Vol.
9 "
I987 Pharmaceutisch Weekblad Scientific Edi~on
POSSIBILITIES
AND L I M I T A T I O N S OF PHYTOTHERAPY
H. Schilcher
It is often difficult t o arrive at a consensus in a debate
on the pros and cons of phytotherapy, because the various
discussion partners merely perceive the possibilities and
limitations of phytotherapy from their own viewpoint. Other
viewpoints are frequently overlooked, or are often unknown.
The following subjects should play a role in the debate on
the pros and cons of phytotherapy:
national and international legislation;
historical background and development;
- phytotherapeutical experience and folk medicine;
- pharmacology;
- pharmacy;
- clinical medicine.
A common opinion on the possibilities and limitations of
phytotherapy can only be reached when all viewpoints are
given due consideration.
-
-
Institute of Pharmacognosy and Phytochemistry, Free
University Berlin, Khnlgln-Lulse Strasse 2 + 4,
I000 Berlin 33 (F.R.G.)
CULTIVATION
AND BREEDING OF MEDICINAL PLANTS 1987
215
ON THE BIOCHEMISTRY AND BIOLOGY OF STRESS REACTION:
ADAPTOGENS AS MEDICINES FOR HEALTHY PEOPLE
R. H~nsel
Stress response is characterized by a series of biochemical
and physiological changes. It is associated with an activation of a neural and several neuroendoerine pathways In
the body. The stress-lnduced changes may have beneficial
effects and lead to enhanced resistance towards pathogens.
So, the stress of an infection increases the resistance
against ether pathogens. Likewise, the stress induced by
endotoxins increases the physical efficiency. On Dhe other
hand, stress may also lead to metabolic disarrangements.
In recent years, adaptogens have been subject of several
studies, mainly in the USSR and Japan. Adaptogens are defined as substances possessing the property of raising the
body to a higher level of physiological adaptation and of
enhancing its resistance towards stress injuries and illnesses. However, they have only effect when they are administered prior to stress influences. Thus, adaptogens act
as prophylactics, and they are unable to suppress any
pathological processes.
In ancient times, considerably more attention was paid to
maintain health rather than to treat illnesses. At present,
only few people use prophylactics trying to maintain their
health or to prolong their lives. Although there are many
preparations claimed to be useful as prophylactics, western
medicine is extremely sceptical about pharmacologically
unspecific protective medicines.
Special attention will be paid to three comprehensive
publications concerning adaptation, stress and u n s p e c i f l
prophylaxis from the standpoint of western science.
Some crucial experiments will be discussed to give a betler
insight in the problems of adaptogens.
Institute of Pharmacognosy and Phytochemistry, Free
University Berlin, K6nigln-Luise Strasse 2 + 4,
[000 Berlin 33 (F.R.G.)
Abstracts of short lectures
D. P a l e v i t c h
The integration of modern cultural practices with breeding
of improved cultivars is an appropriate approach for the
development of medicinal plants as economic crops.
T h e potential u s e of agrotechnical techniques as irrigation,
fertilization, selective herbicides, plant spacing, sowing
dates and growth regulators for increasing the biomass'and/
or phytochemlcals yield will be discussed.
Examples of the potential contribution of suitable genotypes for specific environmental and 6griculturel conditions will be shown.
Some effects of pre- and post-harvest treatments on phytochemicals yield will he presented.
Department of Medicinal, Aromatic a n d Spice Crops,
Agricultural Research Organization, Volcani Center,
P.O. Box 6, Bet Dagan 50 250 (Israel)
NEW HYDROXYALOINS: THE 'PERIODATE-POSITIVE SUBSTANCE'
CAPE ALOES ANn CINNAMOYL ESTERS FROM CURACAO ALOES
H.W. Rauwald
FROM
This report deals with the isolation and structure elucidation of two hydroxyaloins from aloes which are interesting
due to their pharmaceutical history (1,2). ghhme et a~. (2)
described a 'periodate-positive substance' in Cape aloes
showing a violet spot at daylight in PC, similar to
'isobarbaloln' (= 7-hydroxyaloins) from Curacao aloes. Its
presence was confirmed by H~rhammer et aZ. using TLC (3).
We identified this extremely labile 'periodate-positive
substance' as 5-hydroxyaloln A, a characteristic compound
in Cape aloes. Furthermore, we analyzed additional hydroxyaloins from Curacao aloes corresponding to a violet band
just above aloin in TLC, and identified a pair of C-10
diastereomerie 6'-0-p-coumeroyl-7-hydroxyaloins
A and B.
The importance of these compounds for identification of
officinal aloes will be discussed. DCCC was successfull
in separating the two stereoisomers of the 10-glucosylanthrone derivatives. The a~plication of soft ionization
MS (FAB, DCI) and [H- and IoC-NMR gave highly valuable
information for the structure determination of this group
of labile natural substances (4).
I. W. Awe, H. Auterhoff and C. Waehsmuth-Melm, Arzneim.Forsch., 4 (1958) 725
2. H. B~hme and L. Kreutzlg, Dtsch. Apoth.-Ztg., IO3 ([963)
505
3. L. H~rhammer, H. Wagner and G. Bittner, Arzneim.-Forsch.,
13 (1963) 537; Pharm. Ztg., t08 ([963) 259
4. H.W. Rauwald and K. Roth, Arch. Pharm., 3[7 ([984) 362
Institute of Pharmaceutical Biology, University of Frankfurt, Georg-Voigt-Strasse 16, 6000 Frankfurt/M. (F.R.G.)
216
COYRdENTS ON THE SPECIFIC ORIGIN OF KENYAN ALOES
J. Conner*, A. Gray*, T: Reynolds O and P. Waterman*
Commercial
aloes is commonly named after its place of
origin. In some cases, e.g. Natal aloes, the actual species
from which it is produced is unknown, and may in fact be a
mixture of species growing locally. As in Cape aloes, the
commercially available Kenyan aloes is reported to be
obtained from Agoe ferox and its hybrids (I). However,
examination of three samples of Kenyan aloes by TLC,
developing the chromatograms with Fast Blue B reagent,
showed no trace of the bright orange aloereslns characteristic of Cape aloes (2). These are widely distributed
through the genus Aloe, including the e x u d a t e of AZoe
Ierox (3).
We i n v e s t i g a t e d
the exudates of several East African
s h r u b b y AZoe s p e c i e s and o b s e r v e d t h e e x u d a t e o f Aloe
rabas
R e n d l e t o be s i m i l a r t o o u r s a m p l e s o f Kenyan
aloes. However, given the chemical similarit~ between many
ALoe species (3) and the fact that one species can occur
as a number of chemovars, prevents any pos~tlve assertion
on the true identity of Kenyan aloes. The chemical investigation of Kenyan aloes and its relevance to possible
specific origins will be presented.
I. G. E. Trease and W. C. Evans, Pharmacognosy,
[983,
Balliere Tindsll, 404-408
2~ L. HSrhammer, H. Wagner and G. Bittner, Pharm. Zig.,
108 41963) 259-262
3. T. Reynolds, Bot. J. Linn. Soc. 90 (1985) 179-199
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
APPLICATION OF THE ROTACHROM FOR THE ANALYSIS AND ISOLATION
OF NATURALLY OCCURRING COMPOUNDS
Sz. Nyiredy and O. Stlcher
The Rotaehrom rotation planar chromatograph (1)(Petazon AG,
Zug, Switzerland) includes several analytical and on-llne
preparative techniques, llke normal chamber centrifugal
layer chromatography (NCLC), micro chamber centrifugal
layer chromatography (MCLC), ultra-micro chamber centrifugal layer chromatography (UCLC), sequential centrifugal
layer chromatography (SCLC) and centrifugal planar-column
chromatography (CPCC) (2).
The mobile phase may he transferred from TLC plates to
analytical MCLC or UCLC, depending whether the TLC preassays have been carried out in saturated or insaturated
chambers. For the analysis of plant extracts various
development modes may be employed according to the separation problem. A direct up-scaling is possible from analytical MCLC or UCLC to preparative MCLC or UCLC, considering the layer thickness and the flow-rate of the mobile
phase. Both methods may he combined with the sequential
technique for a fast elution of t h e separated compounds
in a smaller elutlon volume. For the CPCC all stationary
phases without binder may he used in finer particle sizes.
The separation may he started with a dry or equilibrated
system, which h a s to be considered for the optimization
and transfer of the mobile phase.
The efficiency of the different separation techniques will
be demonstrated for several naturally occurring compounds,
e.g. anthraquinones, furocoumarins, irldoid glycosides,
flavonoid glycosides, saponins and alkaloids.
I. Sz. Nyiredy, S.Y. M~sz~ros, K. Nyiredy-Mikita, K.
Dallenbach-Telke and O. Sticher, GIT Suppl. Chromatogr.,
3 ( 1 9 8 6 ) 51
2. Sz. Nyiredy, S.Y. M6szgros, g. Nyiredy-Miklta, g.
Dallenbach-T~ike and O. Stlcher, HRC & CC., 9 (19861 605
* Phytochemistry Research Laboratories, Department of
Pharmacy, University of Strathclyde, Glasgow G1 IXW
(Scotland)
0 Jodrell Laboratory, Royal Botanic Gardens, Kew,
Richmond, Surrey, TW9 3DS (England)
Pharmazeutlsches
schule Z ~ r l c h ,
THE ROLE OF THE N-OXIDES IN BIOSYNTHESIS AND ACCUMULATION
OF PYRROLIZIDINE ALKALOIDS
T. Hartmann, R.-D. Adolph, A. Ehmke, H. Sander, G: Toppel
and L. Witte
SEPARATION OF PHOKBOL ESTERS BY TWO-DIMENSIONAL TLC AND
RAPID PURIFICATION USING LPLC AND SUBSEQUENT REVERSEDPHASE OPLC
C.A.J. Erdelmeier*, P.A.S. van Leeuwen 0 and A.D. Kinghorn*
Pyrrolizidine alkaloids (PA) are a fascinating group of
biologically active plant consCltuents. They are effectively synthesized in root cultures of Seneoio v~gay~s (l)
and other species. In vi~o tracer experiments revealed that
the pyrrollzidine nucleus is derived from arginlne via
agmatlne, putresclne and homospermldine, Seneclonine Noxide is the primary product of blosynChesls, iC accumulates as an end product. Comparative studies indicate that
presumably in most PA plants the PA are accumulated as Noxides. The tertiary PA are easily formed spontaneously,
from the respective N-oxides during alkaloid extraction.
The PA N-oxldes are selectively translocated into and
stored within the cell vacuoles (2). In CPotalaPia
scasseZlatis (Fabaceae) the tertiary alkaloids are stored
as tertiary PA. However, during germination t h e y are rapidly oxidized and translocated into the growing shoots and
roots exclusively as N-oxides. The role of the PA N-oxide
compounds in translocatlon, storage and accumulation of
alkaloids will be discussed.
i. T. Hartmann and G. Toppel, Phytochemistry, 1987, in
press
2. A. Ehmke, K. v. Boratel and T. Rartmann, In: Nato Adv.
Sol. Workshop, Sophla-Antlpolls (France). Plenum Press,
i n press
Fourteen known phorbol diester constituents of CPoton
tig~s
L. seed oil (croton oil) have been separated by
two-dimenslonal Chin-layer chromatography (2D-TLC) employing two different sorbent layers (i). Two 2D-TLC approaches
were attempted: separation with a coupled, two-phase plate,
coated slde-by-side with silica and RP-18 material, and a
chromatogram transfer technique, which allows "flipping"
of spots from a developed normal-phase place into a reversed-phase layer for farther separation.
After a preliminary purification step using low-pressure
liquid chromatography, reversed-phase overpressure layer
chromatography (2) has been shown to be potentially useful
for a rapid purification of phorbol dlesters. The identity
of the major croton, oll phorbol diesters was established
by co-chromatography with authentic compounds and/or by
off-line chemical ionization mass spectrometry.
I. M~ Zakaria, M.-F. Gonnord and G. Gulochon, J. Chromatogr., 271 41983) 127
2. Z. Witxlewicz and J. Bladek, J. Chromatogr., 373 4[986)
III
Institut fHr Pharmazeutlsche Biologic der T.U.,
Mendelssohnstr. I, 3300 Braunschweig (F.R.G.)
Institut, Eidgen~ssische Technlsche
8092 Zfirlch (Switzerland)
Hoch-
* Program for Collaborative Research in the Pharmaceutical
S c i e n c e s , and Department o f Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illlnois st Chicago, Chicago, IL 60612 (U.S.A.)
0 Clinical Pharmacoklnetics Laboratory, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612
(U.S.A.)
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
APPLICABILITY OF VARIOUS ANALYTICAL AND PREPARATIVE
CHROMATOGRAPHIC TECHNIQUES FOR THE SEPARATION OF THE
MAIN COMPONENTS OF SILYMARIN
K. Dallenbaeh-TSlke, Sz. Nyiredy and O. Stlcher
217
HYDROXYCINNAMIC
ACID ESTERS IN TISSUE CULTURES OF
Leontopodium a~pinwr#, "Edelweiss"
Deirdre Hennessy,
Ingrld Hook and Helen Sheridan
is available on the phytochemistry of
Cass., Compositae, and there have been
no literature reports on the establishment or phytochemical
analysis of its tissue cultures. In this study callus
cultures of L. a l p s
were initiated from sterile micropropagated plants. Callus was maintained on a modified
Murashige and Skoog medium. Light grown callus became deep
red in colour due to the formation of an anthocyanln
identified as cyanidin. Dark grown cultures lacked this
pigmentation but yielded a series of hydroxycinnamic acid
esters. Suspension cultures were developed and maintained
in the above medium. Phytochemical analysis of these cultures led to the isolation and identification of two
hydroxycinnamic acid esters present in major concentration
(HPLC indicating I and 2% of totsl dry biomass).
Results will be presented of a time course experiment set
up to determine growth rates and ester production in batch
suspension cultures. Also the results of analysis on the
effects of cell immobilization, medium supplementation and
biological and chemical elicitors will be reported.
Little information
In recent years several new analytical and on-line preparative chromatographic methods were developed and applied
in phytochemlstry. In planar chromatography the forcedflow techniques llke overpressure
layer chromatography
(OPLC) (I) and the various rotation planar chromatographic
(RPC) (2) methods gave new possibilities for the analysis
of p~ant extracts and on-llne isolation of compounds. In
column chromatography the various preparative pressure LC
(3) methods have become more efficient using finer particle
sizes of the normal and reversed phase stationary phases.
Until now only few publications give a systematic comparison of the advantages and application of the various analytical and preparative techniques. Silymarin, an extract
of SiZybum mGPianum L. Gaertner, containing flavonollgnan
isomers llke silyhfn, silydianln and silychristln was used
to compare the efficiency of the various forced-flow planar
and column chromatographic methods. The compounds were
separated on silica TLC plates with water saturated diethyl
ether and this mobile phase was transferred to analytical
and preparative OPLC, various RPC methods and "to MPLC.
The mobile phase was also optimized for RF-HPLC and transferred to LPLC. The advantages of the various development
modes (linear, circular) and chromatographic techniques
will be discussed.
I. Sz. Nyiredy, C.A.J. Erdelmeier, K. Dallenbach-TSlke,
K. Nyiredy-Mikita and O. Sticher, J.Nat. Prod., 49
(1986) 885
2. Sz. Nyiredy, S.Y. M~sz~ros, K. Nylredy-Miklta, K.
Dallenbach-TSlke and O. Sticher, GIT Suppl.Chromatogr.,
3 (1986) 51
3. K. Hostettmann, M. Hostettmann and A. Marston, Preparative Chromatography Techniques, 1986, Springer, Berlin
Leontopodiw~ agplnw,
Pharmazeutlsches
schule ZQrich,
Department of Pharmacognosy, School of Pharmacy, Trinity
College Dublin, 18, Shrewsbury" Rd., Dublin 4 (Ireland)
Institut, EidgenSssische Technische Hoch8092 ZHrieh (Switzerland)
A RADIOIPR4UNO ASSAY FOR QUINOLIZIDINE
WITH PLANT CELL CULTURES
M. Wink
ALKALOIDS
FOR STUDIES
A R I A has been developed for the detection of quinolizidine
alkaloids, which is specific for lupaoioe, [3-hydroxylupanine and 13-hydroxylupanine esters. Other alkaloids, such
as sparteine, lupinine or cytisine do not crossreact. The
detection limit is 0.05 ng. Parallel to capillary GLC and
GC-MS the RIA was employed for cell culture studies.
Lupanine formation can be induced in cell suspension cultures of lupins after induction with low-molecular weight
effeetors, i.e. biogenetically different alkaloids, cAMP
or polyamines. Under stress conditions traces of lupanine
can also be detected in cell cultures of plants which are
not known to contain quinolizidine alkaloids. It is assumed
that the genes of lupin alkaloid biosynthesis are widely
distributed, however "silent", in the plant kingdom.
Universitgt MQnchen, Institut fur Pharmazeutische
Karlstr. 29,
8000 MOnchen 2 (F.R.G.)
Biologie,
FORMATION OF FORSKOLIN, AN ADENYLATE CYCLASE ACTIVATING
DITERPENOID, BY SUSPENSION CULTURES OF CoZeu8 forsko~ii
R. Mersinger*, H. Dornauer O and E. Reinhard*
Suspension cultures of COZeu8 fO~SkO~ii, Lamiaceae, were
established and cultivated on s B5 medium, containing the
phytohormones 2,4-D and kinetin: no forskolin or other
labdan diterpenoids were formed. To start the biosynthesis
of forskolin, the cell aggregates were brought into an
inauctlon medium containing above all indole-butyric-acid
instead of 2,4-D.
Within two cultivation periods of 14 days the amount of
forskolin increased to 0.2 - I g/kg dry cell weight. During
this period, the producing cells often showed differentiation, i.e. formation of roots or big bowl aggregates.
They could he subcultlvated for continuation of the production process for more than 2 months. Forskolin was
isolated from the freeze dried cell material and its identity verified by IH-NMR spectroscopy.
The scale up of the procedure was performed in an 20 lairlift-bloreaetor after precultivating the cells 2 weeks
on induction medium in I 1-flasks. The result was a maximum
forskolin content of 729 mg/kg dry weight after 19 days of
fermentation.
For the assay of forskolin and 1,9-didesoxyforskolin,
the
main components, an analytical method was established,
consisting of purification of the crude extract by minicolumn chromatography on silica gel and NPLC on reversed
phase material.
* Pharmaceutical Institute, University
(F.R.G.)
O Hoechst AG, Frankfurt a. M. (F.R.G.)
of TQbingen
218
REGULATORY ENZYMES IN SHIKONIN BIOSYNTHESIS
L. Heide* and M. Tabata O
Cell c u l t u r e s of Lithospelmllan erythrorhizon a r e c a p a b l e
o f p r o d u c i n g l a r g e amounts o f n a p h t h o q u i n o n e s , via.
s h i k o n i n d e r i v a t i v e s . T h i s p r o c e s s has become t h e f i r s t
example of the industrial application of plant cell
culture for the production of a useful secondary metabnllte.
Many chemical and physical parameters stimulating or repressing shlkonln production have been investigated (I),
but enzymatic studies of shlkonln formation have not been
possible so far, due t o the presence of very potent enzyme
Inhlbltors in the shlkonin-produclng cultures.
In the present s t u d y / methods were developed co obtain
active enzyme extracts from the cultures by removal of
shikonln with a two-layer culture method and the use of
enzyme-protectlve reagents. Using these methods, a key
enzyme of shlkonin biosynthesis, 91z. p-hydroxybenzoic
acid geranyltransferase, was found. Some characteristics
of this enzyme will be presented.
The role of this aad other secondary metabolic enzymes,
especially phenylalanlne ammonia lyase and a p-hydroxybenzoic acid glucosyltransferase, in the regulation of
shlkonln biosynthesis, in light, will be discussed.
I. M. Tabata and Y. FuJita, In: Biotechnology in Plant
Science, P. Day, M. Zaltlin and A. Hollaender, ads.,
]985, Academic Press, Florida, pp. 207-2]8.
* Inscitut for P h a r m a z e u c i s c h e Biologie, Universit~t
Bonn, Nussallee 6, 5300 Bonn I (F.R.G.)
0 Department of Pharmacognosy, Faculty of Pharmaceutical
Sciences, Kyoto University, Kyoto
(Japan)
ON THE SYSTEM ALLIIN-ALLIINASE-ALLICIN
K. Knobloch, g.'Jansen and B. M~ller
Garlic bulbs from mediterranlan origin were used for the
isolation of alliin and allicin as well as for the purification of the alllinase protein system. In addition,
alllin and alllcin were synthesized.
The alliinase was isolated from the ammonium sulfate
f r a c t i o n , 0 t o 35% s a t u r a t i o n , pumped on a h y d c o x y l a p a t i t e
column f o l l o w e d by f u r t h e r s e p a r a t i o n on columns w i t h Con
A-Sepharose 4B and B i o - G e l A-O.5m. The i s o l a t e d p r o t e i n
revealed allilnase activity with synthetic or native
alliin as the enzyme substrate. Allicin, the reaction
product, was determined quantitatively by HPLC (I). The
enzymatic reaction occurred at an optimum rate at pH 6:5.
There was no relevant modulation in the reaction when
divalent cations were added, although it had been reported
that the addition of EDTA caused an increase in the turnover rate (2).
The molecular weight was determined by using gel filtration
177 000; SDS gel electrophoresis yielded two subunlts of
different molecular masses: 80 000 and 45 000, or smaller.
These data were not in full agreement with results obtained
by others using garlic from California (3).
