molecules
Article
Isolation, Characterization and In Silico Studies of Secondary
Metabolites from the Whole Plant of Polygala inexpectata
Peşmen & Erik
Ayşe Ünlü 1, *, Kerem Teralı 2 , Zübeyde Uğurlu Aydın 1 , Ali A. Dönmez 1 , Hasan Soliman Yusufoğlu 3
and İhsan Çalış 4
1
2
3
4
*
Citation: Ünlü, A.; Teralı, K.;
Uğurlu Aydın, Z.; Dönmez, A.A.;
Yusufoğlu, H.S.; Çalış, İ. Isolation,
Characterization and In Silico Studies
of Secondary Metabolites from the
Whole Plant of Polygala inexpectata
Peşmen & Erik. Molecules 2022, 27,
684. https://doi.org/10.3390/
molecules27030684
Academic Editor: Natalizia Miceli
Received: 24 December 2021
Accepted: 15 January 2022
Department of Biology, Faculty of Science, Hacettepe University, Ankara 06800, Turkey;
zubeydeugurlu@hacettepe.edu.tr (Z.U.A.); donmez@hacettepe.edu.tr (A.A.D.)
Department of Medical Biochemistry, Faculty of Medicine, Girne American University, Kyrenia 99428, Cyprus;
keremterali@gau.edu.tr
Department of Pharmacognosy & Pharmaceutical Chemistry, College of Dentistry & Pharmacy, Buraydah
Private Colleges, Buraydah 51418, Saudi Arabia; yusufoglu@psau.edu.sa
Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia 99138, Cyprus;
ihsan.calis@neu.edu.tr
Correspondence: ayseunlu@hacettepe.edu.tr; Tel.: +90-548-821-89-69
Abstract: Polygala species are frequently used worldwide in the treatment of various diseases,
such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative
diseases, due to the large number of secondary metabolites they contain. The present study was
performed on Polygala inexpectata, which is a narrow endemic species for the flora of Turkey, and
resulted in the isolation of nine known compounds, 6,3′ -disinapoyl-sucrose (1), 6-O-sinapoyl,3′ O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3′ -O-(O-methyl-feruloyl)-sucrose (3), 3′ -O(sinapoyl)-sucrose (4), 3′ -O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3′ -O-feruloyl-sucrose
(sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8),
and 7,4′ -di-O-methylquercetin-3-O-β-rutinoside (ombuin 3-O-rutinoside or ombuoside) (9). The
structures of the compounds were determined by the spectroscopic methods including 1D-NMR
(1 H NMR, 13 C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC), and HRMS. The isolated
compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding
pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through
hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously
established anti-inflammatory properties of some Polygala-derived phytochemicals may be due, in
part, to the modulation of pro-inflammatory enzyme activities.
Keywords: liriodendrin; molecular docking; ombuoside; sucrose esters; syringin
Published: 21 January 2022
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1. Introduction
published maps and institutional affil-
Polygala L. is the largest genus of the family Polygalaceae with more than 700 recognized
species and is distributed in all continental areas except the Arctic and New Zealand [1–3].
The genus is represented by 16 native and one cultivated species in Turkey [4–7]. Recently,
two new species in the subgenus Polygala have been described from the Eastern part
of Turkey [8,9]. Among Polygala taxa, P. inexpectata Peşmen & Erik, which is subject
to this study, is a narrow endemic species and is known from only the type locality,
Ermenek, Turkey.
The Polygala species represent a rich molecular diversity in terms of plant phytochemical constituents, and they have widely been used in folk medicine for a long time to treat
chronic asthma, bronchial asthma, and whooping cough as an expectorant and stimulant
in many countries such as China, Japan, Thailand, India, North-South America, Brazil, and
Turkey [10–16]. For instance, the roots of P. tenuifolia Willd. and P. senega L. are the two
iations.
Copyright: © 2022 by the authors.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
Molecules 2022, 27, 684. https://doi.org/10.3390/molecules27030684
https://www.mdpi.com/journal/molecules
Molecules 2022, 27, 684
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species with the most important plant components in Traditional Chinese Medicine (TCM).
Both are species included in the German (DAB), European (Ph. Eur.), Austrian (ÖAB),
Indian Ayurvedic, British Herbal, Swiss (Ph. Helv.), Japanese Pharmacopoeias and ESCOP,
Commission E, and WHO Monographs. Besides, more than 140 compounds have been
isolated from the Polygala species [17]. These compounds are mostly xanthones [18–29],
saponins [30–33], and oligosaccharides [34–41]. Some other secondary metabolites include
coumarins [42–44], flavonoids [45–51], sterols [52], and lignans [53–58]. Many pharmacological studies have revealed the important pharmacological properties of the roots of
Polygala tenuifolia [59]. Its effects on the cardiovascular and nervous systems [60–63] are well
known. It has also been reported to have adjuvant [64,65], anti-inflammatory [66,67], antifungal [68], antitumor [69], antinociceptive [70], and pain-reducing [71] effects. Last but not
least, various studies have demonstrated that it has anxiolytic and sedative-hypnotic [72]
and analgesic [73] effects.
Both cell culture and mouse models of inflammatory disease have demonstrated that
Polygala crude extracts and pure compounds are likely to exert their anti-inflammatory effects
by blocking major inflammation-related signaling pathways, reducing pro-inflammatory
mediators and/or down regulating the expression of inducible enzymes [59]. However,
the direct inhibitory effects of Polygala secondary metabolites on pro-inflammatory enzyme
systems have not been evaluated before.
