Rhamnellosides A and B, ω-phenylpentaene fatty acid amide diglycosides from the fruits of rhamnella franguloides

Article abstract of DOI:10.3390/molecules23040752

Two new ω-phenylpentaene fatty acid amide diglycosides, rhamnellosides A (1) and B (2), were isolated from the fruits of Rhamnella franguloides (Rhamnaceae). These compounds were prioritized using LC-MS/MS molecular networking dereplication based on our p

Full text of DOI:10.3390/molecules23040752

molecules
Article
Rhamnellosides A and B, ω-Phenylpentaene Fatty
Acid Amide Diglycosides from the Fruits of
Rhamnella franguloides
Kyo Bin Kang 1,†, Ming Gao 2,†, Geum Jin Kim 2, Hyukjae Choi 2,* and Sang Hyun Sung 1,
*
1
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University,
Seoul 08826, Korea; kbinkang@gmail.com
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Korea; gaoming104@hotmail.com (M.G.);
2
kimgeumjin@naver.com (G.J.K.)
*
Correspondence: h5choi@yu.ac.kr (H.C.); shsung@snu.ac.kr (S.H.S.);
Tel.: +82-53-810-2824 (H.C.); +82-2-880-7859 (S.H.S.)
†
These authors equally contributed to this manuscript.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 March 2018; Accepted: 23 March 2018; Published: 24 March 2018
Abstract: Two new
ω-phenylpentaene fatty acid amide diglycosides, rhamnellosides A (
1) and B
(
2
), were isolated from the fruits of Rhamnella franguloides (Rhamnaceae). These compounds were
prioritized using LC-MS/MS molecular networking dereplication based on our previous discovery
of 2-acetoxy- -phenylpentaene fatty acid triglycosides berchemiosides A C from a phylogenetically
ω
−
related species, Berchemia berchemiifolia. The structures of the isolated compounds were elucidated
by spectroscopic analyses in combination with chemical derivatization. The pentaene groups of
1
and 2 were found to have (6E, 8E, 10Z, 12Z, 14E)-geometry, which is the same as that found in
berchemioside A.
Keywords: Rhamnella franguloides; Rhamnaceae; ω-phenylpentaene fatty acid amide; dereplication
1. Introduction
The genus Rhamnella (family Rhamnaceae) comprises approximately 10 species of evergreen
shrubs and small trees, which are found in central China, Japan, Korea, and the Himalayas [
those species, R. gilgitica, has been used for the treatment of rheumatism, swelling, and pain in
traditional Chinese medicines [ ], but the other species are not known to have any commercial uses.
Little is known about the phytochemicals in the Rhamnella species; only a few flavonoids and fatty
acids have been reported from R. gilgitica [ ] and R. inaequilatera [ ]. In this study, we report the
targeted isolation and structural elucidation of two -phenylpentaene fatty acid amide diglycosides,
rhamnelloside A ( ) and B ( ), along with a previously known flavonoid di-C-glycoside ( ), from the
fruits of R. franguloides, the only Rhamnella species found in Korea (Figure 1). Based on our previous
discovery of 2-acetoxy- -phenylpentaene fatty acid triglycosides berchemiosides A–C ( ) from the
fruits of Berchemia berchemiifolia (Rhamnaceae) [ ], we hypothesized that phytochemicals with similar
1]. One of
2
3,4
5
ω
1
2
3
ω
4–6
6
structures would be present in species that are genetically similar to B. berchemiifolia. R. franguloides was
selected for the follow-up study because plastid DNA sequence-based analysis revealed that Rhamnella
is phylogenetically close to Berchemia [7]. Fruits of R. franguloides look similar to those of B. berchemiifolia;
they are 0.8- to 1.5-cm long drupes and are yellow when immature. However, unlike those of
B. berchemiifolia, the fruits of R. franguloides turn black when they are fully mature. For dereplication
and prioritization, MS/MS molecular networking, an emerging tool for the exploration of MS/MS
data from natural products [
8,
9], was applied to prioritize the
ω-phenylpentaene fatty acid derivatives
Molecules 2018, 23, 752; doi:10.3390/molecules23040752
www.mdpi.com/journal/molecules

Molecules 2018, 23, 752
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in R. franguloides. This method establishes an MS/MS spectral similarity network, which allows an
efficient investigation of structurally similar metabolites [10].
