HPLC analysis of ammonium glycyrrhizate listed in the European, United States, and Japanese Pharmacopoeias under reported and modified conditions: revision of the peak assignment for 18α-glycyrrhizin in the European and United States Pharmacopoeias.

We examined ammonium glycyrrhizate listed in the monographs of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) as well as in the reagents and solutions used in the general test of the Japanese Pharmacopoeia by performing HPLC on their sample standards or reference reagents under reported and modified conditions. Comparative experiments involving five authentic samples, namely, 18ß-glycyrrhizin (1), 18α-glycyrrhizin (2), licorice-saponin G2 (3), licorice-saponin H2 (4), and galacturonic acid-replaced glycyrrhizin (the 4″-epimer of 18ß-glycyrrhizin) (5), led us to propose the revision of the peak assignment of 18α-glycyrrhizin (2) and postscript a possible co-existence of galacturonic acid-replaced glycyrrhizin (5) as a hidden component in the EP and USP. We also proposed that the α-configuration used in the nomenclature of the glycosidic bond between aglycone and the sugar units of ammonium glycyrrhizate and impurities in the EP and USP should be revised to the ß-configuration.

Is 18α-Glycyrrhizin a real natural product? Improved preparation of 18α-Glycyrrhizin from 18ß-Glycyrrhizin as a positive standard for HPLC analysis of licorice extracts.

18α-Glycyrrhizin is an epimer of 18ß-glycyrrhizin, a major component of licorice (Glycyrrhiza sp.), which is widely used as a traditional medicine. Whether 18α-glycyrrhizin is a real natural product has been debated in the long history of glycyrrhizin chemistry because 18ß-glycyrrhizin is epimerizable to a more thermodynamically stable 18α-glycyrrhizin under aqueous alkali conditions. We improved the preparation of 18α-glycyrrhizin from 18ß-glycyrrhizin by successive epimerization reactions of 18ß-glycyrrhizin, trimethyl esterification of the resulting epimerized mixture, and alkaline hydrolysis of a purified 18α-glycyrrhizin trimethyl ester. Approaches to the possible presence of 18α-glycyrrhizin in licorice extracts by HPLC using synthetic 18α-glycyrrhizin as a positive standard strongly suggested that 18α-glycyrrhizin could naturally exist as a minor congener of glycyrrhizin derivatives in Glycyrrhiza species.

Quercetin 3,5,7,3',4'-pentamethyl ether from Kaempferia parviflora directly and effectively activates human SIRT1.

Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a crucial regulator that produces multiple physiological benefits, such as the prevention of cancer and age-related diseases. SIRT1 is activated by sirtuin-activating compounds (STACs). Here, we report that quercetin 3,5,7,3',4'-pentamethyl ether (KPMF-8), a natural STAC from Thai black ginger Kaempferia parviflora, interacts with SIRT1 directly and stimulates SIRT1 activity by enhancing the binding affinity of SIRT1 with Ac-p53 peptide, a native substrate peptide without a fluorogenic moiety. The binding affinity between SIRT1 and Ac-p53 peptide was enhanced 8.2-fold by KPMF-8 but only 1.4-fold by resveratrol. The specific binding sites of KPMF-8 to SIRT1 were mainly localized to the helix2-turn-helix3 motif in the N-terminal domain of SIRT1. Intracellular deacetylase activity in MCF-7 cells was promoted 1.7-fold by KPMF-8 supplemented in the cell medium but only 1.2-fold by resveratrol. This work reveals that KPMF-8 activates SIRT1 more effectively than resveratrol does.

Quantification of terpene trilactones in Ginkgo biloba with a 1H NMR method.

Ginkgo biloba L. has been used as a herbal medicine in the traditional treatment of insufficient blood flow, memory deficits, and cerebral insufficiency. The terpene trilactone components, the bioactive agents of Ginkgo biloba L., have also been reported to exhibit useful functionality such as anti-inflammatory and neuroprotective effects. Therefore, in the present research, we attempted to analyze quantitatively the terpene trilactone components in Ginkgo biloba leaf extract, with quantitative 1H NMR (qNMR) and obtained almost identical results to data reported using HPLC. Application of the qNMR method for the analysis of the terpene trilactone contents in commercial Ginkgo extract products, such as soft gel capsules and tablets, produced the same levels noted in package labels. Thus, qNMR is an alternative method for quantification of the terpene trilactone components in commercial Ginkgo extract products.