There is reason to assume that the allllnase system we
isolated is associated with a flavine moiety.
i. H. Jansen, B. MOIler and K. Knobloch, Allicln Characterization and its Determination by HPLC. Planta Med.,
submitted
2. M. Maze~is and L. Crews, Biochem. J., 108 ([968) 725730
3. L.P. Nock and M. Mazells, Arch. Biochem. Biophys.,
249 ( 1 9 8 6 ) 27-33
Instltur for Botanik und Pharmazeutlsche Biologle der
UniversltAt Erlangen-NOrnberg, Staudtstr. 5,
8520 Erlangen (F.R.G.)
V o l . 9 - 1987
Pharmaceutisch Weekblad Scientific Edition
CHROMATOGRAPHIC ANALYSIS OF POLYPHENOLS
~y~8
species
T. Adzet~ R. Vila and S. C a ~ g u e r a l
OF SOME IBERIAN
The taxonomical complexity of the genus Thymus as well as
the pharmaceutical applications of its species, make Its
phytochemlcal study interesting. Moreover, the West Medlterranean area is considered to be the original region of
the genus with many endemlsms.
In this study, a comparative chromatographic analysis of
flavonoids and phenolic acids of some Iberian Thy~a~8 species of t h e sections Paeudothymb~, Thynms and SerpyZZum
was performed by means of MPLC and TLC.
The purified leaf extracts were analyzed and compared with
various authentic samples, some of which had been isolated
previously from Thymus moroderi (I).
Among the substances detected were 15 flavnnes, one
flavonol, four flavanones, two dlhydroflavonols, and caffeic and roamarlnie acids.
The results will be discussed as well as some analytical
remarks concerning the UV detectlon in HPLC.
I. T. Adzec, R. Vila and S. Canigneral, Planta Mad.,
(1986) 526
Laboratory of Pharmacology and Pharmacognosy, Faculty of
Pharmacy, University of Barcelona, Zona Unlversltaria de
Pedralbes, 08028 Barcelona (Spain)
AROMATIC ACID DERIVATIVES
L. (URTICACEAE)
J. Budzlanowski
FROM Pomietomia officir~Zi8
PaP~etoJ~a officinalis L. is known as medicinal plant for
centuries. Previous chemical investigations (I) showed
the presence of ten flsvonol glycosides. Further studies
on phenolic compounds lad to the isolation of three other,
main constituents, water-very-soluble acids. They were
identified mainly on the basis of 13C- and IH-NMR, as
caffeoylmalic acid, a polyhydroxyphenylaliphatic
acid aster
of caffaic acid, and dlhydroxybenzenediearboxyllc
acid.
The mixture of these acids showed no diuretic activity
in rats and no activity against bacteria causing infections
of the urinary system.
IC seems that the d i u r e t i c activity of Parietaria may be
caused by flavonoids and potassium nitrate; a large ~mount
of this salt was isolated.
The isolation and structure elucidation of the above-mentioned acids will be presented.
I. J. Budzlannwskl, L. Skrzypczak and D. Walkowlak,
J. Nat. Prod., 48 (1985) 336
Department of Pharmaceutical Botany, K. Marcinkowski Medical Academy, ul. Wlenlawsklego I, 61-712 Pozna6 (Poland)
Vol. 9- I987 Pharmaceutisch Weekblad Scientific Edition
TRANSFORMATION OF Oubolsia myoporoidea WITH Agrobacteri~m
rhizogenes AND INVESTIGATION OF THE ALKALOIDS IN THE
TRANSFORMED TISSUES
P. Baehmann and F.-C. Czygan
Agrobacterium rhizogenes i s a p h y t o p a t h o g e n i c
b a c t e r i u m of
the soil, which is able to integrate a part of the Riplasmid, the so-called T-DNA, into the genom of the plant.
The result is a transformed cell, which develops into a
root. These roots were easily cultivated in media'without
any phytohnrmones and they were capable to regenerate shootE
So, transformation with A. rhiaogenes seems to be a powerful tool for studying secondary products synthesized in
roots,e.g, tropane alkaloids, l~bolsla ~oporoidee, an
important tropane alkaloid-produclng plant, was chosen for
the transformation experiments, because roots are difficult
to cultivate ~n vitpo, and regenerates from tissue cultures
show no spontaneous formation of roots.
Transformation of the "alkaloid free", rootless shoots is
elegant, because all alkaloids found in the tissue after
transformation are newly synthesized and/or newly accumulated.
The alkaloid content and the alkaloid spectrum of the roots
from specific transformed organs were analyzed by GLC and
GC-MS.
The results will be discussed.
Lehrstuhl fOr Pharmazeutische Biologie der Universit~t
WOrzburg, Mittlerer Dallenbergweg 64, 8700 W~rzburg (F.R.G.)
METABOLISM OF I"C-RHEIN
~
AND I~.~C_RHEIN ANTHRONE IN RAT
P. de Wltte and J. Lemll
In order to study the metabolism of rhein and rhein
anthrone, we synthesized the 14-C radiolabelled compounds
(I). Both radio-active compounds were administered intracaecally to rats; the recovery rates amounted to 90.4 and
97.2% for 14-C rhein (6 days) and 14-C rhein anthrone (~
days) respectively. Only a small amount of 14-C rheln
anthron was excreted by urine in contrast with 14-C rhein
(37.3%). All organs and tissues showed for both 14-C compounds a significant clearance of radlo-activlty with
exception of the kidneys where high levels were found even
after 5 days. Four urinary metabolites could be detected
after administration of |4-C rhein. After oxidative hydrolysis of the metabolites of 14-C rhein anthrone, 78% was
recovered as 14-C rhein. The extracted metabolites from
faeces seem to be the same for 14-C rhein as for 14-C rhein
anthrone as could be demonstrated by autoradiographlc
techniques. Also 14-C rheln anthrone was detected as a
metabollte of 14-C rhein in faeces. In vitro metabolism
studies of the caecal content of 14-C rhein and I4-C rhein
anthrone confirmed the in uiuo results.
I. P. de Witte and J. Lemli, J. Labelled Comp. & Radioph.,
submitted
Laboratorium voor Farmaceutische giologie sn Fytofarmacologic, K.U. Leuven, Van Evenstraat 4, 3000 Leuven (Belgium)
219
ANALYSIS OF TWO FUNCTIONALLY DIFFERENT IM~UNOMODOLATORS
FROM Aloe v e ~ GEL
L.A. 't Hart, P.H. van Enekevnrt and R.P. Labadie
The r e c o g n i t i o n o f A~oe v e r a g e l ( L . ; Burm. f . ) f o r t r e a t ment of infections h a s initiated investigations of its
clinical use, biochemistry and working mechanism (recently
reviewed in I). Although the results of these experiments
show little correlation, data on the treatment of infections with the crude gel in animal models suggest that it
affects thv immune system. Therefore, we analysed the gel
on the basis of its activity in immunological assays; i.e.
the activation of the human complement cascade and the
zymosan-induced oxydative burst in human peripheral blood
polymorph nuclear cells (PMN). The parenchymnus tissue of
AZoe v e ~ leaves from Sri Lanka was carefully isolated and
freeze-dried. To obtain an aqueous extract the gel was
swollen in Hanks balanced salt solution under gentle stirring. The extract was subjected to ultra-filtratlon (Amico~
YM-10) to obtain a low (I-MW, less than I0 kD) and a high
(h-MW, more than I0 kD) molecular weight fraction. Both
fractions were further analysed with ion-exchange and gelfiltration chromatography. The h-MW fraction, consisting
predominantly of acid polysaecharides, inhibited only the
activation of the complement cascade via the classical or
alternative pathway. TLC analysis of the hydrolysed pnlysaecharides showed the presence of glucose, mannose and
glucuronic acid, of which only the latter could abolish the
inhibitory effect. The I-MW fraction, consisting among
others of anthraquinone glycosides, was inactive in the
complement assay but inhibited the production of oxygen
metabolites by activated PMN, whereas the phagocytosis of
radlo-labelled bacteria remained unaffected. Preliminary
results showed that the I-MW components interfered with the
activation of protein klnase C, but not with the influx of
calcium.
[. D. Grlndlay and T. Reynolds, J. Ethnopharmacol., 16
( 1 9 8 6 ) I17
Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, Catharij~eslngel 60, 3 5 1 1 G H Utrecht
(The Netherlands)
ELICITATION OF SECONDARY METABOLISM IN A SUSPENSION CULTURE
OF Tabernaemontana divaricata
R. van der HeiJden*, H.H.M. de Keyzer*, E.R. Verhey*, J.
Schripsema*, P.A.A. Harkes O, R. Verpoorte* and A. Baerheim
Svendsen*
T r e a t m e n t o f a Tabernaemontana d s
(Apocynaceae)
suspension culture with biotic elicitors like cellulase,
AspergiZ~us niger o r Candida aZblean8 r e s u l t e d i n a r a p i d
de-novo p r o d u c t i o n of t r i t e r p e n e s
of t h e u r s e n e c a r b o x y l i c
acid type. For Candida albicans as elicitor the influence
on alkaloid and triterpene production was studied in detail
Triterpene production proved to be time and elicitor-dose
dependant. Triterpenes were detected within 18 h after
addition of 5 mg (dry weight) C. albioans elicitor preparation to 60 ml of a I0 days old T. dluarieata suspension
culture. The production of the alkaloids vallesamine and
O-aeetyl-vallesamine was inhibited. No triterpenes were
detected in the media. The alkaloids were not excreted
from the cells after addition of an elicitor. The elicited
suspension cultures became yellow, probably because of
the formation of flavonolds. The experiments show the
possibilities of elicitors as tools for studying secondary
metabolism.
* Division of Pharmacognosy, Center for Bio-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories,
P.O.Box 9502, 2300 RA Leiden (The Netherlands)
O Department of Plant Molecular Biology, Leiden University, Nonnensteeg 3, 2 3 1 1 V J Leiden (The Netherlands)
220
PRODUCTION OF ARTEMISINI~
E.M. Croom, Jr.
V O I . 9 - 1987
(QINGHAOSU)
IN Artemlsiu annua L.
Pharmaceutisch Weekblad Scientific Edition
THE CHEMICAL DISCRIMINATION
OF Eohinuoea angustlfolia AND
E. pallida
R. Bauer*, V. Wray O and H. Wagner*
The sesqulterpene peroxide artsmlsinin (Qinghaosu) is the
p a r e n t molecule of the moat promising new class of antimalarial agents. One of the major limits for this class of
compounds is the cost of obtaining artemislnin from the
major plant source A. ann~. The variation in isolated
yield of artemislnin (0.02 - 0.8%) is a major problem for
a constant, economical supply of this antimalarial agent.
Basic environmental and agronomic influences on the production of artemisinin in A. a n n ~ will be presented.
Research Institute of Pharmaceutical
Pharmacy, University of Mississippi,
Sciences, School of
MS 38677 (U.S.A.)
VARIATION OF YIELD AND ESSENTIAL OIL COMPONENTS OF
CHAMOMILE BY GENETIC, ONTOGENETIC AND ECOLOGICAL FACTORS
W. Letchamo and A. V~mel
Examination of polyploid forms of chamomilla recutita (L..)
Rausch, was performed to obtain varieties with larger
flower heads than diploids and improved chemical characteristics (l). The main aim, however, was to develop
varieties which could not cross spontaneously with wild
chamomile.
For selection, the flower heads were analyzed at different
stages of development and under various ecological conditions. Field and pot experiments were carried out with
varieties of different ploidy grades in a temperate zone
of medium rainfall (610 mm/a) and a low altitude (220 m~.
After steam distillation the essential oil amounts were
found go vary from 0.4-1.1% in diploid and 0.6-1.3% of
the crude drug in tetraploid varieties, with highest yield
in the "medium stage" of the pedantium. GC analysis indicated that the composition of the essential oll also varied
according to the ontogenetic stage. There was, however,
no significant difference neither in the amount of sssentlal oll nor in the yield of crude drug at the three dates
of harvest.
Nitrogen application had a positive influence on the yield
of crude drug and on the essential oil content of the
flower heads in all varieties analyzed. These experiments
are part of a study to establish minimum variation due to
genotype by environmental interaction under extreme ecological infruences.
I. Ch. Franz et aS., Dtsch. Apoth.-Ztg., 125, Suppl. I,(1985)
20-23
Institut for Pflanzenbau und ZQchtung der JL-Unlversit~t
Giessen,Versuchsstation Rauischholzhausen, 3557
Ebsdorfergrund 4 (F.R.G.)
In our search for immunologically
active compounds
in
Eehinaeea species, recently we investigated E. angustifoZia
and E. paZZi~a. There was evidence that both species are
mixed up because of similarity in morphological characters,
especially of the roots. However, they can be distinguished
by their chemical constituents.
In a previous paper (I) we reported on two new polyacetylanes in dried E. paZlioh roots, which were suspected to be
artifacts. Analysis of fresh roots showed the presence of
five main constituents, which can be considered as the precursors of these artifacts. Three of them were identified
(mainly by NMR) as tetradeca-g-ene-ll,13-diyne-2-ons,
pentadeca-g,13-dlene-ll-yne-2-one
and pentadeea-8,11-dleue2-one. Another compound, g-hydroxy-pentadeca-9,13-diene-llyne-2-one, obtained from dried roots, was also shown to be
an artifact. The formation of these artifacts takes place
via oxidation during the drying and grinding process.
TLC and HPLC analyses of the roots showed the absence of
these polyacetylenes in E. ~ g u s t i f o Z ~ and the presence of
echinacoslde in both species. E. angustifoZia contained
also cynarin which was not found in E. paIZida. Chromatographic methods for the differentiation of drug preparations from both species will be presented.
I. R. Bauer, I. Khan, V. Wray and H. Wagner, Phytochemlstr~
26 ([987) 1198
* Institut f~r Pharmazeutlsche Biologle der Universit~t
MQnchen, Karlstr. 29, 8000 M~nchen 2 (F.R.G.)
0 Gesellschaft f~r Biotechnologische Forschung mbH,
Mascheroder Weg I, 3300 Braunschwelg (F.R.G.)
NOVEL FUNGICIDAL POLYPNENOLICS
A. Marston and K. Hostettmanu
FROM CoPdia goetzei
The tree Cordia goetsei G~rke (Boraginaceae), used for the
treatment of leprosy and malaria in Tanzanian traditional
medicine, was investigated for the presence of antifungal
constituents. The yellow layers of the bark were found to
contain four compounds which were active against spores of
the fungus CZadospoPi~m o u o u ~ , ~ 2 ~ .
These compounds are all polyphenols which can be considered
to derive from two chalcone moieties. They were isolated
from a methanolic extract of the bark by a combination of
DCCC (chloroform-methanol-water 43:37:20, descending mode)
and low-pressure LC (methanol-water mixtures).
While two of the chalcone dlmers have previously been isolated from B ~ c k e ~ i d g e a a ~ e b a ~ c a
(Ochnaceae) in which
they are the yellow pigments responsible for the colour
of the hark, the two other polyphenols were new compounds.
Their structures were elucidated by mass spectrometry
(FAB-MS), hlgh-resolutlon NMK~ (including COSY, HETCOR and
COLOC techniques) and chemical methods.
Instltut de Pharmacognosie et Phytochlmie,
Lausanne, Lausanne (Swltzerland)
Unlverslt~ de
V o l . 9 - 1987
Pharmaceutisch Weekblad Scientific Edition
BIOLOGICAL ACTIVITY OF SOME ISOLATED SAPONINS AND
SAPONIN-LIKE COMPOUNDS FROM STARFISH AND BRITTLE STARS
L. Andersson*, L. Bohlln*, M. Iorizzl D, R. Ricclo O,
L. Minale 0 and J. Moreno-Lopez V
Twentyfour saponins and saponin-like compounds, isolated
from warm and c o l d water species of starfish and warm
water species of brittle stars, have been tested in five
different bioassays.
In the guinea-pig ileum test, one out of seven ~ompounds
tested showed contracting activity, whereas six compounds
showed inhibiting activity. All twentyone compounds tested
in the antibacterial test were inactive against the Gramnegative bacteria E. eoZi. However, the Gram-posltlve
bacteria S. auPeu8 was affected by the polyhydroxylated
steroidal glycosides, polyhydroxylated sterols and disulfated sterols. In a cytotoxlcity test, activity was
observed for all twentyone compounds tested at the higher
dose of I00 Ng/ml, while the cells were not affected at
the lowest concentration of I Ug/ml. In the antitumor test
sixteen compounds were tested for two lymphoma cell lines.
Inhibiting activity at the lower concentration could be
seen for three compounds, all polyhydroxylated sterols.
* Department of Pharmacognosy, Uppsala Biomedical Center,
Uppsala University, Box 579, 75[ 23 Uppsala (Sweden)
0 Dipartimento dl Chimlca delle Sostanze Naturali, Unlversita di Napoll, Via L.godino 22, 80 138 Napoli (Italy)
V Department of Veterinary Microbiology, Section of
Virology, Uppsala Biomedical Center, Box 585, 751 23
Uppsala (Sweden)
221
AN ANTITUMOR ACTIVE
8-1,3/I,6-GLUCAN FROM
Phytophthora
paraaitica
J. Kraus, W. Blaschek and G. Franz
The antitumor activity of a newly found fl-l,3/l,6-glucan
isolated from the cell walls of PhytophthcPa pa~sit~ea has
recently been described (i). The water-soluble glucan
exhibited a prominent antltumor activity against the
allogenelc solid Sarcoma-180 in CDI mice. The antltumor
effect of the glucan was dose dependant, with an optimum
activity at [ mg/kg. The physiological activity was further
s h o w n t o be dependant on the chain length of the polymer.
The glucan with the highest molecular weight (200.000) had
optimum activity. Polysaccharlde treatment prior to tumor
inoculation proved to have comparable effectiveness as
treatment affer tumor inoculation. In vigPo studies demonstrafed that the glucans had no direct cytotoxic effects.
In mice, treated simultaneously with the glocan and trypan
blue, a macrophage inhibiting agent, t h e antltumor activity
was only slightly reduced. On the other hand, treatment
with eiclosporin A, an inhibitor of the specific cellular
immunity, abolished the antitumor effect of the glucan.
The results indicate that the antltumor activity of the
B-I,3/[,6-glucan from Ph~tophthora parasitica is hostmediated mainly by stimulation of the T-cell population of
the immune system.
1. M. Bruneteau, I . Fabre, J . Perret, G. Michel, P. Riecl,
J.P. Joseleau, J. Kraus, M. Schneider, W. Blaschek and
C. Franz, Carbohydr. Res., in press
Institute o f Pharmaceutical Biology, University of
Regensburg, Universit~tsstr. 31, 8400 Regensburg (F.R.G.)
Abstracts of posters
A SIMPLE METHOD FOR THE ISOLATION OF PLANT VOLATILES
T.A. van Beek and H. van Bokhoven
Volatiles play an important biological role in both the
animal and plant kingdom. Usually the concentration in
which these mediators occur in air are too low for direct
measurements by today's analytical techniques. Therefore
the compounds have to be concentrated from a large volume
of air (headspace) around the plant. This can be done by
coldtrapplng or absorption on porous polymers or charcoal.
The latter method has some advantages r The desorptlon is
usually carried out by heating the material. The method ha~
as drawbacks that a complex apparatus is needed, that uo
second analysis can be carried out and that thermal degradation can occur. Desorption by an organic solvent does not
suffer from these problems. Although this method has been
used for the collection of air pollutants and pheromones,
not much research has been carried out concerning the
influence of the different steps of the concentration process on the recovery percentage and the type of compounds
recovered.
With ten model compounds, several variables in the concentration process were studied. Finally one method was chosen
which gave satisfactory results. This method consisted of
four steps: 1) flushing the plant material with highly
purified air; 2) leading this air through a Tenax tube; 3)
desorblng the Tanax with pentane-acetone in a micro Soxhlet
and 4) concentrating the solvents to a small volume with a
Vigreux column at low temperature. Compounds with a boiling
point between [30 and 300"C and occurring in the headspace
in a concentration of ca. I ug/l air could be recovered for
90% in the organic solvents in a concentration of 0.5 ~g/ui
solvent. This solution can he studied by means of GC, GCMS, HPLC, bioassays or, in case of a large quantity of
plant material, after evaporation even by NMR.
A practical result of the method will be shown.
Department of Organic Chemistry, Agricultural University,
De DreiJen 5, 6703 BC Waganlngen (The Netherlands)
EFFECTS OF LIGHT ON ANTHRACENE DERIVATIVE PRODUCTION [N
SUSPENSION CULTURES OF Rhcom~us purshiana
A.J.J. van den Berg, M.H. Radema and R.P. Labadie
The dried bark of Rhamnua purshs
is applied medicinally
as laxative. The main active principles are glycosides of
1,8-dlhydroxyanthracene derivatives.
When suspension cultures of R. purahiana had been established, it was shown that the pattern of accumulating
"antbra-derlvatives" (~.e. [,8-dlhydroxyanthraquinones,
-anthrones and/or -dlanthrones) differed from that in the
intact bark. Suspension cultures grown in the dark accumulated predominantly glycosides of mainly physelon (and/or .
its reduced forms), whereas in the original bark emodln and
aloe-emodln glycosides dominated (1,2).
A daily photoperlod of 12 h beneficially affected the accumulation of anthra-derivatives in the suspension cultures.
The emodln (and to a lesser extent chrysophanol) content of
the cultures increased significantly by a [2 h llght/dark
cycle, whereas the levels of physclon were much less affected. The formation of anthra-derlvatlves, however, was
strongly suppressed when the cultures were continuously
illuminated. Thus, an alternation of light and dark periods,
in a daily cycle, was of slgnfflcant importance to enhance
the production of anthra-derlvatlves In suspension cultures
o f R. puPahs
[ . A . J . J . v a n d e n B e r g and R . P . L a b a d i e , P l a n t a M e d . , 50
([984) 449-451
2. A.J.J. van den Berg, Production of anthraqulnones,
anthrones and dianthrones by plant cell cultures of
Rhamnua pupahiano and Rhsmnua fpanguZa, Thesis, 1987,
State University of Utrecht
Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, Catharijneslngel 60, 3 5 1 1 G H Utrecht
(The Netherlands)
222
V o l . 9 - I987
Pharmaceutisch Weekblad Scientific Edition
ANTIVIRALLY ACTIVE CONSTITUENTS OF Spondias mombin L.
