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S404 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
Evaluation of antidiabetic, antioxidant and antiglycating
activities of the Eysenhardtia polystachya
Rosa Martha Perez Gutierrez, Efren Garcia Baez
1
Laboratory of Research on Natural Products, School of Chemical Engineering and Extractive Industries, Av. Instituto Politécnico Nacional S/N,
Unidad Profesional Adolfo Lopez Mateos, Zacatenco, Mexico D.F.,
1
Laboratory of Research Organic Chemistry. UPIBI‑IPN, Col. San Pedro
Zacatenco, Mexico D.F.
Submitted: 18‑10‑2013 Revised: 20‑11‑2013 Published: 28‑05‑2014
ORIGINAL ARTICLEPHCOG MAG.
Address for correspondence:
Dr. Rosa Martha Perez Gutierrez,
Laboratory of Research on Natural Products,
School of Chemical Engineering and Extractive Industries,
Av. Instituto Politécnico Nacional S/N, Unidad Profesional Adolfo
Lopez Mateos, Zacatenco, cp 07708, Mexico D.F.
E‑mail: rmpg@prodigy.net.mx
Background: Many diseases are associated with oxidative stress caused by free radicals. The aim
of the present study was to evaluate the antidiabetic, antioxidant and antiglycation properties
of Eysenhardtia polystachya (EP) bark methanol-water extract. Materials and Methods: The
antioxidant capacities were evaluated by studying in vitro the scavenging of DPPH and ABTS
free radical, reactive oxygen species such as RO
2
, O
2
·
‑
, H
2
O
2
, OH
.
, H
2
O
2
, ONOO-, NO, HOCl,
1
O
2
,
chelating ability, ORAC, β-carotene-bleaching and lipid peroxidation. The antiglycation activities
of EP were evaluated by haemoglobin, bovine serum albumin (BSA)-glucose, BSA-methylglyoxal
and BSA-glucose assays. Oral administration of EP at the doses of 100 mg/kg, 200 mg/kg and
400 mg/g was studied in normal, glucose-loaded and antidiabetic effects on streptozotocin-induced
mildly diabetic (MD) and severely diabetic (SD) mice. Results: EP showed Hdonor activity, free
radical scavenging activity, metal chelating ability and lipid peroxidation Antioxidant activity may
be attributed to the presence of phenolic and avonoid compounds. EP is an inhibitor of uorescent
AGE, methylglyoxal and the glycation of haemoglobin. In STZ-induced diabetic mice, EP reduced
the blood glucose, increased serum insulin, body weight, marker enzymes of hepatic function,
glycogen, HDL, GK and HK while there was reduction in the levels of triglyceride, cholesterol,
TBARS, LDL and G6Pase. Conclusions: Eysenhardtia polystachya possesses considerable
antioxidant activity with reactive oxygen species (ROS) scavenging activity and demonstrated an
anti-AGEs and hepatoprotective role, inhibits hyperglycemic, hyperlipidemic and oxidative stress
indicating that these effects may be mediated by interacting with multiple targets operating in
diabetes mellitus.
Keywords: Antidiabetic, antiglycation, antioxidant, Eysenhardtia polystachya
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Website:
www.phcog.com
DOI:
10.4103/0973‑1296.133295
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INTRODUCTION
It is increasingly being realised that many of today’s
diseases are due to the “oxidative stress” that results from
an imbalance between the formation and neutralisation of
pro‑oxidants. Oxidative stress is initiated by free radicals,
that seeks stability through electron pairing with biological
macromolecules such as proteins, lipids and DNA in
healthy human cells and cause protein and DNA damage,
along with lipid peroxidation.
[1]
All human cells protect
themselves against free radical damage by enzymes such as
superoxide dismutase (SOD) and catalase or compounds
such as ascorbic acid, tocopherol and glutathione.
[2]
Sometimes, these protective mechanisms are disrupted by
various pathological processes and antioxidant supplements
are vital to combat oxidative damage. Recently, much
attention has been directed towards the development of
ethnomedicines with strong antioxidant properties but low
cytotoxicities. Antioxidants are compounds that can delay
or inhibit the oxidation of lipids and other molecules and by
doing so inhibit the initiation and propagation of oxidative
chain reactions. They act by one or more of the following
mechanisms: reducing activity, free radical‑scavenging,
potential complexing of pro‑oxidant metals and quenching
of singlet oxygen.
Diabetes mellitus is the most prevalent metabolic disorder
that is principally characterized by insulin resistance
(IR) and elevated blood glucose levels.
[3]
Prolonged
hyperglycemia contributes importantly to the pathogenesis
of diabetic complications by increasing protein glycation,
ABSTRACT
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S405
leading to the gradual buildup of advanced glycation end
products (AGEs) in body tissue.
[4]
The complex, uorescent
AGE molecules formed during Maillard reaction can lead to
protein cross‑linking, which contributes to the development
and progression of various diabetic complications.
[5]
Many
researchers have discussed the pathological features of
diabetes, that are caused to a great extent by the accelerated
formation of AGEs promoted by hyperglycaemia in
tissues.
[6]
Inhibition of the formation of AGEs has been
shown to be an effective way of retarding the full range of
diabetes complications, such as nephropathy, neuropathy,
retinopathy and vasculopathy.
The tree Eysenhardtia polystachya, (Ortega) Sarg, belonging to
the Leguminosae family, is known as “palo azul” and has
wide use for the treatment of nephrolithiasis, as a blood
depurative, diuretic and anti‑rheumatic and bladder disorders
developing during diabetes.
[7]
Phytochemical studies indicate
that E. polystachya contains polyphenols, beside these
compounds, 7‑hydroxy‑2’,4’,5’‑trimethoxyisoavone was
isolated as the principal uorescent phenolic constituent
of the heartwood.
[8]
In another study, (3S)‑7‑hydroxy‑2’,
3’, 4’, 5’, 8‑pentamethoxyisoavan, (3S)‑3’, 7‑dihydroxy‑2’,
4’, 5’, 8‑tetramethoxyisoavan and soduartin displayed
moderate cytotoxic activity against KB cell lines.
[9]
Further studies isolated chalcones coatline A, B and
(αR)‑α,3, 4, 2’, 4’‑penta‑hydroxydihydrochalcone,
( α R)‑3’‑C‑D‑xilopyranosyl‑α , 3, 4, 2’,
4’‑pentahydroxy‑dihydro chalcone and
( α R)‑3’‑C‑
β
‑ D‑xylopyrano‑ syl‑α , 3, 4, 2’,
4’‑pentahydroxydihydrochalcone from the bark and
trunks.
[10,11]
The methanolic extract of branches
displayed hypoglycaemic activity and their chemical
analysis allowed the isolation of 3‑O‑acetyl‑11α, 12α–
epoxy‑oleanan‑28, l3β‑olide, (+)‑catechin and (+)‑catechin
3‑O‑β‑D‑galactopyranoside.
[12]
In another report,
methanolic bark extract was further separated by
column chromatography, yielding four known
substances: (‑)‑epicatechin, (+)‑afzelechin, eriodictyol,
(+)‑quercetin‑3‑O‑p‑D‑galacto‑pyranoside, all of which
showed scavenging properties against DPPH. Subcoriacin,
displayed strong antioxidant activity in pancreatic
homogenate.
[13]
Other Eysenhardtia as E. platycarpa,
E. punctate and E. subcoriaceae protected the pancreas and
displayed antioxidant activity.
[12]
3‑O‑acetyloleanolic acid
identied as the major constituent of E. platycarpa, showed
a signicant decrease (31 mg/kg of body weight, P < 0.05)
in the glucose level of STZ‑induced diabetic rats.
[14]
This study was performer to evaluate the hypoglycemic,
antioxidant potential and AGEs inhibition capacity of the
methanol‑water extract from the bark of E. polystachya in
in vitro assays and also using diab etes‑induced oxidative
damage in the liver, kidney and pancreas.
MATERIALS AND METHODS
Plant material and preparation of extracts
Fresh bark of E. polystachya was collected in Mexico
State. A voucher specimen (No. 7345) was deposited
in the Herbarium of the UAM‑Xochimilco, for further
reference. Bark was dried at room temperature and
powdered (300 g). The powdered material was extracted
using 900 ml of methanol‑H
2
O 1:1 v/v consecutively using
soxhlet apparatus. These extracts (EP) were ltered and
concentrated by rotary vacuum evaporator and kept in a
vacuum desiccator for the complete removal of solvent.
Estimation of total phenolic content
Total soluble phenolics of the extracts were determined
with Folin‑Ciocalteau reagent using gallic acid as the
standard.
[15]
Antioxidant activity in vitro
1, 1‑Diphenyl‑2‑picrylhydrazyl (DPPH) Assay
The ability of the extract to scavenge DPPH radicals
was assessed as described by Gyam et al.
[16]
using one
of the most extensively used antioxidant assays for plant
samples. This method is based on the scavenging of DPPH
radicals by the antioxidants, which produces a decrease in
absorbance at 517 nm. When a solution of DPPH is mixed
with a substance that can donate a hydrogen atom, the
reduced form of the radical is generated, accompanied by
a loss of colour. This delocalisation is also responsible for
the deep violet colour, characterized by an absorption band
in ethanol solution at about 517 nm. A 50 µl of aliquot of
extract or control was mixed with 450 µl PBS (10 mM/l,
pH 7.4) and 1.0 ml of methanolic DPPH (0.1 mM/l)
solution. After a 30 min reaction, the absorbance was
recorded at 517 nm.
Trolox equivalent antioxidant capacity assay
The antiradical properties of the extracts were
determined using the TEAC assay. The TEAC
assay is based on the reduction of the 2, 2’‑azinobis
(3‑ethylbenzothiazoline‑6‑sulfonic acid (ABTS)
radical‑cation by antioxidants and was adapted with minor
modications.
[17]
The ABTS
+.
radical‑cation was prepared
by mixing ABTS stock solution (7 mM in water) with
2.45 mM potassium persulphate (K
2
S
2
O
8
). This mixture was
left for 12 to24 h in the dark until the reaction was complete
and the absorbance was stable [Abs
734 nm
to 0.700 (±0.030)].
