A new phenolic glycoside from Saprosma merrillii

Article abstract of DOI:10.1080/14786419.2016.1195382

A new phenolic glycoside derivative, saproglucoside (1), along with five known phenolic glycoside derivatives (2–6) were isolated from the stems of Saprosma merrillii. The structure of the new compound 1 was determined by 1D and 2D NMR as well as by HRESIMS and hydrolysis. The inhibitory activities of all compounds against seven pathogenic bacteria and two cancer cell lines were evaluated.

Full text of DOI:10.1080/14786419.2016.1195382

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new phenolic glycoside from Saprosma merrillii
Xue-Ming Zhou, Cai-Juan Zheng, Xiao-Ping Song, Guang-Ying Chen, Yu-Hua
Cao, A-Qian Huang, Shao-Hui Su, Wen-Xing Chen & Hui Yang
To cite this article: Xue-Ming Zhou, Cai-Juan Zheng, Xiao-Ping Song, Guang-Ying Chen, Yu-Hua
Cao, A-Qian Huang, Shao-Hui Su, Wen-Xing Chen & Hui Yang (2016): A new phenolic glycoside
from Saprosma merrillii, Natural Product Research, DOI: 10.1080/14786419.2016.1195382
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1195382
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Date: 12 June 2016, At: 18:35

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2016.1195382
A new phenolic glycoside from Saprosma merrillii
Xue-Ming Zhoua, Cai-Juan Zhenga, Xiao-Ping Songa, Guang-Ying Chena, Yu-Hua Caoa,
A-Qian Huanga, Shao-Hui Sua, Wen-Xing Chenb and Hui Yanga
aKey laboratory of tropical Medicinal Plant chemistry of Ministry of education, college of chemistry and
b
chemical engineering, hainan Normal university, haikou, china; Jiangsu Key laboratory for Pharmacology
and safety evaluation of chinese Materia Medica, Nanjing university of chinese Medicine, Nanjing, Jiangsu,
china
ARTICLE HISTORY
ABSTRACT
received 21 March 2016
A new phenolic glycoside derivative, saproglucoside (1), along with
accepted 6 May 2016
five known phenolic glycoside derivatives (2–6) were isolated from the
stems of Saprosma merrillii. The structure of the new compound 1 was
determined by 1D and 2D NMR as well as by HRESIMS and hydrolysis.
The inhibitory activities of all compounds against seven pathogenic
bacteria and two cancer cell lines were evaluated.
KEYWORDS
antibacterial activity;
Saprosma merrillii; phenolic
glycoside derivatives
1. Introduction
The genus Saprosma (Rubiaceae) consists of about 30 species, which are mainly distributed
in the tropical Asian region, and has been used as a traditional medicine for the treatment
of fever (Ling et al. 2002; Lu et al. 2010; Quang et al. 2002; Singh et al. 2006; Wang et al. 2011,
2014). Saprosma merrillii is an endemic medicinal plant in Hainan, P.R. China. Our group recently
reported the isolation of three new ursane-type triterpenoids and many anthraquinone
derivatives, which showed antibacterial and cytotoxic activity (Li et al. 2015; Zhang et al.
2014, 2013). In our ongoing investigation of natural antibacterial products from the S. merrillii,
a new phenolic glycoside derivative, saproglucoside (1), along with five known phenolic
glycoside derivatives (Figure 1), isotachioside (2) (Inoshiri et al. 1987), tachioside (3) (Inoshiri
et al. 1987), canthoside D (4) (Kanchanapoom et al. 2002), canthoside C (5) (Kanchanapoom
et al. 2002) and koaburaside (6) (Ogawa et al. 1973; Kosuge et al. 1994) were isolated from
the stems of S. merrillii. Known compounds 2–6 were isolated from Saprosma for the first
CONTACT Guang-Ying chen
chgying123@163.com
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2016.1195382.
© 2016 Informa uK limited, trading as taylor & Francis Group

