Indian Journal of Experimental Biology
Vol. 53, June 2015, pp. 371-379
Phlorotannins from Brown Algae: inhibition of advanced glycation end products
formation in high glucose induced Caenorhabditis elegans
Ganeshan Shakambari1, Balasubramaniem Ashokkumar2* & Perumal Varalakshmi1*
1
Department of Molecular Microbiology; 2Department of Genetic Engineering, School of Biotechnology,
Madurai Kamaraj University (MKU), Madurai-625 021, Tamil Nadu, India.
Received 20 November 2014; revised 26 November 2014
Advanced Glycation End products (AGE) generated in a non enzymatic protein glycation process are frequently
associated with diabetes, aging and other chronic diseases. Here, we explored the protective effect of phlorotannins from
brown algae Padina pavonica, Sargassum polycystum and Turbinaria ornata against AGEs formation. Phlorotannins were
extracted from brown algae with methanol and its purity was analyzed by TLC and RP-HPLC-DAD. Twenty five grams of
P. pavonica, S. polycystum, T. ornata yielded 27.6±0.8 µg/ml, 37.7 µg/ml and 37.1±0.74 µg/ml of phloroglucinol equivalent
of phlorotannins, respectively. Antioxidant potentials were examined through DPPH assay and their IC50 values were
P. pavonica (30.12±0.99 µg), S. polycystum (40.9±1.2 µg) and T. ornata (22.9±1.3 µg), which was comparatively lesser
than the control ascorbic acid (46±0.2 µg). Further, anti-AGE activity was examined in vitro by BSA-glucose assay with the
extracted phlorotannins of brown algae (P. pavonica, 15.16±0.26 µg/ml; S. polycystum, 35.245±2.3 µg/ml; T. ornata,
22.7±0.3 µg/ml), which revealed the required concentration to inhibit 50% of albumin glycation (IC50) were lower for
extracts than controls (phloroglucinol, 222.33±4.9 µg/ml; thiamine, 263 µg/ml). Furthermore, brown algal extracts
containing phlorotannins (100 µl) exhibited protective effects against AGE formation in vivo in C. elegans with induced
hyperglycemia.
Keywords: AGEs inhibition, Hyperglycemia, Padina pavonica, Sargassum polycystum, Turbinaria ornata
Advanced glycation end products (AGEs) are
a complex, heterogeneous group of irreversiblymodified adducts and products of proteins generated
through non-enzymatic glycation reactions between
the free amino groups of proteins and the carbonyl
group of reducing aldose or pentose sugars. Such a
non-enzymatic early glycation reaction often results
in the formation of Schiff bases and Amadori
products, which further undergo a series of
rearrangements, dehydration, and condensation to
generate AGEs1. AGE formation has been recorded
not only from the reducing sugars, but also from
carbonyl compounds derived from the autoxidation
of sugars and other metabolic pathways2. Reactive
carbonyls including methylglyoxal and glyoxal
are generally rich in various foods, including
sugar-sweetened beverages, high lipid content
foods and heat processed foods, which propel AGEs
accumulation in tissues. AGEs are known to cause
cellular damage by interaction with specific receptors
like RAGE. AGEs may fluoresce, produce reactive
——————
*Correspondence:
Ph.: 91 452 2458273 (PV); 2459105 (BA) Fax: 91 452 2459115
E-Mail: [email protected] (PV); [email protected] (BA)
oxygen species (ROS), bind to specific cell surface
receptors, and form cross-links. AGEs that are
commonly found and best characterized in the
humans are pentosidine and carboxyl methyl lysine
(CML).
Protein glycation and AGEs accumulation in vivo
play a crucial role in the pathogenesis of diabetes,
Alzheimer’s disease, aging, renal failure and
other chronic diseases3,4. In diabetic patients, AGEs
accumulation has been noticed in organs such
as kidney, retina, and vascular system prior to
development of complications. The presence of
RAGE has been demonstrated in all cells relevant
to the atherosclerotic process including monocytes,
macrophages, endothelial cells, and smooth muscle
cells, which do not express RAGE significantly
under physiological conditions but induced to express
RAGE due to AGEs accumulation5. Consequently,
the interaction of AGEs with RAGE triggers
inflammatory responses, platelet and macrophage
activation and modulates gene expression of
growth factors and cytokines in vascular cells by
oxidative stress, thus contributing significantly
in the pathogenesis of vascular complications
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INDIAN J EXP BIOL, JUNE 2015
during diabetes1,6. The molecular understanding of
pathophysiology of vascular complications associated
with diabetes, have not only evidenced the severity
of AGEs in the disease progression, but also have an
impact on discovery of novel therapeutics using
pharmacological compounds with anti-AGE activities.
