Screening of Luzula species native to the Carpathian Basin for anti-inflammatory activity and bioactivity-guided isolation of compounds from Luzula luzuloides (Lam.) Dandy & Wilmott

Article abstract of DOI:10.1016/j.fitote.2016.12.004

The present study focused on the anti-inflammatory screening of Luzula species native to the Carpathian Basin and bioactivity-guided isolation of compounds of Luzula luzuloides (Lam.) Dandy & Wilmott. The anti-inflammatory properties of extracts with different polarity prepared from Luzula species were determined. Among them, the CH₂Cl₂-soluble fraction of L. luzuloides possessed strong inhibitory effects on superoxide anion generation (99.39 ± 0.37%) and elastase release (114.22 ± 3.13%) in fMLP/CB-induced human neutrophils at concentration of 10 μg/mL. From this fraction, six compounds (1–6) were isolated by the combination of different chromatographic methods. The structures of the compounds were determined by means of MS, 1D and 2D NMR spectroscopy. The results allowed the identification of the new 1,6-dihydroxy-2-keto-1,7-dimethyl-8-vinyl-1,2-dihydrophenanthrene (1) from the plant, named luzulin A. Chiral HPLC and HPLC-ECD analysis revealed that 1 possesses low enantiomeric excess and TDDFT-ECD calculations afforded the configurational assignment of the separated enantiomers. Three known phenanthrenes [juncuenin B (2), dehydrojuncuenin B (3) and juncusol (4)] and two flavonoids [apigenin (5) and luteolin (6)] were also isolated. The anti-inflammatory activity of the isolated compounds was tested and IC₅₀ values were determined. This was the first time that phenanthrenes were detected in a Luzula species. The oxidative transformation of juncuenin B (3) led to the isolation of its possible biometabolites, namely luzulin A (1), dehydrojuncuenin B (4), and juncuenin D (7). The isolated compounds (1–4) confirm that besides flavonoids, phenanthrenes could also serve as chemotaxonomic markers for Luzula species and prove the close relationship of Juncus and Luzula genus.

Full text of DOI:10.1016/j.fitote.2016.12.004

Screening of Luzula species native to the Carpathian Basin for anti-
inflammatory activity and bioactivity-guided isolation of compounds from
Luzula luzuloides (Lam.) Dandy & Wilmott
´
Barbara To´th, Fang-Rong Chang, Tsong-Long Hwang, Ada´m Szappanos,
Attila Ma´ndi, Attila Hunyadi, Tibor Kurta´n, Guszta´v Jakab, Judit Hohmann,
Andrea Vasas
PII:
DOI:
Reference:
S0367-326X(16)30663-3
doi:10.1016/j.fitote.2016.12.004
FITOTE 3541
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
28 October 2016
1 December 2016
3 December 2016
Please cite this article as: Barbara T´oth, Fang-Rong Chang, Tsong-Long Hwang,
´
Ad´am Szappanos, Attila Ma´ndi, Attila Hunyadi, Tibor Kurt´an, Guszta´v Jakab, Ju-
dit Hohmann, Andrea Vasas, Screening of Luzula species native to the Carpathian Basin
for anti-inflammatory activity and bioactivity-guided isolation of compounds from Luzula
luzuloides (Lam.) Dandy & Wilmott, Fitoterapia (2016), doi:10.1016/j.fitote.2016.12.004
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ACCEPTED MANUSCRIPT
Screening of Luzula species native to the Carpathian Basin for anti-inflammatory activity
and bioactivity-guided isolation of compounds from Luzula luzuloides (Lam.) Dandy &
Wilmott
Barbara Tóth,a Fang-Rong Chang,b,c,d Tsong-Long Hwang,e,f,g Ádám Szappanos,h Attila Mándi,h
Attila Hunyadi,a Tibor Kurtán,h Gusztáv Jakab,i Judit Hohmann,a,j Andrea Vasasa*
a Department of Pharmacognosy, University of Szeged, 6720 Szeged, Hungary
b Graduate Institute of Natural Products, Kaohsiung Medical University, 807 Kaohsiung, Taiwan
c Research Center for Environmental Medicine, Kaohsiung Medical University, 807 Kaohsiung,
Taiwan
d Department of Marine Biotechnology and Resources, National Sun Yat-sen University, 804
Kaohsiung, Taiwan
e Graduate Institute of Natural Products, Chang Gung University, 333 Taoyuan, Taiwan
f Research Center for Industry of Human Ecology, Research Center for Chinese Herbal
Medicine, and Graduate Institute of Health Industry Technology, Chang Gung University of
Science and Technology, 333 Taoyuan, Taiwan
g Department of Anesthesiology, Chang Gung Memorial Hospital, 333 Taoyuan, Taiwan
h Department of Organic Chemistry, University of Debrecen, 4032 Debrecen, Hungary
i Institute of Environmental Sciences, Faculty of Water and Environmental Management, Szent
István University, 5540 Szarvas, Hungary
jInterdisciplinary Centre of Natural Products, University of Szeged, 6720 Szeged, Hungary
*Corresponding author: Dr. Andrea Vasas, email: vasasa@pharmacognosy.hu; Tel: +36 62
546451; Fax: +36 62 545704
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ABSTRACT
The present study focused on the anti-inflammatory screening of Luzula species native to the
Carpathian Basin and bioactivity-guided isolation of compounds of Luzula luzuloides (Lam.)
