions was pipetted into 4 test tubes. To each test tube was added 1 drop of ferric chloride and I drop of lead acetate. The mixtures were observed for colour change and presence of precipitate.
Test for terpenes
0.5 ml of F5, F6, F7 and control solutions was pipetted into 4 test tubes. To each test tube was added 0.5 ml acetic anhydride. The mixtures were observed for colour change and presence of precipitate.
Test for saponin
0.2 ml of F5, F6, F7 and control solutions was pipetted into 4 test tubes. To each test tube was added 2 ml distilled water and shaken vigorously. The mixtures were observed for frothing.
6. 5 RESULTS
6.5.1 Phytochemical analysis of crude MEST
The result of the phytochemical analysis of the crude MEST showed the presence of carhohydrate, starch, glycosides, alkaloids, flavonoids, terpenes, tannins and saponins
(Table 13). 145
6.5.2 Phytochemical analysis of MEST fractions
The fractions were soluble in distilled water as well as Tween 20. To make solutions were first dissolved with Tween 20 and distilled water was added to make up the required volume. The results of the phytochemical analysis of F5, F6 and F7 revealed the presence of alkaloids flavonoids and saponins (Table 14).
146
Table 13: Phytochemical tests results of MEST
Test Observation Inference 1 Alkaloid Filtrate + Mayer’s reagent White ppt. seen Alkaloid present Filtrate + Wagner’s reagent Reddish-brown ppt. seen Alkaloid present Filtrate + Dragendorrf’s reagent Brownish solu. seen. Alkaloid present 2 Glycoside Filtrate + Fehling’s I and II solu. Brick red ppt. seen Glycoside present 3 Starch Filtrate + iodine solution Solution turned bluish- Starch present black. 4 Flavonoids Filtrate + NaOH Solution turned yellowish Flavonoid present Filtrate + NaOH + N Hcl Solution turned yellowish Flavonoid present 5 Tannins
Filtrate + ferric chloride Brownish ppt. seen Tannins present
6 Saponins Filtrate + olive oil Brownish emulsion seen Saponins present Filtrate + distilled water Honey comb like foam Saponins present seen 7 Terpenes Filtrate + acetic acid + H2SO4 Reddish violet colour Terpene present seen between the 2 liquid layers with an upper layer 8 Artroquinone Filtrate + ammonia solution No colour change Artroquinone absent 9 Polyuronides Ethanol + filtrate No colour change Polyuronide absent
147
Table 14: Phytochemical tests results of MEST fractions
Tests Observation Inference
1 Alkaloids F5, F6, F7 + Dragendorrf’s Brick red ppt. seen Alkaloid present reagent Control + Dragendorrf’s reagent Solution turned Alkaloid absent yellowish F5, F6, F7 + Mayer’s reagent Solution turned cloudy Alkaloid present white Control + Mayer’s reagent Clear solution seen Alkaloid absent 2 Tannins F5, F6, F7 + ferric chloride No colour change Tannins absent Control + ferric chloride No colour change Tannins absent 3 Saponins F5, F6, F7 + distilled water Foaming seen Saponins present Control + distilled water No foaming seen Saponins absent 4 Flavonoids F5, F6, F7 + NaOH + N Hcl Solution turned light Flavonoids present yellow Control + NaOH + N Hcl No colour change Flavonoids absent 5 Terpenes F5, F6, F7 + acetic anhydride No colour change Terpenes absent Control + acetic anhydride No colour change Terpenes absent
148
6.6DISCUSSION
The result of the phytochemical analysis of the crude MEST and its fractions suggests that the analgesic activity of this plant may be resident in the alkaloids, flavonoids and saponins contained in the plant. Tropane alkaloids compete with the acetylcholine and block transmission of nerve signals (Anon, 2009b). These alkaloids have been identified in medicinal plants such as Hyoscyamus niger, Erythroxylum coca and Datura stramonium (Jimson weed) which are known to have analgesic properties. Also isoquinoline alkaloids such as tetrahydropalmatine (THP), stepholidine, tetrandrine, cycleanine and higenamine have been shown to have analgesic activity (Anon, 2009b).
