Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received: October 28, 2015
Accepted : December 18, 2015
Published online : March 31, 2016

Original Article

Formulation of five curry spice mixtures and investigation of their effect on
advanced glycation endproduct formation
Moniruzzaman Mohammad, Wakako Takabe, Masayuki Yagi, Yoshikazu Yonei
Anti-Aging Medical Research Center & Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objectives: Curry is a common food containing numerous spices known to have anti-oxidative or anti-glycative actions,which prevent advanced glycation endproduct (AGE) formation. In order to develop an anti-glycative ready-to-serve curry,
we formulated five combinations of curry spice mixtures and investigated their effect on AGE formation.
Methods: Test samples were five mixtures of spices to make curry. Hot water and 50% ethanol extractions were used andthe polyphenol content was measured by the Folin-Ciocalteu method. Using the in vitro human serum albumin/glucose model,
percent inhibition of AGE formation was evaluated by measuring fluorescent AGE, pentosidine, 3-deoxyglucosone (3-DG),
glyoxal (GO) and methylglyoxal (MGO). Fluorescent AGE was measured by fluorescence spectroscopy at excitation 370 nm
and emission 440 nm. Pentosidine, 3-DG, GO, and MGO were measured by high performance liquid chromatography (HPLC).
Results: This is the first report of formulating curry spice mixtures with both hot water and 50% ethanol extraction fromthe anti-glycation point of view. The inhibitory activity of 50% ethanol extracted curry formulation against fluorescent AGE,
pentosidine, and reactive carbonyl species (3-DG, GO and MGO) was better than that of the hot water extracted formulation.
The carbonyl trapping activity of curry mixtures is correlated with their polyphenol content.
Conclusion: This study provides information on spice mixture formulation from the point of view of anti-glycation. Further
study will be necessary to measure the color, taste, moisture and pH of curry prepared from the formulation to make it a readyto serve curry.

KEY WORDS: advanced glycation endproducts (AGEs), pentosidine, 3-deoxyglucoson, glyoxal, methylglyoxal

Introduction

medicinal herbs, are known to possess a variety of antioxidant effects and properties 5, 6). Phenolic compounds inthese plant components are closely associated with their antioxidant activity 4). They have been reported to offer diverse
bioactivities, including anti-inflammatory, anti-allergic,
anti-viral/anti-bacterial, anti-mutagenic/anti-carcinogenic
properties, and protective effects against diverse diseases 7).
Many of these biologically significant functions are believed
to be mediated by phenolics’ free radical scavenging capability,
which combats the oxidative stress induced by reactive oxygen
species 8).
It has been reported that spices with anti-oxidative andradical scavenging activity were found to possess an in vitro
anti-glycation activity 9,10). The proposed anti-glycation
mechanism includes scavenging of free radical and reactive
carbonyl species 8). Even though intensive studies on the
bioactive compounds and anti-oxidant and anti-glycation