The main aim of this study was to isolate and structurally elucidate the secondary
metabolites from P. inexpectata and predict the inhibitory activities of the isolated phytochemicals on pro-inflammatory enzyme systems using computational methods. Accordingly, nine compounds (1–9) were isolated and subsequently characterized through
NMR and HRMS interpretations. Each of the identified compounds was then docked onto
myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase
(iNOS) in an in-silico setting.
2. Results
The aerial parts of P. inexpectata (150 g) were extracted using methanol (MeOH). After removing its lipophilic constituents using differential extraction in water (H2 O) and
dichloromethane (DCM), successive chromatographic techniques were applied for the
fractionation and isolation of the secondary metabolites, as detailed in Section 4. In total,
nine compounds were isolated, including six sucrose esters, namely 6,3′ -disinapoyl-sucrose
(1) [74], 6-O-sinapoyl,3′ -O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2) [51,75], 3′ O-(O-methyl-feruloyl)-sucrose (3) [76], 3′ -O-sinapoyl-sucrose (4) [77], 3′ -O-trimethoxycinnamoyl-sucrose (glomeratose) (5) [78], and 3′ -O-feruloyl-sucrose (sibiricose A5) (6) [79].
Additionally, a monomeric phenylpropane glycoside, sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7) [80,81], a tetrahydrofurofuran-type lignan diglycoside, liriodendrin (8) [82–84], and a flavonol glycoside, and 7,4-di-O-methylquercetin-3-O-β-rutinoside
(ombuin 3-O-rutinoside or ombuoside) (9) [47,85] were isolated (Figure 1). The structure
elucidation of the compounds 1–9 was based on the 1D- and 2D-NMR experiments (1 H-,
13 C-NMR, COSY, HSQC, HMBC, and NOESY) and were confirmed by the HRMS analysis
(see Supplementary Materials). The 1 H- and 13 C-NMR spectral data of compounds 1–6
were presented in Tables 1 and 2, respectively. Compounds 4 and 6 have been isolated as
a mixture of other sucrose esters. The 1 H- and 13 C-NMR data based on 1D and 2D-NMR
measurements as well as HRMS supported the proposed structures of 4 and 6. The corresponding spectroscopic data presented in the experimental as well as in Tables 1 and 2
were in good accordance with those reported [47,51,74–85].
Table 1. 1 H-NMR data of sucrose esters (1–6) (δH 500 MHz, CD3 OD).
H–Atom
1
2
1
2
3
4
5
6
δH , ppm
5.53 d (3.8)
3.51 dd (3.8/9.7)
δH , ppm
5.52 d (3.8)
3.51 dd (3.8/9.7)
δH , ppm
5.45 d (3.4)
3.46 (3.4/9.8)
δH , ppm
5.45 d (3.5)
3.48 dd (3.5/9.5)
δH , ppm
5.44 d (3.6)
3.45 dd (3.6/9.5)
δH , ppm
5.46 d (3.6)
3.49 dd (3.6/9.5)
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Table 1. Cont.
3
4
5
6
1′
2′
3′
4′
5′
6′
Acyl→Glu-6(OH)
2′′
6′′
7′′
8′′
3′′ &5′′ -OMe
Acyl→Fru-3′ (OH)
2′′′
5′′′
6′′′′
7′′′
8′′′
3′′′ &5′′′ -OMe
4′′′ -OMe
1
2
3
4
5
6
3.70 t (9.2)
3.34 t (9.3)
4.29 gdd
(9.3/7.3)
3.69 t (9.2)
3.34 t (9.3)
4.28 gdd
(9.3/7.3)
3.68 t (9.3)
3.43 t (9.3)
3.68 t
3.43 t (9.5)
3.67 t (9.5)
3.42 t (9.5)
3.70 t (9.5)
3.45 t (9.5)
3.92 m
3.92 m
3.92 m
3.94 m
4.23 dd
(11.7/7.3)
4.69 gd (11.7)
4.23 dd
(11.7/7.3)
4.69 gd (11.7)
3.84 †
3.78 dd
(12.2/4.4)
3.85/3.79
3.65 d (12.2)
3.61 d (12.2)
5.53 d (8.1)
4.52 t (8.1)
4.00 ddd
(3.1/8.1/10.0)
3.91 dd
(12.1/6.9)
3.84 †
SA
6.88 s
6.88 s
7.57 d (15.7)
6.44 d (15.7)
3.85 s
SA
6.84 s
6.84 s
7.65 d (15.7)
6.42 d (15.7)
3.82 s (6H)
-
3.65 d (12.2)
3.60 d (12.2)
5.54 d (8.1)
4.52 t (8.1)
4.00 ddd
(3.1/8.1/10.0)
3.90 dd
(12.1/6.9)
3.84 †
SA
6.84 s
6.84 s
7.56 d (15.9)
6.44 d (15.9)
3.82 s
TMC
6.90 s
6.90 s
7.67 d (15.9)
6.53 d (15.9)
3.84 s (6H)
3.77 s (3H)
3.68 d (12.3)
3.61 d (12.3)
5.49 d (7.8)
4.40 t (7.8)
3.64 d (12.1)
3.60 d (12.1)
5.49 d (7.8)
4.40 t
3.84 dd
(12.0/2.1)
3.78 dd
(12.0/4.6)
3.66 d (12.2)
3.60 d (12.2)
5.49 d (7.8)
4.39 t (7.8)
3.69 d (12.2)
3.62 d (12.2)
5.50 d (7.8)
4.41 t (7.8)
3.97 m
3.97 m
3.95 m
3.97 m
3.85 †
3.85 †
3.86 †
3.86 †
MFA
7.22 d (2.0)
6.95 d (8.0)
7.21 dd (8.0/2.0)
7.71 d (15.9)
6.47 d (15.9)
3.85 s (3H)
3.86 s (3H)
SA
6.93 s
6.93 s
7.69 d (15.9)
6.44 d (15.9)
3.87 s (6H)
-
-
FA
7.22 d (2.0)
6.8 d (8.2)
7.14 dd (8.2/2.0)
7.71 d (15.9)
6.44 d (15.9)
3.89 s (3H)
-
TMC
6.96 s
6.96 s
7.72 d (16.0)
6.54 d (16.0)
3.88 s (6H)
3.80 s (3H)
3.86 †
3.81 †
SA = sinapic acid; TMC = trimethoxycinnamic acid; MFA = 3-O-methylferulic acid; FA = ferulic acid; † Signal
patterns unclear due to overlapping.
