Interaction of the Flos Lonicerae Japonicae (FLJ) with protein was studied by microdialysis coupled with HPLC-DAD-MS. Eight compounds were identified by comparing their tR, UV data and MS data with reference compounds. Microdialysis recoveries and binding degrees of compounds in FLJ with bovine serum albumin (BSA) were determined. Recoveries of microdialysis sampling ranged from 51.3 to 73.2% with relative standard deviation (RSD) below 3.1%, and the binding degrees of those compounds to BSA ranged from 4.8 to 61.2 (0.3 mM BSA) and from 11.1 to 76.2% (0.6 mM BSA), respectively. The results showed that the binding properties of compounds in FLJ were influenced by pH. Furthermore, the binding degrees of five reference compounds were determined separately under the same conditions, the binding degrees of chlorogenic acid, luteolin-3-O-glucoside and 4,5-di-O-caffeoyl quinic acid was lower in FLJ than in single compound solution, on the contrary the binding degree of caffeic acid and rutin was higher in FLJ, which indicated that a significant effects of the interaction of compounds with each other on their binding degrees to BSA. The results showed that there were ten compounds had interaction with BSA, eight of them were the proven active compounds, and the other two compounds had similar binding degree with the proven active compounds, so the ten compounds might possess potential activities.
The ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL) tagged with a histidine cluster was overexpressed, amounting to as much as 1—2% of total membrane proteins in Drosophila cell line S2. TAPL was effectively solubilized from membranes by Triton X-100, NP-40 and n-dodecyl-β-D-maltoside. Solubilized TAPL bound ATP-agarose and adenosine 5′-diphosphate (ADP)-agarose but not adenosine 5′-monophosphate (AMP)-agarose. The binding was competed for by excess free ATP, ADP, guanosine 5′-triphosphate (GTP) and dATP but not by AMP. Pyrimidine nucleotides such as uridine 5′-triphosphate (UTP) and cytidine 5′-triphosphate (CTP) were less effective competitors, suggesting that purine nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg2+, and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.
Macrophage-derived foam cells are formed as a result of the accumulation of cholesteryl ester (CE) not only in cytoplasm where CE is produced by the reesterification of free cholesterol derived from oxidized low density lipoprotein (OxLDL) undergoing hydrolysis, but also in lysosomes where the remaining CE of OxLDL is deposited. We examined the possible involvement of cytosolic phospholipase A2s (cPLA2s) in the production of CE through the reesterification and in the formation of foam cells. In [3H]oleic acid-labeled human acute monocytic leukemia (THP-1) cell-derived macrophages (THP-M) and mouse peritoneal macrophages (MPM), which possessed at least cPLA2α and cPLA2γ, stimulation with OxLDL induced the production of [3H]cholesteryl oleate ([3H]CE).The production was suppressed by an inhibitor of cPLA2s. However, the inhibitor tended to slightly decrease total intracellular levels of CE, and did not affect the formation of foam cells, as estimated by staining with Oil Red O. In cPLA2α-knockout MPM, OxLDL-induced increases in [3H]CE and total CE did not differ from those in wild-type MPM. Our results suggest that cPLA2s other than cPLA2α contribute to the supply of fatty acids, which are utilized for the production of CE through the reesterification, in OxLDL-stimulated macrophages. However, the formation of foam cells could not be inhibited only by the suppression of cPLA2-mediated CE production.
Proteins destined for the peroxisome matrix or membrane possess distinct targeting signals that engage signal sequence receptors to drive their transport to their final subcellular destination. Peroxisomal targeting signal 1 (PTS1) and 2 (PTS2) are well characterized as signaling the import from the cytosol into the peroxisomal matrix of soluble peroxisomal enzymes. The expression vectors of the enhanced green fluorescent protein (eGFP)-tagged alanine:glyoxylate aminotransferase (AGT) and deletion mutants were introduced into HeLa cells to identify the peroxisomal targeting signal of the AGT. Two sites of the targeting signals into peroxisome, an amino acid sequence containing 59—66 a.a. (8 amino acids) and 389—392 a.a. (4 amino acids, KKKL, but not KKL) on the AGT were clarified. In a three-dimensional structural model, their sites were separately located on the surface of the AGT protein.
