Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 25, 2022
Accepted : May 29, 2022
Published online : June 30, 2022
doi:10.24659/gsr.9.2_106

Original Article

Cleavage of glycated protein cross-linking and cleavage of AGEs cross-linkingof substances contained in vegetables and herbs
Yurim Kim, Saki Yokota, Masayuki Yagi, Chieko Sakiyama, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

Glycative stress-induced production and accumulation of advanced glycation end products (AGEs) in the body contributeto aging and the development of various diseases. PTB cleaves α-diketone bonds and cleaves cross-links associated withthe formation of AGEs in glycated proteins (cleavage of AGE cross-linking; CAC). However, since glucospan, α-diketone,
and lysine-dihydropyridinium-lysine are involved in the formation of protein cross-links by glycation, the usefulness of thecross-link cleavage effect must be verified using glycated proteins. Whereas, measurement of cleavage of glycated-protein
cross-linking (CGPC) using glycated lysozyme as a model has been reported. In this study, CGPC and CAC of 12 substances
contained in vegetables and herbs were measured to examine the possibility of degradation of protein cross-links polymerized
by glycation. 12 substances were measured for CGPC and CAC, and a high positive correlation was observed between CGPC
and CAC in 6 substances (50 %), indicating that α-diketone bond cleavage may be involved in the degradation of glycatedprotein cross-links. In contrast, only CGPC was observed for the three substances, and it was possible that they cleaved
cross-links different from the α-diketone structure; the CGPC measurement had the potential to evaluate glycated protein
cross-link cleavage actions other than α-dicarbonyl bond cleavage.

KEY WORDS: advanced glycation end products (AGEs), glycated lysozyme, cleavage of glycated-protein cross-linking

		Introduction(CGPC), cleavage of AGE cross-linking (CAC)

The production and accumulation of advanced glycation
end products (AGEs) in the body due to glycative stress arefactors in aging and the development of various diseases 1, 2).
In particular, the formation of protein cross-links associatedwith the generation of AGEs leads to tissue stiffening, which
is a factor in the deterioration of biological functions. The
components involved in the formation of protein cross-links
by glycation include glucospan, α-diketone, and lysinedihydropyridinium-lysine 3). N-phenacylthiazolium bromide(PTB) cleaves α-diketone bonds and cleaves cross- links
associated with the AGE formation in glycated proteins(cleavage of AGE cross-linking: CAC) 4). The CAC of asample is determined by measuring the amount of benzoic
acid released when the sample breaks down the intramolecular
α- dicarbonyl bond using 1-phenyl-1, 2-propanedione (PPD)

as a model compound for α-diketone bonding. CAC active
substances include rosmarinic acid 5), elagitannin 6), andflavonoid 7), which are found in plants. On the other hand,in a study in which diabetic rats were treated with PTB, a
substance with CAC, the glycated protein cross-links in skinand tail tendon collagen were not degraded 8). Therefore, theusefulness of CAC in cleaving glycated protein cross-links
must be verified using glycated proteins.
When lysozyme is glycated, it forms dimers andtrimers by generating cross-linking AGEs, e.g., pentosidine,vesperlysine, pyrropyridine, and crossline 9). Polyacrylamide
gel electrophoresis (SDS-PAGE) can be used to verify thepolymerization of glycated lysozyme. Cleavage of glycatedprotein cross-linking (CGPC) using glycated lysozyme as amodel has been reported in a 50 % ethanol extract of edible
purple chrysanthemum 10).

Contact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3 Tatara Miyakodani, Kyotanabe, Kyoto, 610- 0394 Japan
TEL & FAX: +81-774-65-6394 e-mail: myagi@mail.doshisha.ac.jp
Co-authors: Kim Y, cgsc2036@mail4.doshisha.ac.jp; Yokota S, ctug2031@mail4.doshisha.ac.jp;
Sakiyama C, csakiyam@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 10 6 _Glycative Stress Research 2022; 9 (2) : 106 -111(c) Society for Glycative Stress ResearchGlycative Stress Research

In the present study, CGPC and CAC of 12 substancesin vegetables and herbs were measured to verify thepossibility of degradation of protein cross-links polymerized
by glycation.