Macroalgae, from marine environment abundantly
found in the coastal areas are a proficient source for
bioactive natural substances which exhibit inhibitory
activity against AGEs formation7. Phlorotannins, one
such bioactive secondary metabolite are polymers of
phloroglucinol (1,3,5-trihydroxybenzene) as monomeric
units formed biosynthetically by the acetate-malonate
pathway. They are hydrophilic in nature with a wide
range of sizes, 126-650 kDa8,9. Phlorotannins from
several species of brown algae have been found to
exhibit remarkably high antioxidant activity, anti
inflammatory, and pro-SIRT1 activities in vitro10-12.
Phlorotannins have also been reported to possess
superior antioxidant properties with 2-10 folds
increased free radicals scavenging activities than
catechin, α-tocopherol and ascorbic acid13.
Furthermore, phlorotannins have also been shown to
possess anti-obesity, anti-inflammatory, anti-bacterial,
anti-allergic, anti-cancer and anti-HIV activities14.
The present study focuses on isolation and
purification of phlorotannins from the brown algal
species Padina pavonica, Sargassum polycystum and
Turbinaria ornata and assessment of their antioxidant
potential and inhibition of AGEs formation in vitro
and in vivo using C. elegans as an animal model by
inducing hyperglycemic conditions.
Materials and Methods
Algal sample Collection—The brown algae samples
were collected from Gulf of Mannar, Keelakarai,
Tamil Nadu, India. The collected samples were
identified (Sargassum polycystum, Padina pavonica
and Turbinaria ornata) based on the morphology as
given in Dhargalkar & Kavlekar15, washed with sea
water and air dried at room temperature (27±1 °C)
followed by drying at 50 °C in hot air oven for
60 min.
Extraction and estimation phlorotannin—The dried
samples (25 g) were ground and extracted with 45 ml
of methanol at room temperature (27±1 °C) for 24 h.
The extracts were passed through the filter paper and
filtrate was concentrated to dry at 61 °C followed by
evaporation and the dried extracts were further
suspended in distilled water. The aqueous suspension
was partitioned sequentially in three different solvents,
hexane (25 ml), ethanol (15 ml), and n-butanol (10 ml),
to fractionate the polar and non-polar compounds. The
final extraction was made in methanol (25 ml) and the
concentration was estimated and used in all further
assays. Total phenolic content in each fraction was
determined by the Folin-Ciocalteu method16 with some
modifications, using phloroglucinol (20-120 µg/ml) as
a standard. The extract of all the three brown algae
(1 ml) was mixed with 5 ml of the 10% Folin-Ciocalteu
reagent and incubated for 5 min, after the incubation
4 ml of 7.5% (w/v) Na2CO3 was added. Post incubation
at room temperature (27±1 °C) for 2 h in the dark, the
absorbance was read at 740 nm on visible spectrophotometer (Eppendorf, Germany) and concentration
of the polyphenols was expressed as phloroglucinol
equivalents (PGE) in µg/ml of extract.
TLC of methanolic extract of phlorotanins—Thin
layer chromatography was performed as a screening
method for detection of phlorotannins in the extracts of
the brown algae and spotting an aliquot of each extract
on the silica gel plate with a solvent system of chloroform/
ethanol/acetic acid/water (98:10:2:2 v/v). The air dried
chromatogram was visualized under UV light wavelength
365 nm for the detection spots in phloroglucinol
standard17. Further, the phlorotannins spots were also co
visualized by spraying with 1% solution of FeCl3 in
water18,19. The presence of blue colored spots indicated
that the fractions had phenolic compounds.
RP-HPLC of methanolic extracts of phlorotannins—
The analytical method used was reversed phase HPLC
(RP-HPLC) (Shimadzu, Japan) in a binary mode (dual
pump), coupled to a diaode array detector where 2 µl
sample solution was directly injected into C-18
column. The mobile phase of HPLC grade methanol:
water (0.2% v/v) at a ratio of 85:15 was delivered at a
flow rate of 0.4 ml/min. and PDA set at 254 nm.
