Two new aromatic glycosides, elengiosides A and B, from the flowers of Mimusops elengi

Article abstract of DOI:10.1007/s11418-017-1160-z

Two new aromatic glycosides, elengiosides A (1) and B (2), were isolated from the methanolic extract of the flowers of Mimusops elengi (Sapotaceae) together with 26 known compounds. Their stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Among the isolates, a phenylethanoid glycoside, undatuside C (14), was found to exhibit hyaluronidase inhibitory activity.

Full text of DOI:10.1007/s11418-017-1160-z

Journal of Natural Medicines
https://doi.org/10.1007/s11418-017-1160-z
NOTE
Two new aromatic glycosides, elengiosides A and B, from the fowers
of Mimusops elengi
Toshio Morikawa1,2 · Yoshiaki Manse1 · Mika Koda1 · Saowanee Chaipech3 · Yutana Pongpiriyadacha4 ·
Osamu Muraoka1,2 · Kiyofumi Ninomiya1,2
Received: 7 November 2017 / Accepted: 29 November 2017
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2017
Abstract
Two new aromatic glycosides, elengiosides A (1) and B (2), were isolated from the methanolic extract of the fowers of
Mimusops elengi (Sapotaceae) together with 26 known compounds. Their stereostructures were elucidated based on their
spectroscopic properties and chemical evidence. Among the isolates, a phenylethanoid glycoside, undatuside C (14), was
found to exhibit hyaluronidase inhibitory activity.
Keywords Mimusops elengi · Elengioside · Hyaluronidase inhibitor · Sapotaceae
Introduction
Snuf made from the dried fowers of M. elengi is supposed
to be useful in treating high fever, headache, and body pains
[1, 5]. Pillow stufng made from the dried fowers induces
nasal discharge and relieves headache [5]. Regarding the
phytochemical investigation of this plant, several tritepenes
[1, 2, 6–8] and triterpene saponins have been extracted from
the bark and seeds [1, 2, 9–12], while steroidal glycosides
[13, 14] and gallic acid derivatives have been extracted from
the stem bark [15]. In addition, the pharmacological proper-
ties, such as antioxidant, antibacterial, anti-cariogenic, anti-
fungal, gastroprotective, hypotensive, antidiabetic, diuretic,
antineoplastic [1], and anti-infammatory activities [16], of
the extracts obtained from bark, leaves, seeds, and fruits
have been investigated. Recently, a neuroprotective efect
of the fower extract against stroke-like conditions has been
reported [17]. However, studies on the chemical constituents
from the fowers of M. elengi are rare. In the course of our
characterization studies on the bioactive constituents used in
Thai natural medicine [18–35], two new glycosides, elengi-
osides A (1) and B (2), were isolated from the methanolic
extract of the fowers of M. elengi originating in Thailand
along with 26 known compounds (3–28). Here, we discuss
the isolation and structural elucidation of these new com-
pounds (1 and 2).
Mimusops elengi L. (known as Bakul in Bengali, Pikul
in Thai, and Spanish cherry, Medlea, and Bullet wood in
English), a Sapotaceae plant, is an evergreen tree widely
distributed in India, Sri Lanka, Malaysia, Myanmar, and
Thailand [1–3]. M. elengi is considered a sacred plant by
Hindus and occupies an important place in religious texts
as well as in ancient Sanskrit literature [2, 4]. In Ayurveda,
a form of traditional medicine, almost all the morphological
parts of this plant are used for the management of a wide
range of bodily disorders [1–5]. The fowers of M. elengi
exert a cooling and astringent efect on the bowels and are
reported to possess immunomodulatory, hepatoprotective,
analgesic, anti-asthmatic, and wound healing activities [4].
