Column Chromatography and Preparative TLC for
Isolation and Purification of Coumarins from Peucedanum
verticillare L. Koch ex DC
Małgorzata Kozyra*, Kazimierz Głowniak, Andrzej Zabża, Grażyna Zgórka, Tomasz Mroczek, Tomasz Cierpicki,
Joanna Kulesza, and Iwona Mudło
Key Words:
Peucedanum verticillare L. Koch ex DC
Preparative TLC
Column chromatography
RP HPLC
Furanocoumarins
Pyranocoumarins
Pteryxin
Epoxypteryxin
Summary
Preparative separation and isolation of coumarins from petroleum
ether and methanol extracts of the fruits and roots of Peucedanum
verticillare L. Koch ex DC (Umbelliferae = Apiaceae) have been
achieved by column chromatography and normal- and reversed-
phase TLC with gradient elution. Isolated coumarins were identi-
fied by comparison with standards in analytical TLC and in RP
HPLC with gradient elution, by melting point (m.p.) measurement,
by UV spectroscopy, by MS, and by 1H and 13C NMR spectroscopy.
Esculin, umbeliferone, bergapten, xanthotoxin, isoimperatorin,
imperatorin, psoralen, and cis-kellactone were isolated for the first
time from P. verticillare L. Koch ex DC. Two angular-type pyra-
nocoumarins isolated from the roots and fruits of Peucedanum ver-
ticillare L. Koch ex DC. were identified as pteryxin and epoxy-
pteryxin by MS and 1H and 13C NMR.
1 Introduction
In the Department of Pharmacognosy and Medicinal Plant Lab-
oratory, Skubiszewski Medical University, several investiga-
tions have been conducted on the extraction, chromatographic
separation, and isolation of coumarins, furanocoumarins, and
pyranocoumarins from different Apiaceae species (Angelica,
Pastinaca, Heracleum, Libanotis, Peucedanum). The Apiaceae
family is a very well known source of biologically active com-
pounds such as coumarins, furanocoumarins, and pyra-
nocoumarins [1]. Coumarins (very common secondary metabo-
lites) play a protective role in plant defense, because of their
antibacterial, antiviral, and antifungal activity [2].
M. Kozyra, K. G³owniak, G. Zgórka, T. Mroczek, J. Kulesza, and I. Mud³o,
Department of Pharmacognosy with Medicinal Plant Laboratory, Skubiszewski
Medical University, 1 Chod¿ki St., 20-093 Lublin, Poland; and A. Zab¿a and
T. Cierpicki, Institute of Organic Chemistry, Biochemistry and Biotechnology,
Technical University, 27 Wyb. Wyspiañskiego St., 50-370 Wroc³aw, Poland.
224 VOL. 18. MAY/JUNE 2005
DOI: 10.1556/JPC.18.2005.3.11
Peucedanum verticillare L. Koch ex. DC family Umbelliferae
(Apiaceae) grows naturally in southeastern Europe [3] and is
cultivated in Poland. For a long time P. verticillare root has been
used as a diuretic and diaphoretic drug, and for relief of gas-
trointestinal disorders, e.g. intestinal colic, flatulence, etc. [4]. It
has recently been reported that a human leucocyte culture treat-
ed with aqueous or methanolic extracts of P. verticillare signif-
icantly increased production of viral-activated interferon.
Extracts from related P. officinale and P. ostrutium species did
not have such properties, however [5]. Previous studies on the
chemical composition of alcoholic extracts from P. verticillare
leaves have revealed the presence of flavonoids, coumarins, and
furanocoumarins [4]; bergapten, osthol, and oxypeucedanin
have been found in root extracts [5]. The variety of coumarin
compounds found in Peucedanum species and promising results
from analysis of P. verticillare extracts has prompted further
detailed studies of coumarins from P. verticillare species.
Separation of natural compounds from plants is a very impor-
tant problem in phytochemical analysis. Because of their very
similar chemical structures furanocoumarins are very difficult
to separate [2, 6, 7]. Owing to their biological activity, however,
there has been much interest in the compounds and they have
been widely investigated by TLC [6, 8–14 ] and HPLC [10, 15,
16]. Separation and isolation of coumarins from extracts of
fruits and roots were carried out in two steps – optimization of
TLC and HPLC systems and analysis of fractions isolated by
column chromatography by TLC, HPLC, melting point mea-
surement, and UV spectroscopy. Two angular-type pyra-
nocoumarins have been isolated from the roots and fruits and
identified as pteryxin and epoxypteryxin by MS and 1H and 13C
NMR.
In this work separation and isolation of the components of
Peucedanum verticillare L. Koch ex DC fruits and roots in
petroleum ether extracts and sediment have been performed by
Journal of Planar Chromatography

