Visconata: A rare flavonol having long chain fatty acid from Dodonaea viscosa which inhibits Human neutrophil elastase (HNE)

Article abstract of DOI:10.1016/j.tetlet.2017.05.059

A series of flavonoids were isolated from Dodonaea Viscosa and tested for inhibition of human neutrophil elastase (HNE), enzyme involved in inflammatory disorders. Isolated compounds were identified as a novel flavonol (1) along with eight known flavonoids (2–9). Novel flavonol, visconata (1) has a very rare skeleton having odd numbered long chain (C19) fatty acid, which was completely identified by mass fragmentation and 2D NMR analysis. All compounds (1–9) inhibited HNE in dose dependent manner with IC₅₀s ranging between 2.4 and 150?μM. Visconata (1) emerged to be the most potent compound with 2.4?μM of IC₅₀. In kinetic studies, compound (1) was observed to be reversible, noncompetitive inhibitor having K_i?=?1.8?μM, whereas other flavonoids (2–9) displayed mixed type inhibition.

Full text of DOI:10.1016/j.tetlet.2017.05.059

Tetrahedron Letters 58 (2017) 2507–2511
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Visconata: A rare flavonol having long chain fatty acid from Dodonaea
viscosa which inhibits Human neutrophil elastase (HNE)
⇑
Zia Uddin, Zuopeng Li, Yeong Hun Song, Jeong Yoon Kim, Ki Hun Park
Division of Applied Life Science (BK21 plus), IALS, Gyeongsang National University, Jinju 52828, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of flavonoids were isolated from Dodonaea Viscosa and tested for inhibition of human neutrophil
elastase (HNE), enzyme involved in inflammatory disorders. Isolated compounds were identified as a
novel flavonol (1) along with eight known flavonoids (2–9). Novel flavonol, visconata (1) has a very rare
skeleton having odd numbered long chain (C19) fatty acid, which was completely identified by mass frag-
mentation and 2D NMR analysis. All compounds (1–9) inhibited HNE in dose dependent manner with
Received 10 April 2017
Revised 11 May 2017
Accepted 16 May 2017
Available online 17 May 2017
IC50s ranging between 2.4 and 150
2.4 M of IC50. In kinetic studies, compound (1) was observed to be reversible, noncompetitive inhibitor
having Ki = 1.8 M, whereas other flavonoids (2–9) displayed mixed type inhibition.
Ó 2017 Elsevier Ltd. All rights reserved.
lM. Visconata (1) emerged to be the most potent compound with
Keywords:
Visconata
Odd numbered long chain fatty acid
Dodonaea viscosa
l
l
Novel flavonol
Human neutrophil elastase (HNE)
Introduction
tissues degradation and inflammation.8 The uncontrolled
elastolytic activity has been implicated in several diseases such
Dodonaea Viscosa (L.) Jacq is an ever green medicinal plant,
belonging to Sapindaceae family and is widely distributed in trop-
ical and subtropical countries including India and Pakistan.1 The
aerial parts of this plant has been used in folk medicine to suppress
diabetes, bacterial and fungal infections, skin diseases and a variety
of inflammatory disorders.2,3 Previous phytochemical studies of D.
viscosa have resulted in isolation of flavonoids, triterpenoids, lab-
dane diterpenoids, and saponines.4 Several researchers have
explored the biological effectiveness of the whole extract and indi-
vidual phytochemicals of D. viscosa such as gastroprotective, hep-
atoprotective, biofilm suppression, antiviral and antibacterial
activities.4–6 But still much work is needed to disclose its further
biological effects especially based on enzyme inhibition. During
our ongoing research on exploration of human neutrophil elastase
(HNE) inhibitors from natural sources, we found that the methanol
extract of aerial parts of D. viscosa showed potent inhibition against
HNE.
