Molecular mechanism of action of antioxidant biomolecules from higher plants

Jyothi M, Veigas (2009) Molecular mechanism of action of antioxidant biomolecules from higher plants. PhD thesis, University of Mysore.

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Abstract

Oxidative stress is characterized by an altered antioxidant defense and is associated with human pathologies. High intake of fruits and vegetables has reduced risk of degenerative diseases due to the presence of antioxidant phytochemicals that react with reactive species which otherwise might lead to damage in the body. The present study investigated the potentials of anthocyanins and carotenoids derived from Syzygium cumini fruits and Delonix regia flowers respectively, as antioxidants, evaluated through chemical methods and animal cell models by monitoring antioxidant enzymes and the respective mRNAs. The fruits of S. cumini contain anthocyanins (230mg/100g DW) which are identified to be glucoglucosides of malvidin, petunidin and delphinidin. The anthocyanin extract of S. cumini (SCA) shows strong antioxidant activities in vitro at very low concentrations up to 5ppm. The flowers of D.regia are a unique combination of hydrophilic (anthocyanins) and lipophilic (carotenoids) compounds. Fresh petals of D. regia contain 825mg and the oven dried petals contained 580mg/100g DW of total anthocyanins and 660mg/kg DW of β-carotene. The major carotene in the carotenoid fraction (CF) was β-carotene (50% of total) whereas the xanthophyll fraction (XF) was identified to have lutein, zeaxanthin, β- cryptoxanthin and astaxanthin. The carotenoids and anthocyanins from the two sources showed varying degrees of antioxidant activities depending on the assay system used. The purified fraction of S. cumini contains solely mixture of anthocyanins and gives intense hue in solutions of low pH. It is highly colored at pH 2.0 with the color intensity reducing with an increase in pH, which however is stable up to pH 5.0 with no further color loss during the 45 days of study. The incorporation of the pigment in the pharmaceutical syrup gives it a pinkish hue which remains so with a minimal loss at the end of 8 weeks suggesting its potential to be a stable natural color apart from its antioxidant activity. The antioxidant activity of the extracts of S. cumini and D. regia were studied using a biological model such as the isolated rat hepatocytes to further establish their usefulness as effective oxidative stress neutralizers. Epigallocatechin gallate (EGCG) and quercetin were used as standard antioxidants for comparison. Independent exposure of isolated rat hepatocytes to carbon tetrachloride (CCl4) and tert-butyl hydroperoxide (TBH) brought about significant cellular injury marked by cell death, increased lipid peroxidation and decreased glutathione (GSH) content. Cells show differential response to oxidative stress. While antioxidant enzyme activities were radically reduced by CCl4 it was increased in 6 TBH-exposed cells with a consequent increase in mRNA expression. In CCl4 model, SCA acts chiefly via the glutathione redox system by elevating the cellular glutathione and activity of glutathione peroxidation by 2-fold while having no significant effect on catalase and superoxide dismutase. In TBH model SCA reversed the TBH-induced increase in enzyme activities and their mRNA expression with little effect on glutathione peroxidase activity at higher concentrations. Carotenoids of D. regia were protective at low concentrations of up to 100ppm beyond which they had no significant effect. The CCl4-induced reduction in superoxide dismutase and catalase activities were further reduced by CF while XF had no effect. EGCG and CF further reduced the CCl4-induced reduction in CAT and SOD activity while effect of XF on these two enzymes is not significant. The carotenoids extracts and EGCG confer protection mainly by increasing cellular GSH content and reducing lipid peroxidation. Carotenoid fractions, EGCG and quercetin increased TBH-induced increase in enzyme activity but reduced their mRNA expression suggesting a post transcriptional regulation of the enzymes. Lipid peroxidation was significantly abrogated by carotenoids fractions as well as EGCG and quercetin. These observations indicate that while SCA has a direct effect on AOEs, carotenoids and standards appear to act via mechanisms independent of these enzymes. The antioxidant activity of the pigment extracts were further elucidated using a continuously growing stable cell line, Hep3B. In addition to the carotenoids extracts, anthocyanin rich fraction of D. regia (DRA) was also assessed in this study. TBH caused a significant increase in superoxide dismutase activity while reducing catalse and glutathione peroxidase activity in Hep3B cells and caused cytotoxicity via apoptosis associated with reduced Bcl-2/Bax ratio and increased caspase-3 activation. All the extracts and standards protected from TBH-induced cell death, the order of protection being SCA=DRA>EGCG>quercetin>XF>CF as measured by MTT assay and LDH leakage. The extracts alleviated the TBH-induced reduction in GSH content and increase in lipid peroxidation. In vitro DNA damage was prevented by the anthocyanin extracts as well as EGCG and quercetin while no significant protection was offered by carotenoids extracts, which may be attributed to their low solubility in aqueous medium. The anthocyanin extract of S. cumini significantly reversed the stress induced alterations in antioxidant enzyme chiefly via modulation of the transcription of the same. Among the two carotenoids fractions of D. regia, XF was more effective compared to the CF in preventing the cell death in Hep3B cells exposed to TBH. The anthocyanin extract also brought about significant protection in terms of cell viability, lipid peroxidation and GSH 7 content. The carotenoids extracts and the standards (EGCG) reversed the TBH-induced increase in antioxidant enzyme activity and their expression. The activity of GPx appears to be post transcriptionally regulated in Hep3B cells pretreated with quercetin, EGGC (10μM) and SCA since an increase in activity was not associated with an increased expression of its mRNA. A transcriptional regulation of CAT and SOD was obvious in cells pretreated with extracts as well as standards except DRA (100ppm) and quercetin (10μM). Since SCA was found to act via modulation of AOEs, its effect on the stability of transcribed mRNA of these enzymes was studied by inhibiting the transcription followed by measuring the steady state mRNA level in a time dependent manner. TBH caused a radical reduction in steady state mRNA level of CAT and GPx. GPx was most susceptible to degradation by TBH and was almost completely degraded one hour after the addition of the transcription inhibitor, actinomycin D. SCA provided protection to CAT mRNA by almost completely inhibiting the action of TBH on the same. Pretreatment of SCA delayed the decay of GPx mRNA by about 3 h, as compared to 1 h in TBH treated cells. The results of the present study suggest that SCA provides protection to AOEs by increasing their stability and hence making them available for translation. In addition to being cytoprotective under oxidative stress conditions, the carotenoids and anthocyanins were also inhibitory to cell growth on long term exposure indicating antiproliferative or growth inhibitory activity in carcinoma cells. Antiproliferative activity appeared to be mediated via induction of apoptosis as evidenced by microscopic examination using propidium iodide stain. The overall results suggest that the extracts of S. cumini and D. regia offer protection to cells by improving cellular defense systems such as the antioxidant enzymes either directly or by enhancing their turn over by extending their mRNA half life; and also by directly quenching free radicals and probably reversing the damage by prooxidants and other toxins through regulation of transcription factors and signaling cascades involved in cell survival.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Anthocyanins; Antioxidant enzymes; Biomolecules; Oxidative stress; S. cumin
Subjects:	500 Natural Sciences and Mathematics > 10 Plants 600 Technology > 08 Food technology > 32 Antioxidants
Divisions:	Plant Cell Biotechnology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	06 Apr 2010 03:59
Last Modified:	14 Dec 2016 10:18
URI:	http://ir.cftri.res.in/id/eprint/9407

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