The search for limonoids started long back when scientists started looking for the factor responsible for bitterness in citrus which has negative impact on citrus fruit and juice industry worldwide. The term limonoids was derived from limonin, the first tetranortriterpenoid obtained from citrus bitter principles. Compounds belonging to this group have exhibited a range of biological activities like insecticidal, insect antifeedant and growth regulating activity on insects as well as antibacterial, antifungal, antimalarial, anticancer, antiviral and a number of other pharmacological activities on humans. Although hundreds of limonoids have been isolated from various plants but, their occurrence in the plant kingdom is confined to only plant families of order Rutales and that too more abundantly in Meliaceae and Rutaceae, and less frequently in Cneoraceae and Harrisonia sp. of Simaroubaceae. Limonoids are highly oxygenated, modified terpenoids with a prototypical structure either containing or derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton. All naturally occurring citrus limonoids contain a furan ring attached to the D-ring, at C-17, as well as oxygen containing functional groups at C-3, C-4, C-7, C-16 and C-17. The structural variations of limonoids found in Rutaceae are less than in Meliaceae and are generally limited to the modification of A and B rings, the limonoids of Meliaceae are more complex with very high degree of oxidation and rearrangement exhibited in the parent limonoid structure. To counter the problem of bitterness in citrus juice and products genetic engineering of citrus to maximize the formation of limonoid glucosides for reducing limonoid bitterness is the focus of recent and future research. Regarding the biological activities of limonoids the investigations are to be directed towards detailed characterization, quantification, and designing a simple as well as versatile synthetic route of apparently important limonoids. Extraction methods too should be optimized; evaluation and establishment of pharmaco-dynamic and kinetic principles, and structure activity relationships should be a key goal associated with limonoids so that they can be safely introduced in our arsenal of pharmaceuticals to safeguard the humanity from the wrath of disease and its discomfort.
Catecholamines (CAs) are important hormones in regulating blood pressure both in centrally and peripheral sympathetic nerve endings. Production of CAs, release and inactivation are three components to regulate CAs level. We have reported that the inactivation of CAs by catechol-O-methyltransferase (COMT) in the liver is important in high blood pressure in spontaneously hypertensive rats (SHR). In the present study, we investigated central role of COMT in hypertension. We investigated COMT activities in cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus of SHR and Wistar-Kyoto (WKY) rats. COMT activities were assessed by measuring normetanephrine with the use of norepinephrine as an endogenous substrate. Membrane-bound COMT activities in cerebral cortex were significantly reduced in SHR (19.1±1.8 pmol/min/mg protein) compared with WKY rats (25.0±3.3 pmol/min/mg protein). The ratio of concentrations of normetanephrine/norepinephrine in cerebral cortex was also lower in SHR than in WKY rats. Our results suggest that there is an association between MB-COMT in cerebral cortex and blood pressure regulation.
Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induced chromosomal aberration. Estrogen receptors (ER) participate in some of these genotoxic effects by estrogen. In this study, we showed the effects of bisphenol A (BPA), an endocrine-disrupting chemical eliciting weak estrogenic activity, and of 17β-estradiol (E2), on DNA damage in ER-positive MCF-7 cells by Comet assay. Higher concentrations of BPA, more than 1000 times of E2, were needed to induce the same levels of effects by E2. Immunofluorescence microscopy showed that γH2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in MCF-7 cells. γH2AX foci colocalized with Bloom helicase, which is considered to be responsible for the repair of DNA damage after treatment with E2 or BPA. Interestingly, DNA damage was not as severe in ER-negative MDA-MB-231 cells as in MCF-7 cells. The ER antagonist ICI182780 blocked E2 and BPA genotoxic effects on MCF-7 cells. These results together suggest that BPA causes genotoxicity ER dependently in the same way as E2.
As an attempt to find bioactive medicinal herbs exerting anti-asthmatic activity, the effects of an ethanol extract from the parts of Saururus chinensis were evaluated in both in vitro and in vivo. The ethanol extract of S. chinensis (ESC) inhibited generation of the cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 in bone marrow-derived mast cells in a concentration-dependent manner with an IC50 value of 14.3 μg/ml. ESC also inhibited leukotriene C4 production with an IC50 value of 0.3 μg/ml. This demonstrates that ESC has COX-2/5-lipoxygenase dual inhibitory activity. In addition, this compound inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 1.3 μg/ml. An ovalbumin induced mouse asthmatic animal model was used to determine its in vivo anti-asthmatic activity. The oral administration (50—200 mg/kg) of ESC reduced the number of infiltrated eosinophil in a bronchoalveolar lavage fluid. Furthermore, ESC (100 mg/kg) inhibited the eotaxin and IL-4 mRNA expression levels. These results suggest that the anti-asthmatic activity of S. chinensis might in part occur via the inhibition of eicosanoid generation, degranulation as well as the down regulation of IL-4 and eotaxin mRNA expression.
Glucokinase (GK) is known to be the critical glucose sensor of pancreatic B-cells. However, the localization and functional role of GK in the brain remains to be elucidated. In this study, we measured both the activity and mRNA level of GK in the hypothalamic nuclei and the cortex of rats injected intraperitoneally with streptozotocin or vehicle. GK activity was measured by a fluorometric assay; and the GK mRNA level, by use of the real-time reverse transcription polymerase chain reaction. GK activity in vehicle-treated rats was high in the arcuate nucleus, moderate or low in the ventromedial nucleus, lateral hypothalamic area, and paraventricular nucleus, and very low in the cortex. The order of GK mRNA level was almost the same as that of GK activity. GK activity and GK mRNA level only in the arcuate nucleus of streptozotocin-treated rats at 7 d, but not at 2 d, after treatment were lower than those of vehicle-treated rats. The results suggest that prolonged hyperglycemia induced by diabetes decreased the activity of GK in the arcuate nucleus.