Two diterpene alkaloids, 3 acetylaconitine (ACC) and aconitine were isolated from the root of Aconitum flavum, a plant used locally in China in the treatment of arthralagia. The analgesic effect of ACC was studied by Tang et al. (1986) using the writing test, hot plate test, formalin test and tail flick test. Their findings showed that the relative analgesic potency of ACC was 5.1 to 35.6 and 1250 to 3912 times that of morphine and aspirin respectively. Also the analgesic action of aconitine at 0.1mg/kg has been shown to be stronger than that of 6mg/kg of morphine. Organic alkaloids such as cinnamic acids
(methyl or ethyl cinnamate), shikimic acid and ferulic acid have been shown to have analgesic activity (Anon, 2009b). It has also been shown that the presence of alkaloids confers local analgesic effect to medicinal plants. For example, the leaves bark and seeds of Erythroxylum coca plant contain the alkaloid cocaine (Henry, 1949). The local analgesic effect of Erythrophyleum guineenses has been attributed to the presence of casssine while an indole alkaloid is the main constituent responsible for the local analgesic effect of Mitragyna spp (Oliver-Bever, 1986). The seeds of Cassia absus L. contain fixed oils and a toxalbumin absin as well as two alkaloids Chaksine and isochaksine. The pharmacologic study of these two alkaloids by Bukhari and Khan (1963) 149 and Khan et al. (1963) showed that both alkaloids had local analgesic action on guinea pig skin when administered intradermally.
Saponins are amphipathic glycosides which are soap like in nature, faoaming when shaken in aqueous solution. Structurally they contain one or more hydrophilic glycoside moieties with a lipophilic triterpene derivative (Hostettmann and Marston,
1995). Most analgesic glycosides are terpene glycosides. Glycosides in Cynanchum species and Clematis henryi are strongly analgesic (Anon, 2009b). Sapanion glycosides
(triterpenes) from bupleurum have been shown to have analgesic and anti- inflammatory effects. Also monoterpene glycosides of Paeonia species such as paeoniflorin have anti- spasmodic, anti-inflammatory and analgesic properties (Anon, 2009b).
The crude extract was also seen to contain flavonoids and tannins which are known to confer anti inflammatory and analgesic activities to medicinal plants (Duke,
1992; Ahmadiani et al., 2000; Usman et al., 2005). A variety of in vitro and in vivo experiments have shown that flavonoids isolated from medicinal plants possess antiallergic, anti inflammatory, anti viral and antioxidant properties (Musa et al., 2007).
Some flavonoids have been shown to have potent inhibitory effect on a wide range of enzymes such as protein kinase, protein tyrosine and phospholipase A2 (Middleton,
1998). Experiments have also shown that flavonoids also target prostaglandins which are pro inflammatory molecules (Manthey, 2000; Rajnarayana et al., 2001). These studies were able to prove that this effect of flavonoids was due to inhibition of key enzymes such as lipoxygenase, phospholipase and cycloxygenase involved in prostaglandin synthesis. Thus it can be concluded that the anti inflammatory and anti nociceptive effect of the crude MEST may be due to the presence of flavonoids and tannins.
150
CHAPTER SEVEN
GENERAL DISCUSSION AND CONCLUSION
151
7.1 DISCUSSION
The various experiments carried out showed that the crude MEST possesses peripheral analgesic, anti-inflammatory and local analgesic properties. The fractions were also shown to possess local analgesic property of different degrees.
The present study demonstrated the ability of the crude MEST to produce anti- inflammatory and anti-nociceptive activities in the experimental animals. These results authenticate the use of the bark, shoots and seeds of Sterculia tragacantha in the preparation of ethno medicines in the treatment of joint diseases such as arthritis, rheumatism, gout and whitlow. The ability of the crude MEST to show anti nociceptive activity by reducing the number of contortions induced by acetic acid suggests peripheral analgesic properties (Gene et al., 1998; Chakraborthy et al., 2004). The inhibition of acetic acid induced contortions as well as the late phase of carrageenan induced edema by the extract suggests that MEST mediated anti nociceptive and anti inflammatory activities involved inhibition of cyclo oxygenase action and prostaglandin synthesis (Damas et al.,
1986).
