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Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients



Nonenzymatic glycation, the reaction of glucose and other reducing sugars with protein, reversibly produces Amadori products and over a long period irreversible advanced glycation end products. In diabetes, these reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid colorimetry after hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products. The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite, selenium yeast, and carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE formation (P < 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects, 7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured Vitamin C® for a period of 4 weeks. Serum protein glycation was decreased an average of 46.8% (P < 0.01). These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use of these nutrients for the prevention of diabetic complications.
Inhibition of protein glycation and
advanced glycation end products by
ascorbic acid and other vitamins
and nutrients
Joe A. Vinson and Thomas B. Howard III
From the Department of Chemistry, University of Scranton, Scranton, PA USA
Nonenzymatic glycation, the reaction
glucose and other reducing sugars with protein, reversibly produces
Amadori products and over a fang period irreversible advanced glycation end products. In diabetes, these
reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.
In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose
was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid calorimetry after
hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins
and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products.
The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The
inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite. selenium yeast, and
carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE
formation (P i 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects,
7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured
Vitamin C@ for a period of 4 weeks. Serum protein glycation was decreased an average of
46.8% (P < 0.01).
These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use oj
these nutrients for the prevention of diabetic complications.
0 Elsevier Science Inc. 1996 (J. Nutr. Biochem.
7:6.59-663. 1996.)
Keywords: glycation; advanced glycation end products; vitamins; nutrients: ascorbic acid
protein + sugar # Schiff # Amadori 4 + Advanced Glycation
Although there have been important advances in the control
of the hyperglycemia of diabetes by means of diet, hypo-
glycemic drugs, insulin, the insulin pump and islet trans-
plantation, the long-term complications of diabetes are still
leading causes of death.’ These complications are a direct
result of protein alterations which result in irreversible tis-
sue damage. One of the consequences of hyperglycemia is
the excessive nonenzymatic glycation of proteins known as
the Maillard reaction, which is shown schematically below.
Base Products
(AR End Products (AGE)
Equilibrium levels of Schiff base and Amadori products
(AP) are reached quickly, in hours and weeks, respectively.’
The levels of these early glycation products change in re-
sponse to blood glucose and are reflected in the analysis of
glycated hemoglobin (GHb) and glycated albumin to moni-
tor average blood glucose control over several months and
several weeks, respectively, in diabetic patients. However
the irreversibly formed advanced glycation end products
(AGE) do not return to normal when hyperglycemia is cor-
rected and continue to accumulate over the lifetime of the
Address correspondence and reprint requests to Dr. J. Vinson at Depart-
ment of Chemistry, University of Scranton, Scranton, PA 18510-4626,
Received February 20, 1996; accepted August 27, 1996.
protein. These substances can form covalent bonds with
amino groups on other proteins and thus cause protein
cross-linking.3 Glycation and AGE modifications result in
pathological changes to the protein such as enzyme activa-
Nutritional Biochemistry 7:659463, 1996
0 Elsevier Science Inc. 1996
655 Avenue of the Americas, New York, NY 10010 0955-2863/96/$15.00
PII SO955-2863(96)00128-3
Research Communications
tion of aldose reductase,4 deactivation of superoxide dismu-
tase,5 increased atherogenicity of LDL,6 increased basement
membrane permeability7 and decreased insulin binding to
insulin receptors.8 All of these modifications contribute to
diabetic complications such as cataracts, nephropathy, vas-
culopathy, proliferative retinopathy, and atherosclerosis.
A major effort has been launched to find a therapeutic
means of mediating protein glycation. The most studied and
successful agent has been aminoguanidine which reacts
with AP and early glycation products and limits the forma-
tion of AGE.9 Aspirin, acetaminophen, and ibuprofen have
been shown to decrease glycation of lens proteins and pre-
vent diabetic cataracts in rats.” In this report we investigate
vitamins and nutrients as possible inhibitors of protein gly-
cation and AGE formation.
Methods and materials
All biochemicals were obtained from Sigma Chemical Company
(St. Louis, MO USA). Other chemicals were reagent grade. Sele-
nium yeast (containing 1% selenium) and Re-Natured Vitamin C@
(25.1% ascorbic acid, 10.0% bioflavonoids, and a minimum 30%
carbohydrates and 15% protein) were gifts from Grow Company,
Inc. (Hackensack, NJ USA). Dubelco’s Formula Buffered Saline,
pH 7.4, was obtained from Flow Laboratories (McLean, VA
Bovine serum albumin (BSA) solutions were made in the buffer
with 0.02% sodium azide to prevent degradation. Fructose and
glucose were made together in 0.02% sodium azide. Inhibitors
were freshly prepared in water or, if lipid soluble, in Tween 80.
Aliquots of BSA, sugar, inhibitor, and distilled water or Tween 80
as a control, were added to 5-cm screw-capped test tubes to give
final solutions of 7 mg/mL BSA, 25 mM glucose, 25 mM fructose,
and the inhibitors at concentrations in the physiological range. All
incubations were done in quadruplicate and the tubes were de-
gassed with nitrogen before being placed in a constant temperature
bath at 37°C for 3 to 30 days.
