Salacia oblonga root decreases cardiac hypertrophy in Zucker diabetic fatty rats: inhibition of cardiac expression of angiotensin II type 1 receptor.

AIMS: We investigated the effect of the water extract of Salacia oblonga (SOE), an ayurvedic antidiabetic and antiobesity medicine, on obesity and diabetes-associated cardiac hypertrophy and discuss the role of modulation of cardiac angiotensin II type 1 receptor (AT(1)) expression in the effect. METHODS: SOE (100 mg/kg) was given orally to male Zucker diabetic fatty (ZDF) rats for 7 weeks. At the end-point of the treatment, the hearts and left ventricles were weighed, cardiomyocyte cross-sectional areas were measured, and cardiac gene profiles were analysed. On the other hand, angiotensin II-stimulated embryonic rat heart-derived H9c2 cells and neonatal rat cardiac fibroblasts were pretreated with SOE and one of its prominent components mangiferin (MA), respectively. Atrial natriuretic peptide (ANP) mRNA expression and protein synthesis and [(3)H]thymidine incorporation were determined. RESULTS: SOE-treated ZDF rats showed less cardiac hypertrophy (decrease in weights of the hearts and left ventricles and reduced cardiomyocyte cross-sectional areas). SOE treatment suppressed cardiac overexpression of ANP, brain natriuretic peptide (BNP) and AT(1) mRNAs and AT(1) protein in ZDF rats. SOE (50-100 microg/ml) and MA (25 micromol) suppressed angiotensin II-induced ANP mRNA overexpression and protein synthesis in H9c2 cells. They also inhibited angiotensin II-stimulated [(3)H]thymidine incorporation by cardiac fibroblasts. CONCLUSIONS: Our findings demonstrate that SOE decreases cardiac hypertrophy in ZDF rats, at least in part by inhibiting cardiac AT(1) overexpression. These studies provide insights into a potential cardioprotective role of a traditional herb, which supports further clinical evaluation in obesity and diabetes-associated cardiac hypertrophy.

Modifying effects of naturally occurring products on the development of colonic aberrant crypt foci induced by azoxymethane in F344 rats.

Modifying effects of dietary exposure of seven naturally occurring products on the development of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM) were investigated in male F344 rats. The effects of these compounds on proliferation biomarkers such as the number of silver-stained nucleolar organizer region protein, ornithine decarboxylase activity, and polyamine concentration in the colon were also estimated. The naturally occurring products tested included four terpenoids (rebaudioside A, oleanolic acid, costunolide, and soyasaponin A2), one flavonoid (liquiritin), and two isocoumarins (phyllodulcin and hydrangenol). Animals were given 3 weekly s.c. injections of AOM (15 mg/kg body weight) to induce ACF. These rats were fed the diet containing 200 ppm of each test chemical for 5 weeks, starting 1 week before the first dosing of AOM. All rats were sacrificed 2 weeks after the last AOM injection to estimate their modulatory effects on the occurrence of ACF and the cell proliferation biomarkers in the colon. In groups of rats given AOM and hydrangenol, oleanolic acid, or costunolide, the frequencies of ACF/colon were significantly lower than that of AOM alone (P < 0.05, P < 0.005, and P < 0.05, respectively). In groups of rats given AOM and costunolide and those treated with AOM and soyasaponin A2, both ornithine decarboxylase activity and polyamine concentration of the colonic mucosal tissue were significantly decreased compared with those in rats given AOM alone (P < 0.05 and P < 0.001 for costunolide and P < 0.001 and P < 0.05 for soyasaponin A2, respectively). In groups of rats given AOM and liquiritin, oleanolic acid, or costunolide, the numbers of silver-stained nucleolar organizer regions/nucleus were significantly lower than that of AOM alone (P < 0.05, P < 0.01, and P < 0.05, respectively). Costunolide decreased four AOM-induced biomarkers, such as the frequencies of ACF/colon, ornithine decarboxylase activity, polyamine concentration level, and silver-stained nucleolar organizer region number in the colon. These results indicate that, among the test chemicals, costunolide has blocking effects against rat colon carcinogenesis and is a possible chemopreventive agent against colon tumorigenesis. Also, the short-term model described here could be a very useful prescreening tool for chemopreventive agents against colon cancer.

