Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : January 19, 2016
Accepted : March 24, 2016
Published online : June 30, 2016

Original article

Effect of iridoid (containing plants) on AGE formation and degradation
Yumi Abe 1, 2), Masayuki Yagi 1), Akemi Uwaya 2), Fumiyuki Isami 2), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0394, Japan.
2) Quality Assurance & Research, Morinda Worldwide, Inc., 3-2-2 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.

Abstract

Objectives: Glycation is a non-enzymatic reaction between reducing sugars and the amino groups of proteins and lipids. This
reaction produces advanced glycation end products (AGEs). AGEs are involved in diabetic complications, and plants containing
iridoids are reported to prevent diabetes complications. However, it is unknown if these increase AGE degradation. In thisstudy, the abilities of extracts from iridoid containing plants (Morinda citrifolia, Cornus officinalis and Olea europaea) toprevent AGE formation were tested with three model proteins (Human serum albumin, collagen and keratin). Further, theAGE degrading activity of these extracts was determined by examining their impact on the α-diketone structure of 1-phenyl-1,
2-propanedione (PPD).
Method: To measure the inhibition of AGE formation, each model protein was incubated with glucose and samples at 60℃.
Changes in detectable fluorescent AGEs (excitation wavelength 370 nm, detection wavelength 440 nm) were used to determine
the percent inhibition of AGE formation of each sample. Determination of AGE degradation activity involved reacting PPD
solution with samples at 37℃, for 8 h. Changes in measured benzoic acid content (by HPLC), produced from cleavage of the
α-diketone structure, was used to calculate AGE degrading activity.
Result: Morinda citrifolia seed extract was the most active sample in preventing AGE formation in all model proteins. The
other extracts were also active. But inhibition intensity differed by protein type. At 1 mg/mL, Cornus officinalis fruit juice
extract was the strongest AGE breaker (degradation rate; 29.8%), followed by Olea europaea leaf extract (26.5%) and Morinda
citrifolia seed extract (18.2%).
Conclusion: The results demonstrate that iridoid containing plants may prevent AGE formation, as well as degrade existing
AGEs.

KEY WORDS: Morinda citrifolia, Cornus officinalis, Olea europaea, advanced glycation end products (AGEs),
anti-glycation, cross-link break, iridoids

Introduction

Glycation is a non-enzymatic reaction between reducing
sugars and the amino groups of proteins, lipids, and nucleic
acids. Advanced glycation end products (AGEs), which are
irreversible glycation products, trigger “glycative stress”
and play an important role in the pathogenesis of diabetic
complications 1, 2), atherosclerosis 1, 2) and osteoporosis 3), andin aging 4, 5). There has been a great deal of interest in glycative
stress inhibitors. Researchers have developed some approaches
such as: (1) managing blood sugar levels, (2) inhibiting AGE
formation, and (3) breaking preexisting AGE derived crosslinks. Inhibition of AGE formation has been investigatedwidely. One well-known antiglycating agent is aminoguanidine(AG), which prevents the development of diabetic complicationin vivo by inhibiting the formation of AGEs 6). On the one
hand, It has been reported that N-phenacylthiazolium bromide(PTB) may have therapeutic value in metabolizing AGEs
due to its ability to cleave C-C bonds of alpha-diketone
structures 7) . But studied less intensively are mechanisms of
involving the breaking of preexisting AGE derived crosslinks. There is limited evidence for the clinical effectivenessand safety of AG 8) . But plant-based AGE inhibitors, whose
safety profiles are supported by long consumption histories,
are also expected to reduce glycative stress.
Noni (Morinda citrifolia), cornelian cherry (Cornus
officinalis) and olive (Olea europaea) are traditional foodsources and have been also been used as traditional
medicines. French Polynesians and other indigenous groups
throughout the tropics have used noni fruits, leaves, seeds,
and flowers for general health problems 9). Cornelian cherry isa major herb in Traditional Chinese Medicine, with dried fruit

Corresponding author:
Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0394, JAPANTEL: 0774-65-6382 FAX: 0774-65-6394 E-mail: yyonei@mail.doshisha.ac.jp_ 56 _

Glycative Stress Research 2016; 3 (2): 56 - 64(c) Society for Glycative Stress ResearchGlycative Stress Research

powders being used for nutritive and analeptic purposes 10).
Olive leaf was used medicinally in Mediterranean countries,
and multiple researchers report various health benefits 11).
These herbs contain a common phytonutrient group,
iridoids. These include deacetylasperulosidic acid (DAA) in
noni, loganin in cornelian cherry and oleuropein in olive 12-14).
Iridoids in noni, cornelian cherry, and olive are reported tomitigate diabetes induced liver and kidney damage 15 -18).
Further, cornelian cherry inhibits AGE formation andimproves diabetic conditions 18) . The object of this study wasto evaluate the ability of iridoid containing plants to inhibition
AGE formation and reduce preexisting AGE cross-links.

