I

THE HAEMA TOLOGICAL AND BIOCHEMICAL EFFECTS OF

I CONCOMITANT ADMINISTRATION OF COTRJMOXAZOLE AND

Ageratum conyzoides () IN WISTARRATS

BY

OCAKA KENETH

REG. NO: BPH/0004/101/DU

SUPERVISOR : MR. EZEONWUMELU JOSEPH.O.C

(MPHARM)

A RESEARCH REPORT SUBMITTED TO THE SCHOOL OF PHARMACY IN

PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A

BACHELOR OF PHARMACY DEGREE OF KAMPALA INTERNATIONAL

UNIVERSITY

AUGUST, 2014 . - -~.:- __-___ -~- -~_:_ __ --- -_ _- ..:...... : . -~ __ -_ -- ---=::.. -- _-_-_-- _·--~- __--;-_:_--:. .:-.=-~- -:· ___ _:: ______------,

DECLARATION

I OCAKA KENETH, a fourth year Pharmacy student hereby declare to the best of my

knowledge that this report is a true representation of my efforts. It is my original work and has

never been presented to any other institution of higher learning for approval.

.. J .?. .~.l. . o . q.J. 7.: 9..1. .t......

Signature Date

OCAKA KENETH

BPHJ0004/101/DU

Mr. EZEONWUMELU JOSEPH.O.C 1 9 /;;. Oif ~ . 2f.do ..' ...... Signature Date DEDICATION

This work is dedicated to all persons that have IDV/AIDS and whose quality of life has thus been impaired by the condition.

11 ACKNOWLEDGEMENT

I would like to thank God for the wisdom, guidance and mental stability He gave me all through

the time.

I am also grateful to my lovely mother and wonderful sister for the support and the

encouragement they have invested in my education.

I would love to appreciate all my relatives, friends and colleagues for the different forms of

support they accorded me during this period. God bless you.

I would like to acknowledge my supervisor Mr Ezeonwumelu Joseph.O.C for the academic support, guidance and time that he spent guiding and correcting me.

Ill TABLE OF CONTENTS TITLE PAGE ...... 1

DECLARATION ...... i

DEDICATION...... ii

ACKNOWLEDGEMENT ...... iii

LIST OF FIGURES ...... viii

CHAPTER ONE: ...... 1

1.0 INTRODUCTION: ...... 1

1.1 BACKGROUND ...... 1

1.2 PROBLEM STATEMENT ...... 3

13 PURPOSE OF THE STUDY/GENERAL OBJECTIVE ...... 4

1.4 SPECIFIC OBJECTIVES ...... 4

1.5 STUDY HYPOTHESIS ...... 4

1.6 STUDY JUSTIFICATION ...... 5

CHAPTER TWO : ...... 6

2.0 LITERATURE REVIEW: ...... 6

2.1 conyzoides. (An Overview) ...... 6

2.1.1 Summarized Description/Morphology ...... 8 2.1.2 Scientific classification ...... 12 2.1.3 Geographical distribution ...... 12 2.1.4 Some studies already done on the ...... 13 2.2 Cotrimoxazole ...... 17

2.2.1 Uses of the Drug ...... 17 2.2.2 Extent of use ofCotrimoxazole to manage HIV/AIDs problems e.g. Oral lesions and Pneumocystis carinii pneumonia ...... 17 2.2.3 Cotrimoxazole-Drug and Herb interactions ...... 18 2.2.4 Drug Interactions ...... 18

IV 2.2.5 Nutrition/Dietary supplement/Herb Interactions ...... 20 CHAPTER THREE: ...... 21

3.0 METHODS AND MATERIALS: ...... 21

3.1 Study Design ...... 21

3.2 Study Area/setting ...... 21

3. 3 Study Population ...... 21

3.4 Sample Size ...... 21

3 .4.1 Inclusion Criteria ...... 22 3 .4.2 Exclusion Criteria ...... 22 3. 5 Sampling Techniques ...... 22

3.6 Data Collection Procedures ...... 22

3.7 Data Analysis Procedures ...... 30

3. 8 Ethical Considerations ...... 31

CHAPTER FOUR: ...... 32

4.0 RESULTS: ...... 32

4 .1. Plant identification: ...... 3 2

4.2. Phytochemical Analysis: ...... 32

4.3 . Percentage Yield calculations: ...... 33

4.4. Acute toxicity studies using Lorkes Modified Method: ...... 34

4.5. Weight Change of animals during the study: ...... 35

4.6. Effects of treatment on some Biochemical Parameters at different time points: ...... 37

4. 7. Effects of treatment on some haematological Parameters at different time points: ...... 39

4.8. Effect oftreatment on other haematological parameters i.e. (on Counts ofLeukocyte

differentials and on their percentages ...... 44

4.9. Effect of treatment on electrolytes ...... 48

CHAPTER FIVE: ...... 52

v 5.0 DISCUSSION, CONCLUSION, RECCOMENDATIONS: ...... 52

5.1. DISCUSSION...... 52

5.2. CONCLUSION...... 58

5.3. RECCOMENDATIONS ...... 58

CHAPTER SIX: ...... 59

6.0 REFERENCES ...... 59

CHAPTER SEVEN: ...... 67

7.0 APPENDICES ...... 67

7.1 SOME TABLES: ...... 67

7.2 WORK PLAN: ...... 79

7.3 BUDGET ...... 80

7.4 THE MAP OF BUSHENYI DISTRICT ...... 81

VI LIST OF TABLES

Tablel: Phytochemicals in Ageratum conyzoides. L

Table 2: Traditional medicina//Ethno-pharmacological uses Table 3: Phytochemical analysis

Table 4: Acute toxicity results

Table 5: Mean body weights and S.E.M values ofrats (%)

Table 6: Effects of treatment on some biochemical parameters

Table 7: Effects of treatment on some haematological parameters

Table 8: Effects of treatment on other haematological parameters

Table 9: Effects of treatment on electrolytes

Table 10: Summary of the number of statistically significant changes

produced in haematological parameters

Tables I I -12: Summary of the most probable nature of the statistically

significant changes produced in haematological parameters

Table 13: Work plan

Table 14: Budget

VII LIST OF FIGURES

Figure I : Flowers of Ageratum conyzoides. Figure 2 : Ageratum conyzoides. L Herbarium specimen. Figure 3 : Graph showing change in AST over time Figure 4: Graph showing change in AL T over time Figure 5: Graph showing change in ALBUMIN over time Figure 6: Graph showing change in ALP over time Figure 7: Graph showing change in RBC count over time Figure 8: Graph showing change in MPV over time Figure 9: Graph showing change in HGB over time Figure 10: Graph showing change in HCT over time Figure II: Graph showing change in WBC over time Figure 12: Graph showing change in MCV over time Figure 13: Graph showing change in MCH over time Figure 14: Graph showing change in MCHC over time Figure 15: Graph showing change in RDW over time Figure 16: Graph showing change in PLTS over time Figure 17: Graph showing change in EOSINOPHIL count over time Figure 18: Graph showing change in BASOPHIL count over time

Figure 19: Graph showing change in MONOCYTE% over time

Figure 20: Graph showing change in EOSINOPHIL % over time

Figure 21: Graph showing change in Neutrophil count over time

Figure 22: Graph showing change in Monocyte count over time

Figure 23: Graph showing change in Neutrophil % over time

Figure 24: Graph showing change in Lymphocyte% over time

Figure 25: Graph showing change in Basophil % over time

Figure 26: Graph showing change in Na+ over time

Vlll Figure 27: Graph showing change inK+ over time

Figure 28: Graph showing change in cr overtime

Figure 29: A graph of number of statistically significant changes versus time points

Figure 30: Graphical representation of possible activity of the three agents

Figure 31 : Map ofBushenyi District.

IX ABBREVIATIONS

ADRs Adverse Drug Reactions

AIDs Acquired immune deficiency syndrome

ALB Albumin

ALP Alkaline Phosphatase

ALT Alanine Amino Transferase

ANOVA Analysis of Variance

AST Aspartate Amino Transferase

BA Basophil

CK Creatinine kinase I cr Choride

CNS Central Nervous System I I EDTA Ethylene diamine tetra acetic acid I EO Eosinophil I GLU Glucose I

GP General Practitionear

HCN Cyanic acid

HCT Haematocrit

HDL High Density Lipoprotein

HGB Haemoglobin

HIV Human Immuno deficiency Virus

K+ Potassium

X KIU Kampala International University

LDso Mean Lethal Dose

LDH Lactate Dehydrogenase

LDL Low Density Lipoprotein

MCH Mean corpuscular Haemoglobin

MCHC Mean Cell Haemoglobin Concentration

MCV Mean Cell Volume

MCV Mean Corpuscular volume

MO Monocyte

MPV Mean platelet volume

Na+ Sodium

NE Neutrophil

NIH National Institute of Health

NSA!Ds Non Steroidal Anti Inflammatory Drugs

OTC Over The Counter PCP Pneumocystis carinii Pneumonia I PLT Platelets I RDW Red blood cell distribution width I

S.E.M Standard Error of Mean

TGS Triglycerides

TIBC Total iron binding capacity

XI ABSTRACT

Introduction: Ageratum conyzoides, a member of the family of"Asteraceace" is increasingly

being used in traditional medicine for the treatment of diverse ailments (Abiodun, 2009).

Objectives: The objective of this study was to determine the haematological and biochemical effects of concomitant use of cotrimoxazole and Ageratum conyzoides leaf aqueous extract in

Wistar Rats.

Methods: Extraction of phytochemicals was performed by decoction by boiling in a beaker inside a water bath. After the extraction process, three groups (A, B, C) of rats irrespective of sex were treated as follows. The first group (A) of 5 rats was administered only cotrimoxazole orally

(trimethoprim (TMP) (5mg/kg of body weight) and sulphamethoxazole (SMX) (25mg/kg of body weight)) orally 6 hourly for 13 consecutive doses (equivalent to 3 days of conventional

Pneumocystis carinii pneumonia dosing of20mglkg/day trimethoprim and 1OOmg/kg/day sulphamethoxazole), the next group (B) of 5 rats was administered a combination of cotrimoxazole and Ageratum conyzoides orally, the third group( C) of5 animals was treated with only Ageratum conyzoides orally. All the treatments were for three days. (I.e.l3 doses which is the dose of cotrimoxazole that would establish steady state concentration within the stated period). Blood samples were collected from the rats at specific time points at 0 minute immediately after sacrificing the rats, I hour, 6 hours, 12 hours, 24 hours, 48hours and 72 hours after completing the 13 doses.

The animals were dissected from the abdominal region; blood was collected from the heart through cardiac puncture using suitable syringe and needle into EDT A vacuitaner tubes for haematological analysis and non-EDT A vacuitaner tubes for biochemical analysis.

Xll Data Analysis Proced01·es

The results obtained were expressed as numerical values of mean± S .E.M. The data was analyzed using one way analysis of variance SPSS version 12.0 (ANOVA). Differences between two means were detected using the Student's t-test. Data at P :S 0.05 was considered significant in all cases. SPSS was used to produce tables, charts and graph as a way of analyzing quantitative data. In the analysis of qualitative data, the researcher described the data collected using descriptive statement in words and some part of empirical test was done using SPSS.

