Antihypertensive substance in seeds of Areca catechu L.

Life Sciences, Vol. 38, pp. 1375-1382 Printed in the U.S.A.

Pergamon Press

ANTIHYPERTENSIVE SUBSTANCE IN SEEDS OF ARECA CATECHU L.

Jin-ichi Inokuchi*, Hikaru Okabe*. Tatsuo Yamauchi*, Atsuo Nagamatsu *I, Gen-lchlro Nonaka** and Itsuo Nishioka**

*Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma 8-19-1, Jonan-ku, Fukuoka 814-01, Japan and **Faculty of Pharmaceutical Sciences, Kyushu University, Maldashl 3-I-I, Higashi-ku, Fukuoka 812, Japan (Received in final form February 6, 1986) Summary

Among various tannins tested, Areca II-5-C, a fraction isolated from seeds of Areca catechu L., showed the most potent anglotensinconverting enzyme (ACE) inhibitory activity in vitro. Its antihyper" tensive activity was therefore investigated in normotensive and spontaneous hypertensive rats (SHR) after both oral and intravenous (i.v.) administration. The activity was compared with that of captoprll (D-3-mercapto-2-methylpropanoyl-L-prollne), a potent ACE inhibitor. Oral administration of Areca II-5-C to SHR produced a lasting, dose-related antihypertenslve effect, and the responses obtained with doses of I00 and 200 mg/kg were comparable to those of captoprll at doses of 30 and I00 mg/kg. Intravenous administration of Areca II-5-C to SHR produced a rapid and marked reduction in blood pressure at doses of I0 and 15 mg/kg. The maximum antihypertensive effect of Areca II-5-C in SHR, at an i.v. dose of 15 mg/kg, was about 5 times as large as that of captoprll at the same dose. Although the vasopressor response to norepinephrlne and vasodepressor responses to bradyklnln and acetylchollne were not appreciably changed by i.v. treatment with Areca II-5-C at a dose of 5 mg/kg, it did produce dose-related inhibition of the pressor responses to angiotensln I and II. It is suggested that Areca II-5-C has favorable properties as a hypotensive drug through its ability to inhibit the pressor responses to both anglotensln I and II.

We previously reported that preparations from eight plants were potent and specific inhibltors of angiotensin-converting enzyme (ACE) in vitro (1,2). All of the active fractions from these preparations have been shown to consist of condensed tannins of high molecular weight, consisting exclusively of chains of flavan-3-ols (procyanidins) (I). The purity, molecular weights, and structures of the active materials remain to be determined. Among them, Areca II-5-C, the tannin fraction of Areca catechu L. seeds, showed the most potent ACE inhibitory activity (2). Nishioka et al. (3) have isolated and characterized several dlmerlc, trimerlc, and higher oligomeric flavan-3-ols from seeds of this plant. Therefore, we have compared the ACE inhibitory activity of Areca II-5-C to I To whom reprint requests should be addressed. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.