Dandy & Wilmott. The anti-inflammatory properties of extracts with different polarity prepared
from Luzula species were determined. Among them, the CH2Cl2-soluble fraction of L. luzuloides
possessed strong inhibitory effects on superoxide anion generation (99.39 ± 0.37%) and elastase
release (114.22 ± 3.13%) in fMLP/CB-induced human neutrophils at concentration of 10 μg/mL.
From this fraction, six compounds (1–6) were isolated by the combination of different
chromatographic methods. The structures of the compounds were determined by means of MS,
1D and 2D NMR spectroscopy. The results allowed the identification of the new 1,6-dihydroxy-
2-keto-1,7-dimethyl-8-vinyl-1,2-dihydrophenanthrene (1) from the plant, named luzulin A. Chiral
HPLC and HPLC-ECD analysis revealed that 1 possesses low enantiomeric excess and TDDFT-
ECD calculations afforded the configurational assignment of the separated enantiomers. Three
known phenanthrenes [juncuenin B (2), dehydrojuncuenin B (3) and juncusol (4)] and two
flavonoids [apigenin (5) and luteolin (6)] were also isolated. The anti-inflammatory activity of
the isolated compounds was tested and IC50 values were determined. This was the first time that
phenanthrenes were detected in a Luzula species. The oxidative transformation of juncuenin B (3)
led to the isolation of its possible biometabolites, namely luzulin A (1), dehydrojuncuenin B (4),
and juncuenin D (7). The isolated compounds (1-4) confirm that besides flavonoids,
phenanthrenes could also serve as chemotaxonomic markers for Luzula species and prove the
close relationship of Juncus and Luzula genus.
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Keywords
Juncaceae, Luzula luzuloides, phenanthrenes, flavonoids, anti-inflammatory activity, electronic
circular dichroism
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1. Introduction
The family Juncaceae includes more than 500 species, which are distributed worldwide.
Juncus and Luzula are the two largest genera of the family Juncaceae, comprising approximately
340 and 115 species, respectively [1,2]. Among them up to 45 species are native to the
Carpathian Basin. Although traditional use of plants belonging to the genus Luzula has not been
reported yet, several Juncus species (e.g. J. effusus and J. acutus) are used in oriental folk
medicine for the treatment of cold, fever, anxiety or insomnia [3-5].
Despite of the comprehensive research focused on Juncaceae family, until now only
species belonging to the genus Juncus were investigated extensively [6-9]. There is a significant
lack of information with regards to chemical constituents of the plants belonging to the genus
Luzula. Hitherto only the flavonoid components of Luzula species were studied. Luteolin and
luteolin-7-O-glucoside were isolated by the use of preparative paper chromatography from L.
sylvatica [10]. Williams et al. identified several flavonoids by using TLC, comparing their Rf
values and colors with standards, and luteolin and luteolin 7-O-glucoside were detected from all
of the investigated Luzula samples (L. arcuata, L. campestris, L. confusa, L. forsteri, L. lactea, L.
multiflora, L. nivea, L. nodulosa, L. nutans, L. pilosa, L. spicata and L. sylvatica) [11]. Quercetin,
procyanidin and other luteolin derivatives (luteolin 5-methyl ether, luteolin 5-methyl ether 7-O-
glucoside) were also identified in some species [11]. Therefore, flavonoids are considered to be
the main secondary metabolites, and one of the most characteristic components of Luzula species.
Previously benzocoumarins, diterpenoids, cycloartane triterpenes, carotenoids,
phenanthrenes, flavonoids, and other phenolic compounds were isolated from Juncus species [12-
16]. Apart from flavonoids, phenanthrenes and 9,10-dihydrophenanthrenes are the most specific
chemical constituents of the family. To date more than 90 novel monomeric and 8 dimeric
phenanthrenes have been identified from Juncaceae species [6-9]. Phenanthrenes are considered
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to be important taxonomic markers, because of their limited occurrence. Moreover, vinylated
phenanthrenes have been reported only from species of the family Juncaceae [6-9].
Phenanthrenes occurring in plants have drawn considerable interest from the aspect of natural
product drug discovery because of the wide range of their potentially valuable biological
activities, e.g. cytotoxic, antimicrobial and anti-inflammatory activities [6-9].
Juncus effusus has been used in the traditional oriental medicine as a remedy for
inflammation, therefore, isolated phenanthrenes from the plant were also tested for their anti-
inflammatory activity by the inhibition of LPS-induced NO production on RAW 264.7 cells and
compounds showed promising effect [17-20]. By the use of this assay a strong anti-inflammatory
activity was detected in case of effususin B (IC50 = 7.42 ± 0.50 μM), compared to the positive
control quercetin (IC50 = 6.63 ± 0.28 μM) [17]. 8-Hydroxymethyl-2-hydroxy-1-methyl-5-vinyl-
9,10-dihydrophenanthrene (IC50 = 14.42 ± 2.82 μM), 5-(1-methoxyethyl)-1-methylphenanthrene-
2,7-diol (IC50 = 11.10 ± 1.67 μM), effusol (IC50 = 15.13 ± 0.54 μM), dehydroeffusol (IC50
12.69 ± 1.68 μM), dehydroeffusal (IC50 = 10.50 ± 0.90 μM), 2,7-dihydroxy-5-hydroxymethyl-1-
methyl-9,10-dihydrophenathrene (IC50 16.06 1.18 μM), 5-hydroxymethyl-1-
=
=
±
methylphenanthrene-2,7-diol (IC50 = 16.30 ± 2.41 μM), dehydrojuncusol (IC50 = 15.57 ± 1.08
μM), and the mixture of 2,7-dihydroxy-1,8-dimethyl-5-vinyl-9,10-dihydrophenanthrene and
juncusol (IC50 = 13.65 ± 1.40 μM) also had remarkable activities in comparison with the positive
control quercetin [18].
