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Bionano Frontier, Vol.

7 Special Issue -12, January - 2014, 25-30

ROLE OF NATURAL COMPOUNDS IN THE PREVENTION OF DNA AND PROTEINS DAMAGE BY


GLYCATION
AHMAD ALI, RAJEEV SHARMA, SUBRAMANIAN SIVAKAMI
University Department of Life Sciences, University of Mumbai, Vidyanagari,
Santacruz (E), Mumbai
Email: - ahmadali95@gmail.com
ABSTRACT : Glycation is a non-enzymatic process which involves an interaction between the carbonyl groups of reducing
sugars and amino groups of proteins, lipids, nucleic acids resulting in the formation of Amadori products (early glycation
products). These products then rearrange themselves and get converted to more stable and irreversible advanced end glycation
products (AGEs). Glycation products have been implicated in various diseases like cataract, diabetes mellitus, Alzheimers etc. In
the present workthe effect of glycation on DNA and proteins was studied. When plasmid DNA (pBR 322) was incubated with
lysine andmethylglyoxal in the presence of metal ion a conformational change was observed on the agarose gel electrophoresis
which indicated the damage to DNA by glycation. Different sugars were also incubated with plasmid DNA to check their
comparative deleterious effects. In the presence of Lysine and metal ion, pentose sugar and sugar phosphates caused maximum
damage to DNA as compared to other sugars. Some natural antioxidants like vitamin B6 derivatives,curcumin and
jasadbhasmawere used to check their effect and mechanism in reversing the glycation induced damage to DNA. SDS-PAGE was
performed to analyze the glycation-induced damage to proteins and it was found that methyl glyoxal caused more damage than
glucose. Curcumin and jasadbhasma prevented the damage to protein in the presence of methyl gloxal. It can be concluded from the
results obtained in this study that the mechanism of damage due to glycation is different for different biomolecules. Free radicals
were found to play important role in damaging DNA structure. On the hand aggregation and cross-linking by AGEs were found to
be the major cause of structural alteration of proteins.
Keywords :AGE,Antioxidants, Dicarbonyls, DNAdamage, Glycation, Protein aggregation
Amadori products and AGES and this includes superoxide
anions (O.2-), hydrogen peroxide (H2O2), alkoxyl (RO.),
.
.
peroxyl (ROO ), hydroxyl radicals ( OH), and hypochlorous
acid (HOCl).
Dicarbonyls are formed during various metabolic
activities and react with proteins faster than sugars and lead to
formation of AGEs. Glyoxal, methylglyoxal (MG), 3deoxyglucosone (DG), and malondialdehyde are few such
dicarbonyl compounds which have been characterized well
and linked in the pathophysiology of several diseases. Various
strategies have been developed to control or delay the damage
due to the accumulation of AGEs. One of them is based on the
use of inhibitors of AGE formation. There are several drugs
which are available commercially: aminoguanidine, Dpenicillamine, D-lysine, diclofenac, vitamins and there
analogues, desferoxamine and diaminophenazine.
Aminoguanidine, a dicarbonyl scavenger, is a powerful
inhibitor of AGE formation (Jakus, 2000). Some natural
compounds such as curcumin and resveratrol also inhibit the
AGE formation. Vitamins are carbonyl trapping AGE
inhibitors that inhibit the post-Amadori stage in the glycation
cascade. All these inhibitors may be either dicarbonyl
scavengers, metal ion chelators or radical scavengers.
DNA damage has been shown to be oxygen
dependent and mediate through ROS and RNS reactive
nitrogen species). Damage to DNA by ROS/ RNS appears to
occur naturally, in that low steady-state levels of base damage
products have been detected in nuclear DNA from human cells
and tissues (Halliwell and Dizdaroglu, 1992). Transition
metals, Fe+3 and Cu+2, enhanced the DNA damage by lysine
with MG and indicated the role of free radicals. Oxidative
DNA damage from ROS has been hypothesized to play a
critical role in several diverse biological processes including

