Phytomedicine Vol. 4 (1), pp.

41-45, 1997
© 1997 by Gustav Fischer Verlag

Anti-depressant activities of Areca catechu fruit extract

A. DAR, S. KHATOON, G. RAHMAN and ATIA-UR-RAHMAN

H. E. J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.

Summary

The hexane and aqueous fractions of Areca catechu (A. catechu) have demonstrated anti-de-
pressant properties in screens used to detect such activity. Similar properties had previously been
detected in the plant's aqueous ethanolic extract. The aqueous ethanolic extract (F1) , and the hex-
ane (F2 ) and aqueous (Fs ) fractions inhibit monoamine oxidase (MAO) in rat brain homogenates.
The aqueous fraction seems to be the most potent inhibitor of MAO and its effect is similar to
that of clorgyline (a specific MAO-A inhibitor). The extract and fractions from A. catechu, there-
fore, merit further research in the quest for new anti-depressants.

Key words: Areca catechu, hexane and aqueous fractions, immobility time, rat brain, mono-
amine oxidase inhibition.

Introduction

Areca catechu (Palmae), commonly known as "Chalia"
in the Indo-Pakistan subcontinent, has been shown to pos-
sess stimulant properties (Nadkarni, 1976). We have re-
cently reported that the aqueous ethanolic extract of this
plant possesses anti-depressant properties (Dar et al., 1995;
Dar and Khatoon, 1996).
Forced swim (Porsolt, et aI., 1977 a; Porsolt, et aI., 1977 b;
1978) and tail suspension tests (Steru et aI., 1985) have
gained considerable acceptance as reliable screens for de-
pression. The duration of the immobility time in both tests
was reduced after pre-treatment with a wide variety of anti-
depressant agents that included tricyclics, monoamine oxi-
dase inhibitors (MAOI) (Porsolt, et aI., 1977 b; Van der Hey-
den, et aI., 1987) as well as the aqueous ethanolic extract of
A. catechu (Dar et aI., 1995; Dar and Khatoon, 1996).
Monoamine oxidases (EC 1.4.3.4) (MAO-A and MAO-
B) are mitochondrial enzymes responsible for the degrada-
tion of the biogenic amines and xenobiotics (Sau-Wah
Kwan, et al., 1992). The inhibitors of MAO are often the
drugs of choice for depressions associated with anxiety, ag-
itation or phobias (Murphy et aI., 1985).
In this investigation, the tail suspension test (TST), the
forced swim test (FST) and the locomotion test were used
to assess the anti-depressant properties in the hexane and
aqueous fractions of A. catechu. The effects of Fl , F2 and Fs
on the amine oxidase activities in rat brain homogenates
were also determined.
Material and Methods
Plant material. The nuts (10 kg) of A. catechu were powe-
dered and the powdered material was extracted with 70 %
ethanol in water. The evaporated aqueous ethanolic extract
(F1) was either used directly for pharmacological screening
or partitioned between water and hexane. Evaporation of
the hexane extract produced a gum (F2 ) . The aqueous layer
was further partitioned successively with dichloromethane
(F3 ) and then with ethylacetate (F4 ) to afford the aqueous
fraction (Fs )' The extraction procedure is presented in Fig. 1.
Pharmacological testing was conducted on Fl , F2 and Fs. The
studies on F3 and F4 are under investigation.
Drugs. Adrenaline bitartrate, ascorbic acid, clorgyline
[N-methyl-N-propargyl-3(2,4-dicholorophenoxy)propyla-
mine HCI], horseradish peroxidase (type II) and 5-hydrox-
ytryptamine, were purchased from Sigma USA and dis-
solved in distilled water. Fl , F2 and F, were suspended in 0.9
% saline and administered as a clear solution or in suspen-
sion, respectively. All other chemicals were of analytical
grade.
42 A. Dar et al.