Andrographolide has been reported to possess a variety of pharmacological activities. In this study, we have investigated the effect of andrographolide on the production of TNF-α and IL-12 (Interleukin-12) in murine peritoneal macrophages. Andrographolide decreased TNF-α, IL-12a and IL-12b at mRNA level, and reduced the production of TNF-α and IL-12p70 proteins in a concentration-dependent manner. Furthermore, we have found that addition of andrographolide inhibited the activation of ERK1/2 MAP kinase, but not that of JNK, p38 or NF-κB. These results suggested that andrographolide inhibit LPS-induced production of TNF-α via suppression of the ERK1/2 signaling pathway.
DAP kinase-related apoptosis-inducing kinase 2 (DRAK2), a member of the DAP kinase family, is a serine/threonine kinase capable of inducing apoptosis. Here we studied the relationship between DRAK2 intracellular localization and apoptosis, and found that UV light acts as a stimulus for apoptosis induced by DRAK2. The intracellular location of DRAK2 depended on the cell line: DRAK2 was found primarily in the nuclei of NRK, NIH3T3, and Caco-2 cells while it was present primarily in the cytoplasm of ACL-15, HeLa, and WI-38 cells. Overexpression of Myc-tagged DRAK2 led to apoptosis-like cell death in NRK cells, but not in ACL-15 cells. A GFP fusion protein of DRAK2 was spontaneously localized to the nucleus of ACL-15 cells and resulted in cell death. Nuclear localization and cell death were also observed with DRAK2(1—293) fused to the NLS of SV40 but not with DRAK2(1—293) alone. These results suggested that nuclear accumulation of DRAK2 and the resulting increase in the endogenous level of its kinase activity are required for cell death. UV irradiation caused nuclear accumulation of endogenous DRAK2 in ACL-15 cells, which was followed by apoptosis-like cell death. Knockdown of DRAK2 expression by siRNA partially suppressed UV-induced apoptosis. These results suggest that DRAK2 plays a role in UV induced apoptosis.
L-Arginine is a precursor of polyamine, nitric oxide (NO), creatine, and agmatine and is essential for the differentiation and proliferation of blood cells, although the precise biological role of L-arginine is unclear. We have recently reported that the depletion of L-arginine in cultured medium prevented both proliferation and differentiation of blood cells (Shima et al., Blood First Edition Paper, October 6, 2005; DOI 10.1182). Since one of metabolic products of L-arginine in the cells is polyamine that associates with cell differentiation and proliferation, the effects of L-arginine on the human K562 cell line and human cord blood-derived CD34 positive cells were investigated by focusing on polyamines such as putrescine, spermidine, and spermine in the present study. When polyamines were added to the culture medium in the absence of L-arginine, the cells did not grow or differentiate well. However, when intracellular polyamines were depleted using ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), the proliferation and differentiation of K562 cells to erythrocytes were reduced even in the presence of L-arginine. Moreover, in the presence of DFMO, cell differentiation and proliferation were recovered by the addition of putrescine or spermidine in the presence of L-arginine. Accordingly, it was demonstrated that polyamines are essential for the proliferation and differentiation of the blood cells as the metabolites of L-arginine and the externally added polyamines are also effective by being taken up through polyamine transporter.
Candida albicans water soluble fraction (CAWS), water soluble fraction of Candida albicans mainly composed of mannoprotein–β-glucan complex, has various biological effects, such as anaphylactoid shock and coronary arteritis. These toxicological effects fit CAWS as one of PAMPs, pathogen-associated molecular patterns. Acute anaphylactoid reaction is known to be induced by lipopolysaccharide from Escherichia coli O9 (O9 LPS), which possesses the mannose homopolysaccharide as the O-antigen region. In the present study, we compared immunotoxicological and immunochemical similarity between CAWS and O9 LPS. CAWS strongly reacted with Candida serum factors, and the reactivity was found to be partially competed with O9 LPS. CAWS induced lethal toxicity was inhibited by pretreatment of mice with i.v. injection of CAWS. The lethality was found to be inhibited by i.v. injection of O9 LPS. Vice versa, O9 LPS induced acute lethal toxicity was also inhibited by pretreatment of mice with CAWS. These results suggested that CAWS, fungal PAMPs, and O9 LPS from Gram-negative bacteria share, at least in part, immunochemical and immunotoxicological activities.
Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. We studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-β-D-ribofuranosylbenzimidazole (BDCRB). Na+-dependent uptakes of [3H]-inosine and [3H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [3H]-inosine and [3H]-adenosine (60—70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10—30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [3H]-inosine and [3H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80—90% for adenosine and 60—80% for inosine). The amino acid site of attachment (3′- and 5′-monoesters) and stereochemistry (L- and D-amino acid esters), did not significantly affect the uptake of [3H]-inosine and [3H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs.
