Volume 13. Issue 8. Pages 919-1096. 2018ISSN 1934-578X (printed); ISSN 1555-9475 (online)
www.naturalproduct.us

NPC

Natural Product Communications

EDITOR-IN-CHIEF
DR. PAWAN K AGRAWAL
Natural Product Inc.
7963, Anderson Park Lane,
Westerville, Ohio 43081, USA

agrawal@naturalproduct.us
EDITORSPROFESSOR MAURIZIO BRUNO
Department STEBICEF,
University of Palermo, Viale delle Scienze,
Parco d’Orleans II - 90128 Palermo, Italy
maurizio.bruno@unipa.it
PROFESSOR CARMEN MARTIN-CORDERO
Department of Pharmacology, Faculty of Pharmacy,
University of Seville, Seville, Spain
carmenmc@us.es
PROFESSOR VLADIMIR I. KALININ
G.B. Elyakov Pacific Institute of Bioorganic Chemistry,Far Eastern Branch, Russian Academy of Sciences,Pr. 100-letya Vladivostoka 159, 690022,
Vladivostok, Russian Federation
kalininv@piboc.dvo.ru
PROFESSOR YOSHIHIRO MIMAKI
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
mimakiy@ps.toyaku.ac.jp
PROFESSOR STEPHEN G. PYNE
Department of Chemistry, University of Wollongong,
Wollongong, New South Wales, 2522, Australia
spyne@uow.edu.au
PROFESSOR MANFRED G. REINECKE
Department of Chemistry, Texas Christian University,
Forts Worth, TX 76129, USAm.reinecke@tcu.edu
PROFESSOR WILLIAM N. SETZER
Department of Chemistry, The University of Alabama in Huntsville,
Huntsville, AL 35809, USAwsetzer@chemistry.uah.edu
PROFESSOR PING-JYUN SUNG
National Museum of Marine Biology and Aquarium
Checheng, Pingtung 944
Taiwanpjsung@nmmba.gov.tw
PROFESSOR YASUHIRO TEZUKA
Faculty of Pharmaceutical Sciences, Hokuriku University,
Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japany-tezuka@hokuriku-u.ac.jp
PROFESSOR DAVID E. THURSTON
Institute of Pharmaceutical Science
Faculty of Life Sciences & Medicine
King’s College London, Britannia House
7 Trinity Street, London SE1 1DB, UK
david.thurston@kcl.ac.uk

HONORARY EDITOR
PROFESSOR GERALD BLUNDENThe School of Pharmacy & Biomedical Sciences,
University of Portsmouth,
Portsmouth, PO1 2DT U.K.
axuf64@dsl.pipex.com

ADVISORY BOARD
Prof. Giovanni Appendino
Novara, Italy
Prof. Norbert Arnold
Halle, Germany
Prof. Yoshinori Asakawa
Tokushima, Japan
Prof. Vassaya Bankova
Sofia, BulgariaProf. Roberto G. S. Berlinck
São Carlos, Brazil
Prof. Anna R. Bilia
Florence, Italy
Prof. Geoffrey Cordell
Chicago, IL, USA
Prof. Fatih DemirciEskişehir, Turkey
Prof. Francesco Epifano
Chieti Scalo, Italy
Prof. Ana Cristina Figueiredo
Lisbon, Portugal
Prof. Cristina Gracia-Viguera
Murcia, Spain
Dr. Christopher GraySaint John, NB, Canada
Prof. Dominique GuillaumeReims, France
Prof. Duvvuru Gunasekar
Tirupati, India
Prof. Hisahiro Hagiwara
Niigata, Japan
Prof. Judith Hohmann
Szeged, Hungary
Prof. Tsukasa Iwashina
Tsukuba, Japan
Prof. Leopold Jirovetz
Vienna, Austria
Prof. Phan Van Kiem
Hanoi, Vietnam

Prof. Niel A. Koorbanally
Durban, South Africa
Prof. Chiaki Kuroda
Tokyo, Japan
Prof. Hartmut Laatsch
Gottingen, Germany
Prof. Marie Lacaille-Dubois
Dijon, France
Prof. Shoei-Sheng Lee
Taipei, Taiwan
Prof. M. Soledade C. Pedras
Saskatoon, Canada
Prof. Luc Pieters
Antwerp, Belgium
Prof. Peter Proksch
Düsseldorf, Germany
Prof. Phila Raharivelomanana
Tahiti, French Polynesia
Prof. Stefano Serra
Milano, Italy
Dr. Bikram Singh
Palampur, India
Prof. Marina Stefova
Skopj, Republic of Macodenia
Prof. Leandros A. Skaltsounis
Zografou, Greece
Prof. John L. Sorensen
Manitoba, Canada
Prof. Johannes van Staden
Scottsville, South Africa
Prof. Valentin Stonik
Vladivostok, Russia
Prof. Winston F. Tinto
Barbados, West Indies
Prof. Sylvia Urban
Melbourne, Australia
Prof. Karen Valant-Vetschera
Vienna, Austria

INFORMATION FOR AUTHORS
Full details of how to submit a manuscript for publication in Natural Product Communications are given in Information for Authors on our Web site
http://www.naturalproduct.us.
Authors may reproduce/republish portions of their published contribution without seeking permission from NPC, provided that any such republication isaccompanied by an acknowledgment (original citation)-Reproduced by permission of Natural Product Communications. Any unauthorized reproduction,transmission or storage may result in either civil or criminal liability.
The publication of each of the articles contained herein is protected by copyright. Except as allowed under national “fair use” laws, copying is not permitted byany means or for any purpose, such as for distribution to any third party (whether by sale, loan, gift, or otherwise); as agent (express or implied) of any third
party; for purposes of advertising or promotion; or to create collective or derivative works. Such permission requests, or other inquiries, should be addressedto the Natural Product Inc. (NPI). A photocopy license is available from the NPI for institutional subscribers that need to make multiple copies of single
articles for internal study or research purposes.
To Subscribe: Natural Product Communications is a journal published monthly. 2018 subscription price: US$2,595 (Print, ISSN# 1934-578X); US$2,595(Web edition, ISSN# 1555-9475); US$2,995 (Print + single site online); US$595 (Personal online). Orders should be addressed to Subscription Department,
Natural Product Communications, Natural Product Inc., 7963 Anderson Park Lane, Westerville, Ohio 43081, USA. Subscriptions are renewed on an annual
basis. Claims for nonreceipt of issues will be honored if made within three months of publication of the issue. All issues are dispatched by airmail throughout
the world, excluding the USA and Canada.

