A fatty acid glycoside from a marine-derived fungus isolated from mangrove plant Scyphiphora hydrophyllacea

Article abstract of DOI:10.3390/md10030598

To study the antimicrobial components from the endophytic fungus A1 of mangrove plant Scyphiphora hydrophyllacea Gaertn. F., a new fatty acid glucoside was isolated by column chromatography from the broth of A1, and its structure was identified as R-3-hydroxyundecanoic acid methylester-3- O-α-L- rhamnopyranoside (1) by spectroscopic methods including 1D and 2D NMR (HMQC, 1H-1H COSY and HMBC) and chemical methods. Antimicrobial assay showed compound 1 possessed modest inhibitory effect on Saphylococcus aureus and methicillin-resistant S. aureus (MRSA) using the filter paper disc agar diffusion method.

Full text of DOI:10.3390/md10030598

Mar. Drugs 2012, 10, 598-603; doi:10.3390/md10030598
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Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
A Fatty Acid Glycoside from a Marine-Derived Fungus Isolated
from Mangrove Plant Scyphiphora hydrophyllacea
Yan-Bo Zeng 1,2,†, Hui Wang 1,2,†, Wen-Jian Zuo 1,2, Bo Zheng 1,2, Tao Yang 1, Hao-Fu Dai 1,2,
*
and Wen-Li Mei 1,2,
*
1
Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine,
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural
Sciences, Haikou 571101, Hainan, China; E-Mails: zengyanbo@163.com (Y.-B.Z.);
wanghuilily2000@163.com (H.W.); zuowenjian88@163.com (W.-J.Z.);
zhengbofootball@163.com (B.Z.); yangtaoyy3@126.com (T.Y.)
2
Haikou Key Laboratory for Research and Development of Tropical Natural Products,
Haikou 571101, Hainan, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: daihaofu@itbb.org.cn (H.-F.D.);
meiwenli@itbb.org.cn (W.-L.M.); Tel./Fax: +86-898-6696-1869 (H.-F.D.);
+86-898-6698-7529 (W.-L.M.).
Received: 16 January 2012; in revised form: 14 February 2012 / Accepted: 1 March 2012 /
Published: 6 March 2012
Abstract: To study the antimicrobial components from the endophytic fungus A1 of
mangrove plant Scyphiphora hydrophyllacea Gaertn. F., a new fatty acid glucoside was
isolated by column chromatography from the broth of A1, and its structure was identified as
R-3-hydroxyundecanoic acid methylester-3-O-α-L-rhamnopyranoside (1) by spectroscopic
1
methods including 1D and 2D NMR (HMQC, H-1H COSY and HMBC) and chemical
methods. Antimicrobial assay showed compound 1 possessed modest inhibitory effect on
Saphylococcus aureus and methicillin-resistant S. aureus (MRSA) using the filter paper
disc agar diffusion method.
Keywords: marine endophyte; secondary metabolite; Scyphiphora hydrophyllacea;
R-3-hydroxyundecanoic acid methylester-3-O-α-L-rhamnopyranoside

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599
1. Introduction
Marine-derived fungi have proved to be a rich source of structurally unique and biologically active
secondary metabolites [1]. Marine natural products have been the focus of discovery for new products
of chemical and pharmacological interest. Over the last decade, many new metabolites have been
isolated and identified from marine-derived fungi and their biological activities have been
evaluated [2,3]. Previously, phytochemical studies showed that mangrove plant Scyphiphora
hydrophyllacea Gaertn. F. is a rich source of new iridoids, which have been found to possess cytotoxic
activity [4–7]. Therefore, endophytic fungi isolated from S. hydrophyllacea were studied [8]. During
our ongoing investigation on new natural antibacterial agents from endophytic fungi isolated from
mangrove plant, the fungus A1 isolated from S. hydrophyllacea attracted our attention because the
EtOAc extract of the fungus culture showed inhibitory activity against methicillin-resistant
Saphylococcus aureus (MRSA). Further investigation on the secondary metabolites from the culture
broth of the fungus A1 led to the isolation of a new fatty acid glycoside by using column
chromatography, and its structure was unambiguously elucidated as R-3-hydroxyundecanoic acid
methylester-3-O-α-L-rhamnopyranoside (1) (Figure 1). Herein we report the isolation, structural
elucidation, and biological activity of this compound.
Figure 1. The structure of compound 1.
2. Results and Discussion
2.1. Structural Elucidation
Compound 1 was obtained as colorless oil. The IR spectrum of 1 showed absorption bands for
OH (3404 cm−1), C=O (1711 cm−1), and C–O (1050 cm−1) functional groups in the molecule. The
[M + Na]+ at m/z 385.2201 in the HR-ESI-MS spectrum corresponds to the molecular formula
C18H34O7 (calc. 385.2202), indicating two degrees of unsaturation. The 13C-NMR (Table 1) along with
the DEPT experiments revealed the presence of 18 carbon atoms including one carbonyl carbon,
six methines, eight methylenes, and three methyl groups. Five carbon signals (δC 98.1, 72.9, 71.6, 71.4,
68.4) resonated in the region 60–100 ppm and a methyl group (δC 17.3) were ascribed to be a rhamnose
unit. The anomeric proton of rhamnose at 4.87 (1H, d, J = 1.2 Hz) suggested that the rhamnose was in
α-orientation. The HMQC spectrum allowed all carbon resonances to be unambiguously assigned to
the resonances of their directly attached protons. In the 1H-1H COSY spectrum, the correlations of H-1′

