Recovery effect of phenylpropanoid glycosides from Magnolia obovata fruit on alloxan-induced pancreatic islet damage in zebrafish (Danio rerio)

Article abstract of DOI:10.1016/j.carres.2015.07.010

Investigation of phytochemicals from Magnolia obovata fruit led to the isolation of three novel phenylpropanoid glycosides: obovatoside A-C (1-3) and two known phenylpropanoids, syringin (4) and pavonisol (5). The structures of 1-5 were determined by NMR,

Full text of DOI:10.1016/j.carres.2015.07.010

Accepted Manuscript
Title: Recovery effect of phenylpropanoid glycosides from Magnolia obovata
fruit on alloxan-induced pancreatic islet damage in zebrafish (Danio rerio)
Author: Kyeong-Hwa Seo, Youn-Hee Nam, Dae-Young Lee, Eun-Mi Ahn,
Tong-Ho Kang, Nam-In Baek
PII:
S0008-6215(15)00207-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carres.2015.07.010
CAR 7036
To appear in:
Carbohydrate Research
Received date: 8-6-2015
Revised date: 13-7-2015
Accepted date: 16-7-2015
Please cite this article as: Kyeong-Hwa Seo, Youn-Hee Nam, Dae-Young Lee, Eun-Mi Ahn,
Tong-Ho Kang, Nam-In Baek, Recovery effect of phenylpropanoid glycosides from Magnolia
obovata fruit on alloxan-induced pancreatic islet damage in zebrafish (Danio rerio),
Carbohydrate Research (2015), http://dx.doi.org/doi:10.1016/j.carres.2015.07.010.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service
to our customers we are providing this early version of the manuscript. The manuscript will
undergo copyediting, typesetting, and review of the resulting proof before it is published in its
final form. Please note that during the production process errors may be discovered which could
affect the content, and all legal disclaimers that apply to the journal pertain.

Carbohydrate Research – Full Papers
Recovery effect of phenylpropanoid glycosides from Magnolia
obovata fruit on alloxan-induced pancreatic islet damage in
zebrafish (Danio rerio)
Kyeong-Hwa Seoa, Youn-Hee Nama, Dae-Young Leeb, Eun-Mi Ahnc, Tong-Ho
Kanga, Nam-In Baeka,*
aGraduate School of Biotechnology and Department of Oriental Medicine
Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea
bDepartment of Herbal Crop Research, National Institute of Horticultural and
Herbal Science, RDA, Eumseong 369-873, Republic of Korea
cDepartment of Herbal Foodceutical Science, Daegu Haany University,
Gyeongsan 712-715, Republic of Korea
Email: nibaek@khu.ac.kr
Tel.: +82-31-201-2661
Fax: +82-31-204-8116
1
Page 1 of 31

Highlights
 Three new phenylpropanoid glycosides were isolated from Magnolia
obovata fruit.
 Recovery effect on alloxan-induced islet damage in zebrafish were
evaluated.
 All compounds increased the size of injured pancreatic islets 0.60-1.14
fold.
 The isolated phenylpropanoids increased the glucose absorption ability in
zebrafish.
Graphical abstract
2
Page 2 of 31

ABSTRACT
Investigation of phytochemicals from Magnolia obovata fruit led to the
isolation of three novel phenylpropanoid glycosides: obovatoside A-C (1-3) and
two known phenylpropanoids, syringin (4) and pavonisol (5). The structures of 1-
5 were determined by NMR, HRMS, IR and CD spectroscopic analyses. All
compounds were evaluated for their effects on recovery from alloxan-induced
pancreatic islet damage in zebrafish. All compounds increased the size of the
injured pancreatic islet from 0.60 to 1.14-fold. Compounds 1 and 3-5 significantly
increased glucose absorption in zebrafish.
Keywords: alloxan; anti-diabetic; Magnolia obovata; obovatoside; pancreatic
islet; zebrafish
3
Page 3 of 31

1. Introduction
The incidence of diabetes is growing rapidly worldwide. The Republic of
Korea has the fifth highest diabetes death rate of the OECD countries, behind
Mexico, Turkey, Israel and Portugal.1 Diabetes is classified into type 1 (insulin-
dependent) and type 2 (non insulin-dependent). Type 1 is characterized by an
absolute deficiency of insulin caused by massive β-cell necrosis of the pancreatic
islets of Langerhans of the pancreas. The destruction of these cells leads to failure
of the pancreas to respond to glucose and classic symptoms of insulin deficiency.
Most people with diabetics have type 2, which is characterized by hyperglycemia
in the context of insulin resistance and a relative lack of insulin.2 Type 2 diabetes
can result in diabetic retinopathy, nephropathy, mastophathy, cardiovascular and
microvascular complications.3,4 Diabetes is not curable, but proper control of
blood glucose is possible.5 Diabetes can be regulated with insulin preparations or
hypoglycemic drugs, which can prevent morbidity and reduce diabetic mortality.
Various medicinal plants have also been reported as potential sources of anti-
diabetic treatments.6-8 These plants contain secondary metabolites such as lignans,
flavonoids, alkaloids, and terpenoids, that can be assessed for the ability to
alleviate diabetes symptoms. Our preliminary experiments showed that a
4
Page 4 of 31