Figure 1. Chemical structures of compounds
isolated from B. berchemiifolia.
1–3 isolated from R. franguloides and 4–8 previously
2. Results and Discussion
Contrary to our expectation, none of the spectral nodes from the R. franguloides extract
were clustered with compounds in the molecular network, which was established based on
4–6
UHPLC-Q/TOF-MS data from the B. berchemiifolia and R. franguloides extracts (Figure 2). Only two
spectral nodes from R. franguloides could be putatively annotated by a clustering with nodes from
B. berchemiifolia; nodes in a cluster with compounds
5-O-diglycosides based on their spectral similarity to and
two species was unexpected, so we decided to focus on the dissimilarity instead. Spectral clusters
7
and
8 were suggested to be flavanone
7
8. This kind of dissimilarity between the

Molecules 2018, 23, 752
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that formed nodes with compounds only from R. franguloides were carefully investigated. A cluster
containing three nodes of precursor ions at m/z 533.127 and 563.140 was initially examined; tandem MS
spectra of those nodes exhibited characteristic neutral losses of m/z 60, 90, 120, and 150 Da, suggesting
that these nodes correspond to flavonoid di-C-glycosides [11]. This was confirmed by the targeted
isolation of compound
comparison with a reference sample [12]. Afterward, two spectral nodes, both with precursor ions
of m/z 728.3287 ([M
H]−, calcd for C38H50NO13, 728.3288), were prioritized because their even m/z
values suggested they were alkaloids. These spectral nodes exhibited two chromatographic peaks with
similar retention times to those of (Figure S1, Supplementary Materials). The characteristic broad
3, which was identified as apigenin 6,8-di-C-α-L-arabinopyranoside by spectral
−
4–6
UV absorption spectra of these peaks suggested that they are derivatives of conjugated phenylpolyene
fatty acids. The targeted isolation of these prioritized peaks yielded previously unknown compounds
1 and 2.
Figure 2. MS/MS molecular network of R. franguloides and B. berchemiifolia fruit extracts. Red nodes
represent ions from R. franguloides; blue nodes represent ions from B. berchemiifolia. Only clusters
containing at least two nodes are shown.
Compound
data suggested its molecular formula was C38H51NO13. The MS/MS spectrum of
fragment ions at m/z 434.2336 ([M C11H18O9
H]−, calcd for C27H32NO4, 434.2337) and 130.0865
([C6H12NO2]−, calcd 130.0874). The former was thought to correspond to the loss of a hexose unit
and a pentose unit, and the latter suggested that is a fatty acid amide derivative with a leucine
1
was isolated as a yellow, amorphous solid. As described above, the HRESIMS
1
exhibited
−
−
1
or isoleucine moiety. Acid hydrolysis followed by thin layer chromatography (TLC) and HPLC
analysis after arylthiocarbamoyl-thiazolidine derivatization [13] confirmed the assignments of a
D-glucosyl (Glc) unit and a D-xylosyl (Xyl) unit. Using C3 Marfey’s method [14] on the hydrolysate
1
confirmed the presence of an L-leucine unit. The H NMR spectrum of
1 confirmed that 1 is a
ω
-phenylpentaene fatty acid derivative based on the presence of an aromatic A2B2 spin system
δH 7.41 (2H, d, J = 8.7 Hz, H-17 and H-21) and 7.02 (2H, d, J = 8.7 Hz, H-18 and H-20)) and 10
conjugated polyene protons at δH 5.75–6.85 (Table 1). However, the C-1–5 spin systems were different
from those of the corresponding carbons in ; two olefinic protons at δH 6.58 (1H, m, H-3) and
6.01 (1H, d, J = 15.3 Hz, H-2) exhibited HMBC correlations with the amide carbonyl carbon at
δC 164.5, which suggested a trans- -unsaturation in (Figure 3). Two methylenes at δH 2.24 (4H,
br s, H-4, H-5) were also observed, and those resonances were attributed to H-4 and H-5 based
(
4
α
1

Molecules 2018, 23, 752
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on 1H-1H COSY and HMBC experiments. Coupling constants of the anomeric protons of the two
sugars (δH 4.80 (1H, d, J = 7.6 Hz, Glc H-1) and 4.18 (1H, d, J = 7.5 Hz, Xyl H-1)) suggested that the
D-glucose and D-xylose units were in
Xyl H-1 and Glc C-6 (δC 82.0) protons suggested a 1
correlation between Glc H-1 and C-19 (δC 157.0) confirmed the
fatty acid amide moiety. For compounds , the geometries of the conjugated double bonds were
established from the two-dimensional (2D) J-resolved NMR spectrum [ ]. Interestingly, compound
was suggested to have (6E, 8E, 10Z, 12Z, 14E)-geometry, which is identical to that of (Figure S8,
, rhamnelloside A, was defined
β
-configurations. The HMBC correlation between the anomeric
6 interglycosidic linkage, and the HMBC
-glycosylation at the phenylpentaene
→
ω
4–6
6
1
4
Supplementary Materials). Consequently, the structure of compound
1
as 15-(4-O-
hexaenoic acid L-leucinamide.