Materials and Methods

Santa Cruz Biotechnology (Heidelberg, Germany); 4', 5,7trihydroxyisoflavone (Genistein) from LC Laboratories(Woburn, Massachusetts, USA); rosemary extract (AGE
Breaker) 5) from A2P Science (Lyon, France); Other reagentswere purchased from Fujifilm Wako Pure Chemical Industries
or Nacalai Tesque (Kyoto, Japan) and used as special or
HPLC grade reagents.

1) Reagents

2) Samples

The reagents used were purchased from the following
manufacturers; lysozyme hydrochloride ( from egg white),rosmarinic acid, 1-phenyl-1, 2-propanedione (PPD), and benzoic
acid from Fujifilm Wako Pure Chemical Industries (Osaka,
Japan); 2- 40 % Mini-Protean TGX Precast gel from Bio-Rad(Hercules, California, USA); N-phenacylthiazolium bromide(PTB) from Sigma-Aldrich Japan (Meguro-ku, Tokyo, Japan).
(-)-epicatechin and (-)-epigallocatechin gallate from Nagara
Science (Gifu, Japan); 7-hydroxyflavone, 7,4'-dihydroxyflavone,
7,4'-dihydroxyflavanone (Liquiritigenin), 5,7-dihydroxyflavone
7-glucuronide (Chrysin), 3-(4-hydroxyphenyl) -1-(2,4,6 trihydroxyphenyl) propan-1-one (Phloretin), rosmarinic acid
(ester of caffeic acid and 3, 4-dihydroxyphenyllactic acid),
3, 3', 4', 5,7-pentahydroxyflavone 3-rutinoside (Rutin) from
Extrasynthese (Genay Cedex, France); punicalagin (2,3-(S)hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose) from

Samples were collected from 12 substances known
to be present in vegetables, herbs and other plants;
flavone,7-hydroxyflavone,
7,4'-dihydroxyflavone,
7, 4'-dihydroxyflavanone, 5,7-dihydroxyflavone 7-glucuronide,
3, 3', 4', 5,7-pentahydroxyflavone 3-rutinoside, (-)-epicatechin,(-)-epigallocatechin gallate, 4', 5,7-trihydroxyisoflavone,
3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1one, rosmarinic acid, and punicalagin ( Table 1 ). PTB wasused as a positive control for CGPC and CAC.

3) Preparation of glycated lysozyme
With reference to a previous report 10 -12), glycatedlysozyme was prepared by the reaction of 2 mg/mL lysozyme
with 0.1 mol/L phosphate buffer (pH 7.4) containing 0.2 mol/L
glucose at 60 ℃ for 40 hours, followed by the removal of

Table 1. Relationship between CGPC and CAC of substances contained in vegetables and herbs.
No

Substances

Typical herbs containing ingredients

CGPC (%)

CAC (%)

1flavonechamomile, green tea, parsley

ND

ND

3

7, 4'- dihydroxyflavone

broad beans, fenugreek

16.6 ± 4.7ND

47, 4'- dihydroxyflavanone(Liquiritigenin)

nuts, winged beans, leafy vegetables

8.3 ± 1. 3

ND55, 7-dihydroxyflavone 7- glucuronide(Chrysin)passion fruit, honey, propolis

54.7 ± 4.9ND

63, 3', 4', 5, 7-pentahydroxyflavone 3-rutinoside(Rutin)apple, buckwheat, green tea

13.6 ± 1.9

2.1 ± 0.9(-) -epicatechingreen tea, apple, berries, cacao
green tea, cacao

24.6 ± 5.4
96.8 ± 2.1

42.0 ± 3.994', 5, 7-trihydroxyisoflavone(Genistein)soy, broad beans

ND

ND

10

3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one(Phloretin)apple, pear

19.8 ± 1.3

1.1 ± 0.1

11

rosmarinic acid
(ester of caffeic acid and 3,4-dihydroxyphenyllactic acid)rosemary, lemon balm, perilla

33.6 ± 0.6

27.4 ± 0.1

12punicalagin(2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose)pomegranate