Juncutol displayed noteworthy anti-inflammatory activity by the inhibition of the iNOS
protein expression in LPS-stimulated RAW 264.7 cells. The iNOS level was reduced to 11.2 ±
13.1% from 100% after treating with 10 μM juncutol [19]. Dehydrojuncusol and its dimer, 8,8’-
bidehydrojuncusol were also assayed by the abovementioned method. According to the
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immunoblot analysis, the effect of dehydrojuncusol on the iNOS protein (41.0 ± 8.0% inhibition
at 10 μM) decreased dramatically after dimerization (< 12.0% inhibition at 10 μM) [20].
The anti-inflammatory activity of flavonoids is well-studied, but in some cases the
mechanism of action of these compounds is not completely established [21,22]. Apigenin and
luteolin inhibited the LPS induced NO-production on RAW 264.7 macrophages with IC50 values
of 23 μM and 27 μM, respectively. While luteolin reduced the iNOS expression and interestingly
increased the enzyme activity in a dose-dependent manner, apigenin inhibited the iNOS and
COX-2 expression in LPS-activated RAW264.7 cells, but had no effect on the iNOS enzyme
activity [23,24]. Both apigenin and luteolin inhibited significantly the TNF α-induced NF-κB
activation on KF 8 cells. Moreover, their anti-inflammatory activity was confirmed in vivo by the
inhibition of carrageenan-induced paw inflammation on mice [25].
The purposes of the present study were to investigate the anti-inflammatory properties of
Luzula species, and to perform an activity-guided fractionation of Luzula luzuloides in order to
isolate the compounds responsible for this effect.
2. Experimental
2.1. General
Normal phase vacuum liquid chromatography (VLC) was carried out on silica gel
(Kieselgel GF254, 15 µm, Merck). LiChroprep RP-18 (40-63 µm, Merck) stationary phase was
used for reversed-phase VLC. Column chromatography (CC) was performed on polyamide (MP
Biomedicals). Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemicals) was used for gel
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filtration. Preparative thin-layer chromatography (preparative TLC) was carried out on silica gel
60 F254 plates (Merck).
NMR spectra were recorded in CD3OD and DMSO-d6 on a Bruker Avance DRX 500
spectrometer at 500 MHz (1H) and 125 MHz (13C). The signals of the deuterated solvents were
taken as reference. Two-dimensional (2D) experiments were performed with a standard Bruker
software. In the COSY, HSQC and HMBC experiments, gradient-enhanced versions were
applied. The high resolution MS spectra were acquired on a Thermo Scientific Q-Exactive Plus
orbitrap mass spectrometer equipped with ESI ion source in positive ionization mode. The
resolution was over 1 ppm. The data were acquired and processed with the MassLynx software.
ECD spectra was recorded on a J-810 spectropolarimeter.
HPLC-ECD analysis: Chiral HPLC separation was performed with a JASCO HPLC
system using Chiralpak IA column with 250 mm × 4.6 mm i.d., 5 m (Daicel, Chemical
Industries, Ltd.), and n-hexane–propan-2-ol eluent at a flow rate of 1.0 mL/min was used. HPLC-
UV and OR chromatograms were recorded with JASCO MD-910 multiwavelength and OR-
2090Plus chiral detector, respectively. The on-line ECD and UV spectra were measured
simultaneously by stopping the flow at the UV absorption maximum of each peak. The values of
the ECD ellipticity () were not corrected for the concentration. Three consecutive scans were
recorded and averaged for an HPLC-ECD spectrum with standard sensitivity, 2 nm bandwidth
and 1 sec response. The background HPLC-ECD spectrum of the eluent was recorded in the same
way.
2.2. Plant material
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Plants (Luzula campestris (L.) DC., L. forsteri (Sm.) DC., L. luzuloides (Lam.) Dandy &
Wilmott, L. sudetica (Willd.) Schult., L. sylvatica (Huds.) Gaudin) were collected during the
flowering period between June and September 2014, in several regions of the Carpathian Basin
(Hungary, Croatia and Romania). Botanical identification of the plant material was performed by
Gusztáv Jakab (Institute of Environmental Sciences, Szent István University, Szarvas, Hungary)
and voucher specimens (No. 869-873) have been deposited at the Department of Pharmacognosy,
University of Szeged, Szeged, Hungary.