The mode of vasorelaxant action of 2-aminoisoquinoline, 1.3 (2H.4H)-dione, a novel 'intracellular calcium inhibitor'.

In rabbit aorta, pretreatment with 2-aminoisoquinoline, 1.3 (2H.4H)-dione (AQ, 10(-5) M and 10(-4) M) shifted the concentration-response relationship to noradrenaline (NA, 10(-9) M to 10(-4) M) in a parallel manner whereas the agent (10(-4) M) failed to affect the response to potassium and only slightly depressed Ca2+-induced contractions in a Ca2+-free medium in the presence of K+ (40 mM). Ca2+-entry blockers such as nifedipine and diltiazem (10(-6) M and 10(-5) M) had very weak or no apparent effects on the response to NA but markedly attenuated or abolished the K+- and Ca2+-induced contractions. Following incubation of tissues for 15 min in a Ca2+-free medium with low EGTA (0.01 mM) and methoxyverapamil (D600, 10(-5) M), NA (3 X 10(-7) M) caused a phasic (transient) contraction and the subsequent application of Ca2+ (2mM) resulted in a tonic contraction. This NA-induced, Ca2+-dependent, D600-insensitive contraction was inhibited by AQ (10(-5) M and 10(-4) M) in a concentration-dependent manner. This suggests that the inhibitory action of AQ may be related to Ca2+ entry through specific receptor activated pathways. Following incubation of tissues for 30 min in a Ca2+-free medium with high EGTA (2.0 mM), NA (10(-5) M) caused a contraction of rabbit aorta which is dependent upon release of intracellular Ca2+, but the response was 50% to 60% less than that in a normal medium. This contraction was inhibited by AQ (10(-5) M and 10(-4) M) and nitroglycerin (10(-5) M) but not by nifedipine or diltiazem. The inhibitory action of combined treatment with AQ and nitroglycerin (10-5 M) on the response to NA was not different from that of either agent alone. 5 These results suggest that AQ may have inhibitory actions on the release of intracellular Ca2+ and also on Ca2+-entry through D600-insensitive, receptor-activated Ca2+ pathways in rabbit aorta.

Chemopreventive effect of costunolide, a constituent of oriental medicine, on azoxymethane-induced intestinal carcinogenesis in rats.

Modifying effects of costunolide, a constituent of oriental medicines, on intestinal carcinogenesis were examined in a rat model using azoxymethane (AOM). A total of 115 male F344 rats, 6 weeks old, were divided into four groups. Group 1 (30 rats) was fed a diet containing costunolide at a concentration of 0.02% for 4 weeks during which time three s.c. injections of AOM (15 mg/kg) were applied, and then kept on a basal diet until the end of the experiment (33 weeks). Group 2 (30 rats) was given AOM as in group 1 and fed the basal diet throughout, without costunolide. Group 3 (25 rats) was administered costunolide at the start of the experiment, but not given the carcinogen. Group 4 (30 rats) received the basal diet alone throughout the experiment and served as a control. Intestinal tumors were seen in groups 1 and 2, their incidence in group 1 (17%) being significantly lower than in group 2 (50%) (P < 0.05). Furthermore, the number of aberrant crypt foci/cm2 of colon in rats of group 1 at the termination of the experiment was significantly smaller than in group 2 (P < 0.01). BrdU labelling indices of S-phase mucosal cells in the colon of rats in group 1 were significantly lower than those in group 2 (3.9 +/- 3.1 vs. 9.1 +/- 4.5, P < 0.005). The present results suggest that the natural sesquiterpene contained in plants which have been used as oriental drugs, could be a promising chemopreventive agent for human intestinal neoplasia.

Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal carcinogenesis in rats.