Results

On phytochemical analysis, Ageratum conyzoides aqueous leaf extract was found to contain saponins, reducing sugars, flavonoids, steroids, terpenoids, diterpenes, resins, anthraquinones and essential oils. No mortality was recorded during the acute toxicity studies in rats. There was no general change in body weight of test rats; however, most of them gained weight. There was no statistically significant change in serum levels of AL T, AST, ALP and ALB enzymes at all time points when compared with the control. However, there were statistically significant decreases in MCV, RDW, MCH at 48 and 72-hr points, HCT, PLTS at 0-mins, NE count, NE% and cr while there were statistically significant increases in MCH at 24-hrs, MCHC, PL TS at 48 and 72-hr time points, MO count, L Y %, BA %, K +, and cr at the 24-hr time point.

XIII Conclusion

Ageratum conyzoides contains active secondary metabolites/phytochemicals. They could be behind the medicinal property of Ageratum conyzoides as explored in traditional medicine.

Ageratum conyzoides aqueous leaf extract has a high safety margin.

Concomitant administration of cotrimoxazole and Ageratum conyzoides aqueous leaf extract has no effects on the biochemical parameters studied and as such does not cause any significant biochemical changes with regard to the studied parameters.

Concomitant administration of cotrimoxazole and Ageratum conyzoides aqueous leaf extract has some effects on haematological parameters studied and as such causes some significant changes in the haematology of Wistar rats, notably anaemia and thrombocytopenia. However, these effects and other statistically significant effects are higher at 0-mins when drug levels in circulation are high but they disappear/wane with progressive time, upon withdrawal of the drugs.

XIV ~V> "~~---'''''''"""~'='"~

CHAPTER ONE :

1.0 INTRODUCTION:

I.! BACKGROUND

Drug-herb iuter-actions

Herbs are crude plant material such as leaves, flowers, seeds, stems or other plant parts, which may be entire, fragmented or powdered.

Study shows a behavioral pattern of concomitant use of herbs and conventional medicines

(Kaufman, eta!., 2002).

14 to 16% of American adults and 49.4% oflsraeli users often take herbs with prescription drugs

(Kaufman, eta!., 2002; Giveon, eta!., 2004)"

Many dangerous interactive actions of herbs result when co-administered with other drugs or herbs e.g. increased bleeding tendencies (Stephen, 2004).

Virtually all herbal remedies can cause allergic reactions and several can be responsible for photosensitization (Ernest, 2000).

Although herbs are often believed to be "natural" and therefore safe, many dangerous and lethal side effects have recently been reported, including direct toxic effects, allergic reactions, effects from contaminants and interactions with drugs and other herbs. (Stephen, 2004).

I According to Frederick (2004), herbal medicines and nutritional supplements for example chamomile, canthaxanthine, Echinacea pmpurea, Ginkgo biloba, liquorice, niacin, and vitamin

A are all associated with clinically significant ocular side effects.

Niggeman and Gruber (2003) also reported that organ toxicity had been observed associated with various herbal preparations involving the liver, kidneys and the heart. The authors further stated that herbs and drugs could also interact the same way drug/drug interactions occurred.

A study done in UK revealed that 26% of patients using both conventional OTCs and herbal remedies would consult their GP for a serious ADR to OTC medicines but not a similar ADR to a herbal remedy, whereas 0.8% of respondents would consult their GP for a serious ADR to a herbal remedy but not a similar ADR to conventional OTC medicine (Barnes, et al., 1998)

Acetaminophen may interact with ginkgo and possibly with some other herbs e.g. ginseng, ginger and feverfew to increase the risk ofbleeding. Further, the incidences of hepatotoxicity and nephrotoxicity may be augmented by acetaminophen when concomitantly used with the potentially hepatotoxic herbs Echinacea and kava, and with herbs containing salicylate (willow, meadowsweet), respectively. The concomitant use of opioid analgesics with the sedative herbal supplements, valerian, kava and chamomile, may lead to increased central nervous system (CNS) depression. The analgesic effect of opioids may also be inhibited by ginseng (Abebe, 2002).

Concurrent use of herbs may mimic, magnify, or oppose the effect of drugs (Adriane, 2000).

An important surgical interaction is unanticipated excessive bleeding associated with garlic, ginkgo biloba and ginger. The peri-operative use of NSAIDs is increasing. The use of NSAIDs in patients taking herbal medicines such as garlic, gingko and ginger may cause increased peri­ operative bleeding (Kam, 2002).

2 Wide spread use of herbal medications among pre-surgical population may have negative impact on perioperative patient care (Michael, eta!., 200 1).

Morbidity and mortality associated with herbal medications may be more likely in the perioperative period because of complications due to polypharmacy and physiological alterations that occur. Such complications include myocardial infarction, stroke, bleeding, inadequate oral anticoagulation, prolonged or inadequate anesthesia, organ transplant rejection, and interference with medications indispensable for patient care (Michael, et al., 2001 ).

1.2 PROBLEM STATEMENT

According to the above recorded information, it is clear that co-administration of a drug with a herb can have either positive or negative effects on some or all body systems. The above drawn conclusions were made after careful drug-herb interaction studies. However, the biochemical and I haematological effects of co-administration of cotrimoxazole and Ageratum conyzoides is not known because no such a study has been undertaken before and yet some HIVI AIDS patients I who concomitantly use cotrimoxazole for prophylaxis of pneumocystis carinii pneumonia and extract of Ageratum conyzoides for treatment of resistant and persistent oral lesions do so f without experiencing adverse effects. It therefore presents a knowledge gap that needs to be filled. It is therefore the aim of this research to determine any effects of co-administration of cotrimoxazole and Ageratum conyzoides leaf aqueous extract e.g. magnified effects or opposed effects of the two if any is present.

3 1.3 PURPOSE OF THE STUDY/GENERAL OBJECTIVE

To ascertain the biochemical and hematological effects of concomitant administration of

Ageratum conyzoides leaf extract and cotrimoxazol e on some parameters in Wistar rats.

1.4 SPECIFIC OBJECTIVES

To collect, identity and authenticate the leaves of Ageratum conyzoides.

To obtain a hot aqueous leaf extract of Ageratum conyzoides

To determine its phytochemical constituents.

To determine its acute toxic effects.

To determine the biochemical and hematological effects of concomitant administration of

Ageratum conyzoides leaf aqueous extract and cotrimoxazole in Wistar rats

1.5 STUDY HYPOTHESIS

Null Hypothesis:

When concomitantly given, cotrimoxazole and Ageratum conyzoides leaf aqueous

extract would not interact and the combination would produce no changes in any

biochemical or haematological parameter in Wistar rats.

Alternative Hypothesis:

When concomitantly given, cotrimoxazole and Ageratum conyzoides leaf aqueous extract

would interact to cause changes in some biochemical and hematological parameters in

Wistar rats.

4 1.6 STUDY JUSTIFICATION

This study would provide scientific evidence that would:

yield information that could expand the global scientific body of knowledge.

enable the general public to know whether concomitant use of Ageratum conyzoides leaf

aqueous extract and cotrimoxazole is safe.

support or discourage the concomitant use of cotrimoxazole and Ageratum conyzoides

extract.

5 CHAPTER TWO :

2.0 LITERATURE REVIEW:

2.1 Ageratum conyzoides. (An Overview)

Ageratum was derived from the Greek words 'a geras', meaning non-aging, referring to the longevity of the whole plant. Conyzoides on the other hand was derived from 'konyz' the Greek name of Inula helenium which the plant resembles. (Kissmann and Groth, 1993)

Ageratum conyzoides L. belongs to the family Asteraceae tribe Eupatoriae. This family is well marked in their characteristics and cannot be confused with any other. A large majority ofthe in this family are herbaceous while trees and shrubs are comparatively rare. The genus

Ageratum consists of approximately thirty species but only a few species have been phytochemically investigated (Burkill, 1985).

The synonyms of A. conyzoides include A. album Stend, A. caeru.leum Hort.ex Poir., A. coeruleum Des£, A. cordifolium Roxb., A. hirsutum Lam., A. humile Salisb, A. latifolium Car.,

A. maritimum H.B.K., A. mexicanum Sims., A. obtusifolium Lam., A. odoratum Vilm. and

Cacalia mentrasto Yell. (Jaccoud, 1961).

In Thailand, local names of A. conyzoides L. were listed as follow: Tapsuealek (Sing Buri),

Thiam mae haang (Loei), Saap raeng saap kaa (Chiang Mai), Yaasaap haeng (Chiang Mai) and

Yaa saap raeng (Ratchaburi) (Smitinand, 1980).

Johnson (1971) classified A. conyzoides into two subspecies, Latifolium and conyzoides. Subsp. latifolium is found in the entire USA continental and subsp.conyzoides has a pantropical distribution. The basic chromosome number is 2n = 20 but natural tetraploids are found.

A. conyzoides subsp. latifolium is diploid while A.conyzoides subsp. conyzoides is tetraploid.

6 Backer and van den Brink (1968) described the genus Ageratum L. as an erect herb whose Lower

leaves arrangement are opposite, petioled, serrate-crenate above the entire base, penninerved or

subtrinerved, higher ones 5 alternate, petiolate, ovate, dentate or serate. Heads a~e corymbose or loosely peniculate, homogamous, discoid, with many flowered. They also have an Involucre campanulate, imbricate, bracts 2-3 seriates, linear, acute to acuminate, sub equal; receptacle flat or nearly so, naked or with caduceus scales. Also, Corollas are all tubular, equal, regular and limb 5-fid. Their anthers have an apical appendage, the base entire and obtuse. They also have long style-arms which is slender, obtuse and pubescent at the apex. Their achenes are oblong and

5-angular. Their pappus is uniseriate, of 5 short free or connate scales, or of I 0-20 narrow acuminate unequal scales.

Ageratum conyzoides L. is an annual branching erect herb which grows to approximately I m. in height. The stems and leaves are covered with fine white hairs, the leaves are petiolate, ovate up to 7.5 em long, the apex acute, the base truncate to rounded, rarely cordate, the margins crenate.

The inflorescence is purple to white head, less than 6 mm across and arranged in close terminal corymbs of 8-15 heads. Involucres are campanulate, the bracts are 2-3 seriate, linear, sub equal, acute, sparsely pilose outside; corollas are all tubular, 1-1.5 mm long, the limb 5-cleft. The fruits are linear-oblong black achene with 5-angled and are easily dispersed while the seed are photoblastic and often lost within 12 months; pappus are 5 short scales, the scales are often serrate below ending in a long awn (Backer and van den Brink, 1968).

A. conyzoides L. are native to tropical America. It is now found in all warm and subtropical areas of the world that is very common in West Africa and some parts of Asia and South America. It is usually found in waste places, rice fields, gardens, old cultivations, low secondary growth

7 forests, forest-edges, roadsides, water courses etc., where there is ample exposure to sunlight

(Dung, eta!., 1996).

It has a particular odour likened in Australia to that of a male goat and hence its name 'goat weed' or billy goat weed' (Okunade, 2002).

A. conyzoides L. was beneficial in conventional medicine all over the tropical areas. The biological activities of the plant have been undertaken. Some of the significant ethno­ pharmacological applications of A. conyzoides L. are shown in a subsequent Table.

2.1.1 Summarized Description/Morphology.

Ageratum conyzoides L., Asteraceae, is an annual herbaceous plant that grows about

60 em high and produces small pretty pink flowers at the top of its hairy stems.

According to Medicinal Plants Information of Orissa (India), Ageratum conyzoides is:

-a herb or undershrub that normally flowers in October/November.

-Its flowers are normally white in colour and the plant fruits in November/December.