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those of the structurally determined flavan-3-ol oligomers in Areca catechu L. and have investigated the antihypertensive effect of Areca II-5-C to establish whether the tannins are of potential use as hypotensive drugs. Methods Animals. Male spontaneous hypertensive rats (SHR) 14-16 weeks old and age-matched Wistar-Kyoto normotensive rats (WKY) from Charles River Japan were used for measuring systolic blood pressure in the conscious state. Male Wistar rats weighing 300-400 g were utilized for measuring direct arterial blood pressure under anesthetized conditions. All rats were fed laboratory diet and water ad llbitum. The experiments were begun following acclimation in a 22=C animal colony with 12 h light and dark cycles. Chemicals. Angiotensin I, angiotensin II, bradykinin and Bz-Gly-His-Leu were from the Protein Research Foundation, Minoh, Japan and dl-norepinephrine and acetylcholine were from Sigma Chemical Co. Captopril (D-3-mercapto-2methylpropanoyl-L-proline) was from Squibb Pharmaceutical Co. Areca II-5-C was isolated from dry Areca seeds according to the procedure reported previously (I). Fresh Areca seeds were used as the source in the isolation (3) of the flavan-3-ols, (-)-epicatechin and (+)-catechin, and their polymers (see Table I). Captopril was dissolved and Areca II-5-C was suspended in distilled water (5 ml/kg body weight) and administered orally to the animals. For intravenous (i.v.) administration, captopril was dissolved in 0.9% saline (1 ml/kg body weight) and Areca II-5-C was dissolved in 5% EtOH (I ml/kg). ACE assay. Inhibitory effects of tannin samples on ACE activity (Bz-GlyHis-Leu as substrate) were measured as described previously (i). Measurement of systolic blood pressure by tail-cuff method. The SHR and WKY were trained to give stabilized blood pressures. The systolic blood pressures were measured by the tail-cuff method (4) with a Narco PE-300 programmed electrosphygmomanometer after keeping each animal at 38 + 1.5°C for 20 min. Their pressures were 175-210 mm Hg (SHR) and 115-135 mm Hg--(WKY). The pressures were measured again after a single oral administration of Areca II-5-C (i00 or 200 mg/kg) or captopril (30 or I00 mg/kg), or after i.v. administration of Areca II-5-C (I0 or 15 mg/kg) or captopril (15 mg/kg). Direct measurement of arterial blood pressure. Male Wistar rats were anesthetized intraperitoneally with urethane (1.2 g/kg). Each anesthetized rat was placed in a supine position, and a carotid artery and jugular vein were exposed surgically. A i0 cm length of polyethylene tubing (PE 50) was placed in the carotid artery for the measurement of direct arterial blood pressure, and a 15 cm length of polyethylene tubing (PE I0) was placed in the jugular vein for i.v. injections. The blood pressures and heart rates were measured with the polygraph 360 system (SAN-E1 Co., Ltd.; P 231D pressure transducer, 1257 blood pressure amplifier, 1321 tachometer and Rectigraph-8K). When the blood pressure stabilized, a hypertensive or hypotensive agent was injected i.v. and the blood pressure was monitored. The procedures and dose levels described by Rubin et al. (5) and Ondetti et al. (6) were used to evaluate the influence of captopril and Areca II-5-C on the pressor responses to angiotensin I (310 ng/kg), angiotensin II (100 ng/kg), and noreplnephrlne (200 ng/kg), and the depressor responses to bradykinin (10 ~g/kg) and acetylcholine (300 ng/kg). The other conditions are described in each figure. Results and Discussion The inhibitory effects of various tannins and their precursors in Areca seeds on ACE were determined at four concentrations (Table I). Because of the

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Antihypertensive

Effect of Tannin

1377

limited number of available tannins, correlation between the compound structures and their ACE inhibitory activities is unclear at present. However, the present data strongly suggest that the ACE inhibitory activity of these tannins is proportional to their degree of polymerization. Among them, Areca II-5-C, a highly polymerized tannin (I), showed the most potent ACE inhibitory activity The 150's for the two most active inhibitors, the pentamer and Areca II-5-C, were 7.5 and 1.0 ~g/ml, respectively.

TABLE 1 Inhibitory Effects of Various Tannins and their P r e c u r s o r s

on ACE Activity

Inhibition (%) Sample Inhibitor conc:

20 ~g/ml

I0 ~g/ml

2 ~g/ml

I ~g/ml

Monomer:

A B

3 4

0 0

0 0

0 0

Dimer:

C D

II 20

6 8

0 0

0 0

Trimer:

E F G

7 14 8

4 I0 7

0 0 0

0 0 0

Tetramer:

H

38

19

6

0

Pentamer:

I

69

58

14

0

96

93

78

50

Areca II-5-C

A:

(-)-epicatechin

B:

(+)-catechin

C:

A-~8~B--B

D:

A-k~6--B

E :

A~-S~--A~-S~--S

F:

A-~-8-8- A-k~- -B

G:

A-9-B~-- B -~-~-8-- B

H:

A-9-B-8--A-~-B~--A-~-B-8--B

I:

A-k-B~--A-4-B~-- A-4-B-8--A -k-B-8-- B

The data presented in Fig. 1 show that a single oral administration of Areca II-5-C to SHR produced a long lasting and dose-related decrease in systolic blood pressure. The responses obtained at doses of I00 and 200 mg/kg were comparable to those of captopril at doses of 30 and 100 mg/kg. On the other hand, single oral doses of Areca II-5-C to WKY (i00 and 200 mg/kg) did not affect their blood pressures (data not shown).