* Toshio Morikawa
morikawa@kindai.ac.jp
1
Pharmaceutical Research and Technology Institute, Kindai
University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502,
Japan
2
Antiaging Center, Kindai University, 3-4-1 Kowakae,
Higashi-osaka, Osaka 577-8502, Japan
3
Faculty of Agro-Industry, Rajamangala
University of Technology Srivijaya, Thungyai,
Nakhon Si Thammarat 80240, Thailand
4
Faculty of Science and Technology, Rajamangala
University of Technology Srivijaya, Thungyai,
Nakhon Si Thammarat 80240, Thailand
Vol.:(0123456789)
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Journal of Natural Medicines
Fig. 1 Structures of 1–28 isolated from the fowers of M. elengi
43], 2-phenylethyl α-l-rhamnopyranosyl-(1 → 6)-β-d-
glucopyranoside (13, 0.0009%) [40], undatuside C (14,
0.0006%) [45], rosin (15, 0.0135%) [46], vimalin (16,
0.0010%) [46], 2′-O-β-d-glucosylrosine (17, 0.0119%) [47],
chavicol diglycoside (18, 0.0049%) [48], 2-methoxy-4-(2-
propenyl) β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside
(19, 0.0005%) [49], (2S,3S)-1-phenyl-2,3-butanediol 3-O-β-
d-glucopyranoside (20, 0.0018%) [50], and 4-methylphenyl
β-d-glucopyranoside (21, 0.0040%) [51]. From the EtOAc-
soluble fraction, seven known compounds, 4-hydroxyben-
zyl alcohol (22, 0.0009%)1, 4-(methoxymethyl)phenol (23,
0.0012%) (see footnote 1), p-hydroxybenzaldehyde (24,
0.0158%) (see footnote 1), p-anisic acid (25, 0.0021%) (see
footnote 1), 4-hydroxyacetophenone (26, 0.0010%) (these
compounds were identifed by comparison of their physical
and spectral data with those of commercially available sam-
ples), 4-hydroxybenzoic acid phenethyl ester (27, 0.0005%)
(see footnote 1), and trans-cinnamic acid (28, 0.0014%) (see
footnote 1), were isolated (Fig. 1).
Results and discussion
Isolation
The dried fowers of M. elengi were extracted with metha-
nol under refux to give a methanolic extract (31.15% from
the dried material). The extract was partitioned into a
mixture of ethyl acetate (EtOAc) and water to produce the
EtOAc-soluble fraction (3.41%) and the aqueous phase. The
latter was subjected to Diaion HP-20 column chromatog-
raphy (H2O → MeOH) to yield H2O- and MeOH-eluted
fractions (21.39% and 4.61%, respectively). The MeOH-
eluted fraction was subjected to normal-phase silica gel
and reversed-phase octadecylsilyl (ODS)-column chroma-
tographies and fnally HPLC to give two new glycosides,
elengiosides A (1, 0.0248%) and B (2, 0. 0132%) along
with 19 known compounds, benzyl β-d-glucopyranoside (3,
0.0191%) [36, 37], gastrodin (4, 0.0101%) [38], 4-methoxy-
benzyl β-d-glucopyranoside (5, 0.0206%) [36], benzyl β-d-
glucopyranosyl-(1 → 6)-β-d-glucopyranoside (6, 0.0175%)
[37], 2-phenylethyl β-d-glucopyranoside (7, 0.0963%)
[39, 40], viridoside (8, 0.0017%) [41], 2-phenylethyl β-d-
glucopyranosyl-(1 → 2)-β-d-glucopyranoside (9, 0.4252%)
[42], 2-phenylethyl β-d-glucopyranosyl-(1 → 6)-β-d-
glucopyranoside (10, 0.0989%) [39, 43], 2-(4-hydroxyphe-
nyl)ethyl β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside
(11, 0.0017%) [44], icariside D1 (12, 0.0008%) [39,
Structure determination of elengiosides A (1) and B (2)
Elengioside A (1) was obtained as a white powder with nega-
tive optical rotation ([α]2D5–21.1 in MeOH). A quasimolecular
1
These compounds were identiied by comparison of their physica-
land spectral data with those of commercially available samples.