Zymosan activates phagocytes through the innate immune system and causes inflammatory responses in animals. Because of the complexity of the active substances included in Zymosan preparations, simplifying the active moiety actually responsible for innate immune recognition is needed. One way to remove possible active substances from commercially available Zymosan preparations is to wash then with pyrogen-free water to obtain a ZWIS (Zymosan water insoluble fraction), ethanol insoluble (EtIS), or chloroform/methanol insoluble (CMIS) preparation. The effects of various washed Zymosan preparations on nuclear factor (NF)-κB activation and binding to β-glucan recognition protein were examined. Significant NF-κB activation by Toll-like receptor (TLR) 2-expressing HEK293 cells and enhanced NF-κB activity via the co-expression of TLR2 and Dectin-1, a functional β-glucan receptor, was only observed in response to ZWIS. However, the ability of Zymosan preparations to bind Dectin-1 protein was not altered even after treatment with the organic solvents by which the TLR2-mediated NF-κB activity was abolished. Another NF-κB activation pathway involving CARD9/Bcl10 was triggered by these Zymosan preparations in the presence of Dectin-1. The results suggest that the β-glucan-dependent characteristics of Zymosan were not affected by the washing with chloroform/methanol or ethanol, and that TLR2-mediated activity was easily eliminated with these organic solvents. This treatment might be useful for distinguishing natural ligands for TLR2 and β-glucan receptors when studying the innate immune response to fungal macromolecules.
Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Tumor cells are exposed to higher oxidative stress compared to normal cells. Numerous reports have demonstrated that the intracellular redox (oxidation/reduction) state is closely associated with the pattern of VEGF expression. Electrolyzed reduced water (ERW) produced near the cathode during the electrolysis of water scavenged intracellular H2O2 and decreased the release of H2O2 from a human lung adenocarcinoma cell line, A549, and down-regulated both VEGF transcription and protein secretion in a time-dependent manner. To investigate the signal transduction pathway involved in regulating VEGF expression, mitogen-activated kinase (MAPK) specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (ERK1/2 inhibitor) and JNKi (c-Jun N-terminal protein kinase inhibitor) were applied. The results showed that only PD98059 blocks VEGF expression, suggesting an important role for ERK1/2 in regulating VEGF expression in A549 cells. As well, ERW inhibited the activation of extracellular signal-regulated kinase (ERK) in a time-dependent manner. Co-culture experiments to analyze in vitro tubule formation assay revealed that A549 cell-derived conditioned medium significantly stimulated the formation of vascular tubules in all analyzed parameters; tubule total area, tubule junction, number of tubules, and total tubule length. ERW counteracted the effect of A549 cell-conditioned medium and decreased total tube length (p<0.01). The present study demonstrated that ERW down-regulated VEGF gene transcription and protein secretion through inactivation of ERK.
Since the first report about cytoplasmic nucleophosmin (NPM) in acute myelogenous leukemia with a normal karyotype was announced, the shuttling activity of NPM and its proper subcellular localization have drawn many attentions. Mechanisms that regulate nucleocytoplasmic transport of proteins may provide novel opportunities for drug development. Here we show that, in Jurkat cells, strong fluorescence density of NPM prevails in the nucleus, while, some key nucleoporins: Nup88 and Nup214 localize mainly in the cytoplasm. Deguelin, a natural occurring rotenoid, presents powerful anti-leukemia effects through proliferation inhibition and apoptosis induction in Jurkat cells. Deguelin downregulates the expression of NPM, Nup88 and Nup214 in a dose-dependent manner and reverts the localization of Nup88 and Nup214 to nuclear rim. These results suggest that deguelin exhibit its strong anti-leukemia effects might through the regulation of some nucleoporins, thus influence the subsequent abnormal expressions or localizations of some key proteins involved in proliferation and/or apoptosis, such as: NPM.
Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. However, the effects of ASA on melanogenesis are not well known. Therefore, we investigated the effect of ASA on melanin production using B16 murine melanoma cells and demonstrated a new biological effect of ASA. In the presence of α-melanocyte stimulating hormone (α-MSH), B16 melanoma cells are stimulated to enhance melanin synthesis. ASA (2 mM) inhibited α-MSH-enhanced melanin synthesis in melanoma more strongly than other well-known anti-melanogenic agents such as arbutin (2 mM) and kojic acid (200 μM). Interestingly, ASA did not inhibit the catalytic activity of mushroom tyrosinase (concentration range 0.5—4.0 mM). To clarify the target of ASA action in melanogenesis, we performed Western blotting for tyrosinase, which is a key melanogenic enzyme. ASA inhibited tyrosinase expression in a dose-dependent manner. Therefore, the depigmenting effect of ASA might be due to inhibition of tyrosinase expression or enhancement of tyrosinase degradation. This study suggests that ASA is a candidate anti-melanogenic agent and it might be effective in hyperpigmentation disorders.
