A new iridoid glycoside and a new cinnamoyl glycoside from Scrophularia ningpoensis Hemsl

Article abstract of DOI:10.1080/14786419.2017.1306704

A new iridoid glycoside, namely 8-O-(threo-2, 3-dihydroxyl-3-phenyl-propionoyl)-harpagide (1), along with a new cinnamoyl glycoside named as cis-sibirioside A (2), were isolated from Scrophularia ningpoensis Hemsl. Their chemical structures were completely established by spectroscopic methods and comparison with related literatures. Compound 1 exhibited moderate antifouling effect against the settlement of Balanus amphitrite larvae with IC₅₀ being 13.5?μg/mL and LC₅₀?>?25?μg/mL.

Full text of DOI:10.1080/14786419.2017.1306704

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new iridoid glycoside and a new cinnamoyl
glycoside from Scrophularia ningpoensis Hemsl
Jun Zhang, Fanny C. F. Ip, Yan Liang, Nancy Y. Ip, Ba-Lian Zhong, Chun-Wang
Lai & Shi-Hai Xu
To cite this article: Jun Zhang, Fanny C. F. Ip, Yan Liang, Nancy Y. Ip, Ba-Lian Zhong,
Chun-Wang Lai & Shi-Hai Xu (2017): A new iridoid glycoside and a new cinnamoyl
glycoside from Scrophularia ningpoensis Hemsl, Natural Product Research, DOI:
10.1080/14786419.2017.1306704
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Date: 10 April 2017, At: 05:44

Natural Product research, 2017
http://dx.doi.org/10.1080/14786419.2017.1306704
A new iridoid glycoside and a new cinnamoyl glycoside from
Scrophularia ningpoensis Hemsl
Jun Zhanga,b,c, Fanny C. F. Ipb, Yan Lianga,b, Nancy Y. Ipb, Ba-Lian Zhonga,
Chun-Wang Laia and Shi-Hai Xuc
aNational engineering research center of Navel orange, Gannan Normal university, Ganzhou, P.r. china;
bdivision of life science, the hong Kong university of science andtechnology, hong Kong, P.r. china; ccollege
of science and engineering, Jinan university, Guangzhou, P.r. china
ABSTRACT
ARTICLE HISTORY
received 18 January 2017
accepted 5 March 2017
A new iridoid glycoside, namely 8-O-(threo-2, 3-dihydroxyl-3-phenyl-
propionoyl)-harpagide (1), along with a new cinnamoyl glycoside
named as cis-sibirioside A (2), were isolated from Scrophularia
ningpoensis Hemsl. Their chemical structures were completely
established by spectroscopic methods and comparison with related
literatures. Compound 1 exhibited moderate antifouling effect against
the settlement of Balanus amphitrite larvae with IC50 being 13.5 μg/mL
and LC50 > 25 μg/mL.
KEYWORDS
Scrophularia ningpoensis
hemsl; iridoid glycoside;
cinnamoyl glycoside;
antifouling
1. Introduction
The genus Scrophularia of the family Scrophulariaceae, versatile in metabolising varieties of
iridoid glycosides being commonly considered as its chemotaxonomic markers (Ramunno
et al. 2006; Venditti et al. 2016), encloses more than 300 species widely distributed in China.
Scrophularia ningpoensis Hemsl., commonly known as‘Xuanshen’in Chinese, has been used
in traditional Chinese medicine practice for over 2000 years, which is frequently utilised in
combination with other herbs as a nutrient and a health strengthening agent (Sagare et al.
2001).
Previous chemical investigations have identified more than 30 iridoid glycosides and
sugar esters from S. ningpoensis (Kajimoto et al. 1989; Qian et al. 1992; Li et al. 1999, 2000a,
CONTACT Jun Zhang
junyan@jnu.edu.cn
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1306704.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
J. ZHANG ET AL.
Figure 1. chemical structures of compounds 1–2.
2009; Zou & Yang 2000; Nguyen et al. 2005; Chen et al. 2007, 2008; Niu et al. 2009). Some of
these compounds were found to exhibit antibacterial (Li et al. 2009), antioxidant (Li et al.