INTRODUCTION:
Glycation is a spontaneous non-enzymatic amino-carbonyl
reaction between sugars and long-lived proteins, lipids and
nucleic acids and it is also one of the post translational
modification processes between the reducing sugars and free
amino groups of proteins (Monnier and Carami., 1981). In the
early stage of glycation the synthesis of intermediates lead to
the formation of Amadori compounds and in late stage
advanced glycation end products (AGEs) are irreversibly
formed as a result of a complex cascade of reactions. Among
the amino groups lysine is a preferred group for non-enzymatic
sugar attachment and subsequent formation of AGEs (Arai et
al., 1987).
The process of glycation occurs continuously in the
body even at normal glucose levels and the damage caused by
the Amadori and AGE products keep accumulating slowly
overtime. The deleterious effects of these products are more
significantly observed when the blood glucose level increases
above the normal level in conditions like hyperglycemia.
Glycation has been linked to various diseases such as diabetes,
cataract, Alzheimer's, dialysis related amyloidosis (DRA),
atherosclerosis, Parkinson's as well as physiological aging
(Thorpe and Bayens, 1996).
The complex process of glycation alters the biological
activities of proteins, their metabolic processes and inhibits the
specific functions of proteins through cross linking,
aggregation, precipitation and produce reactive oxygen species
(ROS) (Suji and Sivakami, 2004). Protein cross-linkage results
in protein aggregates, which then form intracellular proteaseresistant and ubiquitin-proteasome-resistant deposits and
inhibits the intracellular transport of materials. A large amount
of ROS is produced as a result of subsequent modifications of

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remove Schiff base and free sugar.

mutagenesis, aging and carcinogenesis (Ames, 1983).


In this study, DNA cleavage induced by the glycation
reaction of MG with lysine in the presence of Fe+3 was
investigated. This system was found to produce hydroxyl
radicals. The ability of vitamins and their corresponding
coenzymes such as pyridoxine, pyridoxal-5-phosphate, and
pyridoxamineto prevent oxidative damage to DNA was
evaluated. Some other antioxidants like sodium azide,
curcumin and bhasma were used and their preventive effect on
DNAdamage was analyzed.
Sugars or sugar metabolites glycate the proteins and
change the integrity of the proteins. In the present study,
glucose and MG, representative molecule of sugar and
dicarbonyl, were used to examine the structural alteration of
BSA. MG caused severe aggregation of BSA which was
prevented significantly by curcumin and bhasma and partially
by AG. All these results indicate that the DNA damage is
mediated by the free radicals formed as a result of
accumulation and modification of AGEs. On the other hand the
structural alteration to the proteins was due to the AGEs
themselves and adducts. The contribution of free radicals in the
damage to proteins was not very significant.

Measurement of Browning:Browning was measured at 420


nm by using Nano-Drop 2000.
Analysis of Glycated Proteins by SDS-PAGE: The Glycated
and dialyzed sample (15 l) was mixed with 2X SDS loading
dye (15 l) and boiled at 100 C for 5 minutes. 10 l
supernatant were loaded onto SDS-PAGE (10% resolving gel
and5% stacking gel) and electrophoresed at 20 mA constant
current. The gel was stained in Coomassie brilliant blue R-250
staining solution for 3 hour. Gel was destained with destaining
solution until excess stain was removed. Gel was observed
under white light.
RESULTS AND DISCUSSIONS :
Plasmid DNA Cleavage during Glycation of Lysine by
Methyl Glyoxal (MG)
The incubation of DNA with MG and lysine caused strand
breaks and resulted in decreased amount of form I and
concomitant increase in open circular form II (Fig. 1; Lane 6).
However there was no damage to DNA by lysine and MG
alone (Fig. 1; Lane 2 and 3). Presence of Fe3+in the system
enhanced DNA strand breakage (100% open circular) (Fig. 1;
Lane 7). Strand breakage was inhibited by Sodium azide(Fig.
1; Lane 8).On the other hand sodium azide had no effect on its
own (Fig. 1; Lane 5).

MATERIALS AND METHODS :


In vitro Glycation of DNA : 0.5 g of pBR 322 plasmid DNA
in 100mM potassium phosphate buffer at pH 7.4 was incubated
for 3h at 37 0 C with lysine (20 mM) and MG (20 mM) in the
3+
presence and absence of Fe (100 M), sugar/sugar
phosphates, vitamins and antioxidants. The reaction was
stopped byfreezing (-20 0 C) (Suji and Sivakami, 2007).

