ii

EVALUATION OF PAIN ALLEVIATING, ANTI-INFLAMMATORY AND ANTI-

RHEUMATIC PROPERTIES OF SELECTED KENYAN MEDICINAL PLANTS

A proposal submitted in fulfillment of requirements for Doctor of Philosophy degree of

University of Nairobi, Physiology.

Stanley Nderitu Wambugu, BVM, MSc

Department of Veterinary Anatomy and Physiology

Signature…………………………. Date……………

iii

This proposal has been submitted with our approval as university supervisors.

1. Prof. Titus I. Kanui, PhD.

Signature…………………………... Date…….

Department of Vet. Anatomy and Physiology,

2. Dr. Stephen G. Kiama, PhD.

Signature…………………………... Date…….

Department of Vet. Anat. and Physiology,

Faculty of Veterinary Medicine, U.o.N.

3. Dr. Peter Mbaabu Mathiu, PhD,

Signature…………………………... Date…….

Department of Vet. Anat. and Physiology,

Faculty of Veterinary Medicine, U.o.N.

4. Dr. Daniel W. Gakuya, PhD.

Signature…………………………... Date…….

Department of Clinical Studies,

All of the Faculty of Veterinary Medicine, University of Nairobi

Date of submission…………………………………………………………..

iv

TABLE OF CONTENTS

CHAPTER ONE ............................................................................................................................. 1

1. INTRODUCTION ................................................................................................................... 1

CHAPTER TWO ............................................................................................................................ 2

2. Literature review ...................................................................................................................... 2

2.2 Objectives ....................................................................................................................... 4

2.2.1 Specific objectives: ..................................................................................................... 4

CHAPTER THREE ........................................................................................................................ 5

3.0 MATERIALS AND METHODS ........................................................................................ 5

3.1 Ethnobotanical survey .................................................................................................... 5

3.2 Sample preparation ......................................................................................................... 6

3.3 Evaluation of the anti-nociceptive activity in the Tail-flick test ..................................... 6

3.4 The Evaluation of the Analgesic and Anti-Inflammatory Activity in the Formalin Test 7

3.5 Evaluation of Anti-Arthritic Effect in Adjuvant-Induced Arthritis in Rats ..................... 8

3.6 Induction of RA Rats Using the Collagen Induced Arthritis (CIA) ................................ 9

3.6.1 Macroscopic scoring system: .................................................................................... 10

3.6.2 Hind paw diameter .................................................................................................... 10

3.6.3 Wetting with acetone ................................................................................................. 11

3.6.4 Biochemical assay of pro-inflammatory and anti-inflammatory cytokines .............. 11

3.7 Statistical analysis ......................................................................................................... 12

References ..................................................................................................................................... 12

Budget .............................................................................................................................................. i

Work plan ........................................................................................................................................ ii

Appendix 1: Data acquisition questionnaire .................................................................................. iii

Appendix 2: Histological grading of arthritis ................................................................................ vi

1

CHAPTER ONE

1. INTRODUCTION

Rheumatoid arthritis (RA) is a complex chronic inflammatory condition with a systemic

autoimmune disorder that manifests with pain, inflammation and tissue damage in joints,

together with anemia. The disease has idiopathic etiology and a rather unusual pathogenesis

which makes it difficulty to identify effective therapies (Wood, 2004; Hultqvist et al., 2006;

Lindquist 2002). Osteoarthritis (OA) on the other hand is a degenerative joint disease

characterized by damage to the articular cartilage, changes in subchondral and marginal bone,

synovitis and capsular thickening, usually affecting the weight bearing joints (Bendele, 2001;

Schaller et al., 2005).

Conventionally, the treatments of intense and unrelenting pain, such as that resulting from

rheumatoid arthritis, joint pain, neuropathic pain or injury is based on opioids, non-steroidal anti-

inflammatory drugs (NSAIDs) and drugs inhibiting inflammatory cytokines despite their well

documented side effects and prohibitive cost. On the contrary, natural products offer a more

accessible and cheaper alternative, and they have even contributed enormously to the

development of current therapeutic agents (Setty and Sigal 2005; Lipsy and Tao, 2007).