Glycation analysis
Glycated proteins were measured in the form of the AP from BSA
reacting with both glucose and fructose. Sugars were removed
from the solution by extensive dialysis in the cold and the proteins
precipitated with trichloroacetic acid and hydrolyzed with oxalic
acid. The product was reacted with thiobarbituric acid and the
chromogen measured using hydroxymethylfurfural as a standard.
Serum samples were treated similarly, except that sugars were
removed by precipitating with trichloroacetic acid and washing the
protein precipitate with water.
AGE analysis
Fluorescence of the samples was measured at the excitation and
emission maxima of 350 and 450 nM, respectively versus an un-
incubated blank containing the protein, sugar and inhibitor.
Supplementation study
Seven healthy college students, three females and four males, aged
18 to 22, and 11 healthy normoglycemic subjects, five females and
six males, aged 39 to 76, participated with informed consent. Fast-
ing blood samples were drawn before and after 3 weeks supple-
mentation with 1000 mg of ascorbic acid/day as Re-Natured Vi-
tamin C@ (4 g/day). The blood was converted to serum and ana-
lyzed for glycated proteins.
Albumin was chosen as the protein for the in vitro reactions
because it is the most abundant protein in serum. Fructose
was included in the incubation because it is present in the
tissues at comparable concentrations to that of glucose and,
as Suarez has pointed out, reacts with proteins at faster rates
than glucose and produces about 10 times more protein-
bound fluorescence than glucose.” Fructose is also elevated
in those tissues where the polyol pathway is active. The
AGE product in the present study was a mixture of both
protein-glucose and protein-fructose reaction products.
Ascorbic acid, one of the inhibitors tested, was incubated at
20 p,M with BSA, glucose, and fructose over a period of 30
days. The inhibition of glycation relative to the control re-
mained constant; 3 day-70.4%, 15 days-75% and 30
days-73%. The AGE inhibition gradually increased; 3
days-56.6%, 15 days-67.7%, and 30 days-80.4%.
The results of 15-day incubations of various vitamins
and nutrients and the inhibition of glycation and AGE are
shown in Table 1. All substances showed a dose-response
inhibition of both glycation and AGE at concentrations in
the physiological range. An example of the inhibition at
different concentrations is shown for ascorbic acid in Figure
1. In comparison to the control with no inhibitor, all nutri-
ents at the physiological concentration except selenite pro-
duced a significant inhibition of glycation and AGE by the
ANOVA test, P < 0.05. Niacinamide and pyridoxal almost
completely inhibited glycation and AGE at physiological
concentrations. For example, niacinamide at 70 pm inhib-
ited glycation 100% and AGE 99.7%; pyridoxal at 1.1 p,M
inhibited glycation 100% and AGE 87.2%. The order of
effectiveness for both glycation and AGE inhibition is se-
lenium yeast > ascorbic acid > niacinamide > carnosine >
tocopherol > pyridoxal > sodium selenite. The combination
of Vitamins C and E were examined as inhibitors at physi-
ological concentrations (20 PM) and the inhibition was
95.2% for glycation and 85.2% for AGE formation. The
Table 1 Concentration of nutrients for 50% inhibition (I&,) of in
vitro protein glycation and AGE formation
IC& Albumin
Glycation AGE
of Nutrient
Ascorbic acid 11.8 uM 13.3 uM 20 uM
Tocopherol 19.5 uM 219 uM 20 uM
Niacinamide 16.0 uM 17.8 uM 41
Pyridoxal 417 uM 458 uM uM
649 nM
Sodium selenite 652 LIM 791 uM 380 nM
Selenium yeast
Carnosine 5.3 uM 6.2 LM
20.6 uM 16.3 uM 380 nM
660 J. Nutr. Biochem., 1996, vol. 7, December
Inhibition of protein glycation by nutrients: Vinson and Howard
O- 0.w 0.P P
Ascorbic Acid Concentration
Figure 1 Dose response of % inhibition of protein glycation and
AGE after 15 days of incubation with ascorbic acid at concentra-
tions near physiological (P), 0.1 P = 2 uM, 0.5 P = 10 PM, P = 20 PM
relative to control.
combination was a significantly greater inhibitor than either
vitamin alone at that concentration, ANOVA
P <
The 15 day data for all the nutrients was pooled and the
inhibition of glycation and AGE formation was found to be
highly correlated
< 0.001). The regression line was %
AGE inhibition = 0.852% glycation inhibition + 2.96 with
Pearson’s correlation coefficient of 0.988 (Figure 2).
Vitamin C was chosen for a supplementation study since it
is a very inexpensive and non-toxic nutrient. Re-Natured
Vitamin C@ was used for supplementation because it has
been found to be more bioavailable to animals and humans
than ascorbic acid alone.L2313 The supplementation pro-
duced a significant decrease in serum protein glycation of
both the college age and middle age groups,
P < 0.05
by a
paired t test (Table 2). Five of the 7 college students and all
11 of the middle-age subjects experienced a decline in gly-
cation as a result of the Vitamin C regimen.
