1979, Vol. 37, pp. 37M78, O Hippokrates Verlag GmbH
Wnta
medica
=
pbntPeseach
Metabolic Activation of Sennoside A in Mice K. Sasaki, K. Yarnauchi and S. Kuwano
Key Word Index: Sennoside A; lntraluminal Active Substance; Mice; Rhein anthrone. Abstract
,
The purgative action of rhein anthrone in the alimentary canal was more intense and rapid in onset than that of sennoside A or of its other possible degradation products, when administered directly into the cecum in mouse. Furthermore, rhein anthrone was detected prominently in the large intestine, a t the site of action of orally administered sennoside A. By pretreatment of mice with chloramphenicol, the purgative action of sennoside A was largely suppressed and concurrently the formation of rhein anthrone in the large intestine was reduced. Accordingly, it is concluded that rhein anthrone, formed mainly by intraluminal bacterial action, is the real active substance of sennoside A in mice.
Introduction
Numerous workers have suggested that sennosides, the active principles of senna or rhubarb, are intrinsically in-
active in the form of glucosides and are converted into the real active substances in the intestine by bacterial action. STRAUBand TRIENDL [I] and OKADA [2] postulated that the free oxyanthraquinones such as emodin should cause the purgative action in cats or rabbits, et al. [3, 41 assumed whereas FUJIMURA the aglucone sennidin A as the active substance of sennoside A in mice. SCHMID [5] ascribed the purgative activity of the anthracene glucosides t o the anthrones transported through thesmall intestine and formed in situ. After the investigation on the relative purgative activities of the 1,8-dihydroxyanthraand cene derivatives in mice, FAIRBAIRN Moss [6] suggested that the anthrone form is the active substance in situ and that the sugar moiety prevents absorption of the glucosides from the alimentary canal and further the dianthrone structure itself is an important factor in protecting the anthrone form from and oxidation en route. HARDCASTLE WILKINS [7] found that the motility of the human colon was not changed by intraluminal senna concentrate; but the
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Japan.
Metabolic Activation of Sennoside
Experimental
SIICI, in acidic medium according to the method of AUTERHOFP and SCHERFF[18]. These substances were identified by comparing their m.p., analytical results for carbon and hydrogen, ultraviolet spectra and infrared spectra with those in the literature and by the Rf values on thin-layer chromatograms. Chloramphenicol was purchased from Sankyo Co., Ltd., Tokyo. Diarrheal test in mice The experimental procedure used here was virtually identical with that for senna assay based on an "all-or-none" response in mice [ I l l with described by MILLERand ALEXANDER slight modifications, that is, purgation was observed 8 hour's after dosing and during the test period animals were allowed access to MF pellets of commercial mouse diet supplied by Oriental Yeast Co., Ltd., Tokyo and tap Tvater. The albino mice used were all females of the ICR-JCL strain obtained from CLEA, Japan, Inc.,. Tokyo weighing 20 to 35 g, and were kept at an ambient temperature of 22 to 25O. The anthracene derivatives tested were dissolved in 2010 NaHCO, aqueous solution. These solutions were kept under nitrogen at all times before dosing to protect against oxidation, except for rhein alkali solution, and administered at a dose of 10 mllkg body weight. In preliminary experiments, we ascertained that 2OIo NaHCO, solution not containing anthracene derivatives had no purgative activity but the feces remained solid and dry when it was introduced into the animal's stomach or cecum. The purgative response was usually expressed in the ratio of the number of animals responded to the number of animals tested. As the relative purgative potency, the 50010 purgative dose (ED,,) was determined by the probit method. Concurrently the intensity of purgative effect was also estimated by observing the number of soft or wet feces and the extent of staining on filter paper placed beneath the cages.
