European Journal of Fharmaco~o~,
191 (1990) 97-99
97
Elsevier EIP 20737
Short communication
erfusion of rat colon with sennosides, rhein an eina ~is~a~i~e~~~e~e Giuseppina
Autore, Giuseppe
Caliendo t , Anna Pepe and Francesco
e. Capasso
Department of Experimental Pharmacology and ’ Department of Pharmaceutical Chemisrry_ Via Domenico Montesano 49. 80131 Naples. lraly
Received 18 September 1990, accepted 2 October 1990
Rat colon perfused intraluminally in vitro and in vivo released histamine into the perfusates. Histamine release was increased by rhein 0.1-10 &ml and much more by rheinanthrone 0.1-10 pg./ml but not by sennosides A or B l-10 pg/ml. The effect of rhein and rheinan~rone was reduced by t~to~u~ine 20 mg/kg. This raises the possibility that
laxation by senna and its derivatives involves histamine formation. Sennosides; Rhein; ~~nanthrone:
2. Materials and methods
1. Introduction
Histamine is present in large amounts in the intestinal submucosa and its parenteral administration causes copious intestinal secretion (Lee and Silverberg, 1976), mainly trout vascular dilatation and increased permeability. Excess intestinal histamine release has been also reported to cause secretion in various diarrhoeal diseases. The presence of histaminases in the intestinal mucosa, predominantly at the villous tip, suggests that histamine might have roles in mucosal function and intestinal secretion (Shakir et al., 1977). These findings led to research on the possibility that laxatives act by stimulating histamine biosynthesis. We now describe further studies on histamine formation by rat colon perfused both in vitro and in vivo after stimulation with sennoside A, sennoside B, rhein and rheinanthrone.
Correspondence to: F. Capasso, Department of Experimental Pharmacology. Via Dome&o Montesano 49, 80131 Naples, Italy. 0014-2999/~/$03.50
histamine; Colon (rat)
0 1990
Male Wistar rats (230-250 g). fasted for 24 h but allowed access to water, were killed by cervical dislocation. The colon was removed and rinsed through with Tyrode solution at room temperature. A segment 8 cm long was cannulated at both ends with soft polyethylene tubes and perfused with Tyrode solution (1 ml/min, Watson-Marlow peristaltic pump) with and without sennoslde A, sennoside B, rhein or rheinanthrone. Both the perfusate and the bath fluid were kept at 37°C and equilibrated with Oz/CO, (95 : 5). After 30 min, perfusate (2 ml) was collected and its histamine content was measured ~u~~rnet~~ally (Anton and Sayre, 1969). In other experiments the colon of rats anaesthetized with urethane (10 ml/kg of 10% solution se.) was cannulated in situ. A soft polyethylene tube (about 3 mm outer diameter, 6 cm long) was inserted through a midline incision into the colon about 2 cm distal to the caecum, and the abdomen was closed. The animals were kept warm (23°C) with a heat lamp.
Elsevier Science Publishers B.V. (Biomedical Division)
on was ~~sbed with Tyrode solution until B ~~~~~nt \ws clear. .A second ~I~ethy1ene tube was then inserted t 3 cm into the rectum. and the colon was sed as above for 30 min before collecting 2 ml ~~er~~s~tefor analysis. In two ~xpe~irnel~ts a colon segment was perfused ii~t~~~lumit~ally with Tytode solution in vitro ing added histamine flO0 ng/ml). After 30 histamine in the perfusate was measured as above. Experiments were also carried out in vivo in rats pretreated 2 h earlier with tritoquahne (Hypostamine, Zyms. 20 mg/kg s.c.). a drug known to inhibit histidine decarboxylase (Von Hahn et al.,
TABLE 1 Outputs of histamine into colon perfused with sennoside A, sennoside B. rhein and rhein~throne. The results are means& S.E. The number of observations is given in parentheses. Compounds
Histamine output in vitro, ng/ml
None
30.5 +4.0 (18)
Perfusate in vivo 44.8 * 5.1(12)
Rheinatdrone 0.01 il
0.1 1.0 5.0 10.0
33.1 * 3.3 39.8 & 8.0 61.6k7.1 85.4 + 7.0 F0.3 f 6.0
(4) (6) b (7)’ (6) d (5) d
44.7 rt 7.0 67.5 + 4.4 100.1 f 10.3 145.9 + 10.1 135.5 rt 9.3
(3) (4) (5) (5) (5)
= c d d
31.4*4.9 36.7 + 6.9 51.4+ 7.8 71.45 5.8 60.8 = 8.9
(4) (6) (6) ’ (8) d (4) d
44.3 + 39 58.3& 8.1 87.3+ 7.1 110.6& 7.5 99.7 rc_ 9.5
(4) (4) (6) (5) (4)
h d d d
Rhein 0.01
0.1 1.0 5.0 10.0 Sennoside A
Sennosides A and B were purified by high performance liquid chromatography (HPLC) from crude sennosides available co~ercjally. Rhein was prepared by refluxing pure sennosides A and l3 in a solution of iron-III-hex~ydrate in water overnight (Leng-Peschlow, 1988). Rheinanthrone was prepared by the reduction of rhein with stannous chloride. 2.3. Siatisrics
The results are shown as means + S.E. and were analysed statistically by means of Student’s t-test for paired data (btailed test).
