Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Received 22 May 2001; in revised form 17 July 2001; accepted 30 July 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reduction of ethanol absorption is also thought to be a convenient and useful means of preventing alcoholic disorders. Our group has reported on many bioactive saponins from herbal medicines with inhibitory effects on blood-ethanol elevation (Yoshikawa and Yamahara, 1996; Yoshikawa et al., 1996a
,b
,c
,d
, 1997
). Recently, sesquiterpenes isolated from the leaves of Laurus nobilis (bay leaf, laurel) such as costunolide (Fig. 1
) and dehydrocostus lactone were found to potently inhibit blood-ethanol elevation in ethanol-loaded rats (Matsuda et al., 1999b
; Yoshikawa et al., 2000
). In addition, costunolide is known to be present in the roots of Saussurea lappa, which are used as a stomachic, and the leaves of Magnolia grandiflora ( el-Feraly and Chan, 1978
; Matsuda et al., 2000
). Investigation of structureactivity relationships revealed that an
-methylene-
-butyrolactone (
-MGBL) moiety of the active constituents was essential for activity. However, it remains unclarified whether the inhibitory effects of the sesquiterpenes and
-MGBL on blood-ethanol elevation depend on either inhibition of alcohol absorption from the digestive tract or acceleration of alcohol metabolic enzyme activity in the liver. In the present study, we examined the basic inhibitory mechanism of costunolide and
-MGBL on blood-ethanol elevation in ethanol-loaded rats.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
Costunolide was isolated from the extract of the leaves of Laurus nobilis using the method reported previously (Matsuda et al., 1999b; Yoshikawa et al., 2000
).
-MGBL and pepsin (from porcine stomach mucosa) were purchased from Sigma Co. Ltd (St Louis, MO, USA). NAD+ was purchased from Oriental Yeast Co. Ltd and other reagents were purchased from Wako Pure Chemical Industries Co., Ltd (Osaka, Japan).
Measurement of blood-ethanol elevation in several routes of ethanol-loaded rats
Test samples were given orally to starved (2022 h) rats (120150 g, body wt). Thirty minutes later, 20% (v/v) ethanol (5 ml/kg) was either given orally (p.o.), injected into the abdominal cavity (i.p.), or injected into duodenum (i.d.) after laparotomy under ether anaesthesia. Blood samples were collected from infra-orbital venous plexus at 15 (i.p.) and 30 min after loading of ethanol (p.o., i.p, i.d.). Blood was immediately mixed with a 10-fold volume of 0.33 M perchloric acid and centrifuged (4°C, 800 g, 10 min). Ethanol in supernatant was determined by an enzymatic method (F-kitTM ethanol; Boehringer, Mannheim, Germany).
Measurement of blood-ethanol elevation in pylorus-ligated rats
Starved (2022 h) rats (120150 g, body wt) were laparotomized under ether anaesthesia, and the pylorus was ligated with suture. Twenty per cent (v/v) ethanol was administered immediately (p.o.) to the pylorus-ligated rats, and blood samples were collected 30 min later. Test samples were given orally 30 min before the operation.