J. Corthout*, L. PietersP, M. Claeys*, D. Vanden Eerghe O
and A.J. Vlietinck*
A RAPID SCREENING METHOD FOR HELENALIN DERIVATIVES IN
FLOWERS OF A~iea mo~tcrna
F.C. Fischer*, P.W.J. Caspers* and M.J.M. GiJbels O
The results of a broad screening program for various chemotherapeutical and antiviral properties of higher plants
showed that an ethanollc extract from the l e a v e s and stems
of Spondias mombin L. (Anacardiaceae) exhibited a pronounced sntiviral sctlvlty against poliomyelitis, coxsackie
B 4 and herpes simplex 1 viruses. The ethanollc solution was
c o n c e n t r a t e d u n d e r reduced pressure and the residue acidified with HCI and then extracted wlth carbon tetraehlorid~
dlethyl ether and a mixture of butanol-methylethylketone
(III), respectively. Fraction III showed an antl-herpes
activity (Rf : 103); it was subjected to column chromatography (CC) on Sephadex LH-20 (gradient elution), to CC
on silanized silica gel (gradient elutlon), and to DCCC in
the descending mode.
One of the fractions obtained was further purified by
preparative TLC and at last by CC on Sephadex LH-20 which
resulted in the isolation of chlorogenic acid and its butyl
ester.
Their spectroscopic data as well as data of their ~n Pi~Po
antivlral properties will be presented. Several antlvirally
active polyphenols were isolated from other fractions. The
structures of t h e s e substances i s currently determined and
will also be discussed.
Preparations of arnica flowers are used externally for
their antiinflammatory activity. The pharmacological action
is attributed to the sesquiterpene lactones helenalln,
dihydrohelenalin and their esters (1,2). Arnica flowers
from different origins, cultlvated or collected in the wild,
are commercially available. The content of helenalin and
derivatives is found to vary to a large extent. I n o r d e r to
control a constant quality of the pharmaceutical preparations, a fast and simple screening method for the active
sesquiterpene lactones was needed. These compounds, containing cyclopentenone and/or ~-methylene y-lactone moieties, are apt to react with nucleophilic groups like -NH2,
-SH and -SO2H. By incorporating these groups in polymeric
materials it was possible to remove the lactones from
extracts (3). The amount of reactive compounds can be
deduced from the difference between UV spectra taken before
and after column passage. A number of polymers were tested.
Sulflnated polystyrene, amlnomethylated polystyrene and
(di)aminopropylsilica were unsatisfactory, but a basic
polyacrylic resin, Serdolit AW 14, yielded good results.
I. G. Willuhn, Pharm. u. Z., i0 (1981) I
2. l.N. Hall et aZ., Planta Mad., (1987) 153
3. A. Cheminat et aZ., Can. J. Chem., 59 (1981) 1405
* Department
* Department of Pharmaceutical Sciences, University of
Antwerp (UIA), 2610 Wllrijk (Belgium)
O Department of Medicine, University of Antwerp (UIA),
2610 Wilrijk (Belgium)
REMOVAL OF PYRROLIZIDINE ALKALOIDS FROM Symphyt~ TINCTURES
THEIR TOXICITY TOWARDS ISOLATED REPATOCYTES
P.M.v.d. Dungen*; N.P.E. Vermeulen*, F.C. FischerQ, L.A.
't Hart 0 and M.J.H. GiJbels V
Root extracts of Symphytum offic~nale L. are ingredients
of pharmaceutical pCeparations for external use. Symphytum
extracts have a serious drawback: their content of pyrrolizldine alkaloids (PA). PA are very toxic and have no known
beneficial effect (I). It would be desirable to have them
removed from the extracts. Using ion exchange resins we
succeeded in removing PA from the extracts and in retaining
the useful compounds. Doing so we obtained a crude PA Noxide fraction, which was purified and reduced to a mlxture
of PA, that was used for a quantitative assessment of
cytotoxicity towards isolated hepatocytes. However, the
release of lactic acid dehydrogenase, a feature of the
dying cells used in the test, was unobservable because the
PA effectively inhibited its activity. Then, cellular death
was determined by microscopic observation using trypan blue
vital staining. The PA proved to be very toxic to
hepatocytes.
From the root extract we also isolated a polysaccharide
fraction, consisting mainly of fructose and glucose, which
showed immunological properties, e.g. it inhibited the
classical pathway of the complement system.
I. A.R. M a t t o c k s , Chemistry and Toxicology of Pyrrolizldlne
Alkaloids, 1986, Academic Press, London
* Department of Pharmacochemistry-Molecular
Toxicology,
Free University, Amsterdam (The Netherlands)
0 Department of Pharmacognosy, Faculty of Pharmacy, State
University o f Utrecht, CatharlJneslngel 60, 3 5 1 1 G H
Utrecht (The Netherlands)
V Laboratorium Biohorma, Elburg (The Netherlands)
o f Pharmacognosy, Faculty o f Pharmacy, State
Unlversity of Utrecht, Catharljnesingel 60, 3 5 1 1 G R
Utrecht (The Netherlands)
O Laboratorlum Biohorma, P.O. Box 33, 8080 AA Elburg
(The Netherlands)
SCREENING OF SECONDARY PLANT SUBSTANCES IN SMALL ERMINE
MOTHS, Yponomeuta SPP (LEPIDOPTERA:YPONOMEUTIDAE)
S.Y. Fung and W.M. Herrebout
Larvae of European small ermine moths, Yponomeuta spp.,
feed on plants of the families Celastraceae, Rosaceae,
Salicaceae and Grassulaceae. Results of preliminary feeding
trials showed that some small ermine moths were unpalatable
to birds. As most insects acquire their unpalatability by
storage of specific secondary substances from their foodplants, we decided to screen small ermine moths and their
host plants on substances which could account for the
moth's distatefolness. Y. maha~ebeZ~s, the larvae of which
feed on Prunus n~haZeb, a rich source of coomarins, was
found to contain coumarin, umbelliferone and herniarin (l).
A simple butenolide, siphonodin, was isolated from
Euonymus euPopaeus, host plant of several Yponomeuta
species (2). An isomer of siphonodin, isosiphonodin, was
detected in extracts ef seven small ermine moths (2).
I. S.Y. Fung and W.M. Herrebout, Coumarins in PPunus
mahaZeb and its herbivore, the small ermine moth
Yponomeuta maha~ebe~Zus, J. Chem. Ecol., in press
2. S.Y. FunK, W.H. Herrebout, R. Verpoorte and F.C.
Fischer
Butenolldes in small ermine moths, Yponomeuta
spp. (Lepidoptera:Yponomeutidae),
and spindle-tree,
E~onymus europaeus ( C e l a s t r a c e a e ) , J . Chem. Ecol.,
i n press
Department of Population Biology, University of Leiden,
Sehelpenkade 14a, 2313 ZT Leiden (The Netherlands)
V o l . 9 - I987
Pharrnaceutisch Weekblad Scientific Edition
223
SIMULTANEOUS FLAME-IONISATION AND NITROGEN-SPECIFIC
DETECTION AND RETENTION INDICES IN GAS CHROMATOGRAPHY OF
STEROIDAL ALKALOIDS
W.M.J. van Gelder
PRODUCTION OF SOME ASPIDOSPERMATAN INDOLE ALKALOIDS BY A
SUSPENSION CULTURE OF Tc~bez,v~temont.a~ diuo.rs
R. van der HeiJden, G.M. van der Graaf, E.J.M. ~ennings
and R. V e r p o o r t e
Capillary GC for analysis of the composition of steroidal
alkaloids (SA) showed that several unidentified compounds
were present in Solansceae plant material, in addition to
SA which w e r e identified using reference substances ( I , 2 ) .
In the screening for SA in plant breeding programmes,
characterization of each unknown peak in a chromatogram by
costly spectrometric techniques is not feasible. Therefore,
a method was developed to distinguish peaks of unknown SA
and peaks of non-alkaloldal constituents. The method applies
a dual detector system, consisting of an FID and a nitrogen/
phosphorus-speclflc detector (NPD), connected to a twochannel system for integrated processing of FID and NPD
data. Calibration curves constructed for the FID and the
NPD were linear for all SA tested. NPD/FID response ratios
appeared to be useful as an aid in identification and
characterization of compounds. A number of SA, sterols and
steroidal sapogenlns were separated by capillary GC. The
combined NPD/FID system enabled unambiguous differentiation
between the SA and the non-nltrogen containing compounds.
Retention indices were sufficiently reproducible for identification of these compounds, even for the closely eluting
45 and 5e-eglycone pair solanidine and demissldine.
[. W.M.J. van Gelder, Steroidal alkaloid composition of
tubers of exotic So~anu.m species of value in potato
breeding determined by high-resolution gas chromatography. In: Potato Research for Tomorrow, A. Beckman,
ed., 1986, PUDOC, Wageningen, pp. 166-170
2. W.M.J. van Gelder and O.M.B. de Pontl, Euphytica, 36
([987)
Suspension cultures of PoJ>er'~u~emontar~z dlvaPieata are
capable of producing a wide variety of indole alkaloids
belonging to different classes, such as ibogan, plumeran,
aspldospermatan and corynanthean. By screening of several
cell lines we obtained a cell llne which produced the
aspidospetmatan alkaloids valleeamine and O-acetyl-vallesamine as major products. Their biosynthesis in a batch
culture was studied by means of HPLC in combination with
diode array detection and cell free incubation experiments
of their precursors stemmadenine and etemmadenlne-N4-oxlde.
Diode array ffetectlon proved to be a powerful tool in this
study, because substrata, intermediates and products showed
different UV maxima or even completely different chromophores. Chromatograms obtained for the cell free incubation
experiments showed great similarity with those obtained for
the extracts of the batch culture.
The results indicated a non-enzymatlc conversion of
stemmadenlne to eondylocarplne during lag and growth phases,
while during the stationary phase vellesamlne was produced
and acetylated.
Foundation for Agricultural Plant Breeding (SVP), P.O. Box
117, 6700 AC Wagenlngen (The Netherlands)
ANIONIC POLYMERS WITH ANTI-COMPLEMENTARY ACTIVITY FROM
AZoe vePa GEL
L.A. 't Hart, P.H. van Enckevort and R.P. Labadle
The mucilagenous gel of AZoe uera leaf (L.; Burm. f.) is
used among others for t h e treatment of infections (I).
Preliminary studies on the incubation of human serum with
an aqueous extract of the gel showed an antl-complemen~ary
activity. The present study deals with a definition of this
particular activity. The results suggested that the inhibition of the classical and alternative pathway activity was
due t o the consumption of a complement factor(s). The
extract was enriched as to its anti-complementary activity
using ion-exchange and gelfiltration chromatography. The
results indicated that the anti-complementary components
were anionic macromolecules.
In accordance with literature
data (2) analysis of the polymer fraction of the extract
revealed the presence of polysaccbarldes,
probably glucomannans.
The role of the anionic groups in the molecules, most
likely glucuronlc acid, will be discussed.
I. D. Grindlay and T. Reynolds, J. Ethnopharmacology,
16
(1986) 117
2. C.G. Gowda, g. Neellslddalah and Y. AnJaneyalu,
Cerbohydr. Res., 72 (1979) 201
Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, Catharljneslngel 60, 3 5 1 1 G H Utrecht
(The Netherlands)
Division of Pharmacognosy, Center for Blo-Pharmaceutlcal
Sciences, Leiden University, Gorlaeus Laboratories, P.O.
Box 9502, 2300 RA Leiden (The'Netherlands)
FROM OPIUM TO ILLICIT HEROIN
H. Huieer*, K. van Zuilen O and A. Baerhelm Svendsen 0
A major part 0$ the opium produced world-wlde is used for
the production of illicit heroin. The production comprises
isolation of the crude morphine (and purification), foll~wed by acetylatlon of the morphine and purification of
the heroin formed. The quality of the illicit heroin depends on the origin of the opium and the method of heroin
preparation. In spite of the varying composition of the
crude opium used and the rather primitive circumstances for
the heroin preparation, the old "Hong Kong Heroin" was
quite pure, whereas other types of illicit heroin originating from south-western Asia, contain a number of other
opium alkaloids and related compounds. Special attention
will be paid to the influence of the morphine isolation
procedure both on the quality of the crude morphine and on
the purity of the heroin. The influence of the acetylation
procedure on other opium alkaloids will also be discussed.
Several minor constituents of illicit heroin, originating
directly or indirectly from opium, often give 'fingerprints'
most useful for comparative analyses of illicit heroin
samples.
* Forensic Science Laboratory, Ministry of Justice,
Volmerlaen 17, 2288 GD RiJswlJk (The Netherlands)
0 Division of Pharmacognosy, Center for Bio-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories,
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
224
ISOLATION OF TWO COMPONENTS FROM LATEX OF Jatropha
muZtifida L., WHICH INHIBIT CLASSICAL PATHWAY COMPLEMENT
ACTIVITY in vitro
S. Kosasi*, L.A. 't Hart O, F.C. Fischer O and R.P. Labadie 0
In Indonesia the use of traditional medicinal plants has
become increasingly popular. However, only few laboratory
evaluations have been carried out. One of the plant products which is widely used as first aid antiseptic in Java,
is the latex of JatPopha muZtifida L., called "Jodium".
Two components were isolated from the l a t e x on the guidance
of the inhibition of the classical pathway complement activity. One was a proanthocyanidin. Isolation and identification of proanthocyanidins was performed as described by
Haslam (I). It was found that the inhibitory activity was
based on selective Cs ++ binding. Ca ++ is an essential element in the first step of t h e classical pathway complement
activity, namely for the binding of C 1 to sensitized sheep
erythrocytes. The second compound was a diterpene derivative. For the isolation a method developed by Evans (2) was
used. The diterpene also showed a strong antl-complementary
activity. The mechanism of the inhibition is subject t o
further investigations.
1. E. Haslam, Proceeding of Fourth Hungarian Bioflavanoid
Symposium, Kezthely 1973
2. F.J. Evans, Lloydia, 41 (1978) 3
* Graduate School, University of Santo Tomas, Manila
(Phillipplnes)
O Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, CathariJneslngel 60, 3 5 1 1 G B
Utrecht (The Netherlands)
V o l . 9 " z987
Pharmaceutisch Weekblad Scientific Edition
TWO FUNCTIONALLY DIFFERENT IMMUNOMODULATORS FROM AN AQUEOUS
BARK EXTRACT OF Aaadipachta indies A. JUSS. (MELIACEAE)
J.M. van der Nat, L.A. 't Hart, W.G. van der Sluls and
R.P. Labadie
Aqueous bark extracts of A. indica have been used f o r
centuries in traditional medicine in various Asian countries
because of t h e i r anti-rheumatic, anti-inflammatory and skinhealing properties. Recently, we.reported that bark snd
leaf extracts of the tree had been shown to possess antiinflammatory and anti-tumour activities (I). These data led
us to the assumption that components in A. indies bark
might affect the human immune system. Using in nitro tests,
we were able to demonstrate an anti-complementary activity
in s crude aqueous bark extract. Furthermore, we observed
an inhibition of the production of oxygen radicals by
activated polymorphonuclear leukocytes (PMN) and an increase
in the production of mlgratlon inhibition factor by lymphocytes (2,3). This prompted us to fractionate the crude
extract on the guidance of the inhibitory effects on activation of the complement cascade (alternative pathway) and
PMN. Using methanol extraction, ethanol precipitation,
ultra-filtration, ion-exchange and gel-permeation chromatography two functionally different fractions were obtained.
A methanol-insoluble, high molecular weight ( > 10 kD)
saccharide-containing highly enriched fraction showed a
strong inhibition of the alternative pathway complement
activation. A methanol-soluble, low molecular weight (< I0
kD) fraction showed a strong inhibitory effect on the
production of oxygen metabolites by activated PMN.
I. J.M. van der Nat, W.G. van der Sluis, A.H.J.M. de Haan,
K.T.D. de Silva and R.P. Lsbadie, Planta Mad., (1986) 552
2. J.M. van der Nat, J.P.A.M. Klerx, H. van DiJk, K.T.D. de
Silva and R.P. Labadie, Plants Med., (1986) 428-9
3. J.M. van der Nat, J.P.A.M. Klerx, H. van DiJk, K.T.D. de
Silva and R.P. Labadie, J. Ethnopharmacol., in press
Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, CathariJnesingel 60, 3 5 1 1 G H Utrecht
(The Netherlands)
INFLUENCE OF L-TRYPTOPHAN AND SOME OF ITS PRECURSORS ON
GROWTH OF CELL SUSPENSION CULTURES OF Cinchona ledgeriana
T. van der Leer*,' S.M. ten Hoope*, P.A.A. Harkes0, 9R.
Verpoorte* and A. Baerheim Svendsen*
HPLC ASSAY OF STRICTOSIDINE SYNTHASE FROM PLANT CELL
SUSPENSION CULTURES
E.J.M. Pennings, R. v.d. Bosch, J.A. Duine, R. v.d. HeiJde~
T. v.d. Leer, J. Schripsema and R. Verpoorte
In previous e x p e r i m e n t s , L - t r y p t o p h a n p r o v e d t o be extremely toxic for a flne cell suspension culture of Cinchona
ledgeriana Moens.
F u r t h e r experiments were performed to study the metabolism
of L-tryptophan and its precursors in more detail, in connection with the possible link with the biosynthesis of
indole acetic acid. The cell cultures were grown in the
presence of indole, anthranllic acid, L-tryptophan,
tryptamine and combinations thereof. In none of the cultures any quinoline alkaloids were produced.
Growth was promoted in the presence of both tryptamine
and indole.
The growth inhibiting effect of L-tryptophan could partly
be diminished by addition of anthranilic acid.
Anthraquinone production was inhibited by addition of
indole.
Strictosidine synthase (SSS) is the enzyme catalyzing the
stereospecific condensation of tryptamine and secologanin.
This reaction may be the committing step in the formation
of monoterpenoid d e r i v e d indole alkaloids. As we a r e
studying the regulation of activities of the enzymes of the
indole alkaloid pathway, we have developed a rapid method
for assaying SSS based on UV detection (280 nm) of strictosidine after BPLC on a LiChrosorb RP-8 Select g column.
Incubation was carried out in 0.I M phosphate buffer pH 6.8
containing 0.5 mM tryptamine, 2 mM secologanin, and 3 mM
dithlothreitol. After 30 min at 35 ~ the reaction was
stopped by adding methanol and cooling the tubes in ic~.
A known amount of codeine was added as internal standard.
The HPLC eluent consisted of methanol-water (67:33 v/v),
pH 6.0, containi.ng "7 mM sodium dodecylsulfonic acid and
lO mM potassium dihydrogenphosphate. The retention time for
strletosidine was 4.8 min and the detection limit was about
3 ng. The geactlon was linear for at least 3 h, and the
amount of ~trictosidine formed was equal to the amount of
tryptamlne disappeared. Some properties of the enzyme will
be presented.
* Division of Pharmacognosy, C e n t e r for Bin-Pharmaceutlcal
Sciences, Leiden University, Gorlaeus Laboratories,
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
O Department of Plant Molecular Biology, Leiden University,
Nonnensteeg 3, 231[ VJ Leiden (The Netherlands)
Division of Pharmacognosy, Center for Bin-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories,
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
RAPID ASSAY OF TRYPTOPHAN DECARBOXYLASE FROM PLANT CELL
CULTURES
E . J . M . P e n n i n g s , R. v . d . Bosch, R. v . 6 . H e i J d e n , T. v . d .
Leer and R. V e r p o o r t e
Tryptophan decarboxylase (TDC) is the enzyme generating
tryptamlne in plant cells. The enzyme may function as a
key enzyme for the production of indole alkaloids. The aim
of the present study was to obtain a rapid assay of TDC as
the activity of the enzyme rapidly declines during its
purification. The assay was based on the fluorometrlc
detectlon of tryptamlne in HPLC. Incubation was carried out
in 0.I M Tris-BCl buffer, pB 7.4, in the presence of 2.0
mM L-tryptophan, 0.2 mM pyrldoxal-5'-phosphate,
3 mM
dithiothreitol and 50 ~I of the crude enzyme preparation.
After an incubation period of 30 min at 35~ t h e reaction
was stopped by addition of methanol. A known amount of 5methyl-tryptamlne was then added as internal standard.
HPLC was carried out on a LiChrosorb RP-8 Select g column.
The eluent contained 7 mld sodium dodeeylsulfonlc acid and
10 mM potassium dlhydrogenphosphate in methanol-water
(67:33 v/v), pH 6.0. The detection limit for tryptamlne
was 0.3 ng. Using this assay we were able to detect as
little as 0.02 pkat of enzyme activity.
Crude TDC is a labile enzyme; at 40C half of its activity
is lost in 4 days; at - 1 5 ~
the half llfe time is about 7
days. Partially
p u r i f i e d p r e p a r a t i o n s showed an i n c r e a s e d
stability, in particular when stored a t -800C. After DEAESephacel chromatography, TDC retained its activity at
-80"C for at least two months. Thanks to this assay we are
able to purify TDC from Cat~z~anthu8 roseu8 within two
weeks using five purification steps.
225
INVESTIGATION OF THE FLAVONOIDS OF BPukenthalia
AND THEIR MOLLUSCICIDAL ACTIVITY
M.M. 8aleh*, R.Z. Abbas* and J.B. Zwavlng 0
apicuZifoZia
Although chemotherapy is one of the most valuable methods
for the control of schistosomiasis, there is a need for
more selective and efficient molluscicides for the control
of the snail vector. The application of chemical
molluscicides is highly expensive and produce toxic effects
on the water fauna. The use of plants with mollusclcidal
propertieg is a simple, inexpensive and appropriate technology.for the snail control.
Two flavonoids were isolated from the methanolic extract of
t h e d e f a t t e d p l a n t powder of Br~kenthaZs spieu~ifo~ia.
Both flavonoids were aglycones and they were identified as
genistein and'dihydrorobinetin. Their identity was confirmed by UV, IR, MS and NMR analyses.
