ANTIPLASMODIALAND

CYTOTOXIC ACTIVITIES OF RWANDAN

MEDICINAL PLANTS USED IN THE

TREATMENT OF MALARIA

Muganga, R., Angenot, L., Tits, M., Frédérich, M.

Journal of Ethnopharmacology

128 (2010)

Eduardo X. Rodríguez

Background

Background

• Value of medical ethnobotany

• increasing resistance to

current drugs

• artemisinin was discovered

from 青蒿素 (qīng hāo sù),

Artemisia annua, used in

traditional Chinese medicine

• different disease etiology

paradigms, can lead to novel

approaches in cosmopolitan

biomedicine

Background

• Great Lakes region

• located in Great Rift Valley, plateau region with some mountains

• modern nations of:

• Uganda

• Rwanda

• Burundi

• western Tanzania

• eastern DR Congo

• troubled history

• ruinous European rule

• genocides, civil wars

• Great African War

Background

• Malaria in Sub-Saharan Africa

• 45 countries endemic

• highest mortality in children

under 5

• Malaria in Rwanda

• prevalence reduced (ITNs,

ACT, IRS)

• high endemicity persists

• high morbidity/ mortality in

every province

• one of leading causes of

outpatient attendance

Details of study

Details of study

• Partnership between two universities

• National University of Rwanda Butare, Rwanda

• University of Liège Liège, Belgium

• Thirteen medicinal plants

• source: Rwandan traditional healers and literature review

• from east, south, and southwest (highest Pf endemicity)

• Extracts

• dichloromethane

• methanol

• aqueous

Scientific name of the plant

(family), date

Plant parta Voucher number Rwanda Voucher number Meise Botanical

Garden, Belgium

Place of collection (altitude)

Aristolochia elagans Mast.

(Aristolochiaceae), 2007

Seed 2007V39 BR0000005093953 Huye (1684 m)

Conyza aegyptiaca (L.) Aiton

(Asteraceae), 2007

L 2007V38 BR0000005093878 Huye (1684 m)

Markhamia lutea K. Schum.

(Bignoniaceae), 2008

L 2008R17 BR0000005093979 Nyaruguru (1753 m)

Microglossa pyrifolia (Lam.)

Kuntze (Asteraceae), 2007

L 2007R1 BR0000005093977 Bugesera (1413 m)

Mitragyna rubrostipulata (K.

Schum.) Havil. (Rubiaceae), 2008

L and SB 2008R19 BR0000005093595 Nyaruguru (1648 m)

Fuerstia africana T.C.E. Fr.

(Lamiaceae), 2008

L and S – BR0000005088850 Huye (1719 m)

Rumex abyssinicus Jacq.

(Polygonaceae), 2008

R 2008R15 BR0000005093670 Huye (1670 m)

Rumex bequaertii De Wild.

(Polygonaceae), 2008

R 2007R3 BR0000005094059 Huye (1600 m)

Solanecio mannii (Hook.f.) C.

Jeffrey (Asteraceae), 2008

L 2007R2 BR0000005093472 Musanze (2033 m)

Terminalia mollis M.A. Lawson

(Combretaceae), 2007 and 2008

L, SB and RB – BR0000005087167 Ndego (1592 m)

Tithonia diversifolia (Hemsl.) A.

Gray (Asteraceae), 2008

F and L 2007R12 BR0000005093793 Huye (1680 m)

Trimeria

grandifolia subsp. tropicaSleumer

(Flacourtiaceae), 2008

L 2008R16 BR0000005093496 Nyaruguru (1722 m)

Zanthoxylum chalybeum Engl.

(Rutaceae), 2007 and 2008

SB and RB – BR0000005087266 Ndego (1377 m)

Table 1. Selected species, their scientific names, parts used, voucher number and place of collection (altitude).a F, flower; L, leaves; R, root; RB, root bark; S, stem; SB, stem bark.

Details of study

• Antiplasmodial assays

• IC50 growth inhibition evaluated

• lactate dehydrogenase (pLDH) activity

• controls: infected (+) & uninfected (-) erythrocytes

• Cytotoxicity assays

• human fetal lung fibroblast cell line (WI-38)

• IC50 mean of at least two independent assays

• mitochondrial enzyme activity

• control: camptothecin (+)

Details of study

• Two-step process

• antiplasmodial assay of Pf 3D7 (CQ-sensitive)

• antiplasmodial assay of Pf W2 (CQ-resistant) & cytotoxicity assay

• WHO guidelines for IC50 for antiplasmodial activity

• inactive > 50 μg/ mL

• moderate 15- 50 μg/ mL

• promising 5- 15 μg/ mL

• highly active < 5 μg/ mL

Results

Table 2. In vitro antiplasmodial and cytotoxic activities, and selectivity index of the selected samples.

a F, flower; L, leaves; R, root; RB, root bark; S, stem; SB, stem bark.b IC50 values shown in bold express promising antiplasmodial activity.c For chloroquine, n = 11.d nd = not determined.e Root bark collected in August.