83.3 ± 2.0

22.8 ± 0.4

ref

PTB
(N-phenacylthiazolium bromide)—

5.6 ± 0.4

23.3 ± 0.2278berries, green tea, citrus

7-hydroxyflavone(-)-epicatechin gallate

ND

ND

6.9 ± 0.2

Data are shown as mean ± SD, n = 3; Sample concentration; 0.5 mmol/L (CGPC), 2.5 mmol/L (CAC); ND, less than 1 %; CGPC, cleavage of glycated-protein
cross-linking; CAC, cleavage of AGE cross-linking, ref; positive control; SD, standard deviation.,_ 10 7 _Substances with Cleavage of Glycated Protein Cross-linking and AGE Cross-linking

low molecular weight substances by 3kDa ultrafiltration
membrane (Amicon Ultra -0.5 mL centrifugal filters Ultracel3K; Merck, Darmstadt, Germany).

4) CGPCCGPC was calculated as the cleavage ratio of the ratioof glycated lysozyme dimer to monomer upon addition of thesample, referring to a previous report 10). To measure CGPC,
a 1 : 1 solution of 2 mmol/L sample mixed with 0.05 mol/L
phosphate buffer containing 0. 5 mg/mL glycated lysozyme;(S), and a solution of only sample lysate added instead ofsample in S; (R) were prepared and incubated at 37 °C for 16
hours.
The gel was stained with CBB Stain One (Nacalai
Tesque), the gel images were imaged with the Pharos FX
System (Bio-Rad, California, USA), and the band intensity ofthe monomer (M) and dimer (D) were analyzed with ImageJ(NIH, Maryland, USA) 13, 14). For CGPC, an glycated-protein
cross-link cleavage rate of 1% or more was considered to beactionable.
Glycated protein cross-link cleavage rate (%)= [ 1 − {(DS /MS) / (DR /MR )}] × 100
S; Lysozyme band intensity at the time of sample addition,
R; Band intensity of lysozyme when only sample solution isadded, M; monomer band, D; Dimer band.

5) CACCAC was measured by using PPD as a cross-link model
substance and measuring the cleavage rate of α-diketone
bonds of PPD by samples according to a previous report 4, 6, 15).
Briefly, 5 mmol/L sample, 10 mmol/L PPD, and 0.2 mol/L
phosphate buffer (pH 7.4) were mixed at a ratio of 5 : 1 : 4
and allowed to react at 37 °C for 8 hours. After the reaction
was stopped by adding 0.7 N HCl, the amount of benzoicacid; (A) released by the cleavage of the α-diketone bondof PPD by the sample was measured under the same HPLC
conditions as previously reported 6). The AGE cross-linkcleavage rate was calculated using the formula below, basedon the fact that 1 mol of PPD in the reaction solution yields 1
mol of benzoic acid when the α-diketone bond of PPD; (C)is cleaved. For CAC, an AGE cross-link cleavage rate of 1%
or more was considered to be actionable.
AGE cross-link cleavage rate (%) = { ( A − B ) / C } × 10 0
A; Amount of benzoic acid in the reaction solution (mol),
B; Amount of benzoic acid in the sample (mol),C; Amount of PPD used in the reaction (mol).

Statistical analysis
Measurements were expressed as the mean ± standard
deviation (SD) of triplicate measurements. Pearson's productrate correlation coefficient was used to analyze the correlation
between measurements. Statistical analysis results wereconsidered significant at a risk rate of less than 5% (p < 0.05).
Statistical analysis was performed using the statisticalanalysis software BellCurve for Excel (Shakai Joho Service,
Tokyo).

Results

1) CGPCCGPC was found in 9 out of 12 (75 %) of the samples
at a concentration of 0. 5 mmol/L in the reaction solution(Table 1 ). The percentage of glycated protein cross-linkcleavage of the 9 CGPC-containing substances was 35.7 ±
30.4% (mean ± SD) with (-)- epigallocatechin gallate (96.8 ±
2.1%) having the highest value and 7, 4'-dihydroxyflavanone(8.3 ± 1.3 %) showed the lowest value. An 11.7-fold difference
observed for the nine CGPC substances. (-)-Epigallocatechingallate showed a 17.3-fold stronger effect than PTB, a
positive control.