2
X.-M. ZHOu ET AL.
H
HO
O
O
1
4
O
O
O
1''
3
H
O
O
O
O
6''
O
O
6'
HO
HO
HO
O
OH
HO
HO
O
O
1'
6
3''
HO
O
5''
OH
OH
8
OH
OH
HO
OH
OH
HO
OH
2
3
OH
HO
1a
1
O
O
O
O
O
O
HO
HO
HO
HO
HO
O
O
O
O
O
HO
O
O
OH
OH
HO
HO
OH
OH
HO
OH
OH
OH
O
OH
OH
4
6
5
Figure 1. structures of compounds 1–6 and 1a.
time. The isolation and structure elucidation of 1 and the inhibitory activities of all com-
pounds against six pathogenic bacteria and two cancer cell lines are reported herein.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder with a molecular formula of
C22H30O12 (8° of unsaturation), as deduced from HRESIMS (m/z 485.1668 for 1 [M − H]-; calc’d
485.1659) and 13C NMR data. In the 1H NMR spectrum, three phenyl proton signals δH 6.74
(d, J = 8.0 Hz, H-8), δH 6.76 (d, J = 8.0 Hz, H-9) and δH 6.90 (br s, H-5), one methoxy group at
δH 3.84 (s), two singlet methyl groups at δH 1.13 and 1.25 (s), together with many protons
arising from sugar moiety were observed. The 13C NMR data showed 22 resonances, including
two ester carbonyl carbons (δC 178.0 and 176.8), six olefinic carbons (δC 149.0, 129.3, 121.9,
116.3, 112.8 and 104.4), nine O-bearing carbons, one methoxy carbon (δC 56.4), one methyne
carbon (δC 55.5), one sp3 quaternary carbon (δC 41.9) and two methyl carbons (δC 23.0 and
19.6). These spectroscopic features suggested that 1 comprised of one 6-methoxyl-7-hy-
droxytropic acid (Li & Kuo 1998; Watson &Youngson 1972) subunit, one glucose subunit and
one pantolactone subunit. The location of the glucose at C-2′′ and C-3 was confirmed by the
HMBC correlation from H-1′ to C-2′′ (as well as H-2′′ to C-1′) and H-6′ to C-3. The location of
the methoxy group at C-6 was confirmed by the HMBC correlation between 6-OMe, H-5, H-8
and C-6 (Figure S1). The glycone part of 1, a glucose moiety, was identified and characterised
by the anomeric proton doublet at δH 4.45 (1H, d, J = 7.6 Hz, H-1′). These data suggested that
the glycosyl moiety was β-glucose. The acidic hydrolysis of 1 afforded d-(-)-pantolactone
([α]25D -49.6, c 0.1, CH3OH). Further basic hydrolysis of the remainder afforded glucose and
6-methoxyl-7-hydroxytropic acid (1a). The glucose as the sole sugar identified on the basis
of thin-layer chromatography (TLC) by comparing with an authentic sugar sample. The glu-
cose isolated from the hydrolysate gave a positive optical rotation, [α]24D + 19.2 (c 0.1, MeOH)
and indicated that they are d-glucose. The 1a ([α]25 −21.2, c 0.1, MeOH) has the (S)-form
D
because of the negative specific rotation based onY.C. Li confirmation on the S-configuration
of the compound ficusol (Li & Kuo 1998). Thus, compound 1 was identified as an uncommon
phenolic glycoside derivative which comprised of (2S)-6-methoxyl-7-hydroxytropic acid,
β-d-glucose and d-(-)-pantolactone. And we named compound 1 as saproglucoside.