The acute anti inflammatory effects of the plant were found to be similar to those of indomethacin a known non steroidal anti inflammatory drug. The plant extract was however more effective than indomethacin in the suppression of chronic inflammation.
Thus since the repeated use of the NSAIDs such as indomethacine for pain relief in chronic and recurrent joint pain leads to unacceptable side effects (Insel, 1990), the use of this plant extract in the management of inflammatory conditions may be preferred with the hope that its use may not only suppress the inflammatory pain but also produce eventual elimination of the cause of inflammation and pain.
The advantage of the combined anti inflammatory and analgesic effects of the plant was also seen following its use in West African dwarf goats for local analgesia 152 before castration. The goats pretreated with MEST had lower total pain scores at 2 and 4 hr compared to goats in the two other experimental groups. The animals in the MEST treated group were observed to be standing without hunching and eating as early as two hours post castration which was an indication that they felt less pain at this time period.
This observation shows that the use of MEST for local analgesia may be more advantageous than the use of lignocaine.
The crude MEST showed local analgesic activity following intradermal and subcutaneous injection in guinea pigs and goats respectively (Experiments 1 and 3). This prompted the fractionation of the crude extract and the fractions obtained were also tested for local analgesic activity. Intra dermal injection of the fractions in guinea pigs showed that all the fractions had local analgesic activity although of varying degrees. These results suggest that both the crude extract and fractions of Sterculia tragacantha were able to inhibit nerve impulse conduction in the skin of both guinea pigs and goats. Thus it can be concluded that S. trgacantha leaves contain some active principle(s) which possesses local analgesic property.
Phytochemical analysis of the crude extract and fractions showed that they contained alkaloids and saponins. Various alkaloids such as tropane, isoquinoline, diterpene and indole alkaloids have been shown to have analgesic properties (Tang et al.,
1986). Also saponins such as triterpenes and monoterpenes have been shown to confer local analgesic effect to some medicinal plants (Hostettmann and Marston, 1995). There is however, need to further purify the fractions obtained to enable the isolation and identification of the active compound responsible for the local analgesic activity of
Sterculia tragacantha leaves.
153
7.2 CONCLUSION
The various experiments carried out during this study were able to show that the crude MEST possessed peripheral analgesic, anti inflammatory and local analgesic properties. The crude extract was found to be effective as a local analgesic for castration in West African Dwarf goats. The advantage of the combined anti-inflammatory and analgesic effects of the plant was also seen during its use in West African dwarf goats for local analgesia before castration. On fractionation of the MEST, the fractions obtained also exhibited remarkable local analgesic activity with some fractions being more potent than lignocaine. It was suggested that the local analgesic activity of MEST and its fractions may be due to the presence of alkaloids and saponins. However further work will be carried out to isolate the active compound responsible for these activities.
154
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Appendix 1: Blood glucose (mmol/l) of orchidectomized goats Time(hours) Experimental groups GP 1 GP 2 GP 3 Gp 4 0 4.9 ± 0.4a 4.5± 0.2 a 4.9±0.5 a 4.8±0.4 a 30 5.2 ± 0.3a 4.3± 0.2 b 4.7±0.3 c 4.7±0.1 c 120 5.1±0.4a 4.4±0.2b 4.2±0.3 b 4.6±0.1 c 240 5.8±0.5 a 4.2±0.2 b 4.4±0.2 b 4.4±0.2 a 480 4.6±0.2 a 4.7±0.3 a 4.1±0.2 b 5.1±0.2 b 720 4.5±0.2 a 4.9±0.2 a 4.7±0.2 a 4.7±0.1 a GP1: Non anaesthesized orchidectomized., GP 2: LIG + orchidectomy., GP 3: MEST + orchidectomy., Gp 4 = Non anaesthesized non orchidectomized.