Plant extracts (1 ml) were allowed to react with 1ml of the
ABTS solution and the absorbance was taken at 734 nm
after 7 min using a spectrophotometer. Appropriate solvent
blanks were run in each assay. The scavenging capacity
of the extract was compared with that of α‑tocopherol
and percentage inhibition was calculated as ABTS radical
scavenging activity.
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S406 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
Oxygen radical absorbance capacity (ORAC) assay
The assay was performed as per the method described
by Cao et al.
[18]
All solutions were prepared in 75 mM
phosphate buffer (pH 7.4). Samples were diluted by a
factor of 30 in 5% randomly methylated cyclodextrin
solution before being assayed. Twenty microlitres of diluted
extracts were manually pipetted into sample wells of the
microplate which was then placed in the microplate reader
and incubated for 15 min at 37°C. During the rst cycle,
120 µl of uorescein solution (disodium salt, 20 µM) was
injected into each well using the rst automated reagent
injector; each injection was followed by a 1’s mixing cycle.
During the second cycle, 60 µl of 45 mM of AAPH
(2, 2’‑azobis‑2‑methyl‑propanimidamide, dihydrochloride)
was injected into each well using the second automated
reagent injector, followed by a 1 s mixing cycle. Following
mixing, the initial uorescence was read; uorescence
readings were then taken every 30’s. with a total assay
time of 30 min. A standard curve was prepared with
α‑tocopherol using a concentration range of 12.5‑150 µM.
The relative uorescence versus time graph of each sample
was recorded from which the area under curve (AUC) of
each sample was calculated. The AUC of each sample was
used along with the standard curve to calculate the oxygen
radical absorbance capacity (ORAC) of each extract,
expressed as µmol α‑tocopherol equivalents/g extract
equivalent. Fluorescence lters with excitation and emission
wavelengths of 485 and 520nm, respectively, were used;
these conditions correspond to the uorescence properties
of uorescein.
Ferrous ion chelating ability
The method of Decker and Welch was used to investigate
the ferrous ion chelating ability of extracts.
[19]
Brief1y, a
given volume of the extract (0.1222 mg/ml), ascorbic
acid (0.1564 mg/ml), or butylatedhydroxytoluene (BHT)
(0.1890 mg/ml) was added to 50 µl of 2.0 mM aqueous
FeSO
4
in 5.0 ml test tube, then ethanol was added to
4.0 ml. After 5 min incubation, the reaction was initiated by
1.0 ml of 5.0 mM ferrozine. After 10 min of equilibrium,
the absorbance at 562 nm was recorded. The controls
contained all reaction reagents except the extract or positive
control substance.
Nitric oxide radical scavenging assay
This assay was performed according to the method
described by Sreejayan et al.
[20]
Nitric oxide generated
from sodium nitroprusside in aqueous solution at a
physiological pH interacts with oxygen to nitrite ions, that
was measured by Griess reagent. The reaction mixture
(3 ml) containing 10mM nitroprusside in phosphate
buffered saline and the fractions or the extracts at different
concentrations (50‑800 µg/ml) were incubated at 25°C
for 150 min. Aliquots of 0.5 ml of incubated sample were
removed at 30 min intervals and 0.5 ml Griess reagent
was added. The absorbance of the chromosphore formed
was measured at 546 nm. Inhibition of the nitric oxide
generated was measured by comparing the absorbance
values of control and extracts.
Peroxynitrite scavenging
Peroxynitrite (ONOO‑) was synthesised by the method
described by Beckman et al.
[21]
An acidic solution
(0.6 M HCl) of 5ml H
2
O
2
(0.7 M) was mixed with 5 ml
0.6M KNO
2
on an ice bath for 1’s and 5 ml of ice‑cold
1.2 M NaOH was added. Excess H
2
O
2
was removed
by treatment with granular MnO
2
prewashed with
1.2 M NaOH and the reaction mixture was left overnight
at ‑20
o
C. Peroxynitrite solution was collected from the top
of the frozen mixture and the concentration was measured
spectrophotometrically at 302nm (ε = 1670 M
‑1
cm
‑1
).
An Evans Blue bleaching assay was used to measure
peroxynitrite scavenging activity. The reaction mixture
contained 50 mM phosphate buffer (pH 7.4), 0.1 mM
DTPA (diethylene triamine pentaacetic acid), 90 mM
NaCl, 5 mM KCl, 12.5 µM Evans Blue, various doses of
plant extract (0‑200 µg/ml) and 1mM peroxynitrite in a
nal volume of 1ml. After incubation at 25
o
C for 30 min,
the absorbance was measured at 611nm. The percentage
scavenging of ONOO‑ was calculated by comparing
the results of the test and blank samples. All tests were
performed six times. Gallic acid was used as the reference
compound.
β
‑Carotene‑linoleic acid assay
A solution of β‑carotene was prepared by dissolving 2 mg of
β‑carotene in 10ml chloroform and 1.0 ml of this solution
was then pipetted into a ask containing 20 mg of linoleic
acid and 200 mg of Tween‑40 emulsier. Chloroform was
completely evaporated using a vacuum evaporator. Aliquots
of 5.0 ml of this emulsion were transferred into a series of
tubes containing various concentrations of the fractions
(25‑400 μg/ml) or tocopherol. The absorbance of the
extracts and the standard was measured immediately (t = 0)
and after 90 min at 470 nm. The tubes were incubated
at 50°C in a water bath during the test. The antioxidant
activities (AA) of the samples were evaluated in terms of
bleaching of β‑carotene.
[22,23]
Superoxide radical scavenging assay
The scavenging activity against chemically generated
superoxide radicals (O
2
·
-
) of the crude extracts was
measured by means of spectrophotometric measurement
of the product following the reduction of nitroblue
tetrazolium (NBT). Superoxide anions were generated
in a non‑enzymatic PMS/NADH system.
[24]
The
reaction mixture contained 1ml of test solution, 1.9 ml
of 0.1M phosphate buffer (pH 7.4), 1 ml of 20 µM
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S407
phenazine methosulphate (PMS), 156 µM nicotine adenine
dinucleotide (NADH) and 25 µM NBT in phosphate
buffer (pH‑l 7.4). After 2 min of incubation at 25°C,
the colour was read on a spectrophotometer at 560 nm
against blank samples that contained no particle mass
spectrometry (PMS).
Hydroxyl radical scavenging
This was assayed as described by Elizabeth and Rao.
[25]
with
a slight modication. The assay is based on quantication
of the degradation product of 2‑deoxyribose by
condensation with TBA. Hydroxyl radical was generated
by the Fe
3+
‑ascorbate‑EDTA‑H
2
O
2
system (the Fenton
reaction). The reaction mixture contained, in a final
volume of 1ml, 2‑deoxy‑2‑ribose (2.8mM), KH
2
P0
4
‑KOH
buffer (20 mM, pH 7.4), FeCl
3 (
100 µM), EDTA (100 µM);
H
2
O
2
(1.0 mM), ascorbic acid (100 µM) and various
concentrations (0‑200 µg/ml) of the test sample or
reference compound. After incubation for 1 h at 37
o
C,
0.5 ml of the reaction mixture was added to 1 ml 2.8%
TCA, then 1 ml 1% aqueous TBA was added and the
mixture was incubated at 90
o
C for 15 min to develop the
colour. After cooling, the absorbance was measured at
532 nm against an appropriate blank solution. All tests were
performed six times. Mannitol, a classical OH‑scavenger,
was used as a positive control. Percentage inhibition was
evaluated by comparing the test and blank solutions.
Hydrogen peroxide scavenging
An aliquot of 50 mM H
2
O
2
and various concentrations
(0‑2 mg/ml) of samples were mixed (1:1 v/v) and incubated
for 30 min at room temperature. After incubation, 90 µl of
the H
2
O
2
‑sample solution was mixed with 10 µl methanol
and 0.9ml FOX reagent was added (prepared in advance
by mixing 9 volumes of 4.4 mM BHT in methanol with
1 volume of 1 mM xylenol orange and 2.56 mM ammonium
ferrous sulphate in 0.25 M H
2
S0
4
). The reaction mixture
was then vortexed and incubated at room temperature
for 30 min. The absorbance of the ferric‑xylenol orange
complex was measured at 560 nm. All tests were carried out
six times and sodium pyruvate was used as the reference
compound.
[26]
Singlet oxygen scavenging
The production of singlet oxygen (
1
O
2
) was determined
by monitoring N, N‑dimethyl‑4‑nitrosoaniline (RNO)
bleaching, using a previously reported spectrophotometric
method.
[27]
Singlet oxygen was generated by a reaction
between NaOCl and H
2
O
2
and the bleaching of RNO
was monitored at 440 nm. The reaction mixture contained
45 mM phosphate buffer (pH 7.1), 50 mM NaOCl,
50 mM H
2
O
2
50 mM histidine, 10 µM RNO and various
concentrations (0‑200 µg/ml) of sample in a nal volume
of 2 ml. It was incubated at 30°C for 40 min and the
decrease in RNO absorbance was measured at 440 nm.
The scavenging activity of sample was compared with that
of lipoic acid, which was used as a reference compound.
All tests were performed six times.
Hypochlorous acid scavenging
Hypochlorous acid (HOCl) was prepared immediately
before the experiment by adjusting the pH of a 10% (v/v)
solution of NaOCl to 6.2 with 0.6 M H
2
SO
4
and the
concentration of HOCl was determined by measuring the
absorbance at 235 nm using the molar extinction coefcient
of 100 M
‑1
cm
‑1
. The assay was carried out as described
by Aruoma and Halliwell.
[28]
with minor changes. The
scavenging activity was evaluated by measuring the decrease
in absorbance of catalase at 404 nm. The reaction mixture
contained, in a nal volume of 1 ml, 50mM phosphate
buffer (pH 6.8), catalase (7.2 µM), HOCl (8.4 mM) and
increasing concentrations (0‑100 µg/ml) of plant extract.
The assay mixture was incubated at 25
o
C for 20 min and
the absorbance was measured against an appropriate blank.
All tests were performed six times. Ascorbic acid, a potent
HOCl scavenger, was used as a reference.