Panax japonicus is an important medicinal plant. The aim of this study was to develop species-specific molecular markers for P. japonicus. Amplified fragment length polymorphism (AFLP) was compared among P. japonicus, P. ginseng and P. quinquefolius. A clear species-specific AFLP marker for P. japonicus was generated. After isolation and sequencing of the AFLP fragment, a DNA sequence (293 bp) was obtained and named JG14. Oligonucleotide primer (23 mer) was designed for amplifying 191 bp of the sequence of JG14. PCR analysis revealed a clear amplified band for P. japonicus but not in 3 other Panax species (P. ginseng, P. quinquefolius and P. notoginseng). This sequence characterized amplified regions (SCAR) marker will be used for rapid authentication of P. japonicus among other related Panax species. This is the first report of species-specific SCAR marker development in P. japonicus.
Korean red ginseng saponins (ginsenosides) have been reported as having various biological properties, but the antifungal effects and the mode of action of ginsenosides remain mostly unknown. In this study, saponins were isolated from Korean red ginseng, and the antifungal effects of ginsenosides were investigated. Ginsenosides showed fungicidal effects toward pathogenic fungi tested. To elucidate the antifungal mode of action of ginsenosides, flow cytometry analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest that ginsenosides may exert antifungal activity by disrupting the structure of cell membrane. The present study indicates that ginsenosides have considerable antifungal activity, deserving further investigation for clinical applications.
Gastric mucosal cell-derived L-lactic acid strongly enhances proliferation of Helicobacter pylori, and may contribute to the long-term colonization of H. pylori in the stomach. Therefore it is assumed that inhibitory substances active against L-lactic acid-dependent growth of H. pylori will be useful candidates as novel therapeutic agents for H. pylori infection. In this study, we developed a new assay system for screening anti-H. pylori substances, and baicalein and glycyrrhetinic acid were found as potent inhibitory substances against L-lactic acid-dependent H. pylori growth but not L-lactic acid-independent growth. The newly developed assay system described in this study also may facilitate the development of novel therapeutic agents for H. pylori infection.
Deep-sea water is rich in minerals, e.g., Mg, Ca, and K which have been considered to be associated with prevention of cardiovascular disease. We investigated the effect of deep-sea water on cardiovascular hemodynamics in Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits. Deep-sea water was pumped in the offing of Cape Muroto in Japan and the mineral constituents were refined to a degree of hardness of 1000. Twenty four 4-month-old KHC rabbits were given refined deep-sea water (n=12) and tap water (n=12) for 6 months. Pressure and flow waves at the ascending aorta were recorded under pentobarbital anesthesia. Systolic, diastolic, pulse and mean arterial pressures and total peripheral resistance were significantly lower in the deep-sea water group than in the control group. There were no significant differences in changes in serum lipid levels, plasma renin and angiotensin converting enzyme activities and electrolyte levels except for Mg2+ after the feeding of the water between the two groups. A slight increase in serum Mg2+ level in the deep-sea water group may not account for the inhibition of mild hypertension. From our results, we conclude that deep-sea water could improve cardiovascular hemodynamics, even though the factors which affect the blood pressure are still unknown.
A safe and effective delivery system with a submicron emulsion for puerarin was studied. Puerarin submicron emulsion was prepared by a novel complex-phase inversion-high press homogenization technology. The mechanism to reduce the hemolysis side effect of puerarin was studied by blood cell counts in rabbits. The average diameter, zeta potential and entrapment efficiency of the emulsion prepared was 198.14±8.61 nm, −29.45±1.47 mV, 87.32±0.34%, respectively. Compared with control group, the red blood cell values, packed cell volume, plasma hemoglobin level, haptoglobin level and osmotic fragility of puerarin i.v. group was significantly different (p<0.05) at 42, 43 d, respectively. The blood cell parameter values of puerarin submicron emulsion group were not significantly different (p>0.05) in contrast to control group. Such observations indicated that the intravascular hemolysis occurred at 42, 43 d in puerarin i.v. group rabbits, the hemolysis did not occur for puerarin emulsion group rabbits. As an explanation for these results, it was proposed that the puerarin was either incorporated into the lipophilic core or intercalated between the phospholipid molecules at the interface. It could be concluded that puerarin submicron emulsions prepared markedly reduced the hemolysis effect of puerarin.
