The Protective Activity of Linear Furanocoumarins from Angelica dahurica against Glucose-mediated Protein Damage

Hyun Young Kim · Ki Ho Lee · Dong Gu Lee · Sanghyun Lee

Received: 16 February 2012 / Accepted: 18 April 2012 / Published Online: 30 June 2012

© The Korean Society for Applied Biological Chemistry and Springer 2012

Abstract Advanced glycation end products (AGEs) are known

to be directly involved in diabetes mellitus and aging. Therefore,

protective activities of isoimperatorin, imperatorin, byakangelicin,

and oxypeucedanin hydrate from Angelica dahurica on the

formation of AGEs were examined using an in vitro glycation

reaction. Isoimperatorin showed strong inhibitory activity against

the formation of AGEs. The inhibitory activity of isoimperatorin

was more potent than that of the positive control, aminoguanidine.

These results suggest that isoimperatorin from A. dahurica may be

a promising agent for the treatment of glycation-associated diseases.

Keywords advanced glycation end products · Angelica dahurica ·

coumarin · diabetes mellitus · Umbelliferae

Introduction

Advanced glycation endproducts (AGEs), the nonenzymatic

modification of proteins by reducing sugars, play an important

role in the development of chronic diabetic complications and

aging (Ulrich and Cerami, 2001; Ahmed, 2005). Glycation and

oxidative stress are closely linked, with all glycation steps

generating oxygen-free radicals (Gillery, 2001). Oxidative damage

to proteins is directly involved in the pathogenesis of many

diseases. Free radicals can induce protein modifications, which

can cause the loss of protein function, including enzyme activity,

membrane transporter activity, and the sensitivity of receptors

(Davies and Goldberg, 1987; Meucci et al., 1991), resulting in

biological dysfunction. Proteins are also modified by glucose

through the glycation reaction. This reaction produces AGEs,

characterized by fluorescence, a brown color, and intra- or inter-

molecular cross-linking. The accumulation of AGEs has been

observed in Alzheimer’s disease (Monnier and Cerami, 1981;

Smith et al., 1994; Vlassara, 1997) and diabetic complications,

such as retinopathy, neuropathy, and nephropathy (Baynes, 1991;

Ahmed, 2005). In addition, AGEs accumulate slowly in the body

with age and more rapidly in individuals with diabetes mellitus.

An abnormally elevated blood glucose level in diabetes mellitus

causes the formation of AGEs. Furthermore, oxidative reactions

contribute significantly to the formation of AGEs (Fu et al., 1994;

Yaylayan and Huyghues-Despointes, 1994), indicating that

biological proteins are susceptibly modified in vivo to form AGEs

under oxidative stress.

Angelica dahurica (Umbelliferae) is a perennial herb distributed

all across Korea, and its root has been most frequently prescribed

as a sedative and an analgesic in Chinese medicine (Soka, 1985).

A number of studies have reported on the isolation of

phytochemicals (Shin et al., 1994; Choi et al., 2005), content

analysis (Shin et al., 1990), and the biological activities (Shin et

al., 1988; Choi et al., 2005; Liu et al., 2011) of A. dahurica.

Several coumarins such as coumarin, scopoletin, psoralen,

xanthotoxin, bergapten, and imperatorin, which are the

constituents of A. dahurica, have been studied extensively for

their chemical structures (Saiki et al., 1971; Wang et al., 2001) and

pharmacological effects (Kimura et al., 1982; Kim et al., 1992;

Kwon et al., 1997). Coumarin derivatives are the main biological

constituents in Angelica species (Shin et al., 1988; 1994; Lee et

al., 2003; Choi et al., 2005). Therefore in the present paper, the

protective activity of four furanocoumarins from A. dahurica

against protein damage (the formation of AGEs), using in vitro

model systems are reported.

K. H. Lee · D. G. Lee · S. Lee (�)Department of Integrative Plant Science, Chung-Ang University, Anseong456-756, Republic of KoreaE-mail: slee@cau.ac.kr

H. Y. KimDepartment of Food Science, Gyeongnam National University of Scienceand Technology, Jinju 660-758, Republic of Korea

ORIGINAL ARTICLE

J Korean Soc Appl Biol Chem (2012) 55, 355−358

DOI 10.1007/s13765-012-2035-3

356 J Korean Soc Appl Biol Chem (2012) 55, 355−358

Materials and Methods

Plant materials. Dried roots of A. dahurica Bentham et Hooker

(Umbelliferae) were purchased from Kyoung Dong Market,

Seoul, Korea. A voucher specimen was deposited at the

Herbarium of Department of Integrative Plant Science, Chung-

Ang University, Korea.