Antioxidant activity—Total antioxidant activities of
the methanolic fraction of all three samples were
determined according to the modified method of
Prieto et al.19,20. Briefly, 0.3 ml of sample was mixed
with 3 ml of reagent (0.6 M sulfuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate).
The reaction mixture was incubated at 95 °C for
90 min in a water bath. To determine the total
antioxidant activity the absorbance of all the sample
extracts was measured at 695 nm and ascorbic acid
(0.125-1.25 mg/ml) was used as standard. The total
antioxidant activity of the extracts were determined
and expressed as per mg ascorbic acid equivalent to
per ml of the extract.
SHAKAMBARI et al: INHIBITION OF AGEs FORMATION BY PHLOROTANNINS FROM BROWN ALGAE
The free radical scavenging activity of all the three
extracts was evaluated by DPPH according to17-21. In
brief, 0.16 mM solution of DPPH in methanol was
prepared and 2 ml of this solution was further added
to 2 ml of each extracts with different concentrations
(5, 10, 20, 30, 40 and 50 µg/ml). The mixture was
vortexed and incubated at room temperature (27±1 °C)
for 30 min. Then, the absorbance was measured at
517 nm using a visible spectrophotometer (Eppendorf,
Germany). The free radical scavenging activity was
calculated by the following equation:
Percentage inhibition of DPPH activity =
([(A0 – A1) / (A0)] × 100%
where A0 is the absorbance value of the control
sample and A1 is the absorbance value of the test
sample. A curve of percent inhibition or percent
scavenging effect against samples concentrations was
plotted and the concentration of the sample required
for 50% inhibition was determined. The assay was
carried out with ascorbic acid as a standard
antioxidant.
AGEs inhibition in vitro—Methanolic extracts of
Sargassum sp., Padina sp. and Turbinaria sp. were
used to screen for their AGEs inhibition by BSAglycation inhibition assay22. Briefly, bovine serum
albumin (100 mg/ml) and glucose (188 mg/ml) were
dissolved in phosphate buffer (100 mM, pH 7.4)
separately. One ml of the BSA solution was mixed
with 1 ml of the glucose solution to which 1 ml of
concentrated extracts was added. The mixtures were
incubated at 37 °C for 7 days. Sodium azide (0.2 g/L)
was used as an aseptic agent to avoid bacterial
contamination. Phosphate buffer was used as blank.
Phloroglucinol was used as positive controls. After
8 days of incubation, fluorescence of the samples
was measured using an excitation of 330 nm and an
emission of 410 nm. The percentage inhibition of
AGE formation was calculated as follows:
% inhibition =
[1 − (fluorescence of the test group/
fluorescence of the control group)] × 100%.
Nematode culture conditions—The wild type
C. elegans (N2, Bristol) was grown on Nematode
Growth medium (NGM) with E. coli OP50 at 20 °C for
3 days till most of the worms grew into gravid adults23.
Synchronized worm culture—Gravid adult
population was treated with 5 ml of fresh bleach
/NaOH solution incubated in dark for 5 min until
the worms broke open. The effect of bleach was
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neutralized by adding 5 ml of sterile M9 buffer. The
eggs washed in M9 buffer were centrifuged at 2500
rpm for 2 min and resuspended in fresh 50 ml of
S-complete medium, and allowed to hatch overnight
and was enumerated to 100 larvae per ml24.
To prepare feed bacteria, overnight grown culture
of E. coli OP50 in 100 ml of LB broth was
centrifuged at 8000 rpm for 10 min and the pellet was
weighed in a 50 ml capacity pre-weighed sterile
centrifuge tube. The pellet was suspended in S-complete
medium to maintain the concentration as 170 mg cells
dry weight in 10 ml of the medium25.
Experimental design: Seeding 24-well tissue culture
plate—On day 1, all the wells were seeded with L1
stage worms to contain 50 larvae each, 6 mg/ml of
E. coli OP50 in complete nutrient S medium.
Hyperglycemic conditions were maintained by
inducing the worms with glucose in the medium
at final concentrations of 100 mmol/l glucose and
200 mmol/l glucose. The worms with the feed bacteria
were incubated overnight at 20 °C.
