Antiplasmodial Activities and in Vivo Safety of Extracts and Compounds of Seven Indigenous Kenyan Medicinal Plants Used Traditionally to Treat Malaria

ANTIPLASMODIAL ACTIVITIES AND IN VIVO SAFETY OF EXTRACTS AND COMPOUNDS OF SEVEN INDIGENOUS KENYAN MEDICINAL PLANTS USED TRADITIONALLY TO TREAT MALARIA

ONYOYO SAMWEL GUYA I56/8873/99

A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF

SCIENCE IN BIOCHEMISTRY, KENYATTA UNIVERSITY

OCTOBER, 2020

DECLARATION AND DEDICATION

I declare that this thesis is my original work and it has not been presented for a degree in any other University or for any other award.

I dedicate this work to my late father mzee Festus Onyoyo Ouya who was the source of my aspiration as he had a special aspiration towards acquiring knowledge.

Onyoyo Samwel Guya Reg/No: 156/8873/99

Signature______Date______

We confirm that the work reported in this thesis was carried out by the candidate under our supervision Supervisors

Prof Eliud NM Njagi Department of Biochemistry, Microbiology and Biotechnology Kenyatta University P.O Box 43844, Nairobi

Signature______Date______

Prof Anastasia N Guantai Department of Pharmacology and Pharmacognosy University of Nairobi P.O Box 19676, Nairobi

Signature______Date______

Prof Caroline C Langat-Thoruwa Department of Chemistry Kenyatta University P.O Box 43844, Nairobi

Signature______Date______

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TABLE OF CONTENTS

DECLARATION AND DEDICATION ...... ii TABLE OF CONTENTS ...... iii LIST OF TABLES ...... vi LIST OF FIGURES ...... viii ACKNOWLEDGMENTS ...... ix ABBREVIATIONS AND ACRONYMS ...... x ABSTRACT ...... xvi CHAPTER ONE ...... 1 INTRODUCTION ...... 1 1.0 Background information ...... 1 1.1 Malaria the disease ...... 1 1.2 Control and treatment of malaria ...... 2 1.3 Drug discovery for malaria ...... 3 1.4 Phytotherapeutics ...... 4 1.5 In vivo lethality test (Brine Shrimp Bioassay) ...... 4 1.6 Statement of the problem ...... 5 1.7 Justification of the study ...... 7 1.8 Null hypotheses ...... 8 1.9 Study objectives ...... 9 1.9.1 General objective ...... 9 1.9.2 Specific objectives ...... 9 CHAPTER TWO ...... 10 LITERATURE REVIEW ...... 10 2.1 Control and treatment of malaria ...... 10 2.1.1 Vector control and malaria prophylaxis ...... 10 2.1.2 Antimalarial chemotherapy using conventional drugs ...... 11 2.2 Plants as sources of antimalarial agents ...... 16 2.2.1 Extraction and purification of compounds ...... 21 2.2.2 Liquid-solid (adsorption) chromatography ...... 21 2.2.3 Gelfiltration (exclusion) chromatography ...... 24 2.2.4 Preparative thin layer chromatography (PTLC) ...... 24 2.2.5 Structural elucidation of isolated compounds ...... 25

2.3. Ethnomedicine survey ...... 26 2.3.Reasons for conducting ethnomedicine survey in Kilifi and Homa Bay ...... 27 2.3.1 Kilifi County ...... 27 2.3.2 Homa-Bay County ...... 28 2.3.3 Geographical distributiom and phytochemistry of selected medicinal plants .. 30 2.4. Evaluation of safety of crude extracts ...... 38 2.4.1 Overview ...... 38 2.4.2 Safety screening methods ...... 39 CHAPTER THREE ...... 43 MATERIALS AND METHODS ...... 43 3.1. Materials ...... 43 3.1.1 Test organisms ...... 43 3.1.2 Medicinal plants (Test articles) ...... 43

3.1.3 Reagents ...... 43 3.1.4 Equipments ...... 43 3.2.0 Methods ...... 44 3.2.1 Preparation of in vitro Plamodium test ...... 44 3.2.2 Preparation of the plant extracts ...... 46 3.2.3 Preparation of parasite culture system ...... 47 3.2.4 Preparation of the working drug solutions for the tests ...... 49 3.2.5 Harvesting the malaria parasites ...... 49 3.2.6 Calculation of percentage parasite growth inhibition ...... 50 3.2.7. Safety screening ...... 51 3.3.0 Isolation and characterization of active chemicals/compounds from plants .... 58 3.3.1 Isolation of compounds ...... 58 3.3.2. Characterization of isolated compounds ...... 65 CHAPTER FOUR ...... 67 RESULTS ...... 67 4.0 Pant extracts ...... 67 4.1 Yield of plant extracts ...... 67 4.3 Results of the Brine shrimp lethality test ...... 68 4.3 In vitro antiplasmodial activity of the plant extracts ...... 69 4.4 In vivo subacute toxicity test of DCM extract of C limon root in rabbits ...... 70

4.3 Results on Histology ...... 73 4.6 Results for isolation of compounds ...... 74 4.6.1 Compounds isolated from the DCM root of C. limon (CLR 2)...... 74 4.6.1.1 Isolation of compounds from the DCM root of C. limon (CLR 2) ...... 74 4.6.2 Compounds isolated from Bridelia cathartica methanolic leaf extract (BCL-3)75 4.6.3 Compounds isolated from M. pyrifolia hexane leaf extract: Adsorption chromatography ...... 76 4.6.4 Structures of isolated pure compounds ...... 78 4.6.5 NMR, IR and MS results for compound CLR 2F12(b) (Xanthyletin) ...... 82 4.6.6 NMR data for compound MPL-1F37 (a) (Spinasterol) ...... 86 4.6.7 Antiplamodial activity of the isolated compounds ...... 87 CHAPTER FIVE ...... 89 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ...... 89 5.1 Discussion ...... 89 5.2 Conclusions ...... 100 5.3 Recommendations ...... 102 5.3.1 Recommendations for further studies ...... 102 REFERENCES ...... 104 APPENDICES ...... 115 Appendix 1: Compound information...... 115 Appendix 2: NMR Spectra for pure compounds ...... 116 Appendix 3: IR spectra ...... 127

LIST OF TABLES

Table 1: The Principal Classes of Conventional Antimalarial Dugs in use ... 11 Table 2: Plant families and species exhibiting antimalarial activity and the

IC s of pure compounds isolated from them (Nkunya, 1992) ...... 18 50 Table 3:List of Plants Collected and their Characteristics ...... 45 Table 4: Parameters used to assess safety of Citrus limon root DCM

extract ...... 53

Table 5: Compatibility Model CLR 2F12 (a) ...... 66 Table 6: Compatibility model CLR 2f12 (b) 66 ...... Table 7: Yield of the plants extracts ...... 68 Table 8: LC s (µg/ml) of crude plant extracts against brine shrimps 50 calculated at 95% confidence interval using probit ...... 69 Table 9: In vitro antiplasmodial activity of plants extracts against V1/S

Strain ...... 70 Table 10:Effect of subcutaneous administration of DCM root extracts on haematological, immunological and biochemical parameters,in

rabbits (N=5)...... 72 Table 11: Adsorption chromatography of DCM extract of C. limon

rootLR ...... 75 Table 12: Column adsorption results for compounds isolated from

Bridelia cathartica ...... 76 Table 13: Antimalarial test results for fractions of Bridelia cathartica leaf

methanolic extract ...... 76 Table 14: Adsorption chromatography results of Microglossa pyrifolia

hexane leaf extract ...... 77 Table 15: Weights of fractions of Microglossa pyrifolia hexane leaf

extract ...... 77 Table 16: 13C (75 MHz) 1H (360 MHz) data of Suberosin (CDCL , 3 CD3OD, δ in ppm) in Hz ...... 80

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Table 17: 1H (360 MHz) and 13C (360 MHz) data of Xanthyletin

(CDCl , δ in ppm) in Hz ...... 85 2 Table 18: Pharmacological and chemical data of pure compounds and

reference drugs ...... 88

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LIST OF FIGURES

Figure 1:County Governments". Kenya Law Reform Commission. 29

August 2012 ...... 27

Figure 2: A photograph of a specimen of Achiranthes aspera leaves ...... 31

Figure 3: A photograph of a specimen of Heinsia crinite leaves ...... 32

Figure 4: A photograph of a specimen of Bridelia cathartica leaves ...... 33

Figure 5: A photograph of a specimen of Citrus limon plant ...... 34

Figure 6: A photograph of a specimen of Microglossa pyrifolia leaves ...... 35

Figure 7: A photograph of a specimen of Vernonia glabra leaves ...... 36

Figure 8:A photograph of a specimen of Carissa edulis plant ...... 38

3.2.7.2.2.4.1 Single radial immunodiffusion (SRID) ...... 55

3.2.7.2.2.5.1 Determination of weight ...... 56 Figure 9: A1-E2 Photographs of Experimental & Control Rabbit Tissues

compared using x 100 objective ...... 74

Figure 10: MS retention time of compound CLR- 2F12 (a) (Suberosin) ...... 81 Figure 11: MS spectra, structure and molecular weight of compound CLR

2F12 (a) (Suberosin) ...... 82

Figure 12: MS retention time of compound CLR 2F12 (b) ...... 85

Figure 13: Chemical structure of compound CLR 2F12 (b) (xanthyletin) ...... 86

Figure 14: Proposed molecular structure of Spinasterol from NMR data ...... 87

(Detailed primary data is shown inAppendices 1C and 2I-L)...... 87

ACKNOWLEDGMENTS

I hereby thank the Kenya Agricultural Research Organization (KALRO) Director

General, Dr Eliud Kireger for granting me study leave and for providing materials that made this work possible. I am grateful to the Biotechnology Research Institute

Director Dr Raymond Mdachi for suggestions on the doses of the crude extract used in toxicity studies and the Biotechnology Deputy Director Dr Judy Chemulit for facilitating the writing of this thesis by providing time and the necessary materials.

I am greatly indebted to the Multilateral Initiative on Malaria (MIM) Project number

990096, a WHO funded project for sponsoring this work.. I am greatly indebted to

Prof Anastasia N Guantai, Dean Faculty of Pharmacy who apart from being the

MIM Project coordinator was also my local supervisor for availing funds when it was necessary. I would like to thank the Department of Chemistry, University of

Nairobi for collaboration and more so to Prof Abiy Yenesew who facilitated the isolation and NMR identification of the isolated pure compounds through expert advice. I would like to thank Mr Hoseah Akala of KEMRI Kisumu for giving guidance on antimalarial tests. I would like to thank my University supervisors Prof

Eliud NM Njagi, Department of Biochemistry, Microbiology and Biotechnology, and Prof Caroline C Langat-Thoruwa, Department of Chemistry both of Kenyatta

University for keeping me on my toes and making sure that I completed this project.

Much thanks go to Prof Caroline C Langat-Thoruwa who facilitated MS analysis of the two pure compounds isolated from Citrus limon at ICIPE.

ABBREVIATIONS AND ACRONYMS

AAL Achyranthes aspera leaves

ABO blood groups

ACT artemisinin base combination therapy

ACTs artemisinin-based combination therapies

AFLP amplified fragment length polymorphsm

ANOVA analysis of variance

ASAT aspartate aminotransferase

ASTM American Society for Testing and Materials

B cell antibody producing cell

BCL Bridelia cathartica leaves

BCL-1, 2, 3 & 4 Bridelia cathartica leaf hexane, DCM, methanolic & aqueous

extracts

13C carbon 13

CG cycloguanil

CSP circumsporozoite protein

CAMAG the world leader in instrumental Thin-Layer Chromatography

CDC centre for disease control

CDCl2 dichloromethane

CER Carissa edulis roots

CHCl3/MeOH chloroform/methanol solvent system

CLR Citrus lemon root extract

CLR-1, CLR-2, CLR-3, CLR-4 hexane, DCM, methanolic & aqueous extracts

CMS complete medium with serum

CNS central nervous system

CO2 carbon dioxide

COSSY a useful method for determining which signals arise from

neighbouring protons

CPD citrate phosphate dextrose

CQ chloroquine

D6 CQ sensitive strain of P. faciparum

Da dalton

DCM dichloromethane

DILI drug induced liver injury

DMSO dimethylsulphoxide

DNA di-oxy nucleic acid

DTPE Department of Training Plans and Evaluation

DHS Demographic and Health Survey

DHFR dihydrofolate reductase

ECG electrocardiogram

ED50 effective dose for 50 percent of the group tested

EDTA ethylene diamine tetraacetic acid

EI electron impulse

FY fiscal year

F1, F2-F100 fractions 1, 2-100

FDA Food and Drug Administration

FIND Foundation for Innovative New Diagnostics

FTIR Fourier transform infrared spectroscopy g gram

GHz gigahertz

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GSK GlaxoSmithKline

GoK Government of Kenya

G6PD glucose-6-phosphate dehydrogenase

GR growth rate

GRAFIT data analysis and scientific graphing package for Windows

[3H] labeled proton

1H proton

HBsAg hepatitis B surface antigen ,

Hb hemoglobin

HCL Hensia crinita leaves

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HETCOR heteronulear correlation

HRP2 histidine-rich protein 2

HYPOGuaRD advance glucose test strip at american diabetes wholesale

Hz hertz (cycles per second)

IRS indoor residual spraying

IPTp intermittent preventive treatment of pregnant women

IC50 half maximal inhibitory concentration

IgA, D, E, G, M five immunoglobin classes

IR infra red

K7 Coastal kenya and Eastern

KARI-TRC Kenya Agricultural Research Institute-Trypanosomiasis

Research Centre

KALRO Kenya Agricultural and Livestock Research Organisation

Kbr potassium bromide

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kHz kilohertz

KMHFL Kenya Master Health Facility List

LAMP loop mediated isothermal amplification of DNA

LDL low density lipoproteins

LD50 median lethal concentration

LDH lactate dehydrogenase

LH-20 a type of sephadex for gel filtration chromatography

MLEM Model List of Essential Medicines

MoH Ministry of Health

MeOH methanol

MHz megahertz

MIM multilateral initiative on malaria

MPL 1F37 fraction 37 of hexane extract from a column

MPL Microglossa pyrifolia leaves

MS mass spectrometry

MICC Malaria Interagency Coordination Committee

MSP merozoite surface protein

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAD nicotinamide dinucleotide

NADH reduced form of NAD

NHS normal human serum

NMR nuclear magnetic resonance

NMCP National Malaria Control Program

60 PF254 silica gel powder to be visualized under UV254

PABA para-aminobenzoic acid

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PfPI3K P. falciparum phosphatidylinositol-3-kinase

PABA para-aminobenzoic acid

PanLAMP plasmodium LAMP

PCR polymerase chain reaction

PCV packed cell volume

Pf-LAMP Plasmodium falciparum LAMP

PLT platelet

PMS phenylmethosulphate

PTLC preparative thin layer chromatography

QBC quantitative buffy coat

RAPD randomly amplified DNA

RBC,WBC red and white blood cells

Rf the distance travelled by the isolate divided by the distance

travelled by the solvent.

RFLP restriction fragment length polymorphsm

RPMI 1640 medium for culturing plasmodium

SRID single radial immunodiffusion

SM&E surveillance monitoring and evaluation

TWGs technical working groups

TNs treated mosquito nets

TDR tropical diseases research

TEQ toxic equivalents

TLC thin layer chromatography

TNF tumour necrosis factor

UPR unfolded protein response

UV ultra violet

VGL Vernonia glabra leaves

VLDL very low density lipoproteins

VI/S an international multidrug resistant strain of P.falciparum

originally from a patient in Vietnam

W2 chloroquine-resistant strain of P.falciparum

WHO world health organisation

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ABSTRACT

Among human parasitic diseases, malaria is the most important and it has global incidence of 300 to 500 million cases per year with about 1.5 million deaths among African children. Recently the disease has been subjected to massive control efforts with varying degrees of success. The development of parasite and vector resistance to conventional drugs and insecticides has complicated both treatment and control today. The main goal of this project was to identify viable phytomedicines traditionally employed for the treatment of malaria in Kenya that could be developed into antimalarial agents. Seven medicinal plants used by herbalits in Kilifi and Homa-Bay Counties were examined: Achyranthes aspera, Heinsia crinita, Bridelia cathartica, Citrus limon, Microglossa pyrifolia, Vernonia glabra and Carissa edulis. Both organic and aqueous plant extracts were obtained and tested for antiplasmodial activity against CQ sensitive and resistant strains of P.falciparum in vitro. Active extracts were subjected to bioactivity guided fractionation and isolation of compounds. Structural elucidation of isolated compounds was determined using standard spectroscopic techniques (IR, NMR & MS). The most active plant extracts against P. falciparum had the following IC50 activities: DCM extract of Citrus limon roots (7.017 μg/ml), aqueous extract of Carissa edulis roots (8.054 μg/ml), DCM and methanolic extracts of Bridelia cathartica leaves (11.537 μg/ml, 15.647 μg/ml) respectively and DCM extract of Heinsia crinita leaves (13.336 μg/ml). The others were as follows: hexane extract of Achyranthes aspera leaves (18.087 μg/ml), hexane extract of Microglossa pyrifolia leaves (21.376 μg/ml), methanolic extract of Heinsia crinita leaves (24.805 μg/ml), aqueous extract of Bridelia cathartica leaves (25.985 μg/ml), and DCM extract of Carissa edulis roots (30.074 μg/ml). Toxicity tests on crude DCM root extract of C. limon, indicated that 70% of the biochemical parameters studied using the rabbit model were not affected. Two pure coumarin compounds, suberosin (IC50 53.1415μg/ml ά D6 strain of P. falciparum, 26.732μg/ml ά W2 strain) and xanthyletin (IC50 1580μg/ml ά W2 strain) were isolated from C. limon. Spinasterol (IC50 43.169 μg/ml ά V1/S strain) was isolated for the first time from hexane leaf extract of Microglossa pyrifolia. Histology of the liver, heart, kidney and brain did not reveal any damage. Citrus limon is a potential antimalarial drug and owes its activity to the presence of suberosin and other compounds with which it works in synergy against falciparum. In conclusion, there were no significant toxic effects on rabbit tissue observed upon treatment with the crude DCM root extract of C. limon in the subchronic toxicity studies as about 90% of the parameters examined were not affected. Therefore a drug that can cure malarial infection can be synthesized modelled upon the structure of suberosin. Quinine and mefloquine have structures which are closely related to this compound. Further studies on C. limon required to be carried out in order to isolate and identify more compounds and test them against falciparum. In addition, compound isolation from aqueous extracts of C. edulis which also demonstrated high activity against P. falciparum requires to be carried out. This study has verified the use of these plants for the treatment of malaria by the traditional communities.

CHAPTER ONE

INTRODUCTION

1.0 Background information

1.1 Malaria the disease

Malaria a parasitic disease transmitted by female anopheles mosquitoes has a global

incidence of three hundred to five hundred million cases per year (malaria report

2020) and significant morbidity and mortality particularly among the under five year

children (CDC, 2015). It is a disease of public health interest affecting more than

107 tropical countries and more than 3.2 billion people, which is 40% of the world’s

population (Malaria-A global epidemic (2008, 2015) and The European Alliance

Against Malaria (2008, 2015). The estimated number of malaria deaths stood at

405000 in 2019 (WHO malaria report 2019). Malaria is one of Africa’s biggest

obstacles to socio-economic development that causes more than one million deaths

each year (Gallup et al., 2001).

Plasmodium falciparum malaria is the most prevalent in Kenya with an incidence of

6.7 million and mortality of four thousand per year, with a high risk among Western

Kenya around Lake Victoria, Coast Province and other areas including the

highlands (Kenya MOH 2015). If not treated promptly it may progress to the

severest form celebral malaria associated with kidney failure, seizures, mental

confusion, coma and death (Idro et al., 2010). It also causes abortion, premature

deliveries, growth retardation, low birth weight and anaemia (Hay et al., 2003;

Minakawa et al., 2002) and overburdening of health care faclities (Ochola, 2003).

The symptoms and signs of malaria are non-specific making clinical diagnosis, unreliable. Microscopy, is the confirmatory test, but it is costly in terms of personnel and resource requirements and hence not widely used. Rapid tests among other diagnostic methods have been developed though their use is restricted due to lack of sensitivity (for P. vivax, malariae and ovale) and specificity in high transmission areas and its prohibitive cost (Murray et al, 2008); however, these tests are useful for detection of P. falciparum (Guerin et al., 2002; FIND, 2013). Quality diagnosis improves health outcomes, reduces emergence of drug resistance and infection transmission (Guerin et al., 2002). The study of the impact of malaria is challenged by the lack of high quality data due to poor epidemiological data (Guerin et al.,

2002).

1.2 Control and treatment of malaria

Use of prophylactic drugs, mosquito eradication and the prevention of mosquito bites are the methods used to prevent the spread of the disease, or to protect individuals in areas where malaria is endemic. Development of vaccine to prevent malaria is an active field of research (Glazer and Nakaido, 1996; MacGregor, 2005).

