Samin-derived flavonolignans, a new series of antidiabetic agents having dual inhibition against α-glucosidase and free radicals

Article abstract of DOI:10.1080/14786419.2018.1553169

A series of novel flavonolignans were synthesized by the reaction between a lignan named samin (1) and a range of flavonoids. This simple and rapid approach allowed direct assembly of these two bulky motifs in good yields without the formation of byproducts. Upon evaluation of antidiabetic activity of the synthesized products, epicatechinosamin (β-2g) was the most active α-glucosidase inhibitor toward maltase and sucrase. The kinetic study indicated that β-2 g inhibited the enzymes in a mixed manner of competitive and noncompetitive inhibition.

Full text of DOI:10.1080/14786419.2018.1553169

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Samin-derived flavonolignans, a new series of
antidiabetic agents having dual inhibition against
α-glucosidase and free radicals
Wisuttaya Worawalai & Preecha Phuwapraisirisan
To cite this article: Wisuttaya Worawalai & Preecha Phuwapraisirisan (2019): Samin-derived
flavonolignans, a new series of antidiabetic agents having dual inhibition against α-glucosidase and
free radicals, Natural Product Research, DOI: 10.1080/14786419.2018.1553169
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1553169
Samin-derived flavonolignans, a new series of
antidiabetic agents having dual inhibition against
a-glucosidase and free radicals
Wisuttaya Worawalai and Preecha Phuwapraisirisan
Center of Excellent in Natural Products, Department of Chemistry, Faculty of Science, Chulalongkorn
University, Bangkok, Thailand
ABSTRACT
ARTICLE HISTORY
Received 10 September 2018
Accepted 25 November 2018
A series of novel flavonolignans were synthesized by the reaction
between a lignan named samin (1) and a range of flavonoids.
This simple and rapid approach allowed direct assembly of these
two bulky motifs in good yields without the formation of byprod-
ucts. Upon evaluation of antidiabetic activity of the synthesized
products, epicatechinosamin (b-2g) was the most active a-glucosi-
dase inhibitor toward maltase and sucrase. The kinetic study indi-
cated that b-2 g inhibited the enzymes in a mixed manner of
competitive and noncompetitive inhibition.
KEYWORDS
Flavonolignan; diabetes;
glucosidase; antioxidant;
Friedel-Crafts; oxocarbenium
1. Introduction
Prolonged postprandial hyperglycemia commonly detected in Diabetes mellitus (DM)
patients has been recognized to trigger glucose autoxidation and free radical overproduc-
tion, eventually leading to oxidative stress and inducing b-cell dysfunction or failure (Rahimi
et al. 2005). Therefore, type 2 DM therapy and treatment of complications can be successfully
achieved by simultaneously attenuating hyperglycemia and suppressing glucose autoxida-
tion. However, oral administration of combined antihyperglycemic drugs and antioxidants
CONTACT Preecha Phuwapraisirisan
Preecha.p@chula.ac.th
Supplemental data for this article can be accessed here at https://doi.org/10.1080/14786419.2018.1553169.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
W. WORAWALAI AND P. PHUWAPRAISIRISAN
Figure 1. Structures of synthesized antidiabetic lignans.
must be carefully performed due to Drug-drug interactions and unexpected adverse effects.
To avoid this concern, the idea of ‘One Drug Dual Function (ODDF)’ has been introduced as
an alternative approach (Wang et al. 2012). We adopted the ODDF approach to synthesize
such compounds and assess antihyperglycemic activity through a-glucosidase inhibition as
well as antioxidation through free radical scavenging. The success of this strategy resulted in
the discovery of new ODDFs such as quercitylcinnamates (Rattanangkool et al. 2013) and N-
arylmethylaminoquercitols (Worawalai et al. 2015). To our knowledge, acarbose, a widely
used antihyperglycemic drug possessing a-glucosidase inhibition, has not been reported for
benefit in suppressing DM-associated complications.
In our continuing work on synthesis of new ODDFs, we recently synthesized a new series
of furofuran lignans (Worawalai et al. 2016) by direct coupling between a lignan named
samin and a variety of phenolic compounds. Of twenty products synthesized, lignans encom-
passing at least one free phenolic (Ar-OH) moiety (Figure 1) showed more potent inhibition
against a-glucosidases and free radicals than their parent starters. This observation suggests
that inhibitory potency would largely depend on the number of free phenolics. To further
explore more potent ODDFs and gain insight into the role of the phenolic group on inhibit-
ing a-glucosidase and free radicals, a new series of furofuran lignans containing multiphe-
nolic residues were synthesized. In the present study, we plan to apply flavonoids (Ar-C3-Ar)
as multiphenolics and samin (1) as a more reactive lignan starter (Scheme 1). We expect that
the newly generated products, collectively named flavonolignans (2), would display more
potent inhibition than furofuran lignans previously reported.
2. Results and discussion
2.1. Synthesis and characterization
Samin (1), used as starting lignan, was obtained from sesamolin, a major lignan isolated
from sesame (Sesamum indicum) seed oil, via acid hydrolysis (Scheme S1,
Supplementary material, Worawalai et al. 2016). With starting samin (1) in hand, we first
evaluated the scope of flavonoids capable of undergoing Friedel-Crafts reaction. We
screened six different types of flavonoids, namely dihydrochalcone, chalcone, flavanone,