-It frequently occurs in plains and hilly areas.

-is regarded as prone to becoming a rampant environmental invasive weed when

grown outside of its natural range.

Other sources describe Ageratum conyzoides as: an erect, herbaceous annual, 30 to 80

em tall whose stems are covered with fine white hairs, whose leaves are opposite,

pubescent with long petioles and include glandular trichomes. The inflorescence

contain 30 to 50 pink flowers arranged as a corymb and are self-incompatible (Jhansi

and Ramanujam, 1987; Kaul and Neelangini, 1989; Ramanujam and Kalpana, 1992;

Kleinschimidt, 1993).

The fruit is an achene with an aristate pappus and is easily dispersed by wind. In some

8 countries the species is considered a weed, and control is often difficult (Lorenzi, 1982;

Scheffer, 1990; Kalia and Singh, !993; Lam, eta/., 1993, Paradkar, et al., 1993;

Waterhouse, 1993; Kshatriya, et a/.,1994).

Seeds are positively photoblastic, and viability is often lost within 12 months (Marlks

and Nwachuku, 1986; Ladeira, eta/., 1987).

The optimum germination temperature ranges from 20 to 25°C (Sauerborn and Koch,

!988).

The species has great morphological variation, and appears highly adaptable to different

ecological conditions.

9 Figure 1:

10 Figure 2: Ageratum conyzoides L herbarium specimen.

11 2.1.2 Scientific classification.

Kingdom: Plantae

Class: Angiospermae

Unranked:

Superorder/Subclass: Asteridae

Order:

Family: Asteraceae

Tribe: Eupatorieae

Genus: Ageratum

Species: conyzoides

Binomial name: Ageratum conyzoides

Common name in Uganda (Runyankole): Butabuta

2. 1.3 Geogmphical distl"ibution.

Ageratum ranges from South-eastern North America to Central America, but the

centre of origin is in Central America and the Caribbean. Ageratum is also found in

several countries in tropical and sub-tropical regions, including Brazil. It is also

found in Africa, Australia, and South eastern Asia.

12 2.1.4 Some studies already done on the plant.

Laboratory research has validated several of Ageratum's uses in traditional medicine.

Test tube studies (in vitro) have reported that extracts of the whole plant have an

antibacterial action against Staphylococus aw·eus (Akinyemi, et al., 2005) and

Eschericichia coli (Moody, et a/.,2004).

In animal studies, it demonstrated a muscle relaxing and pain relieving effect,

confirming its popular use for rheumatism (Silva, et al., 2000).

Researchers in Africa confirmed its traditional uses for wound healing in animal

studies with rats in 2003 (Oladejo, eta!., 2003; Durodola, eta!., 1977) Other research in India reported that Ageratum protected mice from radiation damage I and prevented ulcers in mice. Both cellular protection actions were attributed, in part, r I to an antioxidant effect noted for Ageratum (Jagetia, et al., 2003) !

Scientists have also discovered that Ageratum has an insecticidal effect by interfering

with the reproductive cycles of many species of insects (Saxena, eta!., 1992) I

Biochemical, haematological and histopathological studies of the extract of

Ageratum conyzoides.L in Sprague Dawley rats have also been done (Abiodun, eta!.,

2010)

2.1.5 Phytochemical components.

Study of the chemical profile of Ageratum conyzoides showed that they contain

phytochemicals, as illustrated below. These compounds could be behind the medicinal

property of Ageratum conyzoides as explored in traditional medicine. fable 1: Phytochemicals in Ageratum conyzoides. L

13 Phytochemical Example References

Alkaloids Lycopsamine Pyrrolizidine Wiedenfeld and Roder, 199

1,2 desilropirrolizidinic Rorie, eta!., 1993

Caffeic acid Nair, eta!., 1977

Flavonoids Nobiletin Vyas and Mulchandani.1986; Gonzalez, eta!., 1991b

Ageconyflavone B Vyas and Mulchandani.l986

Triterpenes and Friedel in Dubey, eta!., 1989; Horng, eta!., 1976; sterols Hui and Lee, 1971

Dubey et al., 1989; p -Sitosterol Horng eta!., 1976; Hui and Lee, 1971

Mono and sabinene Ekundayo, eta!., 1988

Sesquiterpenes camphene, Dung, et al., 1996 cubebene, elemene farnesol. Chromenes, 7-methoxyageratochromene Dung, eta!., 1996; (Precocene I) benzofurans and Encecalin Ekundayo, eta!., 1988; Gonzalez, eta!., coumanns 1991a

(l.buakhao hongsachum, 2008)

NB) Aside from the phytochemicals/bioactive compounds named in table I, Ageratum

7onyzoides contains other components including essential oils. The main plant chemical found in

14 the plant include: 6, 7-dimethoxy-2,2-dimethylchromene, 6-demetoxyageratochromene, 6-vinyl­ demethoxy-ageratochromene, ageratochromene, alpha-cubebene, alpha-pinene, alpha-terpinene, beta-caryophyllene, beta-cubebene, beta-elemene, beta-farnesene, beta-myrcene, beta-pinene, beta-selinene, beta-sitosterol, cadinene, caryophyllene-oxide, conyzorigin, coumarin, dotriacontene, endo-borneol, endo-bornyl-acetate, ethyl-eugenol, ethyl-vanillin, farnesol, friedelin, HCN, hexadecenoic-acid, kaempferol, kaempferol-3, 7-diglucoside, kaempferol-3-o­ rhamnosylglucoside, linoleic-acid, quercetin, quercetin-3, 7-diglucoside, and quercetin-3-o­ rhamnosylglucoside (Lin chau ming, 1999).

15 2.1.6 Traditional medicinai/Ethno-pharmacological uses.

It differs according to continent, country or ethnicity as illustrated below in Table 2:

ETHNOMEDICAL USES

1 Geological area l Part used Indication Reference . 1 J I .

I Africa, Asia Whole plants ' Folk remedies, purgatives, Githens, 1948 ! and South I i febrifuge, opthalmia, colic, I I America ulcer, wound dressing. I I Kenya Whole plants !Anti asthmatic, 1 Kokwaro, 1976 I I f I ' Antispasmodic, Haemostatic 1 I l effects I 1 Kenya 1 Leaves, roots Stomach ache Geissler eta/, ! I , 2002 I I I I 1 I Tanzania Seeds Treatment of epilepsy Moshi, eta/., I I I l f 2005 j I I I 1 Nigeria Leaves Skin diseases, wound healing, Okunade, 1981 1 I I I I I I 1diarrhea, relieves pain I ! I I I Central Africa , Whole plants Burned wound Durodola, 1977 I 1

1' Central Nigeria Fresh leaves For body infection, neck pain Bhat, e I 1 i 1 I I I I 1 a!., 1990 I i I

16 2.2 Cotrimoxazole.

2.2.1 Uses of the Drug.

According to Uganda Clincal Guidelines (UCG, 2012) cotrimoxazole is used to treat a variety of infections including: Upper respiratory tract infections, lower respiratory tract infections, urinary tract infections, gastro-intestinal infections and skin infections i.e. cotrimoxazole is used in severely compromised patients e.g. IDV patients as prophylaxis against

Pneumocystis Jiroveci Pneumoniae (PCP) according to UCG (2012). Its also used in the treatment ofbrucellosis, typhoid/enteric fever, ulcerations and secondary infections due to

Drancunculiasis (Guinea worm infestation) and super infections that occur in chronic asthma among others.

Cotrimoxazole is reccomended by UCG as the first line drug for the management of the following conditions among children: pneumonia, dysentery due to shigella, cholera, acute ear infections and very severe /complicated measles.

2.2.2 Extent of use of Cotrimoxazole to manage HIVI AIDs problems e.g. Oral lesions and

Pneumocystis carinii pneumonia.

Co-trimoxazole (trimethoprim-sulfamethoxazole) is a widely available, off-patent, low-cost antibiotic that is used in resource-limited settings to treat and prevent community-acquired infections. Although not recommended as malaria prophylaxis, similar to pyrimethamine­ sulfadoxine, it also has antimalarial activity (Bloland, eta/., 1991). In IDV infection, it is highly effective for treatment of and prophylaxis against Pneumocystis jirovecii pneumonia,

Toxoplasma gondii, and Isospora belli (Jones, et al. , 1999). Results of clinical trials and

17 observational studies in IllY-infected, combination antiretroviral therapy (ART)-naive adults and children across Africa have shown that co-trimoxazole prophylaxis reduces mortality, morbidity, and hospital admissions, even in areas of high background bacterial resistance (Anglaret, et al. ,

1999). WHO guidelines recommend that co-trimoxazole prophylaxis is given to all symptomatic adults with CD4 counts lower than 350 cells per~ in resource-limited settings. Cotrimoxazole is thus widely used among IDV patients in Africa (WHO, 2010).

2.2.3 Cotrimoxazole-Drug and Herb interactions

2.2.4 Drug Interactions

According to Stockleys drug interactions book, 8th edition, (Ed) Karen Baxter, the following is true about cotrimoxazole:

Cotrimoxazole is a combination of Sulfamethoxazole (800 mg) and Trimethoprim (160mg)

Sulfamethoxazole: Substrate of CYP2C8/9 (major), 3A4 (minor); Inhibits CYP2C8/9

(moderate)

Trimethoprim: Substrate (major) of CYP2C8/9, 3A4; Inhibits CYP2C8/9 (moderate)

ACE Inhibitors and angiotensin receptor antagonists: May increase the risk of hyperkalemia with sulfamethoxazole/trimethoprim. (pg 3)

Amantadine: Concurrent use with sulfamethoxazole/trimethoprim has been associated with toxic delirium (rare).

18 Cyclosporine: May result in an increased risk of nephrotoxicity when used with sulfamethoxazole/trimethoprim. Sulfonamides may decrease the serum concentrations of cyclosporine.

CYP2C8/9 inducers: May decrease the levels/effects of sulfamethoxazole. Example inducers include carbamazepine, phenobarbital, phenytoin, rifampin, rifapentine, and secobarbital.

CYP2C8/9 substrates: Sulfamethoxazole/trimethoprim may increase the levels/effects of

CYP2C8/9 substrates. Example substrates include amiodarone, fluoxetine, glimepiride, glipizide, nateglinide, phenytoin, pioglitazone, rosiglitazone, sertraline, and warfarin.

Dapsone: Trimethoprim may increase the serum concentration of dapsone.

Diuretics, potassium-sparing: May increase the risk of hyperkalemia with sulfamethoxazole/trimethoprim.

Leucovorin: Although occasionally recommended to limit or reverse hematologic toxicity of high-dose sulfamethoxazole/trimethoprim, concurrent use has been associated with a decreased effectiveness in treating Pneumocystis carinii.

Methotrexate: Sulfamethoxazole/trimethoprim may increase toxicity of methotrexate (due to displacement from binding sites and/or decreased renal secretion).

Phenytoin: Sulfamethoxazole/trimethoprim may increase phenytoin levels/toxicity. Phenytoin may decrease sulfamethoxazole/trimethoprim levels.

Procainamide: Trimethoprim may decrease the excretion of procainamide.

19 Pyrimethamine: Concurrent therapy with pyrimethamine (in doses >25 mg/week) may be at increased risk of megaloblastic anemia.

Sulfonylureas: Sulfamethoxazole/trimethoprim may increase the hypoglycemic effect of sulfonylureas; monitor.

Warfarin: Sulfamethoxazole/trimethoprim may increase the hypoprothrombinemic effect of warfarin; monitor INR closely.

