Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 18, 2016
Accepted : May 24, 2016
Published online : June 30, 2016

Original Article

Inhibitory effect of rosehip (Rosa canina L.) on the formation of advanced
glycation endproducts by scavenging reactive carbonyls
Tomoaki Kawaguchi 1), Akira Takano 2), Tadayuki Tsukatani 1), Rieko Kuroda 1), Kyoko Ueda 1),
Masahito Tsubata 2)1) Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume, Fukuoka
2) Research and Development Division, Toyo Shinyaku Co., Ltd., Tosu, Saga

Abstract

The accumulation of advanced glycation endproducts (AGEs) and carbonyl stress in vivo are associated with diabetic
complications. The objective of this study was to investigate the inhibitory effect of an aqueous extract of rosehip (Rosacanina L.) on the formation of AGEs derived from reactive carbonyl species such as methylglyoxal (MGO). A water extractof rosehip exhibited anti-glycation activity in the MGO-BSA assay and a protective effect against reactive carbonyl species
in a direct MGO-scavenging assay with IC 50 values of 3.7 mg/mL and 2.7 mg/mL, respectively. To determine the active
substances present in the extract, activity-guided fractionation was performed. The extract was divided into water andbutanol layers. The water layer was further fractionated into five subfractions by DIAION HP-20 column chromatography.
The twenty-five percent ethanol eluate had high anti-glycation activity (IC 50 = 1.0 mg/mL) and direct MGO-scavenging
activity (IC 50 = 1.0 mg/mL). HPLC analysis revealed that the eluate was mainly composed of procyanidin glycosides with anumber-average molecular weight of 3,500. These results suggest that this aqueous rosehip extract could be beneficial as anatural source of a glycation inhibitor because procyanidin glycosides present in the extract inhibited the formation of AGEs
through their direct carbonyl scavenging activities.

KEY WORDS: Glycation, methylglyoxal, Rosa canina L., proanthocyanidin, carbonyl scavenger
Introduction

Advanced glycation endproducts (AGEs) are the final
products derived from non-enzymatic glycation and oxidation
reactions of sugars with the amino groups of proteins, lipids
and nucleic acids 1). The accumulation of AGEs has been
implicated as a major pathogenic process in diabetic
complications 2), normal aging 3), and many other chronic
diseases such as Alzheimer’s disease 3) and atherosclerosis 4).
Dicarbonyl intermediates such as methylglyoxal (MGO),
glyoxal, and 3-deoxyglucosone in the glycation reaction are
known as precursors of AGEs because of their high abilities
to crosslink with amino groups of proteins to form irreversible
AGEs 5). Increased levels of MGO have been observed in the
blood or tissues of diabetic patients with complications,and have been reported to be an important factor whichcauses insulin resistance 5). The chronic exposure to highconcentration of MGO has been thought to induce insulin
signaling impairment and apoptosis 5) and to be linked to thedevelopment of diabetic complications 6).

AGEs inhibitors which possess reactive carbonyl
scavenging capacities have been reported to be effective inthe prevention of MGO-associated impairment of insulin
signaling pathways 5) and in the amelioration of diabetic
complications 1). Therefore, inhibiting the formation of AGEs
by scavenging reactive carbonyl species (especially MGO)
could be a promising therapeutic approach to treat diabetes
and prevent diabetic complications.
In traditional medicine, rosehip has been used for theprevention and therapy of the common cold, flu, and diabetes
in many European countries 7). Biological activities of rosehip
which have been reported include anti-oxidative 8), antiobesity 9), anti-inflammatory 10), and melanin production
inhibitory 11) activities. Rosehip is rich in vitamin C,
carotenoids, and phenolic compounds such as quercetin,
ellagic acid, hydroxycinnamic acids, and proanthocyanidin
aglycones 12, 13). Recently, rosehip and its constituent transtiliroside, a major glycosidic flavonoid isolated from rosehip
seed, were reported to exert a blood glucose lowering effectCorresponding author: Tomoaki Kawaguchi, Ph.D.
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center,
1465-5 Aikawa-machi, Kurume-shi, Fukuoka 839-0861, Japan.
Tel: +81-942-30-6215, Fax: +81-942-30-7244 E-mail: t_kawaguchi@fitc.pref.fukuoka.jp
Co-authors: Takano A, takanoa@toyoshinyaku.co.jp ; Tsukatani T, tukatani@fitc.pref.fukuoka.jp ;Kuroda R, kuroda@fitc.pref.fukuoka.jp ; Ueda K, kueda@fitc.pref.fukuoka.jp ;Tsubata M, tsubatam@toyoshinyaku.co.jp_ 91 _