NPC

2018
Vol. 13No. 81007 – 1010

Natural Product Communications

Evaluation of Anti-glycation Activities of Phlorotannins in Humanand Bovine Serum Albumin-glyceraldehyde Models
Shingo Sugiuraa,f, Ryosuke Taniguchia,f, Yoshihiko Nishiokaa, Ryota Iwaseb, Reiji Tanakaa,d,f,Hideo Miyakea,d,f, Tetsushi Moric,d,f, Mitsuyoshi Uedae,f and Toshiyuki Shibataa,d,f*aGraduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan
Faculty of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japanc
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japand
Seaweed Biorefinery Research Center, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japane
Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake, Sakyo-ku, Kyoto 606-8502, Japanf
Japan Science and Technology Agency, CREST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japanbshibata@bio.mie-u.ac.jp
Received: June 1st, 2018; Accepted: June 22nd, 2018

The anti-glycation activities of phlorotannins contained in the Japanese Lessoniaceae (Ecklonia cava, Eck. kurome, Eck. stolonifera, Eisenia arborea, and Eis.
bicyclis) were tested using serum albumin-glyceraldehyde (GA) models. In the human serum albumin (HSA)-GA model and the bovine serum albumin (BSA)GA model, the concentrations of crude phlorotannins at 50% inhibition (IC50) of fluorescent advanced glycation end products (AGEs) formation was in therange of 0.48 to 0.70 mg/mL and 0.52 to 0.75 mg/mL, respectively. In tests using phloroglucinol and purified phlorotannins (eckol, fucofuroeckol A,
phlorofucofuroeckol A, dieckol, and 8,8´-bieckol), dieckol had the highest inhibitory activity (IC50: 5.5 x 102 µM) against fluorescent AGEs formation in HSAGA model and showed about 18 times inhibition compared with aminoguanidine hydrochloride of positive control. In the BSA albumin model, 8,8´-bieckol
had about 27 times AGEs formation inhibitory activity (IC50: 6.2 x 102 µM) against aminoguanidine hydrochloride. In tests on GA scavenging activity, it was
shown that compounds with phloroglucinol tetramer or higher had a scavenging rate of 70%, or more, with a reaction time of 120 minutes. These results
suggest that among the phlorotannins, in particular the dimers of eckol (dieckol and 8,8´-bieckol), there are effective compounds for inhibiting the formation of
AGEs derived from GA.
Keywords: Advanced glycation end products, Anti-glycation, 8,8´-Bieckol, Glyceraldehyde, Dieckol, Phlorotannins, Lessoniaceae.

Advanced glycation end products (AGEs) are a general term forstructures generated by nonenzymatic reactions between proteins
and reducing sugars such as glucose and fructose [1,2]. In previous
studies [3-6], it has been clarified that AGEs are produced not only
from the reducing sugars but also from sugar metabolic
intermediates and intermediates of Maillard reactions. It is known
that dicarbonyl compounds (methylglyoxal, glyoxal, and 3deoxyglucosone) generated from autoxidation, and degradation
products of glucose, have higher blood concentrations in diabetic
patients than in healthy subjects [7,8]. In addition, it has been
considered that the dicarbonyl compounds have high reactivity withproteins because there are two carbonyl groups in the molecule.
From this scientific background, the relationship between AGEs
derived from the dicarbonyl compounds and lifestyle-relateddiseases has been drawing attention. In recent years, according to
Takeuchi et al.’s report [9], it was revealed that α-hydroxy
aldehydes such as glyceraldehyde (GA) and glycolaldehyde are
more reactive with proteins than dicarbonyl compounds. Among
AGEs generated in vivo, it has been reported that AGEs derived
from GA (GA-AGEs) accelerate intracellular oxidative stressthrough its binding to its receptor, and can cause strong cytotoxicity[10-12]. It is also pointed out that the GA-AGEs are involved in theonset and progression of diabetic vascular complications [9,13,14],
Alzheimer’s disease [9,15,16], nonalcoholic steatohepatitis[9,17,18], hypertension [9,14], and cancer [9,19]. Therefore,
suppression of GA-AGEs formation and scavenging of GA can be
regarded as effective for prevention and treatment of these diseases.

HO

a

OH

OH

OH

O
O

HO

c

OH

HO

OH

O

OH

OH

O

e

OH
O

OH

HO

HO

OH

OH

O
O

OH

OH

HO

O
OH

O
O

OH

O
O

OH

HO

OH

O

OH

OH

OH

O
O

OH

OH

OH

HO

OH

O

HO

d

O

O

O

OH

HO

HO

OH

O

O

OH

HO

O

O

OH

O

OH

b

OH

HO

OH

O
OH

Figure 1: Chemical structures of phlorotannins isolated from the Japanese Lessoniaceae. a:eckol, b: fucofuroeckol A, c: phlorofucofuroeckol A, d: dieckol, e: 8,8´-bieckol.

