Journul of Ethnopharmucology, 33 ( 199 I ) I3Elsevier Scientific Publishers Ireland Ltd.

Direct relaxant

I3

I9

effects of garlic juice on smooth muscles M.B. Aqel, M.N. Gharaibah College of Medicine,

University

(Accepted The effects of garlic juice on smooth

and cardiac

and cardiac

and A.S. Salhab

of Jordan, Amman (Jordan)

October

muscles of rabbit

15, 1990) and guinea

pig were tested in vitro using isolated

segments

of

aorta, trachea and intestines and isolated rabbit hearts. Garlic juice inhibited the contractions of rabbit and guinea pig aortic rings Krebs-Henseleit solutions. Also, garlic juice inhibited the contractions induced by norepinephrine in Ca Z+-free and Ca’+-containing of rabbit and guinea pig tracheal smooth muscles induced by acetylcholine and histamine. respectively, in both Ca’+-free and Ca’+-containing Krebs-Henseleit solutions. Furthermore. garlic juice inhibited the spontaneous movements of rabbit jejunum and guinea pig ileum and inhibited the force of contraction of isolated rabbit hearts in a concentration-dependent manner. All inhibitions were reversible. These data suggest that the hypotensive action of garlic juice may be due. at least in part. to a direct relaxant effect on smooth muscles. Key words: garlic juice;

aorta;

trachea;

intestine;

isolated

rabbit

heart.

Malik and Siddiqui, 1981; Foushee et al., 1982). The hypotensive mechanism, however, has not been investigated. Therefore, the aim of this study was to document the in vitro effect of garlic juice on smooth and cardiac muscle preparations.

Introduction

Garlic, Alhn sativum L. (Liliaceae), has been a mainstay in the medicinal arsenal of many past civilizations, including the Egyptian, Hebrew, Chinese, Greek, Indian, Japanese and Roman (Bolton, 1982; Fenwick and Hanley, 1985). The pharmacological effects of garlic are associated with documented activity as a hypoglycemic and hypolipemic agent (Bordia and Bansal, 1973; Chang and Johnson, 1980; Bordia, 1981), anticoagulant (Jain, 1977; Arisa et al., 1981; Srivastava, 1984), antihypertensive (Malik and Siddiqui, 1981; Ruffin and Hunter, 1983), antimicrobial (Nagai, 1973; Tansye and Appleton, 1975; Handy et al., 1983) and, immune system modulator (Kandil et al., 1987). The hypotensive effect of garlic was recognized 68 years ago (Loeper and Debray, 1921) and has been repeatedly confirmed in human subjects (Damrau, 1941; Petkov, 1979) and animals (Chanderkar and Jain, 1973; Banerjee, 1976; Correspondence to: M.B. Aqel, College of Medicine, of Jordan, Amman, Jordan.

0378-8741603.50 0 1991 Elsevier Published and Printed in Ireland

Scientific

Materials Preparation

Ireland

of garlic juice

Fresh garlic was purchased from a local grocery store and garlic juice was prepared according to the method reported by Foushee et al, (1982). Briefly, the peeled garlic cloves were weighed (500 g), finely chopped with a stainless steel meat grinder and the garlic paste squeezed out through double cheesecloth to obtain a pale extract. The process yielded 110 ml of garlic juice with a solid content of 369 mg/ml. The juice was divided into 5-ml aliquots and stored immediately at -20°C until used. Animal preparation

Rabbits of either sex weighing approximately 1.5 kg and guinea pigs of either sex weighing approximately 0.5 kg were used in these experiments.

University

Publishers

and Methods

Ltd.