On the second day, 100 µl of extract of Padina
pavonica, Sargassum polycystum and Turbinaria
ornata was given to 100 mmol/l and 200 mmol/l
induced worms in triplicates. Control groups were
maintained in duplicates as uninduced normal worms,
100 mmol/l glucose induced worms and 200 mmol
glucose induced worms.
High content screening analysis—Live worms
were imaged using High Content Screening System,
Model Operetta (Perkin Elmer) for fluorescence
attributed to accumulation of AGEs in C. elegans
grown under normal conditions, 100 mmol/l and
200 mmol/l induced conditions and treated conditions,
at excitation 360 nm and emission 420 nm26.
Quantitative AGE accumulation analysis—Aqueous
homogenates of the nematodes were prepared by
suspending aliquots of 25 worms from each well in
3 ml of deionized water and subjecting them to repeated
freeze-thaw cycles. Worm disruption was verified by
microscopic observation. The cellular debris was
removed by a short spin and the cell free supernatant
was used for the identification of the fluorescence
associated with AGEs accumulation in these
homogenate which was assayed spectro-fluorometrically
at excitation 365 nm and emission 470 nm on Cary
Eclipse Fluorescence spectro-photometer (Agilent
Technologies, USA)27. Sterile water was used as
blank. A set of triplicate was estimated for fluorescence
spectrum on day 4 and another set was further
incubated and estimated on day 9.
INDIAN J EXP BIOL, JUNE 2015
374
Total reducing sugar assay—The reducing sugar
content in the initial medium of each well, the leftover
medium post incubation (8 days) of worms and
the worm extract (25 adult worms) was assayed by
DNSA method.
Results and Discussion
Extraction and estimation of phlorotannins
in different solvents—The polarity of the compound
in crude extract and solvent in which it is to be
extracted and extraction of different phenolic
compounds from different materials of biological
origin is important for the yield of polyphenols28.
Phenolic compounds are generally soluble in solvents
which are less polar than water, and hence, the choice
of solvent is a mixture of water and methanol, ethanol
or acetone. Moreover, phlorotannins are extremely
hydrophilic components with the molecular sizes
ranging between 126 Da and 650 kDa29. In this study,
phlorotannins were extracted and fractioned from
three brown algae using various solvents like hexane,
ethanol, butanol and methanol. However, the high
yield of polyphenol without impurities was achieved
only in methanolic extraction than the other
solvent extractions. Further, the individual fractions
were tested for its phenolic content and
the phloroglucinol equivalent concentration was
estimated as phloroglucinol is the monomeric unit
of phlorotannins and the final methanolic
fraction resulted that 25 g of each brown algae
Padina pavonica, Sargassum polycystum and
Turbinaria ornata yielded 27.6±0.8, 37.7and
37.1±0.74 µg/ml of phloroglucinol equivalent
respectively (Table 1). Obviously, the yield and
extractability of phenolic compounds from may vary
from species of brown algae30. It is also reported that
the methanolic extract of brown alga had shown
efficient antioxidant activity in RAW 264.7 cells31.
Chromatography for separation and identification
of phlorotannins—The final extracts were subjected
to TLC which revealed the presence of phenolic
compounds that appeared as blue bands after the
application of spraying reagent 1% FeCl3 whereas
monomer phloroglucinol as a standard exhibits a band
with blue illumination under UV exposure (365 nm)
itself. The blue band of the brown algae extracts were
displayed at a higher front than the phloroglucinol
standard. Also, the non availability of a commercial
standard phlorotannins necessitates the use of its
monomer phloroglucinol as the only compound to be
used as a standard28 (Fig. 1). It is noteworthy to
mention that a single band was observed in each
extracts indicating the presence of only one type of
compound in all the three extracts and this was
further confirmed by Reversed Phase-High Pressure
Liquid Chromatography (RP-HPLC). The Rf value
of Padina pavonica (0.87), Sargassum polycystum
(0.86), Turbinaria ornata (0.87) extracts were higher
than that of phloroglucinol (0.73) in TLC. Similarly,
the retention time in RP-HPLC for the extracts
of P. pavonica 19.456 min, S. polycystum 19.729 min
and T. ornata 19.578 min were lesser than
phloroglucinol 23.594 min (Fig. 2). Structural
properties of the compound under analysis play a vital
role in its retention pattern, as branched chain
Fig. 1—Thin Layer Chromatography of the three brown algal
extracts containing phlorotannins: (A) TLC plates under UV
illumination at 365 nm; (B) TLC plates developed with 1% FeCl3.
