Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 3, 2020
Accepted : May 28, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_196

Original article

Evaluation of effectiveness of Prunus mume extract such as anti-glycation
Takaya Mineo, Chihiro Kumagai, Hiroaki Tsutsumi, Haruma Kawaguchi
Holistic Beauty Research Center, Wamiles Cosmetics Inc., Yokohama, Kanagawa, Japan

Abstract

Subjective: Since the skin is constantly exposed to the external environment, it is susceptible to environmental factors. Thismay cause morphological changes due to skin aging, and the apparent age may occasionally exceed the chronological age.
In the glycation reaction, a risk factor of skin aging, abnormal crosslinks are formed in collagen and elastin in the dermis,
resulting in reduction of flexibility and elasticity. It is useful to impart an anti-glycation effect to cosmetics from the viewpointof preventing skin aging. In this study, to develop a cosmetic material targeting inhibition of glycation reactions, we examined
the approach by ume (Prunus mume), which is generally used in foods and health foods.
Methods: The test samples were prepared by extracting from each site including the shoots (young branches), flowers,
branches, leaves, bark, and roots of Ume, the two varieties of Bungo and Shirakaga. Using this extracts, we verified theactivities of SOD (superoxide dismutase)-like action, DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging, elastase
inhibition, and Maillard reaction crosslinking inhibition.
Results: The effects of elastase inhibiting, anti-oxidant, and anti-glycation were observed in ume root extracts of bothvarieties; Bungo and Shirakaga.
Conclusion: It has been found that the beneficial effect is on the plum root, of which extracts can be expected to be a usefulraw material for preventing skin aging.

KEY WORDS: ume (Japanese apricot; Prunus mume), Maillard reaction, anti-oxdation, aging care, skin, root

Introduction
The skin is located in the outermost layer of humanand is the largest organ in the body that covers the internalorgans. The skin protects the inside of the body from any
external environment such as invasion of foreign matter,and thus plays a role in maintaining homeostasis. To exert
these functions, skin is composed of various cells and has acomplex structure equipped with a variety of appendages 1).
However, their functions decline with age and changes occur
in each tissue.
With age, the skin loses its ability to retain water.
Besides, due to the decreased ability of regeneration andsebum secretion, aging phenomena appear in the appearance
induced by drying and roughening 2).
Glycation reaction is one of the factors that accelerate
skin aging. Glycation, also called Maillard reaction, hasrecently attracted attention as it is an important factor foraging care 3). Once glycation reaction produces advanced

glycation end products (AGEs), protein molecules lose
flexibility and become sclerotic. Skin aging caused byglycation also affects the appearance, resulting that theapparent age may exceed the chronological age 4).
The appearance characteristics of aged skin include
changes of color tone changes, i. e. spots (senile pigment
spots) and dullness, and morphological changes, i.e. wrinkles,
sagging.
This morphological change is considered to be due tostructural changes in the skin dermis, which are caused by
the decrease, degradation, or deterioration of collagen andelastin, the main components of the dermis 5). Collagen andelastin form crosslinks via lysine and hydroxylysine residues
during the process of fibrogenesis, thus forming a threedimensional structure and maintaining the skin flexibility.
The amino group of these lysine residues or the guanidylgroup of arginine residues non-enzymatically reacts with thecarbonyl group of reducing sugar 6). In the early stage of thisnon-enzymatic reaction, the reaction product is dehydrated

Corresponding to: Takaya Mineo
Holistic Beauty Research Center, Wamiles Cosmetics Inc.
2-3-12 Konan, Konan-ku, Yokohama, Kanagawa 233-0003, Japan

TEL: +81- 45-847- 0002
FAX: +81-45-847- 0008
e-mail: takaya.mineo@wamiles.co.jp

Glycative Stress Research 2020; 7 ( 2) : 196-203(c) Society for Glycative Stress Research_ 19 6 _Glycative Stress Research