2000b), cytotoxic (Nguyen et al. 2005) and neuroprotective (Kim SR & Kim YC 2000; Kim
et al. 2002; Wang et al. 2009) activities. In view of the herb’s versatility in metabolising struc-
turally and biologically interesting compounds, S. ningpoensis was selected and systemati-
cally explored by us, resulting in the isolation of three unusual zwitterionic alkaloids in our
previous investigation (Zhang et al. 2015). Herein, we reported the isolation, structural elu-
cidation and antifouling effect of a new iridoid glycoside, namely 8-O-(threo-2, 3-dihydrox-
yl-3-phenyl-propionoyl)-harpagide (1), along with a new cinnamoyl glycoside named as
cis-sibirioside A (2) (Figure 1), from the other fraction of the same crude extract of S. ning-
poensis as described previously.
2. Results and discussion
The 70% EtOH/H2O crude extract of the dried root of S. ningpoensis was suspended in water
and extracted subsequently with petroleum ether, EtOAc and n-BuOH. From the n-BuOH
extract, a new iridoid glycoside (1) and a new cinnamoyl glycoside (2) were isolated by means
of macroporous resin column chromatograph, silica gel column chromatography and pre-
parative reversed-phase HPLC.
Compound 1 was obtained as an amorphous grayish powder. The molecular formula of
1 was determined to be C24H32O13 by HR-ESI-MS, displaying ion peak [M + Na]+ at m/z
551.1743 (calcd for C24H32NaO13+ at m/z 551.1741). The IR spectrum of compound 1 showed
absorption bands for hydroxyl group (3373 cm−1), carbonyl group (1719 cm−1) and aromatic
moiety (1648 cm−1). Preliminary inspection of the 1H NMR spectrum of 1 led to the identifi-
cation of the following representative signals: δH 1.70 (3H, s, H-10), 2.09 (1H, dd, J = 15.0,
4.2 Hz, H-7a), 2.69 (1H, d, J = 15.0 Hz, H-7b), 3.76 (1H, s, H-9), 5.17 (1H, d, J = 6.0 Hz, H-4), 5.32
(1H, d, J = 9.0 Hz, H-1″), 6.69 (1H, s, H-1), 6.52 (1H, d, J = 6.0 Hz, H-3), as well as five aromatic
protons δH 7.85 (2H, d, J = 7.8 Hz, H-5′, H-9′), 7.37 (2H, t, J = 7.5 Hz, H-6′, H-8′), 7.26–7.29 (1H,
m, H-7′). On the basis of above information, compound 1 was assumed to be an iridoid
glycoside, similar to harpagoside previously isolated from Scrophularia buergeriana Miq.
(Kitagawa et al. 1967) and Harpagophytum procumbens DC. (Qi et al. 2006). This assumption
was in good agreement with the 13C NMR data of 1, and further clearly supported by the
detailed 2D NMR analyses involving 1H-1H COSY, HSQC and HMBC (Figure S1 in Supporting

NATURAL PRODUCT RESEARCH
3
Information, SI). The 1H and 13C NMR data (Table S1 in SI) of compound 1 were very similar
to those of harpagoside with the major difference being that two olefinic signals of the
cinnamoyl moiety in harpagoside were replaced by the signals of two oxygenated CH at δH
4.80 (1H, d, J = 7.5 Hz, H-2′)/δC (77.2, C-2′) and δH 5.35 (1H, d, J = 7.5 Hz, H-3′)/δC (75.8, C-3′),
respectively, suggesting the presence of 2,3-dihydroxy-3-phenyl-propionoyl moiety in 1,
instead of cinnamoyl one. This assignment was unambiguously corroborated by the 1H-1H
COSY displaying cross peak between H-2′ and H-3′, in combination with the diagnostic
correlations from H-2′ to carbonyl carbon C-1′ (δC 173.5), and the H-3′ to aromatic carbon
C-5′/C-9′ (δC 128.0) in HMBC (Figure S1 in SI). The relative configuration of the two hydroxyl
groups at C-2′ and C-3′, respectively, was tentatively assigned to be threo, by the coupling
constant of J2′,3′ = 7.5 Hz (~3.0/8.0 for erythro/ threo, respectively) (Hada et al. 1988; Sung
et al. 2001). This assumption was consistent with the results expected from the ChemBio3D
software, by which the dihedral angle between H-2′and H-3′ of the energy-minimised con-
figuration was calculated to be 53° for threo vs 75° for erythro (Figure S2 in SI), leading to the
rough assignment that the predicted J2′,3′ for threo was more close to the experimental data
(7.5 Hz) than that for erythro according to Karplus equitation (Minch 1994). Consequently,
compound 1 was assigned as 8-O-(threo-2, 3-dihydroxyl-3-phenyl-propionoyl)-harpagide.