Glycation is the nonenzymatic reaction between aprotein and a reducing sugar, such as glucose and fructose.
The addition of reducing sugars to amine groups in proteinsleads to the formation of a Schiff base, which rearranges toform a more stable Amadori product. Advanced glycation
end products (AGEs) are formed after a series of complex
reactions (e.g; oxidation, phosphorylation) 1).
Spices are common food adjuncts that have been used
as flavoring, seasoning, and coloring agents, and evenpreservatives, throughout the world for thousands of years,
particularly in Bangladesh, India, China, and many othersouthern Asian countries 2). It has been hypothesized that thedevelopment of diabetes and the accumulation of AGEs may
be reduced by intake of natural anti-oxidants through the diet 3).
Spices have been studied for their anti-oxidant properties forat least 50 years 4). Spices, like fruits, vegetables, andContact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences,
Doshisha University
1-3 Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
Phone/Fax: +81-774-65-6394 E-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Moniruzzaman M, sumonbge2009@gmail.com ;Takabe W, wtakabe@mail.doshisha.ac.jp_ 5 _Glycative Stress Research 2016; 3 (1): 5-14(c) Society for Glycation Stress ResearchCurry Spice Mixture and AGE formation
activity of many spices have been carried out, the antiglycation activity of spice mixtures remains to be elucidated.
Spices are fundamental to all curry recipes and there isa huge range of spices, which are used to prepare curry. The
spices are used to flavor the food, making each dish distinct
and wonderfully aromatic. Each spice by itself imparts avery unique flavor, but when used together with other spices,
the combination and permutation of different spices magicallychange the individual characteristics. Curry is a dish withorigins in South and Southeast Asians cuisines. The common
feature is the incorporation of complex combinations of
spices or herbs, usually including fresh or dried hot chilies.
Some limit the use of the term “curry” to dishes prepared ina sauce 11, 12), but curries may be wet or dry. The main spices
found in most South Asian curry powder are chili, onion,
turmeric, ginger, cinnamon, bay leaves, and cloves. A wide
range of additional spices may be included depending on thegeographic region and the foods which are added, such as
white/red meat, fish, lentils, rice and vegetables 13).
In this regard, the present study attempted to formulate
five combinations of curry spice mixtures, investigates
their total phenolic contents, and evaluate their effect on
fluorescent AGEs and some AGEs representatives such as
3-DG, GO, MGO and pentosidine, with the aim to develop
an anti-glycative ready-to-serve curry.

at - 20°C until investigation of their effect on the generationof fluorescent AGEs, formation of AGE intermediates (3-DG,
GO and MGO) and pentosidine in the human serum albumin(HSA)/glucose model 1).

In vitro HSA/glucose reaction model

The glycation of HSA and formation of AGEs wasmodeled by incubating HSA with and without glucose at
60°C for 40 hours as previously reported 1). The glucose (+)
reaction solution contained 0.1 M phosphate buffer (pH 7.4),
40 mg/mL HSA, 2.0 M glucose solution, and distilled waterat a 5:2:1:2 volume ratio. The glucose (-) reaction solution
contained 0.1 M phosphate buffer (pH7.4), 40 mg/mL HSA,
and distilled water at a 5:2:3 volume ratio.

Measurement of fluorescent AGEs

AGE-derived fluorescence was measured as reported
previously using an in vitro HSA/glucose model 1, 14). Briefly,
100 μL of various concentrations of test samples in aqueous
solution were added to 500 μL of 0.1 mol/L phosphate buffer
solution (PBS, pH 7.4), 100 μL distilled water, 200 μL 40
mg/mL HSA (Sigma Chemical Co., Ltd., St. Louis, MO, USA),and 100 μL 2.0 mol/L aqueous solution of glucose. Distilledwater was then added to make up a total volume of 1.0 mL,
and the material was incubated at 60°C for 40 hours (Solution
A). Final concentrations were 8 mg/mL HSA and 0.2 mol/
L glucose. At the same time, a solution including distilledwater, added in lieu of aqueous glucose, was incubated as a
blank for each reaction (Solution B). Samples preparedwithout the addition of test samples were incubated as apositive control (Solution C). And at the same time, a solution
including distilled water, added in lieu of aqueous glucose,
was incubated as a blank for positive controls (Solution D).
Fluorescent AGEs were measured quantitatively in eachsample reaction solution (A, B, C, D) to evaluate inhibitory
activity on AGE formation. Fluorescent AGEs were measured
using an ARVO MX 1420 ARVO series Multilabel Counter(Perkin-Elmer Japan Corp, Yokohama, Japan) microplate
reader at an excitation wavelength of 370 nm and a

Materials and Methods

Preparation of spice mixture extract

Test components were curry spice mixtures, of which thecomponents are listed in Table 1. The spice mixture samples
were dried at 65ºC for 72 hours and ground into fine powders.
Two grams of each type of spice power was extracted with
20 mL of 100% distilled water at 80ºC in a water bath forone hour. Spice power was also extracted with 20 mL of 50%
ethanol at 49ºC in a water bath for 4 hours. The concentrationof each sample was estimated from the weight difference
before and after incubation of 5 mL sub-samples, dried inaluminum trays at 120°C for 1.5 hours. The extracts were kept
Table 1. Component of curry spice mixture.
Spice