Effect of Incubation Time and Different Concentration of


SodiumAzide on Plasmid DNADamage
As can be seen from the Fig. 2 that the extent of DNA damage
increased with an increase in the duration of incubation in the
presence of Lys, MG and Fe3+ (Fig. 2; Lanes 3, 6, 9 and 12).
Sodium azide(250mM) protected the DNA from the damage
during initial period of incubations, i.e., 3, 6 and 12 hrs (Fig. 2;
Lane 4). However a higher concentration (500mM) of sodium
azide was required for more effective prevention of DNA
damage caused due to increased incubation time (Fig. 2; Lanes
5, 8, 11 and 14).

Analysis of Glycated DNA sample by Agarose Gel


Electrophoresis : 20l of samples were mixed with 4l gel
loading dye (6X) and loaded on to 0.8 % agarose gel (320mg of
agarose dissolved in 40 ml of 1X TAE buffer and 1l of
ethidiumbromide added). Agarose gel electrophoresis was
done at 50 -60 V until the dye band ran 2/3rdof gel length.
Subsequently the gel was visualized under UVtransilluminator
and bands analyzed with the help of control.

Effect of Different Sugars and Sugar Phosphates on


Plasmid DNADamage
The effect of different sugars/sugar phosphates in causing the
damage to DNA was analyzed by incubating the DNA with
various sugars/sugar phosphates (250 mM) in the presence of
lysine (20 mM) and FeCl3 (100M) for 24 hrs at 37C. Sugars
nicked the supercoiled form of plasmid DNA and converted it
to either linear or open circular. The sugars damaged the DNA
in the following order: Glucose < Glucose-6-phosphate <
Fructose-1, 6-bisphosphate < Ribose = Ribose-5-phosphate <
Fructose-6-phosphate (Fig. 3). Only one sugar and two sugar
phosphates, Ribose, ribose-5-phosphate and fructose-6phosphate, led to the formation of both linear and open circular
form of the DNA (Fig. 3; Lanes 6, 7 and 8). However the
damage of DNA by MG was much more extensive than sugars
and sugar phosphates (Fig. 3; Lane 3).

Measurement of Hydroxyl Radical: Measurement of


hydroxyl radicals was carried out by measuring thiobarbituric
acid reactive 2-deoxy-D-ribose oxidation products (Halliwell
and Gutteridge, 1981). Reaction mixtures (200 l) containing
MG and lysine in the presence and absence of antioxidants and
2-deoxy-D-ribose (100 mM) were incubated at 37 C for 3 hrs.
The degradation of 2-deoxy-D-ribose was then measured by
adding 300 l PBS, 200 l 2.8 % (w/v) TCA, followed by the
addition of 400 l 0.5 % (w/v) thiobarbituric acid and heating
at 100 C for 10 min. After cooling, the absorbance was
measured at 532 nm.
In Vitro Glycation of Protein: Bovine serum albumin (BSA)
was modified in vitro by glucose and MG and antioxidants
(curcumin, jasadbhasma and aminoguanidine) at 37 C. All the
incubations were carried out in 0.1 M phosphate buffer, pH 7.4
and contained 3 mM sodium azide to prevent bacterial
contamination. Aliquots were taken at various time points and
dialysed at 10 mM phosphate buffer (pH 7.4) for 24 hours to

Assessment of Vitamins in Preventing Plasmid DNA


Damage
Figure 4 shows that Pyridoxine (P) and Pyridoxal-5-phosphate

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the development ofdiabetic complications, atherosclerosis,


