WOUND HEALING POTENTIAL, ANTIMICROBIAL ACTIVITY, PHYTOCHEMISTRY AND SAFETY TESTS OF ASPILIA PLURISETA SCWEINF. (ASTERACEAE). A project for Master of Science.

Presentation on theme: "WOUND HEALING POTENTIAL, ANTIMICROBIAL ACTIVITY, PHYTOCHEMISTRY AND SAFETY TESTS OF ASPILIA PLURISETA SCWEINF. (ASTERACEAE). A project for Master of Science."— Presentation transcript:

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 I NVESTIGATOR Dr James Menni Kuria (BVM) Department of Public Health, Pharmacology and Toxicology University of Nairobi

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

4 I NTRODUCTION 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.

5 O BJECTIVES 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 U TILIZATION 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 J USTIFICATION FOR THE S TUDY 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 F ACTORS 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 S UMMARY OF WOUND HEALING PROCESS

11 H EALING IN RELATION TO TIME

12 M ETHODOLOGY 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 E XTRACTION 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 W OUND O INTMENT 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 O INTMENT P REPARATION S UMMARY

16 W OUND H EALING A SSAY 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 T REATMENT 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 I MMEDIATE POST - WOUNDING TIME

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

20 O BSERVATIONS 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 7 th and 14 th day. Scar tissue taken from all the animals after termination on 21 st day. Tissues processed routinely, blocked with paraffin wax and stained with H&E, Masson’s Trichrome.

21 W OUND SIZE COMPARISONS (D AY 6)

22 A NTIMICROBIAL 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.

23 A NTIMICROBIAL 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* 10 6 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 P ARAMETERS 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 P HYTOCHEMISTRY 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 S KIN S ENSITIZATION A SSAY : B UEHLER N ON - 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 1 st, 7 th, 14 th and 21 st days (induction exposure); Test substance applied on contralateral side on 28 th day (challenge exposure).

27 S KIN S ENSITIZATION A SSAY : B UEHLER N ON - ADJUVANT TEST.

28 EXTRACTION MeOH extract yield: 12.5%.

29 R ESULTS : T IME TO COMPLETE EPITHELIALIZATION

30 R ESULTS : P ERCENT WOUND AREA REDUCTION

31 R ESULTS : MIC VALUES FOR TESTED ORGANISMS

32 R ESULTS : MBC VALUES FOR TESTED ORGANISMS OrganismMBC Bacillus cereus ATCC- 11778800mg/ml Staphylococcus aureus ATCC- 25923800mg/ml Pseudomonas aeruginosa ATCC-27853800mg/ml Escherichia coli ATCC- 25922800mg/ml Streptococcus agalactiae100mg/ml Candida albicans800mg/ml

33 C ANDIDA ALBICANS IN 50 MG / ML EXTRACT CONCENTRATION

34 A NTIMICROBIAL A CTIVITY A SSAY R ESULTS All the concentrations of reference drugs used completely eliminated all the test organisms

35 R ESULTS : SKIN SENSITIZATION ASSAY

36 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