Materials Sennoside A was isolated from senna leaves by the method of MIYAMOTO et al. [13] and further purified by silica gel column partition chroma.tography as described by SCHMID and [14]. Sennidin A was prepared by ANGLIKER the hydrolysis of sennoside A with 8 N H,SO, Surgical operation for cecal tubing in mice according to the method of STOLLet al. [Is]. The operation was carried out as reported Rhein anthrone glucoside was obtained by the previously [19]. method of STOLLet al. [16]. Rhein was isolated from rhubarb by the method of MATSUOKA Large intestinal propulsive activity test This test was made by measuring the speed I171 with slight modifications. Rhein anthrone was prepared by the reduction of rhein with of evacuation of BaSO, injected into the cecum
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
introduction of the concentrate, previously incubated with feces or colibacillus, produced colonic peristalsis which was similar to that caused by et rhein anthrone. Recently LEMMEN~ al. [8-101 indicated that rhein and rhein anthrone equally inhibited the absorption of water and Na+ in the rat colon but the sennosides did not show any effect on the colon, and that after metabolism of sennoside A and B in the rat cecum in vivo no rhein anthrone but rhein dianthrone (i. e. sennidin A) and rhein were formed. In these publications, unfortunately, the correlation of the purgative action of the anthracene glucosides with the formation of the active substances in situ has not been fully established. The purpose of the present investigation is to explore the real active substance of sennoside A a t the site of action in mice. I t has been shown t h a t . the purgative potencies of senna preparations estimated by the biological assay, based on the purgative action in mice, are parallel to the laxative effects 1, in normal and constipated humans [I 121.
Sasaki, Yamauchi. Kuwano
372
Results and Discussion
~ ~ I shows b lthat ~sennoside A was found to have no or little purgative activity when administered parenterally Detection and determination of rhein anthrone to mice a t a dose of 40 mg/kg body in the intraluminal contents weight which causes a diarrheal responThe mice to which sennoside A had been fed se of l00O/o in the case of oral ingestion. orally were killed with chloroform and dissected, all the intestines were taken out and cut These results suggest that sennoside A into three segments, namely, small intestines, absorbed into the circulatory system ceca and colons together with recta after liga- would not produce a purgative action ting the respective segments at both sides. The in mice. Next, the possibility was intraluminal contents in each of the three examined that one of the degradation ligated segments of the intestines were allowed then to react with 0.5 ml of 0.1 010 p-nitrosodi- products of sennoside A in the alimenmethylaniline pyridine solution [20] introduced tary canal (Fig.1) might be the intrinsic by injection, pressed out with dissecting forceps form in the intestine. The doses of and washed into a centrifuge tube with 0.5 ml sennidin A, rhein anthrone glucoside, of pyridine. After centrifuging, the reagentrhein anthrone and rhein corresponding anthrone condensation product in the supernatant, if present, was readily detected by thin- to 40 mg/kg of sennoside A were emlayer chromatography according t o LEMLX and ployed, on the assumption that degraCUVEELE 1211. dation of one mol of sennoside A leads ,For determination of rhein anthrone, the to the formation of one mol of sennidin supernatant obtained in the same way as above A or two mol of rhein anthrone glucowas extracted in 4 ml'of d~loroform.The condensation product content of the chloroform side, rhein anthrone and rhein, respectiextract was determined by means of the ab- vely. When administered parenterally, sorbance at 640 nm. their observed activities were very weak. exceDt that of sennidin A. Statistical analysis Although the purgative responses to In comparison of mean values, statistical rhein anthrone glucoside and rhein sipnificance was determined bv the use of the anthrone given orally were practically Student's t-test.
Table I Purgative Activities of Sennoside A and Its Possible Degradation Products Administered b y Various Routes. Administration
Dose (mg/kg)
p.0.
Purgative response* I.V.
i.p.
40.0 25.0 40.1 25.1 26.3
10110 9/10 8/10 8/10 0110
0116 8/10 3/10 3/10 0110
2/10 9/10 0110 4/10 2/10
-
Sennoside A Sennidin A Rhein anthrone glucoside Rhein anthrone Rhein
:' number of
animals responded/number of animals tested.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
in mouse. This was estimated by the lapse of time from the beginning of the injection of 0.1 ml of 20°/0 BaSO, aqueous suspension through the cecal tubing to the first appearance of white feces involving BaSO,.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Metabolic Activation of Sennoside
373
Sasaki, Yamauchi, Kuwano
374
Table I I Purgative Activities of Sennoside A and Sennidin A Administered by Various Routes.
