Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB.

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells. METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of ß-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs. RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral ß-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1. CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Isolation, Solid-state Structure Determination, In Silico and In Vitro Anticancer Evaluation of an Indole Amino Acid Alkaloid L-Abrine.

BACKGROUND: Abrus precatorius Linn. (Kunch in Bengali) is widely spread in tropical and sub-tropical regions. It is a typical plant species which is well-known simultaneously as folk medicine and for its toxicity. OBJECTIVE: Phytoceutical investigation of the white variety seeds of Abrus precatorius Linn. METHODS: Traditional extraction, separation, isolation, and purification processes were followed. The structure was elucidated by various spectral analyses and the solid-state structure of this indolealkaloid was determined by X-ray crystallographic analysis. Docking interactions of L-abrine had been studied against ten major proteins, responsible for various types of cancers. In silico studies were done by Schrödinger Maestro, AutoDock4, PyMOL and AutoDock Vina. The protein structures were downloaded from Protein Data Bank. Sulforhodamine B (SRB) colorimetric assay was used for in vitro anticancer evaluation against four human cancer cell lines. RESULTS: An indole-containing unusual amino acid alkaloid had been isolated from the white variety seeds of Abrus precatorius Linn. In silico docking studies demonstrated significant antiproliferative activity against four human cancer cell lines. CONCLUSION: The solid-state zwitterion structure of the indole-containing alkaloid (α-methylamino- ß-indolepropionic acid, L-abrine) has been confirmed for the first time by X-ray crystallography. Highly promising in silico and in vitro results indicate that L-abrine may find its space in future anticancer drug discovery research.

Evaluation of mosquito larvicidal activity of fruit extracts of Acacia auriculiformis against the Japanese encephalitis vector Culex vishnui.

The larvicidal potentiality of crude and ethyl acetate extracts of fruits of Acacia auriculiformis was investigated against all the larval instars of JE vector Culex vishnui. The crude extracts showed good results against all the larval instars with highest mortality at 0.09%. Highest mortality was found at 300 ppm of ethyl acetate extract. Lowest LC50 value was obtained at 72 h for third instar larvae. Non target organisms tested, showed no to very less mortality to ethyl acetate solvent extract. Presence of N-H stretching, a C=O stretching, C=C and C-N stretching vibrations of secondary amide or amine group were confirmed from IR analysis. GC-MS analysis revealed the presence of three compounds namely Ethane 2-chloro-1,1-dimethoxy, Acetic acid, 1-methyl ether ester and [4-[1-[3,5-Dimethyl-4[(trimethylsilyl)oxy)phenyl]-1,3-dimethylbutyl)-2,6dimethylphenoxy)(trimethyl) silane, responsible for mosquito larval death.

Glucosinolate from leaf of Solanum nigrum L. (Solanaceae) as a new mosquito larvicide.

The present study was carried out to investigate the biocontrol potentiality of active ingredient isolated from ethyl acetate extract of mature leaves of Solanum nigrum L. (Solanaceae) against the larval form of Culex quinquefasciatus Say. Mortality rate at a concentration of 25 mg/L of the active compound was highest (P < 0.05) amongst all tested concentrations. Result of log-probit analysis (at 95% confidence level) revealed that LC50 and LC90 values are inversely proportional to exposure period of bioassay. A clear dose-dependent mortality was observed, as the rate of mortality (Y) was positively correlated with the concentrations of the compound (X); having regression coefficient value close to 1. The compound was found to be ecofriendly as it did not show any adverse effect to the studied nontarget organisms. Chemical characterization of the active ingredient was also carried out by infrared spectroscopic analysis (IR), mass analyses (GC-MS) and carbon-hydrogen-nitrogen-sulphur analyses (CHNS), that revealed the presence of a glucosinolate compound [1-thio-ß-D-glucopyranose-1-[(R)-3-hydroxy-2-ethyl-N-hydroxysulfonyloxy propanimidate] having the molecular formula of C11H21NO10S2.

Mosquito larvicidal activity of Solanum nigrum berry extracts.

