Isolation of enantiomeric furolactones and furofurans from Rubus idaeus L. with neuroprotective activities

Article abstract of DOI:10.1016/j.phytochem.2019.05.008

A phytochemical study on the fruits of Rubus idaeus L. (Rosaceae) yielded eight pairs of enantiomeric lignans, including one undescribed furolactone named (?)-idaeusinol A and six undescribed furofuran derivatives named (+/?)-idaeusinol B–D. The structures of these isolated compounds were elucidated by spectroscopic analyses and a combination of computational techniques including gauge-independent atomic orbital (GIAO) calculation of 1D NMR data and TD-DFT calculation of electronic circular dichroism (ECD) spectra. Bioactivity screenings suggested that (+)-idaeusinol D exhibited the most significant protective effect against H₂O₂-induced neurotoxicity at the concentration of 25 μM. In contrast, (?)-idaeusinol D, as the enantiomer of (+)-idaeusinol D, showed no effect against H₂O₂-induced neurotoxicity at both 25 and 50 μM concentration.

Full text of DOI:10.1016/j.phytochem.2019.05.008

Phytochemistry164(2019)122–129
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Isolation of enantiomeric furolactones and furofurans from Rubus idaeus L.
with neuroprotective activities
Le Zhoua, Feng-Ying Hana, Li-Wei Lua, Guo-Dong Yaoa, Ying-Ying Zhanga, Xiao-Bo Wangb,
Bin Linc, Xiao-Xiao Huanga,b,∗∗, Shao-Jiang Songa,∗
a School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province,
Shenyang Pharmaceutical University, Shenyang, 110016, China
b Chinese People's Liberation Army 210 Hospital, Dalian, 116021, China
c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Rubus idaeus
Rosaceae
Lignans
Enantiomeric
DP4 plus
A phytochemical study on the fruits of Rubus idaeus L. (Rosaceae) yielded eight pairs of enantiomeric lignans,
including one undescribed furolactone named (−)-idaeusinol A and six undescribed furofuran derivatives
named (+/−)-idaeusinol B–D. The structures of these isolated compounds were elucidated by spectroscopic
analyses and a combination of computational techniques including gauge-independent atomic orbital (GIAO)
calculation of 1D NMR data and TD-DFT calculation of electronic circular dichroism (ECD) spectra. Bioactivity
screenings suggested that (+)-idaeusinol D exhibited the most significant protective effect against H2O2-induced
neurotoxicity at the concentration of 25 μM. In contrast, (−)-idaeusinol D, as the enantiomer of (+)-idaeusinol
D, showed no effect against H2O2-induced neurotoxicity at both 25 and 50 μM concentration.
Calculated ECD
Neuroprotective
1. Introduction
neurodegenerative disorders (Blumenthal, 1998; Garcia et al., 2017;
Ghalayini et al., 2011). Previous phytochemical investigations of this
Lignans are an important class of naturally occurring metabolites
biosynthetically generated from two (or more) units of phenylpropa-
noid monomers by the free-radical coupling reaction. They are widely
spread within the plant kingdom (Teponno et al., 2016). Due to their
structural diversity, lignans exhibit multifarious clinical pharmacolo-
gical activities, such as antitumor (Chen et al., 2013), anti-in-
flammatory (Kim et al., 2012), calcium antagonistic, antioxidant (Lu
et al., 2016; Mei et al., 2009; Min et al., 2004), antiviral (Xue et al.,
2015), and antihyperglycemic effects (Kumar et al., 2009). In recent
years, owing to their fascinating chemical structures and remarkable
biological activities, these compounds have attracted great interest
from the synthetic and pharmacological communities. Such naturally
occurring lignans normally exist as racemates due to their biosynthetic
origin through free-radical coupling reactions. For a long time, they
have rarely been analyzed systematically for their enantiomeric purity.
Rubus idaeus L. (raspberry), taxonomically belonging to the family
of Rosaceae, is widely distributed in temperate and subtropical areas.
Its fresh fruits are edible, and the dried fruits, known as “fu-pen-zi”,
have been traditionally used as a folk medicine in China frequently
prescribed for the treatment of colic pain, diarrhea, kidney disease and
species were mainly focused on the characterization of flavonoids and
phenolic acids derivatives, including quercetin, rutin, pinoresinol, el-
lagic acid, caffeic acid (Ciric et al., 2018; Rao and Snyder, 2010). Apart
from these phenolic derivatives, Rubus species and raspberry also con-
tain other classes of constituents (Mazur et al., 2000; Mcdougall et al.,
2017). To identify more bioactive substances responsible for the ben-
eficial effects of R. idaeus fruits, further chemical investigation was
conducted in this study. As a result, eight pairs of enantiomers (1a/1b-
8a/8b, Fig. 1), including two pairs of enantiomeric furolactones (1a/1b
and 5a/5b), four pairs of enantiomeric furofurans (2a/2b and 6a/6b-
8a/8b), and two pairs of enantiomeric sesquilignans (3a/3b and 4a/
4b) were isolated from the fruits of the plant. The enantioseparations of
these compounds were achieved using chiral HPLC approach. Their 2D
structures and relative configurations were elucidated by extensive
spectroscopic analyses. Notably, ( )-idaeusinol A (1), a furolactone,
its relative configuration was established by employing gauge-including
atomic orbitals (GIAO) NMR chemical shift calculations, with the aid of
the advanced statistical method DP4 plus (Grimblat et al., 2015). Ad-
ditionally, their absolute configurations were determined by compar-
ison of experimental and calculated ECD spectra. Their neuroprotective
∗ Corresponding author.
