N. Krishnakumar

Chemopreventive efficacy of piperine in 7,12-dimethyl benz [a] anthracene (DMBA)-induced hamster buccal pouch carcinogenesis: An FT-IR study

The present study is designed to investigate the effect of piperine in modifying the carcinogenic process, as well as biochemical alterations at the molecular level during DMBA-induced hamster buccal pouch carcinogenesis by FT-IR spectroscopy. Specific changes were noticed in the FT-IR spectral features of DMBA-induced hamster buccal pouch carcinoma. These alterations include structural changes of proteins and possible increase of its content, an increase in the nuclear-to-cytoplasm ratio, an increase in the relative amount of DNA, an enhancement in the phosphorylation of proteins, a loss of hydrogen bonding of the C-OH groups in the amino acid residues of proteins and diminished lipid peroxidation which were accompanied by a significant reduction in the relative amount of lipids compared to untreated control animals. Administration of piperine significantly increased the levels of lipid peroxidation and decreased the levels of proteins and nucleic acid content that were found to increase in oral cancer bearing animals. In conclusion, the results of the present study suggest that piperine may exert its chemopreventive effect by modulating the biochemical changes at the molecular level during DMBA-induced hamster buccal pouch carcinogenesis which can be detected using FT-IR spectroscopic technique.

Morphological changes due to Lead exposure and the influence of DMSA on the gill tissues of the freshwater fish, Catla catla.

Catla catla fingerlings were reared in freshwater and exposed to 15.5 ppm concentration of lead for 60 days. The morphological changes on the gill of the C. catla fingerlings due to lead intoxication and the effect of DMSA (meso 2,3-dimercaptosuccinic acid) on the affected tissues were observed using Scanning Electron Microscope. It has been found that the lead treated gill tissues showed certain marked changes, such as cell hypertrophy, alteration in the lamellar surfaces, epithelial hyperplasia and the fusion of adjacent lamellae. The antidote DMSA treatment reduces the toxic effects and helps the recovery of gill tissue and its return to the level of the control/normal.

Enhanced cytotoxicity and apoptosis-induced anticancer effect of silibinin-loaded nanoparticles in oral carcinoma (KB) cells.

Silibinin (SIL) is a plant derived flavonoid isolated from the fruits and seeds of the milk thistle (Silybum marianum). Silibinin possesses a wide variety of biological applications including anticancer activities but poor aqueous solubility and poor bioavailability limit its potential and efficacy at the tumor sites. In the present study, silibinin was encapsulated in Eudragit® E (EE) nanoparticles in the presence of stabilizing agent polyvinyl alcohol (PVA) and its anticancer efficacy in oral carcinoma (KB) cells was studied. Silibinin loaded nanoparticles (SILNPs) were prepared by nanoprecipitation technique and characterized in terms of size distribution, morphology, surface charge, encapsulation efficiency and in vitro drug release. MTT assay revealed higher cytotoxic efficacy of SILNPs than free SIL in KB cells. Meanwhile, reactive oxygen species (ROS) determination revealed the significantly higher intracellular ROS levels in SILNPs treated cells compared to free SIL treated cells. Therefore, the differential cytotoxicity between SILNPs and SIL may be mediated by the discrepancy of intracellular ROS levels. Moreover, acridine orange (AO) and ethidium bromide (EB) dual staining and reduced mitochondrial membrane potential (MMP) confirmed the induction of apoptosis with nanoparticle treatment. Further, the extent of DNA damage (evaluated by comet assay) was significantly increased in SILNPs than free SIL in KB cells. Taken together, the present study suggests that silibinin-loaded nanoparticles can be used as an effective drug delivery system to produce a better chemopreventive response for the treatment of cancer.

The study of the changes in the biochemical and mineral contents of bones of Catla catla due to lead intoxication.

In the present study, an attempt has been made to analyze the changes in the biochemical and mineral contents of lead‐intoxicated bones of Catla catla at subchronic (15.5 ppm) exposure, and also to determine whether the effects of Pb intoxication can be reversed with the chelating agent meso 2, 3‐dimercaptosuccinic acid (DMSA) on the bones of freshwater fingerlings Catla catla by using Fourier transform infrared (FT‐IR) spectroscopy, X‐ray diffraction (XRD), and atomic absorption spectrophotometer techniques. The FT‐IR spectra of the lead‐exposed bones show significant alteration in the biochemical constituents. The XRD analysis showed a decrease in crystallinity due to lead exposure. Further, the Ca, Mg, and P contents of the lead‐exposed bones were less than those of the control group, and there was an increase in the mineral contents of the bones after DMSA treatment. In conclusion, the present study suggests that the subchronic lead exposure results in severe loss of bone minerals. The overall decrease in the FT‐IR band intensity of Pb‐exposed bones relative to the control indicates a decrease in the biochemical constituents like proteins and lipids. The increase in the band intensity after treatment with chelating agent DMSA indicates increased biochemical constituents, showing that the subchronic effects of lead can be reversed by DMSA. The amide I bands observed at 1654 cm−1 in the present study suggest that the protein is dominated by α‐helical structure.