use of variety of adsorbents, binary and ternary mobile phases,
and combination of LC, TLC, and RP HPLC.
2 Experimental
2.1 Plant Material
The investigation was performed on dried and powdered fruits
and roots (200 g) of Peucedanum verticillare L. Koch ex DC,
collected, in August (fruits) and November (roots) 1999 and
2000, in the Pharmacognostic Garden of the University Medical
Academy of Lublin (Poland).
2.2 Extraction
The plant materials were dried at room temperature, powdered,
macerated (24 h), and extracted exhaustively in a Soxhlet appa-
ratus with petroleum ether (Polish Reagents, Gliwice, Poland;
b.p. 60–70°C) and methanol (Polish Reagents; b.p. 78°C), each
time for 48 h. After routine extraction with petroleum ether the
extract obtained was concentrated under reduced pressure and
stored under refrigeration. A sediment of coumarins precipitated.
2.3 Preparative Column Chromatography
Glass columns filled with Florisil (60–100 mesh; Fluka) and sil-
ica gel 60 (230–400 mesh; E. Merck) suspended in petroleum
ether, and polyamide (0.05–0.16 mm) suspended in methanol
(100 g adsorbent per 200 mL solvent) were used. The coumarin
sediment was dissolved in dichloromethane and eluted with gra-
dients of petroleum ether in dichloromethane, ethyl acetate in
dichloromethane, and methanol in water. Fractions (20 mL)
were collected and monitored by analytical TLC on silica gel
with n-heptane–dichloromethane–ethyl acetate, 40 + 50 + 10
(v/v), as a mobile phase. Separated compounds were detected
under UV light at λ = 366 nm. The fractions obtained were com-
bined in accordance with their TLC similarities and selected for
further separation by preparative TLC.
2.4 Thin-Layer Chromatography
Analytical and preparative thin-layer chromatography was per-
formed on 100 mm × 200 mm glass plates coated with 0.25 mm
layers of silica gel 60 (E. Merck) or Florisil for TLC (Fluka) for
analytical TLC, or with 0.5 mm layers of silica gel 60 for
preparative TLC. Fractions from column LC separation richest
in coumarin compounds were chromatographed by preparative
TLC; the mobile phases used were n-heptane–dichloro-
methane–ethyl acetate, 40 + 50 + 10 (v/v) (mobile phase I),
n-heptane–dichloromethane–ethyl acetate, 30 + 40 + 30 (v/v)
(mobile phase II), and n-heptane–diisopropyl ether–iso-
propanol, 80 + 20 + 12.5 (v/v) (mobile phase III). Thin-layer
chromatography was performed in horizontal Teflon DS cham-
bers (Chromdes, Lublin, Poland) [9–11, 17, 18]. The plates
Journal of Planar Chromatography
Isolation and Purification of Coumarins from Peucedanum verticillare
were conditioned for approximately 10 min in mobile-phase
vapor. The separated bands were scraped from the plates and
extracted with methanol–acetone.
Extracts of the individual compounds were purified by crystal-
lization from methanol and the homogeneity of the coumarins
was examined by analytical TLC. The isolated coumarins were
identified by analytical co-chromatography with standard sub-
stances. In this way five pure compounds from the fruits and
five pure compounds from the roots were isolated in the crys-
talline form. Other compounds were identified by HPLC, again
by comparison with standards. The structures of isolated com-
pounds were elucidated by spectroscopic methods (IR, UV, and
1H and 13C NMR) and mass spectrometry.
2.5 RP HPLC
RP HPLC was performed with a Hewlett–Packard (Palo Alto,
USA) model 1100 liquid chromatograph with variable-wave-
length UV–visible diode-array detection (DAD) and with an
HP 1050 liquid chromatograph with a UV–visible detector.
Both chromatographs were equipped with a 20-µL sample
injector (Rheodyne, Cotati, CA, USA) and fitted with a
250 mm × 4.6 mm i.d., dp = 5 µm, Hypersil AG 1315 A ODS
(C18) column (Shandon, UK). All eluates containing furano- and
pyranocoumarins were separated by use of the 1100 chromato-
graph, with an acetonitrile gradient as mobile phase (Table 1).
The dwell time was 28 min and 10 min was used for equilibra-
tion of the system. The compounds were detected at λ = 254 nm
and λ = 320 nm. The mobile phases used with the 1050 chro-
matograph were MeOH 65% +1% CH3COOH (v/v) and
60% CH3CN. For both chromatographs the flow rate was
1.0 mL min –1.
2.6 Melting Point Measurement
Uncorrected melting points of the isolated crystalline coumarins
from fruits (A–F) and from roots were measured by use of a
Boetius-type microscope (Franz Kûstner, Dresden, Germany).
3 Results and Discussion
The multistep procedure used for extraction, isolation, and