Powdered Nut
(10 kg)

Soaking
Ethanol : water)
( 7:3

Aqueous ethanolic
extract (F1)
(266 g)
Partition Hexane: Water
(I : I)

Aqueous extract Hexane fraction (F 2)

(0.45 g)
Extract with
dichloromethane
( 3 I)

Evaporation
Aqueous extract
Dichloromethane fraction (F3 )
Ethyl acetate extraction (0.78g)
(41)

Aqueous layer Ethyl acetate layer

IFre=W,
Aqueous fraction (Fs)
IE"""'"
Ethyl acetate fraction (F4)
(180g) (8.31 g) Fig.1. Extr action procedure for fractionation of Areca catechu powder.

Doses. In all the experiments described below, the num-
ber of animals used were 3-5/dose. In FST and TST control
animals were given 0.9 % saline (lP: 0.2 ml/20 g, mice or
0.5 ml/lOO g, rats) 1 h before testing with the exception of
the positive control group or treated group, which received
various concentrations of c1orgyline, F2 or Fs' respectively,
60 min before testing.
Forced swim test. Male Wistar rats were treated with sa-
line, clorgyline (2.5, 5 and 10 mg/kg) , F2 (2.5, 5, 10 and
13 mg/kg) or Fs (2.5, 5, 10, 13, 20 and 50 mg/kg) and the
length of their immobility during a 5 min period was re-
corded described previously (Porsolt, et aI., 1977 a; Porsolt,
et aI., 1977 b).
Tail suspension test. Male NMRI mice were treated with
saline, clorgyline (4, 10, 13 and 16 mg/kg) , F2 or F, (0.5,2,
3, 4, 10 and 13 mg/kg) and the length of their immobility
recorded during a 6 min period as described earlier (Stem
et aI., 1985).
Locomotion test. Five NMRI mice of either sex were
treated 1 h before the test with saline. Mice were placed in
Optovarimex minor (Svensson and Thieme, 1969) 15 min
prior to observation. Ten min locomotor counts were re-
corded for a period of 1 h. The mean of 6 (10 min) readings
were noted. The same animals were treated with Fz or Fs
(13 mg/kg) and observed as described above. The counts of
the control and the treated groups were compared.
Monoamine oxidase assay. The animals were sacrificed
by rapid decapitation, and their brains quickly removed
and placed on ice. Monoamine oxidase activity was deter-
mined by the f1uorometric method (Gaddum and Schild,
1934; Guilbault, et aI., 1968; Dar, 1990) using 5-hydroxy-
tryptamine as the substrate. The test substances were pre-
pared in distilled water. The brain tissues were homoge-
nized in 0.1 M potassium phosphate buffer, pH 7.8. The
homogenate (20 Ill) was then incubated with c1orgyline, Fz
or F, for 30 min at 37°C, followed by another incubation
 Anti-depressant activities of Areca catechu 43

for 20 min with 5-HT in a tot al volume of 200 ul, An Ad- 1211
rena linlperoxi dase system was used to form fluorescent ad-
renolut ine, the intensity of the fluor escence was determined

-
Illll
to have an emission wave length of 405 nm with an excita-

.--
tion wave length of 550 nm. Hydrogen peroxide was used
. IJ
as the standard. Clorgyline caused comp lete enzyme inhi bi-
tion at 111M, confirming the presence of MAO in the reac-
tion mixture.
-
:..
00
Th e protein concentra tions of the brai n homogenates :.-.
were estimated by the method of Lowry et al. (195 1) with ~ 40
:..
bovine seru m alb umin as a standard.
The qu anitity of H 2 0 2- produced (in n moles) h- l mg:' :..
tJ 20
:::
L,
pro tein was calculated. The percent inhibition of enzyme
activity was determined by com paring it with the control
values. 0 •
cc 0
N
0
~
<n
~
0
<n
0
\0
0
r-
0
ee 8 8 0
\0
Statist ical analysis. The statistical significance of the dif- l'l l'l