However, most of the commonly used herbal remedies used by the Kenyan TMP have not been

systematically validated. In addition, there’s a rapid disappearance of traditional herbal

knowledge and increased biodiversity degradation all threatening the loss of our economically

valuable cultural heritage (Srithi et al., 2009). All these calls upon for conscious and concerted

efforts to collect, document and scientifically validate medicinal plants use in our communities.

The commonly used medicinal plant preparations for the management of OA and RA are usually

combined plant extracts, and indeed this may the future direction in the development of

2

efficacious, safe and cost effective herbal remedies world over (Lee 2000; Chrubasik et al.,

2007; Park et al., 2007). These herbal remedies have actually been recognized as effective, safe

and cost effective agents and indeed several herbal based traditional medicinal preparations are

patented as phytopharmaceutical products (Darshan and Doreswamy 2004). In Kenya, several

medicinal preparations are used in the management of osteoarthritis and rheumatoid arthritis and

the objective of this study will be to investigate the analgesic, anti-inflammatory and anti-

rheumatoid properties of selected medicinal plant preparations/extracts in Machakos/Makueni

region of Kenya.

CHAPTER TWO

2. Literature review

Osteoarthritis (OA) and rheumatoid arthritis (RA) are among the most common joint ailments

and a leading cause of irreversible joint deformities and functional impairment (Wood, 2004).

Moreover, these conditions often lead to both long term morbidity and early mortality (Kelly et

al., 1997; Doran et al., 2002) and the economic burdens associated with their management are

devastating. RA affects 1-2% of the population is more common in women than in men while

osteoarthritis is a common problems affecting over 60% of all people by the age of 50 years

(Wood 2004; Schaller et al., 2005; Lawrence et al., 2008).

Over the years, there has been a variety of exciting new therapies for the management of OA and

RA with improved patient responses based on clinical signs and symptoms reported (Wood

2004; Setty and Sigal 2005; Caporali et 2008). However, all these therapies have not been

without devastating side effects, some of which are life threatening but remain in use only by the

terminally ill patients where the safety vs. severity of the condition is the driving force towards

their use. For instance, the most commonly prescribed DMARDs are associated with a range of

3

side effects related to liver and bone marrow toxicity (methotrexate, sulfasalazine, leflunomide,

azathioprine, gold salts, D-penicillamine), renal toxicity (cyclosporine A, parenteral gold salts,

D-penicillamine), pneumonitis (methotrexate), allergic skin reactions (gold compounds,

sulfasalazine), autoimmunity (D-penicillamine, sulfasalazine, minocycline) and infections

(azathioprine, cyclosporine A) (Mikuls, 2003; Fleischman et al., 2004; Woods 2004; Majithia

and Geraci, 2007; Caporali et al., 2008). TNF and interleukin antagonists have been implicated

with serious infections, malignancies, heart failure, chronic demyelinating disorders, lupus-like

autoimmune disease, vasculitis, liver abnormalities and hematological abnormalities (Francis

1999; Mohan et al., 2001; Brown et al., 2002; Kroesen et al., 2003; Wood 2004).

Like many other chronic diseases RA and OA are associated a high usage of herbal medicines

(Resch et al., 1997; Lipsy and Tao, 2007; Setty and Sigal 2005). According to World Heath

Organization statistics, more than 80 percent of the population in developing countries continues

to rely on traditional medicine and indigenous knowledge to meet their health needs (Nanyingi et

al., 2008). Owing to the fact that traditional medicine is accessible, affordable, culturally

acceptable, socially sanctioned, and easy to prepare with little or no side effects, most people

prefer it to the exorbitantly priced health care services conventional medicine and practitioners

offer, not to mention the hazardous side effects of these medications and their limitations in the

domain of holistic health, especially in economically impoverished African societies.

Thus, there is a growing need for safe and effective traditional medicine therapies, an important

tool to increase access to health care particularly in poor and marginalized populations Several

plants species are used in the management of these conditions and but very few have been

systemically investigated for their efficacy. This study hopes to come up with a valid anti-

arthritic and anti-rheumatoid herbal formula.

4

2.2 Objectives

The general objective will be to investigate the analgesic, anti-inflammatory and anti-rheumatoid

properties of selected medicinal plants used by some Kenyan TMP.

2.2.1 Specific objectives:

1. To analyze the diversity of medicinal plants used in the management of RA by different

TMP in the selected localities (Machakos/Makueni area). The entire method of

preparation, extraction and formulation will also be analyzed and documented.

