Saravanan Dharmaraj

The application of pattern recognition techniques in metabolite fingerprinting of six different Phyllanthus spp.

FTIR spectroscopy was used together with multivariate analysis to distinguish six different species of Phyllanthus. Among these species P. niruri, P. debilis and P. urinaria are morphologically similar whereas P. acidus, P. emblica and P. myr- tifolius are different. The FTIR spectrometer was used to obtain the mid-infrared spectra of the dried powdered leaves in the region of 400-4000 cm −1 . The region of 400-2000 cm −1 was analyzed with four different pattern recognition methods. Ini- tially, principal component analysis (PCA) was used to reduce the spectra to six principal components and these variables were used for linear discriminant analysis (LDA). The second technique used LDA on most discriminating wavenumber variables as searched by genetic algorithm using canonical variate approach for either 30 or 60 generations. SIMCA, which consisted of constructing an enclosure for each species using separate principal component models, was the third technique. Finally, multi-layer neural network with batch mode of backpropagation learning was used to classify the samples. The best results were obtained with GA of 60 gens. When LDA was run with the six wavenumbers chosen (1151, 1578, 1134, 609, 876 and 1227), 100% of the calibration spectra and 96.3% of the validation spectra were correctly assigned.

Chemometrics of differentially expressed proteins from colorectal cancer patients

AIM To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues for differentiating cancer and normal tissues. METHODS A Proteomic approach was used to identify the differentially expressed proteins between CRC and normal tissues. The proteins were extracted using Tris buffer and thiourea lysis buffer (TLB) for extraction of aqueous soluble and membrane-associated proteins, respectively. Chemometrics, namely principal component analysis (PCA) and linear discriminant analysis (LDA), were used to assess the usefulness of these proteins for identifying the cancerous state of tissues. RESULTS Differentially expressed proteins identified were 37 aqueous soluble proteins in Tris extracts and 24 membrane-associated proteins in TLB extracts. Based on the protein spots intensity on 2D-gel images, PCA by applying an eigenvalue > 1 was successfully used to reduce the number of principal components (PCs) into 12 and seven PCs for Tris and TLB extracts, respectively, and subsequently six PCs, respectively from both the extracts were used for LDA. The LDA classification for Tris extract showed 82.7% of original samples were correctly classified, whereas 82.7% were correctly classified for the cross-validated samples. The LDA for TLB extract showed that 78.8% of original samples and 71.2% of the cross-validated samples were correctly classified. CONCLUSION The classification of CRC tissues by PCA and LDA provided a promising distinction between normal and cancer types. These methods can possibly be used for identification of potential biomarkers among the differentially expressed proteins identified.

Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis.

Candida albicans has become resistant to the commercially available, toxic, and expensive anti-Candida agents that are on the market. These factors force the search for new antifungal agents from natural resources. Cassia spectabilis had been traditionally employed by healers for many generations. The possible mechanisms of the C. spectabilis leaf extract were determined by potassium leakage study and the effect of the extract on the constituents of the cell wall and enzymes as well as the morphological changes on C. albicans cells were studied along with cytotoxicity assays. The cytotoxicity result indicated that the extract is nontoxic as was clearly substantiated by a half maximal inhibitory concentration (IC50) value of 59.10 μg/mL. The treated cells (C. spectabilis extract) demonstrated potassium leakage of 1039 parts per million (ppm) compared to Amphotericin B (AmpB)-treated cells with a released potassium value of 1115 ppm. The effects of the extract on the cell wall proteins illustrated that there were three major types of variations in the expression of treated cell wall proteins: the presence of new proteins, the absence of proteins, and the amount of expressed protein. The activities of two enzymes, α-glucosidase and proteinase, were determined to be significantly high, thereby not fully coinciding with the properties of the antifungal reaction triggered by C. spectabilis. The morphology of C. albicans cells treated with the C. spectabilis extract showed that the cells had abnormalities and were damaged or detached within the microcolonies. Our study verifies C. spectabilis leaf extract as an effective anti-C. albicans agent.