Through Indo-US collaboration a recombinant multistage P. falciparum candidate

DNA vaccine has been developed (Adrian V.S. Hill, 2011 & Paul Muller; 2015 © malariasite.com).

The Plasmodium parasite invades red blood cell by binding to a transmembrane protein known as glycophorin. The parasite`s transmembrane protein found on its surface is responsible for the attachment of the parasite to glycophorin on the erythrocyte`s surface. Interruption of this host- parasite (protein-protein) interaction

that leads to malaria is being researched on (Malacinsky and Freifelder, 1998).

Ordering the malaria specific diagnostic tests that can detect severe anemia, hypoglycemia, renal failure, hyperbilirubinemia, and acid-base disturbances influences the course of treatment ([email protected]). These include Standard

Methods commonly known as Giemsa Stain and Polymerase Chain Reaction such as fluorescence methods based on acridine orange or benzothiocarboxy purine stains, quantitative buffy coat (QBC, Becton Dickson) centrifugal heamatology system, enzyme linked assays for the detection of P. falciparum antigen, and PCR; however, all require a laboratory and expertise and are restricted to large cities and research institutes. For the routine diagnosis of malaria Giemsa-stained blood film still remains the method of choice (Guerin et al., 2002).

1.3 Drug discovery for malaria

This has involved evaluation of crude drugs mainly from plants and semi synthetic derivatives using in vitro methods. The recent introduction of semi-automated in vitro screening method for P. falciparum in blood cultures based on the capacity of drugs to inhibit the incorporation of [3H]-hypoxanthine into the malaria parasite

(Desjardins et al., 1979) has streamlined drug screening search for antimalarials from plants (Desjardins et al., 1979). The drug to be tested is dissolved or micronized in a suitable solvent and is serially diluted as desired. Aliquots of the test solution are disperised into wells of microtitre plate and then diluted with human blood containing P. falciparum at 1% parasitaemia. To each plate is added

[3H]-hypoxanthine and the plates are kept in an incubator containing oxygen, carbon dioxide and nitrogen and against two controls, one containing parasitized blood without drug and another with uninfected erythrocytes. The capacity of the parasites

to take in the [3H]-hypoxanthine is determined by scintillation counting from which the amount of the drug in solution required to retard the growth of 50% of the parasites is calculated. This method is semiautomatic so that an enormous number of samples can be screened simultaneously (Desjardins et al., 1979).

1.4 Phytotherapeutics

Pharmacognosists have come up with sophisticated procedures which results in active principle isolation, resolve their chemical structure, perceive their production in living organisms and demonstrate their effects on people. The study includes chemical, physical, biological and biochemical characteristics of drug substances from living resources. Today it is a branch of chemistry with historical roots in pharmacy. At the present time, random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs) which are DNA fingerprinting techniques are used to authenticate the plants (Ashok Kumar et al., 2013). Ethnopharmacology emphasizes on describing the medicinal properties of remedies used by local people with a focus on how local people carefully choose, concoct and give these curative plants and animals. Ethnopharmacologists are therefore required to divide their time between the laboratory and field as their study combines perspectives from botany, chemistry and anthropology.

1.5 In vivo lethality test (Brine Shrimp Bioassay)

originally proposed by Meyer et al., (1982). It represents any easy way to detect general bioactivity in plant extracts and again a useful means for monitoring bioactivity during seclusion of bioactive constituents.

1.6 Statement of the problem

Plasmodium falciparum malaria remains one of the world’s major killer diseases with 10-40% mortality rate due to its resistance to antimalarial drugs. There has been an emergence of strains of P. falciparum which are resistant to all the widely used antiplasmodials during the last 20 years. In East Africa resistance of P. falciparum to chloroquine was initially reported in 1978. Apparent spreading of resistance was via central regions around 1983, to south West Africa by 1984 and

Cameroon in 1985. By 1987 reports from Nigeria indicated that several isolates had shown resistance in-vitro to chloroquine and mefloquine (Serpa et al., 1988).

Chloroquine is no longer used in Kenya. It was withdrawn as an over the counter drug (Mwai et al., 2009).

Resistance to quinine the first effective antimalarial drug has been reported in East

Africa (Kenya, Tanzania and Zambia), Cameroon, Congo, Guinea, Tanzania,

Nigeria and Senegal (WHO 2001.4). An additional problem encountered is the serious adverse effects of some of the first line drugs such as amodiaquine and fansidar and also the discovery of a resistant strain of P. vivax (Marco et al., 2013).

Primaquine has both prophylactic and curative activities but is seldom used because of its toxicity (Marco et al., 2013). In rare cases, Stevens-Johnson syndrome, toxic epidermal necrosis, urticaria, serum sickness, hepatitis and exfoliative dermatitis have been associated with Sulfadoxine-Pyrimethamine (Marco et al., 2013). It is

therefore important to carry out research aimed at identifying new drugs for the treatment of malaria. The contemporary first line drug for relieving malaria burden is coartem which is the trade name of artemether/lumefantrine, which is artemisinin- based combination therapy (ACT) designated for the alleviation from acute uncomplicated P. falciparum malaria.

Widespread appearance of chloroquine resistant parasites in Kenya and other tropical countries resulted from the use and misuse of chloroquine based drugs for preventing and treating falciparum malaria (Njoroge and Bussmann, 2006). The local communities experiencing high poverty levels, rising costs of non-chloroquine drugs and high prices of insecticide treated nets have turned to tradition remedies

(Guyatt et al., 2002). The fact that there is rising costs of non-chloroquine drugs and high poverty levels in most African countries including Kenya, increases the need to come up with new drugs which can be used to treat malaria (Nuwaha, 2001).

The ability to control infectious disease is constrained by four key factors of medical or veterinary importance with a drug: (i) It can be undermined by drug resistance (ii) Side effects can prevent its use for the period of time required to effect a cure (iii) Inherent efficacy can be limiting; and (iv) Its means/path of entering the body may not be convenient (Gutteridge, 1993). Majority of the currently available antimalarials are subject to one or more of these restraints; some from all of them (Gutteridge, 1993). New drugs as well as vaccines are therefore needed (Guerin et al., 2002).

1.7 Justification of the study

Nature is rich in resources including medicines for use in control and management of various disorders. Natural product extracts, embracing terrestrial and marine plants, animals and microbial cultures are screened for biological activities in many industrial, governmental and university laboratories all over the world (Kinghom,

1998).

The list of drugs from higher plants alone includes the widely used agents; digoxin, digitoxin, reserpine, codeine, atropine, hyoscyamine, scopolamine, quinine, quinidine, pilocarpine and phytostigmine. Plants are still used in Western medicine in the form of potent pharmacologically active extracts, as exemplified by the official drugs Belladona, Ipecac, Opium, Rauwolfia and Digitalis (Kinghom, 1998).

Only about 20% of the approximately two hundred and fifty thousand species of plants have been scrutinized. Notable examples include quinine from Cinchona bark

(such as Cinchona officinalis), febrifugine (Pinter, 1970) from Dichroa febriguga, and more recently, ginghaosu (WHO, 1984, Klayman et al., 1984) from Artemesia annua, and quassinoids abtained from Simarouba spp have been found to possess antimalarial activity (Trager and Polonsky, 1981). The majority of the synthetic drugs for malaria in current use were either secured from quinine and artemisinin or have been replicated on the molecular geometry of quinine or in recent past the peroxide moiety of artemisinin as template (Gessler et al., 1994). High antimalarial activity against P. falciparum has been shown by compounds like pyronaridines and mepacrine (Elueze et al., 1996). Resistance to clinically used drugs such as chloroquine, mefloquine and pyrimethamine by P. falciparum is a grave problem and in some parts of South East Asia the only effective antimalarial drug is

artemisinin or one of its derivatives (Phillipson et al., 1991; Figureit et al., 1992;

Oketch-Rabah, 1996). Africa suffers from high morbidity and mortality due to endemic neglected tropical diseases such as malaria, schistosomiasis and trypanosomiasis and many others, plus infectious diseases and also non- communicable diseases thereby affecting labour productivity, especially in respect to Agriculture (Sindiga et al., 1995). The health infrastructure is also poor and limited and favours urban areas where only a small percentage of the population lives. Some 57% of the households in Kenya must trek more than four km to the nearby health facility; only 30% of the population lives within 2 kilometres of a health facility (Bennett and Maneno, 1986).

This population would benefit immensely from use of safe and efficacious natural remedies that are easier to access and probably cheaper. WHO also very early recognised that eighty percent of the world’s population rely solely on herbal plants for their basic health care needs (Akerele, 1987, Geoffrey, 1996) and has consistently encouraged and supported natural product research through various initiatives such as Tropical Disease Research (TDR). This study seeks to exploit the rich diversity of the Kenyan flora by identifying and screening for safety and antiplasmodial activity of the selected plants through ethno medicine survey carried out in target malaria endemic counties in Kenya hence enhancing chances for good outcomes.

1.8 Null hypotheses

(i) There is no difference in activity between plant extracts and standard/control drugs against Plasmodium falciparum.

(ii) There is no significant change in body weight, hematological and biochemical parameters between the extract treated and the control rabbits.

(iii) All the selected plant extacts do not exhibit activity against brine shrimps,

Artemia salina.

(iv) All the plants extracts and isolated compounds do not demonstrate significant in vitro antiplasmodial activity.

1.9 Study objectives

1.9.1 General objective

To determine the antiplasmodial activity and safety of extracts and isolated compounds of Achyranthes aspera, Heinsia crinita, Bridelia cathartica, Citrus limon, Microglossa pyrifolia, Vernonia glabra and Carissa edulis.

1.9.2 Specific objectives

(i) To determine the in vitro toxicity of extracts of Achyranthes aspera, Heinsia crinita, Bridelia cathartica, Citrus limon, Microglossa pyrifolia, Vernonia glabra and Carissa edulis using brine shrimp, Artemia salina assay.

(ii) To determine the in vitro antiplasmodial activity of extracts of Achyranthes aspera, Heinsia crinita, Bridelia cathartica, Citrus limon, Microglossa pyrifolia,

Vernonia glabra and Carissa edulis against chloroquine resistant and sensitive strains of P. falciparum.

(iii) To determine the bioactivity of crude plant extracts and isolated pure compounds against P. falciparum.

(iv) To determine the in vivo subacute toxicity of the most active crude extract against Swiss white rabbits.

CHAPTER TWO

LITERATURE REVIEW

2.1 Control and treatment of malaria

2.1.1 Vector control and malaria prophylaxis

Malaria control involves long term strategies in which sustained high coverage with malaria prevention and treatment interventions such as use of insecticide-treated mosquito nets (ITNs), indoor residual spraying (IRS), accurate diagnosis and prompt treatment with artemisinin-based combination therapies (ACTs), and intermittent preventive treatment of pregnant women (IPTp). Use of prophylactic treatments would progressively lead to malaria-free zones in Africa, with the ultimate goal of worldwide malaria eradication by 2040-2050. The President’s (US)

Malaria Initiative (PMI) launced in 2005 and implemented in Kenya in 2007 aims to reduce malaria-related mortality by 50% across 15 high-burden countries in sub-

Saharan Africa (US President’s Malaria Initiative (2019)).

Malaria transmission and infection risk in Kenya is determined largely by altitude, rainfall patterns, and temperature. The variations in altitude and terrain create contrasts in the country’s climate, which ranges from tropical along the coast to temperate in the interior to very dry in the north and northeast. The two rainy seasons are the long rains occurring from March to May and the short rains from

October to December. Temperatures are highest from February to March and lowest from July to August. Therefore, malaria prevalence varies considerably by season and across geographic regions.

National Malaria Control Program is responsible for policy development and implementation in key aspects such as vector control, case management, malaria in pregnancy, epidemic preparedness and response, advocacy, communication and social mobilization and surveillance, monitoring, and evaluation (SM&E) and operational research (OR). It is supported by The Malaria Interagency Coordination

Committee (MICC) that includes representatives of relevant government and, non- governmental organizations, community-based organizations, private sector, partners and donors. Kenya has been selected as a site for the RTS,S/AS01 (RTS,S)

Malaria Vaccine Implementation Programme to assess the feasibility, impact on mortality, and safety profile of the RTS,S malaria vaccine.

There have been reports of both parasite & vector resistance to the conventional drugs (CQ, Amodiaquine, Fansidar, Mefloquine, Primaquine, Quinine &

Artemisinin, etc) and insecticides (DDT) (Serpa et al., 1988, WHO 2019, Marco et al., 2013) and hence the need for continued search for more dtugs.

2.1.2 Antimalarial chemotherapy using conventional drugs

Several classes of drugs have been developed and used in treatment of malaria

(Table 1). These include:

Table 1: The principal classes of conventional antimalarial drugs in use Class Drugs Quinoline derivatives Quinine, 4-aminoquinolines (chloroquine and amodiaquine) primaquine, mefloquine Amino acridines Mepacrine Folate synthesis inhibitors Sulphonamides and sulphones such as dapsone, sulphodoxine and sulphalene Dihydrofolate reductase inhibitors Proquanil and pyramethamine Sequiterpene lactones Qinghaosu (Artemisinin)

The drugs have varied modes of of action, levels of efficacy and side effects. It is thought that the structurally related quinoline such as quinine, mepacrine, chloroquine and mefloquine act through disruption of hemoglobin digestion in the blood stage of the malaria paresite leading to inhibition of the spontaneous formation of betahaematin (haemozoin or malaria pigment) which is a toxic product of the digestion of hemoglobin by parasites. Quinine is a very potent antimalarial agent isolated from Cinchona tree in 1820 and has served humanity to date although resistance was first reported in the 1980s and as of 2006, the drug has been reserved on the WHO’s Model List of Essential Medicines (MLEM) for the treatment of severe malaria in cases where artemisinins are not available. Mepacrine itself is no longer used today due to the high chance of undesirable side effects such as toxic psychosis (Drugs.com).

Resistance to CQ was first reported in the 1950s and over the years many strains of malaria have developed resistance. Indeed, resistant strains (K1, 7GB, W2, Dd2, etc.) of the malaria parasite are now used in potency evaluation assays as a way of showing efficacy. Chloroquine is on the MLEM for the treatment of P. vivax in regions where resistance has not developed (Hyde et al., 2007).

Mefloquine is a chiral molecule. It is reported that while the (+) enantiomer primarily mediates an antimalarial activity, the (-) enantiomer contribute to the psychotrophic effects by specifically binding to adenosine receptors in the central nervous system. Its (DrugBank 2020) drug resistance was first reported in 1986.

These drugs are commonly used in combination with a complementary drug (such as mefloquine and artesunate, sold as Artequin™) to reduce the chance of resistance

development to the quinoline family of compounds. It is no longer widely used due to the perception of central nervous system toxicity that has been suggested to affect a large number of its users.

The mechanism of action of Halofantrine may be similar to that of chloroquine, quinine, and mefloquine; by forming toxic complexes with ferritoporphyrin IX that damage the membrane of the parasite. Its use has diminished over time due to a number of undesirable side effects, such as the potential for high levels of cardiotoxicity. It is only used as a curative drug and not for prophylaxis due to the high toxicity risks and its unreliable pharmacological properties. Halofantrine is still used today, under the brand name Halfan™, but only in cases where patients are known to be free of heart disease and where infection is due to severe and resistant forms of malaria (DrugBank 2020).

Artemisinin acts through a number of ways including its activation by haem to generate free radicals, which in turn damage proteins required for parasite survival based around previous studies which identified haem and PfATP6 (Ca2+ transporter) as potential MoAs, up-regulation of the unfolded protein response (UPR) pathways which may be linked to decreased parasite development and also as potent inhibitor of P. falciparum phosphatidylinositol-3-kinase (PfPI3K) (Meshnick SR, 2002).

4-Aminoquinolines depress cardiac muscle, impair cardiac conductivity, and produce vasodilatation with resultant hypotension. They depress respiration and cause diplopia, dizziness and nausea. The mechanism of plasmodicidal action of amodiaquine is not completely certain. It is mainly used for the treatment of

uncomplicated P. falciparum malaria when used in combination with artesunate and is commonly sold under the trade name Camoquine® or Coarsucam™. Similar to chloroquine, amodiaquine’s MoA is thought to involve complexation with haem and inhibition of haemozoin formation.

The mechanism of Piperaquine which involves inhibition of the haem detoxification pathway is unknown but is expected to be similar to that of Chloroquine. Initially used throughout China as a replacement for chloroquine, resistance led to its diminished use as a monotherapy. These days, Piperaquine is used as a partner drug with dihydroartemisinin (commonly sold under the trade name Eurartesim®)

(DrugBank 2020).

The exact mechanism by which Lumefantrine exerts its antimalarial effect is unknown. However, available data suggest that lumefantrine inhibits the formation of β-hematin by forming a complex with hemin and inhibits nucleic acid and protein synthesis. It is currently sold under the trade name Coartem®. The exact MoA of lumefantrine is unknown; however studies suggest that it inhibits nucleic acid and protein synthesis through the inhibition of β-haematin formation by complexation with haemin. Lumefantrine is currently used only in combination with artemether.

Proguanil is a biguanide derivative that is converted to an active metabolite called cycloguanil. It exerts its antimalarial action by inhibiting parasitic dihydrofolate reductase enzyme. It has causal prophylactic and suppressive activity against P. falciparum and cures the acute infection. It is also effective in suppressing the clinical attacks of vivax malaria. However, it has slower action compared to 4-

aminoquinolines. It is used alone and also in combination with atovaquone

(Malarone™). The combination appears to act at the cytochrome bc1 complex

(Complex III) inhibiting mitochondrial electron transport chain and indirect inhibition of several metabolic enzymes. The ultimate metabolic effects of such blockade may include inhibition of nucleic acid and ATP synthesis. The combination has proven to be a very effective antimalarial due to the synergistic effect of the two components due to their different MoAs. Proguanil (when used alone) acts as a dihydrofolate reductase (DHFR) inhibitor through its metabolite,

Cycloguanil (CG) which disrupts deoxythymidylate synthesis. When used in combination with atovaquone, however, proguanil does not act as a DHFR inhibitor but instead reduces the concentration of atovaquone required for treatment. Generic

Atovaquone/Proguanil is still available today for the treatment of chloroquine- resistant malaria.

Pyrimethamine inhibits the dihydrofolate reductase of plasmodia and thereby blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication. This leads to failure of nuclear division at the time of schizont formation in erythrocytes and liver. Sulfadoxine was developed in the early

1960s. Sulfadoxine helps inhibit the enzyme dihydropteroate synthetase which is an enzyme necessary in the conversion of para-aminobenzoic acid (PABA) to folic acid. As folic acid is vital to the synthesis, repair, and methylation of DNA which is vital to cell growth in Plasmodium falciparum. With this vital nutrient lacking, the parasite has difficulty in reproducing.

Sulfadoxine-pyrimethamine combination is used to treat malaria and also as prophylaxis to prevent malaria in people who are living in, or will be traveling to, an area where there is a chance of getting malaria. The combination of pyrimethamine and sulfadoxine was approved for use for the treatment of malaria in 1981 and is now commonly sold under the trade name Fansidar®. In Kenya chloroquine was withdrawn as a first line drug and replaced by fansidar in 2000, hence the need to develop new drugs as resistance to fansidar has also been reported. Like

Lumefantrine, Pyronaridine has been found to act through the inhibition of β- haematin formation.

The active moiety of Tafenoquine, 5, 6 orthoquinone tafenoquine, seems to be redox cycled by P. falciparum which are upregulated in gametocytes and liver stages.

Once inside, the oxidated metabolite produces hydrogen peroxide and hydroxyl radicals. It is thought that these radicals produce leads to the parasite death. On the other hand, Tafenoquine inhibits heme polymerase in blood stage of parasites which explains the activity against blood stages of parasites. The continued use of the above drugs has been challenged by development of resistance of the malaria parasite leading to loss of efficacy (Oketch Rabah, 1996; Kihamia et al., 1982,

Alder 1992; Serpa et al., 1988; Watkins, 1997).

2.2 Plants as sources of antimalarial agents

Systematic assessment of plants with medicinal properties which have been used in the production of traditional medicine has furnished modern medicine with very efficacious drugs against parasitic diseases. Through screening antiplasmodial activity has been demonstrated in many plants using both in vivo and in vitro

systems. Isolation of compounds such as furoquinolines and acridone alkaloids has been carried out from several plants of Rutaceae family (Mitaku et al., 1985; 1986

1987; Nkunya et al., 1991; Joanne Bero et al., 2009). Members of the plant families;

Meliaceae, Rubiaceae and Simaroubaceae, and the genera Artemisia (Compositae) and Uvaria (Annonaceae), have been found to offer antimalarial activity, and several active compounds have been obtained from these plants (Nkunya et al.,

1991). In addition to Qinghaosu, several quassinoids, and limonoids have been isolated from traditionally used plant families for treatment of malaria. Some of the isolated compounds, such as sergeolide, glaucarubinone, and bruceantin, from members of the family Simaroubaceae, have been found to be highly potent against multi-drug resistant and chloroquine sensitive strains of P. falciparum in vitro

(Nkunya et al., 1991) at times with potency which is much higher than that of chloroquine diphosphate. Gedunin is the most active compound in this series and is obtained from Azadirachta indica (Nkunya et al., 1991) (Table 2).

