Protective Effects of 3,4-seco-lupane Type Triterpenes from Acanthopanax

senticosus against Advanced Glycation Endproducts

Hyun Young Kim1, Dong Gu Lee

2, Ki Ho Lee

2, and Sanghyun Lee

2*

1Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Korea

2Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Korea

*Corresponding author: slee@cau.ac.kr

Received March 11, 2012 / Revised April 17, 2012 / Accepted April 17, 2012

Korean Society for Horticultural Science and Springer 2012

Abstract. Advanced glycation end products (AGEs) are thought to be directly involved in diabetes mellitus and aging.

In this study, the protective activities of 3,4-seco-lupane type triterpenes (chiisanogenin and chiisanoside) from Acanthopanax

senticosus against the formation of AGEs were examined using in vitro glycation reactions. Of the two isolated

compounds, chiisanogenin exhibited strong inhibitory activity against the formation of AGEs. The inhibitory activity

of chiisanogenin was similar level in 50 M treatment with the AGE inhibitor aminoguanidine, which was used as a

positive control. These results suggest that chiisanogenin from A. senticosus is a bioactive component that contributes

to glycation-associated diseases.

Additional key words: Araliaceae, extraction, fractionation, isolation, reflux

Hort. Environ. Biotechnol. 53(3):242-246. 2012.

DOI 10.1007/s13580-012-0030-6

Research Report

Introduction

Acanthopanax species (Araliaceae) are native to Asia, the

Malay Peninsula, Polynesia, Europe, North Africa and the

Americas, and about 15 species of Acanthopanax have been

identified in eastern Asia. Among the Acanthopanax species

growing in the Korean Peninsula, A. senticosus, A. chiisanensis

and A. sessiliflorus are the most abundant. Acanthopanax

species have traditionally been used as tonics and sedatives,

as well as in the treatment of rheumatism and diabetes.

Indeed, their regular use has been reported to restore vigor,

appetite, memory, impotence, and increase longevity with

ginseng-like activities (Bae et al., 2001; Huang et al., 2011;

Jung et al., 2005; Lee et al., 2002). A nitrogenous compound,

a furan-containing compound, and an aliphatic alcohol, as

well as quinoid, benzoid, coumarin, phenylpropanoid, lignan,

flavonoid, terpenoids, phytosterols, polyacetylenes, pyrimidine,

cyclitol, and monosaccharide compounds have all been isolated

from Acanthopanax species and have been shown to have

various levels of anti-bacterial, anti-cancer, anti-hepatitis, anti-

hyperglycemic, anti-inflammatory, anti-oxidant, immunostimulatory,

and radioprotectant activities (Bae et al., 2001; Hong et al.,

2011; Kasai et al., 1986; Nhiem et al., 2011; Sithisarn et al.,

2011).

Advanced glycation endproducts (AGEs), which were formed

as the result of nonenzymatic modification of proteins by

reducing various sugars, play an important role in the

development of chronic diabetic complications and aging

(Ahmed, 2005; Ulrich and Cerami, 2001). In addition, glycation

and oxidative stress are closely linked, and each of the steps

in glycation generates free oxygen radicals (Gillery, 2001).

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 glycation reactions, which also produce

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 (Ahmed, 2005; Baynes, 1991). 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. Therefore, the inhibition of AGE for-

mation may be a promising target for therapeutic intervention

in AGE-related disorders. Recent reports have showed that

flavonoids inhibit the formation of AGEs (Kim et al., 2011;

Hort. Environ. Biotechnol. 53(3):242-246. 2012. 243

Sengupta et al., 2006; Urios et al., 2007). However, there

are few studies on the formation of AGEs of triterpene from

plants.

In the present study, we report the protective activity of

triterpenes from A. senticosus against protein damage (the

formation of AGEs) using in vitro model systems.

Materials and Methods

The dried leaves of Acanthopanax senticosus Harm (Araliaceae)

were collected at Gongju Province and verified by Prof. S.

