Rapid Preparative Isolation of Cleistanthin A from the Leaves of Cleistanthus Collinus Using Reverse-Phase Flash Chromatography.

Introduction: Cleistanthin A (CA) is an aryl naphthalene lignan, which has a potent anticancer activity by regulating the tumor microenvironment. The objective was to develop a new technique for the isolation of cleistanthin A from the acetone extract of Cleistanthus collinus utilizing reverse phase flash chromatography. Materials and Methods: Cleistanthus collinus leaves were shade dried, defatted using n-hexane and then macerated to obtain acetone extract which was further subjected to reverse phase flash chromatography for the isolation of cleistanthin A using the gradient mobile phase of 0.1% formic acid (v/v) in water and acetonitrile. Gradient elution of chromatographic run was performed for 80 min. The separated peaks that showed absorbance at λmax 254 nm were collected for the chemical characterization. Cell viability of the isolated cleistanthin A was studied on hepatocellular cancer cell line HePG2 and prostate cancer cell line PC3 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The chemical characteristics of the isolated compound cleistanthin A was further characterized using spectral techniques such as 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and electron spray ionization-tandem mass spectrometry (ESI-MS/MS). Cleistanthin A has decreased the cell viability of the HePG2 cell line to 52.25% at 32 µg/ml and PC3 cell line to 51.82% at 16 µg/ml in a dose-dependent manner. Conclusion: Cleistanthin A was successfully isolated from the natural source using reverse phase flash chromatography and the MTT assay has shown that cleistanthin A has decreased the cell viability in both the HePG2 and PC3 cell lines in a dose-dependent manner.

Synthesis, antibacterial and antimutagenic activity of zerumbone-bicarbonyl analogues.

Synthesis of new zerumbone-bicarbonyl analogues by SeO2 oxidation is reported. Selective oxidation of methyl at C-13 to an aldehyde and a ketone with exo-cyclic double bond between C-13 and C-6 in zerumbone has been recognized. Both these compounds have an additional conjugated-carbonyl-functionality. They exhibited significantly higher antimutagenic activity than zerumbone against Salmonella tester strains. They are more active against Gram positive bacteria than Gram negative bacteria; however zerumbone showed highest activity against E. coli, whereas its derivatives were least effective against E. coli.

Management of chronic luxated central incisor with hopeless prognosis.

Traumatic injuries are the most studied yet most challenging for a dentist. Extrusive luxation injuries are usually managed by repositioning tooth in the original position and pulp sensibility is evaluated at regular periodic intervals. However, when injuries are chronic, tooth is necrotic with compromised periodontal support, treatment is a challenge. Intentional replantation utilizing growth factors in fibrin rich network of platelet rich fibrin can be a treatment modality in such patients where other restorative modalities to establish function-esthetics rehabilitation is not possible.

Antibacterial and antimutagenic activities of novel zerumbone analogues.

Zerumbone, the key constituent of Zingiber zerumbet Smith, is a very important bioactive phytochemical. Two new compounds viz. azazerumbone 1 and azazerumbone 2 were synthesised by ZnCl2-catalysed Beckmann rearrangement of the zerumbone oxime. The structure elucidation of these analogues of zerumbone was carried out by 1D ((1)H NMR and (13)C NMR) and 2D-NMR (COSY, HSQC and NOESY) spectral analysis. Studies on the antibacterial activity established that azazerumbone 2 had better activity than zerumbone. Among the tested bacteria, Bacillus cereus was the most sensitive and Yersinia enterocolitica was found to be the most resistant. These compounds exhibited strong protection against sodium azide induced mutagenicity of Salmonella typhimurium strains TA 98 and TA 1531. Azazerumbone 2 showed better antibacterial and antimutagenic activity than azazerumbone 1. The antibacterial and antimutagenic activities exhibited by zerumbone and its analogues demonstrate their potential for use as nutraceuticals and in food preservation.

Myeloperoxidase staining in the diagnosis of aggressive periodontitis.