∗∗ Corresponding author. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China.
E-mail addresses: xiaoxiao270@163.com (X.-X. Huang), songsj99@163.com (S.-J. Song).
https://doi.org/10.1016/j.phytochem.2019.05.008
Received 14 November 2018; Received in revised form 18 March 2019; Accepted 9 May 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

L. Zhou, et al.
Phytochemistry164(2019)122–129
Fig. 1. Chemical structures of compounds 1a/1b–8a/8b.
effects against H2O2-induced oxidative injury in human neuroblastoma
SH-SY5Y cells were investigated.
(+)-idaeusinol A (Li et al., 2013), was deduced to be (7S,8R,2′R), and
(−)-1b, namely, (−)-idaeusinol A, was deduced to be (7R,8S,2′S).
Compound 2 (2a/2b), obtained as a colorless, light oil, was assigned
the molecular formula C18H18O4, the same as 6, based on its sodiated
molecular ion at m/z 321.1085 [M + Na]+ (calcd 321.1097) in the
HRESIMS. Analysis of the NMR data of 2 indicated that it was an isomer
of 6, due to their closely similar spectroscopic features. However, the
two symmetric oxymethine signals [δH 4.59 (2H, d, J = 4.0 Hz, H-2/6]
of the furofuran ring in 6 were split into a higher field signal with
smaller coupling constant [δH 4.74 (1H, d, J = 5.9 Hz, H-2)] and a
lower field signal with larger coupling constant [4.27 (1H, d,
J = 7.0 Hz, H-6)] in the 1H NMR spectrum of 2. Some of the aliphatic
carbon signals were shifted to a higher field [δC 81.4 (C-6, ΔδC −3.3),
δC 68.8 (C-8, ΔδC −2.0), δC 49.4 (C-1, ΔδC −4.2)], which was likely
due to the anisotropic effect of an aromatic ring. Naturally occurring
furofurans have been reported to possess typical cis-8,8′-fused (Lu et al.,
2015). The chemical shift differences of H2-9 and H2-9′ (ΔδH-9 0.29;
ΔδH-9′ 0.62) of 8-H type furofuran lignans indicated 7-H/8-H trans, 7′-
H/8′-H cis relative configurations (Shao et al., 2018). The smaller value
2. Results and discussion
Compound 1 (1a/1b) was obtained as a pale amorphous gum. Its
molecular formula was deduced as C12H12O4, on the pseudo-molecular
ion peak at m/z 243.0605 [M + Na]+ in the HRESIMS, indicating seven
degrees of unsaturation. The presence of a hydroxy and a benzene ring
were suggested by the IR spectrum, showing absorptions at 3423, 1630
and 1427 cm−1. The 1H NMR data (Table 1) showed the signals for a
1,4-disubstituted phenyl ring due to the aromatic proton signals at δH
7.20 (2H, d, J = 8.5 Hz, H-2/6) and 6.79 (2H, dd, J = 8.5 Hz, H-3/5).
Additionally, the characteristic signals at δH 4.64 (1H, d, J = 6.6 Hz, H-
7), 4.50 (1H, d, J = 9.6, 6.9 Hz, H-9a), 4.32 (1H, dd, J = 9.6, 2.0 Hz, H-
9b), 4.25 (1H, t, J = 8.7 Hz, H-3′a), 4.05 (1H, dd, J = 9.1, 3.4 Hz, H-
3′b), 3.52 (1H, td, J = 8.7, 3.4 Hz, H-2′), and 3.16 (1H, dtd, J = 8.9,
6.7, 2.0 Hz, H-8) indicated the existence of a 3,7-dioxabicyclo[3.3.0]
octan-2-one skeleton. This deduction was further verified by the HMBC
and 1H–1H COSY spectra (Fig. 2). The 1H NMR signal at δH 4.64 (H-7)
showed correlations with C-1 (δC 131.5) and C-2/6 (δC 128.8), in-
dicating that the phenyl group was located at C-7 position.