Table 1 illustrates the output of histamine into perfusates. The control perfusate contained small amounts of histamine after 30 min of perfusion. The output was greater in vivo (44.8 f 5.1) than in vitro (30.5 + 4.0). Rheinanthrone 0.1-5.0 pg/ml usually caused a concentration-related increase of histamine in the colonic perfusate in vitro (30190%) and in vivo (51-226s). The lowest concentration (0.01 ~g/ml) had little (8%) or no significant effect. and the response to 10 pg/ml showed no further increase over the effect of 5
1.0 10.0
34.1 + 5.7 (5) 33626.1(5)
44.3 * 7.0 (5) 53.8 Lt 7.4 (5)
35.8 f 6.0 (5) 38.1 + 7.5 (5)
44.1& 7.1 (4) 52.1 f 6.9 (5)
Sennoside B 1.0
10.0 ” pg/ml
perfusate. h P c 0.05. c P -= 0.01, d P r 0.001.
pg/ml (163-20246). Rhein 0.01-10 pg/ml had an effect similar to but weaker than that of rheinanthrone, whereas sennoside A or B 1 and 10 gg/ml had no significant effect. The increased histamine yield seems unlikely to have resulted from decreased histamine degradation. When exogenous histamine was perfused for 30 min and quantified, only 8% of the histamine was found to be metabolized (two experiments). Tritoqua~ne 20 mg/kg S.C. reduced the mean amount of histamine (44.8 f 5.1) in the control by 8% (41.0 &4.5), but reduced the in viva yields in presence of rheinanthrone or rhein (5 pg/ml) by 66% (48.9 +_5.3, P
191 (1990) 97-99
97
Elsevier EIP 20737
Short communication
erfusion of rat colon with sennosides, rhein an eina ~is~a~i~e~~~e~e Giuseppina
Autore, Giuseppe
Caliendo t , Anna Pepe and Francesco
e. Capasso
Department of Experimental Pharmacology and ’ Department of Pharmaceutical Chemisrry_ Via Domenico Montesano 49. 80131 Naples. lraly
Received 18 September 1990, accepted 2 October 1990
Rat colon perfused intraluminally in vitro and in vivo released histamine into the perfusates. Histamine release was increased by rhein 0.1-10 &ml and much more by rheinanthrone 0.1-10 pg./ml but not by sennosides A or B l-10 pg/ml. The effect of rhein and rheinan~rone was reduced by t~to~u~ine 20 mg/kg. This raises the possibility that
laxation by senna and its derivatives involves histamine formation. Sennosides; Rhein; ~~nanthrone:
2. Materials and methods
1. Introduction
Histamine is present in large amounts in the intestinal submucosa and its parenteral administration causes copious intestinal secretion (Lee and Silverberg, 1976), mainly trout vascular dilatation and increased permeability. Excess intestinal histamine release has been also reported to cause secretion in various diarrhoeal diseases. The presence of histaminases in the intestinal mucosa, predominantly at the villous tip, suggests that histamine might have roles in mucosal function and intestinal secretion (Shakir et al., 1977). These findings led to research on the possibility that laxatives act by stimulating histamine biosynthesis. We now describe further studies on histamine formation by rat colon perfused both in vitro and in vivo after stimulation with sennoside A, sennoside B, rhein and rheinanthrone.
Correspondence to: F. Capasso, Department of Experimental Pharmacology. Via Dome&o Montesano 49, 80131 Naples, Italy. 0014-2999/~/$03.50
histamine; Colon (rat)
0 1990
Male Wistar rats (230-250 g). fasted for 24 h but allowed access to water, were killed by cervical dislocation. The colon was removed and rinsed through with Tyrode solution at room temperature. A segment 8 cm long was cannulated at both ends with soft polyethylene tubes and perfused with Tyrode solution (1 ml/min, Watson-Marlow peristaltic pump) with and without sennoslde A, sennoside B, rhein or rheinanthrone. Both the perfusate and the bath fluid were kept at 37°C and equilibrated with Oz/CO, (95 : 5). After 30 min, perfusate (2 ml) was collected and its histamine content was measured ~u~~rnet~~ally (Anton and Sayre, 1969). In other experiments the colon of rats anaesthetized with urethane (10 ml/kg of 10% solution se.) was cannulated in situ. A soft polyethylene tube (about 3 mm outer diameter, 6 cm long) was inserted through a midline incision into the colon about 2 cm distal to the caecum, and the abdomen was closed. The animals were kept warm (23°C) with a heat lamp.
