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1Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa2Department of Chemistry, University of Ottawa, Ottawa
Plants used by the Q’eqchi’ of Belize to treat epilepsy and susto were
screened using an in vitro GABAA-benzodiazepine (BZD) assay. Most of these
plants were able to displace the test compound, [3H]-flunitrazepam by more than
50% from the BZD site of the GABAA receptor. A statistically significant
correlation was observed between GABAA activity and relative frequency (%RF) of
plant use for epilepsy (r = 0.383, p< 0.05) as well as susto (r = 0.728, p<0.02). The
phytochemical characterization of Gonzalagunia rosea Standl., one of the most
active Q’eqchi’ plants yielded ethyl and isopropyl caffeate as the active compounds.
The relatively high activities of the ethyl ester, IC50 = 25.8 µg/mL, and isopropyl
ester, IC50= 7.90 µg/mL, make them ideal candidates for further development as
antiepileptic or anxiolytic drugs.
GABAGABAAA activity of Qactivity of Q’’eqchieqchi’’ plants for neurological disorders: potential sources of plants for neurological disorders: potential sources of
natural antinatural anti--epileptic and anxiolytic drugsepileptic and anxiolytic drugsFida AhmedFida Ahmed11, Martha Mullally, Martha Mullally11, , AsimAsim MuhammadMuhammad22, , AmmarAmmar SaleemSaleem11, & John T. Arnason, & John T. Arnason11
ABSTRACTABSTRACT
INTRODUCTIONINTRODUCTION
Retention time (min)
Ab
sorb
an
ce (
mA
U)
METHODSMETHODS
Figure 4 (A) G. rosea (B) HPLC chromatogram of ethyl acetate fraction (325 nm
trace). (1) Ethyl caffeate, 1.9 mg/g dry weight (2) Isopropyl caffeate, 0.6 mg/g
dry weight of plant
Figure 6 Comparison of the % displacement activity (+SD) of isolated actives and
related phenolics at 100 µg/mL (n=3). An asterisk (*) denotes significant activity
compared to the solvent control (p < 0.05).
Conventional drugs used to treat these disorders include the classical
benzodiazepines such as diazepam (Valium) which are active at the
benzodiazepine (BZD) site of the GABAA receptor, an ionotropic chloride
channel. These drugs, however, have various side-effects, including amnesia,
decreased alertness, and cognitive impairment5. There is an increased need to
search for alternatives, and natural products such as alkaloids and flavonoids are
of particular interest because of their high activity and reduced side-effects6.
RESULTS & DISCUSSIONRESULTS & DISCUSSION
A B
� GABAA-BZD assay5
� Plant extracts tested at 200 µg/mL
� Isolated compounds tested at 1, 3, 10,
30, and 100 µg/mL
� GABAA in vitro activity measured as
% displacement of [3H]-
flunitrazepam; negative control:
95% ethanol.
� Quantification of active compounds
� The active compounds were
quantified using a validated HPLC-
DAD method using a Waters YMC-
OD-AM column (150 mm x 2 mm).
80.5 (+1.5)PiperaceaePiper sp.
81.5 (+11.3)SelaginellaceaeSelaginella af. stellata
Spring
86.0 (+4.2)RubiaceaeGonzalagunia rosea
Standl.
92.6 (+3.2)PiperaceaePiper tuerckheimii
94.1 (+3.0)PiperaceaePiper amalago
Displacementa
% (+ SD)
FamilyScientific name
Table 1 GABAA activity (% displacement + SD) of the most active plants identified
from the screening process. The extracts were tested at 200 µg/mL (n= 3 for each
plant).
Figure 3 Correlation analysis of GABAA activity (% displacement) and %
relative frequency (RF) of plants used for (A) epilepsy, r = 0.383, r2= 0.147, p<
0.05 (n= 31) and (B) susto, r = 0.728, r2= 0.530, p< 0.02 (n= 10). [%RF = (no.
of healers using a plant for epilepsy or susto/Total no. of healers) x 100%]
Figure 5 Dose response relationships of (A) ethyl caffeate (1), IC50 = 25.8 µg/mL and
(B) isopropyl caffeate (2), IC50 = 7.90 µg/mL. At each concentration, n= 3.
� This study demonstrates the importance of traditional Q’eqchi’ knowledge
as well as the importance of ethnobotany as a route to drug discovery.
Majority of the plants used by the Q’eqchi’ healers to treat epilepsy and
susto are active at the BZD site of the GABAA receptor. Thus, hypothesis 1
is supported. Biological activity is well correlated with healer plant selection
in the case of susto, providing partial support for hypothesis 2. Ethyl and
isopropyl caffeate are simple phenolics with low cytotoxicity8, and
inexpensive synthesis methods, which are advantages over the classical
benzodiazepines as therapeutic drugs.
