The main purpose of this study was to investigate the potential laxative effect of propolis. In the present study, WEP, but not EEP, treatment caused a significant increase in stool weight in normal mice. WEP also ameliorated the clonidine-induced constipation, although had no effect on the loperamide-induced constipation model mice (Figures
). Neither WEP nor EEP affected GI transit (Figure
A). These findings indicate that WEP, but not EEP, had a laxative effect, and indicated that the main constituents found in WEP, but not in EEP, may be responsible for the laxative effect of propolis.
As to the main constituents of WEP and EEP, artepillin C (WEP: 0.59%, EEP: 14%), baccharin (WEP: 0.03%, EEP: 6.8%), and drupanin (WEP: 0.12%, EEP: 1.8%) are relatively little in WEP
-coumaric acid (WEP: 3.7%, EEP: 2.5%), 3,4-di-caffeoylqyinic acid (CQA) (WEP: 6.1%, EEP: 3.5%), and 3,5-di-CQA (WEP: 4.9%, EEP: 2.7%) are almost the same amount level in WEP and EEP
], and chlorogenic acid (WEP: 3.6%, EEP: 0.8%) and other hydrophilic chemical constituents are probably larger in WEP than in EEP. Among other minor chemical constituents, naringenin at 150 mg/kg is reported to have laxative effect in a rat loperamide-induced constipation model
]. Considering the effective dose of naringenin in
], micro amount of naringenin in propolis, and the failure of WEP and EEP to have laxative effects in loperamide-induced model in our study, naringenin would not be active constituents of WEP at least in the present study. Taken together, highly hydrophilic compounds including ferulic acid, and isoferulic acid , but not di-caffeoylquinic acids and its metabolites (caffeic acid and quinic acid), might be active constituents of WEP in the present study, however there are no reports showing the laxative effects of these chemical compounds now.
The laxative effect of WEP was seen in the clonidine-induced constipation model, but not in the loperamide-induced model (Figure
A and B). Clonidine and loperamide are agonists of α2
adrenergic receptor and of μ opioid receptor, respectively. The opioid receptor agonists and the α2
receptor agonists inhibit endogenous acetylcholine release as results of inhibitions of adenyl cyclase via
G protein in myenteric plexus, and chronic treatments of opioid receptor agonist or α2
adrenergic receptor agonist are reported to increase expressions of G proteins in gastrointestinal tracts in guinea pig
]. Among opioid agonists, loperamide is classified into piperidine delivatives as well as fentanyl and pethidine. But unlike fentanyl, pethidine, and othe opioid agonists (heroine, morphine, oxycodone, etc.), only loperamide is non-narcotic because it does not reach central nerve system with difficult solubility in water and little absorption into blood flow. Loperamide is estimated to act directly on the intestinal nerve system to induce constipation because loperamide has little central action. On the other hand, clonidine specifically binds to the α2
adrenergic receptor of the brainstem
], and also binds to peripheral adrenergic receptor via
blood flow to relax intestinal smooth muscle to induce constipation. From these points of view, mechanisms of constipation induced by loperamide and clonidine are analogous to each other, apart from absorption into blood flow and direct interaction to Auerbach’s plexus from gastrointestinal tract. These differences might lead to the result we showed in Figure
Subsequent experiments investigated the direct potency of WEP on the small intestine with the Magnus method (Figure
B, C, and D). WEP significantly increased the intestinal tension in a dose-dependent manner. The acute response to WEP suggests that WEP has a direct effect on the gastrointestinal tract in vivo.
We also evaluated the influence of pre-treatments of GR113808, a 5-HT4 receptor antagonist, and atropine, an acetylcholine receptor antagonist on the WEP-induced increase in intestinal tension (Figure
C and D). No effect was observed for GR113808 on the WEP-induced increase in intestinal tension, while atropine treatment significantly reduced the WEP-induced increase. These findings indicated that the WEP-induced increment of intestinal tension is probably not mediated by the 5-HT4 receptor.
In Auerbach's plexus, activated 5-HT4 receptor and 5-HT3 receptor (M receptor) induce endogenous acetylcholine release from parasympathetic nerve, on the other hand activated 5-HT2 receptor (D receptor) directly induces contraction of intestinal smooth muscle. The failure of GR113808 to affect WEP-induce increment of intestinal tension indicates that the acute effect of WEP on intestinal tract was not be mediated by serotonin or serotonin-induced parasympathetic nerve system. From this point of view, WEP may induce the intestinal contraction via mechanism of direct stimulation of muscarinic receptor, endogenous acetylcholine release, or parasympathetic nerve stimulation, which is not mediated by serotonin.
The results obtained with the Magnus method demonstrated that WEP increased the tension of ileum immediately after administration (Figure
B), indicating that unmetabolized ingredients in WEP induced the intestinal smooth muscle contraction, unlike rhein-anthrone which is active metabolite by intestinal flora from sennoside A in Senna alexandrina.
Many types of treatment are available for chronic constipation, including dietary fiber, fluids, and exercise. Effective pharmacological agents can be divided into several categories, including “bulk forming agents” (psyllium, bran, etc.), “stool softeners” (docusate sodium/potassium, etc.), “osmotic agents” (polyethylene glycol-lactulose, sorbitol, etc.), “stimulants” (senna, castor oil, bisacodyl, etc.), and “chloride channel activators” (Lubiprostone)
]. Traditional and herbal medicines are widely used for constipation, and many of these are classified into stimulant types, such as senna (Senna alexandrina
), aloe (Aloe barbadensis miller
), or castor oil (Ricinus communis
). These stimulant-type agents often induce diarrheas as adverse effects at common doses
The present study shows that WEP stimulated the ileum isolated from guinea pigs (Figure
B), on the other hand WEP did not affect gastrointestinal tract in GI transit test (Figure
A). GI transit test is mainly targeted duodenum and jejunum, meaning not ileum, because charcoal travelled less than 60% of the whole small intestine in any groups in Figure
A. Though there is no clearly-defined distinction between jejunum and ileum, ileum is more sensitive to enteral toxins
] and some herbal medicine
] than jejunum. In our previous study, basal tension of jejunum is 1.5-fold stronger than ileum. The difference between Figure
B may attribute to difference of sensitivity between jejunum and ileum.
WEP did not induce an adverse effect of diarrhea in the present experiments (data not shown), unlike senna or castor oil that are reported to induce diarrhea
]. We hypothesized that this is because of differences in target organs and pharmacological activities. The adverse effect of diarrhea is largely caused by inhibition of water absorption in the large intestine. Among the stimulants, senna (Senna alexandrina
, rhein anthron) and aloe (Aloe ferox
, barbaloin) have been identified to stimulate large intestine, which is a water absorptive organ, whereas olive oil and castor oil stimulate small intestine and also inhibits water and nutrition absorption in the small intestine. The data from the present study indicate that WEP does not inhibit absorption and probably act through a different mechanism from that invoked by senna, aloe, or castor oil.