OH
OH
O
HO
O
O
OH
O
H3CO
OCH3
CH3
OH
O
1
OH
OCH3
OH
HO
O
H3CO
HO
O
O
H3CO
H3CO
H3CO
OCH3
OCH3
OCH3
OH
O
OH
OH
O
2
3
4
OCH3
OCH3
OH
HO
H3CO
O
O
HO
O
HO
O
CH3
CH3
OCH3
H3CO
OCH3
HO
OCH3
CH3
OH
OH
O
OH
O
Human neutrophil elastase (HNE, EC 3.4.21.37) is a serine pro-
tease, belonging to the chymotrypsin family, and is stored in azur-
ophilic granules of neutrophil.7 This enzyme has a broad substrate
specificity, causing enzymatic cleavage of elastin, but also hydro-
lyzes other components of extracellular matrix including proteo-
glycan, collagen and fibronectin, hence leading to connective
5
6
7
OH
OCH3
HO
H3CO
O
HO
H3CO
O
OH
OH
OCH3
OCH3
CH3
CH3
OH
O
OH
O
8
9
⇑
Corresponding author.
E-mail address: khpark@gnu.ac.kr (K.H. Park).
Fig. 1. Chemical structures of isolated compounds (1–9) from Dodonaea viscosa.
http://dx.doi.org/10.1016/j.tetlet.2017.05.059
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

2508
Z. Uddin et al. / Tetrahedron Letters 58 (2017) 2507–2511
as cystic fibrosis, acute respiratory distress syndrome, chronic
obstructive pulmonary disease, acute lung injury, atherosclerosis
and other inflammatory diseases such as rheumatoid arthritis.9,10
Recently several studies has also reported its role as key mediator
of secondary pathogenesis in spinal cord injury, brain ischemia,
obesity and insulin resistance 8,11. Being a multiple player, discov-
ery of novel HNE inhibitors which targets its substrates could be a
promising strategy to contribute in the ongoing research against
inflammatory diseases.
Aim of the present work was to investigate HNE inhibitory
activity of phytochemicals of D. viscosa, in this regard the methanol
extract of aerial parts gave nine phenolic compounds (1–9), espe-
cially a novel compound (1) having a unique odd number long
chain fatty acid (C19). We elucidated compound 1 to be a flavonol
having odd number long chain fatty acid by mass fragmentation
using HREIMS and detailed 1D and 2D NMR analysis. Furthermore
inhibitory mechanisms of isolated inhibitors were ascertained by
kinetic plots.
Results and discussion
In preliminary experiment, the methanol extract of the areal
parts of D. viscosa exhibited a good potential activity against HNE
(100 lg/ml, 75% inhibition). Successive purification of methanol
extract over dianion HP-20, silica gel, Octadecyl-functionalized sil-
ica gel, and Sephadex LH-20 gave nine flavonoids (1–9) (Fig. 1),
which were responsible for HNE inhibition. These compounds were
identified as penduletin (2), 5,6-dihydroxy-3,40,7-trimethoxyfla-
vone (3), viscosine (4), isokaemferide (5), viscosol (6), 5,7-dihy-
droxy-30-(2-hydroxy-3-methylbutenyl)-3,6,40-trimethoxy-flavone
(7), 5,7-dihydroxy-30-(3-hydroxy-methylbutyl)-3,6,40-trimethoxy-
flavone (8), and 5,7,40 -trihydroxy-30-(3-hydroxymethylbutyl)-
3,6-dimethoxyflavone (9), through analysis of spectroscopic data
and comparison with previous studies, (Supplementary
material)12,13,14a
.
Compound 1 was isolated as a pale yellow sticky oil with
molecular formula C41H58O11 by the [M+] ion at 726.3980 (Calcd
Fig. 2. Key 1H-1H COSY and HMBC correlations of compound 1 (A). Proposed fragmentation pattern of compound 1, deduced by EI-MS (B).