Molluseicldal screening tests of the isolated compounds
were made according to the method recommended by the WHO
(I). The LCso, LC98, slope and 95% confidence limits were
calculated according to the method of Litchfield and
Wilcoxon (2). The screening showed that genistein was more
potent than dlhydroroblnetln; the LCs0 were 30 and 89 ppm
respectively for BiomphaZars alexandrEna, and 13 and 70
ppm respectively for Bulinua truncatua snails.
I. WHO Technical Report Series, (1961) 214
2. J.T. Litchfield and F. Wilcoxon, J. Pharm. Exper.
T h e r a p . , 96 (1949) 113
Division of Pharmacognosy, Center for Bio-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories, P.O.
Box 9502, 2300 RA Leiden (The Netherlands)
* National Research Centre, DokkL Cairo, Egypt
O Department of Pharmacognosy, State University of Groningen
Ant. Deusinglaan 2, 9713 HW ~ronlngen (The Netherlands)
VACUUM LIQUID CHROMATOGRAPHY AND QUANTITATIVE IN-NMR
SPECTROSCOPY OF TUMOR-PROMOTING DITERPENE ESTERS
L.A. Pieters, J. Tottg, J. Corthout and A.J. Vlietinek
THE ESSENTIAL OIL OF Calamintha nepeta ( L . ) SAVI SSP.
nepeta GROWN IN TURKEY
E. ~arer*, A. Looman O, J.J.C. Seheffer 0 and A. Baerhelm
Svendsen O
Plants and plant products form a major part of human and
animal intake. Therefore the evaluation of plants for
carcinogenic or tumor-promoting constituents should receive
high priority. A phytochemlcal screening method was de:
veloped for the identification and the quantitative determination of tumor-promoting dlterpene esters.
Tumor-promotlng phorbol-12,13-diesters~ and cryptic tumorpromoting phorbol-12,13,20-trlesters were isolated from
croton oil, the seed oll of CPoton tig~iwn L. (Euphorblaceae), by rotation locular eountercurrent chromatography
(I). The diester fraction, which consisted of three different types of phorbol-12,13-dlesters
(the A- and B-serles,
which contain a long-chaln and a short-chain acyl substltuent, and the short-chain diesters, with two short-chaln
acyl substituents), was separated using vacuum liquid
chromatography. All compounds were identified by 13C_NM R
spectroscopy, and quantified by IR-NMR spectroscopy (1,2).
The same techniques were applied for the analysis of ingenol
esters from Euphorbia ingens E. Mey.
I. L.A. Pieters and A.J. Vlletlnck, Planta Med., (1986)
465-468
2. L.A. Pieters and A.J. Vlietlnck, 13C-NMR Spectroscopy
of Phorbol Esters, Magnetic Resonance in Chemistry,
in press
Department of Pharmaceutical Sciences,
Antwerp (UIA), 2610 WilriJk (Belgium)
University
of
Flowering parts of Calamintha nepeta (L.) Savi ssp. nepeta
wgre collected September 1983 at three locatles in Turkey.
The air-dried material was submitted to hydrodistillatlon
for 3 h. The essential oils were analyzed by capillary
GLC on columns of different polarity. Two of the oll
samples, collected in Adana, were very much alike.
They were characterized by large amounts of plperltone
epoxides (about 50-60%). The third sample, collected in
Hatay, showed a totally different
fingerprint, since it
contained no piperitone epoxides at all, but large amounts
of carvacrol (about 60%). Then we analyzed the herbarium
specimen of the three samples, as well as the isolated oils
by means of headspace GC (HSGC).
Comparison of the composition of the oils with that of the
corresponding herbarium samples showed no large differences
for the samples from Adana. Only plperltenone epoxide
was found in a lower concentration in the herbarium samples
analyzed by BSGC.
The fingerprint of the herbarium specimen collected in
Hatay was totally different from that of the oll and from
the samples collected in Adana.
* Faculty of Pharmacy, Ankara University, Tandogan-Ankara
(Turkey)
O Division of Pharmacognosy, Center for Bio-Pharmaceutlcal
Sciences, Leiden University, Gorlaeus Laboratories,
P.O.Box 9502, 2300 RA Leiden (The Netherlands)
226
VOI. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
DETERMINATION OF THE GROWTH CURVE IN A SINGLE CULTURE FLASK
J . S c h r i p s e m a * , A.H. H o l i e r * , C.R. L a n g e z a a l * , P.A.A.
Harkes O, R. V e r p o o r t e * and A. Baerheim Svendsen*
3-METHOXYFLAVONES, ANTIVIRALLY ACTIVE SUBSTANCES FROM
HIGHER PLANTS
L. Van Hoof*, D.A. Vanden Berghe* and A.J. Vlietinck O
Each point of a growth curve is usually determined by
harvesting the contents of a cell culture flask and determination of the fresh and/or dry weight of the cells. This
method is laborious and inaccurate because of the differences between single flasks (e.g. inoculum, oxygen supply,
light). So, there is a need for another method to determine
growth curves, preferably in a single culture flask.
Because most plant cell cultures are not capable of photosynthesis, a carbon source must be provided, which is converted to blomass and dlssimilated to provide energy (about
50/50). In several experiments a linear relationship was
found between the sugar contents of the medium and the dry
weight of the cells; so, measurement of the sugar concentrations can be used to follow the growth. Although in this
way a single flask experiment is possible, a disadvantage
of the method is the need of frequent aseptic sampling.
However, dissimilation of sugars should cause a loss of
weight of the culture flask, which should be correlated
with growth. The loss of weight due to dissimilation was,
however, relatively small compared to the loss of weight
due to water evaporation. This evaporation depends on the
closure of the flask and of the relative humidity of the
c~iture room. Changes in this humidity can be corrected
for, by using blanks. The closure of the flask was the
critical point. It should be stable and tight enough to
avoid drying, but the oxygen need of the cell culture
should be satisfied. Only silicon stops met all requirements.
Optimization of the different factors resulted in a simple
method for accurate determination of growth curves in
single culture flasks.
In previous studies we examined the antiviral activity of
3-methylquercetin, a flavonoid isolated from Euphorbia
gPoJztii Oliv. (1). The replication of picornaviruses is
inhibited at the level of the RNA synthesis (2,3). The
present study deals with the antivlral activity of other
Euphorbia species. The inhibitory effect could be assigned
to the presence of 3-methoxyflavones. The parent structures
are 3-methylquercetln and 3-methylkaempferol, indicating
the necessity of the 3-methoxy group. Some plants from
other families are showing the same typical antipicornaviral effects. Among them are Fernonia a.,mjgdaZina Delile
(Asteraceae) and Dodonea vlaeosa (L.) Jacq. (Sapindaceae).
The correlation between the use of these plants in traditional medicine, and the detected antipicornaviral activity will be discussed.
I. L. Van Hoof, D.A. Vanden Berths, G.M. Hatfleld and
A.J. Vlietinck, Plants Med.," 50 (1984) 513-517
2. L. Castrillo, D.A. Vanden Berghe and L. Carrasco,
Virology, 152 (1986) 219-227
3. R. VriJsen, L. Everaert, L. Van Hoof, A.J. Vlietinck,
D.A. Vanden Berghe and A. Boey~, Antiviral Res., 7
(1987) 35-42
* Division of Pharmacognosy, Center for Bio-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories,
P.O. Box 9502, 2300 RA Leiden (The Netherlands)
0 Department of Plant Molecular Biology, Leiden University,
Nonnensteeg 3, 2 3 1 1 V J Leiden (The Netherlands)
ANALYSIS OF HYDROPHILIC AND LIPOPHILIC IMMUNOMODULATORY
FACTORS FROM THE ROOTS OF P~cPoPhia~ kuPPoa
J.M. Simons, L.A~ 't Hart and R.P. Labadle
Picrorhiza kurroa Royle ex Benth. (Scrophulariaceae)
is a
small herb which grows in the Himalayas from Kashmir to
Sikkim at an altitude of 2790-5000 meters (I). The subterranean parts are extensively used in the indigenous
systems of medicine of India, Sri Lanka, China, and other
eastern countries as a (hitter) tonic and stomachic. The
drug is mainly used in fever, dyspepsia, liver and lung
diseases, but also in many other conditions of illness.
A possible explanation for the broad medicinal application
of P. ku-~Poa might be an action on the immune system. In
a recent screening of plants used in Srl Lanka for their.
influence on the human complement cascade (2) and on the
production of migration inhibition factor (MIF) by human
lymphocytes (3), P. ku~Poa showed an inhibitory effect in
b o t h assays. The present study was aimed at the characterization of the immunomodulatory components. Therefore
an aqueous and a chloroform extract were tested for anticomplementary activity and for their effect on zymosaninduced chemiluminescence of human polymorph nuclear cells
(PMN). The most p o t e n t antlcomplementary activity was
found for the chloroform extract, whereas both extracts
showed a comparable inhibitory effect on the zymosaninduced production of oxygen metabolites.
I. P.N. Mehra and S.S. Jolly, Res. Bull. Punjab. Univ.,
[9 ([968) 141-156
2. J.M. van der Nat et aZ., Acts Agron. Acad. Sci. Hung.,
34 suppl. "(1985) I03
3. J,M. van der Nat et aZ., Acta Agron. Acad. Sci. Hung.,
34 suppl. (1985) lOl
Department of Pharmacognosy, Faculty of Pharmacy, State
University of Utrecht, CathariJnesingel 60, 351[ GH Utrecht
(The Netherlands)
* Deparfment of Microbiology, University of Antwerp (UIA),
2610 WilriJk (Belgium)
O Department of Pharmacognosy, University of Antwerp (UIA),
2610 Wilrljk (Belgium)
In vitPo CYTOTOXICITY OF SESQUITERPENE LACTONES FROM
Eupatorlum eanno~inum AND SEMI-SYNTHETIC DERIVATIVES FROM
EUPATORIOPICRIN
H.J. Woerdenbag*, H. Hendriks*, K. van der Berg O, R. van
Stralen O, Th.M. Malingr~* and A.W.T. Konings V
Sesqulterpene lactones from Eupatorium eanrtabinum L. p o s s e s s cytoCoxic properties ~n vitro ([). The principal sesquiterpene lactone, eupatorloplcrin (EUP), was proved Co
act as a cytostaCle agent in tumour bearing mice (2,3).
In an attempt to obtain compounds that are more active than
EUP and in order to attain more insight in structure activity relationships, [3 semi-synthetics from EUP were prepared. The compounds were screened for cytotoxlclty ~n
vltPo against the murine cell lines EAT (Ehlich Ascltes
Tumour), LSITSY (lymphoma), P388 (leukemia) and FIO 26
(flbrosarcoma) and the human Hela (cervix uteri) cell line,
using the LAD (Lowest Active Dose) test (4). The cyto(oxic
effects were compared with five sesquiterpene lactones from
E. oa~r~binum (EUPL eupatolide, hiyodorilactone E, eucannab i n o l i d e and eo~pound I ) . O x i d a t i o n of one of the hydroxyl
groups in the ester side chars of EUP, resulting in a mono
a l d e h y d e , enhanced c y t o t o x i c l t y ,
as d i d a c e t a l l s a t i o n
of
b o t h h y d r o x y l g r o u p s w i t h a c e t o n e . I n t r o d u c t i o n of more
bulky grodps via acetallsatlon
r e v e r s e d the enhancement.
F s u b s t i t u t i o n o f a c e t o p h e n o n e as b u l k y group had a p o s l t l v ~
CI of Br substitution a negative influence. Oxidation at
the germacrane ring structure, yielding an alcohol or an
epoxy derivative, affected cytotoxicity adversily. Whether
optimized cytotoxlcity corresponds with better therapeutic
activity, will be determined In the murlne tumour model.
I. H.J. Woerdenbag et aZ., Planta Hod., (1986) [12-4
2. H.J. Woerdenbag et aZ., Plants Med., (1987), in press
3. H.J. Woerdenbag et aZ., Phytotherapy Research, in press
4. H.B. Lamberts et aZ., Oncology, 40 (1983) 30[-4
* Department of Pharmacognosy, State University of
Groningen, Ant. Deusinglaan 2, 9F13 AW Gronlngen
(The Netherlands)
0 Department of Organic Chemistry
V Department of Radiopathology
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
ENHANCED CYTOSTATIC EFFECT OF INTRAVENOUSLY ADMINISTERED
EUPATORIOPICRIN IN TUMOUR BEARING MICE AFTER DEPLETION
OF GLUTATNIONE
H.J. Woerdenba~*, W. Lemstra O, Th.H. Malingr~* and
A.W.T. Konings u
The principal sesquiterpene lactone from Eupatori~ cannabin ~ L., eupatoriopicrln (EUP), exhibits cytostatie activity.
The mechanism of action is not known as yet. It has been observed that EUP affects the level of reduced glutathione
(GSH) in vitro as well as in vivo (I). However, the lowering of GSH content as such may not be considered as the
cause'of cytoetatic activity, especially because depleting
GSH with BSO did not influence
tumour growth at all.
Because EUP can easily react with biological nueleophilic
compounds, it was of interest to investigate whether or not
GSH acts as protective system against cytostatic activity.
For this reason C57B1 mice, bearing a solidly growing Lewis
Lung carcinoma or a FI0 26 fibrosarcoma, were injected i.v.
with EUP (20 or 40 ms/ks). Tumour development was recorded
by measuring the tumour volume with calipers. Compared with
a non-treated control group, tumour growth was delayed
significantly. In a different experiment mice were treated
with BSO 4 mmol/kg, i.p., 6 h before i.v. injection of EUP.
Tumour GSH levels were reduced by the BSO treatment to
about 60% at the moment of injecting EUP. Tumour growth in
these pretreated animals was significantly retarded compared with the group receiving EUP only. It is concluded
that GSH in tumour tissue might play a role in the defense
mechanism against the cytostatic action of EUP.
|. H.J. Woerdenbag, W. Lemstra, Th.M. Malingr~ and A.W.T.
Konings, Pharm. Weekbl (Sci)., 9 (1987) |5g
* Department of Pharmacognosy, State University of
Groningen, Ant. Deusinglaan 2, 9713 AW Groningen
(The Netherlands)
O Department of Radiopathology
FURANOSESQUITER~ENES FROM ECUADORIAN Senecio caneacens
S. Abdo*, M. De Bernard| O, G. Mar|non|*, S. Samaniego*,
G. Mellerio V and G. Vidari 0
The Senecio species have been largely studied. Most of them
contained furanosesquiterpenes. During our research on the
medicinal plants used in Ecuador we investigated the endemic plant Seneclo canescens Humb. ex Bonpl. which is used
against infections.
Some furoeremophilane sesqulterpenes have been isolated.
Their structure, elucidated by spectroscopic methods, will
be discussed. In the formulae some of the compounds are
shown.
O
H
*Escuela P o l i t e c n i c a d e l Chimborazo, Riobamba Ecuador
ODipartimento d i Chimica O r g a n i c a , U n i v e r s i t ~ d e g l i S t u d i ,
Via T a r a m e l l i [0, 27100 P a v i a ( I t a l y )
V L a b o r a t o r i o dt S p e t t r o m e t r i a di Massa C . G . S . , U n i v e r s i t ~
d e g l i S t u d i , Via T a r a m e l l i 10, 27100 P a v i a ( I t a l y )
227
PRELIMINARY PHARMACOLOGICAL SCREENING OF CALYCANTHINE, A
MAJOR ALKALOID IN THE STEM BARK OF PsyehotPia ~orste~a~
Y. AdJibad~*, B. Kuballa*, P. Cabal|on O and R. Anton*
In a previous report, we described the isolation and
identification of calycanthlne, a quinoline dimerlc
tryptophane derived alkaloid from the stem bark of
PaychotPia foreteriarta A. Gray, Rubiaeeae ( 1 ) .
The unusual occurrence of the bls-tetrahydroqulnoline
base in this plant family, its peculiar conformation and
the very few informations available on its activity, drew
our attention.
The pharmacological study of this alkaloid is only
limited to a couple of papers, the most recent one having
been published, to our knowledge, in the early forties (2).
Calycanthine has been reported to be highly toxic in vivo
and to induce violent convulsions.
In order to elucidate its mechanism of action and to
reveal potential new activities, we carried out a large
preliminary biological screening, using more than 50
different test models including the following fields:
acute toxicity, central nervous system, cardiovascular,
inflammation, allergy, immunopharmaeology and
gastrointestinal.
These results will be presented and discussed.
i. Y. AdJlbad~, B. Kuballa, P. Cabal|on and R. Anton,
Acta Agron. Hung., 34, suppl., ([985) 8i
2. A. Ling Chen, C.E. Powell and K.K. Chen, J. Amer. Pharm.
Assoc., 3l (|942) 5|3-516
* Laboratoire de Pharmacognosie, Facult~ de Pharmacie,
Universit~ Louis Pasteur, B.P. 10, 67048 Strasbourg
C~dex (France)
O Office de la Recherche Sclentlfique et Technique OutreMet (ORSTOM),Paris (France)
PHYTOCHEMICAL INVESTIGATION OF Sideritis hyssopifo~ia
VAR. pyrenaica
T. Adzet*, S. Ca~igueral*, C. I b ~ e z O, I. Monasterio*O
and R. Vila*
The polyphenols and the essential oil composition of aerial
p a s t s of siderites h~ssopifoZia L. v a r . pyrenaica B r i q u e t
( L a b i a t a e ) were i n v e s t i g a t e d .
Two a l l o s e c o n t a i n i n g f l a v o n e d i g l y c o a i d e s , 7 - O - ( a c e t y l allosyl-glucosyl)-laoscutellarein
and the c o r r e s p o n d i n g
unacetylated glycoside, and a caffeoylquinic acid
(chlorogenic acid) were isolated from methanol and methanolwater (l:l) extracts by column chromatography, and identified by UV-Vis, H-NMR, 13C-NMR, FAB-MS, and hydrolysis.
The essential oil ( 0 . 0 1 % v/w), obtained by hydrodlstillation, was analyzed by GC and GC-MS using Carbowax 20M
and silicone SE-30 capillary columns. The identification
of the essential oil components was carried out by
comparison of their retention indices and mass spectra
with literature data and authentic samples. The major components were ~-caryophyllene, a-pinene and o -phellandrene.
* Laboratory of Pharmacology and Pharmacognosy, Faculty of
Pharmacy, U n i v e r s i t y of Barcelona, Zona U n i v e r s i t a r i a de
Pedralbes,
08028-Barcelona (Spain)
0 LUCTA S.A., Apartado 1112,
08080-Barcelona (Spain)
228
Vol. 9
" I987
Pharmaceutisch Weekblad Scientific Edition
EFFECT OF DRYING ON YIELD OF LENONGRASS OIL AND ITS
ALDEHYDE CONTENT
S.M. H u s s e i n Ayoub
THE IMPORTANCE OF YIELD COMPONENTS FOR THE SELECTION OF
PupuveP bPaotea~um ON HIGH THEBAINE YIELD PER UNIT AREA
C. Bartsch
The oil and citral content in the leaves of lemongrass
have been reported to be affected by conditions of soil,
climate, number of cuttings and their intervals as well as
methods of distillation (I).
I n a p r e v i o u s p a p e r we r e p o r t e d o u r r e s u l t s on t h e compos i t i o n of the Sudanese lemongrass oll analysed by column
chromatography and GLC (2). The present paper concerns
investigations on the effect of drying on the yield of
lemongrass oil end its aldehyde content. The oll content
in fresh leaves of 6~mbopogon eltPa~us (DC.) Stapf was
determined; yields were higher in the winter than in the
summer. The sun-dried leaves of the Sudanese C. ci~ratu8
gave a maximum yield 0.87 % of oil with a citral content
of 91.8 %, when dried to 50 % of their original weight.
I. E. Guenther, The Essential Oils, vol. IV, 1972, Krleger,
New York
2. S.H. Bussein Ayoub and L.K. Yankov, Fitoterapia,55
(1984) 368
The thebaine yield of Pa~zver br~vteatum Lindl. depends on
the yield of capsules and on their thebaine content. The
capsule yield is determined by the number of capsules and
their mean weight. For the plant breeder, the total
thebaine yield is more difficult to estimate.
The correlation between the capsule yield and the thebaine
yield was calculated based on two years field experiments.
Hoping to find easily detectable morphological characters
correlated with the thebaine content, which should allow
an indirect selection for high contents of thebalne, characters such as form, length, diameter, and number of stigmatic rays were measured for the biggest capsule of each
plant. Afterwards the thebaine content of each examined
capsule was determined. Single as well as multiple correlations between capsule characters and thehalne content were
calculated. Besides the single characters, indices of several c h a r a c t e r s such a s w e l g h t - v e r s u s d i a m e t e r were formed
and i n c l u d e d i n t o t h e c a l c u l a t i o n s .
The r e s u l t s showed t h a t t h e number and w e i g h t o f t h e c a p s u l e s had t h e largest i n f l u e n c e on t h e y i e l d ; t h e i n f l u e n c e
of the thebaine content was of minor importance.
The thebaine content could not be explained by one or more
single characters of the capsules. Only some indices showed
a s l i g h t l y p o s i t i v e t e n d e n c y , b u t t h e y gave o n l y s l i g h t
c l u e s f o r selection.
It could be shown, that there is no interdependence between
the yield characters, so that combinations of the strongest
character developments seem possible.
Department of Pharmacognosy and Natural Products, Faculty
of Pharmacy, Al-fateh University, P.O.Box 13645, Tripoli
(Libya)
Institut ffir Pharmazeutische
Bonn (F.R.G.)