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As shown in Fig. 2A, single i.v. administration at doses of i0 and 15 mg/kg of Areca II-5-C to SHR caused a rapid and marked reduction of the systollc blood pressure. The maximum antihypertensive effects of Areca II-5-C occurred within I h. Comparison of the two drugs at the 15 mg/kg level showed that Areca II-5-C was about 5 times more potent in SHR in terms of the maximum antihypertenslve effects. On the other hand, both Areca II-5-C and captopril decreased the systolic blood pressures of WKY to lesser extents in the same experiment (Fig. 2B). We previously reported that the ACE inhibitory activity of Areca II-5-C, on a weight basis, is about 200 times less potent than that of captoprll (I). The observed difference in relative potencies in vlvo may be due to differences in accessibility to the enzyme or to a second action of Areca II-5-C. Rubin et al. (5) and Ondettl et al. (6) reported that i.v. or oral administration of eaptoprll to normotensive rats produced a graded reduction of the pressor response to i.v. administration of anglotensin I and also a graded augmentation of the hypotenslve action of bradyklnln without affecting the responses to anglotensln II and acetylchollne. Since it has been suggested that ACE and klnlnase II are identical (7,8), these selective actions of captoprll on anglotensln I and bradyklnin in the blood pressure may be due to its specific inhibitory activity of ACE. In order to elucidate the mechanism of the antihypertensive effect of Areca II-5-C, we tested its effects towards several vasoactive compounds (anglotensln I, antlogensin II, bradyklnin, aeetylchollne and norepinephrine) by measuring arterial blood pressure in aortic-llgated anesthetized normotenslve Wistar rats. Typical records of the effects of a single i.v. administration of Areca II-5-C at 5 mg/kg are shown in Fig. 3. When anglotensln I and II were injected, there was the usual rapid rise in pressure. Very soon after the tannin injection, however, the hypertensive responses to both peptldes were somewhat reduced and the effect persisted for over 20 mln. Since the hydrolysis of anglotensin II is not effected by ACE, our observed blockage of the hypertensive action of this peptide by Areea II-5-C is apparently not due to its ability to inhibit ACE. The vasodepressor activities of bradykinin and acetylcholine and the vasopressor activity of noreplnephrine were not appreciably affected by the treatment with Areca II-5-C (Fig. 3). Since bradyklnin is inactivated by ACE, the failure of the tannin to augment the bradyklnin effect is in agreement with our conclusion above. The slight decrease in arterial blood pressure seen in the upper two curves of Fig. 3 after i.v. administration of Areca II-5-C seemed to occur, in these and other instances, when the initial arterial blood pressure before treatment was relatively high. Fig. 4A shows the prompt, graded inhibitory effects of Areca II-5-C on the pressor response to multiple injections of angiotensin I (310 ng/kg). After single i.v. doses of 2, 10 and 15 mg/kg of Areca II-5-C, the half-times for loss of the blocking action of the tannin were I0, 50 and 120 min, respectively. Areca II-5-C also induced a dose-related inhibition on the pressor response to i.v. administration of anglotensin II at a dose of I00 ng/kg (Fig. 4B). None of these treatments significantly affected the heart rates of these animals (data not shown). The results of graphical analysis of Figs. 4A and B are shown in Fig. 5. The maximum inhibitory effects of Areca II-5-C on the pressor responses to i.v. administration of anglotensin I are about two times as strong as those seen

Vol.

38, No. 15, 1986

Tlmeafter

o

10

Antihypertensive

O r a l Admlnisfratlea

20

30

40

60

1379

FIG. I. Blood pressure change in SHR after a single oral dose of Areca II-5-C and captopril. Areca II-5-C ( • , I00 mg/kg; I, 200 mg/kg), captopril ( ~ , 30 mg/kg; o, 100 mg/kg), distilled water as the vehicle (X; 0.5 ml/100 g). Values are means + S.E. of separate determinations with six to eight animals.

(~) S0

Effect of Tannin

70

i

G -20

i

~ -30 Z

!

-4o

]~-50

A Time after

0

1

I.V,

B

2

3

4

l/

0

" E E .q

Time after I.V.

Administretion (hr)

24

0

--

D

:~

1

2

Administration

3

4

(hr) //

0

E -TO

~ -20

|

m -30 ._u o

• -10

/t"---1~1

,fit

~" -2o m -30 " 5'

~

~-~o

-4o

.E

~ -50

Z

-50

e

o

-60

o -60

-70

-70

FIG. 2. Blood pressure changes in SHR (A) and WKY (B) after a single i.v. injection of Areca II-5-C and captopril. Areca II-5-C ( • , I0 mg/kg; Q, 15 mg/kg), captopri~ (o, 15 mg/kg). Saline was the vehicle for captoprIl ( X, 0.I ml/lO0 g); 5% EtOH was the vehicle for Areca II-5-C ([], 0.I ml/100 g). Values are means + S.E. of separate determinations with four to six animals.

24

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Antihypertensive

Effect of Tannin

Vol. 38, No. 15, 1986

mmHg

,5ot'L.

=,oo I

A?eca 'P

I .V. 5 mg/kg

150 jAIl

5Ot t

t

t

t

I'

t

t

t

t

1 g0 t BK I00~

-" 5oj 1501 a~h

llm m m

I00~ t t Im

150 t Norepi.