2. To evaluate the pain alleviating properties of the various plants parts/extracts and

preparations using the tail flick and the formalin tests. Analgesic potency will also be

analyzed in the formalin test.

3. To investigate the anti-arthritic properties of selected plant extract/preparation in the

Complete Freund’s Adjuvant (CFA) arthritis model in rats.

4. To evaluate the anti-rheumatoid properties of the plant extract/preparation in the

Collagen-Induced Arthritis model in rats.

5. To evaluate the immunosuppressive effects of the plant extract/preparation in rats.

6. To study the histopathological effects of the antiiarthritic and anti-rheumatoid plant

extract/preparation.

7. Perform separation of the most active anti-arthritic/rheumatoid extracts/preparation to

elucidate on the biochemical composition of the most active ingredients.

5

2.3 Hypothesis

The commonly used herbal medicine preparation and plant extracts have significant pain

relieving, anti-arthritic and anti-rheumatoid properties and are safe for medicinal usage.

2.4 Justification

Joint pain and rheumatoid arthritis are difficulty and expensive conditions to manage especially

in economically impoverished communities where provision of primary healthcare is inexistent

or inadequate. There is also a great demand for new anti-rheumatic agents that are able to act on

chemical mediators of inflammation, but have fewer toxic effects and cost less than the current

treatments that block the actions of TNF-α, IL-1/6 or those of the Disease Modifying Anti-

Rheumatic Drugs (DMARDs) (Majithia and Geraci, 2007).

The therefore aims to identify and verify the efficacy of locally used medicinal plant

formulations used in the management of the two joint conditions. Individual plant extracts and

the medicinal preparation will also be compared for relative potency. The study hopes to come

validate the hypothesis that the commonly used herbal preparation/plant extracts are safe,

cheaper and effective in the management of rheumatoid arthritis and osteoarthritis.

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Ethnobotanical survey

An ethnobotanical survey will be carried out in Machakos and Makueni districts to identify the

plants and procedures involved in the making of the medicinal preparation used in the

management of RA/OA. Field visits will be done and semi-structured questionnaires

6

administered to registered traditional herbal practitioners (TMP)to obtain data regarding the

diversity of plants used, methods of preparation and formulation, diagnosis, dosage, treatment

outcomes and prognostic indicators, recurrence as well as patient satisfaction. A sample

questionnaire is shown on appendix 1. Data obtained from the questionnaire will be analyzed

using the appropriate methods and the commonly used plants collected for taxonomic

identification. Voucher specimens will be deposited at the national museums of Kenya

herbarium/UoN herbarium.

3.2 Sample preparation

The plants of interest will be harvested and dried under the shade. They will be processed into

powder form before extraction, which will be done in accordance to the methods used by the

TMP. For comparative purposes, methanolic, chloroform and metnanolic: diclorolomethanolic

extracts will also be prepared by cold extraction. Basically the dry powdered plant material will

be macerated in the solvent extractor for 3 days at room temperature and the supernatant filtered

in whatman no.1 paper. The residue from the filtration will also be extracted again twice using

the same procedure. The filtrates obtained will be combined and then evaporated to dryness

under reduced pressure to obtain a lyophilized extract. The percent yield will be calculated as per

the equation % yield = Wcrude extract/Wdried plant × 100 where Wcrude extract = weight of

crude extract and Wdried plant = weight of dried plant material (Phrompittayarata et al., 2007).

3.3 Evaluation of the anti-nociceptive activity in the Tail-flick test

The anti-nociceptive activity of the plant extract/preparation will be assessed using the tail flick

test. This is a phasic pain test that measures tail flick responses to radiant heat stimulation, a

spinally integrated nociceptive reflex (Le Bars et al., 2001). The test is simple to perform,

7

displays minimal inter-animal variability and allows for repeated testing without conditioning

effects, making it a useful tool for screening analgesic drugs (Langerman et al., 1995; Le Bars et

al., 2001).

The mice will be kept in cages in a well ventilated drought free house with a 12 hour light-dark

cycle and provided with food and water ad libitum. The test room will be maintained at equal

ambient temperature to that of the animal house. A randomized block design will be used based

on the sex of the animals. The test stimulus will be preset at a temperature that rises from 35ºC to

55ºC in ten seconds. The heating will be terminated manually when a tail flick response occurs.

