Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : October 19, 2020
Accepted : November 15, 2020
Published online : December 31, 2020
doi:10.24659/gsr.7. 4_287

Original article

Involvement of glycative stress in diabetic nephropathy and effects ofanti-glycation material Kuromoji (Lindera umbellata Thunb.)
Kiriko Akiyama 1), Akihiko Shimode 1), Shigeru Matsumi 1), Tetsuya Maruyama 1),
Wakako Takabe 2), Masayuki Yagi 3), Yoshikazu Yonei 3)
1) Yomeishu Seizo Co. Ltd., Tokyo, Japan
2) Department of Materials and Life Science, Faculty of Science and Technology,
Shizuoka Institute of Science and Technology, Shizuoka, Japan3) Anti-Aging Medical Research Center/Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

Objective: The purpose of this study is to investigate the effects of anti-glycation activity of kuromoji (Lindera umbellata
Thunb.) extract (KE) on diabetic nephropathy.
Methods : Using streptozotocin-induced diabetic rats (STZ rats) and spontaneously diabetic mice (db/db mice), glycative
stress-related indicators and biochemical changes were investigated during continuous administration of KE, and the effectsof KE on inflammatory response in renal tissue was compared between two animal models. Then, mouse macrophage cells(RAW264.7 cells) were used to investigate the effects of glycated substances on the inflammatory response.
Results : KE suppressed the amount of inflammatory cytokines (TNF-α, IL-6) in the renal tissue of STZ rats and db/db mice.
In STZ rats, KE suppressed the increase in renal weight and the amount of autoantibodies against N ε - (Carboxymethyl) lysine(CML). In db/db mice, the elevation of serum total cholesterol was suppressed. Treatment of RAW264.7 cells with CMLHSA (human serum albumin) increased TNF-α production, while CML alone did not.
Conclusion: KE is thought to suppress the development of inflammatory response in renal tissue by reducing glycative stressduring diabetic conditions.

KEY WORDS: kuromoji (Lindera umbellata Thunb.), advanced glycation end products (AGEs),		Introduction

N ε - (Carboxymethyl)lysine (CML), autoantibody, diabetic nephropathy, inflammatory cytokines

Lauraceae Kuromoji (Lindera umbellata Thunb.) is anaromatic plant containing essential oils, i.e. linalool, geraniol
1, 2), and the dried branches are used in medicinal liquor asa crude drug called “ usho ( Lindera umbellatae Ramus)” 3).
Its non-volatile extract has various pharmacological actions
including antioxidant, anti-glycation, anti-ulcer action,
immunopotentiative action and antiviral action, and therefore
it is expected to work as a functional material aimed atpreventing lifestyle-related diseases 4- 8).
We have used the human serum albumin (HSA) - glucose(GLU) glycation reaction system to target more than 500
types of food materials. As a result of evaluating theinhibitory activity of advanced glycation end products (AGEs)
formation, it was clarified that kuromoji extract (KE) shows
strong activity among these food materials 5). KE had astrong inhibitory activity on AGEs formation, not only for
HSA but also for collagen and elastin in the GLU glycation
reaction system. Also, KE has, as an AGEs decomposition
action, an activity enhancing cross-linking cleavage action
and oxidized protein hydrolase (OPH) 6). Furthermore, it

Corresponding author: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tatara Miyakodani, Kyotanabe-shi, Kyoto, 610- 0321 Japan
TEL & FAX: + 81-774 - 65- 6394 e-mail: myagi@mail.doshisha.ac.jp
Co-authors: Akiyama K, k-akiyama@yomeishu.co. jp; Shimode A, a-shimode@yomeishu.co.jp;
Matsumi S, s-matsumi@yomeishu.co.jp; Maruyama T, te-maruyama@yomeishu.co.jp;
Takabe W, takabe.wakako@sist.ac; Yonei Y, yyonei@mail.doshisha.ac.jp_ 287 _

Glycative Stress Research 2020; 7 (4 ) : 287-297(c) Society for Glycative Stress ResearchEffects of Kuromoji (Lindera umbellata Thunb.) Extract on Diabetic Nephropathy

was observed that administration of KE to streptozotocin(STZ) -induced diabetic rats (STZ rats) suppressed increased
levels of inflammatory cytokines, i.e. tumor necrosis
factor- α (TNF-α) and interleukin-6 (IL-6) in renal tissue,
resulting in improvement of renal function 5). It is reported
that infiltration of inflammatory cells, mainly macrophages,
is observed in the renal tissue of diabetic nephropathy.
Additionally, inflammatory cytokines are increased 9), andglycation and oxidative stress are involved as the mechanismof progression of nephropathy 10). In consideration of theabove, it is considered that the suppression of inflammatory
reaction in renal tissue observed in STZ-induced diabetic
rats may be related to the inhibitory action by KE on AGEs
formation and accumulation.
In this study, we measured the amount of anti-CML(N ε - (Carboxymethyl) lysine) autoantibodies in the serum of
STZ rats and spontaneously diabetic mice (db/db mice), andinvestigated the effect of KE on the reduction of glycative
stress and renal inflammation. Concurrently, the involvementof glycated substances in inflammation using macrophagecells (RAW264.7) was investigated.

Methods

1. Subjects
The preparation of KE follows the previously reported
method 5). Briefly, KE was prepared by adding 10 times
the amount of water to the branch after finely chopping
kuromoji (Lindera umbellata Thunb.), then heated at 95 °C
for one hour to obtain extract and centrifuged to remove
insoluble matter, followed by concentration by using avacuum concentrator. Positive control drugs used wereaminoguanidine (AMG) hydrochloride (Fuji Film Wako Pure
Chemical Industries, Ltd., Osaka, Japan), a glycation reaction
inhibitor, and metformin (MET) hydrochloride (Fuji Film
Wako), an antidiabetic drug for reducing insulin resistance.