NATURAL PRODUCT RESEARCH
3
O
O
O
OH
Ar-C3-Ar
*
Amberlyst-15
4Å MS, 70 °C
Ar-C3-Ar
H
H
H
H
flavonoids
O
O
O
O
O
samin (1)
flavonolignans (2)
Scheme 1. Synthesis of flavonolignans.
Figure 2. Flavonoids (a–g) used in synthesis of flavonolignans. The bold arrow indicates where the
bonding between flavonoid and furan moiety was formed.
flavonol, flavone, and flavanol (Figure 2). It was expected that ring A of flavonoids
would be substituted by samin (1) as oxocarbenium ion. When the process was com-
plete, the reactions between samin (1) and three different flavonoids, namely dihydro-
chalcone (a), chalcones (b and c) and flavanol (g), furnished the expected products.
The reaction between samin (1) and panduratin A (a) in an acidic solution (Amberlyst-
15, CH3CN) at 70 ꢀC furnished a single spot of flavonolignan, trivially named panduratino-
samin (a–2a), in 33% yield (Table 1). The connectivity between samin (1) and flavonoid a
in a–2a was proved by HMBC correlations shown in Figure S1a. The relative configuration
of the newly generated chiral center C-2 on the furan motif was determined by analysis
of multiplicity of the neighboring proton H-4 (Worawalai et al. 2016). The a-isomer
revealed doublet of doublet (dd) for both H-4ax and H-4eq while the b-isomer showed the
dd pattern only for H-4ax, but a unique doublet (d) for H-4eq. This observation was fully
proved by NOESY analysis and also provided an effective means for rapid recognition of
a- and b-isomers. We therefore adopted the above observation to predict the C-2 config-
uration of panduratinosamin (a–2a). The diastereomeric methylene protons H-4 dis-
played dd signals at dH 3.83 and 4.57 ppm, thus indicating a-configuration.
With the success on the synthesis of flavonolignan a–2a in hand, we subsequently
examined the reactions between samin (1) and chalcones b and c. Under similar con-
ditions, a pair of diastereomeric products named a-cardamominosamin (a–2b, 40%)