Measurement of liver alcohol dehydrogenase activity
Rats were starved for 40 h to decrease liver glycogen prior to the experiment. Test samples were given orally to the fasted rats (120140 g, body wt), and 30 min thereafter 20% ethanol was given orally (5 ml/kg) to the rats. Thirty minutes later, the abdominal cavity was opened under ether anaesthesia, and the liver was perfused with 10 to 15 ml of 0.25 M ice-cold sucrose solution. The liver was removed and homogenized with a 3-fold volume (liver weight) of a solution (pH 8.4) containing 0.05 M HEPES and 0.33 mM dithiothreitol. The homogenate was centrifuged (4°C, 600 g for 10 min, followed by 8000 g for 10 min). The supernatant was further ultracentrifuged (4°C, 105 000 g, 60 min), and the supernatant (cytosol fraction) was obtained. The activity of alcohol dehydrogenase in the cytosol fraction was determined using a slightly modified version of the method reported by Lumeng et al. (1979). Briefly, the cytosol fraction (20 µl), the protein concentration of which was determined using Lowry's method (Lowry et al., 1951), was added to 180 µl of substrate mixture [the substrate mixture was composed of 1 ml of glycine buffer (0.2 M glycine, 0.4 M NaCl, adjusted to pH 10.0 with 1 M NaOH), 0.1 ml of ethanol (660 mM in water), 0.1 ml of NAD+ (48 mM in water) and 0.7 ml of distilled water] in a 96-well microplate (UV plate, Corning, NY, USA). The mixture was incubated at 25°C for 0 to 5 min and absorbance at 340 nm was measured every 1 min. The enzyme activity (nmol/min/mg of protein) was calculated from the reaction curve plotted from the NADH production and the reaction time.
Measurement of gastric emptying
Test samples were given orally to starved (2022 h) rats (120150 g, body wt). Thirty minutes later, the test mixture consisting of 20% ethanol, 1% CMC-Na, or 20% glucose and 0.05% Phenol Red was given orally (0.6 ml/rat). Thirty minutes later, the stomach was removed, and the quantity of Phenol Red remaining in the stomach was determined according to the method reported previously (Matsuda et al., 1999b).
Measurement of contractile response of pylorus preparation
Rats weighing 400 g were killed by severing both carotid arteries under ether anaesthesia, and the stomach with 2 cm duodenum from the pylorus was removed. The pylorus was carefully cut circularly along the pinched duodenum in Tyrode's solution (138 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 12 mM NaHCO3, 1 mM NaH2PO4, 5.6 mM glucose, pH 7.4). The ring preparation was cut and opened, and the strip obtained (
4 mm wide x 10 mm long) was suspended in an organ bath (6.0 ml) filled with Tyrode's solution and subjected to an initial loading of 1 g. Tyrode's solution was aerated with a 95% O2/5% CO2 gas mixture and maintained at 37°C. Contractions were measured isometrically via a force displacement transducer (Nihon Denki Sanei, Tokyo, Japan) and recorded on a polygraph. After equilibration, 3 M KCl solution was added to the bath (final concentration of K+: 53 mM) and the contractile response was recorded. The strip was washed with Tyrode's solution and this procedure was repeated to confirm a reproducible response.
-MGBL (10 and 20 mM) was added to the bath, and the change in tension was monitored.
Measurement of the gastric fluids in the stomach
Test samples were given orally to starved (2022 h) rats (120150 g, body wt), and 30 min thereafter 20% ethanol (5 ml/kg) was given (p.o.). Thirty minutes later, the stomach was removed and the volume of gastric fluid was measured.
Measurement of gastric fluid factors in pylorus-ligated rats
Rats given samples 30 min earlier were laparotomized under ether anaesthesia, and the pylorus was ligated with a suture. Twenty per cent ethanol was immediately given (p.o.) followed by suturing of the abdominal cavity. Thirty minutes later, rats were killed by cervical dislocation under ether anaesthesia, the stomach removed and the volume of gastric juice measured. Factors in the gastric juice, pH and acidity, were determined using the method reported previously (Matsuda et al., 1999a). Pepsin activity was determined by Anson's method (Anson, 1938
) using a purified pepsin (Sigma) as a standard enzyme. The quantity of hexosamine, an indicator of mucus, was measured by Neuhaus's method using D-glucosamine as a standard (Neuhaus and Letzring, 1957
).