In a preceding report [20], we isolated phlorotannins (eckol,
fucofuroeckol A, phlorofucofuroeckol A, dieckol, and 8,8´-bieckol)(Figure 1) from Japanese Lessoniaceae (Ecklonia cava, Eck.
kurome, Eck. stolonifera, Eisenia arborea, and Eis. bicyclis) andevaluated their anti-glycation properties in the serum albuminmethylglyoxal models. In the report [20], we clarified the following
two facts: (1) phlorofucofuroeckol A and fucofuroeckol A with abenzobisbenzofuran skeleton have inhibitory activities far superior
to aminoguanidine hydrochloride against the formation offluorescent AGEs, and (2) eckols (eckol, dieckol, and 8,8´-bieckol)
have higher methylglyoxal scavenging activity than aminoguanidine

1008 Natural Product Communications Vol. 13 (8) 2018

In both human serum albumin (HSA)-GA and bovine serum
albumin (BSA)-GA models, all the crude phlorotannins tested inthis study inhibited the formation of fluorescent AGEs in a dosedependent manner. Table 1 shows the concentration (IC50) of thecrude phlorotannins at 50% inhibition of fluorescent AGEs
formation. The IC50 values of crude phlorotannins were in the rangeof 0.48 to 0.70 mg/mL in the HSA-GA model and 0.52 to 0.75
mg/mL in the BSA-GA model, respectively (Table 1). In bothmodels, crude phlorotannins prepared from Eis. bicyclis showed thehighest inhibitory activity against fluorescent AGEs formation
among the tested samples (Table 1). Aminoguanidine is a synthetic
glycation inhibitor that inhibits the formation of AGEs andsuppresses crosslinking and polymerization of proteins in vitro [22].
Aminoguanidine is not clinically applied because it has several
adverse side effects on humans, but it is frequently used as apositive control in studies in the search for compounds having antiglycation activity. Since the IC50 values of aminoguanidinehydrochloride obtained in this study were 1.10 mg/mL in HSA-GA
model and 1.93 mg/mL in BSA-GA model, it was found that thecrude phlorotannins of Eis. bicyclis has anti-glycation activity of
about 2.3 times and 3.7 times with respect to aminoguanidinehydrochloride. Currently, Eck. kurome is cultivated as a supply
source of phlorotannins in Kumamoto prefecture, Japan [23]. The
crude phlorotannins were prepared from both naturally occurringand cultured versions of Eck. kurome, and their inhibitory activities
on the formation of fluorescent AGEs were evaluated. As shown in
Table 1, IC50 values of crude phlorotannins from cultured Eck.
kurome in each model were almost the same as those of the natural
plants of Eck. kurome. Therefore, as with the natural plants of
Lessoniaceae, it was confirmed that crude phlorotannins of cultured
Eck. kurome can be utilized as a natural product having an
inhibitory effect against AGEs formation.
Table 1: IC50 values of crude phlorotannins from Lessoniaceae against fluorescent
AGEs formation.
Algae
Specific area of origin HSA-GA (mg/mL)
BSA-GA (mg/mL)
Eck. cava
Mie
0.70
0.75
Eck. kurome
Fukuoka
0.58
0.55
Eck. kurome
Kumamoto
0.61
0.59
Cultured Eck. kurome Kumamoto
0.52
0.58
Eck. stolonifera
Yamaguchi
0.54
0.56
Eis. arborea
Mie
0.51
0.61
Eis. bicyclis
Fukuoka
0.48
0.52
All the data are expressed as the mean of three independent measurements. The IC50 values ofaminoguanidine hydrochloride were 1.10 mg/mL in the HSA-GA model and 1.93 mg/mL in
the BSA-GA model.

Table 2: IC50 values of phloroglucinol and isolated phlorotannins against fluorescent
AGEs formation

isolated compounds (eckol, fucofuroeckol A, phlorofucofuroeckol
A, dieckol, and 8,8´-bieckol). Similar to the results in the albuminmethylglyoxal models obtained in the preceding report [20],phloroglucinol and the isolated eckols inhibited the formation offluorescent AGEs in a concentration-dependent manner in both
serum albumin-GA models (data not shown). As a result of
calculating the IC50 value, dieckol and 8,8´-bieckol showed theeffective activities in both models (Table 2). In the HSA-GA model,
the IC50 value of dieckol was 5.5 x 102 µM (Table 2), which was
found to have about 18 times activity compared withaminoguanidine hydrochloride. The IC50 value of 8,8´-bieckol
obtained in the BSA-GA model was 6.2 x 102 µM (Table 2), and it
had about 27 times inhibitory activity with respect toaminoguanidine hydrochloride. Even compounds with the lowest
inhibition of AGEs also had activity about 9.1 times (eckol) in the
HSA-GA model and about 12 times (fucofuroeckol A andphlorofucofuroeckol A) in the BSA-GA model as compared withaminoguanidine hydrochloride. Therefore, it was strongly suggested
that the phlorotannins contained in Lessoniaceae, in particulardimers of eckol (dieckol and 8,8´-bieckol) have very excellent
inhibitory activity on the formation of AGEs derived from GA.
GA is thought to be caused by three pathways: the glycolytic
pathway, the polyol pathway, and the fructose metabolic pathway invivo [24]. It has also been shown that GA-AGEs are produced more
rapidly in vivo than other AGEs such as AGEs derived from glucose
and AGEs derived from methylglyoxal [4,25,26]. Therefore, it isconsidered that the scavenging of GA may contribute to the
reduction of glycation stress. As a result of experiments at aconcentration of 5 mg/mL, each type of crude phlorotannins
prepared from the five kinds of Lessoniaceae scavenged GA overtime (data not shown). The GA scavenging rate at the reaction timeof 120 minutes was in the range of 62.3% (Eck. stolonifera) to78.2% (Eck. cava). Furthermore, GA scavenging activity wasmeasured for phloroglucinol and five kinds of eckols, and the dataobtained are shown in Figure 2. Except for eckol, the tested
compounds scavenged GA is a roughly linear manner over time
(Figure 2). The GA scavenging rate at a reaction time of 120
minutes was 63.8% for phloroglucinol, 58.1% for eckol, 77.3% forfucofuroeckol A, 70.0% for phlorofucofuroeckol A, 73.9% fordieckol, 75.0% for 8,8´-bieckol, and that of aminoguanidinehydrochloride was 86.2% (Figure 2). Although the GA scavenging
activity of tested compounds was lower than aminoguanidinehydrochloride, it was revealed that compounds having
phloroglucinol tetramer or higher had a scavenging activity of
approximately 70%, or more.
phloroglucinol
eckol