H. Cho, Kong Ju National University of Education, Korea.

A voucher specimen was deposited at the Herbarium of

Department of Integrative Plant Science, Chung-Ang Uni-

versity, Korea.

Electron ionization mass spectrometry (EI-MS) was per-

formed with a JEOL JMS-600W (Tokyo, Japan) mass spec-

trometer. The1H-NMR spectrum was recorded with a Bruker

AVANCE 400 NMR spectrometer in pyridine, using TMS

as an internal standard. Chemical shifts were reported in

parts per million ( ), and coupling constants (J) were expressed

in Hertz (Hz). TLC analysis was conducted with Kiesel gel

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

0.25 mm layer thickness), and compounds were visualized

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

Silica gels (200-400 mesh, Merck, Germany) were used for

open column chromatography. All other chemicals and reagents

were of analytical grade.

Dried and coarsely powdered leaves (1 kg) of A. senticosus

were extracted three times with methanol under reflux for 5

h in a water bath. The methanol extract was concentrated

under reduced pressure and fractionated into MC, EtOAc,

and n-BuOH fractions. A portion of the MC fraction over a

silica gel using a gradient of n-hexane-EtOAc produced com-

pound 1. A portion of the n-BuOH fraction over a silica gel

using a stepwise-gradient elution of CHCl3-MeOH produced

compound 2.

Chiisanogenin (1): EI-MS (rel. int. %): m/z 484 [M]+ (23.1),

396 (100), 368 (21.0), 161 (52.4); IR max (KBr): 3447

(OH), 1718 (COOH) cm-1

;1H-NMR (400 MHz, Pyridine):

5.10 (1H, s, H-23b), 5.00 (1H, s, H-23a), 4.91 (1H, d, J =

2.0 Hz, H-29b), 4.62 (1H, s, H-29a), 4.58 (1H, ddd, J = 9.4,

9.4, 9.4 Hz, H-11), 3.70 (1H, d, J = 8.0 Hz, H-1), 3.47 (1H,

ddd, J = 4.6, 10.8, 10.8 Hz, H-19), 3.09 (1H, d, J = 14.4 Hz,

H-2 ), 2.89 (1H, dd, J = 8.0, 14.4 Hz, H-2 ), 2.72 (1H, d, J

= 9.4 Hz, H-9), 2.55 (1H, H-16 ), 2.22 (1H, H-21 ), 2.17

(1H, H-22 ), 1.85 (3H, s, H-24), 1.76 (1H, H-15 ), 1.68

(3H, s, H-30), 1.66 (1H, dd, J = 10.8, 10.8 Hz, H-18), 1.50

(1H, H-22 ), 1.47 (1H, H-16 ), 1.42 (1H, H-21 ), 1.14 (1H,

H-15 ), 1.05 (3H, s, H-27), 0.98 (3H, s, H-26), 0.97 (3H, s,

H-25); 13

C-NMR (100 MHz, Pyridine): 179.6 (C-28),

173.9 (C-3), 151.4 (C-20), 148.6 (C-4), 114.7 (C-23), 111.5

(C-29), 76.2 (C-11), 71.3 (C-1), 57.2 (C-17), 50.5 (C-5),

50.4 (C-18), 48.7 (C-19), 44.9 (C-9), 44.8 (C-10), 43.1

(C-14), 42.5 (C-8), 39.6 (C-2), 38.2 (C-22), 36.2 (C-13),

34.4 (C-17), 33.5 (C-7), 33.3 (C-12), 31.9 (C-21), 30.5

(C-15), 26.0 (C-6), 23.5 (C-24), 19.8 (C-25), 19.7 (C-30),

18.7 (C-26), 14.6 (C-27).