2. Effect of KE on diabetic model animals

1) Experimental animals
Six-week-old Sprague Dawley (SD) male rat (Nippon
SLC Co., Ltd., Hamamatsu, Shizuoka, Japan), 7-week-old
C57BLKS / J Iar-m +/+ Leprdb (db/+) male mouse, andC57BLKS / J Iar -+ Leprdb / + Leprdb (db/db) male mice
(Nippon SLC Co., Ltd.) were bred for one week in a barrier
facility set at temperature: 24 ± 2 °C, humidity: 50 ± 10%,and lighting time: 12 hours a day (7:00 to 19:00), and thenused for the experiment.
2) Breeding management
Throughout the entire test period, rats were individuallyhoused in polycarbonate cages (W26 x D42 x H18 cm) andmice were housed individually in polycarbonate cages (W11
x D18 x H11 cm). The cage used was equipped with a firmade floor (floor for laboratory animals, soft tip: Nippon
SLC Co., Ltd.).

For feed, Powder Lab MR Stock (Nihon Nosan
Corporation, Yokohama, Kanagawa, Japan) was used, and for
drinking water, well water was freely ingested using a waterbottle. Body weight and food intake were measured once aweek.
3) Preparation and group composition of model animalsa) Streptozotocin -induced diabetic rats (STZ rats)
STZ rats were prepared by intraperitoneallyadministering 60 mg/kg of STZ to 7-week-old rats after
the completion of pre-breeding, followed by measuring theblood GLU level 3 days later to confirm diabetic state (300mg/dL or more). The test subject was administered by gavage
daily for 8 weeks using a disposable syringe and an oral
sonde. The rats were grouped as follows so that there was nobias in blood GLU level and body weight using a grouping
software (StatLight: Yukms Corp, Kawasaki, Kanagawa,Japan); No-treated group (Normal), group in which distilledwater was administered to SD rats (n = 8); Control group(Vehicle), STZ rats treated with distilled water (n = 8); KE
low-dose group (KE100), STZ rats treated with 100 mg/kg/
day of KE (n = 8); KE high-dose group (KE300), STZ rats
administered KE 300 mg/kg/day (n = 8); AMG-administeredgroup (AMG100), STZ rats administered 100 mg/kg/day of
AMG hydrochloride (n = 8).
b) Spontaneous diabetic (db/db) mice
After the completion of pre-breeding, the body weightsof 8-week-old mice were individually measured and groupedusing the above-mentioned software so that the average bodyweight and blood GLU level of each group became almost
uniform. The test subject was administered by gavage dailyfor 12 weeks using a disposable syringe and an oral sonde.
The group composition was as follows: db/+ group, db/+
mice treated with distilled water (n = 10); Control group(Vehicle), db/db mice treated with distilled water (n = 10);
KE low-dose group (KE100), db/db mice treated with 100
mg/kg/day of KE (n = 10); KE high-dose group (KE300),
db/db mice treated with 300 mg/kg/day KE (n = 10); METadministered group (MET350), db/db mice were treated with
350 mg/kg/day of MET hydrochloride (n = 10).
4) Blood sampling and autopsy
Partial blood sampling was performed before and afteradministration every 4 weeks as follows: for blood GLU,
specimen collected from the tail vein without anesthesia;
for hemoglobin A1c (HbA1c), rat specimen collected fromthe jugular vein under isoflurane inhalation anesthesia andmouse specimen from the orbital vein under isoflurane
anesthesia. Whole blood was collected after a 16-hour fastfrom the evening of the end of administration to the nextday. Blood was collected from the abdominal aorta underisoflurane anesthesia for rats and from the heart underisoflurane anesthesia for mice. Serum was centrifuged,
separated and cryopreserved. After total blood collection, theabdominal aorta and posterior vena cava were amputated andexsanguinated, followed by sampling the right kidney andweighing. Radio-immunoprecipitation (RIPA) buffer (Fuji
Film Wako) was added in 4 -fold doses in rats and 5-fold_ 288 _Glycative Stress Researchdose in mice to the kidney, followed by homogenizing andcentrifuge (14,000 g, 30 minutes) to obtain the supernatant,which was then stored frozen until measurement.
5) Measurement
The blood GLU level was measured using a simple
blood GLU meter (One Touch Ultra: Johnson & Johnson Co.,
Ltd. or Precision Exceed: Abbott Japan Co., Ltd., Tokyo,
Japan). HbA1c was measured using the DCA2000 system(Bayer Yakuhin, Ltd., Osaka, Japan). Rat serum totalcholesterol (TC), triglyceride (TG), free fatty acids (NEFA),LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), total
bilirubin (T-BIL), GLU and glycoalbumin (GA) were measured
by an automatic biochemical analyzer (Hitachi Co., Ltd.,Tokyo, Japan).
For mouse serum, Cholesterol E -test Wako, TriglycerideE-test Wako, LDL-C/M E-test Wako and HDL-C E-test
Wako (Fuji Film Wako) was used for measuring respectively
TC, TG, LDL-C and HDL-C, and Levis high-density
adiponectin-mouse/rat (Fuji Film Wako) was used for
adiponectin measurement. Additionally, for the measurementof CML autoantibodies in serum, rats used the CircuLex
Anti-CML rat autoantibody ELISA Kit (CycLex Co., Ltd.,Ina, Nagano, Japan) and mice used CircuLex Anti-CML
mouse autoantibody ELISA Kit (CycLex). For measurementof TNF-α and IL-6 in renal tissue supernatant in thehomogenized renal-tissue supernatant, rats used Rat OptEIA
ELISA Set (Becton, Dickinson and Company, Franklin
Lakes, NJ, USA) and mice used Mouse OptEIA ELISA Set(Becton). The protein amount in the renal-tissue supernatant
was measured using the BCA Protein Assay Kit (Takara Bio
Inc., Kusatsu, Shiga, Japan) for both rats and mice.
6) Ethical standards for the animal study
In conducting this test, "Act on welfare and managementof animals (Act No. 105 of October 1, 1973, revised onJune 22, 2005)" and "Standards for breeding and storageof laboratory animals and reduction of pain" (April 28,
2006, Ministry of the Environment Notification No. 88) was
respected, followed by inspectiom and approval (ApprovalNo. 17117 and 17118) based on the Laboratory Animal
Welfare Regulations (Nippon SLC Co., Ltd.,).