100
fucofuroeckol A

Remaining amount of GA (%)

hydrochloride. Phlorotannins are polyphenols that are specificallycontained in brown algae and have a structure in which
phloroglucinol (1,3,5-trihydroxybenzen) is polymerized [21]. In thisstudy, to further evaluate the effectiveness of phlorotannins as anovel anti-glycation substance derived from natural plants, theirinhibitory activity against the formation of fluorescent AGEs in theserum albumin-GA models and the GA scavenging activity ofphlorotannins were examined.

Sugiura et al.

phlorofucofuroeckol A

80

dieckol
8,8'‐bieckol

60

aminoguanidinehydrochloride

40

Compounds
HSA-GA (µM)
BSA-GA (µM)
Phloroglucinol
3.8 x 103
3.8 x 1033
Eckol
1.1 x 10
1.3 x 1032
Fucofuroeckol A
7.4 x 10
1.4 x 103
Phlorofucofuroeckol A
7.3 x 102
1.4 x 103
Dieckol
5.5 x 1028.7 x 1028,8´-Bieckol
5.7 x 1026.2 x 102
All the data are expressed as the mean of three independent measurements. The IC50 values ofaminoguanidine hydrochloride were 1.0 x 104 µM in the HSA-GA model and 1.7 x 104 µM in
the BSA-GA model.

Figure 2: GA scavenging activity of phloroglucinol, isolated phlorotannins, andaminoguanidine hydrochloride. All the data are expressed as the mean of three independentmeasurements.

In order to further analyze the inhibitory activity of phlorotannins
against fluorescent AGEs formation in the albumin-GA models,
tests were carried out using phloroglucinol and five kinds of

In comparison with polyphenols of terrestrial plants such as
catechins, there are few reports on the toxicity evaluationof phlorotannins to animal cultured cell lines, the influence on cell

20

0
0

30

60

90

120

Reaction time (min)Anti-glycation activities of phlorotannins in albumin-glyceraldehyde models

a HeLa cell
120
phloroglucinol

Cell viability（%）

100
eckol

80
fucofuroeckol A

60
phlorofucofuroeckol A

40
dieckol

20
8,8´-bieckol

0
0

1

10

20

50

100

Concentration（μg/mL）

b 3T3‐L1 cell
120
phloroglucinol

Cell viability（%）

100
eckol

80
fucofuroeckol A

60
phlorofucofuroeckol A

40
dieckol

20
8,8´-bieckol

0
0

1

10

20

50

100

Concentration（μg/mL）

Figure 3: Cytotoxicity assay of phloroglucinol and isolated phlorotannins using MTT assay.
Each value is the average of triplicate cultures.

proliferation, and bioavailability. Using phloroglucinol and theisolated compounds, their effect on the growth of HeLa (cancer cellline) and 3T3-L1 (normal cell line) cells was evaluated by 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT)
assay (Figure 3). As a result of adding each compound to themedium at a concentration of 1 to 100 μg/mL, the survival rate ofboth cell lines was approximately 85%, or more (Figure 3). As aresult of testing at concentrations of 50 and 100 μg/mL, eachcompound showed inhibitory activity in the range of about 10 to
20% against formation of fluorescent AGEs. Therefore, it wassuggested that phloroglucinol and phlorotannins may not exhibit
cytotoxicity at concentrations that exert anti-glycation properties.
To our knowledge, there is no report on the evaluation of the antiglycation properties of plant extracts or natural products in theserum albumin-GA models. The Lessoniaceae used in this study are
the same algae used as a foodstuff in Japan and Korea. The results
obtained in the preceding [20] and present studies strongly suggestthat phlorotannins are superior anti-glycation substances derived
from natural plants and that they may contribute to both alleviationof symptoms and prevention of onset of diseases caused by AGEs
generated in vivo.
Experimental
Materials: For samples of brown algae (Ecklonia cava Kjellman,
Eck. kurome Okamura, Eck. stolonifera Okamura, Eisenia arborea
Areschoug, and Eis. bicyclis Kjellman), the algal plants used in thepreceding report [20] were used. The cultured Eck. kurome waspurchased from the Fisheries Cooperative Association of Amakusa
in Kumamoto Prefecture, Japan, in 2015. The algae used for the
extraction of phlorotannins were washed with filtered seawater, airdried, and pulverized via pulverizing mill (ABS-W, Osaka
Chemical). The algal powders were stored at -30°C until use.
Aminoguanidine hydrochloride, HSA, and GA were purchasedfrom Wako Pure Chemical Industries. BSA was obtained from
Sigma-Aldrich. 1,3-Cyclohexanedione was purchased from Tokyo
Chemical Industry. All other reagents used in this study were ofanalytical grade.