Materials and methods
Plant materials

Morinda citrifolia fruits (MCF), leaves (MCL) and seeds(MCS) were collected in French Polynesia. The fruits wereseparated into flesh and seeds by hand. The flesh was thenfreeze-dried. The leaves and seeds were air-dried. Aqueous
ethanol extracts of noni fruit flesh, leaves and seeds wereprepared at Kinki University, as described previously 19).
Cornus officinalis fruit (COF), grown in China, was extractedwith aqueous ethanol. Olea europaea leaves (OEL), grown in
Morocco, were extracted with ethanol.

Reagents

Deacetylasperulosidic acid (DAA) was purchased fromChengdu Biopurify Phytochemicals Ltd. (Sichuan, China).
Loganin was purchased from Tokyo Chemical Industry Co.,
Ltd. (Tokyo, Japan). Oleuropein was purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). Human serum
albumin (HSA) was purchased from Sigma-Aldrich Japan(Tokyo, Japan). Collagen type I (bovine skin, Pepsinsolubilized) was purchased from Nippi, Incorporated (Tokyo,
Japan). Keratin was purchased from Nacalai Tesque, Inc.
(Kyoto, Japan). Aminoguanidine hydrochloride and quinine
sulfate dihydrate were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). 1-phenyl-1, 2-propanedione(PPD) was obtained from Sigma-Aldrich Japan (Tokyo,
Japan). N-phenacylthiazolium bromide (PTB) was obtained
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Other reagents were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan).

HSA, collagen and keratin glycation tests

Glycation tests were performed according to the methods
described by Hori et al. 20), with minor modifications. Glucose
in phosphate-buffer (PB; pH 7.4) was combined with HSA,
collagen and keratin, as described in Table 1. The test samples
and aminoguanidine (the positive control) were dissolved inTable 1. Preparation of glycation test reaction solution

A. HSA-glucose model
PB (100 mM)

Glucose solution (2M)
Water

HSA solution (40 mg/mL)
Sample (50% DMSO)

B. Collagen-glucose model
PB (100 mM)

Glucose solution (2M)
Water

Collagen solution (3 mg/mL)
Sample (50% DMSO)

C. Keratin-glucose model
PBS (100 mM)

Glucose solution (2M)
Water

Keratin solution (3 mg/mL)
Sample (50% DMSO)

Final conc.

Glucose (+)

Glucose (-)

50 mM

500 μL500 μL−100 μL200 μL

5% DMSO

100 μL100 μL

0.2 M

100 μL

8 mg/mL

200 μL200 μL

Final conc.

Glucose (+)

Glucose (-)

50 mM

500 μL500 μL

0.4 M

200 μL−200 μL

0.6 mg/mL

200 μL

Final conc.

Glucose (+)

Glucose (-)

50 mM

500 μL500 μL100 μL

5% DMSO

0.4 M

200 μL−200 μL

0.6 mg/mL

100 μL

5% DMSO_ 57 _

200 μL100 μL200 μL200 μL100 μLEffect of Iridoid on AGE formation & Degradation
dimethyl sulfoxide (DMSO) and diluted with water to a DMSO
concentration of 50% v/v. The control consisted of only 50%
DMSO in water. After incubation at 60℃, the fluorescenceof reaction products was measured in a microplate reader(SpectraMax Paradigm Malti-Mode Microplate Reader;
Molecular Devise, Sunnyvale, CA, USA) with excitation at
370 nm and emission at 440 nm. Each fluorescence intensity
was calculated as a relative value, comparing emission values(reported by the microplate reader) of samples and controls
to that of 5 μg/mL quinine sulfate in 0.1 N sulfuric acidaqueous solution (this was set at 1000). Inhibition offluorescent AGE formation was calculated by the following
formula:Inhibition (%) = (Control F I* - Sample F I*) / Control F I* × 100.
*FI: fluorescence intensity = (emission value of Glucose (+)– emission value of Glucose (-)) / quinine sulfate × 1000.