3. Effect of glycated substances on mouse
macrophage cells (RAW264.7)

1) Culture cells
RAW264.7 cells derived from mouse macrophagewere purchased from ATCC (American Type Culture
Collection, Manassas, VA, USA). Cells are cultured at 37 °C
in the presence of 5 % CO 2 in Dulbecco’s modified Eagle's
medium (DMEM, Sigma-Aldrich Japan GK, Tokyo, Japan)
supplemented with 10 % deactivated fetal bovine serum(FBS, Equitech-Bio, Inc., Kerrville, TX, USA), 100 units/mL penicillin and 100 µg/mL streptomycin (Nacalai Tesque,Inc., Kyoto, Japan)

2) Solution of glycated materials
CML (Nippi Co., Ltd., Tokyo, Japan) and CML-HSA(CycLex) were diluted with phosphate-buffered saline (PBS)
and used as glycated materials.
3) Measurement
After 4 × 10 5 cells/mL RAW 264.7 cells were seeded on
a 48-well plate, the cells were cultured for 24 hours, washed,
and then 50 µL of CML diluted to an arbitrary concentration
was added so that the total volume became 500 µL, followedby culture for 24 hours. Additionally, 125 µL of CML-HSA
or HSA diluted to an arbitrary concentration and added sothat the total volume became 500 µL, followed by culture for3 hours. Then, the medium supernatant was collected, diluted
to an arbitrary concentration with Assay Diluent (Becton),
and TNF-α was measured using Mouse OptEIA ELISA Set.

4. Data processing
The results are expressed as mean ± standard deviation(SD). For statistical processing, Wilcoxon rank sum test wasused for comparison between two groups, and nonparametricBonferroni multiple comparison test was used for comparison
between multiple groups. When the p values are < 0.05, it isdefined as having a significant difference.

Results

1. Effect of KE on diabetic model animals

1) Body weight, food intake and kidney weighta) Body weight
The transition of body weight is shown in Fig. 1. In
rats, the transition of weight gain seen in Normal was notobserved in the STZ-treated groups. The body weight after
8 weeks was significantly lower in Vehicle (255 ± 37.3 g)than in Normal (419 ± 29.1 g), while there was no significant
difference in the body weights between Vehicle and the test
groups (KE100, KE300, AMG100) (Fig. 1-a).
In mice, the body weight of Vehicle was higher thanthat of db/+ from the start of the test, while the body weightof Vehicle (38.0 ± 5.4 g) was higher than that in db/+ (26.7± 0.8 g) until 12 weeks. There was no difference in bodyweight between Vehicle and the test groups (KE100, KE300,
MET350) (Fig. 1-b).
b) Food intake
The transition of food intake is shown in Fig. 2. In
rats, the mean daily food intake throughout the study
period, compared to STZ Vehicle (37.4 ± 2.29 g/day), wassignificantly lower in Normal (24.9 ± 1.07 g/day) and KE100(34.0 ± 3.62 g/day), while not different with AMG100 ( Fig.
2-a).
In mice, the daily food intake of Vehicle (6.51 ± 0.62 g/day) was higher than that of db/+ (4.35 ± 0.53 g/day), while
no difference noted between Vehicle and the test groups(KE100, KE300, MET350) (Fig. 2-b).

_ 289 _

Effects of Kuromoji (Lindera umbellata Thunb.) Extract on Diabetic Nephropathyc) Kidney weight
Figure 3 shows the kidney weight at autopsy. The rat
renal weight of STZ Vehicle (2.50 ± 0.24 g) was higher thanthat of Normal (2.24 ± 0.15 g). The renal weight of KE100(2.25 ± 0.19 g) was significantly lower than that of Vehicle,
while that in AMG100 was not different from Vehicle (Fig.
3-a).
The mouse renal weight of Vehicle (0.40 ± 0.03 g) was
higher than that of db/+ (0.36 ± 0.02 g), while there was nodifference between Vehicle and the test groups (KE100,
KE300, MET350) (Fig. 3-b).
2) Biochemical test
Table 1-a shows the results of the STZ rat study after
8 weeks. For GLU metabolism-related indicators, GLU,
GA and HbA1c were significantly higher in STZ Vehicle
than in Normal, while there was no difference between
Vehicle and the test groups (KE100, KE300, AMG100). For
lipid metabolism-related indicators, TG and HDL-C weresignificantly higher in Vehicle than in Normal, and tended
to be higher in the KE-treated groups than in Vehicle (TGin KE300, p = 0.088; HDL-C in KE100, p = 0.095). TC andNEFA did not differ in Vehicle compared to Normal. TC in
KE-treated groups was significantly higher than in Vehicle.
NEFA in KE300 and AMG100 was significantly higher thanin Vehicle.
Table 1- b shows the results of the db/db mouse study
after 12 weeks. GLU and HbA1c were significantly higher
in Vehicle than in db/+. GLU tended to be lower in MET350
than in Vehicle ( p = 0.059). HbA1c was significantly lower in
MET350 than in Vehicle. TC, LDL-C and HDL-C showedsignificantly higher values in Vehicle than in db/+. In
KE300, TC was significantly lower than in Vehicle, TG and
LDL-C tended to show lower values than in Vehicle (TG, p= 0.067; LDL-C, p = 0.084). In MET350, TG and LDL-C
were significantly lower than in Vehicle.
3) Anti-CML autoantibodies in serum
The results of anti-CML autoantibodies in serum after
8 weeks in the STZ rat study are shown in Fig. 4-a. The
amount of anti-CML autoantibodies in STZ Vehicle (321
± 171.6 µg/mL) was significantly higher than Normal (108 ±54.4 µg/mL). It was significantly lower in KE-treated groups(KE100, 71 ± 55.8 µg/mL; KE300, 113 ± 89.3 µg/mL) andAMG100 (122 ± 76.3 µg/mL) compared to Vehicle.
The results of anti-CML autoantibodies in serum after
12 weeks in the db/db mouse study are shown in Fig. 4-b.
The anti-CML autoantibody level in Vehicle (37.7 ± 20.9 µg/mL) was significantly higher than that in db/+ (22.6 ± 4.9 µg/mL). There were no significant difference between Vehicle
and the test groups.
4) Inflammatory cytokines in kidney tissue
The results of TNF-α and IL-6 in the renal tissue
after 8 weeks in the STZ rat study are shown in Fig. 5-a, b.
The amount of TNF-α per protein in renal tissue wassignificantly higher in STZ Vehicle (1.72 ± 0.39 ng/mg
protein) than in Normal (1.26 ± 0.22 ng/mg protein). KE100(1.01 ± 0.30 ng/mg protein) and AMG100 (0.85 ± 0.12 ng/mg
protein) showed significantly lower values than Vehicle. IL-6
was significantly lower in KE groups (KE100: 4.54 ± 1.63 ng/
mg protein, KE300: 4.24 ± 1.75 ng/mg protein) and AMG100(4.03 ± 0.51 ng/mg protein) compared with Vehicle (6.39 ±1.27 ng/mg protein). There was no difference between STZ
Vehicle and Normal.
The results of TNF-α and IL-6 in the renal tissue after
12 weeks in the db/db mouse study are shown in Fig. 5-c, d.
TNF-α per protein in the renal tissue was significantly lower
in KE300 (0.47 ± 0.12 ng/mg protein) than in Vehicle (0.70 ±0.19 ng/mg protein). IL-6 was significantly lower in KE300(0.32 ± 0.08 ng/mg protein) and MET350 (0.35 ± 0.10 ng/mg
protein) compared with Vehicle (0.47 ± 0.12 ng/mg protein).
There was no difference in TNF-α and IL-6 between db/+
and db/db Vehicle.