Assay for low‑density lipoprotein (LDL) oxidation
and measurement of lipid peroxidation
LDL was kept at ‑8°C and a working suspension (200 µg
of protein/ml of phosphate‑buffered saline) was prepared
just before use. The method of LDL oxidation by copper
ions was applied, as has been previously described.
[29]
To
measure the resulting lipid peroxidation, we performed
the chromogenic thiobarbituric acid assay. Thiobarbituric
acid‑reactive substances (TBARS) concentrations were
evaluated using a standard curve of standard MDA at
different concentrations versus absorbance at 532 nm. All
experiments were carried out in triplicate. The formation
of conjugated diene (CD), a lipid oxidation product,
in low‑density lipoprotein (LDL) also was determined
according to the method described by Esterbauer et al.
[30]
The
lipid oxidation of an LDL solution containing 200 to 500 µg
of EP was initiated at 37°C by 0.1 mM CuC1
2
. Absorbance
at 234 nm was continuously recorded for 60 min at 37°C by
a Hitachi U‑2001 spectrometer with a constant re‑circulating
temperature. The lag phase, expressed in minutes, was
dened as the period where no oxidation occurred. A longer
lag phase indicated less CD formation.
Hypoglycemic activity assay in vivo
Animals
The study was conducted in male mice, weighing about
30‑35 g. Before and during the experiment, animals were
fed a standard laboratory diet (Mouse Chow 5015, Purina)
with free access to water. Mice were procured from the
bioterium of ENCB and were housed in microloan boxes in
a controlled environment (temperature 25 ± 2
o
C). Animals
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S408 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
were acclimatised for a period of three days in their new
environment before the initiation of the experiment. Litter in
cages was renewed three times a week to ensure hygiene and
maximum comfort for animals. The experiments reported
in this study were performed following the guidelines stated
in Principles of Laboratory Animal Care (NIH publication
85‑23, revised 1985) and the Mexican Ofcial Normativity
(Norma ofcial Mexicana NOM‑062‑Z00‑1999). Food
consumption and weight gain were measured daily.
Streptozotocin‑induced diabetic severe
Severe diabetes mellitus was induced in overnight
fasted male mice by a single intra‑peritoneal injection
of streptozotocin, at a dose of 50 mg/kg body weight
dissolved in cold citrate buffer (pH 4.5).
[31]
The mice with
diabetic symptoms such as polydipsia and polyuria, as
well as fasting blood glucose concentration higher than
13 mmol/l after 7 days of STZ injection, were selected
for use as experimental animals. In some cases, an STZ
injection may trigger massive insulin release and result
in fatal hypoglycaemia. This hypoglycaemic period was
followed by hyperglycaemia which then became permanent.
To prevent death induced by STZ, the mice were fed with
a 3% glucose solution for 24 h.
Induction of mildly diabetes
Mild diabetes was induced in overnight fasted mice by
administering a single dose of freshly prepared solution
of streptozotocin (STZ), 45 mg/kg b. w. i. p) in 0.1 mol/l
cold citrate buffer (pH 4.5), 15 min after the intra‑peritoneal
administration of 120mg/kg nicotinamide (Sigma
Chemical Company, St. Louis, MO, USA). The STZ
treated animals were allowed to drink 5% glucose solution
over night to overcome drug induced hypoglycemia.
After 10 days of development of diabetes, mice with
moderate diabetes having persistent glycosuria and
hyperglycaemia (blood glucose >250 mg/dl) were used
for further experimentation.
[32]
Experimental design in diabetes mice
Effect of single oral administration of extract of
E. polystachya in glucose level in normal, severe
and mild diabetic mice
After the rat had been denied access to food/water
overnight, they were randomly divided into twelve groups
(six rats per group) matched for body weight. The test
groups were orally administered 100, 200 and 400 mg/kg
body weight (b. w.) of EP suspended in Tween 80, 1% via
gavage. Glibenclamide (GB) at the dose of 5 mg/kg b. w.
as standard drug. Blood samples were collected from the
tail vein at 0, 2, 4, 6, 8 and 12 h after the administration.
The plasma glucose concentration was determined by
an enzymatic colorimetric method using a commercial
kit (Sigma Aldrich, USA).
Antidiabetic test in chronic severe and mild
streptozotocine‑induced diabetic mice
In a parallel study seven groups (n = 10) of diabetic mice
were used to determine the chronic effect of methanol:
water 1:1 extract. Each group was submitted to a specic
treatment, as follows. Normal control and severe and
mild diabetic mice, groups, were fed with normal diet and
drinking water ad libitum, and were given saline by gastric
gavage. Severe and mild diabetic mice that received extracts
by gastric gavage (400 mg per kg of body weight) every day
were designated as SD + EP and MD + EP. Two groups
with severe (SD + GB) and mild diabetes (MD + GB) rat
were administered with glibenclamide (GB) 5 mg/kg as
positive control.
Determination of body and food intake
Body weights of mice and the intake of food and water and
were taken prior to the induction of hyperglycemia, at day
0 of treatment, and on a daily basis thereafter for 4 weeks.
Oral glucose tolerance test in diabetic mice
Animals of each group were orally administered EP extract
at doses of 400 mg/kg body weight on a daily basis for
30 days. At the end of the experiment, an oral glucose
tolerance test (OGTT) was performed to assess the animals’
sensitivity to a high glucose load. Overnight‑fasted rat were
fed orally 2 g glucose/kg b.w. Blood samples were collected
from the caudal vein from a small incision at the end of
the tail at 0 min (immediately after glucose load), 30, 60,
90 and 120 min after glucose administration.
Oral glucose tolerance test in normal mice
Oral glucose tolerance test was performed in overnight (16 h)
starved normal mice. The mice were randomly divided
into three groups (n = 6). Glucose 2 g/kg was fed 30 min
after the administration of 400 mg/kg of extract and
glibenclamide. Blood was withdrawn from the tail vein 0,
30, 60, 90 and 120 min, blood glucose level were appraised
by commercial kit (Sigma Aldrich, USA).
Collection of organ tissues
At the end of chronic diabetes experiments all mice
were anesthetized with 1.0% pentobarbital sodium and
blood was obtained from the retro‑orbital plexus of each
animal following the injection of heparin (100 IU kg
‑1
body weight) into a tail vein for 10 min. The liver and
kidney were removed according to dened anatomical
landmarks.
Plasma biochemical analysis
Blood samples were collected from tail vein of the mice
into micro centrifuge tubes containing heparin (10 µl,
1000 IU ml
‑1
). The blood samples were then centrifuged
at 1600 × g for 15 min at 4ºC for the preparation
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S409
of plasma. Concentrations of plasma glucose, total
cholesterol (TC), triglycerides (TG) and HDL‑cholesterol,
were measured with enzymatic assay kit (Genzyme
Diagnostics), LDL‑cholesterol was calculated as the
remaining difference of total cholesterol and HDL.
Blood glucose levels were measured employing the
glucose oxidase‑peroxidase (GOD‑POD) method.
Lipid peroxidation, i.e., thiobarbituric acid reactive
substances (TBARS) was estimated by the method of Fraga
et al.,
[33]
and expressed as µM/g of liver and kidney tissue.
Serum glutamate oxaloacetate transaminase (SGOT),
glutamate pyruvate transaminase (SGPT), serum alkaline
phosphatase (SALP) and total protein, using a commercial
Diagnostic Kit Biocompare, BioVision, Biocompare and
Thermo scientic respectively. Malondialdehyde (MDA)
as thiobarbituric acid reactive substances was measured at
532nm spectrophotometrically.
Antioxidant parameters levels in serum, liver,
pancreas and kidney
Activity of serum superoxide dismutase (SOD) was
measured by the xanthine oxidase method using
commercial kits with the absorbance measured using
spectrophotometer at 550 nm. Serum catalase (CAT) and
glutathione peroxidase (CSH‑Px) activities were measured
by the colorimetric method measuring absorbances
at 405 nm and 412 nm respectively. Glutathione
reductase (GSH) by measuring the rate of NADPH
oxidation at 340 nm. All the assay kits were purchased from
Cayman Chemical (Michigan, USA) and the procedures
were according to the kits instructions. In the pancreas
the protein concentration was determined by the Bradford
method as described in the Bio‑Rad protein assay kit.
Estimation al glucose metabolic enzymes activities
in liver tissues
When the mice were sacriced, the liver tissues were
removed and immediately frozen by liquid nitrogen
and stored at ‑80°C for further study. The activity of
glucokinase and glucose‑6‑phosphatase was assayed by the
color change of therapeutic massage and bodywork (TMB)
substrate using commercial EUSA kits purchased from R
and O system (USA) and the color change was measured
spectrophotometrically at the wavelength of 450 nm.
Protein concentration and liver tissue glycogen were
estimated using commercial kits purchased from Cayman
Chemical (Michigan, USA). All the tests were carried out
according to the kit instructions, respectively.
Anti‑AGES activity assay in vitro
Bovine serum albumin‑glucose assay
The methodology was based on that of Brownlee et al.
[34]
BSA (l0 mg/ml) was incubated with glucose (500 mM)
in phosphate buffered‑saline (PBS) (5 ml total volume,
pH 7.4) and extract containing 0.02% sodium azide at 37°C.
All the reagent and samples were sterilised by ltration
through 0.2 µm membrane lters. The protein, the sugar
and the prospective inhibitor were included in the mixture
simultaneously. Aminoguanidine was used as an inhibitor
positive control. Reactions without any inhibitor were also
set up. Each solution was kept in the dark in a capped
tube. After 15 days of incubation, uorescence intensity
(excitation wavelength of 370 nm and emission wavelength
of 440 nm) was measured for the test solutions.
BSA‑methylglyoxal assay
This assay was modied based on a published method.
[35]
The assay evaluates the middle stage of protein glycation.
BSA and methylglyoxal were dissolved in phosphate buffer
(100 mM, pH 7.4) to a concentration of 20 mg/ml and
60 mM, respectively. Extract or fractions were dissolved
in the same phosphate buffer. One millilitre of the BSA
solution was mixed with 1ml of methylglyoxal solution
and 1 ml of OM extract. The mixture was incubated at
37ºC. Sodium azide (0.2 g/l) was used as an aseptic agent.
