Sterubin: Enantioresolution and Configurational Stability, Enantiomeric Purity in Nature, and Neuroprotective Activity in Vitro and in Vivo

Article abstract of DOI:10.1002/chem.202001264

Alzheimer′s disease (AD) is a neurological disorder with still no preventive or curative treatment. Flavonoids are phytochemicals with potential therapeutic value. Previous studies described the flavanone sterubin isolated from the Californian plant Eriodictyon californicum as a potent neuroprotectant in several in vitro assays. Herein, the resolution of synthetic racemic sterubin (1) into its two enantiomers, (R)-1 and (S)-1, is described, which has been performed on a chiral chromatographic phase, and their stereochemical assignment online by HPLC-ECD coupling. (R)-1 and (S)-1 showed comparable neuroprotection in vitro with no significant differences. While the pure stereoisomers were configurationally stable in methanol, fast racemization was observed in the presence of culture medium. We also established the occurrence of extracted sterubin as its pure (S)-enantiomer. Moreover, the activity of sterubin (1) was investigated for the first time in vivo, in an AD mouse model. Sterubin (1) showed a significant positive impact on short- and long-term memory at low dosages.

Full text of DOI:10.1002/chem.202001264

Accepted Article
Accepted Article
Title: Sterubin: enantioresolution and configurational stability,
enantiomeric purity in nature, and neuroprotective activity in vitro
and in vivo
Authors: Julian Hoffmann, Shaimaa Fayez, Matthias Hofmann,
Matthias Scheiner, Pamela Maher, Tangui Maurice, Gerhard
Bringmann, Michael Decker, Sabrina Oerter, and Antje
Appelt-Menzel
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.202001264
Link to VoR: https://doi.org/10.1002/chem.202001264
01/2020

10.1002/chem.202001264
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Sterubin: enantioresolution and configurational stability,
enantiomeric purity in nature, and neuroprotective activity in vitro
and in vivo
Julian Hofmann,1[a] Shaimaa Fayez,1[b],[c] Matthias Scheiner,[a] Matthias Hoffmann,[a],[d] Sabrina Oerter,[e]
Antje Appelt-Menzel,[e],[f] Pamela Maher,[g] Tangui Maurice,[d] Gerhard Bringmann,*[b] and Michael
Decker*[a]
Abstract: Alzheimer´s disease (AD) is a neurological disorder with Introduction
still no preventive or curative treatment. Flavonoids are
phytochemicals with potential therapeutic value. Previous studies
described the flavanone sterubin isolated from the Californian plant
Eriodictyon californicum as a potent neuroprotectant in several in vitro
assays. Herein, we describe the resolution of synthetic racemic
sterubin (1) into its two enantiomers, (R)-1 and (S)-1, on a chiral
chromatographic phase and their stereochemical assignment online,
by HPLC-ECD coupling. (R)-1 and (S)-1 showed comparable
neuroprotection in vitro with no significant differences. While the pure
stereoisomers were configurationally stable in methanol, fast
racemization was observed in the presence of culture medium. We
also established the occurrence of extracted sterubin as its pure (S)-
enantiomer. Moreover, we investigated the activity of sterubin (1) for
the first time in vivo, in an AD mouse model. Sterubin (1) showed a
significant positive impact on short- and long-term memory at low
dosages.
Alzheimer’s disease (AD) is an age-associated neurodegenerative
disorder and the most common form of dementia (60-80%) among
people aged between 65 and 85 years.[1] Pathological hallmarks of the
disease include the deposition of amyloid-β (Aβ) containing plaques
and tau () containing neurofibrillary tangles.[2] This protein
accumulation is accompanied by the loss of neurotrophic factors, ATP
depletion, oxidative stress, and neuroinflammation,[3] which lead to
neurodegeneration with subsequent cognitive problems and loss of
memory.[4]
Over the past few decades, numerous plant-derived natural products
have been investigated for their activities against neurodegenerative
in vitro hallmarks, including the reduction of oxidative stress, Aβ
aggregation, and neuroinflammation.[5] Among these compounds,
flavonoids and related polyphenols are powerful antioxidants with
potential therapeutic effects.[6] Nevertheless, putative metabolic
instability and a potential lack of blood-brain barrier (BBB) penetration
are considered as drawbacks regarding “druggability”.[7] Recent
studies by Schramm et al.[8] and Gunesch et al.[9] have shown a
remarkable increase in potency of both flavonoids and flavonolignan
derivatives, in vitro and in vivo, by esterification of the hydroxy group
at C-7 with phenolic acids. The resulting esters, however, suffer from
poor water solubility and high molecular weight.[10]
Recently, Fischer et al.[11] have investigated the extract of Eriodictyon
californicum (known as yerba santa) from a new pharmacological and
medicinal perspective. This plant has long been used for medicinal
purposes by native inhabitants of California, where the plant is
indigenous.[12] The leaves contain different flavonoids (Figure 1),
which are known for their anti-inflammatory and anti-microbial
activities against Gram-positive bacteria, and are also used as
ingredients in food and pharmaceuticals as bitter-masking agents.[13]
Fischer et al. tested extracts of E. californicum in a set of age-
associated phenotypic screening assays related to AD. They
assigned sterubin (7-methoxy-3',4',5-trihydroxyflavanone, 1) as the
most active compound in the extract of E. californicum, showing a
remarkably higher in vitro activity than the co-existing flavonoids
eriodictyol (2) or homoeriodictyol (3).[11] The only very minor structural
difference between 1 and 2 demonstrates the “steep” structure-activity
relationships of flavonoids. Nothing, however, has so far been
reported on the activity of 1 in vivo. Moreover, the chirality of sterubin
(1), as a result of the stereocenter at C-2, was not taken into
consideration, making it unclear which enantiomer was responsible
for the activity. Previous reports had shown that flavonoids mainly
exist as their respective (S)-enantiomers in the plants and that they
tend to racemize during the isolation workup procedure.[14] Herein, we
[a]
Mr. J. Hofmann, Dr. M. Hoffmann, Mr. M. Scheiner, Prof. Dr. M.