Category-I(%)

Category-II(%)

Category-III(%)

Category-IV(%)

Category-V(%)

Red chilli

40

20

30

15

10

Onion

20

30

20

30

20

Turmeric

15

15

10

10

15

Ginger

10

20

10

20

30

Garlic

10

5

5

2

3

5

4

2

Cinnamon
Cardamom
Bay leaf

2
5

5

2

5

5

5

Cumin

5

6

10

Coriander

5

3

1

Cloves

5
10

_ 6 _

Glycative Stress Researchfluorescence wavelength of 440 nm. Calibration curves forinhibition of fluorescent AGE formation (%) were constructedby adding individual samples to a reaction solution at three
concentrations (0.1%, 0.01%, 0.001%) and calculating theinhibition of fluorescent AGE formation (%) after the reaction.
The measurement was repeated three times for each sample.

Shimadzu). The HPLC analytical conditions were as follows:
separation column: YMC Triat C18 (column size 150 × 4.6
mm ID, 5 μm/12 nm; YMC); flow rate: 1.0 mL/min; andcolumn temperature: 30ºC. The samples were detectedusing an excitation wavelength of 335 nm and a detection
wavelength of 385 nm. Elution was performed using eluent
I (ACN/Methanol/Water/Perfluobutyric acid [HFBA] =16/4/76/0.2) and eluent II (ACN/HFBA = 100/ 0.2), andthe following gradient conditions: analysis: 0 ~ 20 min with
0% eluent II; washing: 20 ~ 25 min with 100% eluent II; andconditioning: 25 ~ 40 min with 0% eluent II. A standard curve
was generated using standard pentosidine samples. Three
measurements (n = 3) were taken for each sample.

Measurement of 3-DG, GO and MGO

For AGE intermediates, 3-DG, GO and MGO were
measured by HPLC (LC-20AT system equipped with SPD20A UV/VIS detector and LC-solution software; Shimadzu,
Kyoto, Japan). Briefly, 10 μg/mL 3-DG, GO, and MGO
standard solution was prepared by mixing 0.4 mL of 50 μg/
mL 3-DG, 0.4 mL of 50 μg/mL GO and 0.4 mL of 50 μg/mL
MGO with 1.2 mL of distilled water. Standard solutions of
1.0, 0.25, 0.05, 0.025, and 0 μg/mL were prepared by serial
dilutions with distilled water from 10 μg/mL. Then, 200 μL
standard solution, 100 μL of 200 mM phosphate buffer and30 μL of distilled water were added to a microtube and mixed
properly; 200 μL of antiglycation test reagent with/without
spice sample were added to 130 μL of distilled water in a
microtube and mixed properly; 170 μL of 6% perchloric
acid was added separately to both the sample and standard
solutions, and centrifuged at 12,000 rpm for 10 minutes.
After centrifugation 400 μL of supernatant was transferred
into another microtube. This was followed with the additionof 350 μL saturated sodium bicarbonate, and finally 50 μL
of 2, 3- diaminonaphthalene as an internal standard. After
mixing, the reaction mixtures were allowed to incubate at
4ºC for 24 hours. After incubation, the reaction mixtures were
centrifuged at 15,000 rpm for 10 minutes, and the supernatant
was used for analysis by HPLC-UV. The analytical conditions
were as follows: eluent 50mM phosphate : acetonitrile(ACN) = 89:11, YMC-pack CN, S-5 μm, 150 × 4.6 mm ID(YMC, Kyoto, Japan), a flow rate of 1.0 mL/min, column
temperature 40ºC, detection wavelength 268 nm, injection
volume 20 μL. A 3-DG, GO and MGO standard curve wasconstructed by measuring peak areas of the standard solutions.
The 3-DG, GO and MGO inhibitory effect of each spicesample was determined from the standard curve by measuring
the peak areas of the respective samples. Two measurements(n = 2) were taken for each sample.