Design, synthesis, and evaluation of polyamine-memantine hybrids as NMDA channel blockers.

N-Methyl-d-aspartate (NMDA) receptors have been implicated in learning and memory, and may also play a central role in various conditions leading to neuronal degradation. NMDA receptor antagonists could therefore be of therapeutic benefit for a number of neurological disorders. We have designed hybrid compounds of polyamines and memantine, both of which function as NMDA channel blockers. The triamine derivative with a guanidine moiety showed more potent antagonistic activity than memantine.

A simple and effective preparation of quercetin pentamethyl ether from quercetin.

Among the five hydroxy (OH) groups of quercetin (3,5,7,3',4'-pentahydroxyflavone), the OH group at 5 position is the most resistant to methylation due to its strong intramolecular hydrogen bonding with the carbonyl group at 4 position. Thus, it is generally difficult to synthesize the pentamethyl ether efficiently by conventional methylation. Here, we describe a simple and effective per-O-methylation of quercetin with dimethyl sulfate in potassium (or sodium) hydroxide/dimethyl sulfoxide at room temperature for about 2 hours, affording quercetin pentamethyl ether (QPE) quantitatively as a single product. When methyl iodide was used in place of dimethyl sulfate, the C-methylation product 6-methylquercetin pentamethyl ether was also formed. A computational study provided a rationale for the experimental results.

Changes in intracellular copper concentration and copper-regulating gene expression after PC12 differentiation into neurons.

It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation.

Phase II trial of panitumumab with irinotecan as salvage therapy for patients with advanced or recurrent colorectal cancer (TOPIC study).

Little is known about the clinical impact of salvage panitumumab with irinotecan for metastatic colorectal cancer (mCRC) patients. The present study conducted a single-arm, multicenter phase II trial for mCRC with skin toxicity prevention program. The subjects were mCRC patients with wild-type KRAS, who showed resistance to fluoropyrimidine, oxaliplatin and irinotecan. Panitumumab was administered at a dose of 6 mg/kg every 2 weeks by intravenous infusion over 60 min, and irinotecan was administered at a dose of 100-180 mg/m2 every 2 weeks by intravenous infusion over 90 min, depending on the preceding treatment dose. To prevent skin toxicities, a moisturizer was applied and oral antibiotics (100 mg minocycline twice daily) were initiated for 6 weeks. The primary endpoint was the response rate (RR) determined by independent reviewers. Secondary endpoints were the disease control rate (DCR), progression-free survival (PFS) time, overall survival (OS) time and adverse events. A total of 35 patients were enrolled between October 2010 and March 2012. The median age was 61 years (range, 41-76 years), with 25 male and 10 female patients. The initial irinotecan dose was 150 mg/m2 in 19 patients and 180 mg/m2 in 1 patient. The remaining patients were treated with ≤120 mg/m2. A central review indicated a partial response in 8 patients (22.9%) and stable disease in 6 patients (17.1%), with an RR of 22.9% (95% confidence interval, 12.1-39.0) and a DCR of 40%. The RR of the patients with standard-dose irinotecan (150 or 180 mg/m2) was 30%, although that of low-dose irinotecan (100-120 mg/m2) was 13%. The median PFS time was 2.7 months, and the median OS time was 6.3 months. A grade 3 or above acne-like rash developed in 25.7% of patients. In conclusion, panitumumab and irinotecan as salvage therapy for mCRC KRAS wild-type patients with skin toxicity prevention exhibits limited efficacy. In particular, the effect of low-dose irinotecan with panitumumab appears to be clinically insignificant. Routine use of skin toxicity prevention is currently under evaluation.

Unprecedented 8,9'-neolignans: enantioselective synthesis of possible stereoisomers for structural determination.

(+)-Wutaienin (3) and its C-7 methyl ether (4), isolated from Zanthoxylum wutaiense, were found to be unprecedented 8,9'-neolignans containing an (S)-2-(1,1-dimethyl-1-hydroxymethyl)-7-methoxydihydrobenzofuran skeleton. Wutaienin (3) was present in the plant as an inseparable 1:1 mixture of the (7,8)-syn-diastereoisomers. The diastereoisomeric mixture was characterized by comparison with four possible diastereoisomers, which were enantioselectively synthesized from (S)-5-bromo-(1,1-dimethyl-1-hydroxymethyl)-7-methoxydihydrobenzofuran using Evans' oxazolidinone-assisted asymmetric aldol condensation to install the chiral centers at the C-7 and C-8 positions.