Compound 2 was obtained as an amorphous brown powder. Its molecular formula
C21H28O12 was determined by HR-ESI-MS displaying ion peak at m/z 495.1453 [M + Na]+ (calcd
for C21H28NaO12+ at m/z 495.1478). After acid hydrolysis, fructose and glucose were detected
by GC analysis. The IR spectrum suggested the presence of hydroxyl (3377 cm−1) and con-
jugated ester groups (1700 cm−1). The NMR data of 2 was similar to that of sibirioside A first
isolated from the Veronicastrum sibiricum (L.) Pennell (Lin et al. 1995), with the major differ-
ence being that the signals of trans olefinic double bond in sibirioside A was replaced by
the signals of the cis one. The 1H NMR spectrum of 2 (Table S1 in SI) displayed typical signals
arising from a monosubstituted benzene ring at δH 7.60–7.62 (2H, m, H-2/H-6), 7.33–7.36
(3H, m, H-3/H-4/H-5) and a cis CH = CH group at δH 7.04 (1H, d, J = 12.6 Hz, H-7), 6.03 (1H, d,
J = 12.6 Hz, H-8), suggesting the presence of cis-cinnamoyl moiety in 2, which was confirmed
1
by the H–1H COSY correlations, together with the correlations from H-7 to C-9 (δC 167.8)
and H-8 to C-1 (δC 136.2) in HMBC (Figure S1 in SI). In addition, the typical signal at δH 5.36
(1H, d, J = 3.3 Hz, H-1′) ascribing to the anomeric proton of the α-glycopyranose, together
with the 13C NMR spectrum of 2 exhibiting signals indicative of two anomeric carbons at δC
105.2 (C-2″) and 93.4 (C-1′) and three methylene groups at δC 64.6 (C-6′), 64.1 (C-1″) and 63.9
(C-6″), strongly suggested the presence of a sucrose moiety in 2, which was consistent with
the results of GC analyses and further supported by the HMBC correlation from H-1′ to C-2″.
Furthermore, the diagnostic correlation from H-6′ (δH 4.44/4.25) to C-9 in HMBC, clearly
indicated that the cis-cinnamoyl was connected to the sucrose moiety at C-6′ position.
Therefore, compound 2 was elucidated to be 6-O-cis-cinnamoyl β-fructofuranosyl-(2→1)-
O-α-glucopyranoside and named as cis-sibirioside A.
Marine biofouling, an undesirable accumulation of marine microorganisms, algae and
animals on submerged substrates, has caused series of severe problems for maritime and
aquaculture industries worldwide (Wang et al. 2015), being one of major concerns to scien-
tists. With the global banning of organotin, one type of previously widely used antifoulant,
from production and application in antifouling paint due to displaying toxicity to target
and/or non-target marine organisms (Li et al. 2013), the search of effective and environmen-
tally benign antifouling compounds is becoming an urgent need of marine coating

4
J. ZHANG ET AL.
industries. Natural products have been proven to be a rich resource for identifying non-toxic
and effective antifoulants (Huang et al. 2014; Zhang et al. 2014). Keeping this in mind, in this
study, the isolates are evaluated for antifouling effect against the settlement of Balanus
amphitrite larvae. Pleasingly, compound 1, despite less potent than the positive control
(IC50 = 3.2 μg/mL, LC50 > 25 μg/mL), exhibits moderate inhibitory effect with IC50 being
13.6 μg/mL and LC50 over 25 μg/mL, being a potent non-toxic antifoulant. Whereas, com-
pound 2 shows no antifouling effect at the concentrations up to 25 μg/mL.