In the NOESY spectrum (Fig. 2), δH 3.16 (H-8) showed cross-peaks
with δH 3.52 (H-2′), 4.50 (H-9a) and 7.21 (H-2/6) indicated the same
orientation of these protons. On the other hand, correlation between δH
4.26 (H-7) and δH 3.52 (H-9b) was used to place them on the opposite
face. Moreover, the relative configuration at C-7 was further assigned
by employing calculations of shielding tensor values with support from
DP4+ probability analysis. The theoretical calculations of 13C NMR
data of the two possible isomers (7S*,8R*,2′R*)-1 and (7R*,8R*,2′R*)-1
(Fig. 3) were predicted using the GIAO method with the Gaussian 09
software (Frisch et al., 2009) at the B3LYP/6-311 + G(d,p) level uti-
lizing the polarizable continuum model (PCM) in methanol. Compar-
ison of the experimental and calculated 13C NMR data allowed the
determination of the relative configuration of 1 as 7S*,8R*,2′R* with a
DP4+ probability of approximately 98.4%. The absolute configuration
of 1 was determined by comparison of experimental and calculated
electronic circular dichroism (ECD). The calculated ECD of 7S,8R,2′R-1
is consistent with the measured ECD of (+)-1a but opposite of that of
(−)-1b (Fig. 4). Therefore, the structure of (+)-1a, namely,
of the specific rotation {[α]20 −0.3 (c 0.1, MeOH)} compared with
D
that of its stereoisomer indicated that 2 was likely a partial racemic
mixture. Subsequently, the chiral HPLC purification of 2 afforded the
enantiomers 2a and 2b with opposite specific rotation and mirror im-
aged Cotton effects in their ECD spectra. The absolute configurations of
(−)-2 and (+)-2 were determined as (7R,8R;7′R,8′S) (2a) and
(7S,8S;7′S,8′R) (2b), respectively, by comparison of the experimental
and calculated ECD spectra (Fig. 4). Thus, the absolute configuration of
(−)-2a was elucidated as 7R,8R;7′R,8′S and that of (+)-2b as
7S,8S;7′S,8′R, and the compounds 2a and 2b were assigned the names
(−)-idaeusinol B and (+)-idaeusinol B, respectively.
Compound 3 (3a/3b) and 4 (4a/4b) were obtained as a mixture of
light yellow oil and displayed only a single chromatographic peak on
the reversed-phase HPLC. However, the carbon signals at δC 110.6 and
118.3, which were assigned to δH 6.93 (1H, d, J = 1.6 Hz, H-2) and
6.82 (1H, dd, J = 8.4, 1.6 Hz, H-6), respectively, were both split in the
13C NMR spectrum. Subsequently, a series of chiral HPLC resolutions
were performed to understand this distinctive discord. The results re-
vealed that the isolate was a mixture of stereoisomers, which afforded
two pairs of enantiomers 3a/3b and 4a/4b, corresponding to the four
peaks observed from the chiral HPLC (Fig. S19).
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Phytochemistry164(2019)122–129
Table 1
1H (400 MHz) and 13C (100 MHz) data of enantiomers 1–4 in DMSO‑d6 (δ in ppm).
No.
1a
2
3
4
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
1
2
3
4
5
6
7
8
9
131.5
128.8
116.3
158.6
116.3
128.8
87.6
128.8
126.6
115.0
156.3
115.0
126.6
81.4
134.4
110.6
149.7
147.9
115.7
118.4
85.0
134.4
110.6
149.7
147.9
115.7
118.4
85.0
7.20 (d, J = 8.5 Hz)
6.79 (d, J = 8.5 Hz)
7.13 (d, J = 8.3 Hz)
6.73 (d, J = 8.3 Hz)
6.93 (d, J = 1.6 Hz)
6.93 (d, J = 1.6 Hz)
6.79 (d, J = 8.5 Hz)
7.20 (d, J = 8.5 Hz)
4.64 (d, J = 6.6 Hz)
3.16 (dtd, J = 8.9, 6.6, 2.0 Hz)
4.50 (dd, J = 9.6, 6.9 Hz)
4.32 (dd, J = 9.6, 2.0 Hz)
6.73 (d, J = 8.3 Hz)
7.13 (d, J = 8.3 Hz)
4.74 (d, J = 5.9 Hz)
3.30, m
3.66 (t, J = 8.6 Hz)
3.04 (t, J = 8.6 Hz)
6.97 (d, J = 8.4 Hz)
6.82 (dd, J = 8.4, 1.6 Hz)
4.63 (d, J = 4.7 Hz)
3.02, overlap
4.11, overlap
3.72, overlap
6.97 (d, J = 8.4 Hz)
