Transcript Dr. James Kuria - University of Nairobi

WOUND HEALING POTENTIAL,
ANTIMICROBIAL ACTIVITY,
PHYTOCHEMISTRY AND SAFETY
TESTS OF ASPILIA PLURISETA
SCWEINF. (ASTERACEAE).
A project for Master of Science degree in Natural
Products and Bio-prospecting (University of
Nairobi).
INVESTIGATOR
Dr James Menni Kuria (BVM)
Department of Public Health, Pharmacology
and Toxicology
University of Nairobi
SUPERVISORS
Prof J. M. Mbaria
 Prof P. K. Gathumbi
 Prof S. G. Kiama
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INTRODUCTION
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Approx. 50% of drugs in clinical use: natural
products or derivatives (Cowan, 1999);
Africa: 25% of global genetic pool;
Yet, little contribution to NP based industry, and
high rate of biodiversity loss;
Wound healing abnormalities: deformity and
disability (Ashoka et al, 2011): costly, substrate
for infection. Yet, few cost effective remedies;
Repeated mention of A. pluriseta use as wound
healant ethnomedically, hence need for
validation of claims.
OBJECTIVES
General Objective:
• To study the wound healing efficacy of Aspilia
pluriseta, its phytochemistry and safety.
Specific Objectives:
• To evaluate the wound healing activity of Aspilia
pluriseta powder on excision wounds in mice;
• To determine the antifungal and antibacterial
activity of methanolic extract of Aspilia pluriseta
against wound pathogens;
• To screen qualitatively the phytochemicals in the
methanolic extract of Aspilia pluriseta; and,
• To evaluate the safety of topical application of
Aspilia pluriseta.
ASPILIA PLURISETA SCHWEINF.
(ASTERACEAE)
UTILIZATION OF A. PLURISETA
Reports of use in Burundi, Kenya, Rwanda and
Zimbabwe;
 Use ranges from treatment against infections,
infestations, post partum disorders to magic;
 Frequent mention of use as therapy for cuts,
burns, bruises and other dermatologic conditions
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JUSTIFICATION FOR THE STUDY
Wounds: significant impact on QOL of human
and animal patients; present a heavy economic
burden; pose reduction in the efficiency of the
human labor force and productivity of food
animals;
 Natural resources derived remedies are cheaper,
more available and purportedly safer. Other DD
strategies have yielded few wound healants;
 Documentation and validation of traditional
knowledge is key to conservation of shrinking
plant genetic resource base (Neuwinger, 2000).
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FACTORS AFFECTING RATE OF WOUND
HEALING
Local factors: oxygenation, infection, foreign
matter in the wound and blood supply to the
wound;
 Systemic factors: age and gender, ischemia,
concurrent diseases, obesity, medications, drug
and substance abuse, immune suppression and
nutrition
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SUMMARY OF WOUND HEALING PROCESS
HEALING IN RELATION TO TIME
METHODOLOGY
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Collection and preparation of plant material
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Thika Superhighway in Ruiru (1° 9' 0" South, 36° 58'
0" East), January 2012;
Cleaned with water;
Shade dried for 10 days;
Ground to powder;
Packaged, stored till use.
EXTRACTION
Cold maceration, methanol;
 72 hours, regular agitation;
 Course filtration (cotton wool), fine filtration
(Whatman № 1);
 Evaporation in vacuo at 50°C;
 Sand bath (50°C) until constant weight achieved;
 Yield determined.
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WOUND OINTMENT PREPARATION
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Sieving of plant powder (mechanical sieve
shaker);
Formulation of simple ointment (BP): white soft
paraffin, ceto-stearyl alcohol, hard paraffin and
wool fat in 17:1:1:1 ratio melted together then
stir mixed until solid cool;
Trituration of +125µm powder into SO until
uniformly mixed. 10% and 20% ointment made;
Ready ointment packaged.
OINTMENT PREPARATION SUMMARY
WOUND HEALING ASSAY
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Animals: Swiss albino mice, approx. 12-15 wks
old, 28-34gms, room conditions housing at PHPT;
Depilation of dorsum using electric clipper,
sanitized using 5% povidone iodine;
Full thickness excision wounds created using
thumb forceps and Mayo’s scissors, approx.
100mm², under inhalant anesthesia (halothane);
4 groups of mice;
Wound area traced onto tracing paper;
Wounds left open and respective Rx applied.