ference between the contr ol and the test was estimated by

the "Student" t test.
Results
Table 1 shows that clorgyline, F2 and P, cause significant
dose-dependent reduction in the immobility time using FST.
The max imum reduction prod uced by clorgyline was at
10 mglkg, while the same magni tude of reduc tion was ob-
tained with F2 and F, at 13 mg/kg. All of them caused a
more than 70 % reduction in the immobility time.
Clorgyline, F2 and F, caused a significant dose-dependen t
reductio n in the immo bility time using TST (Table 2). Bot h
F2 and Fs showe d about a maximum reductio n of 30 % at
4 mg/kg, whereas clorgyline at same dose caused only an
18 % reduction in the immobility time. F2 and F, showed a
decline in the immobility time at doses of more than
10 mg/kg, whereas clorgyline showed consistent dose-de-
pendent increase, reachi ng a maxim um of 92 %.
Locomotor activity in the control mice (2949.2 ±523 .7)
ug/m l
Fig. 2. Effect of vario us concentrations of aqueous ethanolic ex-
tract F1 (0), hexane FI (0) or aqueous F, (_) frac tio ns on brain
MAO activi ties of th e rat. Samples of the homogena tes were pre-
incubated with Fl' FI or Fs for 20 min at 37·C before the addition
of 5-hydroxytryp tamine (500 J.lM). T he inhibition of enzyme activ-
ities is expressed as a percentage of activity relative to the control
samples pre-incubated without inhibitor. Each point is the mean of
3 determinations, each in dup licate or trip licate .
did not change significantly after the administratio n of
13 mg/kg of either F2 or r;
The aqueous ethano lic extract and the hexa ne and aque-
ous fractions of A. catechu (10-260 ug/ml) were used to as-
sess the MAO activities in the rat brai n hom ogenate using
5-hydroxytryptamine (500IlM) as a substrate. The addi-
tion of clorgyline (0.1 and 111M), Fl , F2 or F, caused dose-
dependent inhibition of enzyme activity. Fig. 2 shows that
the aq ueous ethanolic extract at doses of between
10-80 Ilg/ml did not inhibit the enzyme activity, however,