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Journal of Natural Medicines
Table 1 1H and 13C NMR spectroscopic data of elengiosides A (1) and B (2) and (2S,3S)-1-phenyl-2,3-butanediol 3-O-β-d-glucopyranoside (20)
Position
1 (in pyridine-d5)a
2 (in CD3OD)a
20 (in CD3OD)b
δH
δC
δH
δC
δH
δC
1
139.5
129.6
128.7
126.4
36.7
140.1
131.0
129.1
127.1
39.6
140.6
130.5
129.2
127.0
39.9
2, 6
3, 5
4
7.32 (2H, br d, ca. 7)
7.25 (2H, dd, 7.2, 8.0)
7.14 (1H, br t, ca. 8)
3.08 (2H, m)
7.28 (2H, d, 7.6)
7.24 (2H, dd, 7.3, 7.6)
7.16 (1H, t, 7.3)
2.95 (1H, dd, 5.6, 13.8)
3.01 (1H, dd, 7.0, 13.8)
3.60 (1H, ddd, 5.6, 5.7, 7.0)
7.25 (2H, m)
7.26 (2H, m)
7.16 (1H, br t, ca. 7)
2.71 (1H, dd, 8.3, 13.8)
2.93 (1H, dd, 3.7, 13.8)
3.72 (1H, br ddd, ca. 2, 8, 13)
7
8
3.82 (1H, dt, 7.2, 8.8)
4.28 (1H, dt, 7.2, 8.8)
70.8
86.8
77.0
9
10
3.69 (1H, dq, 5.7, 6.5)
1.18 (3H, d, 6.5)
4.38(1H, d, 7.8)
3.23 (1H, dd, 7.8, 9.3)
3.34 (1H, m)
3.23 (1H, dd, 9.0, 9.6)
3.20 (1H,ddd, 2.5, 5.7, 9.6)
3.61 (1H, m)
69.0
19.6
106.0
75.8
77.9
71.7
77.7
62.8
3.73 (1H, br s)
1.30 (3H, d, 6.3)
4.40 (1H, d, 8.1)
3.22 (1H, m)
3.36 (1H, m)
3.27 (1H, m)
3.24 (1H, m)
3.66 (1H, dd, 5.4, 11.7)
3.85 (1H, m)
80.4
18.6
105.4
75.5
77.9
71.6
77.9
62.7
Glc-1′
-2′
-3′
-4′
-5′
-6′
4.81 (1H, d, 7.2)
102.9
83.9
77.9
71.3
77.0
70.0
4.81 (1H, dd, 7.2, 9.6)
4.22 (1H, dd, 8.8, 9.6)
4.08 (1H, dd, 8.8, 9.6)
3.96 (1H, m)
4.25 (1H, dd, 6.4, 11.2)
4.75 (1H, dd, 1.6, 11.2)
3.78 (1H, dd, 2.5, 11.8)
Glc-1′′
-2′′
-3′′
-4′′
5′′
5.25 (d, 8.0)
105.5
75.2
78.4
71.8
78.7
62.9
4.05 (1H, dd, 8.0, 8.8)
4.19 (1H, dd, 8.8, 9.6)
4.20 (1H, dd, 9.6, 9.6)
3.18 (1H, m)
4.35 (1H, dd, 5.6, 12.0)
4.47 (1H, dd, 2.4, 12.0)
5.01 (1H, d, 8.0)
4.00 (1H, dd, 8.0, 8.8)
4.16 (1H, dd, 8.8, 8.8)
4.16 (1H, m)
-6′′
Glc-1′′′
-2′′′
-3′′′
-4′′′
-5′′′
106.3
76.7
78.2
71.8
78.4
62.9
3.91 (1H, m)
4.32 (1H, 5.6, 12.0)
4.47 (1H, dd, 3.2, 12.0)
-6′′′
a Measured at 800 MHz
b Measured at 500 MHz
ion peak was observed at m/z 631 [M + Na]+ in positive-
ion fast atom bombardment mass spectrometry (FABMS)
and high-resolution fast atom bombardment mass spec-
trometry (HRFABMS) revealed that its molecular formula
is C26H30O11. Its IR spectrum exhibited broad absorption
bands at 3400 and 1074 cm−1, which suggests the presence
of a glycosyl moiety. Treatment of 1 with 1 M hydrochloric
acid (HCl) liberated 2-phenethyl alcohol (these compounds
were identifed by comparison of their physical and spectral
data with those of commercially available samples) together
with d-glucose, which was identifed by HPLC using an opti-
using distortionless enhancement by polarization transfer,
1H-1H homonuclear correlation (COSY), heteronuclear mul-
tiple quantum coherence, and hetero-nuclear multiple-bond
correlation (HMBC) experiments (Fig. 2), showed signals
assignable to two methylenes [ 3.08 (2H,m, H2-7), 3.82 and
4.28 (1H each, both dt, J = 7.2, 8.2 Hz, H2-8)], a monosub-
stituted benzene [ 7.14 (1H, br t, J = ca. 8 Hz, H-4), 7.25
(2H, dd, J = 7.2, 8.0 Hz, H-3,5), 7.32 (2H, br d, J = ca. 7 Hz,
H-2,6)], and three glucopyranosyl moieties [ 4.81 (1H, d,
J = 7.2 Hz, H-1′), 5.01 (1H, d, J = 8.0 Hz, H-1″′), and 5.25
(1H, d, J = 8.0 Hz, H-1′′)]. As shown in Fig. 2, in the HMBC
experiment of 1, long-range correlations were observed
between the following proton and carbons: Glc-H-1′ and C-8
cal rotation detector [18–20, 23–26, 43, 50]. The 1H and 13
NMR spectra (Table 1, pyridine-d5), which were assigned
C
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Journal of Natural Medicines
Fig. 2 1H-1H COSY and HMBC
correlations of 1 and 2
(C 70.8), Glc-H-1′′ and Glc-C-2′ (C 83.9), and Glc-H-1′′′ and
Glc-C-6′ (C 62.9). Thus, the structure of 1 was determined
to be 2-phenethyl O-β-d-glucopyranosyl-(1 → 2)-[β-d-
glucopyranosyl-(1 → 6)]-β-d-glucopyranoside.