JCICM-6, the extract of an anti-arthritic herbal formula composed of medicinal herbs of Sinomenium acutum, Aconitum carmichaeli DEBX., Curcuma Longa L., Paeonia lactiflora PALL., and Paeonia suffruticosa ANDR., was examined in the effectiveness and mechanism in reducing experimentally-induced inflammation and nociception using nine animal models. JCICM-6 was extracted from herbs and purified with Amberlite XAD-7HP adsorbent resin and analyzed with HPLC-fingerprint for quality consistency. In acute inflammatory models, the paw edema of rats was induced by subcutaneous injection of carrageenan or pro-inflammatory mediators, including histamine, serotonin, bradykinin, and prostaglandin E2 (PGE2) into the right hind paws of animals; while the ear edema of mice was induced by applying arachidonic acid or 12-O-tetradecanoylphorbol 13-acetate (TPA) on the ear surface. In nociceptive models, the tail-flick response induced by radiant heat stimulation was measured and the numbers of abdominal writhing episodes of mice induced by intraperitoneal injection of acetic acid were recorded. JCICM-6 orally administered in a range of dosages from 0.438 g to 1.75 g/kg significantly and dose-dependently suppressed the paw edema of rats induced by carrageenan or various pro-inflammatory mediators and the ear edema of mice induced by arachidonic acid or TPA. JCICM-6 also significantly prolonged the reaction time of rats to radiant heat stimulation and reduced the numbers of writhing episodes of mice. These results indicated that JCICM-6 possesses significant anti-inflammatory and analgesic effects, which implies that it would be a potential candidate for further investigation as a new anti-arthritic botanical drug for humans.
The purpose of this study was to characterize the putative anxiolytic-like effects of the aqueous extract of the rhizome of Gastrodia elata along with its phenolic constituents, 4-hydroxybenzyl alcohol (HA) and 4-hyroxybenzaldehyde (HD), using an elevated plus maze (EPM) in mice. The mice were administered either the aqueous G. elata extract orally or received an intraperitoneal injection of the phenolic constituents, 1 h before the behavioral evaluation in the EPM. A single treatment of the aqueous G. elata extract significantly increased the percentage of time spent and arm entries into the open arms of the EPM versus the saline controls. Among the phenolic constituents of G. elata, HA and HD significantly increased the percentage of time spent and arm entries into the open arms of the EPM versus saline controls (p<0.05). Moreover, there were no changes in the locomotor activity and myorelaxant effects in any group compared with the saline controls. In addition, the anxiolytic-like effects of G. elata extract were blocked by both WAY 100635 (0.3 mg/kg, i.p.), a 5-HT1A receptor antagonist, and flumazenil (10 mg/kg, i.p.), a GABAA receptor antagonist. The anxiolytic-like effects of HA were inhibited by WAY 100635 and the effects of HD were antagonized by flumazenil. These results indicate that G. elata is an effective anxiolytic agent, and suggests that the anxiolytic-like effects of G. elatavia the serotonergic nervous system depends on HA and those effects of G. elatavia the GABAergic nervous system depends on HD.
We previously demonstrated that Ginkgo biloba extract (GBE) produced vasodilation via the nitric oxide synthesis and release by increasing the intracellular calcium level in vascular endothelial cells of rats. The present study aimed to clarify the effects of dietary administration of GBE on the blood pressure and vascular tone of hypertensive Dahl salt-sensitive (Dahl) rats in order to evaluate its therapeutic actions and availability. Dahl rats were fed an 8.0% NaCl diet or an 8.0% NaCl plus 0.5% GBE diet for 24 d. The feeding of GBE did not change the heart rate, but significantly decreased systolic blood pressure. After 24 days' administration, the effects of GBE on the atria and aorta isolated from Dahl rats were examined. The GBE-containing diet did not affect the negative and positive actions of isolated atria that were produced by acetylcholine and isoproterenol, respectively. In the aortic preparations, the relaxation in response to acetylcholine was significantly potentiated by a GBE-containing diet. Sodium nitroprusside-induced relaxation was unchanged by GBE-containing diet. These results demonstrated that GBE reduced salt-related elevation of blood pressure and restored the impaired acetylcholine-induced vasodilation in aortic segments.
Several neurological disorders such as Alzheimer's and Parkinson's diseases have been attributed to γ-aminobutyric acid (GABA) depletion in the brain. In order to provide a pharmacological basis for the neuroprotective actions of the enhanced accumulation of GABA in mulberry leaves (ML) against cerebral ischemia in vitro and in vivo, a process was developed to enhance the accumulation of GABA in mulberry leaves (GAML) as a result of the various anaerobic treatments. The GABA concentrations were changed by N2 gas purging, the reaction temperature, reaction time, pH and the leaf size. GABA enhanced the potential of neuroprotection in the PC12 cells damaged by H2O2-induced oxidation. GAML reduced the cytotoxicity in the PC12 cells against oxygen glucose deprivation-induced cerebral ischemic condition. The neuroprotective effect of GAML was further demonstrated in vivo using middle cerebral artery occlusion brain injury model. GAML significantly decreased the infarct volume of the brain compared with than control group. Overall, these results suggest that the anaerobic treatment of ML makes GAML enhance the neuroprotection effect against in vivo cerebral ischemia such as in vitro.
Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF) has been introduced into renal transplant immunosuppressant protocols in combination with calcineurin inhibitors (CNIs) and steroids. This study compared the pharmacokinetic profiles of MPA and its major metabolite MPA glucuronide (MPAG) in combination with tacrolimus (TAC) or cyclosporine (CyA) during the maintenance period (>6 months) following renal transplantation. There was no difference between TAC and CyA-treated groups in MPA plasma concentration before drug administration (C0). MPA C0 in TAC and CyA-treated patients did not differ from that in patients who were not treated with a CNI. In patients treated with a CNI, MPAG C0 was significantly greater in those treated with CyA compared with TAC. The MPAG/MPA ratio in CyA-treated patients was significantly greater than that in the TAC-treated group. We observed that C0 of MPA was negatively correlated with that of TAC and CyA. Positive correlation between MPA C0, MPAG C0 and serum creatinine was stronger in patients treated with CyA compared with TAC. Our study suggests that CyA, but not TAC, inhibits enterohepatic circulation of MPAG as a secondary excretion pathway, and that renal function makes a major contribution to elimination of MPA and MPAG. We indicate that it may be necessary to estimate biliary excretion of MPAG to avoid the risk of intestinal injury in patients receiving combination therapy with TAC during the maintenance period.
The effects of dexamethasone and aminophylline on survival of Jurkat T-lymphocytic leukemia cells and HL-60 promyelocytic leukemia cells were investigated. Dexamethasone (10, 1000 nM) and aminophylline (1, 100 μM) induced apoptosis in Jurkat and HL-60 cells in a concentration-dependent manner. Treatment with a combination of dexamethasone (10 nM) and aminophylline (1 μM) significantly increased the number of apoptotic HL-60 cells, but not that of Jurkat cells, compared with dexamethasone (10 nM) or aminophylline (1 μM) treatment alone. Dexamethasone and aminophylline also increased the number of phospho-histone H2B (Ser14)-positive Jurkat and HL-60 cells. Phospho-histone H2B (pH2B)-positive HL-60 cells were significantly increased by treatment with a combination of dexamethasone (10 nM) and aminophylline (1 μM), although no such effect was observed in Jurkat cells. On the other hand, simultaneous treatment with 10 nM dexamethasone and 1 μM aminophylline activated the 36-kDa MBP kinase, pro-apoptotic protein kinase in HL-60 cells. The activation of 36-kDa MBP kinase by dexamethasone and aminophylline was supported by studies showing an increase in the number of pH2B-positive and apoptotic Jurkat and HL-60 cells upon exposure to these drugs. Thus treatment with a combination of dexamethasone and aminophylline accelerates apoptosis of HL-60 cells via activation of 36-kDa MBP kinase and H2B phosphorylation.
Aggregation of the high affinity receptor for IgE (FcεRI) on mast cells by antigen and IgE complex induces release of chemical mediators, leading to acute allergic inflammation. We recently found that 3-O-(2,3-dimethylbutanoyl)-13-O-decanoylingenol (DBDI), purified from the Euphorbia kansui L., inhibits degranulation in rat basophilic leukemia 2H3 cells upon aggregation of the FcεRI. In the present study, we demonstrated that the DBDI significantly inhibits release of β-hexosaminidase, synthesis of eicosanoids, and mobilization of intracellular Ca2+ in the bone marrow-derived mouse mast cells stimulated with IgE and antigen. Furthermore, we revealed that phosphorylation of Syk, phospholipase C-γ2, and extracellular signal-related kinase 1/2 is significantly suppressed in the DBDI-treated mast cells. These findings suggest that the DBDI may have a therapeutic potential for allergic diseases by inhibiting intracellular signaling pathways for activation and chemical mediator release in mast cells.
The aim of this study was to evaluate the effect of Fructus Ligustri Lucidi (FLL), a kidney-tonifying Chinese herbal medicine, on the biochemical markers of bone turnover, calcium metabolism and balance in osteoporotic rat model developed by ovariectomy. Four weeks after surgical operation, animals were randomly assigned to one of the four treatments for 14 weeks: sham-operated control treated with vehicle (sham, n=8), ovariectomized group treated with vehicle (OVX, n=8), OVX group treated with 17β-estradiol (E2, n=10, 2 μg/kg/d) and OVX group treated with FLL extracts (FLL, n=10, 550 mg/kg/d). Serum osteocalcin and urinary deoxypyridinoline levels were upregulated in rats in response to OVX, suggesting that the bone turnover rate was accelerated in these animals. Treatment of OVX rats with FLL extract could prevent OVX-induced increase in bone turnover by suppression of both serum osteocalcin (p<0.05, vs. OVX) and urinary deoxypyridinoline (p<0.05, vs. OVX) levels. In addition, FLL extract could prevent OVX-induced loss of calcium in rats by increasing the intestinal calcium absorption rate (p<0.01, vs. OVX), suppressing urinary Ca excretion (p<0.05, vs. OVX) as well as increasing bone calcium content (p<0.05, vs. OVX). Our study is the first to report that FLL can modulate bone turnover and calcium balance in OVX rats and it might be a potential candidate for prevention and treatment of postmenopausal osteoporosis.