Decker
Pharmaceutical and Medicinal Chemistry
Institute for Pharmacy and Food Chemistry
University of Würzburg, Am Hubland, 97074 Würzburg, Germany
E-mail: michael.decker@uni-wuerzburg.de
Dr. S. Fayez, Prof. Dr. G. Bringmann
[b]
[c]
Institute of Organic Chemistry
University of Würzburg, Am Hubland, 97074 Würzburg, Germany
E-Mail: bringman@chemie.uni-wuerzburg.de
Dr. S. Fayez
Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams
University, Organization of African Unity Street 1, 11566 Cairo,
Egypt
[d]
[e]
Dr. M. Hoffmann, Dr. T. Maurice
MMDN, University of Montpellier, INSERM, EPHE, UMR-S1198,
34095 Montpellier, France
Dr. S. Oerter, Dr. A. Appelt-Menzel
University Hospital Würzburg, Department for Tissue Engineering
and Regenerative Medicine, Röntgenring 11, 97070 Würzburg,
Germany
[f]
Dr. A. Appelt-Menzel
Fraunhofer Institute for Silicate Research ISC, Translational Center
Regenerative Therapies (TLC-RT), Röntgenring 11, 97070
Würzburg, Germany
[g]
Dr. P. Maher
The Salk Institute for Biological Studies, 10010 North Torrey Pines
Rd., CA 92037 La Jolla, United States
1 These authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.
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report on the synthesis of sterubin (1), the chiral resolution of its
synthetic racemate, the chiroptical analysis and stereochemical
assignment of the (R)- and (S)-enantiomers, (R)-1 and (S)-1, online,
by HPLC coupled to electronic circular dichroism, (ECD) and on the
configurational stability of the two stereoisomers. Furthermore, we
determined the enantiomeric purity of sterubin in the plant E.
californicum. Moreover, we describe the in vitro activity of the isolated
enantiomers against intracellular oxidative stress using the murine
hippocampal neuronal cell line HT22. And, for the first time, we have
conducted in vivo investigations on sterubin (1) in an AD mouse model
with Aβ25-35-induced memory impairment[15] and normal mice.
7
8
7
8
7
2
5
4
2
9
Scheme 1. Key steps in the synthesis of racemic sterubin (1). MOM
= methoxymethyl.
Figure 1. Flavonoids of Eriodictyon californicum (yerba santa): sterubin (1),
eriodictyol (2), homoeriodictyol (3), hesperetin (4), chrysoeriol (5), and luteolin
(6). The structural differences of 2-6 as compared to 1 are underlaid in grey.
Results and Discussion
Synthesis of sterubin (1)
The synthesis of 1 was performed by analogy to methods for the
preparation of similar racemic flavonoids described in the
literature.[16] A key step was the condensation of the known[17]
acetophenone 7 with the likewise known[18] aldehyde 8 to form the
respective chalcone 9 (Scheme 1). Cleavage of the MOM groups
and concomitant ring closure was achieved by heating 9 in 10%
HCl in MeOH, followed by treatment with sodium acetate to give
racemic sterubin (1).
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Resolution of the sterubin enantiomers, (R)-1 and (S)-1
The synthetic racemate of 1 was successfully resolved on a
ChiralPak IA® column (10 x 25 mm, 5 µm) using gradient elution
with initial condition from 32% B to 60% B in 29 min and a flow
rate of 6 mL/min, where B is 90% acetonitrile in water with 0.05%
TFA as a buffer (Figure 2A). Maximum absorption and peak
detection was achieved using a PDA detector at λ = 290 nm.
Figure 2. (A) Enantiomeric resolution of racemic sterubin (1) on a ChiralPak IA® column; (B) ECD trace (recorded at λ = 290 nm); (C) online LC-ECD spectra
of the two sterubin enantiomers; (D) configurational assignment of the two enantiomers by comparison of their online ECD curves with the offline spectrum
reported for the closely related, and configurationally known flavanone glycoside hesperidin (10). Sugar = rutinose.
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Configurational assignment of the sterubin enantiomers,
(R)-1 and (S)-1, by HPLC-ECD coupling
The absolute configuration of the two resolved peaks was
assigned online, by HPLC-ECD coupling.[19] By measurement at
a single wavelength, 290 nm, the chiroptically opposite behavior
of the two peaks was clearly seen (Figure 2B), further supporting
the assumption that the compounds were indeed enantiomers.
This was corroborated by the full online ECD spectra showing a
first, negative couplet at 330 nm (Peak I) and a second, positive
one at 290 nm (Peak II) for the fast enantiomer and an opposite
curve for the slower peak (Figure 2C). The assignment of the
rapidly eluting peak as corresponding to the R-enantiomer of
sterubin, (R)-1, and the slower one as its S-configured isomer,
(S)-1, was achieved by comparison of the ECD spectra of the two
enantiomers with that of the known, closely related, S-configured
flavanone glycoside hesperidin (10) (Figure 2D).[20] The ECD
curve of the second peak (Peak II) showed a good match with the
spectrum of 10, hence the slower enantiomer was S-configured.
For the first peak, by contrast (Peak I), virtually opposite spectra
were detected, so the faster eluting enantiomer was (R)-1.