Glycative Stress Research 2016; 3 (2): 91- 98(c) Society for Glycative Stress ResearchInhibitory Effect of Rosehip on AGE Formation
after glucose loading in mice 14) and hypoglycemic effects
in diabetic rats 15). However, no study has been conducted
to investigate the capacity of rosehip to attenuate carbonyl
stress and protein glycation. The objective of this study
was to evaluate the anti-glycation and carbonyl scavenging
activities of rosehip.

Materials and Methods
Materials

Methylglyoxal (MGO, 4 0 % aqueous solution),aminoguanidine, and polystyrene standards were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum
albumin fraction-V fatty acid-free was from Millipore(Billerica, MA, USA). Procyanidin B2, cyanidin, andpelargonidin were purchased from Extrasynthese (Genay,
France). Girard’s T reagent was purchased from Tokyo
Chemical Industry (Tokyo, Japan). Ammonium ferric sulfate
was obtained from Wako Pure Chemical Industries (Osaka,
Japan). Delphinidin was purchased from Cayman Chemical(Ann Arbor, MI, USA).

Extraction and fractionation of rosehip

Rosehip powder (50 g) was extracted with 500 mL of
distilled water for 1 hour at 95°C after rapid washing with
200 mL of distilled water to remove excess sugar. The extract
was mixed with an equal volume of water-saturated n-butanoland divided into the water and butanol layers. This liquidliquid extraction was repeated three times. Each layer wasdried under reduced pressure. The water layer dissolved inwater was loaded onto a DIAION HP-20 (Mitsubishi Kagaku
Co., Ltd., Tokyo, Japan) column. The column was eluted withwater, 5% ethanol, 25% ethanol, 50% ethanol, and 99.5%
ethanol.

Glycation assay with MGO-BSA

This assay was performed as described previously 16)
with slight modifications. Bovine serum albumin (BSA) and
MGO were dissolved in 100 mM sodium phosphate buffer(pH 7.4) containing 0.2 g/L sodium azide at a concentrationof 20 mg/mL and 60 mM, respectively. BSA solution (50 μL)
was reacted with 50 μL of MGO solution in the presenceof 50 μL of rosehip samples, aminoguanidine (positive
control), or phosphate buffer as a control at 37 °C for 7 days.
To remove unreacted MGO and interfering substances, 17 μL
of 100% w/w trichloroacetic acid (TCA) was added to thereaction mixture. After centrifugation, the TCA precipitatecontaining fluorescent AGEs-BSA was washed with phosphatebuffer and dissolved in 200 μL of alkaline phosphate buffer(pH 10). The fluorescence intensity of this solution wasmeasured using a Synergy H4 microplate reader ( BioTek
Instruments, Inc., Winooski, VT, USA). The excitationand emission wavelengths used were 340 nm and 420
nm, respectively. Phosphate buffer was used as the blank.
The apparent inhibitory effect was obtained from the following
equation: apparent inhibition rate (%) = {1 − (fluorescenceof test sample solution) / (fluorescence of control solution)}× 100.
To subtract the contribution of co-precipitated substanceswith AGEs-BSA from the obtained fluorescence, the quenchingrate was then estimated as follows 17). BSA was reacted with

MGO in the absence of rosehip samples as described above.
The AGEs-BSA solution obtained was mixed with the rosehip
sample for 3 hours at room temperature to allow adsorption
to AGEs-BSA. After TCA treatment, the sample was dissolved
in alkaline phosphate buffer. The fluorescence intensity ofthis solution was measured using the microplate reader. The
quenching rate was calculated using the following equation:quenching rate (%) = {1 − (fluorescence of test sample
solution) / (fluorescence of control solution)} × 100.
The actual inhibition activity was calculated by subtracting
the quenching rate from the apparent inhibition rate.