Phosphate buffer was used as a blank. Aminoguanidine
and phloroglucinol were used as positive controls. After
seven days of incubation, uorescence of the samples was
measured using an excitation of 340nm and an emission
of 420nm, respectively.
Glycation of haemoglobin
Glycosylated haemoglobin (HbA
1c
) was estimated using
a commercial diagnostic kit from Sigma‑Aldrich (Human
haemolysate [glycated haemoglobin (HbA
1c
)] Kit).
Anti‑AGES activity assay In vivo
Thiobarbituric acid‑reactive substance level and
AGE level in kidneys
TBARS levels in serum were determined using the
method of Naito and Yamanaka.
[36]
Mitochondria
were prepared from kidney homogenate by differential
centrifugation (800 × g and 12000 × g, respectively) at
4°C according to the methods of Jung and Pergande
[37]
,
with minor modications. Each pellet was resuspended in
preparation medium and the concentration of TBA‑reactive
substance was determined by the method of Mihara and
Uchiyama.
[38]
The renal AGE level was determined by the method of
Nakagawa.
[39]
Briey, minced kidney tissue was degreasing
with chloroform and methanol (2:1, v/v) overnight. After
washing, the tissue was homogenised in 0.1 N NaOH,
followed by centrifugation at 8000 × g for 15 min at 4°C.
The amounts of AGEs in these alkali‑soluble samples were
determined by measuring the uorescence at an emission
wavelength of 440 nm and an excitation wave length of
370 nm. A native BSA preparation (l mg/ml of 0.1 N
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S410 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
NaOH) was used as a standard and its uorescence intensity
was dened as one unit of uorescence. The uorescence
values of samples were measured at a protein concentration
of 1 mg/ml and expressed in arbitrary units (AU).
The renal glucose level was determined by the method
of Momose.
[40]
with some modications. In brief, frozen
kidney tissue was homogenised with ice‑cold physiological
saline and, after being deproteinised, the content of glucose
was determined using the Wako kit described above.
Minced kidney tissue was delipidated with chloroform
and methanol (2: 1, v/v) overnight. After washing with
methanol and distilled water, the tissue was homogenised
in 0.1 NaOH, followed by centrifugation at 8000 × g for
15 min at 4°C. The amounts of AGEs in these alkali‑soluble
samples were determined by measuring uorescence at
an emission wavelength of 440 nm and an excitation
wavelength of 370 nm. A native BSA preparation (1 mg/ml
of 0.1 N NaOH) was used as a standard and its uorescence
intensity was dened as one unit of uorescence. The
uorescence values of samples were measured at a protein
concentration of 1 mg/ml and expressed in arbitrary units
(AU) compared with the native BSA preparation.
Statistical analysis
All experiments were performed in triplicate (n = 3) and
results were expressed as mean ± SEM. Statistical analysis
was carried out by using OriginPro 7.5 software. One way
ANOVA was applied to data and results were compared
by using Tukey test. A difference was considered to be
statistically signicant when the (P < 0.05).
RESULTS AND DISCUSSION
Due to the complex nature of the extract derived from
EP, it is not possible to evaluate the antioxidant activity of
the extract by employing only a single method. Therefore,
several radical‑scavenging assays were performed, to
determine the abilities of the extract to inhibit oxidation.
The total phenolic (TP) and total avanoid (TF) content
in methanol‑water extract were expressed as chemical
equivalents of gallic acid and catechin, respectively, since
different phenolic compounds contribute differently to the
readings using the Folin‑Ciocalteau reagent. TP and TF
values were 856.50 ± 13.53 and 368.29 ± 16.3, respectively,
indicating that the mean TF value corresponds to 43% of
the mean TP value.
The DPPH
•
radical is considered to be a model of a stable
lipophilic radical. This reaction has been widely used to test
the ability of compounds to act as free‑radical scavengers or
hydrogen donors and to evaluate the antioxidative activity
of plant extracts, In this test, the free radical scavenging
activity of EP was found to be 90.34% at 0.1 mg/ml while
showed 98.32% and ascorbic acid 86.53% at the same
concentrations [Figure 1].
The free‑radical scavenging activity of the extract was
conrmed in the TEAC assay, which is based on the ability
of antioxidants to quench the ABTS
+
radical‑cation. The
methanol/water extract of EP was fast and effective
scavengers of the ABTS radical [Figure 2] and this activity
was comparable to that of BHT. At 0.02 mg/ml, the extract
exhibited higher activity than BHT, but at 0.1 mg/ml the
activity of the extracts was similar to that of BHT. The
percentage inhibition was 98.34% for the extract and
99.42% for BHT at 0.1 mg/ml concentration. Proton
radical scavenging is an important attribute of antioxidants.
Peroxyl radicals are the most common radicals found in the
human body, but ORAC measurements should be more
biologically relevant. EP showed signicant antioxidant
potential and the values represent ORAC
ROO
+ activities of
the tested extract equivalent to Trolox. The results showed
EP with the ORAC
ROO
+ value of 5.12 ± 1.28 µmol
α‑tocopherol [Table 1].
Both assays (TEAC and ORAC) are inhibition methods:
TEAC reects the relative ability of hydrogen or electron
donating antioxidants to scavenge the ABTS radical‑cation
compared with Trolox, while ORAC is a method used
to measure the scavenging activity of peroxyl radicals.
The ORAC, ABTS and DPPH values indicated that EP
possesses signicant radical quenching properties.
Iron is the most important lipid pro‑oxidant. It is known
that Fe
2+
‑accelerates lipid peroxidation by breaking down
hydrogen and lipid peroxides formed by Fenton free radical
reaction. As shown in Figure 3, EP has the best antioxidants
of ascorbic acid and BHT, suggesting that EP is able to
chelate metals. The median inhibitory concentration values
for the EP, ascorbic acid and BHT were 23.67, 50.1 and
Figure 1: Free radical (DPPH) scavenging activity of the methanol/
water extract of the Bark of E. polystachya compared to ascorbic acid
and BHT. Values are mean ± SEM and significantly P< 0.05
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S411
47.2 µg/ml, respectively. EP and the standard antioxidant
ascorbic acid and BHT competes for the metal with ferrozine,
suggesting that they have chelating activity, capturing the
ferrous ion before it can form a complex with ferrozine.
[41]
Table 1, illustrates the decrease in the concentration of
nitric oxide free radicals due to the scavenging ability of EP
and quercetin. A 50 µg/ml solution of EP and quercetin
exhibited 74% and 70.45% inhibition, respectively. NO
scavenging capacity of extract was higher than that
of quercetin. In this study, the ONOO‑ scavenging
ability of EP was also investigated. The result showed
that EP possesses a moderate scavenging activity with
IC
50
=
2.02 ± 0.98 µg/ml compared to penicillamine, with
an IC
50
=
0.45 ± 0.0 µg/ml. Nitric oxide (NO) is a particular
oxygen reactive species; although its excess can produce
a harmful effect in the organism, it is a very important
cell mediator that regulates a number of functions and
cellular processes in the organism.
[42]
As a member of
reactive species, ONOO‑ has been implicated in several
major chronic diseases such as diabetes, Alzheimer’s
disease, rheumatoid arthritis, cancer and atherosclerosis.
In the current research, EP was shown to have a moderate
scavenging activity against ONOO‑.
The antioxidant compounds in the plant extract prevent
the oxidation of β‑carotene [Figure 4]. The extract showed
high antioxidant activity (80.9 ± 9.7%) by inhibiting the
formation of conjugated dienes. It can be concluded
that the methanolic/water extract had a similar activity
to those of the standards BHA (81.3 ± 1.7%) and BHT
(84.2 ± 1.1%) (P < 0.05). EP exhibited the highest
antioxidant activity of β‑carotene remaining after 20 min.
Hydrophobic antioxidants are reported to perform more
efciently than hydrophilic antioxidants in the β‑carotene
bleaching test, because they have an affinity for the
lipid phase, where they can react with peroxyl radicals,
avoiding β‑carotene oxidation. The strong activity of
EP components may be due to their higher level of
hydrophobic antioxidants.
The production of highly reactive oxygen species such as
superoxide anion radicals is catalysed by free iron through
Haber‑Weiss reactions;
[43]
superoxide anions are also
implicated as harmful ROS, as they have a detrimental
effect on the cellular components in a biological system.
Superoxide anions indirectly initiate lipid oxidation by
generating singlet oxygen.
[41]
Table 1 presents the superoxide radical scavenging activity
of 50 µg/ml EP in comparison with the same dose
of quercetin. EP had a superoxide radical scavenging
activity (62.19% inhibition), which was greater than that
of reference antioxidant (52.97% inhibition) at the same
concentrations (P < 0.05).
Another ROS, singlet oxygen, which is a high energy form
of oxygen, is generated in the skin upon UV‑irradiation.
Singlet oxygen induces hyperoxidation, oxygen cytoxicity
and decreases the antioxidant activity.
[44]
ROS scavenging
activities of the extract are shown in Table 1. Methanol/
water extract showed the radical scavenging activities
against HOCl, ONOO
‑
, NO, O
2
‑
, OH, H
2
O
2
and ORAC.
Hydroxyl radical is one of the ROS formed in biological
systems, causing DNA strand breakage, which brings
about carcinogenesis, mutagenesis and cytotoxicity.