ED,, (mglkg) (95OIo confidence limits) p.0.
Sennoside A Sennidin A
11.1 ( 8.8 16.6 (14.6
1.p.
1.v.
N N
13.8) 19.2)
similar to that to sennoside A, the intensity of their purgative effects was much weaker than with sennoside A. On the other hand, Table I1 shows that there is no essential difference among the oral and parenteral EDk,, values of sennidin A. The intensity of the purgative effect of sennidin A, however, was found to decrease in ohe order of oral, intraperitoneal and intravenous dosage effect, and only if the purgative action of sennoside A would be achieved through the systemic action of sennidin A liberated in the alimentary canal and absorbed, the intravenous EDcavalue of sennidin .A would be considerably higher than might be expected from the oral ED,, value of sennoside A. In addition, the intravenous injection of sennidin A caused the death of mice, whereas excessive oral administration of sennoside A failed to kill mice. From these facts, it seems reasonable to assume that sennoside A ingested orally arrives unchanged at the site of action mainly through the alimentary canal in mice. Although the assumption that purgatives are effective by their action on intestinal motility requires further study, we examined whether or not sennoside A might affect intestinal motility as a propulsive agent. Since the promotion of charcoal evacuation and a significant increase in the propulsive rate exclu-
83.2 (69.5 19.0 (16.6
N
--
94.2) 20.4)
49.8 (42.2 13.5 ( 3.8
N
N
58.1) 16.4)
sively in the large intestine caused by sennoside A were observed by techniques similar to those used in the previous report [19],the large intestinal propulsive activity of sennoside A was tested in mice with the aid of BaSO, aqueous suspension. Table 111 shows that the evacuation of BaSO, injected into the cecum through the cecal tubing 2 hours after the oral administration of sennoside A when the glucoside might arrive in the large intestine, was promoted significantly (P
Wnta
medica
=
pbntPeseach
Metabolic Activation of Sennoside A in Mice K. Sasaki, K. Yarnauchi and S. Kuwano
Key Word Index: Sennoside A; lntraluminal Active Substance; Mice; Rhein anthrone. Abstract
,
The purgative action of rhein anthrone in the alimentary canal was more intense and rapid in onset than that of sennoside A or of its other possible degradation products, when administered directly into the cecum in mouse. Furthermore, rhein anthrone was detected prominently in the large intestine, a t the site of action of orally administered sennoside A. By pretreatment of mice with chloramphenicol, the purgative action of sennoside A was largely suppressed and concurrently the formation of rhein anthrone in the large intestine was reduced. Accordingly, it is concluded that rhein anthrone, formed mainly by intraluminal bacterial action, is the real active substance of sennoside A in mice.
Introduction
Numerous workers have suggested that sennosides, the active principles of senna or rhubarb, are intrinsically in-
active in the form of glucosides and are converted into the real active substances in the intestine by bacterial action. STRAUBand TRIENDL [I] and OKADA [2] postulated that the free oxyanthraquinones such as emodin should cause the purgative action in cats or rabbits, et al. [3, 41 assumed whereas FUJIMURA the aglucone sennidin A as the active substance of sennoside A in mice. SCHMID [5] ascribed the purgative activity of the anthracene glucosides t o the anthrones transported through thesmall intestine and formed in situ. After the investigation on the relative purgative activities of the 1,8-dihydroxyanthraand cene derivatives in mice, FAIRBAIRN Moss [6] suggested that the anthrone form is the active substance in situ and that the sugar moiety prevents absorption of the glucosides from the alimentary canal and further the dianthrone structure itself is an important factor in protecting the anthrone form from and oxidation en route. HARDCASTLE WILKINS [7] found that the motility of the human colon was not changed by intraluminal senna concentrate; but the
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Japan.