2.3. Preparation of plant samples
For the anti-inflammatory screening assay, extracts were prepared from 20 g of air-dried
plant materials (whole plant, roots and aerial parts) with 5 × 100 mL of MeOH with the use of
ultrasonic bath (5 × 15 min). After filtration, the solutions were evaporated to dryness under
reduced pressure. The residues were dissolved in 50 mL of 50% aqueous MeOH and subjected to
solvent–solvent partition between n-hexane (3 × 100 mL) (extracts A), dichloromethane (3 × 100
mL) (extracts B), and EtOAc (3 × 100 mL) (extracts C), and the remaining H2O extracts gave
extracts D.
2.4. Extraction and isolation
The air-dried whole plant of L. luzuloides (480 g) was percolated with MeOH (20 L) at
room temperature. The crude methanol extract was concentrated under reduced pressure (76 g)
and subjected to solvent–solvent partitioning with CH2Cl2 (2.5 L). After evaporation, the CH2Cl2
fraction (4.95 g) was chromatographed on a polyamide column with mixtures of MeOH and H2O
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[1:1, 4:1 (1 L and 4 L, respectively); each eluent was collected as a fraction]. The fraction
obtained from the polyamide column with methanol–water 4:1 (1.10 g) was further
chromatographed by VLC on silica gel with a gradient system of cyclohexane–EtOAc–EtOH
[from 9:1:0 to 5:5:1 (300 mL/eluent), and finally with EtOH (200 mL); volume of collected
fractions was 20 mL] to yield twelve major fractions (1-12).
From fraction 1 (9.2 mg) compound 2 (5.2 mg) was crystallized. Fraction 4 (31.3 mg) was
purified by Sephadex LH-20 gel chromatography using MeOH as eluent [(100 mL); volume of
collected fractions was 2 mL] to afford four subfractions (4/1–4). Compound 1 (3.0 mg) was
purified from subfraction 4/1 by preparative TLC using n-hexane–EtOAc (7:3) as solvent system.
Subfraction 4/3 (14.1 mg) was also purified by preparative TLC on silica gel, with cyclohexane–
EtOAc–MeOH (20:10:1) as solvent system to afford compound 3 (5.1 mg). Compound 4 (3.2
mg) was crystallized from subfraction 4/4. Fraction 6 (25.1 mg) was separated by RP-VLC, with
a gradient system of MeOH–H2O [from 4:6 to 9:1 (150 mL/eluent), and finally with MeOH (100
mL); volume of collected fractions was 10 mL] to yield five subfractions (6/1–5). Compound 5
(9.0 mg) was crystallized from subfraction 6/2. Furthermore, compound 6 (7.1 mg) was
crystallized from fraction 7.
2.4.1. Luzulin A (1)
Amorphous solid; []2D6 0 (c 0.1, MeOH); H and 13C NMR data, see Table 2; (+)-
1
HRESIMS m/z 281.1175 [M+H]+ (calcd for C18H17O3, 281.1177). 25% ee with an excess of (S)-
1.
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(R)-1: tR = 4.88 min on Chiralpak IA column (n-hexane–propan-2-ol, 8:2); HPLC-ECD n-
hexane–propan-2-ol, 8:2,  [nm] (): 424 (2.30), 376 (4.49), 312 (3.51), 287 (0.10), 266
(2.52), 244sh (3.92), 229 (5.17), 204 (7.81).
(S)-1: tR = 5.41 min on Chiralpak IA column (n-hexane–propan-2-ol, 8:2); HPLC-ECD n-
hexane–propan-2-ol, 9:1,  [nm] (): 424 (2.96), 374 (4.26), 312 (2.68), 287 (1.23), 268
(1.53), 244sh (7.57), 227 (8.36), 204 (5.46).
2.5. Oxidation of juncuenin B
50.0 mg of juncuenin B (3) was dissolved in CH3CN–H2O (9:1, 50 mL). 2 eq.
[bis(trifluoroacetoxy)iodo]benzene (PIFA) (106.1 mg) was added to the solution in a careful
manner to prevent sudden boiling. The reaction was monitored by TLC using cyclohexane–
EtOAc–EtOH (20:10:1) as solvent system. After 30 min of stirring at room temperature, all of the
starting material, juncuenin B (3) was converted. The mixture was cooled down in an ice bath,
and evaporated at room temperature.
2.6. Separation of the oxidative products of juncuenin B
The concentrated mixture was chromatographed by VLC on silica gel (25 mm × 100 mm)
with a gradient system of CH2Cl2–acetone [from 99:1 to 7:3 (200 mL/eluent), and finally with
MeOH (150 mL); volume of collected fractions was 20 mL] to yield seven major fractions (1-7).
Dehydrojuncuenin B (4) (8.9 mg) was purified from fraction 2 after gel filtration on Sephadex
LH-20 using MeOH–CH2Cl2 (3:1) as eluent [(60 mL); volume of collected fractions was 3 mL].
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Fraction 4 was further separated by gel filtration on Sephadex LH-20 using MeOH as eluent [(50
mL); volume of collected fractions was 2 mL] to afford three subfractions (4/1–3). From
subfraction 4/2 luzulin A (1) (8.1 mg) was obtained by the use of preparative TLC using toluene–
acetone (8:2) as solvent system. Juncuenin D (7) (11.2 mg) was isolated from fraction 6 after gel
filtration on Sephadex LH-20 using MeOH–CH2Cl2 (3:1) as eluent [(50 mL); volume of collected
fractions was 2 mL]. The compounds were identified according to their TLC patterns and the
comparison of their 1H-NMR spectrum with literature data [5].
