molecules

Article
Isolation and Antimicrobial Activity of Coumarin
Derivatives from Fruits of Peucedanum
luxurians Tamamsch
Jarosław Widelski 1 , Simon Vlad Luca 1,2 , Adrianna Skiba 1 , Ioanna Chinou 3 ,
Laurence Marcourt 4 , Jean-Luc Wolfender 4 and Krystyna Skalicka-Wozniak 1, * ID

1 Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Chodzki,
120-093 Lublin, Poland; [email protected] (J.W.); [email protected] (S.V.L.);
[email protected] (A.S.)
2 Department of Pharmacognosy, “Grigore T. Popa” University of Medicine and Pharmacy, 16 Universitatii
Street, 700115 Iasi, Romania
3 Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy,
University of Athens, Zografou, 15771 Athens, Greece; [email protected]
4 School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, 1,
Rue Michel Servet, 1211 Geneva 4, Switzerland; [email protected] (L.M.);
[email protected] (J.-L.W.)
* Correspondence: [email protected]; Tel.: +48-8-1448-7086; Fax: +48-8-1448-7080




Received: 30 April 2018; Accepted: 16 May 2018; Published: 20 May 2018


Abstract: As a continuation of searching for phytoconstituents that act as promising agents for
antimicrobial therapy, rare coumarins were isolated from fruits of Peucedanum luxurians and tested.
In a first step, the content of major compounds in the aerial parts and fruits of P. luxurians were
compared. The results clearly showed that the fruits with dichloromethane as a solvent yielded, in
most cases, higher concentrations of almost all the analyzed coumarins than the aerial parts, with
peucedanin detected as the most abundant compound with a concentration of 4563.94 ± 3.35 mg/
100 g. Under this perspective, the dichloromethane extract from the fruits of P. luxurians was
further submitted to high performance countercurrent chromatography with a mixture of n-hexane,
ethyl acetate, methanol, and water 6:5:6:5 (v/v). Combination of HPCCC and prep-HPLC yielded
60 ,70 -dihydroxybergamottin (1), officinalin (2), stenocarpin isobutyrate (3), officinalin isobutyrate
(4), 8-methoxypeucedanin (5), and peucedanin (6). Isolated compounds were tested against several
Gram-positive and Gram-negative bacteria strains. 60 ,70 -Dihydroxybergamottin, peucedanin, and
officinalin isobutyrate appeared to be the most active against all tested bacteria strains with minimum
inhibitory concentration (MIC) values between 1.20 and 4.80 mg/mL. To the best of our knowledge,
this is the first report about countercurrent isolation of mentioned coumarins, as well as the first
information about their antimicrobial activity.

Keywords: Peucedanum luxurians Tamamsch.; Apiaceae; accelerated solvent extraction; purification;

natural products; countercurrent chromatography

1. Introduction
As stated in the WHO report, in the last few decades, the incidence of microbial infections has
increased dramatically together with emergence of antimicrobial resistant strains [1]. Discovering
some alternatives that can potentially be effective in the treatment of these infectious diseases is a
major challenging issue worldwide and phytoconstituents are considered as promising agents for
antimicrobial therapy [1].

Molecules 2018, 23, 1222; doi:10.3390/molecules23051222 www.mdpi.com/journal/molecules

