PHYTOTHERAPY RESEARCH, VOL. 6,84-88 (1992)

Further Studies on the Antidiarrhoeal Activity of Bisnordihydrotoxiferine, a Tertiary Indole Alkaloid in Rodents

G. Thomas*, M. de F. F. Melo Diniz and R. Mukherjee Laboratbrio de Tecnologia FarmacPutica, Universidade Fcderal da Paraiba, Caixa Postal 5009, 58.059-Joio Pessoa, Paraiba. Brazil

The dimeric tertiary indole alkaloid bisnordihydrotoxiferine isolated from the root bark of Shychnos trinervis (Vell.) Mart. inhibited, on i.p. administration, Escherichia coli-induced diarrhoea and cholera toxin-stimulated intestinal fluid accumulation in mice. The respective EDso values were 10.6 and 20.0mg/kg. The activity of bisnordihydrotoxiferine was unaffected by nalorphine or yohimbine, suggesting that the opioid and a,-adrenoceptors respectively, are not important for its antidiarrhoeal action. In smooth muscle studies, bisnordihydrotoxiferine antagonized in a noncompetitive and reversible manner, the contractions produced by histamine, acetylcholine and substance P in guinea-pig ileum and by 5-hydroxytryptamine and arachidonic acid in rat fundic strip. The respective pD; values (mean k SEM) were 5.21 * 0.29, 5.20 k 0.29, 5.35 k 0.12, in the ileum and 4.95 k 0.13 and 3.66 k 0.12 in the fundic strip. The values of slopes of the regression lines differed significantly from unity in all cases. Bisnor was inactive against PGE,-induced contractions in the ileum. The mechanism of action of the alkaloid may be related to nonspecific antagonism of gastrointestinal smooth muscle stimulant activity of several endogenous substances.

Keywords: bisnordihydrotoxiferine; antidiarrhoeal mechanism; smooth muscle relaxation.

INTRODUCTION MATERIALS AND METHODS

The dimeric tertiary indole alkaloid bisnordihydrotoxi- ferine (bisnor) had been isolated from a number of plants of the family Loganiaceae (Verpoorte et al., 1978; Massiot et al., 1983; Melo et al., 1987). In addi- tion to its broad spectrum antimicrobial and antitumour activities (Verpoorte et al., 1978; Melo et al . , 1987), bisnor isolated from Strychnos trinerois (Vell.) Mart. was also found to have antidiarrhoeal activity (Melo et al., 1988). Thus, on intraperitoneal (i.p.) administ- ration in mice, bisnor inhibited normal defaecation, and diarrhoea induced by castor oil, arachidonic acid and magnesium sulphate. The antidiarrhoeal activity was attributed to the suppression of intestinal transit and of electrolyte and water accumulation. The objec- tives of the present work were (1) to evaluate the inhibitory activity of bisnor in Escherichia cofi (E. coli) induced diarrhoea and cholera toxin (chtox) stimulated intestinal fluid accumulation in mice, (2) to observe whether opioid or a,-adrenoceptors participate in the antidiarrhoeal effect of bisnor and (3) to investigate whether the alkaloid inhibits the gastrointestinal stimu- lant action of endogenous substances such as histamine (Hist.), acetylcholine (Ach), substance P (SP), prostag- landin Ez (PGE,), 5-hydroxytryptamine (5-HT) and arachidonic acid ( AA).

* Author to whom correspondence should be addressed.

09.51-418X/92/020084-05 $0.5.00 0 1992 by John Wiley L Sons, Ltd

Chemicals

Acetylcholine chloride, arachidonic acid sodium salt, castor oil, chtox from Vibrio cholera, clonidine hydro- chloride, diphenoxylate hydrochloride, histamine dihydrochloride, 5-hydroxytryptamine hydrochloride, nalorphine hydrochloride, polyvinylpyrrolidone (MW 10 OOO.OO), prostaglandin E,, substance P, and yohim- bine hydrochloride, were purchased from Sigma Chemical Co., St. Louis, MO, USA. E. Coli, batch ATCC 25.922, was a gift from the Department of Tropical Medicine, Federal University of Paraiba.