methanol extract and solvent fractions of Magnolia obovata fruit significantly
increased the size of alloxan-induced pancreatic islets in the zebrafish (Danio
rerio). This study was performed to identify the anti-diabetic constituents of M.
obovata fruit.
M. obovata (Magnoliacea) is a deciduous tree that is found throughout Korea,
China, and Japan. It has been used in Chinese medicine to treat fever, headache,
and diarrhea and to relieve asthma.9 The fruit of this plant is called Hoo-Bahk-Ja
in Korean traditional medicine. Previous phytochemical research isolated several
phenylethanoids and phenylpropanoids from the Magnolia species.10-12 However,
few studies have been carried out on their phytochemical and pharmacological
potential, including investigations on anti-diabetics from M. obovata fruits.
The zebrafish (Danio rerio) is a small, shoaling freshwater cyprinid fish. It is
a model organism that is widely used in biomedical research because of its small
size, easy maintenance in laboratories, numerous offspring, transparent embryos,
and amenability to genetic and chemical screens.13-14 Zebrafish have been treated
with alloxan to damage the pancreatic islets and observe variation in islet size and
glucose absorption. Flavonoids from Morus alba fruit were used to treat zebrafish
5
Page 5 of 31

to examine recovery from alloxan-induced damage of pancreatic islets in
zebrafish.15 Therefore, this study isolated and identified phenylpropanoid
glycosides in the fruit of M. obovata, and examined the anti-diabetic potential of
the isolated compounds. This paper describes a procedure for isolating and
determining the structure of three new phenylpropanoid glycosides and two
known ones, from the fruit of M. obovata and for evaluating the effect on
recovery from alloxan-induced pancreatic islet damages in zebrafish.
6
Page 6 of 31

2. Results and discussion
2.1. Structural characterization of the compounds.
Methanol extracts of M. obovata fruit were fractionated using EtOAc, n-BuOH,
and water. From the EtOAc and n-BuOH fractions, three new phenylpropanoid
glycosides (1-3) along with two known ones (4-5) were isolated through repeated
SiO2, ODS, and Sephadex LH-20 column chromatography. The two known
compounds were identified to be syringin (4) and pavonisol (5) on the basis of
spectroscopic analysis data such as NMR, IR, MS, and CD by comparison of the
data with those reported in the literatures (Figure 1). 16,17
Compound 1 was isolated as orange needles. The IR spectrum (CaF2, ν)
showed absorption bands for a hydroxyl group (3357 cm-1), CH group (2918 cm-
1), and olefin group (1610 cm-1). The molecular formula was determined to be
C15H22O8 from the high-resolution molecular ion peak [M]+ m/z 330.1328 (calcd.
1
for C15H22O8, 330.1315) in the HREIMS. The H NMR spectrum (400 MHz,
CD3OD, δ) showed four olefin methine proton signals at δ 7.15 (2H, d, J = 8.8 Hz,
H-2,6), 7.01 (2H, d, J = 8.8 Hz, H-3,5) from a para-disubstituted benzene ring,
one oxygenated methine proton signal at δ 3.83 (1H, dddd, J = 7.2, 6.0, 5.6, 4.4
7
Page 7 of 31

Hz, H-8), one germinal coupled oxygenated methylene proton signal at δ 3.50
(1H, dd, J = 11.2, 4.4 Hz, H-9a) and 3.43 (1H, dd, J = 11.2, 6.0 Hz, H-9b) and
one germinal coupled methylene proton signal δ 2.78 (1H, dd, J = 14.0, 5.6 Hz,
H-7a) and 2.65 (1H, dd, J = 14.0, 7.2 Hz, H-7b), which were assigned to the
signals of a phenylpropanoid moiety with three hydroxyl groups. In addition, a
anomer proton signal at δ 5.22 (1H, d, J = 8.0 Hz, H-1'), four oxygenated methine
proton signals at δ 4.14 (1H, dd, J = 2.8, 2.8 Hz, H-4'), 3.77 (1H, m, H-5'), 3.61
(1H, dd, J = 8.8, 8.0 Hz, H-2'), 3.58 (1H, dd, J = 8.8, 2.8 Hz, H-3') and one
oxygenated methylene proton signal at δ 3.87 (1H, dd, J = 12.4, 2.4 Hz, H-6'a),
3.69 (1H, dd, J = 12.4, 5.6 Hz, H-6'b) indicated the presence of an aldohexose
moiety. Taken together, these data suggested that compound 1 was a
phenylpropanoid monoglycoside. The 13C-NMR spectrum showed 15 carbon
signals from phenylpropanoid and hexose moieties. The multiplicity of each
carbon was determined using distortionless enhancement by polarization transfer
(DEPT). In the downfield region, one sp2 oxygenated olefin quaternary carbon
signal at δ 157.7 (C-4), one sp2 olefin quaternary carbon signal at δ 133.8 (C-1)
and four sp2 olefin methine carbon signals at δ 131.2 (C-2, 6) and 117.6 (C-3, 5)
from a para-disubstituted benzene ring with one hydroxyl group were observed.
8
Page 8 of 31