β
-D-xylopyranosyl-(1
→
6)-β-D-glucopyranosylphenyl)-pentadeca-2E,6E,8E,10Z,12Z,14E-
Table 1. The 1H (850 MHz) and 13C (212.5 MHz) NMR spectroscopic data (
and 2 (DMSO-d6).
δ
in ppm) of compounds 1
1
2
Position
a
a
δ
mult. (J in Hz)
δC
δ
mult. (J in Hz)
H
δC
H
c
1
2
3
4
5
164.5
124.9
141.7
31.1
n.d.
6.03, d (15.4) b
6.56, m
6.01, d (15.3)
6.58, m
2.24, br s
2.24, br s
125.7
141.3
31.6
2.23, br s
2.23, br s
31.1
31.6
5.78, dt (15.2, 7.6) b
6.17, dd (15.2, 10.3) b
6.29, dd (15.1, 10.3) b
6.29, dd (15.1, 9.0) b
6.37, dd (9.6, 9.0) b
6.45, dd (9.6, 9.6) b
6.37, dd (9.6, 9.6) b
6.45, dd (10.5, 9.6) b
6.87, dd (15.2, 10.5) b
6.58, d (15.2) b
6
7
8
9
5.75, dt (15.0, 7.1)
6.17, dd (15.0, 9.8)
6.27, dd (16.3, 9.8) b
6.28, dd (16.3, 8.1) b
6.35, dd (9.0, 8.1) b
6.44, dd (9.0, 9.0) b
6.35, dd (9.0, 9.0) b
6.44, dd (9.6, 9.0)
6.85, dd (15.4, 9.6)
6.56, d (15.4)
134.3
131.4
133.6
131.5
132.9
133.1
132.9
133.1
127.7
131.9
131.1
127.6
116.6
157.0
116.6
127.6
134.6
131.4
133.5
131.7
133.3
133.4
133.3
133.4
128.3
132.2
131.6
127.8
116.8
157.3
116.8
127.8
10
11
12
13
14
15
16
17
18
19
20
21
7.41, d (8.7)
7.02, d (8.7)
7.41, d (8.5)
6.98, d (8.5)
7.02, d (8.7)
7.41, d (8.7)
6.98, d (8.5)
7.41, d (8.5)
Leu (
1
)/Ile (2)
c
1
2
3
4
5
6
172.5
52.9
43.0
25.1
22.8
23.7
n.d.
n.d.
c
3.98, m
1.50, m; 1.37, m
1.58, m
0.84, d (2.3)
0.84, d (2.3)
4.01, m
1.59, m
1.36, m; 1.23, m
0.84, d (6.2) b
0.83, t (7.0) b
25.0
29.2
23.5
22.9
Glc
1
2
3
4
5
4.80, d (7.6)
3.23, m
3.27, m
3.14, m
3.53, m
100.4
73.7
76.9
70.2
76.3
68.7
4.95, d (7.6)
3.29, m
3.46, m
3.14, m
3.52, m
100.2
69.7
78.8
70.0
75.9
63.5
6
3.96, d (10.9); 3.55, m
3.36, m; 3.28, m
Xyl
1
2
3
4
4.18, d (7.5)
2.99, m
3.07, m
3.26, m
3.67, m; 2.95, m
104.4
73.9
77.0
70.1
66.1
4.28, d (7.6)
2.99, m
3.11, m
3.27, m
3.76, m; 3.08, m
103.6
74.0
76.9
69.9
65.9
5
a 13C NMR spectra were unavailable because of the scarce number of isolates; hence, chemical shifts were suggested
by HSQC and HMBC spectra.; b J values were approximately identified based on J-resolved NMR spectra.; c Not
determined in HSQC or HMBC spectra due to the scarce amount of purified 2.

