STUDIES ON ELLAGIC ACID AND 4-HYDROXYISOPHTHALIC ACID ISOLATED FROM SWALLOW ROOT (DECALEPIS HAMILTONII)

Objective: The aim of the study was to elucidate the free radical scavenging activity of purified major phenolic acids from swallow root (Decalepis hamiltonii) extract against 2, 2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced oxidation in rat erythrocyte membrane and tissues.Methods: Major phenolic acids, ellagic acid (EA) and 4-hydroxyisophthalic acid (4-HIA) were isolated from aqueous root extract of Decalepis hamiltonii by column chromatography and identified using HPLC, LC-MS, NMR and FT-IR. Free radical scavenging activities of the isolated compounds were estimated. Marker of lipid peroxidation was determined in erythrocyte membrane, liver and brain.Results: The EA showed higher 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging activities, while 4-HIA exhibited potent hydroxyl radical scavenging activity when compared to standard antioxidants. Malondialdehyde (MDA) levels were increased with AAPH treatment in erythrocytes, liver and brain tissues and the treatment with phenolic acids inhibited the formation of MDA. 4-HIA was found to be more profound in attenuating the lipid peroxidation which may be attributed to its high hydroxyl radical scavenging activity.Conclusions: Our study demonstrated the antioxidant properties of the major phenolic acids isolated from Decalepis hamiltonii and further suggests the therapeutic potential of these compounds in treating the oxidative stress disorders.Keywords: AAPH, Brain, Decalepis hamiltonii, Erythrocytes, Liver, MD

Optimization of LC​/MS (APCI)​+ Methods for the Determination of Possible Lutein Oxidation Products in Plasma and Tissues of Adult Rats

In spite of lutein and its isomer zeaxanthin being richly available in natural sources, the role of these components on redn. of age-​related macular degeneration, cancer, and cardiovascular disorders suggested that an update of the anal. procedure is required to det. the oxidative products and to understand their nutritional significance. In the present study, we have standardized and developed an improved method to obtain characteristic ions of lutein, zeaxanthin, and its major oxidative products in vivo (rats) using LC-​MS (APCI)​+. In addn., lutein and zeaxanthin isomer were sepd. on a C30 column with shorter run time with high resoln. and calibrated on the basis of picomolar concn. on HPLC (DAD)​, with the lower detection limit of 0.125 for lutein and 0.128 pmol for zeaxanthin. Characteristic mass spectral ion for lutein is m​/z 568.7 [M]​+ and 551.5 [M + H-​H2O]​+ and for zeaxanthin isomer is m​/z 568.8 [M]​+, 569.8 [M + H]​+. Further, optimized conditions produced structurally characteristic fragmented ions under standardized MS (APCI)​+ conditions. Total ionic chromatogram together with fine UV-​Visible and mass spectra were used to differentiate lutein isomers and its oxidative products, such as 523 [M+ + H+-​3CH3]​, 479 [M+ + H+-​6CH3]​, 551 [M+ + H+-​H2O]​, 276.43 [M+-​C22H19O]​, di-​epoxides and 3'-​oxolutein. The APCI mass spectral characteristics of major oxidative products of lutein in adult rat tissues are reported here for the first time, to our knowledge. These findings could provide new insights into lutein bioavailability and bioconversions with respect to health benefits

Biofunctionality of Carotenoid Metabolites: An Insight into Qualitative and Quantitative Analysis

Epidemiological and clinical studies have shown that dietary intake of carotenoid-rich fruits and vegetables is positively correlated with reduction in age-related eye diseases, atherosclerosis, certain cancers and chronic diseases. Carotenoids consist of unique chemical characteristics and are highly vulnerable to structural modifications, leading to the formation of various derivatives under physiological conditions. The identification of these molecules is necessary before addressing their biological functions. Carotenoid metabolomics is believed to be highly complex to fingerprint due to instability and interference with complex biological matrices. Noteworthy, progress has been made in understanding carotenoid metabolism or its biotransformation in biological samples. In this regard, the chapter highlights the concept of metabolomics and their related bio-analytical techniques pertaining to the detection of carotenoids and their derived products to elucidate their bio-transformation on targeted biological functions. Further, this chapter highlights the various hyphenated analytical tools and their optimization