Langat Thoruwa et-al, (2003) enhanced the antiplasmodial activity of bitter principle quassin by converting it using chemicals to γ-lactone quassilactone. They tested it in vitro against P. falciparum (K1) IC50 -23μg/ml) which they established to be 40 times more active against malaria than that of quassin.

Table 2: Plant families and species exhibiting antimalarial activity and the IC50s of pure compounds isolated from them (Nkunya, 1992)

Plant family Species Class of compoud Compound IC50 (μg/ml) Melianceae Azadirachta indica Limonoids Azadichtin ˃ 4 Nimbinin 0.77 Gedunin 0.72 Nimbolide 1.74. Dihydrogenu in 2.63 Simaroubaceae Picrolemma Quassinoids Sergeolide 0.006 pseudocoffea Eurycomanone 0.27 Simarouba glauca Glaucarubanone 0.006 Soularubinone 0.006 Inhibitors of protein in P. falciparum Simalicalactone D 0.002 D-Simarolide 0.010 Chaparrinone 0.010 Brucea antidysenteric a Bruceatin 0.010 Bruceatinol 0.010 Bruceine A 8.66 Bruceine B Bruceine C 1.95l Brucea javanica Quassinoids Yadanzioside F 5.00l Yadanzioside IF 22.0l Fructus bruceae Quassinoids Brusatol 22 Chaparrin 22 Ailanthus altassima Quassinoids Ailanthone 0.015 Ailanthinon 0.009 Picrasma javanica Alkaloids B1 - Annonacea C*Artabotrys unciatus Sesquiterpenoid Qinghaosu 0.24 Artemisia annua Mono, Sesqui, Di, Arteanuin B - and Triterpenoids Artemisisnin 0.03 Sesquiterpenoid Artemetin 26 Casticin 24 Chrysoplenetin 23

Chrysoplenol D 32 Cirsilincol 36 Eupatorin 65 Annona squamosa Polyketide 11-β-acetogenin 3.11 Uvaria pendensis(Root) Sesquiterpenoid Tricyclic sesquiterpene 1.90 μg/ml U. pendensis 3-Farnesyndole 16.70 3-Farnesyndole derivatives 2.90 Cyconexene 8.35 U. lucida spp Uvaretin 3.49 U. tanzaniae Tanzanene Inactive U. angolensis Uvarisesquiterpene D - Astreraceae Parthenium Parthenin 0.72 hysterophorus Cyperaceae α-Cyperone 5.50 Cyperus rotunus ß-Selinene 5.56 Lamiaceae Hoslunda opposita 3-o- Benzoylhosloppone 0.41 3-o- Cinnamoylhosloppone 3.80 Celastraceae T*celastrus Quinononoid triterpene Pristimerin 200 ng/ml Paniculatus Chenopodiaceae Chenopodium Ascaridole 0.05 ambrosiodes Guttiferae C*Hypericum Phloroglucinols Japonicine A Activity α P. japonicum berghei Japonicine B Activity α P. berghei Berberidaceae Podophylum Ecteinaschidin Species

Many of the medicinal preparations have not been subjected to careful scientific evaluation for efficacy and safety. Any traditional medicine, like all other drugs, must satisfy the following three major criteria for it to be used. These criteria are: identity, safety and efficacy. Conventionally manufactured drugs often meet these criteria through enforcement of law since pharmacopoedial specifications already exist. Such laws are not readily applicable to traditional medicines. However, it should be possible to device suitable methods for identification, safety and efficacy that could be applied to such medicines (Kofi-Tsekpo, 1991).

The evaluation of a traditional medicine is carried out according to the three criteria as follows:-

A. Identity:- This requires the following:

(i) Ethnomedical data, which include pharmaceutical or compounding information.

(ii) Ethnobotanical data: this includes botanical, ecological and pharmacognostical information.

(iii) Chemical data: This includes phytochemistry and chemotaxonomy.

B. Efficacy: This requires as much information as possible from the traditional practitioner, and may involve directly witnessing treatment. Standardised clinical trials should also be carried out after establishing the identity and safety of the medicine.

C. Safety: This can only be derived from the ethnomedical clinical information.

However, when such data are correlated with the ethnobotanical and chemical information, it is possible to determine the safety of the medicine to a high degree of accuracy. Laboratory animal experimentation should be able to confirm the information gathered.

2.2.1 Extraction and purification of compounds

Extracting the plant material (whole plant, stem, bark, leaves or roots), starts with non polar solvents such as hexane or petroleum ether, and followed with solvents of increasing polarity such as ethyl acetate, dichloromethane (DCM), ethanol, and water. Alternatively others extract into alcohol. These extracts are then subjected to column chromatography and preparative TLC. The isolations of a biologically active compound from a plant or animal is rather akin to searching for a needle in haystack. In most cases, only one compound in a natural product extract may be active, and in such a case, it is diluted in the initially tested crude extract by thousands of other plant constituents (Kinghom, 1998). The process involves liquid- solid (adsorption) chromatography, gelfiltration (exclusion) chromatography and preparative thin layer chromatography.

2.2.2 Liquid-solid (adsorption) chromatography

Adsorption is a phenomenon involving the bulk properties of a solid, liquid or gas.

It involves atoms or molecules crossing the surface and entering the volume of the material. As in adsorption, there can be physical and chemical absorption. It makes use of a mobile phase which is either in liquid or gaseous form. The mobile phase is adsorbed onto the surface of a stationary solid phase. Adsorption chromatography involves the analytical separation of a chemical mixture based on the interaction of the adsorbate with the adsorbent. The mixture of gas or liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at different rates.

An adsorbent is a substance which is porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces. Some commonly used

adsorbents are Silica gel H, silica gel G, silica gel N, silica gel S, hydrated gel silica, cellulose microcrystalline, alumina, modified silica gel, etc.

Silica and alumiona packing and a non-polar mobile phase, such as a hydrocarbon, mixed with a more polar solvent are used in liquid solid chromatograpghy

(Kremmer and Boros, 1979; Majors, 1981, Pavia et al., 1990). The hydrocarbons heptanes, hexane, or isooctane is thought to be the weak solvent at the same time the more polar component, isopropanol, diethylether or dichloromethane is thought to be the strong solvent. The polar Si-OH group of the silica attracts the polar sample constituent by hydrogen bonds and additional molecular inter-relationships.. The non-polar movable phase is favoured by the non-polar sample constituent and are therefore eluted before the polar constituents and hence separations. The solute maybe displaced from an adsorption site by a more-polar solvent (Majors, 1981;

Miller and Neuzil, 1980; Miller, 1990).

The solute may be displaced from an adsorption site by a more-polar solvent

(Majors 1981). A description chromatography is the right technique for class portion which is portion based on the type of functional groups. It at the same time partitions multifunctional compounds and isomers as is seen in the separation of mono-di and triglycerides or the oestracal steroids. Similarly, the partition of geometrical compounds with the same formulae but different arrangement of atoms in the molecular and different properties (isomers) for example, cis-trans can be achieved (Majors, 1981). Gradient elution is effected when one increases the more polar solvent concentration with time (Houghton and Raman, 1998).

2.2.2.1 Principle of TLC during monitoring column effluent

Thin-layer chromatography (TLC) is a technique used to separate non-volatile mixtures. It is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, silica gel, aluminium oxide

(alumina), or cellulose. This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved (Miller and

Neuzil, 1980). It may be performed on the analytical scale as a means of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a compound (Miller and Neuzil, 1980; Pavia et al., 1990). To help identify the compounds present, Rf values can be calculated for each spot. A binding agent such as calcium sulphate hemihydrate holds the silica firmly on the support aluminium foil (Touchstone and Dobbins, 1998). Sodium fluorescein as an UV indicator is incorporated and fluoresces when exposed to UV light at 254nm so that substances adsorbing this wavelength will contrast sharply by appearing dark while quenching the greenish-yellow fluorescing background. Sodium salts of hydroxyl-purene- sulphonic acids, another indicator, fluoresces at 336nm and provides a contrasting background for substances that absorb at this frequency. The type of material to be partitioned and the type of absorption material used for separation determines the nature and the relative numbers of the elements that make up the mobile phase

(Touchstone and Dobbins, 1998).

2.2.3 Gelfiltration (exclusion) chromatography

In this method, diffusion of the dissolved substance into and out of the pore in a pervi matrix is assessed. All molecules bigger than the pore size are removed and are washed out in exclusion volume. Molecules less than the smallest pore size are washed out at the total percolation volume. Molecules that are part of the openings but are removed from others can be partitioned. All partitioning happens between the exclusion volume and the percale volume. The washing order is in terms of reducing molecular size (Kremmer and Boros, 1979, Pavia et al., 1990). The molecule used in decreasing order for gel chromatography is sephadex (Pavia et al.,

1990). In chemical terms, sephadex is a polymer of cross-linked carbohydrates whose amount of cross-linking accounts for the size of the pore sizes in the polymer matrix (Kremmer and Boros, 1979, Pavia et al., 1990). Furthermore the hydroxyl group on the repeated cross-linked carbohydrate can absorb water and therefore causing the material to swell. As it swells, pores are generated in the matrix.

2.2.4 Preparative thin layer chromatography (PTLC)

This is basically the same as analytical TLC, the major difference being that the thickness of the sorbent layer is at least twice that used in analytical work ranging between 500µm (0.5mm) and 10000µm (10mm). The commonly used thicknesses are 1000µm (1.0mm) and 2000µm (2.0mm) (Touchstone and Dobbins, 1998). Layer thickness up to 4mm may be employed (Funniss et al., 1994). For non-aqueous solvents, sephadex LH-20 has been developed. Alkylation of a portion of the hydroxyl groups has been accomplished and therefore the material can expand under both water containing or an anhydrous environment (Pavia et al., 1990). It can be acetone, methylene chloride, and aromatic hydrocarbons (Pavia et al., 1990). The

advantage with this method is that 100% of the sample loaded is recoverable and relatively large amounts of material can be loaded and separated substances used for further work such as additional chromatography, infrared analysis, melting point determination, or synthesis (Funniss et al., 1994; Touchstone and Dobbins, 1998).

PTLC has a number of advantages compared with column chromatography, which is also commonly used for preparative separations. Namely: (i) Smaller particle size

(5-40µm) of the PTLC layer results in sharper, distinct separations; (ii) Conditions necessary for development of PTLC may be experimentally determined beforehand by using rapid, analytical TLC. (iii) Separated zones may be easily removed from a

PTLC plate and eluted. The most frequently used immobilized phases are cellulose powder, alumina, silica gel and kieselguhr, many of which are obtainable with a flourescent compound for example, zinc sulphide (Touchstone and Dobbins, 1998;

Pavia et al., 1990; Funniss et al., 1994) .

2.2.5 Structural elucidation of isolated compounds

Among the instrumental methods of structure determination the most prominent of these techniques are ultra-violet (UV-vis), infra-red (IR), proton nuclear magnetic resonance (NMR) and mass spectra (MS) and X-ray crystallography. NMR, IR, and

UV-vis spectroscopy provide complementary and all are useful but among them,

NMR provides the information most directly related to molecular structure (Carey,

1999). The extent of compound characterization is largely dependent on whether the isolate is known or novel compound (Farnsworth, 1966). In NMR spectroscopy, the typical taking in of energy by certain spinning nuclei in a magnetic field when shot by a second and less powerful field at 90o, allows the recognition of atomic

configurations in molecules. Absorption takes place when these nuclei experience transition from one appropriate relative position in the applied field to another one

(Willard et al., 1986). The spectra obtained identify the nuclei, their locations in the molecule, their number, identity of the neighbours and their positions. The tools have metamorphosed the practice of NMR in organic chemistry (Willard et al.,

1986) and therefore for the same reasons this study adopted the technique as the main one due to its availability and cost effectiveness and used MS to confirm structures. MS is used in the identification of unknown compounds, quantification of known compounds and determination of structural and chemical properties of compounds when present in small amounts (10-6-10-8). The technique involves: (i) the production of ions of the materials in sample, (ii) their separation on the basis of their mass: charge (m/e) ratio, and (iii) determination of relative abundance of each ion. MS consists of three components: the source of ion, an analyser, and a detector.

It does not directly measure the molecular mass but detects m/e ratio. Mass is measured in terms of Dalton (Da). One Dalton= 1/12th of mass of a single atom of isotonic carbon (13C+) (Tanaka et al., 1972). A pure organic crystal has a defined sharp melting point range whose range in not supposed to exceed about 0.50oC.

Melting point is therefore a precious measure of purity for an organic compound

(Pavia et al., 1990).

2.3. Ethnomedicine survey

Following an ethnomedicine survey carried out in Kilifi and Homa-Bay Counties among the Chonyi and Luo people under the auspices of WHO funded Maltilateral

Initiative on Malaria (MIM) project number 990096 (Moses et al., 2002), several plants were mentioned by herbalists as having been utilized against malaria (Table

3). Kilifi and Homa-Bay Counties are found in Coast and Nyanza provinces of

Kenya, respectively. They share almost the same climatic conditions due to proximity to Indian Ocean and Lake Victoria, respectively, which favour breeding of mosquitoes and therefore have high incidences of malaria (Figure 1A, B & C).

B C

Figure 1:County Governments". Kenya Law Reform Commission. 29 August 2012

2.3.Reasons for conducting ethnomedicine survey in Kilifi and Homa Bay

2.3.1 Kilifi County

Kilifi is located in the former Coast Province, 60 km north of Mombasa. It shares its borders with Mombasa and Kwale to the south, Tana River to the north, and Taita to the west. The county has six subcounties; namely, Kilifi, Ganze, Malindi, Magarini,

Rabai and Kaloleni. It has 17 divisions, 54 locations, 175 sublocations. The total area is 12,246 km². The population of the county was estimated to be 1,217,892 in

2012 as projected in the Kenya Population and Housing Census 2009, composed of

587,719 males and 630,172 females. The population was projected to rise to

1,336,590 and 1,466,856 in 2015 and 2017, respectively, at growth rate of 3.1 percent per annum.

The average annual rainfall ranges from 300mm in the hinterland to 1,300mm at the coastal belt. Majour occupation is fishing due to its proximity to Indian Ocean. The annual temperatures range between 21oC and 30oC in the coastal belt and between

30oC and 34oC in the hinterland. This is suitable for Mosquito survival and therefore malaria epidemics. Health services include malaria control. Malaria test positivity rate (%) 30.0, malaria cases (per 100,000 people) 21,945 and malaria admission

179,160. Established control measures are no longer working. Resistance against insecticides and antimalarials, with malaria morbidity and mortality rising. There is an increase in the mean age of children admitted with P. falciparum infections from

20.2 months in 1990 to 45.3 months in 2014 (von Seidlein et al., 2016). Though the

Kilifi people of Kenya rely mostly on ethno-medicine to manage human ailments, the indigenous knowledge remains largely undocumented. This study was set to survey, record and report some of the medicinal plant species they use to manage human ailments with particular interest on malaria (Kilifi County Goverment,

(2020) (Figure 1A & B).

2.3.2 Homa-Bay County

It is located in the former Nyanza Province along the south shore of Lake Victoria and its capital and largest town is Homa Bay. The Luo and Basuba who inhabit the area are fishermen. The county has a population of 1,131,950 (2019 census) and an

area of 3,154.7 km². Lake Victoria is a major source of livelihood for its residents.

Administrative Divisions include Asego, Ndhiwa, Nyarongi, Rangwe and Riana.

The climate in Homa Bay is warm and overcast. Over the course of the year, the temperature typically varies from 65°F to 85°F and is rarely below 62°F or above

90°F. Mosquitoes thrive under these conditions. The natural resources include Lake

Victoria, good arable land, game reserves, clean natural beaches, building materials such as sand, rough stones, granite, limestone and permanent rivers.

Malaria test positivity rate (%) 46, cases (per 100,000 people) 58,820 and admission

12,479. The challenges are many in a rural county with only three doctors and 40 nurses per 100,000 persons. For example, in 2015 nearly 59 % of the population had malaria. Forty % of children were not born at health facilities, making it more difficult to track maternal and child health needs. There is therefore improved malaria data and use through Surveillance. Herbal medicines are therefore alternatives to health care in the county (Homa-Bay County Government, (2020)

(Figure 1A & C) whose documentation is lacking. Herbalists from the two counties mentioned numerous plants that were used to treat malaria seven of which were used in this study.

The following seven plants, Achyranthes aspera (Amarathaceae), Heinsia crinita

(Rubiaceae), Bridelia cathartica (Euphorbiaceae), Citrus limon (Rutaceae),

Microglossa pyrifolia (Asteraceae, Compositae), Vernonia glabra (Compositae) and

Carissa edulis (Apocynaceae) were mentioned by the herbalist as being used to treat malaria. They are either used singly or combined in concoction or decoction depending on the severity of illness.

2.3.3 Geographical distributiom and phytochemistry of selected medicinal plants

2.3.3.1 Achyranthes aspera (Amarathaceae)

It is commonly known as Tama Tama (Swahili). It is a perennial or an annual short or long-hairly, herb or weak shrub with ovate lanceolate leaves; flowers reddish when open, 3-7mm long, fruiting flower with spiny sepals and bracheoles, carried away on fur and clothes (Agnew and Agnew, 1994) (Figure 2). It is a very variable plant in habit and colour. This plant is found in all disturbed dry places, or as a field weed, 0-3080m. It is found in the following highlands, Cherengani, Tinderet, Mau

Aberdares, Mount Kenya, Kitale, Mumias, Kisii, Narok, Baringo, Rift Valley,

Magadi, Embu, Machakos, Nairobi and Kajiado (Agnew and Agnew, 1994). The leaves are dried by fire and ground to powder.

In case of ankle sprains, cuts are made with razor and the powder is then applied together with common salt. It is also used for headache. Bleeding stops on chewing the roots and on application on the cuts. Medicine prepared from the roots treats constipation in children and stitch. Venereal diseases are cured by steeping pounded roots in hot water and then drinking the cold extract. The ash of leaves consumed by fire is applied to boils. The plant also protects against evil spirits (Kokwaro, 1993).

Dried aerial parts used for periodic disorders, diabetes, as purgative, abortifacient, antiarthritic, labour inductor, antiasthmatic cough, for snake bite, whooping cough, hydrophobia, rheumatism, intestinal parasites, urinary calculi, boils, pimples, contraceptive, aching back, amenorrhea, chest pain and against plasmodia, viruses, bacteria and fungi. Triterpene and alkaloids have been isolated such as quinolizidine

alkaloid from leaves. Other compounds isolated are alkane, alkene, lipid and saponin (Basu et al., 1957).

Figure 2: A photograph of a specimen of Achiranthes aspera leaves

2.3.3.2 Heinsia crinita (Rubiaceae)

This plant is commonly known as Mfyofyo (Swahili), Mshosho (Giriama), Mushoka

(Duruma) and Dewakiri (Nanya) (Beentjes, 1994). This plant has flowers, which are terminal on branchlets, in dense cymes; corolla with 4-6 lobes, each one ending in a linear acumen. It has fruits with persistent calyx lobes. The leaves are almost glabrous (Beentjes, 1994). It is either a shrub or tree, 1-4.5m (sometimes climbing).

The leaves are shortly narrowing to a slender point, oval or entire, base cuneate and apex acyte, 1.5-4.5 by 0.7-2 cm glabrous except for venation. It has white flowers, which are solitary or in few flowered shortly stalked cymes; corolla tube 18-25mm, long (Beentjes, 1994) (Figure 3). It is found in the K7 area, 1-500m above sea level.

It is found in forest, woodland, bush-land and thickets. Its fruits are edible

(Beentjes, 1994). As a treatment for the injured neck, its ash is massaged into cuts on the neck and shoulders (Kokwaro, 1993). Leaves are used for convulsive fever, against biomphalaria and roots for stomachache in Tanzania, and orally taken for epilepsy and malaria. Decoction is orally-taken for hernia, vomiting and malaria.

Triterpene isolated from the extracts include 3-Beta-o-(alpha-L-triterpene) and quinovic acid isolated from rootbark in Zaire (Bila et al., 1994).

Figure 3: A photograph of a specimen of Heinsia crinite leaves

2.3.3.3 Bridelia cathartica (Euphorbiaceae)

Common name is Mnembe Nembe (Swahili). This plant occurs as a shrub, tree or climber 0.5-7m. The bark is grey brown, fishurred. The leaves are base cuneate to rounded, elliptic to ovate, apex obtuse or subacute, margin shallowly crenate (rarely subentire), 3-8 by 1-4cm, glabrous or pubescent beneath. It has yellow green flowers, in very dense axillary clusters. The fruits are purple or black, round or nearly so, 6-11mm (Figure 4). It is found along the coast of Kenya (K7 area) with an altitude of 1-4.5m. It is found along the forest margins, bush-land and littoral thicket. Its fruit is edible (Mbeentjes, 1994). Boiled root decoction is drunk twice a day for stomach ache (Kokwaro, 1993). The plant has been used to treat rectal prolapse, sterility, asthma, bilharzia, stomachache and as a purgative. It has antitumour, antifungal and antimalarial activity. Inorganic compounds are present together with anthocyanins, quinines and triterpenes (Chhabra et al., 1984).