Alzheimer's disease and the normal agingprocess (Rahbaret
al., 2003). Age-related diseases exhibit increased levels of
glycationand its end products, further supporting the idea that
sugars and their metabolites may act as damaging molecules
especially when they accumulate in the cells and tissues.
Although the mechanism of this damage was first described
almost a century back byMaillard the exact mechanism and
events are yet to be figured out. Major mechanisms by which
AGEs cause damage to the biomolecules include aggregation,
precipitation orthrough ROS formation. In last several
decades some AGEs have been identified andtheir role has
been characterized. Based on these informations several
molecules havebeen designed to prevent the accumulation of
AGEs and related products. The role ofantioxidants especially
natural products in the preventions of damage to biomolecules
has also been reported in literature. However there are still
very few reports regarding theextent of damage to DNA
caused by different sugars and their phosphates and
thepotential of these molecules in the generation of free
radicals. Similarly the mechanism by which natural products
prevent the damage is also not very clear.
In the present study plasmid DNA and BSA,
representative molecules for DNAand proteins, have been
used to analyse the role of sugar and sugar metabolites in
causingstructural damage to these molecules.The plasmid
DNA, pBR 322, is a sensitive indicator of single strand breaks
arising due to damage by glycation and free radicals (Levi and
Werman, 2001). Themodel system of methylglyoxal (MG) and
lysine has been used to study the glycationbetween dicarbonyl
intermediates formed during glycation of MG and free amino
groups of proteins. The results presented in this work suggest
that the incubation of DNA withMG or lysine alone for 3 hrs
did not induce strand breakage. DNA strand breakage was
induced only by co-incubation of lysine and MG. These results
also suggest that thereaction of MG-mediated DNA breakage
may be caused by traces of transition metalswhich undergo
Fenton reaction and lead to the production of free radicals. The
damage of plasmid DNA increased with the duration of
treatment and in the presence of ferric ion (Fe3+). Similar
results have been reported by Ahmad et al. (2011). Trace metal
such as iron, which is present in biological systems may react
with H2O2 to produce hydroxyl radical and then induce DNA
strand breakage.
Radical trapping antioxidants have been known to
inhibit the glycation reaction(Khalifahet al., 1999). When
sodium azide, a hydroxyl radical scavenger, was added in the
reaction mixture it was found that the free radical could not
induce damage to DNAand a higher concentration (500 mM)
was required to prevent the damage due toincreased
incubation with Lys + MG + Fe3+. Therefore it can be
concluded that damageto DNA is mainly due to the generation
of free radicals during the late stage of glycationreactions.
There are several other reports which suggest this mechanism
of metal-induced free radical mediated damage to DNA
(Kang, 2003; Suji and Sivakami, 2007).
Free radical trapping antioxidants have been known
to inhibit the glycationreaction. This study revealed that there
was a strong correlation between inhibition ofproduction of
superoxide and hydroxyl radical and protection of DNA.
Sodium azide, ahydroxyl radical scavenger protected the DNA

(PLP) prevented DNA damage at 5mM concentration (Lane 3


and 5). At higher concentration P and PLP prevented the DNA
damage more effectively (Fig. 4; Lane 6 and 8). However
Pyridoxamine (PM) showed no effect in preventing damage
even at higher concentration (10 mM) (Fig. 4; Lane 4 and 7).
Effect of Preincubation and Postincubation of
Antioxidants on Plasmid DNADamage
The effect of various free radical scavengers was checked on
the DNA damage by adding these scavengers before (pre) and
after (post) incubating the DNAin the presence of Lys, MG and
Fe. As depicted in Fig. 5, NaN3 prevents the DNA damage both
under pre (Lane 7) and also at post (Lane 3) incubation along
with FeCl3. Whereas PLP protects the DNA significantly only
when it is incubated before the addition of FeCl3 (Fig. 5; Lane
10), but does not show any protective effect when added to the
damaged DNA (Fig. 5; Lane 6). Pyridoxine prevented the
DNA damage partially when added before the 2 hrs incubation
with Fe and showed no effect when added after 2 hrs
incubation with Fe (Fig. 5; lane 4 and 8). On the other hand PM
was not able to protect the DNA against the damaging agents in
both pre and post incubation conditions (Fig. 5; Lane 5 and 9).
Effect of Curcumin and Bhasma on Plasmid DNADamage
Curcumin and jasad bhasma prevented the DNA damage by
glycation (Fig. 6; Lanes 3 and 4).However jasadbhasma was
more effective in preventing the damage as compared to
curcumin (Fig. 6, Lane 4)
Effect of Vitamins on Hydroxyl Radical Production
Though all vitamins tested showed abilities to prevent
hydroxyl radical production to different extents, P and PLP
showed maximum abilities (Fig. 7). PM showed minimum
inhibitory activity on hydroxyl radical production even at
higher concentration (10 mM).
Effect of Curcumin and Bhasma on Hydroxyl Radical
Production
It can be seen from the Fig. 8 that jasadbhasma inhibited the
production of hydrogen peroxide (43%) by metal catalyzed
glycation system. On the contrary Curcumin caused enhanced
production of hydroxyl radical formation (almost 2 fold).
Glycation of BSAby Glucose and Methyl Glyoxal (MG)
When BSA was incubated with glucose/MG at 37C for 21
days a characteristic smearing was observed (Fig. 9; Lane 2
and 3). It can also be seen that both the carbonyls differed in the
aggregation of proteins: MG caused more extensive
aggregation.
Effect of Curcumin, Bhasma and Aminoguanidine on
Glycation of BSA
Figure 10 shows that MG glycated and precipitated the BSA
protein (Lane 2). Curcumin and bhasma inhibited the glycation
of protein and prevented the protein aggregation or
precipitation (Fig. 10; Lanes 3 and 4). AG shows partial
inhibitory effect on protein glycation (Fig. 10; Lane 3).
DISCUSSION:
The formation and accumulation of AGEs are major factors in