‘CQ 1 .
0 20
% InhiiYition of G$cation
Ascorbate and its free radical, semihydroascorbate, form
ionic bonds with biological molecules such as proteins.29
The carbonyl group of ascorbate and its oxidation product
may also compete with glucose for protein as seen with in
vitro erythrocyte glycation3’ Vitamin C has been shown to
inhibit in vitro oxidation of low density lipoprotein by a
dual action; scavenging aqueous oxidants and by stable
modification of the protein by its oxidation product dehy-
Figure 2 Correlation between 15 day % inhibition of glycation and
Vitamin E, a-tocopherol, is a potent antioxidant. It pre-
AGE formation by vitamins and nutrients relative to control.
viously has been shown by Ceriello to be an anti-glycating
Table 2 Effect of ascorbate supplementation (1 g/day) on serum
protein glycation of college-age and middle-age subjects
Serum Protein Glycation (FM)
Subjects Before After
Supplementation Supplementation
Normal college age (n = 7)
Middle age (n = 11)
All subjects
a.57 * 6.65
15.70 f 7.53
12.76 zt 7.65
3.38 zt 2.50*
7.89 zt 6.25*
6.04 + 5.44**
*P < 0.05 by a paired t test.
**p < 0.01.
Although the mechanism of inhibition is at present un-
known, all of the substances can bind to sugars or proteins
and/or are proven antioxidants. Binding to sugar or protein
would inhibit AP production and subsequent AGE forma-
tion. The antioxidant function would decrease the concen-
tration of free radicals. Several reports indicate the produc-
tion of radicals and highly reactive oxidants from glycated
proteins under physiological conditions.‘4’6 Free radicals
are known to stimulate AGE production by autoxidation of
sugars.16 Oxidative stress has been linked to diabetic com-
plications17 and diabetic atherogenesis.‘* Recently in vitro
AGE formation from glucose and albumin was found to be
due in part to oxidants and free radicals acting on the lipid
moiety of the albumin.” Bucala and co-workers*’ found a
linear relationship between low density lipoprotein oxida-
tion and AGE in diabetic subjects. A linear correlation
between plasma glycation and AGE fluorescence has re-
cently been reported,21 which our results confirm.
Carnosine is a natural dipeptide and a major brain and
muscle antioxidant2* which can also compete with proteins
for binding with the sugars. Pyridoxal can bind to proteins
via the aldehyde grou
.& .
and thus be a competitive inhibitor
as found by Khataml. This vitamin has been used at high
doses to decrease glycosylated hemoglobin in human dia-
betics. Nicotinamide decreases the severity of streptozoto-
tin-induced diabetes in animals due to its antioxidant ef-
fect.25 Niacinamide supplementation was also found to
prevent the onset on diabetes in children with first-de-
gree relative with type I diabetes.‘6 The greater oxygen
radical scavenging ability of organic forms of selenium27
may explain the much larger inhibition of glycation and
AGE of the selenium yeast compared with selenite. Also,
the yeast proteins contain lysine, which has been shown to
inhibit in vitro protein glycation.*’
J. Nutr. Biochem., 1996, vol. 7, December 661
Research Communications
substance in vitro3* and in vivo as a supplement to diabet-
ics.33 Jain recently demonstrated Vitamin E blocks glyca-
tion of erythrocyte hemoglobin by inhibiting in vitro lipid
peroxidation. The present study confirms these results that
vitamin E is both a glycation and AGE inhibitor.
The fact that the combination of Vitamin C and E more
effectively inhibited glycation and AGE than the single vi-
tamins is evidence for the antioxidant mechanism of inhi-
bition. These vitamins have been shown to ;pergistically
inhibit free radical mediated lipid oxidation.
The present Vitamin C supplementation study (in a bio-
flavonoid mixture) demonstrated an average decrease of
46.8% in protein glycation in normoglycemic subjects in
good agreement with Davie.30 He showed a 33% decrease
in glycated albumin after 3 months with 1 g/day of ascor-
bate and an 18% reduction in GHb. Stolba also found a
significant decrease in fructosamine in insulin-dependent
diabetics given 1.5 g of ascorbate/day.36 The glycation-
lowering effect in the supplement could be due to both the
ascorbate and flavonoids in the mixture. Odetti found that
the flavonoid rutin decreased collagen-linked AGE fluores-
cence in diabetic rats.37
Epidemiological studies have recently found a negative
correlation between vitamin C intake and GHb in normal
subjects.38 Ascorbate either alone or in the form of a citrus
extract has also been found to be an effective agent in low-
ering red blood cell sorbitol in human diabetic subjects at 2
g/day, 39 1 glday,40 and 100 mg/day.41 Vitamin C thus ben-
eficially influences the two mechanisms of diabetic compli-
cations; glycation and the sorbitol pathway.
An important therapeutic factor to consider is that it
would be necessary to administer the inhibitor as soon as
possible after diagnosis of diabetes and regularly throughout
the lifetime of the patient. This is essential, because it has
been proposed that once the progress of excessive glycation
has begun, subsequent remediation of hyperglycemia would
not prevent diabetic complications2 Important attributes of
an ideal inhibitor would be easy absorption and excretion,
little or no toxicity, and the absence of serious side effects.**
Vitamin C should, therefore, be an ideal candidate for dia-
betes supplementation.