to produce a Schiff base. Furthermore, intramolecularrearrangement occurs, thus forming Amadori rearrangement
products 7). In the late stage, amino compounds and carbonylcompounds react with Amadori products to form highly
reactive dicarbonyl compounds, i. e. 3-deoxyglucosone 6, 7).
AGEs are produced in the late stages of the reaction andform abnormal crosslinks in the skin dermal tissue. The
generation of AGEs in skin tissues influences the color tone
and morphological changes of the skin, which causes theapparent age to exceed the chronological age.
Generation of AGEs by glycation involves oxidative
reactions. Oxidation of sugars and Amadori compounds in thelate stages produces compounds such as 3-deoxyglucosone,glyoxal, and methylglyoxal 8, 9). In this process, O 2− (superoxideanion radical), one of reactive oxygen species (ROS), is
generated 10, 11). ROS are physiologically active substances,and concurrently, once excessively produced, exert harmful
effects on the body known as oxidative stress 12, 13).
Further, when abnormal cross-linking of collagen or
elastin is formed by glycation, the cross-linked products
are judged to be foreign substances, therefore increasing
secretion of degrading enzymes, i. e. collagenase, elastase,thus resulting that normal collagen and elastin become easily
decomposed 6). It has been reported that skin aging due toglycatiin is likely to affect tissues that contain abundant
proteins with long half-lives, i. e. collagen, elastin 14, 15).
Ume (Japanese apricot; Prunus mume) is a deciduoustree belonging to the family Rosaceae the genus Prunus,
and is widely cultivated in East and Southeast Asian regions
including China, Japan, South Korea, Taiwan, Thailand,
and Vietnam. Its fruit has a long history of being used as an
ornamental, edible, and traditional Chinese medicine sinceancient times, and is rich in organic acids, i.e. citric acid 16).
In this study, for the purpose to develop a cosmetic
material targeting the prevention of skin aging, by focusing
on the flowers, roots, and branches of ume, which are widelyused in foods and health foods, we elucidated the effects ofanti-glycation, anti-oxidation and the inhibition effects onelastase activity of these extracts.

Methods
Sample preparation
For the two varieties of ume, Bungo and Shirakaga,
each site of the shoots (young branches), flowers, branches,leaves, bark, and roots, adjusted their contents at 5%, wasimmersed in purified water or 95% ethanol as extractants,
and the extraction was performed at 30 °C for 3 days. Each
extract obtained was filtered to remove insoluble mattersand used as a test sample. Further, the samples confirmed
to be effective were subjected to vacuum concentration andlyophilization for the additional test.

Evaluation for superoxide dismutase (SOD)-like
activityat the concentration pre-adjusted with purified water, 25 µL
of 2 mM hypoxanthine (Sigma, St. Louis, MO, USA), 25 µL
of 2.0 mM ethylenediaminetetraacetic acid (EDTA; Dojindo,
Kumamoto, Japan), 25 µL of 0.5 mM nitro blue tetrazolium(NBT; Wako, Osaka, Japan), 100 µL of 0.1 M carbonate
buffer, and 50 μL of 30 mU/mL SOD (Sigma) were added
and then incubated at 37℃ for 30 minutes, followed bymeasuring absorbance at 570 nm with a microplate reader.
The scavenging rate was calculated using purified water as acontrol.

Evaluation for 2,2-Diphenyl-1-picrylhydrazyl(DPPH) radical scavenging effect

DPPH radical scavenging activity was evaluatedaccording to previous reports 19, 20). To the 96 well plate, 15 μL
of test sample at the concentration pre-adjusted using purified
water, 60 μL of 0.2 M 2-(N-morpholino)ethanesulfonic acid
(MES) buffer, 50 μL of ethanol, and 25 μL of 0.6 mM DPPH
radical (Wako) were added and then incubated at 30℃ for
30 minutes, followed by measuring absorbance at 54 0 nm
with a microplate reader. For the DPPH radical scavenging
activity, the scavenging rate was calculated using purified
water as a control.

Evaluation for elastase inhibition activity
The elastase inhibition activity was evaluated according
to previous reports 21). To the 96 well plate, 50 μL of test
sample at the concentration pre-adjusted with purified
water, 35 μL of 0.1 M Tris-HCl buffer, 100 μL of 1 mM
Suc-Ala-Ala-Ala-PNA (Sigma), and 15 μL of 0.25 U/mL
elastase (Wako) was added and incubated at 37 ℃ for 30
minutes, followed by measuring absorbance at 405 nm witha microplate reader. The inhibitory rate was calculated using
purified water as a control.