In the upfield region, one oxygenated methine carbon signal at δ 74.5 (C-8), one
oxygenated methylene carbon signal at δ 66.4 (C-9), and one methylene carbon
signal at δ 40.1 (H-7) were observed. The sugar was identified as a β-
galactopyranose based on 13C-NMR chemical shifts such as a anomer carbon
signal at δ 100.2 (C-1'), four oxygenated methine carbon signals at δ 75.6 (C-5'),
72.9 (C-3'), 72.0 (C-2'), 68.6 (C-4') and one oxygenated methylene carbon signal
at δ 62.8 (C-6') and the coupling constant (J=8.0 Hz) of the anomer proton signal.
To determine the position of the key functional groups, a heteronuclear multiple
bonding connectivity (gHMBC) experiment was performed. In the gHMBC
spectrum, the cross peak between the anomer proton signal of the
galactopyranosyl moiety (δH 5.22, H-1') and the oxygenated olefin quaternary
carbon signal (δC 157.7, C-4) indicated that the galactopyranose was linked to the
hydroxyl of C-4. The structure of 1 was determined to be a new compound, 4-
(2,3-dihydroxypropyl)phenyl-1-O-β-D-galactopyranoside.
The
absolute
configuration of C-8 was confirmed as S by compared the specific optical rotation
value of the aglycone (1a) ([α]D -8.3°), obtained through acid hydrolysis, with the
literature ([α]D -4.0°).18 As a result, the chemical structure of 1 was determined to
be 4-[(2S)-2,3-dihydroxypropyl]phenyl β-D-galactopyranoside and the compound
9
Page 9 of 31

was, named obovatoside A (1).
Compound 2 was isolated as a pale yellow viscous oil. The IR spectrum (CaF2,
ν) showed absorption bands for the hydroxyl group (3352 cm-1), CH group (2923
cm-1), and olefin group (1601 cm-1). The high-resolution pseudomolecular ion
peak [M+Na]+ m/z 369.1251 (calcd. for C15H22O9Na, 369.1264) in the
HRFABMS, which was 16 amu higher than 1, indicated that 2 had one more
hydroxyl group than 1. The 1D and 2D NMR assignments of 2 were based on 1H
13
and C NMR, DEPT, gHSQC and gHMBC and comparison with results for 1.
The NMR data of 2 were similar to 1 with exception of the aglycone moiety. The
NMR spectrum (400 MHz, CD3OD, δ) showed three olefin methine proton
signals at δ 7.09 (1H, d, J = 2.0 Hz, H-2), 6.79 (1H, dd, J = 8.0, 2.0 Hz, H-6), and
6.75 (1H, d, J = 8.0 Hz, H-5) and two oxygenated olefin quaternary carbon
signals at δC 146.6 and 146.7 from a 1,2,4-trisubstituted benzene ring with two
hydroxyl groups. The position of the galactopyranosyl group was determined be
an glycosidation shift in the 13C-NMR spectrum and gHMBC results. Shifts of the
two oxygenated olefin quaternary carbon signals of C-3 and C-4 (δC 146.6 and
146.7) were 1.5 ppm downfield and 1.7 ppm upfield, respectively; the shift of the
olefin quaternary carbon signal of C-1 (δC 133.2) 5.6 ppm upfield compared to
10
Page 10 of 31

demethyleugenol β-D-glucopyranoside.19 Confirmation came from the cross peak
between the anomer proton signal (δH 5.12, H-1') and the oxygenated olefin
quaternary carbon signal (δC 146.6, C-3) in the gHMBC spectrum. The structure
of 2 was determined to be a new compound, 4-(2,3-dihydroxypropyl)-2-
hydroxyphenyl-2-O-β-D-galactopyranoside. The absolute configuration of C-8
was confirmed as S by compared the optical rotation value of the aglycone (2a)
([α]D -13.6°), obtained through acid hydrolysis, with the literature ([α]D -4.0°).18
The chemical structure of 2 was determined to be 4-[(2S)-2,3-dihydroxypropyl]-
2-hydroxyphenyl β-D-galactopyranoside and the compound was, named
obovatoside B (2).
Compound 3 was isolated as a pale brown viscous oil. The IR spectrum (CaF2,
ν) showed absorption bands for the hydroxyl group (3383 cm-1), CH group (2919
cm-1), and olefin group (1586 cm-1). The molecular formula was determined to be
C16H22O7 from the high-resolution molecular ion peak [M]+ m/z 326.1299 (calcd.
1
for C16H22O7, 326.1365) in the HREIMS. The H NMR spectrum (600 MHz,
CD3OD, δ) showed the proton signals of a 1,2,4-trisubstituted benzene ring (δH
7.07, 1H, d, J = 8.4 Hz, H-5; 6.97, 1H, d, J = 1.8 Hz, H-2; 6.86, 1H, dd, J = 8.4,
1.6 Hz, H-6) and a trans-double bond (δH 6.34, 1H, d, J = 15.6 Hz, H-7; 6.20, 1H,
11
Page 11 of 31