2.2.5 Nutrition/Dietary supplement/Herb Interactions

Herbs/Nutraceuticals: Avoid dong quai and StJohn's wort (may also cause photosensitization).

Vitamin K: Several cases of excessive bleeding have been reported in people who take

antibiotics e.g. cotrimoxazole (Suzuki, Fukushima and Meguro, et al., 1999;

Conly and Stein, 1994). This side effect may be the result ofreduced vitamin K

activity and/or reduced vitamin K production by bacteria in the colon.

Potassium : Cotrimoxazole (TMP/SMX) has been reported to increase blood potassium to

levels above the normal range in some patients, particularly those with impaired

kidney function (Alappan, Perazella and Buller, 1996).

PABA: PABA may interfere with the action of sulfamethoxazole. It should not be taken

together with cotrimoxazole.

Folic acid: Cotrimoxazole (TMP/SMX) has, on rare occasions, been associated with anaemia

due to folic acid deficiency (Young and Koda-Kimble, 1988). This effect may be

due to trimethoprim which is a Folic acid antagonist/Analogue (Kahn, Fein and

Brodsky, 1968). TMP/SMX should be used with caution in patient with folic acid

deficiency (Young and Koda-Kimble, 1988).

20 CHAPTER THREE:

3.0 METHODS AND MATERIALS:

3.1 Study Design

It was an experimental study in which laboratory Wistar rats were used. There was a control group and other experimental groups for all the work involving experimental animals.

3.2 Study Area/setting

The study was carried out in the Pharmacognosy and Pharmacology Laboratories ofKampala

International University-Western Campus, Ishaka, Bushenyi District, Uganda and analytical laboratories ofMbarara University of Science and Technology, Mbarara.

3.3 Study Population

81 Wistar rats were used for the study and they were selected from a group of rats bred at School of Pharmacy Laboratory.

3.4 Sample Size

16 Wistar rats were used for acute toxicity test done according to a modified version of a standard method (Lorke, 1983) and 65 Wistar rats were used for the interaction study.

SOOOgms of fresh Ageratum conyzoides leaves will be picked randomly from gardens in

Bushenyi.

21 3.4.1 Inclusion Criteria

Only mature, healthy and adult rats weighing greater than lOOgms were used for the studies.

3.4.2 Exclusion Criteria

Immature rats, those that weighed less than 1OOgms and any rat that had any wound on the body or that manifested any signs of pregnancy or ill health were excluded.

3.5 Sampling Techniques

Selection of animals for the study was by random sampling technique. The rats were chosen randomly by picking without replacement and marking on the tail using a permanent marker.

Randomization of these rats into groups were done by writing the numbers of all these animals on small papers that were folded and placed together into a box. Five papers were picked at random from the box without replacement and these were automatically assigned to a group and that was done for all the remaining groups.

3.6 Data Collection Procedures

The primary source was the observations and recordings of experimental findings in the laboratory during the study period.

Plant material collection and processing

About 5000gms of Ageratum conyzoides leaves were randomly taken from gardens in Bushenyi, dried under shade, then ground in to powder and weighed.

22 Extract preparation

Extraction from powdered leaves was carried out by macerating 1OOg of initially powdered leaves in 500ml ofhot aqueous solvent, and then shaking for 24 hours while placed in boiling water in a water bath.

Filtration was done to get filtrate which was concentrated in boiling water in a water bath and the residue dried in a hot air oven and extract was weighed.

• Percent yield of extraction from hot aqueous extraction was calculated as follows:

% Yield= (weight of extract+container)-weight of container alone X 100

Weight of the initial powder

• Generally, volume to administer was calculated by the formula below

Volume = weight ofthe rat (g) x dose (mg/Kg)

Concentration (mg/ml) x 1000

• The extract was then kept under refrigeration until when required for use/administration.

23 Qualitative phytochemical tests

The extract of Ageratum conyzoides was subjected to qualitative chemical tests for the identification of active phytochemicals. These tests were carried out based on the standard methods described by Trease and Evans (2002) and Harbone (1998).

(a) Test for alkaloids

Mayer's tests:

2 ml of the extract was acidified with 3 drops ofHCL and stirred on a water bath.3 drops of

Mayer's were added to 1 ml. No observable change indicated the absence of alkaloids.

Wagner's test:

To 2 ml of extract, 3 drops of 10% HCL was be added. The mixture was be stirred on a water bath. Then 3 drops of Wagner' s reagent was added to the filtrate. No observable change confirmed the absence of alkaloids.

(b) Test for reducing sugars

Benedict's test:

To 1 ml of extract, 4 drops of distilled water was added. The mixture was filtered. 3 drops of

Benedict's solution was added to filtrate and boiled for 5 minutes. A brick red colour precipitate indicated the presence of reducing sugars.

(c) Test for saponins

Frothing test:

2ml of extract was shaken with 5 ml distilled water and heated to boil. Formation of a layer of froth showed the presence of saponins.

24 ...... _ - -~ - - -~- :::

(d) Test for flavonoids

Shinoda's test:

To 4 ml of extract, 1 ml of 50% methanol solution and metal magnesium was added. The mixture was warmed and 5 drops of concentrated HCL was also added. Presence of a purple colour showed that flavonoids were present.

(e) Test for tannins

To 3 ml of extract, 3 drops offerric chloride solution was added. No observable change showed the absence of tannins.

(f) Test for steroids

3 drops of acetic anhydride was added to 2 ml of extract. The mixture was boiled and cooled. 1 ml of concentrated sulphuric acid was added down the side of each test tube containing the sample. Formation of a brown ring at the junction of the two layers, with the upper layer turning green indicated the presence of steroids.

(g) Test for terpenoids

Salkowski test:

2ml of extract was mixed with 2 ml of chloroform. To the mixture, 3 ml of concentrated sulphuric acid was carefully added to form a layer. A reddish brown colouration at the interface showed the presence of terpenoids in the sample.

25 (h) Test for amino acids

Ninhydrin test:

To 2 ml of extract, 1 ml of Ninhydrin (0.25% w/v) was added and the mixture boiled for 5

minutes. The absence of a blue colouration in the test sample showed the absence of amino acids

in the sample.

(i) Test for phenols

2 ml of distilled water was added to 1 ml of extract. 2 drops of ferric chloride ( 10%) solution was added to the test sample. Absence of a green or blue colour indicated the absence of phenols.

(j) Test for diterpenes

Copper acetate test:

To J ml of extract, 4 drops of copper acetate solution was added. Formation of emerald green colour/precipitate showed the presence of diterpenoids.

(k) Test for phlobatanins

1 ml of extract was dissolved in 2 ml of distilled water and filtered. The filtrate was boiled with 1 ml of2% HCL. No observable change indicated the absence ofphlobatanins in the sample.

(I) Test for anthraquinones

1ml of extract was boiled with 2 drops of 10% HCL for 5 minutes in water bath. A few drops of

10% FeCb and Diethyl ether was then added followed by 1ml ofconc. Ammonia solution. A rose pink colour formed thereby confirming the presence of Anthraquinones.

26 (m) Test for resins

1 ml ofthe extract was dissolved in Alcohol solution and the mixture was added to 10mls of

distilled water. A suspension was observed on addition of water and ethanol. That confirmed the

presence ofResins.

(n) Test for Quinones

lml of extract was mixed with 1ml ofconc. Sulphuric acid. No observable change confirmed the absence of Quinones.

( o) Test for Essential/volatile oils

1ml of extract was mixed with 1ml of an alcoholic solution of Sudan 3 dye. Traces of a red colour indicated the presence of some Essential oils.

(p) Test for proteins.

To lml ofthe extract was added 1ml of a 5% NaOH solution followed by 2 drops ofCuSo4 solution, mixed well in a test tube then heated over a Bunsen burner flame. No purple colour formed denoting absence of proteins.

( q) Test for starch.

To Sml ofthe extract was added 4 drops oflodine solution. The absence of an intense blue colour indicated starch was absent.

Acute Toxicity

The acute toxicity study was conducted in accordance with modified Lorke's method (Lorke,

1983). The study was conducted in two phases using a total of 16 rats. In phase 1, nine rats were divided into 3 groups of 3 rats each. Groups 1, 2 and 3 animals were respectively given 200, 400 and 800 mg/kg body weight of the Ageratum conyzoides leaf aqueous extract orally, and the rats

27 were observed for at least three hours post administration for signs of toxicity. After 24 hours,

they were scored for mortality and general behaviour. In addition, a fourth group of three rats

was set up as control group and animals in the group were given 1OmL /kg of distilled water.

In phase two after 24hours, 4 groups of one rat each were given geometrically increasing doses

ofthe extract based on findings of phase 1 (there was no death recorded), (group 1-2500 mg,

group 2 - 5000 mg, group 3-7500mg and group 4 - 10,000 mg/kg body weight) and then observed as in phase 1 above.

The geometrical mean of the smallest dose that killed a rat and the highest dose that did not kill the rat was taken as the mean lethal dose (LD5o).

Acquisition and maintenance of Wistar rats

81 Wistar rats were:

• acquired from the School ofPharmacy Laboratory

• housed in spacious cages with wood shavings

• at room temp. with adequate ventilation

• 12 hrs each of light and darkness

fed on standard diet

access to clean drinking water ad libitum.

28 - ..,...,...... - ...

Treatment of the animals.

The rats were divided into four groups of A, B, C and D and treatment administered to each

group as follows: .i.e. The first group (A) of 5 rats were given only cotrimoxazole orally

(trimethoprim (TMP) (5mg/kg of body weight) and sulphamethoxazole (SMX) (25mg/kg of

body weight)) orally 6 hourly for 13 consecutive doses which was required to complete 3 days of conventional pneumocystis carinii pneumonia dosing of 20mg/kg/day trimethoprim and

lOOmg/kg/day sulphamethoxazole), the next group (B) of 5 were given a combination of cotrimoxazole and Ageratum conyzoides of a calculated amount in mg/kg orally, the third group

(C) of 5 animals were treated with only Ageratum conyzoides as calculated per mg/kg orally and the fourth or control group D of 5 rats only received normal saline (lOmL/kg) and served the entire 4 sampling times as control. AJJ the treatments lasted for 3 days (i.e. 13 hours which was the dose of cotrimoxazole required to establish steady state concentration within the stated period). Thereafter, animals were sacrificed by euthanasia in a transparent bucket with chloroform and dissected from the abdominal region while blood samples were collected from the from the heart through cardiac puncture using suitable syringe and needles into EDT A vacuitaner tubes for haematological analysis and non EDT A vacuitaner tubes for biochemical analysis. The blood samples were collected from the rats at specific time points i.e. at 0 minute immediately after sacrificing the rats, 24 hours, 48hours and 72 hours after completing the 13 doses.

29 _•'::.._ ~- :"-~...._ __ ~ ..... ------

Biochemical and haematological tests.

The anticoagulated blood was used for haematological analyses ofwhite blood cell count (WBC)

and differentials, red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean

cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin

concentration (MCHC) and platelet count (PLT) while the serum from non-EDT A tubes were

used for biochemical analyses of aspartate aminotransferase (AST), alaninie aminotransferase

(ALT), alanine phosphatase (ALP), K+, Na+, Cr and albumin (ALB).

~ Biochemical and haematological samples were run immediately using the automated

multi-item analyzer and haemato-analyzer.