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Appendix 2: Heart rates (beats/min) of orchidectomized goats Time(hours) Experimental groups GP 1 GP 2 GP 3 0 130.4±7.3 a 128.2±7.4 a 130.8±3.4 a 10 135.6±4.5 a 126.5± 3.0 b 131.8± 2.4 a 30 144.3±1.2 a 130.2±2.3 b 136.8±1.2 b 120 152±6.7 a 125.2±3.3 b 133.7±7.1bc 240 157.6±9.5 a 128.6±8.5 b 148.4±7.7 a 480 179.6±21.6 a 126.0±8.6 b 153.8±5.9 a 720 160.0±13.2 a 121.2±8.3 b 134.8±9.2 b GP1: Non anaesthesized orchidectomized., GP 2: LIG + orchidectomy., GP 3: MEST + orchidectomy.
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Appendix 3: Respiratory rates (breaths/min) of orchidectomized goats Time(hours) Experimental groups GP 1 GP 2 GP 3 0 32.9±3.8 a 34.9±2.5 a 33.2±1.3 a 10 48.2±1.0 a 40.5±1.3 b 45.8±0.7 c 30 58.9±0.5 a 44.2±0.6 b 48.1±0.3 c
120 44±2.3 a 30.42±2.1b 37.2±2.4 c 240 42.4±3.4 a 32.9 ± 3.9 b 33.6±3.0 b 480 37.2±3.3 a 24± 2.4 b 33.2±2.0 a 720 34.8±3.3 a 23.6±3.9 b 33.2±2.0 a GP1: Non anaesthesized orchidectomized., GP 2: LIG + orchidectomy., GP 3: MEST + orchidectomy., Gp= Non anaesthesized non orchidectomized
Appendix 4: Total post operative pain scores of orchidectomized goats Time(hours) Experimental groups GP 1 GP 2 GP 3 2 51 27 21 4 49 30 24 8 45 30 31 12 33 27 27 GP1: Non anaesthesized orchidectomized., GP 2: LIG + orchidectomy., GP 3: MEST + orchidectomy.
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Appendix 5: Duration of anaesthesia Animals GP 1(MEST) GP 2 (LIG) 1 27 63 2 26 72 3 49 76 4 24 70 5 44 70 Mean 34 70.2 SD 11.59 4.71 SE 5.18 2.10
Appendix 6: Distance of diffusion at 2 min. Animals GP 1(MEST) GP 2 (LIG) 1 1.6 2.2 2 2.0 2.8 3 1.6 1.2 4 2.1 2.2 5 2.3 2.0 6 2.5 2.1 Mean 2.02 2.08 SD 0.37 0.52 SE 0.15 0.21
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Appendix 7: Distance of diffusion at 7 min. Animals GP 1(MEST) GP 2 (LIG) 1 3.0 7.0 2 2.5 2.9 3 2.5 3.5 4 4.6 3.0 5 3.5 4.0 6 4.1 2.7
Mean 3.37 3.85 SD 0.86 1.61 SE 0.35 0.66
Appendix 8: Diffusion at 12 min. Animals GP 1(MEST) GP 2 (LIG) 1 4.0 8.0 2 2.5 4.2 3 3.9 3.5 4 4.2 6.0 5 4.5 5.1 6 5.4 2.7 Mean 4.08 4.92 SE 0.95 1.91 SD 0.39 0.78
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Appendix 9: Degree of pain at 2 min. Animals GP 1(MEST) GP 2 (LIG) 1 4.0 4.0 2 4.0 4.0 3 4.0 4.0 4 4.0 4.0 5 4.0 4.0 6 4.0 4.0 Mean 4.0 4.0 SE 0.0 0.0 SD 0.0 0.0
Appendix 10: Degree of pain at 7 min. Animals GP 1(MEST) GP 2 (LIG) 1 3.0 3.0 2 3.0 3.0 3 3.0 4.0 4 4.0 4.0 5 4.0 2.0 6 3.0 4.0 Mean 3.33 3.33 SE 1.03 1.02 SD 0.67 0.67
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Appendix 11: Degree of pain at 12 min. Animals GP 1(MEST) GP 2 (LIG) 1 3.0 3.0 2 3.0 3.0 3 2.0 4.0 4 4.0 4.0 5 2.0 2.0 6 2.0 4.0 Mean 2.67 3.33 SD 1.03 1.03 SD 0.67 0.67