6.82 (dd, J = 8.4, 1.6 Hz)
4.63 (d, J = 4.7 Hz)
3.02, overlap
4.11, overlap
3.72, overlap
49.1
71.8
49.4
68.8
53.5
71.0
53.5
71.0
1′
2′
3′
181.1
47.5
70.8
131.5
127.4
114.9
131.5
127.5
115.1
131.5
127.5
115.1
3.52 (td, J = 8.9, 3.4 Hz)
4.25 (t, J = 8.7 Hz)
7.15 (d, J = 8.6 Hz)
6.72 (d, J = 8.6 Hz)
7.15 (d, J = 8.5 Hz)
6.73 (d, J = 8.5 Hz)
7.15 (d, J = 8.5 Hz)
6.73 (d, J = 8.5 Hz)
4.05 (dd, J = 9.1, 3.4 Hz)
4′
5′
6′
7′
8′
9′
156.9
114.9
127.4
86.9
53.9
70.9
156.8
115.1
127.5
85.0
53.7
71.0
156.8
115.1
127.5
85.0
53.7
71.0
6.72 (d, J = 8.6 Hz)
7.15 (d, J = 8.6 Hz)
4.27 (d, J = 7.0 Hz)
2.78 (m)
4.00 (d, J = 9.2 Hz)
3.71 (dd, J = 9.2, 6.3 Hz)
6.73 (d, J = 8.5 Hz)
7.15 (d, J = 8.5 Hz)
4.61 (d, J = 4.6 Hz)
3.02, overlap
4.11, overlap
3.72, overlap
6.73 (d, J = 8.5 Hz)
7.15 (d, J = 8.5 Hz)
4.61 (d, J = 4.6 Hz)
3.02, overlap
4.11, overlap
3.72, overlap
1″
132.3
127.9
114.5
156.4
70.8
132.3
127.9
114.5
156.4
70.8
2″/6″
3″/5″
4″
7″
8″
7.17 (d, J = 8.4 Hz)
6.68 (d, J = 8.4 Hz)
7.17 (d, J = 8.4 Hz)
6.68 (d, J = 8.4 Hz)
4.71 (d, J = 4.7 Hz)
4.20, m
4.71 (d, J = 4.7 Hz)
4.20, m
85.0
85.0
9″
3.56 (dd, J = 11.5, 3.7 Hz)
3.22 (dd, J = 11.5, 6.3 Hz)
3.77, s
60.1
3.56 (dd, J = 11.5, 3.7 Hz)
3.22 (dd, J = 11.5, 6.3 Hz)
3.77, s
60.1
OCH3
a
55.8
55.8
Measured in CD3OD.
The molecular formula of 3 (3a/3b) was deduced as C28H30O8 by
the HRESIMS ion peak at m/z 517.1854 [M + Na]+ (calcd 517.1838),
corresponding to fourteen degrees of unsaturation. The NMR spectrum
indicated the presence of a 1,3,4-trisubstituted and two symmetrical
1,4-disubstituted aromatic rings, one aromatic methoxyl, three oxy-
methylenes, and six methine (four oxygenated) groups. These data
suggested that 3 was a sesquineolignan. Moreover, comparison of the
NMR data between 3 (Table 1) and ( )-demethoxypinoresinol (7)
indicated that they differed in the presence of resonances attributable to
an additional 4″-hydroxyphenylglycerol-8″-yl moiety in 3. In the HMBC
spectrum (Fig. 2), correlation from H-8″ to C-4 verified the 8″,4-oxy
linkage in 3. On the basis of the similar chemical shift differences of H2-
9 and H2-9′ (ΔδH-9 0.39; ΔδH-9′ 0.39) in the 3,7-dioxabicyclo[3.3.0]oc-
tane moiety, the partial relative configuration of 3 was established as
7R*,8S*,7′R*,8′S*, which was identical to that of 7. The 7″,8″-threo
configuration was elucidated by the larger coupling constant between
H-7″ and H-8'' (J7″,8'' = 8.2 Hz in CDCl3) (Xiong et al., 2011). The ab-
solute (7S,8R;7′S,8′R;7″R,8″R)-configuration of 3a was determined by
Fig. 2. Selected 1H–1H COSY, HMBC and NOESY correlations of compounds 1–4.
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Phytochemistry164(2019)122–129
Fig. 3. Two plausible diastereomers of 1 used for the DP4+ application.
comparison of its experimental and computed ECD curves (Fig. 4).
Thereby, the absolute configuration of 3a and 3b were determined as
(7S,8R;7′S,8′R;7″R,8″R) and (7R,8S;7′R,8′S;7″S,8″S) with the trivial
names of (+)-idaeusinol C and (−)-idaeusinol C, respectively.
(7R,8S;7′R,8′S;7″R,8″R) with the trivial names of (+)-idaeusinol D and
(−)-idaeusinol D, respectively.
The known compounds were identified via comparison of their
spectroscopic data with literature values as (+)-salicifoliol (5a)
(Yamauchi et al., 2004), (−)-salicifoliol (5b) (González et al., 1989),
(+)-ligballinol (6a) (Kobayayashi and Ohta, 1983), (−)-ligballinol
(6b) (Wang et al., 2009), (+)-demethoxypinoresinol (7a) (Cuenca
et al., 1991), (−)-demethoxypinoresinol (7b) (Hosokawa et al., 2004),
(+)-pinoresinol (8a) (Cuenca et al., 1991), (−)-pinoresinol (8b) (Li
et al., 2012).