The present study was performed to examine whether the leaves of Saururus chinensis (LOUR.) BAILL (SC), an herb used for the management of various skin diseases including atopic dermatitis (AD) in Eastern countries, inhibited the development of AD-like skin lesions in NC/Nga mice which was induced by repeated application of picryl chloride (PiCl). The efficacy of SC was judged by measurement of skin severity, itching behavior, histological study, serum IgE levels, IL-4 and IFN-γ in lymph nodes. Oral administration of SC extract to the PiCl-treated NC/Nga mice for 8 weeks (5 d per week) inhibited significantly the development of AD-like skin lesions macroscopically. Histologically, SC inhibited dermatitis changes like hypertrophy, hyperkeratosis, and infiltration of inflammatory cells into epidermis and dermis. The itching behavior and serum IgE level decreased significantly after SC administration. SC administration enhanced IFN-γ mRNA expression but did not have an effect on IL-4 mRNA expression. These results suggest that SC could inhibit the development of AD-like skin lesions in NC/Nga mice possibly through modulating the Th1/Th2 imbalance by the promoting of Th1 cell response. Thus, SC may be an alternative substance for the management of AD patients.
We investigated the combined effect of cyclophosphamide (CPA) and 5-bromo-2′-deoxyuridine (BrdUrd) both in mice bearing L1210 ascites tumors and in L1210 leukemic cells in vitro. Administration of BrdUrd (100 mg/kg) for 5 consecutive days before a single dose (80 mg/kg) of CPA significantly extended the survival of mice by 158%, compared with CPA alone. BrdUrd administered at daily doses of 100 or 200 mg/kg for 5 consecutive days did not extended the survival of mice. An in vitro MTT assay revealed that BrdUrd enhanced the cytotoxic effect of 4-hydroxycyclophosphamide, an active form of CPA, in the L1210 cells. These results indicate that BrdUrd enhanced the antitumor effect of CPA both in vivo and in vitro.
Various mechanisms can influence the intestinal absorption and oral bioavailability of drugs. The barrier effects of efflux transporters may be one of the critical factors limiting the bioavailability of certain drugs. It has been reported that multidrug resistance-associated protein 2 (Mrp2) is expressed in the mucosal membrane of the epithelium of the small intestine and secretes various drugs into the jejunum lumen. However, it is possible that total intestinal secretion of Mrp2 substrates is accounted for the contribution of Mrp2 and other transporter(s) to the intestinal secretion of Mrp2 substrates. In this study, we found that phenolsulfonphthalein and pravastatin, both Mrp2 substrates, are transported by different transport systems in the intestine. These results suggest that contribution of transporters to the drug transport may be a critical factor affecting drug disposition and drug–drug interaction. In addition to evaluating the substrate specificity of a transporter, it is important to be aware of the contribution of a transporter to drug disposition.
Our previous studies reported that methanol extract of Sanguisorbae radix from Sanguisorba officinalis L. (Rosaceae) prevented neuronal cell damage induced by Aβ (25—35) in vitro. The present study was carried out to investigate the effect of gallic acid isolated from Sanguisorbae radix on Aβ (25—35)-induced neurotoxicity using cultured rat cortical neurons. Gallic acid (0.1, 1 μM) showed a concentration-dependent inhibition on Aβ (25—35) (10 μM)-induced apoptotic neuronal death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Pretreatment of gallic acid inhibited 10 μM Aβ (25—35)-induced elevation of cytosolic Ca2+ concentration ([Ca2+]c) and generation of reactive oxygen species (ROS), which were measured by fluorescent dyes. Gallic acid also inhibited glutamate release into medium induced by 10 μM Aβ (25—35), which was measured by HPLC. These results suggest that gallic acid prevents Aβ (25—35)-induced apoptotic neuronal death by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and generation of ROS, and that these effects of gallic acid may be partly associated with the neuroprotective effect of Sanguisorbae radix.