Metabolic Activation of Sennoside
Experimental
SIICI, in acidic medium according to the method of AUTERHOFP and SCHERFF[18]. These substances were identified by comparing their m.p., analytical results for carbon and hydrogen, ultraviolet spectra and infrared spectra with those in the literature and by the Rf values on thin-layer chromatograms. Chloramphenicol was purchased from Sankyo Co., Ltd., Tokyo. Diarrheal test in mice The experimental procedure used here was virtually identical with that for senna assay based on an "all-or-none" response in mice [ I l l with described by MILLERand ALEXANDER slight modifications, that is, purgation was observed 8 hour's after dosing and during the test period animals were allowed access to MF pellets of commercial mouse diet supplied by Oriental Yeast Co., Ltd., Tokyo and tap Tvater. The albino mice used were all females of the ICR-JCL strain obtained from CLEA, Japan, Inc.,. Tokyo weighing 20 to 35 g, and were kept at an ambient temperature of 22 to 25O. The anthracene derivatives tested were dissolved in 2010 NaHCO, aqueous solution. These solutions were kept under nitrogen at all times before dosing to protect against oxidation, except for rhein alkali solution, and administered at a dose of 10 mllkg body weight. In preliminary experiments, we ascertained that 2OIo NaHCO, solution not containing anthracene derivatives had no purgative activity but the feces remained solid and dry when it was introduced into the animal's stomach or cecum. The purgative response was usually expressed in the ratio of the number of animals responded to the number of animals tested. As the relative purgative potency, the 50010 purgative dose (ED,,) was determined by the probit method. Concurrently the intensity of purgative effect was also estimated by observing the number of soft or wet feces and the extent of staining on filter paper placed beneath the cages.
Materials Sennoside A was isolated from senna leaves by the method of MIYAMOTO et al. [13] and further purified by silica gel column partition chroma.tography as described by SCHMID and [14]. Sennidin A was prepared by ANGLIKER the hydrolysis of sennoside A with 8 N H,SO, Surgical operation for cecal tubing in mice according to the method of STOLLet al. [Is]. The operation was carried out as reported Rhein anthrone glucoside was obtained by the previously [19]. method of STOLLet al. [16]. Rhein was isolated from rhubarb by the method of MATSUOKA Large intestinal propulsive activity test This test was made by measuring the speed I171 with slight modifications. Rhein anthrone was prepared by the reduction of rhein with of evacuation of BaSO, injected into the cecum
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
introduction of the concentrate, previously incubated with feces or colibacillus, produced colonic peristalsis which was similar to that caused by et rhein anthrone. Recently LEMMEN~ al. [8-101 indicated that rhein and rhein anthrone equally inhibited the absorption of water and Na+ in the rat colon but the sennosides did not show any effect on the colon, and that after metabolism of sennoside A and B in the rat cecum in vivo no rhein anthrone but rhein dianthrone (i. e. sennidin A) and rhein were formed. In these publications, unfortunately, the correlation of the purgative action of the anthracene glucosides with the formation of the active substances in situ has not been fully established. The purpose of the present investigation is to explore the real active substance of sennoside A a t the site of action in mice. I t has been shown t h a t . the purgative potencies of senna preparations estimated by the biological assay, based on the purgative action in mice, are parallel to the laxative effects 1, in normal and constipated humans [I 121.