All of the substances studied are naturally occurring vi-
tamins and nutrients in the body and several of them are
known to be lowered in the tissues of subjects with diabetes,
such as ascorbate,42 pyridoxal,43 and nicotinamide.25 This
fact and the results of the present study point to the neces-
sity of good nutrition in diabetes and to the possibility of
inexpensive, relatively non-toxic therapies for the preven-
tion and treatment of diabetic complications. Because AGE
and atherogenic oxidized LDL are correlated in vivo,21 in-
hibition of glycation and oxidation by nutrients may also
form the basis of future antiatherogenic strategies in both
diabetic and non-diabetic individuals.
The authors would like to thank Teressa Vacarelli for doing
the initial studies that suggested inhibition, John Gumbs and
Sunah Jung for experimental work, and Debbie Jennings for
invaluable secretarial assistance. I&s Birlouez-Aragon and
Gerard0 Suarez are thanked for helpful discussions. This
662 J. Nutr. Biochem., 1996, vol. 7, December
work is dedicated to the memory of Dr. Pave1 Stolba, a
devoted Czech researcher.
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J. Nutr. Biochem., 1996, vol. 7, December 663
... The consumption of foods high in ascorbic acid (AA) has been associated with lower risk of diabetes. AA status may influence glycemic control, protein glycation and the sorbitol pathway (Kositsawat and Freeman, 2011;Vinson and Howard, 1996). Reduction in protein glycation with AA supplementation is evident from animal and human studies (Gembal et al., 1994;Krone and Ely, 2004). ...
... NEG (13 to 15% decrease, p < 0.05) and 0.11 ± 0.09, 0.12 ± 0.05, 0.19 ± 0.04 unit/mL AGEs levels (7 to 17% decrease; p < 0.05). The data is partially in accordance with the findings of Vinson and Howard (1996). They quantitated amadori product by thiobarbituric acid colorimetry in normal subjects after ascorbic acid supplementation. ...
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Advanced glycation end products (AGEs) formation is increased in diabetes mellitus, leading to microvascular and macrovascular complications. Recently, much attention has been focused on natural and synthetic inhibitors to delay the onset or progression of diabetes and its comorbidities. Ascorbic acid (AA) can react with proteins, including hemoglobin and possibly interfere with protein glycation process. An in vitro glycation model containing plasma from type 2 diabetic and non-diabetic healthy volunteers together with glucose as glycating agent was used to study antiglycation activity of AA. Samples with different concentrations of glucose and AA were incubated for five weeks at 37°C. Nonenzymatic glycation (NEG) was quantitated by thiobarbituric acid calorimetry and AGEs were measured by enzyme linked immuno-sorbent assay (ELISA). The NEG and AGEs levels were reduced by AA. Increasing the AA concentrations greatly diminished protein glycations, indicating dose-dependent effects of AA. Plasma NEG and AGEs were decreased with an average of 20 to 26% (p < 0.05) and 26 to 28% (p < 0.05). A significant correlation was found between the glycation inhibition and the inhibition of AGE formation (p < 0.05). The antiglycation role of AA is evident in the present study and it also indicates the possibility of inexpensive, relatively non-toxic vitamin therapy for the prevention and treatment of diabetic complications. It is plausible that AGEs inhibition by AA may also form the basis for future intervention strategies in both diabetic and non-diabetic individuals.
... One class majorly includes immunochemical techniques such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and autoantibody of AGEs, which are mainly adapted for in vivo studies; and the second class includes fluorescence spectroscopy, affinity chromatography, fluorescent phenylboronate acrylamide gel electrophoresis, liquid chromatography-mass spectroscopy (LC-MS), and high-performance liquid chromatography (HPLC). Additionally, methods such as nitroblue tetrazolium (NBT) assay [105] and thiobarbituric acid [106,107] colorimetric tests are utilized to identify intermediate products of Maillard reactions. Fluorescence spectroscopy is a commonly used method which is adapted to confirm the presence of advanced glycation end product at an excitation wavelength ranging between 335 and 420 nm that covers the band for all types of AGEs, and therefore it is one of the most common methods used to detect AGEs preparation in vitro [63]. ...
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Chronic elevation of sugar and oxidative stress generally results in development of advanced glycation end products (AGEs) in diabetic individuals. Accumulation of AGEs in an individual can give rise to the activation of several pathways that will ultimately lead to various complications. Such AGEs can also be prepared in an in vitro environment. For an in vitro preparation of advanced glycation end products (AGEs), proteins, lipids, or nucleic acids are generally required to be incubated with reducing sugars at a physiological temperature or higher depending upon the protocol optimized for its preparation. Certain other factors are also optimized and added to the buffer to hasten its preparation or alter the properties of prepared AGEs. Through this review, we intend to highlight the various studies related to the experimental procedures for the preparation of different types of AGEs. In addition, we present the comparative study of methodologies optimized for the preparation of AGEs.