Elsevier Science Publishers B.V. (Biomedical Division)
on was ~~sbed with Tyrode solution until B ~~~~~nt \ws clear. .A second ~I~ethy1ene tube was then inserted t 3 cm into the rectum. and the colon was sed as above for 30 min before collecting 2 ml ~~er~~s~tefor analysis. In two ~xpe~irnel~ts a colon segment was perfused ii~t~~~lumit~ally with Tytode solution in vitro ing added histamine flO0 ng/ml). After 30 histamine in the perfusate was measured as above. Experiments were also carried out in vivo in rats pretreated 2 h earlier with tritoquahne (Hypostamine, Zyms. 20 mg/kg s.c.). a drug known to inhibit histidine decarboxylase (Von Hahn et al.,
TABLE 1 Outputs of histamine into colon perfused with sennoside A, sennoside B. rhein and rhein~throne. The results are means& S.E. The number of observations is given in parentheses. Compounds
Histamine output in vitro, ng/ml
None
30.5 +4.0 (18)
Perfusate in vivo 44.8 * 5.1(12)
Rheinatdrone 0.01 il
0.1 1.0 5.0 10.0
33.1 * 3.3 39.8 & 8.0 61.6k7.1 85.4 + 7.0 F0.3 f 6.0
(4) (6) b (7)’ (6) d (5) d
44.7 rt 7.0 67.5 + 4.4 100.1 f 10.3 145.9 + 10.1 135.5 rt 9.3
(3) (4) (5) (5) (5)
= c d d
31.4*4.9 36.7 + 6.9 51.4+ 7.8 71.45 5.8 60.8 = 8.9
(4) (6) (6) ’ (8) d (4) d
44.3 + 39 58.3& 8.1 87.3+ 7.1 110.6& 7.5 99.7 rc_ 9.5
(4) (4) (6) (5) (4)
h d d d
Rhein 0.01
0.1 1.0 5.0 10.0 Sennoside A
Sennosides A and B were purified by high performance liquid chromatography (HPLC) from crude sennosides available co~ercjally. Rhein was prepared by refluxing pure sennosides A and l3 in a solution of iron-III-hex~ydrate in water overnight (Leng-Peschlow, 1988). Rheinanthrone was prepared by the reduction of rhein with stannous chloride. 2.3. Siatisrics
The results are shown as means + S.E. and were analysed statistically by means of Student’s t-test for paired data (btailed test).
Table 1 illustrates the output of histamine into perfusates. The control perfusate contained small amounts of histamine after 30 min of perfusion. The output was greater in vivo (44.8 f 5.1) than in vitro (30.5 + 4.0). Rheinanthrone 0.1-5.0 pg/ml usually caused a concentration-related increase of histamine in the colonic perfusate in vitro (30190%) and in vivo (51-226s). The lowest concentration (0.01 ~g/ml) had little (8%) or no significant effect. and the response to 10 pg/ml showed no further increase over the effect of 5
1.0 10.0
34.1 + 5.7 (5) 33626.1(5)
44.3 * 7.0 (5) 53.8 Lt 7.4 (5)
35.8 f 6.0 (5) 38.1 + 7.5 (5)
44.1& 7.1 (4) 52.1 f 6.9 (5)
Sennoside B 1.0
10.0 ” pg/ml
perfusate. h P c 0.05. c P -= 0.01, d P r 0.001.
pg/ml (163-20246). Rhein 0.01-10 pg/ml had an effect similar to but weaker than that of rheinanthrone, whereas sennoside A or B 1 and 10 gg/ml had no significant effect. The increased histamine yield seems unlikely to have resulted from decreased histamine degradation. When exogenous histamine was perfused for 30 min and quantified, only 8% of the histamine was found to be metabolized (two experiments). Tritoqua~ne 20 mg/kg S.C. reduced the mean amount of histamine (44.8 f 5.1) in the control by 8% (41.0 &4.5), but reduced the in viva yields in presence of rheinanthrone or rhein (5 pg/ml) by 66% (48.9 +_5.3, P