Molecules 2018, 23, 752
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Figure 3. Key 1H-1H COSY and HMBC correlations of compound 1.
Compound
The MS/MS spectrum of
2
had a molecular formula of C38H51NO13, which is the same as that of
was also similar to that of ; it showed a cosine similarity of 0.6378
in the molecular network and exhibited fragment ions at m/z 434.2345 [M C11H18O9
H]− and
130.0852 [C6H12NO2]−. Some regions of the one-dimensional (1D) and 2D NMR spectra of
were
1.
2
1
−
−
2
not clear due to the small amount of
assigned to also be an
2
available (0.2 mg), but compound
2
could be confidently
1
ω
-phenylpentaene fatty acid amide derivative. In its H NMR spectrum,
two methyl group signals at δH 0.84 (3H, d, J = 6.2 Hz, Ile H-5) and 0.83 (3H, t, J = 7.0 Hz, Ile H-6)
suggested that had an isoleucine moiety instead of the leucine moiety found in (Table 1). Acid
hydrolysis and use of C3 Marfey’s method revealed the absolute configuration of the amino acid
residue in as being L-allo-isoleucine, a nonproteinogenic amino acid that naturally occurs in plants,
fungi, and human plasma [15]. The acid hydrolysis of also revealed the presence of a D-glucosyl
unit and a D-xylosyl unit. However, the resonances of Glc H-6 (δH 3.36 and 3.28) indicated that
compound did not have a 1 6 interglycosidic linkage. Otherwise, the downfield-shifted Glc
H-3 (δH 3.46) suggested the presence of a 1 3 interglycosidic linkage, and this assignment was
confirmed by the HMBC correlation between Xyl H-1 (δH 4.28 (1H, d, J = 7.6 Hz)) and Glc C-3 (δC 78.8).
Compound was also identified to have (6E, 8E, 10Z, 12Z, 14E)-geometry based on the J-resolved
NMR spectrum (Figure S16, Supplementary Materials). Thus, rhamnelloside B ( ) was defined
as 15-(4-O- -D-xylopyranosyl-(1 3)- -D-glucopyranosylphenyl)-pentadeca-2E,6E,8E,10Z,12Z,14E-
hexaenoic acid L-allo-isoleucinamide.
Isolated compound and previously isolated compounds
their antimicrobial activities against Bacillus subtilis, Escherichia coli DH5
2
1
2
2
2
→
→
2
2
β
→
β
1
4,
5, and
6 were evaluated for
α
, Pseudomonas aeruginosa,
and Staphylococcus aureus and showed no activities (minimum inhibitory concentration > 250
compound 2 could not be tested because of the scarce amount).
µM;
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a JASCO P-2000 polarimeter using a 1-cm cell. UV and
electronic circular dichroism (ECD) spectra were recorded on a Chirascan CD spectrometer (Applied
Photophysics, Surrey, UK). 1D and 2D NMR spectra were obtained with Bruker AVANCE III HD
850 spectrometers (Bruker, Billerica, MA, USA) at the National Center for Interuniversity Research
Facilities at Seoul National University (NCIRF). UHPLC-Q/TOF-MS analyses were performed on a
Waters Acquity UPLC system (Waters Co., Milford, MA, USA) coupled with a Waters Xevo G2 QTOF
mass spectrometer (Waters MS Technologies, Manchester, UK) that was equipped with an electrospray
interface (ESI). The absolute configurations of the amino acids in compounds
1 and 2 were determined
using an Agilent 6120 quadruple MSD consisting of a 1260 Infinity pump, a 1260 Infinity autosampler,
a 1260 Infinity DAD (Agilent Technologies, Santa Clara, CA, USA), and an Agilent Zorbax SB-C3
column (150
×
4.6 mm, 5 µ
m) at 50 ◦C. Semi-preparative HPLC separations were performed with
a system consisting of a Gilson 321 Pump and a UV/Vis-151 detector (Gilson Inc., Middleton, WI,
USA). Extra-pure grade solvents for extraction, fractionation, and isolation were purchased from Dae
Jung Pure Chemical Engineering Co. Ltd., Siheung, Korea. Deuterated DMSO for NMR analyses was
purchased from Merck (Darmstadt, Germany).