Figure 4: A photograph of a specimen of Bridelia cathartica leaves

2.3.3.4 Citrus limon (Rutaceae)

Vernacular name is Machunga Mar Ndim (Luo) (Figure 5). It is used for stomachache. Its fruit is a treatment against diabetes, coughs, colds, fever and as an antidysenteric. Leaves are used for leprosy and venereal diseases. Roots are used as an antihelmintic. The plant being indigenous in Asia with possible origin in India is widely planted in Italy, Australia and California. Apart from having an elevated vitamin C content responsible for resistance to infection in man therefore worthy for flu and colds, the plant is a known food source as well as remedy. As well as controlling bacterial and fungal infections, the natural lemon flavor has the following attributes; lowers blood pressure, is diuretic, promotes gluconoride detoxification, improves peripheral circulation, improves digestion and reduces inflammation. Used for indigestion, flatulence, diarrhoea, infant colic, and shock. It prevents many conditions including; arteriosclerosis, stomach infections and circulatory problems. The following have been isolated from fruitpeel: adenosine, alkaloid, apigenin-6-8-DI-C-glucoside, flavone, carbohydrate, coumarin, sesquiterpene (Baldi et al., 1995, Oketch-Rabah, 1996).

Figure 5: A photograph of a specimen of Citrus limon plant

2.3.3.5 Microglossa pyrifolia (Asteraceae, Compositae)

Vernacular name is Nyabung` Odide (Luo). This is an erect woody, sparsely hairy herb with oblanceolate, coarsely or hardly toothed, stalk less leaves and it has loose terminal corymbs of cylindrical heads. It has blue or becoming blue florets, 9-13mm long in volucre, phyllaries with marginal comb-like short fine bristles tips which are straw-coloured and dark green, achenes densely silky-hairly, and hardly ridged,

4mm long outer pappus of shorter bristles (Agnew and Agnew, 1994) (Figure 6).

The plant is common in disturbed grassland and shallow impermeable soils, especially near Thika, 1300-2160m (Agnew and Agnew, 1994). The plant is found in the following areas; Aberdares highlands, Mumias, Kisii, Narok, Embu,

Machakos and Nairobi (Agnew and Agnew, 1994). This plant is known by the following names in various dilects; Mukutu or Uvatha mutheke in Kamba, Mutei in

Kikuyu, Kuombereriet or Kwambereit in Kipsigis, Enguu in Luhya, Engokumati or

Ol-ogomati in Maasai (Beentjes, 1994). It is a woody herb, shrub or scrambler, which is 0.5-6 (12) m. The leaves are ovate, its base is cuneate, occasionally slightly unequal with acute apex, dentate margin which is rarely entire, 4-12 by 1.5-5cm,

pubescent or puberulous. It has white, cream or yellow flowers with 2-3mm long rays. It is found both in central and Western Kenya at altitude between 1200-2900m above sea level (Mbeentjes, 1994; Agnew and Agnew, 1994). It is found along forest edges, riverine forest, grass/bush land, and wasteland. The plant grows naturally in the following areas; Tinderet highlands, Mau highlands, Mumias, Kisii,

Baringo, Embu, Machakos and Nairobi (Beentjes, 1994, Agnew and Agnew, 1994).

Roots are pounded, soaked in water and used against headache and colds. An infusion of the leaves is then taken as a remedy for malaria, but it is bitter and acts as an emetic as well. Crushed leaves are also used against limb fractures and malaria (Kokwaro, 1993). Leaf is used in East Africa against malaria; infused leaves are taken orally. Sesquiterpene has been detected in leaf extract in Papua-New

Guinea (Rucker et al., 1994). (E)-β-farnesene and β-caryophyllene yielded as main constituents during a study on essential oil of leaves.

Figure 6: A photograph of a specimen of Microglossa pyrifolia leaves

2.3.3.6 Vernonia glabra (Compositae)

Vernacular name is Akech Madongo (Luo). An aqueous extract of the leaves is taken orally against hepatic diseases by the Haya in Bukoba (Kokwaro, 1993)

(Figure 7). New vernolepin derivatives and biologically active substance have been

isolated and pharmacological activity has been verified in this plant (Achola et al.,

1996).

Figure 7: A photograph of a specimen of Vernonia glabra leaves

2.3.3.7 Carissa edulis (Apocynaceae)

Vernacular name is Ochuoga (Luo). The plant is known as Mtanda-Mboo (Swahili),

Fonkole, Dagams (Boran), Mutimuli (Bonjun), Molowe, Mulolwe (Duruma),

Dagamsa (Gabora), Mokalakalo, Kaka-mchangani (lwana/Malakote), Mukawa

(kamba), Mukawa (Kikuyu), Olamuriaki (Maasai), Legatetwo (Marakwet/Tugen),

Legetetwa, Legetetwet (Nandi, Kipsigis, Tugen), Lokotetwo (Pokot), Lmuria,

Lmiriel (Samburu), Gurura, (Sanya), Kirumba (Taita) and Ekamuria (Turkana)

(Beentjes, 1994). It is a shrub, occasionally scrambling. It is 1-6 (-14m) and the bark is grey. The spines are simple, rarely forked, 0.5-5.5cm long. The leaves are ovate, elliptic or almost round, base rounded or cuneate, apex obtuse or acute, 1.5-7 by

1.5-4cm, glabrous or pubescent. Its flowers are white inside, pink to red outside, in dense cymes; corolla tube 13-20mm, lobes 4-9m long. It has red to black fruits, which are round or ellipsoid, 6-14mm across, sometimes with a sharp apex

(Beentjes, 1994). The fruits are wholesome, delicious, a bit harsh and occasionally taken as treatment for dysentery; aqueous extract of the roots is taken as a painkiller.

It has almost hairless leaves to 4cm long; flowers white within, red on outside,

15mm long (Figure 8). The plant is found along Elgon highlands, Cherengain highlands, Mau highlands, Loita highlands, Aberdares highlands, Mt. Kenya highland along forest edges, Kitale, Mumias, Kisii, Narok, Baringo, Rift Valley,

Nanyuki, Embu, Machakos, Nairobi, Kajiado, along the Kenyan Coast and also around lake Victoria, bush-land, thicket or bushed grassland, especially in rocky places growing wild along altitudes 1-2550m above sea level (Agnew and Agnew,

1994). It is used for indigestion, lower abdominal pains when one is pregnant and also as food (fruit). The washed roots are decocted and the solution drunk warm in small quantities several times in a day. A drink prepared from the roots together with other plants used as herbal drugs is used against chest pains. An aqueous root extract is also a remedy for malaria and polio symptoms (Kokwaro, 1993). Its root is used in East Africa against malaria and to restore virility in Ghana. Decoction is used for malaria, gonorrhea and psyciatric problems and is also used as antigiardiasis activity in Kenya. Dried leaf is used to control diabetes mellitus in

Egypt and it is also used for schistosomiasis in South Africa. Acetophenone-2- hydroxy benzenoid, acetophenone, ortho-hydroxy benzenoid, carinol sesquiterpene and alkaloid are isolated from rootbark (Achenbach et al., 1985).

Figure 8:A photograph of a specimen of Carissa edulis plant

2.4. Evaluation of safety of crude extracts

2.4.1 Overview

By administering the drug to animals in sub-lethal doses for a length of time it is possible to observe whether it causes any ill effects as indicated by loss of weight, a poor condition of the fur, loss of fur, inability to walk steadily, changes in temperament, etc. A battery of testing procedures is employed when new compounds are screened for there is the possibility of missing unique activity.

Today no study of a new or potential drug is complete without animal testing. By carrying out haematological, biochemical and pathological studies it is possible to detect toxic changes in the liver, kidneys, gastro-intestinal tract, lungs, heart and other organs. The success of the toxicity test depends on the choice of dose. Three dose levels are normally chosen. The low dose should be asymptomatic in toxicity terms, but should be a multiple of that required for pharmacological action while the top dose must be chosen to produce toxicity and the intermediate dose should have symptomatic effect but not lethal (Corwin Hansch, 1990).

Drug induced liver injury (hepatotoxity) is a life threatening and growing problem.

It has become the most frequent cause of liver failure in the US, surpassing all other causes put together, including alcoholic liver disease and viral infections. It is also a major reason for removing recommended drugs from the market (William Lee,

2003). During the pre-clinical and non-clinical phases of new drug processing, toxicologists prevent this by diagnosing liver injury in animals and yet those attempts backfire. Akebia trifoliate caulis which contains aristolochic acid precipitates nephrotoxicity and kidney failure. This herb contains aristolochic acid.

Asarum sieboldii herba cum Radix (Xi-Xin) causes kidney toxicity and kidney failure. This herb also carries aristolochic acid. In 2001 the FDA categorized the two drugs as class 1 toxic substances and commodity recalls wre started (William

Lee, 2003).

2.4.2 Safety screening methods

2.4.2.1 Plasma/serum enzyme changes during drug administration

Different tissues contain a great number of different enzymes in various concentrations. Following tissue injury plasma enzyme levels do not necessarily change in proportion to those in the damaged tissue. However, in some cases, the injuries are associated with changes of specific enzymes in the plasma. For example, myocardial infarction is associated with increased plasma lactate dehydrogenase

(LDH) and aspartate transferase (AST) concentrations (Baron et al., 1989). LDH is widely distributed as would be expected from its involvement in glucose metabolism, being found in all organ cells nonetheless it is principally abundant in cardiac and skeletal muscle, kidney, liver and erythrocytes.

The enzyme catalyses the reaction:

- + - + CH3.CHOH.COO + NAD ↔ CH3.CO.COO + NADH + H

Lactate Pyruvate

In human when physician reckon some kind of tissue damage, in most cases, total

LDH measure may be requested together with other tests as an investigating test.

Due to this determination of individual LDH isoenzyme concentrations can be used together with other tests, to help ascertain the disease or disorder causing tissue damage. LDH is found in virtually all body tissues, so its test is done to discover tissue changes and it assists in the diagnosis of cardiac disease. One’s health professional/practitioner may advise him/her to restrain drugs that may interfere with the test. Drugs that can elevate LDH levels include anaesthetics, aspirin and others (www.healthatoz.com/ healthatoz/Atoz/ency/lactate_dehydrogenase_test.jsp

24k, www.henryford health.org/14366.cfm - 66k).

2.4.2.2 Immunotoxicity

Immunotoxicity refers to any adverse effect on an organism`s normal functioning of the immune system that results from exposure to a chemical substance. Dose- response relationships could be demonstrated between immunoglobulin measurements and internal exposure markers. Immune parameters are the following; immunohistopathology, total white blood cell counts, immunophenotyping of peripheral blood leukocytes, challenge with specific antigen, cell-mediated immunity, natural killer cell activity, phagocytosis, host resistance assays, hypersensitivity and autoimmunity and quantification of total serum immunoglobulin levels. Total serum immunoglobulin (Ig) levels (IgG, IgM, and

IgA) can be quantified in rodents (Bondy and Pestka, 1991) and in nonhuman primates (Tryphonas, 2005) using the enzyme-linked immunosorbent assay

(ELISA). However, the determination of total serum Ig levels in experimental animals has not proven useful, since pronounced effects on immune function are required before significant changes in total serum Ig levels can be observed. In single radial immunodiffusion (SRID) use is made of standard antigen concentrations calibration curves constructed to determine unknown concentrations of antigens (Rabbit IgG, IgM and IgA) used to immunize goats (Hudson and Hay,

1979).

2.4.2.3 Chemical pathology

Chemical pathology is the study of changes in the body that occur following administration of a chemical or a drug. These include changes in the normal physiology and biochemistry. When the normal values are altered, this is interpreted to mean there is injury on some tissues of the body leading to changes in specific enzymes, blood sugar and products of immune cells (Baron et al., 1989). The degree of alteration of the normal body parameters caused is used to assess the relative safety of the chemical or drug. It is a standard scientific requirement that the safety assessment tests are done in animals first before recommending administration of the test substance in human beings.

2.4.2.4 Blood sugar

The application of the Supreme Test Strips is for the determination of blood glucose in capillary blood. The test principle is specific for ß-glucose and is a glucose oxidase/peroxidase based reaction. The results obtained equate to capillary whole blood glucose values. Measuring range-visual readings with colour comparison chart 2.2-27.7 mmol/l (40-500 mg/dl). The Supreme strip can be used with Supreme

Plus, Extra and Petit meters. The target area of each Supreme test strip contains reactive ingredients in the following approximate proportions; Glucose oxidase-33.4

µg, peroxidase-0.75 μg & tolidine 8.5 μg (HYPOguaRD, 2005).

CHAPTER THREE

MATERIALS AND METHODS

3.1. Materials

3.1.1 Test organisms

(a) Plasmodium falciparum strains: (i) V1/S, a multidrug resistant strain, (ii) W2- multidrug resistant strain and (iii) D6, CQ sensitive strain

(b) brine shrimp (Artemia salina) eggs, and (c) male Swiss white rabbits.

3.1.2 Medicinal plants (Test articles)

Achyranthes aspera L., (ii) Heinsia crinita (Afz.)G,Tayl, (iii) Bridelia cathartica

Bertol.f., (iv) Citrus limon L., (v) Microglossa pyrifolia (Lam.) O. Kutze, (vi)

Vernonia glabra (Steetz) Vatke and (vii) Carissa edulis (Old name) changed to

Carissa spinarum L.

3.1.3 Reagents

Chloroform, paraffin wax, agarose, coomassie blue, PBS pH 7.2, acetic acid,

formalin, RPMI 1640, ethanol, DMSO, DCM, methanol, giemsa stain, immersion

oil, hexane, double distilled water, CPD-adenine buffer, HEPES 25mmol/μl, sodium

bicarbonate, Rhesus +ve normal human serum, liquid nitrogen, gas (CO2 3%, O2 5%

and N2 95%), hypoxanthine solution, HYPOguaRD & ABO blood, haematoxylin

and eosin stains.

3.1.4 Equipments

Rabbit cages, syringes, needles, culture flasks-250 ml, culture microtitre plates,

syringe filters, microscope slides, coverslips, microscope Axioskope 40 light

microscope, axio-imager zimicroscope (100X), clean sterile sacks,

“Muharata”(hammer grinding machine), mettler weighing balance, safety hood,

freeze drier, NMR machine, IR machine, MS machine, incubator, micropipettes,

heater, Mesh II harvester, liquid scintillator, GRAFIT 3.0 computer, brine shrimp

hatchery box, IBM computer (McLaughlin et al., 1991, probit software, knives,

microtome, scapel blades, QBC II (Clay Adams), QBC blood tubes, glass plates for

SRID & water bath and staining dishes.

3.2.0 Methods

3.2.1 Preparation of in vitro Plamodium test

Plasmodium falciparum standard laboratory-adapted field reference isolates V1S, W2 and D6 were used in this study. The strains were graciously provided to the study by

KEMRI where they are stored as reference stabilate. The viabilities were confirmed before the antiplasmodial test was conducted on them. However, crude plant extracts ere tested against VIS (Table 9) while isolated compounds were tested against strains

W2 and D6 (Table 18). Viability and parasitemia of cultured parasites were calculated by light microscopy analysis of blood smear stained with Giemsa (5000 erythrocytes counted per blood smear) as follows: thin smears were made from the donated parasitized erythrocytes. Thin air-dried blood smears were prepared on glassslides, fixed with methanol for 30 s, and stained with Giemsa’s staining solution for a minimum of 10 min. Parasitemia (percentage of parasite infected-erythrocytes) was determined by counting 5000 erythrocytes using the Axioskop 40 lightmicroscope equipped with a 100X oil-immersion objective (Zeiss, Jena,

Germany). Multiply infected cells were counted as one. Microscopic pictures were taken with an Axio Imager Z1microscope (100 X oil-immersion objective) using

Axio Vision software (Zeiss).

Seven Kenyan medicinal plants were authenticated by botanist from the University of Nairobi (UoN) as a way of confirming their botanical identity before they were collected from Kilifi and Homa Bay Counties. Specimens of these plant parts were then collected, kept individually in well labeled clean and sterile sacks in which they were transported to the laboratory for processing. A voucher specimen of each plant part was deposited at the University of Nairobi`s herbarium (Table 3).

Table 3:List of plants collected and their characteristics Plant Botanical Voucher No. Common/Vernacular Name Name Achyranthes aspera SG2OO1/01 T Tama Tama (Swahili), Prickly Chaff L. flower, Devil`s horsewhip,Sanskrit, Apamarga ama Tama (Swahili), Prickly Chaff flower, Devil`s horsewhip,Sanskrit, Apamarga Heinsia crinita SG2OO1/02 Mfyofyo (Swahili), Mshosho (Afz.)G,Tayl, (Giriama), Mushoka (Duruma) and Dewakiri (Nanya), Bush apple, Jasmine-gardenia, Small false gardenia Bridelia cathartica SG2OO1/03 Mnembe Nembe (Swahili), Blue Bertol.f. sweetberry Citrus limon L SG2OO1/04 Malimau (Swahili) Machunga Mar Ndim (Luo), Lemon Microglossa pyrifolia SG2OO1/05 Nyabung` Odide (Luo) (Lam.) O. Kutze Vernonia glabra SG2OO1/06 Akech Madongo (Luo), Cornflower (Steetz) Vatke vernonia Carissa edulis (Old SG2OO1/07 Ochuoga (Luo) Mtanda-Mboo name) changed to (Swahili) Fonkole, Dagams (Boran), Carissa spinarum L. Mutimuli (Bonjun), Molowe, Mulolwe (Duruma), Dagamsa (Gabora), Mokalakalo, Kaka-mchangani (lwana/Malakote), Mukawa (kamba), Mukawa (Kikuyu), Olamuriaki (Maasai), Legatetwo (Marakwet/Tugen), Legetetwa, Legetetwet (Nandi, Kipsigis, Tugen), Lokotetwo (Pokot), Lmuria, Lmiriel (Samburu), Gurura, (Sanya), Kirumba (Taita) and Ekamuria (Turkana), Egyptian Carissa , Carandas plum, Karaunda (India) For the purpose of this study, the plants were examined indivindually.

3.2.2 Preparation of the plant extracts

Upon collection from the field the plant cuttings were chopped, dried at room temperature (about 300C) in the presence of air for two weeks for adequate drying of roots and then pulverized by "Muharata" (Hammer grinding machine made locally by Kariobangi Light Industries) to powder before storage at room temperature. The dry pulverized powder, approximately 3000g, was divided into two portions, a small one of 100g and a larger portion of (2900g). The larger portion was used to prepare organic extracts by cold percolation for 24 hours with the following purified solvents: n-hexane, dichloromethane and methanol sequentially. The filtrates from each extraction were concentrated in vacuo. After each extraction, the plant material was spread out in the hood and the solvent allowed to evaporate before extraction with the next solvent. The 100g portion of plant material was used to prepare an aqueous extract by boiling in water for 2 hours. The filtrate from the water extract was freeze dried to powder. All dried extracts were stored at 40C. Aqueous and organic extracts were subjected to bioactivity guided fractionation using the in vitro antiplasmodial activity screening method.

The most active crude extract against P. faciparum was tested for its toxicity in adult Swiss white male rabbits. Pure compounds were isolated from the most active crude plant extracts against the Plasmodium using bioactivity-guided fraction. The isolated compounds were then subjected to Nuclear Magnetic Resonance (NMR),

Infra-Red (IR) and Mass Spectroscopy (MS) for structure elucidation and then to in vitro efficacy assays against P. falciparum as shown in scheme 1.

Scheme 1: Screening for antiplasmodial activity

3.2.3 Preparation of parasite culture system

Antiplasmodial test was carried out according to a revised method described by

Desjardins et al., (1979). Plasmodium falciparum laboratory reference isolate; V1/S an international multidrug resistant strain originally from a patient in Vietnam, W2- multidrug resistant strain and D6, CQ sensitive strain

were used. Non-parasitised human O +ve red blood cells (RBC) (12-28 days old)

were infected with the malaria parasites and cultured. Fifty (50) % RBC was

prepared according to Watkins et al., (1987). The O +ve red blood cells in plasma

was collected into 20 ml vacutainers containing citrate phosphate dextrose (CPD)-

adenine buffer and was stored at 40C for 24 hours; it was viable for 3 weeks before

use. Erythrocytes were prepared for use by washing three times in WM (RPMI 1640

containing HEPES (5.94g/L), and sodium bicarbonate (7.5%, 31ml/L)). The

supernatant and the buffy coat containing WBC were removed after each wash.

After the final wash the RBCs were suspended in WM 50% (v/v) which would also be used in parasite cultures. Parasites stored under liquid nitrogen were rapidly thawed at 370C and the isotonicity reconstituted as described by Watkins et al.,

(1987). One ml of complete medium with serum (CMS) containing ten percent normal human serum which has been pooled and heat inactivated, Rhesus +ve,

(NHS) in RPM1 1640 containing HEPES buffer 25mmol/µl and sodium bicarbonate

25 mmol/l was added to the culture, homogenized spun and the supernatant removed.