2) CACCAC actions were found in 6 of 12 substances (50 %)at a concentration of 2.5 mmol/L in the reaction solution( Table 1 ). Six substances with CAC showed a glycatedprotein cross-link cleavage rate of 17.9 ± 14.0 %, with(-)- epigallocatechin gallate (42.0 ± 3.9 %) having the highest
value and 3-(4-hydroxyphenyl)-1- (2,4,6-trihydroxyphenyl)
propan-1-one (1.1 ± 0.1%) showed the lowest value. A 38.2 fold difference was observed in the CACs of the six substances.
(-)-Epigallocatechin gallate was 1.8 times stronger than PTB,
which was compared as a positive control.

3) CGPC measurement of rosemary extract and PTB
The CGPC assay showed an attenuation in the band
intensity of lysozyme. In particular, when 1 ~ 5 mg/mL ofrosemary extract was added, the monomer band intensityof lysozyme attenuated to 88.5 ~ 33.5 % compared to 100 %when no extract was added ( Fig. 1, 2 ). CGPC measurementsof PTB showed an attenuation of the monomer band intensity,
similar to that of rosemary extract. However, the attenuation
was 93.6% at the maximum (5 mmol/L). The intensity of thelysozyme dimer band attenuated with increasing concentrationsof rosemary extract and PTB.

Discussion

1) Relationship between CGPC and CAC
Of the 12 substances tested, 3 (flavone, 7-hydroxyflavone,and 4', 5, 7- trihydroxyisoflavone) were not found to be
associated with either CGPC or CAC. In addition,
3, 3', 4', 5,7-pentahydroxyflavone 3-rutinoside, (-)-epicatechin,(-) - epigallocatechin gallate, 3 - (4 - hydroxyphenyl) -1(2,4,6-(trihydroxyphenyl)propan-1- one, rosmarinic acid,and punicalagin were found to act on both CGPC and CAC.
trihydroxyphenyl)propan-1- one, rosmarinic acid, andpunicalagin shouwed to act on both CGPC and CAC. A highpositive correlation was observed between CGPC and CAC
for the six substances with both of these effects ( p < 0.05 ),suggesting that α-diketone bond cleavage may be involved in
the degradation of protein cross-links ( Fig. 3 ).
In contrast, three substances (7,4'- dihydroxyflavone,
7, 4'- dihydroxyf lavanone, and 5, 7- dihydroxyf lavone
7-glucuronide) were found only in CGPC. In particular, the_ 10 8 _Glycative Stress Research

PTB (mmol/L)
MW

1

2

Rosemary extract (mg/mL)

5

Ref

0.1

0.5

1

2

5

35kDa

Dimer

25kDa

Monomer

11kDa

Fig. 1. CGPC by PTB and rosemary extract and the change of glycated lysozyme band intensity.

Sample and 0.5 mg/mL glycated lysozyme were incubated at 37 ℃ for 16 hours, n = 1; SDS-PAGE wasconducted using 2 ~ 40 % acrylamide gels. Stained with CBB stain one; MW, molecular weight markers; Ref,
incubation without sample (with 50 % ethanol); PTB, N-phenacylthiazolium bromide, a positive control; CGPC,
cleavage of glycated-protein cross-linking.

b)
60000

60000

50000

50000

Band intensity

Band intensity

a)

40000
monomer

30000

dimer

30000
20000

10000

10000

0

1

2

3

4

Concentration (mmol/L)

5

dimer

40000

20000

0

monomer

0

0

1

2

3

4

5

Concentration (mg /mL)

Fig. 2. Band intensity of PTB and rosemary extract on lysozyme dimer in the glycated lysozyme.

a) N-phenacylthiazolium bromide (PTB), b) rosemary extract. Sample and 0.5 mg/mL glycated lysozyme were incubated at 37 ℃ for 16

hours, n = 1. SDS-PAGE was conducted using 2 ~ 40 % acrylamide gels, stained with CBB stain one. MW, molecular weight markers;
Ref, incubation without sample (with 50 % ethanol).

a)

b)

Fig.2
A, N-phenacylthiazolium bromide (PTB), B; rosemary extract; Sample and 0.5 mg/mL glycated lysozyme w
ere incubated at 37! for 16 hours; SDS-PAGE was conducted using 2-40% acrylamide gels. Stained with C_ 10 9 _BB stain one; MW, molecular weight markers; Ref,
incubation without sample (with 50% ethaol).