AIMS: To evaluate neutrophil myeloperoxidase (MPO) staining procedure as a reliable, affordable and easily available diagnostic assay for aggressive periodontitis. MATERIALS AND METHODS: Fifteen subjects were recruited in the study wherein five each were diagnosed as aggressive periodontitis and chronic periodontitis respectively, and five were periodontally healthy. Three millilitres (ml) of venous blood was collected using Vacutainers containing ethylene diamine tetra acetate (EDTA) and was subjected to MPO staining procedure. Histological picture was evaluated using a visual analogue scale (VAS). RESULTS: MPO stained specimen of all the patients showed positive MPO staining of the neutrophils. The intensity of the stain of MPO granules was more in aggressive periodontitis specimen as compared to the chronic periodontitis patient specimen and healthy subject specimen. The staining characteristics were comparable for chronic periodontitis patients and healthy subject. CONCLUSIONS: This study shows that there is a potential and probable place for MPO staining as an economical, relatively convenient and easily available assay in the diagnosis of aggressive periodontitis.

Quantitative evaluation of europium in blue ballpoint pen inks/offset printing inks tagged with europium thenoyltrifluoroacetonate by spectrofluorometry and ICP-AES.

Tagging of writing/printing inks with suitable inorganic taggants such as rare-earth chelates has the potential to help document examiners identify fraud in sensitive written/printed documents. Selection of rare-earth chelates as taggants primarily depends on the satisfactory sensitivity of analytical determination and the absence of the taggants in normal varieties of inks used for document writing/printing. Spectrofluorometric determination of trace amounts of europium in blue ballpoint pen inks and offset printing inks tagged with europium thenoyltrifluoroacetonate was carried out with sodium tungstate solution. Sodium tungstate acts as a specific reagent that enhances the fluorescence intensity of the Eu3+ ion. The excitation and emission wavelengths are 270 nm and 605 nm respectively. The results were compared with the data obtained with ICP-AES. Satisfactory recoveries were observed with precision better than 5% RSD and comparable accuracy. Under the optimized experimental conditions, detection limits and quantitation limits were determined. The detection limits obtained by spectrofluorometry and ICP-AES were 0.01 microg/mL and 0.006 microg/mL respectively whereas the limits of quantitation were about 0.03 microg/mL and 0.018 microg/mL respectively. The spectrofluorometric method is rapid, selective, sensitive and accurate for the determination of europium in blue ballpoint pen ink and offset printing inks and may be suitable for application in the examination of sensitive documents to aid in document related crime investigation. The advantages and limitations of the tagging approach and proposed analytical techniques are discussed.

Bilateral renal artery stenosis presenting as flash pulmonary edema.

Flash pulmonary edema is a condition characterized by sudden and recurrent episodes of dyspnea at rest resulting from acute pulmonary venous congestion in the presence of normal or well-preserved LV systolic function. This is usually associated with bilateral renal artery stenosis or stenosis of a single surviving kidney. We report here a case of a 58-year-old man, a patient of coronary artery disease who later developed bilateral renal artery stenosis and presented with recurrent episodes of flash pulmonary edema. He was successfully treated with stenting of both renal arteries.

The use of ultra filtration in trace metal speciation studies in sea water.