Compound 4 (4a/4b) had the same molecular formula, C28H30O8,
as that of 3. Compounds 3 and 4 were suggested by their identical NMR
spectra to have the same planar structure and relative configurations.
Detailed comparison of the experimental ECD data between 3a and 4a
(or 3b and 4b) indicated that there were obvious differences at the
region of 200–225 nm in their experimental ECD curves (Fig. S34). The
absolute configurations of 4a and 4b were designated as
(7S,8R;7′S,8′R;7″S,8″S) and (7R,8S;7′R,8′S;7″R,8″R) by comparison of
the experimental and theoretical ECD. Thus, the absolute configura-
tions of 4a and 4b were assigned as (7S,8R;7′S,8′R;7″S,8″S) and
2.1. Neuroprotective activity
Lignans are a large group of naturally occurring phenols with great
Fig. 4. Calculated and experimental ECD spectra of 1a/1b-4a/4b.
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Phytochemistry164(2019)122–129
Fig. 5. Neuroprotective effects of compounds against
H2O2-induced cell growth inhibition of SH-SY5Y
cells. In the presence or absence of the tested com-
pounds at different concentrations (25 and 50 μM),
MTT assay was used to examine the cell viability
after H2O2 (300 μM) treatment for 4 h ∗P < 0.05,
∗∗P < 0.01 vs. H2O2-treated group; #P < 0.05 was
considered statistically significant when compared
with its enantiomer.
chemical diversity and multifarious clinical pharmacology activities
(Saleem et al., 2005). The recent decades have witnessed a renewed
interest in biologically active lignans isolated from a plethora of natural
resources (Teponno et al., 2016; Pan et al., 2009). Currently, the neu-
roblastoma SH-SY5Y cell line has been widely used to study the mole-
cular and cellular mechanisms underlying the effects of some of the
neurodegenerative disorders, although it is not classified as purely
dopaminergic neurons cell (Xicoy et al., 2017). All the isolated com-
pounds (1a/1b-8a/8b) were evaluated for their neuroprotective effects
against H2O2-induced oxidative stress in human neuroblastoma SH-
SY5Y cells by the MTT assay (Qi et al., 2018). The results (Fig. 5)
showed that compounds 1a and 4a exhibited significant neuroprotec-
tive activity against H2O2-induced neurotoxicity at 25 μM concentra-
tion, improving cell viability by 13% and 21% compared with the ne-
gative control group, respectively. Compounds 1b, 2a and 3a showed
weak neuroprotective effects at both 25 and 50 μM concentration. In-
terestingly, however, compound 4b, which is the enantiomer of 4a,
showed no effect against H2O2-induced neurotoxicity at the con-
centration of 25 and 50 μM.
To further determine the protective effect of enantiomers 4a and 4b
on the H2O2-treated SH-SY5Y cells, Annexin V/propidium iodide (AV/
PI) staining was performed to quantitatively determine the apoptotic
cell percentage by flow cytometry. As shown in Fig. 6, after treatment
with 25 or 50 μM 4a for 24 h, the percentages of both early and late
apoptotic cells decreased significantly relative to those of the untreated
control, while its enantiomer 4b could not decrease the percent of
apoptotic cells induced by H2O2.
In general, neuronal cell damage is the most important factor in
many neurodegenerative disorders. Epidemiological studies demon-
strated that a dietary intake of phenolics have an inverse relationship
with the risk of various neuronal cell damage-mediated diseases in-
cluding Parkinson's disease, Alzheimer's disease, stroke and other
neurodegenerative diseases (Chen et al., 2008; Kim et al., 2010; Jan and
Ho, 2014). However, clinical evidence regarding the specific role of
dietary lignans in the prevention of neurodegenerative diseases is still
very limited. It should be noticed that the efficacy of a natural com-
pound in vivo depends on not only its intrinsic bioactivity but also its
bioavailability.
According to in vitro studies, when the dietary lignans most com-
monly found in food are incubated with human fecal microflora, they
are transformed to the tetrahydrofuranoid metabolites or enterolignans
(Wan et al., 2006; Pan et al., 2005). Besides, the current studied showed
that these lignans may be incorporated into the liver and then trans-
ported to other tissues (lung, kidney, and brain) via the lymphatic
system after oral administration (Jan et al., 2012). The highest con-
centrations of its conjugated metabolites (glucuronide/sulfate) in liver
and kidney suggested that they were regard as the major tissues of
metabolism (Tomimori et al., 2017; Valdés et al., 2015).