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600
to H-2′, H-2′ to H-3′, H-3′ to H-4′, H-4′ to H-5′ and H-5′ to H-6′ allowed to structure the rhamnose. In
the HMBC spectrum, the correlation between H-1′ and C-3 (δC 74.0) suggested the rhamnose unit link
at the C-3 position. With a shift of 172.2 ppm, the carbonyl carbon C-1 was determined to be a
conjugated ester function. The chemical shift of C-12 (δC 51.7) and the HMBC correlation from H-12
(δH 3.77) to C-1 (δC 172.2) revealed that C-12 was connected with C-1 through an oxygen atom
1
(Figure 2). The HMBC correlations of H-2 to C-1, H-2 to C-3, H-3 to C-1 and H-1H COSY
correlation of H-2 to H-3 suggested that C-1 was connected with C-3 via C-2, as shown in Figure 2.
1
The H-1H COSY correlations of H-3 to H-4 and the HMBC correlations of H-4 to C-3, H-4 to C-2
revealed that C-4 was connected with C-3. The remaining fatty acid chain was assigned by comparing
with the spectral data of R-3-hydroxydodecanoic acid methylester [9]. Compared to known compound
rhamnolipid 3, compound 1 had one more methylene in fatty acid chain and the C-1 hydroxyl was
methylated [10]. Thus, the primary structure of 1 was obtained (Figure 1). The relative configuration
of C1′-C5′ centers of the sugar moiety could be deduced by analysis vicinal coupling constants for the
sugar proton signals. Thus, a H1′/H2′ diequatorial relationship was indicated by the 1.2 Hz coupling
constant observed at H1. Likewise, the big coupling constant observed in the triplet signal for H4′
(δ 3.44, t, J = 9.6 Hz) demonstrated that H3′, H4′, and H5′ were axial. The stereochemistry of the
asymmetric center of 1 at C-3 was proposed to be R by acid hydrolysis, similar to R-3-hydroxydecanoic
acid methylester [9], based on the negative sign of its specific rotation ([α]2D1 −20.5, (c 1.0, CHCl3)).
Meanwhile, the sugar moiety of 1 was determined as L-rhamnose based on the positive sign of its
specific rotation [11]. Thus, the structure of 1 was established as R-3-hydroxyundecanoic acid
methylester-3-O-α-L-rhamnopyranoside.
Table 1. 1H- and 13C-NMR data of compound 1.
No.
1
2
3
4
5
6
7
8
δC
δH
172.2 (s)
40.1 (t)
74.0 (d)
33.2 (t)
24.8 (t)
29.2 (t)
29.7 (t)
29.6 (t)
31.8 (t)
22.6 (t)
14.0 (q)
51.8 (q)
98.1 (d)
71.4 (d)
71.6 (d)
72.9 (d)
68.4 (d)
17.3 (q)
2.51 (2H, m)
4.08 (1H, m)
1.52 (2H, m)
1.25–1.28 (2H, overlapped)
1.25–1.28 (2H, overlapped)
1.25–1.28 (2H, overlapped)
1.25–1.28 (2H, overlapped)
1.25–1.28 (2H, overlapped)
1.25–1.28 (2H, overlapped)
0.88 (3H, t, 6.8 Hz)
9
10
11
12
1′
2′
3′
4′
5′
6′
3.68 (3H, s)
4.87 (1H, d, 1.2 Hz)
3.86 (1H, brs)
3.69 (1H, overlapped)
3.44 (1H, t, 9.6 Hz)
3.60 (1H, m)
1.25–1.28 (3H, overlapped)