Inhibition tests on a Maillard reaction-induced
crosslinking
The Maillard crosslinking inhibition test was evaluatedaccording to a previous report 22, 23). Into sample tubes, 40
μL of 0.5 M ribose (Wako) prepared with 800 μL of 0.1 M
phosphate buffer (pH 7.4), 100 μL of 50 mg/mL lysozyme(Wako), and add 60 μL of test sample at the concentration
pre-adjusted using purified water were added and thensterilized by filtration using a 0.45 μm filter in a clean bench,followed by incubation at 40℃ for 7 days in an incubator.
After the reaction, electrophoresis was performed by SDSPAGE, stained with Coomassie Brilliant Blue R-250, andafter decolorization, the dimer of lysozyme was visually
determined. For judgement, the state when aminoguanidine
sulfate was added was determined as a positive control andthe other in which purified water was used instead of the test
sample was used as a negative control. The band intensityof electrophoresis gel was quantified by performing image
analysis on the same area using NIS-Elements (Nikon,
Tokyo, Japan).

The SOD-like activity was measured according toprevious reports 17, 18). To a 96 well plate, 25 µL of test sample_ 19 7 _Efficacy Evaluation of Prunus mume Extract

Results
Regarding two ume cultivars, Bungo and Shirakaga, eachof the six sites was extracted with water and ethanol (Figs. 1,
2 ). The appearance, such as the color of the extract, differeddepending on the variety, extractant, and site, that indicates thepresence of difference in what was extracted. As a result of theexamination, the anti-oxidant effect (SOD-like activity, DPPH
radical scavenging activity) and the elastase inhibition activity
were confirmed in the extracts from shoots (young branches)and roots ( Tables 1, 2 ).

Among the ume extract liquids, the root-derived extract
was found to have higher anti-oxidant activity and elastase
inhibition activity than others. Particularly in the ethanol
extract of ume roots, IC 50 (the concentration of the sample inwhich 50% inhibition is shown) for active oxygen scavenging
activity was less than 0.1%. Therefore, it was confirmed thatthe root extract has a higher anti-oxidant effect than other sites.
In addition, it was noted that the root ethanol extract had
an elastase inhibition activity with IC 50 values being 0.45% in
Bungo and 0.24% in Shirakaga, which is lower compared toother sites.

a

b

c

d

e

f

Fig. 1. Photographs of each site of ume (Prunus mume).
a) Shoots (young branches). b) Flowers. c) Branches. d) Leaves. e) Bark. f ) Roots.

a

b
S

F

Br

L

Ba

R

c

S

F

Br

L

Ba

R

S

F

Br

L

Ba

R

d
S

F

Br

L

Ba

R

Fig. 2. The appearance of 5% (w/w) extracts of each site by ume variety.
a) Extract with ethanol / Shirakaga. b) Extract with water / Shirakaga. c) Extract withethanol / Bungo. d) Extract with water / Bungo. S, shoots (young branches) ; F, flowers;
Br, branches; L, leaves; Ba, bark; R, roots.