Assignment of the absolute configuration and enantiomeric
purity of sterubin (1) in Eriodictyon californicum
Most naturally occurring flavonoids have so far been isolated as
the respective (S)-enantiomers.[14] To investigate the absolute
configuration and the enantiomeric purity of sterubin (1) in E.
californicum, dried leaves of the plant were extracted by mild
maceration in ethyl acetate assisted by ultrasonication for 30 min
at room temperature. Sterubin (1) and related flavanones were
enriched by precipitation from the ethyl acetate crude extract after
addition of n-hexane. The resulting precipitate was filtered,
dissolved in methanol, and injected on a ChiralPak IA® column
(Figure 3B). Spiking experiments with the synthetic racemate of 1
(Figure 3A) revealed an increase in the peak intensity of the S-
enantiomer (Figure 3C), showing that the plant contained sterubin
(1) in an enantiomerically pure form, as its (S)-enantiomer, (S)-1.
No racemization had occurred during the extraction procedure
described, while extraction under reflux conditions as described
in the literature[13a] obviously can lead to racemization.
Configurational stability of the sterubin enantiomers (R)-1
and (S)-1
Figure 3. Chromatograms on a ChiralPak IA® column: (A) synthetic
racemic sterubin (1); (B) the extract enriched with 1; (C) coelution of 1 with
the extract enriched with racemic 1 showing an increase in the peak
intensity of the (S)-enantiomer and evidencing that in E. californicum,
sterubin (1) is produced in an enantiopure S-form, (S)-1.
For an investigation of the configurational stability of sterubin, its
pure enantiomers, (R)-1 and (S)-1, were kept dissolved in
methanol at room temperature and the solution was monitored for
the formation of the other respective enantiomer by HPLC on a
ChiralPak IA® column after 2, 20, and 44 h. Under the applied
conditions, the two enantiomers proved to be configurationally
fully stable over 2 d and no racemization was observed as seen
in Figure 4.
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enantiomers might possibly undergo racemization upon contact
with cells or even upon exposure to the culture medium, in
contrast to their proven configurational stability in methanol (see
above).
(S)-1
(R)-1
A
after 2 h
10
15
20
25
10
15
20
25
Time [min]
Time [min]
B
after 20 h
10
15
20
25
Time [min]
10
15
20
25
Time [min]
C
after 44 h
10
15
20
25
10
15
20
25
Time [min]
Time [min]
Figure 4. Stability studies on the (R)- and (S)-enantiomers of sterubin,
(R)-1 and (S)-1, in methanol: (A) after 2 h; (B) after 20 h; (C) after 44 h
on a ChiralPak IA® column. The enantiomers were configurationally
stable over the entire time. The arrows indicate the expected sites of the
respective minor enantiomer.
Oxytosis assay
HT22 cells are a murine hippocampal nerve cell line. They are
sensitive to oxidative glutamate toxicity (oxytosis) due to a lack of
21]
ionotropic glutamate receptors.[9-10,
Addition of high
concentrations of extracellular glutamate inhibits the transport of
cystine, the oxidized form of cysteine, via the cystine/glutamate
antiporter, which results in glutathione (GSH) depletion. The
consecutive accumulation of ROS and calcium leads to
intracellular oxidative stress followed by cell death.[22] As GSH
depletion is similarly observed during aging of the brain and is
even accelerated in AD, the oxytosis assay gives information
about the neuroprotective properties of 1 against oxidative stress
in cells.[23] Flavonoids generally have only moderate antioxidant
properties[24] and, as reported by Fischer et al., sterubin (1) is one
of the more potent flavonoids against oxidative stress and
neuroinflammation in vitro.[11] The pure enantiomers of sterubin,
(R)-1 and (S)-1, as well as the synthetic racemic mixture, were
investigated in the oxytosis assay to identify possible differences
in activity between the stereoisomers. The flavonol quercetin at a
high concentration (25 µM) served as
a positive control.
Unexpectedly, no difference in activity was observed between the
racemic mixture and any of the pure enantiomers. All of them
provided significant neuroprotection at concentrations from
2.5 µM to 10 µM, which even exceeded that of the positive control
quercetin at a concentration of 5 µM. The lack of a difference in
bioactivity between the pure enantiomers (and between them and
the racemic mixture) raised the question whether the pure
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100
50
0
Glutamate 5 mM
Quercetin 25 µM
Compound [µM]
1
(S)-1
(R)-1
Figure 5. Oxytosis Assay. Treatment of HT22 cells with 5 mM glutamate (red) induced cytotoxicity. Quercetin (yellow) served as a positive control, 1, (S)-1, and (R)-1
all showed the same neuroprotective efficacy. Data are presented as means ± SEM of three independent experiments and results refer to untreated control cells
(black). Statistical analysis was performed using One-Way ANOVA followed by Dunnett’s multiple comparison posttest using GraphPad Prism 5 referring to cells
treated with 5 mM glutamate. Level of significance: *** p < 0.001.
Cellular uptake and racemization
Vrba et al.[25] and Gunesch et al.[9] observed the formation of
dehydrogenated products of esters combining the flavonoid
taxifolin and polyphenolic acids in RAW264.7 macrophages or
BV-2 microglia. In the case of sterubin (1), this would cause a loss
of the stereogenic center at C-2, which would explain why the
same activities were found for 1, (S)-1, and (R)-1. Another
explanation could be racemization in the cell culture medium.