[Lane 1, phloroglucinol and lanes 2-4, Sargassum, Padina and
Turbinaria extract, respectively].
Table 1—Quantitative Estimation of phlorotannins from various extracts
Algal sample
Turbinaria
Padina
Sargassum
Phloroglucinol equivalent concentration (µg/ml)
Hexane
Ethanol
Butanol
(25 ml)
(15 ml)
(10 ml)
Methanol crude
(45 ml)
Water
(25 ml)
51.2
55.6±3.3
182±3
28.8±2.5
39.6±3.2
85±2
1.6±0.06
6±1
11±1.02
1.2±0.2
5.6±0.1
20.2±0.4
0
0
0
Methanol Final
(25 ml)
37.1±0.74
27.6±0.8
37.7
SHAKAMBARI et al: INHIBITION OF AGEs FORMATION BY PHLOROTANNINS FROM BROWN ALGAE
compounds elute sooner than their corresponding
linear isomers or monomers due to the decreased
overall surface area32. Thus, the findings suggest
the quicker the retention time of the compounds in
the extracts of brown algae imply the presence of
polymers of phloroglucinol.
Antioxidant activities of phlorotannin extracts—
The total antioxidant capacities and 1,1-diphenyl
1,2-picrylhydrazyl DPPH radical scavenging activities
of the extracts of three brown algae were determined
with respect to ascorbic acid as a known antioxidant.
The antioxidant capacity of T. ornata. (37.1 µg/ml)
was found to be maximum among the extracts and it
was found to be equivalent to that of 1.53±0.1 mg/ml
of ascorbic acid in antioxidant activity. While the
antioxidant activity of S. polycystum (37.7 µg/ml)
and P. pavonica (27.6 µg/ml) were corresponding to
0.45±0.09 mg/ml and 0.65± 0.1 mg/ml of ascorbic
acid, respectively (Table 2). Total antioxidant
capacities of the compounds are based on the ability
to reduce molybdenum VI (Mo6+) to form a green
phosphate/Mo5+ complex 20. Previous reports have
indicated that this antioxidant capacity is due to the
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presence of multiple phenolic groups in brown
algae17. It is very important to analyze and validate
the results of antioxidant activity of the three extracts,
thus scavenging of DPPH radical was performed by
the well known DPPH assay. Hence in this study,
DPPH assay was performed to validate the
antioxidant capacity of the three algal extract and the
findings of this experiment revealed the lowest IC50
was attained in T. ornata extract (22.9±1.3 µg/ml)
than other two algal extracts such as P. pavonica. and
S. polycystum 30.12±0.9 µg/ml and 40.9±1.2 µg/ml,
respectively. Interestingly, the aforementioned results
were corroborating with the total antioxidant activity
of all the three algal extracts (Table 2). Several studies
have been reported that phlorotannins from brown
algae exhibit antioxidant activity wherein they protect
from the cellular damage by the action of hydrogen
peroxide33, 34.
Inhibition of AGEs in vitro—The antidiabetic
activity of phlorotannins was explored by performing
an experiment to inhibit the formation of AGE
by inhibition of BSA glycation method. AGE are
described as heterogeneous compounds formed
Fig. 2—RP-HPLC analysis of final extracts of brown algae and phloroglucinol as standard on a C-18 column and mobile phase
methanol:water, flow rate 0.4 ml/min PDA-254 nm. [(a) phloroglcinol, (b-d) methanolic extract of Sargassum, Padina and Turbinaria,
respectively].
Table 2—Antioxidant properties and AGE inhibition values of extracts
Compound
Phloroglucinol
Thiamine (50µg/ml)
Concentration
PGE µg/ml
AGEs inhibition
IC50 µg/ml
Total antioxidant activity
ascorbic acid equvl.