dq, J = 15.6, 6.6 Hz, H-8). In the upfield region, one methoxy proton signal (δH
3.85, 3H, s) and one allyl methyl proton signal (δH 1.84, 3H, d, J = 6.6 Hz, H-9)
were assigned to a phenylpropenyl moiety with one hydroxyl and one methoxy
group. In addition, a anomer proton signal at δ 5.21 (1H, d, J = 8.4 Hz, H-1'), four
oxygenated methine proton signals at δ 4.14 (1H, dd, J = 3.0, 3.0 Hz, H-4'), 3.82
(1H, m, H-5'), 3.61 (1H, dd, J = 9.6, 8.4 Hz, H-2'), 3.59 (1H, dd, J = 9.6, 3.0 Hz,
H-3') and one oxygenated methylene proton signal at δ 3.68 (1H, dd, J = 12.0, 6.0
Hz, H-6a'), 3.61 (1H, dd, J = 12.0, 3.6 Hz, H-6b') indicated the presence of a
aldohexose moiety. These data suggested that compound 3 was a phenylpropenyl
monoglycoside. The 13C-NMR spectrum showed 16 carbon signals for
phenylpropenyl, hexose and methoxy (δC 56.8) moieties. In the downfield region,
two oxygenated olefin quaternary carbon signals at δC 151.0 (C-3) and δC 147.4
(C-4) one olefin quaternary carbon signal at δc 134.7 (C-1) and five olefin
methine carbon signals at δc 131.9 (C-7), δc 125.3 (C-8), δc 120.1 (C-6), δc 118.0
(C-5), and δc 111.1 (C-2) were observed. In the upfield region, a methoxy carbon
signal and one allyl methyl carbon signal at δC 18.6 (C-9) were observed. The
sugar was identified as a β-galactopyranose from chemical shifts such as a
anomer carbon signal at δ 100.9 (C-1'), four oxygenated methine carbon signals at
12
Page 12 of 31

δ 76.0 (C-5'), 73.0 (C-3'), 72.3 (C-2'), 68.8 (C-4') and one oxygenated methylene
carbon signal at δ 63.0 (C-6') as well the coupling constant (J=8.4 Hz) of the
anomer proton signal. In the gHMBC spectrum, the cross peak between the
anomer proton signal of the galactopyranosyl moiety (δH 5.21, H-1') and the
oxygenated olefin quaternary carbon signal (δC 147.4, C-4), and between the
methoxy proton signal (δH 3.85, -OCH3) and the oxygenated olefin quaternary
carbon signal (δC 151.0, C-3) indicated that galactopyranose and the methoxy
were linked to the hydroxyls of C-4 and C-3, respectively. Compound 3 was
determined to be a new compound, 4-(1E-prop-1-enyl)-2-methoxyphenyl β-D-
galactopyranoside and named obovatoside C (3).
The isolated compounds 1-5 were evaluated for their effect on recovery from
alloxan-induced pancreatic islet damage in zebrafish. The zebrafish is a good
model for studying pancreatic change in vivo, because the zebrafish embryo has a
single pancreatic islet during the first stage of development, which allows rapid
analysis of organ changes. Alloxan damage the pancreatic islet and diabetogenic,
decreasing β-cell mass in pancreatic islet.16 Alloxan treatment significantly
decreased pancreatic islet size by 7.88-fold (p<0.01) compared to the normal
group. Treatment with compound 1 led to a significant increase in the size of the
13
Page 13 of 31

injured pancreatic islet by 0.90-fold (p<0.001) compare to the alloxan-induced
group. Compounds 2-5 significantly increased the size of the pancreatic islet by
0.60-fold (p<0.001), 1.14-fold (p<0.001), 0.60-fold (p<0.01), and 0.74-fold
(p<0.001), respectively, compared to the alloxan treated group (Figure 2 A, C).
The ability to absorb glucose was assessed using 2-[N-(7-nitrobenz-2-oxa-1,3-
diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), which is a recently introduced
fluorescent derived from glucose modified with a 2-[N-(7-nitrobenz-2-oxa-1,3-
diazol-4-yl) amino] group at the C-2 position.20 It is used in diabetes studies to
measure cellular ability to absorb glucose. Fluorescence intensity of the
pancreatic islet was analyzed by histograms using Image J software, which
indicates pixel intensity (green) from 0 to 255. Fluorescence intensity of the
alloxan-treated group (pixel mean = 33.70) significantly decreased compared to
the normal group (pixel mean = 114.38), (p<0.001). Although the group treated
with compound 2 (pixel mean = 38.48) showed no increase in the fluorescence
intensity of the pancreatic islet compared to group treated with alloxan (Figure 2
B, C), groups treated with compounds 1 (pixel mean = 81.28, p<0.05), 3 (pixel
mean = 84.42, p<0.01), 4 (pixel mean = 79.71, p<0.01) and 5 (pixel mean = 78.09,
p<0.05) significantly increased fluorescence intensity compared to the alloxan-
14
Page 14 of 31

induced group.
15
Page 15 of 31

3. Conclusion
Three new and two known phenylpropanoids were isolated from M. obovata
fruits. All compounds increased the size of the injured pancreatic islet, and
compounds 1 and 3-5 significantly increased glucose absorption in zebrafish.
Therefore, these results demonstrate the potential for the biomedical use of these
compounds and plant extracts as anti-diabetic agents.
16
Page 16 of 31