Therefore, we investigated cellular uptake experiments in
microglial BV-2 cells and performed stability measurements in cell
culture medium. BV-2 cells were treated with 50 µM of the
respective compounds and incubated for 2 h or 4 h or lysed
immediately. Lysates were analyzed by HPLC on a chiral-phase
column and LC/UV. Reference chromatograms with sterubin (1)
and dehydrosterubin were recorded beforehand. As seen in
Figure 6, sterubin (1) is chemically stable in the BV-2 cells. While
no conversion of sterubin (1) to dehydrosterubin (12) was found,
HPLC on a chiral stationary phase revealed rapid racemization in
the cell culture medium even without the presence of cells (cf. SI).
Figure 6. The chemical stability of (R)-1 in BV-2 cells assigned by HPLC/UV: (A)
Synthesis of dehydrosterubin
at 0 h (black), after 2 h (red), and after 4 h (blue) of incubation, reference
chromatogram of 1 and dehydrosterubin (12) (green); (B) UV spectra of 1; (C) UV
spectra of 12. (R)-1 was chemically stable over the whole time.
Dehydrosterubin (12), also named hydroxygenkwanin, as a
reference compound was synthesized by analogy to a procedure
described by Aft.[26] For this purpose, the triacetate 11[16] of
sterubin (1) was dehydrogenated with N-bromosuccinimide (NBS)
in the presence of catalytic amounts of benzoyl peroxide (BPO) to
give the respective dehydro compound. Deprotection was
accomplished in
6
M
HCl(aq.) in acetonitrile, resulting in
dehydrosterubin (12).
11
12
Scheme 2. Final steps in the synthesis of dehydrosterubin (12).
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Neuroprotection in vivo
A
A
B
B
Recently,
a
number of polyphenols, including synthetic
compounds as well as natural products, have been identified as
11, 25]
potent neuroprotective agents in vitro.[9,
Surprisingly,
sterubin (1) showed a higher activity against oxidative stress and
neuroinflammation than several other flavonoids.[11] To determine
if sterubin (1) also has neuroprotective effects in vivo,
experiments were performed using a mouse model of AD
27]
described previously.[15,
Beforehand, in vitro cytotoxicity
experiments with human induced pluripotent stem cell derived
blood brain barrier endothelial cells were performed (cf. SI) to
exclude toxic effect. The results demonstrate, that 1 did not have
any major toxic effects. For the in vivo studies described in this
work, AD-like neurotoxicity and memory impairments were
induced by intracerebroventricular (ICV) injection of the amyloid β
(Aβ) fragment Aβ25-35 (9 nmol) on the first day of the study. Control
mice received distilled water (V1) ICV. Racemic sterubin (1) was
dissolved in a mixture of 60% DMSO and 40% saline (0.9% NaCl
in milliQ water) and the solutions were injected intraperitoneally
(IP) once per day (o.d.) for the following 7 d at doses between 0.3
and 3 mg/kg of 1. Injections of vehicle (60% DMSO + 40% saline,
V2) were used for the two control groups. Sterubin did not affect
the mouse body weight gain during the period of treatment (cf. SI).
Short-term spatial memory was evaluated in the Y-maze test
(YMT) on day 8 and long-term memory was evaluated on days 9
(training) and 10 (measurement of step-through latency) in the
step-through passive-avoidance assay (STPA). On day 11, the
mice were sacrificed, and the brains were frozen at -80°C.
Sterubin (1) significantly improved the Aβ25-35-induced alternation
deficit in the YMT at doses greater than 1 mg/kg (Figure 7A),
further substantiating the neuroprotective effects observed in
vitro.[11] In agreement with the results obtained in the YMT (Figure
7A), the Aβ25-35-induced deficit in long-term memory was also
compensated at a dose of sterubin (1) of 1 mg/kg or higher (Figure
7B). Sterubin (1) exceeded the activity of previously studied
polyphenols such as silibinin and taxifolin used in the same
mouse model of AD with respect to the dose needed to
compensate for the Aβ25-35 induced effects.[9, 28]
Sterubin [mg/Kg] IP
Sterubin [mg/Kg] IP
Sterubin [mg/kg] IP
Sterubin [mg/kg] IP
Figure 7. Effect of sterubin (1), administered IP, on Aß25-35-induced learning
impairments in mice: (A) spontaneous alternation performance in YMT and (B) step-
through latency in the STPA. Animals obtained distilled water (V1) or Aß25-35 (9 nmol
ICV) on day 1 and received sterubin (0.3-3 mg/kg IP), or DMSO 60% in saline (V2),
o.d. between day 1 and 7. They were examined in the YMT on day 8 and passive
avoidance training was performed on day 9, with retention being tested after 24 h.
Data show mean ± SEM in (A) and median and interquartile range in (B). n = 12-18
per groups. ANOVA: F(4,57) = 3.85, p < 0.01 in (A). Kruskal-Wallis ANOVA: H = 11.6, p
< 0.05 in (B). * p < 0.05 vs. (V+V)-treated group; # p < 0.05, ## p < 0.01, ### p < 0.001
vs. (V+Aß25-35)-treated group; Dunnett's test in (A), Dunn's test in (B).
Effects on memory in normal mice
We also asked if sterubin could improve memory in normal mice
as was previously shown for the flavonol fisetin using the object
recognition test.[29] In this test, mice are presented with two
identical objects during the training period which they explore for
a fixed time period. To test for memory, mice are presented one
day later with two different objects, one of which was presented
previously during the training and is thus familiar to the mice, and
the other that is novel. The better a mouse remembers the familiar
object, the more time it will spend exploring the novel object. To
test the effects of sterubin in this memory task, it was
administered orally to the mice before the start of the training
period. Rolipram, a phosphodiesterase inhibitor that potentiates
memory in this assay[30], requires intraperitoneal injection and was
used as a positive control. As shown in Figure 8, three doses of
sterubin were tested in the object recognition task and 10 mg/kg
showed a strong but not quite significant effect.