Cross-link breakage test

Following the method described by Yagi et al. 21), with amodification, 1-phenyl-1, 2- propanedione (PPD), dissolvedin 50% acetonenitrile, was used as a reactive substrate in theAGE cross-link model. In this test, 100 μL of sample solution
as added to 100 μL PPD (10 mM) and 400 μL PB (200 mM)
and mixed. The test samples and PTB (as a positive control)
were dissolved into 50% DMSO (final concentration 5%
DMSO v/v). The mixture was incubated at 37℃ for 8h. Afterincubation, the reaction was stopped by adding 200 μL
hydrochloric acid (2M), followed by centrifugation at 10000
rpm for 2 min. The supernatant (50 μL) was analyzed byhigh performance liquid chromatography (HPLC: LC-10A;
Shimadzu, Kyoto, Japan) for benzoic acid. HPLC conditions
were: Cadenza CD-C18 column (75 × 4.6 mm ID, Imtakt,
Kyoto, Japan); 40℃ column temperature; mobile phase of
0.2% acetic acid in 2 mM ethyenediamine-N, N, N’, N’tetraacetic acid - disodium salt (EDTA-2Na) - dehydrate/acetonitrile (70/30, v/v); 1.0 mL/min flow rate; and detection
at 270 nm and 3.3 min retention time. Benzoic acid is released
as the alpha-diketone structure of PPD is cleaved 7). Crosslink breaking activity was determined by dividing benzoic
acid concentration by the original PPD concentration of thereaction solution. Preexisting benzoic acid concentrationsof the samples were subtracted from the values measured in
the final reaction solutions.

Statistical analysis

Means and standard deviations (SD) were determinedfor each of experiments described in the figures or tables.
Intergroup differences were evaluated by one-way analysis
of variance (ANOVA) followed by Dunnett’s multiple
comparison test with Statcel Ver. 3 (OMS Publishing Inc.,
Saitama, Japan, 2011).

Results

Inhibition of HSA, collagen and keratin glycation

All herbal extracts (MCF, MCL, MCS, COF, and OEL)
suppressed in vitro formation of fluorescent AGEs in all
three protein models. All activities were in a concentrationdependent manner and statistically significant, compared tothe control ( Table 2-A, B, C ). The HSA model ( Table 2-A
HSA) revealed that all herbal extracts, except MCF, were very

effective inhibitors of AGE formation with IC 50s lower thanAG. In the collagen model, most herbal extracts suppressed
AGE formation more effectively than AG ( Table 2-B ). In
the keratin model, the IC 50 of noni leaf (MCL) could not be
determined due to the weak inhibition while other herbal
samples showed strong activity ( Table 2-C ). These results
supported our hypothesis that iridoid-containing plants (noni,
cornelian cherry, and olive) had anti-glycation properties.
However, their efficacies varied by the type of protein
involved. For example, the IC 50 values of AG in the HSA,
collagen, and keratin tests were, respectively, 196, 150, and516 μg/mL. Protein-dependent results were also observedfor MCL. The IC 50 values were 168 and 134 μg/mL with
HSA and collagen. But no inhibition was observed whenkeratin was the glycation target. MCF and OEL were mosteffective at inhibiting AGE formation in the HSA model.
The COF extract displayed the strongest inhibition activity
in the collagen model. In all protein models, MCS extract
exhibited the greatest anti-AGE activity. Taken together, thedata suggest that anti-AGE efficacy of the iridoid sources
varies by the type of protein involved.

Cross-link breaking activity of MCF, MCL, MCS,
COF, OL and PTB

Glycative stress can be alleviated if accumulated AGEs
were selectively degraded. Reportedly, AGEs that form
intermolecular cross-links, such as pentosidine, are degraded
and reduced by breaking the cross-links 7). The PPD molecule
is an AGE model compound because of its unique alphadiketone structure. Because of this, we evaluated the abilityof iridoid-containing herb extracts to degrade PPD. Analysis
of benzoic acid released during the cleavage of the alphadiketone structure of PPD revealed that cross-link breaking
activities of COF, OEL, and MCS (1mg/mL) were 29.8%,
26.5%, and 18.2%, respectively ( Fig. 1 ).

Cross-link breaking activity of DAA, loganin,oleuropein and PTB

Results from the cross-link breakage tests suggestthat iridoids in the plant extracts cleave AGE cross-links.
To confirm this, we investigated the breaking activity of
individual iridoids, such as loganin in COF, oleuropein in
OEL, and DAA in MCS. DAA and loganin demonstrated
weak cross-link breaking activity, Fig. 2. However, oleuropein
from OEL caused benzoic acid release in a dose-dependent
manner. This seems to indicate that OEL’s cross-link
breaking activity is due in part to oleuropein.

