Jacob Bell
New Member
Jong-Chang TSAI,a Shuli TSAI,b and Weng-Cheng CHANG*,b,c
aDepartment of Physical Education, National Changhua University of Education; Changhua, 500 Taiwan; bDepartment of Physiology, China Medical University; and cDepartment of Sports Medicine, China Medical University; 91 Hsueh-Shih Road, Taichung, 404 Taiwan. Received April 25, 2003; accepted October 10, 2003
The effects of ethanol extracts of three Chinese medicinal plants Dahuang (Rheum palmatum L.), Badou (Croton tiglium L.), and Huomaren (Cannabis sativa L.), on ion transport of the rat intestinal epithelia were stud-ied. Rat intestinal epithelia mounted in an Ussing chamber attached with voltage/current clamp were used for measuring changes of the short-circuit current across the epithelia. The intestinal epithelia were activated with current raised by serosal administration of forskolin 5 mM. Ethanol extracts of the three plants all augmented the current additively when each was added after forskolin. In subsequent experiments, ouabain and bumetanide were added prior to ethanol extracts of these medicinal plants to determine their effect on Na1 and Cl2 move-ment. The results suggest that ethanol extracts of the three medicinal plants may affect the Cl2 movement more directly than Na1 movement in the intestinal epithelial cells. The results provide evidence for the pharmacologic mechanism of the three Chinese medicinal plants on the intestinal tract.
Key words Chinese medicinal plant; ion transport; intestinal epithelia; laxative; Ussing chamber
According to the Chinese medical literature, Dahuang (Rheum palmatum LINN., family Polygonaceae), Badou (Cro¬ton tiglium LINN., family Euphorbiaceae), and Huomaren (Cannabis sativa LINN., family Cannabaceae) have com¬monly been used as laxative agents for a long period.1) De-spite the considerable use of these plants, little is known about their actions on cellular events.
Laxatives act to promote water secretion and facilitate passage of bowel contents. In the intestinal epithelia, water secretion and absorption are fundamental aspects of daily activities.2,3) Water movement across the epithelia is gener-ally driven by an osmotic gradient created by active ion transport.4) Typically, the epithelial cells absorb water by ac¬tive Na1 uptake which is pumped by Na1—K1 ATPase on the serosal side, and secrete water by actively pumping out Cl2 which is powered by the Na1—K1—2Cl2 cotransporter on the serosal side.5,6) The Na1 and Cl2 transport changes and re¬sults in water movement in the intestinal epithelia. This is seen in many cases of diarrhea and is the pharmacologic mechanism of antidiarrheal agents.7–10)
To detect ion transport in epithelia, the Ussing chamber at¬tached with voltage/current clamp for measuring short-cir¬cuit currents across epithelial tissues is a reliable method and has been used for decades. Movement of charged ions across the epithelia generally creates a potential difference between the epithelia.11) When the potential difference is clamped to zero, the short-circuit current is recorded using the voltage-clamp equipment. Fluctuation of the short-circuit current can be monitored and reflects changes of ion across the ep¬ithelia.12,13)
Using this method, our previous work revealed that the ethanol extracts of three Chinese medicinal plants had an-tidiarrheal properties, namely, Qinpi (Fraxini cortex), Kushen (Sophora flavescens, AITON), and Huanglian (Coptis teeta, WALLICH) affect Na1 or Cl2 movement in the rat in¬testinal epithelia.14) Since laxatives and antidiarrheal agents all act primarily on the intestinal tissue, the present study was aimed at discerning the effects of extracts from three Chinese medicinal plants with laxative characteristics on ion transport of the rat intestinal epithelia. The results indicate that the ethanol extracts of the three medicinal plants may affect the Na1—K1—2Cl2 cotransporter more directly than Na1—K1 ATPase on the serosal side of the intestinal epithelial cells. It provides evidence that the three Chinese medicinal plants have laxative properties.
MATERIALS AND METHODS
Chinese Medicinal Plants and Extract Preparation Dried Dahuang (Rheum palmatum L.), Badou (Croton tiglium L.), and Huomaren (Cannabis sativa L.) were pur-chased from the Chinese pharmacy of the China Medical College Hospital. Twenty grams of each dried material was dissolved in 200 ml of ethanol and boiled for 30 min. The su¬pernatants were then collected and concentrated with a vac¬uum evaporator (EYELA N-N SERIAS, Japan) until the vol¬ume was reduced to 5 ml and were stored at 220 °C in a re¬frigerator.