Table 2.
13 C-NMR
C/H
Glu
1
2
3
4
5
6
Fru
1′
2′
3′
4′
5′
6′′
Acyl→Glu-6(OH)
1′′
2′′
3′′
4′′
5′′
6′′
7′′
8′′
9′′
3′′ &5′′ -OMe
Acyl→Fru-3′ (OH)
1′′′
data of sucrose esters (1–6) (δC 125 MHz, CD3 OD).
1
2
3
4
5
6
DEPT
δC , ppm
δC , ppm
δC , ppm
δC , ppm
δC , ppm
δC , ppm
CH
CH
CH
CH
CH
CH2
92.59
72.99
75.00
71.83
72.41
65.60
92.61
72.97
75.01
71.81
72.42
65.58
93.19
72.94
74.86
71.02
74.42
62.20
93.18
72.79
74.87
70.99
74.42
62.20
93.23
73.00
74.93
71.07
74.51
62.28
93.20
72.94
74.42
71.01
74.87
62.18
CH2
C
CH
CH
CH
CH2
63.72
104.78
79.19
74.07
84.18
65.60
SA
126.46
106.89
149.25
139.42
149.25
106.89
147.33
115.65
169.16
56.79
SA
126.51
63.72
104.76
79.32
74.09
84.21
65.58
SA
126.48
106.75
149.24
139.33
149.24
106.75
147.23
115.66
169.10
56.73
TMC
131.34
65.19
104.69
79.59
73.74
83.96
62.84
65.20
104.70
79.52
73.76
83.94
62.82
65.24
104.71
79.70
73.80
84.06
62.85
65.19
104.71
79.54
73.75
83.96
62.84
MFA
128.56
SA
126.49
TMC
131.46
FA
127.60
C
CH
C
C
C
CH
CH
CH
C
CH3
C
Molecules 2022, 27, 684
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Table 2. Cont.
2′′′
3′′
4′′′′
5′′′
6′′′
7′′′
8′′′
9′′′
3′′′ &5′′′ -OMe
4′′′ -OMe
CH
C
C
C/CH
CH
CH
CH
C
CH3
CH3
1
2
3
4
5
6
106.74
149.24
139.30
149.24
106.74
147.97
115.29
168.31
56.84
106.78
154.59
141.10
154.59
106.78
147.29
117.63
167.85
56.79
61.18
111.63
150.47
152.68
112.45
124.10
147.30
115.83
168.17
56.51
61.39
106.95
149.27
139.43
149.27
106.95
147.95
115.32
168.26
56.90
106.85
154.66
141.17
154.66
106.85
147.22
117.74
167.82
56.79
61.20
112.00
150.49
149.21
116.43
124.15
147.73
114.89
168.39
56.88
SA = sinapic acid; TMC = trimethoxycinnamic acid; MFA = 3-O-methylferulic acid; FA = ferulic acid.
′
′
Figure 1. Chemical structures of compounds
1–9: 6,3′ -disinapoyl-sucrose
(1), 6-O-sinapoyl,3′ ′
′
′
′
O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3′ -O-(O-methyl-feruloyl)-sucrose (3), 3′ -Oβ
sinapoyl-sucrose (4), 3′ -O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3′ -O-feruloyl-sucrose
(sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8),
and a flavonol glycoside, 7,4-di-O-methylquercetin-3-O-β-rutinoside (ombuin 3-O-rutinoside or
ombuoside) (9).
In an attempt to better understand the direct inhibitory effects of P. inexpectata secondary
metabolites on pro-inflammatory enzyme systems, we docked nine compounds isolated from
the plant on human counterparts of MPO, COX-2, and iNOS. The results of the redocking
calculations revealed that JAMDA was able to reproduce the crystallographic binding modes
of the bona fide enzyme inhibitors well, with root-mean-square deviations (RMSDs) of less
than 1 Å (Table 3). In cross-docking experiments, all nine compounds tested were found
to be able to occupy the inhibitor-binding pockets of MPO and iNOS, with similar or even
higher docking scores compared to those of the cocrystallized inhibitors. The binding of the
compounds in the inhibitor-binding pockets of MPO and iNOS appeared to be stabilized
mainly by hydrogen-bonding interactions and π-effects (Figure 2). Two comparatively bulky
sucrose esters (compounds 1 and 2) in particular were predicted to be potent inhibitors of
Molecules 2022, 27, 684
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MPO. Besides forming favorable non-covalent interactions with key residues lining the activesite cleft of MPO (e.g., Gln91, His95, and Arg239), they were also able to interact with the
enzyme’s heme prosthetic group through their sinapoyl moieties. Compound 9, a flavonol
glycoside, could also serve as a potent MPO inhibitor. Its best-scoring docking pose was
estimated to engage in multiple non-covalent interactions with the heme. Compounds 1 and 2
were likely to inhibit iNOS as well. They could make interactions with invariant Glu377, other
active-site residues involved in inhibitor binding (e.g., Gln263, Arg266, Tyr347), and the heme
prosthetic group. Compound 8, a tetrahydrofurofuran-type lignan diglycoside, emerged
as a potentially superior inhibitor based on its docking score. It was able to form multiple
non-covalent interactions with both Glu377 and the heme of iNOS through its pyranose
and dimethoxybenzene rings. For COX-2, only compound 7, a monomeric phenylpropane
glycoside, was able to favorably bind to the enzyme, possibly due to its relatively small size.