Molecules 2018, 23, 752
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3.2. Plant Material
R. franguloides was cultivated in the Medicinal Plant Garden, College of Pharmacy, Seoul
National University, Koyang, Korea (GPS N37◦42042.900, E126◦49010.600), and the ripe fruits from
one individual plant were collected in September 2015. The sample was authenticated by Mr. S. I.
Han (The Medicinal Plant Garden, College of Pharmacy, Seoul National University), and a voucher
specimen (SUPH-1509-05) was deposited in the Herbarium of the Medicinal Plant Garden.
3.3. LC-MS/MS Molecular Networking
Separations were conducted on a Waters Acquity UPLC BEH C18 (100 mm
column. The flow rate of the mobile phase was 0.3 mL/min, and the column temperature was
maintained at 30 ◦C. The mobile phase consisted of H2O (A) and MeCN (B) with a linear gradient of
×
2.1 mm, 1.7 µm)
10–90% B (0–20 min). Analyses of the samples (1.0 µL injected into the partial loop with needle overfill
mode) were performed using an optimized data-dependent acquisition (DDA) mode consisting of a
full MS survey scan in the 100–1500 Da range (scan time: 100 ms), followed by an MS/MS scan for the
three most intense ions. The collision energy was applied at a gradient from 20 to 80 V. The molecular
network was created using the Data Analysis workflow 2.0 on the Global Natural Products Social
Molecular Networking (GNPS) platform (http://gnps.ucsd.edu) [9]; the reliability was enhanced by
data preprocessing using MZmine 2 software [16]. Raw LC-MS files were converted into mzXML
using ProteoWizard 3.0.9935 [17] and then imported into MZmine 2.29. The mass detection was
performed with the noise level at 1000 (for MS) and 40 (for MS/MS). The chromatogram was built with
ions showing a minimum time span of 0.01 min, a minimum height of 2500, and an m/z tolerance of
0.001 (or 5.0 ppm). The chromatographic deconvolution was achieved by a baseline cut-off algorithm,
with the following parameters: minimum peak height of 1500, peak duration range of 0.02–0.15 min,
and baseline level of 500. Chromatograms were deisotoped using an algorithm for grouping isotopic
peaks with an m/z tolerance of 0.002 (or 5.0 ppm) and a tR tolerance of 0.1 min. The preprocessed
chromatograms were exported to GNPS for molecular networking. MS/MS spectra were window
filtered by choosing only the top six peaks in the
±
50 Da window throughout the spectrum. A network
was then created where edges were filtered to have a cosine score above 0.60 and more than three
matched peaks. Further edges between two nodes were kept in the network if and only if each of
the nodes appeared in each other’s respective top 10 most similar nodes. The library spectra were
filtered in the same manner as the input data. The molecular network was visualized using Cytoscape
3.5.1 [18]. The MS/MS data are deposited in the MassIVE Public GNPS data set (http://gnps.ucsd.edu,
MSV000081660).
3.4. Extraction and Isolation
Frozen fruits of R. franguloides (27.5 g including seeds) were extracted with MeOH (2
for 3 h each) with ultrasonication at room temperature, and then the extract was concentrated in
vacuo. The crude extract (582.9 mg) was suspended in 100 mL of H2O and partitioned successively
×
100 mL,
(2
a separation funnel. The EtOAc fraction was subjected to semi-preparative reversed-phase HPLC
on a YMC Triart C18 column (10 250 mm, 5 m, YMC Co. Ltd., Kyoto, Japan) eluting with 45%
aqueous MeCN to yield compounds (1.0 mg) and (0.2 mg). The BuOH fraction was purified by
semi-preparative reversed-phase HPLC on a YMC Triart C18 column (10 250 mm, 5 m, YMC Co.