• one of most used medicinal plants for malaria in Rwanda

• traditionally, leaf used for fever/ malaria, hematuria

• “...more clarification regarding this toxicity is needed”

Conyza

aegyptiaca

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf MeOH 22.7 24.7 81.9

MeCl2 36.8

• another study found EtOAc leaf extract IC50 10.2 μg/ mL (plants from

different regions, Pf were different strains)

• also, MeOH leaf extract reported to be active in vivo

Markhamia

lutea

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf MeCl2 29.0 N/A N/A

• traditionally, leaf used to treat fever/ malaria, intestinal worms,

rheumatism, pain relief, diarrhea, gonorrhea, etc.

• cytotoxicity & hepatotoxicity already noted in literature

Microglossa

pyrifolia

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf MeCl2 1.5 2.4 4.7

• traditionally, whole plant (without roots) used to treat fever/ malaria;

leaf used to treat gonorrhea

• compound previously isolated with IC50 1.95 μg/ mL, but cytotoxic

Fuerstia

africana

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf & stem MeOH 6.9 4.2 13.0

• artemisinic acid analogue isolated from stem in another study

• high cytotoxicity

Tithonia

diversifolia

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Flower MeOH 8.1 6.5 5.3

MeCl2 1.1 1.0

aq 24.5 N/A

Leaf MeOH 1.2 1.5 2.5

MeCl2 0.6 0.7

aq 15.6 N/A

• traditionally, root used to treat fever/ mal, gonorrhea, constipation,

poisoning, hepatitis, constipation, sciatic neuralgia; leaf used to treat

coughs and gastric ulcers

• this is first study to find antiplasmodial activity of extract from this

plant, but “...more clarification regarding this toxicity is needed”

Rumex

abyssinicus

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf MeCl2 4.3 3.1 13.3

• other studies confirm anti-fungal, anti-bacterial, anti-viral activities

• another study found MeOH root extract IC50 125- 150 μg/ mL for Pf (plants from different regions, Pf were different strains)

• this is first study to find antiplasmodial activity of extract from this plant

• ***strong potential source***

Terminalia

mollis

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Root bark MeOH 11.7 N/A N/A

aq 33.5 N/A

Stem bark MeOH N/A 18.9 77.2

• traditionally, leaf used for fever/ malaria, burns, abscesses, leprosy,

anthrax, poisoning

• ***strong potential source***

Crassocephalum

(Solanecio)

mannii

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Leaf MeCl2 18.2 12.9 122.9

• another study found aq root bark extract IC50 < 6 μg/ mL for Pf 3D7

• August in dry season, November in rainy season

• ***strong potential source***

Zanthoxylum

chalybeum

Part of plant Extract Pf 3D7 Pf W2 cytotoxicity

Root bark

(Nov)

MeOH 4.2 1.9 40.0

MeCl2 6.2 N/A

Root bark

(Aug)

MeOH 38.3 N/A N/A

Considerations and

critique

Considerations and critique

• Factors affecting efficacy

• season during collection

• locality of the plant (including altitude)

• Extracts with consistently low cytotoxicity, possible

primary prophylaxis

• Vigilant of resistance to new medicines, especially if in

folk distribution

Considerations and critique

• Two research approaches:

• search for lead compounds

• usual route of biomedical research

• e.g., isolation of marchantin A from liverwort species in Iceland

• study of synergistic effects

• crude extracts usually more effective than lead compounds

• also, sometimes better with overall disease condition than with etiologic agent

Considerations and critique

Strengths

• antiplasmodial assays on

strains sensitive and resistant

to CQ

• cytotoxicity assay

• two new plants shown to have

antiplasmodial activity

• two other plants continue to

demonstrate promise

• comparison of same medicinal

plant from two different

seasons

Weaknesses

• assays performed in Belgium, months after collection

• holes in data not explained, possible resource restrictions

• only used polar extracts (value of lipophilic solvents?)

• cytotoxicity assay, hypo- or hyper-sensitivity of cell lines?

• issues with selectivity index

• usual arguments, in vitro human cell lines outside system vs non-human system

Summary and

conclusions

Summary and conclusions

• Many medicinal plants demonstrated antiplasmodial

activity, but many of them also demonstrated cytotoxicity

• Some more active against Pf 3D7, some more active

against Pf W2

• Three were the most promising:

• Terminalia mollis

• Crassocephalum (Solanecio) mannii

• Zanthoxylum chalybeum

THANK YOU

Questions?