2.7. Preparation of human neutrophils
Blood was taken from healthy human donors (20–30 years old) by venipuncture using a
protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital.
Neutrophils were isolated using a standard method as previously described [26,27]. The cells
were obtained from peripheral blood according to the standard method of dextran sedimentation,
followed by centrifugation in a Ficoll-Hypaque gradient and the hypotonic lysis of the
erythrocytes. The purified neutrophils contained >98% of viable cells, as determined by Trypan
blue exclusion, and were suspended in calcium-free HBSS at 4 ˚C before used.
2.8. Measurement of superoxide generation
SOD inhibition was measured by reduction of ferricytochrome c [26,27]. Neutrophils (6 ×
105 cells/mL) in 0.5 mg/mL ferricytochrome c and 1 mM Ca2+ were equilibrated at 37 °C for 2
min and then incubated with drugs for 5 min. The cells were activated using formyl-methionyl-
leucyl-phenylalanine (fMLP, 100 nM)/cytochalasin B (CB, 1 μg/mL) for 10 min. The absorbance
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was continuously monitored at 550 nm using a double-beam, six-cell positioned
spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were
based on the differences in absorbance with and without SOD (100 μ/mL) divided by the
extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).
2.9. Measurement of elastase release
The degranulation of azurophilic granules was determined by elastase release as described
previously [26,27]. Neutrophils (6 × 105/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-
nitroanilide (100 μM), an elastase substrate, at 37 °C for 2 min and then incubated with drugs for
5 min. The cells were activated using fMLP (100 nM) in the presence of CB (0.5 μg/mL), and
changes in the absorbance at 405 nm were continuously measured to monitor elastase release.
The results are expressed as a percentage of elastase release in the fMLP/CB-activated, drug-free
control system.
2.10. Statistical methods
Results of the anti-inflammatory assays were expressed as means ± S.E.M. Statistical
analysis using Student’s t-test was performed with SigmaPlot (Systat Software, San Jose, CA,
USA). A value of P < 0.05 was considered statistically significant.
2.11. Computational section
Mixed torsional/low-frequency mode conformational searches were carried out by means
of the Macromodel 9.9.223 software using the Merck Molecular Force Field (MMFF) with an
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implicit solvent model for CHCl3 [28]. Geometry reoptimizations were carried out at the
B3LYP/6-31G(d) level in vacuo, the B97D/TZVP [29,30] and the CAM-B3LYP/TZVP [31,32]
levels with the PCM solvent model for CHCl3. TDDFT ECD calculations were run with various
functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set as implemented
in the Gaussian 09 package with the same or no solvent model as in the preceding DFT
optimization step [33]. ECD spectra were generated as sums of Gaussians with 3000 cm-1 widths
at half-height (corresponding to ca. 41 nm at 370 nm), using dipole-velocity-computed rotational
strength values [34]. Boltzmann distributions were estimated from the ZPVE-corrected
B3LYP/6-31G(d) energies in the gas-phase calculations and from the B97D/TZVP and CAM-
B3LYP/TZVP energies in the solvated ones. The MOLEKEL software package was used for
visualization of the results [35-37].
3. Results and Discussion
In the course of our screening study, the anti-inflammatory activities of 5 species of the
Luzula genus native to the Carpathian Basin were evaluated. The results of the assays are listed in
Table 1. Extracts were prepared with methanol from whole plants of L. campestris, L. forsteri, L.
luzuloides and L. sudetica. The plant material allowed the separation of L. sylvatica into aerial
and root parts, therefore methanol extracts were prepared from selected plant organs in case of L.
sylvatica. The crude extracts were concentrated and suspended in 50% MeOH, and then solvent–
solvent partitions were made with n-hexane (A), CH2Cl2 (B), and EtOAc (C). The remaining H2O
fractions were signed as D (altogether 24 extracts). The apolar, n-hexane-soluble fractions of L.
forsteri, L. luzuloides, L. sudetica and L. sylvatica showed more than 70% inhibition at the tested
concentration (10 μg/mL) in both anti-inflammatory assays. The fraction A of L. campestris
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exerted the most selective elastase release inhibition. Among the fractions with different
polarities, fractions B (containing CH2Cl2-soluble lipophilic constituents) had the most
remarkable activities (> 93% inhibition) at concentration of 10 μg/mL. All of the CH2Cl2-soluble
fractions displayed significant activities on the inhibition of elastase release, and proved to be
slightly less active on superoxide generation. The EtOAc-soluble fractions of L. campestris, L.
luzuloides, L. sudetica, L. sylvatica (aerial part) possessed noteworthy (> 65%) inhibitory
activities at concentration of 10 μg/mL. The remaining H2O fraction of L. luzuloides also showed
notable anti-inflammatory activities (> 65%) in both methods. On the basis of these preliminary
results, L. luzuloides has been chosen for further investigation.
Dried whole plant material of L. luzuloides (480 g) was powdered and extracted with
methanol at room temperature. After concentration, the extract was dissolved in 50% aqueous
MeOH, and solvent–solvent partition was performed with n-hexane, CH2Cl2, and EtOAc. The
CH2Cl2 fraction was separated and purified with the combination of different chromatographic
methods (CC, VLC, Sephadex LH-20 gel chromatography, prep TLC) to afford 6 compounds (1–
6, Figure 1).