The aim of the present study was to investigate the mechanism of apoptosis in human multiple myeloma cell line, U266, caused by 2-aminophenoxazine-3-one (Phx-3). Flow-cytometrical and morphological analyses showed that Phx-3 increased the population of annexin V-positive cells including early stage apoptotic cells and late stage apoptotic cells and induced DNA fragmentation or apoptotic body formation in U266 cells, indicating that Phx-3 induced the apoptosis of U266 cells. Activity of caspase-3 was extensively increased in U266 cells treated with Phx-3 time-dependently within 24 h, but this Phx-3-stimulated activity of the enzyme in the cells was completely cancelled by the addition of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. The addition of z-VAD-fmk almost blocked the apoptotic effect of Phx-3 against U266 cells, indicating that Phx-3-induced apoptosis of U266 cells was dependent on a caspase signaling pathway. Moreover, the apoptosis of U266 cells occurred after the induction of cell cycle arrest of the cells in the S and G2/M phase, the loss of mitochondrial membrane potential, and activation of caspase-3 reached maximum, which were caused by Phx-3 within 24 h. These results support the views that the apoptosis of U266 cells caused by Phx-3 may be preceded by the cell cycle arrest, depolarization of mitochondria and activation of caspase-3. These results support the view that Phx-3 may be utilized in future as chemotherapeutic agent against multiple myeloma which is extremely refractory to chemotherapy.
We previously demonstrated that Ginkgo biloba extract (GBE) produces an anti-hypertensive effect via enhanced vasodilation responses in young spontaneously hypertensive rats (SHR) and hepatic hypertrophy occurs with increased cytochrome P450 (CYP) mRNA expression in young rats. In the present study, to clarify whether these actions of GBE are observed in older rats, we investigated cardiovascular functions and hepatic CYP protein expressions in aged SHR fed a control diet or a diet containing 0.5% GBE for 4 weeks. In aged SHR, GBE feeding significantly increased liver weight per 100 g body weight without changing the body weight. Furthermore, significant increases in alanine aminotransferase level in serum and marked increase in CYP2B protein expression in the liver were observed in aged SHR fed GBE. On the other hand, GBE feeding did not affect blood pressure, but significantly reduced heart rate and blood flow velocity in tail arteries of aged SHR. Furthermore, GBE feeding did not affect contractile response to phenylephrine, relaxation responses to not only sodium nitroprusside but also acetylcholine, and protein levels of endothelium nitric oxide synthase and soluble guanylate cyclase in aortas of aged SHR. These results suggested that long-term GBE feeding impairs peripheral circulation due to bradycardia and hepatic function in aged SHR. Thus, in the elderly population with hypertension, the use of GBE may need to be assessed for effects on heart rate and liver function.
In this study, we investigated the hypolipidemic effects of Sophora flavescens in poloxamer 407-induced hyperlipidemic and cholesterol-fed rats. The MeOH extract and 4 fractions of S. flavescens were administered at doses of 250 and 100 mg/kg body weight, respectively, once a day for 3 d to the poloxamer 407-induced hyperlipidemic rats. Serum lipid levels such as total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-cholesterol (LDL-C) were markedly elevated in the poloxamer 407-induced hyperlipidemic control rats, while lipid levels were significantly decreased in the rats administered the MeOH extract or 4 fractions of S. flavescens. In addition, serum high-density lipoprotein-cholesterol (HDL-C) was reduced in the poloxamer 407-induced hyperlipidemic control rats. However, oral administration of both the MeOH extract and 4 fractions significantly increased HDL-C levels. Of the tested fractions, the EtOAc fraction showed the strongest lipid-lowering effect, as well as a high antiatherogenic potential with atherogenic index (A.I.) values of less than 1.92. We also investigated the hypolipidemic effects of the main compounds of the EtOAc fraction, kurarinol and kuraridinol, using the hyperlipidemic and hypercholesterolemic animal models. Here, elevated TC, TG, and LDL-C levels in the poloxamer 407-induced hyperlipidemic and cholesterol-fed rats were significantly reduced after oral administration of the compounds, and HDL-C levels had a significant increase. Furthermore, A.I. values were lowered by administering kurarinol and kuraridinol. In particular, kuraridinol exhibited stronger protective activities against hyperlipidemia than kurarinol. These results suggest that S. flavescens and its constituents may be effective cholesterol-lowering agents and useful for preventing hypercholesterolemic atherosclerosis.
We compared anti-proliferative activities of (−)-epigallocatechin gallate (EGCG) and (−)-epigallocatechin (EGC) against HCT116 colorectal carcinoma cells. These catechins inhibited cell growth to nearly the same extent at low cell confluency in plates. However, their inhibitory effect grew weaker as cell confluence increased, and this tendency was more conspicuous for EGC than for EGCG. Both EGCG and EGC activated the phosphorylation of the major MAPKs, ERK, JNK, and p38, in the HCT116 cells as in many other established human cancer cells though to different extents. Cell cycle analyses, DNA fragmentation assays, and TUNEL assays as well as Western blot assays suggested that these catechins inhibited cell growth through mitogen-activated protein kinase (MAPK)-mediated apoptosis rather than cell cycle regulation.