MOLLUSCICIDAL PROPERTIES OF Hyd~ora o~yssinica
S.M. Nussein Ayoub, L.K. Yankov and R.O. Ovtcharov
S tachy s lavandu ~if o Zla VAR . Zavar~u Zif o lia
LAVANDULIFOLIOSIDE,
A.A. Ba~aran*,
In a previous paper (I) we reported that blending of tannin
extracts with other additives which are chemically related
to the tannins would offer plent-derived molluscicides
which are potent arrd cheap. It would also give a practical
solution to the unstability problem of the tannin extracts
of Acacia nilotica. The promising results obtained prompted
us to investigate the molluscicidal factor of HydnoPa
ab~ssiniou A. Braun ( H y d n o r a c e a e ) .
The p l a n t c o l l e c t e d i n c e n t r a l and e a s t e r n p a r t s o f Sudan
y i e l d e d an a c t i v e e x t r a c t on p e r c o l a t i o n w i t h a c e t o n e - w a t e r
(7:3). The e x t r a c t killed I00 % of BuZinus ~runcatue ~nd
Biomphalaria pfeifferi, t h e two snail species which
transmit schistosomiasis in Sudan. Fractionation of the
extract with diethyl ether and ethyl acetate and biological
monitoring of the fractions showed that the activity was
found mainly in the ethyl acetate fraction. Evaluation
of molluseicidal activity was performed according to the
WHO method (2).
I. S.M. Hussein Ayoub and L.K. Yankov, Fltoterapla, 56
(1985) 371
2. WHO Expert Committee on Bilharzia, Bull. N.H.O., 33
(1965) 567
Department o f Pharmacognosy and N a t u r a l P r o d u c t s , F a c u l t y
o f Pharmacy, A l - f a t e h U n i v e r s i t y , P.O.gox 13645, T r i p o l i
(Libya)
Biologic,
Nussallee
A NEW PHENYLPROPANOID
I. Calls*,
6, 5300
GLYCOSIDE FROM
S. Nishibe O and O. Stlcher V
Stachys lavandulifolia Vahl e a r .
lavandulifolia (Lamiaceae)
i s c a l l e d " h a i r y t e a " . I t i s used a s m t e a i n t h e v i c i n i t i e s o f A n t a l y a ( T u r k e y ) . T h e r e f o r e , i t was i n v e s t i g a t e d
for its essential oil (1).
I n v e s t i g a t i o n s o f the w a t e r - s o l u b l e c o n s t i t u e n t s r e s u l t e d
i n t h e i s o l a t i o n o f s e v e r a l i r i d o i d s , f l a v o n o i d s and two
p h e n y l p r 0 p a n o i d g l y c o s i d e s . We w i l l r e p o r t on t h e i s o l a t i o n
and s t r u c t u r e e l u c i d a t i o n o f t h e more p o l a r p h e n y l p r o p a n o i d
g l y c o s i d e , named l a v a n d u l i f o l i o s i d e
(3,4-dihydroxy-8-phenylethoxy-O- a -L-arabinopyranosyl-(l § 2)-a -L-rhamnopyranosyl(l~3)-4-O-caffeoyl-8-D-glucopyrsnoside)
in a d d i t i o n to a
known glycoside, acteoslde. By f r a c t i o n a t i n g the watersoluble constituents over polyamide lavandullfolloside-was
o b t a i n e d . The compound was p u r i f i e d by u s i n g r e v e r s e d - p h a s e
vacuum l i q u i d c h r o m a t o g r a p h y . The c h e m i c a l and the s p e c t r a l
a n a l y s i s (IH- a~d t'3C-NMR), and i t s p e r a c e t a t e e s t a b l i s h e d
the p r o p o s e d s t r u c t u r e . UV, IR and MS d a t a s u p p o r t e d t h e
proposed structure of lavandulifolioside,
C34H44019.
I. A.A. Basaran, Pharmacognostical investigations on
Stachya ZavanduZifoZia Vahl v a t . lavandullfoZia, Ph.D.
Thesis, 1984, Hacettepe University, Ankara
* D i v i s i o n o f Pharmacognosy, F a c u l t y o f Pharmacy, H a c e t t e p e
U n i v e r s i t y , 06100 Ankara (Turkey)
0 F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , H i g a s h i , Nippon
Gakuen U n i v e r s i t y , I s h l k a r i - T o b e t s u , 061-02 Hokkaldo
(Japan)
V Pharmazeutisches Institut,
ETH-Zentrum, 8092 ZOrlch
(Switzerland)
Vol.
9 -
I987 Pharmaceutisch Weekblad Scientific EdiHon
229
STRUCTURAL REVISION OF KNOWN FREGNANE ESTER GLYCOSIDES
FROM CONDURANGO CORTEX AND STRUCTURES OF FOUR NEW COMPOUNDS
S. Berger*, F. Junlor 0 and L. Kopanskl v
ISOLATION AND DENSITOMETRIC DETERMINATION OF HYFOLAETIN-gO-GLUCURONIDE FROM LEAVES OF MaZva 8iZvestPi8
M. Billeter, B. Meier and O. Stiohsr
The pregnane ester glycoside fraction from Condurango
Rchb. f.
cortex, dried bark of Marsden~ c o ~ g o
(Asclepiadaceae), was reinvestigated. Four as yet unknown
g l y c o s i d e s o f the new a g l y c o n e c o n d u r a n g o g e n i n E were
i s o l a t e d by column c h r o m a t o g r a p h y and s u b s e q u e n t HPLC, and
named c o n d u r a n g o g l y c o s i d e (CG) E, EO, E2 and E 3. ~ h e s t r u c t u r e s o f t h e s e compounds were e s t a b l i s h e d by a c o m b i n a t i o n
o f de~gradation o f s u g a r c h a i n s and s p e c t r o s c o p i c methods
(IH-, 13C-NMR, FD-MS). In addition, rsinvestlgation of the
known pregnane ester compounds condurangogenin A, CG A, A O,
and C (1,2) by a selective proton-decoupling technique in
gated decoupled 13C-NtLR spectra showed that a structural
revision of all these and related compounds previously
found in Condurango cortex is needed. The aglyeones of
the A, B, C, D and E series are acylated with acetic acid
at II ~-ON and with clnnamlc acid at 12 B -OH of the
steroid skeleton.
I. R. Tschesche, H. Kohl and P. Welzel, Tstrahedron,
23 (1967) 146
2. K. Nayashl, K. Wada and H. Mitsuhaahl et G~., Chem.
Fharm. Bull., 28 (1980) 1954
Malva si~vestr~a leaves are used in folk medicine for
3550 Marburg
* F a c h b e r e i c h Chemie, Phillipps-Universit~t,
(F.R.C.)
0 Institut ffir Pharmazeutische Biologie der Universitgt
DUsseldorf, 4000 DQsseldorf I (F.R.G.)
V Schaper und Br~mmer GmbH & Co. KG, 3320 Salzgitter 61
(F.R.G.)
treatment of cough and external inflammatory diseases due
to t h e i r high c o n t e n t of m u c i l a g i n o u s s u b s t a n c e s . Therefore
most s t u d i e s on M. s i l v e s t r i e
c o n c e r n p o l y s a c c h a r i d e s (1)
and only few (2,3) low molecular weight substances.
In order to elaborate a satisfactory and simple chromatographic characterization of malva leaves we investigated
the flavonoid spectrum of aqueous and organic extracts. One
of the main flavonolds, hypolaetln-8-O-gluouronide,
was
isolated from a l-butanol extract by column chromatography
on Sephadex LR-20. Its structure was elucidated by means of
UV and IH- and' 13C-NMR spectroscopy.
Since the occurrence of hypolaetln and its glycosides in
plants is rather scarce, it is possible to use this flavohold as a "guide substance' for thequalitatlve and quantitative characterization of the plant, its extracts and
pharmaceutical preparations. After a clean-up of the
extracts by filtration through C]8- and NH2-sample preparation columns, the denaitometric determination of
hypolaetln-B-O-glucuronlde was performed on silica gel
60F-254 TLC plates (solvent: dlethylkstone/sthyl formate/
acetic acid/formic acid/water 50:20:11:5:14) with a Camag
ll-scanner. Examples will be given for the measurement in
the absorption mode and in the fluorescence mode after
treatment of the TLC plates with natural products/polyethyleneglycol-reagent.
I. H. Hsrmann, Thesis, 1977, Ludwlg-Maximillans-Universit~t,
M~nchen
2. M.A.M. Nawwar et a~., Fhytochsmlstry, 16 (1977) 145
3. M.A.M. Nawwar and J. Buddrus, Fhytocbemlstry, 20 ([981)
2442
Fharmazeutisches Instltut, Eidgen~ssisehe Technische
Hochschule Z~rich, 8092 Z~rich'(Swltzerland)
MODULATION OF THE IMMUNE RESPONSE BY POLYMERIC SUBSTANCES
FROM Baptisia tinctoria AND Eehlnacea angus~ifoZia
N. Beuscher and L. Kopanskl
DETERMINATION OF THE FRUCTAN CONTENT IN SYMPHYTI RADIX BY
A GASCHROMATOGRAPRIC AND AN ENZYMATIC METHOD
W. Blaschsk, M. Dhll and G. Franz
Echinaceu angustifolia and Baptisla tinctoria contain, llke
The roots o f Symph~t~m officlnaZe contain allantoin,
tannins, pyrrollzldine alkaloids and high amounts of
f~ctans. A gaschromatographic and an enzymatic method for
the determination of the fructan content in Symphyti radix
were developed.
Fructans were extracted with boiling water from freezedried, homogenized root material of field grown plants.
Tannins were removed by precipitation with lead acetate
and the extract was freeze dried. Fructana were hydrolyzed by mild acid treatment without degradation of sugars.
Glucose and fructose released by hydrolysis were quantified
by GLC on OV-17 columns as their oxlme-silyl-derlvatlves.
The amount of glucose and fructose was also determined
sequentially by the reduction of NADF due to the action of
a hexoklnase, glucose-6-phosphate-dshydrogenase
and phosphoglucose-lsomerase. With both methods, comparable results
were obtained. The fruetan content of Symph~tu2n roots harvested in autumn was 10-15% of fresh weight and 45-55% of
dry weight, respectively.
By methylatlon analysis and GC-MS it was shown, that the
fructans of Symphytl radix are of the branched type containing mainly 2,1-11nked fructose and some branch points
of 2,[,6-11nked fructose.
several other medicinal plants, heteropolyaaccharldes and
glycoprotelns, which possess an Immunomodulating activity
in Vitro and in vivo (I). The aim of our studies was to.
isolate and characterize these compounds. Aqueous ethanollo
extracts from both plants were fraetiuned by ultrafiltratlon, and subsequently subjected to column chromatography on Sephadex G-50 or LH-20. For {urther separation
FFLC was used. The activation of mouse lymphocytes from .
spleen and human lymphoeytes from buffycoat by the plant
polymeres was measured through the incorporation of 6-3H
thymldlne. The antibody response against sheep red blood
cells was determined in the haemolytlc plaque assay, according to Jerne. Our results showed that the glycoprotelns and
polysaccharldes led to a polyclonal E-cell stimulation.
This could be shown particularly in lymphocyte populations
liberated of T-cells by monoclonal antibodies. In addition,
it could be shown that supernatants of stimulated spleenic
lymphoeytes contained a- and 8-interferon. By SDS electrophoresls and immuneblotting, we were able to identify three
main glycoproteins with a molecular weight of 30000, 21000
and 17000 dalton.
It can be concluded that E. angustifogla and B. tinetoPia
contain immunomodulatlng glycoproteins with a considerable
in vitro efficacy.
1. U. Lindequlst and E. Teusoher, Pharmazle, 40 (|985) 10
Schaper and BrQmmer,
(F.R.G.)
Bahnhofstr.
35, 3320 Salzgltter
61
Institute of Pharmaceutical Biology, University of Regensburg, Unlverslt~tsstr. 31, 8400 Regensburg (F.R.G.)
23o
ISOLATION AND STRUCTURE OF AN ANTITUMOR ACTIVE 8-GLUCAN
FROM Ph~tophthora paraaitieu
W. Blaschek, d. Kraus a n d G. Franz
Various polysaccharides are known to exhibit an ant|tumor
activity which is thought to be mediated by stimulation of
the host's immune system (I). A ~-1,3/I,6-glucan isolated
from the cell walls of Ph~tophthoPa paPas|tics (carnation)
exhibited a strong ant|tumor activity against Sarcoma-180
in mice (2).
A similar glucan was isolated from PhytophthoPa ~a~itiea
(Dastur 1828) by extraction with hot water, ion exchange
and ConA-affinity chromatography. An optimum activity
against Sarcoma-180 was achieved at a dose of I mg/kg by
l.p. injections for lO consecutive days.
By methylation analysis it was shown that the ~ -gluean
consisted of 1,3-, 1,3,6- and terminal glucose residues
in a molar ratio of 5:1:i. Three sequential Smith degradations resulted in a linear 8-1,3-glucan indicating that
side chains did not exceed three glucose units. Methylation
analysis after each step of Smith degradation showed the
presence of menu- as well as ~-l,3-1inked di- and
trisaccharldes as side chains in a ratio of about 6:5:3.
A structure of the ant|tumor active 8-I,3/l,6-glucan is
proposed.
I. G. Chihara, EOS Riv. Immunol. Immunofarmacol., 2
. (1984) 85
2. M. Bruneteau, I. Fabre, J. Perret, G.M. Michel, P. Rico|,
J.P. Joselau, J. Kraus, M. Schneider, W. Blaschek and
G. Franz, Carbohydr. Res., in press
Institute of Pharmaceutical Biology, University of
Regensburg, Unlverslt~tsstr. 31, 8400 Regensburg (F.R.G.)
Vol.
9 -
I987 Pharmaceutisch Weekblad Scientific Edition
FLAVONOID PATTERNS IN THE GENUS Sogidago
J. Budzianowski, L. S k r z y p c z a k and M. Wesolowska
Previous studies (1,2) showed that the phenolic glycoside
leiocsrposlde and flavonoids are responsible for the
pharmacological activity of Solldago uirgaurea. This
prompted us to study the flavonoids and leioearposide
presence in other representatives of the genus SoZidago.
More than 30 clones of S. uirgau~ea, S. eanadensis vat.
oa~denags and war. seabr~ and S. gigantea were investigated. The samples of inflorescences were collected in
Poland, Czechoslovakia, Italy, Switzerland and Canada.
Two dimensional TLC on polyamide (3) showed more or less
different patterns for each taxon. About 25 different
flavonoids were isolated; their structure analysis will
be presented.
1. A. Chodera, K. Dabrowska, L. Skrzypczak and J.
Budzianowski, Acta Polon. Pharm., 43 (1986) 499
2. J. Metzner, R. Hlrschelmann and K. Hiller, Pharmazle,
3 9 (1984) 869
3. J. Budzianowski, L. Skrzypc~ak and M. Wesolowska,
Acta Biol. Crac. Ser. But., in press
Department of Pharmaceutical Botany, K. Marcinkowski
Medical Academy, ul. Wienlawskiego I, 61-712 Pozna~
(Poland)
ECOLOGICAL FACTORS INFLUENCING GROWTH AND HYPERICIN
PIGMENTS OF H~pePieum PROVENIENCES
H. Braunewell and A. VSmel
ISOLATION AND STRUCTURE ELUCIDATION OF TWO HIGHLY ACYLATED
IRIDOID DIGLYCOSIDE$ FROM Seroph~Zaria 8copoZli
I. Calls*, G.A. Gross O, T. Winkler V and O. Stlcher O
For the supply and quality of natural products, cultivation of medicinal plants is more appropriate than
collection from the spontaneous flora. Hypericum
perfoY~tum L.has been cultivated more or less succesfully.
Still improvement should be obtained by selection of plants
more suitable to cultivation.
Therefore, plants were collected at about 20 sites in the
F.R.G. Since the genetic variability had to be defined
under ecologically comparable conditions, the various.
eco-types were planted in field experiments at Raulschholzhausen Experimental Station on loamy soil; plants from
five sites were planted at Welhenstephan on humic soil.
Analysis of the hypericin pigments was performed photometrically (I).
Considerable variability of growth, of crude drug yield
and pigments occurred at the natural sites and under
cultivatioN.
The buds were found to contain the largest amounts of
hypericin pigment. Varying from 0.4 to |.0% in H.
perfor~tum, and 0.3 to 1.5% in H. n~cuZatumj whereas dried
leaves contained just 0.1% to 0.2%.
In general the hypericin content of the buds decreased by
cultivation. However, the proportion of the buds within
the plant top was higher in cultivated plants; thus some
of the eco-types produced a high quality crude drug.
Response of the yield of crude drug and hypericln was
similar at thd two experimental stations.
The results indicate that various eco-types of Hyperion',
may be found with positive response to cultivation by
providing higher yield and quality of crude drug.
I. J. Hglzl, personal communication
During our studies on the glycosidic constituents of the
roots of Scrophu~odega scopolis (Hoppe ex) Pets. vat.
8eopoZii (Scrophulariaceae), we obtained five fractions
(A-E) from the methanolic root extract. Fraction D contained phenylpropanoid glycosides (I). TLC of the fractions showed two UV active spots in fraction A. We
describe here the isolation and structure elucidation
of these substances named scropolioside A and B.
Their structures were elucidated, on the basis of chemical
and spectral data, as 6-0-(2",4"-di-O-acetyl-3"-O-p-methoxytrans- cinnamoyl)-~-L-rhamnopyranosyl
catalpol
and 6-0(2"-O-acecyl-Y',4"-O-di-trans-einnamoyl)- ~-L-rhamnopyranosyl catalpol, respectively.
[. I. Calls, G.A. Gross and O. St|chef, Phytochemiscry,
in press
Institut for Pflanzenbau und ZOchtung der JL-Universit~t
Giessen, Versuchsstation Raulschholzhausen, 3557 Ebsdorfergrund 4 (F.R.G.)
* Faculty of Pharmacy, Hacettepe University, 06100 Ankara
(Turkey)
0 Fharmazeutisches Institut, Eidgen~ssische Technlsche
Hochschule Z~rlch, 8092 ZUrich (Switzerland)
V Ciba-Geigy AG, 4002 Basel (Switzerland)
Vol. 9 -
I987 Pharmaceutisch Weekblad Scientific Edition
231
STDDIES ON PLANT CELL CULTURES OF CheZidoniura mo~u8
V
M.L. Colombo*, F. Tome~ N. Crespi-Perellino V, A. Guicciardi
and A. Minghetti ~
NEW COL91ARINS FROM Meaua ferpea L.
M. De Bernardi*, C. Vidari*~ P. Vita-Finzi*, G. Mellerio O
B. Gabetta V and A. Scilingo V
Chelidonium magus L. drew t h e a t t e n t i o n
Mesua ferrea L., Guttiferae, is an indigenous tree very
of s e v e r a l a u t h o r s
f o r its use in folk medicine. Cytotoxic, antiviral, antifungal and antiinflammatory activities have been found in
crude extracts of this plant. Chelldonine, the main alkaroots, is the basis for chemical
lold from C. ~ u 8
synthesis of chelidonine tiophosphorlc acid triaziridlde,
a potential lu%munostlmulant drug in the treatment of
cancer and AIDS.
Tissue cultures originating from C. m~'ua seeds were
initiated under various growth conditions and subcultured
several times. A growth curve of a suspension culture will
be reported and the alkaloid production discussed.
Both callus and cell suspension cultures were able to
produce alkaloids. HPLC analysis of the isoquinoline
alkaloids showed the presence of three major alkaloids;
other alkaloids occurred in traces.
Isoqulnoline alkaloids were also detected in the medium,
and their pattern was comparable to that found in the cells,
also in the early phase (three days) of subculturlng.
In addition the correlation between light exposure, growth
under different hormonal conditions and alkaloid yields
was studied.
* Istltuto Sc. Farmacol., Universit~ di Milano (Italy)
0 Dipart. Biologia, Universit~ dl Milano (Italy)
V Farmitalia C. Erba, Sviluppo Biotecnologico, Milano
(Italy)
A Facolt~ di Farmacia, Universit~ di Bologna (Italy)
PRODUCTION OF DRUGS ACTIVE ON CNS BY CELL CULTURES OF
Euphorbia calyptrata var. invo~uorata BETT.
N. Crespi-Perellino*, A. Guicciardi*, A. Minghetti O, E.
Speronl 0 and F. Vincleri ?
In a previous study (I) the activity on CNS by an extract
from r o o t s o f Euphorbia calyptrata was o b s e r v e d .
Euphorbia aa~yptrata is a very uncommon shrub growing i9
the desert o f Sahara and known hy natives as "Hihouek"
(madness plant).
The difficulties in finding this shrub prompted us to
utilize cell cultures in order to have an easier source
of material for isolation and identification of the prodm
net(s) responsible for its biological activity.
Different pharmacological tests such as the sleeping time
induced by pentobarbltal administration and the spontaneous
motor activity were affected by administration of the
ethanolic extract both of the roots and of the cell cultures. Both extracts also showed similar activities on CNS.
Therefore, cell cultures of E. caZyptrata, starting from
seeds, were produced as callus and as suspension culture.
The activity of the callus, when referred to the dry
weight, was three times higher than that of the cell suspensions. A typical fermentation curve of E. caZyptPata in
liquid medium will be discussed.
i. P. Eiacalone, Thesis, 1982, Unlversit~ di Firenze
* Farmitalia C. Erba, Sviluppo Riotecnologico, Milano
(Italy)
0 Facolt~ dl Farmacia, Universit~ di Bologna (Italy)
V Facolt~ dl Farmacia, Universlt~ dl Firenze (Italy)
common in the Indian and Ceylonese regions, used in the
folk medicine for the treatment of the wounds because of
its antibacterial and antibiotic properties. From previous
chemical investigation, Mesua ferrea is known to contain
xanthones, 4-phenylcoumarins and flavonoids. In order to
evaluate the pharmacological activities, a study on the
chemical composition of the hexane extract of the flowers
was undertaken: three new coumarlns (I-IIl) were isolated
and their structures elucidated by spectroscopic methods.
A number of sesqulterpene hydrocarbons were also identified and thelr'structures will be discussed. Preliminary
antibacterial data will be reported.