Tissue Preparation Healthy male Sprague—Dawley (SD) rats were obtained from the Laboratory Animal Breed¬ing and Research Center of the National Science Council, Taipei, Taiwan. The rats were maintained under temperature control of 23 °C and kept on a 12-h light—dark cycle, with diet and water supplied ad libitum. Rats weighing 250– 300 g were selected and killed by exposure to ether. In each rat, several 2-cm segments ileum free of Peyer's patches were immediately removed and washed in Kreb's solution. Intact and flat sheets of the ileal epithelia were prepared by cutting along the mesenteric border, and the serosal and muscular layers were peeled away under a binocular microscope.12)
Ussing Chamber and Recording of Short-Circuit Cur-rent The epithelia were mounted between the Ussing chambers (CHM6, W.P.I., Sarasota, FL, U.S.A.). The buffers were oxygenated continuously and maintained at 37°C. An automatic voltage clamp (DVC 1000, W.P.I.), was corrected for fluid resistance between the potential difference-sensing Ag/AgCl electrodes. A second pair of Ag/AgCl electrodes monitored the short-circuit current across the tissue continuously. The bathing solution in the serosal and mucosal cham¬bers was composed of (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, NaH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1, pH 7.4. These chemicals were purchased from Merck (Darmstadt, Germany). The buffer was gassed with 95% O2—5% CO2 before filling the chambers.13)
Experimental Procedure To show the effects of the ex-tracts on short-circuit current of the rat ileal epithelia, forskolin (Sigma, St. Louis, MO, U.S.A.) was first added on serosal side (final concentration 5 mM) of the tissue. The cur-rent rose and was stable after 1–3 min. Ethanol extract of the medicinal plant (25ml) was then added on serosal side about 20 min after forskolin. Changes in the short-circuit cur-rent were recorded continuously during the experiment. To discern whether the movement of sodium or chloride was involved in the effects of the extracts on short-circuit cur-rent across the epithelia, ouabain (an inhibitor of Na1—K1 ATPase, purchased from Sigma) and bumetanide (an in¬hibitor of Na1—K1—2Cl2 cotransporter, purchased from Sigma) were added, respectively, (final concentration 100mM) 20 min after forskolin. In both cases, there was a drop in the current and it was stable after 1–3 min. Ethanol extract of each plant (25 ml) was then added on the serosal side about 10 min after ouabain or bumetanide.
Statistical Analysis Data are expressed as mean6S.D. Comparisons between two groups were performed using Stu¬dent's t-test. A difference of p,0.05 was considered statisti¬cally significant.
RESULTS
Dahuang The ethanol extract of Dahuang further raised the short-circuit current across the forskolin-activated ileal epithelia. The increase in current was 14.3 63.1 mA/cm2 (Table 1). When adding ouabain before the ethanol extract of Dahuang, the current increment was attenuated to 7.7 62.0 mA/cm2, which is significantly different from when Dahuang extract was added alone (p50.02). When adding bumetanide before the ethanol extract of Dahuang, the current increment was reduced to 2.761.9 mA/cm2, and the difference is signifi¬cant (p50.00001). The values with ouabain and bumetanide treatments were significantly different from each other (p 5 0.001) (Fig. 1).
Badou The ethanol extract of Badou also increased the short-circuit current across the forskolin-activated epithelia to 14.763.0mA/cm2 (Table 1). When ouabain was added before the ethanol extract of Badou, the current was 7.8 6 2.5 mA/cm2. The value was significantly different from that when Badou extract was added alone (p50.001). Adding bumetanide before the ethanol extract of Badou attenuated the current increment to 4.5 62.3 mA/cm2, which was significantly different from that with Badou extract alone (p 5 0.00006). The values of ouabain and bumetanide treatment were also significantly different from each other (p50.038) (Fig. 2).
Huomaren The ethanol extract of Huomaren likewise increased the short-circuit current across the forskolin-activated epithelia to 13.262.9mA/cm2 (Table 1). Adding ouabain before the ethanol extract of Huomaren attenuated the current increment to 8.2 61.7 mA/cm2, which was signifi¬cantly different from that when Huomaren extract was added
alone (p50.005). Adding bumetanide before the ethanol ex-tract of Huomaren attenuated the current increment to 5.76 2.2mA/cm2, which was significantly different (p 50.0005) compared with treatment of Huomaren extract alone. The values of ouabain and bumetanide treatment, however, did not differ significantly different from each other (p50.051) (Fig. 3).