... The inhibition of AGE formation was evaluated using a modification of a previously published method [32]. To prepare the reaction solution, 10 mg/mL of bovine serum albumin was added to 0.2 M fructose, 0.2 M glucose, and 3 M sodium azide. ...
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Previously, we reported the anti-diabetic effect of Morus alba root bark and the compounds therein. In our continuous study of other parts of this plant, the ability of the branch of Morus alba to inhibit α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), and advanced glycation end products (AGEs) formation was evaluated. Moreover, there are no previous studies that have performed enzyme kinetics and molecular docking analyses, along with assessments of peroxynitrite (ONOO −) inhibitory activities. Since the Morus alba branch exhibited favorable inhibitory effects, repeated column chromatography was performed to obtain eight compounds, including four fla-vonoids (1, 3, 6, 8), one arylbenzofuran (2), one stilbene (5), one Diels-Alder-type adduct (7), and one sterol (4). Among them, compounds 1-3 and 5-7 were mixed-type inhibitors of α-glucosidase, sharing the same catalytic residues with acarbose and the same allosteric sites with (Z)-3-bytyli-denephthalide. On the other hand, kuwanon C (1) and oxyresveratrol (5) interacted with residues of the allosteric site (α3 and α6 helices) of PTP1B, indicating their use as non-competitive inhibitors. Interestingly, kuwanon G (7) directly bound the catalytic site, or interrupted the binding between the substrate and the active site, as a mixed-type inhibitor. Moreover, most of the compounds exhibited greater activity against AGE formation and ONOO − than positive controls. The IC50 values required to inhibit ONOO − using compounds 1, 3, 5, 6, and 7 were reported for the first time, and range from 1.08 to 12.92 μM. Based on the structure-activity relationship, the presence of hydroxyl, resorcinol, and prenyl moieties was important in the prevention of diabetes' pathological mechanisms , and these findings have been further supported by molecular docking analysis. These computational and experimental results will be useful in the development of therapeutic candidates to prevent/treat diabetes and its complications.
... To determine the enzyme inhibitory activities of the extracts against some NCDs, key enzymes that control obesity (lipase), diabetes (α-amylase and α-glucosidase), hypertension (angiotensin-converting enzyme), and Alzheimer's disease (acetylcholinesterase, butyrylcholinesterase and β-secretase) were chosen for inhibitory reactions using the well-established protocols as previously described [11][12][13]22]. The inhibition of glycation reaction induced by D-glucose and methylglyoxal (MG) as a non-enzymatic reaction was also determined for anti-aging property [60]. Enzyme inhibitory assays consisted of an enzyme, a substrate, an indicator from Sigma-Aldrich (St. Louis, MO, USA) and a sample extract as an inhibitor. ...
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Non-communicable diseases (NCDs) are the leading global cause of death. The World Health Organization (WHO) has endorsed the consumption of fruits and vegetables because they are rich in phytochemicals that sustainably ameliorate the occurrence of NCDs. Thai food contains many spices and vegetables with recognized health benefits. Quality control of plant samples encountered a bottleneck in the field and comparative studies of plant control origins including species or cultivar identification, growing area and appropriate harvesting time are limited. To address this issue, all plant samples used in this study were cultivated and controlled by the Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand. The samples were phytochemically screened and determined their health-promoting bioactivities via antioxidant activities and inhibition of NCD-related enzymes including lipase (obesity), α-amylase and α-glucosidase (diabetes), angiotensin-converting enzyme (hypertension), as well as acetylcholinesterase, butyrylcholinesterase and β-secretase (Alzheimer’s disease). The non-enzymatic reaction toward glycation was also evaluated. The results showed that Senegalia pennata subsp. insuavis (Lace) Maslin, Seigler & Ebinger, Citrus hystrix DC. and Solanum melongena ‘Kermit’ extracts exhibited high antioxidant activities. Moreover, Citrus hystrix DC. extract was a potent inhibitor against lipase, angiotensin-converting enzyme and butyrylcholinesterase, while Coriandrum sativum L. and Psophocarpus tetragonolobus (L.) DC. were potent anti-diabetic agents and Senegalia pennata subsp. insuavis (Lace) Maslin, and Seigler & Ebinger was a potent anti-glycation agent. Our data provide a comparative analysis of ten vegetables to encourage healthy food consumption and development to control NCDs in Thailand in the future.
... Menurut Wu & Yen, 2005), usul mekanisme yang mungkin dalam penghambatan glikasi protein oleh senyawa antioksidan adalah penghambatan pembentukan radikal bebas yang berasal dari proses glikasi dan penghambatan modifikasi protein yang dianggap salah satu mekanismenya efek antiglikasi. Data ini didukung oleh beberapa penelitian yang menunjukkan bahwa antioksidan atau nutrisi seperti vitamin B1 (tiamin pirofosfat), vitamin B6 (piridoksamin), vitamin C, vitamin E, niacinamide, karnosin, dan natrium selenit dapat menghambat secara in vivo dan in vitro pembentukan AGEs (Booth dkk., 1996;Vinson & Howard, 1996;Krone & Ely, 2004 Metode spektrofotometer bisa digunakan untuk identifikasi asam fenolat dan secara umum memiliki panjang gelombang antara rentangan 220 sampai 320 nm (Mabry dkk. 1970;Macheix dkk. ...