Molecules 2018, 23, 1222 2 of 12

Plants have been well-known sources of inspiration for developing novel drug compounds since
antiquity [1]. Coumarins, naturally plant-derived compounds with benzopyrone moiety, possess
a wide variety of biological activities. Series of coumarin analogues are being extensively studied
due to their broad spectrum, low toxicity, and lower drug resistance properties [2]. Novobiocin, an
aminocoumarin antibiotic produced by the actinomycete Streptomyces niveus, was launched onto the
market in the mid-1950s as an effective antistaphylococcal agent used in the treatment of MRSA [3].
Different naturally-isolated coumarins, as well as their chemically modified analogs, are active against
numerous bacterial strains, including those which developed multidrug resistance [2].
The genus Peucedanum, comprising between 100 and 120 species, and widely distributed across
the world, is an important group of the Apiaceae family. Plants belonging to Peucedanum have been
widely used to treat various diseases, including sore throat, coughs, colds, headaches [4,5], epilepsy,
rheumatism, and cardiovascular and gastrointestinal problems [6,7]. The first information about
the pharmacological application of Peucedanum plants can be found in “The Canon of Medicine”—an
encyclopedia of medicine compiled by Persian philosopher Avicenna in 1025 [7]. In Central European
medicine, the fruits of P. alsaticum and P. cervaria were found to be applied as expectorant, diuretic,
diaphoretic, sedative, stomachic, and antimicrobial agents [8]. In Austrian traditional medicine,
rhizomes of P. ostruthium have a very long history of use as a remedy for anti-inflammatory ailments [9].
Plants of the Peucedanum genus have been used for centuries as antibacterial agents and for some
of them, the activity was confirmed by biological and pharmacological studies, showing moderate
or high activity against different human pathogens. Such activities have also been reported for
P. paniculatum (leaves and roots), P. alsaticum, P. cervaria, P. graveolens, P. ruthenicum, P. zenkeri
fruits, or P. ostruthium roots [7]. Dichloromethane extracts of P. ostruthium roots have demonstrated
anti-mycobacterial activity against Mycobacterium fortuinum, responsible for respiratory system
infections. Two isolated furanocoumarins, ostruthin and isoimperatorin, were found to be the most
potent, with low minimum inhibitory concentration (MIC) values similar to those of ethambutol and
isoniazid [10]. Oxypeucedanin hydrate, isolated also from rhizomes of P. ostruthium, showed prominent
activity against Bacillus cereus [11]. Imperatorin, bergapten, and isopimpinellin were responsible for
the antimicrobial activity of methanolic extract of P. zenkeri seeds [12].
Due to the versatile and numerous therapeutic traditional uses, a number of phytochemical
analyses were performed on Peucedanum representatives. Comparative phytochemical data are
available for 50 species belonging to genus Peucedanum [7]. Among all the chemical constituents
identified in the plants from Peucedanum genus, the highest impact on pharmacological activity is
assigned to coumarins and essential oils (usually containing coumarin compounds) [7,13]. Coumarins
with different molecular scaffolds have been isolated and identified in different Peucedaum such as:
Simple coumarins and their glycosides; linear and angular furano- and dihydrofuranocoumarins and
their glycosides; and linear and angular dihydropyranocoumarins and their glycosides [7].
Peucedanum luxurians Tamamsch. was found as an endemic plant from Armenia, growing in the
area around Mount Ararat. Preliminary studies showed the presence of rare furanocoumarins with
unique structures in the herb and fruits of this species [14]. For some of them, the immunomodulating
activity was evaluated [15]. The aim of our study is to elaborate the efficient and fast procedure for
their isolation by the use of high-performance countercurrent chromatography, as well as to evaluate
their antimicrobial activity.

2. Results and Discussion

2.1. Preliminary HPLC Analysis and Screening of Activity of Extracts

Previously, few coumarins have been isolated from aerial parts of the P. luxurians, such as
stenocarpin; stenocarpin isobutyrate; officinalin; officinalin isobutyrate; and 8-methoxypeucedanin
together with xanthotoxin, isoimperatorin, bergaptene, peucedanin, and cnidilin [14] where few steps
of column chromatography were applied. Because the fruits of Apiaceae plants are usually the best
Molecules 2018,23,
Molecules2018, 23,1222
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parts and fruits of P. luxurians were compared. Additionally, the influence of different solvents on
source of these compounds, in the first step of the experiment, the major compounds in the aerial parts
extraction efficiency was investigated. The procedure for the extraction (accelerated solvent
and fruits of P. luxurians were compared. Additionally, the influence of different solvents on extraction
extraction) and all the other parameters (temperature, time, amount of plant material) were the same
efficiency was investigated. The procedure for the extraction (accelerated solvent extraction) and all
for each sample.
the other parameters (temperature, time, amount of plant material) were the same for each sample.
Obtained extracts prepared for quantitative analysis were injected to HPLC with diode-array
Obtained extracts prepared for quantitative analysis were injected to HPLC with diode-array
detection (DAD) and in the first step, the area under each peak was compared. Additionally, tentative
detection (DAD) and in the first step, the area under each peak was compared. Additionally,
qualitative analysis was performed using an LC–MS system. Six coumarin derivatives were identified
tentative qualitative analysis was performed using an LC–MS system. Six coumarin derivatives
and their presence was confirmed in each analyzed extract. The results clearly showed that the fruits
were identified and their presence was confirmed in each analyzed extract. The results clearly showed
yielded higher concentrations of almost all analyzed coumarins compared to the aerial parts and
that the fruits yielded higher concentrations of almost all analyzed coumarins compared to the
dichloromethane produced the highest yield for almost all coumarins. At the same time,
aerial parts and dichloromethane produced the highest yield for almost all coumarins. At the same
dichloromethane and methanolic extracts from both the fruits and aerial parts were subjected to
time, dichloromethane and methanolic extracts from both the fruits and aerial parts were subjected
preliminary screening for their antibacterial activity, which showed that those from fruits were more
to preliminary screening for their antibacterial activity, which showed that those from fruits were
active against most of the test pathogens. Under this perspective, the dichloromethane extract of P.
more active against most of the test pathogens. Under this perspective, the dichloromethane extract
luxurians fruits was further submitted to high-performance counter-current chromatography
of P. luxurians fruits was further submitted to high-performance counter-current chromatography
(HPCCC) separation to isolate pure compounds for evaluation of their biological activity, as well as
(HPCCC) separation to isolate pure compounds for evaluation of their biological activity, as well as to
to complete the quantitative analyses (HPLC-DAD chromatogram of the crude extract is presented
complete the quantitative analyses (HPLC-DAD chromatogram of the crude extract is presented in
in Figure 1).
Figure 1).