O~
IIR- C.~OR)Eto
~
R
Ho~
* Dipartimento di Chimica Organica, Universit~ degli Studi,
Via Taramelll lO, 27100 Pavia (Italy)
0 Laboratorlo dl Spettrometria di Massa C.G.S., Universlt~
degll Studl, Via Taramelli [0, 27100 Pavia (Italy)
V Laboratorio di Ricerea Invernl della Beffa, Via Ripamonti 99, 20100 Milano (Italy)
HOLLUSCICIDAL SAPONINS FROM Sesbania sesban
A.C. Dorsaz, M. Hostettmann and K. Hostettmann
The use of plants with mollusclcldal properties appaers
to be a simple and inexpensive means for controlling the
snail vector responsible for the transmission of schistosomiasis. Previous studies showed that dried powdered leaves o f Sesbania sesban M e r r i l ( L e g u m i n o s a e ) w a r e t o x i c to
the intermediate host of schistosomiasis (i). In order to
isolate the mollusclcidal compounds, fractionation of the
MeOH extract of the aerial parts of Sesbania 8esban was
realized by silica gel column chromatography with chloroform-methanol-water (80:30:3 ~ 60:40:10). Further purification by MPLC on RP-8 with methanol-water mixtures enabled
the isolation of three trlterpenoid saponins. Structures
were established by spectroscopic methods (FAB-MS,DCI-MS,
GS-MS, 13C-NMR) and by chemical degradation (acid and alkaline hydrolysis). They were identified as glucuronldes of
oleanolic acid. Following structure activity relationships,
only monodesmosldlc saponlns are mollusclcides. The spermicldal properties of the products were also tested.
1. A, T e k l e , E t h i o p i a n Med. J , , 15 ( 1 9 7 7 ) 131
Institut de Pharmacognosie et Phytochimie, Universit~ de
Lausanne, Lausanne (Switzerland)
232
V o l . 9 - ~987
SESQUITERPENE LACTONES FROM Arnica nevadensis and Arnica
~i8~osa
M. Ebert and G. Willuhn
In p r e v i o u s p a p e r s we r e p o r t e d on the a e s q u i t e r p e n e
l a c t o n e s (S1) of Arnica s p e c i e s b e l o n g i n g to the s u b g e n e r a
Montana and Andropurpurea (I-3). In continuing
our studies on the Sl of the genus Arnica, two species of
the subgenus Austmomon~zna Maguire (4) were investigated.
From leaves of A. nevadensls (section EuZatifo~iae) the
audesmanolides aaperilin, ivalin (main component), 2-0acetylivalin and ll~,13-dihydroivalin (I) were isolated
and identified by IR, MS and I R - N ~ analysis. The natural
occurrence of I is described for the first time. Other Sl
were not detected.
From leaves and flowers of A. viaoosa (section EPod~tae)
the pseudoguaianolides helenalin, lla,13-dihydrohelenalin,
their 6-O-acetylderivatives and the yet unknown 2B-hydroxy6-O-acetyltetrahydrohelenalin
(II) were isolated as the
only S1 of this species. The chemosystematic significance
of these results are discussed with regard to the contrary
view of the arranged subgenus Austromont~ (5,6).
Chcunissons
"~
I
o idoc I o
Pharrnaceutisch Weekblad Scienttfic Edition
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY. ON-LINE AND OFFLINE COUPLING FOR THE ANALYSIS OF SO,hUm GLYCOALKALOIDS
AND AGLYCONE$
A. Ehmke*, H.-M. Schiebel 0 and M. McDowell V
In continuation of our studies on SoZanum alkaloids (1), we
examined the potential of RPLC in combination with fast
atom bombardment (FAg) and thermospray mass spectrometry
for the identification of glycoalkaloids and aglycones in
plant extracts. The positive ion FAg mass spectra of the
glycosides and aglycones examined (solasonine, solamargine,
tomatine and solasodine, soladulcidine, tomatidine,
diosgenin, respectively) showed abundant [M+R] + signals as
observed with potato glycoalkaloids (2). In equimolar mixtures protonation of the aglycones was strongly favoured.
On-line coupling with thermospray LC/MS revealed the same
tendency for the preferential protonation of the aglycones
(alkaloids and sapogenins) and the relative discrimination
of the glycosides. The method seems well suited for a
screening of complex mixtures for a certain steroidal
alkaloid. Up to now, however, the detection of such components in plant extracts was hampered by the limited knowledge about their behaviour under thermospray conditions.
I. A. Ehmke and U. Eilert, Plant Cell Reports, 5 (1986)
31-35
2. K.R. Price e~ a~., Biomed. Mass Spec., 12 (1985) 79-85
II
I. G. Willuhn and H.-D. Herrmann, Planta Med., 37 (1979)
325-332
2. G. Willuhn, I. Junior, J. Kreskan, G. Pretzsch and D.
Wendisch, Planta Mad., (1985) 398-401
3. G. Willuhn, I. Junior and D. Wendisch, Planta Med.,
(1986) 349-351.
4. B. Maguire, Brittonia, 4 (1943) 386-510
5. G.B. Strayley: Ph.D. Dissertation, University of British
Columbia (1980)
6. S.J. Wolf and K.E. Denford, Rhodora, 86 (1984) 239-309
Institut f6r Fharmazeutische Biologic der Universit~t
D~saeldorf, D-4000 D~sseldorf I (F.R.G.)
* Institut for Pharmazeutische Biologic der T.U.,
Mendelssohnstr. I, 3300 Braunschweig (F.R.G.)
O Instltut f6r Organische Chemie der T.U., Ragenring 30,
3300 graunschweig (F.R.G.)
V~ VG Masslab, Tudor Road, Altrincham, Cheshire WAI4 5RZ
(O.K.)
A NEW 2-METHYLBUTANYLGLUCOSIDE FROM B~stropogon pZumosua
D. Economou*, A. Nahrstedt* and V. Wray O
Alisma pZantago-aquatica L. SSP. p~antago-aquatica
Bystropogon plumosus (L.fil.)L'Rer.
E. Eich*, G. Kohaupt*, K. Stopp*, A. Maidhof O, W:E.G.
Mflller O, P. Schade V and K. Mfillen V
(Lamiaceae-Thyminae)
occurs on the Canary Islands where it is used traditionally against colds. The essential oil of the herb was
recently investigated and contained mainly pulegone and
biogenetically related carbonyl compounds (I). The search
for monoterpene glycosides in the lyophilized herb failed,
but a glycosylated substance of slightly higher polarity
was isolated with a yield of 0.01% using an MY-8 column.
The glycoside gave glucose and 2-methylbutane-l-ol upon
hydrolysis with a B-glucosidase.
The IH- and 13C-NMR
spectra established a 2-methylbutane-l-yl-B-D-glucopyranoside as a new hemiterpane glucoside. The [~]zo of the,
new glucoside was -22~
the stereochemistry at C-2 is
currently under investigation, but several attemps to
separate the (S) and (R) active amyl alcohols via formation of diastereomers were not successful. An unsaturated
isomer of the new glucoside, 2-methyl-3-buten-2-yl-~-Dglucopyranoside, was recently isolated from Ferula
Zoscosil (2).
I. A. Nahrstedt, D. Economou and F.-J. Rammerschmidt,
Planta Med., (1985) 247
2. M. Pinar and M. Martin-Loma, Fhytochemistry, 16 (1977)
281
* Institut f o r Pharmazeutiache Biologic und Phytochemie
der Universit~t, Hittorfstr. 56, 4400 MOnster (F.R.G.)
O Gesellschaft fOr Biotechnolische Forschung, Mascheroder
Nag 1, 3300 Braunschweig (F.R.G.)
STRUCTURE AND CYTOTOXIC ACTIVITY OF TRITERPENES
FROM
Rhlzomes of AZisma plantago-aquatica L. ssp. orientals
are used in several traditional medicines of East-Asia
against different diseases. Part of the biological activity is due to the triterpenoidal alisols (1). There is no
information in literature about the secondary metabolites
of the European A. plantago-aqu~iea ssp. plan~ago-aquatica.
From the inflorescences of this subspecies we isolated
four triterpenes. Structural elucidation studies led to
the identification of fl-sltosterol, alisol B, and alisol B
monoacetate which are already known from ssp. orientaZe.
Furthermore we found a new compound for which we propose
the trivial name ll-deoxycycloalisol. Alisols known until
now possess a tetracycllc ring system with a branched C 8
side chain at C-17. Ih the case of ll-deoxycycloalisol this
chain shows C-16/C-23-cyclization resulting in a pentacyclic ring system with a tetrahydropyran partial structure.
Such triterpenoidal structures were found in nature only
in a few ca~es. Alisol g and its monoacetate showed remarkable cytotdxic activities in the L5178y mouse lymphoma cell
system (EDs0 cont.: 5.5 and 4.2 ~M), whereas ll-deoxycycloalisol was less active (EDs0: 11.9 ~M).
I. T. Murata, M. Shinohara, T. Hirata and M. Miyamoto,
Tetrahedr~n Letters (1968) 103-108; 849-854
*Institut
Saaratr.
OInstitut
V Institut
fQr
21,
fQr
for
Pharmazie der Universit~t Mainz,
D-6500 Mainz (F.R.G.)
Physiologiache Chemie der Universit~t Mainz
Organischa Chemie der UniversitKt Mainz
V o l . 9 " I987
Pharmaceutisch Weekblad Scientific Edition
ELICITOR INDUCTION OF S-ADENOSYLMETHIONIN:ANTHRANILIC
ACID
N-METHYLTRANSFERASE ACTIVITY IN SUSPENSION CULTURES OF
233
IRIDOID AND SECOIRIDOIDIIGLUCOSIDES FROM G e n t i ~ olivieri
T. Ers6z*, A.J. Chulia O, J. Garcia 0 and I. Calzs*
Ruta graveoZena
U. Ellert and B. Wolters
Suspension-cultured cells of Ruta graveolens respond with
rapid accumulation of acridone epoxides when treated with
autoclaved culture homogeuate of RhodogoPuZa r~bl~t (1). As
found with similar elicitatlon reactions, induction of biosynthetic enzymes is assumed. The first spaclfic s t e p in
acridone alkaloid biosynthesis was the N-methylation of
anthranillc acid (2). The effect of elicltation on the
a c t i v i t y of this enzyme was investigated. In d a r k - g r o w n and
in a light-grown control during 35 days N-methyltransferase
activity was low (between 1 and 0.1 ~kat/kg protein), which
was close to the detection limit. Addition of elicitor
(lO0 ~i/I00 ml c u l t u r e ) resulted i n a r a p i d increase o f the
enzyme activity. Maximum activity (10-24 ~kat/kg in darkgrown, 8-15 ~ kat/kg in light-grown cultures) was reached
10-15 h after elicitation followed by a slow decline. Time
courses of enzyme activity were similar in dark- and in
light-grown cultures. Depending on the culture age, enzyme
activity resumed to normal 4-7 days after elicitatlon. In
cell cultures of five other species (Atropu belladon~,
Solarium triflorum, Senecio vulgaris, Eschsohol~aia
ca~ifor, nica, Catharanthus roseus), f o r m i n g d i f f e r e n t
alkal o i d s , measurable
SAM:anthranilie acidmN-methyltransferase
activity was neither present nor inducable by elicitatlon.
Addition of actlnomycin D, an inhibitor of transcription,
or cycloheximide, an inhibitor of translation, 30 min
prior to ellcitation almost completely suppressed induction
of enzyme activity, as well as acridone epoxide accumulation. The results sho~ro present clear evidence for an
induction process to take place.
1. Eilert et aZ., Planta Med., 50 (1984) 508-512
2. Baumert et aZ., Planta Med., 48 (1983) 258-262
The genus Gentiar~ (Gentianaceae) r e p r e s e n t s twelve species
in Turkey ([). Some of them are used as folk medicine in
different regions o f Turkey. The crude d r u g " A f a r " ( a e r i a l
p a r t s o f G. oZivier'i G r i s e b . ) has been used as a b i t t e r
t o n i c , s t o m a c h i c o r s t i m u l a n t o f a p p e t i t e i n e a s t e r n and
south-eastern Anatolia. It had not been chemically studied.
In connection with our interest in irldoida and seeniridoids we studied the glycosidic constituents of G.
olivieri.
The water-soluble part of the metbanollc extract was
e x t r a c t e d with ethyl acetate and n-butanol, r e s p e c t i v e l y .
Further fractionations using silica gel column chromatography and finally reversed-phase vacuum liquid chromatography (2) yielded four secolridoid glucosldes, gentiopicroside, swertiamarin, sweroeide and trifloroside, besides
an iridoid glucoside, loganic acid.
The structures of the isolated substances were established
by chemical and spectral methods.
I. P.H. Davis, Flora of Turkey and East Aegean Islands,
Vol. 6, 1965, University Press, Edinburgh
2. S.W. Pelletier, B.a. Joshi and H.K. Desai, Techniques
for isolation of alkaloids. In: Advances in Medicinal
Plant Research, A.J. Vlietinck and R.A. Dom~isse, eds.,
Wissenschaftliehe Verlagsgesellschaft, Stuttgart, 1985,
pp. 153-195
Institut fflr Pharmazeutische Biologic der T.U.,
Mendelssohnstr. l, 3300 Braunschwelg (F.R.G.)
* Division of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, 06[00 Ankara (Turkey)
O Laboratoire de Pharmacognosie, U.E.R. de Pharmacie
Universit~ de Grenoble, Oomaine de la Merci,
38700 La Tronche (France)
ANTITUMOUR ACTIVITY OF EGYPTIAN Cappo~s
N. E1 Tanbouly*, J.P. Beck O, S. Hanna V, T. E1 Alfy V and
R. Anion*
DIASTEREOMERIC 3,11
OXYGENATED CRINANE ALKALOIDS
CPi~um a s i a t i c u m L. EX THAILAND AND OTHER SPECIES
A.W. Frahm and D. Bsutner
Capp~ia spinosa L. v a r . aeg~ptia ( C a p p a r l d a c e a e ) which
Antlviral activity is well documented for lycorine (I).
However it is not restricted to 4,5-ethanophenanthridlnes
bu t also found for 5,10b-ethanophenanthridine alkaloids,
During our studies on Amaryllidaceae alkaloids we investigated several CPinumspecles, all of which contained
different types of 3,1J-oxygenated crinane alkaloids. The
structure elucidation of new constituents from this group,
which were isolated from CP~num asiats
ex Thailand (2)
will be discussed. It was carried out with methods,
capable to discriminate diastereoisomerlsm at the chiral
centres 3, 5, 10b and II. Results of biological activity
testing will be given.
I. M. Ieven, D.A. van den Berghe and A.J. Vlietinck,
Plants Med., 49 (1983) 109
2. A.W. Frahm and D. Beutner, Plants Med., (1986) 450
grows in the Egyptian desert, is used in folk medicine as
antitumour crude drug (1,2). The possibility of finding
new agents with broad or strong antitumour activities as
well as low toxicity led us to examine this plant. We
investigated different extracts of C. spinoaa with regard
to antitumour properties. The plant was extracted with
ethanol followed by solvent fractionation in a liquidliquid extractor with petroleum ether, diethyl ether,
chloroform and ethyl acetate. Total alcohol extract E-I,
aqueous extracts E-Z, total root alcohol extract E-3,
dlethyl e t h e r extract E-4 were examined. Their activity
was determined in oitPo using a human leukemia cell llne
according to (3). E-4 showed a pronounced antitumour
activity. In order to test if E-4 also inihibited tumour
growth in vivo, it was injected into mice already bearing
tumours. Preliminary results confirmed that E-4 is active
against tumours s vivo, too. We are now isolating the
antitumour compound(s) from the diethyl ether e x t r a c t
E-4 of C. spinosa by means of chromatography.
I. Ibn al-Beithar, Leclerc L., "Trait~ des simples par
Ibn el-Beithar", 1877-1883
2. L. Hartwell, Lloydia, 31 ([968} 10[-104
3. J. Minowada, T. Onnuma and G.E. Moore, J.Natl. Cancer
Inst., 49 (1972) 89[-895
* L a b o r a t o i r e de P h a r m a c o g n o s i e , F a c u l t ~ de P h a r m a c i e ,
Oniversit~ Louis Pasteur, B.P. I0, 67048 Strasbourg C~dex
(France)
O Laboratoirs de Culture Cellulaire. Institut de Physlologic, 21 Rue Descartes, 67084 Strasbourg C6dex (France)
V Department of Pharmacognosle, Faculty of Pharmacy, Cairo
University, C a i r o (Egypt)
-
Institut f~r Pharmazeutische Chemle der Universit~t
Bonn, Kreuzbergweg 26, 5300 Bonn I (F.R.G.)
FROM
V o l . 9 - I987
234
Pharmaceutisch Weekblad Scientific Edition
STUDY OF THE VARIATION OF THE ESSENTIAL O I L CONTENT AND
QUALITY IN A LANDRACE OF.Angelioa arehangeZiea FROM BELGIUM
P. Gautier*, D. Shongo*, X. Vekemans*, M. Van Koninckxloo O,
M. Van Haelen V and C. Lef~bvre*
AN ATTEMPT TO CHARACTERIZE E e h i ~ e e a BY MEANS OF
CHROMATOGRAPHIC PATTERNS AND PLANT ANATOMY
E. Giger*, T.W. Baumann*, F. Heinzer O, J.-P. Meusy 0 and
M. Chavanne O
In Belgium, most of the medicinal plant cultures are located in north-western Hainaut, where AngeZiea arc~ngeZioa
L. is among the most cultivated species. The essential oil
is isolated from fresh roots; it is mainly used for flavouring liquors. In order to provide a basis for breeding
this plant,we investigated characteristics linked with oil
production of this landrace. The oil content (~ydrodistillation) in roots and in petioles showed variation between
individuals (0.6-1.9% d.w. and 0.4-1.8% d.w., respectively),
suggesting that breeding for oil content might be successful. No relationship between petioles and root oil contents
(r = -0.073 N.S.) was observed. A significant positive
correlation (r = 0.682***) was found between root weight
and oil content. Data on a field experiment performed to
study the effect of nitrogen fertilizer on oil production
will be presented. A GC analysis will allow us to determine
the extent of variation in oil constituents in roots and
petioles. The genetical make-up of the oll content in roots
and petioles will be discussed from data obtained by analysis of half-sib families.
Plants were grown in a field plot in Boncourt (Switzerland)
from seeds of Eehinaoea p ~ . r e a and of E. an~rustifoZia
from two sources. In the second year of outdoor cultivation
20 plants of each origin were harvested at random on the
same day, pooled and, after separation into roots and
aerial parts, finely homogenized. Aliquots of I00 g corresponding to about 25 g dry weight were processed either by
hydrodistillation or by ethanol extraction. Fingerprints
of the essential oils were obtained by GC on polar (Carbowax 20 M) and non-polar (OV-l) glass capillary columns.
HPLC was performed on 5 ~m ODg-Hypersil (precolumn 2.1 x
20 mm; column 2.1 x 100 mm) using a gradient consisting of
acetonitrile-water.
Plant anatomy was studied with regard to the shape of
epidermal cells of the adaxial surface of the llgule, to
the occurrence of secretory canals, and to sclerotic cells
in stems and roots.
It was found that E. p~.r~_~ea is not only anatomically but
also phytochemically different from E. angu8tifoZia. The
characteristics and the differences between the E. a n ~ s t i fulls plants from two different sources (FRG versus USA)
will be discussed.
* Laboratoire d'Ecologie V~g~tale et de G~n~tique,
Universit~ Libre de Bruxelles, Chauss~e de Wavre 1850,
If60 Bruxelles (Belgium)
O Centre Agronomique de Recherches Appliqu~es du Hainaut
(CARAH), 7800 Ath (Belgium)
V Laboratoire de Pharmacognosle, Institut de Pharmacie,
Universit~ Libre de Bruxelles, 1050 Bruxelles (Belgium)
THE CHEMICAL COMPOSITION OF THE VOLATILE OIL OF
Haronga
madagascariensis
B. Gehrmann and LJ. Kraus
The composition of the volatile oil of Haronga
bark, isolated by steam distillation, was
investigated by different chromatographic methods.
TLC on silica gel showed about six major and seven minor
spots, which were detected by anisaldehyde-sulfuric acid
reagent. Column chromatography as described by Kubeczka (I)
yielded five fractions of different polarity. Some fractions
were obtained by LPLC, using a refractometer for detection,
and subsequently analyzed by capillary GC.
The main components of the oll were ~-pinene and some
alkanes such as undecane and dodecane.
I. K.-H. Kubeczka, Chromatographla, 6 (1973) 106-108
madagaseariensis
Division of Pharmacognosy, University of Hamburg,
Bundesstr. 43, 2000 Hamburg 13 (P.R.G.)
* Institute of Plant Biology, University of ZHrich,
8008 ZOrich (Switzerland)
0 Spagyros SA, 2926 Boncourt (Switzerland)
PHENOL SULFATE ESTERS FROM PHAEOPHYCEAE
K.-W. Glombitza*, W. Kn~ss, B. Deutscher*, G. Piepenbrink*
and J. Peter O
Over 80 different oligomeric dehydropolymerisates or corresponding polymeric compounds (phlorotannlns) have been isolated so far from brown algae.
The traceability of 1,2,3,5-tetrahydroxybenzene-2,5-disulfate in AseophylZum nodos~ as well as the occurrence of
phenols esterified with sulfuric acid in some red algae make
the search for further phenol sulfate esters worthwhile.
The difficulty hereby lies in the enrichment of the very
hydrophillc sulfate esters, the separation of the inorganic
and organic salts and the MS investigation of the isolated
substances.
We isolated several mono- and dlsulfate esters of phlorogluclnols, diphlorethols and difucols from Pleuroph~eus
gardneri, Pterygophora califonnica and Hedophyll~ sessile
by enrichment of the.phenols on a polyvlnylpyrrolidone (PVP)
column, desorptlon ~ith diluted ammonia, separation on a RP
HPLC column with diluted potassium sulfate and desalinization on a second PVP column.
