Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 13, 2023
Accepted : October 10, 2023
Published online : December 31, 2023
doi:10.24659/gsr.10.4_124

Original article

Direct proof of the deglycating effect of rosmarinic acid on a glycated protein
Audrey Picaud, Cédric Barthel, Louis Astier, Daniel Jean
Institut des Substances Végétales, Clermont-Ferrand, FranceAbstract

Purpose: It was shown in a previous work that rosmarinic acid is able to break AGE cross-links. This study focuses on theearly stage of glycation: the objective is to highlight the ability of rosmarinic acid to deglycate glycated lysozyme and see towhat extent the protein is recovered in its original state.
Basic procedures: Purified lysozyme is glycated with ribose. After elimination of ribose by purification, glycatedlysozyme is treated with rosmarinic acid. Glycation and deglycation are monitored over time by ESI-MS analysis.
Main findings: After 168 hours, the glycation leads to the binding of at least three molecules of ribose to the amino radicals
of lysozyme forming Schiff bases. The deglycation test shows that glycation is a naturally reversible reaction. The presence ofrosmarinic acid leads to a faster deglycation reaction.
Principal conclusions: This study shows that rosmarinic acid has an important deglycating effect on a glycated protein.
Further studies are required to better understand the mechanism of the deglycation process involving glycated lysozyme androsmarinic acid.

KEY WORDS: glycation, deglycation, rosmarinic acid, lysozyme

Introduction

Glycation is a non-enzymatic reaction between
carbohydrates and molecules with free amino groups, such asproteins. At first, a Schiff base is produced while a moleculeof water is discarded. Later, the irreversible isomerization of
the Schiff base leads to more stable products: the HeynsCarson products which are formed from ketoses and theAmadori products formed from aldoses 1).
At later stages, additional reactions occur on Amadori
products, such as cleavage or cyclization. Those reactions
lead to the formation of a heterogeneous group of compounds
called Advanced Glycation End products (or AGEs) 1). This
process involves several oxidative phenomena 2).
AGEs can be found in all body tissues and fluids.
They are able to cross-link with other proteins, altering
their functionalities 3). They can also alter gene expression,
intracellular signaling and provoke complications related
to diabetes or to aging such as Alzheimer's disease 4),
cardiovascular disease 5) or osteoporosis 6).
Research in AGE prevention has led to the discoveryof several AGE inhibitors. Some of these inhibitors areantioxidant 7, 8) and carbonyl trapping agents 9, 10). Beyond AGE
inhibitors, other molecules have been claimed to be able
to reverse glycation such as thiazolium compounds 11) orrosmarinic acid 12). Concerning the latter, a previous study

suggested its ability to disrupt AGE cross-links as well as its
potential use in preventing functional and structural damage
to proteins 13).
Lysozyme is an antimicrobial enzyme essential to theinnate immune system 14). Its small molecular weight makes
it better suited to analyze by mass spectrometry. Several
reports evaluated the dissociation of lysozyme fromdimer to monomer as AGE degradation 15). But to our best
knowledge, no one has studied deglycation in the initialstage.
In this work, a mass spectrometry method was developed
to study the ability of rosmarinic acid to reverse glycation
at its early steps and to what extent it could return glycatedlysozyme back to its original state.

Materials and methods

Reagents and materials are as follows: lysozyme fromchicken egg white (Sigma Aldrich, reference 62971),
ribose (Sigma Aldrich, reference R7500), acetic acid (SigmaAldrich, reference 45754), ammonium hydroxide (SigmaAldrich, reference 221228), deionized water, sephadex®
G-50 (Sigma Aldrich, reference G5080) and rosmarinicacid (Sigma-Aldrich, reference 536954).