DISCUSSION
Dahuang, Badou, and Huomaren are categorized as laxa-tives in the Chinese medical literature.1) They are mainly used to induce diarrhea and facilitate defecation. Our data re-veal that ethanol extracts of these three plants all augment the short-circuit current of the rat ileal epithelia activated by forskolin. Forskolin stimulates the production of cellular cyclic AMP and leads to Cl2 movement, thus increasing the short-circuit current across the epithelia.15) Our results con-firm that extracts of the three plants influence ion transport across the rat ileal epithelia. Moreover, the results are con¬trary to our previous study on three Chinese medicinal plants with antidiarrhea properties. Ethanol extracts of Qinpi (Frax
ini cortex), Kushen (Sphora flavescens, AITON), and Huan¬glian (Coptis teeta, WALLICH) reduced the short-circuit cur
rent of the forskolin-activated rat ileal epithelia.14) It appears that extracts of these Chinese medicinal plants with laxative and antidiarrheal properties exert completely opposite effects on the short-circuit current of the forskolin-activated rat ileal epithelia.
The effects of these three medicinal plants on Na1 and Cl2 transport were further studied by adding ouabain or bumetanide before the addition of the extracts. The results with Dahuang, Badou, and Huomaren were similar. Treat-ment with bumetanide prior to the extracts of these plants di¬minished the current increment caused by the three extracts alone. Bumetanide blocks the Na1—K1—2Cl2 cotransporter and hence retards Cl2 movement toward the lumen. Accord¬ingly, we speculate that the ethanol extracts of Dahuang, Badou, and Huomaren may affect the Na1—K1—2Cl2 cotrans¬porter in the intestinal epithelial cells, facilitate Cl2 move¬ment from the serosal side to the lumen, and hence raise the current across the epithelia. On the other hand, adding ouabain attenuated the current increment caused by the three extracts alone; however, the decrement was less than that with bumetanide treatment. The Na1—K1—2Cl2 cotransporter is commonly attributed to secondary active transport, which must act coupled through the primary active transport, namely, the Na1—K1 ATPase, in many epithelia.16) The cur-rent decrease with ouabain treatment may be due to the fact that ouabain inhibits the Na1—K1 ATPase and then prevents Na1 from moving out of the cytosol to the serosal side to cre¬ate an Na1 gradient for the Na1—K1—2Cl2 cotransporter to exert Cl2 movement. The Cl2 movement from the serosal side to the lumen is hindered and hence the current cannot be elevated further by the extracts of these plants.
The contents of the three plants have been described. Dahuang extract may contain rhein, aloe-emodin, sennoside, and others.1) Rhein was reported to act on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. 17) Sennosides increase paracellular permeability of small molecules and stimulate chloride secretion.18) Aloe-emodin anthrone and rhein anthrone promote intestinal water secretion. 19) Badou contains mainly croton oil that consists of phorbol, crotonic acid, and others.1) Phorbol ester is well known for its action on protein kinase C and thus serial cellular events including Ca21 and ion transport.20) The extract of Huomaren may con¬tain trigonelline, betaine, phytin, and others.1)
It should be noted that adding ethanol extracts of these plants has little effect on the short-circuit current of rat ileal epithelia not activated by forskolin (data not shown). This implies that extracts of these plants may not act directly on the Na1—K1—2Cl2 cotransporter. Changes in the short-circuit current in the epithelia reflect disturbance of the ion transport and consequently the water movement in the tissue. There are many potential stimulators of ion transport and water se¬cretion in the intestinal tissue, notably inflammatory mole¬cules and neuropeptides. They act on the signaling molecules on the cell membrane and subsequently on the ion transport apparatus. Cholera toxin, for example, stimulates the G pro¬tein on the membrane of intestinal epithelial cells, and elicits signal transduction leading to Cl2 movement from the serosa to mucosa and hence water secretion into the intestinal lumen.21–23) In addition, intrinsic factors produced by im¬mune or nervous tissues may participate in the molecular events leading to diarrhea.24) Lipopolysaccharide (LPS) of the Gram-negative bacteria may stimulate the immunocytes or neural cells in the intestinal wall to produce nitric oxide or prostaglandin, and these two molecules are involved in vari¬ous cases of diarrhea.25–29) Proinflammatory cytokines, such as interleukin (IL)-1 and IL-3, stimulate Cl2 secretion,30,31) while the antiinflammatory cytokines IL-4, IL-10, and IL-13 promote intestinal uptake of Na1 and Cl2.32,33) In conclusion, this present study confirms that ethanol extracts of Dahuang, Badou, and Huomaren may have more direct effects on Cl2 movement than on Na1 movement in the rat intestinal epithe¬lia. Nevertheless, further study is needed to discriminate the detailed actions in the intestinal epithelia elicited by these Chinese medicinal plants.
Acknowledgments This study was supported by a grant to J. C. Tsai and W. C. Chang from the National Science Council, Taiwan (NSC89-23 1 6-B-039-002), and from the China Medical University (CMC90-M-01).
REFERENCES
1) Yen C. H., "The Pharmacology of Chinese Plants,", 1st ed., Chin-Yin Publishing, Taipei, 1994 (in Chinese).