Fifty percent erythrocytes and CMs were added to the cells and homogenised to produce 6% haematocrit. A mixture of three percent carbon dioxide, five percent oxygen and ninety-five percent nitrogen gas was used to flush the parasites for 2 minutes which were then incubated at 37°C. The supernatant in each flask was renewed after every 24 hours and the cultures mixed by gently rotating the flask on a level surface before re-gassing and re-incubating. Parasitaemia was assessed after every three days on Giemsa stained thin films by counting the parasitised RBC among 10,000 RBC. When the parasitaemia exceeded 2% the culture was diluted to a desired level by adding fresh 50% RBC and CMS, but maintaining the 6% haematocrit. The growth rate (GR) per 48 hours was calculated from the formula

GR= (Pf/Pi) 2/n where Pf = final parasitaemia, and Pi = initial parasitaemia n = number of days in the culture (Chulay et al., 1984). The parasites were considered adapted to the in vitro culture and ready for drug test when they achieved a growth rate of 3 fold or greater in 48 hours.

3.2.4 Preparation of the working drug solutions for the tests

The stock solution of the drug containing 1 mg/20-50 l DMSO was further diluted ten fold to a concentration of 2 mg/ml with medium and was purified by filtering through a 0.22 m filter. Twenty five microlitres (25l) of the working solution was dispensed in duplicates in row B of the test plate and diluted with an equal amount of CMS. A multitipped pippete was used to make two-fold dilutions from one row to the next such that the highest concentration of a drug in row B was x64 that in the last row H. The final concentration after adding 200 l of parasites into the wells was such that row B had a drug concentration of 111.1 g /ml while in row H it was

1.74 g /ml.

3.2.5 Harvesting the malaria parasites

The malaria parasite cultures were incubated for 24 hours and labelled by adding radiolabelled [3H]-hypoxanthine solution per well and plates re-incubated further for

24 hours. The [3H]-hypoxanthine incorporation was measured by liquid scintillation on a Beta counter after drying the filter papers at 60°C for 30minutes.The set up was that each drug concentration was tested in duplicate. The parasites were harvested using a Mesh II harvester on mini mash glass filter (Wittaker M A products) with plenty of distilled water after the second incubation period. The incorporation of

[3H]-hypoxanthine was determined by liquid scintillation counting on a scintillaton counter. The % inhibition was calculated using the formula: % Inhibition = [mean

NTPE-mean DTPE cpm/ mean NTPE-mean NPE] x 100. Where: cpm = count per minute. Mean NTPE = mean cpm for non-treated parasitized erythrocytes, mean

NPE = mean cpm for non-parasitized erythrocytes and mean DTPE = mean cpm for drug treated parasitized erythrocytes.

3.2.6 Calculation of percentage parasite growth inhibition

The percentage inhibition of P. falciparum parasite growth by the drugs was determined using the formula:

Inhibition = [(cpm control-cpm drug)/(cpm control-cpm background)] x 100:

Where: cpm is count per minute; cpm drug = cpm for drug treated parasites; cpm control = cpm for the non-treated parasite; cpm background = cpm for wells containing medium alone (no parasite).

The percentage inhibition data were used to derive the drug concentration causing

3 50% inhibition of [ H]-hypoxanthine incorporation into nucleic acids (IC50). A computer programme (GRAFIT 3.0 designed by Leatherbarrow (1990)) was used where the data was fitted to the equation (a) below. The concentration response curve was plotted with the drug concentration displayed logarithmically on the X- axis and the percentage inhibition on the Y-axis.

Radioimmunoassay equation (a) for the concentration-response curve expressed in terms of IC50 value is thus given as: y = [a/[(1+1/IC50C) x C]] + d where a, is the maximum y range; d, is the background y value; and c, is a slope factor. Where necessary, statistical differences between mean IC50 values were examined by the student’s t-test.

3.2.7. Safety screening

3.2.7.1 Brime shrimp (Artemia salina) lethality test

Since most bioactive plant constituents are toxic at higher doses, a possible approach to developing a useful general bioassay is to screen for plant extracts that are toxic to zoologic systems. For this purpose, the brine shrimp (Artemia salina) lethality test was originally proposed by Meyer et al., 1982). It represents an easy way to detect general bioactivity in plant extracts and is again a handy procedure for tracking the isolation of bioactive constituents. A rectangular plastic double- chambered box with dividing wall and which had 2.3 mm holes (Encia-Italy) was used to hatch brine shrimp eggs from Lake Urmia-Iran. Artificial sea salt water made by dissolving 16g of sea salt in five hundred millilitre of distilled water was used to fill the chamber. Dry yeast (3mg) was added to serve as food for the larvae.

The eggs were sprinkled carefully in the dark compartment while the other compartment was illuminated by natural light through a hole in the lid of the box.

After 48 hours the larvae were collected by using a pipette from the illuminated side to which they moved on hatching due to their phototropism behaviour. They were separated from their shells by the divider wall (Solis et al., 1992).

Dimethylsulphoxide (DMSO) was used as a solvent to dissolve the plant extracts and the drug solution was then diluted with artificial sea salt water so that the

DMSO content did not exceed 0.05%. Ten brine shrimps (Lake Urmia-Iran) were transferred to 1 ml of each plant sample vial containing 125, 250, 500, and 1000

g/ml of plant extract using a Pasteur pipette. The experiment was set in duplicate for each concentration of the drug. The control tube had only sea water and DMSO.

Brine shrimp survivors were enumerated after twenty four hours and the lethality

fifties (LD50 values) were determined by taking average of five assays using a

Finney Probit analysis program on an IBM computer (McLaughlin et al., 1991) or

the ED50 values (µg/ml) calculated using Probit, a computer program (Solis et al.,

1992). The results are shown in Table 5.

3.2.7.2 In vivo toxicity studies

3.2.7.2.1 Experimental design

DCM extract of ground Citrus limon roots was subjected to toxicity studies in adult male rabbits weighing 2 kg which were fed daily on rabbit pellets (UNGA® feeds

Ltd) and tap water ad libitum. The rabbits were chosen for this study for ease of handling during blood collection. The animals were divided into four groups of 5 rabbits each. The fourth group was the control. Hematological parameters such as packed cell volume (PCV), platelets (PLT), granulocytes (neutrophils (NEU), eosinophils (EOS) and basophils (BAS)) and lymphocytes (LYM), and biochemical parameters such as blood glucose (GLU) levels and serum lactate dehydrogenase

(LDH) activity, immunoglobulin such as IgA, IgG and IgM levels, and general health parameters such as weight were determined twice a week for two weeks during which the drug was administered. The administration of the plant extract was as follows: group one rabbits was injected subcutenously with 0.8 g/kg body weight, group two was injected subcutenously with 1.6 g/kg body weight dose, group three was injected subcutenously with 2.4 g/kg body weight while group four were not given any drug except the vehicle (Zhou et al., 2017). The administration of the drug continued on daily basis for 14 days (Louis Maes (2013)). The parameters were monitored six hours after the administration of the drug and twice a week for two

weeks post extract administration. Overall, the health and general well being were observed and recorded on daily basis (Table 4).

Table 4: Parameters used to assess safety of Citrus limon root DCM extract Treatment group Dose (g/kg body Parameters weight) 1 0.8 Body weight Biochemistry: LDH and glucose levels Histology: liver, heart, kidneys, brain, spleen, and pancreas 2 1.6 Body weight Biochemistry: LDH and glucose levels Histology: liver, heart, kidneys, brain, spleen, and pancreas 3 2.4 Body weight Biochemistry: LDH and glucose levels Histology: liver, heart, kidneys, brain, spleen, and pancreas 4 0 Body weight Biochemistry: LDH and glucose levels Histology: liver, heart, kidneys, brain, spleen, and pancreas

3.2.7.2.2 Drug administration and sample collection

3.2.7.2.2.1 Subcutaneous injection (SI)

The crude Citrus limon root DCM extract was dissolved in 0.05 ml 10% analytical grade ethanol in distilled water completely and then injected into the subcutaneous tissues of the rabbits daily for 14 days. The dissolved drug for parenteral injection as a requirement was warmed to rabbit body temperature before administration.

According to Emmanuel B. Thompson, (1985) this volume of ethanol could not have effect the plasma volume as a vehicle on a 2 kg rabbit. This route of injection is frequently used in the laboratory experiments especially in tests for toxicity

(Bantle, 1998; Merina, 2005). Absorption through this route is slow but uniform

(WHO 1993; WHO 2004). A stock of the drug of 3.2 g/kg body weight was prepared.

Body weights of the rabbits and whole blood for blood counts, plasma and serum for biochemistry (plasma glucose, LDH, IgA, IgG and IgM levels) were collected asceptically using syringe and needle as baseline data (day zero) and twice weekly for two weeks (14 days) the period for which the drug was administered.

Throughout the experiment, the animals were observed for any toxic reactions twice daily.

3.2.7.2.2.2 Hematology

Before collecting blood from the marginal ear-vein of the rabbit, the ears were cleaned thoroughly with 70% ethanol analytical grade. Venous blood was drawn with a vacutainer containing anticoagulant ethylenediamine tetraaceticacid disodium salt (EDTA). The blood was then thoroughly mixed with the anticoagulant and analyzed for hematological parameters using the QBC II (Clay Adams®). The Clay

Adams QBC II System is a seven-parameter haematology screening device with the following quantitative values from a centrifuged blood tube: packed cell volume

(PCV), platelet count (PLT), white blood cell count (WBC), granulocyte count (% and number) and lymphocyte-monocyte count (% and number). The QBC II platelet count, white blood cell counts and counts of the granulocyte and lymphocyte/monocyte white cell subpopulations are derived from electro-optical measurements of the packed cell volumes in a specially-designed QBC blood tube.

3.2.7.2.2.3 Biochemistry

Blood glucose levels were determined using the supreme kit (HYPOGuaRD USA

2005). The ears of the rabbits were aseptically prepared using 70% ethanol and 2 ml of blood obtained using a syringe. The blood was applied to the test strip and colour

development matched with the colour comparison chart or the strip was inserted into the reading meter. Serum LDH levels were determined by the standard methods described by Zimmerman and Henry (1989).

3.2.7.2.2.4 Immunology

3.2.7.2.2.4.1 Single radial immunodiffusion (SRID)

This assay was performed according to Mancini et al., 1965.

Briefly the following steps were followed. Agarose was melted in a microwave and transferred to 560C water bath. 240 μl of antisera was each added to 12 ml of agarose at 560C and mixed well. The agarose was carefully layered on to pre-coated glass plate 8x8 cm2 standing on a levelled surface and allowed to set. After the gel had set, use was made of a gel punch to cut 20 wells per plate. The wells were 3mm in diameter and had vertical sides. The agarose plug was removed with a Pasteur pipette attached to a water vacuum pump. Each of the 5 wells on the left hand side was filled with standard solutions of 50, 100, 150, 200 and 250 μg/ml. A measured volume of 10 μl for IgG and 20 μl for the rest was adequate. The remaining 15 wells were filled with the sera from the experimental and control rabbits. The plates were left in a humid chamber to equilibrate.

To measure the precipitin rings, the plates were washed for 24 hours in a few changes of phosphate buffered saline pH 7.2 (PBS) with the aim of removing free protein from the agarose. Good quality, lintfree filter paper was used to cover the plates and then tissue paper was added on top with little weight to dry the gel overnight. The tissue paper and the weight were removed first then the filter paper was damped slightly with distilled water before removing it. The plates were air

dried then stained submerged in the staining mixture (Mancini et al., 1965) in a staining dish for 5 minutes. The staining mixture was prepared as follows: The

Coomassie brilliant blue dye (1.25g) was dissolved in a solution of glacial acetic acid (50ml) and distilled water (185 ml). The plates were stained for five minutes and the same solution without the dye was used for differentiation. The dry, stained plates were placed in a photographic enlarger and the diameter of the precipitation rings was measured with a ruler.

For the standards the diameters of the rings were measured and plotted on a linear scale against the log of the antigen concentration. The concentrations of both experimental and control immunoglobulins were read from the respective curves.

3.2.7.2.2.5 Dermination of general health of animals

3.2.7.2.2.5.1 Determination of weight

Weights of experimental and control rabbits were determined using Shadow-Graph

(Exact Weight Scale Company, Columbus, Ohio), a weighing balance with a wide maximum capacity range. The rabbits were weighed as follows: a rabbit was removed from its holding cage one at a time. The animal was carefully placed on the weighing balance. Weights were removed or added from the scale until balance was achieved. Weight determination of the animal was done by adding up the total numbers on the balance in designated metric units.

3.2.7.3 Statistical analysis

In this study, the collected biochemical, hematological, immunological and health data was initially recorded in the laboratory notebook. This was then entered into the

excel spreadsheet, cleaned for errors and exported into SPSS software. The descriptive statatistics including mean, SEM, median, mode, variance, standard deviation, range, minimum and maximum were generated. Statistical significance within and between means of the measured parameters including weight, packed cell volume (PCV), granulocytes (neutrophils, eosinophils and basophils) (GRAN), lymphocytes (LYM), platelets (PLT), immunoglobulins IgA, IgM, and IgG, lactate dehydrogenase (LDH), and glucose (GLU) at the doses of 0.8, 1.6, and 2.4 g/kg body weight, and control at at time point 14 days were compared using ANOVA followed by Tukeys post ANOVA. An adjusted p-value of less than 0.00833 was considered significant.

3.2.7.4 Histology

3.2.7.4.1 Tissue harvesting

Tissues (brain, heart, liver, spleen and kidney) were harvested immediately after euthanasia to prevent postmortem autolysis and decomposition as changes occur in tissues within minutes of death. More organs and tissues were collected than needed even when the study was limited to just one organ system. Just one tissue was submited for histology and the rest were kept in 10 % formalin indefinitely, just in case the need arose to look at other tissues. One tissue for each organ was trimmed for fixation and the others saved. The fixative, 10 percent formalin, was prepared by dissolving 8.5 g of sodium chloride in 900 ml of distilled water and mixing with 100 ml of 40 percent formaldehyde. The trimmed tissue was fixed in 10 percent formalin using 10 to 20 times its volume.

3.2.7.4.2 Histological tissue embedding, sectioning and staining

Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mould. Since the tissue blocks are very thin in thickness they need a supporting medium in which the tissue blocks are embedded. This supporting medium is called embedding medium. In this process tissue was dehydrated through a series of graded ethanol baths to displace the water, and then infiltrated with wax. The infiltrated tissues were then embedded into wax blocks. Once the tissue was embedded, it was stable for many years. The tissues were then cut or sectioned in the microtome at thicknesses varying from 2 to 50 μm.

Obtained sections from the same site of the same tissue were processed and stained by hematoxylin and eosin. These sections were then microscopically examined using X100 objective and photographs were taken.

3.3.0 Isolation and characterization of active chemicals/compounds from plants

3.3.1 Isolation of compounds

3.3.1.1 Procedure for column chromatography fractionation

Slurry was prepared by mixing a known amount of silica gel with a solvent. The column (80 cm long & 5.5cm diameter) was filled about half-full with solvent and the stopcock was opened to allow solvent to drain slowly into a large beaker. The column was packed with silica gel slurry 60 (0.063-0.2mm/70-230 mesh ASTM for column chromatography-Macherey Nagel-Germany) to a height of 70 cm. The void volume of the column was calculated as follows: the radius of the column (27.5 mm) squared multiplied by pi (3.1416) multiplied by the column length (800 mm), and the resulting volume was divided by 1000 [corrected formula for units]. This afforded the 1900.668 mL. The bed volume (L) was calculated as follows: bed

height (70 cm) x column crossectional area (πr2h) (cm2) / 1000 =. 3.1416 x 102 x

700/1000 = 1663.0845 cm2. Semipurified biologically active extracts, obtained from solvent fractionation were dissolved in a minimum solvent and added to the top of the column to form a layer on top of the adsorbent. Care was taken not to exceed the recommended solute loading capacity for silica gel. The sample was drained into the adsorbent until the top surface just begun to dry. Solvent elution was carried out starting with the solvent in which the sample was extracted until the first fractions were obtained then polarily of the solvent was increased with the addition of the more polar solvents. Different components of the sample charge passed through the column at different rates depending on their individual adsorption coefficients.

These fractions eluted from the column were collected and concentrated.

3.3.1.2 Procedure for TLC monitoring of column effluent

Eluted and concentrated fractions from the previous section were monitored by

TLC. Fractions with similar TLC profiles were combined dried, weighed and were tested for biological activity against plasmodium parasites before proceeding to the next purification procedure which was either gel filtration through sephadex LH-20, preparative TLC or a combination of both. Commercial grade solvents were redistilled before use. The fractions from the column were monitored as follows:

Analytical TLC plate coated with aluminium (CAMAG Ltd) was spotted with column effluents using clean glass capillary tubes. Solvent system was made and poured into a developing chamber. The plate was placed into the tank containing developing solvent with the sample side resting at the bottom of the tank. Care was taken that the spots did not touch the solvent. The chromatogram was developed to the full distance (6cm). They were developed 6 times. Different solvent mixtures

were used on various extracts. After the chromatogram was properly dried, the spots were located as follows: (i) coloured zones were readily located, (ii) some were visualized under UV light, (iii) the chromatogram was stained in iodine vapour, and

(iv) the chromatrogram was sprayed with 5% sulphuric acid in methanol then heated at 1500C for 5 minutes. Only fraction from the adsorption column with the highest activity was purified further in the next section.

3.3.1.3 Procedure for preparative thin layer chromatography

Twenty grams of silica gel powder 60 PF254 was transferred into 500 ml conical flask and then thoroughly mixed with 50 ml distilled water to make homogeneous slurry. The slurry was carefully poured on a scrapulously clean 20x20cm glass plate and then spread evenly to cover the whole plate. The plate was left to dry at room temperature in a dust free environment overnight. The plate was then reactivated at

1100C for 45 minutes in an Oven. Twenty to 100 mg of sample dissolved in appropriate solvent was carefully streaked 1 cm from the end of the plate, it was left to dry and then visualized using ultraviolet (UV) light source 254nm and 366nm

CAMAG limited. The plate was developed, dried and then viewed under UV light for UV active compounds. These were marked on the plate. The bands with codes such as A, B, C and D starting from the top of the plate were scrapped off and kept separately. The samples were separated from the gel using filter paper, Buchner funnel and solvent. Any pure compound which gave a weight of 5 mg and above was subjected to 1H and 13C Nuclear Magnetic Resonance (NMR) and Mass Spectra

(MS) structure elucidation (Tables 15 and 16). Fractions from PTLC with the highest activity were subjected to NMR analysis.

3.3.1.4 Bioactivity guided isolation of compounds from various plant extracts

3.3.1.4.1 Isolation of compounds from DCM root extract of C. limon (CLR 2)

Crude DCM root extract of Citrus limon (CLR-2) showed highest activity against both P. falciparum and brine shrimp and therefore was subjected to bioactivity- guided fractionation. The DCM extract (30g) was packed in hexane, adsorbed on

25g Silica gel using DCM and then extracted sequentially through a silica gel column with hexane, hexane/DCM mixtures, DCM, methanol, ethylacetate and finally acetic acid. Elution profile of plant C. limon root extract using hexane and

DCM as solvents is shown in Table 10. The extract obtained from hexane/DCM mixtures yielded fractions 15 fractions from which fractions F8-15 were further purified as illustrated in scheme 3 below as up to 100% inhibition of growth in P. falciparum was seen with these fractions. The column was eluted with increasing percentage of DCM in hexane and ethylacetate. Heavy crystals that settled without centrifugation were obtained from fractions 12 and 13 as these samples were stored at -200C. The two fractions (12 & 13) after crystalising out at -200C therefore apart from being most active against Plasmodium and Brine Shrimp became easy targets for isolation of compounds on crystallizing out. They gave single spots on analytical

TLCs after further purification. The fractions 12 and 13 (Table 10) were further purified according to scheme 2.

3.3.1.4.2 Further purification of CLR-2 fractions 12 and 13 on preparative TLC

DCM Extract of Citrus limon root (CLR)

Bioactivity Fractions 8-15 Guided inhibited Fractionation Solvent system P.falciparum Hexane/Ethyl growth 100% acetate 1:1

Crystalization of Stored in DCM fractions 12 & 13 Analytical TLC 50% & MeoH in the freeezer 50% at -200C

Fractions 12 & 13 Preparative TLC Solvent system found to be one Hexane/Ethyl &the same with acetate 1:1 little contaminants

Recrystalization at -200C Dissolved in MeOH Washing in 100% hexane

Pure compounds CLR-2 F12(a) and CLR-2 F12(b) sent for structure elucidation

Scheme 2: Further purification of CLR-2 compounds on preparative TLC

Fractions F12 and F13 were the most active against P. falciparum and therefore preparative TLC was performed on them. Solvent and development system containing 50% Ethyl acetate in hexane gave the best separation of the two fractions on analytical TLC plates (Scheme 2). Sixty milligrams of fractions F12 and F13 were subjected to preparative TLC using hexane 1:1 ethylacetate as solvent system.