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from damage.
The conformational modification of plasmid DNA
due to in vitro incubation withseveral sugars or their sugar
phosphates was investigated. Ribose, ribose-5-phosphate
andfructose-6-phosphate damaged the DNA in the presence of
Fe3+ and formed the opencircular as well as linearized the
supercoiled DNA. The extent of damage was differentfor
different sugars and sugar phosphates. It could be due to the
differences in thereactivities of sugars and their phosphates
and also their potential in generating thesuperoxide radicals.
The results showed that the sugar phosphates are highly
reactive and more damaging than sugars. Sugar metabolite
such as MG damaged the DNA faster than sugars. This is
probably due to MG consists two carbonyl groups which
makes it highlyreactive and more damaging than sugars
(Thornalleyet al., 1999).
PLP and P were able to protect DNA damage while
PM lacked DNA protectionability. Those vitamins having high
hydrogen donating ability, show effective antioxidantnature
(Suji and Sivakami, 2007). The weak hydrogen donating
ability of PM makes it amarginally effective antioxidant. The
electron donating para substituents have a stronginfluence on
the radical trapping ability of PM, as they lower the phenolic
O-H bondingdissociation enthalpies (Delange and Glazer,
1989). PLP showed higher hydrogenperoxide radical trapping
capacity. These results indicate that antioxidant property
ofvitamins is probably playing important role in preventing the
damage to DNA.
The effect of various antioxidants was checked on the
DNA damage by addingthese scavengers before (pre) and after
(post) incubating the DNA in the presence of Lys, MG and Fe.
From this study it was seen that NaN3 prevented the DNA
damage bothunder pre and also at post incubation along with
FeCl3. Whereas PLP protected the DNAdamage significantly
only when it was incubated before the addition of FeCl3but it
didnot show any protective effect when added to the damaged
DNA. Pyridoxine prevented the DNA damage partially when
added before the 2 hour incubation with FeCl3and showed no
effect when added after 2 hour incubation with FeCl3. On the
other hand PMwas not able to protect the DNA against the
damaging agents in both pre and postincubation conditions.
Curcumin (Sajithlalet al., 1998) and bhasma
(Pattanaiket al., 2000) have beenknown for their antioxidant
properties and as potent free radical scavengers. In this study,
curcumin and jasadbhasma prevented the DNA damage from
free radicals. Bhasmainhibits DNA damage more effectively
than curcumin. Jasadbhasma inhibited the production of
hydrogen peroxide (43%) by metalcatalyzedglycation system.
On the contrary Curcumin caused enhanced the
hydroxylradical formation (almost 2 fold).
The results presented in this study shows that glucose
and MG can glycate BSAdue to formation of AGEs. Among
these the MG is more reactive in generating browningand in
damaging of BSA than glucose. Curcumin and jasadbhasma
show antiglycatingproperty and prevented the aggregation or
precipitation of the protein.

Figure 1: pBR 322 DNA (0.5 g) was incubated with lysine


(20 mM), MG (20 mM), FeCl3 (100 M) and sodium azide
(250 mM) individually and in combinations (mentioned in
lane description) at 37 C for 3 hrs.

Figure 2: pBR 322 DNA (0.5 g) was incubated with lysine


(20 mM), MG (20 mM), FeCl3 (100 M) with or without
sodium azide (250 mM and 500 mM) at 37 C for various
duration.