MGO-scavenging assay

The MGO-scavenging capacity was evaluated bymeasuring MGO consumption after incubating the rosehip
samples with MGO 18). MGO concentration was measured
using Girard’s T reagent. Phosphate buffer was used as theblank. Twenty-five microliters of 2 mM MGO solution was
mixed with 25 μL of test samples, aminoguanidine (positive
control), or phosphate buffer as a control under the conditionsof 0.1 M sodium phosphate (pH 7.4) at 37 °C for 1 hour,
followed by the addition of 950 μL of 120 mM sodium borate.
An aliquot of the mixture (200 μL) was reacted with 800 μL
of 100 mM Girard’s T reagent for 10 minutes at room
temperature. Absorbance at 326 nm was measured using a
UV-VIS spectrometer Evolution 220 (Thermo Fisher ScientificInc., Waltham, MA, USA). The MGO-scavenging capacity
was calculated using the following equation: MGO-scavenging
capacity (%) = [1 − {(absorbance of test sample solution withMGO) − (absorbance of test sample solution without MGO)}/ (absorbance of control solution)] × 100.

Determination of proanthocyanidin content

To determine the proanthocyanidin content in the rosehip
samples, the acid-butanol assay was performed as described
previously 11). Fifty-eight microliters of the samples was mixed
with 1 mL of 0.6 N HCl-containing butanol and 16 μL of
0.04 M ammonium ferric sulfate solution. Then, the mixture
was incubated for 50 minutes at 95°C. Absorbance at 555
nm was measured. Quantification of proanthocyanidins wasachieved with procyanidin B2 as the standard.

Qualitative analysis of proanthocyanidin

To characterize proanthocyanidins present in the rosehip
samples, HPLC analysis of the acid hydrolysate of the rosehip
sample was performed 19). One milligram of the rosehip sample
dissolved in 1 mL of 1 N HCl was hydrolyzed for 1 hour inboiling water. The hydrolysate was evaporated to dryness
and dissolved in methanol. The type of proanthocyanidin
in the sample was analyzed by an Agilent 1260 HPLC
system (Agilent Technologies, Palo Alto, CA) equipped withan Agilent Poroschell 120 EC-C18 column (4.6 mm × 50
mm, 2.7 μm) and a photodiode array detector at 30°C withanthocyanidins (cyanidin, delphinidin, and pelargonidin) as
standards. The elution was achieved under the conditions:linear gradient of 5% to 40% B in 60 minutes with (A) 0.1%
formic acid in water, (B) acetonitrile at a flow rate of 0.5
mL/min. The detection wavelength was 550 nm.

Determination of total phenolic content_ 92 _

The amount of total phenolics in the rosehip samples

Glycative Stress Research

was determined according to the Folin-Ciocalteu method 20)
with procyanidin B2 as a standard. Five hundred microlitersof a rosehip sample or standard solution, 250 μL of aqueous
solution of Folin-Ciocalteu reagent (1:1, v/v), and 2.5 mL of
0.4 M sodium carbonate were mixed and incubated at 30°C
for 30 minutes. Then, the optical density at 765 nm wasmeasured using a UV-VIS spectrometer.

as a standard 11). A 500 μL amount of samples, 500 μL of
5% phenol, and 2.5 mL of sulfuric acid were mixed andincubated for 30 minutes at room temperature. The total
sugar concentration was determined by spectrophotometry
at a 490 nm wavelength.

Gel permeation chromatography

Data were analyzed by one-way analysis of variance(ANOVA) followed by Tukey’s HSD test for multiple
comparisons using R (The R Foundation for StatisticalComputing, http://www.r-project.org). All p-values < 0.05
were considered as statistically significant.