Correspondences: Dr. Daniel Jean, PhD
Institut des Substances Végétales
19 rue Patrick Depailler 63000 Clermont-Ferrand, France
TEL : +33 4 73 69 15 39 e-mail: daniel.jean@insuveg.com
Co-authors; Picaud A, audrey.picaud@insuveg.com;
Barthel C, cedric.barthel@insuveg.com; Astier L, louis.astier@insuveg.com_ 12 4 _Glycative Stress Research 2023; 10 (4): 124 -144
(c) Society for Glycative Stress ResearchGlycative Stress ResearchPreparation of glycated lysozyme at pH 6Step 1: Lysozyme is purified by Reveleris® PREP
Purification System on Sephadex® G50 in deionized water.
Fractions corresponding to pure lysozyme are detected (seeAnalysis), collected and freeze-dried.
Step 2 : A 2.000 % (w/w) solution of lysozyme purified instep 1 and ribose 0.500 % (w/w) is prepared in ammonium
acetate buffer at pH 6. The mixture is then left for 168 hours(h) in a drying oven at 37 °C.
Step 3 : The glycation reaction is stopped throughthe elimination of excess ribose by Reveleris® PREP
Purification System on Sephadex® G50 in deionized water.
Fractions corresponding to glycated lysozyme are detected(see Analysis), collected and freeze-dried.

Deglycation test
A 1.000 % (w/w) solution of purified glycated lysozyme
in ammonium acetate buffer at pH 6 and a 0.017 % (w/w)
solution of rosmarinic acid in MeOH/ammonium acetate
buffer at pH 6 (8:92) are prepared.
Blank sample: 1 mL of purified glycated lysozyme solution
is added to 1 mL of solvent MeOH/ammonium acetate
buffer at pH 6 (8 :92).
Deglycation sample: 1 mL of purified glycated lysozyme
solution is added to 1 mL of rosmarinic acid. Mixtures areleft for 720 h in a drying oven at 37 °C.

Analysis
Lysozyme glycation and deglycation as well as
purification steps are analyzed by mass spectrometry on
a Q-Tof -2 T M instrument (Micromass, Waters) with anelectrospray ion source. Nitrogen is used as the nebulizer
gas and argon as the collision gas at 7 bars and 2 bars
respectively. Analytical conditions are as follow: The source
temperature is 80 °C and 150 °C for the desolvation. The
collision energy, cone and capillary voltage are set at 10eV, 35 V and 2.5 kV, respectively. The acquisition range is90-3,000 m/z in positive ion mode. Samples are analyzed
by direct injection with a syringe pump (AL-300, WPI) at aflow rate of 15 μL/min. Masslynx v4.0 software is used tocontrol the instrument, for data acquisition and processing.

Results and discussion
After the purification of commercial lysozyme (step 1),
the ESI-MS spectrum obtained by deconvolution of themultiple ions shows a major pic of m/z = 14,302 (theoreticalmass: single-chain 14,300 Da, Mr ~ 14,600) (Fig. S1). Then,
glycation between lysozyme and ribose was carried out asdescribed above. To confirm that the reaction took place,mass spectrometry analysis is performed to detect thepresence of glycated lysozyme. These analyses areperformed over time at 0 h, 24 h, 48 h, 72 h and 168 h (Fig.
S2). The mass spectra show an increase in the number ofpeaks, corresponding to the covalent attachment of ribose to
the different amino sites of lysozyme. After purification step
3, at least three molecules of ribose have attached themselvesto the amino radicals of lysozyme forming Schiff bases(Fig. S3). For more accurate results, the eightfold ion wasused for the calculation of the added mass on the protein.
The average added mass is m/z = 132.1691 (theoreticalmass of ribose: 150.0578 Da, minus 18.0105 Da, H2O =132.0473) (Table 1).
After the elimination of ribose (purification step 3) tostop the glycation reaction, a deglycation test is performed.
In order to assess the impact of rosmarinic acid on glycatedlysozyme, mass spectrometry analyses are carried out overtime and compared with the blank sample (Fig. S4 ). Themass spectrum of the blank sample (Fig. S4-a) generates ion
signals m/z ≈ 1,806, m/z ≈ 1,822 and m/z ≈ 1,839 corresponding
to sites of glycated lysozyme. For more accurate results, theion m/z ≈ 1,806 corresponding to the first site of glycation
was chosen. Table 2 shows the evolution of intensity ratios
between m/z ≈ 1,806 and m/z ≈ 1,789 (which correspond toglycated lysozyme and initial lysozyme respectively).
In both cases, ratios decrease over time. The blank sample
shows that glycation is a naturally reversible reaction. The
presence of rosmarinic acid leads to a deglycation rate whichis 54.24% higher than in the blank sample at 192 h ( Fig. 1 ).
At 168 h, regarding the structure, initial protein state is nearlyrecovered ( Fig. S5 ). The recovery of enzyme activity will be
studied in future works.