The blood−brain barrier is a primary interface between the per-
ipheral fluids and the central nervous system, which plays an important
role in the brain by preventing potentially neurotoxic plasma compo-
nents, pathogens, and blood cells from permeating the brain (Zhao
et al., 2015). Although limited knowledge is known about the capacity
of dietary lignans to cross the blood-brain barrier, relevant researches
Fig. 6. Effects of 4a and 4b on H2O2-induced apoptosis in SH-SY5Y cells. Cells were incubated with indicated concentrations of 4a and 4b for 12 h before exposure to
300 μM H2O2 for 3 h. Representative patterns of flow cytometric distribution of SH-SY5Y cells after Annexin V-PI double staining and the quantitative analysis of
apoptotic cells. Data are presented as means
S.D. (n = 3). #p < 0.05 vs. the control group. **p < 0.01 vs. the H2O2-treated group.
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Phytochemistry164(2019)122–129
demonstrated that some furofuran lignans in sesame seeds, such as
sesamin and sesamolin, showed varying degrees of potential to migrate
across the blood-brain barrier (Liu et al., 2017; Katayama et al., 2016).
Hovever, whether these dietary lignans or their derived metabolites
produced in vivo could actually enter the brain and play a role in the
central nervous system remains unclear. This means that more animal
model studies are necessary to determine the metabolism and tissue
distribution in the body as well as elucidate the underlying molecular
mechanisms for the neuroprotective effects of these polyphenol-derived
metabolites.
4.3. Extraction and isolation
The air-dried fruits of Rubus idaeus L. (20 kg) were extracted by
maceration in 70% aqueous ethanol (3 × 50 L) at room temperature.
The filtrates were concentrated on a rotary evaporator at 45 °C under
reduced pressure, which obtained a dark black residue (1.7 kg). The
crude extract was then suspended in distilled H2O and subjected to
D101 macroporous resin column eluting with a step gradient solvent
system EtOH–H2O (20:80 → 100:0) to give four fractions (I−IV).
Fraction III (176 g) was further submitted to separation over silica gel
column using CH2Cl2–MeOH (8:1 → 1:2), which resulted in seven sub-
fractions (IIIa ∼ IIIg). Fraction IIIb (26.5 g) was separated on a reverse-
phase C18 silica gel column using gradient elution with aqueous EtOH
(20% → 70%), which yielded six pooled subfractions (A−F) on the
basis of HPLC analysis. Next, fractions A (4.1 g) was chromatographed
on silica gel eluted with CH2Cl2–MeOH (9:1 → 1:1) to afford five
subfractions (A1∼A5) on the basis of silica gel TLC analysis.
Consequently, subfraction A1-1 was further isolated by preparative
HPLC on a YMC RP-C18 column with MeOH–H2O (22:78, v/v) as a
mobile phase to obtain five fractions (A1-1 ∼ A1-5). Then, A1-1 was
successively purified by semipreparative HPLC-C18 column chromato-
graphy with solvent system MeCN–H2O (10:90, 3.5 mL/min) to yield 1
(17.4 mg) and 7 (6.6 mg). Compound 2 (5.8 mg) was isolated by
semipreparative HPLC-C18 column chromatography (MeCN–H2O,
12:88, v/v) from Fr.A1-2. Similarly, fractions B (5.4 g) was chromato-
graphed on silica gel with a gradient of CH2Cl2–MeOH system (9:1 →
1:1) as eluents, yielding five fractions (B1 ∼ B5). Fr.B4 was separated
with preparative HPLC (eluted with MeOH–H2O, 22:78, v/v) followed
by purification with Semipreparative HPLC-C18 column chromato-
graphy to obtain compounds 5 (8.2 mg), 6 (30.6 mg) and 8 (7.6 mg). In
the same manner using preparative HPLC (eluted with MeOH–H2O,
30:70, v/v), 3 and 4 (22.8 mg) as a mixture were isolated from fraction
B3.
3. Conclusions
In conclusion, two pairs of enantiomeric furolactones (1a/1b and
5a/5b), four pairs of enantiomeric furofurans (2a/2b, 6a/6b-8a/8b)
and two pairs of sesquineolignans (3a/3b and 4a/4b), were isolated
and identified from 70% aqueous ethanol extract of the fruits of Rubus
idaeus L. (Rosaceae). These racemic mixtures were successfully sepa-
rated by HPLC using Daicel chiral-pak IC and AD-H chiral columns with
various mobile phases. The structure determination was based on ex-
tensive spectroscopic data analyses (NMR, HRESIMS), and the absolute
stereochemistry of the undescribed lignans (1b, 2a/2b-4a/4b) were
determined by comparison between experimental and calculated elec-
tronic circular dichroism (ECD) and DP4 plus probability calculations.
The isolates were tested for their neuroprotective activity using H2O2-
treated human neuroblastoma SH-SY5Y cell line. (+)-idaeusinol A (1a)
and (+)-idaeusinol D (4a) exhibited significant neuroprotective ac-
tivity at 25 μM concentration. Interestingly, however, compound
(−)-idaeusinol D (4b), which is the enantiomer of 4a, showed no effect
against H2O2-induced neuroprotective effect at both 25 and 50 μM
concentrations.