Previously, it was reported that some prenylated flavonoids contained in the dichloromethane fraction of the ethanolic extract of Sophora flavescens, such as kuraridin, sophoraflavanone G, kurarinone, and kushenol F, are tyrosinase inhibitors; however, based on the level of these inhibitors in the extract, its inhibitory effect on tyrosinase activity was higher than expected. This has led us to further investigate other possible constituents that may contribute to the extract's strong inhibitory activity. The results of this study indicate that kurarinol (1), kuraridinol (2), and trifolirhizin (3), from the ethyl acetate fraction of Sophora extract, can inhibit tyrosinase activity. Compared with kojic acid (16.22±1.71 μM), compounds 1—3 possessed potent tyrosinase inhibitory activity with IC50 values of 8.60±0.51, 0.88±0.06, and 506.77±4.94 μM, respectively. These three compounds were further tested for their inhibitory effects on melanogenesis. In cultured B16 melanoma cells, 1—3 markedly inhibited (>50%) melanin synthesis at 50 μM. This is the first study indicating that 1—3 exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, and therefore, are candidates as skin-whitening agents.
Pseudocoptisine, a quaternary alkaloid with benzylisoquinoline skeleton, was isolated from Corydalis Tuber, one of the important medicinal plants in traditional medicine. Pseudocoptisine inhibited acetylcholinesterase (AChE) activity in a dose-dependent manner, and the concentration required for 50% inhibition was 12.8 μM. In further study, the anti-amnesic activities of pseudocoptisine in mice on the learning and memory impairments induced by scopolamine (1.0 mg/kg, i.p.) were examined. This alkaloid (2.0 mg/kg, p.o.) administration significantly reversed cognitive impairments in mice by passive avoidance test (p<0.05). It also reduced escape latencies in training trials and prolonged swimming times in the target quadrant during the probe trial in the water maze task (p<0.05). These results indicate that pseudocoptisine has anti-cholinesterase and anti-amnesic activities that may be useful for cognitive impairment treatment.
The skin disposition and metabolism of topically applied ethyl nicotinate (EN) were evaluated in dual agar gel disc-inserted hairless rats, which have two agar gel discs subcutaneously inserted into the abdominal region as drug receptors, and a topical formulation containing EN placed on either side of the gel disc through the skin. Plasma and agar levels of EN and its metabolite, nicotinic acid (NA), were followed every 2 h over 6 h. EN permeated through the skin barrier and partly metabolized to NA with 89.4% of the metabolite ratio [NA/(EN+NA)] at 6 h. Some EN and NA in the skin moved to the systemic circulation, and the remainder migrated into the agar gel below the formulation. The total amount (EN+NA) in the skin that distributed from the formulation directly to the systemic circulation and the application side of the gel corresponded to 95.2% and 4.8% of the total skin permeation at 6 h, respectively. Only NA was distributed from the systemic circulation to both agar gel discs. The NA fraction in the application side of the gel from the circulation was only 1% of the total amount in the agar gel. The metabolite ratio on the application side of the agar gel was higher than that in the receiver for the in vitro skin permeation using excised hairless rat skin. This difference was probably related to a lower EN ratio in viable skin in situ than in vitro. These results suggest that the present in situ method is useful to evaluate the skin disposition and metabolism of topically applied drugs.
The clinical efficacy of calcineurin inhibitors administered to renal transplant patients is considered to be a strong function of the area under the concentration time curve (AUC). Interestingly, monitoring timings of blood concentrations for two similar calcineurin inhibitors, cyclosporine (CYA; Neoral®) and tacrolimus (TAC; Prograf®) are different. Namely, CYA blood concentration is usually monitored at 2 h after administration (C2) substituted for peak concentration (Cp) and TAC at trough concentration (Ct). In the literature, data describing such characteristics of CYA and TAC have been presented in the past. However, each of these patient groups had different backgrounds. We have attempted to examine the behavior of blood concentration curves simultaneously for both CYA and TAC by establishing controlled groups of renal transplant patients with similar clinical backgrounds. Furthermore, we have analyzed the correlation with Cp and Ctversus AUC implementing area under the trough level (AUTL), or area above the trough level (AATL) as new pharmacokinetic parameters, such that C2 for CYA and Ct for TAC have been verified using controlled clinical data. We have also found distinct differences in the pharmacokinetics between CYA and TAC with the relationships between AUC, Cp, and Ct.
