gaba a activity of q ’eqchi’plants for neurological...

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