The effects of two naphthoquinones, juglone and plumbagin, and an isocoumarin, hydrangenol, on intestinal carcinogenesis in rats were examined by dietary exposure during the initiation phase. Starting at 5 weeks of age, male F344 rats were fed the diets containing either of the test chemicals at a concentration of 200 ppm or the control diet without the compounds. At 6 weeks of age, all animals were treated with s.c. injections of azoxymethane (AOM) (15 mg/kg body weight, once weekly for 3 weeks) or saline alone. Animals fed experimental diets were changed to the control diet 1 week after the last carcinogen treatment. Animals given plumbagin together with the carcinogen had a lower incidence (41%) and smaller multiplicity (0.48 +/- 0.62) of tumors in the entire intestine compared with those exposed to carcinogen alone (68% and 1.04 +/- 0.62) (P < 0.05 and < 0.01, respectively). The incidence and multiplicity of tumors in the small intestine (7% and 0.07 +/- 0.25) and the multiplicity of tumors in the entire intestine (0.60 +/- 0.76) of animals treated with juglone and the carcinogen were significantly less than those of animals treated with carcinogen alone (P < 0.05 in each). Hydrangenol tended to decrease the incidence and the multiplicity of tumors in the entire intestine induced by AOM, but the effect was not statistically significant. The present data suggest that the naphthoquinones, juglone and plumbagin, could be promising chemopreventive agents for human intestinal neoplasia.

Suppression of azoxymethane-induced colonic aberrant crypt foci by dietary exposure to a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone, in rats.

The modifying effect of dietary exposure to a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone (KYN-5) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given weekly s.c. injections of AOM (15 mg/kg body wt.) for 3 weeks to induce ACF. These rats were fed diet containing 100 or 200 ppm KYN-54 for 5 weeks, starting 1 week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ornithine decarboxylase (ODC) activity, mucosal polyamine level, and silver-stained nucleolar organizer regions protein (AgNORs) count per nucleus in the colon. In rats given AOM and KYN-54, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone. ODC activity and polyamine levels, and the mean AgNORs number in the colon of rats given AOM and KYN-54 at both doses were also significantly lower than that of rats treated with AOM alone. These results provide further evidence that KYN-54 could be a chemopreventive agent against rat colon carcinogenesis.

Inhibitory effect of 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone, a novel synthesized retinoid, on azoxymethane-induced intestinal carcinogenesis in rats.

Modifying effects of 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone, a novel synthesized retinoid (KYN-54), on intestinal carcinogenesis were examined in a rat model using azoxymethane (AOM). A total of ninety male F344 rats, 6 weeks old, were divided into 4 groups. Group 1 (20 rats) was fed a diet containing KYN-54 at a concentration of 0.02% for 3 weeks, during which time 2 s.c. injections of azoxymethane (15 mg/kg) were applied and then kept on a basal diet until the end of the experiment (1 year). Group 2 (30 rats) was given azoxymethane as in group 1 and fed the basal diet throughout, without synthetic retinoid exposure. Group 3 (20 rats) was administered KYN-54 at the commencement of the experiment, but not given the carcinogen. Group 4 (20 rats) received a basal diet alone throughout the experiment and served as a control. Intestinal tumors were seen in groups 1 and 2, their incidence and average number in group 1 (74%, 1.07 +/- 0.87) being significantly less than in group 2 (39%, 0.56 +/- 0.78) (P < 0.02 and P < 0.05, respectively). These results suggest that the synthetic retinoid might be a promising chemopreventive agent for intestinal neoplasia.

The inhibitory effects of mangiferin, a naturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats.

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside, is one of xanthone derivatives and C-glucosylxanthones, is widely distributed in higher plants and is one of constituents of folk medicines. Recent studies showed that mangiferin has a potential as an anti-oxidant and an anti-viral agent. In this study, we examined the effects of mangiferin in rat colon carcinogenesis induced by chemical carcinogen, azoxymethane (AOM). We performed two experiments: a short-term assay to investigate the effects of mangiferin on the development of preneoplastic lesions by AOM, aberrant crypt foci (ACF), and the following long-term assay for the influence of mangiferin on tumorigenesis induced by AOM. In the short-term assay, 0.1% mangiferin in a diet significantly inhibited the ACF development in rats treated with AOM compared to rats treated with AOM alone (64.6+/-22.0 vs. 108.3+/-43.0). In the long-term assay, the group treated with 0.1% mangiferin in initiation phase of the experimental protocol had significantly lower incidence and multiplicity of intestinal neoplasms induced by AOM (47.3 and 41.8% reductions of the group treated with AOM alone for incidence and multiplicity, respectively). In addition, the cell proliferation in colonic mucosa was reduced in rats treated with mangiferin (65-85% reductions of the group treated with AOM alone). These results suggest that mangiferin has potential as a naturally-occurring chemopreventive agent.