(mg/ml)
DPPH radical
scavenging activity
(IC50) µg
-
222.33±4.9
-
-
-
263
-
-
37.7
35.245±2.3
0.45±0.095
40.9±1.2
Padina extract
27.6±0.8
15.16±0.26
0.65±0.15
30.12±0.99
Turbinaria extract
37.1±0.74
22.77±0.3
1.53±0.11
22.9±1.3
Sargassum extract
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INDIAN J EXP BIOL, JUNE 2015
when reducing sugars react with amine residues on
proteins, lipids, and nucleic acids and finally leading
to their chemical alteration35. In addition, the
increased formation of AGE could cause diabetic
complications, development of cataracts, uremia,
Alzheimer’s disease etc35. The AGE inhibitory
activity was analyzed in all the three brown algal
extracts and thiamine and phloroglucinol were used as
standards for this study. Among the three algal
extracts, inhibition of AGE formation with the least
IC50 value (15.16±0.26 µg/ml) was obtained from
P. pavonica when compared with the other two algae
S. polycystum (35.245±2.3 µg/ml) and T. ornata
(22.77±0.3 µg/ml) and the standards phloroglucinol
and thiamine (222.33±4.9 µg/ml and 263 µg/ml),
respectively (Table 2). Scavenging of reactive
carbonyls appeared to be a major mechanism of algal
extract to inhibit protein glycation7.
In vivo studies using Caenorhabditis elegans as
model organism—C. elegans has been successfully
used as an animal model to study the glucose toxicity,
in which high glucose conditions limit the life span
by increased formation of reactive oxygen species
(ROS) and AGE modification24. Age synchronized
worms were exposed to different concentrations
of glucose (100 mmol/l and 200 mmol/l) to induce
hyperglycemic conditions in the worms, which were
fed with OP50 bacteria from L1 stage. After 24 h of
incubation, worms were treated with methanolic
extract containing pholorotannins from S. polycystum
(37.7 µg/ml), P. pavonica (27.6 µg/ml) and T. ornata
(37.1 µg/ml). Nine day old adult worms were scored
and examined for AGEs accumulation by observing
under High Content Screening Analyzer for imaging
fluorescence at excitation and emission of 360 nm
and 420 nm, respectively. Glucose concentration
dependent fluorescence intensity was recorded in the
worms induced with 100 mmol/l and 200 mmol/l
of glucose. Whereas, hyperglycemic worms treated
with the extracts of P. pavonica, S. polycystum
and T. ornata had shown prominent decrease
in the fluorescence intensity when compared with
hyperglycemic worms (Fig. 3). No fluorescence
was observed with uninduced control worms.
Spectrofluorimetric analysis of brown algal extracts
containing only phlorotannins in S-complete medium
showed fluorescence at 670 nm . Further, aqueous
extracts of the whole worms -hyperglycemic as well
as worms treated with methanolic extracts were
harvested and its fluorescence intensity was measured
using spectrofluorimeter at excitation and emission of
360 nm and 420 nm, respectively, on day 4 and day 9
Fig. 3—High content analysis of worms exposed to (A) 100 mmol/l of glucose [(a) worms under normal conditions, (b-d) worms received
200 µl of Sargassum, Padina and Turbinaria extract, respectively]; (B) 200 mmol/l of glucose [(a) worms under normal conditions,
(b-d) worms received 200 µl of Sargassum, Padina and Turbinaria extract, respectively].
SHAKAMBARI et al: INHIBITION OF AGEs FORMATION BY PHLOROTANNINS FROM BROWN ALGAE
of cultivation. Day 4 analysis by spectrofluorimetry
revealed the internalization of the compound by the
worms by a second peak at 670 nm. While, on the
day 9, worms treated with the phlorotannins extract
from P. pavonica, S. polycystum and T. ornata had
exhibited considerable inhibition in the fluorescence
intensity corresponding to AGEs accumulation in
the hyperglycemic worms induced with 100 mmol/l
of glucose than in 200 mmol/l of glucose, and
disappearance of the second peak at 670 nm (Fig. 4).
Maximum inhibition of fluorescence intensity (90 %)
was recorded with the extract of S. polycystum at
100 mmol/l glucose induced conditions, whereas
P. pavonica had shown maximum inhibition of 70%
at 200 mmol/l glucose induced conditions. Generally,
it is known that the major autofluorescence observed
at excitation and emission pair 340 nm/430 nm
correlates to biomarkers such as AGEs and lipofuscin
granules37. Hence, accumulation of fluorescence in
this range is an indication for AGEs accumulation.
Thus, the results of anti-AGEs formation studies with
C. elegans in vivo, clearly demonstrated the potential
of methanolic extracts containing phlorotannins
obtained from brown algae.