Several independent lines of evidence indicate the direct impairment by extracellular glucose at high concentrations of different osteoblastic functions with a marked decrease in bone mass toward osteoporosis, while the underlying mechanisms are not well clarified to date. We have previously demonstrated the functional expression of the neural amino acid γ-aminobutyric acid (GABA) signaling system including betaine/GABA transporter-1 (BGT-1) with a temperature-, sodium- and chloride-dependent activity of [3H]GABA accumulation in cultured rat calvarial osteoblasts. In this study, therefore, we attempted to demonstrate the possible involvement of BGT-1 isoform in bone dysfunctions due to impaired mineralization in rat calvarial osteoblasts cultured under hyperglycemic conditions. No significant change was seen in [3H]GABA accumulation in osteoblasts cultured for 7 d in vitro (DIV) under hyperglycemic conditions (glucose=25.5—50.5 mM) compared to those cultured in normoglycemic (glucose=5.5 mM) and hyperosmotic (mannitol=25.5—50.5 mM) conditions. In osteoblasts cultured for 14 DIV under hyperglycemic conditions, however, [3H]GABA accumulation was significantly increased compared to those cultured under normoglycemic and hyperosmotic conditions. Kinetic analysis revealed that hyperglycemic cultivation resulted in a significant increase in Vmax values from 2.85 nmol/min/mg protein for normoglycemic conditions to 4.17 nmol/min/mg protein for hyperglycemic conditions without affecting Km values. However, experimental hyperglycemia did not significantly affect the expression of mRNA for BGT-1 isoform by osteoblasts. These results suggest that GABA transport system may at least in part play a role in pathological malfunctions and abnormalities through a mechanism not directly related to gene expression in osteoblasts under hyperglycemia.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequently prescribed drug for the treatment of inflammation and pain. However, conventional NSAIDs and selective COX-2 inhibitors have shown many side effects such as gastric mucosal damage and cardiovascular problems. Recently, the use of dual acting inhibitors of cyclooxygenases (COX) and lipoxygenase (LOX) has been highlighted for their minimized side effects compared to NSAIDs. The objective of the present study was to examine the efficacy and the gastric side effects of 1-furan-2-yl-3-pyridin-2-yl-propenone (FPP-3), a synthetic dual inhibitor of COX/5-LOX. Indomethacin (1—50 mg/kg, p.o.), a non-selective COX inhibitor, and FPP-3 (0.5—50 mg/kg, p.o.), a dual inhibitor, significantly suppressed the carrageen-induced paw edema with different pharmacological profiles. The concentrations of FPP-3 and indomethacin showing 50% inhibition of the maximum paw edema in rats were 10 mg/kg and 20 mg/kg, respectively. More importantly, there were no gastric ulcers formed in FPP-3-treated rats and mice, whereas indomethacin caused gastric mucosal bleeding in a concentration-dependent manner. In addition, FPP-3 showed an analgesic effect in acetic acid-induced writhing response in mice in a dose-dependent manner. The results suggest that FPP-3 may have a benefit in combatting inflammation and pain by dual inhibition of COX and LOX.
Previous reports have shown that ginseng saponins, the active ingredients of Panax ginseng, induce relaxation of hormone- or high K+-induced blood vessel contraction. We recently demonstrated that 20(R)- and 20(S)-ginsenoside Rg3 epimers regulate ion channel activities in a stereospecific manner. Here, we examined whether ginsenoside Rg3 epimers also exhibit differential effects on swine coronary artery contractions induced by high K+ or 5-HT. We found that treatment with 20(S)- but not 20(R)-ginsenoside Rg3 caused a potent concentration-dependent, endothelium-independent relaxation of coronary artery contraction induced by 25 mM KCl. However, treatment with both 20(S)- and 20(R)-ginsenoside Rg3 induced a significant, concentration-dependent relaxation of 3 μM 5-HT-induced coronary artery contractions in intact samples, while only 20(S)-ginsenoside Rg3 inhibited coronary artery contraction in endothelium-denuded arteries. 20(S)- but not 20(R)-ginsenoside Rg3 inhibited L-type Ca2+ channel currents in a dose- and voltage-dependent manner. These results indicate that 20(S)- and 20(R)-ginsenoside Rg3 epimers might exhibit stereospecific relaxation effects on swine coronary artery contractions caused by high K+ and 5-HT receptor activation.
The effects of combined β-cryptoxanthin and zinc on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vivo were investigated. Rats were orally administered either vehicle, β-cryptoxanthin (5 or 10 μg/100 g body weight), zinc sulfate (0.1 or 0.5 mg Zn/100 g), or their combination once a day for 7 d. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal tissues was not significantly altered by the administration of β-cryptoxanthin (5 μg/100 g) or zinc (0.1 or 0.5 mg/100 g). Combined administration of β-cryptoxanthin (5 μg/100 g) and zinc (0.1 or 0.5 mg/100 g) caused a synergistic increase in calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal tissues. The effect of β-cryptoxanthin (5 or 10 μg/100 g) in increasing calcium and DNA contents in the metaphyseal tissues was significantly enhanced by the combined administration of zinc (0.1 or 0.5 mg/100 g), but did not have a significant effect on the metaphyseal components. The metaphyseal alkaline phosphatase activity was markedly increased by the combination of β-cryptoxanthin (5 μg/100 g) and zinc (0.1 or 0.5 mg/100 g). This study demonstrates that the oral administration of the combination of zinc at lower doses synergistically enhances β-cryptoxanthin-induced anabolic effects on bone components in the femoral tissues of rats in vivo.