The vasorelaxant effect of evocarpine in isolated aortic strips: mode of action.

The effect of evocarpine (EVO), a quinolone alkaloid isolated from Evodiae fructus, on Ca2+-blocking activity has been examined. In the isolated rat thoracic aorta evocarpine significantly inhibited the contraction induced by 60 mM K+ with an IC50 of 9.8 microM, and that induced by external Ca2+ in the depolarized muscle in concentrations of 10-100 microM. The relaxant effect of evocarpine and verapamil was antagonized by Bay K8644. The increase of 45Ca2+-influx induced by 60 mM K+ was significantly inhibited by 100 microM evocarpine. In the isolated rabbit thoracic aorta 100 microM evocarpine had no effect on the norepinephrine-induced contraction in normal medium or on the phasic contraction in Ca2+-free medium or on the transient relaxation induced by activation of the Na+ pump. The content of cyclic AMP or cyclic GMP was unchanged. These results suggest that evocarpine inhibits Ca2+ influx through voltage-dependent calcium channels.

Acceleration of gastrointestinal transit by momordin Ic in mice: possible involvement of 5-hydroxytryptamine, 5-HT(2) receptors and prostaglandins.

Possible involvement of 5-hydroxytryptamine (5-HT), 5-HT receptors and prostaglandins in the acceleration of gastrointestinal transit by momordin Ic was investigated in mice. Accelerative effect of momordin Ic (25 mg/kg, p.o.) on gastrointestinal transit was attenuated by pretreatment with a bolus of DL-p-chlorophenylalanine methyl ester (an inhibitor of 5-HT synthesizing enzyme), but not repeated pretreatment with DL-p-chlorophenylalanine methyl ester. Furthermore, cyproheptadine (a nonselective 5-HT(2) receptor antagonist), ritanserin (a 5-HT(2A/2B/2C) receptor antagonist) and clozapine (a 5-HT(2A/2C) receptor antagonist) also attenuated the effect of momordin Ic, but methiothepin (a 5-HT(1) receptor antagonist), MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) and metoclopramide (5-HT(3) receptor antagonists), tropisetron (a 5-HT(3/4) receptor antagonist), ketanserin and haloperidol (5-HT(2A) receptor antagonists) did not. These results suggested a possible involvement of endogenous 5-HT and 5-HT(2B/2C) over 5-HT(2A) receptors. Attenuation by pretreatment with indomethacin (an inhibitor of prostaglandins synthesis) suggested involvement of prostaglandins. It is postulated that momordin Ic accelerates gastrointestinal transit partially by stimulating synthesis of 5-HT to act through 5-HT(2), possibly 5-HT(2C) and/or 5-HT(2B) receptors, which, in turn, increases synthesis of prostaglandins.

Enhancement by escins Ib and IIb of Mg(2+) absorption from digestive tract in mice: role of nitric oxide.