4. Experimental
Compounds 1–8 were further analyzed by HPLC system equipped
with different types of analytical chiral columns. Among them, 1 and 5
were subjected to chiral HPLC, respectively, using a Daicel IC column
(2-propanol/n-hexane, 20:80, v/v, 0.8 mL/min) to afford 1a (2.5 mg, tR
21.3 min), 1b (2.7 mg, tR 26.5 min), 5a (1.3 mg, tR 19.4 min), 5b
(1.1 mg, tR 28.5 min), respectively. Chiral resolution of 2, 6, 7, 8 were
performed on Daicel chiral-pak AD-H column (eluted with 2-propanol/
n-hexane, 45:55, v/v, flow rate 0.8 mL/min) to give 2a (1.7 mg, tR
20.3 min), 2b (1.9 mg, tR 34.1 min), 6a (1.7 mg, tR 16.5 min), 6b
(1.9 mg, tR 29.8 min), 7a (2.7 mg, tR 25.5 min), 7b (3.0 mg, tR
38.8 min), 8a (3.3 mg, tR 20.8 min), 8b (3.2 mg, tR 29.8 min), respec-
tively. 3 and 4 were obtained together from one peak on the semi-
preparative HPLC, and had been divided into four peaks on Daicel
chiral-pak IC column (eluted with 2-propanol/n-hexane, 20:80, v/v,
0.8 mL/min), subsequently, 3a and 4b were further purified by chiral
HPLC equipped with Daicel chiral-pak AD-H column eluting with 2-
propanol/n-hexane (15:85, v/v, 0.6 mL/min), As a result, optically pure
compounds of 3a (1.2 mg, tR 25.2 min), 3b (1.1 mg, tR 128.3 min), 4a
(1.3 mg, tR 40.8 min), 4b (1.2 mg, tR 28.5 min) were successfully
achieved.
4.1. General experimental procedures
Optical rotations were obtained on a PerkinElmer 341 digital po-
larimeter. UV and IR spectra were recorded on Shimadzu UV-1700
spectrometer and Bruker IFS-55 spectrometer, respectively. CD spectra
were given by Bio-Logic MOS 450 detector. NMR spectra were recorded
on Bruker ARX-400 and AV-600 spectrometers. Chemical shifts were
presented in δ (ppm) using tetramethylsilane (TMS) as an internal
standard, and coupling constants (J) were expressed in Hz. HRESIMS
spectra were taken on a Bruker Micro Q-TOF spectrometer (positive-ion
mode). High-performance liquid chromatography (HPLC) was per-
formed on a Waters 1525 series pumping system equipped with a
Waters 2489 UV–vis detector using an YMC-pack ODS-A column
(250 mm × 10 mm, 5 μm) and Waters preparative RP-C18 column
(19 × 150 mm,
(250 mm × 4.6 mm,
10 μm),
5 μm)
and
the
chiral-pak
chiral-pak
IC
column
column
or
AD-H
(250 mm × 4.6 mm, 5 μm). Column chromatography was performed on
silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine
Chemical, Inc., Qingdao, China), octadecyl silica gel (ODS, 60–80 μm,
Merck, Darmstadt, Germany), macroporous resin (D101, Cangzhou
Baoen Chemistry Ltd, Hebei, China). Thin-layer chromatography (TLC)
was carried out on precoated silica gel GF254 plates.
4.3.1. Idaeusinol A (1)
Pale amorphous gum; [α]20 D +0.1 (c 0.1, MeOH); UV (MeOH) λmax
(log ε) 227 (3.89), 277 (3.10) nm; IR (KBr) νmax 3423, 2924, 1630,
4.2. Plant material
1427, 1006 cm−1 1H and 13C NMR (CD3OD) data, see Table 1; HRE-
;
The fruits of Rubus idaeus L. (Rosaceae) were collected from rasp-
berry planting basement, Dongling district, Shenyang, Liaoning pro-
vince, PR China (N41°41′9.86″, E123°41′35.24″) in June 2015, and
then authenticated by Prof. Jin-Cai Lu, Shenyang Pharmaceutical
University, Shenyang, China. A voucher specimen (No. 20150601) has
been deposited in the Herbarium of Shenyang Pharmaceutical
University for further reference.
SIMS (positive-ion mode) m/z 243.0605 [M +
Na]+ (calcd for
C
12H12O4Na, 243.0633).
(+)-idaeusinol A (1a), pale amorphous gum; [α]20 +24.4 (c 0.1,
D
MeOH); ECD (MeOH) 214 (Δε +6.02) nm; (−)-idaeusinol A (1b), pale
amorphous gum; [α]20 −24.6 (c 0.1, MeOH); ECD (MeOH) 222 (Δε
D
−6.24) nm.