During this work, size fractionation technique "ultra filtration" is used in speciation studies of trace elements in the coastal sea water. Filtration is the most commonly used method to fractionate trace metal species, but often only "dissolved" and "particulate" fraction. The purpose of the present study is to determine colloidal and suspended particulate concentrations of Fe, Zn, Cu, Ni, and Mn in sea water. Suspended particulate matter were separated in three different size groups namely (>2.7 microm, <2.7->0.45 microm and <0.45->0.22 microm) by suction filtration using cellulose acetate and nitrate filter membranes. Thereafter to concentrate the solution with colloidal particle <0.22 microm-1.1 nm (0.5 k Nominal Molecular Weight cut-off Limit {NMWL}), the solution obtained from filtration through <0.22 microm, is sequentially passed through the ultra-filtration membranes having pore diameters of 14 nm (300 k NMWL), 3.1 nm (50 k NMWL), 2.2 nm (30 k NMWL), 1.6 nm (10 k NMWL) and 1.1 nm (0.5 k NMWL) by using Stirred Ultra-filtration Cells, operating in concentration mode. The concentration of Fe, Zn, Cu, Ni, and Mn were measured in suspended and dissolved fraction by ion chromatography, ICP-AES and Atomic Absorption Spectrometer. The salinity of the solution in various dissolved fractions of sequential filtration varies between 30.89-34.22 parts per thousand. The maximum concentrations of colloidal Zn, Cu, Ni and Mn in dissolved fraction were in <2.2->1.6 nm fraction. In case of Fe, colloidal fractions <2.2->1.6 nm and <1.6-<1.1 nm shows higher concentration. The concentration of Zn, Cu, Ni and Mn increase with decrease in size in suspended particulate matter, while the reverse is observed in case of Fe. This size separation data that specifies the partitioning of metals between dissolved and suspended solid phases is necessary for developing physically based models of metal transport in aquatic system.

Inflammatory cell function in young rodents with experimental cholestasis: investigations of functional deficits, their etiology, and their reversibility.

Children with cholestasis are susceptible to infective complications. This may be attributable to impaired host defense. We postulated that cholestasis affects systemic polymorphonuclear leukocyte (PMN) function by impeding chemotaxis, phagocytosis, and superoxide release, which are all critical in eliciting an adequate immune response. Sprague Dawley rats (225 g) were assigned to three groups: bile duct ligated (BDL), sham (SH), and normal control (NC). On day 21 after operation, PMN and sera were isolated. Chemotactic response to C5a and FMLP (formyl-methionyl-leucyl-phenylalanine), superoxide release, and phagocytic uptake of 14C-labeled Staphylococcus aureus were performed on pooled PMN samples. Results were expressed as mean +/- SD. Serum bilirubin at day 21 was 6.3 +/- 2.9 v 0.1 +/- 0.1 and 0.1 +/- 0 mg/dL (P less than .01) in BDL, SH, and NC groups, respectively. Kinetic studies of PMN phagocytosis demonstrated impaired 14C S aureus uptake by BDL neutrophils at 60 (P less than .05), 90 (P less than .05), and 120 minutes (P less than .05) compared with SH and NC groups. No differences in PMN chemotactic response to C5a and FMLP was observed in BDL, SH and NC groups (43 +/- 14 v 40 +/- 12 and 33 +/- 1, and 43 +/- 20 v 43 +/- 14 and 28 +/- 1 cell per field, respectively). Zymosan stimulated superoxide release did not differ between groups (14.3 +/- 3.6 (BDL) v 15.1 +/- 8.7 (SH) and 12 +/- 2.0 (NC) nmol/30 min/mg cell protein, respectively. Thus, cholestasis impairs neutrophil phagocytosis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of hepatocellular function on NK and T cell tumoricidal activity.

Recent studies by this group have demonstrated that hepatocellular integrity is important in the preservation of host cellular immune function. This study evaluated the effect of experimental hepatocellular dysfunction (EHD) on host antineoplastic defense mechanisms. In nonspecific immune studies, we examined the effect of EHD on Wistar Furth (WF) natural killer (NK) cell cytotoxicity; in specific immune studies, we assessed WF C3H/HeJ lymphocytic responsiveness to both T cell mitogen and unmodified syngeneic fibrosarcoma. In concurrent studies, we evaluated the effect of EHD on interleukin-2 (IL-2) synthesis, an important NK and T cell trophic factor. WF rats and C3H/HeJ mice were assigned to three groups: EHD induced by bile duct ligation, sham, and normal control (NC). At day 21 serum bilirubin, WF NK cytotoxicity to YAC-1 tumor cells, WF and C3H/HeJ lymphocytic responsiveness to phytohemagglutinin (PHA) and syngeneic MCA-fibrosarcoma (MCA-F), and WF T-helper IL-2 production were determined in respective groups. Serum total bilirubin was elevated in EHD rats and mice with respect to controls (p less than 0.01). Wistar Furth cytotoxicity to the YAC-1 tumor cells was depressed in EHD animals with respect to sham and NC groups at 12.5:1 (p less than 0.01), 25:1 (p less than 0.05), 50:1 (p less than 0.05), and 100:1 (p less than 0.05) effector/target cell ratios. WF T cell responsiveness to PHA was depressed in EHD with respect to controls (p less than 0.01). C3H/HeJ lymphoproliferative response to MCA-F tumor antigen was also depressed in EHD animals when compared with control groups with the addition of 12.5 X 10(3) (p less than 0.05) and 50 X 10(3) (p less than 0.05) MCA-F cells. These impairments in NK and T cell function in EHD could not be attributed to diminished IL-2 production (EHD vs sham and NC: 112,141 +/- 5232 vs 106,691 +/- 1419 and 120,759 +/- 3248 cpm, respectively). These results demonstrate that hepatocellular failure compromises NK and T cell tumoricidal function, an effect not resultant on diminished T helper IL-2 production.