Figure 1. HPLC-DAD chromatogram of crude dichloromethane extract from fruits of P. luxurians.

Figure 1. HPLC-DAD chromatogram of crude dichloromethane extract from fruits of P. luxurians.
2.2. HPCCC Separation
2.2. HPCCC Separation
In order to achieve an efficient separation, different two-phase solvent systems were tested.
In order
Mixtures to achieve
of n-hexane, ethylan efficient
acetate, separation,
methanol, different
and water two-phase
(HEMWat) were solvent systems
proven to be verywere tested.
efficacious
Mixtures of n-hexane, ethyl acetate, methanol, and water (HEMWat) were proven to be very
Molecules 2018, 23, 1222 4 of 12

in the separation of coumarins, including coumarins from other species of Peucedanum genus [16].
The K value, which is a partition coefficient for the target compounds, were calculated by dividing the
concentration of the analyte in the upper stationary phase by that in the lower mobile phase (reversed
mode) [16]. Among the three HEMWat solvent mixtures tested, only the one with a ratio of 6:5:6:5
(v/v/v/v) gave the most appropriate K values of the analyzed compounds (from 0.19 to 3.61; Table 1).
Furthermore, the separation factor (α = K2 /K1 , K2 > K1 ) was found to be optimal (α > 1.20) for all target
compounds, except compounds 3 and 4 (see Table 1), suggesting these two compounds may co-elute
under HPCCC experimental conditions. In this regard, two other solvent systems, one consisting
of petroleum ether, ethyl acetate, methanol, and water (1:1:1:1 v/v/v/v) and the other of n-hexane,
acetone, and water (3.9:5:1.9 v/v/v) were investigated. Despite the fact that the K values of compounds
3 and 4 were adequate, the separation factor α still remained unsolved in both situations. Nevertheless,
the K values of some of the compounds of interest were either lower than 0.2 (which would give a
low peak resolution with elution near the solvent front), or higher than 5 (which would lead to an
excessive broadening of peak with a longer elution time). Thus, HEMWat 6:5:6:5 (v/v/v/v) was further
selected as HPCCC separation solvent system. With this solvent system, at 1600 rpm and a flow rate of
6 mL/min in the semi-preparative separation mode, the retention of the stationary phase was stable
and was equal to 78%.

Table 1. Partition coefficients (K values) of coumarins from P. luxurians.

Coumarins
Solvent System
1 2 3 4 5 6
HEMWat (5:6:5:6) 0.83 3.01 5.14 5.16 9.49 11.57
HEMWat (1:1:1:1) 0.47 1.59 3.09 3.25 5.31 6.34
PEMWat (1:1:1:1) 0.50 1.95 2.47 2.51 4.38 4.90
HAtWat (3.9:5:1.9) 0.16 1.22 1.15 1.06 0.55 0.33
HEMWat (6:5:6:5) 0.19 0.89 1.57 1.65 2.85 3.61
α2/1 = 4.68 α3/2 = 1.76 α4/3 = 1.05 α5/4 = 1.73 α6/5 = 1.27
HEMWat denotes hexane-ethyl acetate-methanol-water, PEMWat denotes petroleum ether-ethyl acetate-methanol-
water, HAtWat denotes hexane-acetone-water, and α denotes the separation factor.