~.1oo1 50 t

t

t

-10'

t

m

t

t

t

t

imm it= t

t

t

O'

t

+2'

t

t

+5'

t

+I0'

t

+15'

t

t

+20'

T i m e (rain)

3'

FIG. 3. Representative direct blood pressure records from anesthetized normotensive Wistar rats before and after i.v. dosage with Areca II-5-C and vasoactive agents. AI, angiotensin I, 310 ng/kg; All, angiotensln II, I00 ng/kg; BK, bradykinin, i0 ~g/kg; Norepi., norepinephrine, 200 ng/kg. Areca II-5-C was admlnistered i.v. as a 5% solution in EtOH, I ml/kg.

with angiotensin II. This observation suggests that the difference in the pressor responses to angiotensln I and II when Areca II-5-C was administered is due to inhibition of ACE. On the other hand, the inhibitory effect of Areca II-5-C on the pressor response to angiotensin II may be attributed to a receptor antagonistic property or to interference with the action of other vasoconstrictors released by angiotensin (e.g., thromboxane A2). Whatever the mechanisms, the results of these in vlvo experiments show that Areea II-5-C has favorable properties as a hypotensive drug, able to inhibit the pressor responses to both anglotensln I and II. Chemical studies are underway to determine the active components of Areca II-5-C and their chemical structures. This should be useful in elucidating the precise mechanisms of the antihypertenslve action.

Vol. 38, No. 15, 1986

Antihypertensive Effect of Tannin

138]

-,-20

_:

0

0

IBL~

=

-2c -~

/ /

[: I

~.=&- I~/I,.T.TI._..,~? 60

o

00, r

30

2 10

~

7~

Time

+20

9~

~o

150

~0

160

in M i n u t e s

B

o O

m c O C&

-

20

o n,

-40

I e

-

60-

f.

FT

O

c t P

-80

u o

-100

6'~' 210

3o

6~

90

T i m e in M i n u t e s

FIG. 4. Inhibition by Areca II-5-C of pressor responses to i.v. administration of anglotensin I (310 ng/kg) and anglotensln II (!00 ng/kg) in anesthetized normotenslve-Wistar rats. The plotted lines are the mean arterial blood pressures. The Areea II-5-C solution in 5% EtOH ( O , 2 mg/kg; o, 5 mg/kg; e, I0 mg/kg; & , 15 mg/kg) was administered intravenously. The vehicle alone, 5% EtOH, was also injected (X). Values are means + S.E. of separate determinations with four animals.

1382

Antihypertensive Effect of Tannin

Vol. 38, No. 15, 1986

100

=

FIG. 5. Dose-response curves for maximum decreases of pressor responses to i.v. administration of angiotensin I (e) or angiotensin II (o). See Figs. 4A and B. Ai

60 0.

40 Q. C

=

!

2O

/Y I

I

I

I

2

5

10

15

Areca

(mg/kg)

Acknowledgements The valuable comments of Dr. Norman Radin, The University of Michigan, during the preparation of this paper are gratefully acknowledged. We are also grateful to Dr. T. Ueda, The University of Michigan, and Drs. M. Fujiwara and T. Katsuragi, Fukuoka University, for their helpful discussions. This research was supported in part by a Grant-in-Aid for Clinical Research from the Foundation of Fukuoka University Hospital.

References I. 2. 3. 4. 5. 6.

J. INOKUCHI, H. OKABE, T. YAMAUCHI and A. NAGAMATSU, Chem. Pharm. Bull. 32, 3615-3619 (1984). J. INOKUCHI, H. OKABE, T. YAMAUCHI, A. NAGAMATSU, G. NONAKA and I. NISHIOKA, Chem. Pharm. Bull. 33, 264-269 (1985). G. NONAKA, F.-L. HSU and I. NISHIOKA, J. Chem. Soc., Chem. Commun. 1981, 781-783. J.M. PFEFFER, M.A. PFEFFER and E. FROHLICH, J. Lab. Clin. Med. 78, 952962 (1971). B. RUBIN, R.J. LAFFAN, D.G. KOTLER, E.H. O'KEEFE, D.A.DEMAIO AND M.E. GOLDBERG, J. Pharmacol. Exp. Ther. 204, 271-280 (1978). M.A. ONDETTI, B. RUBIN and D.W. CUSHMAN, Science (Washington) 196, 441-444

(1977). 7. 8.

H.Y.T. YANG, E.G. ERDOS and Y. LEVIN, J. Pharmacol. Exp. Ther. 117, 291-300 (1971). E.G. ERDOS, Am. J. M.ed. 60, 749-759 (1976).