14

The animals were of inbred stocks obtained locally. Animals were killed by decapitation under ether anesthesia and the tissues of interest removed immediately and rinsed in Krebs-Henseleit solution. Preparation

of aortic rings

The thoracic aorta was dissected free of the adventitial layer, and cut transversely into rings of 3-4 mm width (Van Breemen, 1977; Meisheri et al., 1981). The aorta was kept well oxygenated and moistened with Krebs-Henseleit solution during the dissection. Twelve rings from six animals were used in these experiments. Each ring was mounted in an organ bath of 50-ml capacity filled with Krebs-Henseleit solution which was kept at 37°C by circulating warm water and continuously bubbled with a 95% 0, and 5% CO2 gas mixture. A passive tension of 1 g was applied (Meisheri et al., 1981) and the rings were allowed to equilibrate for 1 h. Following the eqilibration period, the by adding rings were made to contract norepinephrine (NE) to the organ bath. The contractions of the aortic rings were recorded on a physiological recorder (Physiograph, MK- 1V-P, Narco-Biosystems) using isometric transducers (F-60 myograph, Narco-Biosystems). To determine the effect of garlic juice on the contractions of the aortic rings, 0.5 ml of the juice was added to the organ bath before and after stimulating the aortic rings with NE. Preparation

of tracheal smooth muscle strips

Tracheal tissue was dissected free of the adventitial layer and cut into transverse strips (Silver and Stull, 1984). Trachealis muscle was kept well oxygenated and moistened with Krebs-Henseleit solution during the dissection. Eight strips from four animals were used. Each strip was mounted vertically in an organ bath of 50-ml capacity tilled with Krebs-Henseleit solution kept at 37°C by circulating warm water and continuously bubbled with a 95% O2 and 5% CO2 gas mixture. A passive tension of 1.5 g was applied (Silver and Stull, 1984), and the strips were allowed to equilibrate for 1 h. Following the equilibration period, the by adding strips were made to contract acetylcholine (ACh) or histamine to the organ

bath. The contraction of the trachealis muscle was recorded on the physiological recorder using isometric transducers (as mentioned above). To determine the effect of garlic juice on the contraction of the trachealis muscle, 0.5 ml of the juice was added to the organ bath before and after stimulating the muscle with the proper contractile agent. Preparation

of isolated intestinal segments

Segments of intestine (jejunum or ileum) about 2 cm long were dissected immediately from the sacrificed animal and mounted in an organ bath of 50-ml capacity, tilled with Krebs-Henseleit solution which was kept at 37°C by circulating warm water and was continuously bubbled with 95% O2 and 5% CO, gas mixture. Eight segments from four animals were used. The intestinal segments were allowed to equilibrate for 1 h before starting the experiment. The spontaneous movements of the intestine were recorded on the physiological recorder using isometric transducers. To determine the effect of garlic juice on the spontaneous movements of the intestine, 0.5 ml of the juice was added to the organ bath after the equilibration period. Isolated perfused

rabbit heart

These experiments were performed according to the method of Langendorff (Staff of the Department of Pharmacology, University of Edinburgh, 1968). The heart with at least 1 cm of aorta attached was removed as quickly as possible from 9 animals and placed in a dish containing well oxygenated Ringer-Locke solution at room temperature. The preparation was squeezed several times to remove as much blood as possible. The aorta was dissected free and all other tissues connected to the heart were dissected away. The heart was quickly transferred to the perfusion apparatus where the aorta was tied into a glass cannula. The perfusion fluid was oxygenated Ringer-Locke solution. This solution was applied at a constant pressure from the reservoir and warmed to 37°C by a thermostatically controlled heater. A hook was attached to the ventricle and connected to a lever which was adjusted to record ventricular contractions using a kymograph. Serial dilutions of

dlic!!sb

(0.5ml)

t

t Garlic Juice (0.5 11~1)

(lox,

CP-free

sol.

t

-free ca2+ t

N$ (1U w

sol.