2. Effect of glycated substances on mouse
macrophage cells (RAW264.7)

Figure 6 shows the amount of TNF-α produced byadding glycated substances to RAW264.7.
TNF-α produced in the culture solution 24 hours after
the addition of the CML solution did not change as comparedwith that without the addition (Fig. 6-a). TNF-α produced in
the culture medium 3 hours after the addition of the CMLHSA was observed to increase in a concentration-dependent
manner, and was significantly increased by the treatments of
5 µg/mL and 10 µg/mL. In contrast, no increase in TNF-α
was observed with the treatment of HSA alone (Fig. 6-b).

Discussion

1. Diabetes model animals
STZ rats are used as a model for type 1 diabetes mellitus(T1DM). The administration of STZ induces the productionof reactive oxygen species (ROS) in pancreatic β-cells,causing cell death in β-cells and inhibiting insulin secretion,
resulting in a rapid increase in blood GLU and maintenanceof hyperglycemia 11, 12). On the other hand, db/db mice are used
as a type 2 diabetes mellitus (T2DM) model. Since db/db mice
have a deficiency in the receptor for the appetite-suppressing
hormone “ leptin”, they become obese due to overeating anddevelop hyperglycemia at 6 to 10 weeks of age 13). In thesediabetic models, enlargement of the glomerular mesangium
matrix and thickening of the glomerular basement membrane,
which are characteristic of diabetic nephropathy, wereobserved in the kidney 13), and an elevation in inflammatory
cytokines was recognized in the renal tissues 14, 15). Furthermore,
AGEs are accumulated in the kidney, therefore, it isconsidered that the glycative stress generated by the glycation
reaction of biological proteins is deeply involved in theonset of nephropathy 10, 16, 17). In this study, renal weight wasincreased in both model animals, and elevation of TNF-α
and IL-6 production was observed in the renal tissue of STZ
rats. The increase in renal weight is considered to be due
to renal hypertrophy, which is also observed in the initial_ 29 0 _Glycative Stress Research

Fig. 1a) STZSTZ ratsa)ratsb) db/db
mice miceb) db/db

50

500

40

##
300
200

Body weight (g)Body weight (g)

400

##

30
20
10

100
0
0

1

2

3

4

5

6

7

Time (week)
VehicleKE100
KE300
STZ-induced diabetic rats

Normal0801AMG100

2

3

db/+

4

5

6

7

8

9

10

Time (week)
KE100
KE300
db/db diabetic mice

Vehicle11

12

MET350Fig. 1. Change of weight in the diabetic model animals.
a) STZ rats. Normal, no STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and 100 mg/kg/day
KE treated group; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treated group. Results are
expressed as mean, n = 8, # # p < 0.01 vs. Normal.

b) db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treated group; KE100, db/db

diabetic mice and 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group; MET350, db/db
diabetic mice and 350 mg/kg/day MET treated group. Results are expressed as mean. n = 9 – 10, # # p < 0.01 vs. db/+.

STZ, streptozotocin (i.p.); KE, kuromoji extract (p.o.); AMG, aminoguanidine (p.o.); MET, metformin (p.o.); i.p., intraperitoneal
administration; p.o., oral administration.

Fig. 2STZrats
rats
a) a)
STZ

b) db/db
in miceb) db/db
mice
12

50

Food intake (g/day)Food intake (g/day)

10
40
30
20
10

8
6
4
2

0
0

1
Normal2

34Time (week)
Vehicle
KE100

5

6
KE300

7

0

8

0

AMG1001
db/+

STZ-induced diabetic rats

2

3

4

Vehicle5

6

7Time (week)
KE100

8
KE300

9

10

11

12

MET350db/db diabetic mice

Fig. 2. Change of food intake amount in the diabetic model animals.
a) STZ rats. Normal, No STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and 100 mg/kg/day
KE treated group; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treated group. Results are
expressed as mean, n = 8b) db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treated group; KE100, db/db

diabetic mice and 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group; MET350, db/db
diabetic mice and 350 mg/kg/day MET treated group. Results are expressed as mean, n = 9 - 10.

STZ, streptozotocin (i.p.); KE, kuromoji extract (p.o.); AMG, aminoguanidine (p.o.); MET, metformin (p.o.) ; i.p., intraperitoneal
administration; p.o., oral administration.