To determine the molecular weight profile ofproanthocyanidins in the rosehip sample, gel permeation
chromatography (GPC) was performed with polystyrene
standards and a catechin standard 21). The rosehip sample
dissolved in pyridine-acetic anhydride (1:1, v/v) was
acetylated overnight at room temperature. Excess reagent
was destroyed by adding water. The resulting precipitatecontaining acetylated-proanthocyanidins was recovered bycentrifugation and dried under reduced pressure. The
acetylated-proanthocyanidins were dissolved in tetrahydrofuran(THF) and analyzed by an Agilent 1260 HPLC systemequipped with a Shodex KF-804L column (8.0 × 300 mm,
Showa Denko K.K., Tokyo, Japan) at 40°C. The elution was
isocratic with THF at a flow rate of 0.8 mL/min. The detection
wavelength was 254 nm. The number-average molecularweight (Mn), the weight-average molecular weight (Mw),
and the polydispersity index (Mw/Mn) of the sample were
calculated by a GPC data processing program (SIC μ7 Data
Station, System Instruments Co., Ltd., Tokyo, Japan).

Determination of total sugar content

The total sugar content in the rosehip sample wasmeasured by the phenol-sulfuric acid method with glucose

Statistical analysis

Results

Anti-glycation and carbonyl scavenging activities
of rosehip water extract

To evaluate the anti-glycation activity of rosehip
water extract (RE), the MGO-BSA assay was performed.
Aminoguanidine (AG), which is a scavenger of α, βdicarbonyls that prevents the formation of AGEs, was used
as a positive control. Figure 1A indicates that RE inhibited
MGO-mediated AGEs formation in a concentration dependent
manner (IC 50 = 3.7 mg/mL). Next, we investigated whether
reactive carbonyl species such as MGO interact directlywith constituents in rosehip extract.
A concentration dependent MGO-scavenging activity of
RE with an IC 50 value of 2.7 mg/mL was observed as shownin Fig. 1B. These results suggested that RE possesses aninhibitory effect on the formation of AGEs and has direct
MGO-scavenging capacity.

B
100

100

80

80

Decrease of MGO (%)

Inhibition of AGEs formation (%)

A

60

40
RE
AG

20

0

0

2

4

60

40
RE
AG

20

0

6

Concentration (mg/mL)

0

2

4

6

8

Concentration (mg/mL)

Fig. 1. Anti-glycation (A) and MGO-scavenging (B) activities of rosehip extract.

Results are expressed as the mean ± standard deviation of triplicate experiments. MGO, methylglyoxal;
AG, aminoguanidine; RE, rosehip water extract.

_ 93 _

10

Inhibitory Effect of Rosehip on AGE Formation

Activity-guided fractionation of rosehip extract

To determine the active substances in RE, activityguided fractionation was performed based on two in vitro
assays (the MGO-BSA and direct MGO-scavenging assays).
RE was first partitioned into two layers by the addition ofwater-saturated n-butanol. The water layer showed significantly
higher activities compared to the butanol layer with a 53%
inhibition in the MGO-BSA assay ( Fig. 2A) and a 71%
decrease in the MGO-scavenging assay ( Fig. 2B ). The
butanol layer had only a 16% inhibition in the MGO-BSA
assay and an 11% decrease in the MGO-scavenging assay.

Subsequently, the water layer was further fractionated by
DIAION HP-20 column chromatography. Anti-glycationand MGO-scavenging activities of the 25% ethanol eluate(REW25E) were significantly higher than that of the othereluates, but not significantly higher than that of RE or thewater layer ( Figs. 2A and 2B). The activities of REW25E
were concentration dependent, with IC 50 values of 1.04 mg/
mL and 1.02 mg/mL in the MGO-BSA and direct MGOscavenging assays, respectively ( Figs. 3A and 3B). These
results suggested that active substances are present in
REW25E.

Inhibition of AGEs formation (%)

A
60

a

a

a

50
40
30
20

b

b

c

c

10

c

0

RE

butanol
layer

water
layer

water

5%
EtOH

50%
EtOH

EtOH

c

c

50%
EtOH

EtOH

25%
EtOH

HP-20 eluate

B
Decrease of MGO (%)

80

a

a

a

60

40

20

b

bc

bc

0

RE

butanol
layer

water
layer

water

5%
EtOH

25%
EtOH

HP-20 eluate

Fig. 2. Anti-glycation (A) and MGO-scavenging (B) activities of rosehip fractions.