Tail flick latency will be scored and a cut off time of 40 sec will be adopted. Testing will be done

at -10, 0, 0.5, 1, 2, 3, 4, 5 hours. The average of -10 and 0 (pretreatment scores) will be used as

the base line (control) and test material will be administered at time 0. The extract will be

administered i.p at two predetermined dosages with a saline treated control group. The person

scoring the nocifensive behavior will not be aware of what has been injected into the animals.

3.4 The Evaluation of the Analgesic and Anti-Inflammatory Activity in the Formalin Test

The method will be adapted from that described by Rosland et al (1990). In this tonic pain test, a

20 µL of 0.5% formalin solution will be injected into the dorsal surface of the hind paw in mice.

Scoring will be based on the duration of time spent in nocifensive behavior for 60 min in blocks

of five minutes. In this test, the nocifensive behavior will be defined as licking and biting of the

formalin injected paw. The mean duration of time spent in pain in the first 10 minutes will be

taken as the pain response in the first phase of the formalin test, while the late phase will be

taken as between 20 and 45 minutes after injection of the formalin. The observer will not be

aware of what has been administered in the animals.

8

To evaluate for the antinociceptive potency of the antinociceptive activity of the plant

extracts/preparation, the effects will be compared with those of morphine, aspirin and

dexamethasone. Treatment (with the plant material, vehicle, morphine, naloxone, aspirin and

dexamethasone) will be done i.p at two predetermined dosages 30 min before the formalin

injection. The percentage inhibition of licking/biting will be calculated the formula:

[(C−T)/C] × 100; where C represents the vehicle treated control group value for each phase and

T represents the treated group value for each phase (Dongmo et al., 2005).

3.5 Evaluation of Anti-Arthritic Effect in Adjuvant-Induced Arthritis in Rats

Experimental arthritis will be induced in rats according to the method described by Jia et al

(2003). In this chronic pain model, the stimulus is inflammatory reaction in the joints caused by

the injected material and involves intradermal injection of Mycobacterium butyricum with

Freund’s adjuvant into the tails (Bendele, 2001; Le Bars et al., 2001; Yu et al., 2002). Rat

adjuvant induced arthritis is widely used experimental model of polyarthritis, periarthritis and

periosteitis (Mendele, 2001; Yu et al., 2002; Jia et al., 2003) and the polyarthritis induced is

similar to various inflammatory conditions. Furthermore, results from this test are predictive of

analgesic and anti-inflammatory activity of the test substance (Bendele, 2001; Cho et al., 2008).

The right footpad of each rat will be injected S.Q with 0.1 ml of CFA (Sigma). The plant

material will be administered i.p at three predetermined dose levels, distilled water, and

acetylsalicylic acid at 10 mg/kg will be given daily to the treatment groups, control group, and

reference group, respectively, starting at 8 days after CFA injection for 14 consecutive days. The

edema (paw perimeter) of the left and right hind paws will be evaluated at 2 h, 8, 12, 16, and 24

days post-injection of CFA. Experiments terminated at day 24. The edema inhibition will be

9

calculated as follows: Inhibition rate (%) = Vc - Vt/Vc × 100, where Vc is the edema value of

the control group; and Vt is the edema value of treated group.

3.6 Induction of RA Rats Using the Collagen Induced Arthritis (CIA)

Adult (10-15 wk) male Sprague Dawley rats will be used in these experiments due to their

susceptibility to collagen induced arthritis (CIA), the most commonly used animal models of

rheumatoid arthritis which involves both humoral and cell-mediated immune components

(Trentham, 1982; Cho et al., 2007; Bendele, 2001). The arthritis ensues when rats are immunized

against homologous or heterologous type II collagen, and is characterized by marked bone

destruction associated with deposition of immune complexes on articular surfaces, bone

resorption, periosteal proliferation, synovitis and periarticular inflammation (Mendele 2001;

Maruyama et al., 2006). The rats will be housed in groups of 10 and maintained at a temperature

of 21 ± 2°C on a 12-hour light/dark cycle (7:00 am to 7:00 pm) and provided with food and

water ad libitum.