The effects of escins Ib and IIb isolated from horse chestnuts on Mg(2+) absorption from the digestive tract and the role of endogenous nitric oxide (NO) were investigated in mice. Test samples were given orally to fasted mice 30, 120, 180, 240 and 300 min before administration of 0.5 M MgSO(4) (10 ml/kg, p.o.). The serum Mg(2+) levels were determined 30, 60, 120 and 180 min after administration of MgSO(4). Escins Ib and IIb (12.5 and 25 mg/kg) significantly increased the serum Mg(2+) by 10.0-27.3%, 30, 120 and 180 min after administration of the samples, and 30, 60, 120 and 180 min after administration of MgSO(4). Escins Ib and IIb (12.5 mg/kg) significantly decreased the Mg(2+) content in the small intestinal fluid in MgSO(4)-loaded mice, but did not increase the serum Mg(2+) levels in normal mice. The effects of escins Ib and IIb (12.5 mg/kg) on serum Mg(2+) levels were attenuated in a dose-related manner by the pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 3-20 mg/kg, i.p., an inhibitor of constitutive and inducible NO synthase), but not with D-NAME (10 mg/kg, i.p., the inactive enantiomer of L-NAME) or dexamethasone (0.05 and 0.5 mg/kg, s.c., an inhibitor of inducible NO synthase). The effect of L-NAME was reversed by L-arginine (600 mg/kg, i.p., a substrate of NO synthase), but not by D-arginine (900 mg/kg, i.p., the enantiomer of L-arginine). These results suggest that escins Ib and IIb enhance Mg(2+) absorption from the digestive tract in mice, in which the constitutive, but not the inducible, NO synthase plays an important role.

Effects of escins Ia, Ib, IIa, and IIb from horse chestnuts on gastric emptying in mice.

Inhibitory effects of the saponin fraction and its principal constituents, escins Ia, Ib, IIa, and IIb, from horse chestnuts on gastric emptying were investigated in mice loaded with a non-nutrient or nutrient meal. The saponin fraction and escins Ia-IIb inhibited gastric emptying of a 1.5% carboxymethyl cellulose sodium salt (CMC-Na) meal by 11.1-54.2% (12.5-200 mg/kg). Escins Ia-IIb (50 mg/kg) also inhibited gastric emptying of a 40% glucose meal by 21.1-23.5% except for escin Ia, a milk meal by 18.4-33.1%, and a 30% ethanol meal by 13.5-15.9%. The effects of escins Ia-IIb on gastric emptying of the CMC-Na meal were attenuated by pretreatment with streptozotocin (100 mg/kg, i.v.), capsaicin (75 mg/kg in total, s.c.), or insulin (1 U/kg, s.c.). The effect of insulin was reduced by glucose (2 g/kg, i.v.) which can directly nourish the brain, but not by fructose (2 g/kg, i.v.) which cannot be utilized by the brain. The effects of escins Ia-IIb (50 mg/kg) were overridden in 60% ethanol-loaded mice, in which the central nervous system was suppressed by ethanol. These results suggest that capsaicin-sensitive sensory nerves and central nervous system partly participate in the effects of escins Ia-IIb.

Protective effects of oleanolic acid oligoglycosides on ethanol- or indomethacin-induced gastric mucosal lesions in rats.

We examined the effects of various oleanolic acid oligoglycosides obtained from traditional herbs on ethanol- or indomethacin-induced gastric mucosal lesions in rats and on gastric secretion in pylorus-ligated rats. Test samples were given orally to fasted rats 1 h before absolute ethanol (1.5 ml/rat, p.o.) or indomethacin (30 mg/kg, s.c.) treatment, or ligation of the pylorus. Oleanolic acid 3-O-monodesmosides [oleanolic acid 3-O-glucuronide (1, 20-50 mg/kg), momordin Ic (2, 5-50 mg/kg), and 28-O-deglucosyl-chikusetsusaponins IV (5, 10-50 mg/kg) and V (7, 10-50 mg/kg)] were found to show protective effects on ethanol-induced gastric mucosal lesions, whereas oleanolic acid 3,28-O-bisdesmosides [momordin IIc (3), chikusetsusaponins IV (4) and V (6)], oleanolic acid 28-O-monodesmoside [compound O (8)], and their common aglycon [oleanolic acid (9)] showed no such effects. Oleanolic acid 3-O-monodesmosides (1, 2, and 5) also showed protective effects on indomethacin-induced gastric mucosal lesions. 28-O-Deglucosyl-chikusetsusaponin V (7) did not inhibit the indomethacin-induced lesions, while chikusetsusaponins V (6, 50 mg/kg) had the gastroprotective effect. These active saponins (1, 2, 4-7, 10-50 mg/kg) did not decrease the gastric secretion by oral administration in pylorus-ligated rats.