FT-IR study of the effect of lead and the influence of chelating agents, DMSA and D-Penicillamine, on the biochemical contents of brain tissues of Catla catla fingerlings

Abstract.The goal of this study was to investigate the effect of lead and the influence of chelating agents,meso 2, 3-dimercaptosuccinic acid (DMSA) and D-Penicillamine, on the biochemical contents of the brain tissues of Catla catla fingerlings by Fourier Transform Infrared Spectroscopy. FT-IR spectra revealed significant differences in absorbance intensities between control and lead-intoxicated brain tissues, reflecting a change in protein and lipid contents in the brain tissues due to lead toxicity. In addition, the administration of chelating agents, DMSA and D-Penicillamine, improved the protein and lipid contents in the brain tissues compared to lead-intoxicated tissues. Further, DMSA was more effective in reducing the body burden of lead. The protein secondary structure analysis revealed that lead intoxication causes an alteration in protein profile with a decrease in α-helix and an increase in β-sheet structure of Catla catla brain. In conclusion, the study demonstrated that FT-IR spectroscopy could differentiate the normal and lead-intoxicated brain tissues due to intrinsic differences in intensity.

Raman spectroscopic investigation of the chemopreventive response of naringenin and its nanoparticles in DMBA-induced oral carcinogenesis.

Raman spectroscopy is a vibrational spectroscopic technique that can be used to optically probe the biomolecular changes associated with tumor progression. The aim of the present study is to investigate the biomolecular changes in chemopreventive response of prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) during 7,12-dimethyl benz(a)anthracene (DMBA)-induced oral carcinogenesis by Fourier Transform Raman (FT-Raman) spectroscopy. Oral squamous cell carcinoma (OSCC) was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14weeks. Raman spectra differed significantly between the control and tumor tissues, with tumors showing higher percentage signals for nucleic acids, phenylalanine and tryptophan and a lower in the percentage of phospholipids. Moreover, oral administration of free NAR and NARNPs significantly increased phospholipids and decreased the levels of tryptophan, phenylalanine and nucleic acid contents. On a comparative basis, NARNPs was found to have a more potent antitumor effect than free NAR in completely preventing the formation of squamous cell carcinoma and in improving the biochemical status to a normal range in DMBA-induced oral carcinogenesis. The present study further suggest that Raman spectroscopy could be a valuable tool for rapid and sensitive detection of specific biomolecular changes in response to chemopreventive agents.

Endogenous porphyrin fluorescence as a biomarker for monitoring the anti-angiogenic effect in antitumor response to hesperetin loaded nanoparticles in experimental oral carcinogenesis

The purpose of the present study is to evaluate the antitumor efficacy of hesperetin loaded nanoparticles (HETNPs) in comparison with native hesperetin (HET) for monitoring endogenous porphyrin emission against 7,12-dimethyl benz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. The effort has been further complemented by immunohistochemical analysis in an attempt to provide a correlation between the emission intensity of endogenous porphyrin and changes in the vascular endothelial growth factor (VEGF) expression observed in control and the experimental animals. A total of 60 autofluorescence spectra were successfully acquired from different anatomic locations of the control and the experimental tissues. Significant differences in the autofluorescence spectral signatures between the control and that of experimental animals have been noticed under the excitation wavelength of 410 nm with emission ranging from 440–750 nm. DMBA-induced tumor tissues are associated with increased endogenous porphyrin emissions at ∼630 nm, ∼670 nm and ∼705 nm. Moreover, oral administration of HET and its nanoparticulates restored the status of endogenous porphyrin emission in the buccal mucosa of DMBA-painted animals. On a comparative basis, the treatment of nanoparticulate hesperetin was found to be more effective than native hesperetin in reducing the formation of squamous cell carcinoma and in improving the status of endogenous porphyrins to a near normal range in DMBA-induced hamster buccal pouch carcinogenesis. These endogenous porphyrin emissions may be used as a spectral biomarker for monitoring tumor hypervascularity. The hypervascularity in tumors might be due to angiogenesis. Complementary immunohistochemical analysis of the DMBA-induced tumor tissues showed an abundant expression of VEGF (a marker used for angiogenesis), suggesting that increased endogenous porphyrin intensities may be correlated with angiogenesis. Furthermore, the spectral data are also analyzed using a multivariate data analysis and yielded diagnostic sensitivities of 100%, 76.0%, 93.0% and 80.0% and specificities of 100%, 73.3%, 86.0% and 86.6%, respectively, for classification of control vs. DMBA, DMBA vs. DMBA + HET, DMBA vs. DMBA + HETNPs and DMBA + HET vs. DMBA + HETNPs treated tissues. The present study further suggests that endogenous porphyrin fluorescence could be considered as a reliable spectral marker and a very useful tool for monitoring the anti-angiogenic effect of cancer.