The structure determination was carried out by extensive spectroscopic analysis, using
one- and two-dimensional NMR (1H-1H COSY, HSQC, HMBC) spectroscopy, HRESIMS and
HPLC-ECD experiments and comparison of the spectral data with literature data.
Compound 1 was obtained as an amorphous solid with []2D6 0 (c 0.1, MeOH). Its
HRESIMS provided the molecular formula C18H16O3 through the presence of a peak at m/z
1
281.1175 [M+H]+ (calcd for C18H17O3, 281.1172). The H NMR spectrum (Table 2) displayed
signals of two pairs of ortho-coupled aromatic protons (δH 6.25 d/8.35 d and δH 8.12 d/7.63 d),
and one aromatic proton as a singlet (δH 7.52), two methyl singlets at δH 1.53 and δH 2.35, and a
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vinylic system at δH 5.81, 5.35 and 7.05 (C-14, C-13). In the JMOD spectrum, the presence of 18
1
carbon signals was detected (Table 2). In the H NMR spectrum the presence of ortho-coupled
aromatic protons at δH 8.12 d and 7.63 d could be assigned to the H-9 and H-10 protons of a
1
phenanthrene derivative. In the H,1H-COSY spectrum four correlations were observed between
δH 8.12 d and 7.63 d (H-9/H-10), between δH 6.25 d and 8.35 d (H-3/H-4), between δH 7.05 dd
and 5.81 d (H-13/H-14a), and between δH 7.05 dd and 5.35 d (H-13/H-14b). The two ortho-
coupled doublets at δH 6.25 and 8.35 were assigned to the H-3 and H-4 protons. According to the
13C NMR signals at δC 207.1 one carbonyl group was identified, which was placed to C-2 as
confirmed by HMBC correlations of the 11-CH3 (δH 1.53) and H-4 (δH 8.35 d) with C-2 (δC
207.1) (Table 2, Figure 2). The location of one of the methyl groups (δH 1.53 s) was confirmed at
C-1 on the basis of its HMBC correlation with the quaternary carbons δC 146.6 (C-1a), 78.1 (C-1)
and 207.1 (C-2). The linkage of hydroxy groups to C-1 and C-6 was confirmed by the chemical
shifts of the quaternary carbons at δC-1 78.1 and δC-6 157.0, respectively. In the HMBC spectrum
another methyl group was assigned at C-7 by the correlation of H-12/C-7. The vinyl substitution
at C-8 was indicated by the HMBC cross-peaks between H-14a/C-8 and H-14b/C-8.
The NOESY correlations further confirmed the structure of compound 1. Overhauser
effects were detected between H-3/H-4, H-4/H-5, H-9/H-10, H-9/H-13, H-9/H-14b, H-13/CH3-
12, H-14b/CH3-12. All of the above evidence confirmed the planar structure of 1 named as
luzulin A (Figure 1).
Luzulin A (1) is a chiral natural product but it had a very weak ECD spectrum in
acetonitrile and zero specific rotation, which suggested that it had low enantiomeric excess. Thus
chiral HPLC analysis of 1 was carried out, which resulted in the separation of the two
enantiomers on a Chiralpak IA column and determined the enantiomeric excess as 25%. The
quaternary stereogenic center does not enable inversion of the absolute configuration during the
15

ACCEPTED MANUSCRIPT
isolation and hence the low enantiomeric excess can be attributed to the low enantioselectivity of
the biosynthetic step, which has been observed for other natural phenanthrenes as well [38].
In order to elucidate the absolute configuration of the enantiomers and identify the major
enantiomer, HPLC-ECD spectra of the separated enantiomers were recorded and the solution
TDDFT-ECD protocol was applied on the arbitrarily chosen (R) enantiomer (Figure 3) [36]. The
initial Merck Molecular Force Field (MMFF) conformational search resulted in 5 conformers in a
21 kJ/mol energy window, which were reoptimized at various DFT levels [B3LYP/6-31G(d) in
vacuo, B97D/TZVP PCM/CHCl3 and CAM-B3LYP/TZVP PCM/CHCl3] and ECD spectra were
computed with B3LYP, BH&HLYP, CAM-B3LYP and PBE0 functionals and TZVP basis set.
The computed conformers differed in the orientation of the OH-6 proton and the C-8 vinyl group,
the latter of which influenced the ECD spectra as well. The OH-1 group adopted equatorial
position in all the computed conformers (Figure 4). All the applied combinations for all
conformers reproduced the first two transitions at ca 422 and 375 nm and Boltzmann-weighted
spectra acquired at all applied levels resembled also the high-energy transitions at ca. 227 and
206 nm allowing the elucidation of the absolute configuration as (R) for the first-eluting
enantiomer (minor component) and (S) for the second-eluting enantiomer (major component).
Interestingly, the 312 nm Cotton effect (CE) could not be reproduced by any applied combination
of theoretical levels, which can be attributed to difficulties in estimating the proper orientation of
the conjugating quite freely rotating vinyl chromophore and the overlapping transitions of this
region [37].