All other standard laboratory reagents were obtained locally and were of analytical grade.

Animals

Male albino mice (20-25 g) were used for in uiuo experiments. Unless otherwise stated they had free access to water at all times. However, food was with- drawn from some groups at various times before the test depending on the experimental protocol. For stu- dies with isolated tissues, male or female guinea-pigs (350-500 g) or rats (150-190 g) were used.

Methods

Bisnor was suspended in 0.1% Tween 80 for adminis- tering in dose volumes of 10 mL/kg, for in oitro experi- ments and solubilized with polyvinylpyrrolidone using the method described in WHO technical report (1982) for in oitro tests.

Accepted (revised) 3 July 1991

ANTIDIARRHOEAL ACTIVITY OF BISNORDIHYDROTOXIFERINE 8S

E . coli induced diarrhoea. Various doses of bisnor sus- pended in Tween 80 were injected i.p. to groups of six mice 1 h before the oral administration of 0.1 mL sus- pension of E. coli in 0.9% saline (approximately lo9 bacteria/mL). This dose of E. coli was previously found to produce significant diarrhoea in the mice. The ani- mals were placed separately in polythene cages with filter paper at the bottom and 4 h later, the intensity of the diarrhoea was evaluated on an arbitrary scale, depending on the consistency and the number of faeces on the paper (O=no faeces, l= few normal faeces, 2=few soft faeces, 3 = a large number of soft faeces, 4=large number of soft and some watery faeces, as compared with that occurring in the control group which received saline before the bacteria). The mean score obtained with each treated group was compared with the value in the control group. Results were analysed using analysis of variance and were considered significant when probability (p) was <0.05.

Intestinal fluid accumulation. Intestinal fluid secretion was stimulated in mice by administering chtox using the method of Buchheit (1989). Mice were deprived of food for 24 h but had free access to water which was also removed 1 h before and during the experiment. One hour after the i.p. administration of various doses of bisnor, 200 pg of toxin in 1 mL saline was given orally to groups of seven mice. The animals were killed 4 h later and the fluid was collected from the region duodenum to rectum and weighed after discarding the samples with faecal matter. Mean weights of the sam- ples from the bisnor treated groups and the control group were then compared. Statistical analysis was performed as described for E. coli-induced diarrhoea.

Studies on the role of opioid and a,-adrenoceptors in the inhibitory activity of bisnor in castor oil induced diarrhoea. Routine work in the laboratory has shown that castor oil at a dose of 0.1 mL p.o./mouse produces severe diarrhoea which reaches a peak in 2 h, when assessed as described earlier for E. coli induced response. Using this model the possible morphine-like activity of bisnor was investigated as follows. Doses of diphenoxylate and bisnor that inhibited castor oil induced diarrhoea by about 75% were selected. Next, a dose of nalor- phine which abolished diphenoxylate response by approximately 80% was obtained and the following experimental protocol was followed. Five groups of six mice were used. Two groups received nalorphine 50mg/kg i.p. and the others were given 0.9% saline. Thirty min later, the two nalorphine treated groups were injected i.p. with either diphenoxylate (1.25 mg/ kg) or bisnor (12.5 mg/kg) and two saline treated groups also received either diphenoxylate or bisnor in similar doses. The last control group was given saline only. Sixty min later all the animals were dosed with castor oil and the severity of the diarrhoea assessed as before. The role of the opioid receptor on the antidiarr- hoeal activities of diphenoxylate and bisnor was eva- luated by comparing the percent inhibitions of diarr- hoea caused by the two compounds in the presence and in the absence of nalorphine.