_ 19 8 _Glycative Stress Research

Table 1. SOD-like activity and DPPH radical scavenging effect.
Ume varieties

Extractants

Sites

Bungo

Water

Bungo

SOD-like activity

DPPH scavenging

Effects

IC 50

Effects

IC 50

Shoots

84%

0.05%

88%

0.0 4%

Ethanol

Shoots

62%

0.14%

70%

0.08%

Shirakaga

Water

Shoots

74%

0.09%

63%

0.11%

Shirakaga

Ethanol

Shoots37%> 0. 2%

4 6%> 0. 2%

Bungo

Water

Flowers

3 4%> 0. 2%

5 4%

0.20%

Bungo

Ethanol

Flowers− 11%> 0. 2%− 13%> 0. 2%

Shirakaga

Water

Flowers41%> 0. 2%

53%

0.20%

Shirakaga

Ethanol

Flowers− 31%> 0. 2%13%> 0. 2%

Bungo

Water

Branches4%> 0. 2%− 4 2%> 0. 2%

Bungo

Ethanol

Branches

14%> 0. 2%− 48%> 0. 2%

Shirakaga

Water

Branches11%> 0. 2%− 42%> 0. 2%

Shirakaga

Ethanol

Branches11%> 0. 2%− 16%> 0. 2%

Bungo

Water

Leaves

74%

0.08%

83%

0.11%

Bungo

Ethanol

Leaves1%> 0. 2%− 17%> 0. 2%

Shirakaga

Water

Leaves38%> 0. 2%− 3%> 0. 2%

Shirakaga

Ethanol

Leaves− 3%> 0. 2%− 27%> 0. 2%

Bungo

Water

Bark

− 3%

> 0. 2%− 55%> 0. 2%

Bungo

EthanolBark

9%> 0. 2%− 54%> 0. 2%

Shirakaga

Water

Bark

3%

> 0. 2%− 23%> 0. 2%

Shirakaga

EthanolBark29%> 0. 2%− 10%> 0. 2%

Bungo

Water

Roots

49%

0. 2%

12%

> 0. 2%

Bungo

Ethanol

Roots

75%

0.06%

77%

0.10%

Shirakaga

Water

Roots

37%

> 0. 2%17%> 0. 2%

Shirakaga

Ethanol

Roots

77%

0.07%

83%

0.08%

All tests were performed at 0.2% extract concentration. IC 50 value indicates the concentration that inhibits the SOD-like activity or that eliminates the generated
active oxygen by 50%. SOD, superoxide dismutase; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; Shoots, young branches.

_ 19 9 _Efficacy Evaluation of Prunus mume Extract

Table 2. Elastase activity inhibitory effect.
Ume varieties

Extractants

Sites

Bungo

Water

Bungo

Elastase inhibition
Effects (2.5%)

I C 50

Shoots− 8%> 2.5%

Ethanol

Shoots2%> 2.5%

Shirakaga

Water

Shoots

− 2 4%> 2.5%

Shirakaga

Ethanol

Shoots9%> 2.5%

Bungo

Water

Flowers− 13%> 2.5%

Bungo

Ethanol

Flowers− 43%> 2.5%

Shirakaga

Water

Flowers− 9%> 2.5%

Shirakaga

Ethanol

Flowers− 18%> 2.5%

Bungo

Water

Branches

69%

1.10%

Bungo

Ethanol

Branches

43%

2.53%

Shirakaga

Water

Branches− 7%> 2.5%

Shirakaga

Ethanol

Branches53%

2. 21%

Bungo

Water

Leaves− 35%> 2. 5%

Bungo

Ethanol

Leaves34%

4. 4 7%

Shirakaga

Water

Leaves− 22%> 2. 5%

Shirakaga

Ethanol

Leaves− 2%> 2. 5%

Bungo

Water

Bark

11%

> 2. 5%

Bungo

Ethanol

Bark

4 3%

3.74%

Shirakaga

Water

Bark

77%

0.49%

Shirakaga

Ethanol

Bark

51%

2.13%

Bungo

Water

Roots

53%

2. 35%

Bungo

Ethanol

Roots

84%

0.45%

Shirakaga

Water

Roots

66%

1. 32%

Shirakaga

Ethanol

Roots

83%

0. 24%All tests were performed at 2.5% extract concentration. IC 50 value indicates the concentration that inhibits the elastase activity by 50%; Shoots, young branches.

Moreover, it was confirmed that the extract liquid ofume shoots had an anti-oxidant effect with IC 50 values for
active oxygen scavenging activity being less than 0.14% orless except that of Shirakaga.
Therefore, the root extracts from the root and shoots,
confirmed to be effective, were treated by the vacuum
concentration and freeze- dried to obtain samples of the dry
extracts, of which the effectiveness was then examined. Inorder to verify the inhibitory effect of glycative stress, one of
the factors of skin aging, a Maillard crosslinking inhibition

test was conducted. The test was performed by adjusting
the roots dry extract to 0.15 mg/mL with 95% ethanol, andthe results are shown in Fig. 3. Also, integrated values ofthe optical density were calculated from the electrophoretic
image ( Table 3 ).
From the above measurements, the root extract was
confirmed to have an anti-oxidant effect and an elastase
inhibition activity, and its dry extract showed a Maillard
crosslinking inhibition activity.

_ 200 _Glycative Stress Research

Dimer
Water EtOH

Conc.
(mg/mL)

AG

AG

AG

3.0

1.5

0.75

d.w.
(Control)Water

Shirakaga

EtOH

Bungo

Water

EtOH

Water

Shirakaga

Roots

EtOH

Bungo

ShootsFig. 3. The electrophoresis gel image in the inhibition test of a Maillard reaction crosslinking by ume extract.