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W. WORAWALAI AND P. PHUWAPRAISIRISAN
Table 1. Synthesis of flavonolignans.
Isolated product (% Yield)
Entry
a–2
b–2
1
2
3
4
5
6
7
a–2a (33%)
a–2b (40%)
a–2c (75%)
a–2d (NR)b
a–2e (NR)
a–2f (NR)
b–2a (NI)a
b–2b (34%)
b–2c (NI)
b–2d (NR)
b–2e (NR)
b–2f (NR)
b–2g (52%)
a–2g (NI)
aNot isolated. Due to trace amount, structure characterization and bioactivity evaluation could not be accomplished.
bNo reaction.
and b-cardamominosamin (b-2b, 34%) were produced via the reaction between 1 and
cardamomin (b). The occurrence of both a–2b and b–2b clearly supported the mech-
anistic formation of flavonolignans through a Friedel-Crafts reaction (Figure S2).
Similarly, flavonolignan a-2c, named 3,20-dihydroxy-4,40,60-trimethoxychalconosamin,
was also generated a 75% yield from 1 and c. In addition, epicatechinosamin (b–2g)
with 52% yield was also generated by the reaction between samin (1) and epicatechin
(g). However, the reaction between samin (1) and flavonoids having ring C or the cyc-
lic acyl group, namely flavanone d, flavonol e and flavone f, afforded no desired prod-
uct, thus indicating that the acyl group deactivates aromaticity of ring A.
2.2. a-Glucosidase inhibitory activity
All of the synthesized flavonolignans were evaluated for a-glucosidase inhibition toward rat
intestinal maltase and sucrase, compared with furofuran lignans having one free hydroxy
group on phenolic moiety (Figure 3). Generally, all flavonolignans and furofuran lignans
inhibited maltase more selectively than sucrase. The inhibitory activity of them tends to
increase with the increasing of the number of phenolic hydroxy group except for a–2c.
However, a–2a showed weak inhibition against only sucrase. Of flavonolignans examined,
b–2g, having four phenolic hydroxy groups, was the most potent inhibitor against maltase
and sucrase. We hypothesize that the presence of the catechol group as ring B of the flavon-
oid moiety might participate in chelation with enzymes, thus enhancing inhibitory activity
(Kim et al. 2018). Noticeably, comparable inhibitory effects of two epimeric flavonolignans
a–2b and b–2b suggested that the configuration of C-2 plays no role in bioactivity.
2.3. Antioxidant activity
All of the synthesized flavonolignans were also evaluated for radical scavenging, com-
pared with furofuran lignans having one free hydroxy group on phenolic moiety
(Figure S5). For this assay, phenolic hydroxy group could donate hydrogen radical to

NATURAL PRODUCT RESEARCH
5
maltase
sucrase
20.1
25
20
15
10
5
14.9
18.2
4.01
3.84
3.3
3.13
1.57
0.99
0.18
2.44
1.52
1.31
1.14
0.9
0.77
0.52
0.1
0
compound
4
2
1
No. of phenolic OH
Figure 3. a-Glucosidase inhibitory effects of flavonolignans against maltase and sucrase.
terminate radical propagation, thus preventing the onset of cellular impairment. Of
the lignans examined, b-sesaminol, b-2,6-DMPS, a–2c, and b–2g showed strong inhib-
ition against free radicals in the range of 0.15-0.94 mM. Among them, b–2g, having
four phenolic hydroxy groups, was the most potent inhibitor.
2.4. Kinetic study
In order to gain further insight into how these flavonolignans interact with rat intestinal
maltase and sucrase, the inhibition mode of b–2g, the most active inhibitor chosen as
representative, was analyzed in a kinetic study. The Lineweaver-Burk plot of b–2g against
maltase (Figure S4a) showed a series of straight lines; all of which intersected in the
second quadrant. Kinetic analysis subsequently showed that Vmax decreased with elevated
Km in the presence of increasing concentrations of b–2g. This behaviour suggests that
b–2g inhibited maltase in a mixed-type manner comprising two different pathways: com-
petitive and noncompetitive. The observed result was elaborated by simultaneous forma-
tion of enzyme-inhibitor (EI) and enzyme-substrate-inhibitor (ESI) complexes in
competitive and noncompetitive manners, respectively (Scheme S2).
We further investigated the pathway in which b–2g was preferentially preceded by
determining dissociation constants of EI (Ki) and ESI (K0i) complexes (Table S1).
Apparently, the secondary plots (Figures S6a and S6b) demonstrate Ki and K0i values of
0.15 and 0.40 mM, respectively, thus indicating that b–2g was predominantly bound to
maltase (EI) rather than the formed ESI complex. The putative inhibitory mechanism is
summarized in Scheme S2. The inhibitory mechanism of b–2g against sucrase (Figure
S4b) was also examined using the above methodology. Apparently, b–2g inhibited
sucrase via both competitive and noncompetitive manners (mixed-type inhibition)
with Ki and K0i values of 0.42 and 0.84 mM, respectively (Figures S7a and S7b).
3. Experimental section
3.1. Extraction and isolation of natural flavonoids (a and b) and sesamolin
Panduratin A (a) and cardamomin (b) were isolated from fingerroots (Boesenbergia
rotunda). Sesamolin was obtained from sesame seed oil using the methodology