2) Powell D. W., Binder H. J., Curran P. F., Am. J. Physiol., 223, 531– 537 (1972).
3) Fondacaro J. D., Am. J. Physiol., 250, G1–G8 (1986).
4) Rabbani G. H., Greenough W. B., "Textbook of Secretory Diarrhea," ed. by Lebenthal E., Duffey M. E., Raven Press, New York, 1990, pp. 233–255.
5) Schultz S. G., Frizzell R. A., Gastroenterology, 63, 161–1 70 (1972).
6) Hawker P. C., Mashiter K. E., Turnberg L. A., Gastroenterology, 74, 1241–1247 (1978).
7) Davidson G. P., Gall D. G., Pertic M., Butler D. G., Hamilton J. R., J. Clin. Invest., 60, 1402–1409 (1977).
8) McEwan G. T., Schousboe B., Skadhauge E., Pflugers Arch., 417, 174–179 (1990).
9) Osman N. E., Westrom B., Karlsson B., Scand. J. Gastroenterol., 33, 1170–1174 (1998).
10) Starkey W. G., Candy D. C., Thornber D., Collins J., Spencer A. J., Os-borne M. P., Stephen J., J. Pediatr. Gastroenterol. Nutr., 10, 361–370 (1990).
11) Field M., Fromm D., Mccoll I., Am. J. Physiol., 220, 1388–1396 (1971).
12) Kurkchubasche A. G., Cardona M., Watkins S. C., Smith S. D., Al-banese C. T., Simmons R. L., Rowe M. I., Ford H. R., Shock, 9, 121– 127 (1998).
13) Gabriel S. E., Davenport S. E., Steagall R. J., Vimal V., Carlson T., Rozhon E. J., Am. J. Physiol., 276, G58–G63 (1999).
14) Tsai J. C., Tsai S., Chang W. C., J. Pharmacol. Sci., in press (2004).
15) Sharp G. W. G., Hynie S., Nature (London), 229, 266–269 (1971).
16) Frizzell R. A., Welsh M. J., Smith P. L., Soc. Gen. Physiol. Ser., 36, 137–145 (1981).
17) Raimondi F., Santoro P., Maiuri L., Londei M., Annunziata S., Cicci-marra F., Rubino A., J. Pediatr. Gastroenterol. Nutr., 34, 529–534 (2002).
18) Leng-Peschlow E., J. Pharm. Pharmacol., 45, 95 1–954 (1993).
19) Yagi T., Yamauchi K., Kuwano S., J. Pharm. Pharmacol., 49, 22–25 (1997).
20) Epple H. J., Fromm M., Riecken E. O., Schulzke J. D., Scand. J. Gas-troenterol., 36, 731–737 (2001).
21) Kimberg D. V., Field M., Johnson J., Henderson A., Gershon E., J. Clin. Invest., 50, 121 8–1230 (1971).
22) Petritsch W., Eherer A. J., Holzer-Petsche U., Hinterleitner T. A., Beubler E., Krejs G. J., Gut, 33, 1174–1178 (1992).
23) Jodal M., Holmgren S., Lundgren O., Sjoqvist A., Gastroenterology, 105, 1286–1293 (1993).
24) Lundgren O., Jodal M., Comp. Biochem. Physiol., 118A, 319–329 (1997).
25) Brunsson I., Sjotlqvist A., Jodal M., Lundgren O., Acta Physiol. Scand., 130, 633–642 (1987).
26) Pugin J., Schurer-Maly C. C., Leturcq D., Moriarty A., Ulevitch R. J.,
Tobias P. S., Proc. Natl. Acad. Sci. U.S.A., 90, 2744–2748 (1993).
27) Konturek P. H., Scand. J. Gastroenterol., 33, 69 1–700 (1998).
28) Izzo A. A., Mascolo N., Capasso F., Digest. Dis. Sci., 43, 1605–1620
(1998).
29) Tyler K., Keith J. C., Jr., Lab. Invest., 80, 1269–1280 (2000).
30) Chiossone D. C., Simon P. L., Smith P. L., Eur. J. Pharmacol., 180, 217–228 (1990).
31) Theodorou V., Eutamene H., Fioramonti J., Junien J. L., Bueno L., Gastroenterology, 106, 1493–1500 (1994).
32) Madsen K. L., Tavernini M. M., Mosmann T. R., Fedorak R. N., Gas-troenterology, 111, 936–944 (1996).
33) Zund G., Madara J. L., Dzus A. L., Awtrey C. S., Colgan S. P., J. Biol. Chem., 271, 7460–7464 (1996).