~ Biochemical tests were done to check liver and kidney functions using the Humaster 180

machine. Commercial kits as well as the Humalyte plus human machine were used to

determine electrolytes such as Na+, K+and cr·

~ Haematological tests were also conducted using standard kits such as ammonium oxalate

and normal saline and using the sysmex-Japan, machine to assess the status of

haematological parameters.

3. 7 Data Analysis Procedures

The results were expressed as numerical values of mean ± S.E.M. The data was analyzed using one way analysis of variance SPSS version 12.0 (ANOVA). Differences between two means were detected using the Student's t-test. Data at p ::S 0. 05 was considered statistically significant in all cases. SPSS was used to produce tables, charts and graphs as a way of analyzing

30 quantitative data. In the analysis of qualitative data, the researcher described the data collected

using descriptive statement in words and some part of empirical test was done using SPSS.

3.8 Ethical Considerations

Firstly, approval was sought from the KID/School of Pharmacy Research Committee to

undertake the study. Then, the researcher obtained a letter of introduction from the Dean of

School ofPharmacy. This letter was presented to the Laboratory staff to be permitted to use the

laboratory. Also permission was sought from the head of the Laboratory ofMbarara University

of Science and Technology to be permitted to use their Laboratory for some extra tests.

The animal experiments were conducted according to the National Institute ofHealth Guide for the care and use of laboratory animals (NIH, 1996).

The laboratory animals were taken care of in a humane way as much as possible i.e. in spacious, quiet and stress free housing with access to enough feeds and water ad libitum and kept in an environment with 12 hours of light and 12 hours of darkness.

3.9 Limitations to the Study

• There is variation in metabolism between humans and rats and even between species.

• From above, the data obtained from the rats may not be 100% applicable to humans

(Dybing and Sanner, 1999; Grisham, 1997).

31 -----

CHAPTER FOUR:

4.0 RESULTS:

4.1. Plant identification:

The plant i.e. Ageratum conyzoides was identified and confirmed by a botanist from Mbarara as

Ageratum conyzoides L. and given a reference/collection/voucher number as: Kenneth Ocaka

001.

4.2. Phytochemical Analysis:

It revealed the presence of a number of metabolites in the plant as indicated in the table. Their

uses and references for such usage are also indicated.

Table 3

Phytochemical Relative Properties References for

presence properties.

Saponins ++ Haemolytic, Surfactant, Detergent Trease and Evans,

2002

Tannins -

Quinones -

Reducing + Anti-hypoglycaemic Trease and Evans,

sugars 2002

Flavanoids + Anti-bacterial, Anti-fungal, Anti- Trease and Evans,

inflammatory, Hypoglycaemic, Anti-oxidant 2002

Alkaloids -

32 a-Amino group -

Steroids + Diuretic Trease and Evans,

2002

Terpenoids + Anti-inflammatory, Anti-tumour, Anti-

protozoal, Cytotoxic

Diterpenes + Antifungal, Anti-allergic, Dopaminergic Trease and Evans,

2002

Resins + Antiseptic, Anti-bacterial, Purgative Trease and Evans,

2002

Phenols -

Phlobatanins -

Anthraquinones + Antifungal, Purgative Trease and Evans,

2002

Essential Oils + Antibacterial, Anti-Acaricidal Trease and Evans,

2002

Proteins -

Starch -

KEY: + means some; ++ means a lot

4.3. Percentage Yield calculations:

);;> Percent yield of extraction from hot aqueous extraction was as follows:

% Yield= (weight of extract container)-weight of container alone X 100

Weight ofthe initial powder

33 = ((100.2-86.7) xlOO) -7150 = 9%

4.4. Acute toxicity studies using Lorkes Modified Method:

The two phases of the acute toxicity test of Ageratum conyzoides. L aqueous leaf extract, showed that when orally administered to Wistar rats, even up to a dose of 1O,OOOmg/kg , no death occurred.

However, a few effects were noted as shown below.

Table 4

Doses Signs after

l-2hrs 3hrs 24hrs

1Oml s/kg of Distilled None None None

water (Control)

200mg/kg ofExtract Sedation Piloerection None

400mg/kg ofExtract Sedation Piloerection None

800mg/kg of Extract Sedation, arching Piloerection None

2500mg/kg ofExtract Sedation, Blanching Piloerection, Active None, No death.

5000mg/kg ofExtract Sedation, Blanching Piloerection None, No death.

7500mg/kg ofExtract Sedation, Blanching Piloerection, Active None, No death.

1O ,OOOmg/kg of Sedation, Arching, Piloerection, Active None, No death

Extract Blanching

34 Thus, the LD 5o, which is given by the formula:

LD5o= >/ (Lowest dose x Highest non-lethal dose), was estimated/approximated to be

greater than 10,000mg/kg of body weight.

4.5. Weight Change of animals during the study:

There was no general weight change since some rats gained weight while others lost weight.

Most of the animals gained weight; however, none of the changes in weight were statistically

significant. Cotrimoxazole caused the highest average weight change followed by the extract and

lastly the drug combination.

Table 5: Mean body weight and S.E.M values of rats (%)

IRUG USED DAY WEIGHTS OF ANIMALS IN TIME POINTS

OMINS 24HRS 48HRS 72HRS tMALSALINE 1 150.38±9.03 15 0.3 8±9.03 150.38±9.03 150.38±9.03

TROLGROUP) 2 151.62±8.17 151.62±8.17 151.62±8.17 151.62±8.17 . 3 153.62±8.31 153 .62±8.31 153 .62±8.31 153 .62±8.31

" 4 154.64±7.44 154.64±7.44 154.64±7.44 154.64±7.44 1: e Weight Change 4. 26±2.82 4.26±2.82 4.26±2.82 4.26±2.82

1 134.3±22.44 135.86±19.15 141.22±26. 75 143.6±22.64

'TRINONLY 2 139.56±22.74 140.04±20.39 142.5±25.09 144. 72±22.43 "

3 13 9. 94± 18.27 141.16±19.35 144.42±24.68 144.64±20.93 !' '' 4 140.06±17.18 142.34±19.53 146.42±24.0 145.98±21.01

~Weight Change 5.76±6.52 6.48±7.11 5.2±3.34 2. 38±2.76

35

~

I ~ ·TH SEPTRIN & 1 134.86±18.40 136.12±20.83 142.98±23. 14 143 .06±22.04

EXTRACT 2 133.42±17.67 135.2±18.88 144.5±22.72 143 .28±21.06

3 136.26±16.99 137.62±18.57 144.58±21.0 144. 5±20.91

4 138.2±16.56 138.32±16.92 143.96±18.06 146.12±20.65

ge Weight Change 3.34±2.93 2.2±4.57 0.98±6.07 3.06±2 .20

1 13 5. 58± 18.90 135.98±21.89 144.04±22.82 143.38±25.18

TRACT ONLY 2 136.14±18.20 137.88±22.86 145.38±22.44 143 .9±22.59

3 137.44±17.33 137.44±18.8 146.78±20.33 144.06±22.20

4 139.34±13.59 138.4±17.67 147.38±19.78 144.3±19.95

~e Weight Change 3.76±6.45 2.42±4.62 3.34±4.30 0.92±6.12

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRIN +EXTRACT; E=EXTRACT

NB: S means SEPTRIN used synonymously with cotrimoxazole

. j Results expressed as Mean±SEM. Statistical significance tested with student's t-test, p<0.05. -

*indicates statistical significance. I ~

I ~

k

I'C]

36

~

- 4.6. Effects of treatment on some Biochemical Parameters at different time points:

Figure 3: Graph showing change in AST over time

200 ~so J.GO l------~40

~20 ~00 - CTRL so r_ - s GO ------.c SE 40 --E 20 0 0 - IV11NS 24- HRS 4S-HRS 72-HRS

TIIV1E POINTS

------~ ----

Figure 4: Graph showing change in ALT over time

~20

~00 so /

GO - CTRL - s 40 ---.! SE 20 ~------E 0 0-IV11NS 24-HRS 48-HRS r 72- HRS

TIIV1C POINTS ------l

Figure 5: Graph showing change in ALB over time

30

20

~s - --- - CTRL ----s 10 SE s --.E

0 0 - IV11NS 24-HRS 48-HRS f 72- HRS

TIIV1E POINTS

37 - ~--":'------=--~------==--=------=-~~

Figure 6: Graph showing change in ALP over time

2 50 ------

2 00 • •

.l.SO - CTRL 100 ~------s so - E S - E

0 0 - IVIINS 24- HRS 4 8 - HRS 72- HRS 1 TIIVI E POINTS -- ~------There was no significant change in levels of enzyme AST, ALT, ALB and ALP compared to

their controls. However, the levels of Enzymes were generally below/lower than that of the

control/normal values for rats from 0-mins to 48-hrs after which it rose exponentially. The

changes in enzyme levels did not follow a general trend and were not time dependent. However,

between 48hrs and 72 hours, the levels of all enzymes which were somewhat depressed rose

sharply.

According to graphs plotted, both Extract and Cotrimoxazole showed similar changes in trend of

AST and ALT i.e. enzyme levels were decreased between 0-mins-24-hrs; they leveled off

between 24-48-hrs and increased sharply between 48-72-hrs. The drug combination caused enzyme levels to increase between 0-mins-24-hrs, decrease between 24-hrs-48-hrs and increase sharply between 48-hrs-72-hrs.

Cotrimoxazole, drug combination and Extract all caused a slow but steady increase in ALB levels between 0-mins-48-hrs and a sharp/steep increase in ALB levels between 48-72-hrs

Also, all the drugs administered caused a steady decrease in ALP levels between 0-mins-48-hrs and a sharp/steep increase in ALP between 48-72-hrs.

Refer to table 6 in appendix for statistically significant values.

38 4. 7. Effects of treatment on some haematological Parameters at different time points:

Below are some graphs showing change in some haematological parameters over time. Refer to table 7 in appendix for statistical significant values.

Figure 7: Graph showing change in RBC count over time

10 9 8 7 -- ~ -- ~!1!----- 6 5 -+-CTRL 4 3 ---s ~ SE 2 - E 1 ------0 r 48- HRS O- M INS 24- HRS I 72-HRS

TIME POINTS

Figure 8: Graph showing change in MPV over time

18 16 14 --- 12

1 0 ------+-CTRL 8 ---5 6 -:I! SE 4 .....:-E 2 ------~------0 T O-M INS I 24- HRS 48- H RS f 72-HRS

TIME POINTS

39 Figure 9: Graph showing change in HGB over time

6.4 6.3 6.2 6.1 6 ~ CTRL 5.9 ---5 5E 5.8 ------5.7 '------E 5.6 O-M INS T 24-HR5 48-HRS T 72-HRS T TIME POINTS l There is no general trend in the value/level of any haematological parameters.

However, the following was noted:

The drug combination caused a statistically significant decrease (38.6±2.42*) in the value ofHCT in the 0-minute group compared to the control group value (44.24±4.41) but no statistically significant changes were observed in the subsequent time points.

Figure 10: Graph showing change in HCT over time

60 -----

so ------..e-..,.--- ~ ;:, 40 -- - ! =::a q:;::> 30 ~ CTRL

20 --.,!.--- sES 1.0 - E 0 T r - O-M INS 24-M INS 48-HRS f 72-HRS

T IME POINTS

------~- --

40 -----~-

In the 0-minute and 24-hr category, all the values ofWBC were elevated higher than that of the

control (5.8±0.45) though not significant statistically, while at subsequent time points

(48 and 72-hrs), some WBC values were lower than that of the control.