(%M,

t

+ ylo-94) 0.5ml Garlic Juice

Fig. I. A representative record of the effects of garlic juice on the contractions in Ca’+-containing (A) and Ca?+-free Krebs-Henseleit solutions (B). Arrows

of rabbit aortic rings induced show the time of application.

by norepinephrine

(NE)

t Garlic Juice

wash II II

-

t Garlic Juice (0.5 ml)

-I

t iAcll (loM)

700 mo

1 nln.

tt CA'+-free

ACA (10-4~)

“tt Ca*+ +-free Garlic Juice (0.5 1111)

Fig. 2. A representative (ACh) in Caz+containing

record of the effects of garlic juice on the contractions of rabbit tracealis muscle induced solutions (B). Arrows show the time of application. (A) and Ca *+-free Krebs-Henseleit

by acetylcholine

16

stock garlic solution in 0.5-ml amounts were administered by injection through a rubber cap into the perfusion fluid and the response recorded on the kymograph.

juice was added to the organ bath containing a Ca2+-free Krebs-Henseleit solution (0 Ca2+, 2 mM EGTA) 5 min before stimulating the aortic rings with lo4 M NE, it produced a 70-80% inhibition of NE contraction (Fig. 1B).

Results Effect on aortic rings When the garlic juice was added to the organ bath after stimulating the rabbit or guinea pig aortic rings with a submaximal concentration of NE (lo4 M), it inhibited the contractions induced by NE. Addition of 0.5 ml of the juice produced a 70-80% inhibition of NE contraction and this inhibition was reversible after washing (Fig. 1A). It took 15-20 min for the juice to produce its maximum inhibitory effect. In preliminary experiments, when 1 ml of the juice was used, it produced a 100% irreversible inhibition. No inhibition was observed when 0.1 ml of the juice was used. When 0.5 ml of the juice was added to the organ bath, 10 min before a second exposure to the same concentration of NE (lOA M), it produced a 70-80% inhibition of the contraction induced by NE (Fig. 1A). Furthermore, when 0.5 ml of the

/

Effect on trachealis muscles When 0.5 ml of garlic juice was added to the organ bath after stimulating the rabbit trachealis muscle with a submaximal concentration of ACh (lOA M), it produced a 100% inhibition of the contraction (Fig. 2A) and this inhibition was reversible. It took 10-15 min for the juice to produce its maximal effect. When 0.5 ml of the juice was added before the second exposure to the same concentration of ACh, it produced 90-lOOoh inhibition of ACh contraction (Fig. 2A). Furthermore, when 0.5 ml of the juice was added to the organ bath containing a Ca2+-free KrebsHenseleit solution, 5 min before stimulating the rabbit trachealis muscle with 10m4M ACh, it produced a 100% inhibition of ACh contraction (Fig. 2B). When 0.5 ml of the garlic juice was added to the organ bath containing guinea pig trachealis muscles stimulated with a submaximal concentra-

_I

t

f

Garlic Juice (0.5 ml)

Garlic (0.5

Fig. 3. A representative in Ca2+ -containing

Histamine (lo-4M)

Juice ml)

record of the effects of garlic juice on the contractions

(A) and Ca’+-free

t

Krebs-Henseleit

solutions (B). Arrows

of guinea pig trachealis muscle induced by histamine show the time of application.

17

1

luM*19

wn

mq

histamine, it produced a 90-100% inhibition of the contraction induced by histamine (Fig. 3A). This inhibition was reversible. It took 5-10 min for the juice to produce its maximal effect. Furthermore, when 0.5 ml of the juice was added to the organ bath containing a Ca2+-free KrebsHenseleit solution, 5 min before stimulating the guinea pig trachealis muscle with 10e4 M histamine, it produced a 100% inhibition of histamine contraction (Fig. 3B).

11

Fig. 4. Effects of 0.5 ml of garlic juice in a 50-ml perfusion bath on the spontaneous movements of isolated segments of guinea pig ileum (A) and rabbit jejunum (B).

tion of histamine ( 10m4M), it produced a 100% inhibition of the contraction (Fig. 3A). When 0.5 ml of the juice was added to the organ bath before the second exposure to the same concentration of

Effect on intestine When 0.5 ml of garlic juice was added to the organ bath containing segment of rabbit jejunum or guinea pig ileum, it inhibited the spontaneous movements of these tissues. This inhibition was reversible and occurred within 1 min (Fig. 4). Effect on isolated heart When garlic juice was added to the perfused rabbit heart, it inhibited the ventricular contractions and this inhibition was dose-dependent (Fig. 5). Discussion

100 90

00

0.005

0.05

GARLICJUICE

0.5

( ml 1

Fig. 5. Dose-response curve of the inhibitory effects of garlic juice on the contractions of isolated rabbit heart. Values are expressed as percent of maximum response. Plotted values represent the means + S.E.M. of 9 experiments.