Natural Product Communications Vol. 13 (8) 2018 1009

Extraction and purification of phlorotannins: Extraction ofphlorotannins from algal powder was prepared according to themethod described in the previous report [23]. Phlorotannins were
purified by column chromatography and preparative HPLC using
the same conditions described in the previous reports [27,28]. Eachof the obtained phlorotannins (eckol, fucofuroeckol A,
phlorofucofuroeckol A, dieckol, and 8,8´-bieckol) was confirmed tohave a purity of 98% or more by three-dimensional HPLC (SPDM10AV, Shimadzu) with an Inertsil ODS-3 column (4.6 mm i.d. x
250 mm, GL Science) [28]. The identification of the purified
phlorotannins was carried out using LC/ESI/MS with the analysis
condition reported in the preceding study [23]. The purified
phlorotannins were stored at -30°C until used as samples.
Serum albumin-GA assay: Sample, GA (400 mM), and albumin(HSA or BSA, 20 mg/mL) solutions were prepared separately by
dissolving in a 100 mM phosphate buffer (pH 7.4). The
measurement was carried out using a 96-well black plate(FLUOTRAC600, Greiner) and a microplate reader (Infinite 200,
Tecan). The sample solution (40 µL), GA solution (10 µL), andalbumin solution (50 µL) were added to the well in the plate.
Measurement was carried out with fluorescence intensity at an
excitation of 370 nm and an emission of 440 nm, and the obtained
value was taken as a control value. After incubation at 37°C for 24hours, the fluorescence intensities of each well were measured
under the same measurement conditions. For blank wells, a 100 mM
phosphate buffer was used instead of a sample solution.
Aminoguanidine hydrochloride was used as a positive control. The
inhibition rate (%) of fluorescent AGEs formation was calculatedusing the following formula:
Inhibition rate (%) = {1 – [(fluorescence intensity of sample after incubation for 24hours – fluorescence intensity of control of sample) / (fluorescence intensity of blankafter incubation for 24 hours – fluorescence intensity of control of blank)]} x 100.

The IC50 value was calculated from the logarithmic function
obtained by plotting the inhibitory rate of fluorescent AGEs
formation against the sample concentration.
GA-scavenging assay: The GA-scavenging activity ofphlorotannins was measured using a modification of thederivatization method of GA by Usui et al [29]. Twenty-five mM
GA was prepared using a 200 mM phosphate buffer (pH 7.4). The
crude phlorotannins extracted from each brown algae weredissolved in the phosphate buffer to a concentration of 5 mg/mL.
Purified phlorotannins, phloroglucinol, and aminoguanidinehydrochloride were dissolved in the phosphate buffer to aconcentration of 25 mM each. 1,3-Cyclohexanedione (0.25 g) was
dissolved in a mixture consisting of ammonium acetate (10 g),
acetic acid (5 mL), and ultrapure water (50 mL), and it was used asa derivatization reagent. Each sample solution (50 µL) and a GA
solution (50 µL) were mixed in well of a 96-well microplate(BioLite, Thermo Scientific) and incubated at 37°C for 30, 60, 90,
and 120 minutes, respectively. After incubation, the derivatization
reagent (100 µL) was added to the mixture and maintained at 60°C
for 30 minutes. The amount of GA remaining in the reaction
solution was measured at 370 nm using a microplate reader (Infinite
200, Tecan). For the blank test, the phosphate buffer was usedinstead of a GA solution. Aminoguanidine hydrochloride was used
as a positive control. The scavenging rate of GA was calculatedusing the following formula:
Scavenging rate of GA (%) = {1 – [concentration of GA remaining in the reaction
solution (mM) / 25]} x 100.

Cytotoxicity assay: MTT (Dojindo) was used as an indicator of cell
viability. Briefly, cell lines (HeLa or 3T3-L1) were cultured in 96-

1010 Natural Product Communications Vol. 13 (8) 2018

well microplates (BioLite, Thermo Scientific) at a density of 5 x 103
cells per well. After 24 hours cultivation in a CO2 incubator (CPE2601, Hirasawa) with 5% CO2 at 37°C, the cell lines were washedwith fresh medium (Dulbecco’s modified Eagle’s medium withglucose and pyruvate, containing 10% fetal bovine serum and 1%
antibiotic-antimycotic, Gibco) and then treated with each sample
solution (10 µL) for 24 hours in the incubator. Sample solutions
were prepared by dissolving in Dulbecco’s phosphate bufferedsaline (DPBS) without calcium and magnesium (Gibco). For blankand control wells, DPBS was used instead of a sample solution. The
cell lines were then rewashed with the medium, treated with 10 µL
of MTT solution, and cultured for 4 hours in the incubator at 37°C.
MTT solution was prepared by dissolving MTT (25 mg) in theSugiura et al.

DPBS (5 mL). After removing the medium containing the MTT
solution, 200 µL of the DPBS was added to each well and allowed
to maintain for 1 minute at 37°C. Finally, in order to dissolve theformed formazan salt, dimethyl sulfoxide (200 μL) was added toeach well from which DPBS had been removed. The absorbance ofeach well was measured at 535 nm using a microplate reader(Infinite 200, Tecan). The cell viability (%) was calculated using thefollowing formula:
Cell viability (%) = [(absorbance of sample well – absorbance of blank well) /(absorbance of control well – absorbance of blank well)] x 100.

Acknowledgments - This work was financially supported by theJapan Science and Technology Agency, CREST.