[45]
The
Figure 2: ABTS•+ scavenging activity of the methanol‑water extract of
the leaves of E. polystachya. Values are mean ± SEM and significantly
P< 0.05
,QKLELWLRQ
&RQFHQWUDWLRQPJPO
([WUDFW $VFRUELF$FLG %+7
Figure 3: Formation of the Fe
2+
‑ferrozine complex of methanol‑water
extract of the bark of E. polystachya and EDTA. Values are mean ±
SEM and significantly P<0.05
$EVRUEHQFH
7LPHPLQ
%+$ %+7 (3
Figure 4: Absorbance change of β- carotene at 490 nm in the presence
of EP and standards, BHT and BHA. Values are mean ± SEM and
significantly P<0.05
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S412 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
addition of EP to the reaction mixture removes hydroxyl
radicals and prevents further damage. The EP observed
value indicates that the bark extract is a better hydroxyl
radical scavenger than the standards BHT, Trolox and
mannitol with values of 74.28, 73.59, 64.12 and 69.67%,
respectively. EP is a source of antioxidant compounds,
especially for scavenging the highly reactive hydroxyl
radicals. It has been observed that H
2
O
2
through the Fenton
reaction is an active oxygen species and has the potential
to produce the highly reactive hydroxyls radical which are
often involved in free radical chain reactions known to cause
damage to biological macromolecules. This result suggests
that antioxidants in the methanol/water extract were more
reactive toward O
2
‑
. Butylated hydroxyanisole (BHA),
butylated hydroxyl‑tolueno (BHT), quercetin, penicillamine,
lipoic acid and ascorbic acid were used as standards. The
extract was an effective scavenger of ROS and this activity
was comparable to that of the standards used.
At the sites of inammation, the oxidation of Cl
‑
ions
by the neutrophil enzyme myeloperoxidase results in the
production of another harmful ROS, hypochlorous acid.
[44]
HOCl has the ability to inactivate the antioxidant enzyme
catalase through the breakdown of the heme‑prosthetic
group. From the results obtained, it is anticipated that EP
is a more efcient scavenger than standard ascorbic acid.
Table 2 shows the oxidative modication of LDL by CuSO
4
,
as well as the antioxidant activity of EP. The extract inhibited
the chemically‑induced LDL oxidation. The effect of EP
on LDL may be due to the polyphenol content, since these
compounds have been found to act against LDL oxidation
and their capacity is related to their chemical structures.
[46]
In order to determine the inhibitory effect of the methanol/
water extract from Eysenhardtia polystachya on the formation
of AGEs, several assay methods have been proposed,
including assays based on the inhibition of specific
uorescence generated during the course of glycation
and AGE formation and assays based on the inhibition
of AGE‑protein cross‑linking. Table 3 displays the
inhibitory effects of EP on AGE formation in BSA‑glucose
and BSA‑methylglyoxal models. EP, phloglucinol and
aminoguanidine exhibited higher inhibitory activity
against AGE formation after the incubation at 37°C for
15 days, with IC
50
values of 0.204, 0.070 and 0.323 mg/ml,
respectively. Methylglyoxal‑mediated protein glycation
inhibition was evaluated for EP, which exhibited substantial
activity compared with methylglyoxal; this has received
considerable attention as a mediator of advanced glycation
end‑product formation and are known to react with
lysine, arginine and cysteine residues in proteins to form
glycosylamine protein crosslinks.
[47]
In this study, we found that EP inhibited the formation of
methylglyoxal‑derived advanced glycation end‑products in a
bovine serum‑albumin‑methylglyoxal system and may also
act by blocking the conversion of dicarbonyl intermediates
to advanced glycation end‑products.
Haemoglobin A
1C
, a biomarker for chronic exposure to high
concentrations of glucose, was also signicantly decreased
in STZ‑induced diabetic mice. Table 4 shows the amount
of glycated haemoglobin (%GHb). When haemoglobin
was used alone (NC), the amount of glycated haemoglobin
was 9.5%. This noticeably increased with the addition
of glucose to a 27.6% (PC). Nonetheless, it decreased
significantly with the treatment of EP (23.8%) and
dropped further with the treatment of glutathione (8.1%).
The amount of haemoglobin A
1c
(%HbA
1c
) corresponds to
a specic sub‑fraction of glycated haemoglobin; it is lower
than the amount of glycated haemoglobin. However, it
showed a similar tendency in the percentage of glycation.
This result indicates that EP demonstrates the most potent
glycation inhibition in the early stages of protein glycation
at a concentration of 10 mg/ml. The plant, therefore, can
effectively prevent HbA
1C
formation. The formation and
accumulation of AGEs in various tissues increases rapidly
in chronic diabetes. We found that EP decreased the in vitro
formation of uorescent AGE and HbA
1C
, a kind of AGE.
Table 1: Antioxidant activities of methanol/water (EP) extract of E. polystachya
% inhibition
Sample ORAC Peroxynitrite NO radical H2O2 RO2 Hydroxyl Singlet HOCl De Superoxide
α‑tocopherol IC50 µg/ml scavenging scavenging radical radical oxygen radical radical equivalents
EP 6.12±1.28 µmol 2.32±0.98 74 69.46 28.6 74.28 78.23 73.12 62.19
BHA 75.19
BHT 78.56 73.59
Trolox 72.71 64.12
Mannitol 69.67
Penicillamine 0.45±0.0
Quercetine 70.45 52.97
Ascorbic acid 64.19
Lipoic acid 76.46
α‑tocopherol 27.4
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S413
Thus, EP may possess specic antiglycation properties
that contribute to the reduction in HbA
1C
levels. EP could
directly decrease the formation of glycated haemoglobin,
possibly as a result of the antioxidant activity.
The renal glucose level in diabetic control mice at the end
of the experiment was signicantly increased compared
with normal mice (0.81 ± 0.004 mg/g wet tissue). However,
treatment with 200 and 400 mg of EP led to a slight
increase to 6.96 and 6.49 mg/g wet tissue, respectively
[Table 4]. Additionally, diabetic control mice showed an
increase in the uorescent AGE level compared with
that of normal mice (P < 0.01). However, in mice treated
with EP for 30 days, this was signicantly decreased in
the 400 mg‑treated group [Table 4]. These effects may
be caused by the antioxidant activity possessed by EP,
because we found that EP scavenged free radicals. Another
possible cause may be the direct inhibitory activity of EP
on glycation, resulting in a decrease in free radical release.
Effect of single and repeated oral administration of EP on
blood glucose levels in STZ‑diabetic and normoglycemic
mice are presented in Table 5 and Table 6 respectively. EP,
administered at three different doses of 100, 200 and 400 mg/
kg, to normoglycemic and STZ‑treated diabetic mice (SD and
MD) caused signicant reduction of blood glucose levels
which was related to dose and duration of treatment. STZ
produced signicant loss in body weight as compared to
normal animals during the study. Diabetic control continued
to lose weight till the end of the study while EP at dose of
400 mg/kg, showed signicant improvement (P < 0.05) in
body weight compared to diabetic control [Table 10]. All
the diabetic mice consumed more feed than normal mice
throughout the experiment compared to their respective
control groups, indicating the probable appetite‑enhancing
property or a decreased efciency in feed utilization.
In the oral glucose tolerance test, the blood glucose levels
of glucose treated diabetic mice were increased markedly at
30 min. EP at dose of 400 mg/kg inhibited the increasing
blood sugar level signicantly (P < 0.05) at the 60 min and
120 min when compared with disease control [Table 6].
Effect of EP on oral glucose tolerance test in normal
mice is shown in Table 7. The different doses (100, 200
and 400 mg/kg) produced a signicant reduction in blood
glucose level at 120 min when compared to the vehicle
control.
Free radicals are formed disproportionately in diabetes
mellitus by glucose degradation, non‑enzymatic glycation
of proteins and the subsequent oxidative degradation.
[48]
Increased oxidative stress is involved in diabetes. There is
evidence that glycation itself induces the generation of
oxygen‑derived free radicals in diabetic condition.
[49]
The
generatian of free radicals may lead to lipid peroxidation
in diabetes mellitus.
[48]
Diabetic mice showed a signicant reduction in SOD,
CAT, GSH and GPx in hepatic, pancreatic and renal
tissues. Levels of these enzymes reverted close to normal
values after treatment with EP extract [Table 8]. These
results suggest that EP prevents oxidative stress, acts as
a suppressor of liver, kidney and pancreas cell damage
and inhibits the progression of dysfunction induced by
chronic hyperglycaemia. In total, these results suggest
that the protection shown by EP extract may be due to its
antioxidant properties.
STZ induction of diabetes in mice leads to lipid
peroxidation. TBARS are an indication of endogenous lipid
peroxidation and oxidative stress as a result of intensied
free radical production. Increased TBARS suggests an
increase in oxygen radicals that could be caused either by
increased production or by decreased destruction. These
elevated TBARS levels in diabetic mice might be due to the
stimulation of hepatic triglyceride synthesis as a result of
free fatty acid inux. Daily administration of extract at a
dose of 400 mg/kg to diabetic mice for 30 days signicantly
reduced TBARS levels by 50.4% in the liver and 57.3% in
the pancreas [Table 9].