Synthesis, characterization and in vitro anti-cancer evaluation of hesperetin-loaded nanoparticles in human oral carcinoma (KB) cells

Hesperetin (HET), a naturally occurring plant bioflavonoid present in citrus fruits, possesses potential anti-inflammatory and anti-carcinogenic activities but poor aqueous solubility limits its applications. To improve its applicability in cancer therapy, hesperetin was encapsulated in Eudragit® E (EE) 100 nanoparticles in the presence of polyvinyl alcohol (PVA) as a stabilizer and its anticancer efficacy in oral carcinoma (KB) cells was studied. Hesperetin-loaded nanoparticles (HETNPs) were prepared by nanoprecipitation method and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and x-ray diffraction (XRD). The results thus displayed that the prepared nanoparticles showed a particle size in the range from 55 to 180 nm. The encapsulation efficiency of hesperetin was 83.4% obtained by UV spectroscopy. The in vitro release kinetics of hesperetin under physiological condition show initial rapid release followed by slow and sustained release. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay revealed higher cytotoxic efficacy of HETNPs than native hesperetin in KB cells. Further, it has been found that reactive oxygen species (ROS) generation, DNA damage and apoptotic indices in HETNPs treated cells are greater than those in native hesperetin treatment. Hence these findings demonstrate that HETNPs could be a potentially useful drug delivery system to produce better hesperetin therapeutics of cancers.

Influence of Co doping on combined photocatalytic and antibacterial activity of ZnO nanoparticles

The present work aims to investigate the structural, optical, photocatalyst and antibacterial properties of bare and cobalt doped ZnO nanoparticles (NPs) with different concentrations Zn1−x Co x O (x = 0, 0.03, 0.06 and 0.09) synthesized by co-precipitation method. The XRD patterns confirmed that all samples of cobalt doped ZnO nanostructures revealed the formation of single phase having hexagonal wurtzite structure with crystallite size in the range of 31–41 nm. Further, the decreasing trend in lattice parameters and grain sizes were also seen with increasing doping concentrations which confirms the incorporation of Co ions into the ZnO lattice. This result was further supported by the FT-IR data. HR-TEM images demonstrated the distinct hexagonal like morphology with small agglomeration. The UV–visible absorption spectra exhibits red shift with increase in Co doping concentration in ZnO while corresponding bandgap energy of cobalt doped ZnO NPs decreased with increased Co doping concentration. PL spectra showed a weak UV and visible emission band which may be ascribed to the reduction in oxygen vacancy and defects by cobalt doping. XPS and EDX spectral results confirm the composition and the purity of Co doped ZnO NPs. Furthermore, the Co doped ZnO NPs were found to exhibit lesser photocatalytic activity for the degradation of methyl green dye under UV light illumination in comparison with the bare ZnO NPs. Moreover, anti-bacterial studies reveals that the Co doped ZnO NPs possess more antibacterial effect against gram positive Basillus subtills and gram negative Klebsiella pneumoniae bacterial strains than the bare ZnO NPs.

Monitoring the metabolic response to nanoencapsulated silibinin treatment in DMBA-induced oral carcinogenesis using endogenous fluorescence

Autofluorescence spectroscopy is a very sensitive tool for detecting early metabolic response after cancer treatment. The present work aims to investigate the chemopreventive effect of the prepared silibinin-loaded nanoparticles (SILNPs) relative to the efficacy of free silibinin (SIL) for monitoring the changes in the endogenous fluorophore emission and to quantify the metabolic changes in the redox state during 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch (HBP) carcinogenesis using autofluorescence spectroscopy. Significant differences in the autofluorescence spectral signatures between the control and the experimental animals have been noticed under the excitation wavelength at 320 nm with emission ranging from 350–550 nm. The tumor tissues are characterized by a decrease in the emission of collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) compared to the control tissues. Further, the optical oxidation–reduction (redox) ratio is a measure of cellular metabolism and can be determined by the relative change in the fluorescence emission intensity of NADH and FAD. The optical redox ratio will provide sufficient information on metabolic changes associated with tumor transformation. The results revealed that a significant decrease in the optical redox ratio is observed in DMBA-induced tumor tissues, which indicates increased metabolic activity compared to the control tissues. Moreover, an oral administration of SIL and its nanoparticulates restored the status of endogenous fluorophore emission and led to a higher redox ratio in the buccal mucosa of DMBA-painted animals. On a comparative basis, treatment with nanoparticulate silibinin was found to be more effective than free silibinin for reducing the formation of squamous cell carcinoma and improving the status of endogenous fluorophore emission to a near normal range in DMBA-induced hamster buccal pouch carcinogenesis. Furthermore, the diagnostic algorithms based on principal component analysis followed by linear discriminant analysis (PC–LDA) achieved an overall diagnostic sensitivity of 96.46% and specificity of 93.64% for separating the control from the experimental groups. The results of this study further suggest that the fluorescence spectroscopic technique in conjunction with PC–LDA has a potential for rapid and sensitive detection of specific metabolic alteration and changes in the endogenous fluorophores in response to anti-cancer drug treatments.