Discussion

Many researchers report that the accumulation of AGEs
in tissues cause the progression of age related diseases.
In particular, hyperglycemia induced AGEs are related todiabetic complications and may be partly responsible forthe onset of diabetes. AGE inhibitors have been actively
developed to prevent or alleviate these conditions. Because
noni, cornelian cherry, and olive are reported to improve liveror kidney malfunctions caused by diabetes, we hypothesizedthat they possess anti-AGE properties through inhibition of
AGE formation and by cross-link breaking. Previous studies
have revealed that these herbs possess anti-glycation activities.

_ 58 _Glycative Stress Research

Table 2. Inhibition of AGE formation by MCF, MCL, MCS, COF, OL and AG

A. Glucose-HSA model
SamplesConc.
(μg/mL)F I a)

Inhibition(%)

IC 50 value b)(μg/mL)

Control−

255.4 ± 2.8−−

MCF extract200

174.4 ± 3.0**

32

349

400

123.0 ± 3.5**

52

800

48.6 ± 0.8**

81

100

172.0 ± 0.6**

33

200

111.4 ± 2.4**

56

400

57.1 ± 1.1**

78

13

197.1 ± 7.0**

23

25

149.9 ± 6.5**

4150

102.1 ± 2.9**60

25

209.3 ± 1.1**

1850

155.2 ± 4.8**

39

100

90.1 ± 5.6**

65

100

167.8 ± 28.2**

34

200

112.6 ± 20.1**

56

400

85.2 ± 23.6**

67

100

195.3 ± 19.8**

24

200

131.0 ± 15.5**

49

400

50.4 ± 7.3**

80

MCL extract

MCS extract

COF extract

OEL extractAGa)
FI: fluorescence intensity. b) IC 50 is the concentration of sample required to inhibit 50% of AGE formation.
Each value represents the mean ± SD of 3 experiments. **: p< 0.01, significantly different from the control group.

_ 59 _

168

34

66

181

196

Effect of Iridoid on AGE formation & Degradation

B. Glucose-collagen model
SamplesConc.
(μg/mL)F I a)

Inhibition(%)

IC 50 value b)(μg/mL)

Control−

210.2 ± 2.9**−−

MCF extract

200

100.1 ± 1.6**

52

175

400

55.0 ± 2.5**

74

800

23.5 ± 1.2**

89

200

91.8 ± 6.1**

56

400

69.0 ± 0.8**

67

800

46.0 ± 4.7**

78

6

158.7 ± 1.6**

25

13

113.0 ± 0.1**

46

25

79.4 ± 1.7**

6213

111.1 ± 1.7**

47

25

85.1 ± 1.5**

60

50

59.1 ± 2.0**

7225

138.0 ± 2.9**

3450

95.2 ± 4.7**

55

100

48.2 ± 2.8**

77

100

129.6 ± 1.0**

38

200

87.6 ± 0.6**

58

400

44.8 ± 1.7**

79

MCL extract

MCS extract

COF extract

OEL extractAGa)
FI: fluorescence intensity. b) IC 50 is the concentration of sample required to inhibit 50% of AGE formation.
Each value represents the mean ± SD of 3 experiments. **: p< 0.01, significantly different from the control group.

_ 60 _

134

15

15

42

150

Glycative Stress Research

C. Glucose-keratin model
SamplesConc.
(μg/mL)F I a)

Inhibition(%)

IC 50 value b)(μg/mL)

Control−

233.6 ± 2.5−−

MCF extract50

181.8 ± 0.9**

22

146

100

142.3 ± 1.3**

39

200

96.6 ± 0.5**

5950

145.2 ± 1.1**

38

100

130.5 ± 0.8**

44

200

124.3 ± 1.2**

476

190.7 ± 1.0**

18

13

153.9 ± 3.1**

34

25

101.1 ± 6.2**

5750

158.7 ± 1.1**

32

100

121.2 ± 3.4**

48

200

72.2 ± 2.5**

6950

135.1 ± 2.7**

42

100

89.6 ± 1.1**

62

200

64.5 ± 6.9**

72

200

182.0 ± 1.8**

22

400

136.4 ± 4.4**

42

800

85.5 ± 6.0**

63

MCL extract

MCS extract

COF extract

OEL extractAGa)
FI: fluorescence intensity. b) IC 50 is the concentration of sample required to inhibit 50% of AGE formation.
Each value represents the mean ± SD of 3 experiments. **: p< 0.01, significantly different from the control group.