3. Experimental
3.1. General experimental procedures
Optical rotation was measured on JASCO P-2000 digital polarimeter (Jasco, Tokyo, Japan). UV
spectrum was recorded on a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto, Japan).
IR spectra were recorded on Perkin-Elmer 16 PC FT-IR spectrometer (Perkin–Elmer, Boston, USA).
NMR experiments were measured onVarian MercuryVX 300 spectrometer (Varian, Japan) oper-
ating at 300 and 75 MHz for 1H and 13C, respectively, withTMS as internal standard and chemical
shifts reported in ppm. High resolution mass spectra (HRMS) were performed on QSTAR XL
mass spectrometry system (Applied Biosystems, USA).The preparative HPLC was performed on
Waters systems (Waters, UK) involving Waters 2545 binary gradient module, Waters 2996 PDA
detector, MassLynx V4.1 workstation and XBridge RP18 column (19 x 300 mm, 5 μm). Column
chromatography (CC) was performed with silica gel (230–400 mesh, Merck, Germany) or mac-
roporous absorptive resin D101 (250–300 μm, Tianjin Pesticide Factory, P.R. China). TLC was
performed on aluminium sheet precoated with silica gel G60 (Merck, Germany). Spots were
visualised under UV light or by spraying with 5% H2SO4 in EtOH followed by heating.
3.2. Plant material
Roots of S. ningpoensis were collected in August 2006 in Ningbo, Zhejiang province of China.The
plant was identified by Dr. Guangmiao Fu and a voucher specimen (TCM-203) was deposited in
the Biotechnology Research Institute of The Hong Kong University of Science and Technology.
3.3. Extraction and isolation
Dried roots of S. ningpoensis (4.3 kg) were crushed and extracted with 70% EtOH aqueous
(3 x 10 L) for 2 h each time under refluxing condition. The combined extract solutions were
concentrated in vacuo to give a residue (505 g), which was suspended in water (0.8 L), fol-
lowed by successive extraction using petroleum ether (1 L x 3), EtOAc (1 L x 3) and n-BuOH
(1 L x 3). The concentrated n-BuOH extract (126.4 g) was subject to macroporous absorptive
resin D101 column chromatography (diameter 10 cm, height 60 cm) and eluted with solu-
tions of EtOH/H2O (0, 30%, 60%, 95%, each 3 L) to give 8 fractions (Frs. A-H). Fr. G (8.5 g) was
subject to silica gel column chromatography (diameter 4 cm, height 20 cm), which was eluted
with solutions of CHCl3-MeOH-H2O (5:95:0, 10:90:0.2, 15:85:0.5, 20:80:1, 25:75:2, 30:70:5, each
1 L) to give thirteen fractions (Frs. G1-G13). Fr. G4 (85 mg) was further purified by prep.
RP-HPLC, which was eluted with 30% CH3CN-H2O to give compounds 1 (4.5 mg, tR 13.5 min)
and 2 (3.6 mg, tR 10.2 min).