Z. Uddin et al. / Tetrahedron Letters 58 (2017) 2507–2511
2509
726.3979) in the HREIMS14b. The IR spectrum showed absorption
for OH (3450 cm-1) and two carbonyls (1750 and 1660 cm-1). The
UV spectrum resembled that of flavonol derivatives with kmax
H-1700 (dH 1.24–1.45) and H-1600 (dH 1.24–1.45), as well as HMBC
correlation of H-1900 (dH 3.44) with C-1600 (dC 21.7), and C-1700 (dC
32.3). The attachment of fatty acid chain with main skeleton(flavo-
nol) was proved by a strong HMBC correlation between H-110 (dH
3.89) and carbonyl C-100 (dC 174.2). Base hydrolysis of 1 by LiOH
yielded mother skeleton 9 that was ascertained by UPLC-Q-TOF/
MS analysis with reference to an authentic sample. Specific
270 nm (log
e = 4.0) and 350 nm (log e = 4.2). DEPT experiments
in conjunction with 1H and 13C NMR data indicated the presence
of 41 carbon atoms consisting of the following carbon characteris-
tics: 18 methylenes (sp3), 2 methines (sp3), 6 methines (sp2), 3
methyles and 12 quaternary carbons. The extra three degrees of
unsaturation after counting C-C and C-O double bonds were
ascribed to tricyclic skeleton of flavonol structure. The overall
NMR characteristics suggested that 1 bears a flavonol backbone.
A characteristic hydrogen bonded proton signal of C-5 (dC 152.2)
hydroxyl group was observed at dH 12.94. The 5, 6, 7-trisubstitu-
tion of A-ring was deduced by a singlet of H-8 (dH 6.40) that has
a strong HMBC correlation with oxygenated carbons C-7 (dC
152.4) and C-8a (dC 157.7). The presence of C6-OCH3 was proved
by HMBC correlation of OCH3 (dH 3.78) and C-6 (dC 131.3). The
location of C3-OCH3 in C-ring was pointed by strong correlation
of OCH3 (dH 3.66) with C-3 (dC 137.7). The features of B-ring were
confirmed by ABX coupling between H-20 (dH 7.76, d, J = 2.0 Hz),
H-50 (dH 6.78, d, J = 8.5 Hz), and H-60 (dH 7.71, dd, J = 2.5, 8.5 Hz).
The position of C40-OH was deduced by HMBC correlation
between oxygenated carbon C-40 (dC 158.2) and H-50/H-60 (dH
6.78 / 7.71). The presence of 3-hydroxymethylbutyl motif on C-30
was deduced from successive protons network across H-70 (dH
2.60), H-80a/H-80b (dH 1.36, 1.65), H-90 (dH 1.75), H-100 (dH 0.93),
and H-110 (dH 3.89) in the COSY spectrum (Fig. 2A, supplemental).
The location of this functionality was confirmed by HMBC correla-
tion of H-70 (dH 2.60) with C-20 (dC 130.0), C-30 (dC 129.0), and
C-40 (dC 158.2). Thus the above obtained spectral data showed that
rotation value was measured as [a]D = 6.4 (c 0.56, MeOH). Thus
the compound was identified as (Z)-4-(5-(5,7-dihydroxy-3,6-
dimethoxy-4-oxo-4H-chromen-2-yl)-2-hydroxyphenyl)-2-methyl-
butyl-18,19-dihydroxy-nonadec-10-enoate, named visconata (1).
Spectral data of all the detected carbons, protons, COSY and HMBC
of 1 are given in Table 1 and Fig. 2A. To the best of our knowledge,
flavonoid skeleton having long chain fatty acid has never been
reported yet. As well as occurrence of odd numbered long chain
fatty acid is rare in terrestrial plants15, therefore these results are
striking, which could be a starting for further similar discoveries.
The isolated compounds (1–9) were analyzed for their inhibi-
tory potential toward HNE. The enzyme inhibition was assayed
according to a standard literature procedure by following hydroly-
sis of methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide spectropho-
tometrically16. As shown in Table 2 and Fig. 3A, all isolated
Table 2
Inhibitory effects of compounds 1–9 on HNE activities.