Both F12 and F13 yielded two major compounds named CLR-2 F12 (a) and CLR-2

F12 (b) though with traces of contaminants. Both were recrystalised but now in methanol at -20 0C then washed several times with cold hexane in which the compound did not dissolve in except the contaminants (Sheme 2). This resulted in two pure compounds as seen on analytical TLC plate. The structures of the compounds were determined using the following methods: 1H, 13C NMR and MS

and the respective spectra were compared with what were available in the literature

(Figures 10 - 14).

3.3.1.5 Isolation of compounds from Bridelia cathartica methanolic leaf extract

(BCL-3)

The methanolic crude leaf extract of Bridelia cathartica, which displayed good in vitro antiplasmodial activity (15 μg/ml), was adsorbed on silica gel in methanol and eluted with increasing concentrations of DCM on a gel filtration column (Table 11).

The samples were pooled according to their analytical TLC profiles and then dried.

Four major fractions were obtained which were screened for antiplasmodial activity.

Individual fractions were subjected to antiplasmodial testing and the results are as shown in Table 12. BCL-3 F9 (IC50 13.474 µg/ml) was the most active fraction

(Table 12) and therefore was subjected to further purification which did not give a pure compound.

3.3.1.6 Isolation of compounds from M. pyrifolia hexane extract using adsorption chromatography

Microglossa pyrifolia’s 1,1241g of dried leaves were crushed into powder and then extracted four times with pure hexane. The extract (MPL-1) was tested for in vitro

th antiplasmodial activity to give a mean IC50 of 21.376 μg/ml. MPL-1 ranked 7 overall in terms of activity against P. falciparum. It was subjected to isolation of pure compounds as follows: the weight of the hexane extract was 23.0591g from which 20g was weighed out. This was dissolved in hexane, adsorbed onto 20g of silica (MN Kiesegel 60-MACHEREY-NAGEL) and then dried. The sample was loaded on a gel filtration column (80 cm long, 5.5 cm diameter), packed with silica

gel, eluted using hexane containing increasing amounts of DCM and 100ml fractions were collected (Tables 13 and 14). Fractions 14 to 19 and 39 were oily.

Clear white crystals were obtained as fraction 36 (solvent system: 10% MeOH in

DCM) was being concentrated (BUCHI 110). Fraction 38 also gave white crystals which did not dissolve in methanol. The weights of the concentrates are given in

Table 14. Fraction 1 was the heaviest (2130.1mg) while fraction 40 was the lightest

(10.4 mg) of them all. Fractions F28-F38 had similar analytical TLC profiles and therefore were pooled together and then coded MPL-1F37. This fraction had highest activity against falciparum. The tested combined column effluent coded MPL-1F37 was subjected to further isolation of compounds as shown in the next section.

3.3.1.6.1 Isolation of compound MPL-1F37 (a) (Spinasterol) from hexane leaf extract of Microglossa pyrifolia by crystallization

MPL-1 F37 was then concentrated to 5ml and then kept at -200C overnight for selective crystallization. White crystals formed at the bottom of the glass tube which was spun at 4400 rpm for 5 minutes. The crystals that formed were found to dissolve in DCM and methanol at room temperature. The crystals were dried at

500C. This sample was coded MPL-1F37 (a). Analytical TLC profile of MPL-1F37

(a) developed with 2.5% MeOH in DCM showed that it was a pure compound which gave a single band. This compound was later subjected to antiplasmodial activity and NMR analysis and was proposed to be spinasterol (Figure 14).

3.3.2. Characterization of isolated compounds

3.3.2.1 Gas chromatography examination (GC-analysis)

This was executed on a HP Model 6890A gas chromatograph provided with a

Model 5973 mass selective detector, a split capillary inlet system (split ratio = 1/30), a Model 6890 autosampler. The injection (2 µl) was made at a temperature of

250oC. See Tables 5 and 6 for the compatibility models for compounds CLR 2F12

(a) and CLR 2F12 (b) as given by the MS machine (Figure 15-18).

3.3.2.2 MS instrument control Parameters: ICIPE MSD

C:\MSDCHEM\1\METHODS\DCM 15) Solvent B Washes (PreInj)-0

VOLATILES 35-280 XTD 35 16) Solvent B Washes (PostInj)-3

MINUTES.M 17) Solvent B Volume -8 µL

1) Sun Sep 07 18:01:56 2014 18) Sample Washes -0

2) Control Information 19) Sample Wash Volume -8 µL

3) Sample Inlet: GC 20) Sample Pumps -4

4) Injection Source -GC ALS 21) Dwell Time (PreInj) -0 min

Mass Spectrometer-Enabled Oven 22) Dwell Time (PostInj) -0 min

5) Equilibration Time -0.5 min 23) Solvent Wash Draw Speed -300 µL/min

6) Oven Program - On 35°C for 5 min then 24) Solvent Wash Dispense Speed -6000

10°C/min to 280 °C for 5.5 min µL/min

7) Run Time -35 min 25) Sample Wash Draw Speed -300 µL/min

8) Front Injector 26) Sample Wash Dispense Speed -6000

9) Syringe Size -10 µL µL/min

10) Injection Volume -1 µL 27) Injection Dispense Speed -6000 µL/min

11) Injection Repetitions =1 28) Viscosity Delay -7 sec

12) Solvent A Washes (PreInj) -3 29) Sample Depth Disabled

13) Solvent A Washes (PostInj) -3

14) Solvent A Volume -8 µL

Table 5: Compatibility model CLR 2F12 (a)

Name= C:\gcms\1\data\FI2AS SUP CIR2.D 1= PBM Apex minus start of peak [PBM Apex minus start of peak] Time=Fri Sep 12 13:05:44 2014 Header= PK RT Area Pct Library/ID Ref CAS Qual 1= 1 9.4253 0.1429 2-Butenal, 3-methyl- 1388 000107-86-8 53 2= 2 20.9151 0.4953 36.12 Bisabolol 296 023178-88-3 93 3= 3 25.8648 1.0171 4-Nitro-1-naphthol 49193 000605-62-9 72 2H-1-Benzopyran-2-one, 4= 4 26.3576 95.9367 87992 000581-31-7 94 7-methoxy-6-(3-methyl-2-butenyl)- 5= 5 26.9175 0.2552 Benzoyl chloride, 4-hexyl- 74156 050606-95-6 59 6= 6 27.2086 2.15284,4' -Dimethoxy-2,2'-dimethylbiphenyl 86806 046873-19-2 58

Table 6: Compatibility model CLR 2F12 (b)

count=1 Name= C:\gcms\1\data\1.D 1= PBM Apex minus start of peak [PBM Apex minus start of peak] Time= Fri Sep 12 13:08:15 2014 Header= PK RTArea Pct Library/ID Ref CAS Qual 1= 1 20.915 0.7125 36.17 Bisabolol 302 023089-26-1 91 2= 2 23.849 0.1816Cyclodecasiloxane, eicosamethyl- 190220 018772-36-6 91 3= 3 25.1705 98.6696 47.27 Xanthyletin 1406 000523-59-1 50 4= 4 28.1269 0.4362Benzonitrile, m-phenethyl- 62228 034176-91-5 25

3.3.2.3 Determination of melting points

Melting points were measured on a Gallen Kamp® SANYO MPD 350 BM3.5 UK

capillary melting point apparatus at the Chemistry Department Kenyatta University.

CHAPTER FOUR

RESULTS

4.0 Pant extracts

4.1 Yield of plant extracts

The seven plants in this study were extracted with aqueous and organic solvents.

Table 7 gives weights of ground material, weights of their respective organic and aqueous extracts and the percentage yields per plant. Aqueous extracts had the highest percentage yield in each plant. The highest aqueous extracts percentage yield was obtained from the leaf of M. pyrifolia (39.74 g) followed by V. glabra

(25.66 g). The least aqueous extracts percentage yield was obtained from C. edulis root (8.175). Among the organic extracts the highest percentage yield was seen in the methanolic leaf extract of V. glabra (12.203 g) followed by another methanolic leaf extract of M. pyrifolia (10.43 g). Hexane extracts had the least percentage yields followed by DCM extracts (Table 7).

Table 7: Yield of the plants extracts Plant material Powder (g) Solvent Extract weight % yield (g) Achyranthes aspera leaves (AAL) 116 Hexane 0.3 0.259 DCM 0.8 0.69 Methanol 4.3 3.707 Water 1.387 1.196 Bridelia cathartica leaves (BCL) 152.3 Hexane 4.6 3.020 DCM 2.7 1.773 Methanol 10.7 7.026 Water 0.89 0.584 Hensia crinita Leaves (HCL) 92.2 Hexane 0.9 0.976 DCM 0.7 0.759 Methanol 2.7 2.928 Water 1.119 1.214 Citrus limon roots (CLR) 31 Hexane 0.3 0.968 DCM 0.9 2.903 Methanol 1.0 3.225 Water 0.764 2.465 Microglossa pyrifolia leaves 16.3 Hexane 23.059 2.089 (MPL) DCM 18.225 1.651 Methanol 1.7 10.43 Water 3.974 39.74 Vernonia glabra leaves (VGL) 69 Hexane 0.6 0.870 DCM 1.3 1.884 Methanol 7.2 10.435 Water 2.567 3.720 Carrisa edulis root (CER) 159.5 Hexane 1.1 0.690 DCM 0.6 0.376 Methanol 4.5 2.821 Water 1.635 1.025

4.3 Results of the Brine shrimp lethality test

The results of the brine shrimp lethality test are displayed in Table 8. The DCM extract of Achyranthes aspera leaves was the most active against brine shrimps with an LC50 of 0.460 µg/ml. Bridelia cathartica leaf DCM and methanolic extracts had

LD50s of 6.163 µg/mL and 6.197 µg/mL, respectively, against the brine shrimbs.

Both Citrus limon (CL) hexane (˃ 0.00) and DCM (˃ 0.00) root extracts were too active at the concentrations used against the brine shrimbs and the two killed all the

Shrimps depicted by the values. Citrus limon methanolic root extract had LC50 of

2.195 µg/ml. Microglossa pyrifolia (MP) hexane leaf extract had LC50 of 3.389

µg/ml while its DCM leaf extract had LD50 of 3.260 µg/ml. Vernonia glabra (VG) hexane leaf extract had LC50 of 6.087 µg/ml, while its DCM leaf extract was more

active with an LC50 of 2.449 µg/ml; its methanolic leaf extract was the most actve against brine shrimp in this plant with an LD50 of 0.106 µg/ml second to Citrus limon root hexane and DCM extracts. Achyranthes aspera (AA) hexane and methanolic leaf extracts were not active against brine shrimp larva. Bridelia cathartica (BC) hexane leaf extract was inactive against the brine shrimbs.

Methanolic extracts of MPL did not show any activity against brine shrimps (Table

8).

Table 8: LC50s (µg/ml) of crude plant extracts against brine shrimps calculated at 95% confidence interval using probit Drug Hexane (1) DCM (2) Methanol (3) Achyranthes aspera leaves > 1000 0.460 > 500 (AAL) Bridelia cathartica leaves > 500 6.163 6.197 (BCL) Citrus limon roots (CLR) < 0.00 < 0.00 2.195 Microglossa pyrifolia leaves 3.389 3.260 > 500 (MPL) Vernonia glabra leaves 6.087 2.449 0.106 (VGL)

Three plants including B cathartica, C limon and M pyrifolia were further processed using their DCM and methanolic extracts to obtain pure compounds (Tables 11-17).

4.3 In vitro antiplasmodial activity of the plant extracts

The in-vitro antiplasmodial activities of the extracts against V1/S, multidrug resistant strain of P. falciparum were as indicated in Table 9. Results indicate that the most active crude extract against P. falciparum was a DCM root extract of C. limon with an IC50 of 7.017 μg/mL. The second crude extract in in terms of antiplasmodial activity was an aqueous extract of C. edulis roots with an IC50 of 8

μg/mL. The leaves of B. cathartica DCM extract was the third most active crude extract against P. falciparum with an IC50 of 11.537 μg/mL. The statistical differences between mean IC50 values were examined by the student’s t-test (Table

9).

Table 9: In vitro antiplasmodial activity of plants extracts against V1/S Strain

Plant material Solvent IC50s (μg/ml) Achyranthes aspera leaves (AAL) Hexane 18.087 DCM 86.501 Methanol 111.127 Water 38.990 Bridelia cathartica leaves (BCL) Hexane 32.908 DCM 11.537 Methanol 15.647 Water 25.985 Hensia crinita Leaves (HCL) Hexane 34.223 DCM 13.336 Methanol 24.805 Water 47.203 Citrus limon roots (CLR) Hexane 30.092 DCM 7.017 Methanol 916.997 Water 96.860 Microglossa pyrifolia leaves (MPL) Hexane 21.376 DCM 34.88 Methanol 313.647 Water 203.457 Vernonia glabra leaves (VGL) Hexane 427.40 DCM 53.62 Methanol 112.495 Water - Carrisa edulis root (CER) Hexane 193.599 DCM 30.074 Methanol 69.969 Water 8.054 Chloroquine 49.915 ng/ml

4.4 In vivo subacute toxicity test of DCM extract of C limon root in rabbits

The effects of a daily subcutaneous administration of DCM extract of C. limon root at 0.8, 1.6, and 2.4 g/kg body weight to rabbits for 14 days on body weight, packed cell volume, platelets, granulocytes, lymphocytes, immunoglobulins IgA, IgG, and

IgM, lactate dehydrogenase and glucose is presented in Table 10. Results indicate that all the measured parameters of the extract treated rabbits at all the tested doses

were not significantly affected except those of platelets and IgG when compared to those of the control rabbits. The platelet levels for rabbits treated with the DCM extract of C. limon roots at 0.8 and 1.6 g/kg body weight were similarly decreased when compared to that of the control rabbits. Further, the IgG levels for rabbits treated with the DCM extract of C. limon roots at 1.6 and 2.4 g/kg body weight were similarly decreased when compared to those of the normal control rabbits.

Table 10:Effect of subcutaneous administration of DCM root extracts on haematological, immunological and biochemical parameters,in rabbits (N=5)

Treatme Weight(k PCV PLT GRAN LYM IgG IgA IgM LDH GLU nt g) Control 2.450.35a 44.600.2 510109 1.150.7 1.500.50a 13.131.8 0.730.3 0.380.0 8915a 69.508.5a a a a 0 5 8a 0a 7a Extract dose (g/kg body weight) 0.8 2.680.16a 40.661.75a 31724.6b 0.680.24a 1.120.22a 10.500.75a 0.890.1 0.480.0 75.84.9 97.210.2a 1a 2a 5a 1.6 2.680.24a 40.381.51a 30718.1 0.700.3 1.640.12a 12.00.75b 0.890.1 0.600.10a 85.46.5 85.29.07a b a 5 1a 8a 2.4 2.600.21a 42.641.0 59763.1 0.440.1 1.700.2 12.00.75b 0.560.1 0.500.0 80.06.6 106.29.2 a a a 9 0 0a 1a 0a 7a 4a

Results are expressed as mean ± standard deviation. Statistical analysis was carried out using ANOVA followed by post-ANOVA test. ρ < 0.05 was considered significant. Values with the same small case superscript letters are statistically similar. Key: (a) Weight (kg) (b) PCV (%) (c) PLT (x109/L) (d) GRAN (x109/L) (e) LYM (x109/L) (f) IgG, IgA & IgM (mg/L), (g) LDH (U/L), (h) GLU (mmol/L)

4.3 Results on Histology

Subcutaneous administration DCM rot extracts of C. limon to rabbits at 0.8, 1.6 and

2.4 g/kg body weight daily for two weeks demonstrated no observable

histopathological effects on the liver, kidney, heart, brain, and spleen specimen of

the sacrificed experimental rabbits (Figure 9 A1-E2).

Treatment with drug Non-treated COntrol

Brain tissue

A2 A1

Heart tissue

B2 B1

Liver tissue

C1 C2

Spleen

tissue

D1 D2

Kidney

Tissue

E1 E2 Figure 9: A1-E2 Photographs of experimental & control Rabbit tissues compared using x 100 objective

4.6 Results for isolation of compounds

4.6.1 Compounds isolated from the DCM root of C. limon (CLR 2)

The most active fractions DCM root extract of C. limon (CLR-2), crude methanolic

leaf extract of Bridelia cathartica (BCL-3) and the mildly active hexane leaf extract

M. pyrifolia were subjected to fractionation on column chromatography using

solvents of increasing polarity (hexane, hexane/DCM mixtures, DCM,

DCM/methanol mixtures and methanol) on the plant extracts.

4.6.1.1 Isolation of compounds from the DCM root of C. limon (CLR 2)

Sixtteen fractions were obtained (Table 11) and fractions 12 and 13 were active

against P. falciparum. These fractions 12 & 13 were each subjected to preparative

TLC using 50:50 hexane: ethylacetate.

Table 11: Adsorption chromatography of DCM extract of C. limon root CLR Hexane % DCM % Volume of solvent mixture(ml) Fractions 100 0 700 F1 99 1 500 F2 98 2 500 F3 95 5 500 F4 90 10 500 F5 75 25 500 F6 500 first appearance of a red 50 50 F7 fraction 0 100 500 F8 DCM MeOH 99 1 500 F9 95 5 500 deep yellow fraction appeared F10 90 10 500 deep yellow fraction appeared F11 75 25 1000 F12 50 50 1000 F13 0 100 100 F14 Ethylacetate 0 100 1000 F15 Acetic acid 0 100 100 F16

The two fractions gave single spots on analytical TLCs after further purification by preparative TLC and recrystalization in methanol. This yielded two pure coumarin compounds; (Figures 11 and 13).

4.6.2 Compounds isolated from Bridelia cathartica methanolic leaf extract

(BCL-3)

The NMR spectra for compoundsfrom this plant were not clear for structural elucidation (Table 12).

Table 12: Column adsorption results for compounds isolated from Bridelia cathartica Solvent system Quantity (ml) Fraction(s) 100 % DCM 500 1 1.0% MeOH in DCM 3000 2-3 2.5% MeOH in DCM 700 4-7 5% MeOH in DCM 1000 8-10 7 % MeOH in DCM 1000 11-13 9% MeOH in DCM 1500 14-16 15% MeOH in DCM 1000 17-19 30% MeOH in DCM 1000 20-23 50% MeOH in DCM 1000 25-35 70% MeOH in DCM 1300 36-50 80% MeOH in DCM 500 51-59 100% DCM 900 60-72

Fraction BCL-3F9 was the most active against falciparum (13.5 μg/ml) (Table 13).

Table 13: Antimalarial test results for fractions of Bridelia cathartica leaf methanolic extract

Fraction IC50 μg/ml BCL-3F7 29.1 BCL-3F8 19.4 BCL-3F9 13.5 BCL-3F11 29.9

4.6.3 Compounds isolated from M. pyrifolia hexane leaf extract: Adsorption

chromatography

One hundred ml fractions were collected (Tables 14 and 15) and 40 fractions were obtained. Fractions 14 to 19 and 39 were oily (Table 14).

Table 14: Adsorption chromatography results of Microglossa pyrifolia hexane leaf extract Solvent A Amount (ml) Fractions 100% n-hexane 1250 1-7 10% DCM in n-hexane 500 8-10 20% DCM in n-hexane 500 11-13 30% DCM in n-hexane 500 oily 14-16 50% DCM in n-hexane 500 oily 17-19 70% DCM in n-hexane 500 20-23 100% DCM 1500 24-29 1% MeOH in DCM 500 30-32 2% MeOH in DCM 500 33-35 10% MeOH in DCM 500 36-40 (39 oily)

Clear white crystals were obtained as fraction 36 (solvent system: 10% MeOH in DCM) was being concentrated (Table 15).

Table 15: Weights of fractions of Microglossa pyrifolia hexane leaf extract Fraction W Weight(mg) Fraction Weight(mg) MPL-1F1 2130.1 MPL-1F21 288.2 MPL-1F2 1028.4 MPL-1F22 443.7 MPL-1F3 997.1 MPL-1F23 372.2 MPL-1F4 702.9 MPL-1F24 333.3 MPL-1F5 524.4 MPL-1F25 304.1 MPL-1F6 97.3 MPL-1F26 109.4 MPL-IF7 71.4 MPL-1F27 412.5 MPL-1F8 88.0 MPL-1F28 348.5 MPL-1F9 67.6 MPL-1F29 349.1 MPL-1F10 44.5 MPL-1F30 308.2 MPL-1F11 154.2 MPL-1F31 193.3 MPL-1F12 120.1 MPL-1F32 178.3 MPL-1F13 131.0 MPL-1F33 152.1 MPL-1F14 247.6 MPL-1F34 152.3 MPL-1F15 957.1 MPL-1F35 151.8 MPL-1F16 280.9 MPL-1F36 152.2 MPL-1F17 155.0 MPL-1F37 230.0 MPL-1F18 161.7 MPL-1F38 120.0 MPL-1F19 415.3 MPL-1F39 50.1 MPL-1F20 MPL-1F40 10.4

Fraction1 was the heaviest (2130.1mg) while fraction 40 was the lightest (10.4mg) of them all. Fractions F28- F38 had similar analytical TLC profiles and therefore were pooled together and then coded MPL-1F37. The combined column effluent

coded MPL-1F37 was subjected to further isolation of compounds. Spinasterol was obtained (Table 15 & Figure 14).