allergies and infammation [53–55]. It is known that an
anti-allergic medicine, disodium cromoglycate (DSCG),
exhibits a strong inhibitory efect against hyaluronidase
[53]. Therefore, it has been found that there is a close
relationship between allergic reactions and hyaluroni-
dase inhibitory activity [53, 55]. In the present study, the
methanolic extract of M. elengi fowers was found to show
hyarulonidase inhibitory activity (IC50 = 271 μg/mL). To
characterize the active constituents of this plant material,
the inhibitory efects of the isolates against hyaluronidase
were examined. As the result, a phenylethanoid glycoside,
undatuside C [14, inhibition (%): 42.6 5.8 at 300 μM
(p < 0.05)], was found to possess inhibitory activity. How-
ever, this inhibitory activity was weaker than that of clini-
cally used DSCG [inhibition (%): 47.1 2.7 at 100 μM
and 91.9 1.0 at 300 μM (p < 0.05), IC50 = 87.0 μM]
[53]. The other isolates did not show signifcant inhibitory
activities at a concentration of 300 μM (data not shown).
In conclusion, we found that the methanolic extract of
the fowers of M. elengi inhibits the enzymatic activity
of hyaluronidase (IC50 = 271 μg/mL). From the extract,
two new aromatic glycosides, elengioside A (1) and B (2),
were isolated along with 26 known compounds. Among
the isolates, undatuside C (14) was identifed as the active
constituent. The details of the structural requirements of
these aromatic compounds for hyaluronidase inhibitory
activity need to be further explored.
Elengioside B (2) was obtained as a white powder with a
negative optical rotation ([α]2D7–12.9 in CHCl3). In positive-
ion electrospray mass spectrometry (ESIMS), a quasimo-
lecular ion peak was observed at m/z 351 [M + Na]+ and
high-resolution electrospray mass spectrometry (HRESIMS)
revealed that the molecular formula of 2 is C16H24O7. Acid
hydrolysis of 2 with 1 M HCl liberated d-glucose, which was
identifed by HPLC using an optical rotation detector. The 1H
and 13C NMR spectra (Table 1, CD3OD) of 2 showed signals
assignable to a secondary methyl [δ 1.18 (3H, d, J = 6.5 Hz,
H3-10)], a methylene [δ 2.91 (1H, dd, J = 5.6, 13.8 Hz) and
3.01 (1H, dd, J = 7.0, 13.8 Hz), H2-7], two methines bear-
ing an oxygen function [δ 3.69 (1H, dq, J = 5.7, 6.5 Hz,
H-9) and 3.60 (1H, ddd, J = 5.6, 5.7, 7.0 Hz H-8)], and a
monosubstituted benzene moiety [δ 7.16 (1H, t, J = 7.3 Hz,
H-4), 7.24 (2H, dd, J = 7.5, 7.6 Hz, H-3,5), and 7.28 (2H, d,
J = 7.6 Hz, H-2,6)] together with a glucopyranosyl moiety
[δ 4.38 (1H, d, J = 7.8 Hz, Glc-H-1′)]. These spectroscopic
properties were quite similar to those of (2S,3S)-1-phenyl-
2,3-butanediol 3-O-β-d-glucopyranoside (20) [50]. Enzy-
matic hydrolysis of 2 with β-glucosidase resulted in (2S,3S)-
1-phenyl-2,3-butanediol (2a) [52]. In the HMBC experiment
of 2, a long-range correlation was observed between Glc-H-1′
and C-8 (δC 86.8). Consequently, the absolute stereostructure
of 2 was determined to be as shown.