Source: Effect of Ethanol Extracts of Three Chinese Medicinal Plants with Laxative Properties on Ion Transport of the Rat Intestinal Epithelia
aDepartment of Physical Education, National Changhua University of Education; Changhua, 500 Taiwan; bDepartment of Physiology, China Medical University; and cDepartment of Sports Medicine, China Medical University; 91 Hsueh-Shih Road, Taichung, 404 Taiwan. Received April 25, 2003; accepted October 10, 2003
The effects of ethanol extracts of three Chinese medicinal plants Dahuang (Rheum palmatum L.), Badou (Croton tiglium L.), and Huomaren (Cannabis sativa L.), on ion transport of the rat intestinal epithelia were stud-ied. Rat intestinal epithelia mounted in an Ussing chamber attached with voltage/current clamp were used for measuring changes of the short-circuit current across the epithelia. The intestinal epithelia were activated with current raised by serosal administration of forskolin 5 mM. Ethanol extracts of the three plants all augmented the current additively when each was added after forskolin. In subsequent experiments, ouabain and bumetanide were added prior to ethanol extracts of these medicinal plants to determine their effect on Na1 and Cl2 move-ment. The results suggest that ethanol extracts of the three medicinal plants may affect the Cl2 movement more directly than Na1 movement in the intestinal epithelial cells. The results provide evidence for the pharmacologic mechanism of the three Chinese medicinal plants on the intestinal tract.
Key words Chinese medicinal plant; ion transport; intestinal epithelia; laxative; Ussing chamber
According to the Chinese medical literature, Dahuang (Rheum palmatum LINN., family Polygonaceae), Badou (Cro¬ton tiglium LINN., family Euphorbiaceae), and Huomaren (Cannabis sativa LINN., family Cannabaceae) have com¬monly been used as laxative agents for a long period.1) De-spite the considerable use of these plants, little is known about their actions on cellular events.
Laxatives act to promote water secretion and facilitate passage of bowel contents. In the intestinal epithelia, water secretion and absorption are fundamental aspects of daily activities.2,3) Water movement across the epithelia is gener-ally driven by an osmotic gradient created by active ion transport.4) Typically, the epithelial cells absorb water by ac¬tive Na1 uptake which is pumped by Na1—K1 ATPase on the serosal side, and secrete water by actively pumping out Cl2 which is powered by the Na1—K1—2Cl2 cotransporter on the serosal side.5,6) The Na1 and Cl2 transport changes and re¬sults in water movement in the intestinal epithelia. This is seen in many cases of diarrhea and is the pharmacologic mechanism of antidiarrheal agents.7–10)
To detect ion transport in epithelia, the Ussing chamber at¬tached with voltage/current clamp for measuring short-cir¬cuit currents across epithelial tissues is a reliable method and has been used for decades. Movement of charged ions across the epithelia generally creates a potential difference between the epithelia.11) When the potential difference is clamped to zero, the short-circuit current is recorded using the voltage-clamp equipment. Fluctuation of the short-circuit current can be monitored and reflects changes of ion across the ep¬ithelia.12,13)
Using this method, our previous work revealed that the ethanol extracts of three Chinese medicinal plants had an-tidiarrheal properties, namely, Qinpi (Fraxini cortex), Kushen (Sophora flavescens, AITON), and Huanglian (Coptis teeta, WALLICH) affect Na1 or Cl2 movement in the rat in¬testinal epithelia.14) Since laxatives and antidiarrheal agents all act primarily on the intestinal tissue, the present study was aimed at discerning the effects of extracts from three Chinese medicinal plants with laxative characteristics on ion transport of the rat intestinal epithelia. The results indicate that the ethanol extracts of the three medicinal plants may affect the Na1—K1—2Cl2 cotransporter more directly than Na1—K1 ATPase on the serosal side of the intestinal epithelial cells. It provides evidence that the three Chinese medicinal plants have laxative properties.
MATERIALS AND METHODS
Chinese Medicinal Plants and Extract Preparation Dried Dahuang (Rheum palmatum L.), Badou (Croton tiglium L.), and Huomaren (Cannabis sativa L.) were pur-chased from the Chinese pharmacy of the China Medical College Hospital. Twenty grams of each dried material was dissolved in 200 ml of ethanol and boiled for 30 min. The su¬pernatants were then collected and concentrated with a vac¬uum evaporator (EYELA N-N SERIAS, Japan) until the vol¬ume was reduced to 5 ml and were stored at 220 °C in a re¬frigerator.