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Penelitian ini bertujuan untuk menentukan aktivitas antioksidan dan anti glikasi ekstraks fenolik bebas dan ekstrak fenolik terikat dari tongkol jagung. Tongkol jagung diekstraksi secara sekuensial berbantu ultrasonikasi dengan pelarut etanol dan aseton selama 60 menit yang untuk mendapatkan ekstrak fenolik bebas. Residu dari ekstraksi fenolik bebas dihidrolisis dengan NaOH 2 M dan dinetralkan serta diekstraksi dengan etil asetat untuk mendapatkan ekstrak fenolik terikat. Ketiga ekstrak tersebut dilakukan analisis kandungan total fenolik dan pengujian aktivitas antioksidan dan anti glikasi. Kandungan total fenolik ekstrak ekstrak fenolik terikat (EFT), ekstrak fenolik bebas aseton (EFBA) dan ekstrak fenolik bebas etanol (EFBE) berturut-turut adalah 288,39; 100,32 dan 92,95 μg/mL yang dinyatakan sebagai ekuivalen asam galat. Ekstrak EFT menunjukkan aktivitas penangkalan radikal bebas DPPH dan total antioksidan lebih tinggi daripada ekstrak EFBA dan ekstrak EFBE. Sejalan dengan itu, aktivitas anti glikasi dari EFT, EFBA dan EFBE berturut-turut adalah 61,93; 64,42 dan 66,31%. Hasil penelitian ini menyimpulkan bahwa ekstrak EFT dari tongkol jagung mengandung senyawa yang memilki sifat antioksidan dan berpotensi sebagai anti glikasi. ABSTRACTThis objective of this research was to determine the antioxidant activity and inhibition of AGEs formation of free phenolic and bound phenolic extract from corn cobs. Corn cobs were extracted sequentially assisted by ultrasonication with ethanol and acetone solvents for 60 minutes to obtain free phenolic extract. The residue from free phenolic extraction was hydrolyzed with 2 M NaOH and neutralized and extracted with ethyl acetate to obtain bound phenolic extract. The three extracts were analyzed for total phenolic content and evaluated for antioxidant activity and inhibitory activity of AGEs formation. The total phenolic content of bound phenolic extract (EFT), acetone-free phenolic extract (EFBA) and ethanol-free phenolic extract (EFBE) were 288,39; 100,32 and 92,95 μg/mL expressed as gallic acid equivalents, respectively. EFT extract showed higher DPPH free radical scavenging activity and total antioxidants more than EFBA extract and EFBE extract. Anti-glication of EFT, EFBA and EFBE were 61,93; 64,42 dan 66,31%, respectively. These result concluded that the EFT extract from corn cobs contains compounds having antioxidant properties and potential as anti-glication.
... At 20 µM, thymoquinone inhibited 39% of hemoglobin glycation. Earlier, in vitro glycation was studied by Vinson and Howard (1996) with BSA (bovine serum albumin) with glucose or fructose as glycating agents. Eighteen normal subjects were supplemented with 1000 mg of ascorbic acid for a period of 4 weeks. ...
... Inhibitory capacities of AGEs formation were measured by the method of Vinson and Howard [57]. The extent of fluorescent AGEs formed was determined with a fluorescent spectrometer (F-7100, Hitachi, Tokyo, Japan): excitation wavelength, 330 nm; emission wavelength, 410 nm. ...
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We compared the efficacy for protein extraction of water versus enzymatic extraction. The amino-acid composition, inhibitory activity against enzymes α-amylase and α-glucosidase, and anti-glycation activities of silk protein extract (SPE) were determined. We used water extraction (100 °C, six hours) and protease-enzymatic extraction. The microstructure of silk fibers was obviously different after extraction. The results showed that enzymatic extraction gave the greater values of protein content, amino acids, total phenolic content (TPC), and total flavonoid content (TFC), as well as all biological activities parameters tested, but it also provided a more bitter taste in the extract—contributing amino acids of 51% (arginine, phenylalanine, histidine, valine, tryptophan, isoleucine, and leucine) and less sweet and umami taste contributing amino acids than did water extraction, which could be more suitable to be used as concentrated nutraceuticals.
... The inhibitory capacities of AGEs formation of the silk protein and its fractions were measured using the method of Vinson and Howard [40]. The total volume of glycation reaction solution (2.5 mL) was prepared by mixing 500 µL of SPE, 500 µL of 20 mg/mL BSA in phosphate buffer, 500 µL of 0.5 M glucose in phosphate buffer and 1 mL of 0.1 M phosphate buffer at pH 7.4 containing 0.02% (w/v) sodium azide. ...
