Molecular mechanism of action of antioxidant biomolecules from higher plants

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
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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
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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.