Diallyl disulfide (DADS), an important component of garlic (Allium sativam) has been demonstrated to exert a potential chemopreventive activity against human cancers. DADS inhibits proliferation of both androgen dependent and independent prostate cancer cells in vitro. However there is no report available on the role of DADS on prostate cancer initiation in in vivo model. So the present chemoprevention study was conducted to evaluate the activity of diallyl disulfide as an anticancer agent in prostate carcinogenesis of male Sprague Dawly rats. Testosterone and N-Methyl N-Nitroso Urea (MNU) were used to induce prostate carcinogenesis that involves a multi step process like, hyperplasia, dysplasia and prostatic intraepithelial neoplasia (PIN). The rats were induced prostate carcinogenesis by injection of testosterone and single dose of MNU and again the testosterone was continued throughout the experimental period. Forty percentage of animals carried PIN in dorsolateral prostate, while dysplasia and hyperplasia (55 to 65%) were common in ventral as well as dorsolateral prostates of the hormone and carcinogen treated rats. Rats treated with hormone and carcinogen along with DADS developed PIN at incidence of 10% in the ventral and dorsolateral prostates about 20 to 10%. Dysplasia and hyperplasia were less common in these rats. The results of this study provide evidence that DADS may have chemopreventive activity in rat prostate carcinogenesis.
The methanol extract of Sophora flavescens showed a potent glycosidase inhibitory activity. Active components were identified as well-known flavonoid antioxidants: kushenol A (1), (−)-kurarinone (2), sophoraflavanone G (3), 2′-methoxykurarinone (4), kurarinol (5), 8-prenylkaempferol (6), isoxanthohumol (7), kuraridin (8) and maackian (9). All flavonoids were effective inhibitors of α-glucosidase and β-amylase. Interestingly, lavandulylated flavanones 1—5 had strong α-glucosidase inhibitory activities, with IC50 values of 45 μM, 68 μM, 37 μM, 155 μM and 179 μM, respectively. Kushenol A (1) which does not bear a 4′-hydroxy group showed selective α-glucosidase inhibitory activity. Lavandulylated chalcone, kuraridine (8), exhibited IC50 value of 57 μM against β-glucosidase, which is the first report of a chalcone displaying glycosidase inhibition. Results showed that 8-lavandulyl group in B-ring was a key factor of the glycosidase inhibitory activities. The inhibition pattern was noncompetitive for α-glucosidase, whereas mixed inhibition was observed for β-amylase.
Highly inhibitory effects of hybrid liposomes on the growth of human breast tumor cells in vitro were obtained. It is worthy to note that induction of apoptosis through activation of caspases by hybrid liposomes was clearly observed.
The protective effect of a 30 kDa glycoprotein (GF-AS) isolated from the stem bark of Acanthopanax senticosus against acute and chronic alcohol-induced hepatotoxicity were studied. N-terminal amino acid sequence of GF-AS showed NH2-Val-Ala-Tyr-Pro-Trp-Ala-Gly-Phe-Ala-Leu-Ser-Leu-Glx-Pro-Pro-Ala-Gly-Tyr-. GF-AS significantly increases the activities of alcohol-metabolizing enzymes, including alcohol dehydrogenase, microsomal ethanol metabolizing system, and acetaldehyde dehydrogenase in rats acutely treated with alcohol, resulting in decreased plasma alcohol levels. GF-AS also increases the activities of antioxidant enzymes and glutathione level. Markers of liver injury induced by alcohol: elevated serum levels of aspartate aminotransferase, alanine aminotransferase, triglyceride and cholesterol, are reduced by GF-AS in both acutely and chronically treated rats. The activities of lipogenic enzymes including malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphoglucuronic acid dehydrogenase in chronic alcohol-treated rats are significantly decreased by GF-AS. Furthemore, GF-AS improves histological change in fatty liver and hepatic lesions induced by alcohol. Collectively, GF-AS may alleviate alcohol-induced hepatotoxicity through increasing ethanol and lipid metabolism, as well as antioxidant defense systems in livers injured by acute- and chronic-alcohol treatment.
Dried rhizomes of five species of Atractylodes (A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana), Compositae, have been used as crude drugs mainly for the treatment of stomach disorders and for their diuretic properties in Chinese and Japanese traditional medicines. The identification of the botanical origins of these crude drugs is generally difficult from their morphological and chemical features only. In this study, for identification with more reliable, nuclear ribosomal DNA (nrDNA), internal transcribed spacer (ITS) regions of five species of medicinal Atractylodes were sequenced. As a result, specific ITS genotypes were recognized by each species. The four species (A. japonica, A. macrocephala, A. lancea, and A. chinensis) prescribed in Chinese and Japanese Pharmacopoeias as botanical origins of crude Atractylodes drugs could be distinguished by their ITS sequences because they had difference genotypes on the ITS sequences. However, the genotype of A. koreana was the same as that of A. chinensis. Additionally, hybrids between A. lancea and A. chinensis were also recognized as nucleotide additives on their ITS sequences. In this study, several morphological characteristics were researched by their genotype, too. As this result, the hybrids recognized from the genetic analysis had intermediate morphological characteristics between A. lancea and A. chinensis. It was also recognized that A. lancea and A. chinensis except for their hybrids were significant differences. It is therefore suggested that ITS sequences of nrDNA would be useful for the identification of the crude drugs derived from Atractylodes species and their interspecific hybridizations.
Acyl-CoA: cholesterol acyltransferase (ACAT) catalyzes the acylation of cholesterol to cholesteryl ester with long chain fatty acids and ACAT inhibition is a useful strategy for treating hypercholesterolemia or atherosclerosis. Pentacyclic triterpenes, ursolic acid (1), oleanolic acid (2), and betulinic acid (3) were isolated from the methanol extracts of the leaves of Lycopus lucidus TURCZ. by bioassay-guided fractionation. The structures of compounds 1—3 were elucidated by their spectroscopic data analysis. Among them, betulinic acid (3) exhibited more potent human ACAT-1 and ACAT-2 inhibitory activities with IC50 values of 16.2±0.6 and 28.8±1.3 μM, respectively.