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Silk proteins have many advantageous components including proteins and pigments. The proteins—sericin and fibroin—have been widely studied for medical applications due to their good physiochemical properties and biological activities. Various strains of cocoon display different compositions such as amino-acid profiles and levels of antioxidant activity. Therefore, the objectives of this study were to find a suitable silk protein extraction method to obtain products with chemical and biological properties suitable as functional foods in two strains of Bombyx mori silk cocoon (Nangsew strains; yellow cocoon) and Samia ricini silk cocoon (Eri strains; white cocoon) extracted by water at 100 °C for 2, 4, 6 and 8 h. The results showed that Nangsew strains extracted for 6 h contained the highest amounts of protein, amino acids, total phenolics (TPC) and total flavonoids (TFC), plus DPPH radical-scavenging activity, ABTS radical scavenging capacity, and ferric reducing antioxidant power (FRAP), anti-glycation, α-amylase and α-glucosidase inhibition. The longer extraction time produced higher concentrations of amino acids, contributing to sweet and umami tastes in both silk strains. It seemed that the bitterness decreased as the extraction time increased, resulting in improvements in the sweetness and umami of silk-protein extracts.
Advanced glycation end products (AGEs) are a group of complex compounds generated by nonenzymatic interactions between proteins and reducing sugars or lipids. AGEs accumulate in vivo and activate various signaling pathways closely related to the occurrence of various chronic metabolic diseases. In this paper, we describe the process through which AGEs are formed, the classification of AGEs, and biological effects of AGEs on human health. Most importantly, we review recent progress in natural compound-based AGE formation inhibitors. Major classes of natural inhibitors, including polyphenols, polysaccharides, terpenoids, vitamins and alkaloids, have been described. Their mechanisms of action have been summarized as scavenging free radicals, chelating metal ions, capturing active carbonyl compounds, protecting protein glycation sites, and lowering blood glucose levels. Although these natural compounds have good antiglycation activity, to date, they are not widely used in the clinic, likely because of their low content levels. However, these natural compounds and their molecular frameworks will play a valuable role in inspiring drug discovery.
The anti-glycation effects of polysaccharides from Ecklonia cava were examined according to extraction method—hot buffer (HP), ultrasound (UP), enzyme (EP), or a combination of ultrasound and enzyme (UEP). The physicochemical properties, monosaccharide compositions, and structural characteristics of the polysaccharides were determined. UP, EP, and UEP had higher fucose and galactose compositions than HP. The FT-IR spectra of samples showed the presence of sulfate esters and 4-sulfate galactose. ¹H NMR indicated that alginate was removed by purification. UP, EP, and UEP possessed higher sulfate contents than HP. UEP presented with the highest extraction yield and lowest protein and uronic acid contents. The levels of AGE formation, as well as fructosamine, α-dicarbonyl, and protein carbonyl contents were determined during a 3-week incubation in a BSA/fructose system. UEP and UP effectively inhibited AGE, although the inhibition effect was lower than that of aminoguanidine. However, UP and UEP showed higher inhibition of fructosamine, α-dicarbonyl, and protein carbonyl than aminoguanidine. AGE formation was negatively correlated with sulfate content and some monosaccharide compositions (fucose, galactose, and glucose), but positively correlated with molecular weight. Overall, the present study suggests that UEP is a suitable extraction method for obtaining anti-glycation agents from E. cava.
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Nonenzymatic glycation by glucose (glucation) was compared with glycation by fructose (fructation). The rate and extent of protein-bound fluorescence generation upon fructation was about 10 times that upon glucation. In contrast, nonenzymatically glucated bovine serum albumin (BSA) released about twice as much formaldehyde upon periodate oxidation as did nonenzymatically fructated BSA. However, the rate of blocking of amino groups was similar in both proteins. Periodate oxidation of borohydride-reduced glycated BSA led to regeneration of amino groups with preservation of fluorescence. From the ratio between the decrease in formaldehyde-releasing ability and the regenerated amino groups, formaldehyde molar yields of 0.47 and 0.8 were computed for fructose- and glucose-derived Amadori groups, respectively. This is consistent with participation of both carbon 1 and carbon 3 in the Amadori rearrangement from fructose. The formaldehyde releasing ability of nonenzymatically fructated BSA attains asymptotic maximum values earlier than that of nonenzymatically glucated BSA. Thus, the higher rate of fluorescence generation in nonenzymatically fructated BSA could be explained by a faster conversion of its Amadori groups. Since fluorescence generation through the Maillard reaction has been correlated with long term complications of diabetes mellitus, the participation of nonenzymatic fructation in this pathological state deserves further exploration. This is especially relevant in tissues where fructose levels increase in diabetes as a result of the operation of the sorbitol pathway.