Compounds
Human neutrophil elastase
IC50
(l
M)a
Type of inhibition (Ki b, lM)
1
2
3
4
5
6
7
8
2.4 0.2
Noncompetitive (1.8 0.1)
Mixed (44.9 0.3)
Mixed (23.2 0.2)
NTc
Mixed (74.6 0.4)
Mixed (8.0 0.5)
NTc
Mixed (22.0 0.1)
Mixed (54.8 0.6)
NTc
65.4 0.1
25.4 0.4
150.2 1.2
93.9 0.6
10.9 0.3
114.7 0.2
33.4 0.5
74.7 0.3
67.7 0.9
1
has 5,7-dihydroxy-2-(4-hydroxy-3-(4-hydroxy-3-methylbutyl)
phenyl)-3,6-dimethoxy-4H-chromen-4-one moiety. As shown in
Fig. 2B, odd numbered long chain (C19) fatty acid motif was eluci-
dated by the fragment ions at m/z 399 [M-327] and 416 [M-310],
which were selected as main diagnostic product ions in EI-Mass
analysis (Fig. 2B). The position of double bond in C19 chain was
determined by the fragment ion at m/z 595 [M-131], which was
9
Caffeic acidd
a
a
-cleavage of alkene at a position between C1200
All compounds were examined as set of experiments repeated three times; IC50
formed by the
values of compounds represent the concentration that caused 50% enzyme activity
loss.
and C1300 (Fig. 2B). The J value of 10.5 Hz pointed that there is a
cis-configuration between H-1000 and H-1100. Terminal diol func-
tionality was confirmed by successive protons connectivity
between oxygenated atom H-1900 (dH 3.44), H-1800 (dH 3.40),
b
Values of inhibition constant.
NT: not tested.
Positive control
c
d
Table 1
1H and 13C NMR data of new Compound 1.
Position
dH J (Hz)
dC
m
Position
dH J (Hz)
dC m
2
3
4
5
6
7
8
156.9 s
137.7 s
178.8 s
152.2 s
131.3 s
152.4 s
93.7 d
157.7 s
104.8 s
121.1 s
130.0 d
129.0 s
158.2 s
114.6 d
127.6 d
27.0 t
100
200
300
174.2 s
33.8 t
24.7 t
28.7 t
28.8 t
36.8 t
36.6 t
28.7 t
26.7 t
129.2 d
129.5 d
26.7 t
29.3 t
25.5 t
28.8 t
21.7 t
32.3 t
70.8 d
61.5 t
59.1 q
59.5 q
2.20 t, (7.5)
1.48 t, (7.5)
1.14–1.17 m
1.14–1.17 m
1.24–1.29 m
1.32–1.36 m
1.14–1.17 m
1.84–1.92 m
5.17 m (10.5)
5.22 m (10.5)
1.84–1.92 m
1.14–1.17 m
1.24–1.45 m
1.14–1.17 m
1.24–1.45 m
1.24–1.45 m
3.40 m
b
400
b
500
600
700
6.40 s
b
8a
4a
10
800
900
1000
1100
1200
20
7.76 d (2.0)
30
b
40
1300
50
6.78 d (8.5)
7.71 dd (2.5, 8.5)
2.60 m
1.36,1.65 m
1.75 m
0.93 d (7.0)
3.89 dd (1.0, 6.0)
12.94 s (CDCl3)a
1400
b
60
1500
7 ’
80a, 80b
90
100
110
5-OH
1600
33.0 t
1700
32.2 d
15.8 q
68.6 t
1800
1900
3.44 t (6.5)
3.66 s
3.78 s
C3-OCH3
C6-OCH3
a
CDCl3 solvent was used only to detect 5-OH signal at 12.94 ppm.