purification of coumarins from the fruits and roots of
Peucedanum verticillare L. Koch ex DC. is illustrated schemat-
Table 1
Acetonitrile gradient used as mobile phase for RP HPLC analysis.
Time [min] Acetonitrile [%] Water [%]
0 50 50
8 50 50
25 70 30
28 50 50
VOL. 18. MAY/JUNE 2005 225
Isolation and Purification of Coumarins from Peucedanum verticillare
Figure 1
Procedures used for extraction and isolation of coumarins from Peucedanum
verticillare (L.) Koch. ex DC fruits and roots.
ically in Figure 1. The petroleum ether (60–70°C) extracts were
found to be abundant in UV-absorbing coumarins (preliminary
TLC analysis, UV detection). Extensive chromatography of
coumarin fractions by combining two column chromatographic
separations on Florisil and a third separation on silica gel in gra-
dient mode, in each case followed by preparative TLC and RP
HPLC analysis, led to the isolation of seven coumarins from the
fruits (compounds A, B, C+D, E, F, and G – from the methano-
lic extract, dichloromethane fraction) and nine coumarins from
the roots (compounds I, II, III, IV+V, VI, VII, VIII, and IX –
from the methanolic extract, dichloromethane fraction).
Two of the compounds isolated in the crystalline form from the
fruits (E, epoxypteryxin, and F, pteryxin) were identified by
MS and 1H and 13C NMR. For compounds analyzed by RP
HPLC the on-line UV spectra in the 200–400 nm range were
obtained on line by use of the DAD detector of the HP 1100 liq-
Table 2
Details of the UV spectra (λ = 200–400 nm) of the compounds.
Compound
Compounds A and VIII and umbeliferone standard
Compounds D and I and isoimperatorin standard
Unknown compound E
Unknown compound F
Compound IV and xanthotoxin standard
Compound V and bergapten standard
Compound present in petroleum ether extract from fruits and imperatorin standard
Compound present in petroleum ether extract from roots and psolaren standard
Compound present in petroleum ether extract from roots and cis-kellactone standard
226 VOL. 18. MAY/JUNE 2005
uid chromatograph and compared with UV spectra of standards.
Details of the UV spectra of all the isolated compounds are list-
ed in Table 2. Table 3 shows average (from replicate measure-
ments) RF and tR values and the colors of the spots observed in
analytical TLC. The uncorrected melting points of isolated crys-
talline compounds were: compound A = compound VIII,
222.5–224°C (umbeliferone); compound D = compound I,
109–110°C (isoimperatorin); compound E, 126–128°C
(unknown); compound F, 85–86.5°C (unknown); compound G,
204–205°C (esculin); and compound III 174–176°C (cis-kellac-
tone). (Literature m.p. for the compounds were obtained from
Ref. [1]).
Five compounds were isolated from Peucedanum verticillare L.
Koch ex DC fruits in the crystalline form – umbeliferone,
isoimperatorin, esculin, and two unknown compounds. Five
compounds – isoimperatorin, cis-kellactone, xanthotoxin,
bergapten, and umbeliferone – were isolated from Peucedanum
verticillare L. Koch ex DC roots in the crystalline form. Eight of
the crystalline compounds isolated were identified by analytical
TLC, RP HPLC, m.p. measurement, and UV spectroscopy. Two
crystalline compounds from the fruits – pteryxin and epoxy-
pteryxin – were identified by MS and NMR analysis.
All these compounds have been isolated from Peucedanum ver-
ticillare L Koch ex DC for the first time. Only bergapten has so
far been reported in the literature. [1]
Sample preparation and extraction of compounds from biologi-
cal matrices is an important stage for further successful quanti-
tative analysis. Selectivity depends on the nature of the adsor-
bent and the composition of eluent. The preparative TLC
method described is simple and convenient and guarantees good
separation of compounds examined. LC and TLC on silica gel
with multicomponent mixtures as mobile phases enabled isola-
tion and purification of coumarin compounds from polar ballast.
Column liquid chromatography, and normal- and reversed-
phase TLC, both followed by RP HPLC with gradient elution
(acetonitrile in water), can be effectively used for separation,
purification, and isolation of furano- and pyranocoumarins.
Preparative chromatography on Florisil and silica columns and
Spectrum λmax
205, 225, 325
203, 225, 250, 260, 310
205, 215, 245, 255, 295, 320
205, 215, 245, 255, 295, 320
203, 220, 250, 265, 300
200, 225, 245, 255, 265, 310
195, 220, 255, 265, 300, 355
204, 245, 235, 325
200, 220, 245, 255, 325
Journal of Planar Chromatography
 Isolation and Purification of Coumarins from Peucedanum verticillare