_ 2 91 _Effects of Kuromoji (Lindera umbellata Thunb.) Extract on Diabetic Nephropathy

Table 1. The values of blood markers after treatment in the diabetic model animals.

a) STZ rats

STZ (-)

NormalVehicleKE100

GLU (mg/dL)

243 ± 37.3

496 ± 66.3 ##GA (%)

1.81 ± 0.22

HbA1c (%)

STZ(+)

KE300

AMG100480 ± 42.6

464 ± 63.5

53 0 ± 53.6

8.41 ± 0.81 ##8.85 ± 2.71

7.94 ± 2.38

8.79 ± 0.68

3.53 ± 0.14

8.06 ± 0.95 ##8.53 ± 1.95

7.98 ± 1.71

8. 53 ± 1.14

TC (mg/dL)

77 ± 9.0

96 ± 37.8

171 ± 65.4 **145 ± 40.1 *10 9 ± 17.4

TG (mg/dL)

99 ± 21.7

481 ± 385.2 #868 ± 529.2

1101 ± 881.0

5 4 4 ± 211.4

NEFA (μEq/L)

376 ± 63.3

457 ± 138.3

644 ± 123.3

847 ± 307.1 **655 ± 129.8 *LDL-C (mg/dL)

8.3 ± 1.2

16.6 ± 12.8

38.4 ± 29.7

34.6 ± 23.2

14. 5 ± 3.4

28.1 ± 2.0

35.0 ± 9.1 #45.0 ± 11.6

38.8 ± 6.3

41. 3 ± 6.6

KE300

MET350HDL-C (mg/dL)b) db/db micedb/+

Vehicle

KE100

db/db

GLU (mg/dL)

102 ± 36.3

428 ± 6 4.4 ##419 ± 79.7

381 ± 101. 3

330 ± 109.6

HbA1c(%)

3.87 ± 0.12

9.53 ± 1.18 ##9.61 ± 1.09

8.88 ± 1.6 2

7.99 ± 1.71 *TC (mg/dL)

103 ± 12.0

132 ± 15.4 ##118 ± 12.6

117 ± 10. 0 *120 ± 16.8

TG (mg/dL)

109 ± 25.4

133 ± 27.1

111 ± 25.3

1 01 ± 2 6. 4

77 ± 18.4 **LDL-C (mg/dL)

39.2 ± 6.4

48.5 ± 12.5 #42.2 ± 6.4

37. 9 ± 4. 3

27.6 ± 3.4 **HDL-C (mg/dL)

62.6 ± 9.6

86.4 ± 13. 2 ##84.6 ± 11.1

84.4 ± 6. 9

88.8 ± 15.5

Adipo (μg/mL)

7.39 ± 3.94

7.78 ± 1.24

6.96 ± 1.95

6.60 ± 1.87

9.38 ± 7.35a) STZ rats. Normal, no STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and 100 mg/kg/day KE treatedgroup; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treated group. Results are expressed as mean ±SD, n = 8, # p < 0.05, # # p < 0.01 vs. Normal; * p < 0.05, ** p < 0.01 vs. Vehicle.

b) db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treated group; KE100, db/db diabetic miceand 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group; MET350, db/db diabetic mice and 350 mg/
kg/day MET treated group. Results are expressed as mean ± SD, n = 9 - 10), # p < 0.05, # # p < 0.01 vs. db/+; ** p < 0.01, * p < 0.05 vs. Vehicle.
STZ, streptozotocin (i.p.); KE, kuromoji extract (p.o.); AMG, aminoguanidine (p.o.); MET, metformin (p.o.); GLU, glucose; GA, glycoalbumin;
HbA1c, hemoglobin A1c; TC, total cholesterol; TG, triglycerides; NEFA, non-esterified fatty Acid; LDL-C, low-density lipoprotein cholesterol;
HDL-C, high-density lipoprotein cholesterol; Adipo, adiponectin; SD, standard deviation; i.p., intraperitoneal administration; p.o., oral
administration.

_ 292 _Glycative Stress Research

Fig. 3STZrats
ratsa)a)STZb) db/db
micemiceb) db/db

0.5

3.5
3.0

#

0.4

2.5

Kidney wight (g)Kidney weight (g)

*

#

2.0
1.5
1.0

0.3

0.2

0.1

0.5
0.0

0.0
NormalVehicle

KE100

KE300

db/+

AMG100

Vehicle

KE100

KE300

MET350db/db diabetic mice

STZ-induced diabetic rats

Fig. 3. Kidney weight after treatment in the diabetic model animals.
a) STZ rats. Normal, No STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and 100 mg/kg/day
KE treated group; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treated group. Results are
expressed as mean ± SD, n = 8, # p < 0.05 vs. Normal; * p < 0.05 vs. Vehicle.

b) db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treated group; KE100, db/db

diabetic mice and 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group; MET350, db/db
diabetic mice and 350 mg/kg/day MET treated group. Results are expressed as mean ± SD, n = 9 – 10, # p < 0.05 vs. db/+.

STZ, streptozotocin (intraperitoneal administration, i.p.); KE, kuromoji extract (oral administration, p.o.); AMG, aminoguanidine (p.o.);
MET, metformin (p.o.); SD, standard deviation; i.p., intraperitoneal administration; p.o., oral administration.

Fig. 4
a)a)
STZ
rats
STZ
rats

b) db/dbb) mice
db/db mice

600

#

**

500

*

70

*
Anti-CML autoantibody (µg/mL)

Anti-CML autoantibody (µg/mL)

700

400
300
200
100
0
NormalVehicle

KE100

KE300

#

60
50
40
30
20
10
0

AMG100db/+

STZ-induced diabetic rats

Vehicle

KE100

KE300

MET350db/db diabetic mice

Fig. 4. The values of blood anti-CML autoantibody after treatment in the diabetic model animals.
a) STZ rats. Normal, No STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and 100 mg/kg/day
KE treated group; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treated group. Results are
expressed as mean ± SD, n = 8, # p < 0.05 vs. Normal; * p < 0.05, ** p < 0.01 vs. Vehicle.

b) db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treated group; KE100, db/db

diabetic mice and 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group; MET350, db/db
diabetic mice and 350 mg/kg/day MET treated group. Results are expressed as mean ± SD, n = 9 – 10, # p < 0.05 vs. db/+.

STZ, streptozotocin (i.p.); KE, kuromoji extract (p.o.); AMG, aminoguanidine (p.o.); MET, metformin (p.o.); CML, N ε- (Carboxymethyl)
lysine; SD, standard deviation; i.p., intraperitoneal administration; p.o., oral administration.