Results are expressed as the mean ± standard deviation of triplicate experiments. Concentrations assayedwere based on the yields of fractions and were 5, 0.37, 4.49, 3.01, 0.21, 0.91, 0.04 and 0.46 mg/mL for
RE, butanol layer, water layer, water, 5% ethanol (EtOH), 25% EtOH, 50% EtOH and EtOH, respectively.
Different letters above the columns represent a statistically significant difference (p < 0.05) by Tukey’s test.
AGEs, advanced glycation endproducts; MGO, methylglyoxal; RE, rosehip water extract.

_ 94 _Glycative Stress Research

B
100

100

80

80

Decrease of MGO (%)

Inhibition of AGEs formation (%)

A

60

40

REW25E
AG

20

0

0

2

4

60

40

AG

20

0

6

REW25E

02

Concentration (mg/mL)

4

6

8

10

Concentration (mg/mL)

Fig. 3. Anti-glycation (A) and MGO-scavenging (B) activities of REW25E.

Results are expressed as the mean ± standard deviation of triplicate experiments. AGEs, advanced
glycation endproducts; MGO, methylglyoxal; AG, aminoguanidine; RE, rosehip water extract;
REW25E, active fraction of RE.

Compositional properties of the REW25E fraction

Active substances in REW25E were assumed to behydrophilic phenolic compounds such as proanthocyanidin
because of their non-extractability in butanol and adsorption
to the aromatic non-polar surface of HP-20 resin that adsorbs
aromatic molecules by van der Waal’s forces. To clarify
the substances, we measured the total phenolics andproanthocyanidins in the fraction ( Table 1 ). REW25E
contained 61.6% phenolic compounds and 4 8%
proanthocyanidins, indicating that most of the phenolic
compounds in R EW25E were proanthocyanidins(proanthocyanidins/total phenolics = 78%), as expected. Total
sugar analysis showed that REW25E contained 38.8% sugars.
These results indicated that the constituents in REW25E
are mainly composed of proanthocyanidins and sugars.

Table 1. Constituents of each fraction obtained from RE.
total phenolics(w/w%)

proanthocyanidins(w/w%)

RE

19.9

7.0

butanol layer

ND

ND

water layer

17.5

3.4

water eluate

ND

ND

5% EtOH eluate

57.3

34.1

25% EtOH eluate

61.6

48.0

50% EtOH eluate

58.2

46.8

EtOH eluate

28.8

10.9

Results are expressed as the mean of triplicate experiments. ND, not detected; RE, rosehip water extract; EtOH, ethanol.

_ 95 _

total sugar(w/w%)

38.8

Inhibitory Effect of Rosehip on AGE Formation
proanthocyanidins in REW25E were procyanidin-type, i.e.
(+)-catechin and/or (−)-epicatechin derivatives.
The molecular weight profiles of REW25E were obtainedby GPC. As shown in Fig. 5, REW25E had a molecular weight
distribution ranging from about 1,000 to 20,000. The average
molar mass (Mw, Mn) and polydispersity (Mw/Mn) of the
REW25E proanthocyanidins were 6,000 (Mw), 3,500 (Mn),
and 1.7, respectively.

Qualitative analysis of proanthocyanidins in REW25E

To characterize the proanthocyanidins in REW25E,
HPLC analysis of the HCl-hydrolysate of REW25E wasperformed. HPLC profiles of the hydrolysate (upper) andanthocyanidin standards (lower) are shown in Fig. 4. The
single peak observed in the hydrolysate was identified as
cyanidin by comparison of the retention time with that ofanthocyanidin standards. This result indicated that the

A

12

13

14

B

12

13

15

16

17

18

15

20

21

Cy

Dp

14

19

16

17

18

19

22

23

24

25

26

23

24

25

26

Pg

20

21

22

Time (min)Fig. 4. HPLC profiles of acid hydrolysate of REW25E (A) and anthocyanidin standards (B).

Dp, delphinidin; Cy, cyanidin; Pg, pelargonidin. HPLC, high performance liquid chromatography; RE,
rosehip water extract; REW25E, active fraction of RE.

6

Absorbance at 254 nm

5

3

Log M

4

2
1

6

7

8

9

10

11

12

13

14

15

16

0

Time (min)Fig. 5. Chromatogram of acetylated REW25E and molecular weight calibration curve.