Antineovascular therapy (ANET), which eradicates angiogenic endothelial cells by specifically delivered anticancer drugs to tumor cells to obtain complete cutoff of blood supply, is an effective modality for cancer treatment. Since the expression of vascular endothelial growth factor (VEGF) in hypoxic tumor cells is known to fluctuate in a circadian oscillation, we investigated the chronopharmacologic treatment of tumors with ANET. Adriamycin-encapsulated liposomes modified with the Ala-Pro-Arg-Pro-Gly (APRPG) peptide (APRPG-LipADM) were prepared, after the APRPG peptide had been shown to have affinity to angiogenic sites. Colon 26 NL-17 tumor-bearing mice were injected three times with APRPG-LipADM at Zeitgeber time (ZT) 2, 8, 14, and 20 where ZT 0 was the time lights were turned on, and tumor growth was monitored. Tumor growth suppression changed with dosing time and was significantly (p<0.01) more potent at ZT 14 compared with ZT 20. The VEGF concentration in the plasma of the tumor-bearing mice was higher in the light phase compared with that in the dark phase, and this circadian oscillation was related to dosing time dependency with ANET. These results indicate that tumor growth suppression is correlated to some extent with the VEGF concentration in the plasma, and that chronopharmacologic treatment of cancer with ANET may enhance the therapeutic efficacy and reduce the side effects.
Electrets are polymeric discs that carry semi permanent electrostatic charge. These provide electrostatic potentials in the range of 500 to 3000 V. In the current work, the effect of electret exposure on the skin permeability was investigated. Transdermal transport studies were carried out across porcine epidermis in Franz diffusion cells. Salicylic acid, fluorescein labeled dextrans (FD) and propofol were used as test diffusants. The ability of electret to enhance the transdermal permeation of salicylic acid was studied in vivo in Sprague Dawley rats. Electret enhanced the permeability of porcine epidermis to salicylic acid. The enhancement factor increased with the surface voltage, however it was independent of the nature of charge (+ or −). The enhancement by electret was cut-off at 1 kDa, as interpreted by studying the transport of FD. The electrets decreased the permeability of skin to propofol, a lipophilic diffusant. Pretreatment of porcine epidermis enhanced the iontophoretic transport of salicylic acid, whereas the same did not enhance the transport of salicylic acid by electroporation. It is most likely that electret exposure renders the lipid domains of stratum corneum more permeable to polar molecules and in turn hampers the diffusion of nonpolar diffusant.
Aconityl-doxorubicin (ADOX) was synthesized by the modified method of Shen and Ryser. Two isomers of cis-ADOX (cis-configuration) and trans-ADOX (trans-configuration) were generated in the reaction of DOX and cis-aconitic anhydride. These products were separated completely by using HPLC and analyzed by TOF-MS spectroscopy and 1H- and 13C-NMR experiments. The yields of cis-ADOX and trans-ADOX were 36.3 and 44.8%, respectively. The free γ-carboxylic group of ADOX molecule was coupled to poly(vinyl alcohol) (PVA) via ethylenediamine spacer, resulting the macromolecular conjugates of PVA–cis-ADOX and PVA–trans-ADOX, respectively. The DOX content of the conjugates estimated by the hydrolysis method detected the aglycone of DOX which can be estimated as the PVA-bound DOX selectively was 4.4 w/w% which was similar to 4.6 w/w% by the ordinary UV method. Both PVA–cis-ADOX and PVA–trans-ADOX were very stable at neutral pH, but the release of DOX was increased markedly under acidic conditions. Half-life of the release of DOX from PVA–cis-ADOX at pH 5.0 was 3 h which was 4.7-fold shorter than that from PVA–trans-ADOX (14 h). The cytotoxicities of PVA–cis-ADOX and PVA–trans-ADOX were evaluated by using J774.1 cells employing a [3H]uridine incorporation assay as a measure of RNA synthesis. A significant difference in antitumor activity between PVA–cis-ADOX and PVA–trans-ADOX was observed where the former was much active than the later. It was suggested that the conjugate enters the cells and reaches the lysosomal/endosomal compartment, and that the aconityl spacer releases DOX from the conjugate in the acidic compartment of lysosomes/endosomes due to the participation of a free carboxylic group.