Measurement of polyphenols

As previously reported 15), the polyphenol content in eachtest sample was measured using the Folin-Ciocalteu (FC)method in conjunction with the "Commercial Product Test
Results for Polyphenol-Containing Foods" produced by the
National Consumer Affairs Center of Japan (NCAC).
Specifically, 100 μL of test sample solutions was added to 50
μL of a 2-fold dilution of FC reagent (Wako Pure Chemical
Industries) and 500 μL of 0.4 M aqueous Na2CO3 to makeup a total volume of 650 μL; the material was left to standat ambient temperature for 30 minutes, and absorbency at
660 nm was then measured. A (+)-catechin solution (Wako
Pure Chemical Industries) was used as a standard. Multiple(+)-catechin solutions were prepared in 11 steps as 1.0, 0.5,
0.25, 0.1, 0.05, 0.025, 0.0125, 0.01, 0.00625, 0.001, and 0
mg/mL concentrations; 100 μL of each concentration of(+)-catechin solution was added to 50 μL of the 2-fold diluted
FC reagent and 500 μL of 0.4 M aqueous Na2CO3 to makeup a total volume of 650 μL, the material was left to stand at
ambient temperature for 30 minutes, and absorbency at 660
nm was then measured. Using the (+)-catechin calibration
curve prepared, total polyphenol content per solid unit of eachtest sample solution was calculated as a catechin equivalent(mg catechin Eq/mg solid content). Measurement was repeatedthree times for each sample.

Results

Effect of spice mixture on fluorescent AGE formation

Measurement of pentosidine

Fig. 1. shows the inhibitory effects of five curry spicemixture formulations on the fluorescent AGE formation in
the in vitro HSA/glucose model. Each sample showed actionsof preventing fluorescent AGE formation. In the hot water
extracts, mean values of % inhibition varied from 56.5% to157.5%, while in the 50% ethanol extracts, values were stablebetween 84.6% and 91.6%.

200 mL and 100 mL of extra for elute I and elute II
respectively were prepared ( Table 2 ). The evaporated residue
was dissolved in 300 μL of elute-I, mixed by vortex,
centrifuged and then transferred into HPLC vials. Pentosidine
was measured by HPLC (LC-20AT system equipped with
FS-8020 fluorescent spectrometer and LC-solution software;
Table 2. Component of elute- I and - II
Reagent

Elute-I

Elute-II (ratio)

Acetonitrile (ACN)

16

100

Methanol4−

Distilled water76−

Perfluoro butyric acid (HFBA)

0.2

0.2

_ 7 _

Curry Spice Mixture and AGE formation

120

100

% Inhibition

80

Hot water

60

50% Ethanol
40

20

0
Category-1

Category-2

Category-3

Category-4

Category-5

Fig. 1. Inhibitory effects of curry spice mixtures on the formation of fluorescent AGEs in
HSA/glucose model.

Results are expressed as means ± standard deviation, n = 3. AGEs, advanced glycation endproducts;
HSA, human serum albumin.

Discussion

Effect of spice mixture on 3-DG, GO and MGO
formation

Effect of spice mixture on fluorescent AGE formation

The HSA/glucose model adopted in this study provides
a useful tool for assessing the effects of curry spice mixture
on the formation of fluorescent AGEs with both hot waterand 50% ethanol extractions. The first study displays theinhibitory effects of five curry spice mixture formulations
on the fluorescent AGE formation in this model. The results
revealed that 50% ethanol-extracted curry mixture showed
higher fluorescent AGE inhibition activity than water extracted curry mixture. The 50% ethanol extracts of all
curry spice mixtures examined showed significant inhibitory
activities above 90% on the generation of fluorescence AGE.