Sasaki, Yamauchi. Kuwano
372
Results and Discussion
~ ~ I shows b lthat ~sennoside A was found to have no or little purgative activity when administered parenterally Detection and determination of rhein anthrone to mice a t a dose of 40 mg/kg body in the intraluminal contents weight which causes a diarrheal responThe mice to which sennoside A had been fed se of l00O/o in the case of oral ingestion. orally were killed with chloroform and dissected, all the intestines were taken out and cut These results suggest that sennoside A into three segments, namely, small intestines, absorbed into the circulatory system ceca and colons together with recta after liga- would not produce a purgative action ting the respective segments at both sides. The in mice. Next, the possibility was intraluminal contents in each of the three examined that one of the degradation ligated segments of the intestines were allowed then to react with 0.5 ml of 0.1 010 p-nitrosodi- products of sennoside A in the alimenmethylaniline pyridine solution [20] introduced tary canal (Fig.1) might be the intrinsic by injection, pressed out with dissecting forceps form in the intestine. The doses of and washed into a centrifuge tube with 0.5 ml sennidin A, rhein anthrone glucoside, of pyridine. After centrifuging, the reagentrhein anthrone and rhein corresponding anthrone condensation product in the supernatant, if present, was readily detected by thin- to 40 mg/kg of sennoside A were emlayer chromatography according t o LEMLX and ployed, on the assumption that degraCUVEELE 1211. dation of one mol of sennoside A leads ,For determination of rhein anthrone, the to the formation of one mol of sennidin supernatant obtained in the same way as above A or two mol of rhein anthrone glucowas extracted in 4 ml'of d~loroform.The condensation product content of the chloroform side, rhein anthrone and rhein, respectiextract was determined by means of the ab- vely. When administered parenterally, sorbance at 640 nm. their observed activities were very weak. exceDt that of sennidin A. Statistical analysis Although the purgative responses to In comparison of mean values, statistical rhein anthrone glucoside and rhein sipnificance was determined bv the use of the anthrone given orally were practically Student's t-test.
Table I Purgative Activities of Sennoside A and Its Possible Degradation Products Administered b y Various Routes. Administration
Dose (mg/kg)
p.0.
Purgative response* I.V.
i.p.
40.0 25.0 40.1 25.1 26.3
10110 9/10 8/10 8/10 0110
0116 8/10 3/10 3/10 0110
2/10 9/10 0110 4/10 2/10
-
Sennoside A Sennidin A Rhein anthrone glucoside Rhein anthrone Rhein
:' number of
animals responded/number of animals tested.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
in mouse. This was estimated by the lapse of time from the beginning of the injection of 0.1 ml of 20°/0 BaSO, aqueous suspension through the cecal tubing to the first appearance of white feces involving BaSO,.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Metabolic Activation of Sennoside
373
Sasaki, Yamauchi, Kuwano
374
Table I I Purgative Activities of Sennoside A and Sennidin A Administered by Various Routes.
ED,, (mglkg) (95OIo confidence limits) p.0.
Sennoside A Sennidin A
11.1 ( 8.8 16.6 (14.6
1.p.
1.v.
N N
13.8) 19.2)
similar to that to sennoside A, the intensity of their purgative effects was much weaker than with sennoside A. On the other hand, Table I1 shows that there is no essential difference among the oral and parenteral EDk,, values of sennidin A. The intensity of the purgative effect of sennidin A, however, was found to decrease in ohe order of oral, intraperitoneal and intravenous dosage effect, and only if the purgative action of sennoside A would be achieved through the systemic action of sennidin A liberated in the alimentary canal and absorbed, the intravenous EDcavalue of sennidin .A would be considerably higher than might be expected from the oral ED,, value of sennoside A. In addition, the intravenous injection of sennidin A caused the death of mice, whereas excessive oral administration of sennoside A failed to kill mice. From these facts, it seems reasonable to assume that sennoside A ingested orally arrives unchanged at the site of action mainly through the alimentary canal in mice. Although the assumption that purgatives are effective by their action on intestinal motility requires further study, we examined whether or not sennoside A might affect intestinal motility as a propulsive agent. Since the promotion of charcoal evacuation and a significant increase in the propulsive rate exclu-
83.2 (69.5 19.0 (16.6
N
--
94.2) 20.4)
49.8 (42.2 13.5 ( 3.8
N
N
58.1) 16.4)
sively in the large intestine caused by sennoside A were observed by techniques similar to those used in the previous report [19],the large intestinal propulsive activity of sennoside A was tested in mice with the aid of BaSO, aqueous suspension. Table 111 shows that the evacuation of BaSO, injected into the cecum through the cecal tubing 2 hours after the oral administration of sennoside A when the glucoside might arrive in the large intestine, was promoted significantly (P