Following a single HPCCC injection of the dichloromethane extract of P. luxurians fruits (300 mg),
five fractions were collected: FR I (min 15–16), FR II (min 25–26), FR III (min 28–30), FR IV (min 44–46)
and FR V (min 54–59) (Figure S1). All five fractions were collected within 60 min, evaporated, and
dissolved in methanol prior to HPLC-DAD analysis. FR I, II, and IV contained compounds 1, 2, and 5,
respectively, with purities higher than 80% but lower than 95%. A mixture of compounds 3 and 4 was
found in FR III, whereas FR V gave compound 6 with 99.7% purity. In order to obtain higher amounts
of the target compounds, separation was repeated two times more (each time with 300 mg of extract)
and all similar fractions were pooled together with the previously collected ones, to yield 10 mg of FR
I, 5 mg of FR II, 15 mg of FR III, 5 mg of FR IV, and 68 mg of FR V (compound 6, 99.7% purity).
Fractions I-IV were subjected to semi-preparative HPLC separation, separately. Five compounds
were obtained with purity above 97% (as measured by HPLC-DAD): Compound 1 (6 mg, 98.4%
purity), compound 2 (2 mg, 97.1% purity), compound 3 (6 mg, 99.7% purity), compound 4 (7 mg, 97.8%
purity), and compound 5 (1.5 mg, 99.7% purity). The structures of compounds were elucidated by
complementary spectroscopic methods, such as: HPLC-DAD connected to electrospray-ionisation
quadrupole time-of-flight mass spectrometer (HPLC-DAD-ESI-Q-TOF-MS) and 1D- and 2D-NMR
(Supplementary Material). The compounds were determined as 60 ,70 -dihydroxybergamottin (1),
officinalin (2), stenocarpin isobutyrate (3), officinalin isobutyrate (4), 8-methoxypeucedanin (5), and
peucedanin (6). These data were in agreement with those available in the literature [14,17,18]. Their
chemical structures are presented in Figure 2.
Griseb [22]. Moreover peucedanin (6) was purified from the petroleum ether-diethyl ether extract of
P. officinale L. fruits [20] and the diethyl ether, methyl-tert-butyl ether, and ethyl acetate extracts of P.
morisonii Bess. aerial parts [23]. In addition, stenocarpin isobutyrate (3) was isolated from the
methanol and ethyl acetate extracts of P. morisonii Bess., aerial parts [23]. Although these coumarins
were identified in Peucedanum genus (except 6′,7′-dihydroxybergamottin), their isolation through
Molecules 2018, 23, 1222 5 of 12
HPCCC technique was not previously reported.

Figure 2. Chemical
Chemical structures
structures of
of coumarins
coumarins isolated
isolated from
from P. 60 ,70 -dihydroxybergamottin (1),
P. luxurians: 6′,7′-dihydroxybergamottin (1),
officinalin (2), stenocarpin isobutyrate (3), officinalin isobutyrate (4), 8-methoxypeucedanin (5), and
peucedanin (6).

2.3. Quantitative
Except for 6HPLC-DAD Analysis
0 ,70 -dihydroxybergamottin (1), which is a well-known cytochrome P450 3A4 inhibitor
fromThegrapefruit
purifiedjuice that waswere
compounds not reported in Peucedanum
further used to completegenus [17], allanalysis,
quantitative the otherascoumarins were
well to evaluate
identified in the aerial parts of P. luxurians
their potential antibacterial activity. Parameters of the calibration curves and both the limitand
[14,15]. Officinalin (2), officinalin isobutyrate (4), of
peucedanin (6) were also isolated from the dichloromethane fraction of the essential oil of P. tauricum
Bieb. fruits [18], while 8-methoxypeucedanin (5) was previously reported in the chloroform extract of
P. ruthenicum M.B roots [19] and the hexane-diethyl ether-methanol extract of P. longifolium Waldst. and
Kit. roots [20]. Peucedanin (6) and officinalin isobutyrate (4) were isolated also from the etheric extract
of the roots of Opopanax chironium (L.) Koch. (Apiaceae) [21], while peucedanin (6) and officinalin (2)
were isolated from the chloroform extract of the aerial parts of Opopanax hispidus (Friv.) Griseb [22].
Moreover peucedanin (6) was purified from the petroleum ether-diethyl ether extract of P. officinale L.
fruits [20] and the diethyl ether, methyl-tert-butyl ether, and ethyl acetate extracts of P. morisonii Bess.
aerial parts [23]. In addition, stenocarpin isobutyrate (3) was isolated from the methanol and ethyl
acetate extracts of P. morisonii Bess., aerial parts [23]. Although these coumarins were identified in
Peucedanum genus (except 60 ,70 -dihydroxybergamottin), their isolation through HPCCC technique was
not previously reported.
Molecules 2018, 23, 1222 6 of 12