Table 2. The effect of clorgyline and the hexane and aq ueous frac-
Table 1. The effect of clorg yline and the hexane and aqueous frac- tions of Areca catechu on percent reduction in the immobility time
tions of Areca catechu on percent reduction in the immobility time of mice using the tail suspension test .
of rats using the forced swim test .
Dose mg/kg Clorgy line
Do se rng/kg Clorgy line
0.5 2: OAn.5 o
2.5 19::!: 4.6 " " * 7: 2.Y ' 6::!: 2.5 * 2 14 : 1.6 18 : 2.3
5 44 ::!: 2.8 " *" 36::!:3 .9 *** 33 ::!:2 *** 3 27: 6.6 19: 4.2
10 71 : 1.7 *** 69::!: 1.7* ** 68 ::!:2.8*** 4 18: 2.6 35: 5.5 30 : 3.8
13 74 ::!: 3.8 ** * 73 : 2. 7* ** 10 32 : 3.7 29: 2.9 21 : 6.3
20 57 ::!: 3.2 *** 13 63 : 2.7 22: 5 17 : 3.7
50 51 : 4.8 *** 16 92: 1
Immobility time of co ntro l = 174.5 s ::!: 3.2 (N umber of animals Immobility time of con tro l = 121 s::!: 1.7 (N umber of anima ls used
used =45 ). =51).
Values are mean : SEM . Values are mean : SEM.
Asterisks indicate significant percent red uctio n (P < 0.05 *, P < n.s = non-significant and all th e other values sho wed significant
0.01 ,'* and P < 0.005** *] in th e immobility time as compared to percent reduction (P < 0.005 ) in the imm obility time as compared
control. to control.
F2 and Fs represent hexane and aqueous fractions respectively. F2 an d Fs represent hexane and aqueous fractions respectively.
44 A. Dar et al.
enzyme inhibition was evident at doses of between
100-26011g/ml. The enzyme activity remained unchanged
in the presence of up to 4011g/ml Fl , while the enzyme inhi-
bition was obtaind at doses between 45-260 ug/rnl.
50 ug/ml Fl and F5 produced more than 60 % enzyme inhi-
bition, while the same dose of Fj had no effect on the en-
zyme activity. Fl , Fl and F, caused complete enzyme inhibi-
tion at 260, 200 and 6011g/ml, respectively.
Discussion
The current investigation has been extended to see if the
ant i-depressant property of A. catechu we reported earlier
(Dar and Khatoon, 1996) can be impro ved after furthe r
fractionation of the aqueous ethanolic extract. For this pur-
pose the hexan e and aqueous fractions were obtained and
subjected to anti-depressant screens. It has been previously
suggested that the reduction in immobility in rodents signi-
fies behavioral despair, resembling a sta te of mental depres-
sion (Porsolt et aI., 1977 a; Porsolr, et aI., 1977 b). Addi-
tionally am ine ox idase inhibitors have also been reported
to possess anti-depressant properties, therefore, F l , Fl and
Fs of A. catechu were evaluated for their anti-depressant
and MAO-inhibiting activities.
The present study showe d that the Fl and F, fractions of A.
catechu caused a clear and dose-dependent reduction in the
dur at ion of the immo bility time using either TST or FST.
These findings are in accordance with earlier reports of
F 1 (Dar and Khatoon, 1996 ) and clorgyline (Van der Hy-
den, et aI., 198 7; Dar and Khatoon, 1996 ) having anti-d e-
pressant activity. However, when Fl and F, were tested for
anti-depressant properties, they prod uced a significantly
greater reduction in the immobility time than F l (Dar and
Khat oon, 1996), suggesting th at the anti-depressant prop-
erty becomes more pronounced after Fj is processed. Phy-
socostimulants like d-amphetam ine and caffeine (Porsolt,
et aI., 1977 b; Porsolt et aI., 1978) have also been show n to
reduce immobility times, but their effect appeared to be due
to a motor stimulant rath er than to persistant attempts to
escape from the stress situation. In cont rast, the present
st udy showe d that the reduction in immobility time does
not correlate with the spont aneous activity in the open
field. This lack of correlation is an indication that the dif-
ferences in the immobility time are due to the anti-depress-
ant activity of A . catechu extr act and its fraction s and not
to motor activity. A similar corr elat ion has been previously
reported by us in regard s to F, (Dar and Khato on, 1996) as
well as by other researchers using a variety of anti-depress-
ants (Porsolt et al., 1978 ). 5-HT is known to be a preferred
substrate for type A (Kanazawa, 1994 ). Moreover, both en-
zymes (MAO-A and -B) coexist in the rodent brain (John-
ston, 1968). Therefore, the reduction of MAO activity with
5-HT in the presence of Fl , Fl and Fs ma y reflect the pre-
ferred inhibition of type A.
The findings of the in vitro studies show that F, is a mor e
potent inhibitor of MAO-A than F1 or Fl , as is clearly re-
flected by its lowest ICso value (20 ug/rnl). The IC so values
for F1 and Fl were 113 and 47).1g/ml, respectively. MAO-A
plays a crucial role in the treatment of depression by main-
taining the concentrations of biogenic amines (Laux, 1993)
and our results indicate that further fractionation of Fj of
A. catechu clearly improved the anti-depressant activity.
The alkaloids viz arecaine, arecaidine and arecoline have
been show n to be present in the A. catechu nut with only
arecoline having MAO inhibiting pro perties (Sirsi, et aI.,
1966) . The possibility that the MAO inhibition in the
present report may be due to arecoline and some other un-
known compounds needs furth er investigation. In the light
of these observations it may be suggested that A. catechu
qualifies as an important cand idate in the search for new
ant i-depressants.
References
Borsini, F. and Meli, A.: Is the forced swimming test a suita ble
model for revealing anti-depressant activity? Psychopharmacol-
ogy 94: 147- 160, 1988.
Dar, A., Khatoon, 5., Rahman, G. and Atta -ur-Rahman: Anti-de-
pressant activity in the extracts of A reca catechu. Second annu -
al symposium on " Basic and applied research in health care and
social development ". Aga Khan University Karachi Pakistan,
1995.
Dar, A.: An in vitro study of pulmonary vascular reactivity and
amine oxidase activity in the horse. PhD. dissertat ion Cam-
bridge University, 1992.
Dar, A. an d Khatoon, S.: Anti-depressant effects of Ar eca catechu
in Rodents. Phytotherap y Res. (submitted), 1996 .
Gaddum, J. H. and Schild, H.: A sensitive physical test for nora-
drenaline.]. Physiol. 80: 9P, 1934.
Guilbault, G. c., Brignac, P.]., Juneau, M .: New substrates for the
fluorometric determination of oxidative enzymes. Anal.
Chem. 40: 1256-1263, 1968 .
Kanazawa, I.: Short Review on monoamine oxidase and its inhib-
itors. Eur. N eurol. 34: 36-39, 1994 .
Johnston, J. P.: Some observations upon a new inhibitor of mono-
amine oxidase in brain tissue. Biochem. Pharmacal. 17:
1285-1297, 1968.
Laux, G.: Pharmacology and clinical use in neurodegenerative dis-
orders (I. Szelenyi; Eds) Chap. 17, pp. 319-326, 1993 .
Lowry, O. H., Rosebrough, N.J., Farr, A. I.. and Rand all, R.].:
Protein measurement with the Folin phenol reagent. j. Bioi.
Chem. 193: 265-275, 1951.
Mu rphy, D. 1.., Sunderland, T., Campbell, I. and Cohen, R. M. :
" Monoamine oxidase inhibitors as anti-depressants. In pharma-
cotherapy of affective disorders: Theory and pract ice"
(W. G. Dewhurst and G. B. Baker; Eds) Croom Helm Lond on,
pp.328-362, 1995.
Nad karni, K. M.: Indian Materia Medica, Popular Book, Bombay;
pp. 130-1 33, 1976.
Porsolt, R. D., Pichon, M. Le., Jalfre, M.: Depression: a new ani-
mal model sensitive to anti-depressant treatments. Nature 266 :
730-732, 1977 a.
Porsolt, R. D., Bertin, A., JaHre, M. : Behavioral despair in mice:
A primary screening test for anti-depressants. Arch. Int. Phar-
macody n. 229: 327-336, 1977b.

