Transcript In vitro Antimicrobial Activity of Aloe turkanensis, an Ethnomedical

Z. M. RUKENYA1, J. M. MBARIA1, P.M. MBAABU2, S. G.
KIAMA2 R.O.ONZAGO1
Introduction
Five common species
AT…Important ethnomedical remedy.
Sap used for wound management & eye diseases.
Boiled roots juice in soup induce vomiting relieve
persistent headaches.
The roots are used to flavour beer (Bosch, 2006).
Natira community aloe-working group herbalist
 Treat malaria
 Wounds
 Stomachaches
 Pain
 Ringworms
 Poultry diseases.
Justification
 Despite wide usage, there is inadequate technical specifications on
quality, safety and efficacy (no available microbial load standards--KEBS).
Culture on BA
 Scanty literature on AT…Probably following presidential
degree in 1986 …
 Study done by Schmelzer (Protabase), et al 2010…Knowledge
gaps in quality control standards for exudates of Aloe
secundiflora and Aloe turkanensis
 Repercussions:
 Hindrance to international trade
 Why Aloe turkanensis (land rehabilitation, drug discovery, public health,
livelihoods-value chain)
 Good news…C & M of Aloes L.N 403 2007 gazette…80%
ASAL..CBS 1999.
Hypothesis
 Aloe-working group herbalists recognize that wounds, eye
infections, fungal infections, stomach upsets and skin
infections involve pathogenic micro-organisms and that the
plant Aloe turkanensis has metabolites capable of inhibiting or
killing these microbes without causing adverse effects on the
herbal consumer.
Am 95% safe if consumed
Objectives
Determine the bioactivity of the plant’s extract (both
naturally occurring and cultivated) on the growth of
Bacillus cereus, Staphylococcus aureus, Pseudomonas
aeroginosa, Escherichia coli, Candida albicans and
laboratory cultured Artemia salina leach
Materials and methods
Collection of the Plant Material
Whole plant was harvested from Natira community aloe
garden…attended mainly by local women.
Natira sub-location
 Collection was done during a dry season
Kakuma town
 Aided by 10 people from Natira community Aloe-working group herbalists who identified the
plant by the local name and voluntarily provided information on uses Aloe turkanensis as
medicinal plant
AILMENT
No. Of people who have used
within the last 10 years
Part used
Method of disease
diagnosis
Method of reconstituting
the medicine
Route of administration
Malaria
10
WP
Clinical signs
Boiled leaf extract when
dry
Oral
Wounds
9
Leaf
Clinical signs
Aloe juice
Topical
Pain
8
Whole
Clinical signs
Boiling when dry
Oral
Stomach ache
8
Leaf
Clinical signs
Boiling when dry
Oral
Tiredness
9
Leaf
Clinical signs
Boiling when dry
Oral
Detoxify
6
Whole
Clinical signs
Boiling when dry
Oral
Laxative
10
Leaf
Clinical signs
Boiling when fresh
Oral
Emetic
3
Leaf
Clinical signs
Boiling when fresh
Oral
Cosmetic
10
Leaf
Clinical signs
Freshly harvested sap & gel
Topical
Ringworms
7
Leaf
Clinical signs
Freshly harvested sap
Topical
Health Drink
10
Leaf & roots
Clinical signs
Gel
Oral
Removal RAB in
cows
2
Leaf
Clinical signs
Sap
Intrauterine
Chicken disease
2
Leaf
Clinical signs
Freshly harvested sap
Drinking water
A plant specimen was identified at KFRI, stored and young ones propagated
Preparation of plant samples
Washed, chopped dried hot air oven 800C 72 hours then
milled to powder using a laboratory grinder. Sap screened.
Extraction process
Cold maceration
Rota-Evaporation to dryness
Lyophilized to powder using
Christ Beta lyophilizer.
The aqueous extract yielded 13.32g while methanolic
14.96g. Stored in a refrigerator pending further analysis.
Testing for antimicrobial activity of the
extracts
Broth dilution assays on 4 standard cultures & 1 clinical
isolate
 Escherichia coli (ATCC 25922), Pseudomonas aerugenosa
(ATCC 27853), Staphylococcus aureus (ATCC25923) &
Bacillus cereus (ATCC 11778) while human clinical isolate
of Candida albicans
Minimal inhibitory concentrations (MIC) determined.
Reference bacterial obtained from cooked meat media
 Subcultured in blood agar (Oxoid®) for 24 hours at
370C …except for Candida albicans incubated at rt.
 Using a sterile loop, colonies picked & put into 3ml sterile
physiological buffer saline (PBS).
 Serial dilution…Macfarland opacity No. 6
 Stock solution of the plant extracts
 4 grams of powder dissolved 10ml of MHB to make a
concentration of 400mg/ml…then two fold serial dilutions
made in sterile Muller Hinton Broth (MHB) using culture
tubes.
 One ml of the test organisms’ suspension dispensed into the
culture tubes containing the plant extracts.
 Controls
 1ml of the test organisms’ suspension in sterile MHB…-ve
 Benzyl penicillin, Gentamycin and Amphotericin B …+VE
 Each tube containing the test organism and plant extract was
the incubated for 24 hours at 370C…Candida albicans RT 48
hrs.
The inhibition of test organisms was evaluated by
culturing 1ml of the suspension into Muller Hinton
Agar for 24hour at 370C for bacteria and for 48 hours at
room temperature for Candida albicans.
Brine Shrimp Lethality Test:
Lethality testing was done according to McLaughlin
(1991). Three dilutions were prepared by transferring
500µl, 50µl and 5µl of plant extract to a set of five
graduated tubes.
Ten shrimp were transferred into each of the vial using
Pasteur pipette and marine salt added up to 5ml mark
to make dilution of 1000µg/ml, 100µg/ml and 10µg/ml.
After 24 hours exposure, live nauplii were counted.
Results/Discussion
Table 3: Minimum Inhibitory Concentration (MIC mg/ml) of Aloe
turkanensis extracts after incubation with microbial cultures
Test material
Extract
Part used
P. aeroginosa
E. coli
S. aureus
B. cereus
C. albinans
A.
Aqueous
WP
-
-
<3.125
200
-
Methanol
WP
200
-
100
100
-
Ag
Sap
125
500
62.5
62.5
-
Met
Sap
-
-
62.5
62.5
-
-
-
0.625
0.625
turkanensis
Benzyl penicillin
-
Gentamycin
-
-
0.0049
0.0049
-
-
-
Amphotericin B
-
-
-
-
-
-
0.0125
Conclusion
 Based on interviews conducted in the field and the
subsequent in vitro laboratory testing of the sampled
plant, it was concluded that the plant has metabolites
that inhibit the growth of some bacterial organisms and
is a relatively safe herbal remedy when used for human
consumption. It is probably due to the antibacterial
activity and safety of Aloe turkanensis that Natira
community Aloe-working group herbalists use the plant
products for treatment of wounds, eye infections , skin
infections, stomach upsets, livestock diseases and as
cosmetics.
Pending work
Phytochemistry
What is the significance of the noted biological
activity?...
ACKNOWLEDGEMENTS
RISE-AFNNET (SIG)
UON
AUDIENCE
THANK YOU