NATURAL PRODUCT RESEARCH
5
3.3.1. 8-O-(threo-2, 3-Dihydroxyl-3-phenyl-propionoyl)-harpagide (1)
Amorphous grayish powder; [ꢀ]D25 − 10.4 (c = 0.19, CH3OH); IR (film) vmax: 3373, 2929, 1719,
1648, 1235, 1077 cm−1; UV (CH3OH): λmax (log ε): 206 (2.45), 271 (1.86) nm; 1H NMR (300 MHz,
pyridine-d5), δH: 6.69 (1H, s, H-1), 6.52 (1H, d, J = 6.0 Hz, H-3), 5.17 (1H, d, J = 6.0 Hz, H-4), 4.08
(1H, d, J = 4.2 Hz, H-6), 2.09 (1H, dd, J = 15.0, 4.2 Hz, Ha-7), 2.69 (1H, d, J = 15.0 Hz, Hb-7), 3.76
(1H, s, H-9), 1.70 (1H, s, H-10), 4.80 (1H, d, J = 7.5 Hz, H-2′), 5.35 (1H, d, J = 7.5 Hz, H-3′), 7.85
(2H, d, J = 7.8 Hz, H-5′, H-9′), 7.37 (2H, t, J = 7.5 Hz, H-6′, H-9′), 7.26–7.29 (1H, m, H-7′), 5.32
(1H, d, J = 9.0 Hz, H-1″), 3.99–4.04 (1H, m, H-2″), 4.28–4.29 (1H, m, H-3″), 4.27–4.29 (1H, m,
H-4″), 3.99–4.02 (1H, m, H-5″), 4.53 (1H, d, J = 11.7 Hz, Ha-6″), 4.37 (1H, dd, J = 11.7, 4.8 Hz,
Hb-6″); 13C NMR (75 MHz, pyridine-d5), δC: 93.9 (C-1), 141.6 (C-3), 108.4 (C-4), 73.2 (C-5), 77.0
(C-6), 44.9 (C-7), 88.2 (C-8), 55.0 (C-9), 22.3 (C-10), 173.5 (C-1′), 77.2 (C-2′), 75.8 (C-3′), 143.3
(C-4′), 128.0 (C-5′, C-9′), 128.1 (C-6′, C-8′), 127.5 (C-7′), 98.1 (C-1″), 74.6 (C-2″), 78.3 (C-3″), 71.3
+
(C-4″), 78.7 (C-5″), 62.3 (C-6″); HR-ESI-MS: m/z 551.1743 [M + Na]+ (calcd for C24H32NaO13
:
551.1741).
3.3.2. cis-Sibirioside A (2)
Amorphous brown powder;[ꢀ]2D5 + 50.0(c = 0.08, CH3OH); IR (film) vmax: 3377, 2930, 1700,
1
1633, 1056 cm−1; UV (CH3OH): λmax (log ε): 203 (2.62), 216 (2.55), 279 (2.37) nm; H NMR
(300 MHz, CD3OD), δH: 7.60–7.62 (2H, m, H-2, H-6), 7.33–7.36 (3H, m, H-3, H-4, H-5), 7.04
(1H, d, J = 12.6 Hz, H-7), 6.03 (1H, d, J = 12.6 Hz, H-8), 5.36 (1H, d, J = 3.3 Hz, H-1′), 3.38
(1H, dd, J = 9.6, 3.3 Hz, H-2′), 3.67–3.71 (1H, m, H-3′), 3.23–3.27 (1H, m, H-4′), 3.99–4.05
(1H, m, H-5′), 4.44 (1H, d, J = 11.7 Hz, Ha-6′), 4.25 (1H, dd, J = 11.7, 4.8 Hz, Hb-6′), 3.59–3.61
(1H, m, H-1″), 4.10 (1H, d, J = 8.4 Hz, H-3″), 3.96–4.02 (1H, m, H-4″), 3.77–3.93 (1H, m,
H-5″), 3.73–3.79 (2H, m, H-6″); 13C NMR (75 MHz, CD3OD), δC: 136.2 (C-1), 130.9 (C-2, C-6),
129.1 (C-3, C-5), 130.1 (C-4), 144.8 (C-7), 120.1 (C-8), 167.8 (C-9), 93.4 (C-1′), 73.1 (C-2′),
74.5 (C-3′), 71.5 (C-4′), 71.9 (C-5′), 64.6 (C-6′), 64.1 (C-1″), 105.2 (C-2″), 79.1 (C-3″), 75.9
+
(C-4″), 83.9 (C-5″), 63.9 (C-6″); HR-ESI-MS: m/z 495.1475 [M + Na]+ (calcd for C21H28NaO12
:
495.1478).