Collagen II from bovine articular cartilage will be dissolved overnight at 4◦C in 0.1M acetic acid

at a concentration of 2.5mg/mL. The solution will be emulsified with 1.2 times volume of

Complete Freund’s Adjuvant (CFA), and 100 µL of the emulsion administered subcutaneously at

the base of the tail of the rats for immunization on day 1 (see figure 1). Booster injection of 100

µL of the emulsion will be given on day 15. The rats will be given the plant extract/preparation

orally by galvaging at predetermined dosages from day 0 to 21, 5 days per week. Control rats

will be given an equal volume of 2.5% DMSO solution. Their weight and paw diameters will be

measured 2 days each week from day 0 to 39.

10

Fig.1: Schematic diagram of the main activities during the analysis of RA in CIA rats.

Scoring will be done by monitored by assessment of several different parameters at various time

points before and after induction of arthritis.

3.6.1 Macroscopic scoring system:

The extent of paw swelling and edema shall be scored as the method of Paola and Cuzzocrea

(2008), with a score ranging from 0 to 3 (0, no swelling; 1, slight swelling and/or erythema; 2,

pronounced edematous swelling and 3, severe arthritis and joint rigidity). Each limb shall be

graded, thus allowing a maximum score of 12. The arthritic score for each mouse will be the sum

of the scores of all four paws (Paola and Cuzzocrea, 2008).

3.6.2 Hind paw diameter

To investigate the effects of treatments on edema in arthritic joints, tibiotarsal joint

circumferences will be measured using a tape measure and the change in paw size after

treatments expressed as percent change in paw diameter (Chillingworth and Donaldson 2003).

11

3.6.3 Wetting with acetone

This test is used to measure cold allodynia in animal models of pain (Kim et al., 2008). Acetone

will be applied five times on the dorsal surface of the hind paw at intervals of more than 2

minutes at room temperature. Brisk withdrawals will be measured and the data expressed as paw

withdrawal latency (PWL), which is the number of responses divided by number of trials

expressed as a percentage (Kim, 2008). PWL = Number of responses/ Number of trials * 100.

3.6.4 Biochemical assay of pro-inflammatory and anti-inflammatory cytokines

Cytokines, a diverse group of polypeptides that are generally associated with inflammation,

immune activation, and cell differentiation or death, include Interleukins (IL), Interferon’s (IFN),

tumor necrosis factors (TNF), Chemokines and growth factors (Dinarello, 2000; Wong et al.,

2008). Moreover, the balance between the effects of pro-inflammatory and anti-inflammatory

cytokines thought to be the key determinant in the outcomes of an inflammatory reaction as well

as therapeutic efficacies of agents (Wong et al., 2008).in this study, The levels of TNF-ά, IL-1 β

(pro-inflammatory) and INF-ά (anti-inflammatory) will be analyzed from blood samples

collected at different levels of RA (as shown in figure 1) using a rat cytokine ELISA kit (BioPlex

cytokine kit, BD, UK) in accordance with the manufacturers instructions. Rat paw tissue

exudates collected immediately after sacrificing the animals will also be assayed.

3.6.5 Histopathological grading of Rheumatoid Arthritis

This will be based on the three main features of chronic arthritis (Chillingworth and Donaldson

2003; Krenn et al., 2006). These are enlargement of synovial lining, cellular density of synovial

stroma and leukocyte infiltration. Each will be assigned a score of 0-3, with 0 being no change

and 3 the high grade synovitis. The arthritic score for each rat will be the sum of the scores of all

12

four paws. Hind ankle joints will be dissected on day 35, fixed in 10% neutral buffer formalin,

decalcified in 10% formic acid-formalin, and embedded in paraffin. The hind ankle joints will be

sectioned at 4 µm and stained with haematoxylin and eosin and blind scored for arthritis as

shown in appendix 2.

3.7 Statistical analysis

All the data will be expressed as mean ± standard error of the mean (SEM) and statistically

compared using one-way analysis of variance (ANOVA). Post hoc comparisons of the

differences between the drug-treated groups and controls will be performed using Dunnett’s t-

test and P values < 0.05 will be considered significant.

References

Bendele A (2001). Animal models of osteoarthritis. Journal of muscusloskeletal neuron

interactions, 1:363-376.

Brown L, Greene H, Gershon K, Edwards T and Braun M (2002). Tumor necrosis factor

antagonist therapy and lymphoma development: twenty-six cases reported to the Food

and Drug Administration. Arthritis and rheumatism, 46:3151-3158.