We do not, however, exclude the possibility that Polygala-derived phytochemicals may bind
at an alternative site on COX-2 other than the coxib-binding site.
Table 3. Results of cross-docking calculations for Polygala-derived phytochemicals, showing the
JAMDA scores of the best docking solutions. MPO: myeloperoxidase; iNOS: inducible nitric oxide
synthase; COX-2: cyclooxygenase-2.
Protein
MPO (PDB ID: 5QJ2)
iNOS (PDB ID: 3E7G)
COX-2 (PDB ID: 5KIR)
Ligand
JAMDA Score
All-Atom RMSD
PDB chemical ID: JXS
−2.32663
0.632 Å
Compound 1
−3.78280
Compound 2
−3.57348
Compound 3
−2.90840
Compound 4
−3.13930
Compound 5
−2.97990
Compound 6
−2.83140
Compound 7
−2.50225
Compound 8
−3.03640
Compound 9
−3.52060
PDB chemical ID: AT2
−2.62362
Compound 1
−2.85444
Compound 2
−3.14240
Compound 3
−2.66980
Compound 4
−2.34210
Compound 5
−2.54230
Compound 6
−3.02600
Compound 7
−2.67380
Compound 8
−3.47560
Compound 9
−2.78550
PDB chemical ID: RCX
−2.52030
Compound 1
Positive
Compound 2
Positive
Compound 3
Positive
Compound 4
Positive
Compound 5
Positive
Compound 6
Positive
Compound 7
−2.56960
Compound 8
Positive
Compound 9
Positive
0.682 Å
0.532 Å
Molecules 2022, 27, 684
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Figure 2. Results of cross-docking calculations showing (A) the top-ranking predicted binding pose
of compound 2 within the active-site cleft of MPO, (B) the top-ranking predicted binding pose of
compound 9 within the active-site cleft of MPO, (C) the top-ranking predicted binding pose of
compound 2 within the active-site cleft of iNOS, (D) the top-ranking predicted binding pose of
compound 8 within the active-site cleft of iNOS, and (E) the top-ranking predicted binding pose
of compound 7 in the coxib-binding pocket of COX-2. The images were rendered using Discovery
Studio Visualizer, v16.1.0 (Dassault Systèmes BIOVIA Corp., San Diego, CA, USA). A color-coding
scheme was added to distinguish between the different types of non-covalent interactions, in which
the maximum distance between two interacting centers was included in parentheses.
Molecules 2022, 27, 684
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A glimpse at the superposed structures of docked compound 2 and the cocrystallized
triazolopyridine compound revealed that the sinapoyl moiety of compound 2 and the
heterocyclic core of the triazolopyridine compound stack on the heme prosthetic group of
MPO, with their aromatic rings and polar functionalities (i.e., the oxygen atoms of sinapic
acid and the nitrogen atoms of triazolopyridine) coinciding with each other (Figure 3).
Similar correspondences in the positions of polar functionalities also exist between docked
compound 2 and the bona fide iNOS inhibitor aminopyridine compound. These findings
further validate the reliability of our molecular docking calculations.
Figure 3. (A) Ribbon representation of the biological assembly of human MPO (PDB entry: 5QJ2),
with docked compound 2 shown as pink sticks. (B) Close-up view of the superposed structures of
compound 2 (pink) and the bona fide triazolopyridine-type inhibitor (green) in the active-site cavity
of human MPO. The heme prosthetic group is shown as yellow sticks. (C) Ribbon representation
of the biological assembly of human iNOS (PDB entry: 3E7G), with docked compound 2 shown as
pink sticks. (D) Close-up view of the superposed structures of compound 2 (pink) and the bona fide
aminopyridine-type inhibitor (green) in the active-site cavity of human iNOS. The heme prosthetic
group is shown as yellow sticks. All images were rendered using the PyMOL Molecular Graphics
System, v1.8 (Schrödinger LLC, Portland, OR, USA).
Molecules 2022, 27, 684
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3. Discussion
P. inexpectata is a narrow endemic taxon in Turkey. The phytochemistry of this species
was studied in depth for the first time in this study. It is worth noting that most of the
secondary metabolites isolated were sucrose ester derivatives. Additionally, a monomeric
phenylpropane glycoside, a flavonol glycoside, and a tetrahydrofurofuran-type lignan
diglycoside were also isolated from the whole plant of P. inexpectata. These sucrose esters,
or, more specifically, phenylpropanoid sucrose esters, have a sucrose core linked to one or
more Ph–CH=CH–CO– moieties (Ph: phenyl) via an ester bond. The ester-forming moieties
can among others be substituted or unsubstituted sinapic, cinnamic, and ferulic acids, as
observed in the present study. Polygala spp. are rich sources of phenylpropanoid sucrose
esters in both mono- and disubstituted forms. Apart from the Polygalaceous plants, plant
species of the Polygonaceae and Liliaceae families also represent major sources of sucrose
mono- and diesters [86].