The molecular weight and the number of free OH-groups were
determined by FAB-MS after acetylation with acetic anhydride
* Institut fur Pharmazeutische Biologie, Universitgt Bonn,
Nussallee 6, 5300 Bonn (F.R.G.)
O Institut f~r Physiologische Chemie, Universit~t Bonn,
Nussallee II, 5300 Bonn (F.R.G.)
Vol. 9 -
*987 Pharmaceutisch Weekblad Scientific Edition
235
METABOLISM OF QUINOLIZIDINE ALKALOIDS IN THE GENUS Laburnum
(LEGUMINOSAE)
R. Grelnwald, W. Schultze and F.-C. Czygan
INVESTIGATION ON THE VARIABILITY OF APIGENIN AND SELECTION
OF APIGENIN TYPES OF TETRAPLOID CHAMOMILE
J. Hblzl and S. Hess
Quinolizidlne alkaloids are mainly distributed in the
family Legumlnosae. We studied the influence of seasonal
changes on the alkaloid pattern and concentration in the
genus Labu/,num. Whereas in spring a strong methylation of
the tricyclic alkaloid cytisine was observed, in autumn a
large increase of the tetracyclic quinollzidine alkaloid
baptlfoline, which is new for Labu~ko~, was found. The
investigations were carried out by TLC and GC. The key
intermediate baptifollne, isolated from L~bur~tan species,
is not identical with baptifoline from Cau~ophyZZl~71 tha~ictro~des (Berberidaeeae), which was described by Flom et aZ.
(1). We isolated baptifollne from both species. The absolute configuration of these two alkaloids should be determined by spectroscopic methods (IR, MS, and |H- and |3CNMR).
I. M.S. Flom, R.W. Doskotch and J.L. geal, J. Pharm. Sci.,
56 (1967) 1515-1517
The pharmacologically active substances of chamomile are
bisabolol, chamazulene and matrlcln, the spire ethers and
the flavonoids apigenin CA) and apigenln-7-glucoslde (ATg).
The most powerful spasmolytic substance is A: three times
more active than papaverlne and six times more than A7g.
Due to plant breeding, different types of di- and tetraploid chamomiles exist with high contents of blsabolol and
chamazu~ene. No type exists with a high content of A.
We analysed the variability of the A- and A7g-contents of
five different blsabolol-chamazulene types and we selected
single plants with a high A-content for cultivation.
For pre-evalua.tion, TLC of ligula flower extracts was performed on silica gel buffered with sodium acetate. The
quantitative analysis of the flavonoids was performed by
RP-HPLC.
The content of A had a high variability, being lower than
I mg up to 95 mg/100 g flowers. From the ten plants with
the highest A-content, seeds were produced and from each
seed sample 70 single plants were cultivated.
All plants originating from a high A-content parent plant
showed also a high A-content. Comparing the A-content at
three different cultivation periods - during winter in the
greenhouse, and during spring and summer - the highest Acontent was found in plants cultivated in the summer.
Therefore, it may be concluded that the A-content depends
upon light and temperature.
Lehrstuhl fur Pharmazeutische Biologie der Universit~t
W~rzburg, Mittlerer Dallenbergweg 64, 8700 W~rzburg
(F.N.G.)
Institut for Pharmazeutlsche Biologie der Unlversit~t,
Deutschhausstrasse 17 I/2, 3550 Marburg (F.R.G.)
ANTIFERTILITY AND ABORTIFACIENT ACTIVITIES OF MYRSINACEAE
SPECIES
Mazuru Gundidza
APPLICATION OF HEADSPACE GC IN ESSENTIAL OIL ANALYSIS. Xl.
ANALYSIS OF SILYLATED SAMPLES
Y. Holm, E. Aho and R. Hiltunen
Embelia schimpepi, Myrsine africana and Maesa lanceolata
Thymol and carvacrol in thyme ell and thyme herb differ
significantly from the other compounds in the mixture as to
their vapour pressures. In single run headspace analysis
the two compounds gave relatively small peak areas. In
order to increase the proportion of these compounds in the
he~dspace gas phase, thymol and thyme herb samples were
silylated in the headspace vial simultaneously with the
equilibration. As a result, the peak areas of thymol as
well as those of the alcohols in thyme herb increased significantly. It was possible to obtain a complete silylation
under optimized headspace conditions. The precision was the
same in the analyses of silylated and non-siiylated sample~
Rellable determination of the thymol content of thyme herb
was obtained using the sllylation technique in a single
run instead of the tlme-consuming multiple headspace extraction (MHE). However, the MHE technique proved to be
the best method also for the analysis of silylated samples.
(Myrslnaceae) used traditionally for fertility control in
Zimbabwe, were tested for antlfertility and abortlfacient
activities. The tests were carried out on female albino
rats of proven fertility in the pro-oestrus phase of thd
oestrus cycle. Ethanol extracts of the barks and leaves of
the plants were prepared using a Soxhlet apparatus. The
dried extracts were reconstituted in water and the rats
dosed with 200 mg/kg body weight daily for five days.
For infertility tests the rats were dosed before pregnancy and for abortlfacient tests after pregnancy. After
dosing, the rats were allowed to go to term and the size
of litter was noted. All the plants exhibited significant
antifertility and abortifaclent activities; the bark
extract of E. 8chimperi (67% antifertility activity and
64% abortifacient activity) was the most active one.
Department of Pharmacy, University of Zimbabwe, P.O.Box
MP 167, Mount Pleasant, Harare (Zimbabwe)
Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, Fabianinkatu 35, 00170 Helslnki (Finland)
236
SESQUITERPENE LACTONES OF FINNISH TANSY
M. Holopeinen, R. Hiltunan, K. J~rvel~, T. Sepp~nen and
M. yon Schantz
Tar~ce~um vulgate L. is well known in Europe, Asia and
North America. Tansy has been reported to contain a wide
variety of sesquiterpene lactones (I), some of which have
been shown to be biologically active, cytotoxic, antibacterial, antifungal or spasmolytic (2).
The plant mataria] consisted of different tansy types. The
dried plant samples were extracted with chloroform. After
evaporation of the chloroform, acatonltrile-water (l:l)
was added. The samples were analysed by HFLC on an RP-18
column using UV-detection at 225 nm.
We found six main sasquiterpane lactones in different
Finnish tansy types. One of them was parthenolide, which is
also present in Tanacetum parthenlum. Parthenollde seems
to be of therapeutic value; it is used for treating
migraine (2).
I. G.M. Nano, G. Appendino, C. gicchl and C. Frattlni,
Fitoterapia, 51 (1980) 135
2. E.S. Johnson, P.J. Hylands and D.M. Hylands, Pharmaceuticals containing sesqulterpene lactones in extracts
from Tanaeet~m parthenium, Eur. Pat. Appl. EP 98,041,
11 Jan 1984, GB Appl. 82/14,572, 19 May 1982, 93 pp
Division of Pharmacognosy, School of Pharmacy, University
of Helsinkl, Fabianinkatu 35, 00170 Relsinki (Finland)
A NEW I ~ / N O A S S A Y APPROACH FOR THE C a t ~ r ~ n g h u s r o s e u e
ALKALOID VINDOLINE
A. Huhtikangas*, S. LapinJoki*, H. Ver~j~nkorva*,
J. Heiskanen*, V. Kauppinen O and M. Lounasmaa V
The biosynthesis of antibodies against vlndoline, a m o n o merit indole alkaloid of Catharanthus roseu8 (L.) G. Don.
was induced in rabbit lymphocytes by an immunogan featuring
the dimaric alkaloid vlnblastine (VBL) as the haptenic
group attached to bovine serum albumin (BSA). The result
can be attributed to the diazo-coupling involved in the
VBL-BSA immunogen synthesis, and to the fact that the bisindollc molecular assembly of VBL consists of monomerlc
"vlndoline and catharanthine moieties". Significantly, the
VBL-BSA conjugate in question is characterized by an intact
terminal "vindollne moiety". This is achieved by reacting
the coupling agent, diazotized p-amlnobenzoic acid, with
the C-10' position of the "catharanthlne moiety" in VBL,
and linking the newly introduced carboxyl group with a free
amino group in BSA. In addition to the C-10' position, the
C-12' position in the "catharanthine moiety" and the C-12
position in the "vindoline moiety" of VBL are theoretically
susceptible to react with dlazonium ions. However, rapid
and apparently exclusive coupling to the sterlcally least
hindered C-]0' position is obtained by careful pH regulation of the process.
V o l . 9 - I987
FAST ATOM BOMBARDMENT MASS SPECTROMETRY IN THE
CHARACTERIZATION OF LICHEN DEPSIDES
K. Huovinen*, P. H~rm~l~*, P. Ottoila O, R. Hiltnnen* and
M. yon Schantz*
Fast atom bombardment-mass spectrometry (FAg-MS) has been
used in the study of biochemical samples which are not
suitable for conventional electron impact (El) or chemical
ionization (CI) mass spectrometry. Lichen depsldee, some of
them with inhibitory effects against prostaglandln biosynthesis (|), are formed from two phenol carboxylic acid
units (A- end B-ring) by asterificatlon. As e result of
this weak bond, they rarely exhibit molecular ions in
positive ion El or CI spectra. Although FAg-MS mainly
provides informetien about molecular weight, structural
information can also be obtained. Positive and negative ion
FAg spectra were recorded for ten depsldes in a preliminary
trial. The base peak in the positive ion spectra gave information about the size of the A-ring moiety, and about
the molecular weight of the protonated molecule, MH +.
Information on the size of the B-ring moiety was obtained
from the base peak in the negative ion spectra, which also
gave a prominent (M-H)-. This technique in combination with
N ~ techniques was used for the identification of an unknown depside which was isolated by gPLC from Cladonia
peZtas~ioa (Nyl.) MQII. Arg., Venezuela. The depside was
identified as 2-O-methylseklkaic acid, a compound that was
not previously isolated from the genus CZadonda.
I. Sankawa et aZ., Prostaglandins, 24 (1982) 21-34
* Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, Fabianinkatu 35, 00170 Helsinki (Finland)
0 Orion Pharmacautlca, Research Centre, P.O. Box 65,
0 2 1 0 1 E s p o o (Finland)
FLAVONOIDS FROM FLOWERS OF Hetero~heca inuloides
Chr. Jerga, I. Merfort and G. Willuhn
The flowers of Heterotheca inuloides Cass. (Asteraceae,
tribe Astereae) are used in the folk medicine of Mexico,
named as "Arnica" (I). They can be found as falsification
of the officlnal "Arnicae flos DAB 9", originating from
Ar~s
montana or A. chamissons subsp, foZiosa (2). The
examination of purity of the officinal flowers is based
on the flavonoid profiles by TLC.
The present paper reports on the identification of the
flavonolds of "Heterotheeae flos". The following 15 aglycones and 4 glycosides were isolated and identified by UV,
MS and IH-NMR: kaemPferol , quercetin, the methyl ethers
I-9, the quercetagatin-3,6,7,4'-tetramethyl
ether and
-7,3'-dimethyl ether, astragalin, hyperin, Isoquercitrin
and turin.
Flavonoids methylated at position 3 are characteristic for
species of the tribe. Astereae (3) and have not been found
in A M { c a species ontil now (4,5).
ORI
OH 0
I:
~:
_3:
i.
* Department of Pharmaceutical Chemistry, University of
Kuopio, P.O. Box 6, 70211Kuopio (Finland)
0 Biotechnical Laboratory, Technical Research Center of
Finland, 02150 Espoo (Finland)
V Laboratory for Organic and Bioorganic Chemistry, Technical University of Helslnkl, 02150 Espoo (Finland)
Pharmaceutisch Weekblad Scienttfic Edition
OH 0
RI=H ; R2= H
4-9_: RI=H or CH3
RI= CH3;" R2= H
R2= H o r CH 3
R l= H ; R 2- OCH 3
R3= H or CH 3
M. Zurita Esquivel and C. Zolla, Bol. Sanit.panam., I01
(1986) 339-347
2. G. Willuhn and P.-M. RSttger, Dtsch.Apoth. Ztg., 120
(1980) 1039-1042
3. E. Wollenweber, I. Schober, W.D. Clark and G. Yatskievych, Phytochemistry,24 (1985) 2129-2131
4. I. Merfort, Planta Med., (1985) 136-138
5. I. Merfort, Chr. Marcinek and A. Eggert, Phytochemistry,
25 (1986) 2901-2903
Institut f~r Pharmazeutische Biologic der Unlversit~t
D~ssaldorf, 4000 D6sseldorf I (F.R.G.)
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
QUANTITATIVE DETERMINATION OF BORJATRIOL IN
HERB
A. Jim~nez*, S. Ma'bez* and A. Villar O
Sideriti8
mugronensi8
gorJatriol (7S,14R,15-trlhydroxy-8a-13-epoxy-labdane) is a
d i t e r p e n o i d isolated from Sideritis mugronensis B o r j a
( L a m i a c e a e ) w h i c h i s c o n s i d e r e d t o be one o f t h e main a c t i v e principles of this species. Its antiinflammatory activity has been verified in several models (1,2). The
present study deals with the application of a spectrophotometric technique for determining the borJatriol content in
plant samples covering the complete annual cycle.
Aerlal parts collected in Bonete (province of Albacete;
Spain) were dried and extracted with hexane. Preparative
TLC of the extract on silicagel with ethyl acetate-methanol
(8:2) led to the isolation of borJatrlol. Its concentration
was determined after adding anisaldehyde/diluted sulfuric
acid and heating at 60~ by measuring the absorbance at
600 nm and using a calibration curve. The relative amount
of borjatriol seemed to be roughly related to the maximum
plant growth.
I. A. Villar, R. Salom and M.J. Alcaraz, Pharmazie, 38
(1983) 566
2. A. Villar, R. Salom and M.J. Alcaraz, Planta Mad., 50
(1984) 90
* Laboratorio de Farmacognosia y Farmacodinamia, Departamento de Farmacologla y Farmacotecnia, Facultad de Farmacia,
Universitat de Val~ncla. Av. Blasco I b ~ e z 13, 46010
Valencia (Spain)
O Laboratorio de Farmacognosla y Farmacodinamia, Departamento de Farmacologla, Facultad de Farmacia, Unlversidad
Complutense de Madrid. Ciudad Universitarla, 28040 Madrid
(Spain)
FORMATION OF COMPOUNDS IN CULTURES OF THE FUNGUS
Inonotus obZiquus
K. Kahlos and R. Niltunen
The fungus Inonotus obZlquus (Pars. ex Ft.) Pilat, used
as a folk medicine, is rich in closely related triterpane
derivatives, the main and active triterpene being inotodiol
(3~,22-dihydroxy-lanosta-8,24-diene) (l). The production
of fungal compounds on different culture media was inveStigated.
A range of nutrient solutions and agars such as bacto,
nutrient, sabouraud dextrose, potato dextrose and malt
agars were used as culture media. Mixtures of different
agars and birch, rowan or alder sawdust were also used.
The compounds extracted from the fungal mycelia were investigated by TLC, GLC and GC-MS.
The highest yield of fungal triterpenes was obtained from
the cultures grown on mixtures of malt agar and alnus and
5itch sawdust. GC-MS was used to identify the compounds
isolated from the cultures.
I. K. Kahlos, L. Kangas and R. Biltunen, Antltumour
activity of some compounds and fractions of an n-hexane
extract of Inonotus obZiquua, Acts Pharm. Fenn., 96
(1987) in press
Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, Fabianinkatu 35, 00170 Helsinkl (Finland)
237
EFFECTS OF ONION AND GARLIC EXTRACTS ON SPONTANEOUSLY
HYPERTONIC RATS
J. Kiviranta*, T. Sepp~nen*, H. Karppanen O, H. Huovinen*
and R. Hiltunen*
Onion and garlic consumption is supposed to have a positive
effect on hypertension and hyperlipidemia in man (I).
The effects of ethanollc onion and garlic extracts on blood
pressure and plasma lipid composition were investigated.
The test animals were spontaneously hypertonlc rats which
were divided into four groups:
I. comparison group;
2. comparison group, salt addition;
3. test group, salt and onion extract addition;
4. test group, salt and garlic extract addition.
Blood pressure levels in the different groups were followed
over an eight-week period. No statistically significant
differences were found between the groups, although in the
onion group the blood pressure, on average, seemed to be
slightly lower than that in the comparison group 2.
After the test, the plasma lipid composition of the rats
was analysed by GLC. The relative increase of linolelc acid
in the onion and the garlic group was statistically significant compared to group 2. On the other hand, the relative amount of arachidonic acid decreased in both test
groups compared with group 2.
I. D.B. Louria et aZ., Curr. Therap. Res., 37 (1985) 127-130
* Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, 00170 Helsinkl (Finland)
0 School of Pharmacology, University of Helsinkl, 00170
Helslnki (Finland)
A METHOD FOR ESTIMATION OF ANTHRACENE DERIVATIVES USING
THE CAMAG AMD SYSTEM
A. Koch
Numerous methods for the coiorimetric estimation of
anthracene derivatives in medicinal plants have been
p~bllshed. Most of them are determinations of the total
amount of anthracene derivatives. So, it is difficult to
correlate the total amount to the amount of single anthrace,e components and their pharmacological activity.
In order to improve the determination of the individual
compounds, two-dimenslonal TLC in combination with the
Camag AMD System was used.
After hydrolysis of the glycosides on the plate and development of the aglycones, densitograms allowed quantitative estimations of the free anthraquinone aglycones.
The results will be presented.
Division of Pharmacognosy, University of Hamburg,
Bundesstrasse 43, 2000 Hamburg 13 (F.R.G.)
238
CARDIAC GLYCOSIDE GLUCOSYLTRANSFERASE AND GLUCOHYDROLASE
ACTIVITIES IN PLANTS ANU CELL CULTURES OF THREE Olgi~Zis
SPECIES
W. Kreis, U. May and E. Reinhard
V o l . 9 - I987
Pharmaceutisch Weekblad Scienufic Edition
ESSENTIAL OIL OF Vitez agnus-castu8 L. [VERBENACEAE)
i. Laakso*, O. Ekundayo O, g. OguntimelnV and R. Hiltunen*
Vitez agnus-castus is a medicinal plant with pharmacological effects on the human endocrine system. Irldoids
and flavonoids have been found to be the main compounds
in leaves and fruits (I). Analysis of the essential oil
of east European origin showed it to contain ~- and
8-pinene (40Z) and 1,8-clneole(25Z) as major constituents
in addition to seven other components (2).
The volatile oil of V. agnug-o~stu8 grown in Nigeria was
investigated in the present study. Rydrodistilled leaf
oil was fractionated into n-pentane (hydrocarbons) and
ethyl acetate fractions (oxygenated compounds) on a silica
gel column (SEP-PAK). The fractions and the total oil were
submitted to OC and GC-MS.
The essential oll was characterized by [,8 cineole (55Z),
Q-pinene and sabinene (10-13Z) and terpinen-4-ol (SZ).
The most abundant sesqulterpene hydrocarbons included
s
8-caryophyllene, ~-elemene and germacrene-D. Caryophyllene e p o x l d e a n d T-cadlnol were identified as the major oxygenated sesqulterpenes.
I. Th. Kartnig, Z. Phytoth., 57 (1986) ]19
2. S.S. Mishurova, T.A. Malinovskaya, I.g. Akhmedov and
D.G. Mamedov, Rastlt. Resur., 22 (1986) 526
In leaves of Digi~aZis lana~a most of the cardenolides are
primary glycosides. During isolation enzymatic hydrolysis
may occur, leading to the secondary glycosides. Both types
of cardenolides may be present in an acetylated (D. ~anata,
D. ~ o o d i l )
or in a non-acetylated form (O. pMY~uPea).
The scope of our present investigations was to elncidate,
on the enzymatic level, the conversion of primary glycosides to secondary ones and ~ioe uePg~. We supposed that
these two enzymes play important roles in the storage and
transport of cardeno~ides (I). Both enzymes were supposed
to be present in leaves with active cardenollde biosynthesis. One aim was to establish experimental systems where
both enzymes could be studied independently.
We found that a specific glucosldase, hydrolyslng primary
glycosides to the respective secondary ones, was not present in cell cultures of D. Zany[a, D. h ~ o o d i i , and
D. pu2"pumea, whereas high glucosyltransferase activity was
found in selected cell lines (2). Wlth the cell culture
system, an ~n u{tPo assay was developed for a cardiac
glycoside glucosyltransferase. This soluble enzyme, located
in the cytosol, was detected in all DigitaZi8 cell cultures
examined, but not in cell cultures from other plant specle&
It was characterized with regard to subs[rate specificity,
pH-optlmum, and co-factor requirement.
A specific glucohydrolase was obtained from young Digltalis
leaves and a suitable.ln uitPo assay established. The propertles of the cardiac glycoside glucosyltransferases and
the related glucohydrolases will be discussed with regard
to differences found among the three Digitalis species.
I. W. Krels and E. Reinhard, J. Plant Physiol., 128 (1987)
311-326
2. W. Kreis, U. May and E. Reinhard, Plant Cell gep. 5,
(1986) 442-445
Pharmazeutlsches Instltut, Unlverslt~t T~bingen, Auf der
Morgenstelle 8, 7400 TObingen (F.R.G.)
* Division of Pharmacognosy, School of Pharmacy, University
of Nelsinki, Fabianlnkatu 35, 00170 Helslnki (Finland)
O Dep~r~ment of Chemistry, University of Ibadan, Ibadan
(Nigeria)
V Department of Pharmacognosy, Faculty of Pharmacy,
University of Ife, lle-lfe (Nigeria)
ANTIMICROBIAL ACTIVITY OF Vitez agnus-castu8
D. Ku~trak*. S. 2epeljnJak*. A. Antoli6* and N. Bla~evi60
HERBAL PRODUCTS - A PHARMACOGNOSTICAL INVESTIGATION
genre Lorentzen and Anne Marie R~rdam
V{teZ agnus-oagtus L. has been the subject of several
studies intended prlmarely to determine the chemical composition and its phaxmacologlcal properties.