Antidepressant-like effect of Butea superba in mice exposed to chronic mild stress and its possible mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Butea superba (BS) is a Thai medicinal plant that has been used as a folk medicine to improve physical and mental conditions and to prevent impaired sexual performance in middle-aged or elderly males. We have previously reported that this plant extract could improve cognitive deficits and depression-like behavior in olfactory bulbectomized mice, an animal model of dementia and depression. AIM OF THE STUDY: In this study we examined the effect of BS on depression-like behavior in mice subjected to unpredictable chronic mild stress (UCMS) to clarify the antidepressant-like activity of BS and the molecular mechanism underlying this effect. MATERIALS AND METHODS: UCMS mice were administered BS daily (300 mg of dried herb weight/kg, p.o.) or a reference drug, imipramine (IMP, 10 mg/kg, i.p.), 1 week after starting the UCMS procedure. We employed the sucrose preference test and the tail suspension test to analyze anhedonia and depression-like behavior of mice, respectively. Serum and brain tissues of mice were used for neurochemical and immunohistochemical studies. The UCMS procedure induced anhedonia and depression-like behavior, and BS treatment, as well as IMP treatment, attenuated these symptoms. UCMS caused an elevation of serum corticosterone level, an index of hyper-activation of the hypothalamic-pituitary-adrenal (HPA) axis, in a manner attenuated by BS and IMP treatment. BS treatment also attenuated UCMS-induced decrease in the expression levels of brain-derived neurotrophic factor (BDNF) mRNA, cyclic AMP-responsive element binding protein (CREB) and a phosphorylated form of N-methyl-d-aspartate receptor subunit NR1, synaptic plasticity-related signaling proteins. Moreover, the UCMS procedure reduced doublecortin-positive cells in the dentate gyrus region of the hippocampus. BS administration reversed these UCMS-induced neurochemical and histological abnormalities. CONCLUSION: These results suggest that BS can ameliorate chronic stress-induced depression-like symptoms and that the effects of BS are mediated by restoring dysfunctions of the HPA axis and synaptic plasticity-related signaling systems and neurogenesis in the hippocampus.

Butea superba-induced amelioration of cognitive and emotional deficits in olfactory bulbectomized mice and putative mechanisms underlying its actions.

This study investigated the effects of alcoholic extract of Butea superba (BS) on cognitive deficits and depression-related behavior using olfactory bulbectomized (OBX) mice and the underlying molecular mechanisms of its actions. OBX mice were treated daily with BS (100 and 300 mg/kg, p.o.) or reference drugs, tacrine (2.5 mg/kg, i.p.) and imipramine (10 mg/kg, i.p.) from day 3 after OBX. OBX impaired non-spatial and spatial cognitive performances, which were elucidated by the novel object recognition test and modified Y maze test, respectively. These deficits were attenuated by tacrine and BS but not imipramine. OBX animals exhibited depression-like behavior in the tail suspension test in a manner reversible by imipramine and BS but not tacrine. OBX down-regulated phosphorylation of synaptic plasticity-related signaling proteins: NMDA receptor, AMPA receptor, calmodulin-dependent kinase II, and cyclic AMP-responsive element-binding protein. OBX also reduced choline acetyltransferase in the hippocampus. BS and tacrine reversed these neurochemical alterations. Moreover, BS inhibited ex vivo activity of acetylcholinesterase in the brain. These results indicate that BS ameliorates not only cognition dysfunction via normalizing synaptic plasticity-related signaling and facilitating central cholinergic systems but also depression-like behavior via a mechanism differing from that implicated in BS amelioration of cognitive function in OBX animals.

A rare complication: misdirection of an indwelling urethral catheter into the ureter.

We report 3 patients with the rare complication of an indwelling urethral catheter misdirected into the ureter. This is the largest series to date. Patients were referred to us for a variety of reasons following exchange of their chronic indwelling urinary catheters. CT in all cases demonstrated the urinary catheters residing in the left ureter. The ages of the patients were 37, 67 and 81 years old. All patients suffered from neurogenic bladder. Two patients were female, one was male, and 2 of the 3 had a sensory disorder inhibiting their pain response. The catheters were replaced with open-end Foley catheters. Extensive follow-up CT scans were obtained in one case, demonstrating improvement of hydronephrosis and no evidence of ureteral stenosis. Cystoscopy in this patient demonstrated normally positioned and functioning ureteral orifices. Although the placement of an indwelling urethral catheter is a comparatively safe procedure, one must keep in mind that this complication can occur, particularly in female patients with neurogenic bladder. CT without contrast is a noninvasive, definitive diagnostic tool.