Figure 11 : Graph showing change in WBC over time

1 2

1 0

8

- ~> ---CTRL 6 _.._. s 4 ~ ES 2 ------e

0 0-IVIINS 24- IVIINS r 4 8 - HRS r 72-HR S r T IIVI E POINTS

There was a statistically significant decrease in the value ofMCV by the drug combination

(51.2±2.28*) and Extract (51.6±1.67*) in the 0-minute category, by cotrimoxazole (51.2±1.10*),

drug combination (52.8±1.48*) and Extract (51.6±1.52*) at 24hrs, by drug combination

(54.0±2.12*) at 48hrsand by the Extract (53.6±0.55*) at 72hours, all compared to the control value (56.4±1.14).

Figure 12: Graph showing change in MCV over time

57 56 55 5 4

53 --.-.CTRL 52 5 1 ---5ES 5 0 49 -E 4 8 l O - M INS 2 4 - MINS 48- HRS 72-HR S

T I M E P OINTS

41 There was a no statistically significant increase in the levels ofMCH at 0-mins, however, there

was a statistically significant increase in its levels by the drug combination (19.06±0.56*) at 24-

hrs, a statistically significant decrease by the drug combination (18.22±0.49*) at 48-hrs, and by the Extract (18.12±0.25*) at 72-hrs, all compared to the control value (18.76±0.64).

Figure 13: Graph showing change in MCH over time

~9 .2 19 l.8.8

~8.6 :1.8. 4 --..- CTRL

l.8.2 ---5

l.S ES J. 7.8 - E :1.7.6 O-M INS 24-M INS r 48-HRS I 72-HRS

TIME POINTS

Also, there was a statistically significant increase in the values of MCHC by cotrimoxazole

(35.78±1.01 *), the drug combination (36.06±1.24*) and by the Extract (36.2±0.73*) at 0-mins, and by cotrimoxazole (36.4±0.58*) and the Extract (36.14±0.22*) at 24-hrs compared to the control (33.32±0.57) but no significant changes at 48 and 72-hrs.

Figure 14: Graph showing change in MCHC over time

37 36.5 36 35.5 ,- 35 - -- 34.5 - CTRL 34 33.5 ~ s 33 ----:.: ES 32.5 - E 32 3l..5 I" O - M INS 24-M INS r 48-HRS l 72-HRS

TIME POINTS --- I-- I 42 Furthermore, there were statistically significant decreases in the values ofRDW by the drug

combination (10.48±0.69*) and Extract (9.98±0.32*) at 0-mins and by cotrimoxazole

(9.82±0.50*), the drug combination (9.82±0.66*) and by the Extract (10.24±0.95*) at 24-hrs, all

compared to the control (11.08±0.43). Changes at 48 and 72-hrs were not statistically significant.

Figure 15: Graph showing change in RDW over time

1.1.2 11 10.8 10.6 1.0.4 1.0.2 --+--CTRL 10 9.8 ---s 9.6 .-..,: ES 9.4 - E 9.2 9 O-M INS 24-M INS r 48- HRS 72-HRS

TIME POINTS

Additionally, statistically significant changes were noted in the number ofPLTS as follows: a statistically significant decrease by the drug combination (504.8±54.13*) and the Extract

(311.8±100.04*) was noted at 0-mins, statistically insignificant decreases were noted at24-hrs while a statistically significant increase was noted due to the Extract (790.2±69.23*) and drug combinations (885±70.35*) at 48-hrs and due to the Extract (803 .6±71.61 *) and drug combinations (863.2±70.6*) at 72-hrs, all compared to the control (648.6±78.71).

Figure16: Graph showing change in PLTS over time

1.000 900 BOO 700 600 : 500 - CTRL

400 ~ s 300 --..: ES 200 - E :JI.OO

0 ~- j 0-IVIINS 24-IVIINS 48-HRS T 72-HRS r T I IVIE POINTS ------43 4.8. Effect of treatment on other haematological parameters i.e. (on Counts of Leukocyte differentials and on their percentages.

Below are some graphs showing changes in other haematological parameters over time. Refer to table 8 in appendix for statistically significant values.

Figure 17: Graph showing change in EOSINOPHIL count over time

7

6

5

4 - CTRL 3 .-s 2 SE

1 - E

0 O - M INS 24-HRS 48-HRS

TIME POINTS

Figure 18: Graph showing change in BASOPHIL count over time

0.45 0.4 0 .35 0.3 0.25 ~ CTRL 0.2 ---s 0.15 SE 0.1 I - E 0.05 z::_ 0 O-M INS 24-HRS 48-H RS I 72-H RS

T IME POINTS

-~------

44 Figure 19: Graph showing change in MONOCYTE % over time

10 ------9 8 7 6 5 ~ CTRL 4 ---5 3 SE 2 _..._ E :1 0 l O-M INS I 24-HRS 48-HRS r 72-HRS I TIME POINTS ------l

Figure 20: Graph showing change in EOSINOPHIL % over time

2 1.8 1.6 1.4 1.2 1 ~ CTRL 0.8 ---s 0 . 6 SE 0 .4 - E 0 .2 0 O - M INS 24-HRS 48- HRS 72-HRS

TIME POINTS

There was noted a statistically significant decrease in Neutrophil count, due to the Extract

(0.35±0.14*) at time point 0-mins. Also, statistically significant decreases were noted due to the drug combination (0.41±0.16*) and the Extract (0.32±0.12*) at the 24-hr time point after administration ofthe last dose, all compared to the control values (0.66±0. 16).

45 Figure 21 : Graph showing change in Neutrophil count over time

~ 0 .9 0 .8 0.7 0 .6 0.5 - CTRL 0 .4 _.._s 0.3 ES 0 . 2 ------E 0-~ 0 .,. 0 - IVIINS 2 4 - IVIINS r 48- HRS I 72- HRS l TIME POINTS

Monocyte count was statistically significantly increased (1.00±0.53*) by cotrimoxazole at 0- mins time point when compared to the value of the control (0.44±0. 14).

Figure 22: Graph showing change in Monocyte count over time

1 . 2

1

0 .8 - ~= ~-

- CTRL 0 .6 -...:.__ -~ - -- --~-c=?;;; _.._s 0 .4 ~-se - -a - ES 0.2 --E

0 O - M INS 24- M INS 48- HRS T 7 2-HRS I TIME POINTS

Neutrophil percentage was statistically significantly decreased by the Extract (5.96±1.74*) at

0-mins and by the Extract (4.56±0.74*) at the 24-hr time points when compared with the control value (1 1.42±2.96). Other changes within those time points were statistically insignifi cant.

46 Figure 23: Graph showing change in Neutrophil %over time

1 6 ------14 1 2 10 B --CTRL

6 __._s 4 ~ ES 2 - E 0 0 - rviiN S 2 4 - rviiNS T 48-HRS T 72- HRS

TlrviE POINTS ------1 --- - l

Lymphocyte percentage was statistically significantly increased by the Extract (85.78±2.93*) at

0-mins and by cotrimoxazole (87.42±2.20*) and the Extract (89. 78±2.62*) at 24-hr time points

after administration of the last dose of the drugs, when compared with the control value

(77.88±5.07).

Figure 24: Graph showing change in Lymphocyte% over time

1 00 9 0 80 - e ; "' o - 70 6 0 so --CTR L 40 I _.._s

3 0 ES 2 0 - E 10 0 O - M INS 2 4 - MINS r 48- HRS r 72 - HRS l TIME POINTS

Basophil percentage was statistically significantly increased (1.68±0.13*) by the Extract when compared to the value for the control (1.22±0.28) at time point 0-mins.

47 Figure 25 : Graph showing change in Basophil % over time

6

5

4 ------+-CTRL 3

2 ---s ES 1 - E

0 O-M INS 24-M INS 48-HRS 72-HRS

T IME POINTS

-~~---- -

4.9. Effect of treatment on electrolytes.

Figure 26: G raph showing change in Na+ over time

200 180 160 140 1 120 100 --+-CTRL 80 ------5 60 ES 40 -E 20 0 O-M INS 24-MINS "l 48-HRS r 72-HRS r TIME POINTS

48 There were insignificant statistical increases in the levels ofK+ at 0-mins, 24-hrs and at 72-hrs.

However, there was a statistically significant increase (7.01±1.10*) inK+ due to cotrimoxazole

at 48-hrs when compared to the control value (5.85±0.97).

Figure 27: Graph showing change inK+ over time

8 7 -- - ~ 6 5 4 --+-CTRL 3 ---sES 2 1 ------E 0 r O-M INS 24- M INS ~ 48-HRS ! 72-HRS l TIME POINTS

Also, cotrimoxazole caused a statistically significant increase (118.38±1.80*) in the levels ofCr

'at 0-mins and a statistically significant decrease (109.3 2±5. 14*) in cr at 24-hrs when

compared to the control value (115.28±2.13). No such significant changes were noted at48 and

72-hr time-points after drug withdrawal.

Figure 28 : Graph showing change in Cr over time

160 ----- 140 120 Z!l - 100 so --CTRL 60 ---s 40 ~ ES 20 -E

0 ~ 0 - IVIINS 24-IV11N S r

49 Analysis of all the 22 haematological parameters studied revealed the following tabulated

numbers of statistically significant changes produced at the different time points by the

drugs used.

Table 10

ME POINTS NUMBER OF STATISTICALLY SIGNIFICANT CHANGES IN SOME

HAEMATOLOGY PARAMETERS PRODUCED AFTER TREATMENT WITH

SEPTRIN ONLY COMBINATION EXTRACT ONLY (E)

(S) (ES)

0-MINS 2 6 8

24-HRS 5 4 6

48-HRS 1 3 1

72-HRS 0 1 3

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRIN +EXTRACT; E=EXTRACT

S means SEPTRIN and is synonymously used for cotrimoxazole

Figure 29: A graph of number of statistically significant changes versus time points

9 8 7 6 s - 5 -+-SEPTRIN O NLY (S}

4 ---COMBINATION (ES} 3 EXTRACT ONLY (E) 2 1 0 0-MINS 24- HRS 48-HRS 72-HRS

50 Figure 30

Based on the above table, the activity of the three agents can be represented graphically as:

Number of

Statistical Curve for Extract

Effects/Change

Curve for Combination

Curve for Cotrimoxazole

Time Point.

The curve for overall effect i.e. of the combination is expected to be higher than that of both

cotrimoxazole and extract but since overall effect is lower than predicted; the two most likely

interact antagonistically (Williamson, 2001).

51 CHAPTER FIVE:

5.0 DISCUSSION, CONCLUSION, RECCOMENDA TIONS:

5.1. DISCUSSION.

According to the phytochemical study done, Ageratum conyzoides ("Buta buta") leaf aqueous extract contains a wide variety of phytochemicals e.g. flavanoids, resins and essential oils, some ofwhich are antibacterial in property. It also contains diterpenes, anthraquinones and some flavanoids which are antifungal in property. They could most likely be behind the medicinal property of Ageratum conyzoides as explored in traditional medicine e.g. wound healing (Trease and Evans, 2002; I. buakhao hongsachum, 2008)

According to the acute toxicity study done using Lorkes modified method, no animal died. This is because the Ageratum conyzoides leaf aqueous extract has a high LDso i.e. > 10, 0000. It therefore has a very high safety margin when used alone to treat animals.