Materials and methods
Inhibitory efects on hyaluronidase
General
Hyaluronidase is a mucopolysaccharide related to infam-
mation by the histamine released from mast cells. Hya-
luronidase inhibitors seem to be efective in suppressing
The following instruments were used to obtain spectro-
scopic data: specifc rotation, JASCO P-2200 polarimeter
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Journal of Natural Medicines
(JASCO Corporation, Tokyo, Japan, l = 5 cm); UV spec-
tra, Shimadzu UV-1600 spectrometer; IR spectra, IRAfn-
ity-1 spectrophotometer (Shimadzu Co.); 1H NMR spectra,
JNM-ECA800 (800 MHz), JNM-LA500 (500 MHz), and
JNM-ECS400 (400 MHz) spectrometers; 13C NMR spectra,
JNM-ECA800 (200 MHz), JNM-LA500 (125 MHz), and
JNM-ECS400 (100 MHz) spectrometers with tetramethylsi-
lane as an internal standard; FABMS and HRFABMS, JEOL
JMS-SX 102A mass spectrometer; ESIMS and HRESIMS,
Exactive Plus mass spectrometer (Thermo Fisher Scientifc
Inc., MA, USA); HPLC detectors, Shimadzu RID-6A refrac-
tive index and SPD-10A UV–VIS detectors and a Shodex
OR-2 optical rotation detector; HPLC columns, Cosmosil
5C18-MS-II (Nacalai Tesque, Inc., Kyoto, Japan, 4.6 mm
i.d. × 250 mm and 20 mm i.d. × 250 mm) for analytical
and preparative purposes, respectively. A Kaseisorb LC
NH2-60-5 (Tokyo Kasei Co., Ltd., Tokyo, Japan, 4.6 mm
i.d. × 250 mm) was used to identify the sugar part.
H2O-eluted (387.4 g, 21.39%) and MeOH-eluted (83.4 g,
4.61%) fractions. An aliquot (73.4 g) of the MeOH-eluted
fraction was subjected to normal-phase silica gel CC
[2750 g, CHCl3-MeOH-H2O (20:3:0.3 → 10:3:0.4 → 6:4:1,
v/v/v) → MeOH] to yield eight fractions [Fr. 1 (0.91 g),
Fr. 2 (5.42 g), Fr. 3 (2.26 g), Fr. 4 (9.83 g), Fr. 5 (22.13 g),
Fr. 6 (8.87 g), Fr. 7 (10.80 g), and Fr. 8 (6.15 g)]. Frac-
tion 2 (5.41 g) was subjected to reversed-phase silica gel
CC [300 g, MeOH-H2O (30:70 → 50:50, v/v) → MeOH]
to yield four fractions [Fr. 2-1 (2.60 g), Fr. 2-2 (2.15 g),
Fr. 2-3 (363.5 mg), and Fr. 2-4 (217.6 mg)]. Fraction 2-1
(427.5 mg) was purifed by HPLC [detection: RI, MeOH-
1% aqueous AcOH (30:70, v/v)] to yield benzyl β-d-
glucopyranoside (3, 50.0 mg, 0.0191%), 4-methoxybenzyl
β-d-glucopyranoside (5, 41.9 mg, 0.0169%), and 2-pheny-
lethyl β-d-glucopyranoside (7, 10.7 mg, 0.0041%). Frac-
tion 2-2 (439.3 mg) was purifed by HPLC [detection: RI,
MeOH-1% aqueous AcOH (30:70, v/v)] to yield elengi-
oside B (2, 40.1 mg, 0.0123%), 5 (15.1 mg, 0.0046%), 7
(294.7 mg, 0.0906%), viridoside (8, 5.5 mg, 0.0017%), rosin
(15, 15.3 mg, 0.0047%), and (2S,3S)-1-phenyl-2,3-butane-
diol 3-O-β-d-glucopyranoside (20, 5.8 mg, 0.0017%). Frac-
tion 2-3 (363.5 mg) was purifed by HPLC [detection: RI,
MeOH-1% aqueous AcOH (30:70, v/v)] to yield 2 (13.1 mg,
0.0009%), 7 (26.5 mg, 0.0017%), 15 (139.5 mg, 0.0088%),
and vimalin (16, 16.6 mg, 0.0010%). Fraction 4 (9.83 g) was
subjected to reversed-phase silica gel CC [330 g, MeOH-
H2O (20:80 → 40:60, v/v) → MeOH] to yield fve fractions
{Fr. 4-1 (3.44 g), Fr. 4-2 (567.2 mg), Fr. 4-3 [= 2-pheny-
lethyl β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside
(9, 3.40 g, 0.2135%)], Fr. 4-4 (622.4 mg), and Fr. 4-5
(667.1 mg)}. Fraction 4-4 (392.8 mg) was purifed by HPLC
[detection: RI, MeOH-1% aqueous AcOH (40:60, v/v)] to
yield 2′-O-β-d-glucosylrosine (17, 102.1 mg, 0.0102%), 9
(25.3 mg, 0.0025%), icariside D1 (12, 7.8 mg, 0.0008%),
2-phenylethyl α-l-rhamnopyranosyl-(1 → 6)-β-d-
glucopyranoside (13, 9.0 mg, 0.0009%), undatuside C
(14, 5.8 mg, 0.0006%), and 2-methoxy-4-(2-propenyl)
phenyl β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside
(19, 5.5 mg, 0.0005%). Fraction 5 (22.13 g) was sub-
jected to reversed-phase silica gel CC [715 g, MeOH-
H2O (20:80 → 70:30, v/v) → MeOH] to yield nine frac-
tions [Fr. 5-1 (1.91 g), Fr. 5-2 (1.86 g), Fr. 5-3 (9.66 g),
Fr. 5-4 (68.7 mg), Fr. 5-5 (160.1 mg), Fr. 5-6 (725.5 mg),
Fr. 5-7 (249.8 mg), Fr. 5-8 (714.2 mg), and Fr. 5-9
(6.30 g)]. Fraction 5-3 (501.6 mg) was purifed by HPLC
[detection: RI, MeOH-1% aqueous AcOH (40:60, v/v)]
to yield 9 (162.5 mg, 0.197%) and 2-phenylethyl β-d-
glucopyranosyl-(1 → 6)-β-d-glucopyranoside (10, 46.8 mg,
0.0566%). Fraction 5-5 (160.1 mg) was purifed by HPLC
[detection: RI, MeOH-1% aqueous AcOH (30:70, v/v)]
to yield 17 (27.0 mg, 0.0017%). Fraction 5-6 (337.1 mg)
was purifed by HPLC [detection: RI, MeOH-1% aqueous
The following experimental conditions were used for col-
umn chromatography (CC): highly porous synthetic resin,
Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan);
normal-phase silica gel CC, silica gel 60 N (Kanto Chemical
Co., Ltd., Tokyo, Japan; 63–210 mesh, spherical, neutral);
reversed-phase ODS CC, Chromatorex ODS DM1020T
(Fuji Silysia Chemical, Ltd., Aichi, Japan; 100–200 mesh);
TLC, pre-coated TLC plates with silica gel 60F254 (Merck,
Darmstadt, Germany, 0.25 mm) (normal-phase) and silica
gel RP-18 WF254S (Merck, 0.25 mm) (reversed-phase);
reversed-phase HPTLC, pre-coated TLC plates with silica
gel RP-18 WF254S (Merck, 0.25 mm). Detection was carried
out by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 fol-
lowed by heating.
Plant material
The fowers of M. elengi were collected from the Nakhon-
sithammarat Province of Thailand in September 2006. The
plant material was identifed by one of the authors (YP). A
voucher specimen (2006.09. Raj-05) of this plant is on fle
in our laboratory.
Extraction and isolation
The dried fowers of M. elengi (2.00 kg) were extracted
four times with methanol under refux for 3 h. Evaporation
of the combined extracts under reduced pressure yielded
an aqueous acetone extract (622.9 g, 31.15%). An aliquot
(564.1 g) was partitioned in an EtOAc-H2O (1:1, v/v) mix-
ture to furnish an EtOAc-soluble fraction (61.8 g, 3.41%)
and an aqueous phase. The aqueous phase was subjected
to Diaion HP-20 CC (3.0 kg, H2O → MeOH) to yield
1 3

Journal of Natural Medicines
AcOH (30:70, v/v)] to yield 17 (48.9 mg, 0.0066%), chavi-
col diglycoside (18, 35.8 mg, 0.0048%), and 10 (47.5 mg,
0.0064%). Fraction 6 (8.87 g) was subjected to reversed-
phase silica gel CC [300 g, MeOH-H2O (20:80 → 30:70,
v/v) → MeOH] to yield six fractions [Fr. 6-1 (3.09 g), Fr.