Statistics
Values are expressed as means ± SEM. For statistical analysis, one-way analysis of variance followed by Dunnett's test was used. P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ethanol given orally is partly oxidized in the upper digestive tract (Baraona et al., 2000). Ethanol is then absorbed slowly from the stomach and rapidly from the small intestine (Holt, 1981
). In the present study, blood-ethanol elevation in pylorus-ligated rats was lower than that in normal rats (7.3% against normal rats) in agreement with data from previous studies. Costunolide and
-MGBL did not elicit any significant effects on blood-ethanol elevation in the pylorus-ligated rats. These findings suggested that the compounds block absorption of ethanol from the intestine but not from the stomach and may delay the excretion of ethanol from the stomach to the duodenum. However, both compounds lacked significant effects on blood-ethanol elevation after i.d. injection of ethanol; this finding suggests that they delay the excretion of ethanol from the stomach to the duodenum, but do not inhibit ethanol absorption at the small intestine.
Moreover, using the Phenol Red method, in ethanol-loaded rats, both costunolide and -MGBL dose-dependently delayed gastric emptying, although the effect of
-MGBL was more potent than that of costunolide. This difference appears to be reflected in the intensity of inhibitory activity on blood-ethanol elevation. Costunolide and
-MGBL suppressed gastric emptying in CMC-Na-loaded rats at a high dose (50 mg/kg). In contrast,
-MGBL, but not costunolide, inhibited gastric emptying in glucose-loaded rats. We previously reported that
-MGBL suppressed both the increase of blood-ethanol and glucose in both ethanol- and glucose-loaded rats, but costunolide inhibited only blood-ethanol elevation (Matsuda et al., 1999b
; Yoshikawa et al., 2000
). These findings indicate that the inhibition of blood-ethanol elevation by costunolide and
-MGBL is due to their inhibition of gastric emptying; moreover, the inhibitory effect of costunolide on gastric emptying appears to be more selective than that observed in rats administered glucose.
Low concentrations of ethanol, HCl and high concentrations of NaCl are known to be mild irritants, thus demonstrating the adaptive cytoprotection of gastric mucosa. Moreover, the cytoprotective mechanism of low concentrations of ethanol (20% ethanol) is reported to differ with respect to those of other mild irritants (0.3 M HCl and 5% NaCl), since only ethanol suppresses gastric emptying (Ko et al., 1995). Since this cytoprotection is known to be inhibited by pretreatment with atropine and lidocaine and vagotomy, the vagal nerve is thought to participate in cytoprotection of ethanol (Ko and Cho, 1996
). In the present study, no prominent difference was observed between the rates of gastric emptying in the control groups given 20% ethanol (mild irritant), 1% CMC-Na (non-nutritional meal) and 20% glucose (nutritional meal). However, costunolide and, in particular,
-MGBL showed strong inhibition against ethanol-induced gastric emptying. Costunolide showed a potent protective effect on acidified ethanol-induced gastric lesions (Matsuda et al., 2000
). Several vanilloid analogues such as capsaicin and resiniferatoxin, originated from plants, have been reported to act as irritants (Szallasi and Blumberg, 1989
) and show cytoprotective action against gastric lesions at very low concentrations (Abdel-Salam et al., 1999
). A similar partial structure can be observed in costunolide,
-MGBL (
-lactone) and resiniferatoxin (
-lactol). Costunolide may, therefore, stimulate the gastric mucus as a mild irritant and be capable of enhancing the inhibition of gastric emptying by ethanol in rats. Since costunolide selectively inhibited gastric emptying in ethanol-loaded rats, the inhibitory mechanisms of costunolide on gastric emptying (e.g. role of capsaicin-sensitive vagal afferent nerves etc.) should be further studied.
However, to investigate the direct effects of -MGBL, an active component of costunolide, on pylorus muscle constriction, the effect of the compound on a pylorus strip was investigated using the Magnus method. As a result, a high concentration of
-MGBL (20 mM), estimated according to the effective dose of
-MGBL used (12.5 mg/5 ml/kg), proved to be equal to that required for inhibition of gastric emptying, and elicited contraction of the pylorus strip. This finding suggested that
-MGBL directly constricted the pylorus muscle and delayed gastric emptying as one of the mechanisms of action involved.
