Diferuloylmethane augments the cytotoxic effects of piplartine isolated from Piper chaba

Natural compound based anticancer drug discovery is gaining interest against a wide variety of tumors. E-piplartine (trans-piplartine), a natural compound isolated from Piper chaba roots is examined against rat histiocytoma (BC-8), mouse embryonal carcinoma (PCC4), mouse macrophages (P388D1 and J774), and human neuroblastoma (IMR32) tumor cells. While Z-piplartine (cis-piplartine) failed to induce cytotoxicity (even at higher concentrations, 50 μM), E-piplartine induced a dose-dependent cytotoxicity (2–24 μM) in different tumor cells. The combinatorial treatment of piplartine with diferuloylmethane (curcumin), an anti-inflammatory and anticancer agent, significantly enhanced the piplartine induced cytotoxicity in tumor cells. Diferuloylmethane itself is not cytotoxic at 15 μM concentration; however, potentiated the piplartine induced cytotoxicity. The tumor cell killing with piplartine is preceded by G1 cell cycle arrest, and surpassed diferuloylmethane induced G2/M arrest when used in combination. In PCC4 cells, piplartine inhibited the cell cycle progression by inactivating cdk2 and destabilizing cyclin D1, whereas diferuloylmethane combination inhibited the ERK1/2 and Raf-1 signaling in addition to the inhibition of cell cycle progression. The over expression of heat shock protein 70, Hsp70 in rat histiocytic tumor cells interfered with piplartine induced cytotoxicity, hence, a cross talk between stress response and anticancer agents is presented. Our data demonstrates the biological and medicinal importance of piplartine isolated from the roots of P. chaba, and indicates that E-piplartine may be a promising candidate to use in combinatorial treatments to combat cancer.     

Antemortem diagnosis and prevention of human rabies

Human rabies still continues to be a significant health problem in India and other developing countries where dogs are the major vectors of transmission. Rabies in humans can present in two clinical forms, i.e., furious and paralytic. While diagnosis of furious rabies can be made based on the typical symptoms and signs, paralytic rabies poses a diagnostic dilemma to the neurologists who may encounter these cases in their practice. Although there are certain clinical features that distinguish this disease from other forms of Guillain-Barre syndromes, confirmation of diagnosis may require laboratory assistance. Conventional techniques such as antigen detection, antibody assays and virus isolation have limited success. The recently introduced molecular techniques show more promise in confirming the cases of paralytic rabies. There has not been much success in the treatment of confirmed rabies cases and recovery from rabies is extremely rare. Therefore, preventive measures of this dreaded disease after an exposure become extremely important. The present article reviews the current status of human rabies with regard to antemortem diagnosis, disease management and post-exposure prophylaxis.     

Development and evaluation of a latex agglutination test for rabies antibodies.

BACKGROUND: The presently recommended tests for estimation of rabies neutralising antibodies like Rapid Fluorescent Focus Inhibition test (RFFIT) and Mouse Neutralisation test (MNT) are laborious, time consuming and not cost-effective for routine use. Simple, rapid and economical tests need to be developed for routine use. OBJECTIVES: The main objective of the present study was to develop and evaluate a rapid Latex Agglutination Test (LAT) to detect rabies specific antibodies. METHODS: Latex beads were coated with purified rabies glycoprotein at a concentration of 1 mg/ml followed by coating with 0.3% bovine serum albumin (BSA). These sensitised beads were used to detect antiglycoprotein antibodies in sera of 152 people who had taken a course of post exposure rabies vaccination with different cell culture vaccines and whose antibody titers were pre determined by MNT. Sera from 52 normal healthy people without any detectable levels of rabies antibodies were included as controls. The test was carried out on glass slides by mixing 20 micro l of sensitised beads and 20 micro l serum. RESULTS: Preliminary evaluation with rabbit serum of known potency indicated that for clear agglutination of sensitised beads, a minimum of 2 IU/ml of rabies antibody should be present in the serum samples. Visible agglutination was noticed in positive sera with a titer > or =2 IU/ml within 3-5 min after mixing. Seven Sera whose MNT titers were less than 2 IU/ml did not show agglutinati or n. None of the negative control sera showed agglutination. Thus the specificity of the test was 100% and sensitivity was 95.4%. CONCLUSIONS: The LAT described here detects rabies specific antibodies > or =2 IU/ml and can be used to screen large number of sera from vaccinated people to know the protective status after vaccination. This simple and rapid test may be used routinely in antirabies treatment centres to monitor sero conversion in vaccinated people.     

Economical multi-site intradermal regimen with purified chick embryo cell vaccine (Rabipur) prevents rabies in people bitten by confirmed rabid animals.

OBJECTIVE: To determine the efficacy of a cost-effective multi-site intradermal regimen with purified chick embryo cell vaccine (PCECV, Rabipur) in preventing rabies in people bitten by confirmed rabid dogs. METHODS: Thirty-two people of different age groups who were severely bitten by confirmed rabid dogs were immunized with PCECV using the WHO recommended multi-site intradermal regimen of 0.1 mL of vaccine at eight sites on day 0, at four sites on day 7, and at one site each on days 28 and 90. In addition, passive immunization with human or equine rabies immunoglobulin was administered to 22 of these people before administering vaccine. They were followed for up to 3 years with periodic estimation of neutralizing antibody levels in their serum by mouse neutralization test (MNT). RESULTS: There was an excellent immune response with more than protective titers (>0.5 IU/mL) on all days tested up to the end of the 3-year observation period. More significantly, protective titers were seen in all subjects by day 7. Only minimal side effects were observed. All the patients were doing well at the end of the 3-year observation period, which is generally considered to be the maximum incubation period for rabies in humans. CONCLUSIONS: It can be concluded that this multi-site regimen with or without passive immunization has prevented the development of rabies encephalitis in these people bitten by confirmed rabid dogs. This should encourage more such studies, so that this cost-effective economical regimen with safe and potent cell culture vaccines can replace highly reactogenic neural tissue-derived Semple vaccine in developing countries such as India.     

Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay.

OBJECTIVES: The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. METHODS: Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). RESULTS: A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. CONCLUSIONS: The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.     

Simulated post-exposure rabies vaccination with purified chick embryo cell vaccine using a modified Thai Red Cross regimen.

OBJECTIVES: Currently, two intradermal regimens for the administration of cell culture rabies vaccines are approved by the WHO for rabies post-exposure prophylaxis: the two site Thai Red Cross regimen (TRC) and the eight site regimen. For the TRC regimen the volume of vaccine recommended per dose is 0.1 ml of purified Vero cell rabies vaccine (PVRV) and 0.2 ml of purified chick embryo cell vaccine (PCEC). The objective of the present study was to evaluate comparatively the immune response to PCEC and PVRV vaccines administered by the TRC regimen using a uniform dose of 0.1 ml of vaccine. METHODS: Forty-two subjects received TRC regimen (2-2-2-0-1-1) with 0.1 ml of PCEC vaccine and 38 subjects received the same regimen with PVRV. The rabies neutralizing antibody response in these subjects on days 10, 28, 90 and 180 was determined by the standard mouse neutralization test (MNT). RESULTS: There was adequate antibody response with both the vaccines and 100% seroconversion was observed by day 10. Furthermore, the antibody titers obtained with PCEC did not differ significantly from those obtained with PVRV on all days tested (p > 0.05). CONCLUSIONS: It can be concluded from the results that an adequate antibody response can be obtained with PCEC vaccine when administered by the TRC regimen even after reducing the quantity of vaccine from 0.2 ml to 0.1 ml per intradermal dose. The feasibility of using this regimen in true post-exposure cases needs to be further evaluated.     

A passive haemagglutination test for rabies antibodies using rabies glycoprotein coupled erythrocytes

The presently recommended tests for assaying rabies antibodies like mouse neutralization test (MINT) and rapid fluorescent focus inhibition test (RFFIT) are either time consuming or expensive and are generally performed in reference laboratories. There is a need to develop a specific and rapid method for detection of rabies antibodies that can be used to monitor sero-conversion after pre-or post-exposure vaccination. In this study, we have developed a passive haemagglutination (PHA) using purified rabies virus glycoprotein coupled to sheep erythrocytes using chromium chloride (0.04%) as a coupling agent. Two hundred and fifty five serum samples from people vaccinated with different rabies vaccines, 16 paired serum and CSF samples from autopsy confirmed cases of paralytic rabies, and serum samples from 65 normal healthy controls were tested and evaluated in comparison to standard MNT. Among the vaccinees, 250 samples were positive both by MNT and PHA but 5 samples were negative by PHA and positive by MNT. The titres obtained by PHA were lower compared to MNT, but there was significant correlation between the two (r=0.885). The specificity of the test was 99.7% and sensitivity was 100% as compared to MNT. Thus this PHA test promises to be a rapid and specific test for assaying rabies antibodies and may be useful in screening large number of serum samples for sero conversion after vaccination. It may also assist in rapid laboratory confirmation of paralytic rabies cases, based on detection of antibodies in CSF and serum.     

Surface characterization of 2-hydroxypyrimidine sulphate by inverse gas chromatography

The net retention volumes, V _N , of n-alkanes and polar probes on 2-hydroxypyrimidine sulphate (2-HPS) drug surface are determined at 323.15, 328.15 and 333.15 K using inverse gas chromatography. The dispersive surface free energy, $ \gamma_{S}^{d} $ of 2-HPS are evaluated by applying Schultz method as well as Dorris–Gray method and found that $ \gamma_{S}^{d} $ values are higher by 8 per cent in the latter method. The $ \gamma_{S}^{d} $ values are decreasing in both the methods with increase of temperature. The specific component of the free energy of adsorption, $ \Updelta G_{a}^{S} $ , for polar probes are obtained by three methods proposed by Schultz et al., Saint Flour–Papirer and Sawyer–Brookman. The Lewis acid–base parameters, K _a and K _b , are calculated using $ \Updelta G_{a}^{S} $ values. The surface character value, S = (K _b /K _a ) according to the Schultz et al., is found to be 3.9 whereas the S value in the other two methods are found to be 6.2 and 5.6. The results demonstrate that the 2-HPS powder surface contain relatively more basic sites and can interact strongly in the acidic media.     

Synthesis and insect antifeedant activity of plumbagin derivatives

Abstract  

Napthaquinones have received considerable interest in agricultural chemistry because of a novel action mode, extremely high activity against a broad spectrum of insects, low acute toxicity to mammals, and environmentally benign characteristics. A series of plumbagin derivatives (3a–3o) were synthesized under mild esterification conditions in straightforward procedure. The structures of all new compounds were confirmed by NMR, IR, MS, and HREIMS analyses. The plumbagin derivatives were screened for their insecticidal activities against tobacco caterpillar (Spodoptera litura) and castor semilooper (Achaea janata) using a no-choice laboratory bioassay. The results show that some of the title compounds exhibit excellent antifeedant activities against 3rd instar larvae of A. janata and S. litura. The improvement in antifeedant activity requires a reasonable combination of substituents in the parent structure, which provides some hints for further investigation on structure modification.