Tissue Preparation Healthy male Sprague—Dawley (SD) rats were obtained from the Laboratory Animal Breed¬ing and Research Center of the National Science Council, Taipei, Taiwan. The rats were maintained under temperature control of 23 °C and kept on a 12-h light—dark cycle, with diet and water supplied ad libitum. Rats weighing 250– 300 g were selected and killed by exposure to ether. In each rat, several 2-cm segments ileum free of Peyer's patches were immediately removed and washed in Kreb's solution. Intact and flat sheets of the ileal epithelia were prepared by cutting along the mesenteric border, and the serosal and muscular layers were peeled away under a binocular microscope.12)
Ussing Chamber and Recording of Short-Circuit Cur-rent The epithelia were mounted between the Ussing chambers (CHM6, W.P.I., Sarasota, FL, U.S.A.). The buffers were oxygenated continuously and maintained at 37°C. An automatic voltage clamp (DVC 1000, W.P.I.), was corrected for fluid resistance between the potential difference-sensing Ag/AgCl electrodes. A second pair of Ag/AgCl electrodes monitored the short-circuit current across the tissue continuously. The bathing solution in the serosal and mucosal cham¬bers was composed of (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, NaH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1, pH 7.4. These chemicals were purchased from Merck (Darmstadt, Germany). The buffer was gassed with 95% O2—5% CO2 before filling the chambers.13)
Experimental Procedure To show the effects of the ex-tracts on short-circuit current of the rat ileal epithelia, forskolin (Sigma, St. Louis, MO, U.S.A.) was first added on serosal side (final concentration 5 mM) of the tissue. The cur-rent rose and was stable after 1–3 min. Ethanol extract of the medicinal plant (25ml) was then added on serosal side about 20 min after forskolin. Changes in the short-circuit cur-rent were recorded continuously during the experiment. To discern whether the movement of sodium or chloride was involved in the effects of the extracts on short-circuit cur-rent across the epithelia, ouabain (an inhibitor of Na1—K1 ATPase, purchased from Sigma) and bumetanide (an in¬hibitor of Na1—K1—2Cl2 cotransporter, purchased from Sigma) were added, respectively, (final concentration 100mM) 20 min after forskolin. In both cases, there was a drop in the current and it was stable after 1–3 min. Ethanol extract of each plant (25 ml) was then added on the serosal side about 10 min after ouabain or bumetanide.
Statistical Analysis Data are expressed as mean6S.D. Comparisons between two groups were performed using Stu¬dent's t-test. A difference of p,0.05 was considered statisti¬cally significant.
RESULTS
Dahuang The ethanol extract of Dahuang further raised the short-circuit current across the forskolin-activated ileal epithelia. The increase in current was 14.3 63.1 mA/cm2 (Table 1). When adding ouabain before the ethanol extract of Dahuang, the current increment was attenuated to 7.7 62.0 mA/cm2, which is significantly different from when Dahuang extract was added alone (p50.02). When adding bumetanide before the ethanol extract of Dahuang, the current increment was reduced to 2.761.9 mA/cm2, and the difference is signifi¬cant (p50.00001). The values with ouabain and bumetanide treatments were significantly different from each other (p 5 0.001) (Fig. 1).
Badou The ethanol extract of Badou also increased the short-circuit current across the forskolin-activated epithelia to 14.763.0mA/cm2 (Table 1). When ouabain was added before the ethanol extract of Badou, the current was 7.8 6 2.5 mA/cm2. The value was significantly different from that when Badou extract was added alone (p50.001). Adding bumetanide before the ethanol extract of Badou attenuated the current increment to 4.5 62.3 mA/cm2, which was significantly different from that with Badou extract alone (p 5 0.00006). The values of ouabain and bumetanide treatment were also significantly different from each other (p50.038) (Fig. 2).
Huomaren The ethanol extract of Huomaren likewise increased the short-circuit current across the forskolin-activated epithelia to 13.262.9mA/cm2 (Table 1). Adding ouabain before the ethanol extract of Huomaren attenuated the current increment to 8.2 61.7 mA/cm2, which was signifi¬cantly different from that when Huomaren extract was added
alone (p50.005). Adding bumetanide before the ethanol ex-tract of Huomaren attenuated the current increment to 5.76 2.2mA/cm2, which was significantly different (p 50.0005) compared with treatment of Huomaren extract alone. The values of ouabain and bumetanide treatment, however, did not differ significantly different from each other (p50.051) (Fig. 3).