Lysozyme dimers are formed by the Maillard reaction-induced crosslinking. Sample No.1-3, aminoguanidine sulfate (AG) as
positive control, concentrations (Conc.) at 3.0, 1.5, 0.75 mg/mL, respectively; No.4, distilled water (d.w.) as control; No. 5 - 8,
Fig. root
3 extracts; No. 9 -12, shoot (young branches) extracts. Two varieties, Shirakaga (No. 5, 6, 9, 10) and Bungo (No.7, 8, 11, 12),
were tested. Extractants were water in No. 5, 7, 9, 11 or 95% ethanol (EtOH) in No. 6, 8. 10, 12.

Table 3. The optical band intensity of electrophoresis gel images in the inhibition testof a Maillard reaction crosslinking by ume extract.
The sample
number in Fig. 3

Concentration(mg/mL)

Integrated values ofthe optical density

3. 0542. 2 ± 7. 5

1. 5604. 2 ± 6.4

0.75

681.8 ± 6.4−−

881. 3 ± 7. 3

Water

0.15

583.0 ± 3.4

95% ethanol

0.15

567. 3 ± 6.6

Water

0.15

587.4 ± 6. 5

95 % ethanol

0.15

555.6 ± 6.8

Water

0.15

768.6 ± 0. 3

95% ethanol

0.15

733.1 ± 6. 5

Water

0.15

630.1 ± 0. 3

95 % ethanol

0.15

659.0 ± 5. 4

Samples

Sites

1
2

Water

Aminoguanidine sulfate

3
4
5
6
7
8
9
10
11
12

Purified water
Roots (Shirakaga)
Roots (Bungo)
Shoots (Shirakaga)
Shoots (Bungo)

Results are expressed as mean ± standard deviation by repeated measurement (n = 3). The optical band intensity of electrophoresis gel is quantified by using
NIS-Elements (Nikon, Tokyo, Japan). Shoots, young branches.

Discussion
Regarding the efficacy of ume, it has been revealed
that phenolic compounds prepared from ume vinegar, a byproduct when making salting ume fruit (ume-boshi; saltedume), have anti-bacterial activity against intestinal bacteria 24).
Besides, the anti-viral effects of HSV-1 and HSV-2 havebeen confirmed in ume products 25).
Ume has been confirmed to be effective in clinical
trials. In the comparison study of oral ammonia
concentration between the group in which saliva secretion
was induced by ume extracts and the group without them,ammonia concentration rise was reported to be suppressed
to about half 26). This suggests that ume extract can be aneffective method for maintaining the oral hygiene. I t has
also been reported that substances such as mumeose which
is isolated and purified from ume flower buds, as well as
fruits, inhibit aldose reductase 27). This aldose reductase has
a function of converting surplus glucose, i. e. generated indiabetes, into sorbitol and fructose (polyol metabolism),thus the accumulation of sorbitol causes neuropathy. On thecontrary, the inhibition of aldose reductase can be expected
to moderate the neuropathy.

_ 2 01 _Efficacy Evaluation of Prunus mume Extract

As described above, the Maillard reaction has a nonoxidative reaction route and an oxidative reaction route.
Concurrently through these two routes, the reaction proceeds.
It remains unclear which reaction pathway the root extract
is involved in, however, it can be inferred that the inhibitionof O 2− -activity is involved in the anti-glycation effect sincea high anti-oxidant effect was observed in the root extract.
It is already mentioned that abnormal cross-linking ofcollagen and elastin increases the secretion of degrading
enzymes. Also the root extract had an inhibitory effect onelastase as well as on the crosslinking formation by theMaillard reaction. Therefore, ume extract is expected to be auseful raw material for suppressing skin aging such as
wrinkles, sagging and dullness which are caused by reductionof skin firmness and elasticity.

effect (SOD-like effect, DPPH radical scavenging effect)and elastase inhibition activity were confirmed in the root
extract. Additionally, the root extract was confirmed tohave a Maillard crosslinking inhibition activity. From thesefindings, the ume root extracts are expected to have an
inhibitory effect on the manifestation of senescence due toglycative stress. Furthermore, the results suggest that thatume root could be a useful raw material for cosmetics withvarious effects. We are planning to further study, among
various ingredients contained in ume roots, which ingredient
the confirmed effect is derived from.

Conflict of Interest Statement
Inc..

Conclusion

The present study was supported by Wamiles Cosmetics

The effectiveness of each site of shoots, flowers, branches,leaves, bark, and roots was evaluated for two Ume varietiesof Bungo and Shirakaga. Among them, the anti-oxidant

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