Neolignans and phenylpropanoids from the roots of Piper taiwanense and their antiplatelet and antitubercular activities.

Bioassay-guided fractionation of roots from Piper taiwanense led to isolation of three neolignans, diallylcatechol (1) and neotaiwanensols A, B (2, 3), two diphenylpropanoid ethers, taiwandimerols A, B (4, 5), with one phenylpropanoid, 2,3-diacetoxy-1-methoxy-5-allylbenzene (6), previously unknown in nature, together with 18 known compounds (7-24). Their structures were elucidated by spectroscopic evidence. Among the isolates, hydroxychavicol acetate (7), and 4-allylcatechol (8) showed potent inhibitory activities against platelet aggregation induced by collagen, with IC50 values of 2.1, and 5.3 µM, respectively. Hydroxychavicol acetate (7), 4-allylcatechol (8), and trans-caffeicaldehyde (9) showed antitubercular activities against Mycobacterium tuberculosis H37Rv, with MIC values of 30.3, 27.6, and 25.5 µg/mL, respectively.

Enantioselective formal synthesis of (-)-podophyllotoxin from (2S,3R)-3-arylaziridine-2-carboxylate.

Meyers' 4-aryl-1-tetralone-lactone and ent-Zhang's 2-diarylmethyl-4-oxobutanoate were synthesized in the formal synthesis of (-)-podophyllotoxin from (2S,3R)-3-arylaziridine-2-carboxylate, via 3,3-diarylpropanoate as a common intermediate, in an overall 42% yield through 10 steps and 31% yield through 6 steps, respectively. The key steps in the synthesis were regio- and diastereoselective ring opening with an aromatic nucleophile, samarium iodide promoted reductive C-N bond cleavage, and Stille coupling for introducing the vinyl functionality. The starting aziridine was enantioselectively prepared from 3,4,5-trimethoxybenzaldehyde by guanidinium ylide mediated asymmetric aziridination. All nitrogen components used in the reaction sequence are reusable as the starting guanidinium source.

Quantitative analysis of miroestrol and kwakhurin for standardisation of Thai miracle herb 'Kwao Keur' (Pueraria mirifica) and establishment of simple isolation procedure for highly estrogenic miroestrol and deoxymiroestrol.

Quantitative analysis of miroestrol (1) and kwakhurin (3) by HPLC, leading to standardisation of commercially available Thai miracle herb 'Kwao Keur' which has been identified with Pueraria mirifica, was established using independent solvent systems. The simple isolation procedure of highly estrogenic miroestrol (1) and deoxymiroestrol (2) from P. mirifica was also proposed.

Enantioselective construction of a polyhydroxylated pyrrolidine skeleton from 3-vinylaziridine-2-carboxylates: synthesis of (+)-DMDP and a potential common intermediate for (+)-hyacinthacine A1 and (+)-1-epi-australine.

We report an enantioselective synthesis of the polyhydroxylated pyrrolidine alkaloid (+)-DMDP. The key steps in the synthesis were guanidinium ylide mediated asymmetric aziridination, stereospecific ring opening of trans-3-vinylaziridine-2-carboxylate with an oxygen nucleophile, iodine-mediated 5-endo-trig amino cyclization, and Prévost displacement. In addition, a potential common intermediate for the polyhydroxylated pyrrolizidine alkaloids (+)-hyacinthacine A(1) and (+)-1-epi-australine was synthesized from a diastereoisomeric cis-aziridine coformed in the asymmetric aziridination using the same strategy. A rationale for the diastereoselectivity observed for the iodine-mediated amino cyclization reactions is proposed on the basis of the heats of formation of the products.

Mechanism of nanoparticle formation from ternary coground phenytoin and its derivatives.