DISCUSSION
Dahuang, Badou, and Huomaren are categorized as laxa-tives in the Chinese medical literature.1) They are mainly used to induce diarrhea and facilitate defecation. Our data re-veal that ethanol extracts of these three plants all augment the short-circuit current of the rat ileal epithelia activated by forskolin. Forskolin stimulates the production of cellular cyclic AMP and leads to Cl2 movement, thus increasing the short-circuit current across the epithelia.15) Our results con-firm that extracts of the three plants influence ion transport across the rat ileal epithelia. Moreover, the results are con¬trary to our previous study on three Chinese medicinal plants with antidiarrhea properties. Ethanol extracts of Qinpi (Frax
ini cortex), Kushen (Sphora flavescens, AITON), and Huan¬glian (Coptis teeta, WALLICH) reduced the short-circuit cur
rent of the forskolin-activated rat ileal epithelia.14) It appears that extracts of these Chinese medicinal plants with laxative and antidiarrheal properties exert completely opposite effects on the short-circuit current of the forskolin-activated rat ileal epithelia.
The effects of these three medicinal plants on Na1 and Cl2 transport were further studied by adding ouabain or bumetanide before the addition of the extracts. The results with Dahuang, Badou, and Huomaren were similar. Treat-ment with bumetanide prior to the extracts of these plants di¬minished the current increment caused by the three extracts alone. Bumetanide blocks the Na1—K1—2Cl2 cotransporter and hence retards Cl2 movement toward the lumen. Accord¬ingly, we speculate that the ethanol extracts of Dahuang, Badou, and Huomaren may affect the Na1—K1—2Cl2 cotrans¬porter in the intestinal epithelial cells, facilitate Cl2 move¬ment from the serosal side to the lumen, and hence raise the current across the epithelia. On the other hand, adding ouabain attenuated the current increment caused by the three extracts alone; however, the decrement was less than that with bumetanide treatment. The Na1—K1—2Cl2 cotransporter is commonly attributed to secondary active transport, which must act coupled through the primary active transport, namely, the Na1—K1 ATPase, in many epithelia.16) The cur-rent decrease with ouabain treatment may be due to the fact that ouabain inhibits the Na1—K1 ATPase and then prevents Na1 from moving out of the cytosol to the serosal side to cre¬ate an Na1 gradient for the Na1—K1—2Cl2 cotransporter to exert Cl2 movement. The Cl2 movement from the serosal side to the lumen is hindered and hence the current cannot be elevated further by the extracts of these plants.
The contents of the three plants have been described. Dahuang extract may contain rhein, aloe-emodin, sennoside, and others.1) Rhein was reported to act on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. 17) Sennosides increase paracellular permeability of small molecules and stimulate chloride secretion.18) Aloe-emodin anthrone and rhein anthrone promote intestinal water secretion. 19) Badou contains mainly croton oil that consists of phorbol, crotonic acid, and others.1) Phorbol ester is well known for its action on protein kinase C and thus serial cellular events including Ca21 and ion transport.20) The extract of Huomaren may con¬tain trigonelline, betaine, phytin, and others.1)
It should be noted that adding ethanol extracts of these plants has little effect on the short-circuit current of rat ileal epithelia not activated by forskolin (data not shown). This implies that extracts of these plants may not act directly on the Na1—K1—2Cl2 cotransporter. Changes in the short-circuit current in the epithelia reflect disturbance of the ion transport and consequently the water movement in the tissue. There are many potential stimulators of ion transport and water se¬cretion in the intestinal tissue, notably inflammatory mole¬cules and neuropeptides. They act on the signaling molecules on the cell membrane and subsequently on the ion transport apparatus. Cholera toxin, for example, stimulates the G pro¬tein on the membrane of intestinal epithelial cells, and elicits signal transduction leading to Cl2 movement from the serosa to mucosa and hence water secretion into the intestinal lumen.21–23) In addition, intrinsic factors produced by im¬mune or nervous tissues may participate in the molecular events leading to diarrhea.24) Lipopolysaccharide (LPS) of the Gram-negative bacteria may stimulate the immunocytes or neural cells in the intestinal wall to produce nitric oxide or prostaglandin, and these two molecules are involved in vari¬ous cases of diarrhea.25–29) Proinflammatory cytokines, such as interleukin (IL)-1 and IL-3, stimulate Cl2 secretion,30,31) while the antiinflammatory cytokines IL-4, IL-10, and IL-13 promote intestinal uptake of Na1 and Cl2.32,33) In conclusion, this present study confirms that ethanol extracts of Dahuang, Badou, and Huomaren may have more direct effects on Cl2 movement than on Na1 movement in the rat intestinal epithe¬lia. Nevertheless, further study is needed to discriminate the detailed actions in the intestinal epithelia elicited by these Chinese medicinal plants.
Acknowledgments This study was supported by a grant to J. C. Tsai and W. C. Chang from the National Science Council, Taiwan (NSC89-23 1 6-B-039-002), and from the China Medical University (CMC90-M-01).
REFERENCES
1) Yen C. H., "The Pharmacology of Chinese Plants,", 1st ed., Chin-Yin Publishing, Taipei, 1994 (in Chinese).