Figure 8. Effect of sterubin (1) on memory in normal mice. Sterubin
moderately enhances long term memory in mice. The effect of different oral doses
of sterubin on object recognition over a 10 min test period. Rolipram, injected
intraperitoneally at 0.1 mg/kg, served as a positive control. Data represent the
mean ± SEM of 11 mice/treatment group. Data were analyzed by one-way
ANOVA followed by post-hoc comparisons with Fisher's test.
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C), 128.1 (+, HC=CH), 123.8 (+, Ar-C) , 116.3 (+, Ar-C), 116.0 (+, Ar-C),
113.8 (Cq, Ar-C), 95.6 (-,CH2OCH3), 95.2 (-,CH2OCH3), 95.1 (2x +, Ar-C),
94.6 (2x, -,CH2OCH3), 56.5 (+,CH2OCH3), 56.4 (+,CH2OCH3), 56.3 (2x
+,CH2OCH3), 55.6 ppm (+, OCH3); ESI-MS: m/z calcd for C24H30O10+H+:
479.19; found 479.2.
Conclusions
By HPLC on a chiral phase column, resolution of synthetically
prepared racemic sterubin (1) into its pure enantiomers, (R)-1 and
(S)-1 was achieved for the first time. Although in methanol
configurational stability was observed, racemization took place in
the cell culture medium. These findings explain why no difference
in the neuroprotective activity in HT22 cells was found between
racemic sterubin (1) and its pure enantiomers. Importantly, in vivo
experiments revealed the high potency of sterubin as a
neuroprotective agent against Aβ25-35-induced AD-like memory
loss in mice. The effects were observed in both short-term and
long-term memory assays. Lesser effects on cognition were seen
in normal mice suggesting that the cognitive improvements were
not simply symptomatic in nature. It can be concluded that 1
exhibits strong neuroprotective properties during the 7-day
treatment, leading to improved memory in the behavioral tests
after the treatment was stopped. Hence, these findings strongly
support that sterubin (1) holds significant potential as a disease-
modifying neuroprotectant in AD.
Sterubin (1). A solution of chalcone 9 (1.10 g, 2.32 mmol) in 10%
methanolic HCl was stirred for 30 min at 50 °C. NaOAc (3.80 g, 46.4 mmol)
was added and the mixture was heated to reflux for 3 h, cooled, then water
was added and the mixture was extracted with ethyl acetate. The
combined organic layers were dried over Na2SO4 and the solvent was
removed under reduced pressure. The crude product was purified by silica
gel column chromatography using a mixture of dichloromethane and
methanol (40/1) as the eluent. The product was obtained as a white solid
in 55 % yield (391 mg). The analytical data were consistent with those
reported in the literature.[13a]
1H NMR (400 MHz, DMSO-d6): δ 12.11 (s, 1H, OH), 9.03 (m, 2H, OH),
6.91-6.86 (m, 1H, Ar-H), 6.78-6.71 (m, 2H, Ar-H), 6.10 6.06 (m, 2H, Ar-H),
5.42 (dd, 3J = 12.6, 3.0 Hz, 1H), 3.79 (s, 3H, OCH3), 3.24 (dd, 2J = 17.2, 3J
= 12.6 Hz, 1H), 2.72 (dd, 2J = 17.2, 3J = 3.1 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 196.9 (Cq, C=O), 167.4 (Cq, Ar-C), 163.1 (Cq, Ar-C), 162.8
(Cq, Ar-C), 145.7 (Cq, Ar-C), 145.1 (Cq, Ar-C), 129.2 (Cq, Ar-C), 117.9 (+,
Ar-C), 115.3 (+, Ar-C), 114.3 (+, Ar-C), 102.6 (Cq, Ar-C), 94.5 (+, Ar-C),
93.7 (+, Ar-C), 78.6 (+, Ar-C), 55.8 (+, CH3, OCH3), 42.1 (-, CH2). ESI-MS:
m/z calcd for C16H15O6+H+: 303.09; found 303.15.
Experimental Section
General: All reagents were bought from Sigma Aldrich, Munich, Germany,
unless otherwise noted, and were used without further purification. Thin-
layer chromatography was performed using Merck Silica Gel 60 F254 plates.
For column chromatography, Silica Gel 60 (particle size 0.040−0.063 mm)
(Sigma Aldrich, Munich, Germany) was used. Nuclear magnetic
resonance (NMR) spectra were recorded with a Bruker AV-400 NMR
instrument (Bruker, Karlsruhe, Germany) in CDCl3 or DMSO-d6. Chemical
shifts are expressed in ppm relative to CDCl3 (7.26 ppm for 1H and 77.16
For the preparation of 11, see the Supporting Information.
Tri-O-acetyldehydrosterubin: To a solution of tri-O-acetylsterubin (11)
(160 mg, 0.374 mmol) and NBS (67 mg, 0.374 mmol) in chloroform (5 mL)
benzoyl peroxide (6 mg, 26 µmol) was added and the reaction mixture was
heated to reflux for 2 h. Further chloroform was added, and the mixture
was washed with water and brine. The organic layer was dried over
Na2SO4 and the solvent was removed under reduced pressure. The crude
product was purified by silica gel chromatography using an eluent of
cyclohexane and ethyl acetate (2:1  pure ethyl acetate) and the product
was obtained as a white solid in 63% yield (100 mg). 1H NMR: (400 MHz,
1
ppm for 13C) or DMSO-d6 (2.50 ppm for H and 39.52 ppm for 13C). The
purity of the synthetic products was determined by HPLC (Shimadzu,
Duisburg, Germany), containing a DGU-20A3R degassing unit, an LC-
20AB liquid chromatograph, and an SPD-20A UV/vis detector. UV
detection was done at 254 nm. Mass spectra were obtained by an LCMS-
2020 device (Shimadzu, Duisburg, Germany). As a stationary phase, a
Synergi 4U fusion-RP column (150 mm x 4.6 mm) was used, and as a
mobile phase, a gradient of methanol/water with 0.1% formic acid.