Porsolt, R. D., Anton, G., Blavet, N. and [alfre, M.: Behavioral
despair in rats: A new model sensitive to anti-depressant treat-
ments. Eur. J. Pharmacol. 47: 379-391, 1978.
Sau-Wah Kwan, Bergeron, J. M. and Abell, C. W.: Molecular
properties of monoamine oxidases A and B. Psychopharmacol-
ogy 106: 51-55, 1992.
Sirsi, M., Lalithakumari, H. and Jayaram, H. N.: Peripheral and
central actions of Areca catechu. Symposium on CNS drugs.
C.S.I.R. New Delhi, p. 249, 1966.
Steru, L., Chermat, R., Therry, B. and Simon, P.: The tail suspen-
sion test: A new method for screening anti-depressants in mice.
Psychopharmacology 85: 367-370,1985.
Anti-depressant activities of Areca catechu 45
Svensson, T. H., Thieme, G.: An investigation of a new instrument
to measure motor activity of small animals. Psychopharmacolo-
gia 14: 157-163, 1969.
Van der Hyden, J. A. M., Molewik, E. and Olivier, B.: Strain differ-
ences in response to drugs in the tail suspension test for anti-de-
pressant activity. Psychopharmacology 92: 127-130, 1987.
Address
A. Dar, RE.]. Research Institute of Chemistry, University
of Karachi, Karachi-75270, Pakistan.