127

L. Zhou, et al.
Phytochemistry164(2019)122–129
4.3.2. Idaeusinol B (2)
4.6. Neuroprotection bioassays
Colorless, light oil; [α]20 −0.3 (c 0.1, MeOH); UV (MeOH) λmax
D
(log ε) 226 (4.16), 277 (3.45) nm; IR (KBr) νmax 3417, 2963, 1428,
All isolates were evaluated for in vitro neuroprotective activitiy
against H2O2-induced toxicity in human neuroblastoma cells with the
MTT assay. Briefly, SH-SY5Y cells were pretreated with various of
concentrations (25 and 50 μM) of tested samples for 1 h before H2O2
(300 μM) treatment for another 4 h. Then, 20 μL MTT (5 mg/mL) was
added to each well for 4 h, and the crystals were dissolved in DMSO.
Optical density at 490 nm was determined by using a plate microreader
(Thermo, USA).
1266, 1007 cm−1 1H and 13C NMR (DMSO-d6) data, see Table 1;
;
HRESIMS (positive-ion mode) m/z 321.1085 [M + Na]+ (calcd for
C
18H18O4Na, 321.1097).
(−)-idaeusinol B (2a), colorless oil; [α]20 −36.3 (c 0.14, MeOH);
D
ECD (MeOH) 218 (Δε −5.81), 229 (Δε +2.21), 278 (Δε −0.95) nm;
(+)-idaeusinol B (2b), colorless oil; [α]20 D +38.1 (c 0.14, MeOH); ECD
(MeOH) 214 (Δε +6.41), 226 (Δε −0.55), 275 (Δε +0.60) nm.
4.7. Annexin V-FITC/PI staining
4.3.3. Idaeusinol C (3)
Light yellow oil; UV (MeOH) λmax (log ε) 228 (3.91), 278 (3.35) nm;
IR (KBr) νmax 3480, 1626, 1430, 986 cm−1; 1H and 13C NMR (DMSO-d6)
data, see Table 1; HRESIMS (positive-ion mode) m/z 517.1854 [M +
Na]+ (calcd for C28H30O8Na, 517.1838).
Annexin V-FITC and PI double-staining assay was applied to eval-
uate apoptotic ratio according the manufacturer's instructions
(Azadmehr et al., 2015). The treated cells were stained with Annexin V-
FITC followed by PI at room temperature for 15 min. Early and late
apoptotic changes in different cells were analyzed by FACScan flow
cytometry (Becton Dickinson, USA).
(+)-idaeusinol C (3a), light yellow oil; [α]20 D +31.7 (c 0.1, MeOH);
ECD (MeOH) 207 (Δε +6.83), 222 (Δε +3.19), 237 (Δε −7.56), 282
(Δε −1.55) nm; (−)-idaeusinol C (3b), light yellow oil; [α]20 D −30.6 (c
0.1, MeOH); ECD (MeOH) 201 (Δε −7.27), 219 (Δε −4.54), 236 (Δε
+6.86), 279 (Δε −0.44) nm.
4.8. Statistical analysis
All results and data were confirmed in at least three separate ex-
4.3.4. Idaeusinol D (4)
periments. Data are expressed as means
SD. Statistical comparisons
Light yellow oil; UV (MeOH) λmax (log ε) 228 (3.95), 278 (3.37) nm;
were analyzed by student's t-test using GraphPad Prism. P < 0.05 was
considered statistically significant.
IR (KBr) νmax 3422, 2926, 1630, 1426, 1006 cm−1 1H and 13C NMR
;
(DMSO-d6) data, see Table 1; HRESIMS (positive-ion mode) m/z
517.1854 [M + Na]+ (calcd for C28H30O8Na, 517.1838).
Conflicts of interest
(+)-idaeusinol D (4a), light yellow oil; [α]20 D +29.5 (c 0.1, MeOH);
ECD (MeOH) 211 (Δε −6.51), 234 (Δε −10.82), 282 (Δε −3.42) nm;
The authors declare no conflict of interest.
Acknowledgments
(−)-idaeusinol D (4b), light yellow oil; [α]20 −28.3 (c 0.1, MeOH);
D
ECD (MeOH) 214 (Δε +3.78), 235 (Δε +9.62), 294 (Δε −0.26) nm.
This research was financially supported by Cancer Development
Support for Young and Middle-aged Teachers in Shenyang
Pharmaceutical University (ZQN2018006) and the Project of
Innovation Team (LT2015027) of Liaoning of P. R. China.
4.4. ECD and NMR computational details
4.4.1. ECD calculations
Conformational analyses were initially performed using CONFLEX
software (Goto and Osawa, 1989) with the using Merck Molecular Force
Field (MMFF94) with 10 kcal/mol upper energy limit. Then, the pre-
dominant conformers were initially optimized at the quantum me-
chanical (QM) level using the B3LYP functional and the 6-31G(d,p)
basis set. Subsequently, the theoretical calculation of ECD was per-
formed using time dependent Density Functional Theory (TDDFT) at
B3LYP/6-311 + G(d,p) level in CH3OH with PCM model. The ECD
spectra of compounds 1a/1b–4a/4b were obtained by weighing the
Boltzmann distribution ratio of each geometric conformation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.05.008.
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