Caporali, R, Caprioli M, Bobbio-Pallavicini F and Montecucco C. (2008). DMARDs and

infections in rheumatoid arthritis. Autoimmunity reviews, 8: 139-143.

Chillingworth N and Donaldson L (2003). Characterization of a Freund’s complete adjuvant-

induced model of chronic arthritis in mice. Journal of Neuroscience Methods, 128:

45-52.

Cho Y, Cho M, Min S and Kim H (2007). Type II collagen autoimmunity in mouse model of

human rheumatoid arthritis. Autoimmunity reviews, 7: 65-67.

13

Chrubasik J, Roufogalis B and Chrubasik S (2007). Evidence of effectiveness of herbal

antiinflammatory drugs in the treatment of painful osteoarthritis and chronic low back

pain. Phytotherapy research, 21, 675-683.

Darshan S and Doreswamy R (2004). Patented antiinflammatory plant drug development

from traditional medicine. Phytotherapy research, 18: 343-357.

Dinarello A (2000). Proinflammatory cytokines. Chest, 118: 503-508.

Dongmo B, Nguelefack B and Lacaille-Dubois A (2005). Anti-nociceptive and anti-

inflammatory activities of Acacia pennata wild (Mimosaceae). Journal of

ethnopharmacology, 98: 201-206.

Doran F, Crowson S, Pond R, O'Fallon M and Gabriel E (2002). Frequency of infection in

patients with rheumatoid arthritis compared with controls: a population-based study.

Arthritis and rheumatism, 46: 2287-2293.

Francis S (1999). TNF-alpha and heart failure: the difference between proof of principle and

hypothesis testing. Circulation, 99:3213-3124.

Jia W, Gao W, Cui N and Xiao P (20003). Anti-inflammatory effects of an herbal medicine

(Xuan-Ju agent) on carrageenan- and adjuvant-induced paw edema in rats. Journal of

ethnopharmacology, 89: 729-752.

Kim Y, Kim H, Hwang Y, et al. (2003). Oral administration of proteoglycan isolated from

Phellinus linteus in the prevention and treatment of collagen-induced arthritis in mice.

Biological and Pharmaceutical Bulletin, 26:823-831.

Kroesen S, Widmer F, Tyndall A and Hasler P (2003). Serious bacterial infections in patients

with rheumatoid arthritis under anti-TNF-alpha therapy. Rheumatology, 42:617-621.

14

Langerman I, Zakowski B, Piskown and Grant J (1995). Hot plate versus tail flick:

evaluation of acute tolerance to continuous morphine infusion in the rat model.

Journal of pharmacology and toxicology methods, 34: 23-28.

Lawrence R, Felson D, Helmick C, Arnold L, Choi H, Deyo R et al., (2008). Estimates of

the prevalence of arthritis and other rheumatic conditions in the United States: Part II.

Arthritis and rheumatism, 58, 26-35.

Lee H (2000). Research and future trends in the pharmaceutical development of medicinal

herbs from Chinese medicine. Public Health and Nutrition, 3:515-522.

Lindquist A, Backermann R, Johasson A, Nandakumar K, Johannesson M and Holmadahl R

(2002). Mouse models for rheumatoid arthritis. Trends in genetics, 18, S7-S13.

Lipsky E and Tao L (1997). A potential new treatment for rheumatoid arthritis: thunder god

vine. Seminars in arthritis and rheumatism, 26: 713-723.

Majithia V, Geraci SA (2007). Rheumatoid arthritis: diagnosis and management. American

Journal of Medicine, 120: 936-939.

Maruyama N, Ishibashi H., Hu W, Morufuji S, Inouye S, Yamaguchi H and Abe S (2006).

Suppression of carrageenan- and collagen II-induced inflammation in mice by

geranium oil. Mediators of inflammation, 2006: Article ID 62537, 7 pgs.

Maundu P, Berger J, Ole-Saitabau C, Nasieku J, Kipelian M, Mathenge G, Morimoto Y and

Höft R (2001). Ethnobotany of the Loita Maasai: Towards Community Management

of the Forest of the Lost Child- Experiences from the Loita Ethnobotany Project.

People and Plants working paper 8. UNESCO, Paris.

