RASOANAIVO Plants based immunotherapy and aromatherapy

Natural Products Discovery:
Focus on ever increasing African health care needs

Khartoum, Sudan
7- 10 December 2013

Plants-based immunotherapy and aromatherapy:
From research to therapeutic applications

Philippe Rasoanaivo
Polytechnics Schools, University of Antananarivo:
Institut Malgache de Recherches Appliquées

Content
1- Burden of infection diseases in Africa
2- Basic background of biodiversity
and the search of medicines
3- From the search of antiplasmodial compounds
to immunotherapy
4- Neuroprotection against diseases affecting
the brain

Evolution of the malaria epidemiology

1900

Source: Nature 2010, 465, 342.

2007

So what?
Where to find solutions
for increasing African health care needs?
African biodiversity

One answer:

African
indigenous knowledge

Biodiversity and the search for new medicines
Culture
diversity

Ecosystem
diversity

Ethnomedical
diversity

Species
diversity

Gene
Diversity

Chemical
diversity

Oriented biological tests

Biological screening

Drugs; phytomedicines

Reverse pharmacology

UP

Synergy

Bio-informatics
In silico

Virtual screening

In enzymo Combinatorial chemistry
HTS
In cellulo
In vitro (isolated organs)
Traditional
Medicinal chemistry
In vivo

Polypharmacology

In homo

BOTTOM

TOP

REDUCTIONIST

Today drug discovery

Chronology

Drug discovery approaches

Observational studies
Traditional knowledge

HOLISTIC

DOWN

Target
Identification,
Validation

Lead
identification

Lead
Optimization

Pre-clinical
studies

Clinics

Modern drug discovery: from laboratory to clinics

Field
work

In vivo tests

Open clinical
trials

Randomized
clinical trials

Chemical and
biological
investigations

Reverse pharmacology: from clinics to laboratory

Nature Reviews Microbiology, Vol. 11, December 2013, 849

20 year research in malaria chemotherapy
in our Institute,
based on indigenous knowledge

Investigation of Tabernaemontana sp
Serendipity Discovery (2011)
Death

% parasitaemia

All control mice died

Unexpected drop
In parasitaemia ?

Monocyte
proliferation

How to prevent
recrudescence?

N° days

Starting point for lessons and new thinking in malaria

Lesson learned: going beyond the boundaries of
standard antiplasmodial tests.

In the in vivo 4-day suppressive test:
D-1

D-4

Infestation

Curative

% parasite inhibition

100-90: very good to good
90-50: good to moderate
50-10: moderate to weak

Post 4th day events: Neglected
100% parasite inhibition never achieved
with nearly all plant extracts:
ÄParasite recrudescence,
ÄDrug resistance.

Protective

Parameters for consideration
h Number of days of mice survival;
h % inhibition at D-6, 8, 10;
h Appearance/proliferation of lymphocytes;

Lesson learned: limitations of the in vitro tests
to investigate antimalarial plants

HIDEX Chameleon
Multiplate Reader
Donation from IFS/PRISM

IC50 > 100 at 10 microgram/ml

Recalling the past
IFS Award Lecture, 2001

Peaceful coexistence ?
Resistance

Host

Peaceful cohabitation

?

Parasites

Drugs

New directions ?

“Thinking in both sides of the coin”
Parasite
Killing the parasite

Curative

Host
Innate immunity

Protective
Adaptive immunity

“Getting outside the box:
Synergistic approach:
+

What curative drug to use?
We made a critical analysis of standard antimalarials:
range of activities, cost
Kill parasites by blocking haem polymerization,
preventing thus hemozoin formation

Chloroquine
Immuno-modulatory activities

Used to treat HIV-AIDS, Cancer, Chikunguya
Co-administered with vaccines
to enhance protective immunity

4-day suppressive test with
Aqueous extract + Chloroquine (CQ)

% parasitaemia

Death
Death

Asymptomatic infection

Parasite clearance

Number of days

Blood smear

Lymphocytes

Monocytes,
Probably lymphocytes T)

“Frozen” parasite

After 30 days: second turn of 4-day suppressive test with
Aqueous extract + Chloroquine (CQ)