Endotoxemia and cholestasis.

Endotoxemia has been incriminated as a major cause of morbidity and mortality in patients with obstructive jaundice. It has been postulated that absence of gastrointestinal bile salt flow in cholestasis enhances portal absorption of bacterial endotoxin from the intestine, thereby predisposing the host to endotoxemia and its complications. This study re-evaluates this pathologic mechanism, using new quantitative chromogenic and conventional qualitative limulus techniques for the detection of bacterial endotoxin. Female Sprague-Dawley rats underwent either ligation of bile duct or sham operation. Serum total bilirubin, serum bile acid and intestinal bile acid concentrations were determined seven, 14 and 21 days after operation. Chromogenic and conventional qualitative limulus lysate endotoxin determinations were simultaneously performed on post-operative days two, seven, 14 and 21. Serum total bilirubin and bile acid concentrations were elevated and intestinal bile acid levels depressed at days seven, 14 and 21 (p less than 0.05). Results of quantitative and qualitative limulus studies failed to demonstrated the coexisting development of portal or systemic endotoxemia in rats with the bile duct ligated after diminution of flow of gastrointestinal bile salt. These data refute the hypothesis that flow of gastrointestinal bile salt enhances portal absorption of intestinally derived endotoxin and suggest alternative mechanisms are involved in the pathogenesis of endotoxemia in obstructive jaundice.

The protective effect of hepatic dysfunction on vascularized allograft survival.

Recent studies have demonstrated that hepatic dysfunction, induced by experimental biliary ligation (EBL), impairs lymphocytic responsiveness to PHA stimulation in vitro and to cellular antigens in vivo. This suppression appears to be selective for T-cell mechanisms while B-cell-mediated functions remain intact. The purpose of this study was to determine whether coexisting hepatic insufficiency could exert a protective effect on vascularized or nonvascularized allograft survival in the transplanted recipient. Female Wistar-Furth (Rtlw) 225 g rats were assigned randomly to three groups: EBL, sham operation (Sham) and normal control (NC). Fourteen days following operation animals received heterotopic cardiac or skin allografts from Buffalo (Rtlb) donors. Cardiac and skin graft survival was determined daily, rejection was confirmed histologically, and technical failures were omitted from analysis. Allograft survival was expressed as median survival time +/- SEM. Serum total bilirubin (mean +/- SEM) was significantly elevated at Day 14 in EBL animals compared to Sham and NC groups (15.1 +/- 1.0 vs 0.1 +/- 0 and 0.2 +/- 0.1 mg/dl, respectively, P less than 0.01). Median cardiac allograft survival time by Probit was 10.6 +/- 2.6 vs 5.6 +/- 0.7 and 6.0 +/- 0.9 days, respectively (P less than 0.03). Skin graft survival (mean and range) was similar in all groups. These results demonstrate that EBL in the rat suppresses T-cell function and significantly prolongs vascularized allograft survival, but not skin allograft survival across the Rtl histocompatibility barrier. The mechanism whereby coexisting hepatic dysfunction exerts a protective effect on vascularized allograft survival warrants further investigation.