Conventional and Non-conventional Approach towards the Extraction of Bioorganic Phase

Natural products such as natural food are the richest bio-resource of bioorganic compounds for modern medicines, nutraceuticals, food supplements and pharmaceutical applications. The research and application on natural food started with the extraction techniques that play an important role to the extraction quantity (Yield), quality (extracted phytochemicals) and also to the subsequent analyses accomplished to evaluate the biological and chemicals activities. Various types of technologies with different principles of extraction of bioorganic compounds are available today. Based on the literature the conventional extraction methods show better recoveries of bioorganic substances of natural food. Also, conventional extraction methods facilitate the extraction of high concentration of bioorganic substances with the safe solvents system such as pure ethanol. Moreover, conventional extraction methods is still widely used due to its simplicity. However, the conventional extraction methods is not always suitable for industrial uses due to long extraction time and large consumption of harmful solvents systems such as methanol. Therefore, modern non-conventional extraction methods could be an alternative extraction method. Hence, in spite of good results achieved with the conventional extraction methods, modern non-conventional extraction methods was established to search for a faster and better extraction method consuming less solvent, especially those that are unattractive in food industry. This chapter is intended to provide insights on conventional and non-conventional extraction methods with their advantages and disadvantage or limitation.

Hepcidin TH1-5 Induces Apoptosis and Activate Caspase-9 in MCF-7 Cells.

Breast cancer is the most commonly diagnosed and leading cause of cancer deaths among women globally. In continuation of our investigation into the cytotoxicity of the antimicrobial peptide, hepcidin TH1-5, on human breast adenocarcinoma cell line (MCF-7), we further affirm the apoptosis induction effect of the cysteine-rich antimicrobial peptide in the present study. Annexin V-fluorescein isothiocyanate and propidium iodide (annexin V-FITC/PI) apoptosis assay was carried out after treatment of the cells. In the determination of caspase activity and pathway of apoptosis, luminescence assay was also performed where caspase-3/7, caspase-8 and caspase-9 were evaluated at time 12, 24 and 48 hours. Results of annexin V-FITC/PI staining showed that 44.33%, 34.33%, 9.67% of the cell were in the early apoptosis, late apoptosis and necrotic stages respectively after 72 hours of treatment. Based on the data from the luminescence test, hepcidin TH1-5 activates caspases-3/7 and -9 which suggests that the apoptosis induced may be due to the peptide treatment. Hepcidin TH1-5 may have induced apoptosis in MCF-7 cells via the activation of caspase-9 of the intrinsic pathway. These results support our previous findings of the cytotoxicity of hepcidin TH1-5 and indicated that the peptide may be a potential agent for breast cancer therapy.

In vitro α-amylase and α-glucosidase inhibition and increased glucose uptake of Morinda citrifolia fruit and scopoletin

Diabetes mellitus is a metabolic disorder and management of blood glucose level is an important strategy in the control of the disease and complications associated with it. Therefore, components that cause uptake of glucose from the bloodstream and inhibitors of carbohydrate hydrolyzing enzymes can be useful in treatment of diabetes and medicinal plants are often used to achieve this aim. Morinda citrifolia fruit (MCF) is used in various countries for treatment of diabetes and the purpose of this study was to investigate the effect of MCF extract and its biomarker scopoletin on glucose uptake in HepG2 cells as well as its inhibitory effect on α-amylase and α-glucosidase. The safe doses for MCF extract and scopoletin were at 1 mg/ml and 0.2 μM, respectively as assessed by MTT assays and these were used for the assays. The extract had glucose uptake of 59.5% which was comparable to the standard metformin whereas the value for scopoletin was 30.6%. The extract had mild inhibitory activity on α-amylase and α-glucosidase with percentage of inhibition at 43.5% and 57%. The biomarker scopoletin showed lower activities at 23.9% and 35.7% for α-amylase and α-glucosidase respectively. Hence, these three activities may possibly be the mechanisms for MCF to exert its antidiabetic activity.