Figure 3: pBR 322 DNA (0.5 g) was incubated with lysine


(20 mM), MG (20 mM), FeCl3 (100 M), various sugars and
sugar phosphates (250 mM) at 37 C for 24 hrs.

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Fig. 7: Effect of Vitamins on Hydroxyl Radical Production


Figure 7: 2-deoxy-D-ribose (100 mM) incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M) and different concentrations of
vitamins (5 mM and 10 mM) at 37 C for 3 hrs. The degradation of 2deoxy-D-ribose was then measured by adding 300 l PBS, 200 l 2.8
% (w/v) TCA, followed by the addition of 400 l 0.5 % (w/v) TBA
and heating at 100 C for 10 minute. After cooling, the absorbance
was measured at 532 nm.

Figure 4: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M) and different
concentrations of various vitamins (5 mM and 10 mM) at 37 C
for 3 hours.
1

10

Fig. 8: Effect of Curcumin and Bhasma on Hydroxyl


radical production

Figure 5: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M) (Lanes 2 to 6) at 37 C for
2 hours each, then added antioxidants and further incubated at
37 C for 3 hrs. In lanes 7 to 10 first pBR 322 DNA (0.5 g) was
incubated with lysine (20 mM), MG (20 mM), antioxidants at
37 C for 2 hours, then added FeCl3 and further incubated at 37
C for 3 hrs.

Figure 8: 2-deoxy-D-ribose (100 mM) incubated with lysine (20


mM), MG (20 mM), FeCl3 (100 M), curcumin (200 g) and bhasma
(200 g) at 37 C for 3 hrs. The degradation of 2-deoxy-D-ribose was
then measured by adding 300 l PBS, 200 l 2.8 % (w/v) TCA,
followed by the addition of 400 l 0.5 % (w/v) TBA and heating at
100 C for 10 minute. After cooling, the absorbance was measured at
532 nm.

Figure 6: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M), curcumin (200 g) and
bhasma (200 g) at 37 C for 3 hours.

Figure 9: BSA(10 mg/ml)was incubated with glucose (90 mg/ml),


MG (250 mM) and sodium azide (3 mM) at 37 C for 21 days and
dialysed extensively with 10 mM phosphate buffer (pH 7.4) for 24
hours. Dialysed samples were analysed on SDS-PAGE (10 %
resolving and 5 % stacking gel).

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Figure 10: BSA(10 mg/ml)was incubated with MG (250 mM),


curcumin (200 g), bhasma (200 g), AG (10 mM) and sodium azide
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CONCLUSION:
The effect of glycation is more prominent when there is an
accumulation ofsugars and sugars metabolites in the cells and
tissues. These molecules differ in theirpotential in causing
damage to DNA and proteins. In the present study it can be seen
that sugar phosphates cause more extensive damage to DNA
than their corresponding sugars.It can be mainly because of
their reactivities and potential in production of free
radicals.Fructose-6-phosphate is the most effective in
disturbing the supercoiled structure ofplasmid DNA. This can
be due to the amount of hydrogen peroxide produced which
was more for this sugar phosphate as compared to other sugars.
Antioxidants and natural products varied in their
efficacy in preventing the damage to DNA and proteins. There
can be several reasons for this behaviour. These compounds
differ from each other with respect to their capability and
specificity of scavenging the free radicals and at different
concentrations. Among the compounds tested pyridoxamine
showed least preventive activity. This could be due to its low
hydrogen donating capacity (Delange and Glazer, 1989; Suji
and Sivakami, 2007).
It can be concluded from the results presented in this
study that damage to DNA is free radicals mediated and in case
of protein a mechanism based on the accumulation of AGEs
seems to play a critical role in damaging the structures. There
are several other reports in the literature which suggests a
similar mechanism for the structural alteration of biomolecules
(Kikuchiet al.,2003). It can be also suggested from the results
presented in this study that naturalcompounds are very
important for preventing the damage to DNA due to
productaccumulation of early and advanced reactions of
glycation. However, further studies are required to characterize
the exact mechanism of the action of the sugars and
theirmetabolites.

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BIONANO FRONTIER

Vol. 7 Special Issue - 12, January - 2014

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