Accumulated data over the last decade suggest that the anti-inflammatory potential of the genus Polygala forms part of the basis for its widespread use in traditional
medicine [17,59]. In a previous study, where the inhibitory activities of bioactive compounds from P. tenuifolia against lipopolysaccharide (LPS)-stimulated pro-inflammatory
cytokine production in bone marrow-derived dendritic cells were tested, all isolated sucrose mono- and diesters were found to possess anti-inflammatory properties [33]. In
another study on P. japonica, two sucrose esters, namely, tenuifolioside B and β-D-[3-O-(3,4,5trimethoxycinnamoyl)]-fructofuranosyl-α-D-[6-O-(4-methoxybenzoyl)]-glucopyranoside,
were shown to attenuate the release of pro-inflammatory cytokines in LPS-stimulated
BV2 microglial cells [87]. These two studies provide direct evidence to support the notion
that Polygala sucrose esters serve as natural products of significant therapeutic value, which
can be exploited in anti-inflammatory therapy to particularly treat neuroinflammatory
conditions. In fact, the important role played by sucrose esters in the fight against inflammation has been demonstrated not only for Polygala-derived phytochemicals but also
for bioactive compounds extracted and purified from other plant species. For example,
mono- and disubstituted sucrose esters from Bidens parviflora (Compositae) have been
found to be effective in attenuating the release of histamine by rat mast cells stimulated by
antigen–IgE antibody reaction and in suppressing the production of prostaglandin E2 by
macrophages [88].
Our computational analyses reveal that secondary metabolites isolated from the whole
plant of P. inexpectata, including phenylpropanoid sucrose esters with relatively high docking scores, may hold the potential to directly inhibit the heme-dependent pro-inflammatory
enzymes iNOS and MPO. While intermittently increased inflammation is known to be
required for survival during physical injury and infection, aberrant or chronic inflammation has been demonstrated to be associated with a number of diseases such as such as
cardiovascular disease, cancer, diabetes mellitus, and autoimmune and neurodegenerative
disorders [89]. iNOS is a normally silenced enzyme whose expression can be induced
in a myriad of cells and tissues by certain cytokines and other pro-inflammatory factors.
It catalyzes the production of nitric oxide (· NO) from L-arginine. · NO is a gaseous free
radical that, along with its oxidation products, can cause damage to biomolecules (lipids,
DNA, and proteins) and tissues and induce necrosis or apoptosis [90]. MPO is an abundant enzyme expressed by activated immune cells of the myeloid lineage, particularly
macrophages, monocytes, and neutrophils. It interacts with hydrogen peroxide (H2 O2 )
to generate highly reactive species, such as hypochlorite (OCl– ), superoxide (O2 ·− ), and
peroxynitrite (ONOO– ), that can covalently modify lipids and thus lead to tissue injury [91].
MPO-generated free radicals can also induce apoptotic cell death and protein nitrotyrosination [92]. Furthermore, it has been shown that MPO increases the catalytic activity of iNOS
by preventing · NO feedback inhibition at sites of inflammation [93]. Therefore, both iNOS
and MPO are engaged in a complex cascade of inflammatory events involving various cells
and molecules. We believe that the previously established anti-inflammatory effects of
Molecules 2022, 27, 684
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Polygala extracts or components in animals and cell culture systems could be partly due to
the modulation of pro-inflammatory enzyme activities.
4. Materials and Methods
4.1. General Experimental Procedures
Buchi® rotavapor (R-210) with the heating bath (B-491), vacuum pump (V-700), and
vacuum controller (V-850) was used for evaporation under low pressure. Christ® Alpha
1–4 LD plus was used for the lyophilization of the samples. For medium pressure Liquid
chromatography (MPLC), a Buchi Sepacore® Chromatography system with Buchi borosilicate 3.3 columns (36 mm x 230 mm) packed with LiChroprep RP-18 (Merck, Darmstadt)
was used. Reverse phase material LiChroprep C18 was used for vacuum liquid chromatography (VLC). Column chromatography was performed on silica gel 60 (0.063–0.200 mm;
Merck, Darmstadt), and Sephadex™ LH-20 (GE Healthcare, Sweden) was also used for
open column chromatography (CC) studies. Thin Layer Chromatography (TLC) analyses
were performed on aluminum plates (Merck, Darmstadt) coated with silica gel 60 F254
and RP TLC. 1% Vanillin in MeOH and 5% H2 SO4 relative in EtOH were used for spot
detection on TLC plates. For NMR spectroscopy experiments, measurements were performed on a Bruker DRX 500 spectrometer operating at 500 MHz for 1 H and 125 MHz for
13 C, respectively.
4.2. Plant Material
The whole plant of P. inexpectata was collected from Karaman, around Ayrancı dam,
steppe, 41◦ 33′ 10.6′′ N, 36◦ 56′ 56.8′′ , 1265 m, May 2019, A. A. Dönmez 20373-Z. Aydın. The
voucher specimen has been deposited at the Herbarium of the Faculty of Biology, Hacettepe
University (HUB).