Results of the effect of spice mixture on 3-DG, GO andMGO formation are presented in Figs. 2 and 3. The hot water
extracts showed inhibitory effects, except for Category-4 on
the formation of MGO (70.4% - 82.0%) and GO (56.8% 62.9%). The effect of 3-DG was low (12.9% - 36.6%).
All of the samples of the 50% ethanol extract showed
inhibition of formation of GO (64.6% - 67.8%) and 3-DG
(39.1% - 54.2%). The 50% ethanol extract from Category-4
showed no inhibition of MGO formation, however, in the restof samples, % inhibition was from 42.0% to 54.8%.

Effect of spice mixture on pentosidine formation

Relationship between total polyphenol contents andanti-glycation activities of curry spice mixture

The results of the effect of spice mixture on pentosidine
formation are presented in Fig. 4. The category- I sample
showed marked inhibition on pentosidine formation in thein vitro HSA/glucose model. The % inhibition value reachedalmost 100% in the hot water extract and was 59.4% in 50%
ethanol extract. In the other category samples, % inhibition
was less than 32.2%. The values varied depending on the
category.

Recently, various phenolic anti-oxidants from plant extracts
have been found to inhibit the formation of AGEs 14, 15), andtheir inhibition of free radical generation in the glycation
process and subsequent inhibition of the modification ofproteins have been considered as the major mechanisms
which mediate their anti-glycative activities. Thus, in thefollowing experiments, total polyphenol contents of thefive curry spice mixtures with both water and 50% ethanol
extraction were determined using a calorimetric method.
It was found that 50% ethanol-extracted spice mixtures
had higher polyphenol contents than water-extracted spicemixtures. Anti-glycative activities of 50% ethanol-extracted
spice mixtures were correlated with their total polyphenol
contents (R2 = 0.188) ( Fig. 6 ), suggesting that the phenolicingredients play a major role in the anti-glycative activitiesof spice mixtures.

Polyphenol content

Total polyphenol contents in each sample are presented
in Fig. 5. Values varied from 0.07 mg cathechin Eq/mL to
0.09 mg cathechin Eq/mL in hot water extracts and from 0.08
mg cathechin Eq/mL to 0.12 mg cathechin Eq/mL in 50%
ethanol extracts. Polyphenol content tended to be higher in
50% ethanol extract than in hot water extract.
_ 8 _Glycative Stress Research

100

80

3-DG

% Inhibition

60

GO
40

MGO

20

0

-20
Category-1

Category-2

Category-3

Category-4

Category-5

Fig. 2. Effects of hot water extracts from curry spice mixtures on the formation of 3-DG,
GO, and MGO.

In vitro HSA/glucose model. Results are expressed as means of duplicate measurements. HSA, humanserum albumin; 3-DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal.

100% Inhibition

80

60
3-DG
40

GO
MGO

20

0
Category-1

Category-2

Category-3

Category-4

Category-5

Fig. 3. Effects of 50% ethanol extracts from curry spice mixtures on the formation of
3-DG, GO, and MGO.

In vitro HSA/glucose reaction model. Results are expressed as means of duplicate measurements.
HSA, human serum albumin; 3-DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal.

_ 9 _Curry Spice Mixture and AGE formation

100

80

% Inhibition

60
Hot water
40

50% Ethanol

20

0

-20
Category-1

Category-2

Category-3

Category-4

Category-5

Fig. 4. Effect of curry spice mixtures on the formation of pentosidine in the in vitro HSA/glucose model.
Results are expressed as means of duplicate measurements. HSA, human serum albumin.

0.16
0.14

Polyphenol (mg/mL)

0.12
0.10
Hot water
0.08
50% Ethanol
0.06
0.04
0.02
0.00
Category-1

Category-2

Category-3

Category-4

Category-5

Fig. 5. Total polyphenol contents of both hot water and 50% ethanol extracted from five curry spice mixtures.
Results are expressed means ± standard deviation, n = 3.