A molecular weight calibration curve (dashed line) was obtained with six standard polystyrenes (50,000, 30,000, 17,500, 9,000, 5,000,
and 2,500 g/mole) and catechin (290 g/mole). M, molecular weight; RE, rosehip water extract; REW25E, active fraction of RE.

_ 96 _Glycative Stress Research

Discussion
The accumulation of AGEs and carbonyl stress havebeen implicated in the pathogenic process of diabetic
complications. AGEs inhibitors discovered so far exert
their activities through scavenging reactive oxygen species,
carbonyl scavenging, metal ion chelating, or AGEs crosslinks breakage 22). Anti-oxidant capacity has been highly
correlated with AGEs inhibitory activity in vitro 23). However,recent clinical studies were not able to provide clear evidence
for the efficacy of natural anti-oxidant therapy in diabetic
patients 24). In contrast, AGEs inhibitors possessing a carbonyl
scavenging capacity demonstrated their efficacy for thetreatment of diabetic complications in a clinical report 1).
The main objective of this study was to evaluate whether
an aqueous rosehip extract inhibits MGO-mediated protein
glycation and scavenges MGO directly and to explore its
active substances possessing anti-glycation and MGOscavenging activities.
The MGO-BSA assay, which is a non-enzymatic protein
glycation model, revealed that our aqueous rosehip extract
could inhibit MGO-mediated protein glycation. Furthermore,
the extract directly trapped MGO in an MGO-scavenging
assay. The substances contributing to these activities wereidentified as procyanidin-type proanthocyanidins. Procyanidins
are the most abundant type of proanthocyanidins in plants,
while propelargonidins and prodelphinidins are lesscommon 25). GPC analysis showed that the number-averagemolecular weight (Mn) of the proanthocyanidins was about
3,500, corresponding to heptamer as the acetylated procyanidin
unit equivalent 26). The active proanthocyanidin-rich (61.6%)
fraction also contained a high amount of sugars (38.8%).
The free glucose was found only after acid hydrolysis of thefraction (data not shown), indicating that sugars in the fraction
were a component of the proanthocyanidin glycosides. Thus,
we clarified that the active substances in the fraction wereprocyanidin glycosides. This finding is consistent with results
reported by Fujii et al. 11), who found that the active fractionof an aqueous rosehip extract rich in sugar content, exerting
inhibitory effect against melanogenesis in mouse melanoma
and guinea pig skin, was composed of procyanidin glycosides.
The building blocks for proanthocyanidin such as catechin
and epicatechin have been reported to react with MGO 24).
In addition, procyanidin B2 and epigallocatechin gallate(EGCG) were also reported to bind to MGO stoichiometricallyat an equimolar ratio 24, 27). Based on structural similarity
between procyanidin B2 and proanthocyanidin, polymericproanthocyanidin has been proposed to react with MGO ina similar manner 24). Our results conducted in this study
support this proposal, whereas the structure in detail remains
unclear.
There is considerable interest in the bioavailabilityof rosehip proanthocyanidins and their bioactivity invivo. However, little is known about the bioavailability ofproanthocyanidins present in rosehip. Proanthocyanidin
polymer cannot be absorbed from either the stomach or
small intestine, whereas procyanidin dimer B2, epicatechin,and catechin have been detected in human plasma after
consumption of a flavanol-rich cocoa 28). Spencer et al.
reported that procyanidin in a range of trimer to hexamer
were hydrolyzed to mixtures of epicatechin monomers
and dimers 29). The absorbed catechin, epicatechin, andprocyanidin dimers resulting from the hydrolysis of rosehip

proanthocyanidins by intestinal microflora may act as
glycation inhibitors and/or carbonyl scavengers in vivo.

Conclusions

Our findings demonstrated that an aqueous rosehip
extract and its procyanidin glycosides were effective ininhibiting the formation of AGEs. Their abilities to react with
MGO were, at least in part, responsible for the inhibition ofprotein glycation.

Acknowledgments

This study was in part supported by a “ Fukuoka
Prefectural Support Fund for Creating New Product or New
Technology” to authors TK, AT, and MT.

Conflicts of Interest Statement

The present study was partly supported by Toyo Shinyaku
Co., Ltd. The company was not involved in the statisticalanalysis of the results.

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Inhibitory Effect of Rosehip on AGE Formation

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