Besides the new compound, luzulin A (1), two dihydrophenanthrenes [juncusol (2) and
juncuenin B (3)], one phenanthrene [dehydrojuncuenin B (4)] and two flavonoids [apigenin (5)
and luteolin (6)] were also isolated from L. luzuloides. Their structures were determined by
analyses of MS, 1D and 2D NMR data, and by comparison with literature data [38-40].
16

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All compounds were isolated for the first time from L. luzuloides. The methyl group at C-
1, a hydroxyl group at C-2 and vinyl, methyl and hydroxyl substitution on ring B are
characteristic features of the isolated phenanthrenes. In compound 1, a carbonyl group can also
be found in the molecule. The absolute configuration of luzulin A (1) was determined by HPLC-
ECD measurements and TDDFT-ECD calculations.
So far more than 90 novel phenanthrenes have been reported from 6 Juncus species (J.
acutus, J. effusus, J. inflexus, J. roemerianus, J. setchuensis and J. subulatus) [5-7,38]. The first
example of nitrogen-free alkylated phenanthrenes was juncusol (2) isolated from J. roemerianus
[41]. Until now phenanthrenes have not been isolated from the genus Luzula. This was the first
time when extensive phytochemical and pharmacological investigations were performed with the
genus Luzula and with L. luzuloides. Our results allowed the identification of four phenanthrenes
(1–4) substituted with vinyl, hydroxyl, methyl and methoxy groups, one of them (1) is new
natural product. The other phenanthrenes (2–4) were isolated previously only from other
Juncaceae species. All of the compounds were isolated for the first time from L. luzuloides.
In the course of our previous investigations considerable amount of juncuenin B (3) (850
mg) and its unsaturated form, dehydrojuncuenin B (4) have been isolated from J. inflexus [38].
Luzulin A (1) from L. luzuloides and juncuenin D (7) from J. inflexus have obvious structure
similarity with the abovementioned two phenanthrenes [juncuenin B (3) and dehydrojuncuenin B
(4)], suggesting that both phenanthrenequinones are possibly derived from the oxidation of
juncuenin B (3). In the case of luzulin A (1), ring A is oxidized, and in the case of juncuenin D
(7) ring C has changed. In order to confirm their possible biosynthetic connection, juncuenin B
(3) was subjected to an oxidative reaction by the use of hypervalent iodine reagent (PIFA). 50 mg
juncuenin B (3) was dissolved in CH3CN–H2O (9:1, 1 mg/mL), and a reaction with 2 eq. of PIFA
was performed at room temperature for 30 min (Scheme 1). After purification by NP-VLC,
17

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Sephadex LH-20 gel filtration and preparative TLC, three major compounds were isolated from
the mixture. The compounds were identified as juncuenin D (7, 22%), dehydrojuncuenin B (4,
1
18%) and luzulin A (1, 16%) by comparison of their H-NMR spectrum with literature data [5].
These findings confirm that luzulin A (1) and juncuenin D (7) can form by oxidation of juncuenin
B (3), and most probably similar process may be performing during their biosynthesis. Although,
several phenanthrenes and flavonoids have been reported to exhibit remarkable anti-
inflammatory activity, there were no previous data for the anti-inflammatory effects of
metabolites from the genus Luzula. In the performed assay, effects against superoxide anion
production and elastase release by human neutrophils in response to fMLP/CB were measured
according to previously published methods [26,27]. Among the isolated compounds, noteworthy
anti-inflammatory activities were measured for juncusol (2), juncuenin B (3), dehydrojuncuenin
B (4), apigenin (5) and luteolin (6) (Table 3). Although great deals of phenanthrenes were
assayed for their anti-inflammatory properties, this was the first time when the anti-inflammatory
activity of the isolated phenanthrenes (1–4, 7) was evaluated [5,6].
In the superoxide anion generation assay, significant inhibitory activities were recorded
for juncusol (2) (IC50 = 3.11 µM), juncuenin B (3) (IC50 = 4.92 µM), dehydrojuncuenin B (4)
(IC50 = 3.17 µM), apigenin (5) (IC50 = 6.12 µM), and luteolin (6) (IC50 = 4.73 µM). Furthermore,
juncuenin B (3) and luteolin (6) inhibited effectively the elastase release with IC50s 5.47 µM and
6.91 µM, respectively, comparable to that of LY294002 (IC50 = 4.79 µM). These results suggest
that compounds 2–6 display promising anti-inflammatory activity, juncusol (2),
dehydrojuncuenin B (4) and apigenin (5) were considered to be active only in case of inhibition
of superoxide generation, while luzulin A (1) and juncuenin D (7) were proved to be inactive in
both assays. With its unsaturation at ring B, juncuenin B (3) lost its effect on the elastase release,
but dehydrojuncuenin B (4) inhibited the superoxide generation more effectively than juncuenin
18

ACCEPTED MANUSCRIPT
B (3). The two phenanthrenequinones, luzulin A (1) and juncuenin D (7), presumably derived
from juncuenin B (3) showed significantly lower anti-inflammatory activities compared to their
possible biosynthetic precursor.
In conclusion, phenanthrenes are the most characteristic compounds of Juncaceae species.