6-2 (1.24 g), Fr. 6-3 (559.9 mg), Fr. 6-4 (1.81 g), Fr. 6-5
(410.5 mg), and Fr. 6-6 (1.03 g)]. Fraction 6-1 (658.2 mg)
was purifed by HPLC [detection: RI, MeOH-1% aque-
ous AcOH (10:90, v/v)] to yield gastrodin (6, 34.2 mg,
0.0108%). Fraction 6-2 (496.7 mg) was purifed by HPLC
[detection: RI, MeOH-1% aqueous AcOH (20:80, v/v)]
to yield elengioside A (1, 43.0 mg, 0.0067%), benzyl
β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (6,
100.6 mg, 0.0158%), and 2-(4-hydroxyphenyl)ethyl β-d-
glucopyranosyl-(1 → 6)-β-d-glucopyranoside (11, 10.8 mg,
0.0017%). Fraction 6-3 (559.9 mg) was purifed by HPLC
[detection: RI, MeOH-1% aqueous AcOH (20:80, v/v)] to
yield 1 (197.3 mg, 0.0124%), 6 (27.1 mg, 0.0017%), and 10
(64.8 mg, 0.0041%). Fraction 6-4 (548.1 mg) was purifed
by HPLC [detection: RI, MeOH-1% aqueous AcOH (20:80,
v/v)] to yield 1 (27.1 mg, 0.0056%), 9 (61.1 mg, 0.0127%),
and 10 (153.5 mg, 0.0318%). An aliquot (51.8 g) of the
EtOAc-soluble fraction was subjected to normal-phase
silica gel CC [2.00 kg, n-hexane–EtOAc (40:1 → 20:1 →
10:1 → 2:1 → 1:1, v/v) → EtOAc → MeOH] to yield six
fractions [Fr. 1 (7.34 g), Fr. 2 (5.36 g), Fr. 3 (3.20 g), Fr.
4 (4.93 g), Fr. 5 (4.26 g), and Fr. 6 (25.79 g)]. Fraction
3 (3.20 g) was subjected to reversed-phase silica gel CC
[105.0 g, MeOH-H2O (80:20 → 90:10, v/v) → MeOH] to
yield four fractions [Fr. 3-1 (172.5 mg), Fr. 3-2 (76.0 mg),
Fr. 3-3 (35.3 mg), and Fr. 3-4 (2.04 g)]. Fraction 3-1
(172.5 mg) was purifed by HPLC [detection: UV (230 nm),
MeOH-1% aqueous AcOH (30:70, v/v)] to yield 4-(meth-
oxymethyl)phenol (23, 18.7 mg, 0.00123%), p-hydroxyben-
zaldehyde (24, 18.4 mg, 0.00211%), and p-anisic acid (25,
32.1 mg, 0.00211%). Fraction 3-2 (76.0 mg) was purifed by
HPLC [detection: UV (230 nm), MeOH-1% aqueous AcOH
(60:40, v/v)] to yield 4-hydroxybenzoic acid phenethyl ester
(27, 7.4 mg, 0.0005%). Fraction 4 (4.93 g) was subjected
to reversed-phase silica gel CC [460 g, MeOH-H2O (20:8
0 → 30:70 → 50:50 → 60:40 → 70:30 → 80:20 → 90:10,
v/v) → MeOH] to yield eight fractions [Fr. 4-1 (630.0 mg),
Fr. 4-2 (415.1 mg), Fr. 4-3 (60.6 mg), Fr. 4-4 (850.0 mg),
Fr. 4-5 (262.2 mg), Fr. 4-6 (204.6 mg), Fr. 4-7 (638.2 mg),
and Fr. 4-8 (478.6 mg)]. Fraction 4-1 (150.7 mg) was puri-
fed by HPLC [detection: UV (230 nm), MeOH-1% aqueous
AcOH (20:80, v/v)] to yield 24 (75.4 mg, 0.0137%). Frac-
tion 4-3 (60.6 mg) was purifed by HPLC [detection: UV
(230 nm), MeOH-1% aqueous AcOH (20:80, v/v)] to yield
4-hydroxyacetophenone (26, 15.5 mg, 0.0010%). Frac-
tion 4-5 (262.2 mg) was purifed by HPLC [detection: UV
(230 nm), MeOH-1% aqueous AcOH (30:70, v/v)] to yield
trans-cinnamic acid (28, 20.8 mg, 0.0014%). Fraction 4-6
(204.6 mg) was purifed by HPLC [detection: UV (230 nm),
MeOH-1% aqueous AcOH (20:80, v/v)] to yield 4-hydroxy-
benzyl alcohol (22, 14.0 mg, 0.0009%).