The purpose of this study is to assess orally-disintegrating (OD) tablet of clonidine hydrochloride (CL) for a pre-operative sedation in pediatric surgery. Sedation score and plasma CL concentration of OD formulation was compared with original preparation, CL lollipop, in pediatric patients. Fourteen patients (age: 3.9±2.3 years, weight: 16.9±5.0 kg) for OD group and 9 patients (age: 4.4±3.1 years, weight: 17.2±7.0 kg) for lollipop group received 4 μg/kg of CL preparation. Pre-operative sedation was evaluated by 5-point scoring systems at entering the operating room. Plasma CL concentrations were determined 120 min after administration of CL preparation. The changes in systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were also assessed before and after administration of CL preparation. Every patients in OD group had satisfactory sedation (sedation score: 2 and 3), whereas, 3 (33%) in lollipop group had unsatisfactory sedation (sedation score: 0 and 1). Plasma CL concentration in OD group was significantly higher than those in lollipop group (0.75±0.15 vs. 0.42±0.21 ng/ml, p<0.01). There was no significant difference in hemodynamic parameters (SBP, DBP and HR) between before and after administration of CL preparation in both OD and lollipop group. We conclude that OD is superior preparation of CL for pre-operative sedation in pediatric surgery.
Recently, a silent polymorphism of C3435T of the MDR1 gene, encoding the multidrug resistant transporter MDR1/P-glycoprotein, has been found to be associated with susceptibility to ulcerative colitis (UC), but this remains controversial. This study was conducted to find a possible reason for the discrepancies, and it was suggested that the age of onset was important for the association, namely, C3435T was predictive of susceptibility to later onset UC, but not for early onset UC. Linkage disequilibrium of C3435T with T-129C, C1236T and G2677A, T was suggested to be altered in UC, but the analysis of their haplotype provided no advantage in terms of prediction over that with only C3435T. The effect of C3435T on susceptibility could not be explained by that on mRNA expression in rectal mucosa, but it was greater in the C3435-noncarriers in the early onset group, allowing the individualization of steroid-based pharmacotherapy.
We previously reported that the fatty base suppository containing sodium laurate (C12) and taurine (Tau) (C12-Tau suppository) could enhance the colonic absorption of rebamipide, a poorly water-soluble and poorly absorbable drug, without any serious mucosal damages in rats. In the preset study, in order to make C12-Tau suppositories available for practical use, the scaling-up studies of animal and formulation size were performed, compared with the suppositories containing sodium caprate (C10) (C10 suppository) at the same amounts as those contained in the commercial products. Twenty-mg C12 improved the dissolution of rebamipide from suppository remarkably and the addition of 30-mg Tau only slightly decreased the dissolution rate. The absorption of rebamipide from rabbit rectum was more markedly improved by suppositories containing C12 than C10 suppositories. Although Tau tended to attenuate the absorption-enhancing effect of C12, several C12-Tau suppositories kept high bioavailability values, which were much higher than control. Histopathological studies showed that Tau exerted the cytoprotective action and that C12-Tau suppositories were better than C10 suppositories in safety. Considering the balance between efficacy and safety, the suppository containing 10- or 20-mg C12 with 30-mg Tau is better than C10 suppositories as commercial products and could be promising for practical use in human.
The clinical significance of cyclosporine (CsA) concentration 2 h postdose (C2) monitoring is widely recognized in organ transplantation, because C2 value is considered to be a predictable surrogate marker of full area under the concentration–time curve (AUC), and/or a peak concentration value exhibits potent inhibition of calcineurin activity. However, the pharmacological advantage of absorption profile (AP) has not been fully elucidated. In a rat skin allotransplantation model, the authors evaluated the efficacy of AP by different dosage regimens (20, 25 or 30 mg/kg/d, once or twice daily) and routes (p.o. or i.v.), and examined whether high C2 or AUC0—4 is intrinsically valuable for effective immunosuppression. Graft survival was CsA dose-dependent and correlated with full AUC0—24, rather than AP. The difference between the once and twice daily administrations did not influence full AUC0—24 or immunosuppressive effect. Continuous intravenous infusion with flat pharmacokinetics also produced adequate immunosuppression as was observed in enteral administration at the same level of total exposure. The impact of high peak concentration in AP on immunosuppressive effect could not be found. It was suggested that AP would not have intrinsic pharmacodynamic value. However, absorption profiling was considered to be clinically useful in that C2 value is a good surrogate marker of total exposure (AUC0—24).
Dilute solutions of pectin containing complexed calcium ions form gels when these ions are released in the acidic environment of the stomach. The aim of this study was to examine the influence of a variation of gastric pH and the addition of a taste masking agent on the gelation of the pectin solutions and on the in vitro and in vivo release of acetaminophen from the gels. Increase of pH above 2.5 and addition of 10% (w/v) D-sorbitol significantly affected the ability of 1.5% (w/v) pectin solutions to form coherent gels in vitro. Gelation of sorbitol-free formulations was observed at pH 1.2 and in vitro release of acetaminophen from the gels followed diffusion-controlled kinetics; in vitro gelation of these formulations, however, was incomplete at pH 3.0 resulting in poor sustained release characteristics. Inclusion of 10% (w/v) D-sorbitol in the formulations inhibited the in vitro gelation of the 1.5% (w/v) pectin sols and poor sustained release properties were noted from these formulations even at pH 1.2. The bioavailability of acetaminophen from gels formed in the stomach of gastric-acidity controlled rabbits following oral administration of the liquid formulations was not, however, significantly affected either by the inclusion of 10% (w/v) D-sorbitol or increase of pH to 3.6. Visual observation showed in situ gelation of 1.5% (w/v) pectin formulations containing D-sorbitol at pH 4.3 suggesting that normal variations of gastric acidity in the fasting state will have no effect on the bioavailability of acetaminophen when delivered using these formulations.