Phytochemicals are widely used as biocontrol agent against vector mosquitoes. The present study was undertaken to isolate and evaluate the mosquitocidal activity of various extracts of berries of S. nigrum against Culex quinquefasciatus. Crude and chloroform: methanol (1:1, v/v) extracts of fresh, mature, green berries of S. nigrum were tested against Cx. quinquefasciatus. The lethal concentration was determined and the chemical nature of the active substance was evaluated. A qualitative phytochemical analysis of chloroform: methanol (1:1, v/v) extract was performed in search of the active ingredient. The appropriate lethal concentrations at 24 h for chloroform: methanol (1:1, v/v) extract was also studied on non-target organisms. In a 72 h bioassay experiment with crude extract, the highest mortality was recorded in 3 per cent extract. In the chloroform: methanol (1:1, v/v) solvent extract, the maximum mortality was recorded at a concentration of 120 µg/ml. The log probit analysis (95% confidence level) recorded lowest LC 50 value at 72 h of exposure. Both crude and chloroform: methanol (1:1, v/v) extracts showed good larvicidal activity against Cx. quinquefasciatus. The isolated active ingredient may be tested as a potential larvicide after determination of its structure.

Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: histological, biochemical and immunological perspectives.

We have evaluated the toxicity profile of a unique immunomodulator, neem leaf glycoprotein (NLGP) on different physiological systems of Swiss mice and Sprague Dawley rats. NLGP injection, even in higher doses than effective concentration caused no behavioral changes in animals and no death. NLGP injection increased the body weights of mice slightly without any change in organ weights. NLGP showed no adverse effect on the hematological system. Moreover, little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Histological assessment of different organs revealed no alterations in the organ microstructure of the NLGP treated mice and rats. Histological normalcy of liver and kidney was further confirmed by the assessment of liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine. NLGP has no apoptotic effect on immune cells but induces proliferation of mononuclear cells collected from mice and rats. Number of CD4(+), CD8(+) T cells, DX5(+) NK cells, CD11b(+) macrophages and CD11c(+) dendritic cells is upregulated by NLGP without a significant change in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Type 1 cytokines, like IFNγ also increased in serum with a decrease in type 2 cytokines. Total IgG content, especially IgG2a increased in NLGP treated mice. These type 1 directed changes help to create an anti-tumor immune environment that results in the restriction of carcinoma growth in mice. Accumulated evidence strongly suggests the non-toxic nature of NLGP. Thus, it can be recommended for human use in anti-cancer therapy.

Efficacy of Limonia acidissima L. (Rutaceae) leaf extract on larval immatures of Culex quinquefasciatus Say 1823.

OBJECTIVE: To investigate the role of leaf extract of Limonia acidissima L. (Rutaceae) as a biocontrol agent against the larval form of Culex quinquefasciatus, and characterization of bioactive component responsible for larvicidal activity. METHODS: Larval mortality of mosquito species was observed after 24, 48 and 72 hours of exposure to different concentrations of aqueous extract, solvent extract and subsequently bioactive compound. The bioactive compound was subjected to IR and GC-MS analysis. RESULTS: Mortality rate at 3% concentration of crude extract were highest (90%) amongst all concentrations tested and subsequently highest (95%) mortality was achieved in chloroform: methanol extract at 100 ppm concentrations. IR and GC-MS analysis of bioactive compound revealed the presence of steroid compound which may act as larvicide. CONCLUSIONS: The chloroform: methanol extract of mature leaves of Limonia acidissima was found to exhibit considerable mosquito larvicidal activity against Culex quinquefasciatus.

Identification and interaction of amino acids with leucine-anthracene reagent by TLC and spectrophotometry: experimental and theoretical studies.

A new reagent has been synthesized by coupling anthracene moiety to L-leucine. The reagent is characterized by different analytical techniques. It is capable for easy identification of various amino acids on thin-layer chromatography plates by developing distinguishable colors (detection limit between 0.1-0.5 µg at cold condition and 0.1-0.4 µg after heating). This reagent also binds with different amino acids very strongly in solution (methanol). Estimation of equilibrium binding constants of this new reagent with different amino acids has also been carried out. The values of the binding constants are lowest for L-Tyrosine (6.86 × 10³ dm³ mol(-1)) and highest for L-Arginine monohydrochloride (8.86 × 105 dm³ mol(-1)) at 25°C. A theoretical study (Hartree-Fock) has been performed to investigate the interaction of the reagent with a representative amino acid, glycine.