_ 61 _

−

20

101

67

516

Effect of Iridoid on AGE formation & Degradation

60**

Rate of breakage (%)

50

40

**

**

30

20

*

**

**

1

0.2

**

**

**

10

0

Conc.

0.2

(mg/ml )

1

0.2

1

0.2

1

0.2

1

Control
MC F

Samples

1.4( 5 mM )

MC L

MCS

COF

OE L

PTB

Fig. 1. Cross-link breaking activity of MCF, MCL, MCS, COF, OL and PTB

Each value represents the mean ± SD of 3 experiments. *: p<0.05, **: p<0.01, significantly different from the control
group.

**

60

Rate of breakage (%)

50

**

40

**

30

20

**

**

10

**
**

0

Conc.
(mg/ml )Samples0.04 0.2

15

0.04 0.2

15

0.04 0.2

1

5

0.2

1

5

Control
DAA a)

Loganin

Oleuropein

Fig. 2. Cross-link breaking activity of MCF, MCL, MCS, COF, OL and PTBPTBa) DAA; deacetylasperulosidic acid. Each value represents the mean ± SD of 3 experiments. **: p<0.01, significantly
different from the control group.

_ 62 _Glycative Stress Research

Kusirisin et al. reported noni (fruits and leaves) ethanol
extracts had strong anti-glycation activity (IC 50; 0.01 μg/mL) 22). Park et al. reported that an aqueous ethanol extractof cornelian cherry (25 μg/mL) inhibited AGE formation by
12.8% 22) . Kontogianni et al. demonstrated that an aqueous
methanol extract of olive leaf reduced AGE formation
significantly 24). Previous studies have shown that AGE
formation depends on the reaction condition and source ofprotein 20, 25) . In the present study, we also demonstrated thatthe degree of inhibition was affected by protein type. As
such, a single method may not enough to evaluate overall
anti-glycation activity, and the target protein has to becarefully selected to align with testing purposes. Further
study is also important to evaluate non-fluorescent AGEs,
such as N ε-(carboxymethyl) lysine (CML).
Even though there are many reports of botanical extracts
reducing AGE formation, there are very few which report
both inhibition of formation and breaking of existing crosslinks. Here we developed two approaches for testing anti-AGE
capacity. The first involved a more typical anti-glycation
assay. The second approach involved the evaluation of crosslink breaking activity as determined by the release of benzoic
acid after incubation of PPD with COF, OEL, MCF, or MCS.
Our findings indicate that olueropein is an active
component of OEL that is capable of breaking AGE crosslinks. Yagi et al. investigated the breaking activity ofellagitannin compounds in Punica granatum and reported
that trihydroxybenzenes and dihydroxybenzene exhibitedthe strongest effects 21). Oleuropein, unlike other iridoids,
has the dihydroxybenzene structure ( Fig. 3 ). This chemical
structure may contribute the observed cleaving activity. This
study is an important early step towards understanding how
iridoid sources may treat glycative stress through the breakingof AGE cross-links.

A

Conclusion

In all protein models, MCS suppressed AGE formation
more significantly than the other extracts. MCF and MCS
inhibited AGE formation in three protein models but did notexhibit substantial cross-link breaking properties. But MCS
did have modest cross-link breaking activity. The anti-AGE
properties of COF and OEL involved both inhibition of AGE
formation as well as cross-link breaking activity. These results
suggest that MCS, COF, and OEL are potential therapeuticmaterials which may help prevent and treat glycative stress.

Acknowledgments

A part of this study was presented at “the 9th Meetingof the Society for Glycative Stress Research” on September
5th, 2015, at Kyoto, Japan. A part of this study was supportedby the Japanese Council for Science, Technology andInnovation, SIP (Project ID 14533567), “Technologies forcreating next-generation agriculture, forestry and fisheries”(funding agency: Bio-oriented Technology Research
Advancement Institution, NARO).

Conflict of interest

This research was mainly supported by Morinda
worldwide Inc..

B
HO

OH

O
H

H
O

H

HO

O

O

O

HO

CH3

O
H 3C

H

Glu

O

Glu

C
HO
HO

O

O

O

CH3

O
O

H 3C
O

Glu

Fig. 3. Chemical structure of deacetylasperulosidic acid(A), loganin (B), oleuropein (C)_ 63 _

Effect of Iridoid on AGE formation & Degradation

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