_ 2 93 _Effects of Kuromoji (Lindera umbellata Thunb.) Extract on Diabetic Nephropathy

Fig. 5a) TNF-αin STZa) TNF-α
inrats
STZ ratsb)b)IL-6
in STZ
ratsrats
IL-6
in STZ

3.0

10

**
**

#

*

8
IL-6 (ng/mg protein)

TNF-α (ng/mg protein)

2.5
2.0
1.5
1.0

**

*

6

4

2

0.5
0.0

0
NormalVehicle

KE100

KE300

AMG100NormalVehicleSTZ-induced diabetic rats

KE100

KE300

AMG100STZ-induced diabetic rats

Fig.
Fig.55c)c)TNF-α
TNF-αininin
db/db
db/db
mice
mice
c)
TNF-α
db/db
mice

d)d)IL-6
IL-6inin
db/db
mice
mice
d)
IL-6
indb/db
db/db
mice

1.0
1.0

0.8
0.8

****

****

0.8
0.8

**

IL-6 (ng/mg protein)IL-6 (ng/mg protein)

TNF-α (ng/mg protein)
TNF-α (ng/mg protein)

0.6
0.6

0.6
0.6

0.4
0.4

0.4
0.4

0.2
0.2

0.2
0.2
0.0
0.0

0.0
0.0
db/+
db/+

VehicleVehicle

KE100
KE100

KE300
KE300

MET350
MET350

db/+
db/+

db/db
diabetic
mice
db/db
diabetic
mice

VehicleVehicle

KE100
KE100

KE300
KE300

MET350
MET350db/db
diabetic
mice
db/db
diabetic
mice

Fig. 5. The values of kidney cytokine after treatment in the diabetic model animals.
a) TNF-α, b) IL-6 in STZ rats. Normal, no STZ-treat and vehicle treated group; Vehicle, STZ and vehicle treated group; KE100, STZ and
100 mg/kg/day KE treated group; KE300, STZ and 300 mg/kg/day KE treated group; AMG100, STZ and 100 mg/kg/day AMG treatedgroup. Results are expressed as mean ± SD, n = 8, # p < 0.05 vs. Normal; * p < 0.05, ** p < 0.01 vs. Vehicle.

c) TNF-α, d) IL-6 in db/db mice. db/+, non-diabetic mice and vehicle treated group; Vehicle, db/db diabetic mice and vehicle treatedgroup; KE100, db/db diabetic mice and 100 mg/kg/day KE treated group; KE300, db/db diabetic mice and 300 mg/kg/day KE treated group;
MET350, db/db diabetic mice and 350 mg/kg/day MET treated group. Results are expressed as mean ± SD, n = 9 – 10, ** p < 0.01, * p < 0.05
vs. Vehicle.
STZ, streptozotocin (intraperitoneal administration, i.p.); KE, kuromoji extract (oral administration, p.o.); AMG, aminoguanidine (p.o.);
MET, metformin (p.o.); TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; SD, standard deviation; i.p., intraperitoneal administration; p.o.,oral administration.

_ 294 _Glycative Stress Researcha) CMLb) CML-HSA
##

##

##

##

Fig. 6. The values of TNF-α production by treatment with glycated materials in RAW264.7 cells.
a) CML treatment. b) CML-HSA treatment. The culture supernatant was collected 24 hours (a) or 3 hours(b) after the application and measured for TNF-α content by ELISA. Results are expressed as mean ± SD, n =3, ## p < 0.05 vs. 0 µg/mL (control). CML, N ε-(Carboxymethyl)lysine; TNF-α, tumor necrosis factor-α; HSA,
human serum albumin; ELISA, enzyme-linked immuno sorbent assay: SD, standard deviation.

symptoms of diabetic nephropathy. Persistent hyperglycemia
in both models may be involved in the development of theinflammatory response in the kidney.
AGEs are said to increase with age in human and animal
tissues and be associated with diabetic nephropathy 18), chronic
renal failure 19), atherosclerotic lesions of the arterial wall 20)
and Alzheimer's disease 21). In addition, AGEs are known tohave antigenicity and produce autoantibodies. Autoantibodies
against AGEs are detected in high concentrations in thesera of diabetic patients, and are thought to be associatedwith the various diseases mentioned above 22). In particular,
autoantibodies against AGEs are known to be associated withthe severity of diabetic nephropathy, and higher concentrations
are detected in patients with renal failure who require dialysis
than in patients with mild microalbuminuria 23). Furthermore,
CML autoantibodies, which have an epitope common tovarious AGEs, increase about 5 times in T2DM patients withblood GLU levels of 200 mg/dL or higher compared withhealthy subjects22). Taking these findings into consideration,
the amount of autoantibodies to CML reflects the formation
and accumulation of AGEs in vivo, and is considered toplay an important role in the early detection of secondary
complications of diabetes. In this study as well, a marked
increase in anti-CML autoantibodies was observed in thesera of both model animals. The increase in antibody level
was remarkable particularly in STZ rats, and this increase
was suppressed by administration of AMG. This indicatesthat glycative stress with increased production of AGEs isstrongly involved in the renal inflammatory response in bothmodel animals, and is effect appears to be more pronounced
in STZ rats that is the T1DM model.
As one of the mechanisms of elevation of inflammatory
cytokines by AGEs, it has been reported that they are involvedin the AGEs/RAGE inflammatory signal transduction system
via RAGE (Receptor for AGEs) present in macrophages. In
patients with nephropathy, there are marked infiltration of
inflammatory cells mainly macrophages, in the glomerulus
and interstitium 10, 24), and increased RAGE in the renal tissueof STZ rats and db/db mice 15, 25, 26). However, contradictory
discussions continue on each AGE regarding the inductionof inflammatory reactions via RAGE, and some reports
insist that CML does not have the necessary structure tobind to RAGE 27, 28). On the contrary, it has been shown thatmodification of proteins to AGEs is important for binding
to RAGE. Co-treatment of CML alone did not conflict withthe binding of CML-BSA to RAGE 29). Also, treatment with
CML alone did not induce TNF-α production from humanmonocyte-derived cells, whereas it was produced by casein
glycation metabolites, which were co-treated with an antiCML antibody or a RAGE antagonist, resulting suppressionof TNF-α production 27). We also confirmed that TNF-α
production was not observed with CML alone of RAW264.7
cells but was increased in a dose-dependent manner with
CML-HSA treatment. This is a result supporting theimportance of protein modification to AGEs for the inductionof inflammation of macrophages.