4. Experimental
4.1. General methods
The resins used for column chromatography (c.c.) were SiO2 (Kiesel gel 60,
Merck, Darmstadt, Germany), octadecyl silica gel (ODS) (LiChroprep RP-18, 40-
60 μm, Merck), and Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala,
Sweden). Thin layer chromatography (TLC) analysis was carried out using a
Kiesel gel 60F254 and a RP-18 F254s plates (Merck), and the spots on the TLC
plates were detected using a UV lamp Spectroline Model ENF-240 C/F
(Spectronics Corporation, Westbury, NY, USA) and spraying a 10% H2SO4
1
solution followed by heating. H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were recorded on a Varian Unity Inova AS-400 FT-NMR spectrometer
1
(Palo Alto, CA, USA). H NMR (600 MHz) and 13C NMR (125 MHz) spectra
were recorded on a Bruker Avance 600 MHz spectrometer (Bruker, Billerica, MA,
USA). Melting points were determined using a Fisher-John’s Melting Point
apparatus (Miami, FL, USA) with a microscope and the values obtained were
uncorrected. Optical rotation was measured using a JASCO P-1010 digital
polarimeter (Jasco, Tokyo, Japan). IR spectra were obtained using a Perkin Elmer
17
Page 17 of 31

Spectrum One FT-IR spectrometer (Buckinghamshire, England). HRFAB-MS
and HREI-MS were conducted on a JEOL JMSAX-700 mass spectrometer
(Tokyo, Japan). Alloxan monohydrate and sea salts were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)amino)-2-deoxyglucose (2-NBDG) was obtained from Invitrogen (Eugene, OR,
USA). Fluorescence microscopy was used by an Olympus 1X70 microscope
(Olympus, Tokyo, Japan). To gain the image analysis of pancreatic islet in
zebrafish, Focus Lite (Focus Co, Daejeon, Korea) and Image J (National
Institutes of Health, Bethesda, MD, USA) were used.
4.2. Plant materials
The fruits of Magnolia obovata were collected at Kyung Hee University,
Yongin, Republic of Korea in September 2010 and identified by Prof. Seung-Woo
Lee, Department of Horticultural Biotechnology, Kyung Hee University. A
voucher specimen (KHU-NPCL-201009) has been deposited at the Laboratory of
Natural Products Chemistry, Kyung Hee University.
18
Page 18 of 31

4.3. Extraction and isolation
The dried fruits (11 kg) were chopped and extracted with 80% MeOH (40 L ×
4) at room temperature for 24 hr, filtered and concentrated in vacuo. The MeOH
extracts (740 g) were successively partitioned with water (3.5 L), EtOAc (3.5 L ×
4), and n-BuOH (3 L × 3). Each solvent layer was concentrated to yield EtOAc
(MOE, 238 g), n-BuOH (MOB, 97 g) and water (405 g) fractions, respectively.
The n-BuOH fraction (MOB, 97 g) was applied to a SiO2 c.c. (Φ 8.0 × 12 cm)
and eluted with CHCl3-MeOH-water (12:3:1 → 10:3:1 → 7:3:1 → 6:4:1, 3 L of
each) with monitoring by TLC to provide 21 fractions (MOB-1 to MOB-21).
MOB-5 [1.01 g, elution volume/total volume (Ve/Vt) 0.014-0.025] was subjected
to the SiO2 c.c. (Φ 4.5 × 15 cm) and eluted with CHCl3-MeOH-water (20:3:1, 3.5
L), yielding 15 fractions (MOB-5-1 to MOB-5-15). Fraction MOB-5-6 (21 mg,
Ve/Vt 0.180-0.020) was subjected to the ODS c.c. (Φ 1 × 8 cm) and eluted with
EtOH-water (1:2, 45 mL), yielding nine fractions (MOB-5-6-1 to MOB-5-6-9)
and along with a purified compound 3 [MOB-5-6-5, 2.5 mg, Ve/Vt 0.410-0.500,
TLC (RP-18 F254s) Rf 0.50, MeOH-water (2:1)]. Fraction MOB-10 (5.16 g, Ve/Vt
0.151-0.216) was subjected to the SiO2 c.c. (Φ 6.5 × 14 cm) and eluted with
CHCl3-MeOH-water (16:3:1 → 14:3:1 → 12:3:1 → 10:3:1 → 8:3:1 → 6:4:1, 3.8
19
Page 19 of 31