TREATMENT
Group 1 (10% ointment);
 Group 2 (20% ointment);
 Group 3 (negative control, SO);
 Group 4 (Silverex Cream® (Ranbaxy) 1% silver
sulfadiazine and 0.2% chlorhexidine gluconate);
 Daily treatment for 21 days;
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IMMEDIATE POST-WOUNDING TIME
PARAMETERS
Percent wound area reduction;
 Subjective observations for extent of
inflammation, exudation, extent of granulation
and scab formation;
 Time to complete epithelialization;
 Histopathology.
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OBSERVATIONS
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Wounds observed daily before application of
ointment and after cleaning with Savlon®;
Area traces taken every three days. Percent area
reduction computed with area on day 0 as
reference;
Histopathology samples taken from a satellite
group on 7th and 14th day.
Scar tissue taken from all the animals after
termination on 21st day. Tissues processed
routinely, blocked with paraffin wax and stained
with H&E, Masson’s Trichrome.
WOUND SIZE COMPARISONS (DAY 6)
ANTIMICROBIAL ACTIVITY ASSAY
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Methanol extract used, prepared to a 1600mg/ml
stock solution;
Broth micro-dilution method used;
5 bacterial, 1 fungal species used;
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Staphylococcus aureus ATCC-25923
Bacillus cereus ATCC-11778
Pseudomonas aeruginosa ATCC-27853
Streptococcus pyogenes
Escherichia coli ATCC-25992
Candida albicans
Gentamicin Sulphate and Amphotericin B used
as positive controls.
ANTIMICROBIAL ACTIVITY ASSAY
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Starting concentration of extract in Mueller
Hinton broth was 800mg/ml. Diluted serially up
to 3.125mg/ml;
Bacterial/fungal innoculum concentration used:
1* 106 CFU/ml;
Incubated for 24 hrs and 48hrs at 37° C and room
temp. for the bacteria and fungi respectively;
Contents from tubes not showing growth grossly
subcultured onto MH agar plates.
PARAMETERS
Minimum Bactericidal Concentration: lowest
concentration of test substance that does not
permit growth. Lowest concentration to
completely eliminate all organisms after 24 hr
incubation in presence of extract;
 Minimum Inhibitory Concentration: lowest
concentration of test substance that does not
permit visible growth after 24/48 hr incubation.
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PHYTOCHEMISTRY
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Published protocols used to screen for presence of
phytochemicals in methanol extract:
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Alkaloids: Wagner’s test;
Flavonoids: Alkaline Reagent test;
Glycosides: Modified Borntrager’s test;
Phytosterols: Salkowski’s test;
Phenols: Ferric Chloride test;
Tannins: Gelatin test.
SKIN SENSITIZATION ASSAY: BUEHLER
NON-ADJUVANT TEST.
Carried out alongside OECD TG 406 with
modification on number of animals;
 Plan to sequentially test small groups of animals
and only proceed to another group if the
preceding group gave a negative result;
 Test substance applied to clipped, marked area
an left rump on 1st, 7th, 14th and 21st days
(induction exposure);
 Test substance applied on contralateral side on
28th day (challenge exposure).
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SKIN SENSITIZATION ASSAY: BUEHLER
NON-ADJUVANT TEST.
EXTRACTION
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MeOH extract yield: 12.5%.
RESULTS: TIME TO COMPLETE
EPITHELIALIZATION
RESULTS: PERCENT WOUND AREA
REDUCTION
RESULTS: MIC VALUES FOR TESTED
ORGANISMS
RESULTS: MBC VALUES FOR TESTED
ORGANISMS
Organism
MBC
Bacillus cereus ATCC- 11778
800mg/ml
Staphylococcus aureus ATCC- 25923
800mg/ml
Pseudomonas aeruginosa ATCC-27853
800mg/ml
Escherichia coli ATCC- 25922
800mg/ml
Streptococcus agalactiae
100mg/ml
Candida albicans
800mg/ml
CANDIDA ALBICANS IN 50MG/ML EXTRACT
CONCENTRATION
ANTIMICROBIAL ACTIVITY ASSAY RESULTS
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All the concentrations of reference drugs used
completely eliminated all the test organisms
RESULTS: SKIN SENSITIZATION ASSAY
RESULTS: SKIN SENSITIZATION ASSAY
Day 28: Score 2-moderate and confluent
erythema
 Day 35: Score 2-moderate and confluent
erythema
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DISCUSSION
Wound healing activity: appreciable activity
 Antimicrobial activity assay: marginal and nonspecific activity
 Skin sensitization test: moderate sensitizer
 Further tests:
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Testing of aqueous and organic extracts’ wound
healing activity;
 Screening of the extracts for other bioactivity;
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ACKNOWLEDGEMENTS
UoNbi, Supervisors
 RISE-AFFNET
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Thank you very much