� Future studies could involve the phytochemical characterization of some of
the most active plants, in vivo studies using ethyl caffeate, and the extraction
of indole alkaloids from G. rosea using acid-base fractionation9.
a. All values listed are significantly greater than the solvent control (p< 0.001)
CONCLUSIONSCONCLUSIONS
A B
REFERENCESREFERENCES
A B
The Q’eqchi’ Maya, a distinct cultural group who inhabit regions of
Southern Belize have a rich tradition of plant use for treatment of neurological
disorders1 (Figure 1). The disorders recognized and treated by the Q’eqchi’
healers include among others, epilepsy and susto. Susto is a culture-bound
syndrome; it occurs as a result of a frightening event and is considered to be
similar to post-traumatic anxiety disorders3. These two disorders share a common
underlying mechanism- low levels of γ-aminobutyric acid (GABA) in the CNS4.
Figure 1 (A) Sites of plant collection in Southern Belize (B) Q’eqchi’ healers
collecting plants.
1. Amiguet et al. (2005) Economic Botany, 59: 29-42.
2. Bourbonnais-Spear et al. (2007) Journal of Ethnopharmacology, 109:
380-387. 3.American Psychiatric Association (APA). (1994). American
Psychiatric Association, Washington, pp. 848-849.
4.Goddard et al. (2001) Archives of General Psychiatry, 58: 556-561.
5.Sarup et al. (2003) Current Drug Targets - CNS & Neurological Disorders,
2: 269-277.
6.Tsang, S.Y. and Xue, H. (2004) Current Pharmaceutical Design, 10: 1035-
1044. 7. Marder et al. (1996) Phytomedicine, 3: 29-31.
8.Chiang et al. (2005) British Journal of Pharmacology, 146: 352–363.
9.Niño et al. (2006) Brazilian Journal of Microbiology, 37: 566-570.
� Objectives: 1) To screen the plants used by the Q’eqchi’ for epilepsy and
susto using the GABAA-BZD assay, 2) To determine if there is a correlation
between frequency of plant use by healers and GABAA activity and 3) To
carry out bioassay-guided fractionation of Gonzalagunia rosea and identify
its GABAA-active principles.
� Hypotheses: 1) Active extracts or compounds exert anxiolytic/antiepileptic
effects by binding to the BZD site, and 2) Healers select plants based on their
pharmacological activity.
0 20 40 60 80 100
0
20
40
60
80
100
0 20 40 60 80
0
20
40
60
80
100
% d
isp
lace
men
t
of
3H
-flu
nit
raze
pam
% d
isp
lace
men
t
of
3H
-flu
nit
raze
pam
%RF (epilepsy)
%RF (susto)
A
B Eth
yl c
affe
ate
Isop
ropyl
caf
feat
eC
affe
ic a
cid
para-
coum
aric
aci
d
Chlo
roge
nic a
cid
Ros
mar
inic
aci
d% d
isp
lace
men
t of
[3H
]-F
lun
itra
zep
am
-60
-40
-20
0
20
40
60
80
100
**
Control
Acknowledgements:Rosalie Awad, Zul Merali, Tony Durst, Belize Indigenous
Training Institute (BITI)
Photography by Rosalie Awad and Nathalie Bourbonnais-
Spear
OBJECTIVES & HYPOTHESESOBJECTIVES & HYPOTHESES
G. rosea extract
Hexane
Hexane
extract
Sub-fractions
EtOAc
extract
Column
chromatography
Prep-HPLC
Compounds
Active fractions
GABAA assay
EtOAc
Figure 2 Bioassay-guided fractionation of G.
rosea
� Screening of 31 Q’eqchi” plants for GABAA activity led to the identification
of 23 active plants with more than 50% displacement activity. Members of
the Piperaceae (pepper), Selaginellaceae (spikemoss) and Rubiaceae
(coffee) families are very active (greater than 80% displacement activity)
(Table 1).
1 2
� There is a weak positive correlation between GABAA activity and % RF
of plants used for epilepsy treatment and a strong positive correlation
for susto suggesting that GABAA activity may be a better predictor of
anxiolytic activity than antiepileptic activity (Figure 3).
� HPLC analysis of G. rosea active compounds (Fig. 4) showed that
ethyl caffeate is three times more concentrated in the plant compared
to isopropyl caffeate, although the latter has a lower IC50 (Fig.5). Thus,
ethyl caffeate is the major active compound from G. rosea.
� Ethyl caffeate isolated from G. rosea is a known BZD agonist and a potent
antioxidant with neuroprotective properties7,8. It has significantly greater
GABAA activity compared to other simple phenolics found in plants such as
caffeic acid and chlorogenic acid (Figure 6).
Big Falls
Jalacte
Indian
Creek
TOLEDO
CARRIBEAN
SEA
GUATEMALA