Chiisanoside (2): FAB-MS: m/z 955 [M + 1]+; IR max

(KBr): 3432 (OH), 1718 (C = O), 1637 (C = C), 1068 cm-1

;1H-NMR (400 MHz, Pyridine): 6.31 (1H, d, J = 8.2 Hz,

inner Glc H-1), 5.81 (1H, s, Rha H-1), 5.12 (1H, s, H-23b),

5.00 (1H, s, H-23a), 4.91 (1H, d, J = 7.6 Hz, outer Glc H-1),

4.83 (1H, s, H-29b), 4.64 (1H, s, H-29a), 4.52 (1H, ddd, J =

9.0, 9.0, 9.0 Hz, H-11), 3.65 (1H, d, J = 7.9 Hz, H-1), 3.35

(1H, ddd, J = 4.6, 10.7, 10.7 Hz, H-19), 3.04 (1H, d, J =

14.6 Hz, H-2), 2.67 (1H, d, J = 9.0 Hz, H-9), 1.87 (3H, s,

H-24), 1.67 (3H, d, J = 5.8 Hz, Rha H-6), 1.62 (3H, s, H-

30), 1.08 (3H, s, H-26), 0.99 (6H, s, H-27 and -25); 13C-

NMR (100 MHz, Pyridine): 174.8 (C-28), 172.8 (C-3),

149.8 (C-20), 147.4 (C-4), 113.6 (C-23), 110.4 (C-29),

104.8 (Glc-1 ), 102.5 (Rha-1), 95.1 (Glc-1), 78.4 (Glc-4 ),

78.0 (Glc-3), 77.7 (Glc-5 ), 76.8 (Glc-5), 76.2 (Glc-3 ), 75.0

(C-11), 74.9 (Glc-2 ), 73.8 (Rha-4), 73.7 (Glc-2), 72.4 (Rha-

3), 72.3 (Rha-2), 70.5 (Glc-4), 70.2 (C-1, Rha-5), 70.0 (Glc-

6), 61.0 (Glc-6 ), 56.5 (C-17), 49.4 (C-5), 49.3 (C-18), 47.3

(C-19), 43.8 (C-10), 43.7 (C-9), 41.9 (C-14), 41.4 (C-8),

38.5 (C-2), 36.7 (C-22), 34.9 (C-13), 33.2 (C-12), 32.0

(C-7), 31.9 (C-16), 30.7 (C-21), 29.3 (C-15), 25.1 (C-6),

23.4 (C-24), 18.9 (C-25), 18.6 (C-30), 18.2 (Rha-6), 17.7

(C-26), 13.5 (C-27).

According to the method of Vinson and Howard (1996),

bovine serum albumin (10 mg mL-1

) in 50 mM phosphate

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

growth, glucose (25 mM) and fructose (25 mM) were added

to the solution. This reaction mixture was then mixed with

different concentrations of test samples of chiisanogenin (1)

and chiisanoside (2). Four concentrations (1, 5, 25, and 50 M)

were prepared for the experiments. Briefly, after incubating

the reaction mixture with the test samples at 37 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 in terms of

Hyun Young Kim, Dong Gu Lee, Ki Ho Lee, and Sanghyun Lee244

1 2

Fig. 1. Chemical structures of chiisanogenin (1) and chiisanoside (2).

Table 1. Effects of chiisanogenin (1) and chiisanoside (2) on the

formation of AGEs.

the percent inhibition, calculated from a control measurement

of the fluorescence intensity of the reaction mixture with no

test sample.

The results are expressed as mean ± S.E. of five deter-

minations.

Results and Discussion

Chromatographic separation of MeOH extracts of A.

senticosus led to the isolation of 3,4-seco-lupane type triterpenes

(Fig. 1). The typical structure of a 3,4-seco-lupane type

triterpene skeleton was observed in the 1H-NMR spectra of

compounds 1 and 2. Their structures were elucidated as

chiisanogenin (1) and chiisanoside (2) by NMR analysis

(Hahn et al., 1984; Jung et al., 2005; Kasai et al., 1986; Lee

et al., 2003). We next examined the effects of chiisanogenin

and chiisanoside on the formation of AGEs. The percent

inhibition of AGE formation by these compounds was deter-

mined at a concentration of 50 M and was 14.3 and -5.2,

respectively (Table 1). Of the two isolated compounds,

chiisanogenin showed potent inhibitory activity and inhibited

AGE formation by 14.3% at a concentration of 50 M. The

inhibitory activity of chiisanogenin was similar level in 50 M

treatment with the AGE inhibitor aminoguanidine, which

was used as positive control.