References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]

McPherson JD, Shilton BH, Walton DJ. (1988) Role of fructose in glycation and cross-linking of proteins. Biochemistry, 27, 1901-1907.
Singh R, Barden A, Mori T, Beilin L. (2001) Advanced glycation end-products: a review. Diabetologia, 44, 129-146.
Thornalley PJ, Langborg A, Minhas HS. (1999) Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose.
Biochemical Journal, 344, 109-116.
Takeuchi M, Makita Z, Bucala R, Suzuki T, Koike T, Kameda Y. (2000) Immunological evidence that non-carboxymethyllysine advanced
glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo. Molecular Medicine, 6, 114-125.
Takeuchi M, Makita Z. (2001) Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo. Current
Molecular Medicine, 1, 305-315.
Sato T, Iwaki M, Shimogaito N, Wu X, Yamagishi S, Takeuchi M. (2006) TAGE (toxic AGEs) theory in diabetic complications. Current
Molecular Medicine, 6, 351-358.
McLellan AC, Thornalley PJ, Benn J, Sonksen PH. (1994) Glyoxalase system in clinical diabetes mellitus and correlation with diabetic
complications. Clinical Science, 87, 21-29.
Odani H, Shinzato T, Matsumoto Y, Usami J, Maeda K. (1999) Increase in three α,β-dicarbonyl compound levels in human uremic plasma: specificin vivo determination of intermediates in advanced maillard reaction. Biochemical and Biophysical Research Communications, 256, 89-93.
Takeuchi M. (2012) Participation of toxic AGEs (TAGE) in a variety of diseases. Folia Pharmacologica Japonica, 139, 193-197.
Takeuchi M, Bucala R, Suzuki T, Ohkubo T, Yamazaki M, Koike T, Kameda Y, Makita Z. (2000) Neurotoxicity of advanced glycation endproducts for cultured cortical neurons. Journal of Neuropathology and Experimental Neurology, 59, 1094-1095.
Yonekura H, Yamamoto Y, Sakurai S, Petrova RG, Abedin MJ, Li H, Yasui K, Takeuchi M, Makita Z, Takasawa S, Okamoto H, Watanabe T,
Yamamoto H. (2003) Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells andpericytes, and their putative roles in diabetes-induced vascular injury. Biochemical Journal, 370, 1097-1109.
Iwamoto K, Kanno K, Hyogo H, Yamagishi S, Takeuchi M, Tazuma S, Chayama K. (2008) Advanced glycation end products enhance theproliferation and activation of hepatic stellate cells. Journal of Gastroenterology, 43, 298-304.
Takeuchi M, Takino J, Yamagishi S. (2010) Involvement of TAGE-RAGE system in the pathogenesis of diabetic retinopathy. Journal of
Opthalmology, 2010, 170393.
Takeuchi M, Takino J, Yamagishi S. (2010) Involvement of the toxic AGEs (TAGE)-RAGE system in the pathogenesis of diabetic vascularcomplications: a novel therapeutic strategy. Current Drug Targets, 11, 1468-1482.
Choei H, Sasaki N, Takeuchi M, Yoshida T, Ukai W, Yamagishi S, Kikuchi S, Saito T. (2004) Glyceraldehyde-derived advanced glycation end
products in Alzheimer’s disease. Acta Neuropathologica, 108, 189-193.
Koriyama Y, Furukawa A, Muramatsu M, Takino J, Takeuchi M. (2015) Glyceraldehyde caused Alzheimer’s disease-like alterations in diagnostic
marker levels in SH-SY5Y human neuroblastoma cells. Scientific Reports, 5, 13313.
Takino J, Kobayashi Y, Takeuchi, M. (2010) The formation of intracellular glyceraldehyde-derived advanced glycation end-products andcytotoxicity. Journal of Gastroenterology, 45, 646-655.
Sakasai-Sakai A, Takata T, Takino J, Takeuchi M. (2017) Impact of intracellular glyceraldehyde-derived advanced glycation end-products onhuman hepatocyte cell death. Scientific Reports, 7, 14282.
Takata T, Ueda T, Sakasai-Sakai A, Takeuchi M. (2017) Generation of glyceraldehyde-derived advanced glycation end-products in pancreaticcancer cells and the potential of tumor promotion. World Journal of Gastroenterology, 23, 4910-4919.
Sugiura S, Minami Y, Taniguchi R, Tanaka R, Miyake H, Mori T, Ueda M, Shibata T. (2017) Evaluation of anti-glycation activities ofphlorotannins in human and bovine serum albumin-methylglyoxal models. Natural Product Communications, 12, 1793-1796.
Ragan MA, Glombitza KW. (1986) Phlorotannins, brown algal polyphenols. Progress in Phycological Research, 4, 1-241.
Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A. (1986) Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking.
Science, 232, 1629-1632.
Shibata T, Nagayama K, Sugiura S, Makino S, Ueda M, Tamaru Y. (2015) Analysis on composition and antioxidative properties of phlorotannins
isolated from Japanese Eisenia and Ecklonia species. American Journal of Plant Sciences, 6, 2510-2521.
Takeuchi M, Yamagishi S. (2004) Alternative routes for the formation of glyceraldehyde-derived AGEs (TAGE) in vivo. Medical Hypotheses, 63,
453-455.
Takeuchi M, Makita Z, Yanagisawa K, Kameda Y, Koike T. (1999) Detection of noncarboxymethyllysine and carboxymethyllysine advanced
glycation end products (AGE) in serum of diabetic patients. Molecular Medicine, 5, 393-405.
Takeuchi M, Yanase Y, Matsuura N, Yamagishi S, Kameda Y, Bucala R, Makita Z. (2001) Immunological detection of a novel advanced glycation
end-product. Molecular Medicine, 7, 783-791.
Shibata T, Ishimaru K, Kawaguchi S, Yoshikawa H, Hama Y. (2008) Antioxidant activities of phlorotannins isolated from Japanese Laminariaceae.
Journal of Applied Phycology, 20, 705-711.
Fujii Y, Tanaka R, Miyake H, Tamaru Y, Ueda M, Shibata T. (2013) Evaluation for antioxidative properties of phlorotannins isolated from thebrown alga Eisenia bicyclis, by the H-ORAC method. Food and Nutrition Sciences, 4, 8A, 78-82.
Usui T, Yoshino M, Watanabe H, Hayase F. (2007) Determination of glyceraldehyde formed in glucose degradation and glycation. Bioscience,
Biotechnology, and Biochemistry, 71, 2162-2168.

