1. Evaluation of fractions of selected African plants for antimicrobial activity
against Mycobacterium tuberculosis
Richin John Koshy, Nadir Khalil, Marie Larson, Henrik Andersson, Johanna
Raffetseder, Elsje Pienaar, Daniel Eklund, Asaad Khalid and Maria Lerm
Masters in Medical Biosciences
Degree Project (45 ECTS)
,

2. Historical Background
 Neolithic Time
 2400 BC - Egyptian mummies
spinal columns
 460 BC
 Hippocrates, Greece
 First clinical description:
Phthisis / Consumption
(I am wasting away)
 500-1500 AD
 Roman occupation of Europe it
spread to Britain
 1650-1900 AD
 White plague of Europe,
causing one in five deaths
2

3. Discovery of Mycobacterium
tuberculosis  24th March 1882 (Robert
Koch) TB Day
 Discovery of staining
technique that identified
Tuberculosis bacillus
 Definite diagnosis made
possible and thus treatment
could begin
 1890 (Robert Koch)
 Tuberculin discovered
 Diagnostic use when injected
into skin
3

4. Mycobacteria  Small, rod-shaped, aerobic bacilli
 Present in the environment, soil
and water
 High concentration of lipids and
wax in the cell wall
 M tuberculosis grows quite
slowly and colonies do not appear
on solid media up to 2.5 -5 week
www.lookfordiagnosis.com
4

5. Transmission
The spread of M. tuberculosis involves a 3-step process:
Transmission of bacteria,
Establishment of infection, and
Progression to disease.
Netter atlas of Human Anatomy
5

6. Pharmacological discoveries
• 1946 p-aminosalicylic acid
• 1948-1950
– Combination of Strep. and
PAS
• 1952 Isoniazid.
– Replaces sanatorium as major
treatment
– Patients can be treated as out-
patients
• 1954 Pyrazinamide. (PZA)
6

7.  1956-1960
 Combination therapy
of INH and PZA
cures TB
 1955 Cycloserine
 1962 Ethambutol
 1963 Rifampicin
 1970-1977
 Combination of
Rifampicin and
Isoniazid adopted as
International regime
for treatment of TB
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8. World Health Organization (WHO) and the
International Union Against Tuberculosis and
Lung Diseases
WHO and other agencies replaced the term primary resistance by the term
“drug resistance among new cases” and acquired resistance by the term
“drug resistance among previously treated cases
8

9.  Drug resistance in mycobacteria is defined as a decrease in sensitivity to a
sufficient degree that the strain concerned is different from a sample of
wild strains of human type
 Multi-drug resistant tuberculosis is resistance to Isoniazid and Rifampicin.
It is incorrect to classify a patient has having multi-drug resistant disease if
they have an infection with a bacterium susceptible to Rifampicin but
resistant to many other drugs
 XDR TB is resistant to all first line drugs namely; Isoniazid and Rifampin
and Three or more second line drugs (SLD’S) that are used to treat MDR-
TB
 No drugs available to treat resistant strains since none has been developed
in the last 40 years
9

10. Epidemiology of MDR TB
• 490,000 new cases of MDR-TB
each year, with >110,000 deaths1
• Accounts for 5% of 9 million new
cases of TB2
• MDR-TB rates higher than ever
(up to 22.3%), particularly in
former Soviet Union countries
• XDR-TB reported by as many as
49 countries
• Recent WHO/IUATLD Global
Surveillance report indicated 7.5%
(301/4012) of MDR TB to be XDR4
• Around 40,000 XDR-TB cases
emerge every year1
10

11. Mechanisims of Drug Resistance in
Tuberculosis
Lipincott Textbook of Pharmacology
11

12. Reasons identified for resistance
 Deficient or deteriorating TB control programmes resulting in
inadequate administration of effective treatment
 Poor case holding, administration of sub-standard drugs, inadequate or
irregular drug supply and lack of supervision
 Massive bacillary load, illiteracy and low socio-economic status of the
patients, the epidemic of HIV infection, laboratory delays in
identification and susceptibility testing of M. tuberculosis isolates
12

13. Impact of resistance
Huge individual as well as public health consequences in terms of
 Increased mortality
 Prolonged periods of infectiousness with increased risk of
transmission of resistant pathogens to others
 Indirect costs (prolonged absence from work, etc)
 Increased direct cost (longer hospital stay, use of more expensive 2nd
or 3rd line drugs
13

