Repeated psychological stress, chronic vicarious social defeat stress, evokes irritable bowel syndrome-like symptoms in mice.

Increasing evidence has demonstrated that emotional states and intestinal conditions are inter-connected in so-called "brain-gut interactions." Indeed, many psychiatric disorders are accompanied by gastrointestinal symptoms, such as the irritable bowel syndrome (IBS). However, the functional connection remains elusive, partly because there are few useful experimental animal models. Here, we focused on a highly validated animal model of stress-induced psychiatric disorders, such as depression, known as the chronic vicarious social defeat stress (cVSDS) model mice, which we prepared using exposure to repeated psychological stress, thereafter examining their intestinal conditions. In the charcoal meal test and the capsaicin-induced hyperalgesia test, cVSDS model mice showed a significantly higher intestinal transit ratio and increased visceral pain-related behaviors, respectively. These changes persisted over one month after the stress session. On the other hand, the pathological evaluations of the histological and inflammatory scores of naive and cVSDS model mice did not differ. Furthermore, keishikashakuyakuto-a kampo medicine clinically used for the treatment of IBS-normalized the intestinal motility change in cVSDS model mice. Our results indicate that cVSDS model mice present IBS-like symptoms such as chronic intestinal peristaltic changes and abdominal hyperalgesia without organic lesion. We therefore propose the cVSDS paradigm as a novel animal model of IBS with wide validity, elucidating the correlation between depressive states and intestinal abnormalities.

The rodent model of impaired gastric motility induced by allyl isothiocyanate, a pungent ingredient of wasabi, to evaluate therapeutic agents for functional dyspepsia.

Functional dyspepsia (FD) is thought to be mainly based on gastric motility dysfunction and chronic hypersensitivity, yet FD animal models has been reported a few. We studied to establish the mouse model of impaired gastric motility induced by a pungent ingredient of wasabi allyl isothiocyanate (AITC), which is reliable to evaluate prokinetic agents. Male ddY mice were used. Gastric motility was measured by 13C-acetic acid breath test in conscious mice. AITC (80 mM) was given 60 min before the measurement of motility. Prokinetic agents including itopride (30, 100 mg/kg), mosapride (0.1-1 mg/kg), neostigmine (30 µg/kg), acotiamide (10-100 mg/kg), and daikenchuto (100-1000 mg/kg) were given 40 min before the measurement. AITC impaired gastric motility without mucosal damages, which reverted 24 h after AITC treatment. The decreased motility induced by AITC was restored by prokinetic agents such as itopride, mosapride, neostigmine, and acotiamide. In separate experiment, daikenchuto recovered the decreased motility induced by AITC, although daikenchuto had no effect on motility in normal condition. In conclusion, it is considered that the AITC-induced impaired gastric motility mouse model is useful to develop new prokinetic agents for treatment of FD, and to re-evaluate traditional Japanese herbal medicines.

Orally active opioid µ/δ dual agonist MGM-16, a derivative of the indole alkaloid mitragynine, exhibits potent antiallodynic effect on neuropathic pain in mice.

(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active constituent of the traditional herbal medicine Mitragyna speciosa, is an indole alkaloid that is structurally different from morphine. 7-Hydroxymitragynine induces a potent antinociceptive effect on mouse acute pain through µ-opioid receptors. In this study, we developed dual-acting µ- and δ-opioid agonists MGM-15 and MGM-16 from 7-hydroxymitragynine for the treatment of acute and chronic pain. MGM-16 showed a higher potency than that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo assays. MGM-16 exhibited a high affinity for µ- and δ-opioid receptors, with K(i) values of 2.1 and 7.0 nM, respectively. MGM-16 showed µ- and δ-opioid full agonistic effects in a guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding assay and in a functional test using electrically elicited guinea pig ileum and mouse vas deferens contractions. Systemic administration of MGM-16 produced antinociceptive effects in a mouse acute pain model and antiallodynic effects in a chronic pain model. The antinociceptive effect of MGM-16 was approximately 240 times more potent than that of morphine in a mouse tail-flick test, and its antiallodynic effect was approximately 100 times more potent than that of gabapentin in partial sciatic nerve-ligated mice, especially with oral administration. The antinociceptive effect of MGM-16 was completely and partially blocked by the µ-selective antagonist ß-funaltrexamine hydrochloride (ß-FNA) and by the δ-selective antagonist naltrindole, respectively, in a tail-flick test. The antiallodynic effect of MGM-16 was completely blocked by ß-FNA and naltrindole in a neuropathic pain model. These findings suggest that MGM-16 could become a class of a compound with potential therapeutic utility for treating neuropathic pain.

Dietary agonists of TRPV1 inhibit gastric acid secretion in mice.