Overlapped signals.
b

2510
Z. Uddin et al. / Tetrahedron Letters 58 (2017) 2507–2511
(A)
(B) 10
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
[I] = 0 uM
[I] = 0.86 uM
[I] = 1.73 uM
[I] = 3.47 uM
8
6
4
2
0
0.00 0.01 0.02 0.03 0.04 0.05
0.1
1
10
100
1000
[Inhibitor, µM]
E (Unit/ml)
(C)
(D)
[I] = 0 µM
[S] = 0.66 mM
[S] = 0.33 mM
[S] = 0.17 mM
[I] = 1.21 µM
[I] = 2.42 µM
[I] = 4.87 µM
0.3
0.2
0.1
0.3
0.2
0.1
-2
-1
0
1
2
3
4
-4
-2
0
2
4
6
8
Compound 1 (µM)
1/S (mM)
Fig. 3. (A) Dose-dependent inhibitory effects of isolated compounds (1–9) on HNE. (B) Determination of the reversible inhibitory mechanism of visconata (1). (C) Lineweaver-
Burk plot for HNE inhibition by compound 1. (D) Dixon plot for HNE inhibition by compound 1.
flavonols inhibited HNE activity dose-dependently with IC50 values
of 2.4–150 M. The potencies of flavonols were affected by subtle
changes in compounds structures. It appears that better inhibition
is observed when the flavonol B-ring bears methoxy group and
prenyl group. This is apparent from the comparison of compounds
manner. The novel compound, named visconata, has an odd num-
ber long chain fatty acid (C19). This is the first report on flavonoid
attached long chain fatty acid, which has never been reported so
far. In evaluation of HNE inhibition, the most active inhibitor was
l
found to be compound 1 (IC50 = 2.4
pound 1 emerged to be a reversible, noncompetitive inhibitor with
(Ki = 1.8 M), whereas rest of flavonoids (2–9) displayed mixed
lM). In kinetic study com-
6 (IC50 = 10.9 lM), 3 (IC50 = 25.4 lM) and 4 (IC50 = 150.2 lM).
The most striking aspect of this result is the long chain fatty acid
l
which is pivotal in HNE inhibition, due to which compound 1
type inhibition.
(IC50 = 2.4
(IC50 = 74.7
l
l
M) was 30-fold more potent than mother molecule 9
M) shown in Table 2. Further kinetic analysis showed
Acknowledgments
that compound 1 exhibited reversible noncompetitive inhibitory
behavior, because increasing inhibitor concentrations resulted in
a family of straight lines with different slopes but a common x-axis
intercept (Fig. 3B,C). Dixon plot unveiled the inhibitory constant to
This work was done with research funds from the [Ministry of
Agriculture, Food and Rural Affairs] (No. 315032-04-2-SB010)
and
[Ministry
of
Education,
through
(NRF)]
be Ki = 1.8 lM, given in Fig. 3D. Whereas other flavonols exhibited
(2015R1A6A1A03031413), Republic of Korea. BK21 plus program
supported scholarships for all students.
mixed inhibition kinetics. For example, flavonol 6 showed that
with increasing concentration of inhibitors resulted in a family of
lines which shared a common intercept on left side of the vertical
axis and above the horizontal axis (Supplementary material). The
inhibitory constants (Ki) values of tested compounds were also
determined by Dixon plots which are given in Table 1.
Supplementary data
Supplementary data (UV, IR, MS, NMR and kinetic data) associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.tetlet.2017.05.059.
In conclusion, we isolated a new compound along with eight
known flavonoids (1–9) which inhibited HNE in dose dependent

Z. Uddin et al. / Tetrahedron Letters 58 (2017) 2507–2511
13. Yang AM, Guo WJ, Zeng Y, Gong HF, WR. Adv Mater Res. 2014;852:8–11.
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References and notes
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University of Swat, KPK, Pakistan. Plant material (1.2 kg) was extracted with
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