NATuRAL PRODuCT RESEARCH
3
The structures of known compounds 2–6 were identified by comparison with their 1H/13C
NMR spectra and [α]25D with those in the literature.
The isolated compounds were evaluated for cytotoxic activities against the mouse mel-
anoma cell line (B16F10) and the human lung adenocarcinoma cell lines (A549). However,
the IC50 values of all compounds were higher than 10 μM in the assay
The antibacterial activities of all compounds were determined against seven terrestrial
pathogenic bacteria Micrococcus tetragenus, Escherichia coli, Staphylococcus albus, Bacillus
cereus, Saphylococcus. aureus, Micrococcus luteus and Bacillus subtilis. However, the MIC values
of all compounds were higher than 20 μg/mL in the assay.
3. Experimental
3.1. General
IR spectra were recorded on a Nicolet 6700 spectrophotometer. uV spectra were recorded
on a Beckman Du 640 spectrophotometer. Optical rotations were measured on a JASCO
P-1020 digital polarimeter. NMR spectra were recorded on a Bruker AV spectrometer (400 MHz
1
for H and 100 MHz for 13C). TMS was used as an internal standard. HRESIMS spectra were
measured on a Q-TOF ultima Global GAA076 LC mass spectrometer. Silica gel (Qing Dao Hai
Yang Chemical Group Co.; 200–300 mesh) and octadecylsilyl silica gel (YMC; 12 nm–50 μM)
were used for column chromatography (CC). Precoated silica gel plates (YanTai Zi Fu Chemical
Group Co.; G60, F-254) were used forTLC. Semi-preparative HPLC was performed on an Agilent
1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm,
5 μM).
3.2. Plant material
The stems of S. merrillii were collected from Sanya, Hainan Province, China, in June 2014 and
identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal university. A
voucher specimen (No. GFM20140615) has been deposited at the Key Laboratory of Tropical
Medicinal Plant Chemistry of Ministry of Education, Hainan Normal university.
3.3. Extraction and isolation
The air-dried and powdered stems (4.5 kg) of S. merrillii were extracted three times with
85% EtOH (30 L) at room temperature (3 × 3d). After concentrated under reduced pressure,
the water soluble residue was partitioned successively by petroleum ether and EtOAc and
then fractionated to yield an ethyl acetate extract (95 g), petroleum ether extract (60 g)
and aqueous layer (100 g). The aqueous layer was subjected to silica gel CC (EtOAc, MeOH
v/v, gradient) to generate five fractions (Frs. 1–5). Frs. 4 (12 g) was applied to silica gel CC
eluted with EtOAc–MeOH (from 20:1 to 1:1) to afford three subfractions (3a–3c). Subfraction
3c was further purified by using octadecylsilyl silica gel (10% MeOH/H2O) to obtain 1
(31 mg) and 6 (22 mg). Subfractions 3b was further separated by semi-preparative HPLC
(CH3CN/H2O, 7:93 v/v) to obtain 2 (8 mg), 3 (7 mg), and 4 (8 mg) and 5 (6 mg),
respectively.