Mar. Drugs 2012, 10
Figure 2. Key 1H-1H COSY (bold lines) and HMBC (arrows) correlations of compound 1.
601
2.2. Biological Activities
Antibacterial tests demonstrated that compound 1 showed modest inhibitory effects on
Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), diameters of inhibition zones of
which were 9.8 and 10.7 mm, respectively. The diameters of inhibition zones of the positive control,
kanamycin sulfate, were 22.8 and 18.2 mm, respectively.
3. Experimental Section
3.1. General
The IR spectrum was obtained on a Nicolet 380 FT-IR instrument from KBr pellets. The UV spectrum
was measured on a Shimadzu UV-2550 spectrometer. Optical rotation was recorded using a Rudolph
Autopol III polarimeter (USA). The NMR spectra were recorded on a Bruker AV-400 spectrometer,
using TMS as an internal standard. The HR-ESI-MS spectrum was measured with an API QSTAR
Pulsar mass spectrometer. Column chromatography (CC) was performed with silica gel (Marine
Chemical Industry Factory, Qingdao, China) and Sephadex LH-20 (Merck, Darmstadt, Germany). TLC
was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, China).
3.2. Fungal Material and Fermentation
The marine-derived fungus A1 (unidentified) was isolated from the leaves of mangrove plant
Scyphiphora hydrophyllacea Gaertn. F. (No. SH20081205), which were collected in Wenchang county
in Hainan Province (China) in December, 2008. The strain was deposited in the Institute of Tropical
Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China,
and maintained on potato dextrose agar (PDA) slant at 4 °C. The marine-derived fungus A1 was grown
on PDA at room temperature for 5 days. Three pieces of mycelial agar plugs (0.5 × 0.5 cm2) were
inoculated into 1 L Erlenmeyer flasks containing 300 mL potato dextrose broth. The cultivation was
shaken at 120 rpm at room temperature for 7 days, and then kept in still at room temperature for 45 days.

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3.3. Extraction and Isolation
The culture broth (130 L) was filtered to give the filtrate and mycelia. The filtrate was evaporated
in vacuo to small volume and then partitioned in succession between H2O and petroleum ether, EtOAc,
n-Butanol. The EtOAc solution was evaporated under reduced pressure to give a crude extract (10.5 g),
which was separated into 11 fractions (Fr.1–Fr.11) on a silica-gel column using a step gradient elution
of CHCl3/MeOH (1:0 → 0:1). Fraction 10 (427.1 mg) was purified by column chromatography over
Sephadex LH-20 with CHCl3/MeOH (1:1) as eluent, yielding 3 subfractions. Subfraction 1 (90.8 mg)
was submitted to chromatography on a silica-gel column with petroleum ether/acetone (3:1) as eluent,
yielding compound 1 (14.5 mg).
3.4. Acid Hydrolysis
Compound 1 (5 mg) was dissolved in MeOH (2 mL) and 5% H2SO4 solution (2 mL) and hydrolyzed
under refluxed at 80 °C for 3 h. The mixture was diluted twofold with distilled water, and partitioned
between CHCl3 and H2O. The aqueous layer was neutralized with aqueous NaHCO3 solution (1 M) and
evaporated to afford the residue. The TLC analysis (CHCl3–MeOH 8:2, v/v, Rf = 0.43) with authentic
sample suggested the presence of L-rhamnose, and its identity was confirmed after preparative TLC in
the same solvent and by measurement of its optical rotation dispersion as L-rhamnose ([α]2D1 +7.7,
(c 0.15, H2O)). The CHCl3 extract was subjected to CC (silica gel, petroleum/acetone 3:1), yielding
R-3-hydroxydecanoic acid (2.3 mg; identified by 1H-NMR), 1H-NMR (400 MHz, CD3OD): 3.96–3.98
(1H, m, H-3), 2.44 (1H, dd, J = 5.0, 15.5 Hz, H-2a), 2.36 (1H, dd, J = 8.1, 15.0 Hz, H-2b), 1.23–1.48
(14H, m, H-4, 5, 6, 7, 8, 9, 10), 0.90 (3H, d, J = 7.0 Hz, H-11).
3.5. Antibacterial Activity
Compound 1 was tested for antibacterial activity against S. aureus and MRSA strains (obtained
from Food and Drug Administration of Hainan Province, Haikou, China) using the filter paper disc
agar diffusion method [12]. The strains were cultured using nutrient agar. Fifty microliters (28 mM) of
the compound were impregnated on sterile filter paper discs (6-mm diameter), and then, aseptically
applied to the surface of the agar plates. Ten microliters (0.08 mg/mL) of kanamycin sulfate were used
as positive control. Then the diameters of inhibition zones were measured after 24 h incubation at
room temperature. Experiments were done in triplicate, and the results presented as mean values of the
three measurements.
4. Conclusions
In summary, one new secondary metabolite was characterized from a marine-derived fungus A1
isolated from mangrove plant S. hydrophyllacea and its chemical structure was solved by
spectroscopic and chemical analysis. Antibacterial tests demonstrated that compound 1 showed modest
inhibitory effects on Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), diameters of
inhibition zones of which were 9.8 and 10.7 mm, respectively.

Mar. Drugs 2012, 10
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Acknowledgments
This work was financial supported by National Natural Science Foundation of China (No. 20862020).
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