Natural Product Communications Vol. 13 (8) 2018
Published online (www.naturalproduct.us)

Anti-Melanogenic Effect of Chestnut Spike Extract through Downregulation of Tyrosinase-Related Proteins and Activationof ERK 1/2
Jung-Hee Byeon, Md Badrul Alam, Ki-Chan Kim, Sangsun Heo, Ji-young Lim, Yoon-Gyung Kwon, Peijun Zhao, Yeong-Ho Cha,
Hee-Jeong Choi and Sang-Han Lee
Analysis of the Volatile Components of Pouteria sapota (Sapote Mamey) Fruit by HS-SPME-GC-MS
Candelario Rodríguez, Armando A. Durant-Archibold, Ana Santana, Enrique Murillo and Carlos M. Franco Abuín
An Analysis of Volatile Components of the Liverworts Dumortiera hirsuta subsp. hirsuta and Dumortiera hirsuta
subsp. nepalensis (Dumortieraceae) from Panama and Taxonomic Observations on the Species
Armando A. Durant-Archibold, Noris Salazar Allen, Anette Garrido, Jose Gudiño Ledezma and Mahabir P. Gupta
Terpenes and n-Alkanes in Needles of Pinus cembra
Biljana Nikolić, Marina Todosijević, Mihajlo Ratknić, Iris Đorđević, Jovana Stanković, Mirjana Cvetković, PetarD. Marin andVele TeševićMorphologic and Essential oil Profiles of Three Species from Asteraceae
Melda Dolarslan and Tugba Gurkok
Composition and Chemical Variability of Needle and Berry Oils from Corsican Juniperus communis var. communis
Joséphine Ottavioli, Ange Bighelli, Joseph Casanova and Félix Tomi
Antifungal and Insecticidal Properties of Juniperus thurifera Leaves
Meryem El Jemli, Naima Khattabi, Khadija Lachqer, Driss Touati, Yousra El Jemli, Ilias Marmouzi, El Mahdi Wakrim,
Yahia Cherrah and Katim Alaoui
Antimicrobial Activity of two Mentha Species Essential Oil and its Dependence on Different Origin and Chemical Diversity
Mária Pľuchtová, Teresa Gervasi, Qada Benameur, Vito Pellizzeri, Daniela Gruľová, Luca Campone, Vincent Sedlák and Nicola Cicero
Seasonal Study of Methyleugenol Chemotype of Ocimum campechianum Essential Oil and Its Fungicidal and Antioxidant
Activities
Pablo Luis B. Figueiredo, Sebastião G. Silva, Lidiane D. Nascimento, Alessandra R. Ramos, William N. Setzer, Joyce Kelly R. da Silva
and Eloisa Helena A. Andrade
Evaluation of Antipneumonic Effect of Philippine Essential Oils Using Broth Microdilution Volatilization Method andTheir Lung Fibroblasts Toxicity
Marketa Houdkova, Ivo Doskocil, Klara Urbanova, Ea Kristine Clarisse B. Tulin, Johana Rondevaldova, Anabella B. Tulin,
Tomas Kudera, Edgardo E. Tulin, Vaclav Zeleny and Ladislav Kokoska

1023
1027

1031

1035
1039
1043

1047
1051

1055

1059

Accounts/Reviews
The Roles of Natural Compounds in Epigenetics
Yanhong Yang, Zuohua Chi, Ruiping Gao and Zili Lei
Secondary Metabolites, Dietary Fiber and Conjugated Fatty Acids as Functional Food Ingredients Against Overweightand Obesity
Kamila Kasprzak, Karolina Wojtunik-Kulesza, Tomasz Oniszczuk, Maciej Kuboń and Anna Oniszczuk
The Sulfated Polysaccharides of Brown Algae and Products of Their Enzymatic Transformation as Potential Vaccine Adjuvants
Tatyana A. Kuznetsova, Elena V. Persiyanova, Svetlana P. Ermakova, Maxim Yu. Khotimchenko and Natalya N. Besednova