For evaluating the role of a,-adrenoceptors, a similar protocol as above was employed, except that the anta- gonist yohimbine (3 mg/kg, i.p.) and a, receptor stimu- lant clonidine (0.5 mg/kg, p.0.) were used, instead of

nalorphine and diphenoxylate, respectively. The effect of yohimbine was evaluated by comparing the anti- diarrhoea1 activity of clonidine and bisnor in groups pretreated with or without the antagonist. Results were analysed using Students t-test, and were considered significant when probability (p) was <0.05.

Isolated tissue experiments. The composition (mM) of salt solutions used for isolated tissues were as follows.

Tyrode solution: NaCl 136.7, NaHCO, 11.9, NaH2P04 0.4, MgS04 7H20 10.5, KC1 2.6, CaCI, 1.8 and glucose 5.5. Kreb’s solution: NaCl 113.0, NaHC03 25.0, KH,P04 1.2, MgS04 7H20 0.60, KC14.8, CaCI, 2.5 and glucose 11.1.

The solutions were bubbled with a 95% O2 + 5% CO, gas mixture. The responses of tissues suspended in 10 mL organ baths under a resting load of 1 g were recorded using isotonic levers, (7-fold magnification) kymographs and smoked drums. Five or six tissues were used for each series of experiments.

Guinea-pig ileum (ileum) was suspended in Tyrode solution at 33-34°C. After a resting period of 30min, two cumulative concentration-response (C/R) curves of similar magnitude were obtained for Hist. A third C/R curve was then obtained in the presence of a concentration of bisnor added 10 min before. The pro- cedure was repeated with different concentrations of bisnor. Antagonism was measured by calculating the pD; values (Ariens and Van Rossum, 1957) defined as the -log concentration to produce a 50% reduction of the maximum. In a similar manner attempts were made to obtain pD; values for agonists Ach, SP and PGE,.

To determine the antagonistic activity of bisnor against AA and 5 HT, rat fundic strips (Vane, 1957) suspended in Kreb’s solution at 37°C were employed. The procedure was similar to that used for the ileum.

RESULTS

Inhibition of diarrhoea and intestinal fluid accumulation

In all the animals of the control group, E. coli caused diarrhoea which peaked in 4 h and persisted for another 2 to 3 h. Previous administration of bisnor significantly reduced the diarrhoea at 4 h in a dose-dependent man- ner. For example, in a typical experiment the diarr- hoeal score of the control group of five mice was 3.1 kO.l (mean k SEM), out of a maximum score of 4. The values were significantly reduced to 2.2 f 0.2, 1 .4 f0 .2 and 0.0 for groups that received 0.25, 12.5, and 25.0 mg/kg bisnor, respectively. The correspond- ing inhibitions were 29.0%, 54.8% and 100.0%. The mean values obtained in four experiments are pre- sented in Table 1.

Bisnor was also effective in chtox induced intestinal fluid accumulation in the mice. Thus, while in the control group of five mice, the weight (mean f SEM) of the fluid was 2.23+0.20g, it was reduced to 2.02f0.20, 1.39k0.14 and 1.03f0.11 g in groups treated with 6.25, 12.5 and 25.0 mg/kg bisnor, respecti- vely. The results were significant for the last two doses

86 G . THOMAS ET AL.

Table 1. Inhibitory activity of bisnor on E. cofi-induced diarr- hoea (experiment 1) and chtox-induced intestinal fluid accumulation (experiment 2) in mice. Inhibitory values are mean k SEM of 4 experiments

Experiment Agent Bisnor % EDw (mglkg i.p.) inhibition lmg/kg)

1 E. coli 6.25 28.6 k 4.3” 12.50 57.1 k4.0” 10.6 25.00 98.0f 2.1“

12.50 34.5 k 4.1” 20.0 25.00 59.0k 3.9”

2 chtox 6.25 8.6 f 3.0

ap<0.05; significantly different from control; analysis of vari- ance.

response by only 12.6% as the percentage reductions of diarrhoea in the control and treated groups were 76.4 f 6.1 and 66.8 f 3.0, respectively. These values were not significantly different.