In people with diabetes, glycation of apolipoproteins correlates with other indices of recent glycemic control, including HbA1. For several reasons, increased glycation of apolipoproteins may play a role in the accelerated development of atherosclerosis in diabetic patients. Recognition of glycated LDL by the classical LDL receptor is impaired, whereas its uptake by human monocyte-macrophages is enhanced. These alterations may contribute to hyperlipidemia and accelerated foam-cell formation, respectively. Glycation of LDL also enhances its capacity to stimulate platelet aggregation. The uptake of VLDL from diabetic patients by human monocyte-macrophages is enhanced. This enhancement may be due, at least in part, to increased glycation of its lipoproteins. Glycation of HDL impairs its recognition by cells and reduces its effectiveness in reverse cholesterol transport. Glycation of apolipoproteins may also generate free radicals, increasing oxidative damage to the apolipoproteins themselves, the lipids in the particle core, and any neighboring macromolecules. This effect may be most significant in extravasated lipoproteins. In these, increased glycation promotes covalent binding to vascular structural proteins, and oxidative reactions may cause direct damage to the vessel wall. Glycoxidation, or browning, of sequestered lipoproteins may further enhance their atherogenicity. Finally, glycated or glycoxidized lipoproteins may be immunogenic, and lipoprotein-immune complexes are potent stimulators of foam-cell formation.
Glucose chemically attaches to proteins and nucleic acids without the aid of enzymes. Initially, chemically reversible Schiff base and Amadori product adducts form in proportion to glucose concentration. Equilibrium is reached after several weeks, however, and further accumulation of these early nonenzymatic glycosylation products does not continue beyond that time. Subsequent reactions of the Amadori product slowly give rise to nonequilibrium advanced glycosylation end-products which continue to accumulate indefinitely on longer-lived molecules. Excessive formation of both types of nonenzymatic glycosylation product appears to be the common biochemical link between chronic hyperglycemia and a number of pathophysiologic processes potentially involved in the development of long-term diabetic complications. The major biological effects of excessive nonenzymatic glycosylation include: inactivation of enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated proteins and trapping of soluble proteins by glycosylated extracellular matrix (both may progress in the absence of glucose); decreased susceptibility to proteolysis; abnormalities of nucleic acid function; altered macromolecular recognition and endocytosis; and increased immunogenicity.
Serum pyridoxal and folate concentrations were measured in 518 diabetics. There were 185 males and 333 females. The level of pyridoxal was significantly lower in the diabetics when compared with 371 ‘healthy’ controls and 25% had levels below the lower limit of the normal range. No significant difference was observed between diabetics being treated by diet alone, oral hypoglycaemics or insulin. Only 20 patients had a reduced serum folate level and in 6 this was accompanied by a low pyridoxal concentration. The results suggest that diabetics may have an increased demand for pyridoxal.
Fluorescence (excitation 360 nm, emission 454 nm) generation in glycated albumin was investigated. Antioxidants and the metal chelator desferrioxamine (DFX) were used to study the mechanism of fluorescence generation. Delipidation studies, reverse phase chromatography and scanning fluorimetry were performed to examine the nature of this fluorescence. The mechanism of action of aminoguanidine, a compound which has been shown to inhibit the formation of visible fluorescence in proteins in vitro and in vivo was investigated in relation to glycation and by comparison with compounds with structural similarities. We conclude that hydrogen peroxide, metal ions and hydroxyl radicals are involved in fluorescence generation in glycated albumin, which is largely lipid in nature, and arises through glycation, amino acid oxidation and changes in bound lipid. Our results suggest that the action of aminoguanidine is not specifically related to blocking of ketoamine groups on glycated proteins as previously suggested.
Evidence from epidemiological, in vitro and animal studies has accumulated to support the idea that aspirin, ibuprofen and paracetamol protect against cataract. In this study rats made diabetic with streptozotocin were given these drugs in their drinking solution for up to 160 days. All three drugs delayed cataract formation assessed by slit-lamp examination for a large part of this time. Blood glucose levels were a little lower in diabetic rats treated with aspirin and ibuprofen than in untreated diabetic rats although all groups remained diabetic. Similarly, the increased glycation (non-enzymic glycosylation) of lens proteins caused by diabetes was less in the diabetic rats treated with aspirin and ibuprofen. The fall in glutathione induced by diabetes was also alleviated by aspirin and ibuprofen. Paracetamol appeared to afford similar protection against the biochemical changes but its effect was not statistically significant. The decrease in glutathione and increase in glycation were related to the progression of lens opacification. The greatest loss of glutathione occurred at an early stage, whereas glycation had its greatest change at the later stages--nuclear and mature cataract. These results encourage the view that ibuprofen, aspirin and paracetamol could protect against cataract in man: a hypothesis that could be tested in a properly-conducted clinical trial.
Aminoguanidine-HCl inhibits the formation of advanced glycosylation end products (AGEs) in vitro and in vivo, but the mechanism by which this occurs has not been determined. Aminoguanidine inhibited glucose-derived AGE formation on RNase A by 67-85% at aminoguanidine-glucose molar ratios of 1:5 to 1:50 without affecting the concentration of Amadori products. Fast-atom-bombardment mass spectrometry of RNase peptides incubated with glucose alone or with glucose plus aminoguanidine showed that aminoguanidine inhibited the formation of AGEs without forming an adduct with glycosylated peptide. These data suggest that the primary mechanism of aminoguanidine action is reaction with Amadori-derived fragmentation products in solution. These findings are relevant to the potential clinical use of aminoguanidine in the prevention of diabetic complications.