Ltd., Kyoto, Japan) eluting with 20% aqueous MeCN to yield compound 3 (1.5 mg).
×
100 mL for each solvent) into CHCl3 (43.0 mg), EtOAc (15.1 mg), and BuOH (67.8 mg) using
×
µ
1
2
×
µ
Rhamnelloside A (
): Yellow, amorphous solid; [α]2D0 + 34.5 (c 0.10, MeOH); UV (MeOH) λmax (log
(3.1), 370 (2.2), 351 (3.2), 336 (3.0) nm; ECD (MeOH) λmax ) 366 ( 0.6), 334 (0.1), 309 ( 0.1), 282 (0.1),
229 (
1.0), 212 (0.0) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 728.3287 [M H]− (calcd for
C38H50NO13, 728.3288).
1
ε) 391
(
∆
−
−
−
−

Molecules 2018, 23, 752
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Rhamnelloside B (
391 (3.3), 369 (3.3), 351 (3.2), 337 (3.0) nm; ECD (MeOH) λmax
278 (0.0), 229 ( 1.0), 211 (
0.3) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 728.3298 [M
2
): Yellow, amorphous solid; [α]D20
−
42.6 (c 0.10, MeOH); UV (MeOH) λmax (log
ε
)
(
∆
) 375 (
−
0.3), 354 (
−
0.1), 327 (
−
0.3),
−
−
−
H]−
(calcd for C38H50NO13, 728.3288).
3.5. Acid Hydrolysis and Determination of the Resulting Sugars
◦
Approximately 0.5 mg of
for 2 h. The hydrolysates were extracted with EtOAc (2
1
and 0.2 mg of
2
were hydrolyzed with 1 N HCl (200
200
µL) at 80 C
×
µ
L) to remove the aglycones.
The aqueous fractions were concentrated and compared to authentic D-glucose (Rf 0.65) and
D-xylose (Rf 0.78) standards (Sigma-Aldrich, St. Louis, MO, USA) on silica gel TLC plates with
BuOH-acetone-pyridine-H2O (2:2:1:1) visualized with p-anisaldehyde. The absolute configurations of
the sugars in the hydrolysates were determined by HPLC analysis after arylthiocarbamoyl-thiazolidine
derivatization. The aqueous fractions of the hydrolysates were dissolved in pyridine (100
containing L-cysteine methyl ester hydrochloride (0.5 mg), and the mixtures were heated at 60 C
µ
◦L)
for 1 h. o-Tolyl isothiocyanate (100
added, and the mixtures were heated at 60 C for an additional 1 h. The reaction mixtures were directly
analyzed on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA).
µ
L, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was then
◦
The separation was performed on a Waters XBridge C18 column (250
×
4.6 mm, 5 µm) eluting with
a mixture of H2O (A) and MeCN (B) with a linear gradient of 20–40% B (0–30 min). The column
temperature was maintained at 30.0 ◦C. Authentic D-glucose, L-glucose, D-xylose, and L-xylose were
also prepared and analyzed using the same procedure. The retention times of the hydrolysates
and sugar derivatives were as follows: D-glucose derivative (tR 14.59 min), L-glucose derivative
(tR 13.87 min), D-xylose derivative (tR 15.81 min), and L-xylose derivative (tR 15.24 min).
3.6. Determination of the Absolute Configurations of the Amino Acids in Compounds 1 and 2
The hydrolysates (50 µg) of compound 1 were added to 1 M NaHCO3 (20 µL) with 1% L-FDAA
(Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide, Tokyo Chemical Industry Co., Ltd.) in acetone (40
The reaction vials were incubated and stirred for 1 h at 40 C. The reactions were then quenched
µL).
◦
with 1 N HCl (20
Agilent Zorbax SB-C3 column (150
MeOH+5% MeCN (1% formic acid)) (0–75 min) in A (H2O). The retention times of the hydrolysates
and authentic leucine derivatives were as follows: hydrolysate derivative-L-FDAA (tR 40.746 min),
µ
L) and prepared for LC-MS analysis. The samples were analyzed by LC-MS on an
×
4.6 mm, 5
µ
m) at 50 ◦C with a gradient of 25% B to 65% B (95%
L-Leu-L-FDAA (tR 40.306 min), and D-Leu-L-FDAA (tR 53.233 min). The leucine unit in compound
was confirmed to be L-Leu.