Instruments and Reagents. The 1H-NMR spectrum was

recorded with a Bruker AVANCE 300 NMR spectrometer

(Germany) in CDCl3, using tetramethylsilane (TMS) as an

internal standard. Chemical shifts were reported in parts per

million (δ), and coupling constants (J) were expressed in Hertz

(Hz). Thin-layer chromatography (TLC) analysis was conducted

with Kiesel gel 60 F254 (Art. 5715, Merck Co., Germany) plates

(silica gel, 0.25 mm layer thickness), with compounds visualized

by spraying with 10% H2SO4, followed by charring at 60oC. Silica

gel (200–400 mesh, Merck Co.) was used for the open column

chromatography. All other chemicals and reagents were of

analytical grade.

Isolation and Identification of linear furanocoumarins. As

reported in the previous papers (Shin et al., 1994; Choi et al.,

2005), the dried and coarsely powdered roots (1 kg) of A.

dahurica were extracted three times with methanol (MeOH) under

reflux for 5 h in a water bath. The MeOH extract was concentrated

under a reduced pressure and fractionated into n-hexane, ethyl

ether (Et2O), and ethyl acetate (EtOAc) fractions. A portion of the

n-hexane fraction over a silica gel using a gradient of n-hexane-

Et2O gave 13 sub-fractions (sub-fr.). Sub-frs. 9 and 12 gave

isoimperatorin (1) and imperatorin (2), respectively. A portion of

the Et2O fraction over a silica gel using a gradient of benzene-

Et2O-EtOAc gave byakangelicin (3). A portion of the EtOAc

fraction over a silica gel using the stepwise-gradient elution of n-

hexane-EtOAc gave 12 sub-fractions, among which Sub-fr. 10

eluted with n-hexane-EtOAc-acetone, gave oxypeucedanin hydrate

(4).

Measurement of AGEs. According to the method of Vinson and

Howard III (1996), bovine serum albumin (10 mg/mL) in 50 mM

phosphate buffer (pH 7.4), with 0.02% sodium azide to prevent

bacterial growth was added to the glucose (25 mM) and fructose

(25 mM) solutions. These reaction mixtures were mixed with

different concentrations of test samples, including isoimperatorin

(1), imperatorin (2), byakangelicin (3), and oxypeucedanin hydrate

(4). Four concentrations (1, 5, 25, and 50 µM) were prepared for

the tested samples. After incubating the reaction mixture with the

test samples at 37oC for 2 weeks, the fluorescent reaction products

from the glycated albumin were assayed on a fluorescence

spectrophotometer with an excitation wavelength of 350 nm and

an emission wavelength of 450 nm. The data were expressed as

percent of inhibition, calculated based on a control measurement

of the fluorescence intensity of the reaction mixture with no test

sample.

Statistical analysis. The results are expressed as means ± SE of

five determinations.

Results and Discussion

A chromatographic separation of the MeOH extracts from A.

dahurica led to the isolation of compounds 1−4 (Fig. 1). A typical

structure of α-pyrone in the coumarin skeleton at δ 6.25-8.16 was

observed in the 1H-NMR spectra of compounds 1−4. The

compounds isolated were identified by the 1H-NMR data (Table

1). Their structures were elucidated as isoimperatorin (1),

imperatorin (2), byakangelicin (3), and oxypeucedanin hydrate (4)

by 1H-NMR analysis (Hata et al., 1963; Shin et al., 1994). The

effects of isoimperatorin, imperatorin, byakangelicin, and

oxypeucedanin hydrate on the formation of AGEs were examined.

The inhibition percentages on the formation of AGEs by these

compounds were determined at 50 µM as follows: imperatorin,

byakangelicin, and oxypeucedanin hydrate were 16.3, 3.3, −43.4,

and −185.7, respectively (Table 2). Among the four compounds,

isoimperatorin showed potent inhibitory activity of 16.3%. This

activity was higher than that of aminoguanidine, which showed

14.1% inhibition.

AGEs are irreversible end products of the protein glycation

reaction that occurs in the body, leading to the accumulation of

AGEs in the plasma and tissues of aging patients, as well as in

patients with diabetes and renal failure. AGEs cause various types

of protein modification, resulting in structural and functional

alterations, including intra- and inter-mediate cross-linking,

absorption, fluorescence at a specific wavelength, and change in

enzymatic activity (Bucala and Cerami, 1992; Fu et al., 1994;

Yaylayan and Huyghues-Despointes, 1994). Therefore, to evaluate

the inhibitory effects of isoimperatorin, imperatorin, byakangelicin,

and oxypeucedanin hydrate from A. dahurica against the

formation of AGEs in vitro, their fluorescence intensities as

proposed by Monnier and Cerami (1981) were measured.

Fig. 1 Chemical structures of isoimperatorin (1), imperatorin (2),byakangelicin (3), and oxypeucedanin hydrate (4).

