Sushil Kumar Mandal

A New Half-Condensed Schiff Base Compound: Highly Selective and Sensitive pH-Responsive Fluorescent Sensor

A new probe, 3-[(3-benzyloxypyridin-2-ylimino)methyl]-2-hydroxy-5-methylbenzaldehyde (1-H) behaves as a highly selective fluorescent pH sensor in a Britton-Robinson buffer at 25 °C. The pH titrations show a 250-fold increase in fluorescence intensity within the pH range of 4.2 to 8.3 with a pK(a) value of 6.63 which is valuable for studying many of the biological organelles.

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells.

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.

A synthetic coumarin (4-Methyl-7 hydroxy coumarin) has anti-cancer potentials against DMBA-induced skin cancer in mice

Scopoletin, an alkaloid separated from ethanolic extract of the medicinal plant, Gelsemium sempervirens (Fam: Loganiaceae) has been reported to have anti-cancer potentials. The synthetic coumarin (4-Methyl-7 hydroxy coumarin) derived from resorcinol and ethyl aceto-acetate in presence of concentrated sulphuric acid is structurally close to scopoletin, being a coumarin derivative. Whether this synthetic compound also has anti-cancer potentials has been evaluated in vivo on DMBA (7,12-Dimethylbenz[a]anthracene) induced skin cancer in mice by analyzing results of several cytogenetic endpoints, Comet assay, and fluorescence activated cell sorting (FACS). Further, expressions of signal proteins like Aryl hydrocarbon receptor , p53, PCNA, Akt, Bcl-2, Bcl-xL, Bad, Bax, NF-kappaB Apaf, IL-6, Cytochrome-c, Caspase-3 and Caspase-9 were studied by immunoblot analysis along with histology of skin and immuno-histochemical localization of Aryl hydrocarbon receptor and PCNA in DMBA treated mice vis-a-vis carcinogen treated synthetic coumarin fed mice. Feeding of this synthetic coumarin induced positive modulations in expression of all biomarkers in DMBA administered mice, giving clues on its possible signaling pathway(s) - primarily through down-regulation of Aryl hydrocarbon receptor and PCNA and up-regulation of apoptotic proteins like Bax, Bad, Cytochrome c, Apaf, Caspase-3 and Caspase-9, resulting in an appreciable reduction in growth of papilloma in mice. Therefore, this synthetic coumarin shows promise for use in cancer therapy, particularly in skin cancer.

Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells

Crude ethanolic extract of Thuja occidentalis (Fam: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C10H16O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.

(6)-Gingerol induces caspase 3 dependent apoptosis and autophagy in cancer cells: Drug-DNA interaction and expression of certain signal genes in HeLa cells

[6]-Gingerol, a pharmacologically important bioactive component of ginger, has been reported to have anti-hyperglycemic, anti-cancer and anti-oxidative properties, but mechanisms through which these are achieved are largely unclear. The present study focuses on apoptosis and autophagy, two key events of anti-cancer activity, in HeLa cells treated with [6]-gingerol. The treated cells showed several morphological changes, including externalization of phosphatidyl serine, degradation of DNA and increase in TUNEL positivity. Furthermore, there was depolarization of mitochondrial membrane potential, providing evidence of mitochondria mediated apoptosis. The expression of caspase 3 and PARP was increased in cells exposed to [6]-gingerol. Circular dichroism study for testing drug-DNA interaction with both calf thymus and nuclear DNA as target revealed that the drug had potential to bind with the nuclear DNA and induce conformational changes of DNA. The over-expression of NFkβ, AKT and Bcl2 genes in cancer cells was down-regulated by [6]-gingerol treatment. On the other hand the expression levels of TNFα, Bax and cytochrome c were enhanced in [6]-gingerol treated cells. Thus, overall results suggest that [6]-gingerol has potential to bind with DNA and induce cell death by autophagy and caspase 3 mediated apoptosis.

