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).

INVESTIGATORDr James Menni Kuria (BVM)Department of Public Health,

Pharmacology and ToxicologyUniversity of Nairobi

SUPERVISORS Prof J. M. Mbaria Prof P. K. Gathumbi Prof S. G. Kiama

INTRODUCTION• 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.

OBJECTIVESGeneral 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

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).

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

SUMMARY OF WOUND HEALING PROCESS

HEALING IN RELATION TO TIME

METHODOLOGY Collection and preparation of plant material

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.

WOUND OINTMENT PREPARATION

• 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• 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;

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.

OBSERVATIONS • 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• Methanol extract used, prepared to a

1600mg/ml stock solution;• Broth micro-dilution method used;• 5 bacterial, 1 fungal species used;

– 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• 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.

PHYTOCHEMISTRY Published protocols used to screen for

presence of phytochemicals in methanol extract: 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).

SKIN SENSITIZATION ASSAY: BUEHLER NON-ADJUVANT TEST.

EXTRACTION 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 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

DISCUSSION Wound healing activity: appreciable activity Antimicrobial activity assay: marginal and

non-specific activity Skin sensitization test: moderate sensitizer Further tests:

Testing of aqueous and organic extracts’ wound healing activity;

Screening of the extracts for other bioactivity;

ACKNOWLEDGEMENTS UoNbi, Supervisors RISE-AFFNET

Thank you very much