J Korean Soc Appl Biol Chem (2012) 55, 355−358 357

Aminoguanidine, a well-known AGE inhibitor, as a positive

control was employed. Results showed that isoimperatorin

effectively inhibited the formation of AGEs and that this activity

was higher than that of aminoguanidine. The reaction of amino

groups of proteins with reducing sugars leads to the formation of

Schiff bases and Amadori products. These early products undergo

further rearrangements to generate AGEs. It is now apparent that

the protein glycation reaction occurs in the biological tissues. Its

contribution to some pathological conditions, including diabetic

complications, aging, and Alzheimer’s disease, has received

considerable interest in recent years (Monnier and Cerami, 1981;

Smith et al., 1994; Vlassara, 1997).

The protein glycation reaction can be broadly divided into the

early-phase reaction (in which Amadori rearrangement products

are produced) and the late-phase reaction (in which these early

products further undergo various rearrangements to generate

AGEs) (Hata et al., 1963; Vlassara et al., 1994). It has been

proposed that no oxidation reaction is involved in the formation of

Amadori rearrangement products, whereas oxidation plays a role

in the formation of both fluorescence and a molecular bridge,

characteristic features of AGEs (Sakurai and Tsuchiya, 1988;

Smith and Thornalley, 1992; Fu et al., 1994).

Recent reports have shown that flavonoids inhibit the formation

of AGEs (Sengupta et al., 2006; Urios et al., 2007; Jang et al.,

2009, Kim et al., 2011). However, only few studies have been

performed on the formation of AGEs of furanocoumarins.

Therefore, the inhibitory effect of the four furanocoumarins from

A. dahurica against the formation of AGEs was determined.

Among the four compounds, isoimperatorin showed strong

inhibitory activity against the formation of AGEs, whereas

imperatorin, byakangelicin, and oxypeucedanin hydrate showed

no such activity. Because the structural characteristics of

isoimperatorin inhibit AGE formation, this inhibition could be

useful for developing a novel anti-oxidant. There are several

reports on the anti-oxidative activity of isoimperatorin. Isoimperatorin

was reported to have a dual cyclooxygenase-2 selective/5-

lipoxygenase inhibitory activity (Moon et al., 2008). In particular,

Piao et al. (2004) reported on the anti-oxidative activity of

furanocoumarins isolated from A. dahuricae, including

isoimperatorin, using 2,2'-azobis(2-methylpropionamidine)

dihydrochloride (AAPH) to generate peroxyl radicals, and their

results are in agreement with our findings. Therefore, the anti-

oxidative effects of isoimperatorin are, at least in part, involved in

AGE-inhibitory mechanisms.

In conclusion, the present study provides scientific evidence of

the promising therapeutic potential of isoimperatorin for pathological

conditions associated with glycation. Further investigation using

in vivo models and mechanistic studies involving isoimperatorin

should be carried out.

Table 1 The 1H-NMR data of isoimperatorin (1), imperatorin (2), byakangelicin (3), and oxypeucedanin hydrate (4)

No. 1 2 3 4

3

4

5

8

2'

3'

1''

2''

4''

5''

OMe

6.25 (d, 9.7)

8.14 (d, 9.7)

-

7.10 (s)

6.87 (d, 2.0)

7.61 (d, 2.0)

4.90 (d, 7.0)

5.51 (tq, 7.0)

1.70 (s)

1.82 (s)

-

6.34 (d, 9.6)

7.74 (d, 9.6)

7.35 (s)

-

6.79 (d, 2.0)

7.66 (d, 2.0)

4.98 (d, 7.0)

5.60 (tq, 7.0)

1.72 (s)

1.72 (s)

-

6.27 (d, 9.7)

8.12 (d, 9.7)

-

-

7.00 (d, 2.3)

7.64 (d, 2.3)

4.14−4.36 (m)

4.60 (dd)

1.27 (s)

1.31 (s)

4.17 (s)

6.29 (d, 9.7)

8.16 (d, 9.7)

-

7.12 (s)

6.99 (d, 2.4)

7.61 (d, 2.4)

3.91−4.45 (m)

4.55 (dd)

1.11 (s)

1.18 (s)

-

Table 2 Effects of isoimperatorin (1), imperatorin (2), byakangelicin (3),and oxypeucedanin hydrate (4) on the formation of AGEs

Compound Concentration (µM) Inhibition (%)

1

1 3.0±2.1

5 -0.8±3.8

25 -2.0±1.0

50 16.3±5.2

2

1 0.2±2.8

5 10.1±1.6

25 6.2±0.7

50 3.3±0.6

3

1 -5.6±1.0

5 -18.4±4.4

25 -43.6±19.1

50 -43.4±2.5

4

1 1.0±4.7

5 -26.5±6.5

25 -102.9±4.5

50 -185.7±6.6

Aminoguanidine*

1 9.2±1.9

5 8.7±2.4

25 13.3±1.9

50 14.1±2.0

*Aminoguanidine was used as a positive control.

358 J Korean Soc Appl Biol Chem (2012) 55, 355−358

Acknowledgments This research was supported by the Chung-Ang

University Research Scholarship Grants in 2012, Korea. We thank the

National Center for Inter-University Research Facilities for the measurement

of spectroscopic data at Seoul National University, Korea.

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