Mikuls R. (2003). Co-morbidity in rheumatoid arthritis. Best practice in clinical

rheumatology, 17, 729-752.

15

Mohan N, Edwards T, Cupps R, Oliverio P, Sandberg G, Crayton H, Richert J and Siegel J

(2001). Demyelination occurring during anti-tumor necrosis factor alpha therapy for

inflammatory arthritides. Arthritis and rheumatism, 44: 2862-2869.

Nanyingi M, Mbaria J, Lanyasunya A, Wagate C, Koros K, Kaburia H, Munenge R and

Ogara W (2008). Ethnopharmacological survey of Samburu district, Kenya. Journal

of ethnobiology and ethnomedicine, 4: 4-14.

Nguemfo E, Dimo T, Azebaze A, Asongalem E, Alaoui K, Dongmo A, Cherrah Y and

Kamtchouing P (2007). Anti-inflammatory and anti-nociceptive activities of the stem

bark extracts from Allanblackia monticola STANER L.C. (Guttiferae). Journal of

ethnopharmacology, 114: 417-424.

Paola R and Cuzzocrea S (2008). Predictivity and sensitivity of animal models of arthritis.

Autoimmunity Reviews, 8: 73-75.

Park E, Rhee H, Jung H, Ju S, Lee S, Hong S, Yang H, Yoo M and Kim K (2007).

Antiinflammatory effects of a combined herbal preparation (RAH13) of

Phellodendron amurense and Coptis chinensis in animal models of inflammation.

Phytotherapy research, 21: 746-750.

Phrompittayarata W, Putalunc, Tanakad H, Jetiyanon K, Wittaya-areekul S and Ingkaninan K

(2007). Comparison of various extraction methods of Bacopa monnieri. Naresuan

University Journal, 15: 29-34.

Resch L, Hill S and Ernest E (1997). Use of complementary therapies by individuals with

‘arthritis’. Clinical rheumatology, 16: 391-395.

Schaller S, Henriksen K, Hoegh-Andersen P, Søndergaard P, Sumer E, Tanko L, Qvist L and

Karsdal M (2005). Assay and drug development technologies, 3: 553-580.

16

Setty A and Sigal L (2005. Herbal medications commonly used in the practice of

rheumatology: Mechanisms of action, efficacy and side effects. Seminars in arthritis

and rheumatism 34:773-784.

Srithi K, Balsley H, Wangpakapattanawong P, Srisanga P and Trisonthi C (2009). Medicinal

plant knowledge and its erosion among the Mien (Yao) in northern Thailand. In

press.

Tits M, Damas J, Quetin-Leclercq J and Angenot L (1991). From ethnobo tanical uses of

Strychnos henningsii to antiinflammatories, analgesics and antispasmodics. Journal of

Ethnopharmacology, 34: 261-267.

Trentham E (1982). Collagen arthritis as a relevant model for rheumatoid arthritis. Arthritis

and rheumatism, 25; 911-916.

Wong Y, Zhou H, Wang J, Xie Y, Xu H and Liu L (2008). Anti-inflammatory and analgesic

effects and molecular mechanisms of JCICM-6, a purified extract derived from an

anti-arthritic Chinese herbal formula. Phytomedicine, 15: 416-426.

Wood A (2004). New drugs for rheumatoid arthritis. The New England journal of medicine,

350: 2167-2179.

Yu Y, Koo S, Kim C, Lyu Y, Grady J and Chung J (2002). Two variables that can be used as

pain indices in experimental animal models of arthritis. Journal of neuroscience

methods, 115:107-113.

i

Budget

ITEM Unit Cost

(KShs)

Quantity Total (KShs)