Enhanced cytotoxic and apoptosis inducing activity of lycopene oxidation products in different cancer cell lines

Currently, upon understanding the metabolomics of carotenoids, it is important to address the key role of carotenoid derived products. In this regard, aim of the study was to elucidate and explore the role of lycopene (LYC) oxidative products generated through autoxidation (AOL) or chemical (KMnO4) oxidation (COL) against proliferation of selected cancer cells. Preliminary, we investigated the effect of LYC on cell viability of various cancer cell lines (PC-3, MCF-7, A431, HepG2, HeLa and A549). Based on the results of LYC treatment on cell cytotoxicity levels, MCF-7, PC-3 and HeLa cell lines were further tested with AOL and COL products. The decreased cell viability with depleted GSH and increased MDA levels were observed when treated with COL products than control, LYC and AOL. In addition, COL products increased ROS levels and percent apoptosis. The typical morphological changes and nuclear condensations showed that COL products have anti-proliferation and apoptosis inducing activity. Based on results, we hypothesized that ROS generation by LYC oxidation products may be one of intermediate step involved in apoptosis. The redox status and therapeutic approach of COL products in modulating ROS and induction of apoptosis in cancer cells were reported for the first time, to our knowledge. To conclude, COL products involves in cancer growth inhibition efficiently than intact LYC and AOL. Hence, there is a great potential for synthesizing or producing such carotenoid oxidation products to augment cancer complication

Distinct begomoviruses closely related to cassava mosaic viruses cause Indian Jatropha mosaic disease

A begomovirus was recently shown to be causing Jatropha mosaic disease (JMD) on Jatropha for the first time in India. A typical begomovirus-like symptoms characterized by chlorotic specks on leaves, curling and malformation of leaves, severe reduction in leaf size, partial or complete sterility were seen on infected plants. In Karnataka state, South India, JMD caused significant yield losses by affecting the growth of the infected plant and by disease incidences of up to 47%. The putative Jatropha mosaic India virus (JMIV) was successfully transmitted through grafting, the dodder Cuscuta subinclusa and the whitefly, Bemisia tabaci. The JMIV was detected in infected plants and individual B. tabaci by polymerase chain reaction tests using two sets of begomovirus-specific degenerate primers. The core coat protein (CP) sequences of ~575 bases were obtained from two isolates collected at Bangalore and Dharwad, South India. Phylogenetic analysis of the core CP sequences with those of selected begomoviruses grouped JMIV in a separate cluster close to Indian cassava mosaic virus and Sri Lankan cassava mosaic virus and shared highest nucleotide identities (90-95%) with them. The two JMIV isolates were 94% similar to each other. The begomoviruses causing JMD in the Americas grouped separately from JMIV and shared only 72.8-75.2% core CP nucleotide identities thus they are distinct. These results further confirm that JMD in India was caused by begomoviruses and they were most closely related to cassava mosaic viruses from the Indian sub-continent

Low-dose doxorubicin with carotenoids selectively alters redox status and upregulates oxidative stress-mediated apoptosis in breast cancer cells