The percentage yield of extraction from Ageratum conyzoides leaves was good i.e. 9%. This is probably because many of the phytochemicals it contains are water soluble/polar and yet its yield from polar solvents is higher than from non polar solvents (Onuoha, et al).

Though the changes in weight of the animals were not statistically significant, most animals gained weight, probably because of an increase in their appetite by the extract or the carbohydrate/reducing sugar component ofthe extract, excesses of which was probably metabolised to proteins/fats for storage (Guyton,2006).

Though changes in Biochemical parameters studied were not statically significant, levels of

Enzymes were depressed at time points 0-mins-48-hrs, but they "shot-up" thereafter. This depression in levels of Enzymes could have been because of a slight impairment in any one of

52 the enzyme biosynthetic pathways, or increased enzyme degradation, upon formation. The spike/rise in enzyme levels after 48-hrs could have been a result of positive feedback stimulation by the enzyme owing to their low levels (Guyton, 2006)

Analysis of blood parameters is relevant in risk evaluation as changes in the haematological system have higher predictive value for studies (Olson et al., 2000).

In the study done, many haematological parameters were statistically significant. Probable predictions accounting for such statistically significant changes are as follows:

The number of statistically significant changes in animals treated with the drug combination

(Cotrimoxazole +Ageratum conyzoides leaf aqueous extract) was high at the beginning i.e. 6 at

0-mins, and progressively decreased with time i.e. 4, 3, 1 at 24hrs, 48-hrs and 72-hrs, respectively. This is because, of the high amount/levels of drug concentration at the beginning i.e. at 0-mins when a steady state concentration has been reached. Progressive decline is because ofwithdrawal of the drug combination and subsequent decreased levels/concentrations ofthe drug in the blood circulation due to clearance in/from the body (Paul, 2014)

The statistically significantly reduced value ofMCV indicates microcytic anaemia whose most common cause is an iron deficiency (either due to inadequate dietary intake, or a lack of absorption, or an increased loss resulting from poor binding (Mayo clinic, 2014; Stanley, 2011).

This effect manifested at all time points but more at the 24-hrs. The drug combination also seemed to cause anaemia noted at time points 0-mins, 24 and 48-hrs but it resolved at the 72-hr time point i.e. it was not noted, probably due to sub-therapeutic levels ofthe drugs administered in to their blood circulation.

The statistically significant decreases in the values ofRDW denote either macrocytic anaemia or microcytic anaemia. However, the above noted Low MCV combined with apparent low RDW in

53 this paragraph confirms iron deficiency which causes mostly microcytic anaemia (Mosby, 2012;

Chronolab, 2014; Norman Beck, 2008)

The statistically significant increase in MCH at 24-hrs suggests a macrocytic anaemia, which may have been caused by rapid red cell turnover, folate or vitamin B 12deficiency, or inhibition/poisoning of DNA replication by the administered drug (Drug combination and extract which might possess unknown DNA inhibiting properties

The statistically significant decreases in MCH noted are probably due to either, Iron deficiency anaemia, folate deficiency or Vitamin B12 deficiency and it denotes microcytic anaemia (Mayo clinic, 2014)

The statistically significant increase in the value of MCHC is either because of Macrocytic anaemia, Lipemia, cellular dehydration syndromes, vitamin B 12 or folic acid deficiency in the body. However, thi s value could have been falsely elevated. This is possible because the blood sample drawn could have been exposed to temperatures lower than 37 degrees centigrade and it therefore agglutinated making it contain cold agglutinins which causes complications by making

Haemoglobin to clump together thereby appearing more concentrated/dense. MCHC is mainly used as a guideline for analyser function i.e. ifMCHC is elevated it gives us a good indicator the analyser is in need of attention, i.e. if it is averaging high. However ifthe result is true, it is a good indicator of Cold agglutination taking place in the sample (Wallach, 2007; Rifkind, 2002;

Sam Easterbrook, 2014). However, low MCHC denotes microcytic anaemia (Mayo, clinic)

The statistically significant reduction in the HCT in the 0-min category by the drug combination denotes anaemia, caused by either nutritional deficiencies (iron, vitamin Bl2, or folate) or bone marrow suppression by the drug combination (Braunwald, eta/., 2008).

54 The statistically significant decrease ofPLTS at 0-mins was caused by either low platelet production (due to bone marrow suppression by the drugs administered or deficiency of vitamin

B12 or folic acid), by increased platelet destruction or consumption due to the sulphonamide component ofthe drugs administered which normally causes an immunologic reaction against platelets, called drug-induced thrombocytopenia or by increased splenic sequestration (capturing of circulating platelets in the spleen). Cotrimoxazole and anaemia are common cause of thrombocytopenia (BNF, 2009; Siamak, 2014)

The statistically significant increases in subsequent PLT count could be due to the cells within the bone marrow producing more platelets due to positive feedback owing to its low levels, or less platelet matter being removed from the blood by the spleen or anaemia (too few red blood cells), or polycythemia vera (too many Red blood cells.), or iron deficiency anaemia or haemolytic anaemia. (Guyton, 2006; Mayo clinic, 2014)

From the observed high values ofWBC at 0-mins, when drug levels are at a steady state concentration/high, it is clear that an increase in the number ofWBC occurs which is a normal reaction of rats to foreign substances, which alter their normal physiological processes. The observed slight increase in WBC count though not statistically significant, in the treated groups could be as a result of their body defense acting in response to toxic environment (Teguia et al.,

2007). It's observed that WBC values reduce at subsequent time points probably due to clearance of the drugs from the circulatory system (Paul, 2014)

The statistically significant decreases in Neutrophil count could be because of Vitamin deficiencies, aplastic anaemia or the administered drugs which may be able to destroy neutrophils or damage bone marrow affecting its production, or because of Hypersplenism, (a premature destruction of blood cells by the spleen) (Mayo clinic).

55 The statistically significant increase in Monocyte levels suggests either a haematopoietic stem cell disorder caused by the drugs or a recovery phase of neutropenia (Marshall eta!., 2007;

Fernandez, 2013). It's observed that monocyte count decreases/normalizes with respect to the control values at subsequent time points probably due to clearance of the causative drugs from the circulatory system (Paul, 2014).

Neutrophil %was also statistically significantly decreased, probably due to the drugs administered, nutritional deficiencies, decreased production of precursor cells in the bone marrow or increased egress from blood circulation in to body tissues (Marshall eta!., 2007;

Mosby, 2005).

The% of Lymphocytes, which are the main effector cells ofthe immune system (McKnight et al., 1999) were statistically significantly increased, suggesting a challenge to the immune system of the animals or an adverse reaction to the herbal agent.

A statistical significant Increase in % of Basophils at 0-mins suggest either an iron deficiency or that the animals experienced allergic reactions brought about by the drugs administered, which they viewed as foreign (Hoffman, 2008)

The statistically significant increase inK+, following cotrimoxazole administration and testing at

0-mins is because at steady/sufficient concentrations in the blood stream, its Trimethoprim component interferes with K + urinary excretion or it may be a case of pseudohyperkalemia because of EDT A in the vaccutainers or because of haemolysis that occurs shortly after blood draw due to an excessive vacuum of the draw blood or by a collection needle that is of too fine a bore. Such an effect disappeared at subsequent time points probably because of decreased levels ofthe drug in circulation (Mayo clinic, 2011; Sevastos, 2006; Don, 1990). Cotrimoxazole causes hyperkalemia as noted in BNF (2009)

56 The levels of Na+ were not statistically significantly increased; when compared to those of the control animals at 0-mins and 24-hrs. This is probably because of mild dehydration but with resultant concentrating effects leading to the rise in cr ions whose levels were statistically significantly increased at time point 0-mins. Dehydration is usually brought about by a reduction of body water content by fluid loss (most likely excessive sweating, prolonged vomiting or diarrhoea, in this case, caused by Cotrimoxazole (BNF, 2009)) resulting in hyponatremia which causes hyperchloremia hence metabolic acidosis/respiratory alkalosis (Cambier et al. , 1998).

Severe compensatory reductions in chloride ion content occurs at subsequent time points e.g. by increased water retention or hypoventilation in order to correct the above respiratory alkalosis, leading to statistically significant lowering in cr levels noted at the subsequent time point, 24- hrs. (Monica, 2009; Levitin et al, 1958)

57 5.2. CONCLUSION.

Ageratum conyzoides contains active secondary metabolites/phytochemicals. They could be

behind the medicinal property of Ageratum conyzoides as explored in traditional medicine.

Ageratum conyzoides aqueous leaf extract has a high safety margin.

Concomitant administration of cotrimoxazole and Ageratum conyzoides aqueous leaf extract has no effects on the biochemical parameters studied and as such does not cause any significant biochemical changes with regard to the studied parameters.

Concomitant administration of cotrimoxazole and Ageratum conyzoides aqueous leaf extract has some effects on haematological parameters studied and as such causes some significant changes in the haematology of Wistar rats, notably anaemia and thrombocytopenia. However, these effects and other statistically significant effects are higher at 0-mins when drug levels in circulation are high but they disappear/wane with progressive time, upon withdrawal of the drugs.

5.3. RECCOMENDATIONS

Chronic and sub-chronic studies should be carried out on the drug combinations to determine their long term effect on animals.

TlBC and other tests should be carried out to further confirm the type of anaemia that the drug combination causes.

Studies should be carried out on the drug combination, but for which an organic solvent is used to make extractions instead ofwater.

Effects on other biochemical parameters e.g. LDH, GLU, CK, TGS, LDL, etc should be done.

58 .- = ~ - ~ . + - ~- -- ~ ------

CHAPTER SIX:

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66 CHAPTER SEVEN:

7.0 APPENDICES

7.1 SOME TABLES:

A) Effects of treatment on some Biochemical Parameters at different time points:

Table 6 TIME POINTS GROUP AST ALT ALBUMIN ALP

CTRL 92±99.42 69.2±62. 14 24.37±41 .78 212.2±185±32

s 58.4±69.39 8±8.46 2.00±1.51 47.6±37.02

0-MINS ES 27±39.87 10.6±4.51 1.98±1.57 109.8±73.74

E 48.2±23.53 14±6.82 2.34±1.22 58±96.3 6

s 11.8±8.29 3.2±1.92 2.41±1.94 30. 8±35.53

24-HRS ES 56.8±41.73 24.6±29.73 2.28±1.86 36.2±25.94

E 5.4±5.46 5.00±6.75 3.18±1.45 36.2±31.96

s 6.6±4.28 2.8±2.59 1.76±0.66 24±7.25

48-HRS ES 7.98±11.42 2.00±0.71 3.10±1.01 40.4±29. 10

E 3.4±3.13 3.40±1.82 3.92±1.12 33.4±29. 05

s 179.6±133.28 79.2±54.14 17.17±25 .92 209±156. 88

72-HRS ES 173.8±24.17 106.2±25.81 26.86±28.08 222.4±58.68

E 98±86.90 68.8±60.09 11.58±12.42 127.2±103.23

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRIN + EXTRACT; E=EXTRACT

NB: S means SEPTRIN used synonymously with cotrimoxazole

67 Results expressed as Mean±SEM. Statistical significance tested with student's t-test, p<0.05.

*indicates statistical significance.