Tuition fees 150, 000 per yr 3 years 450,000

Ethnobotanical survey 45,000 2 90,000

Mice 100 1000 100,000

Dichloromethane 3000 per 5 lits 20 lits 12,000

Dimethyl-sulphoxide 3000 per 5 lits 20 lits 12,000

Methanol 3000 per 5 lits 20 lits 12,000

Chloroform 3000 per 5 lits 20 12,000

Collagen Induced Arthritis

(CIA) kit

50,000 1 50,000

Cytokine Assay kit 68,000 1 68,000

Harvesting of plants 25,000 2 50,000

Transport costs 20,000 5 100,000

Data storage, handling and

analysis

30, 000

Na-pentobarbitone100ml 5000 1 5000

Syringes, Needles, slides,

cover slips and other

consumables

30,000

Sub-total 1, 021,000

10% Contingency 102,100

GRAND TOTAL 1,123,100

ii

Work plan

Activity Year 1 Year 2 Year 3

Month 1- 4 5-8 9-12 1-4 5-8 9-12 1-4 5-8 9-12

Literature review, PhD

registration and

ethnobotanical survey

Plant collection and

preparation of extracts

Antinociceptive and

analgesic testing

Induction of CIA in

mice and testing for

anti-rheumatic

properties of herbal

extracts

In-vitro analysis and

histological analysis of

arthritis

Data entry and cleaning

Data analysis

Determination of

dosage

Standardization

Thesis writing,

submission

Dissemination of

research

findings/publication

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Appendix 1: Data acquisition questionnaire

Data acquisition questionnaire on plant extract/preparation usage in OA and RA

QUESTIONNAIRE

PART 1: RESPONDENTS DETAILS

Name............................................................... Sex....M/F Age...........Years.

Occupation........................................................

Registered ………….. (Yes/No). If yes, note the registration number/details……………..

Level of education........................................

Location/Residence..........................................................................................................................

Efficacy/Availability/Toxicity Data

Name and part(s) of Plant (Local name)......................................................used in OA........ RA.....

Availability of the plant (abundant/scarce/endangered) .................................................................

Preparation method(s)..........................................................................................

Formulation used (mixed with)………………………………………………… in OA…..RA…..

Administration form (s)..........................................................................................

Route(s) of application ..........................................................................................

Diagnosis OA ………………………………………… RA ……………………………………

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Approximate dosage..........................................................................................

Response of Patient Good.........................Fair........................ Poor.........................

Duration of response: Minutes....... Hours.......... Days …….. Months…… Years……..

Complication……………………………………………………………………………….

Shelf life of the formulation …………………………………………………………….

Treatment outcomes and recurrence ……………………………………………………

Prognostic indicators …………………………………….………………………………

Patient satisfaction………………………………………………………….……………..

Proportion of initial case vs. cases initially reported (and treated) else where ……….. …

………………………… ………………………………………………………….….

PART 2: RESPONDENTS CONSENT AGREEMENT

I.......................................................................................Hereby agree to participate in this study

with my full consent and conscious and declare that to the best of my Knowledge the information

that I have provided is true, accurate and complete.

Signature/Thumb print...........................................Date.............

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PART 3: RESEARCHER'S DECLARATION

1. The following research will be undertaken with respect to the indigenous knowledge and

intellectual proprietary of the herbal practioners.

2. We will at no given time initiate or conduct practices that are deemed to obtain

information from the respondents by intimidation, coercion or false pretence.

3. The respondents will be informed of the intended project prior to questionnaire

administration and in confidential to eliminate any degree of conspiracy.

4. We will be no under any obligation to edit or tamper the information provided by the

respondents.

5. The information collected will be used for the described research purpose and not any

undisclosed intentions.

Signatory Researchers:

1. Dr. Wambugu S.N..................................... Prof. Kanui T.I..........................

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Appendix 2: Histological grading of arthritis

1. Enlargement of the synovial lining cell layer (at X 200).

0 points The lining cells form one layer.

1 points The lining cells form 2-3 layers.

2 points The lining cells form 5-4 layers, few multinucleated cells may occur.

3 points The lining cells form more than 5 layers, lining might be ulcerated and

multinucleated cells may occur.

2. Density of the resident cells (at X200)

0 points The synovial stroma shows normal cellularity

1 point The cellularity is slightly increased

2 points The cellularity is moderately increased, multinucleated cells might occur

3 points

The cellularity is greatly increased, multinucleated giant cells, pannus

formation and rheumatoid granulomas might occur

3. Inflammatory infiltrate (at X400)

0 points No inflammatory infiltrate

1 point Few mostly perivascular situated lymphocytes or plasma cells

2 point Numerous lymphocytes or plasma cells, sometimes forming follicle-like aggregates

3 points Dense band-like inflammatory infiltrate or numerous large follicle-like aggregates

Sum 0 or 1 = No synovitis

Sum 2–4 = Low-grade synovitis

Sum 5–9 = High-grade synovitis