% parasitaemia

Death

Parasite clearance

No death
Number of days

After another 30 days: infestation without treatment

% parasitaemia

Death

Parasite clearance

No death
Number of days

OMAPI Patent, Sept. 20012

15th NAPRECA Symposium in Madagascar:
Good opportunity to share results

Malaria sterile immunity
9 Jul 2012

9 Aug 2012

Infestation
+
treatment

Infestation
+
treatment

19 Jan 2013

20 Oct 2012

Challenge

Challenge

10 June 2013
Challenge

NAPRECA Summer School
(8-15 Sept 2012)

13 Sep 2012
Checking
parasitemia

9 Sept 2012
Challenge
with P. yoellii

10 Sept 2013
Challenge

How could we interpret these results?
Unexpected active constituents

Unexpected single
constituent(s) may be
present in the crude extract.

“Every healing herbal
has its quintessence”

Synergy between constituents

Complex molecular interactions
produce effects that may not be
achieved by single component.

“Nature does nothing without
purpose or uselessly”

T. Efferth, E. Koch. Complex interactions between phytopharmaceuticals:
the multi-target therapeutic target of phytotherapy. Current Drug Targets,
2012, 12, 122.

The Yin and Yang
Plasmodium infection
Immunopathology
(Inflammatory process;
Oxidative stress)

of the immune system
Immune response
Antimalarial
Excess or failure
of the immune response

Adequate treatment

We need to ensure adequate balance, like a thermostat, on complementary opposite
parameters that are interconnected and interdependent in malaria.

African antimalarial plants and
antimalarial drugs
Artemisinin

Quinine

?

« The absence of an African antimalarial compound with nanomolar
antiplasmodial activity should not be understood as a failure of the
pharmacopeia, but rather an indicator that the answer may lie in a
different direction »
Source: The ScientificWorld Journal
Volume 2012, Article ID 978913, 9 pages

Phytochemical investigation
Mechanism of immune protection
Clinical trials

“The majority (59%) of Rx innovation come not from
drug companies, but from doctors trying to help
patients for whom standard therapy had failed”.
Bernard Munos

■ Moving rapidly to therapeutic applications.
OMAPI Patent, 2013

Traditional knowledge and vaccination
Benjamin Jesty, farmer, is one of the
first persons to have experimented
vaccination in 1774.

Dr Edward Jenner is recognized as

the

founder of immunology.
His interest in the protective effects of cowpox
against smallpox came from a common belief
that dairymaids were in some way protected from
smallpox..
Source: Google

Trials on humans: between ethics and life saving

Louis Pasteur
On July 6, 1885, Louis Pasteur tested for the first
time his rabies vaccine on Joseph Meister,
a nine year old boy who had been badly bitten
by a rabid dog.

Open clinical trial:
Broadening the studies
- Asymptomatic malaria;
- HIV-AIDS;
- Sexually communicable infections
- Chikunguya.
A case report: a 21 year old boy infested with HIV and malaria
refractory to standard antimalarials,
successfully treated with the recipe.

Dr Potier is a CNRS Gold Medal Winner,
and the discoverer of the anticancer
drug Taxotere®

Brain is one challenging
problem of the 21st century

(

Source: L’EXPRESS, 1998

Madagascar: hilly island

Dry season
Rainy season

One vernacular name:
Maimbelona: dry but alive
maina = dry
velona = alive

Botanical aspect
Myrothamnaceae family
One genus: Myrothamnus
Two species
M. flabellifolia

M. moschatus

Well documented;
Well investigated.

Poorly documented;
Not investigated

Myrothamnus: resurrection plant (inselberg)

Day 0

Day 1

Day 2

Traditional uses
Used to expel bad spirit
Vernacular name:
Maharoake (which can expel)
Used to treat cough

Dried plant is smoked,
and smoke is pooled in a plastic bag,
Smoke is then inhaled by patients.