Effects of alismol isolated from Alismatis Rhizoma on calcium-induced contraction in the rabbit thoracic aorta.

We examined the inhibitory effects of alismol, a sesquiterpenoid isolated from Alismatis Rhizoma, on vascular contractions induced by high concentrations of K+ and Ca2+, and on 45Ca2+ retention in normal and in high K+-containing medium. Alismol affected neither the resting tension nor the 45Ca2+ retention in normal medium, but it inhibited sustained contraction and increased 45Ca2+ retention induced by high K+ concentrations. Alismol did not affect norepinephrine-induced contractions in normal medium, nor phasic contractions in Ca2+-free medium. These results suggested that alismol inhibited mainly Ca2+ influx through a voltage-dependent Ca2+ channel.

Anticholinergic action of Swertia japonica and an active constituent.

The anticholinergic action of Swertia japonica, used in Japan as a bitter stomachic, was examined using in vivo experiments in rats in order to substantiate the presence or absence of antispasmodic properties. The methanol extract of Swertia japonica was found to be an effective anticholinergic given orally. Fractionation and purification of the methanol extract through column chromatography revealed that swertiamarin, found in the methanol extract in amounts of about 30%, was an active constituent with an anticholinergic action.

Anti-ulcer action of Panax japonicus rhizome.

Using HCl/ethanol-induced ulcer in rats as a screening model, an anti-ulcer effect was observed for a methanol extract of Panax japonicus rhizome, saponin fractions and chikusetsusaponin III. Results suggest that the gastric mucous membrane-protective effect of the methanol extract is likely to be due to the crude saponin fraction and chikusetsusaponin III.

Vascular dilatory action of Artemisia capillaris bud extracts and their active constituent.

In perfusion experiments, the acetone extract of Artemisia capillaris buds significantly inhibited the response to norepinephrine of helical strips of rabbit thoracic aorta. The acetone extract was fractionated by column chromatography to identify the active constituent. Kinetic experiments using rabbit thoracic aorta showed that 6,7-dimethoxycoumarin (scoparone) has a marked inhibitory effect on the contractions induced by norepinephrine, 5-hydroxytryptamine, histamine and angiotensin II. Like nitroglycerin, scoparone appeared to be a competitive antagonist of norepinephrine.

The anti-ulcer effect in rats of ginger constituents.

The effects of ginger, a pungent stomachic natural medicine, on HCl/ethanol-induced gastric lesions in rats, were examined. The orally administered acetone extract at 1000 mg/kg and zingiberene, the main terpenoid from acetone extract, at 100 mg/kg significantly inhibited gastric lesions by 97.5 and 53.6%, respectively. 6-Gingerol, the pungent principle, at 100 mg/kg significantly inhibited gastric lesions by 54.5%. These results suggest that zingiberene, the terpenoid and 6-gingerol are important constituents in stomachic medications containing ginger.

Antianoxic action of evodiamine, an alkaloid in Evodia rutaecarpa fruit.

In order to determine the antianoxic potential of evodiamine, its effects were compared to those of vinpocetine (VPT), using a series of animal models of anoxia. In mice, evodiamine was equivalent to VPT in the KCN-induced anoxia model but was greater than VPT in the low-pressure-induced anoxia model. Its effectiveness was increased by combined treatment with physostigmine, suggesting the involvement of a cholinergic mechanism in the antianoxic action of evodiamine.

Effects of crude drugs on experimental hypercholesterolemia. I. Tea and its active principles.

The effect of methanol extractives from tea leaves on hypercholesterolemia was examined in animal models. It was found that orally administered (-)-epicatechin gallate and (-)-epigallocatechin gallate from tea leaves lowered the serum cholesterol level in mice fed a high fat emulsion. Quantitation of tissue cholesterol and examination of liver tissues in mice fed a high cholesterol diet revealed that these constituents also significantly lowered the amount of cholesterol crystallization. These results support the reputed effectiveness of tea use in hypercholesterolemia.