In the course of gastric emptying, remarkable stagnations of gastric fluid were observed in normal rats given costunolide and -MGBL. Increases in gastric fluid are thought to dilute concentrations of ethanol and to slow down ethanol absorption. We therefore measured the volume of gastric fluid in rats both with and without ethanol administration. In normal rats, costunolide and
-MGBL increased the gastric fluid volume, and the effect of
-MGBL was almost 2-fold stronger than that of costunolide. Increasing effects of both compounds were also observed in ethanol-loaded rats and these effects were well correlated with those in normal rats. Finally, various gastric factors, such as pH, acidity, pepsin activity and the quantity of hexosamine in pylorus-ligated rats were determined. The pH and acidity did not significantly change in rats given costunolide and
-MGBL. However, pepsin activity and hexosamine contents in rats given costunolide were increased in accordance with the increase in the volume of gastric fluid.
-MGBL also increased both factors, although the increasing effect on hexosamine was not significant. These findings indicate that both compounds accelerate gastric fluid secretion with increases in pepsin and gastric mucus.
In conclusion, the inhibitory effects of costunolide and -MGBL on blood-ethanol elevation are due to inhibition of gastric emptying and dilution of the ethanol concentration by the increased gastric fluid.
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anson, M. L. (1938) The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. Journal of General Physiology 22, 7989.
Baraona, E., Abittan, C. S. and Lieber, C. S. (2000) Contribution of gastric oxidation to ethanol first-pass metabolism in baboons. Alcoholism: Clinical and Experimental Research 24, 946951.[ISI][Medline]
Colombo, G., Agabio, R., Lobina, C., Reali, R., Morazzoni, P., Bombardelli, E. and Gessa, G. L. (1999) Salvia miltiorrhiza extract inhibits alcohol absorption, preference, and discrimination in sP rats. Alcohol 18, 6570.[ISI][Medline]
el-Feraly, F. S. and Chan, Y. M. (1978) Isolation and characterization of the sesquiterpene lactones costunolide, parthenolide, costunolide diepoxide, santamarine, and reynosin from Magnolia grandiflora L. Journal of Pharmaceutical Sciences 67, 347350.[ISI][Medline]
Holt, S. (1981) Observations on the relation between alcohol absorption and the rate of gastric emptying. Canadian Medical Association Journal 124, 267277.[Abstract]
Keung, W. M. and Vallee, B. L. (1993) Daidzin and daidzein suppress free-choice ethanol intake by Syrian Golden hamsters. Proceedings of the National Academy of Sciences of the United States of America 90, 1000810012.[Abstract]
Ko, J. K. and Cho, C. H. (1996) The antilesion actions of anticholinergic agents on ethanol-induced injury in rat stomachs: the importance of gastric vascular integrity and toxicity. Journal of Autonomic Pharmacology 16, 117124.[ISI][Medline]
Ko, J. K., Cho, C. H., Lam., S. K. and Ching, C. K. (1995) The importance of gastric emptying and mucosal folds in the adaptive cytoprotection of mild irritants in rats. Inflammation Research 44, 512522.
Lowry, O. H., Rosebrough, W. J., Farr, A. L. and Randall, R. J. (1951) Determination of protein with the Folin phenol reagent. Journal of Biological Chemistry 193, 265275.
Lumeng, L., Bosron, W. F. and Li, T. K. (1979) Quantitative correlation of ethanol elimination rates in vivo with liver alcohol dehydrogenase activities in fed, fasted and food-restricted rats. Biochemical Pharmacology 28, 15471551.[ISI][Medline]
Matsuda, H., Li, Y. and Yoshikawa, M. (1999a) Gastroprotections of escins Ia, Ib, IIa, and IIb on ethanol-induced gastric mucosal lesions in rats. European Journal of Pharmacology 373, 6370.[ISI][Medline]
Matsuda, H., Shimoda, H., Uemura, T. and Yoshikawa, M. (1999b) Preventive effect of sesquiterpenes from bay leaf on blood ethanol elevation in ethanol-loaded rat: Structure requirement and suppression of gastric emptying. Bioorganic and Medicinal Chemistry Letters 9, 26472652.[Medline]
Matsuda, H., Kageura, T., Inoue, Y., Morikawa, T. and Masayuki, Y. (2000) Absolute stereostructures and syntheses of saussureamines A, B, C, D, and E, amino acidsesquiterpene conjugates with gastroprotective effect, from the roots of Saussurea lappa. Tetrahedron Letters 56, 77637777.