Clonidine completely (l00.0Y0) abolished the castor oil induced diarrhoea, which in the presence of yohim- bine was reduced significantly to 16.7 f 3.0%. Thus, yohimbine antagonized the clonidine response by 83.3%. On the other hand, yohimbine antagonized the bisnor activity by only 11 .O% as the percent inhibitions of diarrhoea in the control and treated groups were 77.0 k 4.0 and 68.4 f 4.0, respectively. These values were not significantly different.

of bisnor. The percent inhibitions of the control res- ponse by the three doses of bisnor were 9.4%, 37.7%, and 53.8% respectively. The mean inhibition obtained in four experiments are also shown in Table 1 in which it is apparent that bisnor was less potent in chtox than in E. coli induced diarrhoea, when the EDSo values obtained graphically were compared.

Role of opioid and a,-adrenoceptors in the action of bisnor

While in the control group, diphenoxylate reduced the severity of the castor oil induced diarrhoea by 87.0 k 3.0 (mean f SEM), in the nalorphine treated group the value was significantly reduced to 17.5 f 3.0. Nalorphine, thus reversed the diphenoxylate activity by 79.9%. However, nalorphine antagonized the bisnor

Isolated tissue studies

In the ileum, bisnor inhibited C/R curves to Hist, Ach and SP in a concentration dependent manner as shown in Figure 1A and B and in Figure 2 respectively. Bisnor was inactive against PGEz induced contractions at con- centrations up to 1 0 - 4 ~ . The alkaloid antagonized C/R curves to 5 HT and AA in the rat fundic strips (Figure 3A and B, respectively). The values of pD$, slope (regression of Yo reduction of maximum against con- centration of bisnor), and correlation coefficient ( r ) , obtained against the above agonists are summarized in Table 2. As the values for slope differed significantly from unity the antagonism by bisnor was noncompeti- tive in nature. The alkaloid was least active against AA while it was approximately equipotent against the other stimulants. The effect of bisnor was reversible after repeated washings in all cases.

ul c 0 n ul Q) L I E

5

I-

% m

% - #

7.5- 6.5 5.5 4.5 7.5 6 5 5.5 45

- log(H is t ) M -log (Ach) M Figure 1. Effect of bisnor on cumulative CIR curves to Hist (A) and Ach (B) in the ileum. Values are mean f SEM of 6 experiments. Control (0 ) ; Bisnor ( ~ ) : 2 . 2 5 x ( A ) , 4.5 x (A), and 9 . 0 ~ (0).

ANTIDIARRHOEAL ACTIVITY OF BISNORDIHYDROTOXIFERINE 87

50.0-

0.0-

0)

C 0 Q

v)

v) a L

1 100.0

8.5 7:5 6.5 5.5

-lOg(SP)M Figure 2. Effect of bisnor on cumulative C / R curves to SP in the ileum. Values are m e a n f SEM of 6 experiments. Control (0) ; Bisnor (~ ) :1 .25X10- ' ( [7 ) , 2 . 2 5 x l O ~ ' ( A ) . 4 . 5 x 1 0 - ' ( A ) , and 9.ox (0).

100.0

50.0

0.0

A

DISCUSSION

Bisnor was shown to be effective in various models of diarrhoea induced by castor oil, magnesium sulphate and arachidonic acid (Melo et al. , 1988). The present results indicate that the alkaloid is also effective when the causative agents are bacteria or their toxins as exemplified by E. coli and chtox respectively. Thus bisnor with its known broad spectrum antimicrobial activity (Melo et al., 1987) may not only eliminate the infectious agent but also control the symptoms, such as diarrhoea, caused by the microorganism.

Since nalorphine failed to antagonize the actions of bisnor, it may be assumed that the alkaloid does not act like morphine, which is understandable as benzoquino- line and indole structures are chemically different. Further, bisnor has neither central nervous system activity including analgesic effects, nor does it antago- nize the activity of morphine in the mice tail flick test (Melo, 1986).