1
The hydrolysates of compound
2 were characterized using the same method as that used for
compound . The LC-MS conditions were as follows: 25% B to 45% B (95% MeOH + 5% MeCN (1%
1
formic acid)) (0–75 min) and 100% B at 77 min, where A is H2O. The retention times of the hydrolysates
and authentic isoleucine derivatives were as follows: hydrolysate derivative-L-FDAA (tR 59.468 min),
L-allo-Ile-L-FDAA (tR 59.383 min), L-Ile-L-FDAA (tR 61.362 min), D-allo-Ile-L-FDAA (tR 78.819 min),
and D-Ile-L-FDAA (tR 79.172 min). The isoleucine unit in compound
2 was determined to be L-allo-Ile.
3.7. Antimicrobial Assay
The broth microdilution method was used to evaluate the antimicrobial activities of the isolated
compounds [19]. Four bacterial strains (Bacillus subtilis, Escherichia coli DH5 , Pseudomonas aeruginosa,
α
and Staphylococcus aureus) were inoculated in Muller-Hinton (MH) agar plates and suspended in
MH broth with 0.5 McFarland turbidity equivalents. The prepared microorganism suspensions were
diluted with MH broth. The concentrations of the positive control (gentamicin) and tested compounds
were 20 mM in DMSO. The stock solution of the compounds diluted with MH broth medium (50
µL) were mixed with 50
µL of microorganism suspensions diluted to concentrations between 250
and 0.5 M using serial two-fold dilutions in 96-well plates. The experiments were performed in
µ

Molecules 2018, 23, 752
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triplicate. The 96-well plates were incubated for 14 h at 27 ◦C for B. subtilis and at 37 ◦C for E. coli
DH5 , P. aeruginosa, and S. aureus. The minimum inhibitory concentrations were determined as the
α
lowest concentrations that visually inhibited the growth of the microorganism.
4. Conclusions
In summary, the discovery of two new
rhamnellosides A ( ) and B ( ) from R. franguloides is described in this work. Based on our previous
discovery of 2-acetoxy- -phenylpentaene fatty acid triglycosides ( ) from a phylogenetically close
species B. berchemiifolia, metabolites in the fruits of R. franguloides were chemically screened using
an LC-MS/MS molecular networking dereplication strategy. Although compounds and were
not directly annotated by molecular networking, they were prioritized based on LC-MS/MS spectra
and isolated. The structures of and were identified by analyses in combination with chemical
derivatization. Interestingly, compounds
in their pentaene groups, which is the same as
ω-phenylpentaene fatty acid amide diglycosides,
1
2
ω
4–6
1
2
1
2
1
and
2
4
have unprecedented (6E, 8E, 10Z, 12Z, 14E)-geometry
from B. berchemiifolia. Although these compounds did
not show significant activity in the biological evaluation for antibacterial activity, these unprecedented
metabolites would have biological or ecological roles, which should be revealed by further studies.
Supplementary Materials: The following are available online, LC-MS BPI chromatograms of the R. franguloides
and B. berchemiifolia fruit extracts; raw HRESIMS (MS and MS/MS), NMR, UV, and ECD data for compounds
and 2.
1
Acknowledgments: This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF), which was funded by the Ministry of Science, ICT and Future Planning
(NRF-2015M3A9A5030733). We would like to thank S. I. Han (The Medicinal Plant Garden, College of Pharmacy,
Seoul National University) for kindly providing the plant material and Y.-J. Ko (National Center for Interuniversity
Research Facilities) for the NMR experiments.
Author Contributions: K.B.K., H.C., and S.H.S. conceived and designed the experiments; K.B.K., M.G.,
and G.J.K. performed the experiments; K.B.K. and M.G. analyzed the data; H.C. and S.H.S. contributed
reagents/materials/analysis tools; K.B.K. wrote the paper with discussion and help from all authors.
Conflicts of Interest: The authors declare no conflict of interest.
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