4.3. Extraction and Isolation
The air dried and powdered whole plants (leaves, stems, flowers, and roots) of P.
inexpectata (150 g) were extracted two times with 95% MeOH by a refluxing process at
45 ◦ C. The resulting extracts were combined, filtered, and concentrated under reduced
pressure at 45 ◦ C. The concentrated extract was diluted with water and partitioned with
dichloromethane (DCM) to remove lipophilic compounds. The H2 O phase was concentrated to 50 mL and subjected to column chromatography using polyamide G (Fluka)
as a stationary phase and eluting with H2 O and H2 O–MeOH mixtures with an increasing amount of MeOH to afford six main fractions: A (16.70 g), B (596 mg), C (690 mg),
D (314 mg), E (161 mg), and F (127 mg). Fraction A (16.70 g) was fractionated on a RP_VLC
(Reversed Phase (LiChroprep C18, Vacuum Liquid Chromatography) column and eluted
with increasing concentrations of MeOH in H2 O (0%, 10%, 20%, and 100%) to obtain sixteen
fractions (A1–A16). Fraction A was almost pure sucrose.
Fr. C (690 mg) was fractionated by MPLC with increasing concentrations of i-PrOH
in H2 O (up to 30%) to obtain nine subfractions (C1–C9). Fr. C3 was obtained as pure
compound 1 (111 mg). Fr. C5 was obtained as pure compound 2 (51 mg).
Fr. B (596 mg) was fractionated by MPLC with increasing concentrations of i-PrOH
in H2 O (up to 30%) to obtain 11 subfractions (B1–B11). Fr. B3 was obtained as pure
compound 1 (40 mg). Fr. B8 was obtained as pure compound 2 (54 mg). We found same
result from NMR for chemical compound (1) 6,3′ -disinapoyl-sucrose and compound (2)
6-O-sinapoyl-3′ -O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C).
Fr. A7 (360 mg) was subjected to a silica gel column using DCM-MeOH-H2 O mixture
(80:20:2→70:30:3) to afford compound (3) 3′ -O-(O-methyl-feruloyl)-sucrose (41 mg).
Fr. A6 (277 mg) and Fr. A7d was subjected to a silica gel column using DCM-MeOHH2 O mixture (80:20:2) to afford compounds (7) sinapyl alcohol 4-O-glucoside (syringin or
eleutheroside B) (6 mg) and (4) 3′ -O-(sinapoyl)-sucrose (114 mg).
Molecules 2022, 27, 684
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Fr. A8 (342 mg) was subjected to a silica gel column using DCM-MeOH-H2 O mixture
(80:20:1→80:20:2) to afford compounds (5) 3′ -O-trimethoxy-cinnamoyl-sucrose (glomeratose) (42 mg) and (8) liriodendrin (45 mg).
Fr. A13 (862 mg) was separated by gel chromatography (Sephadex LH-20), eluting
with MeOH–H2 O (1:1) to yield a total of six subfractions (Fr. A13a-f). Fr. A13c (70 mg) was
purified by silica gel using DCM-MeOH-H2 O (60:40:4) to obtain compound (9) 7,4′ -di-Omethylquercetin-3-O-β-rutinoside (ombuin 3-O-rutinoside or ombuoside) (4 mg).
Fr. A5 (255 mg) was subjected to a silica gel column using DCM-MeOH-H2 O mixture
(80:20:1) to afford compound (6) 3′ -O-feruloyl-sucrose (sibiricose A5) (16 mg).
Fr. A10 (531 mg) was rich in compound (9) 7,4′ -di- O-methylquercetin-3-O-β-rutinoside
(ombuin 3-O-rutinoside or ombuoside) and used for the re-isolation of 9.
4.4. Physical and Spectral Data of Isolated Compounds 1 to 9
6,3′ -disinapoyl-sucrose (1): Chemical formula C34 H42 O19 ; Mol. Wt. 754.6910; 1 H-NMR
(CD3 OD, 500 MHz, δ [ppm]): Table 1; 13 C-NMR (CD3 OD, 125 MHz, δ [ppm]): Table 2;
Positive ion HR-MS: m/z 777.2192 [M + Na]+ ; Negative ion HR-MS: m/z 753.2246 [M–H]− .
6-O-sinapoyl,3′ -O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2): Chemical formula C35 H44 O19 ; Mol. Wt. 768.2477; 1 H-NMR (CD3 OD, 500 MHz, δ [ppm]): Table 1; 13 CNMR (CD3 OD, 125 MHz, δ [ppm]): Table 2. Positive ion HR-MS: m/z 791.2362 [M + Na]+ ;
Negative ion HR-MS: m/z 767.2393 [M–H]− .
3′ -O-(O-methyl-feruloyl)-sucrose (3): Chemical formula C23H32O14; Mol. Wt. 532.4950;
1 H-NMR (CD OD, 500 MHz, δ [ppm]): Table 1; 13 C-NMR (CD OD, 125 MHz, δ [ppm]): Table 2;
3
3
Positive ion HR-MS: m/z 555.1677 [M + Na]+; Negative ion HR-MS: m/z 531.1709 [M–H]− .
3′ -O-(sinapoyl)-sucrose (4): Chemical formula C23 H32 O15 ; Mol. Wt. 548.4890: 1 HNMR (CD3 OD, 500 MHz, δ [ppm]): Table 1; 13 C-NMR (CD3 OD, 125 MHz, δ [ppm]): Table 2;
Positive ion HR-MS: m/z 571.1624 [M + Na]+ ; Negative ion HR-MS: m/z 547.1727 [M–H]− .