14. Drug Discovery
 Traditionally used plants are a valuable source for the discovery of
modern drugs
 Screening of Chinese herbal medicine led to introduction of
artemisinin from the leaves of Artemisia annua which is used
worldwide for treatment against malaria
 Shifting the screening strategy from a single enzyme targets to a
whole bacterial cell level have been showing successful results as it
has a holistic approach and only gives hits that can enter the bacterium
through the cell wall
14

15. Preliminary studies
 Rosmarinus officinalis L (RO)
 (Flowers/Leaves)
 Antimicrobial
 Antiviral
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16. Preliminary studies
 Khaya senegalenesis leaves
(KSL)
 Khaya senegalenesis barks
(KSB)
 Fever remedy
 Antimicrobial
 Antiprotozoal
16

17. Hypothesis
 Fractions of Khaya Senegenalenesis and Rosimarinus Officinalis L
possess anti-microbial properties
AIM
 To identify fractions of plant extracts used traditionally to treat
infectious diseases in African countries for possible antimicrobial
activity against M tuberculosis
17

18. Materials and methods
Mycobacterial
culture
Infection of
human Fractions of Plant
macrophages with Extracts prepared
M tuberculosis with DMSO
Cytotoxicity with
Analysis of
hMDM and
bacterial growth
THP-1 cells
by lumometry
THP-1 cell culture Determination of
CFU plating
Separation and
culture of
primary
monocytes
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19. 19

20. Monocytes were cultured in Monocytes are
complete DMEM medium and diffrentiated into
incubated at 37ºC. macrophages.
20

21. Bacteria cultured and OD Monocytes were cultured in Macrophages infected with
measured complete DMEM medium and bacteria at MOI 10 and
incubated at 37ºC. Monocytes fractions of extracts
diffrentiated into macrophages
21

22. Results:
Assesing Mycobacterial growth using Luminometry
in the presence of RO fractions (0.00625 mg/ml)
22

23. Assesing Mycobacterial growth using Luminometry in the
presence of Khaya senegalenesis bark and leaves
[KSB/KSL] (0.00625 mg/ml)
23

24. Viability of Macrophages treated with RO and KSB fractions
(10, 1 and 0.1 mg/ml)
10 mg/ml 1 mg/ml 0.1 mg/ml
24

25. Viability of THP-1 macrophages treated with RO and KSB
fractions (10, 1 and 0.1 mg/ml)
10 mg/ml 1 mg/ml 0.1 mg/ml
25

26. Viability of infected macrophages treated with RO and KSB
fractions (0.1 mg/ml)
26

27. Assessing intracellular Mycobacterial growth using Luminometry
in the presence of Rosimarinus officinalis [RO] (0.1mg/ml)
27

28. Assessing intracellular Mycobacterial growth using Luminometry
in the presence of selected Khaya senegalenesis bark [KSB]
(0.1mg/ml)
28

29. Conclusion
 Plant extract fractions of K Senegalenesis and R Officinalis were
analysed for anti-mycobacterial activities which yielded four fractions
to have distinct anti-mycobacterial properties.
 High-throughput screening using luciferase determines mycobacterial
growth giving conclusive results.
 Inhibition of growth by these extracts was observed in both systems
giving inference about their antituberculous activity which appears to
be meaningful.
 Biologic actions of K Senegalenesis have been attributed to a
complicated concert of compounds for its synergestic and antagonistic
activity. This has enabled the pharmacological and biological
properties of the plant in exerting its anti-microbial properties.
 Further phytochemical studies need carried out in order to find the
active compound with inhibition.
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30. Acknowledgement
 First I will like to give my greatest appreciation to my supervisor
Maria Lerm for her direction, encouragement and support throughout
the period of my project. I am happy that I had the opportunity to
work under her supervision and ever thankful for all that she taught
me
 A big thanks to Nadir for everything and keeping good company
 I also express my gratitude to Johanna Raffetseder, Marie
Larsson, Elsje Pienaar and Daniel Eklund for introducing me to
most of the methods i used in this project and more and also for
always being there to answer numerous questions I had.
 Finally but not the least, I will like to say a big thanks to all the staff
of the department
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31. Thank You
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