2.3. Quantitative HPLC-DAD Analysis

The purified compounds were further used to complete quantitative analysis, as well to evaluate
their potential antibacterial activity. Parameters of the calibration curves and both the limit of detection
(LOD) and limit of quantification (LOQ) values are presented in Table S1 (Supplementary Materials).
Relative standard deviation (RSD%), which is a measure of precision, was lower than 6.05% in fruit
extracts and 5.97% in aerial part extracts (Table 2). Selection of the appropriate extraction methods is
an important issue in phytochemistry. An appropriately selected method should ensure the maximum
recovery of the requisite active substances in the shortest possible time and at the lowest financial cost.
As pressurized liquid extraction (accelerated solvent extraction; ASE) was previously shown to give
the highest yield of simple coumarins and furanocoumarins from plants from Apiaceae family [24,25],
this technique was used to elaborate the optimal conditions for extraction of target compounds. Due to
the better penetration of the sample by the solvent, ASE ensures complete extraction in short time with
a small volume of organic solvent [25]. A temperature of 100 ◦ C and 10 min static time were selected
for comparison of extraction efficiency in currently presented experiments. The results clearly showed
that fruits yielded higher concentrations of all analyzed coumarins compared to those observed in the
aerial parts of P. luxurians. With respect to the extraction solvent, dichloromethane gave, in most cases,
a more efficient extraction of the compounds of interest from the fruits as compared to methanol and
petroleum ether (Table 2). Peucedanin (6) was detected as the most abundant compound in the crude
dichloromethane extract with a concentration of 4563.94 ± 3.35 mg/100 g.

Table 2. Quantitative analysis results of coumarins detected in different extracts of P. luxurians.

Coumarins Plant Material Solvent Yield (mg/100 g d.w.) RSD%

1 Aerial parts PE 13.72 ± 0.41 2.99
DCM 24.71 ± 0.66 2.67
MeOH 30.61 ± 1.83 5.97
Fruits PE 83.17 ± 5.04 6.05
DCM 442.23 ± 2.45 0.55
MeOH 465.74 ± 1.66 0.36
2 Aerial parts PE 29.60 ± 0.84 2.84
DCM 51.70 ± 2.71 5.24
MeOH 53.51 ± 0.39 0.73
Fruits PE 229.12 ± 0.62 0.27
DCM 1336.26 ± 0.80 0.06
MeOH 1309.43 ± 1.31 0.10
3 Aerial parts PE 23.61 ± 0.02 0.09
DCM 33.49 ± 0.19 0.58
MeOH 47.84 ± 0.95 1.99
Fruits PE 56.86 ± 0.03 0.05
DCM 101.72 ± 0.10 0.10
MeOH 93.88 ± 0.59 0.63
4 Aerial parts PE 8.39 ± 0.12 0.13
DCM 8.56 ± 0.16 0.12
MeOH 8.47 ± 0.41 1.06
Fruits PE 89.93 ± 0.12 0.13
DCM 191.78 ± 0.02 0.01
MeOH 170.06 ± 0.09 0.05
5 Aerial parts PE 62.55 ± 0.03 0.05
DCM 79.68 ± 0.29 0.37
MeOH 66.21 ± 0.07 0.11
Fruits PE 341.91 ± 0.17 0.05
DCM 1652.15 ± 0.87 0.05
MeOH 1622.91 ± 0.56 0.03
6 Aerial parts PE 28.94 ± 0.11 0.38
DCM 22.60 ± 0.11 0.49
MeOH 21.49 ± 0.25 1.16
Fruits PE 3689.91 ± 1.07 0.03
DCM 4563.94 ± 3.35 0.07
MeOH 4538.09 ± 1.13 0.02
PE—petroleum ether; DSM—dichloromethane; MeOH—methanol.
Molecules 2018, 23, 1222 7 of 12

2.4. Antimicrobial Activity

The isolated compounds were subjected to further antimicrobial evaluation and results
were compared to reference drug, netilmicin. All coumarins revealed broad diversity regarding
growth-inhibitory activity (Table 3) with 60 ,70 -dihydroxybergamottin (1) as the most active against
all tested bacteria strains (inhibition zone between 16 to 17 mm and MIC values between 1.20 and
2.10 mg/mL). Similar activity was noticed for peucedanin (6) and officinalin isobutyrate (4) (inhibition
zone between 16 to 13 mm and MIC values between 1.40 and 4.80 mg/mL), while the rest of the tested
coumarins showed moderate activity (inhibition zone between 12 to 14 mm and MIC values between
3.90 and 5.75 mg/mL).

Table 3. Activity of P. luxurians extracts and isolated coumarins against tested bacteria (zone of
inhibition (mm)/MIC values (mg/mL)).