Asimafoetidnol: a new sesquiterpenoid coumarin from the gum resin of Ferula assa-foetida.

Chemical investigation of the gum resin of Ferula assa-foetida L. resulted in the isolation of a new sesquiterpenoid coumarin, 7-(((E)-5-((1S,3S,6S)-3,6-dihydroxy-2,2,6-trimethylcyclohexyl)-3-methylpent-2-en-1-yl)oxy)-2H-chromen-2-one (asimafoetidnol), together with several other known compounds. The structure of asimafoetidnol was established on the basis of spectroscopic analyses. Geometry optimization of the compound has been carried out using a DFT/B3LYP/3-21G* method.

n-alkane profile of Argemone mexicana leaves.

An n-hexane extract of fresh, mature leaves of Argemone mexicana (Papaveraceae), containing thin-layer epicuticular waxes, has been analysed for the first time by TLC, IR and GLC using standard hydrocarbons. Seventeen long-chain alkanes (n-C18 to n-C34) were identified and quantified. Nonacosane (n-C29) was established as the n-alkane with the highest amount, whilst octadecane (n-C19) was the least abundant component of the extracted wax fraction. The carbon preference index (CPI) calculated for the hydrocarbon sample with the chain lengths between C18 and C34 was 1.2469, showing an odd to even carbon number predominance.

Determination of the n-alkane profile of epicuticular wax extracted from mature leaves of Cestrum nocturnum (Solanaceae: Solanales).

An n-hexane extract of fresh, mature leaves of Cestrum nocturnum (Solanales: Solanaceae) containing thin layer epicuticular waxes was analysed by thin-layer chromatography, infrared and gas liquid chromatography using standard hydrocarbons. Seventeen long chain alkanes (n-C(18) to n-C(34)) were identified and quantified. Hentriacontane (n-C(31)) was established as the major n-alkane, while nonadecane (n-C(19)) was the least abundant component of the extracted wax fraction. The carbon preference index calculated for the sample was 1.30, showing an odd to even carbon number predominance.

Neem leaf glycoprotein directs T-bet-associated type 1 immune commitment.

Neem leaf glycoprotein (NLGP)-mediated immune activation and associated immune polarization was studied. NLGP-induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-gamma and a lower amount of the Th2 cytokine interleukin (IL)-4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor -alpha and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3(+) T-Reg cells. A type 1-specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.

Mosquito larvicidal and antimicrobial activity of protein of Solanum villosum leaves.

BACKGROUND: Mosquitoes are associated with the transmission of malaria, dengue, Japanese encephalitis, filariasis and other viral diseases throughout the globe, apart from being a nuisance pest. Biological control alone or as a part of integrated vector management stands to be a better alternative to the chemical controls aimed against pest mosquitoes. At the same time it is necessary to control bacteria by synthetic or natural means (plant products). Hence the present study was designed to screen the effect of mosquito larvicidal and antimicrobial activitiy of protein isolated from matured leaves of Solanum villosum against mosquito immatures and some pathogenic bacteria. METHODS: Aqueous solvent extract of fresh mature leaves of S. villosum was tested against 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus and Stegomyia aegypti mosquitoes and against four pathogenic bacteria. The protein fraction was isolated and tested for mosquitocidal and antibacterial activities. Amino acid analysis was performed on isolated protein using PICO.TAG amino acid system. SDS-PAGE was also done to detect the bands of amino acid on the basis of their molecular weights. RESULTS: Proteins isolated from mature leaves of S. villosum were found to have larvicidal and antimicrobial properties. Analysis of the isolated protein identified fifteen amino acids of which eight were essential amino acids. SDS-PAGE detected seven bands corresponding to different molecular weights in the range of 69-109 KDa. CONCLUSION: Proteins of mature leaves of S. villosum exhibited moderate larvicidal and antimicrobial activities. The study provides considerable scope in exploiting local indigenous resources for isolation of antimicrobial and mosquito larvicidal proteins.

Neem leaf glycoprotein helps to generate carcinoembryonic antigen specific anti-tumor immune responses utilizing macrophage-mediated antigen presentation.