Substances with Cleavage of Glycated Protein Cross-linking and AGE Cross-linking

50
8

CAC (%)

40

30

11
12

20

10
7
6

0
0

10
20

40

60

80

100

120

CGPC (%)

Fig. 3. Correlation of six substances with both CGPC
and CAC.

Each point indicates average value in triplets of CGPC
and CAC. Statistical analysis, Spearman's rank correlation
coefficient, p < 0.05; Sample concentration, 0. 5 mmol/L(CGPC), 2.5 mmol/L (CAC). Numbers in the graph indicate
substance number in Table 1. CGPC, cleavage of glycatedprotein cross-linking; CAC, cleavage of AGE cross-linking,protein cross-link cleavage rate of 5,7- dihydroxyflavone
7-glucuronide (64. 5 ± 17.7 %) was the third most potent
among the nine substances with CGPC. No relevance wassuggested between the presence or strength of CGPC/CAC
and substance structure for the 12 substances. In addition to
α-diketone bonds, protein cross-links by glycation include
those by glucospan and lysine-dihydropyridinium-lysine 3).
The cross-links that are cleaved by these three substances
may differ from the α-diketone structure. These results
suggest that the CGPC assay may be able to evaluate theeffect of glycated protein cross-link cleavage other thanα-dicarbonyl bond cleavage. However, the structure of theprotein cross-link formed in the glycated lysozyme dimer is
not known, and the mechanism could not be inferred.

2) Challenges in CGPC measurement
In the CGPC assay, the intensity of the monomericlysozyme band in SDS-PAGE attenuated to 33.5 % at the
maximum (5 mg/mL) when rosemary extract was added at
1 mg/mL or more, compared to 100 % when no extract wasadded. Protein staining with CBB Stain One is a staining
method in which the dye is adsorbed to the amino group of
the protein by electrostatic interaction 16). Insufficient staining
was presumably due to a charge change at the dye bindingsite caused by the coexistence of glycated lysozyme androsmarinic acid, or other unknown substances, contained inrosemary extract.

In the cross-link formation by protein glycation, reactive
aldehyde groups of glucose react with reactive amino groups
of protein chains to form glycation intermediates throughthe formation of Schiff bases 1, 2). These intermediates,
subsequently, attack the lysine-amino groups of Amadori
products and proteins to form stable cross-links that bindprotein chains together. However, when the α-dicarbonyl
linkage is cleaved, the protein does not return to its original
protein structure, but becomes a single protein molecule withmodifications 9, 17). This may have weakened the electrostaticinteraction between the protein and the dye, resulting ininsufficient CBB staining.
Since CGPC is calculated as the cleavage rate as theratio of lysozyme polymerized by protein glycation back tomonomer when the sample is added to the reaction system,
insufficient staining of lysozyme is a cause of measurement
error. The CGPC assay of edible purple chrysanthemum
extract calculated the protein cross-link cleavage rate basedon the reduction rate of only the dimer band intensity ofglycated lysozyme 10). In this case, no attenuation of thelysozyme monomer band was observed. Comparing theCGPC of rosemary extract, which attenuates the lysozyme
monomer band, between the previous and present method,the values by the previous methods 10) was calculated to be
1.4 -3. 5 % higher than the present results.
The SD of the CGPC values measured in this experiment
ranged from 0.4 to 5.4%, therefore, the difference in analysis
methods did not mislead as to the presence or absence of
the action. However, in the CGPC measurement of samples
attenuating lysozyme staining, care should be taken to use aformula adapted to this verification so that the reduction inband intensity does not become a factor in overestimating theprotein cross-link cleavage effect.