The combination of carotenoids and doxorubicin (DOX) selectively alters oxidative stress-mediated apoptosis in breast cancer cells. Primarily, cytotoxic efficiency of carotenoids (β-carotene, BC; lutein, LUT; astaxanthin, AST; or fucoxanthin, FUCO) either with or without a minimal cytotoxic dose of DOX was evaluated in MCF-7 (0.12 μM) and MDA-MB-231 cells (0.28 μM). The higher cell growth inhibition of BC and/or LUT with DOX was selected for testing in further cell-based assays. Low-dose DOX significantly enhanced cytotoxicity in carotenoid (1 μM) or carotenoid (20 μM) treatment alone. Depleted glutathione, increased lipid peroxides and increased ROS levels in cells confirmed the cytotoxic effect. Furthermore, mitochondrial dysfunction, cell growth arrest at G0/G1 phase and caspase cascades as well as up- and down-regulated expression levels of related proteins (p21, p27, Bax, p53, Bcl-2, and cyclin D1) revealed the synergistic effect of carotenoid and DOX treatment on ROS-mediated apoptosis. These observations demonstrated increased apoptosis in BC + DOX/LUT + DOX-treated cells due to the pronounced pro-oxidant action. Interestingly, normal breast epithelial cells (MCF 10A) exposed to similar treatments resulted in non-significant cytotoxicity. These newly observed mechanistic differences of anticancer drugs on the mitigation of toxicity with carotenoids may provide insight into the targeting of cancer therapy

Development of silverleaf assay, protein and nucleic acid-based diagnostic techniques for the quick and reliable detection and monitoring of biotype B of the whitefly, Bemisia tabaci (Gennadius)

The aim of this study was to develop and optimize silverleaf bioassay, esterase analysis and PCR-based techniques to distinguish quickly and reliably biotype B of the whitefly, Bemisia tabaci (Gennadius), from Indian indigenous biotypes. Zucchini and squash readily develop silverleaf symptoms upon feeding by the B biotype, but they are not readily available in Indian markets. A local pumpkin variety ‘Big’ was, therefore, used in silverleaf assay, which developed symptoms similar to those on zucchini and squash and can be used reliably to detect B biotype. Analysis of non-specific esterases of B and the indigenous biotypes indicated both quantitative and qualitative differences in esterase patterns. Two high molecular weight bands were unique to B biotype and they occurred in abundance. These esterases were used to develop quick and field-based novel detection methods for differentiating B from the indigenous biotypes. Development of these simple and cost-effective protocols has wider application as they can be potentially used to identify other agricultural pests. Mitochondrial cytochrome oxidase I gene sequences and randomly amplified polymorphic DNA (RAPD) polymorphisms, generated using the primer OpB11, were also found useful for detecting B. tabaci biotypes. A B biotype-specific RAPD band of 800 bp was sequenced, which was used to a develop sequence characterized amplified region (SCAR) marker. The SCAR marker involved the development of B biotype-specific primers that amplified 550 bp PCR products only from B biotype genomic DNA. Silverleaf assay, esterases, RAPDs or a SCAR marker were used in combination to analyse whitefly samples collected from selected locations in India, and it was found that any of these techniques can be used singly or in combination to detect B biotype reliably. The B biotype was found in southern parts of India but not in the north in 2004–06

Fractionation and Characterization of Lycopene-Oxidation Products by LC-MS/MS (ESI)(+): Elucidation of the Chemopreventative Potency of Oxidized Lycopene in Breast-Cancer Cell Lines

Lycopene (LYC) has been correlated with the reduction of certain cancers and chronic diseases. However, the existence and biofunctionality of degraded, oxidized, and biotransformed LYC products in vivo have not been revealed. Therefore, this study aimed to screen and elucidate the potential bioactive lycopene-derived products in breast-cancer and noncancerous cells. LYC-oxidation or -cleavage products were generated using KMnO4. These oxidation products were separated as fractions I-III by silica column chromatography using gradient solvent systems. Further, LC-MS/MS (ESI)(+) was used to elucidate their possible fragmentation patterns and structures. Fraction II showed higher cytotoxicity (IC50 value of 64.5 mu M), cellular uptake, and apoptosis-inducing activity in MCF-7 cells. This fraction consists of major peak m/z 323, identified as apo-8,6'-carotendial. The cytotoxicity-inducing activity may be due to partial ROS generation with mitochondrial dysfunction. Further, the role of apo-8,6'-carotendial in the induction of apoptosis is demonstrated for the first time. These results illustrated that LYC-oxidation derivatives or metabolites are involved in growth inhibition of cancer cells. Exploration of specific oxidized-carotenoid products will give further insight into the field of nutritional biochemistry