The effects of EP on TBARS levels were also evaluated in
serum and renal mitochondria. As shown in Table 9, the
serum TBARS level in diabetic control mice was signicantly
Table2: Effects of EP on LDL oxidation induced-
CuSO4 in vitro
Groups (µg) TBARS (nmol MDA) CD
Control 5.67±1.28 8.2±2.60
200 8.19±2.21
a
27.5±5.35
a
300 14.34±4.51
a
21.6±8.52
a
500 26.87±5.76
a
15.2±3.41
a
Values are expressed as mean±SD of three independent experiments;
a
Signicantly (P<0.05) dierent from control, where the signicance was performed by
oneway ANOVA followed by post hoc dunnett's test. EP: Eysenhardtia polystachya;
LDL: Low-density lipoprotein; MDA: Malondialdehyde; TBARS: Thiobarbituric acid-
reactive substance
Table3: Effects of EP on glyco-oxidative
damage to BSA by glucose and methylglioxal
Inducer Treatment AGEs IC
50
(mg/ml)
Glucose Extract (EP) 0.204±0.032
Aminoguanidine 0.323±0.081
Phloroglucinol 0.070±0.0049
Methylglyoxal EP 0. 87±0.029
Aminoguanidine 0.195±0.021
Phloroglucinol 0.060±0.0072
Data are mean±standard deviation of triplicate tests. EP: Eysenhardtia polystachya;
AGEs: advanced glycation end products
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S414 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
elevated compared to that of normal (2.3‑times) and the oral
administration of EP at concentrations of 200 and 400 mg
signicantly inhibited this increase compared to the diabetic
control group (P < 0.05). The renal mitochondrial TBARS
level was also signicantly increased in diabetic control mice
(1.97 ± 0.03 nmol/mg protein) compared with normal
mice (1.55 ± 0.05 nmol/ml), but levels in groups treated with
EP were signicantly reduced to the normal level (1.59 ± 0.08
Table4: The inhibitory activities of EP on glycation protein and hemoglobin. Effect of EP on glucose,
weight and ages levels in kidney
Groups GHb HbA
1c
Glycosylated protein
(nmol/mg protein)
Renal
weight (g)
Age (AU) Renal glucose
(mg/g wet tissue)
Negative control 8.9±0.06 7.9±0.98 15.3±1.47 - - -
Positive control 27.6±5.34 17.5±4.10 23.7±2.19 - - -
Methanol extract 23.8±5.53
a
15.3±3.19
a
20.6±2.04
a
- - -
Glutathione 8.1±1.08
a
9.0±0.67
a
- - - -
Aminoguanidine - - 20.2±1.87
a
- - -
Normoglucemic - - - 0.75±0.074
a
16.03±2.19
a
0.81±0.004
a
Diabetic - - - 1.09±0.065 24.25±3.28 6.49±1.67
Diabetic+EP 200 mg/kg - - - 0.94±0.048
a
12.87±1.74
a
6.96±1.07
a
Diabetic+EP 400 mg/kg - - - 0.87±0.025
a
14.90±4.19
a
6.78±0.89
a
Negative control: Incubation with hemoglobin (30 mg/ml), positive control: Incubation with hemoglobin (30 mg/ml) + glucose (0.278 mM), extract: Incubation with
hemoglobin (30mg/dl) + glucose (0.278 mM) + extract (10 mg/ml), glutathione: Incubation with hemoglobin (3 mg/dl) + glucose (2.7 mM) + glutathion (0.5 mM). Data as
expressed as±SD;
a
P<0.05 vs positive control values
Table5: Effect of a single oral administration of EP on fasting blood glucose level of in non-diabetic
and diabetic mice
Groups/dose
(mg/kg)
(mg/dL blood glucose)
0h 2h 4h 6h 8h 12h
Normal 106.2±3.47 104.4±4.32 104.5±3.49 103.8±1.51 104.3±2.40 103.5±1.81
Normal+EP 100 104.3±2.81 98.7±4.65 90.4±2.40
a
80.5±6.78 77.43±3.98
a
102.9±6.81
Normal+EP 200 98.7±5.49 92.3±4.39
a
81.6±3.76
a
70.1±5.72 76.2±3.07
a
96.7±6.12
a
Normal+EP 400 109.8±4.76 94.5±6.33
a
83.2±8.0
a
66.2±3.88 70.6±5.39
a
87.3±6.28
a
MD control 202.6±2.17 201.8±1.64 202.4±2.54 201.0±2.0 201.4±2.07 200.8±1.92
MD+EP 100 265.4±9.45 260.3±7.43 250.2±5.68
a
238.6±7.42 241.4±5.78
a
247.6±8.46
a
MD+EP 200 263.8±5.52 252.4±6.38 245.0±4.39
a
221.1±2.87 236.3±1.30
a
236.2±1.78
a
MD+EP 400 272.7±10.21 253.2±9.58
a
223.5±8.90
a
201.3±8.90 180.2±6.72
a
198.4±7.34
a
SD control 278.3±2.32 275.9±2.86 276.2±2.07 280.6±2.68 279.5±2.28 278.7±1.64
SD+EP 100 222.7±3.65 202.3±5.86 190.0±4.91 183.3±7.53 174.1±6.78
a
186.7±6.51
a
SD+EP 200 252.4±4.52 214.3±3.19
a
194.7±7.39
a
181.6±8.92 172.6±5.91
a
190.3±4.28
a
SD+EP 400 235.2±7.57 203.5±8.50 182.3±6.68
a
169.1±2.88 179.5±9.52
a
161.2±2.89
a
SD+GB 201.5±0.99 186.3±3.11
a
221.4±3.38
a
139.3±1.78 143.8±2.30
a
156.2±1.92
a
MD+GB 277.8±1.97 250.8±2.86
a
152.0±2.23
a
185.3±2.88 191.2±1.64
a
210.6±1.51
a
Each values represent mean±SD (n=6).
a
P<0.05 compared to normal group (ANOVA) followed by Dunnett's test.
b
P<0.05 compared to diabetic group (ANOVA) followed by
Dunnett's test. GB: Gibenclamide; EP: Eysenhardtia polystachya; SD: Standard deviation
Table6: Effect of EP on blood glucose level and oral glucose tolerance test in diabetic mice after
30 day treatment
Group
(mg/kg)
Fasting blood glucose level (mg/dl) week of supplements Blood glucose levels (mg/dl) minutes
0 1 2 3 4 0 30 60 90 120
No-diabetic
control
100.7±5.80 101.3±6.29
a
103.2±1.87
a
106.6±1.67
a
107.3±1.37
a
109.3±1.37
a
177.8±168 166.5±1.21 123.6±2.00
a
102.2±1.30
a
SD control 378.3±2.32 383.6±1.15
b
375.6±1.67
b
383.7±1.48
b
381.6±1.18
b
372.8±6.91 423.6±6.98
a
430.5±4.87
a
397.6±7.49
a
372.6±5.89
a
MD control 202.2±1.68 210.1±1.18
b
232.4±2.01
b
243.9±1.89
b
250.3±2.48
b
223.3±5.87 276.3±8.95
a
265.3±7.84
a
219.2±5.65
a
159.2±10.55
a
SD+EP 348.8±5.71 330.3±6.23
c
324.5±6.21
c
299.3±2.34
c
276.5±3.87
c
337.75±8.21 394.2±7.19
a
440.4±5.17
c
429.3±5.31
c
360.4±11.03
c
MD+EP 211.5±4.18 180.2±1.69
c
161.3±5.19
c
139.4±6.43
c
121.0±2.78
c
259.6±8.38 290.1±9.63
a
290.1±7.37
c
229.0±7.29
c
126.7±3.59
c
SD+GB 377.8±1.97 360.4±1.48
c
333.1±1.11
c
300.9±2.09
c
288.6±2.02
c
368.6±4.39 339.1±3.88
a
398.3±7.38
c
387.6±9.04
c
364.8±4.96
c
MD+GB 212.5±0.99 192.8±1.15
c
177.4±1.67
c
158.6±1.43
c
136.3±2.48
c
246.3±3.98 282.3±7.07
a
158.1±6.97
c
116.5±8.11 107.9±6.23
c
Each value represents mean±S.E.M. (n=10), ANOVA followed by multiple two-tail “t” test. In each vertical column, mean with dierent superscripts (a, b, c) dier from “t” each
other signicantly, <0.05. Glibenclamide (GB) at doses 5mg/kg. EP: Eysenhardtia polystachya; SD: Standard deviation
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S415
Table7: Effect of EP on oral glucose tolerance test in normal mice
Groups Time (min)
0 30 60 90 120
Glucose control 92.6±7.45 123.7±8.92 120.7±6.21 102.8±9.44 98.5±7.49
Glucose+EP (100 mg/kg) 88.7±5.98 110.1±8.99
a
102.6±10.23
a
97.3±12.42
b
87.1±10.31
c
Glucose+EP (200 mg/kg) 90.3±8.67 102.7±11.42
a
98.0±13.17
b
87.7±8.67
c
82.5±9.33
c
Glucose+EP (400 mg/kg) 87.8±9.12 101.4±10.23
b
96.3±12.54
c
84.6±7.45
c
79.5±9.33
c
Glucose+GB (5mg/kg) 89.0±7.59 99.2±6.87
b
85.4±5.88
c
80.6±12.64
c
72.6±8.74
c
All values represent mean±
a
P<0.05;
b
P<0.01;
c
P<0.001; ANOVA, followed by Dunnett's multiple comparison test. EP: Eysenhardtia polystachya; GB: Glibenclamide
Table8: Effect EP on antioxidant enzyme activities in liver, pancreas and kidney in diabetic mice
Parameters Normal
control
Diabetic
control
Diabetic+EP
(200 mg/kg)
Diabetic+EP
(400 mg/kg)
Diabetic+GB
(5 mg/kg)
SOD-liver 7.65±2.54 3.29±1.57
a
5.04±2.29
c
6.85±2.09
b
6.82±2.18
c
SOD-kidney 14.03±0.36 8.04±2.49
a
12.26±4.37
b
13.74±2.58
b
12.97±1.26
b
SOD-pancreas 54.1±3.54 35.76±5.15 42.21±6.42
b
49.89±6.52
c
51.78±4.77
b
CAT-liver 82.10±1.79 43.35±4.94
a
75.31±5.47
c
80.07±3.80
b
74.67±5.49
b
CAT-kidney 34.85±2.65 21.49±1.58
a
30.35±3.43
b
33.99±4.60
c
34.32±1.76
b
CAT-pancreas 59.6±3.17 25.41±3.12
a
46.29±7.80
c
51.49±9.21
c
51.29±4.48
c
GSH-liver 46.48±2.34 23.72±1.80
a
40.39±6.54
b
44.87±8.73
b
42.87±3.31
b
GSH-kidney 24.11±0.73 5.78±0.84
a
21.63±8.41
b
23.12±6.68
b
19.86±1.13
b
GSH-pancreas 11.9±1.23 6.58±0.91
a
8.89±1.39
c
10.48±3.87
c
10.98±1.54
c
GPx-liver 7.43±2.17 4.56±0.24
a
5.90±1.52
c
6.65±2.27
b
5.90±0.75
b
GPx-kidney 5.89±0.78 3.49±0.18
a
4.38±0.36
b
5.01±1.17
b
4.53±0.90
b
GPx-pancreas 4.12±1.09 2.18±0.63
a
3.20±0.53
c
3.89±0.82
b
3.89±0.68
c
All values are expressed as mean±SEM, n=6 values.