_ 10 _Glycative Stress Research

50% EtOH extraction(%) Inhibition of f luorescent AGEs

a)

92
91
90

y = 22.045x + 86.135
R 2 = 0.01822

89
88
87
86
85
84
0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Total phenolic content (mg/mL)

Water extraction(%) Inhibition of f luorescent AGEs

b)

180
160

y = 593.54x + 42.544
R 2 = 0.03711

140
120
100
80
60
40
20
0
0.00

0.02

0.04

0.06

0.08

0.10

Total phenolic content (mg/mL)

Fig. 6. Linear relationship between % inhibition of fluorescence AGE formation and total
polyphenol contents of five curry spice formulations.
a) 50% ethanol extraction, b) Hot water extraction.

Inhibitory effects of curry spice mixtures on theformation of 3-DG, GO and MGO

Several aldehydes, such as 3-DG 16), GO 17), and MGO 16)
are generated in the Maillard reaction and contribute further
to the formation of AGEs.
All curry spice mixtures with 50% ethanol extraction
exhibited strong inhibitory effects on the formation of 3-DG,
GO, and MGO. Mixture category-1 exhibited the strongest
inhibition with 4 9.1% for 3-DG, 66.1% for GO, and 54.8%
for MGO. It is interesting to note that the 50% ethanolextracted curry spice mixture predominantly inhibited theformation of GO (nearly 66%). Correlative analysis revealed
that 3-DG inhibition activity is highly correlated with thepolyphenol content (R2 = 0.870) ( Fig. 7-a). MGO inhibition
activity was poorly correlated with the polyphenol content(R2 = 0.160) ( Fig. 7-c), and GO inhibition was not correlatedwith polyphenol content (R2 = 0.015) ( Fig. 7-b). We predictthat the higher carbonyl trapping activity of the 50%
ethanol-extracted spice mixture is brought about by both thehydrophobic and hydrophilic phenolic compounds.
As seen in Fig. 6. the carbonyl trapping activity of thewater-extracted curry spice mixture is comparatively lower
than the 50% ethanol-extracted mixture. Among the waterextracted mixture category-3 exhibited the highest inhibition
with 25.4% for 3-DG, 63.0% for GO, and 82.0% for MGO.
It is noteworthy that the curry spice mixture primarily
inhibited the formation of MGO with water extraction. It waspreviously reported that water-soluble cathechin, epicatechin,and procyanidin B2 from cinnamon more markedly inhibit
methylglyoxal (about 93%) than aminoguanidin (73%) 18). We
assume that the higher MGO inhibitory effects of waterextracted curry spice mixture is brought about by the watersoluble polyphenols, such as catechin, epicatechin, andprocyanidin B2 from cinnamon, cloves, bay leaf, and turmeric.
Correlative analysis showed that the carbonyl-trapping activityof water-extracted curry spice mixture was not correlatedwith the polyphenol content (data not shown).
A 50% ethanol extracted curry spice mixture is best
suited to prepare the real curry. In real curry oil, ghee as aclarified butter, groundnut oil, and sunflower oil with water
are used as the medium to carry the spices, which makes thecurry a suitable environment for hydrophobic and hydrophiliccomponents in spices.

_ 11 _

Curry Spice Mixture and AGE formation

3-DG vs polyphenol
Total phenolic content (mg/mL)

a)
0.14
0.12
0.10
0.08

y = -0.0028x + 0.2288
R 2 = 0.87327

0.06
0.04
0.02
0.00
0

10

20

30

40

50

60

(%) Inhibition of 3-DG f ormation

GO vs polyphenol
Total phenolic content (mg/mL)

b)
0.14
0.12
0.10
0.08

y = -0.0014x + 0.1928
R 2 = 0.01333

0.06
0.04
0.02
0.00
64

65

65

66

66

67

67

68

68

(%) Inhibition of GO f ormation

MGO vs polyphenol
Total phenolic content (mg/mL)

c)
0.14
0.12
0.10
0.08

y = -1E-04x + 0.1011
R 2 = 0.0162

0.06
0.04
0.02
0.00

0

10

20

30

40

50

60

(%) Inhibition of MGO f ormation

Fig. 7. Relationship between: (a), % Inhibition of 3-DG formation and total polyphenol content;(b), % Inhibition of GO formation and total polyphenol content; (c), % Inhibition of MGO
formation and total polyphenol content of 50% ethanol extracted curry spice mixtures.
3-DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal.