3′ -O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5): Chemical formula C23 H35 O15 ;
Mol. Wt. 562.5210; 1 H-NMR (CD3 OD, 500 MHz, δ [ppm]): Table 1; 13 C-NMR (CD3 OD,
125 MHz, δ [ppm]): Table 2; Positive ion HR-MS: m/z 585.1781 [M + Na]+ .
3′ -O-feruloyl-sucrose (sibiricose A5) (6): Chemical formula C22 H30 O14 ; Mol. Wt.
518.4680; 1 H-NMR (CD3 OD, 500 MHz, δ [ppm]): Table 1; 13 C-NMR (CD3 OD, 125 MHz, δ
[ppm]): Table 2; Positive ion HR-MS: m/z 541.1523 [M + Na]+ ; Negative ion HR-MS: m/z
517.1561 [M–H]− .
Sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7): Chemical formula
C17 H24 O9 ; Mol. Wt. 372.1420; 1 H-NMR (CD3 OD, 500 MHz, δ [ppm]): Sinapyl alcohol
moiety: 6.58 (s, 2H, H-2 and H-6); 6.50 (d, 1H, J = 11.9 Hz, H-7); 5.82 (dt, 1H, J = 11.9 and
6.5 Hz, H-8); 4.35 (dd, 2H, J = 1.1 and 6.5 Hz, H-9); 3.87 (s, 6H, 3/5-OMe); glucose moiety:
4.91 (overlapped, H-1′ ); 3.49 (dd, 1H, J = 7.8 and 9.0 Hz, H-2′ ); 3.50 (t, 1H, J = 9.0 Hz,
H-3′ ); 3.45 (t, 1H, J = 9.0 Hz, H-4′ ); 3.45 (m, 1H, H-5′ ); 3.80 (dd, 1H, J = 12.0 and 2.3 Hz,
H-6′ a); 3.70 (dd, 1H, J = 12.0 and 5.3 Hz, H-6′ ).13 C-NMR (CD3 OD, 125 MHz, δ [ppm]):
Sinapyl alcohol moiety: 134.73 (C-1); 107.96 (C-2/C-6); 153.97 (C-3/C-5); 134.44 (C-4);
132.50 (C-7); 131.46 (C-8); 59.78 (C-9); 57.06 (OCH3 × 2); glucose moiety: 105.22 (C-1′ );
75.66 (C-2′ ); 77.56 (C-3′ ); 71.24 (C-4′ ); 78.29 (C-5′ ); 62.47 (C-6′ ). Positive ion HR-MS: m/z
395.1306 [M + Na]+ .
Liriodendrin (8): Chemical formula C34 H46 O14 ; Mol. Wt. 742.7240; 1 H-NMR (CD3 OD,
500 MHz, δ [ppm]): Pinoresinol moiety: 6.67 (s, 4H, H-2, H-2′ , H-6, H-6′ ); 4.66 (br s, 2H,
H-7, H-7′ ); 3.09 (br s, H-8, H-8′ ); 4.21 (dd, 2H, H-9a, H-9′ a); 3.84 (dd, 2H, H-9b, H-9′ b); 3.77
(12 H, OCH3 × 4). Glucose moieties: 4.88 (d, 2H, J = 7.8 Hz, H-1′′ /H-1′′′ ); 3.21 (m, 4 H,
H-2′′ /H-2′′′ and H-3′′ /H-3′′′ ); 3.15 (m, 2H, H-4′′ /H-4′′′ ); 3.05 (m, 2H, H-5′′ /H-5′′′ ); 4.21
(dd, 2H, J = 11.4 and 1.8 Hz, H-6′′ a/H-6′′′ a); 3.42 (dd, 2 H, J = 11.4 and 6.0 Hz, H-6′′ b/H6′′′ b). 13 C-NMR (CD3 OD, 125 MHz, δ [ppm]): Pinoresinol moiety: 137.60 (C-1/C-1′ );
104.61 (C-2/C-2′ and C-6/C-6′ ); 153.08 (C-3/C-3′ and C-5/C-5′ ); 134.09 (C-4/C-4′ ); 85.53
(C-7/C-7′ ); 54.07 (C-8/C-8′ ); 71.83 (C-9/C-9′ ); 56.88 (OCH3 × 4). Glucose moieties: 103.11
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(C-1′′ /C-1′′′ ); 74.61 (C-2′′ /C-2′′′ ); 76.94 (C-3′′ /C-3′′′ ); 70.34 (C-4′′ /C-4′′′ ); 77.63 (C-5′′ /C-5′′′ );
61.33 (C-6′′ /C-6′′′ ). Positive ion HR-MS: m/z 765.2576 [M + Na]+ .