L of each) to obtain 27 fractions (MOB-10-1 to MOB-10-27) and a pure
compound 4 [MOB-10-14, 55 mg, Ve/Vt 0.147-0.155, TLC (Kiesel gel 60 F254)
Rf 0.47, CHCl3-MeOH-water (65:35:10)]. Fraction MOB-10-22 (659 mg, Ve/Vt
0.612-0.742) was subjected to an ODS c.c. (Φ 4.5 × 5 cm) and eluted with
MeOH-water (1:3, 1.1 L) to obtain 14 fractions (MOB-10-22-1 to MOB-10-22-
14) and a pure compound 1 [MOB-10-22-2, 188 mg, Ve/Vt 0.038-0.058, TLC
(Kiesel gel 60 F254) Rf 0.51, CHCl3-MeOH-water (6:4:1)]. Fraction MOB-15
(7.38 g, Ve/Vt 0.593-0.633) was subjected to the ODS c.c. (Φ 6 × 8 cm) and
eluted with MeOH-water (1:2 → 1:1, 3.4 L of each) to obtain 18 fractions (MOB-
15-1 to MOB-15-18). Fraction 15-22 (556 mg, Ve/Vt 0.042-0.051) was subjected
to a Sephadex LH-20 c.c. (Φ 3 × 30 cm) and eluted with MeOH-water (4:1, 0.7
L) to obtain 10 fractions (MOB-15-2-1 to MOB-15-2-10). Fraction MOB-15-2-2
(155 mg, Ve/Vt 0.245-0.296) was subjected to the SiO2 c.c. (Φ 3 × 19 cm) and
eluted with CHCl3-MeOH-water (10:3:1, 7.5 L) to obtain 11 fractions (MOB-15-
2-2-1 to MOB-15-2-2-10) and a pure compound 2 [MOB-15-2-2-4, 21 mg, Ve/Vt
0.382-0.485, TLC (Kiesel gel 60 F254) Rf 0.35, CHCl3-MeOH-water (65:35:10)].
The EtOAc fraction (MOE, 238 g) was applied to the SiO2 c.c. (Φ 12 × 15 cm)
and eluted with n-hexane-EtOAc (5:1 → 2:1 → 1:2, 2.8 L of each) to provide 15
20
Page 20 of 31

fractions (MOE-1 to MOE-15). MOE-7 (1.56 g, Ve/Vt 0.451-0.574) was
subjected to the SiO2 c.c. (Φ 4.5 × 19 cm) and eluted with CHCl3-MeOH-water
(20:3:1, 1.3 L), yielding six fractions (MOE-7-1 to MOE-7-6). Fraction MOE-7-2
(607 mg, Ve/Vt 0.101-0.183) was subjected to the ODS c.c. (Φ 3 × 8.5 cm) and
eluted with MeOH-water (3:2 → 2:1, 1.8 L of each), yielding 12 fractions (MOE-
7-2-1 to MOE-7-2-12) and along with a pure compound 5 [MOE-7-2-1, 7.1 mg,
Ve/Vt 0.000-0.032, TLC (RP-18 F254s) Rf 0.70, MeOH-water (2:1)].
4.4. Compound characterization
4.4.1. Obovatoside A (1)
Obovatoside A (1) orange needles; m.p. 98-100 °C; [α]D -62.0° (c 0.11, MeOH);
IRν (KBr, cm-1) 3357, 2918, 1610; HREI-MS m/z 330.1328 (calcd. for C15H22O8,
330.1315) [M]+; 1H NMR (CD3OD, δH) 7.15 (2H, d, J = 8.8 Hz, H-2, 6), 7.01 (2H,
d, J = 8.8 Hz, H-3, 5), 5.22 (1H, d, J=8.0 Hz, H-1'), 4.14 (1H, dd, J=2.8, 2.8 Hz,
H-4'), δ 3.87 (1H, dd, J=12.4, 2.4 Hz, H-6'a), 3.83 (1H, dddd, J=7.2, 6.0, 5.6, 4.4
Hz, H-8), 3.77 (1H, m, H-5'), 3.69 (1H, dd, J=12.4, 5.6 Hz, H-6'b), 3.61 (1H, dd,
J=8.8, 8.0 Hz, H-2'), 3.58 (1H, dd, J=8.8, 2.8 Hz, H-3'), 3.50 (1H, dd, J=11.2, 4.4
Hz, H-9a), 3.43 (1H, dd, J=11.2, 6.0 Hz, H-9b), 2.78 (1H, dd, J=14.0, 5.6 Hz, H-
21
Page 21 of 31