Table 1. Calculation showing the addition of ribose on lysozyme after 168 hours.
1789.4940

1806.0155

1822. 5253

1806.0155

1822.5253

1839.0574

Mass difference (Da)

16.5215

16.5098

16.5321

Deconvolution (ion octuple) (Da)132.1720

132.0784

132.2568Average (Da)132.1691

Theoretical mass132.0473

Mass from Fig. S3 (Da)Analyzed by mass spectrometry on a Q-Tof-2TM instrument (Micromass) with an electrospray ion source. For measurement
conditions, see the "Analysis" section of the text.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Table 2-a. Deglycation test: peak intensity evolution over time for blank sample.
Time (hours)

Intensity of m/z ≈ 1789

Intensity of m/z ≈ 1806

Ratio

01813

1856

1.02

2418621275

0.68

722291994

0.43489151752

168

192

2325

609

2218

476

2156

240
336

1911

408

552

0.26

520

0.24

357

0.19

2487

424

1476

211

0.21
0.17

323

2015

720

0. 52

0.16
0.14

Analyzed by mass spectrometry on a Q -Tof-2TM instrument (Micromass) with an electrospray ion source. For measurement
conditions, see the "Analysis" section of the text.

Table 2-b. Deglycation test: peak intensity evolution over time for deglycation sample.
Time (hours)

Intensity of m/z ≈ 1789

Intensity of m/z ≈ 1806

Ratio

01480

1510

1.02

1735

606

2161

265

24

947

0. 55

517

0.252084

230

0.11

1749

174

0.10

169

0.11

1709

48
72

2068

168

192

240
408

552

1415

159

647

86

1473

720

0.12

181

1623

336

0.35

0.11
0.11

0.13

Analyzed by mass spectrometry on a Q-Tof-2TM instrument (Micromass) with an electrospray ion source. For measurement
conditions, see the "Analysis" section of the text.
1.20

Blank sample
Deglycation sample

1.00

Intensity

0.80
0.60
0.40
0.20
0.00

0100

200

300

400

500

600

700

800

Time (hours)

Fig. 1. Deglycation test: Ratios evolution between m/z ≈ 1789 and m/z ≈ 1806 over time.

Analyzed by mass spectrometry on a Q-Tof-2TM instrument (Micromass) with an electrospray ion source.
For measurement conditions, see the "Analysis" section of the text.

_ 12 6 _Glycative Stress Research

On the deglycation mass spectra with rosmarinic acid,the ion m/z ≈ 1,834 appears ( Fig. S4-k). By subtracting theeightfold ion m/z ≈ 1,789 from m/z ≈ 1,834, it leads to m/z≈ 45. Multiplying this mass by 8 leads to m/z ≈ 360 whichcorrespond to the rosmarinic acid molecular weight. Thereby,
the mass m/z ≈ 1,834 might be the eightfold ion of a complex
or an adduct between lysozyme and rosmarinic acid. Further
studies using chromatographic methods are required toexplore this hypothesis.

Conflicts of interest statement
Part of this work was supported by A2P Sciences sas,
Lyon, France, as part of its "Glycation and Healthy
Longevity" research project.

Conclusion
Using a new simple mass spectrometry method weprovide a direct proof of a deglycation reaction on a glycatedprotein. Indeed, our analysis clearly showcases a reversionof glycation reaching a stationary level after 720 h.
Moreover, the impact of rosmarinic acid on the glycatedlysozyme is also highlighted: rosmarinic acid leads to afaster deglycation reaction than what naturally occurs.
This suggest that rosmarinic acid might play a major rolein AGE regulation, going further than previously
discovered AGE inhibitors. Our future work will focus on
the interaction between glycated lysozyme and rosmarinicacid to better understand the mechanism underlying the
deglycation process involving this compound, and on therecovery of enzyme activity.

References
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disease. PLoS One. 2015; 10: e0143345.
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glycation end products in cardiovascular disease. World J
Cardiol. 2012; 4: 90-102.
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critical link between advanced glycation end products,osteoporosis and type 2 diabetes mellitus. Regenerative
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7) Ramkissoon JS, Mahomoodally MF, Ahmed N, et al.
Antioxidant and anti-glycation activities correlates withphenolic composition of tropical medicinal herbs. AsianPac J Trop Med. 2013; 6: 561-569.
8) Daiponmak W, Senakun C, Siriamornpun S. Antiglycation
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Thai rice. International Journal of Food Science andTechnology. 2014; 49: 1805-1810.