Parameters: A = water, B = methanol, V(B)/[(V(A)+ V(B)] = from 5% to 90%
over 10 min, V(B)/[(V(A)+V(B)] = 90% for 5 min, V(B)/[(V(A) + V(B)] = from
90% to 5% over 3 min. The method was performed with a flow rate of
1.0 mL/min. Compounds were used for biological evaluation only if the
purity was 95% or higher.
3
4
4
CDCl3): δ 7.73 (dd, J = 8.5, J =2.2 Hz, 1H, Ar-H), 7.70 (d, J = 2.1 Hz,
1H, Ar-H), 7.35 (d, 3J = 8.5 Hz, 1H, Ar-H), 6.87 (d, 4J = 2.5 Hz, 1H, Ar-H),
4
6.62 (d, J = 2.4 Hz, 1H, Ar-H), 6.55 (s, 1H, C=CH), 3.92 (s, 3H, OCH3),
2.44 (s, 3H, CH3COO), 2.35 (s, 3H, CH3COO), 2.33 (s, 3H, CH3COO);
13C NMR (100 MHz, CDCl3): δ 176.3 (Cq, C=O), 169.7 (Cq, CH3COO),
168.1 (Cq, CH3COO), 167.9 (Cq, CH3COO), 163.7 (Cq, Ar-C), 160.3 (Cq,
C=CH), 158.9 (Cq, Ar-C), 150.7 (Cq, Ar-C), 144.7 (Cq, Ar-C), 142.7 (Cq, Ar-
C), 130.2 (Cq, Ar-C), 124.5 (+, Ar-C), 124.3 (+, Ar-C), 121.6 (+, Ar-C),
111.3 (Cq, Ar-C), 109.0 (+, C=CH), 108.6 (+, Ar-C), 99.2 (+, Ar-C), 56.1 (+,
OCH3), 21.2 (CH3COO), 20.8 (CH3COO), 20.7 (CH3COO); ESI-MS: m/z
calcd for C22H18O9+H+: 427.10; found 427.20.
For the preparation of acetophenone 7 and aldehyde 8, see the Supporting
Information.
Dehydrosterubin (12): A solution of tri-O-acetyldehydrosterubin (97 mg,
0.227 mmol) in acetonitrile (3 mL) and conc. aqueous HCl (3 mL) was
heated to reflux for 1.5 h. Yellow precipitant was formed, which was filtered
off, washed with water and dried under vacuum. The product was obtained
as a yellow solid in 50% yield (34 mg). 1H NMR (400 MHz, DMSO-d6):
δ 12.97 (s, 1H, OH), 9.96 (s, 1H, OH), 9.37 (s, 1H, OH), 7.44 (m, 2H, Ar-
H), 6.90 (d, 3J = 8.1 Hz, 1H, Ar-H), 6.72 (s, 1H, C=CH), 6.71 (d, 4J = 2.5 Hz,
Chalcone 9. A mixture of acetophenone 7 (650 mg, 2.16 mmol) in EtOH
(10 mL) and a saturated solution of KOH in EtOH (15 mL) was stirred at
4 °C for 15 min. A solution of 8 (490 mg, 2.16 mmol) in EtOH (5 mL) was
added dropwise and the mixture was allowed to stir overnight (16 h) at
room temperature. The reaction was quenched with water and extracted
with ethyl acetate. The combined organic layers were dried over Na2SO4
and the solvent was removed under reduced pressure. The crude product
was purified by silica gel chromatography using a mixture of cyclohexane
and ethyl acetate (3/1). The product was obtained as a yellow solid in 85%
yield (1.11 g). The analytical data were consistent with those reported in
the literature.[31] 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H, Ar-H), 7.26 (d,
2J = 16.0 Hz, 1H, HC=CH), 7.13 (s, 2H, Ar-H), 6.86 (d, 2J = 16.0 Hz, 1H,
HC=CH), 6.43 (s, 2H, Ar-H), 5.25 (s, 2H, CH2OCH3), 5.22 (s, 2H,
CH2OCH3), 5.11 (s, 4H, CH2OCH3), 3.82 (s, 3H, OCH3), 3.51 (s, 3H,
CH2OCH3), 3.50 (s, 3H, CH2OCH3), 3.39 ppm (s, 6H, CH2OCH3); 13C NMR
(100 MHz, CDCl3): δ 194.4 (Cq, C=O), 162.1 (Cq, Ar-C), 156.0 (2x Cq, Ar-
C), 149.4 (Cq, Ar-C), 147.5 (Cq, Ar-C), 144.7 (+, HC=CH), 129.4 (Cq, Ar-
4
1H, Ar-H), 6.37 (d, J = 2.2 Hz, 1H, Ar-H), 3.87 (s, 3H, OCH3); 13C NMR
(100 MHz, DMSO-d6): δ 181.7 (Cq, C=O), 165.0 (Cq, Ar-C), 164.2 (Cq, Ar-
C), 161.1 (Cq, C=CH), 157.1 (Cq, Ar-C), 149.8 (Cq, Ar-C), 145.7 (Cq, Ar-C),
121.3 (Cq, Ar-C), 119.0 (+, Ar-C), 115.9 (+, Ar-C), 113.5 (+, Ar-C), 104.6
(Cq, Ar-C), 103.0 (+, C=CH), 97.9 (+, Ar-C), 92.5 (+, Ar-C), 56.0 (+, OCH3);
ESI-MS: m/z calcd for C16H12O6+H+: 301.07; found 301.15.