Ombuin 3-O-rutinoside (ombuoside) [7,4-di-O-methylquercetin-3-O-β-rutinoside] (9):
Chemical formula C29 H34 O16 ; Mol. Wt. 638.5750; 1 H-NMR (DMSO-d6 , 500 MHz, δ [ppm]):
Flavonol moiety: 6.36 (br s, 1H, H-8); 6.67 (br s, 1H, H-6), 7.55 (br s, 1H, H-2′ ); 7.04 (d,
J = 8.7 Hz, H-5′ ); 7.73 (br d, J = 8.7 Hz, H-6′ ); 3.85 (s, 3H, 7-OMe); 3.86 (s, 3H, 4′ -OMe);
12.56 (s, 5-OH); glucose moiety: 5.39 (d, 1H, J = 7.0 Hz, H-1′′ ); 3.26 (m, 1H, H-2′′ ); 3.27 (m,
1H, H-3′′ ); 3.51 (m, 1H, H-4′′ ); 3.30 (m, 1H, H-5′′ ); 3.72 (br d, 1H, J = 12.0 Hz, H-6′′ a); 3.35
(m, 1H, H-6′′ b); rhamnose moiety: 4.40 (br s, 1H, H-1′′′ ); 3.41 (br d, 1H, J = 3.2 Hz, H-2′′′ );
3.10 (dd, 1H, J = 3.2 and 9.2 Hz, H-3′′′ ); 3.08 (t 1H, J = 9.2 Hz, H-4′′′ ); 3.29 (m, 1H, H-5′′′ ); 0.97
(d, 3H, J = 5.8 Hz, H-6′′′ ); 13 C-NMR (DMSO-d6 , 125 MHz, δ [ppm]): Flavonol moiety: 157.21
(C-2); 134.27 (C-3); 178.13 (C-4); 161.43 (C-5); 98.56 (C-6); 165.75 (C-7); 92.84 (C-8); 157.02
(C-9); 105.61 (C-10); 122.13 (C-1′ ); 116.38 (C-2′ ); 146.46 (C-3′ ); 150.63 (C-4′ ); 111.92 (C-5′ );
123.00 (C-6′ ); 56.67 (7-OCH3 ); 56.21 (4′ -OCH3 ); glucose moiety: 101.76 (C-1′′ ); 74.63 (C-2′′ );
76.97 (C-3′′ ); 70.42 (C-4′′ ); 76.40 (C-5′′ ); 67.49 (C-6′′ ). rhamnose moiety: 101.38 (C-1′′′ ); 70.94
(C-2′′′ ); 71.17 (C-3′′′ ); 72.39 (C-4′′′ ); 68.84 (C-5′′′ ); 18.32 (C-6′′′ ); Positive ion HR-MS: m/z
639.1909 [M + H]+ , m/z 661.1726 [M + Na]+ ; Negative ion HR-MS: m/z 637.1761 [M–H]− .
4.5. Protein-Ligand Docking
The energy-minimized 3D conformers of P. inexpectata secondary metabolites were
generated based on the corresponding SMILES strings using myPresto programs available
at https://demo1.biomodeling.co.jp/, accessed on 11 June 2021. The X-ray crystallographic
structures of (i) human myeloperoxidase (MPO) in complex with a potent triazolopyridine
compound [94], (ii) human cyclooxygenase-2 (COX-2) in complex with a selective inhibitor
of the coxib type [95], and (iii) human inducible nitric oxide synthase (iNOS) in complex
with a selective aminopyridine compound [96] were retrieved from the RCSB Protein Data
Bank [97] available at https://www.rcsb.org/, accessed on 23 July 2021. These enzymes
not only engage in inflammatory processes but also in the generation of free radicals and
the oxidation of nucleic acids, lipids, and proteins (please see Section 3 for details). Ligandbound (holo) conformational states of pro-inflammatory enzymes in complex with potent
and selective inhibitors were selected rationally as target structures for docking. They were
also chosen based on their acquisition technique (X-ray crystallography), resolution (less
than 3 Å), and structure completeness. The structures were prepared using the Dock Prep
utility of UCSF Chimera version 1.11.2 [98]. For atoms with alternate locations, only the
highest-occupancy set was retained. Additionally, each truncated side chain was replaced
with a complete side chain of the same residue type using the Dunbrack rotamer library.
Missing hydrogen atoms were added to the structures using Protoss [99,100], available at
https://proteins.plus/, accessed on 23 July 2021. The ligands were docked in the presence
of cofactors and structurally relevant water molecules into the inhibitor-binding pockets
of the proteins using JAMDA [101–103], available at https://proteins.plus/, accessed on
23 July 2021. Each docking site was defined by the cocrystallized inhibitor, with a site
radius of 6.5 Å. Protein–ligand docking was executed with high precision.
5. Conclusions
Overall, the present study attempted to explore the phytochemical composition of
the endemic taxon P. inexpectata from Turkey. The plant appears to be comparable to
other Polygala species with medicinal uses in that it has a rich content of sucrose esters.
Liriodendrin and ombuoside are the isolated lignin and flavone glycosides, respectively,
and are normally rare in this genus. The findings of the molecular docking calculations
highlight the obtained compounds’ potential to inhibit the pro-inflammatory enzymes
iNOS and MPO. The very encouraging in silico results indicate that these phytochemicals
may have a future role in anti-inflammatory drug development research. Further in vitro
and/or in vivo studies, however, are required to demonstrate their efficacy. These data
Molecules 2022, 27, 684
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could prove valuable in extending the knowledge of the phytochemistry and medicinal
properties of the Polygala taxa.
Supplementary Materials: The following are available online. The HRMS and NMR spectra of
compounds 1–9 are deposited as Supplementary Figures S1–S61.
Author Contributions: Conceptualization, İ.Ç., A.Ü., A.A.D. and K.T.; plant material collection and
identification, A.A.D. and Z.U.A.; isolation and identification of the compounds, İ.Ç., A.Ü. and
H.S.Y.; molecular docking studies, K.T.; writing—review and editing, A.Ü., İ.Ç., K.T., Z.U.A. and
A.A.D.; project administration, A.A.D. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available upon
reasonable request from the authors.
Acknowledgments: We are grateful to TÜBİTAK (Project No. 118 Z 708) for the financial support.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: The samples of compounds 1–9 are available upon reasonable request from
the authors.
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