The tight junction formation in Caco-2 cell monolayers was compared after 9 and 13 d of culture. Four different sized paracellular markers were simultaneously applied to the apical side of the monolayers. The transepithelial resistance and permeability coefficient of mannitol for the monolayers cultured for 13 d were 17.4 and 0.095 times those after 9 d, respectively. The tight junction structure developed during that period. The pore permeation model involving two different sizes was used to quantitatively evaluate the paracellular pathways. The results suggest that there were two-different sized (large and small) pathways in the monolayers cultured for 13 d, while there was only a single large pathway in those cultured for 9 d. The small pathway in the monolayers cultured for 13 d might be a major permeation pathway for the paracellular permeation of urea and the equivalent cylindrical pore radius of the small pathway was less than 0.4 nm, suggesting development of the tight junctions after 13 d.
The radioprotective effects of water-soluble derivate of propolis (WSDP) collected in Croatia, and single flavonoids, caffeic acid, chrysin and naringin in the whole-body irradiated CBA mice were investigated. Irradiation was performed using a γ-ray source (60Co), and absorbed doses were 4 and 9 Gy. The efficiency of test components was evaluated when given intraperitoneally (i.p.) at dose of 100 mg kg−1 for 3 consecutive days before and/or after irradiation. Moreover, possible genotoxic effects of all test components were assessed on non-irradiated animals. The higher efficiency of test components was observed when given preventively. The results suggest that propolis and related flavonoids given to mice before irradiation protected mice from lethal effects of whole-body irradiation and diminish primary DNA damage in their white blood cells as detected by the alkaline comet assay.
Basidiomycetes-X (BDM-X) is a novel edible mushroom recently identified as a new fungi species and registered to the database of the NPO organization for International Patent Organism Depositing (IPOD) in the Industrial Technology Institute of Japan (PCT/JP2004/006418). In the present study, we examined anti-oxidant activity of hot water extract of this novel fungus both in vitro and in vivo together with Agaricus Blazei Murill (ABM), a commercially available medicinal mushroom, and other reference antioxidants. As results, the hot water extract of BDM-X showed more potent anti-oxidative actions compared with that of ABM, when evaluated by 1,1′-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, Ferric Reducing Potential (FRP), and also the inhibition of 2,2′-azobis(2-amidino-propane) dihydrochloride (AAPH)-induced lipid peroxidation of rat liver homogenates. Further, the protective activity of BDM-X extract towards lipopolysaccharide (LPS)-induced hepatic oxidative damage was compared with ABM and α-lipoic acid in the mice pre-administered with these antioxidants. It was revealed that all of these antioxidants inhibited the LPS-induced oxidative tissue damage but the hot water extract of BDM-X showed the strongest protection among them. For example, the dose for 50% inhibition of carbonyl formation in liver was 0.53 g dry weight/kg body weight/d for BDM-X and the value corresponded to 16 mmol of α-lipoic acid as an antioxidant reference/kg body weight/d.
A new self-microemulsifying drug delivery system (SMEDDS) has been developed to increase the solubility, dissolution rate and oral bioavailability of vinpocetine (VIP), a poor water-soluble drug. The formulations of VIP-SMEDDS were optimized by solubility assay, compatibility tests, and pseudo-ternary phase diagrams analysis. The optimal ratio in the formulation of SMEDDS was found to be Labrafac : oleic acid : Cremophor EL : Transcutol P=40 : 10 : 40 : 10 (w/w). The average particle diameter of VIP was less than 50 nm. In vitro dissolution study indicated that the dialysis method in reverse was better than the ultrafiltration method and the dialysis method in simulating the drug in vivo environment. Comparing with VIP crude drug power and commercial tablets, (−)VIP-SMEDDS caused a 3.4- and 2.9-fold increase in the percent of accumulated dissolution at 3 h. Further study on the absorption property of VIP-SMEDDS employing in situ intestine of rats demonstrated that VIP in SMEDDS could be well-absorbed in general intestinal tract without specific absorption sites. In addition, the developed SMEDDS formulations significantly improved the oral bioavailability of VIP in rats. Relative bioavailability of (−)VIP-SMEDDS and (+)VIP-SMEDDS increased by 1.85- and 1.91-fold, respectively, in relative of VIP crude powder suspension. The mechanisms of enhanced bioavailability of VIP might contribute to the improved release, enhanced lymphatic transport, and increased intestinal permeability of the drug.