4.6.4 Structures of isolated pure compounds

The pure compounds that were isolated from the plants were the following; (1)

CLR-2 F12 (a), (2) CLR-2 F12 (b) from DCM root extract of C. limon and (3) MPL

-1F37 (a) from hexane leaf extract of M. Pyrifolia (Figures 11, 13 and 14 respectivly). The first two compounds were very closely related as they were moving together as one and the same on analytical TLC with most solvent developers except when developed with hexane- ethylacetate 1:1 mixture which separated them as two distinct compounds. The structures of the compounds were arrived at after comparing their NMR, IR data with data available in literature

(Figures 10, 11 and 13), and confirmed by MS analysis which gave their molecular weights (Figures 11 and 13) while structural elucidation for Figure 14 was proposed by NMR analysis only.

4.6.4.1 NMR, IR and MS Results for Compound CLR 2F12 (a) (Suberosin)

The structure proposal of HSCCC peak fractions was carried out by 1H-NMR and

13C-NMR (University of Nairobi, Department of Chemistry) and IR (Jomo

Kenyatta University of Agriculture & Technology, Department of Chemistry).

NMR spectra were run on RKCM.07.27.06 360 (1H: 360 MHz; 13C: 212 MHz) spectrometer in CDCl3 using TMS as internal standard or by reference to the solvent signal (CHC3 at δH 7.25. EIMS were obtained at 70 eVona Shimadzu QP-2000 spectrometer. Its IR spectrum exhibited absorptions typical for 7-oxygenated coumarins. The 1H NMR spectrum showed a pair of doublets at d 7.57 and 6.20 (J ¼

9.5 Hz), characteristic of H-4 and H-3 in a coumarin nucleus. The pair of doublets at d 5.70 and 6.86 (J ¼ 10 Hz), beside the singlet at d 1.45 (6 H, s) are typical for the dimethylchromene ring. From the IR and UV it was deduced that 2 is a 7- oxygenated coumarin. The 1 H NMR spectrum showed two doublets at d 6.20 and

7.59 (J ¼ 9.5 Hz), and two singlets at d 6.75 (H-8) and 7.15 (H-5) corresponding to

6,7-disubstituted coumarin The presence of a doublet at d 3.28 (2 H, d, J ¼ 7.5, H-9) coupled with a multiplet at d 5.26 (1 H, m, H-10) and two methyl signals at d 1.68 and 1.74 indicated a prenyl function. The singlet at d 3.87 was attributed to the methoxyl group. The MS showed [Mþ] at m/z 244, a base peak at 229 and a fragmentation pattern similar to that of suberosin. By comparison of the obtained data with those reported for suberosin, compound 2 was identified as suberosin. The

13C NMR spectral data showed 20 carbon signals confirming structure 2. Their assignments were addressed herein for the first time on the basis of several NMR experiments (DEPT, COSY and HETCOR). Compound 3 was identified as xanthyletin by comparison with an authentic sample (.m.p., co-TLC and IR) and with literature data (m.p., IR, UV, MS, 1 H and 13C NMR). The above data therefore prompted proposal of compound CLR 2F12 (a) to be suberosin. Compound CLR

2F12 (b) and xanthyletin had similar infrared spectra and therefore they were identical. For 1H NMR, 13C NMR (Table 15 and Appendices 1A, 2A-D including IR in Appendix 3A). The data in Table 36 prompted proposal of compound CLR 2F12

(a) to be suberosin as they compared well with literature information.

4.6.4.1.1 Physical and spectral data of compound CLR 2 F12 (a) (Suberosin)

Compound CLR2F12 (a) was isolated as the major compound of DCM root extract of C. limon with dazling bluish- violet-whitish appearance under UV light. The

compound appeared as colourless crystals with a melting point of 1190 C and Rf equivalent to 0.8 (50% hexane in ethyl acetate) (Figure. 21). 1H NMR δH 7.61 (d, j=

9. 5Hz,1H, H-4), 7. 58 (d, 1H, H-5), 6. 25 (s ,1H, H-8), 6. 16 (d, j=9. 5Hz, 1H, H-

3,), 6. 76 (t, 1H, H-2’), 3.88 (s, 3H, OCH3), 3. 31 (m. 2H, H- 1’), 1.76 (s, 3H, H-3 ’-

CH3), 1. 69(s, 3H, H-3 ’CH3). 13C NMR (100MHz; CDCl3; ppm) δc 161.39 (C-2),

160. 57 (C- 9), 154. 40 (C- 7), 143. 50 (C-4), 133. 53 (C-3’), 127. 40 (C- 10), 127.

32 (C-5), 121. 28 (C-2’), 112. 68 (C-3), 111. 82 (C- 6), 98. 42 (C-8), 55. 76 (C-7

OCH3), 27. 69 C-1’), 25. 70 (C- 3’ –CH3), 17.65 (C-3’- CH3) (Table 16).

Table 16: 13C (75 MHz) 1H (360 MHz) data of Suberosin (CDCL3, CD3OD, δ in ppm) in Hz Atom Number Carbon -13 1H 1 - - 1’ 27.69 3.31 (m,2H) 2 161.39 - 2’ 121.28 6.76.28 (t, 1H) 3 112.68 6.16 (d, 9.5Hz, 1H) 3’ 133.53 - 4 143.50 7.61 (d, 9.5Hz, 1H) 5 127.32 7.58 (s, 1H) 6 111.82 - 7 154.40 - 8 98.42 6.25 (s, 1H) 9 160.57 - 10 127.40 - C3’- CH3 17.65 1.76 (s, 3H) C3’ – CH3 25.70 1.69 (s, 3H) C-7 – OCH3 55.76 3.88 (s, 3H)

IR spectra were recorded in KBr on a Shimadzu FTIR-8201PC IR spectrometer. The

IR spectra of compound CLR 2F12 (a) corresponded to the IR spectra of suberosin, the IR spectrum of which had a frequency at KBr disk) v = 1693 (c = 0) cm-1. These were consistent with what is reported in literature on suberosin (Appendices 2A-D

& 3A).

4.6.4.2 MS retention time of compound CLR 2F12 (a) (Suberosin)

A single peak was obtained confirming the purity of the compound (Figure 10).

Figure 10: MS retention time of compound CLR- 2F12 (a) (Suberosin)

4.6.4.3 MS Spectra and structure of compound CLR 2F12 (a) (Suberosin)

The above data were in agreement with those for suberosin (Figure 11). The molecular formula of compound CLR 2 F12 (a) was C15H16O3 and therefore its molecular weight was 245. The NMR and IR data, the structure and the molecular weight (245.1) as given by the MS suited that of a coumarin known by the name suberosin. The purity of suberosin was estimated at 98 % with Gas Chromatography

Analysis instrument.

Figure 11: MS spectra, structure and molecular weight of compound CLR 2F12 (a) (Suberosin)

4.6.5 NMR, IR and MS results for compound CLR 2F12(b) (Xanthyletin)

Structural analysis was rooted on NMR data. The 1H NMR spectrum demonstrated two doublet signals at δH 7.57 (1H; J = 9.5 Hz) and 6.20 (1H; J = 9.5 Hz). These signals were due to the presence of hydrogen atoms of conjugated double bond with carbonyl group. Two doublet signals at δH 6.33 (1H; J = 9.9 Hz) and 5.68 (1H; J =

9.9 Hz) were also due to alkenyl hydrogen atoms on vicinal carbon atoms. The singlet signals at δH 7.02 (1H) and 6.70 were assigned to aromatic hydrogen atoms distant from others. The singlet signal at δH 1.45 (6H) was assigned to hydrogen atoms of two methyl groups. 13C NMR spectrum showed signals at δC 156.76,

155.38, 118.41, 112.95, and 77.63 which equated to non-hydrogenated carbon atoms. The signals at δC 143.19, 131.12, 124.65, 120.69, 113.41, and 104.31 equated to single- hydrogenated carbon atoms. The signal at δC 28.25 was assigned to two carbon atoms of the methyl groups. The 1D NMR spectra are typical of pyranocoumarin construction (Steck and Mazurek 1972).

The two hydrogen signal at 7.02 (H-5) was linked to the carbon signals at δC 156.76

(C-7), 155.38 (C-9), 143.19(C-4), and 120.69 (C-6). The hydrogen signal at δH 6.70

(H-8) was linked to the carbon signals at δC 156.76 (C-7), 155.38 (C-9), and 118.41

(C-6). The hydrogen signal at δH 6.34 (H-4’) was linked to carbon signals at δC

156.76 (C-7), 124.66 (C-5), and 77.63 (C-2’). The hydrogen signal at δH 6.20 (H-3) was linked to the carbon signals at δC 161.2 (C-2) and 112.95 (C-10). The hydrogen signal at δH 5.68 (H-3´) was linked to the carbon signals at δC 118.41 (C-6), 77.63

(C-2’), and 28.25 (C-1”/2”). The hydrogen signal at δH 1.45 (H- 1”/2”) was linked to the carbon signals at δC 131.12 (C-3’) and 77.63 (C-2’). These suited xanthyletin

(Lee et al., 2006), a coumarin hitherto separated from Brosimum gaudichaudii

(Okahara 1936). The NMR spectrum of xanthyletin in CdCl3 at 360 MHz (Figure

13) (CLR F12 (b)) showed clearly the presence of two methyl groups at = 1,45 pmm

(singlet), two signals corresponding to two protons which can be attributed to a double bond conjugated to a carbonyl group. Two further doublets (j = 10 Hz) as well as two singlets of one proton each at 6,70 and 7,02 pmm suggested the presence of a dimethyl chromene unit on an aromatic ring possessing two protons in paraposition. The above data strongly favoured as structure coumarin with an annelated dimethyl chromene ring. NMR spectra were run on RKCM.07.26.06

360(1H: 360 MHz; 13C: 212 MHz) spectrometer in CDCl3 employing TMS as internal standard or by remission to the solvent signal (CHC3 at δH 7.25 (Table 16).

IR spectra were secured using KBr disks on a Shimadzu FTIR 8000, [default] FTIR

8400 Japan. The IR spectrum of 2 displayed peaks for an α,β- unsaturated carbonyl group that was reaffirmed and by comparing its physical properties with spectroscopic data (IR, 1H NMR), the substance xanthyletin is reported here (Table

17, Appendices 1B, 2E-H & 3B).

4.6.5.1 Physical and spectral data of compound CLR 2 F12 (b) (Xanthyletin)

Compound CLR2F12(b) was isolated as the major compound of DCM root extract of C. limon with dazling bluish- violet-whitish appearance under UV light. The compound was isolated as yellow white crystals, melting point: 122-124.0 C, Rf =

0.6 (hexane-ethyl acetate (1:1)),230Rf = 0.51 (hexane-ethyl acetate (2:1)). It was soluble in ethyl acetate, chloroform, ethanol, and methanol but not in water (Figure.

13). 1H NMR δH (7.57, d, J=9.5Hz, 1H), 7.02 (s, 1H, H-5), 6. 72 (s, 1H, H-8), 6. 70

(d, 9.9Hz, 2H-4’), 6. 33 (d,J= 9. 5Hz, 1H, H-3) 5. 68 (d, J= 9. 9Hz, 1H, H-3’), 1.45

(s, 6H, H-1’). 13C NMR (100MHz; CDCl3; ppm), 156.76 (C-7), 155.38 2(C-9), 143.

19 (C-4), 131. 12 (C- 3’), 124. 66 (C-5), 120. 69 (C-4’), 118. 41 (C-6), 112.95 (C-

3), 112. 64 (C-10), 104. 31 (C-8), 77. 28 (C-2), 28.25(C-3’ -CH3) (Table 17).

Molecular Formula is C14H12O3 and its Chemical Name is 8, 8 - dimethyl pyro (3, 2 - g) chromen-2-one.

Table 17: 1H (360 MHz) and 13C (360 MHz) data of Xanthyletin (CDCl2, δ in ppm) in Hz Atom Number Ca Carbon -13 1H 1 - - 2 77.28 -- 2’ 77.63 - 3 112.95 6.33 (d, 9.5Hz, 1H) 3’ 131.12 5.68 (d, 9.9Hz, 1H) 4 143.19 7.57 (d, 9.5Hz, 1H) 4’ 120.69 6.70 (d, 9.9Hz, 1H) 5 124.66 7.02(s,1H) 6 118.41 - 7 156.76 - 8 104.31 6.72 (s, 1H) 9 155.38 - 10 112.64 - CH3/CH3 28.25 1.45 (s, 6H)

The structure of xanthyletin (Figure 13) was ascertained by comparison of its

physical data (mp, 1H- and 13C- NMR) (Table 17) with reported values.

4.6.5.2 MS retention time of compound CLR 2F12 (b) (Xanthyletin)

Only one peak was obtained showing that the compound was pure (Figure 12).

Figure 12: MS retention time of compound CLR 2F12 (b)

4.6.5.3 Chemical structure of compound CLR 2F12 (b) (Xanthyletin)

The NMR and IR data, the structure and the molecular weight (228.1) suited that of a coumarin derivative known by the name xanthyletin.The molecular formula was found to be C14H12O3 and therefore formula weight was 228.1 (Figure 13).

O O O

Figure 13: Chemical structure of compound CLR 2F12 (b) (xanthyletin)

4.6.6 NMR data for compound MPL-1F37 (a) (Spinasterol)

The 1H-NMR spectrum of compound MPL-1F37 (a) specified vibrational harmony for free olefinic proton at α 5.16 (dd, J=8, 8,) 15.2 Hz), δ 5.15 (br s), and δ 5.02 (dd,

J=8.4, 15.2 Hz); a carbinyl proton at δ 3.59; and six methyl protons at δ 1.03 (d,

J=6.8 Hz), 0.85 (d, J=6.4 Hz), 0.84 (d, J=6.0 Hz), 0.81 (t, J=7.2 Hz), 0.80 (s), and

0.55 (s). The J-mod 13C-NMR spectral data of MPL-1F37 (a) 9 indicated same vibrational quality for twenty-nine carbons with the following functionalities: four olefinic carbons, seven methine carbons, nine methylene carbons, a carbonyl carbon, two quaternary carbons, and six methyl carbons. These are characteristic resonances of a sterol with an alcohol and two olefinic bonds. NMR Spinasterol:

Semisolid, Identity confirmed by 1H NMR, 13C NMR and co-TLC. Spinasterol eluates when freed of the solvent provided 3, identified by co-TLC, 1H NMR

(Figure 14).

4.6.6.1 Physical and spectral data of compound MPL-1F37 (a) (Spinasterol)

Compound MPL-1F37(a) had the following physical properties; white needle-like crystals. This pure compound was found to be a phytosteroid (Figure 14). 1H NMR

(CDCI3, (ppm), (400 MHz) δ 5.22 (1H, d, j = 7. 2 Hz), δ 3.52 (m), δ 1.03 (3H, s), δ

0.94 (3H, d, j =8. 4 Hz), δ 0.86 (9H, m), δ 0.70 (3H, s); 13C NMR (CDCI3, (ppm),

100 MHz). 7δ 11.8 (C-29), 12.0 (C-18), 18.7 (C-26), 19.0 (C-19), 19.4 (C-21), 19.7

(C-27), 21.1 (C-11), 23.1 (C-28), 24.3 (C-15), 26.1 (C-23), 28.2 (C-16), 29.2 (C-

25), 31.7 (C-7), 31.9 (C-2), 31.9 (C- 22), 34.0 (C-8), 36.1 (C-10), 36.5 (C-20), 37.3

(C-1), 39.8 (C-12), 42.3 (C-4), 42.3 (C-13), 45.8 (C-24), 50.1 (C-9), 56.1 (C-17),

56.8 (C-14), 71.8 (C-3), 121.7 (C-6), 140.8 (C-5) (Figure 14).

Figure 14: Proposed molecular structure of Spinasterol from NMR data (Detailed primary data is shown inAppendices 1C and 2I-L).

4.6.7 Antiplamodial activity of the isolated compounds

Table 18 below summarises the IC50s for the activity of the isolated compounds against falciparum strains. The standard drugs CQ, Mefloquine and Quinine were all more active than isolated compounds.

Table 18: Pharmacological and chemical data of pure compounds and reference drugs IC μg/mL against falciparum strain Compound F Formula 50 W2 D6 1.CLR-2F12(a) Suberosin C15H16O3 26.7 53.1 2.CLR-2F12(b) Xanthyletin C14H12O3 1580.0 ND 3.MPL-1F37(a) Spinasterol ND 43.2 4. STDS (a) CQ C6H13Cl2NO 0.040 0.011 b) Mefloquine C17H16F6N2O 0.012 0.040 © Quinine C20H24N2O2 0.103 0.031 Key: CQ-Chloroquine, ND-Not done, W2-multidrug resistant strain; D6 was CQ sensitive strain of P.faciparum

CHAPTER FIVE

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion

The resistance of P. falciparum to antimalarials and mosquitoes to insecticides, has necessitated search for new compounds against malaria making use of leads from ethnopharmacology studies. To those populace relying on medicinal plants against malaria, it is vitally important that the safety and efficacy of such medicines be determined, reproducible dosage forms be developed and made available for use and their active components determined (Phillipson et al., 1991; Vlietinck et al., 1991).

It is against this strong back ground that this project was undertaken.

Resistance to chloroquine is stated as an IC50 less than 100nM (approximately 0.052

μg/mL) (Basco et al., 1991). However, K39 and V1 strains of falciparum fell far below this cut-off concentration in this study for reasons which could not be explained (IC50 of CQ was 0.040 μg/mL for W2 & 0.011 μg/mL for D6, IC50 of

Mefloquine was 0.012 μg/mL for W2 and 0.040 μg/mL for D6 and IC50 of Quinine was 0.103 μg/mL for W2 & 0.031 μg/mL for D6). Most researchers consider IC50 values above 100 g/mL to be inactive (Basco et al., 1991) and that values ranging between 20-100 g/mL as moderate activity. Researchers have grouped plants with the following activities against malaria parasites as follows; Group A (greater than 1

g/mL), B (1 to 5 g/mL) and C (6 to 10 g/mL) (Basco et al., 1991). This study has come up with two coumarins: suberosin with an IC50 of 26.7 g/mL for W2 and

53.1 g/mL for D6 strains as the most active compound and xanthyletin with an

IC50 of 1580 g/mL for W2 strain both from C. limon DCM root extract and

spinasterol with an IC50 of 43.2 g/mL for D6 from M. pyrifolia leaves with a moderate activity.

Most of the plant extracts in this study except DCM root extract of C. limon with an

IC50 of 7.017 μg/mL, aqueous extract of C. edulis with an IC50 of 8.054 μg/mL and

DCM and methanolic extract of B. cathartica with an IC50 of 11.537 μg/mL and

15.647 μg/mL, respectively, DCM extract of H. crinita with an IC50 of 13.336

μg/mL and hexane extract of A. aspera with an IC50 of 18.087 μg/mL are considered to be within the mild or moderate activity range. Values less than 20 g/mL are considered to be in the high activity range for crude plants extracts.

Citrus aurantiifolia is frequently used against malaria in Brazil (Milliken, 1997) and also M. pyrifolia, also studied here, has been used in Ghana against malaria. The present study also established the presence of some very popular herbal antimalarial plant species in Nyanza and the Coastal region that may not be very popular in other regions. However, though B. carthatica has been used in Zimbabwe against malaria, its crude extracts did not exhibit significant antiplasmodial activity in this study probably because of geographical varieties. The parts utilized by the traditional healers may also not necessarily contain the most active compounds for the choice may depend on the convenience of preparation. Most of the antimalarial concoctions are obtained from roots, leaves and at times the entire plant (Njoroge and

Bussmann, 2006).

None of the crude extracts fell in the 1st or 2nd groups. The DCM extract of C. limon roots with an IC50 of 7.017 g/mL, and an aqueous extract of C. edulis roots with an

IC50 of 8.054 g/mL were the only crude extracts that fell within group C. The rest had lower activities with IC50s greater than 11 g/mL; for example, DCM extract of

BCL had an IC50 of 11.537 g/mL, DCM extract of HCL had an IC50 of 13.336

g/mL, methanolic extract of BCL had an IC50 of 15.647 g/mL and hexane extract of AAL had an IC50 of 18.087 g/mL. The remaining extracts had antimalarial activities above 20 g/mL and up to 916.997 g/mL. Contrary to work by Jurg et al., (1991) who demonstrated that unprocessed aqueous and ethanolic extracts of the root and the ethanolic stem extract of B. cathartica resulted in a 50% growth inhibition of P. falciparum when kept at 0.05 μg/mL, the present study showed that the extracts from this plant were generally active. The difference could have been due to the different localities and therefore different soil textures and climatic conditions. Out of the 28 crude extracts tested, only five had IC50s greater than 100

g/mL and thus 82% were active. This can reflect some accuracy in the part played by the herbalist and the authenticating authority at the University of Nairobi.