In Vitro Studies Demonstrate Anticancer Activity of an Alkaloid of the Plant Gelsemium sempervirens

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing 1H nuclear magnetic resonance (NMR), 13C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C10H8O4 having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

Lycopodine from Lycopodium clavatum extract inhibits proliferation of HeLa cells through induction of apoptosis via caspase-3 activation.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimers disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

A water soluble FRET-based ratiometric chemosensor for Hg(II) and S2− applicable in living cell staining

A new highly sensitive and selective Hg(II) probe, 2-(rhodamine-b-hydrazido)-N-(quinolin-8-yl)acetamide (L1) was developed and characterized. L1 specifically binds to Hg(II) in the presence of a large excess of other competing ions with visually observable changes in both electronic and fluorescence spectral behaviour to make possible the naked eye detection of Hg(II) at a very low level (up to 4.5 × 10−7 M) through a fluorescence resonance energy transfer (FRET) process in HEPES buffer (1 mM, pH 7.4; 2% EtOH) at 25 °C. The theoretical and experimental kinetic study also support the binding of Hg(II) ion to induce the opening of the spirolactam ring in L1 for enabling the FRET process. Further studies reveal that the selective dissociation of the L–Hg complex in the presence of sulphide anions to restore the native structure of L1 is also useful in the detection of sulfide anions with a detection limit of a submicromolar range in the same medium of HEPES buffer (1 mM, pH 7.4; 2% EtOH) at 25 °C. L1 could be employed as a FRET based time dependent reversible chemosensor for imaging Hg(II) in living cells and whole bodies, and also could be used as an imaging probe for the detection of sulfide anions in HeLa cells.

A highly selective and biocompatible chemosensor for sensitive detection of zinc(II)

2-Formyl-4-methyl-6-(2-benzoimidazolyliminomethyl)phenol (HL1) has been synthesized via Schiff-base condensation between 4-methyl-2,6-diformylphenol and 2-aminobenzimidazole in a 1:1 ratio in acetonitrile and characterized using elemental analysis and different spectroscopic methods. HL1 has been found to be a selective fluorescence sensor for Zn2+ ions. The emission intensity of HL1 at 528 nm in 10 mM HEPES buffer in water:methanol (1:9, v/v) (pH = 7.2) increases in the presence of Zn2+ when it is excited at 445 nm. Other metal ions can induce a slight increment or lowering of emission intensity. The spectral properties of HL1 and 2-formyl-4-methyl-6-(2-benzoimidazolylmethyliminomethyl)phenol (HL2) have been compared. It has been found that the presence of the methylene group in HL2 can have a significant effect on the absorption and fluorescence peak positions of the Schiff-base molecule and its zinc complex. Some theoretical calculations have been done to get a better view into the different spectral transitions. HL1 and HL2 have been found to be highly sensitive towards the detection of Zn2+ ions with very low LOD values. Excitation in the visible region and the effect of pH on the emission intensity of HL1 encourage us to carry out biological studies. HL1 has been used for human lung cancer cell (A549) imaging without cytotoxicity.

A FRET-based ‘off–on’ molecular switch: an effective design strategy for the selective detection of nanomolar Al3+ ions in aqueous media

A new water-soluble rhodamine-based Al3+ ion-selective probe (L1) was synthesised and characterized by physico-chemico and spectroscopic tools. In the presence of a large excess of other competing ions, L1 specifically binds Al3+ ions with a concurrent visually observable change from colorless to pink in electronic spectral behavior, making it possible to detect the presence of Al3+ ions with the naked eye. The addition of Al3+ ions to a solution of L1 in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 °C, results in a decrease in the weak fluorescence intensity at λem = 470 nm, while a new peak (at λem = 588 nm) increases gradually through a fluorescence resonance energy transfer process. This ratiometric enhancement helps to detect Al3+ ions at a very low concentration of 33 nM. The detection limit of L1 for Al3+ ions was estimated to be 6.19 × 10−9 M using the 3σ method. This probe is also useful for imaging Al3+ ions in HeLa cells.