Table 3

Average (from replicate measurements) RF and tR values and colors of spots in analytical TLC.

Compound UV254 RFa) tR [min] gradient tR [min] isocraticb)

I II III (Table 1) a b c

A and VIII (umbeliferone) Blue 0.06 0.04 – 3.38 12.52 – –

B (unidentified) Violet 0,25 0.22 – – 11.09 –
D and I (isoimperatorin) Yellow–orange 0.62 0.55 – 17.67 – – –
E (unknown compound) Violet 0.14 0,14 – 11.03 – – –
F (unknown compound) Violet 0.18 0.19 – 18.38 – 8.85 –
G (esculin) Blue – – 0.32 – – – 4.87
IV (xanthotoxin) Yellow 0.58 – 0.65 5.67 – 3.80 –
V (bergapten) Orange 0.58 0.45 0.65 6.59 – 4.42 –
Imperatorin Yellow – – – 13.78 – – –
III (cis-kellactone) Violet 0.69 – – 3.58 – – –
Psoralen Yellow – – – 5.47 – – –
a)Mobile phase I is n-heptane–dichloromethane–ethyl acetate, 40 + 50 + 10 (v/v), mobile phase II is n-heptane–dichloromethane–ethyl acetate,

30 + 40 + 30 (v/v), mobile phase III is n-heptane–diisopropyl ether–isopropanol, 80 + 20 + 12.5 (v/v)

b)Mobile
phase a is CH3CN–H2O, 16 + 84 + 1% CH3COOH, mobile phase b is CH3CN–H2O, 60 + 40, mobile phase c is MeOH–H2O, 25 + 75 + 1%
CH3COOH, λ = 345 nm

silica layers is useful for chemotaxonomic studies of umbellif-
erous plants containing pyranocoumarins. It provides informa-
tion on the presence of the compounds and can be applied in
screening studies of medicinal plants containing pyra-
nocoumarins.
References
[1] R.D.H. Murray, J. Mendez, and S.A. Brown. The Natural
Coumarins: Occurrence, Chemistry and Biochemistry, Wiley,
Chichester, 1982.
[2] K. G³owniak and A. Gawron, Appl. Biol. Commun.1/2 (1991)
85–90.
[3] G. Hegi, Illustrierte Flora von Mitteleuropa Band V Teil 2. Berlin,
Hamburg, 1975.
[4] W. Cisowski, Herba Pol. 30, 3–4 (1984) 159–163.
[5] J. Zieliñska-Jenczylik, A. Sypula, and W. Cisowski, Arch.
Immunol. Ther. Exp. Warsaw 32(5) (1984) 577–582.
[6] K. G³owniak and L. Bieganowska, J. Chromatogr. 370 (1986)
281–292.
[7 ] K. G³owniak, T. Mroczek, A. Zab¿a, and T. Cierpicki, Pharm. Biol.
38(4) (2002) 308–312.
[8] W. Cisowski, E. Pa³ka–Gudyka, M. Krauze-Maranowska, Z.A.
Królicki, and W. Cisowski, J. Planar Chromatogr. 4 (1991) 471–474.
[9] M. Waksmundzka-Hajnos and T. Wawrzynowicz, J. Planar Chro-
matogr. 5 (1992) 169–174.
[10] M. Waksmundzka-Hajnos, Chromatographic Disertation No 2,
Medical University, Lublin, Poland, 1999.
[11] M. Waksmundzka-Hajnos and T. Wawrzynowicz, J. Planar Chro-
matogr. 7 (1994) 58–62.
[12] T. Wawrzynowicz, M. Waksmundzka-Hajnos, and L.M. Bie-
ganowska, Chromatographia 28 (1989) 161–166.
[13] K. G³owniak, E. Soczewiñski, and T. Wawrzynowicz, Chem. Anal.
(Warsaw) 32 (1987) 797–811.
[14] K. G³owniak, Dissertation, Medical Academy, Lublin, Poland,
1988.
[15] H. Vuorela, K. Dallenbach-Tölke, S. Nyiredy, K. Hiltunen, and
O. Sticher, Planta Med. 55(2) (1989) 181–184.
[16] P. Härmälä, H. Vuorela, P. Lehtonen, and R. Hiltunen, J. Chro-
matogr. 507 (1990) 367–380.
[17] T.H. Dzido and E. Soczewiñski, J. Chromatogr. 561 (1990)
461–466.
[18] L.M. Bieganowska and K. G³owniak, Chromatographia 25(1)
(1988) 11–16.
[19] M. Takata, S. Shibata, and T. Okuayama, Planta Med. 56(3) (1990)
307–311.
Ms received: August 3, 2004
Accepted by SN: February 24, 2005

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