Research limitations
In this study, CGPC and CAC were verified using
12 substances found in herbs and other plants as samples.
Further comparison and validation of a larger number ofsubstances is needed to determine the relevance of CGPC
and CAC of various substances and their effect on protein
staining in the evaluation of CGPC.

Conclusion
CGPC and CAC of 12 substances in vegetables andherbs were measured. Six substances (50%) showed a highpositive correlation between CGPC and CAC, indicating thatα-diketone bond cleavage may be involved in the degradationof glycated protein cross-links. Whereas, only CGPC wasobserved for the three substances, and it was possible thatthey cleaved cross-links different from the α-diketone
structure. The CGPC assay may be able to evaluate glycatedprotein cross-link cleavage actions other than α-dicarbonylbond cleavage.

_ 110 _Glycative Stress Research

Acknowledgments
A part of this study was presented at the 23rd Meetingof Society for Glycation Stress Research (Kumamoto, Japan,
August 29, 2021). Support for publication of this study
was provided by the Isyoku-Dogen Research Foundation(IDF#22006).

Declaration of conflict of interest
There are no conflicts of interest in the conduct of thisstudy.

Reference1) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stressand photo-aging in skin. Anti-Aging Med. 2011; 8: 23-29.
2) Yagi M, Yonei Y. Glycative stress and anti-aging: 1. Whatis glycative stress? Glycative Stress Res. 2016; 3: 152-155.
3) Furber JD. Extracellular glycation crosslinks: prospects
for removal. Rejuvenation Res. 2006; 9: 274-278.
4) Vasan S, Zhang X, Zhang X. et al. An agent cleaving
glucose-derived protein crosslinks in vitro and in vivo.
1996; Nature. 382: 275-278.
5) Jean D, Pouligon M, Dalle C. Evaluation in vitro of AGEcrosslinks breaking ability of rosmarinic acid. Glycative
Stress Res. 2015; 2: 204-207.
6) Yagi M, Mitsuhashi R, Watanabe A, et al. Cleaving effectof pomegranate (Punica granatum) extract on crosslink
derived from advanced glycation endproducts. Glycative
Stress Res. 2015; 2: 58-66.
7) Takabe W, Mitsuhashi R, Parengkuan L, et al. Cleaving
effect of melatonin on crosslinks in advanced glycation
end products. Glycative Stress Res. 2016; 3: 38-43.
8) Yang S, Litchfield JE, Baynes JW. AGE-breakers cleave
model compounds, but do not break Maillard crosslinks
in skin and tail collagen from diabetic rats. Arch Biochem
Biophys. 2003; 412: 42- 46.
9) Aldini G, Vistoli G, Stefek M, et al. Molecular strategies
to prevent, inhibit, and degrade advanced glycoxidation
and advanced lipoxidation end products. Free Radic Res.
2013; 47(Suppl 1): 93-137.
10) Yagi M, Hara H, Mifuru R, et al. Suppression of glycatedprotein cross-linking formation and cross-linking cleavage
reaction of edible purple Chrysanthemum flower extract.
Glycative Stress Res. 2022; 9: 7-14.
11) Perera HKI, Ranasinghe HASK. A simple method todetect plant based inhibitors of glycation induced protein
cross-linking. Asian J Med Sci. 2015; 6: 28-33.
12) Perera HKI, Handuwalage CS. Analysis of glycationinduced protein cross-linking inhibitory effects of some
antidiabetic plants and spices. BMC Complement Altern
Med. 2015; 15, 175.
13) Rasband WS. Image J. U.S. National Institutes of Health,Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 19972012.

14) Schneider CA, Rasband WS, Eliceiri KW. NIH Image to

ImageJ: 25 years of image analysis. Nat Methods. 2012; 9:
671-675.
15) Yagi M, Sakiyama C, Miyata Y, Kamiya S, Yonei Y.
Antiglycative effect of genipin and crocetin. Glycative
Stress Res. 2021; 8: 156-161.
16) Steinberg TH. Protein gel staining methods: An introduction
and overview. Methods Enzymol. 2009; 463: 541-563.
17) Furber JD. Extracellular glycation crosslinks: Prospects
for removal. Rejuvenation Res. 2006; 9: 274-278.

_ 111 _