a
P<0.01 when compared to normal control group;
b
P<0.01 and
c
P<0.05 compared to diabetic control group; where the
signicance was performed by oneway ANOVA followed by post hoc Dunnett's test. Glibenclamide (GB). The values are given in U/mg of protein. SOD: Superoxide dismutase;
CAT: Catalase; GSH: Glutathione reductase; STZ: Streptozotocin
Table9: Effect of EP on TBA-reactive substance levels in liver, pancreas, serum and renal Mitochondrial
in experimental mice
Group TBARS (mmol/mg protein)
Liver Pancreas Serum Renal mitochondrial
Normal control 1.15±0.35 0.484±0.001 1.62±0.06 1.55±0.05
Diabetic control 2.18±0.61
a
2.87±0.054
a
3.64±0.08 1.97±0.03
Diabetic+EP (200 mg/kg) 1.65±0.12
b
1.43±0.061
b
1.71±0.03 1.59±0.08*
Diabetic+EP (400 mg/kg) 1.10±0.23
b
1.25±0.036
b
1.65±0.09 1. 57±0.02*
Diabetic+GB (0.5 mg/kg) 1.03±0.40
b
1.22±0.029
b
1.62±0.05 1. 53±0.02*
All values are expressed as mean±SEM, n=6 values.
a
P<0.01 when compared to normal control group;
b
P<0.01 compared to diabetic control group; Glibenclamide (GB). The
values are given in mmol/mg of protein. EP: Eysenhardtia polystachya
Table10: Effect of EP on lipid profile, renal and hepatic functions. Body weight, water and food intake
in streptozotocin-induced diabetic mice
Biochemical parameter Normal
control
SD
control
MD
control
SD+ 400
mg/kg
MD+ 400
mg/kg
SD+GB MD+GB
Fasting plasma insulin (µU/ml) 14.3±4.56 4.1±0.62 6.21±1.43 9.32±2.05 10.28±1.76 12.7±3.11 13.8±2.54
Hexokinase (µg/mg of tissue) 148.3±6.98 91.5±4.11 98.9±4.10 129.1±5.32 137.4±5.31 139.2±3.19 143.7±4.23
Glucose-6-phosphatase (unit/mg of tissue) 9.1±1.26 14.6±2.18 13.1±1.97 11.3±2.87 10.5±1.76 9.8±2.24 8.6±1.42
Total cholesterol (mg/dl) 79.0±4.67 129.4±2.67 123.5±6.71 111.1±2.43 95.7±2.40 90.1±3.58 86.7±2.54
Triglycerides (mg/dl) 82.5±1.65 135.8±4.73 129.0±2.93 114.2±3.29 103.6±1.98 94.6±2.41 90.8±4.18
HDL cholesterol (mg/dl) 54.8±1.39 27.1±4.80 29.6±1.89 41.5±1.39 47.2±5.17 51.2±1.68 53.6±1.42
LDL cholesterol (mg/dl) 9.0±3.26 74.28±2.87 68.5±3.9 35.8±5.28 32.17±6.63 19.3±2.78 13.2±2.65
Weight variation (g) 30.7±1.45 36.2±1.39 34.1±1.37 33.2±2.18 32.4±2.07 32.5±2.19 31.1±1.45
Water intake (ml/day) 41.3±6.8 139.8±8.2 131.8±6.3 120.6±5.9 113.4±8.7 110.0±8.6 103.5±6.4
Food intake (9/day) 22.3±5.8 31.4±1.7 29.5±2.8 28.4±1.2 26.7±0.7a 24.6±6.3a 23.5±7.5a
Each values represent mean±SD (n=6).
a
P<0.05 compared to normal group (ANOVA) followed by Dunnett's test.
b
P<0.05 compared to diabetic group (ANOVA) followed by
Dunnett's test. GB: Gibenclamide; SD: Standard deviation; STZ: Streptozotocin
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S416 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
and 1. 53 ± 0.02 nmol/mg protein, respectively (P < 0.05).
Therefore, we hypothesised that EP might mitigate oxidative
stress in the liver, pancreas and kidney in STZ‑induced diabetic
mice. The present investigation showed that the methanol/
water extract of bark of EP contains a high amount of
avonoids and phenolics, possesses considerable antioxidant
activity with ROS scavenging activity and has the ability to
reduce lipid peroxidation. It also has iron chelating, TEAC
and DPPH activities and we also have proven that the oral
administration of EP could decrease oxidative stress associated
with diabetes mellitus in the liver, pancreas and kidney,
enhancing the generation of typical antioxidant enzymes.
To evaluate the effect of EP on lipid prole level, the
level of cholesterol, triglyceride and LDL (low density
cholesterol) were increased and the level of HDL (high
density cholesterol) was decreased in the STZ‑induced
diabetic mice. Oral administration of EP was showed
reduction in cholesterol, triglyceride and LDL compared
to the diabetic control mice group and the level of HDL
was increased compared to the diabetic control mice in a
signicant manner (P < 0.001) [Table 10].
Insulin deciency is associated with hypercholesterolaemia
and hypertriglyceridaemia. STZ‑induced diabetes showed
increased plasma levels of cholesterol, triglyceride, free
fatty acid and phospholipids. Insulin deciency or insulin
resistance could be responsible for dyslipidaemia because
insulin increases fatty acid as well as triglyceride synthesis in
adipose tissue and liver.
[50]
Insulin deciency leads to fall in
lipoprotein lipase activity. In our study, STZ‑mice showed
hypercholesterolaemia and hypertriglyceridaemia and the
treatment with EP signicantly decreased both cholesterol
and triglyceride levels.
There was a signicant (P < 0.05) decrease in the level
plasma insulin in untreated diabetic mice compared
to normal control mice. Oral administration of EP
(400 mg/kg) daily for a period of 30 days to diabetic mice
signicantly (P < 0.05) increased the level of serum insulin
compared to diabetic mice [Table 10].
Table 10 shows the effect of the methanol extract on
G6Pase, GK, HK activity and glycogen content of liver and
skeletal muscle. Administration of EP at 400 mg/kg body
weight, increased the content of hepatic glycogen, GK and
HK in diabetic rat while G6Pase decreased. Biochemical
parameters like SGOT, SGPT, SALP and proteins in the
STZ control group were signicantly (P < 0.001) elevated
as compared with the normal control group. Treatment
with EP at the dose of 400 mg/kg b.w. signicantly
(P < 0.001) brought the SGOT, SGPT, SALP and serum
protein toward the normal values [Table 11]. In increase
in activities of these enzymes might be mainly due to the
leakage from the liver cytosol into blood stream which
gives an indication of the hepatotoxic effect of STZ.
[51]
Reductions in the activity of these enzymes in EP
treated diabetic mice indicated the hepato protective role
in preventing diabetic complications.
CONCLUSION
Our results in this experiment showed that E. polystachya
has an antidiabetic, antihyperlipidemic, a significant
ability to reduce the formation of AGEs and antioxidant
activities, which are considered to play important roles in
the development of diabetes complications. Therefore, this
plant may have relevance in the prevention and treatment
of diseases in which oxidants or free radicals or AGEs
are implicated. As a result, chemical studies are now being
undertaken to characterise these bioactivities.
REFERENCES
1. Ames BN. Dietary carcinogens and anticarcinogens: Oxygen
radicals and degenerative diseases. Science 1983;221:1256‑64.
2. Sumino M, Sekine T, Ruangrungsi N, Igarashi K, Ikegami F.
Ardisiphenols and other antioxidant principles from the fruits of
Ardisia colorata. Chem Pharm Bull 2002;50:1484‑7.
3. Annadurai T, Muralidharan AR, Joseph T, Hsu MJ, Thomas PA,
Geraldine P. Antihyperglycemic and antioxidant effects of a
avanone, naringenin, in streptozotocin‑nicotinamide‑induced
experimental diabetic rats. J Physiol Biochem 2012;68:307‑18.
4. Zhang Y, Liu D. Flavonol kaempferol improves chronic
Table11: Effect of EP on glycogen level and hepatic enzyme activities in non-diabetic and diabetic mice
Group Glycogen (mg/g) (IU/l) ALP (g/dl) Total
protein
Liver Skeletal muscle SGOT SGPT
Normal control 20.72±4.45 10.98±3.21 20.9±3.2 24.3±5.8 170.3±10.4 7.9±2.1
SD control 9.04±6.72
a
4.89±1.34
b
39.2±8.9
a
42.3±7.1
b
245.1±11.6
b
4.3±1.5
b
MD control 11.65±3.75
a
5.27±1.56
b
38.7±7.7
a
42.9±8.6
a
239.8±9.7
a
4.0±1.2
b
SD+EP d14.87±6.89
b
d8.65±3.40
b
18.5±8.0
b
21.7±5.3
b
210.4±10.4
b
5.7±1.6
b
MD+EP d16.32±7.63
b
d9.96±3.73
b
20.3±5.7
b
23.5±4.1
b
201.0±8.5
b
6.2±2.0
b
GB (5 mg/kg) 17.78±2.30
b
11.01±1.96
b
23.1±8.0
b
25.1±5.7
b
192.1±11.8
b
7.2±3.8
b
Each values represent mean±SD, (n=10); ANOVA followed by multiple two tail “t” test. In each vertical column, mean with dierent superscripts (a, b) dier from each other.
Signicant dierence of diabetic control from normal control
a
P<0.001. Signicant dierence of treated groups from diabetic control
b
P<0.01,
c
P<0.05,
d
P<0.01 when compared
with glibenclamide 5mg/kg treated group. GB: Gibenclamide; SD: Standard deviation; EP: Eysenhardtia polystachya
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2) S417
hyperglycemia-impaired pancreatic beta-cell viability and insulin
secretory function. Eur J Pharmacol 2011;670:325‑32.
5. Lin CY, Ni CC, Yin MC, Lii CK. Flavonoids protect pancreatic
beta-cells from cytokines mediated apoptosis through the
activation of PI3‑kinase pathway. Cytokine 2012;59:65‑71.
6. Rahbar S, Figarola JL. Novel inhibitors of advanced glycation
endproducts. Arch Biochem Biophys 2003;419:63‑79.
7. Perez RM, Vargas R, Perez GS, Zavala S. Antiurolithiatic activity
of Eysenhardtia polystachya aqueous extract on rats. Phytother
Res 1998;12:144‑5.