Plant material: Leaves of Eriodictyon californicum Hook.
& Arn.
(Boraginaceae) were collected by Ms. Kyra Bobine in May, 2019.
Plant extraction: Dried leaves of E. californicum (18.6 g) were soaked in
ethyl acetate (3 x 100 mL), ultrasonicated for 30 min, then shaken
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10.1002/chem.202001264
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overnight (~16 h) at room temperature. The crude extract was filtered, and
the filtrate was concentrated in vacuo. The obtained residue (2.0 g) was
re-dissolved in 90% aqueous methanol. By addition of n-hexane,
chlorophyll and non-polar residues were removed and sterubin (1) and
related flavones were precipitated. After filtration, the precipitate (0.6 g)
was dissolved in methanol and directly subjected to HPLC on a ChiralPak-
IA column.
Animals. Male Swiss mice 6 weeks old, body weight 30-40 g, obtained
from JANVIER (Saint Berthevin, France) were housed in the animal facility
of the University of Montpellier (CECEMA, Office of Veterinary Services
agreement # B-34-172-23) with access to food and water ad libitum
(except during behavioral tests). The humidity and temperature were
controlled, and the mice were kept at a 12 h light / 12 h dark cycle (lights
off at 7:00 p.m.). All animal procedures were conducted in strict adherence
to the European Union directives of September 22nd, 2010 (2010/63/UE)
and to the ARRIVE guidelines. The project was authorized by the French
National Ethics Committee (APAFIS #1485-15034). Animals were
assigned to different treatment groups randomly.
Chiral resolution of racemic sterubin: An HPLC-UV guided resolution
of the enantiomers of 1 was performed on a Jasco system equipped with
a DG-2080 degassing unit, a PU-1580 ternary pump, an MD-2010 plus
multiwavelength detector, and an AS-2055 autosampler. Separation of the
enantiomers was done on a ChiralPak IA® (10 x 25 mm, 5 µm, Daicel
Chemical Industries) column using a gradient system with initial conditions
32% B (B: 90% MeCN in water + 0.05% TFA) to 60% B in 29 min. The (R)-
and (S)-enantiomers of sterubin, (R)-1 and (S)-1, had retention times of
17.8 min and 20.2 min, respectively.
Preparation of sterubin injections. Sterubin (1) was dissolved in 100%
DMSO at a concentration of 6 mg/mL to give a stock solution, which was
diluted with saline (0.9% NaCl in milliQ water) and DMSO to the final test
concentration and a final percentage of 60% DMSO. 60% DMSO in saline
served as the vehicle (V2). After compound injections, the behavior of the
mice in their home cage was checked visually. Weight was examined once
per day. As demonstrated in Figure SI 1, a tendency was observed that
weight gain was facilitated with an increasing dose of 1. Nevertheless, the
difference in weight gain remained insignificant compared to Aβ + V2
treated mice in Dunnett's multiple comparison test.
Online LC-ECD analysis of the sterubin enantiomers: ECD
spectroscopic analysis was performed using
spectropolarimeter. Measurements were done at room temperature and
the spectra were processed using the SpecDis software.[32]
a
Jasco J-715
Amyloid peptide preparation and ICV injection. All experiments
followed previously described protocols.[15, 25, 32] The Aβ25-35 peptide was
prepared according to Maurice et al.[15] Mice were anesthetized with 2.5%
isoflurane. Then, oligomerized Aβ25-35 peptide (9 nmol in 3 µL/mouse) was
injected ICV. Bidistilled water was used as a vehicle (V1).
Oxytosis assay: HT22 cells were grown in Dulbecco’s Modified Eagle
Medium (DMEM, Sigma Aldrich, Munich, Germany) supplemented with
10% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin-streptomycin.
5 x 103 HT22 cells per well were seeded into sterile 96-well plates and
incubated overnight (~16 h). Aqueous glutamate solution (5 mM)
(monosodium-L-glutamate, Sigma Aldrich, Munich, Germany) together
with 2.5, 5.0, 7.5, or 10 µM of the respective compound was added to the
cells and incubated for 24 h. Quercetin (25 µM) (Sigma Aldrich, Munich,
Germany) together with glutamate (5 mM) served as a positive control.
After 24 h incubation cell viability was determined using a colorimetric 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma
Aldrich, Munich, Germany) assay. MTT solution (5 mg/mL in PBS) was
diluted 1:10 with medium and added to the wells after removal of the old
medium. Cells were incubated for 3 h and then lysis buffer (10% SDS) was
added. The next day, absorbance at 560 nm was determined with a
multiwell plate photometer (Tecan, SpectraMax 250). Results are
presented as percentage of untreated control cells. All data are expressed
as means ± SEM of three independent experiments. Analysis was
accomplished using GraphPad Prism 5 Software applying Oneway
ANOVA followed by Dunnett’s multiple comparison posttest. Levels of
significance: * p < 0.05; ** p < 0.01; *** p < 0.001.
Spontaneous alternation performance in a Y-maze. On day 8 of the
study, the spatial working memory of all mice was evaluated in the Y-maze.
[15, 25, 32]. The Y-maze is made from grey polyvinylchloride and has three
identical arms (length 40 cm, height 13 cm, bottom width 3 cm, top width
10 cm (walls converge at an equal angle). For evaluation of memory, every
mouse was placed into one arm and was allowed to explore the maze for
8 min. All entries into an arm (including the return into the same arm) were
counted and the number of alternations (mouse entered all three arms
consecutively) was calculated as percentage of total number of arm entries
[alternations / (arm entries-2)*100].