The hypotensive action of garlic has been documented in human subjects and animals (Damrau, 1941; Chanderkar and Jain, 1973; Banerjee, 1979; Petkov, 1979; Foushee et al., 1982) but no studies have been done to investigate if this is due to a direct action on the vascular smooth muscles. In the present study, garlic juice has been demonstrated to have a direct action on vascular smooth muscles. It inhibits the contractions of rabbit and guinea pig aortic rings induced by NE in both Ca2+-free and Ca2+-containing KrebsHenseleit solutions. This inhibition takes 15-20 min and is reversible by washing since the tissues then regain their normal sensitivity to NE. This inhibitory effect of garlic juice is not specific for vascular smooth muscles since it also inhibits tracheal and intestinal smooth muscles. Garlic juice inhibits the contractions of rabbit tracheal smooth muscle induced by ACh in both Ca2+-free and Ca2+-containing Krebs-Henseleit solutions. It also inhibited the contractions of guinea pig tracheal smooth muscles induced by histamine in both Ca2+-free and Ca2+-containing

perfusion media. Furthermore, garlic juice completely abolishes the spontaneous movements of rabbit jejunum and guinea pig ileum. The effect of garlic juice on isolated heart was also investigated, and it inhibited the contractions of isolated rabbit heart in a dose-dependent manner. Contraction of smooth and cardiac muscles is dependent upon an increase in the concentration of cytoplasmic free Ca2+ which activates the contractile elements. The source of activator Ca2+ may be extracellular or intracellular. The relative contribution of Ca 2+ from these two sources to contraction, however, depends largely upon the type of muscle tissue and the contractile agent in question, the concentration of the agent and the component (phasic or tonic) of the contractile response being examined (Bolton, 1979; Van Breemen et al., 1982; Ganong, 1985). It seems that garlic extract interferes with the contraction process beyond the cell membrane. This contention is supported by the following observations: (i) it antagonizes the effects of adrenergic, cholinergic and histaminic receptors; (ii) it inhibits the tonic contractions of aorta and trachealis muscle, both of which are dependent on extracellular Ca2+ influx, (iii) it inhibits the phasic contractions of aorta and trachealis muscle in Ca2+-free solution which are dependent on intracellular Ca’+ release; (iv) it inhibits the spontaneous movements of intestines which are dependent on extracellular Ca’+ influx; and (v) it inhibits the contractions of isolated heart, a tissue dependent on extracellular Ca” influx and intracellular Ca’+ release. The extract is known to contain essential oils (Block, 1985) and such oils should have the capacity to penetrate cell membranes. This time for penetration may explain the time that garlic extract needs to produce its maximal effect. Further experiments are needed to identify the active ingredients(s) of garlic and to determine their mechanisms of action. Acknowledgements

This work is supported by a grant from the Deanship of Scientific Research, University of Jor-

dan, Amman. The authors would like to thank Mrs. Suhad Abu Khalaf for her technical assistance and Mrs. Wafaa Al-Absi for typing this manuscript. References

Arisa,T., Oshiba,S. and Tamada, tion inhibitor

in garlic.