Clonidine prevents diarrhoea in rats and mice by stimulating a,-adrenoceptors located presynaptically on postganglionic parasympathetic nerves in the intes- tine and on pacemaker neurones of the enteric nervous system; the antidiarrhoeal activity is abolished by a2-adrenoceptor blocking drugs (Ruwart et al., 1980; Doherty and Hancock, 1983; Megens and Niemegeers, 1984; Dettmar et al., 1986). In the present studies, yohimbine in a dose that significantly reduced the clonidine effect, failed to modify the activity of bisnor,

7k 6 5 5:s 4:5

-log(AA)M Figure 3. Effect of bisnor on cumulative CIR curves to 5-HT (A) and AA (B) in the fundic strip. Values are meanfSEM of 5 experiments. A=Control (01, Bisnor ( ~ ) : 2 . 2 5 x l O - ' ( A ) , 4.5xlO-' ( A ) , and 9 . 0 ~ l o - ' (0). B=Control (O) , B i s n o r ( ~ ) : 3 . 0 x l O ~ ~ ( A ) , 6 . 0 x l O ~ ~ ( A ) , a n d 1 . 2 ~ 1 0 - ~ (0).

88 G . THOMAS E T A L .

Table2. The values of antagonistic potency (pD;), slope (regression of ‘/o reduction of maximum against concentration), and correlation coefficient ( r ) obtained for bisnor against Hist, Ach, and SP in the ileum and against 5-HT and AA, in the fundic strip. Results are mean k SEM of 5-6 experiments

Agonist Poi Slope r

Hist 5.21 k0.19 0.93 f 0.20 0.98 k 0.08 Ach 5.20 f 0.29 0.60 k 0.10 0.99 +_ 0.06 SP 5.352 0.12 0.67 k 0.08 0.95 f 0.02 5-HT 4.95 f 0.13 0.82 k 0.10 0.92 f 0.02 AA 3.66? 0.12 0.45 k 0.05 0.99 k 0.07

indicating that a,-adrenoceptors are not important in the antidiarrhoeal actions of bisnor.

One of the ways by which bisnor may prevent diarr- hoea becomes evident from the studies on isolated smooth muscle tissues, as the alkaloid antagonized in a noncompetitive and reversible manner the contractions produced by a number of endogenous substances other than PGE, that are known to control gastrointestinal functions. The antagonism was nonspecific as the pD; values obtained for different agonists were similar except for AA in the rat stomach, even though previous results (Melo et al., 1988) showed that bisnor was most active against AA induced diarrhoea (ED,, 3.3 mg/kg i.p.) in mice. Further studies on intracellular second messengers such as Ca2+, cyclic nucleotides, phosphoi- nositides and diacylglycerol (Watson and Godfrey, 1988; Abdel-Latif, 1989; Kamm and Stull, 1989; Rana

and Hokin, 1990) are needed to clarify the mechanism of action and the reasons for the greater specificity of bisnor in the gastrointestinal smooth muscles when compared with reproductive, respiratory and vascular smooth muscles (Melo, 1986). It may be relevant to report that another tertiary indole alkaloid, longicauda- tine isolated from Strychnos trinervis produces spasmo- lytic effects by modifying calcium metabolism at an intracellular site while it has no effect on voltage dependent or receptor operated calcium channels (Medeiros et al . , 1991).

Diarrhoea is a major cause of child mortality in tropical countries and is treated with antimuscarinics, natural or synthetic opioides, electrolyte solutions and adsorbant chemicals. The therapy is aimed at con- trolling the symptoms but not the causative agent and most of the drugs are not recommended for use in children because of side effects (Feldman and Pickering, 1981). Therefore, novel antidiarrhoeal com- pounds without central effects such as bisnor which also has antimicrobial activity, may be worth studying as prototypes of more useful drugs in the treatment of diarrhoea.

Acknowledgements

We thank Professor Delby F. de Medeiros for encouragement, Mr J . C. Duarte for technical help and Mr 0. V. Leite for typing the manuscript. Financial support was obtained from CNPq and CAPES, Brazil.

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