Extraction of the essential oil of M. moschatus
We assumed that active constituents are volatile compounds

Portable alembic
2500 litres alembic

Essential oil composition of M. moschatus

trans‐pinocarveol  
(35.6%,)

α‐pinene 
(10.5%, )
pinocarvone 
(20.0%,)

p‐cymene 
(1.6%)

limonene 
(2.3%)

O

perillyl acetate 
trans‐p‐mentha‐1(7),8‐dien‐2‐ol 
(6.0%)
(3.3%)

β‐selinene 
(8.5%)

cis‐p‐mentha‐1(7),8‐dien‐2‐ol
(2.1%)

P. Rasoanaivo et al. Journal of Essential Oils Research 2012, 24, 299

trans‐pinocarveolo

pinocarvone

β‐selinene
α‐pinene
perillyl acetato

trans‐pinocarveolo

α‐pinene
pinocarvone
β‐selinene

perillyl acetato

Biological effects of the essential oil
Effects of the essential oil in pentylenetetrazole
(PTZ)-induced convulsions in mice

Control mice
Treated mice

Collecting different samples of M. moschatus

Abundance

Chameleonic behaviour of essential oils of M. moschatus
TIC
: 130912-FG
-04.D

2200000

24.43

2000000

1800000

Caryophyllene oxide.

1600000

1400000

1200000

25.94

1000000

18.78

800000
19.52
21.5
2
7
2.8
0
2
4.21
24.13
25.81

600000

19.33
20.27
19.89

400000

Confueing ethnomedical
information

28.33

26.92

19.82
18.19

24.95
28.09
2
6
.5
4
2
5
.7
3
23.47
2
6
.2
8
25
.1
8
2
0
.7
2
8
1
.8
9
2
5
2
.6
6
3
.4
1
2
1
.7
3
2
1
.3
7
23
.5
4
2
5
.0
6
2
2
.5
1
2
0
.5
6
2
6
.1
2
2
7
.1
9
2
1
.4
5
2
4
.5
5
2
6
.1
9
2
8
.2
1
2
2
.1
1
2
3
.0
6
2
4
.6
7
2
2
.4
0
2
3
.0
1
2
1
.0
6
2
6
.4
7
2
4
.6
2
2
7
.8
7
2
2
3
3
.3
.6
7
7
2
3
.7
4
17.4
0
2
1
.2
7
2
3
.9
0
2
7
.6
3
2
6
.7
4
2
9
.6
8
0
.9
2
1
8
.7
02
9
.7
4
2
5
.3
9
2
0
.9
8
2
7
.0
1
2.6
3
.1
2
4
.7
5
2
9
.9
1
2
7
.2
6
2
9
.8
4
2
1
.2
0
2
5
.5
3
2
7
.4
6
1
8
.5
5
2
2
12
2
7
.7
5
3
0
.1
1
2
82
.8
9
3
0
.7
3
0
.2
16
15.49

200000
3.75
6.27
5.2
1
6
.1
5.6
86

9.06

5.00

10.00

15.00

20.00

25.00

33.37

30.00

35.00

Tim
e-->

Abundance
TIC
: 130912-FG-05.D
22.84

2200000

δ-cadinene,
Caryophyllene oxide.

2000000

1800000

24.41

1600000

1400000

1200000
25.94
1000000

21.90
22.52

800000

2
6.4
25
.7
42
21.74 24.21

600000

18.19

24.96
25.82
21.27
26.12
17.40
23.0
2
2
4.55
18.78
26.91
21
.57
20.5
1
18.70
28.09
2
2.11
20.7
8
25.1
9.28
26
28.32
19.51
23.31
2
3
.9
4
26.54
28.21
12
90
.8
2
31
.2
7.3723
.4
8
2
4
.1
2
2
5
.3
4
29.67
2
6
.7
6
2
5
.0
5
2
3
.5
5
2
2
.2
4
2
2
.4
0
2
9
.4
43
2
5.6
2
2
7
.1
8
2
7
.1
3
2
7
.0
0
7
.2
8
19.33
2
7
.9
2
22
8
2
8
.9
0
2
2
7
7
.7
.7
2
1
6
9
.0
2
23
.13
2
3
9
0
.9
.1
36
2
7
.4
7
2
8
2
.5
9
.0
7
9
3
0.7
2
9
.2
9
21.0
7.1

400000

200000
3.75
6.27
5.00

10.00

15.00

20.00

25.00

30.00

35.00

Tim
e-->

Abundance
TIC
: 130912-FG-03.D
24.42
2000000

1800000

1600000

Caryophyllene oxide.