_ 12 _Glycative Stress Research

Total phenolic content (mg/mL)

Pentosidine vs polyphenol
0.14
0.12
0.10
0.08
0.06

y = -3E-05x + 0.098
R 2 = 0.00208

0.04
0.02
0.00
-10

0

10

20

30

40

50

60

70

(%) Inhibition of pentosidine f ormation

Fig. 8. Relation between (%) inhibition of pentosidine formation and total polyphenol content.

No correlation between (%) inhibition of pentosidine formation and total polyphenol content (mg catechin Eq/mL).

Inhibitory effects of curry spice mixture on
pentosidine formation

Conclusion

The 50% ethanol extracted curry spice mixtures exhibited
pentosidine inhibitory effect except for Category-4 ( Fig. 4 ).
Among the water-extracted curry spice mixtures, only
Category-5 exhibited an effect on pentosidine formation.
It has been reported that 3-DG, fructose, ascorbate, andAmadori compounds act as precursors for the formationof pentosidine 1, 19). As seen from Fig. 2, among the waterextracted spice categories, the 3-DG inhibitory effect ofCategory-5 was higher. This high 3-DG inhibitory effectof Category-5 might affect the pentosidine formation. The
inhibitory effects of 50% ethanol-extracted spice categories
on pentosidine formation are 59.4% for Category-1 and 32.2%
for Category-3. Figure 8 showed that pentosidine inhibitory
effect of 50% ethanol-extracted curry spice mixtures was notcorrelated with their polyphenol contents.
On the basis of the anti-glycation, and anti-oxidant
activity, and polyphenol content, we assume that the antiglycation activity of spice mixtures is contributed by the antioxidant activity of cinnamon, ginger, cloves, bay leaf, andturmeric. It has been reported that boiling and steaming causes
significant decreases in phenolic content and anti-oxidant
power 20). Peng et al. reported that although fortification ofbread with grape seed extract (GSE) enhances the anti-oxidant
capacity, baking leads to a 30 - 40% decrease in GSE’s antioxidant capacity 21). An increase in anti-oxidant activity
with thermal processing has also been observed in studies
of pressure-streaming yellow beans, cooking tomato, andhigh-pressure processing of tomato and carrot purees; thisphenomenon was correlated with an increase of particular
phenolic contents 20, 22, 23). It is possible to prepare a bettercurry from the viewpoint of anti-glycation with vegetables,such as tomatoes, carrots, and beans, those by which the antioxidant activities increase with thermal processing.

This is the first report of formulating curry spice mixturesfrom the viewpoint of anti-glycation. The present study
revealed that 50% ethanol-extracted curry spices mixtures
showed a higher fluorescent AGE inhibition, and carbonyl
trapping inhibition activity than hot water-extracted curry
spice mixtures. The fluorescent AGE inhibition activity andcarbonyl trapping activity of 50% ethanol-extracted curry
spice mixtures are correlated with their polyphenol content.
Further investigation will be necessary to prepare curry fromour formulations to achieve ready-to-serve curry to combat
glycative stress.

Acknowledgements

This study was presented at “the 9th Meeting of theSociety for Glycative Stress Research” on September 5th,
2015, at Kyoto, Japan. This work was supported by theJapanese Council for Science, Technology and Innovation,
SIP (Project ID 14533567), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (funding
agency: Bio-oriented Technology Research Advancement
Institution, NARO).

Conflict of Interest Statement

The authors state that performance of this study entailed
no issues representing a conflict of interest.

_ 13 _

Curry Spice Mixture and AGE formation

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