Tested Extract/Compound S. aureus S. epidermidis P. aeruginosa E. cloacae K. pneumoniae E. coli

P. luxurians aerial parts DCM 15/1.90 16/1.88 13/2.40 12/3.50 12/3.10 12/3.35
P. luxurians aerial parts MeOH 17/0.90 17/0.92 13/2.80 12/3.50 12/2.75 12/2.50
P. luxurians fruits DCM 17/0.84 17/0.90 14/3.00 13/3.45 13/2.77 13/2.60
P. luxurians fruits MeOH 18/0.95 18/0.85 14/2.84 14/2.75 14/2.50 14/2.25
(1) 60 ,70 -Dihydroxybergamottin 17/1.20 17/1.35 17/1.37 16/1.75 16/2.10 17/1.45
(2) Officinalin 13/4.50 12/5.50 12/5.00 12/5.75 13/4.80 13/4.90
(3) Stenocarpin isobutyrate 12/5.25 14/4.00 13/5.00 13/4.80 14/3.90 14/4.50
(4) Officinalin isobutyrate 14/3.50 15/2.70 14/3.50 15/2.75 15/2.25 13/4.80
(5) 8-metoxypeucedanin 12/5.25 14/4.00 13/5.00 13/4.80 14/3.90 14/4.50
(6) Peucedanin 16/1.50 16/1.75 17/1.40 16/2.10 16/2.50 16/2.75
Netilmicin 21/0.004 25/0.004 20/0.088 23/0.008 22/0.008 24/0.010

Officinalin isobutyrate (4) was more active than officinalin (2) itself (MIC values 2.25–4.80 and
4.50–5.75 mg/mL, respectively), except for the inhibition E. coli growth, where both compounds had
almost same activity. According to previously published data, the stronger activity was a result of the
aliphatic chain substituted in C-7 position, e.g., an isoprenyl group to the furanocoumarin skeleton
resulted in an increase in lipophilicity of the molecule, what helped to penetrate through the thick
membrane of these bacteria [10,26]. This aliphatic chain substituted at C-5 position shared responsibility
for activity of 60 ,70 -dihydroxybergamottin (1) which was the most active in our experiments. When a
methoxy group was attached to C-8 position in stenocarpin isobutyrate (3), the compound exhibited
a weak antibacterial activity, as compared to its structural analogue, officinalin isobutyrate (4).
Similarly, the presence of methoxy group in the ring of 8-methoxypeucedanin (5) led to a decrease
in activity when compared to peucedanin (6). However previously published reports suggest that
additional methoxygroup in the C-8 position for another furanocoumarin-phellopterin may enhance
the antimicrobial activity [26]; thus, further experiments are required. To the best of our knowledge,
this is the first report concerning the antimicrobial activity of isolated compounds.

3. Materials and Methods

3.1. Chemicals
Analytical-grade petroleum ether, n-hexane, dichloromethane, ethyl acetate, acetone, and
methanol were purchased from POCh (Gliwice, Poland). Methanol, acetonitrile, ammonium formate,
and formic acid used for HPLC and LC-MS analyses were of chromatographic grade and obtained
from J.T. Baker (Deventer, The Netherlands). Deuterated chloroform (CDCl3 , 99.8 atom %D) was
acquired from Armar Chemicals (Döttingen, Switzerland). A Simplicity® water purification system
(Millipore, France) was used to purify the water.

3.2. Plant Material

The plant material (mature fruits and herb of Peucedanum luxurians Tamamsch.) used in the
experiments was obtained in 2016 from the Botanical Garden of Adam Mickiewicz University in
Molecules 2018, 23, 1222 8 of 12

Poznań, Poland (plant material identified by Grażyna Naser). The voucher specimen (7973_S003) was
kept in the Department of Pharmacognosy, Medical University of Lublin, Poland. The air-dried fruits
and herbs were dried, grounded and pulverized.

3.3. Accelerated Solvent Extraction

A Dionex ASE 100 instrument (Dionex, Sunnyvale, CA, USA) was used for solvent extraction.
Exactly 1 g of dried plant material was placed into a 10-mL stainless-steel extraction cell. In order
to optimize the extraction parameters, the aerial parts and fruits were extracted with three different
solvents (petroleum ether, dichloromethane and methanol) at 100 ◦ C for a static time of extraction of
10 min in one cycle. The extracts that resulted were evaporated to dryness, dissolved in methanol in
25-mL calibration flasks, and subjected to quantitative HPLC-DAD analysis.

3.4. HPCCC Separation

3.4.1. HPCCC Apparatus

A spectrum high-performance counter-current chromatograph (HPCCC) instrument (Dynamic
Extractions Ltd., Slough, Berkshire, UK) equipped with two multilayer polytetrafluorethylene coils, one
for analytical (22 mL) and the other for semi-preparative purpose (136 mL), was used. The temperature
was maintained at 30 ◦ C and maximum revolution speed (1600 rpm) was set up to obtain the maximum
centrifugal force (240× g). The HPCCC system was connected to an Alpha 10 pump and a Sapphire
UV detector (ECOM, Prague, Czech Republic).