Lancer

T. (1981) Platelet 1(8212), 15&151.

aggrega-

Banerjee, A. (1979) Effect of aqueous extract of garlic on arterial blood pressure of normotensive and hypertensive rats. Artery 2, 369-373. Block, E. (1985) The chemistry of garlic and onions. Scienrific American 252, 114-l 19. Bolton, S., Null, G. and Troetel, W.M. (1982) The medical uses of garlic - Fact and fiction. American Pharmacy 22 (8), 4cL-43. Bolton, T. (1979) Mechanisms of action of transmitters and other substances on smooth muscle. Physiological Reviews 59, 6067 18. Bordia, A. (1981) Effect of garlic on blood lipids in patients with coronary heart disease. American Journal of Clinical Nutrition 34, 210&2103. Bordia, A. and Bansal, H. (1973) Essential oil of garlic in prevention of atherosclerosis. Lancer 2, 1491-1492. Chanderkar, A. and Jain, P. (1973) Analysis of hypotensive action of Allium sarivum (garlic). Indian Journal of Physiology and Pharmacology 17, 132- 133. Chang, M. and Johnson, M. (1980) Effect of garlic on carbohydrate metabolism and lipid synthesis in rats. Journalof Nurririon 110, 931-936. Damrau, F. (1941) The use of garlic concentrate in vascular hypertersion. Medical Record 153, 249-25 1. Fenwick, G. and Hanley, A. (1985) The genus Allium: Part 1. Critical Reviews in Food Science and Nutrition 22, 199-27 1. Foushee, D., Ruffm, J. and Banerjee, U. (1982) Garlic as a natural agent for the treatment of hypertension: A preliminary report. Cyrobios 34, 145-152. Ganong, W. (1985) Review of Medical Physiology, Middle East Edition, Librairie du liban, Beirut, pp. 55-58. Hamdy, E., Ahmed, T. and Amin, F. (1983) The role of food additives in the control of some parasites contaminating vegetables. Journal of Egyprian Sociq of Parasitology* 13. 539-545. Jain, R.C. (1977) Effect of garlic on serum lipids, coagulability and tibrinolytic activity. American Journal of Clinical Nurririon 30, 138&1381. Kandil, O.M., Abdulleh, T.H. and Elkadi, A. (1987) Garlic and the immune system in human: Its effect on natural killer cells. Fcderarion Proceedings 46. 44 I. Loeper, M. and Debray, M. (1921) Hypotensive action of tincture of garlic. Progress Medical 36, 391-392. Malik, S. and Siddiqui, S. (1981) Hypotensive effect of freezedried garlic sap in dogs. Journal of Pakistan Medical Association 31, 12-13. Meisheri, K., Hwang, 0. and Van Breemen, C. (1981) Evidence

19 for two seperate Ca*+ pathways in smooth muscle plasmalemma. Journal of Membrane Biology 59, 19-25. Nagai, K. (1973) Experimental studies on the preventive effect of garlic extract against infection with influenza virus. Japanese Journal of Infectious Disease 47, 11 l-l 15. Petkov, U. (1979) Plants with hypotensive, antiatheromatous and coronarodilating action. American Journal of Chinese Medicine 7, 197-236.

RufIin, J. and Hunter, S. (1983) An evaluation of the side effects of garlic as an antihypertensive agents. Cytobios 73, 85-89.

Silver, P.J. and Stull, J.T. (1984) Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KC1 and carbachol. Molecular Pharmacology 25, 261-274.

Srivastava, K. (1984) Aqueous extract of onion, garlic and ginger inhibit platelet aggregation and alter arachidonic acid metabolism. Biomedica et Biochimica Acta 43, 335-346. Staff of the Department of Pharmacology, University of Edinburgh (1968) Pharmacological Experiments on Isolated Preparations. E; & S. Livingstone Ltd., Edinburgh/London. pp 108-111. Tanyse, M.R. and Appleton, J.A. (1975) Inhibition of fungal growth by garlic extract. Mycopathologia 61, 409413. Van Breemen, C. (1971) Calcium requirement for activation of intact aortic smooth muscle. Journal of Physiology 272, 317-329.

Van Breemen, C., Aaronson, P. Loutzenhiser, B. and Meisheri, K. (1982) Calcium fluxes in isolated rabbit aorta and guinea pig taenia cob. Federation Proceedings 41. 2891-2897.

Direct relaxant effects of garlic juice on smooth and cardiac muscles.

The effects of garlic juice on smooth and cardiac muscles of rabbit and guinea pig were tested in vitro using isolated segments of aorta, trachea and ...
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