Investigator

1. 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).

2. Investigator Dr James Menni Kuria (BVM) Department of Public Health, Pharmacology and Toxicology University of Nairobi

3. Supervisors • Prof J. M. Mbaria • Prof P. K. Gathumbi • Prof S. G. Kiama

4. 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 (Ashokaet al, 2011): costly, substrate for infection. Yet, few cost effective remedies; • Repeated mention of A. pluriseta use as wound healantethnomedically, hence need for validation of claims.

5. 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.

6. ASPILIA PLURISETA SCHWEINF. (ASTERACEAE)

7. 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

8. 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).

9. 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

10. Summary of wound healing process

11. Healing in relation to time

12. 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.

13. 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.

14. 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.

15. Ointment Preparation Summary

16. 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.

17. 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;

18. Immediate post-wounding time

19. Parameters • Percent wound area reduction; • Subjective observations for extent of inflammation, exudation, extent of granulation and scab formation; • Time to complete epithelialization; • Histopathology.

20. 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.

21. Wound size comparisons (Day 6)

22. 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 • GentamicinSulphate and Amphotericin B used as positive controls.

23. 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.

24. 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.

25. 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.

26. 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).

27. Skin Sensitization Assay: Buehler Non-adjuvant test.

28. extraction • MeOH extract yield: 12.5%.

29. Results: Time to complete epithelialization

30. Results: Percent wound area reduction

31. Results: MIC values for tested organisms

32. Results: MBC values for tested organisms

33. Candida albicans in50mg/ml extract concentration

34. Antimicrobial Activity Assay Results • All the concentrations of reference drugs used completely eliminated all the test organisms

35. Results: skin sensitization assay

36. Results: skin sensitization assay • Day 28: Score 2-moderate and confluent erythema • Day 35: Score 2-moderate and confluent erythema

37. 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;

38. acknowledgements • UoNbi, Supervisors • RISE-AFFNET

39. Thank you very much