4
X.-M. ZHOu ET AL.
3.3.1. Saproglucoside (1)
White amorphous powder; [α]25 -21.8 (c 0.15, MeOH); uV (MeOH) λmax (log ε) 207 (4.2)
D
nm; IR (KBr) νmax−1 3258, 1733, 1610, 1448 and 1288 cm−1; HR-ESI-MS m/z 485.1668 [M − H]-
(C22H29O12; calc’d 485.1659); 1H NMR (MeOD-d4, 400 MHz) δ: 6.90 (1H, br s, H-5), 6.76 (1H,
d, J = 8.0 Hz, H-9), 6.74 (1H, d, J = 8.0 Hz, H-8), 4.58 (1H, s, H-2′′), 4.45 (1H, d, J = 7.6 Hz, H-1′),
4.04 (1H, dd, J = 9.2, 8.0 Hz, H-1α), 4.02 (1H, d, J = 10.4, H-4′′a), 4.00 (1H, d, J = 10.4, H-4′′b),
3.87 (1H, dd, J = 5.6, 3.2, H-6′β), 3.84 (3H, s, 6-OMe), 3.70 (1H, dd, J = 5.6, 3.6 Hz, H-6′α), 3.67
(1H, d, J = 9.2 Hz, H-1β), 3.63 (1H, d, J = 8.0 Hz, H-2), 3.37 (1H, m, H-3′), 3.35 (1H, m, H-4′),
3.30 (1H, m, H-5′), 3.29 (1H, m, H-2′), 1.25 (3H, s, H-5′′), 1.13 (3H, s, H-6′′); 13C NMR (400 MHz,
MeOD-d4) δ: 178.0 (C-1′′), 176.8 (C-3), 149.0 (C-6), 147.0 (C-7), 129.3 (C-4), 121.9 (C-9), 116.3
(C-8), 112.8 (C-5), 104.4 (C-1′), 81.1 (C-2′′), 78.3 (C-5′), 77.8 (C-3′), 77.4 (C-4′′), 74.7 (C-2′),
71.2 (C-4′), 65.4 (C-1), 62.6 (C-6′), 56.4 (6-OMe), 55.5 (C-2), 41.9 (C-3′′), 23.0 (C-5′′), 19.6
(C-6′′).
3.3.2. (2S)-6-methoxyl-7-hydroxytropic acid (1a)
White amorphous powder; [α]25D -21.2 (c 1.0 × 10−3, MeOH); 1H NMR (MeOD-d4, 400 MHz)
δ: 7.05 (1H, d, J = 1.2 Hz, H-5), 6.79 (1H, dd, J = 8.0, 1.2 Hz, H-9), 6.70 (1H, d, J = 8.0 Hz, H-8),
4.06 (1H, dd, J = 11.6, 9.2 Hz, H-1α), 3.85 (3H, s, 6-OMe), 3.72 (1H, dd, J = 11.6, 3.6 Hz, H-1β),
3.58 (1H, dd, J = 9.2, 3.6 Hz, H-2); 13C NMR (400 MHz, MeOD-d4) δ: 177.2 (C-3), 147.9 (C-6),
147.2 (C-7), 129.1 (C-4), 122.6 (C-9), 116.5 (C-8), 112.6 (C-5), 64.9 (C-1),56.5 (6-OMe) 54.8
(C-2).
3.4. Hydrolysis of Saproglucoside (1)
Acid hydrolysis of Saproglucoside (1): a solution of 1 (20 mg) in 5% H2SO4 (2 mL) was heated
under reflux for 3 h and then evaporated to dryness. The mixture was separated by the
semi-preparative HPLC to obtain a -(-)-pantolactone (4 mg) and the remainder (13 mg). The
d
remainder further basic hydrolysis in 20% KOH (1.5 mL) was heated under reflux for 2 h. The
mixture was neutralised with 1 N HCl and then evaporated to dryness. The mixture was
extracted with ethyl acetate. After evaporation, the residue was purificated by the semi-pre-
parative HPLC to give 6-methoxyl-7-hydroxytropic acid (1a) (7 mg). The water soluble residue
evaporated to dryness (contained glucose). The contained glucose was purified by the
semi-preparative HPLC.
3.5. Biological assays
Cytotoxic activity was evaluated by the MTT method as described previously (Scudiero et
al. 1988). Two cancer cell lines, ECA-109 and A549 cell lines, were used. Epirubicin was used
as a positive control. Antibacterial activity was determined by the conventional broth dilution
assay (Pierce et al. 2008). Seven terrestrial pathogenic bacteria M. tetragenus, E. coli, S. albus,
B. cereus, S. aureus, M. luteus and B. subtilis were used, and ciprofloxacin was used as a positive
control.
Disclosure statement
No potential conflict of interest was reported by the authors.

NATuRAL PRODuCT RESEARCH
5
Funding
This work was supported by the National Natural Science Foundation of China [grant number 21162009],
[grant number 31360069]; 973 Program, Ministry of Science and Technology of China [grant number
2014CB560714]; The Key Technology Project of Hainan Province [grant number ZDXN20130059]; and
The Provincial Natural science Foundation [grant number 212016].
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