The following ranges of IC50s were observed per plant regardless of the chemical used for extraction: AAL with an IC50 of 18.087-111.127 μg/mL, HCL with an IC50 of 13.336-47.203 μg/mL, CLR with an IC50 of 7.017-916.997 μg/mL, MPL with an

IC50 of 21.376-313.647 μg/mL, VGL with an IC50 of 53.62-427.40 μg/mL, CER with an IC50 of 8.054-193.599 μg/mL and BCL with an IC50 of 11.537-32.908

μg/mL. The same plants that showed high activities with IC50s of 7.017-11.537

μg/mL in category C against P. falciparum, had also significant bioactivity against brine shrimp, Artemia salina. It therefore shows that these plant extracts were generally active.

Arrey Tarkang et al., (2014) found a low antimalarial activity of an aqueous extract of C. sinensis. Various workers have claimed C. limon to have the following attributes: antiperiodic, astringent, antibacterial, antiscorbutic, carminative, refrigerant, stimulant, miscellany, rubifacient and stomachic. Lemons being the source of the most active crude extract with an IC50 of 7.017 μg/mL is an extremely good prophylactic medicine for most ailments and has many uses at home. Vitamin

C in which the fruit is rich in aids the body in the fight against infections and again protects or treats scurvy infections (Chopra et al., 1986; Grieve, 1984); it has also been employed as a replacement for quinine against malaria and other fevers

(Grieve, 1984).

Subacute toxicity studies and other repeat-dose studies are done to, determine the nature of toxic events, determine the target organ for toxicity (liver, kidney, etc), establish if a dose-response relationship exists, identify differences in species response, sensitivity between the sexes, investigate accumulative effects, any tolerance and correlate findings with any other known effects. The most important is determination and identification of the target (s) for toxicity and measurement of hematology and clinical chemistry parameters during dosing and observation at autopsy and during histopathological examination of preserved tissues. In this study, the experimental and control animal did not have significant differences in the PVC values. The normal PCV range for the rabbit is between 30 and 45 % (Kaneko et al.,

1997). The PCV fell within this range during and after treatment. This would probably mean that the drug did not affect the erythropoietic system in rabbits.

The functions of platelets are in hemostasis, in maintenance of vascular integrity and also in blood coagulation. Reference value for platelet counts in rabbits is in the range of 300-800 x109/L (Rowan and Fraser, 1982). The various drug levels (0.8 and 1.6 g/kg body weight) had a negative effect on the PLT levels except in level 3

(2.4 g/kg body weight) where PLT shot up at the end of the experiment. Among cell counts, that of PLT is the most inconsistent implying that the drugs might cause bone marrow damage (Lewis et al., 2001). Granulocytes were not affected by the administration of the test crude extract. In the control rabbits, results for lymphocytes did not significantly differ from the experimental groups though there were differences among experimental animals.

The LDH levels were not significantly affected among the different groups. This enzyme is of enormous distribution in mammalian tissues, with high concentrations in the heart, liver, kidney and muscle (Bernard, 1989). Spectrophotometric, fluorimetric, and colorimetric methods of determination have been applied to the assay of this enzyme. In the analysis the optimum pH with pyruvate as substrate is

6.8-7.5 but with lactate used in this study, it is appreciably higher at 9-10. Lactate has the advantage of being more stable than pyruvate and NAD+, which has been used in this study, and is cheaper than NADH. Both rate of reaction and colorimetric techniques have been used. In the former the increase/decrease in extinction of

NADH is read. However, a colorimetric technique using the reaction which was introduced in 1960 and used by Babson and Phillips, (1965) appears to have come increasingly into favour. The method using MTT and PMS with lactate as substrate was used in this study with a few modifications. It may be concluded that the administration of this crude DCM extract of C. limon did not interfere with cardiac,

hepatic and pancreatic cells in any way as the LDH levels were normal during and after treatment. The leakage of LDH from even a small amount of damaged tissue can effectively increase its activity in serum as its tissue concentrations are approximately five hundred times higher than its normal concentration in serum

(Burtis et al.,1999).

The characteristically prolonged periods of elevated LDH values in infarction with an increase in LDH isoenzymes, that is, LDH1 higher than LDH2 yields a pattern that is useful in the laboratory diagnosis of myocardial infarction (Galen, 1975).

High LDH levels are also found in the following drug related conditions, toxic hepatitis, cirrhosis and hepatic necrosis. The drug administered at various levels; 1

(0.8 g/kg body weight), 2 (1.6 g/kg body weight) & 3 (2.4 g/kg body weight) did not have the above listed negative effects and the levels ranged between 80 and 89 U/L which is still within the LDH normal range (94.3±28.8 U/L) (Kaneko et al., 1997) in rabbits.

The increase in the serum glucose might mean ineffectiveness of the pancreas, renal system and cardiac system which might have been caused by the administration of the drug though in some quarters it has been found to support all these organs

(www.ningxiared.org.UK/index). However, the method used in this study to determine glucose gave a range of 69.5-106.2 mg/dL in rabbit which is quite close to the normal range as reported by Kaneko et al., (1997). Pamukcu et al., (2004) while working with listeriosis exposed rabbits, established a normal glucose concentration of 97.5±8.4 mg/dL in control rabbits which compares very well with

97.2±10.2 mg/dL determined in rabbits injected with 0.8 g/kg body weight of the

crude drug in this study. This supports statistical analysis results and histological results which showed that hematological, immunological and biochemical parameters in control and experimental rabbits were not significantly different and that tissues were not affected in the treated animals, respectively.

Work by Miyake et al., (2000) showed that lemon flavonoids or eriocitrin and heparidin taken in the diet are effective antioxidant in-vivo. Several workers have used strips that employ the glucose oxidase test with some variations in their findings but with the majority finding they tend to overestimate glucose levels. It is not clear whether the same applied to these results but even though, the control glucose would not have been different. The strips clearly distinguish between hypo and hyperglycaemia but opinions as to their value in other circumstances vary considerably (Varley et al., 1980). Glucose oxidase is, however, highly specific for

β-D-glucose, and any glucose present in the α-form must be converted to the β-form before reacting. One of the chief advantages of glucose oxidase method, that also made this resourse limited study possible, is its inexpensiveness.

Humoral immunity involves the production of circulating immunoglobulin (IgG,

IgA & IgM) by plasma cells which are derived from lymphocytes. These lymphocytes arise largely from the lymphoid tissue of the gastrointestinal tract and are known as B cells. Immunochemical methods used in this study and radioimmunologic methods allow for specific identification and quantitation of individual globulins. Out of the three immunoglobulins subjected to scrutiny in this study, it is only the IgG levels which were affected by the administration of the drug but not in a dose depedent way. One of the two methods used for determining the

levels is either radial immunodiffusion for most samples, or enzyme-linked immunosorbent assay for very low-level samples. For this study it was the radial immunodiffusion (SRID) test which was used because of its simplicity, accuracy and economic viability. The purpose of the determination was to aid in the diagnosis of immunoglobulin deficiency or increased levels during drug administration as is found in cirrhosis and hepatitis. Since it was only the IgG levels which were affected significantly, it would not be advisable to draw strong conclusions for more information may be required to do so. In a land mark pre-clinical study published in the Journal of American Nutraceutical Association, scientists showed that

Wolfberry (Citrus limon) juice, and Ningxia Red TM, formerly Berry Young are immune boosters. According to a study carried out at a hospital in Beijing in 2002,

Wolfberry was found to stimulate interleukin-2 and/or interferon, the two of which are anti-inflammatory substances in supporting a healthy immune system. A study by Gharagozloo and Ghaderi (2001) concluded that concentrated juice of Citrus aurantifolia possessed principles with immunomodulatory effect which probably was due to the presence of the protein component in the extract.

Weight as a parameter was very significantly increased after administration of crude drug at concentrations of 0.8, 1.6 and 2.4 g/kg body weight. The drug may have induced an increase in appetite. The study shows that there was no toxic effect observed in the subchronic toxicity studies conducted in rabbits. This study is in conformity with the Costus pictus D Don extract study (C) 2006 Pharmainfo.net

Joomla). In general one major observation towards the end of the study was that the general health of the experimental rabbits improved as opposed to the controls.

The kidney was not affected in the drug treated rabbits. Work by Bertelli et al.,

(2002) showed the protective effect of resvertrol in the reduction of ischemia reperfusion of rat nephritic tissue damage both by antioxidant and anti-inflammatory mechanisms. C. limon is also known to have the following attributes: supporting cardiovascular health, protecting and supporting the pancreas and liver and supporting eye health (www.ningxared.org.UK/index). All organs were not affected in this study probably because C. limon has supportive attributes. The isolated coumarins may have medicinal value at least at the administered dosages in the crude DCM extract.

Although Miller Peter (1990) gives an exhaustive list within subacute and chronic toxicity studies; the areas of these observations can be grouped together under the categories (1) mortality, (2) clinical symptoms, (3) body weight, (4) food consumption, (5) water consumption, (6) physical examinations such as (esg), (7) urinalysis, (8) hematology, (9) clinical chemistry, (10) organ weights, (11) gross pathology and (12) histopathology. This study only concentrated on the following categories 1, 2, 3, 8, 9 and 12, thus half the number due to time constraint.

Suberosin, a simple coumarin isolated from C. limon was the most active antiplasmodial (26.7 g/ml  W2 and 53.1 g/ml  D6 strains) whereas xanthyletin

(1580 g/ml  W2 strain) a pyranocoumarin also from the same plant had no activity. Spinasterol from M. pyrifolia leaves with a moderate activity (43.200

g/ml  D6). Köhler, (2002) while working on M. Pyrifolia isolated sinapyl diangelate and acetyl-6E-geranylgeraniol-19-oic acid as new compounds. The most active components in their test system were two diterpenes acetyl-6E

geranylgeraniol-19-oic [IC50, 12.9 μmol/L (PoW), 15.6 μmol/L (Dd2)] and E-phytol

[IC50 8.5 μmol/L (PoW), 11.5 μmol/L (Dd2)]. Other compounds that have been isolated from C. Limon are bergapten, bergamottin, yakangelicin, citropten, imperatorin, isoimperatorin, isopimpinellin, phellopterin, prangol, scoparon, scopoletin, umbelliferone, umbelliprenin and xanthyletin (Jaspreetkaur et al., 2013).

Suberosin is structurally related to drugs such as propranolol, osthol, quinine, chloroquine and primaquine, and it inhibits anti-inflammatory activity and prevents growth of human peripheral blood mononuclear cells by means of modulating the transcription factors NF-Kb and NF-AT (Chen et al., 2007). It is forms yellowish crystals with melting point of 88-89oC (Nurhayat et al., 2016) as established in this present study. It has earlier been also isolated from the root bark of C. nobilis var.

Sunki (Tian-Shung, 1987), the roots of Citrus sinensis (Rutaceae) (Bayet et al.,

2007), Citropsis articulate (Lacroix et al., 2011), Citrus grandis (Che-Ming Teng et al., 1992) and from the bark of Xanthoxylum suberosum (Goodwin and Mercer,

2003). Inhibition of aggregation and ATP release of rabbit platelets induced by arachidonic acid collagen, ADP, platelet activating factor (PAF) or U46619

(athromboxane A analog) was characteristic of all the coumarins except xanthyletin

(Che-Ming et al., 1992).

Xanthyletin is structurally related to a drug named spectinomycin, and was also found to inhibit (100%) fungal growth and was isolated from the DCM extract of a plant known as Pilocarpus riedelianus, two shrubs found in North America, India and Bhutan; Zanthoxylum alatum and Zanthoxylum americanum, Stauranthus perforatus root, S. perforatus roots (Marizeter et al., 2004) and also from the bark of

Xanthoxylum americanum, the roots of X. ailanthoides, the fruit of Luvunga

scandens, the wood of Chloroxylon swietenia, and Citrus aurentifolia (all

Rutaceae). Also it is found in the wood of Brosimum spp. (Moraceae). A closely related compound; xanthoxyletin with 100% inhibition on fungal growth has also been isolated from a methanolic extract of C. limon. It has antitumour and antibacterial activities and an efficient inhibitor of Phytophthora citroohthora in vitro and also its synergistic effect was observed with other phenolics of Citrus

(Akhtar et al., 1985). As per Ojala (2001), existence of coumarins in the roots may protect the plant against microbial intrusion. Seselin, suberosin and xanthoxyletin have been characterized by 1HNMR, 13CNMR, IR and UV spectra as standard methods (de Melo et al., 2009).

Spinasterol, a phytosteroid, isolated from Microglossa pyrifolia leaves for the first time in this study, was the only active principle from this plant (MPL-1F37(a) had an IC50 of 43.169 μg/ml). It was found found to structurally resemble adrenocortical antagonists; hydrocortisone, prenisolone, betamethasone, triamcinolone, 7- dehydrocholesterol and gonadal hormones. It has also been isolated from the stems of the flowers of Cucurbita maxima Duch (Consolacion et al., 2005). Microglassa pyrifolia was documented for use as antiplasmodial remedy by Cameroon’s traditional medicine users. Schmidt et al., (2003) worked on Microglossa pyrifolia and found new dihydrobenzofurans and triterpenoids from roots but not spinasterol.

Work by Jean et al. (2006) indicated that the content of M. pyrifolia leaf oil was predominantly gemacrene D (17.4%), careen (15.3%) or (E)-B-ocimene (13.4%), α- humulene (27.1-36.4%) and α-piriene (18.7%) as opposed to the findings in this study.

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Spinasteryl glucocide and spinasterol have been separated as the main sterols from cell suspension cultures of Saponaria officinalis and determined by 1HNMR, 13C-

NMR, MS spectral data. Phytosterol are soluble in most organic solvents and contain alcohol functional group but are insoluble in water.

5.2 Conclusions

The conclusions associated with this study include:

(i) Hexane extracts of Citrus limon roots (CLR), Microglossa pyrifolia leaves

(MPL), and Vernonia glabra leaves (VGL), dichloromethane (DCM) extracts of

Citrus limon roots (CLR), Achyranthes aspera leaves (AAL), Bridelia cathartica leaves (BCL), Microglossa pyrifolia leaves (PML), and Vernonia glabra leaves

(VGL), and methanolic extracts of Bridelia cathartica leaves (BCL), Citrus limon roots (CLR), and Vernonia glabra leaves (VGL) were active against the brine shrimp, Artemia salina. Dichloromethane (DCM) root extract of Citrus limon roots

(CLR) showed the highest activity in vitro against brine shrimp, Artemia salina based on IC50.

(ii) Dichloromethane (DCM) extract of Citrus limon roots (CLR) (7.017 μg/mL), aqueous extract of Carrisa edulis roots (CER) (8.054 μg/mL), DCM extract of

Bridelia cathartica leaves (BCL) (11.537μg/mL), DCM extract of Hensia crinita leaves (HCL) (13.336 μg/mL), methanolic extract of BCL (15.647 μg/mL), and hexane extract of Achyranthes aspera leaves (AAL) (18.087 μg/mL) demonstrated high antiplasmodial activity based on IC50. Hexane extract of Microglossa pyrifolia leaves (MPL) (21.376 μg/mL), methanolic extract of Hensia crinita leaves (HCL)

(24.805 μg/mL), aqueous extracts of Bridelia cathartica leaves (BCL) (25.985

μg/mL), DCM extract of Carrisa edulis roots (CER) (30.074 μg/mL), hexane extract

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of Citrus limon roots (CLR) (30.092 μg/mL), hexane extract of Bridelia cathartica leaves (BCL) (32.908 μg/mL), hexane extract of Hensia crinita leaves (HCL)

(34.223 μg/mL), DCM extract of Microglossa pyrifolia leaves (MPL) (34.88

μg/mL), aqueous extract of Achyranthes aspera leaves (AAL) (38.99 μg/mL), aqueous extract of Hensia crinita leaves (HCL) (47.203 μg/mL), DCM extract of

Vernonia glabra leaves (VGL) (53.62 μg/mL), methanolic extract of Carrisa edulis roots (CER) (69.969 μg/mL), DCM extract of Achyranthes aspera leaves (AAL)

(86.501 μg/mL), and aqueous extract of Citrus limon roots (CLR) (96.86 μg/mL) demonstrated moderate antiplasmodial activity based on IC50. DCM extract of

Citrus limon roots (CLR) (7.017 μg/mL) demonstrated the highest antiplasmodial activity based on IC50.

(iii) Dichloromethane (DCM) extract of Citrus limon roots (CLR) owes is antiplasmodial activity to the presence of suberosin which together with other compounds synergistically works against Plasmodium falciparum. Further,

Xanthyletin a compound without demonstratable antiplasmodial activity was also isolated from DCM extracts of Citrus limon roots. In addition, Spinasterol, a compound without demonstratable antiplasmodial activity was isolated from the

Microglossa pyrifolia leaves (MPL).

(iv) Dichloromethane (DCM) extract of Citrus limon roots (CLR) subcutenously administered to rabbits for 14 days at a dose of 0.8, 1.6 and 2.4 g/kg body weight appears safe since rabbit weight, hematological, biochemical, and immunological parameters, and histopathology of the studied organs were normal.

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5.3 Recommendations

The observation that all the seven studied plants parts extracts including

Achyranthes aspera leaves, Bridelia cathartica leaves, Microglossa pyrifolia leaves,

Vernonia glabra leaves, Hensia crinita leaves, Carrisa edulis roots, and Citrus limon roots demonstrated high to moderate antiplasmodial activity, supports their continued use as antimalarial drugs in Kilifi and Homabay. The isolation of the antiplasmodial compound suberosin in the the DCM extracts of Citrus limon roots and confirmation of safety of its DCM extract supports the continued safe use of this plant as an antimalarial drug.

5.3.1 Recommendations for further studies

(i) The seven studied plants aqueous and organic extracts which demonstrated high to moderate antiplasmodial activity can further be subjected to toxicity studies to confirm their safety or otherwise in the rabbit model. Further, the compounds contributing to the antiplasmodial activity in these aqueous and organic extracts can be isolated, and characterized and identified using spectroscopic techniques based on bioassays.

(ii) Suberosin, the antiplasmodial compound isolated in DCM extracts of Citrus limon roots in this study should be subjected to in vivo antimalarial activity in rodent models such as infecting mice with Plasmodium berghei and treating the infected mice with varying doses of suberosin to confirm its antimalarial activity. If it has moderate antimalarial activity, a drug that can cure malarial infection can be synthesized modeled upon its structure to confer it with a high in vivo antimalarial activity in addition to conferring it with a high in vitro activity against P. falciparum.

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(iii) More work should be done on active antiplasmodial hexane, aqueous and methanolic extracts of C. limon roots in order to isolate, characterize and identify more compounds.

(iv) In addition, further work for example MS needs to be done on compound MPL-

1 F37 (a) (Spinasterol) to confirm its identity.

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APPENDICES Appendix 1: Compound information

A. CLR 2F12a: Suberosin

Synonyms for suberosin are; 6-Isopentenyl-7- methoxy-2H-1-benzopyran-2-one, 7- methoxy-6-(3-methyl-2-butenyl)-2H-1-benzopyrane-2-one, Methoxy-6-(3- methylbut-2-enyl)chromen-2-one;

B. CLR 2F12b: Xanthyletin

2-dimethylpyrano [3, 2-g] chromen-8-one

C. MPL1 F37: Spinasterol

IUPAC name: (3β, 5α, 22E)-Stigmasta-7, 22-dien-3-ol

Other names α-Spinasterin; Bessisterol; Hitodesterol

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Appendix 2: NMR Spectra for pure compounds A. NMR Spectra for compounds CLR 2F12a: RKCM.07.27 CLR 2F12 (a) L-360 MHz Instrument H-1 in chloroform-d

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B. NMR Spectra for compounds CLR 2F12(a): RKCM.07.26 CLR 2F12 (a) C-212 scans C-13 NMR Spectrum in chloroform-d

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C. NMR Spectra for compounds CLR 2F12a: RKCM.07.26 CLR 2F12 (a) C-212 scans C -13 NMR Spectrum in chloroform-d

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D. NMR SPECTRA FOR COMPOUNDS CLR 2F12A: RKCM.07.27 CLR 2F12(A) L-360 MHZ

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E. NMR Spectra for compounds CLR 2F12a: RKCM.07.27 CLR 2F12(a) L-360 MHz Instrument H-1 in chloroform-d

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F NMR Spectra for compounds CLR 2F12 (b): RKCM.07.26 CLR 2F12(b) L-360 MHz Instrument H-1 in chloroform-d

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G. NMR Spectra for compounds CLR 2F12(b): RKCM.07.26 CLR 2F12(b) L- L- 360 MHz

123 H . NMR Spectra for compounds CLR 2F12 (b): C-212 L-360 MHz Instrument C13NMR in chloroform-d

124 I . NMR Spectra for compounds CLR 2F12 (b): RKCM.07.26 CLR 2F12(b) C-212 scans C-13 NMR Spectrum in chloroform-d

I. NMR spectrum for MPL 1F37(a)

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J. NMR Spectrum for MPL 1F37(a)

K. NMR Spectrum for MPL 1F37(a)

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L. NMR spectrum for MPL 1F37(A)

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Appendix 3: IR spectra

A. IR spectrum for Compounds CLR- 2F12 (a)

B. IR spectrum for Compounds CLR- 2F12(b)