Step-through passive avoidance test. STPA was performed on day 9
and day 10 in a two-compartment box [(width 10 cm, total length 20 cm
(10 cm per compartment), height 20 cm] consisting of polyvinylchloride.
One of the compartments was white and illuminated with a bulb (60 W,
40 cm above the center of the compartment), the second compartment
was black, covered, and had a grid floor. A guillotine door separated the
compartments. On day 9 (training), each animal was placed in the white
compartment and was left to explore for 5 s. Then, the door was opened,
which allowed the mouse to enter the black compartment. After it had
entered, the door was closed, and a foot shock was delivered (0.3 mA) for
3 s generated by a scramble shock generator (Lafayette Instruments,
Lafayette, USA). The step-through latency (time the mouse spent in the
white compartment after the door was opened) and the level of sensitivity
(no sign = 0, flinching reactions = 1, vocalization = 2) were recoded.
Treatment with sterubin (1) did not affect the measured parameters. On
the next day (day 10), each mouse was placed in the white compartment
and was allowed to explore for 5 s. Then, the door was opened allowing
the mouse to step over into the black compartment. The step-through
latency was measured for up to 300 s.
Cellular uptake and racemization experiments: 2 x 106 BV2 cells were
grown in sterile 100 mm dishes overnight and 4 mL 50 µM (S)-1 or (R)-1
diluted in cell culture medium were added. Cells were incubated for the
indicated time periods, after which the supernatant was removed, and cells
were washed twice with PBS. Further PBS (1 mL) was added, cells were
scraped and transferred to Eppendorf tubes. The samples were
centrifuged and resuspended in 200 µL of MeOH. The cells were frozen in
liquid nitrogen and thawed at 37 °C (10 times). Cell debris was pelleted by
centrifugation and the supernatant was collected for HPLC analysis.
Neuroprotection studies in vivo: The in vivo behavioral experiments
33]
were performed as established and published previously.[15,
Neurotoxicity was induced by ICV injection of oligomerized Aβ25-35 peptide,
and sterubin (1) was evaluated for its neuroprotective properties. Sterubin
was dissolved in 60% DMSO and 40% saline (0.9% NaCl in milliQ water)
and was injected once per day IP on days 1-7 to give doses of 0.3, 1, and
3 mg/kg. The oligomerized Aβ25-35 peptide was injected ICV on day 1 of
the study. The behavior of the mice was evaluated on day 8 (YMT) and
days 9 and 10 (STPA). On day 11, the mice were sacrificed, and the brains
were collected. Samples were frozen at -80 °C for further biochemical
analysis.
Sacrifice and brain collection. All animals were sacrificed on day 11. The
brains were collected, hippocampus and cortex were isolated, and the
samples were frozen at -80 °C.
Statistical analysis. Weight gain and results from the YMT were analyzed
by the software GraphPad Prism 5.0 using one-way ANOVA, followed by
Dunnett's post-hoc multiple comparison test. STPA had a maximum step-
through latency of 300 s. Therefore, a Gaussian distribution could not be
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10.1002/chem.202001264
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FULL PAPER
assumed. The results were analyzed using
a
Kruskal-Wallis non-
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parametric ANOVA, followed by a Dunn's multiple comparison test. p <
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Novel object recognition test. Male C57Bl/6J mice were used
and the testing was done by Scripps Research. All mice were
acclimated to the colony room for at least 2 weeks prior to testing
and were tested at an average age of 8 weeks. Mice were
randomly assigned across treatment groups with 11 mice in each
group. For each dose tested, a solution of sterubin in corn oil was
prepared. The vehicle was corn oil alone. All were administered
orally 60 min prior to the training session at a volume of 10 ml/kg
body weight. Rolipram was dissolved in 10% DMSO and
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Day 1 mice were habituated to a circular open field arena for one
hour in cage groups of four. 24 hr later, individual mice were
placed back in the same arena which now contained two identical
objects for a 15 min training trial. On Day 3, vehicle-, sterubin- or
rolipram-treated mice were individually placed back in the same
arena in the presence of both the familiar object (i.e. previously
explored) and a novel object. The spatial positions of the objects
were counter-balanced between subjects. Each animal's test trial
was recorded and the first 10 min of each session were scored.
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with novel object x 100)/ Total time spent exploring both objects.
Data were analyzed by a one-way ANOVA followed by post-hoc
comparisons with Fisher's test.
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Acknowledgements
Financial support by the Bavaria California Technology Center to
P.M. and M.D. under project number 3 [2018-2] is gratefully
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Campus France (PHC Procope), and the German Academic
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Conflict of interest
The authors declare no conflict of interest.
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Keywords: Eriodictyon californicum • flavonoids • sterubin • chiral
resolution • circular dichroism • Alzheimer´s disease
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The Californian plant Eriodictyon
californicum produces the flavanone
sterubin as its (S)-enantiomer. Fast
racemization takes places in cell
culture medium, whereas
J. Hofmann, S. Fayez, M. Hoffmann,
M.Scheiner, S. Oerter, A. Appelt-
Menzel, P. Maher, T. Maurice, G.
Bringmann, M. Decker.
Page No. – Page No.
configurational stability was observed
in methanol. Sterubin was a potent
neuroprotectant in vitro and displayed
significant beneficial effects on short-
and long-term memory in an AD
mouse model.
Sterubin: enantioresolution and
configurational stability, enantiomeric
purity in nature, and neuroprotective
activity in vitro and in vivo
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