Neuhaus, O. W. and Letzring, M. (1957) Determination of hexosamines in conjunction with electrophoresis on starch. Analytical Chemistry 29, 12301233.[ISI]
Szallasi, A. and Blumberg, P. M. (1989) Resiniferatoxin, a phorbol-related diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience 30, 515520.[ISI][Medline]
Yoshikawa, M. and Yamahara, J. (1996) Inhibitory effect of oleanene-type triterpene oligoglycosides on ethanol absorption: the structure activity relationships. In Saponins Used in Traditional and Modern Medicine, Waller, G. R. and Yamasaki, K. eds, pp. 207218. Plenum Press, New York.
Yoshikawa, M., Murakami, T., Harada, E., Murakami, N., Yamahara, J. and Matsuda, H. (1996a) Bioactive saponins and glycosides. VI. Elatosides A and B, potent inhibitors of ethanol absorption, from the bark of Aralia elata seem. (Araliaceae): The structure-requirement in oleanolic acid glucuronidesaponins for the inhibitory activity. Chemical and Pharmaceutical Bulletin 44, 19151922.
Yoshikawa, M., Murakami, T., Matsuda, H., Ueno, T., Kadoya, M., Yamahara, J. and Murakami, N. (1996b) Bioactive saponins and glycosides. II. Senegae Radix. (2): Chemical structures, hypoglycemic activity, and ethanol absorption inhibitory effect of E-senegasaponin c, Z-senegasaponin c, and Z-senegins II, III and IV. Chemical and Pharmaceutical Bulletin 44, 13051313.
Yoshikawa, M., Murakami, T., Matsuda, H., Yamahara, J., Murakami, N. and Kitagawa, I. (1996c) Bioactive saponins and glycosides. III. Horse chestnut. (1): The structures, inhibitory effects on ethanol absorption, and hypoglycemic activity of escins Ia, Ib, IIa, IIb, and IIIa from the seed of Aesculus hippocastanum L. Chemical and Pharmaceutical Bulletin 44, 14541464.
Yoshikawa, M., Murakami, T., Yoshizumi, S., Murakami, N., Yamahara, J. and Matsuda, H. (1996d) Bioactive saponins and glycosides. V. Acylated polyhydroxyolean-12-ene triterpene oligoglycosides, camelliasaponins A1, A2, B1, B2, C1, and C2, from the seeds of Camellia japonica L.: Structures and inhibitory activity on alcohol absorption. Chemical and Pharmaceutical Bulletin 44, 18991907.
Yoshikawa, M., Shimada, H., Morikawa, T., Yoshizumi, S., Matsumura, N., Murakami, T., Matsuda, H., Hori, K. and Yamahara, J. (1997) Medicinal foodstuffs. VII. On the saponin constituents with glucose and alcohol absorptioninhibitory activity from a food garnish Tonburi, the fruit of Japanese Kochia scoparia (L.) Schrad.: Structures of scoparianosides A, B, and C. Chemical and Pharmaceutical Bulletin 45, 13001305.
Yoshikawa, M., Shimoda, H., Uemura, T., Morikawa, T., Kawahara, Y. and Matsuda, H. (2000) Alcohol absorption inhibitors from bay leaf (Laurus nobilis): Structure-requirements of sesquiterpenes for the activity. Bioorganic and Medicinal Chemistry 8, 20712077.[ISI][Medline]