B) Effects of treatment on some haematological Parameters at different time points:

Table 7

GROUP WBCx RBCx HGB HCT MCV MCH MCHC RDW PLTx MPV

3 6 , 10 J.11 10 J.1l (g/dl) (%) (fl) (pg) (g/dl) (%) 103 Ill (fl)

CTRL 5.8± 7.89± 14.74± 44.24± 56.4± 18.76± 33.32± 11.08± 648.6± 6.24±

0.45 0.86 1.24 4.41 1.14 0.64 0.57 0.43 78.71 0.41

s 9.74± 6.15± 14.38± 41.26± 52.8± 18.78± 35.78± 10.08± 522.8± 6.2±

4.56 3.45 0.79 3.02 1.92 0.79 1.01 * 0.86 73.68 0.56

ES 10.68± 7.56± 13 .92± 38.6± 51.2± 18.48± 36.06± 10.48± 504.8± 6.32±

5.63 0.54 0.96 2.42* 2.28* 1.41 1.24* 0.69* 54.13* 0.33

E 5.92± 7.05± 13 .22± 36.66± 51.6± 18.74± 36.2± 9.98± 311.8± 5.96±

1.6 1.95 3.67 10.64 1.67* 0.41 0.73* 0.32* 100.04* 0.36

s 7.68± 7.96± 14.8± 40.8± 51.2± 18.64± 36.4± 9.82± 557.8± 5.86±

5.62 0.58 1.02 3.24 1.10* 0.39 0.58* 0.50* 39.05 0. 15

ES 8.04± 7.66± 14.62± 40.38± 52.8± 19.06± 36.20± 9.82± 545.2± 5.88±

4.73 0.36 0.38 1.27 1.48* 0.56* 0.40 0.66* 43.47 0.15

E 6.74± 7.40± 13.8± 38.14± 51.6± 18.64± 36.14± 10.24± 558.2± 5.88±

1.67 0.59 1.17 3.23 1.52* 0.50 0.22* 0.95* 136.30 0.21

s 4.94± 8.53± 15.46± 46.16± 54.0± 18.1± 33.5± 10.5± 703.2± 6.3±

68 1.19 0.72 1.42 4.64 2.00 0.54 0.51 0.62 59.33 0 .32

ES 5.14± 8.48± 15.44± 45.98± 54.0± 18.22± 33.66± 11.04± 885± 6 .06±

1.98 0.67 1.36 4.51 2.12* 0.49* 0.48 1.13 70.35* 0 .17

E 5.46± 8.62± 16.1± 48.06± 55.8± 18.64± 33.52± 10.94± 790.2± 6.26±

2.19 0.74 1.66 5.21 1.64 0.45 0.39 0. 72 69.23* 0 .15

s 5.04± 8.49± 15.34± 45.68± 54.0± 18.1± 33.6± 10. 58± 762.8± 5.96±

1.54 0.65 1.13 3.51 1.58 0. 34 0.55 0. 57 68.49 0.24

ES 5.02± 8.24± 15.14± 45.18± 55.0± 18.36± 33.5± 10.48± 863.2± 5.88±

1.09 0.26 0.3 0.84 1.23 0.32 0.41 0.49 70.6* 0. 13

E 6.86± 8.62± 15.8± 46.88± 53 .6± 18.12± 33.54± 10.76± 803.6± 5.96±

2.15 0.3 8 0.86 2.27 0.55* 0.25* 0.39 0.62 71.61* 0.3

KEY: S= SEPTRIN; ES= Drug combination i.e. SEPTRIN + EXTRACT; E=EXTRACT

NB: S means SEPTRIN used synonymously with cotrimoxazole

Results expressed as Mean±SEM. Statistical significance tested with student's t-test, p

*indicates statistical significance.

69 C) Effect of treatment on other haematological parameters i.e. (on Counts of Leukocyte differentials and on their percentages.

Table 8 !: GROUP NEx LYx MO EO BA NE LY MO EO BA rs 103/~tl 103/~tl 103/~tl 103/~tl 103/~tl (%) (%) (%) (%) (%)

CTRL 0.66± 4.50± 0.44± 0.10± 0.07± 11.42± 77.88± 7.76± 1.72± 1.22:!

0.16 0.46 0.14 0.12 0.02 2.96 5.07 2.46 1.90 0.28

s 0.90± 7.89± 0.53± 0.02± 0.41± 7.54± 80.82± 5.6± 0.14± 5.9±

0.94 3.69 0.23 0.02 0.34 4.80 4.79 1.13 0.15 6.23

~ ES 0.87± 8.66± 1.00± 0.01± 0.14± 10.34± 78.64± 9.42± 0.2± 1.4±

0.59 5.12 0.53* 0.02 0.07 10.73 11.07 2.27 0.28 0.64

E 0.35± 5.06± 0.42± 0.008± 0.1± 5.96± 85.78± 6.48± 0.1± 1.68:!

0.14* 1.17 0.30 0.17 0.03 1.74* 2.93* 3.28 0.12 0.13~

s 0.51± 6.78± 0.3± 0.002± 0.07± 7.48± 87.42± 4.02± 0.02± 1.06:!

I I 0.25 5.13 0.21 0.004 0.04 1.69 2.20* 1.64 0.04 0.44

ES 0.41± 7.12± 0.38± 0.06± 0.09± 5.8± 87.14± 4.92± 1.18± 0.96:!

0.16* 4.37 0.24 0.12 0.1 2.87 6.14 1.84 2.53 0.42

E 0.32± 6.05± 0.32± 0.002± 0.06± 4.56± 89.78± 4.74± 0.02± 0.9±

0.12* 1.48 0.21 0.004 0.03 0.74* 2.62* 2.66 0.04 0.17

s 0.622± 3.88± 0.36± 0.04± 0.06± 12.76± 78.22± 7.22± 0.66± 1.14:!

0.15 0.91 0.21 0.06 0.03 2.51 2.30 3.21 0.10 0.40

ES 0.56± 4.01± 0.42± 0.08± 0.08± 12.22± 77.46± 7.64± 1.10± 1.58:!

70 0.08 1.60 0.23 0.12 0.04 4.66 2.25 3.17 1.46 0.56

E 0.59± 4.37± 0.38± 0.04± 0.08± 11.16± 80.56± 6.28± 0.62± 1.38::1

0.25 1.6 0.33 0.05 0.08 3.27 4.52 2.57 0.69 0.74

s 0.74± 3.71± 0.48± 0.02± 0.06± 14.48± 74.52± 9.38± 0.44± 1.18::1 s 0.35 1.01 0.25 0.02 0.03 4.02 5.27 3.19 0.34 0.44

ES 0.74± 3.80± 0.36± 0.07± 0.05± 14.22± 76.76± 6.88± 1.22± 0.92::1

0.44 0.63 0.16 0.14 0.03 5.51 8.95 1.89 2.16 0.34

E 0.71± 5.07± 0.63± 0.12± 0.2± 12.02± 78.28± 8.98± 1.76± 0.92::1

0.23 1.87 0.26 0.10 0.28 2.44 4.89 3.12 2.30 0.46

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRIN +EXTRACT; E=EXTRACT

Results expressed as Mean±SEM. Statistical significance tested with student's t-test, p<0.05.

*indicates statistical significance.

" l NB: S means SEPTRIN used synonymously with Cotrimoxazole

"

ly - -

...,

71

:- D) Effect of treatment on electrolytes.

Table 9 TIME POINTS GROUPS K+ Na+ cr

CONTROL 5.85±0.97 151. 02±2. 04 115 .28±2.13

SEPTRIN (S) 6.47±0.14 155.04±2.4 118.38±1.80*

0-MINUTES BOTH (ES) 6.55±0.49 153.32±3.20 11 6.52±3.24

EXTRACT(E) 5.71±0.50 151.3±2.86 111.68±3.91

s 6.18±0.16 146.74 ±6.31 109.32±5.14*

24-HOURS ES 6.51±0.89 154.46±10.15 115.68±7.96

E 7.54±2.77 185. 06±85. 68 135.16±56.6

s 7.01 ±1.10* 150.28±0.69 115.06±0.92

48-HOURS ES 7.37±1.03 148.9±2.44 114.76±1.69

E 6.98±1.77 151.68±2.55 115.36±2.24

s 7.06±1.11 150.46±1.64 115.2±1.40

72-HOURS ES 6.86±0.85 151. 08± 1. 12 114.8±1.02

E 5.55±0.61 151.9±1.35 116.9±2.32

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRfN + EXTRACT; E=EXTRACT

Results expressed as Mean±SEM. Statistical significance tested with student's t-test, p<0.05.

*indicates statistical significance.

72 E) Summary tables showing the most probable nature/reasons behind the

statistically significant changes in haematological parameters that occurred in the

study.

Table 11

'IME GROUP HCT MCV (fl) MCH MCHC RDW(%) PLT X lOj Jd

HNTS (%) (pg) (g/dl)

CTRL

s Lipemia

.fiNS Or

Cellular

dehydration

Syndrome

Or

Falsely

elevated

MCHC

ES Anaemia Anaemia Anaemia ~ PLT

(Microcytic) "" Most likely production or

Microcytic '2destruction

or

Anaemia

73 E Anaemia

(Microcytic) "" "" "" s Anaemia

IHRS (Microcytic) "" ""

ES Anaemia Anaemia

(Microcytic) '"'

E Anaemia

(Microcytic) "" "" s

HRS ES Anaemia Anaemia

(Microcytic) ""

E

"" s

HRS ES

""

E Anaemia Anaemia

(Microcytic) '"'

74 Table 12 riME GROUP NEx LYx MO NE(%) LY(%) EO BA (%)

'OINTS 103 /Ill 103 /Ill I 03/111 (%)

CTRL s

ES Haematopoietic

)MINS stem cell

disorder or

Recovery

phase of

Neutropenia.

E Vit B 12 D pdcn of Challenge Allergic

deficiency precursor to reaction

Or cells or rmmune or iron

Aplastic "\! egress system deficiency

anaemia from

or blood

Drug circulation

which

suppresses

bone

marrow

75 s 24HRS """

ES

"'"'

E ,,,,, ,,,, '""' s

18HRS ES

E s

'2HRS ES

E

76 Table 13

TIME POINTS GROUPS K+ Na+ cr

CONTROL

SEPTRIN (S) Dehydration

0-MINUTES BOTH (ES)

EXTRACT(E)

s Severe

24-HOURS compensatory

reduction in

levels by

hyperventilation

ES

E

s Interfered

48-HOURS excretion by

Trimethoprim

or Pseudo

hyperkalemia

due to

haemolysis

ES

E s

77 72-HOURS ES

E

KEY: S=SEPTRIN; ES= Drug combination i.e. SEPTRIN +EXTRACT; E=EXTRACT

78 7.2 WORK PLAN:

Table 13

ACTIVITY MONTHS

May June July August

Proposal development and presentation.

Plant collection and identification.

Extraction and treating animals.

Heamatological and biochemical testing.

Report/Dessertation writing

Wrapup and Defence of research report.

Total 4 months

79 7.3BUDGET

Table 14 ------·--- - - r ~ ------~- -· ITEM No I UNIT COST TOTAL COST ! Rats/Animals 81 8,000 648,000 Plant collection and 30,000 30,000 identification Extraction, Acute toxicity tests 130,000 130,000 and phytochemical screening Biochemical and 1, 500,000 1, 500,000 heamatological tests Printing 4 booklets and ' ' 200,000 ~

stationery ~ Transport 100,000 Lab assistant 300,000 Miscellaneous 100,000 TOTAL 3, 008,000

80 7.4 THE MAP OF BUSHENYI DISTRICT

RUBIRIZI

...... _ Oiltrict 8cundaty Oo

Figure 31

81