3.4.2. Selection of Two-Phase Solvent System

In order to calculate the partition coefficients (K) of the target compounds, approx. 1 mg of crude
extract was mixed in the test tubes with 2 mL of each phase of selected solvent system. After shaking, it
was left to stand at room temperature until complete separation of the layers. Equal volumes (0.5 mL)
of the upper and lower phases were taken, evaporated to dryness, redissolved in methanol (1 mL),
and analyzed by HPLC-DAD.

3.4.3. Separation Procedure

The HEMWat mixture selected for HPCCC separation was equilibrated at room temperature
in a separatory funnel and the two phases were separated before analysis and degassed for 10 min.
The multilayer semi-preparative coil was firstly filled with the stationary phase (upper). The rotation
of the column was set to 1600 rpm, then the mobile phase (lower) was pumped into the column at
a flow rate of 6 mL/min. After the hydrodynamic equilibrium was attained (as indicated by the
absence of the stationary phase flowing from the column), the sample solution, containing 300 mg of
the crude dichloromethane extract of P. luxurians fruits dissolved in 3 mL of each phase of the selected
solvent system, was injected. The effluent from the column was monitored at 254 nm and the collected
fractions were evaporated and dissolved in methanol (1 mL) before HPLC-DAD analysis. The HPCCC
separation was repeated two more times and the fractions containing target compounds were pooled
together and evaporated to dryness.

3.5. Semi-Preparative HPLC Separation

Four fractions obtained after HPCCC separation (FR I–IV) were dissolved in methanol (10 mg/mL)
and further purified by semi-preparative HPLC, which was carried out on a Hitachi LaChrom 7000
HPLC system (Hitachi Ltd., Tokyo, Japan) equipped with a D-7000 interface, an L-7150 pump, an
L-7420 DAD detector, and an Advantec SF-3120 fraction collector. Separation was conducted on a
Cosmosil C18-AR-II (250 mm × 10 mm, 5 µm) by injecting 50 µL of sample; the mobile phase consisted
of 60% methanol for fraction FR I, 50% methanol for fractions FR II and FR III and 65% methanol for
fraction FR IV; the flow rate was 4 mL/min and the effluent was monitored at 254 nm.
Molecules 2018, 23, 1222 9 of 12

3.6. HPLC-DAD Analysis

Analytical HPLC-DAD analysis was performed using Shimadzu HPLC equipment (Shimadzu,
Tokyo, Japan) coupled with an automatic degasser (DGU-20A 3R), a quaternary pump (LC-20AD), an
auto-sampler (SIL-20A HT), a DAD detector (SPD-M20A), and an Agilent Zorbax Eclipse XDB-C18
(250 mm × 4.6 mm, 5 µm) column. Water (A) and methanol (B) with the following gradient was used:
0 min, 50% B; 5 min, 60% B; 25 min, 80% B; and 30–35 min 100% B. The flow rate was 1 mL/min; the
column temperature was 25◦ C, the injection volume was 10 µL and the detection wavelength was set
at 254 nm.
All purified compounds, identified as 60 ,70 -dihydroxybergamottin (1), officinalin (2), stenocarpin
isobutyrate (3), officinalin isobutyrate (4), 8-methoxypeucedanin (5), and peucedanin (6), were
dissolved in methanol (0.1 mg/mL) and the stock solutions were diluted in a series of appropriate
concentrations (20–100 µg/mL) for the construction of calibration curves. The results of the quantitative
analysis were expressed as mean ± standard deviation mg compound/100 g of dry weight plant
material. The LOD and LOQ values were calculated from the standard deviation of y-intercepts
(σ) and the slopes (S), according to the following formulas: LOD = 3.3 × σ/S and LOQ = 10 × σ/S.
All experiments were repeated three times.

3.7. HPLC-DAD-ESI-Q-TOF-MS Analysis

HPLC-DAD-ESI-Q-TOF-MS experiments were performed on an Agilent 1200 HPLC (Agilent
Technologies, Santa Clara, CA, USA) coupled to an auto-sampler (G1329B), a binary pump (G1312C),
a column oven (G1316A), and a DAD detector (G1315D). The Agilent G6530B ESI-Q-TOF-MS was
equipped with a nitrogen generator, a compressed air generator, and a compressed air cylinder.
Separation was carried out on a Phenomenex Gemini C18 (100 × 2 mm, 3 µm) column with 60%
acetonitrile, 10 mM ammonium formate, and 0.1% formic acid in water (solvent A) and 95% acetonitrile,
10 mM ammonium formate, and 0.1% formic acid in water (solvent B); under the following conditions:
0−10 min, 40% B; 30 min, 80% B; and 35 min, 90% B. The total analysis time was 35 min, post-time
was 12 min, injection volume was 10 µL, flow was 0.2 mL/min, and DAD spectra were recorded
from 200 to 400 nm. ESI-Q-TOF-MS analysis was performed in positive mode, as follows: mass range
100–1000 m/z, gas temperature 350 ◦ C, nitrogen flow 10 L/min, nebulizer pressure 40 psig, skimmer
65 V, capillary voltage 4000 V, fragmentor 120 V, and collision energies 10 and 40 eV.