The mechanism of drug nanoparticle formation of phenytoin (DPH) and its derivatives monomethylphenytoin (MDPH) and dimethylphenytoin (DMDPH) was investigated. The drug, polyvinylpyrrolidone K17 (PVP), and sodium dodecyl sulfate were coground to obtain the ground mixture (GM). The DPH GM was amorphous; however, MDPH and DMDPH GMs contained drug crystals. Spectral changes in infrared and (13)C solid-state nuclear magnetic resonance were observed in the DPH GM, partially observed in the MDPH GM, and hardly observed in the DMDPH GM. Mean particle sizes of the DPH, MDPH, and DMDPH GM nanosuspension were almost the same; however, stability after storage differed in the order of DPH > MDPH > DMDPH. The intermolecular interaction between the drug and PVP reflected not only the crystallinity of the drug in the GM but also the stability of the GM suspension. The size and stiffness of drug nanoparticles were evaluated using atomic force microscopy. Crystallization of the amorphous GM and agglomeration of the primary nanocrystals were observed in the DPH GM suspension. In contrast, primary nanocrystals were observed in the DMDPH GM suspension. The size of the drug nanocrystals formed from the different molecular states of the drug in the GM reflects the agglomerated states in water and stability.

New 2-aryl-1,4-naphthoquinone-1-oxime methyl ether compound induces microtubule depolymerization and subsequent apoptosis.

In this study, we describe the antitumor activity of QO-1, one of the new 2-aryl-1,4-naphthoquinone-1-oxime methyl ether derivatives. QO-1 is a derivative of macarpine, a natural occurring product from Rutaceae plant. It could potently inhibit cell growth when tested on 19 cancer cell lines. To investigate its mechanism, two cell lines (HeLa and MCF-7) sensitive to QO-1 were selected. Based on flow cytometry, it was found to induce G(2)/M-phase arrest. Moreover, it could cause microtubule depolymerization both in vitro and in vivo. On the other hand, QO-1 activated spindle assembly checkpoint (SAC) proteins. Expression of Bub1, one of the SAC, was gradually increased, reaching a peak after 16 - 20 h, and then gradually decreased. Instead, QO-1 increased the sub-G(1) population, which suggested a cell death population. Actually, expression of Bcl-2 family proteins and activation of caspase-3/7 were evidences of apoptosis. Consistent with these results, cells with DNA fragmentation and multinucleated cells were increased time-dependently after QO-1 exposure. In conclusion, QO-1 has promising antitumor effects via microtubule depolymerization.

Effects of restricted feeding on fetal and placental development in pregnant rabbits.

In our previous study on the effects of restricted feeding on pregnant rabbits (Matsuoka et al., 2009), animals given 20 g/day of diet on and after gestation day 6 (GD 6) showed significant changes in blood coagulation-related parameters suggesting a tendency to bleed and a decrease in serum concentration of progesterone, an important factor to maintain pregnancy, on GD 22, and a half of them showed serum progesterone concentrations less than 4.0 ng/ml which resulted in abortions on and after GD 23. In the present study, the effects of restricted feeding of 20 g/day from GD 6 to GD 22 on embryo-fetal and placental development on GD 23 as well as on blood coagulation-related parameters and serum progesterone concentrations on GD 22 were examined in pregnant rabbits. As compared with the non-restricted feeding (Not-treated, NT) group, the restricted feeding (RF) group showed lower values of platelets, fibrinogen, activated partial thromboplastin time (APTT) and antithrombin III (ATIII) and a longer prothrombin time (PT), reflecting an inhibition of blood coagulation, and a decrease in serum progesterone concentration on GD 22. Cesarean section performed on GD 23 revealed that the RF group showed a tendency towards an increase in the embryo-fetal death index and lower body weights and placental volumes compared with the NT group. Histological examination of the placenta in the RF group revealed that the labyrinth zone was thin and many glycogen-containing cells still remained in the basal zone, suggesting a delay in placental growth.

Angiogenesis inhibitors identified by cell-based high-throughput screening: synthesis, structure-activity relationships and biological evaluation of 3-[(E)-styryl]benzamides that specifically inhibit endothelial cell proliferation.

Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active antiangiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human umbilical vein endothelial cell (HUVEC) proliferation inhibitors from a cell-based high-throughput screening (HTS), we eliminated those compounds which showed cytotoxicity against HCT116 and vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitory activity. Evaluations in human Calu-6 xenograft model delivered lead compound 1. Following extensive lead optimization and alteration of the scaffold we discovered 32f and 32g, which both inhibited the proliferation and tube formation of HUVEC without showing inhibitory activity against any of 25 kinases or cytotoxicity against either normal fibroblasts or 40 cancer cell lines. Upon oral administration, 32f and 32g had good pharmacokinetic profiles and potent antitumor activity and decreased microvessel density (MVD) in Calu-6 xenograft model. Combination therapy with a VEGFR inhibitor enhanced the in vivo efficacy. These results suggest that 32f and 32g may have potential for use in cancer treatment.