1400000
25.93
1200000

1000000

800000

19.33
24.21

600000
21.57
19.89
400000

3.80

9.04

5.00

26.42
25.81
26.92

24.92
56.48
9.29
22.80 25.1
2
6
2
6.55
2
3
.4
8
2
4
.5
2
4
.1
24
20.22
71.77
2
3
.7
4
2
3
.6
7
3
.9
0
28.09
2
6.1
22
3
.5
22
.4
0
21
1.3
.4
5
2
3
.0
6425
.6
39
2
7
18.19 20
2
2
.2
4
2
4.6
.6
86 27.63
2
3
.3
7
2
4
2
2
5
.0
.7
8
2
3
.1
2
2
.0
9
2
1
.6
5
15
90
21
.2
2
0
2
.6
12
18
.7
2
7
.0
1
2
.3
1
26
.1
2
1.4
6.0
2
2
6
7
.8
.1
2
8
2
7
.2
6
2
6
.7
2
1
98
.5
1
2
2
9
9
.8
.9
4
0
0
.9
2
2
7
.4
2
6
8
.8
3
9
0
.1
1
1
8
.8
5
2
1
.0
5
3
0
.7
6
2
1
2
.2
1
.8
69
.4
0
2
9
.7
5
2
7
.9
2
5
9
3
.4
0
5
.2
1
2
7
2
.7
8
.2
6
2
2
9
.6
8
3
1
.65
11
77
.0
1
22
0
.5
2

200000

10.00

15.00

20.00

25.00

30.00

Ignored by local
communities

35.00

Tim
e-->

Abundance
TIC
: 130912-FG-01.D
21.62

2600000

b-selinene,
Limonene,
trans-p-mentha-1(7),8-dien-2-ol,

2400000
2200000
6.27

2000000
1800000
1600000

11.95
1400000
13.39
1200000
1000000
9.47
10.01

800000
600000

12.3
6
1
3.87
13.05

400000

20.27

6.12
8.37

200000 3.75

5.00

13.48

10.90
24.36
25
.8
45
.8
83
2
69
.72
.3
1
2
.25 11
11
.2
1
2
7
.15
1
0
.1
1
38 22.7
2
5
.1
8
2
4
.1
9
1
0
.7
3
1.3
9.8
2
4
2
.9
2
5
5
6
.7
.2
8
7
8
.6
8
1
0
.6
6
9
.0
3
12.83
5
2
2
6
6
.4
.9
0029.36
1
0
.5
4
16.48 19
23
.6
4
10.00

15.00

20.00

25.00

30.00

38.79
40.93
35.00

cis-p-mentha-1(7),8-dien-2-ol.

Convulsions,
migraines

40.00

Tim
e-->

trans-pinocarveol, Pinocarvone,
β-selinene,
trans-p-mentha-1(7),8-dien-2-ol.

Asthma, cough,
Convulsions
Chem. Biodiv., Nov. 2013

OH

trans-pinocarveol

Viljoen, et al. South African Journal of Botany, 2002, 68, 100-105.

Migralepsy

Migraine aura
(headache)

Migralepsy
Migralepsy

Seizures

Both chronic
neurologic disorders
with episodic attacks

Migraine and epilepsy
Common symptoms

Source: Prof. A.V. Srinivasan. Three pronged approach to migraine, epilepsy
and neuropathic pain. PPT presentation 2004, Kerala.

Effects of essential oil on neurites outgrowth

Effect of different concentrations of the essential oil on the
neurite outgrowth of neuronal culture SHSY5Y cells.
OMAPI Patent N° 2012036, 19/10/2012

Growth of neurons against epilepsy

La Recherche,N°437, 2010, p. 69

Open clinical trials
Simple inhalation against:
- Convulsions,
- Migraines,
- Alcohol addiction,
- Depressive states.
One case report: a young person having seizures in the street