9) Navarro, Marta & Fiore, Alberto & Fogliano, Vincenzo &Morales, Francisco. Carbonyl trapping and antiglycative
activities of olive oil mill wastewater. Food Funct. 2015; 6:574-583.
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glycation end product formation by trapping methylglyoxal
and glyoxal. J Agric Food Chem. 2014; 62: 12152-12158.
11) Vasan S, Zhang X, Zhang X, et al. An agent cleaving
glucose-derived protein crosslinks in vitro and in vivo.
Nature. 1996; 382(6588): 275-278.
12) Jean D, Pouligon M, Dalle C. Evaluation in vitro of AGEcrosslinks breaking ability of rosmarinic acid. Glycative
Stress Res. 2015; 2: 204-207.
13) Alrubaye A, Motovali-Bashi M, Miroliaei M. Rosmarinicacid inhibits DNA glycation and modulates the expressionof Akt1 and Akt3 partially in the hippocampus of diabetic
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14) Ragland SA, Criss AK. From bacterial killing to immune
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_ 127 _Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular DrynessFig. S1. ESI-MS spectra (octuple ion) and deconvolved ESI-MS spectra of lysozyme after purification.
Molecular weight: 14302.

_ 12 8 _Glycative Stress Research

Fig. S2-a. ESI-MS spectra of lysozyme after reacting with ribose for 0 hour.

Fig. S2-b. ESI-MS spectra of lysozyme after reacting with ribose for 24 hours.

_ 12 9 _Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S2-c. ESI-MS spectra of lysozyme after reacting with ribose for 48 hours.

Fig. S2-d. ESI-MS spectra of lysozyme after reacting with ribose for 72 hours.

_ 13 0 _Glycative Stress ResearchFig. S2-e. ESI-MS spectra of lysozyme after reacting with ribose for 168 hour.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S3. ESI-MS spectra (octuple ion) and deconvolved ESI-MS spectra of glycated lysozyme after purification.

_ 132 _Glycative Stress Research

Fig. S4-a. ESI-MS spectra of deglycation test on glycated lysozyme at 0 hour:(A) blank sample and (B) deglycation sample.

_ 133 _

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S4-b. ESI-MS spectra of deglycation test on glycated lysozyme at 24 hours:(A) blank sample and (B) deglycation sample.

_ 13 4 _Glycative Stress Research

Fig. S4-c. ESI-MS spectra of deglycation test on glycated lysozyme at 48 hours:(A) blank sample and (B) deglycation sample.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S4-d. ESI-MS spectra of deglycation test on glycated lysozyme at 72 hours:(A) blank sample and (B) deglycation sample.

_ 13 6 _Glycative Stress Research

Fig. S4-e. ESI-MS spectra of deglycation test on glycated lysozyme at 168 hours:(A) blank sample and (B) deglycation sample.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S4- f. ESI-MS spectra of deglycation test on glycated lysozyme at 192 hours:(A) blank sample and (B) deglycation sample.

_ 13 8 _Glycative Stress Research

Fig. S4-g. ESI-MS spectra of deglycation test on glycated lysozyme at 240 hours:(A) blank sample and (B) deglycation sample.

_ 13 9 _Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S4-h. ESI-MS spectra of deglycation test on glycated lysozyme at 336 hours:(A) blank sample and (B) deglycation sample.

_ 14 0 _Glycative Stress Research

Fig. S4-i. ESI-MS spectra of deglycation test on glycated lysozyme at 408h:(A) blank sample and (B) deglycation sample.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S4-j. ESI-MS spectra of deglycation test on glycated lysozyme at 552 hours:(A) blank sample and (B) deglycation sample.

_ 14 2 _Glycative Stress Research

Fig. S4-k. ESI-MS spectra of deglycation test on glycated lysozyme at 720 hours:(A) blank sample and (B) deglycation sample.

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Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Fig. S5. Deconvolved ESI-MS spectra of deglycation test on glycated lysozyme at 168 hours:(A) blank sample and (B) deglycation sample.

_ 14 4 _