7a), 2.65 (1H, dd, J=14.0, 7.2 Hz, H-7b); 13C NMR (CD3OD, δC) 157.7 (C-4),
133.8 (C-1), 131.2 (C-2, 6), 117.6 (C-3, 5), 100.2 (C-1'), 75.6 (C-5'), 74.5 (C-8),
72.9 (C-4'), 72.0 (C-2'), 68.6 (C-3'), 66.4 (C-9), 62.8 (C-6'), 40.1 (C-7).
4.4.2. Obovatoside B (2)
Obovatoside B (2) a pale yellow viscous oil; [α]D -51.8° (c 0.12, MeOH); IRν
(KBr, cm-1) 3352, 2923, 1601; positive HRFAB-MS m/z 369.1251 (calcd. for
1
C15H22O9Na, 369.1264) [M+Na]+; H NMR (CD3OD, δH) 7.09 (1H, d, J=2.0 Hz,
H-2), 6.79 (1H, dd, J=8.0, 2.0 Hz, H-6), 6.75 (1H, d, J=8.0 Hz, H-5), 5.12 (1H, d,
J=8.0 Hz, H-1'), 4.16 (1H, dd, J=2.8, 2.8 Hz, H-4'), 3.89 (1H, dd, J=12.0, 2.4 Hz,
H-6'a), 3.84 (1H, m, H-8), 3.77 (1H, m, H-5'), 3.71 (1H, dd, J=12.0, 5.6 Hz, H-
6'b), 3.63 (1H, dd, J=8.8, 8.0 Hz, H-2'), 3.61 (1H, dd, J=8.8, 2.8 Hz, H-3'), 3.50
(1H, dd, J=11.2, 4.4 Hz, H-9a), 3.44 (1H, dd, J=11.2, 6.0 Hz, H-9b), 2.73 (1H, dd,
13
J=13.6, 5.6 Hz, H-7a), 2.61 (1H, dd, J=13.6, 6.8 Hz, H-7b); C NMR (CD3OD,
δC) 146.7 (C-4), 146.7 (C-3), 131.7 (C-1), 125.4 (C-6), 119.8 (C-2), 116.7 (C-5),
102.1 (C-1'), 75.9 (C-5'), 74.5 (C-2'), 72.8 (C-4'), 72.2 (C-8), 68.5 (C-3'), 66.5 (C-
9), 62.8 (C-6'), 40.2 (C-7).
22
Page 22 of 31

4.4.3. Obovatoside C (3)
Obovatoside C (3) a pale brown viscous oil; IRν (KBr, cm-1) 3383, 2919, 1568;
[α]D -22.8° (c 0.20, MeOH); HREI-MS m/z 326.1299 (calcd. for C16H22O7,
1
326.1365) [M]+; H NMR (600 MHz, CD3OD, δH) 7.07 (1H, d, J=8.4 Hz, H-5),
6.97 (1H, d, J=1.8 Hz, H-2), 6.86 (1H, dd, J=8.4, 1.8 Hz, H-6), 6.34 (1H, d,
J=15.6 Hz, H-7), 6.20 (1H, dq, J=15.6, 6.6 Hz, H-8), 5.21 (1H, d, J=8.4 Hz, H-1'),
4.14 (1H, dd, J=3.0, 3.0 Hz, H-4'), 3.85 (3H, s, -OCH3), 3.82 (1H, m, H-5'), 3.68
(1H, dd, J=12.0, 6.0 Hz, H-6'a), 3.61 (1H, dd, J=9.6, 8.4 Hz, H-2'), 3.61 (1H, dd,
J=12.0, 3.6 Hz, H-6'b), 3.59 (1H, dd, J=9.6, 3.0 Hz, H-3'), 1.84 (3H, d, J=6.6 Hz,
13
H-9); C NMR (125 MHZ, CD3OD, δC) 151.0 (C-3), 147.4 (C-4), 134.7 (C-1),
131.9 (C-7), 125.3 (C-8), 120.1 (C-6), 118.0 (C-5), 111.1 (C-2), 100.9 (C-1'), 76.0
(C-5'), 73.0 (C-4'), 72.3 (C-2'), 68.8 (C-3'), 63.0 (C-6'), 56.8 (-OCH3), 18.6 (C-9).
4.4.4. Syringin (4)
Syringin (4) white crystals; m.p. 193-194 °C; IRν (KBr, cm-1) 3390, 2989, 1588;
[α]D -15.8° (c 0.21, MeOH); EI-MS m/z 372 [M]+.
23
Page 23 of 31

4.4.5. Pavonisol (5)
Pavonisol (5) colorless crystals; m.p. 140 °C; IRν (KBr, cm-1) 3450, 3310, 1605,
1520; [α]D +89.0° (c 0.15, CHCl3); EI-MS m/z 242 [M]+.
4.5. Acid hydrolysis of 1-2 and isolation of aglycones
A solution of compound 1 (10 mg) in 1M HCl (3 ml) was heated at 80 °C on a
heating block for 3 h. The reaction mixture was added to 2 ml MeOH and
extracted with EtOAc (5 ml). The EtOAc layer was concentrated and purified by
Supeleclean LC-Si SPE tube (n-hexane-EtOAc = 2:1) to obtain the aglycone [1a,
1.8 mg, Rf 0.45 TLC (RP-18 F254s), MeOH-water (2:3)].
A solution of compound 2 (10.8 mg) in 1M HCl (3 ml) was treated in the same
manner as compound 1 to obtain the aglycone (2a, 2.2 mg, Rf 0.50 TLC (RP-18
F254s), MeOH-water (2:3)].
24
Page 24 of 31