2. Actions of Kuromoji
KE strongly suppressed increasing anti-CML
autoantibody in STZ rat serum and decreased renal weightand inflammatory cytokines in renal tissue. Our previous
study showed that KE suppresses the production offluorescent AGEs, CML, 3-deoxyglucosone (3DG), glyoxal(GO), and methylglyoxal (MGO) in the GLU-glycation_ 2 95 _Effects of Kuromoji (Lindera umbellata Thunb.) Extract on Diabetic Nephropathy

reaction system for HSA, collagen and elastin, and especiallyfor CML which was strongly inhibited 6). Also, it has thecleaving effect of AGE cross-linking and the enhancing
effect of the activity of oxidative proteolytic enzyme (OPH),
thus exerting an action of degrading AGEs 6). Furthermore,
the kuromoji branches contain flavan-3-ol derivatives,
i. e. procyanidin B1, and procyanidin B2, and flavonoid
compounds, i.e. kaempferol, quercetin, hyperoside, andpinocembrin, which are reported to have an inhibitory effecton AGEs production, a trapping effect on MGO and GO, andan inhibitory effect on the AGEs-induced apoptosis 30 - 36).
The amount of autoantibodies to CML, which has an
epitope common to various AGEs, may reflect the formation
and accumulation of AGEs in vivo. It is considered that thestrong anti-glycation activity of KE reduces the production
and accumulation of AGEs, thereby suppressing theactivation of inflammatory cells, i.e. macrophage, via theAGEs/RAGE inflammatory signal transduction system
and reducing the progression of the inflammation in thekidneys. The influence of glycative stress was stronger in
STZ rats than in db/db mice, however the effect of KE wasmore obvious in STZ rats. It is considered that the action of
KE was more remarkable in the T1DM model in which the
production and accumulation of AGEs progressed earlier.
On the other hand, in db/db mice, the increase in TC was
suppressed by KE administration. Generally, the main sitesof cholesterol synthesis are the liver and small intestine. It
has been reported that, in STZ rats, cholesterol is mainly
synthesized in the small intestine, whereas in db/db mice,cholesterol is highly synthesized in the liver 37). There aredifferences in cholesterol synthesis between insulin-dependent
diabetic animals, i.e. STZ rats and non-insulin-dependent
diabetic animals, i.e. db/db mice. Concurrently, procyanidin
B1 and procyanidin B2 have been reported to suppress theincrease in TG and TC in db/db mice 25), but no reports havebeen found in STZ rats. The inhibitory effect of KE oncholesterol elevation, which was observed only in db/db mice
this time, is considered to be influenced by the difference inthe mechanism of lipid metabolism in each diabetes model.

Conclusion
In the diabetes model, it was considered that the increased
AGEs in vivo caused renal inflammation by stimulating
inflammatory cells such as macrophages. KE, which has ananti-glycation effect, suppressed renal inflammation in STZ
rats and db/db mice, but the suppressive effect was more
pronounced in STZ rats, a T1DM model in which the effect ofglycative stress is large. It is possible that the strong inhibitory
action of KE on glycative stress is involved in the suppressionof the inflammatory response in renal tissues during diabetic
conditions.

Acknowledgment
This work was supported by the Japanese Council
for Science, Technology and Innovation, SIP (Project ID
14533567), “ Technologies for creating next-generation
agriculture, forestry and fisheries” (funding agency: Biooriented Technology Research Advancement Institution,
NARO).

Conflict of interest declaration
In carrying out this research, we received support from
Yomeishu Seizo Co. Ltd. as a SIP cooperating company.

Reference
1) Furuhata M, Horiguchi T, Kato M. Pharmaceutical studies

on Chinese drug "Wuzhang". I : On the seasonal variationof essential oil in Lindera Umbellata THUNB. Shoyakugaku
Zasshi. 1966; 86: 683-687. (in Japanese)
2) Hayashi N, Komae H. The chemical ecology of Kuromoji(Lindera umbellata). The Koryo. 1976; 115: 31-40. (inJapanese)
3) Ezaki N, Sakai S, Kojima S, et al. Effect of continuous
taking of medicinal tonic liqueur ( Yomeishu® ) on
sensation of cold syndrome and peripheral bodytemperature in women: Preliminary study on open design.
Jpn Pharmacol Ther. 2007; 35: 335-341. (in Japanese)
4) Ezaki N, Kato M, Takizawa N, et al. Pharmacologicalstudies on Linderae umbellatae Ramus: IV. Effects ofcondensed tannin related compounds on peptic activity
and stress-induced gastric lesions in mice. Planta Med.
1985; 51: 34 -38.

5) Yagi M, Takabe W, Matsumi S, et al. Screening andselection of anti-glycative materials: Kuromoji (Lindera
umbellata). Glycative Stress Res. 2017; 4: 317-328.
6) Yagi M, Takabe W, Matsumi S, et al. Biochemistry ofkuromoji (Lindera umbellata) extract: Anti-oxidative andanti-glycative actions. Glycative Stress Res. 2017; 4: 329340.
7) Matsumi S, Maruyama T. The effect of kuromoji (Lindera
umbellata Thunb.) on the immune function of stressloaded mice. Proceedings of The 24th Meeting of theJapan Mibyo System Association. 2017: F1-3. (abstract inJapanese)
8) Igase M, Yonei Y, Matsumi S, et al. Effectiveness ofkuromoji (Lindera umbellata Thunb.) extract in theprevention of influenza infection after vaccination: A
randomized, double-blind, placebo-controlled, parallelgroup study. Glycative Stress Res. 2019; 6: 75-81.