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W. WORAWALAI AND P. PHUWAPRAISIRISAN
described elsewhere with minor modification (Reshma et al. 2010). The detail of isola-
1
tion and purification of flavonoids (a and b) and sesamolin as well as their H NMR
data were supplied as supplementary material.
3.2 Chemistry
3.2.1. Synthesis of samin (1)
To a solution of sesamolin (0.3 mmol) in a mixture of acetonitrile-H2O (9.5:0.5, 10 mL)
was added Amberlyst-15 (1 mg/0.005 mmol of sesamolin). After stirring at 70 ꢀC for 5 h,
the reaction mixture was filtrated, evaporated to dryness and purified by silica gel
chromatography using 1:1 EtOAc-hexane as mobile phase to afford samin (1, 95%) as
colorless oil. NMR data of 1 were nearly identical to those reported previously (Maiti
et al. 1995).
3.2.2. General procedure for synthesis of flavonolignans 2
To a solution of samin 1 (1 equiv) in acetonitrile (1.0 mL/0.1 mmol of 1) was reacted
with flavonoids (1.5–2 equiv), Amberlyst-15 (1 mg/0.005 mmol of 1) and 4 Å molecular
sieve. After stirring at 70 ꢀC for 8 h, the reaction mixture was evaporated to dryness
and purified by flash or Sephadex LH-20 chromatography to yield flavonolignans 2.
The NMR data and spectra of flavonolignans 2 were supplied as supplemen-
tary material.
3.3. a-Glucosidase inhibitory activity
a-Glucosidase inhibitory activity against rat intestinal maltase and sucrase was deter-
mined according to our previous report (Ramadhan and Phuwapraisirisan 2015). For
details of the experiment, see suplementary material.
3.4. Kinetic study of a-glucosidase inhibition
For kinetic analyses of maltase by the active compound, enzyme and active compound
were incubated with increasing concentrations of maltose (2–20 mM). The type of
inhibition was determined by the Lineweaver-Burk plot. For calculation of Ki and Ki0
values, slope and intercept from the Lineweaver-Burk plot were replotted vs. [I], which
gave the secondary plot.
3.5. Antioxidant activity
Radical scavenging activity was validated using DPPH colorimetric method (Hirose
et al. 2013).
4. Conclusion
In conclusion, we first synthesized a series of novel flavonolignans via a single-step
reaction between samin (1) and a variety of flavonoids in the presence of acidic resin.
Of the synthesized flavonolignans, b–2g possessed the strongest inhibition against

NATURAL PRODUCT RESEARCH
7
a-glucosidase (maltase and sucrase) and free radicals. The preliminary structure-activity
relationships displayed two free hydroxyl groups on ring A and a catechol moiety on
ring B in the newly introduced flavonoid unit enhanced activities. The highly potent
antioxidant activity and a-glucosidase inhibition of b–2g should be beneficial in dia-
betes treatments as well as in preventing the onset of its complications.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This project was financially supported by the Thailand Research Fund (RSA5880027) and Faculty
of Science, Chulalongkorn University (Sci-Super IV_61_003). W.W. is grateful to the Graduate
School of Chulalongkorn University for a Postdoctoral Fellowship (Ratchadaphisek Sompot
Fund). The Center of Excellent in Natural Products was subsidized by Chulalongkorn University.
We thank Mr. Thammatee Potipiranun for technical assistance in the isolation and purification of
natural flavonoids.
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