8. Burns DT, Dalgarno BG, Ggargan P, Grimshaw J. An isoavone
and a coumestan from Eysenhardtia polystachya-robert boyle’s
uorescent acid‑base indicator. Phytochemistry 1984;3:167‑9.
9. Alvarez L, Rios MY, Esquivel C, Chávez MI, Delgado G,
Aguilar MI, et al. Cytotoxic isoavans from Eysenhardtia
polystachya. J Nat Prod 1998;61:767‑70.
10. Beltrami E, Bernardi M, Fronzat G, Mellerio G, Vidari G, Vita‑nzi
P. Coatline A and B, two C-glucosyl-α-hydroxydihydrochalcones
from Eysenhardtia polystachya. Phytochemistry 1982;21:2931‑3.
11. Alvarez L, Delgado G. C- and O-Glycosyl-cx-
hydroxydihydrochalcones from Eysenhardtia polystachya.
Phytochemistry 1999;50:681‑7.
12. Narváez-Mastache JM, Soto C, Delgado G. Antioxidant
evaluation of Eysenhardtia species (Fabaceae): Relay synthesis
of 3-O-Acetyl- 11α,12α-epoxy-oleanan-28,13β-olide isolated
from E. platycarpa and Its Protective Effect in Experimental
Diabetes. Biol Pharm Bull 2007;30:1503‑10.
13. Narvaez-Mastache JM, Novillo F, Delgado G. Antioxidant
aryl‑prenylcoumarin, avan‑ 3‑ ols and avonoids from
Eysenhardtia subcoriacea. Phytochemistry 2008;69:451‑6.
14. Narvaez-Mastache JM, Garduño-Ramírez ML, Alvarez L,
Delgado G. Antihyperglycemic activity and chemical constituents
of Eysenhardtia platycarpa. J Nat Prod 2006;69:1687‑91.
15. Kahkonen MM, Kylli P, Ollilainen V, Salminen J, Heinonen M.
Antioxidant activity of isolated ellagitannins from red raspberries
and cloudberries. J Agric Food Chem 2012;60:1167‑74.
16. Gyam MA, Yonamine M, Aniya Y. Free‑radical scavenging
action of medicinal herbs from Ghana: Thonningia sanguinea
on experimentally induced liver injuries. Gen Pharmacol
1999;32:661‑7.
17. Mahakunakorn P, Tohda M, Murakami Y, Matsumoto K,
Watanabe H. Antioxidant and free radical-scavenging activity
of Choto-san and its related constituents. Biol Pharm Bull
2004;27:38‑46.
18. Cao G, Alessio HM, Cutler RG. Oxygen-radical absorbance capacity
assay for antioxidants. Free Radic Biol Med 1993;14:303‑11.
19. Yildrim A, Oktay M, Bilaloglu V. The antioxidant activity of the
leaves of Cydonia vulgaris. Turk J Med Sci 2001;31:23‑7.
20. Sreejayan N, Roa MN. Nitric oxide scavenging by cucurminoids.
J Pharm Pharmacol 1997;49:105‑7.
21. Beckman S, Chen H, Ischiropulos H, Crow JP. Oxidative
chemistry of peroxynitrite. Methods Enzymol 1994;233:229‑40.
22. Singh N, Rajini PS. Antioxidant-mediated protective effect of
potato peel extract in erythrocytes against oxidative damage.
Chem Biol Interact 2008;173:97‑104.
23. Ozen T. Investigation of antioxidant properties of Nasturtium
ofcinale (Watercress) leaf extracts. Acta Pol Pharm Drug Res
2009;66:187‑93.
24. Yu L, Haley S, Perret M, Harris J, Wilson J, Qian M. Free radical
scavenging properties of wheat extracts. J Agric Food Chem
2002;50:1619‑24.
25. Elizabeth K, Rao MN. Oxygen radical scavenging activity of
curcumin. Int J Pharm 1990;58:237‑40.
26. Floriano-Sánchez E, Villanueva C, Medina-Campos ON,
Rocha D, Sánchez-González D, Cárdenas-Rodríguez N, et al.
Nordihydroguaiaretic acid is a potent in vitro scavenger of
peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion
and hypochlorous acid and prevents in vivo tyrosine nitration in
lung. Free Radic Res 2006;40:523‑33.
27. Pedraza-Chaverri J, Barrera D, Maldonado PD, Chirino YI,
Macías-Ruvalcaba NA, Medina-Campos ON, et al.
S-allylmercaptocysteine scavenges hydroxyl radical and singlet
oxygen in vitro and attenuates gentamicininduced oxidative and
nitrosative stress and renal damage in vivo. BMC Clin Pharmacol
2004;4:5‑9.
28. Aruoma OI, Halliwell B. Action of hypochlorous acid on the
antioxidant protective enzymes superoxide dismutase, catalase
and glutathione peroxidase. Biochem J 1987;248:973‑6.
29. Menzel EJ, Sobal G, Staudinger A. The role of oxidative stress in
the long‑ term glycation of LDL. Biofactors 1997;6:111‑24.
30. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid
peroxidation and antioxidants in oxidative modication of LDL.
Free Radic Biol Med 1992;13:341‑90.
31. Maghrani M, Zeggwagh NA, Lemhadri A. Study of the
hyperglycemic activity of Fraxinus excelsior and Silybum
marianum in an animal model of type 2 diabetes mellitus.
J Ethnopharmacol 2004;91:309‑16.
32. Tahara A, Matsuyama-Yokono A, Nakano R. Hypoglycaemic
effects antidiabetic drugs in streptozotocin-nicotinamide-induced
mildly diabetic and streptozotocin-induced severely diabetic
rats. Basic Clin Pharmacol Toxicol 2008;103:560‑8.
33. Fraga CG, Leibovitz BE, Tappel AL. Lipid peroxidation measured
as thiobarbituric acid‑reactive substances in tissue slices:
Characterization and comparison with homogenates and
microsomes. Free Radic Biol Med 1988;4:155‑61.
34. Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A.
Aminoguanidine prevents diabetes-induced arterial wall protein
cross‑linking. Science 1986;232:1629‑32.
35. Peng X, Zheng Z, Cheng KW, Shan F, Ren GX, Chen F, et al.
Inhibitory effect of mung bean extract and its constituents vitexin
and isovitexin on the formation of advanced glycation end
products. Food Chem 2008;106:475‑81.
36. Naito C, Yamanaka T. Lipid peroxides in atherosclerotic
diseases. Nippon Ronen Igakkai Zasshi 1978;15:187‑91.
37. Jung K, Pergande M. Inuence of cyclosporin A on the respiration
of isolated rat kidney mitochondria. Fed Eur Biochem Soc Lett
1985;183:167‑9.
38. Mihara M, Uchiyama M. Determination of malonaldehyde
precursor in tissues by thiobarbituric acid test. Anal Biochem
1978;86:271‑8.
39. Nakagawa S. Immunochemical detection of advanced glycation
end products in lens crystallins from streptozotozin-induced
diabetic rats. Diabetes 1993;42:345‑50.
40. Momose T, Yano Y, Ohashi K. Organic analysis. XLIV. A new
deproteinizing agent for determination of blood sugar. Chem
Pharm Bull 1963;11:968‑72.
41. Podrez EA, Schmitt D, Hoff HF, Hazen SL. Mye1o-
peroxidase-generated reactive nitrogen species convert LDL
into unatherogenic form in vitro. J Clin lnvest 1999;103:1547‑60.
42. Akaike T, Suga Maeda H. Free radicals in viral pathogenesis:
Molecular mechanism, involving superoxide and NO. Proc Soc
Esp Biol Med 1998;21:64‑73.
43. Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant
action of N‑acetylcysteine: Its reaction with hydrogen peroxide,
hydroxyl radical, superoxide and hypochlorous acid. Free Radic
Biol Med 1989;6:593‑7.
44. Kochevar IE, Redrnond RW. Photosensitized production of
Gutierrez and Baez: Antioxidative, antiglycative and antidiabetic effects of Eysenhardtia polystachya
S418 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 (Supplement 2)
singlet oxygen. Methods Enzymol 2000;319:20‑8.
45. Esterbauer H. Cytotoxicity and genotoxicity of lipid‑oxidation
products. Am J Clin Nutr 1993;57 Suppl 5:779S‑85.
46. Janisch K, Hippeli S, Dornisch K, Kern S, Elslner EF.
Determination of the antioxidative potential of human plasma
after supplementation with pycnogenol and whey. Food Res lnt
2002;35:257‑66.
47. Matsuura N, Aradate T, Sasaki E, Kojima H, Ohara M,
Hasegawa J, et al. Screening system for the Maillard
reaction inhibitor from natural product extracts. J Health Sci
2002;48:520‑6.
48. Mahboob M, Rahman MF, Crover P. Serum lipid peroxidation
and antioxidant enzyme levels in male and female diabetic
patients. Singapore Med J 2005;46:322‑4.
49. Senthilkumar GP, Arulselvan P, Sathishkurnar D,
Subramanian SP. Antidiabetic activity of fruits of Termina/ia
chebula an streptozotacin induced diabetie rats. J Health Sci
2006;52:283‑91.
50. Gohil T, Pathak N, Jivani N. Treatment with extract of Eugenia
jambolana seed and Aegle marmelos leaf extracts prevents
hyperglycemia and hyperlipidemia in alloxan-induced diabetic.
Afr J Pharm Pharmacol 2010;4:270‑5.
51. Kasetti RB, Rajasekhar MD, Kondeti VK, Fatima SS, Kumar EG,
Swapna S, et al. Antihyperglycemic and antihyperlipidemic
activities of methanol: Water (4:1) fraction isolated from aqueous
extraer of Syzygium altemifolium seeds in streptozotocin induced
diabetic rats. Food Chem Toxicol 2010;48:1078‑84.
Cite this article as: Gutierrez RP, Baez EG. Evaluation of antidiabetic,
antioxidant and antiglycating activities of the Eysenhardtia polystachya. Phcog
Mag 2014;10:404‑18.
Source of Support: Nil, Conict of Interest: None declared.