Elengioside A (1)
White powder, [α]D25 –21.1 (c 0.13, MeOH); IR (KBr) vmax
:
3400, 1074, 1043 cm−1; 1H (800 MHz, pyridine-d5) and 13
C
NMR (200 MHz, pyridine-d5): shown in Table 1; positive-
ion FABMS m/z: 631 [M + Na]+; HRFABMS m/z: 631.2214
[M + Na]+ (calcd for C26H40O16Na, 631.2207).
Elengioside B (2)
White powder, [α]D27 –12.9 (c 0.41, CHCl3); IR (KBr) vmax
:
1
3400, 1074, 1034 cm−1; H (800 MHz, CD3OD) and 13C
NMR (200 MHz, CD3OD): shown in Table 1; positive-ion
ESIMS m/z: 351 [M + Na]+; HRESIMS m/z: 351.1409
[M + Na]+ (calcd for C16H24O7Na, 351.1414).
Acid hydrolysis of 1 and 2
A solution of 1 (7.0 mg) in 1 M HCl (1.0 mL) was stirred at
80 °C for 1 h. After the solution cooled down, it was neu-
tralized with Amberlite IRA-400 (OH− form) and the resin
was removed by fltration. After removal of the solvent from
the fltrate under reduced pressure, the residue was parti-
tioned in EtOAc-H2O (1:1, v/v), and the solvents removed
in vacuo from the EtOAc-soluble fraction and an aqueous
phase, respectively. The EtOAc-soluble fraction was puri-
fed by HPLC [Cosmosil 5C18-MS-II, MeOH-H2O (80:20,
v/v)] to furnish 2-hydroxyphenethyl alcohol (1.3 mg, 86%)
(see footnote 1). In turn, the aqueous layer was subjected
to HPLC analysis under the following conditions: HPLC
column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm
(Tokyo Kasei Co., Ltd.); detection, optical rotation [Shodex
OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase,
CH3CN-H2O (85:15, v/v); fow rate 0.8 mL/min]. Identifca-
tion of d-glucose present in the aqueous phase was carried
out by comparing the retention time and the optical rotation
with that of the authentic sample [tR: 13.9 min (positive opti-
cal rotation)]. Using a similar procedure, d-glucose was also
identifed to be the sugar part of 2.
Enzymatic hydrolysis of 2
A solution of 2 (10.0 mg) in 0.2 M acetate buffer (pH
5.0, 2.0 mL) was treated with β-glucosidase (10 mg from
Almond, Oriental Yeast Co., Ltd., Tokyo, Japan) and the
solution was stirred at 37 °C for 24 h. After treating with
EtOH, the reaction mixture was fltered and evaporated to
dryness under reduced pressure. Then the residue was puri-
fed by normal-phase silica gel chromatography [500 mg,
1 3

Journal of Natural Medicines
CHCl3-MeOH-H2O (20:3:0.3 → 10:3:0.4, v/v/v)] to yield
(2S,3S)-1-phenyl-2,3-butanediol [2a, 4.3 mg, 90%, [α]D23
–31.6 (c 0.13, CHCl3), lit. [α]2D4 –32.58 (c 1.074, CHCl3)]
[52].
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The experiment was performed according to a previously
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Briefy, the assay was performed in 96-well microplates.
Pre-incubation of 10 μL of hyaluronidase enzyme (Type
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Acknowledgements This work was supported by the MEXT-Sup-
ported Program for the Strategic Research Foundation at Private Uni-
versities, 2014–2018, Japan (S1411037, TM) as well as the JSPS KAK-
ENHI, Japan [Grant Numbers 15K08008 (TM), 15K08009 (KN), and
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Scholarship Foundation, Japan, is also acknowledged (TM).
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