1067

1073
1083

Natural Product Communications
2018
Volume 13, Number 8
Contents
Original Paper

Page

Synthesis and Cytotoxic Evaluation of Artemisinin Derivatives Containing an Aminopropanol GroupLe Nhat Thuy Giang, Doan Duy Tien, Dang Thi Tuyet Anh, Nguyen Tien Dung, Ngo Hanh Thuong, Luc Quang Tan,
Nguyen Ha Thanh, Le Thi Tu Anh, Nguyen Van Tuyen and Phan Van Kiem
Biotransformation of Bicyclic Sesqui- and Diterpene 1,2-dials and Their Derivatives by the Fungus, Aspergillus niger
Yoshinori Asakawa, Masako Sekita and Toshihiro Hashimoto
Synthesis of Ester-linked Taxol-oligosaccharide Conjugate and Its Drug Delivery System Using Bio-nanocapsules andHybrid-bio-nanocapsules
Hiroki Hamada, Shouta Okada, Noriyoshi Masuoka, Yuya Fujitaka, Kei Shimoda, Shouta Doi and Katsuhiko Mikuni
Monoaminergic Involvement in Decreased Locomotor Activity of Mice Treated with α and β-amyrin from Protium heptaphyllum
Gislei F. Aragão, Manoel O. de Moraes Filho, Paulo N. Bandeira, Antônio P. Frota Junior, Yasmin Ingrid S. de Oliveira,Claudina F. Alves Balacó and Maria Elisabete A. de Moraes
Cytotoxic Evaluation of Compounds Isolated from the Aerial Parts of Hedyotis pilulifera and Methanol Extract of Inonotus obliquus
Hoai Thi Nguyen, Duc Viet Ho, Phu Dinh Quynh Nguyen, Hung Quoc Vo, Thao Thi Do and Ain Raal
Production of the Anticancer Compound Withaferin A from Genetically Transformed Hairy Root Cultures of Withania Somnifera
Zeynab Yousefian, Behnaz Hosseini, Hassan Rezadoost, Javier Palazón and Mohammad Hossein Mirjalili
New Oxygenated Steroid from the Marine-Derived Fungus Aspergillus flavus
Meng-Yue Yang, Jian-Kun Yang, Jin-Kai Yang, Lian-Dong Hu, Hua-Jie Zhu and Fei Cao
Sulfated Glycosides from the Sea Cucumbers Block Ca2+ Flow in Murine Neuroblastoma Cells
Evgeny A. Pislyagin, Ekaterina S. Menchinskaya, Dmitry L. Aminin, Sergey A. Avilov and Alexandra S. Silchenko
New Sesquiterpene Pyridine Alkaloids from Hippocratea excelsa
Megumi Furukawa, Masakatsu Furukawa, Mitsuko Makino, Taketo Uchiyama, Yasuo Fujimoto and Keiichi Matsuzaki
Flavonoids from Milletia leucantha and Their Cytotoxicity
Uraiwan Sriphana, Chavi Yenjai, Siriporn Tungnoi, Jongjai Srirapa and Auemporn Junsongduang
Inhibitory Effect of Pelargonidin on Secretory Group IIA Phospholipase A2
In-Chul Lee and Jong-Sup Bae
Skin Anti-aging Assays of Proanthocyanidin Rich Red Rice Extract, Oryzanol and Other Phenolic Compounds
Supachai Yodkeeree, Pilaiporn Thippraphan, Wanisa Punfa, Jatupol Srisomboon and Pornngarm Limtrakul (Dejkriengkraikul)
Identification of Plant Origin of Propolis from Thailand Stingless Bees by Comparative Analysis
Eriko Ishizu, Sari Honda, Boonyadist Vongsak and Shigenori Kumazawa
Leaves of Eugenia brasiliensis Used as a Folk Medicine Contain Cyclooxygenase Enzyme and Lipid Peroxidation Inhibitory
Compounds
Alessandra C. Dametto, Nivaldo Boralle, Chuan-Rui Zhang, Dulce H. S. Silva and Muraleedharan G. Nair
Difficulties to Determine the Absolute Configuration of Guaiaretic Acid
Alfredo R. Ortega, Eleuterio Burgueño-Tapia and Pedro Joseph-Nathan
Comparison of Chemical Constituents in Magnoliae Officinalis Cortex Processed by “Sweating” and “Non Sweating”
based on Ultra Fast Liquid Chromatography-Triple Quadrupole-Time of Flight Mass Spectrometry and Gas
Chromatography-Triple Quadrupole Mass Spectrometry Combined with Multivariate Statistical Analysis
Hui Zhao, Ying Yan, Cheng-cheng Wang, Li-si Zou, Xun-hong Liu, Shu-yu Chen and Jing-jing Shi
Potent α-Glucosidase Inhibitors from the Roots of Aruncus sylvester
Zhang-Peng Li, Meng Que, Wen-Yuan Gao and Yan-Fang Su
Cytotoxic Compounds from the Seeds of Sophora alopecuroides
Ping Song, Hao Chen, Yanzhang Wen, Yibing Lv, Shihao Deng and Xinzhou Yang
Antibacterial and Antibiofilm Effects of Zanthoxylum bungeanum Leaves against Staphylococcus aureus
Shi-Yuan Chang, Kai Xiao, Jia-Qi Zhang, Kai Zhong, Elena Grosu, Zhen Gao, Yan-Ping Wu and Hong Gao
Evaluation of Anti-glycation Activities of Phlorotannins in Human and Bovine Serum Albumin-glyceraldehyde Models
Shingo Sugiura, Ryosuke Taniguchi, Yoshihiko Nishioka, Ryota Iwase, Reiji Tanaka, Hideo Miyake, Tetsushi Mori,
Mitsuyoshi Ueda and Toshiyuki Shibata
Stereoselective Total Synthesis of 1,4-Dideoxy-1,4-imino-L-ribitol by an Intramolecular Ring Opening of Epoxide with aTethered Amide
Dhudmal Chaya N, Dhanraj O Biradar, Maddipatla V. Satyanarayana and Basi V Subba Reddy
Impact of Melittin on Microalgae Cell Wall: A Monolayer Study
Magda Vargas-Perez, Gerardo Sierra-García, Hugo Luna Olvera, Abelardo Chavez-Montes and Azucena Gonzalez-Horta
Phytochemical Profile and Anti-lipase Activity of Balkan Endemic Jurinea tzar-ferdinandii
Antoaneta Trendafilova, Milka Todorova, Nikolina Kutova and Maya Guncheva
The Chaenomeles sinensis Extract has the Potential to Exhibit Antioxidant Activity or Attenuate Liver Damage
Young-Ji Choi, Young-Moo Choo, Seung-Il Jeong, Kang-Yeol Yu and Jiyoung Kim
Continued inside backcover

919
923

933

935
939
943
949
953
957
961
963
967
973

977
981

987
993
997
1001

1007

1011
1013
1017
1021