3.4. Acid hydrolysis
The acid hydrolysis of the glycoside was performed according to the literature (Nord & Kenne
1999). Briefly, The isolate 2 (1.0 mg) were hydrolysed in 2 M CF3COOH (0.5 mL) at 120 °C for
1 h, respectively. Then the solvent was removed by blowing with N2 to give a residue, to
which 1 M NH4OH (0.2 mL) was added, followed by the addition of the solution of NaBH4
(4 mg) in 1 M NH4OH (0.3 mL). The mixture then was reacted at 40 °C for 1 h. Excess NaBH4
was quenched with 0.5 mL AcOH and the boric acid formed was removed by co-distillation
with 10% AcOH in MeOH (3 x 0.5 mL) followed by MeOH (3 x 0.5 mL). The resulting alditols
were acetylated using Ac2O-Pyridine (1:1, 0.6 mL) at 120 °C for 40 min. Then the solvent was
evaporated by blowing with N2, the resulting residue was extracted with DCM (0.5 mL). The
obtained DCM solution was subject to GC analysis under the following conditions:
HP 6850 gas chromatograph equipped with an FID detector. Column: HP-1 column
(30 m x 0.25 mm x 0.25 μm). Column temperature: 170 °C. Carrier gas: H2 (2.0 mL/min).
Injector and detector temperature: 220 °C/300 °C. Injection volume: 1μL. GC analysis showed
the presence of glucose (tR 12.44 min) and fructose (tR 11.93 min and 12.44 min) in 2 with a

6
J. ZHANG ET AL.
ratio of 1:1. Retention time of the authentic samples after being treated with the same pro-
cedure as that of 2, were detected at 12.44 min for D-glucose, 11.93 min and 12.44 min for
D-fructose.
3.5. Antifouling assay
The antifouling assay was evaluated according to the procedure reported previously (Zhang
et al. 2014). Briefly, Fresh cyprids of the barnacle Balanus amphitrite were used in the testing.
Larval settlement assays were examined using 24-well polystyrene plates with Sea-Nine
211TM (4,5-dichloro-2-n-octyl-4-iso-thiazolin-3-one) being served as positive control. The
tested samples and Sea-Nine 211TM were dissolved with small amount of dimethyl sulfoxide
(DMSO) and then diluted with 0.22 μm filtered seawater (FSW) to achieve the final concen-
trations of 25.0, 12.5, 6.25, 3.13 and 1.56 μg/mL. About 15−20 competent larvae were added
to each well containing 1 mL of test solution in triplicate, with wells having only FSW, DMSO
and larvae being served as negative control. The plates were incubated at 25 °C for 48 h,
followed by being examined under a microscope to count settled, unsettled larvae and
where appropriate, potential toxic effects were recorded. The number of settled larvae was
expressed as a percentage of the total number of larvae per well. The EC50 was calculated as
the concentration where 50% of the larval individuals were inhibited to settle as compared
to the control, while LC50 was calculated as the concentration where 50% of the larval pop-
ulation was dead.
4. Conclusions
In summary, as part of our searching for bioactive metabolites from natural resources, the
versatile herb S. ningpoensis was selected and chemically investigated, leading to the iden-
tification of one new iridoid glycoside, namely 8-O-(threo-2, 3-dihydroxyl-3-phenyl-propi-
onoyl)-harpagide (1), along with a new cinnamoyl glycoside named as cis-sibirioside A (2).
Structurally, compound 1, containing a threo-2, 3-dihydroxyl-3-phenyl-propionoyl group
which is unusual within natural products, is interesting, to the best of our knowledge, this
is the first report of natural products containing this kind of group. Biologically, compound
1 exhibits moderate antifouling effect against the settlement of Balanus amphitrite larvae
with IC50 being 13.5 μg/mL and LC50 > 25 μg/mL, being a potential lead compound for further
investigation.
Disclosure statement
No potential conflict of interest was reported by the authors
Funding
This work was financially supported in part by the National Natural Science Fund of China [grant
number 41506156]; Natural Science Fund of Guangdong Province [grant number 2016A030310095];
Research Foundation from Department of Education of Jiangxi Province [grant number 460911]; and
Hong Kong Research Grants Council Theme-based Research Scheme [grant number T13-607/12R].

NATURAL PRODUCT RESEARCH
7
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