Capsaicin and 6-gingerol, pungent components of chilli pepper and ginger, are known as dietary agonists of transient receptor potential vanilloid-1. Transient receptor potential vanilloid-1 nerve fibers are recognized to play a role in gastric mucosal integrity in rats. In the present studies, we examined the acute effects of peroral administration of capsaicin and 6-gingerol on gastric acid secretion in conscious mice. These agents were given p. o. 30 min before the pylorus was ligated. Oral administration of capsaicin (1.0-100 mg/kg) or 6-gingerol (1.5-50 mg/kg) significantly and dose-dependently inhibited basal acid secretion. Pretreatment with BCTC, a transient receptor potential vanilloid-1 antagonist, significantly reversed the reduced basal acid secretion by capsaicin or 6-gingerol. The combination of the lowest doses of capsaicin and 6-gingerol markedly inhibited basal acid secretion in conscious mice and this was also significantly reversed by BCTC. Moreover, the combination of the maximal dose of capsaicin and 6-gingerol inhibited basal acid secretion only to the level of a single administration of the maximal dose of capsaicin. These results suggest that the combination of capsaicin and 6-gingerol has an additive effect on the inhibition of gastric acid secretion through activation of transient receptor potential vanilloid-1. In separate experiments, intraduodenal administration of either capsaicin (30 mg/kg) or 6-gingerol (15 mg/kg), whose doses were observed to have a significant inhibitory effect by oral administration, tended to inhibit basal acid secretion compared with the vehicle. These results suggest that the combination of capsaicin and 6-gingerol has an additive effect on inhibition of gastric acid secretion through activation of transient receptor potential vanilloid-1, and oral administration of transient receptor potential vanilloid-1 agonists directly stimulates transient receptor potential vanilloid-1 in the gastric lumen, resulting in a potent reduction of gastric acid secretion.

Decrease of guanylyl cyclase ß1 subunit and nitric oxide (NO)-induced relaxation in mouse rectum with colitis and its reproduction on long-term NO treatment.

Nitric oxide (NO) influences motility in the colon in patients with ulcerative colitis, but the exact mechanism involved remains unknown. Colitis was induced in mice by the oral administration of 2.5% dextran sodium sulfate (DSS), and the motility in longitudinal preparations from rectum and distal colon and expression of ß1 subunit of soluble guanylyl cyclase (sGCß1) were analyzed. Electrical stimulation (ES) caused a transient relaxation via the NO pathway in both rectum and colon from control mice. Stimulation with sodium nitroprusside (SNP) caused relaxation in the two regions, and the half-time (T (1/2)) of the maximal relaxation induced by 100 µM SNP was 8.1 ± 1.0 s in rectum. DSS treatment (1) abolished the ES-induced relaxation, but not dibutyryl cyclic GMP-induced response, in both regions, (2) decreased the maximal response to SNP accompanied by a loss of immunoreactive sGCß1 protein in rectum, but did not affect the amplitude of the relaxant response or the protein in distal colon, and (3) caused an increase in the T (1/2) value in response to SNP in both regions. Pretreatment of both preparations from control mice with 600 µM SNP for 30 min decreased both ES- and SNP-induced relaxation, SNP-induced cyclic GMP formation, and immunoreactive sGCß1 levels. NO-mediated relaxation was impaired by a dysfunctional sGC with and without a loss of immunoreactivity to sGCß1 in rectum and colon from DSS-treated mice, respectively. Long-term exposure of the tissues with an excess amount of NO changes the sGC-mediated relaxation.

Inhibitory effect of Iboga-type indole alkaloids on capsaicin-induced contraction in isolated mouse rectum.

Voacanga africana (Apocynaceae) is used as an anti-diarrheal medicine in West Africa. In the present study, we investigated the effect of an extract of V. africana and its constituents on smooth muscle contraction induced by capsaicin in mouse rectum, where transient receptor potential vanilloid type 1 (TRPV1)-immunoreactive fibers are abundant. Methanol and alkaloid extracts of the root bark of V. africana were found to inhibit capsaicin-induced contraction in a dose-dependent manner (30-300 µg/ml). Major constituents isolated from the alkaloid extract were then studied for their effects on the capsaicin-induced contraction. The main active constituents were found to be Iboga-type alkaloids, including voacangine (1), 3-oxovoacangine (2), voacristine (3), and (7α)-voacangine hydroxyindolenine (4). The voacangine concentration dependently (3-100 µM) inhibited the capsaicin-induced contraction. The capsaicin-induced contraction was almost completely inhibited by the TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). On the other hand, the Iboga-type alkaloids did not inhibit the contractions induced by 3 µM acetylcholine and 300 µM nicotine. These results suggest that Iboga-type alkaloids isolated from V. africana inhibit capsaicin-induced contraction in the mouse rectum, possibly via the inhibition of a TRPV1-mediated pathway. This inhibition may be involved in the anti-diarrheal effect of V. africana.

MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate], a derivative of the indole alkaloid mitragynine: a novel dual-acting mu- and kappa-opioid agonist with potent antinociceptive and weak rewarding effects in mice.

Mitragynine is a major indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that has opium-like properties, although its chemical structure is quite different from that of morphine. We attempted to develop novel analgesics derived from mitragynine, and thus synthesized the ethylene glycol-bridged and C10-fluorinated derivative of mitragynine, MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate]. We hypothesized that a dual-acting mu- and kappa-opioid agonist could produce potent antinociceptive effects with fewer rewarding effects compared with mu agonists. In this study, MGM-9 exhibited high affinity for mu- and kappa-opioid receptors with Ki values of 7.3 and 18 nM, respectively. MGM-9 showed a potent opioid agonistic effect, and its effects were meditated by mu- and kappa-opioid receptor mechanisms in in vitro assays. Subcutaneous and oral administration of MGM-9 produced potent antinociceptive effects in mouse tail-flick, hot-plate, and writhing tests. When administered orally, the antinociceptive effect of MGM-9 was seven to 22 times more potent than that of morphine. The antinociceptive effects of MGM-9 were mediated by both mu- and kappa-opioid receptors. Subcutaneous administration of MGM-9 twice daily for 5 days led to antinociceptive tolerance. In the gastrointestinal transit study, MGM-9 inhibited gastrointestinal transit, but its effect was weaker than that of morphine at equi-antinociceptive doses. Furthermore, MGM-9 induced less hyperlocomotion and fewer rewarding effects than morphine. The rewarding effect of MGM-9 was blocked by a mu antagonist and enhanced by a kappa antagonist. Taken together, the results suggest that MGM-9 is a promising novel analgesic that has a stronger antinociceptive effect and weaker adverse effects than morphine.

Chemical constituents of Aristolochia constricta: antispasmodic effects of its constituents in guinea-pig ileum and isolation of a diterpeno-lignan hybrid.

Twenty constituents were isolated from the n-hexane and chloroform extracts of Aristolochia constricta, a plant whose aerial parts have been used empirically in folk medicine for various purposes. The inhibitory effects of these constituents on smooth muscle contraction in isolated guinea-pig ileum were studied in order to observe their antispasmodic effects. 3,4-Dibenzyldihydrofuran-type lignans [(-)-cubebin, (-)-hinokinin, and (-)-pluviatolide] and a kaurene-type diterpene [(-)-kaur-16-en-19-oic acid] were isolated as active principals. They inhibited electrically induced and acetylcholine-induced contraction in the isolated guinea-pig ileum. In addition, 9- O-[(-)-kaur-15-en-17-oxyl]cubebin was isolated as a new diterpeno-lignan hybrid, although this constituent did not exhibit antispasmodic activity.

Involvement of mu-opioid receptors in antinociception and inhibition of gastrointestinal transit induced by 7-hydroxymitragynine, isolated from Thai herbal medicine Mitragyna speciosa.

7-hydroxymitragynine, a constituent of the Thai herbal medicine Mitragyna speciosa, has been found to have a potent opioid antinociceptive effect. In the present study, we investigated the mechanism of antinociception and the inhibitory effect on gastrointestinal transit of 7-hydroxymitragynine, and compared its effects with those of morphine. When administered subcutaneously to mice, 7-hydroxymitragynine produced antinociceptive effects about 5.7 and 4.4 times more potent than those of morphine in the tail-flick (ED50=0.80 mg/kg) and hot-plate (ED50=0.93 mg/kg) tests, respectively. The antinociceptive effect of 7-hydroxymitragynine was significantly blocked by the mu1/mu2-opioid receptor antagonist beta-funaltrexamine hydrochloride (beta-FNA) and the mu1-opioid receptor-selective antagonist naloxonazine in both tests. Thus, 7-hydroxymitragynine acts predominantly on mu-opioid receptors, especially on mu1-opioid receptors. Isolated tissue studies further supported its specificity for the mu-opioid receptors. Further, 7-hydroxymintragynine dose-dependently (ED50=1.19 mg/kg, s.c.) and significantly inhibited gastrointestinal transit in mice, as morphine does. The inhibitory effect was significantly antagonized by beta-FNA pretreatment, but slightly antagonized by naloxonazine. The ED50 value of 7-hydroxymitragynine on gastrointestinal transit was larger than its antinociceptive ED50 value. On the other hand, morphine significantly inhibits gastrointestinal transit at a much smaller dose than its antinociceptive dose. These results suggest that mu-opioid receptor mechanisms mediate the antinociceptive effect and inhibition of gastrointestinal transit. This compound induced more potent antinociceptive effects and was less constipating than morphine.

Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum.

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.

Antinociception, tolerance and withdrawal symptoms induced by 7-hydroxymitragynine, an alkaloid from the Thai medicinal herb Mitragyna speciosa.

7-Hydroxymitragynine is a potent opioid analgesic alkaloid isolated from the Thai medicinal herb Mitragyna speciosa. In the present study, we investigated the opioid receptor subtype responsible for the analgesic effect of this compound. In addition, we tested whether development of tolerance, cross-tolerance to morphine and naloxone-induced withdrawal signs were observed in chronically 7-hydroxymitragynine-treated mice. Subcutaneous (s.c.) administration of 7-hydroxymitragynine produced a potent antinociceptive effect mainly through activation of mu-opioid receptors. Tolerance to the antinociceptive effect of 7-hydroxymitragynine developed as occurs to morphine. Cross-tolerance to morphine was evident in mice rendered tolerant to 7-hydroxymitragynine and vice versa. Naloxone-induced withdrawal signs were elicited equally in mice chronically treated with 7-hydroxymitragynine or morphine. 7-Hydroxymitragynine exhibited a potent antinociceptive effect based on activation of mu-opioid receptors and its morphine-like pharmacological character, but 7-hydroxymitragynine is structurally different from morphine. These interesting characters of 7-hydroxymitragynine promote further investigation of it as a novel lead compound for opioid studies.

Inhibitory effect of mitragynine, an analgesic alkaloid from Thai herbal medicine, on neurogenic contraction of the vas deferens.

The effect of an indole-alkaloid mitragynine isolated from the Thai medicinal herb kratom (Mitragyna speciosa) on neurogenic contraction of smooth muscle was studied in guinea-pig vas deferens. Mitragynine inhibited the contraction of the vas deferens produced by electrical transmural stimulation. On the other hand, mitragynine failed to affect the responses to norepinephrine and ATP. Mitragynine did not reduce KCl-induced contraction in the presence of tetrodotoxin, prazosin and alpha,beta-methylene ATP. Mitragynine inhibited nicotine- or tyramine-induced contraction. By using the patch-clamp technique, mitragynine was found to block T- and L-type Ca2+ channel currents in N1E-115 neuroblastoma cells. In the Ca2+ measurement by a fluorescent dye method, mitragynine reduced KCl-induced Ca2+ influx in neuroblastoma cells. The present results suggest that mitragynine inhibits the vas deferens contraction elicited by nerve stimulation, probably through its blockade of neuronal Ca2+ channels.

Indole alkaloids of a Thai medicinal herb, Mitragyna speciosa, that has opioid agonistic effect in guinea-pig ileum.

Recently, we found that mitragynine, a major constituent of Mitragyna speciosa, has an opioid agonistic activity, but its weak potency could not explain the opium-like effect of this plant. In the present study, bioassay-guided fractionation of the crude extract of the leaves of M. speciosa was carried out to search for potent opioid agonists other than mitragynine. Opioid agonistic activities were evaluated using twitch contraction induced by electrical stimulation in guinea-pig ileum. The crude extract of M. speciosa inhibited the twitch contraction in a concentration-dependent manner. The inhibition was reversed by naloxone. The opioid effect was detected only in the crude base fraction, which was followed by the isolation of five indole alkaloids. Among these alkaloids, 7-hydroxymitragynine showed the most potent opioid effect on the electrically-stimulated contraction (pD (2) = 8.38 +/- 0.12). The potency, calculated using pD (2) values, was 30- and 17-fold higher than that of mitragynine and morphine, respectively. Antagonism of naloxone on concentration-response curves for 7-hydroxymitragynine confirmed its opioid effect. These results suggest that the opioid effect of M. speciosa is mostly based on the activity of 7-hydroxymitragynine.

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa.

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.

Studies on the synthesis and opioid agonistic activities of mitragynine-related indole alkaloids: discovery of opioid agonists structurally different from other opioid ligands.

Mitragynine (1) is a major alkaloidal component in the Thai traditional medicinal herb, Mitragyna speciosa, and has been proven to exhibit analgesic activity mediated by opioid receptors. By utilizing this natural product as a lead compound, synthesis of some derivatives, evaluations of the structure-activity relationship, and surveys of the intrinsic activities and potencies on opioid receptors were performed with guinea pig ileum. The affinities of some compounds for mu-, delta-, and kappa-receptors were determined in a receptor binding assay. The essential structural moieties in the Corynanthe type indole alkaloids for inducing the opioid agonistic activity were also clarified. The oxidative derivatives of mitragynine, i.e., mitragynine pseudoindoxyl (2) and 7-hydroxymitragynine (12), were found as opioid agonists with higher potency than morphine in the experiment with guinea pig ileum. In addition, 2 induced an analgesic activity in the tail flick test in mice.