_ 296 _Glycative Stress Research9) Shikata K. Inflammatory mechanism in pathogenesisof diabetic nephropathy. Journal of the Japan Diabetes
Society. 2005; 48: 673-676. (in Japanese)10) Okada S, Shikata K, Yokono H. Inflammatory mechanism
involved in the onset and progression of diabetic
nephropathy. Journal of Okayama Medical Association.
2005; 117: 9-15. (in Japanese)
11) Sato H, Ishii H. Evolving diabetes model animals.
The Dental Journal of Health Sciences University of
Hokkaido. 2015; 34(1): 47. (in Japanese)
12) Takeda Y, Shimomura T, Wakabayashi I. Immunologicaldisorders of diabetes mellitus in experimental rat models.
Japanese Journal of Hygiene. 2014; 69: 166 -176. (inJapanese)13) Kitada M, Ogura Y, Koya D. Rodent models of diabetic
nephropathy: Their utility and limitations. Int J NephrolRenovasc Dis. 2016; 9: 279-290.
14) Palsamy P, Subramanian S. Resveratrol protects diabetic
kidney by attenuating hyperglycemia-mediated oxidative
stress and renal inflammatory cytokines via Nrf2-Keap1
signaling. Biochim Biophys Acta. 2011; 1812: 719-731.
15) Park CH, Yokozawa T, Noh JS, et al. Oligonol, a lowmolecular-weight polyphenol derived from lychee fruit,attenuates diabetes-induced renal damage through theadvanced glycation end product-related pathway in db/db
Mice. J Nutr. 2014; 144: 1150-1157.
16) Forbes JM, Cooper ME, Thallas V, et al. Reduction of theaccumulation of advanced glycation end products by ACE
inhibition in experimental diabetic nephropathy. Diabetes.
2002; 51: 3274-3282.
17) Sohn E, Kim J, Kim CS, et al. The extract of Litsea
japonica reduced the development of diabetic nephropathy
via the inhibition of advanced glycation end products
accumulation in db/db mice. Evid Based Complement
Alternat Med. 2013; 769416.
18) Imai N, Nishi S, Suzuki Y, et al. Histological localizationof advanced glycosylation end products in the progressionof diabetic nephropathy. Nephron. 1997; 76: 153-160.
19) Yamada K, Miyahara Y, Hamaguchi K, et al. Immunohistochemical study of human advanced glycosylation endproducts (AGE) in chronic renal failure. Clin Nephrol.
1994; 42: 354 -361.
20) Kume S, Takeya M, Mori T, et al. Immunohistochemical
and ultrastructural detection of advanced glycation end
products in atherosclerotic lesions of human aorta with anovel specific monoclonal antibody. Am J Pathol. 1995;
147: 654-657.
21) Vitek MP, Bhattacharya K, Glendening J M, et al.
Advanced glycation end products contribute to amyloidosis
in Alzheimer disease. Proc Natl Acad Sci USA. 1994; 91:
4766- 4770.
22) Ashraf JM, Abdullah SMS, Ahmad S, et al. Prevalenceof autoantibodies against 3-DG-glycated H2A protein in
type 2 diabetes. Biochemistry (Mosc). 2017; 82: 579-586.
23) Shibayama R, Araki N, Nagai R, et al. Autoantibody
against N ε-(carboxymethyl) lysine: An advanced glycation
end product of the Maillard reaction. Diabetes. 1999; 48:
1842-1849.
24) Furuta T, Saito T, Ootaka T, et al. The role of macrophages
in diabetic glomerulosclerosis. Am J Kidney Dis. 1993; 21:480-485.

25) Noh JS, Kim HY, Park CH, et al. Hypolipidaemic andantioxidative effects of oligonol, a low-molecular-weight
polyphenol derived from lychee fruit, on renal damage intype 2 diabetic mice. Br J Nutr. 2010; 104: 1120-1128.
26) Muthenna P, Raghu G, Anil Kumar P, et al. Effect of
cinnamon and its procyanidin-B2 enriched fraction
on diabetic nephropathy in rats. Chemico-BiologicalInteractions. 2014; 222: 68-76.
27) van der Lugt T, Weseler AR, Gebbink WA, et al. Dietaryadvanced glycation endproducts induce an inflammatory
response in human macrophages in vitro. Nutrients. 2018;10(12): 1868.
28) Buetler TM, Leclerc E, Baumeyer A, et al. N ε carboxymethyllysine-modified proteins are unable to bindto RAGE and activate an inflammatory response. Mol
Nutr Food Res. 2008; 52: 370-378.
29) Kislinger T, Fu C, Huber B, et al. N ε-(Carboxymethyl)
lysine adducts of proteins are Ligands for receptor
for advanced glycation end products that activate cellsignaling pathways and modulate gene expression. J Biol
Chem. 1999; 274: 31740-31749.
30) Yang R, Wang WX, Chen HJ, et al. The inhibition ofadvanced glycation end-products by five fractions andthree main flavonoids from Camellia nitidissima Chi
flowers. J Food Drug Anal. 2018; 26: 252-259.
31) Jiang W, Wang, Liu D, et al. Protective effects of
Kaempferitrin on advanced glycation end products induce
mesangial cell apoptosis and oxidative stress. Int J Mol
Sci. 2018; 19(11): 3334.
32) Morimitsu Y, Yoshida K, Esaki S, et al. Protein glycation
inhibitors from thyme (Thymus vulgaris). Biosci Biotechnol
Biochem. 1995; 59: 2018-2021.
33) Li X, Zheng T, Sang S, et al. Quercetin inhibits advanced
glycation end product formation by trapping methylglyoxal
and glyoxal. J Agric Food Chem. 2014; 62: 12152-12158.
34) Zhang Z, Sethiel MS, Shen W, et al. Hyperosidedownregulates the receptor for advanced glycation end
products (RAGE) and promotes proliferation in ECV304
cells via the c-Jun N-terminal kinases (JNK) pathway
following stimulation by advanced glycation end-products
in vitro. Int J Mol Sci. 2013; 14: 22697-22707.
35) Potipiranun T, Adisakwattana S, Worawalai W, et al.
Identification of pinocembrin as an anti-glycation agent
and α-glucosidase inhibitor from fingerroot (Boesenbergia
rotunda): The tentative structure-activity relationshiptowards MG-trapping activity. Molecules. 2018; 23(12):3365.
36) Borriello M, Iannuzzi C, Sirangelo I. Pinocembrin
protects from AGE-induced cytotoxicity and inhibits nonenzymatic glycation in human insulin. Cells. 2019; 8(5):385.
37) Feingold KR, Lear SR, Moser AH. De novo cholesterolsynthesis in three different animal models of diabetes.
Diabetologia. 1984; 26: 234-239.

_ 297 _