3.8. NMR Analysis

A Bruker Avance III HD 600 MHz NMR spectrometer equipped with a QCI 5 mm Cryoprobe and
a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany) was employed for
1D-NMR (1 H and 13 C-NMR in CDCl3 ) and 2D-NMR (correlation spectroscopy, COSY; heteronuclear
multiple-bond correlation, HMBC; heteronuclear single-quantum correlation, and HSQC) spectroscopy.
Chemical shifts are reported in parts per million (δ) using the residual CDCl3 signal (δH 7.26; δC 77.2)
as internal standards for 1 H and 13 C NMR, respectively; coupling constants (J) are given in Hz.

3.9. Antimicrobial Activity

The antimicrobial activities of extracts and isolated coumarins were determined using the
diffusion and dilution techniques and by measuring their MICs against two Gram-positive bacteria,
Staphyloccocus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228), as well as four
Gram-negative Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Enterobacter
cloacae (ATCC 13047), and Klebsiella pneumoniae (ATCC 13883), according to a previously published
method [27].
The extracts and test compounds were dissolved before the assay in methanol (5 mg/mL). For each
experiment, control disks with pure solvent were used as a blind control. Petri dishes had been
previously inoculated with the tested microorganisms to give a final cell concentration of 107 cells/mL.
Molecules 2018, 23, 1222 10 of 12

Ten µL volumes of the above solutions were required to wet the test paper discs. The incubation
conditions used in experiments were 24 h at a temperature of 37 ◦ C. Standard antibiotic netilmicin
(4–88 µg/mL) was used as reference drug. For each experiment, any pure solvent used was also applied
as a blind control. The results were reported as the diameter of the zone of inhibition around each disk
(in mm) and MIC values were calculated. The MIC values were determined using the dilution method
in 96-well plates. The experiments were repeated three times and the results were expressed as average
values. Netilmicin was used as reference compound.

4. Conclusions
An efficient strategy based on elaboration of optimal extraction parameters as well as one
step HPCCC for the rapid separation, and purification of rare coumarins from the nonpolar
extract of P. luxurians, was presented. This was the first report concerning the efficient isolation
of coumarin derivatives such as: 60 ,70 -dihydroxybergamottin, officinalin, stenocarpin isobutyrate,
officinalin isobutyrate, 8-methoxypeucedanin, and peucedanin with HPCCC. Additionally, 60 ,70 -
dihydroxybergamottin was reported for the first time in Peucedanum genus. The isolated compounds
were evaluated for their antibacterial activity for the first time and possible structure–activity relation
was discussed.

Supplementary Materials: The following are available online. Figure S1: HPCCC chromatogram of the
dichloromethane extract of Peucedanum luxurians fruit, Figure S2: HPLC-DAD chromatograms and UV spectra
of isolated compounds, Table S1: Parameters of calibration curves of quantitative HPLC-DAD analysis, and
Spectroscopic data of compounds 1–6.
Author Contributions: J.W., K.S.-W. designed experiments and prepared manuscript; S.V.L., A.S. performed
the isolation and optimization of extraction; J.W., K.S.-W. and S.V.L. performed LC-MS analysis; I.C. designed,
performed and analyzed the biological experiments; and L.M. and J.-L.W. performed structural elucidation of
pure compounds, analyzed and described the results. All the authors approved the final manuscript and declare
no conflicts of interests.
Funding: This research was financed from grant No 4/POLTUR-1/2016.
Acknowledgments: Authors would like to acknowledge Botanical Garden of Adam Mickiewicz University in
Poznań, Poland for providing plant material. The School of Pharmaceutical Sciences of the University of Geneva
(J-L. Wolfender) is thankful to the Swiss National Science Foundation for the support in the acquisition of the
NMR 600 MHz (SNF R’Equip grant 316030_164095).
Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds 60 ,70 -dihydroxybergamottin, officinalin, stenocarpin isobutyrate,
officinalin isobutyrate, 8-methoxypeucedanin, and peucedaninare available from the authors.

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