4.6. Zebrafish maintenance
Adult zebrafish were maintained in a zebrafish system S type [1500(W) ×
400(D) × 2050(H) mm] (Daejeon, Korea) on a cycle of 14 h light and 10 h dark at
28.5 °C. To obtain zebrafish larvae, two pairs of adult zebrafish were put in a
spawning box overnight and induced to spawn by providing light for 30 minutes.
Zebrafish embryos were collected at 3 h post-fertilization (hpf) for experiments
and incubated in a petridish in 0.03% sea salt solution. Embryos were maintained
under a 14 h light and 10 h dark cycle in an incubator at 28.5 °C.
4.7. Recovery effect of compounds 1-5 on alloxan-induced pancreatic islet
damage in zebrafish larvae
Zebrafish larvae were divided into seven groups: normal, alloxan-induced
(control), and alloxan-induced with treatment with compounds 1-5. Wild-type
zebrafish larvae at 5 days post-fertilization (dpf) were placed into 96-well plates.
Larvae were exposed to 25 μM 2-NBDG for 12h and rinsed for 10 min. To induce
pancreatic islet damage, alloxan was added at 100 μM for 15 min. At 4 h and 45
min after alloxan treatment, larvae were stained for 1 h with 25 μM 2-NBDG and
25
Page 25 of 31

mounted in 96-well plates and observed using a fluorescence microscope. To
determine compound effects, alloxan-induced larvae were treated with 10 μM
compounds for 1 h and restained for 1 h with 25 μM 2-NBDG and observed
under a fluorescence microscope. All images were analyzed for pancreatic islet
size and histograms of fluorescence intensity were generated using Focus Lite
and Image J software.
4.8. Statistical analysis
Statistical analysis was performed using Graphpad Prism (version.5). Data were
expressed as mean ± standard error of the mean (SEM). Significance was
determined using repeated one-way ANOVA followed by Tukey’s test.
Probability levels for statistical significance were p<0.05, 0.01 and 0.001.
Acknowledgments.
This work was carried out with the support of “Cooperative Research Program for
Agriculture Science & Technology Development (Project No. PJ01133301)”
Rural Development Administration, Republic of Korea.
26
Page 26 of 31

27
Page 27 of 31

References
1. Chang, Y. S.; Ham, S. Y. Korea Institute Health Social Affairs, 2014, 257, 1-8.
2. Kumar, V.; Fausto, N.; Abbas, A. K.; Cotran, R. S.; Robbins, S. L. Robbins
and Cotran Pathologic Basis of Disease, 7th ed; Schmitt, W., Eds; Academic
Press: Philadelphia, 2005; 1, pp 1194-1195.
3. Logan, W. W.; Hoffman, N. Y. Radiology, 1989, 172, 667-670.
4. Byrd, B. F. Jr.; Hartmann, W. H.; Graham, L. S.; Hongle, H. H. Ann. Surg.,
1987, 205, 529-532.
5. Sharma, A. K. Diabetes mellitus and its complications: An update, 1st ed;
Sharma, A.K., Eds; Academic Press: Macmillan India, 1993; 1, pp 58-61.
6. Lee, H. J.; Kim, A. R.; Lee, J. J. J. Korean Soc. Appl. Biol. Chem., 2014, 57,
639-645.
7. Ahn, H. C.; Kim, J. H.; Kim, J. I.; Auh, J. Y.; Choe, E. O. Food Sci.
Biotechnol., 2014, 23, 1287-1293.
8. Kumar, K.; Issac, A.; Ninan, E.; Kuttan, R., Maliakel, B. J. Func. Food, 2014,
28
Page 28 of 31

10, 54-64.
9. Seo, K. H.; Lee, D. Y.; Jeong, R. H.; Yoo, K. H.; Chung, I. S.; Kim, G. S.; Seo,
W. D.; Kang, H. C.; Ahn, E. M.; Baek, N. I. J. Appl. Biol. Chem., 2013, 56,
181-183.
10. Li, J.; Tanaka, M.; Kurasawa, K.; Ikeda, T.; Nohara, T. J. Nat. Med., 2007, 61,
222-223.
11. Nakazawa, T.; Yasuda, T.; Ohsawa, K. J. Pharm. Pharmacol., 2003, 55,
1583-1591.
12. Hasegawa, T.; Fukuyama, Y.; Yamada, T. Chem. Pharm. Bull., 1988, 36,
1245-1248.
13. Spence, R.; Gerlach, G.; Lawrence, C.; Smith C. Biol. Rev., 2008, 83, 13-34.
14. Paciorek, T.; Mcrobert, S. Curr. Zool., 2012, 58, 129-137.
15. Seo, K. H.; Nam, Y. H.; Kim, Y, E.; Hong, E. K.; Hong, B. N.; Kang, T. H.;
Baek, N. I. J. Appl. Biol. Chem., 2015, 58, 51-54.
16. Ferri, P. H.; Barata, L. E. Phytochem., 1998, 31, 1375-1377.
29
Page 29 of 31