Although phenanthrenes have been identified from taxonomically distant families [6], vinyl
substituted derivatives can be considered as chemotaxonomic markers for plants belonging to
Juncaceae family, since these specifically substituted phenanthrenes were only reported from
Juncus species, and this was the first time when they were isolated from the genus Luzula. The
chemical characterization of L. luzuloides, and the presence of vinylated phenanthrenes in the
plant further confirm the close botanical relationship between the genera Luzula and Juncus.
This was the first time when the anti-inflammatory activity of species belonging to the
genus Luzula was evaluated. Along with the extracts prepared from different species of the
genus, several isolated compounds showed promising anti-inflammatory activities. The present
study highlights that phytochemical and pharmacological studies of the previously uninvestigated
genus Luzula are very promising.
Acknowledgements
Financial support from the Hungarian Scientific Research Fund (OTKA K109846) is
gratefully acknowledged. A.V. acknowledges the award of a János Bolyai scholarship of the
Hungarian Academy of Sciences. T.K. thanks the Hungarian National Research Foundation
(OTKA K105871) for financial support and the National Information Infrastructure Development
Institute (NIIFI 10038) for CPU time. T.L.H. thanks the Chang Gung Memorial Hospital
(CMRPD1B0281~3, CMRPF1D0442~3, CMRPF 1F0011~3, CMRPF1F0061~3 and BMRP450)
19

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for financial support. The authors would like to thank to Attila Csorba (Department of
Pharmacognosy, University of Szeged) for the HRMS measurements.
Appendix A. Supplementary data
1D and 2D NMR and ECD spectra of compound 1 are available in the Supplementary
data.
Conflict of interest
The authors declare no competing financial interest.
20

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Table 1. Inhibitory effects of extracts with different polarity from Luzula species on superoxide
anion generation and elastase release by human neutrophils in response to fMLP/CB at
concentration 10 μg/mL.
Yield
(w/w %)
8.06
Superoxide anion
inhibition (%)
Elastase inhibition
(%)
Species
Fraction
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
50.04 ± 5.08 ***
96.06 ± 0.91 ***
77.45 ± 6.44 ***
32.52 ± 5.43 ***
71.53 ± 7.75 ***
93.14 ± 6.14 ***
35.24 ± 1.43 ***
32.80 ± 3.40 ***
70.65 ± 6.65 ***
99.39 ± 0.37 ***
62.84 ± 3.50 ***
68.80 ± 5.92 ***
72.85 ± 6.35 ***
109.25 ± 6.01 ***
77.95 ± 0.22 ***
37.92 ± 5.39 **
85.27 ± 4.20 ***
96.09 ± 1.92 ***
106.98 ± 6.35 ***
112.19 ± 6.16 ***
84.22 ± 4.09 ***
45.18 ± 6.47 ***
71.88 ± 7.07 ***
113.51 ± 1.07 ***
37.95 ± 6.88 **
54.52 ± 0.99 ***
92.50 ± 3.58 ***
114.22 ± 3.13 ***
68.39 ± 5.74 ***
77.71 ± 4.99 ***
76.08 ± 5.45 ***
115.21 ± 4.14 ***
91.41 ± 1.84 ***
8.59 ± 3.19
3.55
Luzula campestris (L.)
DC.
7.42
54.84
10.54
6.67
L. forsteri (Sm.) DC.
46.67
22.67
23.44
4.69
L. luzuloides (Lam.)
Dandy & Wilmott
49.89
11.18
17.86
7.14
L. sudetica (Willd.)
Schult.
17.86
47.62
8.57
89.10 ± 6.41 ***
111.90 ± 2.99 ***
L. sylvatica (Huds.)
Gaudin (aerial part)
5.71
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C
D
A
B
C
D
18.10
43.81
7.96
73.32 ± 2.91 ***
26.90 ± 7.01 **
89.18 ± 6.58 ***
95.28 ± 2.48 ***
53.62 ± 7.13 ***
41.60 ± 6.19 ***
54.82 ± 4.11 ***
48.83 ± 6.51 **
107.88 ± 3.96 ***
104.23 ± 5.67 ***
54.13 ± 3.03 ***
43.02 ± 2.96 ***
4.69
L. sylvatica (root part)
46.12
12.65
A = n-hexane fraction, B = CH2Cl2 fraction, C = EtOAc fraction, D = remaining H2O fraction
Results are presented as mean ± S.E.M. (n=3~5). ** P < 0.01, *** P < 0.001 compared with the
control value (DMSO).
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Table 2. NMR spectroscopic data for luzulin A (1) (500 MHz, CD3OD)
1
position
δH (J in Hz)
δC, type
HMBC (H→C)
1
78.1, C
1a
2
146.6, C
207.1, C
123.1, CH
141.9, CH
122.6, C
103.3, CH
132.2, C
157.0, C
126.3, C
138.3, C
127.7, C
129.8, CH
121.0, CH
32.1, CH3
13.4, CH3
3
6.25, d (10.2)
1, 4a
1a, 2
4
8.35, d (10.1)
4a
5
7.52, s
4a, 6, 8a
5a
6
7
8
8a
9
8.12, d (8.5)
7.63, d (8.7)
1.53, s
1a, 5a
4a, 8a
1, 1a, 2
6, 7, 8
10
11
12
13
14a
14b
2.35, s
7.05, dd (17.9, 11.5) 135.7, CH
5.81, d (11.5)
5.35, d (18.0)
122.2, CH2
8
8
29