Furthermore, the intracellular levels of reducing
sugars were estimated by DNSA analysis with
the aqueous extracts collected from the whole
377
hyperglycemic worms as well as worms treated with
methanolic extracts. The levels of sugar concentration
in the hyperglycemic worms induced with 100 mmol/l
and 200 mmol/l glucose were higher than the
uninduced control (5.0±0.7 mmol/l).The glucose
concentration estimated with the worm groups
induced with 100 mmol/l glucose and treated
with extracts of P. pavonica (9.44±0.6 mmol/l)
S. polycystum (17.9±0.8 mmol/l) and T. ornata
(28.4±0.6 mmol/l) showed less glucose concentration
than the hyperglycemic worms (30.5±0.31 mmol/l)
(Fig. 5). Similarly, the level of glucose concentration
in the medium in which the worms grown for 8 days
were estimated and it was observed that the
induced groups treated with the extracts had shown
elevated levels (P. pavonica 50.6±2.52, S. polycystum
38.4±1.2 and T. ornata 47±1.5 mmol/l glucose
than the medium of the untreated worms (25.1±7.4).
The same way the level of glucose concentration of
the worm extract was observed in groups induced
with 200 mmol/l glucose and treated with the extracts
of brown algae had shown lesser level P. pavonica
(24.2±0.3 mmol/l); S. polycystum (26.06±0.6 mmol/l);
and T. ornata (32.2±0.9 mmol/l) of glucose in worm
extract than the hyperglycemic control (44.1 mmol/l).
In addition, the glucose concentration in the
leftover medium (after the harvesting of worms) was
Fig. 4—Plot of fluorescence intensity emitted vs. excitation wavelength for quantitative estimation of AGEs accumulation. The 100 and
200 refers to glucose concentration in mmol/l. (A) Spectrum of fluorescence emission of the phlorotannins in the brown algal extract in
S-complete medium at 670 nm; (B) Spectrum of fluorescence emission by phlorotannins at 670 nm and AGE accumulation at 360 nm and
420 nm in C. elegans treated with macroalgal extract and control on day 4; (C) Spectrum of fluorescence emission by AGE accumulation
at 360 nm and 420 nm in C. elegans treated with macroalgal extract and control and absence of phlorotannins at 670 nm on day 9.
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INDIAN J EXP BIOL, JUNE 2015
emphasize the effect of phlorotannins for treating
hyperglycemic conditions in higher animals and
phlorotannins may pave a new path to inhibit the
AGE formation, being one of the major complications
in diabetes. Thus, phlorotannins from brown algae
may ameliorate a new candidate as antidiabetic
compound in pharmocological studies.
Acknowledgements
Authors thank the SERB, India (Ref. No:
SB/EMEQ-128/2013) for the financial support to
PV; CSIR-UGC fellowship to GS (No.17-06/2012
(i) EU-V) and DST-PURSE program of MKU for
instrumentation facility.
Fig. 5—DNSA analysis of initial and leftover medium
and aqueous worm extract for reducing sugar. [C, Control; CN,
Normal; S, Sargassum; P, Padina; T, Turbinaria. The 100 and
200 refer to the glucose concentration in mmol/l].
estimated and the results revealed higher glucose level
in the medium where in the worms treated with brown
algae extracts P. pavonica (66.27±5.3 mmol/l);
S. polycystum (66.3±1.9.6 mmol/l); and T. ornata
(63.94± 1.5 mmol/l) than the medium of hyperglycemic untreated conditions (48.62±13.8) (Fig. 5).
However, the reason for the increased level of glucose
concentration in the left out medium where the worms
are provided with the brown algal extract remains
unclear. Furthermore, none of the study has been
reported for the antioxidant activity and inhibition of
AGE formation in C. elegans so far. Thus, further
study would be required to elucidate the mechanism
of glucose uptake and accumulation in the medium
and the inhibition AGE formation in C. elegans.
Conclusion
In this study, methanolic extracts of phlorotannins
from the brown algae Padina pavonica, Sargassum
polycystum and Turbinaria ornata were tested for
their antioxidant and anti-AGE formation activities.
The findings revealed that phlorotannins of all the
three algal extracts showed potent antioxidant
capability while Turbinaria ornata showed maximum
activity. However, the maximum AGEs inhibition
activity was observed with the phlorotannins from
Padina pavonica compared to other two extracts.
Further, anti-AGE activity of phlorotannins was tested
and confirmed in vivo, for the first time with
hyperglycemic C.elegans as a model organism.
Consequently, the results obtained in this study would
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