In an objective to generate effective carcinoembryonic antigen (CEA) specific anti-tumor immune response in Swiss mice, CEA was presented using macrophages with adjuvant help from neem leaf glycoprotein (NLGP). Such vaccination generates significantly higher antibody (IgG2a) and T cell response than immunization protocol without NLGP. NLGP controls the function of both B cells and macrophages by altering the expressions of various regulatory molecules, like, CD19, CD11b, etc. NLGP also directs CEA vaccination towards Th1 bias, by modulating cytokine secretion. This NLGP-generated anti-CEA immune response would be effective as a vaccine to lyse CEA(+) tumors in vitro and in vivo.

Neem leaf glycoprotein restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance.

Interaction between CXCL10 and CXCR3 is dysregulated in head and neck squamous cell carcinoma (HNSCC) and hampers chemotaxis of cytotoxic cells at tumor site. In continuation of the demonstration of significant immunomodulatory effects of neem leaf preparation (NLP), the active ingredient of NLP is characterized as a glycoprotein (NLGP). NLGP is responsible for in vivo immunomodulation to restrict the growth of mice tumors. Effect of NLGP in rectification of the dysregulated IFN gamma dependent chemokine and its receptor CXCR3 splice variants was investigated. Upregulated expression of CXCR3B in HNSCC-PBMC were downregulated following in vitro NLGP treatment. Unchanged expression of CXCR3A+B by NLGP with downregulation of the CXCR3B indirectly suggests the upregulation of the CXCR3A, responsible for cellular migration. However, stimulation of healthy-PBMC with NLGP maintains physiological homeostasis of CXCL10 and increases IFN gamma secretion. The suppressed chemotaxis of HNSCC-PBMC could be restored either by in vitro treatment with NLGP or during use of NLGP stimulated PBMC supernatant as a chemoattractant. Neutralization studies confirmed that the chemoattraction process is guided by both receptor (CXCR3A) and its ligand (CXCL10). Neutralization of the IFN gamma in PBMC culture in presence of NLGP unexpectedly increases the intracellular release of CXCL10, suggesting the NLGP mediated IFN gamma independent release of CXCL10. Interestingly, downregulation of the CXCL10 release was detected after IFN gamma neutralization in absence of NLGP and IFN gamma receptor neutralization in presence of NLGP. Efficacy of NLGP in restoration of the dysregulation of the chemokine signaling may be utilized to design new immunotherapeutic protocol.

Antibody response against neem leaf preparation recognizes carcinoembryonic antigen.

An immune serum generated in Swiss mice against an aqueous preparation from neem leaf was reactive with carcinoembryonic antigen (CEA) and a peptide sequence derived from it. Using ELISA, we have demonstrated that CEA reactive antibody titer (chiefly IgG2a) was significantly decreased after absorption of the immune sera with CEA. Neem leaf preparation (NLP) generated immune sera was also reactive with CEA in immunoblotting and CEA reactive component in the NLP sera can be immunoprecipitated. Identical recognition of CEA expressed on human colorectal cancer specimens, by anti-CEA monoclonal antibody and NLP sera was documented by immunohistochemistry. NLP generated sera could also react with NLP in ELISA and this reactivity was decreased after absorption of the NLP with anti-CEA antibody. Estimation of protein in NLP revealed the presence of it, at least 10% of the total dry weight. In addition, existence of flavone and quercetin was also evidenced from LC-MS analysis. Further studies are needed to identify the antigenic component may have some homology with CEA molecule. This unique property of neem may be utilized for the immunotherapy of CEA positive tumors.

Study of electron donor-acceptor complexes of tri-n-octyl amine with [60]- and [70]fullerenes and some other electron acceptors by absorption spectrophotometric method.

Electron donor-acceptor (EDA) complexes of tri-n-octylamine (TOA) with [60]- and [70]fullerenes and some other electron acceptors have been studied in chloroform medium by absorption spectrophotometric technique. Charge transfer (CT) absorption bands are observed in the visible region. Vertical ionization potential of TOA was determined utilizing CT transition energy. Oscillator strengths, transition dipole strengths and resonance energies for all the complexes have been calculated. [60]Fullerene/TOA and [70]fullerene/TOA complexes are found to decay slowly with time. Kinetics of these reactions have been studied and activation energies for such processes have been estimated. Ab initio calculations suggest that complexation of [70]fullerene with TOA is enthalpy favoured.