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 or renal failure. AGEs cause

various types of protein modification, resulting in structural

and functional alterations, including intra- and inter-mediate

cross-linking, absorption, changes in specific fluorescence

wavelengths, and changes in enzymatic activity (Bucala and

Cerami, 1992; Fu et al., 1994; Yaylayan and Huyghues-Despointes,

1994). Therefore, to evaluate the inhibitory effects of

chiisanogenin and chiisanoside from A. senticosus against

the in vitro formation of AGEs, we measured the fluores-

cence intensity as proposed by Monnier and Cerami (1981).

We employed aminoguanidine, a well-known AGE inhibitor,

as a positive control. In this study, we found that chiisanogenin

effectively inhibited the formation of AGEs at a level higher

than that of aminoguanidine.

The reactions between amino groups of proteins and reducing

sugars lead to the formation of Schiff bases and Amadori

products. These early products undergo further rearrange-

Hort. Environ. Biotechnol. 53(3):242-246. 2012. 245

ments to generate AGEs. It is now apparent that protein

glycation reactions occur within biological tissues, which in

turn contribute to various pathological conditions including

diabetic complications, aging, and Alzheimer’s disease. Thus,

AGEs have received considerable interest in recent years

(Monnier and Cerami, 1981; Smith et al., 1994; Vlassara, 1997).

Protein glycation reactions can be broadly divided into

early-phase reactions (in which Amadori rearrangement pro-

ducts are produced) and the late-phase reactions (in which

these early products further undergo various rearrangements

to generate AGEs) (Bucala and Cerami, 1992; Vlassara et

al., 1994). It has been proposed that no oxidation reactions

are involved in the formation of Amadori rearrangement

products, whereas oxidation plays a role in creating the

characteristic fluorescence changes and molecular bridges

of AGEs (Fu et al., 1994; Sakurai and Tsuchiya, 1988;

Smith and Thornalley, 1992).

Recent reports have showed that flavonoids inhibit the

formation of AGEs (Kim et al., 2011; Sengupta et al., 2006;

Urios et al., 2007). However, there are few studies on the

formation of AGEs of triterpene. Therefore, we determined

the inhibitory effects of the two triterpenes from A. senticosus

against the formation of AGEs. Of the two isolated com-

pounds, chiisanogenin showed strong inhibitory activity

against the formation of AGEs, whereas chiisanoside had no

such activity. The mechanism of the AGE inhibitor aminoguanidine,

which was used as a positive control has been shown to

involve in trapping of reactive dicarbonyl species (Thornalley

et al., 2000), antioxidant activity by transition metal chelation

(Price et al., 2001), and other antioxidant activity including

hydroxyl radical scavenging (Giardino et al., 1998; Price et

al., 2001). Therefore, potent antioxidant activity is expected

to play an important role in AGE inhibition. There are

several reports on the anti-oxidative activity of chiisanogenin

(Jung et al., 2005; Won et al., 2005). Therefore, the anti-

oxidative effects of chiisanogenin appear to be involved, at

least in part, in AGE-inhibitory mechanisms.

In conclusion, these results show that chiisanogenin may

contribute to the prevention of glycation-related diseases,

suggesting the possibility of in vivo inhibition of AGE for-

mation.

Acknowledgements: This research was supported by the

Chung-Ang University Research Scholarship Grants in 2012.

We thank the National Center for Inter-University Research

Facilities for the measurement of spectroscopic data.

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