2) Powell D. W., Binder H. J., Curran P. F., Am. J. Physiol., 223, 531– 537 (1972).
3) Fondacaro J. D., Am. J. Physiol., 250, G1–G8 (1986).
4) Rabbani G. H., Greenough W. B., "Textbook of Secretory Diarrhea," ed. by Lebenthal E., Duffey M. E., Raven Press, New York, 1990, pp. 233–255.
5) Schultz S. G., Frizzell R. A., Gastroenterology, 63, 161–1 70 (1972).
6) Hawker P. C., Mashiter K. E., Turnberg L. A., Gastroenterology, 74, 1241–1247 (1978).
7) Davidson G. P., Gall D. G., Pertic M., Butler D. G., Hamilton J. R., J. Clin. Invest., 60, 1402–1409 (1977).
8) McEwan G. T., Schousboe B., Skadhauge E., Pflugers Arch., 417, 174–179 (1990).
9) Osman N. E., Westrom B., Karlsson B., Scand. J. Gastroenterol., 33, 1170–1174 (1998).
10) Starkey W. G., Candy D. C., Thornber D., Collins J., Spencer A. J., Os-borne M. P., Stephen J., J. Pediatr. Gastroenterol. Nutr., 10, 361–370 (1990).
11) Field M., Fromm D., Mccoll I., Am. J. Physiol., 220, 1388–1396 (1971).
12) Kurkchubasche A. G., Cardona M., Watkins S. C., Smith S. D., Al-banese C. T., Simmons R. L., Rowe M. I., Ford H. R., Shock, 9, 121– 127 (1998).
13) Gabriel S. E., Davenport S. E., Steagall R. J., Vimal V., Carlson T., Rozhon E. J., Am. J. Physiol., 276, G58–G63 (1999).
14) Tsai J. C., Tsai S., Chang W. C., J. Pharmacol. Sci., in press (2004).
15) Sharp G. W. G., Hynie S., Nature (London), 229, 266–269 (1971).
16) Frizzell R. A., Welsh M. J., Smith P. L., Soc. Gen. Physiol. Ser., 36, 137–145 (1981).
17) Raimondi F., Santoro P., Maiuri L., Londei M., Annunziata S., Cicci-marra F., Rubino A., J. Pediatr. Gastroenterol. Nutr., 34, 529–534 (2002).
18) Leng-Peschlow E., J. Pharm. Pharmacol., 45, 95 1–954 (1993).
19) Yagi T., Yamauchi K., Kuwano S., J. Pharm. Pharmacol., 49, 22–25 (1997).
20) Epple H. J., Fromm M., Riecken E. O., Schulzke J. D., Scand. J. Gas-troenterol., 36, 731–737 (2001).
21) Kimberg D. V., Field M., Johnson J., Henderson A., Gershon E., J. Clin. Invest., 50, 121 8–1230 (1971).
22) Petritsch W., Eherer A. J., Holzer-Petsche U., Hinterleitner T. A., Beubler E., Krejs G. J., Gut, 33, 1174–1178 (1992).
23) Jodal M., Holmgren S., Lundgren O., Sjoqvist A., Gastroenterology, 105, 1286–1293 (1993).
24) Lundgren O., Jodal M., Comp. Biochem. Physiol., 118A, 319–329 (1997).
25) Brunsson I., Sjotlqvist A., Jodal M., Lundgren O., Acta Physiol. Scand., 130, 633–642 (1987).
26) Pugin J., Schurer-Maly C. C., Leturcq D., Moriarty A., Ulevitch R. J.,
Tobias P. S., Proc. Natl. Acad. Sci. U.S.A., 90, 2744–2748 (1993).
27) Konturek P. H., Scand. J. Gastroenterol., 33, 69 1–700 (1998).
28) Izzo A. A., Mascolo N., Capasso F., Digest. Dis. Sci., 43, 1605–1620
(1998).
29) Tyler K., Keith J. C., Jr., Lab. Invest., 80, 1269–1280 (2000).
30) Chiossone D. C., Simon P. L., Smith P. L., Eur. J. Pharmacol., 180, 217–228 (1990).
31) Theodorou V., Eutamene H., Fioramonti J., Junien J. L., Bueno L., Gastroenterology, 106, 1493–1500 (1994).
32) Madsen K. L., Tavernini M. M., Mosmann T. R., Fedorak R. N., Gas-troenterology, 111, 936–944 (1996).
33) Zund G., Madara J. L., Dzus A. L., Awtrey C. S., Colgan S. P., J. Biol. Chem., 271, 7460–7464 (1996).
Source: Effect of Ethanol Extracts of Three Chinese Medicinal Plants with Laxative Properties on Ion Transport of the Rat Intestinal Epithelia