CpG-oligodeoxynucleotide (CpG-ODN) plays a critical role in immunity via the augmentation of Th1 and suppression of Th2 responses. We examined here the effect of CpG-ODN on the immune response to mite antigen sensitized through barrier-disrupted skin of human atopic dermatitis (AD) model mouse. Although sensitization with mite antigen induced Th2-dominant immune response, co-administration of CpG-ODN elicited Th1-predominant immune response. In terms of antigen-specific antibody production, the level of IgG2a was increased by CpG-ODN, but not in IgE. These results suggested that administration of CpG-ODN via skin is a simple strategy for patients with diseases like AD, which is characterized by Th2-dominated inflammation.
Inducible nitric oxide synthase (iNOS) and NO have been suggested to be involved in acute radiation response in tissues such as the liver, intestine, colon, and brain. However, direct measurement of NO and iNOS in ionizing radiation-induced skin inflammatory reactions is not reported yet. We show here for the first time, by in vivo experiments, that X-ray irradiated mouse skin generates NO with concomitant expression of iNOS at both the mRNA and protein levels. When irradiated at 50 Gy, iNOS mRNA appeared at day 8 post-irradiation, whereas iNOS protein could be detected only at day 14. No iNOS protein was detectable however for the mice receiving 5 or 15 Gy irradiation, even at day 14. Skin inflammatory reactions were observed at day 8 post-irradiation as an increase in skin thickness, which increased further by day 14. Histological observations showed acute inflammatory responses. The parallel relationship between iNOS induction and the onset of skin inflammatory reactions suggests the involvement of iNOS and NO in the skin damage. Immunohistochemical staining showed the localization of iNOS at skin erosion areas, exudate and infiltrating cells. Taken together, these findings suggest that iNOS induction and NO production in X-irradiated skin are relatively early events in skin inflammatory reactions and are probably secondary rather than primary reactions of irradiation.
Water extract (WE) of Cordyceps militaris has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not been known. In this study, we investigated whether water extract of C. militaris induces the phenotypic and functional maturation of dendritic cells (DC). It profoundly increased CD40, CD54, CD80, CD86, and MHC class II expression in murine bone marrow (BM)-derived myeloid DC. Endocytosis was assessed by the uptake of FITC-dextran and FITC-albumin. The ability of unstimulated DC (UT-DC) to uptake dextran and albumin was higher than that of WE- or LPS-stimulated DC (LPS-DC). Also, UT-DC secreted a low concentration of IL-12, while WE- or LPS-DC secreted higher levels of IL-12 than UT-DC. WE not only formed morphologically mature DC and clusters, but also induced predominantly functional maturation. Moreover, WE is shown to promote the cytotoxicity of specific-cytotoxic T lymphocyte (CTL) induced by DC which were pulsed with P815 tumor-lysate during the stage of antigen presentation. These results suggest that DC maturation by WE can play a critical role in the improvement of the immunoregulatory function in patients with impaired host defense.
The morphology, structure, and antigenicity of the cells and the cell wall mannans of the Candida guilliermondii IFO 10279 strain cultivated at 33 and 34 °C for 48 h in yeast extract-added Sabouraud liquid medium (YSLM) were compared with those cultivated at 27 °C and 33 °C and then at 27 °C (33—27 °C). This strain showed little growth at higher than 35 °C. The density of the yeast formed cells decreased, with dry weights of about 50% at 33 and 34 °C, and only the cells at 34 °C revealed a failure of cytokinesis. The structure of the mannans revealed by 1H-NMR analysis that the mannans obtained at both 33 and 34 °C had drastically decreased two consecutive β-1,2-linked mannopyranose units at the nonreducing terminal of the α-linked oligosaccharides and increased one β-1,2-linked mannopyranose unit at the nonreducing terminal attached to the α-1,3-linked mannose unit and the non-reducing terminal α-1,3- and α-1,2-linked mannopyranose units. The enzyme-linked immunosorbent assay (ELISA) showed that the mannans obtained at 33 and 34 °C had decreased reactivity against the factor serum 9 and increased its reactivity against the factor serum 4, in the commercially available factor serum kit ‘Candida Check’.
5α-Reductase inhibitory activity-guided fractionation of the EtOH extract of the fruiting body of Ganoderma lucidum (LEYSS.:FR.) KARST. (Ganodermataceae), which is called Reishi, or Mannentake in Japan and Lingzhi in China, led to the isolation of two active compounds which were ganoderic acid DM and 5α-lanosta-7,9(11),24-triene-15α,26-dihydroxy-3-one with an IC50 of 10.6 μM and 41.9 μM respectively. A carboxyl group of side chain of ganoderic acid DM is essential to elicit the inhibitory activity because of much less activity of its methyl ester.