Our investigation concerns the antimicrobial activity of
the essential oil, ethanollc and dlethyl ether extracts and
the irldoids of V. agnug-oastus which have been hardly
studied until now.
Leaves, flowers and fruits of V. agnug-oastus were subjected to steam distillation in order to obtain the essential
oil, which was analyzed by TLC and GC.
Agnuside and aucubln were isolated from the leaves of
V. agnua-oastus by a modified method (1).
The antlmlcroblal activity was determined by an agar
dilution and diffusion method. The micro-organisms used
Candida aZbicans, ShigeZ~a sones
were Bacillus subtiZs
and Escherichia coll. The essential oil, the extracts and
iridoids were active against the micro-organisms tested.
1. E. Winde and R. H~nsel, Arch. Pharm., 293 (1960)
556-67
In [976, a new Danish consolidate medicines act decided
that products produced from purely natural remedies having
no harmful effect for the userj were excepted from the
general quality requirements previously in force, and
allowed for sale outside pharmacies. The act led to a boom
of more or less serious products of variable quality.
This initiated the National Food Agency of Denmark to start
an investigation of the different products. An investigation of 27 blends of tea with the purpose of controlling
their contents in relation to the declaration showed that
only in three blends all the declared drugs were present,
and no undeclared ones were found. However, none of these
three blends carried an unambiguous declaration (1).
As consumers expect to buy herbal products of good quality
at pharmacies and to get proper information, too, the
Association of Danish Licensed Pharmacists and the Danish
Health Product Assoclatlon made an agreement concerning a
quality evaluation of the herbal products available at the
pharmacies. The products were also submitted to pharmacognosticgl investigations, the results of which will be
given. Positive evaluated products are published in a
catalogue.
I. A. Adsersen, L. Gudlksen and B. Lorentzen, Arch. Pharm.
Chem., 90 (1983) 677-682
* Faculty of Pharmacy and Biochemistry, University of
Zagreb, P.O. Box I56, 4[000 Zagreb (Yugoslavia)
O Chromos-Aroma/Mirlsl, Zagreb (Yugoslavia)
Department of Pharmacognosy, Royal Danish School of
Pharmacy, Unlversltetsparken 2, 2lOO Copenhagen (Denmark)
Pharmaceutisch Weekblad Scientific Edition
V o l . 9 - I987
ALTHAEAE RADIX MUCILAGE POLYSACCHARIDES,
CHARACTERIZATION AND STABILITY
A. Madaus, W. Blaschek and G. Franz
ISOLATION,
The r o o t s o f Althaea o f f l c i n a ~ i s c o n t a i n l a r g e amounts o f
starch and water-soluble mucilage polysaccharides. The
mucilage was analyzed by GLC, HPLC, ion exchange chromatography and viscosity m e a s u r e m e n t s .
Althaeae radix was extracted with water at temperatures
below 40~C in order to avoid contamination with starch, and
the extract was clarified by centrifugation. For removal of
low molecular weight components the solution was subjected
to ultrafiltratlon and freeze dried, yielding the crude
mucilage.
Ion exchange chromatography showed that the crude mucilage
was composed mainly of acidic polysaccharides, the sugar
composition of which was determined (after hydrolysis with
mineral acids) by HPLC and by GLC of the acetylated sugar
derivatives. The mucilage was composed of galacturonlc
acid, glucuronic acid, rhamnose, galactose and arabinose
in a molar ratio of 2.5:[.2:2.5:2:1.
The influence of physical and chemical stress on the apparent molecular weight of the polysaecharides in Althaeae
radix extracts was examined by rotation viscosity measurements. It was shown that various parameters such as concentration, temperature, pH and ionic strength, considerably
influenced the viscosity.
Institute Of Pharmaceutical Biology, University of Regensburg, Universlt~tsstr. 31, 8400 Regensburg (F.R.G.)
A GERMACRANOLIDE, GUAIANOLIDES AND XANTHANOLIDES FROM
FLOWERS OF Arnica moZlls
Chr. Marcinek and G. Willuhn
Up to now pseudoguaianolides, xanthanolides, and eudesmanolides have been found in species of the genus Arnica
(1,2). In the present paper we report on the isolation of
a germacranollde (I), two gualanolldes (II,III), and a new
xanthanolide (IV) in addition to xanthalongln and l ~ , "13dlhydroxanthinosin from the flowers of Arnica mollis Book.
(subgenus C ~ s s o n i s ) ,
which were identified by spectroscopic methods (1H-NMR, L3C-NMR).
I is the 2-0-acetylderivative
of balleyin;
the baileyin
c o n f i g u r a t i o n a t C -I and C-10 ha s been d i s c u s s e d c o n t r a d i c t o r y ( 3 , 4 ) . According to our i n v e s t i g a t i o n
I is a germacrolide (l,10-tPans),
whic h was e s t a b l i s h e d by X - r a y s t r u c ture analysis.
I I and I I I were found to be 1 , 5 - o l s - g u a i a nolides, which is in agreement with the postulated biosynthetic pathway for xanthanolides (5).
RO H ~/OH
~ , 0
I
"0
0
ii
R : He
II| R
IV
; hg
I. G. Willuhn and H.-D. Herrmann, Planta Mad., 37 (1979)
325-332
2. G. Willuhn, I. Junior, J. Kresken, G. Pretzsch and
D. Wendisch, Planta Med., (1985) 398-401
3. T.G. Waddell and T.A. Gelssman, Phytochemistry, g (1969)
2371-2379
4. W. Berz, E. Murari and J.F. Blount, J.Org. Chem., 44
(1979) 1873-1875
5. F . C . Seaman, B o t . R e v . , 48 ( 1 9 8 2 ) 1 2 1 - 5 9 5
Institut for Pharmazeutische Biologle d e r Unlverslt~t
DUsseldorf,
4000 D~sseldorf I (F.R.G.)
239
CHARACTERIZATION OF Ribes n i g n ~ ANTHOCYANINS IN EXTRACTS
BY GC, MS AND HPLC
E.M. Martinelli*, R. Seraglia*, A. Scillngo*, M." Briquez O
and P. Reglnault 0
The extract of fruits of R. nigr~m contains a number of
anthocyanin mono- and diglycosldes (l). Derlvatization with
hexamethyldisilazane, transforms flavylium cations of
anthocyanins into the corresponding quinoline-llke derivatives (2) which are suitable for capillary GC. The EI mass
spectra showed both aglycone and sugar characteristic fragments, 9but the molecular ions (range 1000-1400 m/z).appeared
as very small peaks, whereas the CI mass spectra, obtained
using ammonla as reagent gas, afforded very intense molecular ions. The results allowed the identification of the
main anthocyanlns, i.e. cyanidln and delphlnldin as 3-0monoglucosldes and 3-O-rhamnoglucosldes. Capillary GC-MS
revealed the presence of minor anthocyanins, up to now undescribed. HPLC did not allow the online recording of mass
spectra due to the use of strongly acid eluents necessary
to avoid chemical transformation of the anthocyanlns. HPLC
with UV-VIS detection is the method of choice for a specific detection of the anthocyanlns in the extract due to the
high wavelength absorbance of these compounds (530 nm),
because it allows an identification of the individual anthocyanins without derlvatlzation.
I. J. Le Lous, B. Majole, J.L. Morlniere and E. Wulfert,
Ann. Pharm. F r a n ~ a i s e s , 33 ( 1 9 7 5 ) 3 9 3 - 3 9 9
2. E. B o m b a r d e l l i , A. B o n a t i , B. G a b e t t a , E.M. M a r t i n e l l i
and G. M u s t i c h , J . C h r o m a t o g r . , 139 ( 1 9 7 7 ) I l l - 1 2 0
* Invernl della Beffa, Research and Development Laboratories, Via Ripamonti 99, 20141 Milan (Italy)
O Laboratolres Fournier, 42 Rue de Longvic, 2[300 Chengve
(France)
COMPARATIVE STUDY OF THE TABERSONINE CONTENT OF
Amsonia AND
Rhaaya SPECIES
I. M~th~ Jr.*, I. M~th~* and A. M~th~ O
The evaluation of the alkaloid content of the Eu~sonia
species Amsonia tabez~aemontana Air., A. ilZustris Woodson,
A, angustifogia (Air.) Michx. (1), was extended to two further species A. rlg~da Shuttlew. and A. eILiptica (Thunb.)
Scem. et Schult. Seeds of species from the subgenus
Sphinctoss
A. kaerneyana Woodson, A. paLmeri Gray, and
t h e s u b g e n u s Longiflor~, A. grandlfLora A l e x a n d e r , and from
t h e s u b g e n u s hrticuLaria, A. breus
( G r a y ) and A.
tomentosa T o r t . and T r a m . , were t e s t e d f o r l n d o l e a l k a l o i d s .
The species mentioned contained relatively high amounts of
the s
alkaloid tabersonine in their seeds (0.56 1.17%, dry wt.).
A study of the variation of the tabersonlne content, regarding both the tabersonine percentage and the blomass,
i n d i c a t e d t h a t A. ~ZlustPis and A. an~rustifol~a n o r m a l l y
c o n t a i n e d h i g h e r v a l u e s t h a n A. taber'naemontana.
Based on t h i s s t u d y i t became p o s @ i b l e t o c h a r a c t e r i z e n o t
o n l y t h e s p e c i e s o f t h e s u b g e n u s Euamsonia g r o w i n g i n
H u n g a r y , b u t a l s o t o compare them w i t h Rhazya orientaZia.
The latter yields less seed and', therefore, also less
tabersonlne in comparison with the former species, although
R. oPiental~s seeds contain tabersonine in similar percentages (dry wt.).
I. I. M~th~ Jr., A. Vad~sz, T. Rave, I. M~th@ and A. M~th~,
Acta Horticult., 132 (1983) I01-IIO
* Institute of Ecology and Botany of the Hungarian Academy
of Sciences, 2163 V~cr~t6t (Hungary)
O Institute of Plant Breeding of the University Horticultur,
Budapest (Hungary)
240
Vol.
FORMATION OF 5-METHYLDIGINATIN
VIA BIOTRANSFORMATION
BY
Digitalis Zanata CELL cuLTURES
P. Meiss and E. Relnhard
9 -
I987 Pharmaceutisch Weekblad Scientific Edition
A NEW ACYLATED AND SOME OTHER FLAVONOID GLYCOSIDES OF
ARNICAE FLOS
I. Merfort
We reported previously that Digitalis cell cultures are
capable to form 12~,16B-dihydroxylated eardenolides via
biotransformation (I).
As diginatigenin glycosides occur only to s small extent in
nature, we tried to optimize the production of ~-methyldiginatin in I i Erlenmeyer flasks with 300 ml culture medium.
Primarily cell strain AI/10 was tested, both in the dark
and in the light.
After administration of a single dose of B-methylgitoxln
(I00 mg/l), both cultures showed similar hydroxylation
rates. After administ~atlon of several doses of substrata,
the cells cultivated in the light yielded higher amounts of
B-methyldiglnatin. Therefore, further experiments were made
with the cells cultured in the light.
In a further series of experiments an 8% glucose solution
was used as a production medium (2). Nigher yields of Bmethyldlginatin were observed under these conditions. The
substrata was added on the second and on the sixth day
after transfer of the cells into the 8% glucose solution.
After nine days of incubation with 420 mg/l 6-methylgltoxin,
in total only 20-30 mg/l of the substrate were not hydroxyls.ted, whereas B-methyldiginatin reached a concentration
up to 350 mg/l in the culture medium.
[. P. Melss, J. Sepasgosarian and E. Relnhard, Plants Med.,
(1986) 511
2. U. B a r t h l e n , D i s s e r t a t i o n ,
1983, U n i v e r s i t y of THhingen
In continuation of our studies on flavonoid glycosides in
Arnicas flos DAB 9 (A. montana L. and A. chamisso~is Less.
ssp. foZiosa (Nutt.) Maguire) this paper describes the
isolation and identification of further flavonoid glycosides (I). The flavonoid glycoside pattern is used in DAB 9
for examination of the purity by TLC.
From the ethyl acetate extract of flowers of A. chamissonis
sap. foliosa var. i n c a n a a new compound was isolated by
column chromatography (Sephadex LN-20, Polyclar AT) and
RLCC. Its structure was confirmed by UV spectroscopy, MS,
IH- and 13C-NMR, and hydrolysis as patuletin 3-(6"-0acetyl)-6-D-glucopyranoside,
a new natural compound. Together with quercetin 3-(6"-O-acetyl)-~-D-glucopyranoside
it can be used to distinguish the flowers of both Arnica
species by TLC (1).
Additionally the 7-O-glucosides of hispidulio, i, Jaceosidin, ~, pectolinarigenin, !, and eupafolin, 4, were
isolated and identified. ! has already been found in A.
montana (I), 4 in Arnica species of subgenus Austromontana
(2); 2 and 3 are new for the genus Arnica.
From the flowers of A. montana the first glucuronide in the
genus Arnica was isolated by column chromatography
(Amberlite XAD-2, Polyclar AT, Sephadex LH-20), RLCC, and
TLC. It was identified by the methods mentioned above as
patuletin 3-O-6-glucuronide.
The structure elucidation of further flavonold glycosides
is in progress.
I. I. Merfort and D. Wendisch, Planta Mad., in press
2. S.J. Wolf and K.E. Denford, Biochem. Syst. Ecol., 12
(1984) 183
Pharmaceutical Institute, University of TObingen,
Morgenstelle g, 7400 TObingen (F.R.C.)
Institut for Pharmazeutische Biologie der Universitgt
DUsseldorf, 4000 D0sseldorf I (F.R.G.)
A DITERYENOID
Auf der
WITH A NEW CARBON SKELETON FROM
P~gmaeopremna he~baoea
S E P A R A T I O N OF NON-POLAR COMPONENTS OF ANISI FRUCTUS BY DCCC
H. Miething, V. Seger and R. Hgnsel
Qlngchang Meng* V, NalJue Zhu O and Welxln Chen*
Pygmaeopremna herbacea (Roxb.) Moldenke
(Verbenaceas) is
used to diminish inflammations and to cure malaria as a
folk remedy in the Yunnan Province of China. No
Pygmaeopremna species has been described as to its
chemical constituents to date. From the dlethyl ether
extracts of the roots of P. herbaoea seven crystalline
compounds were isolated. One of them, pygmaeocine E, is
reported upon in this paper. Its structure was determined
by spectroscopic methods, and it was verified by X-ray
diffraction: [,l,10-trimethyl-7-isopropyl-3-hydroxyanthran2,5,6-trl-one, a diterpenoid with a new carbon skeleton.'
* Chengdu Institute of Biology, Chinese Academy of Sclence~
Chengdu, Sichuan (People's Republic of China)
O Institute of Chemistry, Chinese Academy of Sciences,
Beljing (People's Republic of China)
V Division of Pharmacognosy, Center for Blo-Pharmaceutical
Sciences, Leiden University, Gorlaeus Laboratories, P.O.
Box 9502, 2300 RA Leiden (The Netherlands) - present
address
Droplet counter current chromatography (DCCC) is a widely
used technique for the separation of natural compounds.
Because of the requirement to form droplets, especially
water-contalnlng solvent systems were developed. Also some
experiences wlth water-free systems have been made.
During our studies on the analysis of Anlsi fructus (fimpi~ella anisum) we tested water-free solvent systems using
methanol and n-hexane for the enrichment and separation of
the non-polar components.
The application of these systems is demonstrated for
anethole, anisaldehyde, foeniculin, pseudoisoeugenyl 2methylbutyrate and some other compounds. Additionally mixtures of other non-polar natural products were separated
using these solvent systems.
Institute of Pharmacognosy and Phytochemistry, Free
University of Berlin, K~nigin-Luise-Str. 2-4, I000 Berlin
33 (F.R.G.)
Vol. 9 "
I987 Pharmaceutisch Weekblad Scientific EdiSon
24I
THE EFFECT OF ALKALOIDS ON THE GROWTH OF PHYTOPATROGEN
BACTERIA OF THE GENUS Agrobaoterium
H. Neuh~fer, G. Lurz, P. Bachmann, R. Greinwald and
F.-C. Czygan
SELECTION OF Hyoecyamua muticua PLANTS WITH HIGH TROPANE
ALKALOID CONTENTS
K.-M. Oksman-Caldentey*7, H. Vuorela O, M. Isenegger*,
A. Strauss* and R. Hiltunen O
Alkaloids may represent a part of the plant defense system
against pathogens and herbivores. From the investigations
of Wink (I), it is known that qulnolizidlne alkaloids are
very effective in this respect. These alkaloids occur in
high concentrations (up to 200 a~M) in the cells of the
epidermis of plants belonging to the family Fabaceae.
Scopolamine is primarily extracted on an industrial scale
from plant sources, mainly from Duboiaia but also from
Datu_ra and Hyogc~amus species. The alkaloid content in
Egyptian henbane, Hyoac~am~a n~tioua L., has been shown
to be dependant on the development stage, ploidy level and
environmental factors. We have earlier shown that there is
great internal variation on the scopolamine as w e l l a s in
the hyoseyamlne content among the same plant population
( 1 ) . In the present study it was confirmed that such variation was also present in selected plant populations.
The quantitative alkaloid determination was performed by
radio or enzymeimmunosssay.
Selection for high alkaloid containing plants (selfed)
resulted in an increased production of these alkaloids in
their progenies. The alkaloid content in the F[ generation
was increased by a factor three, and in the F 4 generation
by a factor 20 compared to the P generation, respectively.
The mean byoscyamine contents were higher than the mean
scopolamine contents for all the populations. When crossing
the high and low scopolamine producing plants, the scopolamine content of the progenies was found to be intermediate
between that of the two parents.
I. K.-M. Oksman-Caldentey, H. Vuorela, A. Strauss and
R. Hiltunen, Planta Med., in press
However, somephytopathogenbacteria can overcome the
plant defense system. Some of the most prominent ones
belong to the genus Ag~obactePi~'n, which cause the "crown
gall" and "hairy root" diseases on a wide range of
dicotyledons.
Therefore we tested the effect of different alkaloids on
the growth of various strains of AgrobactePium. The
experiments showed, that the resistance against the alkaloids must be localized on the chromosome and not on the
plasmlds of Agrobacteri~.
I. M. Wink, Z. Naturforseh., 39 (1984) 548-552
Lehrstuhl f~ir Pharmazeutische Blologie der Unlversit~t
WUrzburg, Mittlerer Dallenbergweg 64, 8700 WUrzburg
(F.R.G.)
* Ciba-Geigy Ltd., Department of Biotechnology, 4002 Basel
(Switzerland)
O Division of Pharmacognosy, School of Pharmacy, University
of Helslnki, Fablaninkatu 35, 00170 Helsinki (Finland)
V Present address, see O
SELECTION OF Valeriana officinalia L. BY EVALUATION OF
GROWTH AND CHEMICAL COMPOSITION
F. Noller and A. V~mel
FURANODITERPENE GLYCOSIDE AS A BITTER PRINCIPLE OF
YinospoPa cpispa MIERS
P. Pachaly and A.Z. Adnan
From former screening experiments of Valeriana officina~is
It can be concluded that besides valeranone also valerenal
and valerenic acid (VA) have central depressive and/or
spasmolytic properties (1). The goal of our investigations
was to retain highly valid covarieties from populations. VA
and valerenal in the roots of filial generations from cross
pollinated inbred lines were analysed by means of reversedphase HPLC; estimation of valeranone was performed qualitatlvely by TLC. From each of the F1-plants half of the"
root and rhizome was used for determination of yield and
chemical analysis. Results:
l) Compared to plants of a clone, variability of FI- and
F2-generations was high. Valerenal and VA varied from
0 to 150 mg/[00 g and from 0 to 550 mg/lO0 g respectively in the roots;
2) Absence of VA in filial generations from VA-free parents
seems to indicate recessive heredity;
3) Slight correlation between the percentages of valerenal
and VA existed in F2-generations of some of the
crossings.
While the present data enable us to select high valid types
from F2-plants, further studies about influence on the
variability of the active compounds have to be performed.
I. H. Hendrlks, R. Bos, H.J. Woerdenbag and A.SJ. Roster,
Planta Med., (1985) 28-31
Stems of Tinospora crispa are one of the most popular
traditional drugs in Indonesia.
Th~ drug infused in boiling water gives a bltter tasting
drink with aperltive, digestive, tonic and antimalarial
properties, which is used against stomach trouble, dlarrhoea and rheumatism as well.
Known ingredients of Tinospopa orlspa are the quarternary
alkaloids berherlne, palmatine and tembetarine (]).
In our relnvestlgatlon of the drug we isolated a very
bitter tasting new dlterpene glycoside tlnocrlsposide.
Its structure I was established by chemical and spectroscoplc methods.
H]C~O
,
I. N.G. Bisset and J. Nwaiwu, Planta Med., 48 (1983)
Institut f~r Pflanzenbau und ZUchtung der JL-Universit~t
Giessen, Versuchsstation Rauischholzhausen, 3557 Ebsdorfergrund 4 (F.R.G.)
275
Pharmazeutisches Instltut der Universit~t Bonn, Kreuzbergwen 26, 5300 Bonn (F.R.G.)
242
V o l . 9 - z987
SELINANE SESQUITERPENOIDS AND A CI8-POLYACETYLENE FROM
At~actyLis koreana (ASTERACEAE)
P. Pachaly*. A. Lansing* and K.S. Sin 0
The medicinal plant Atractyli8 koPeana is often used
against fever, rheumatism, dysentery and apoplexy.
The first investigation of the rhizomes of Atz~aot~i8
koreans led to the isolation of two sesquiterpenoids and
one polyacetylene from a petrolether/ether extract.
The sesquiterpenoids I and 2 were identified as atractylone
and sellna-4(14),7(l|)-dlen-g-one from spectroscopic and
chemical data. These substances are knotm as components o f
the essential oil of AtractyZi8 japonica (1,2).
The structure of the polyacetylene 3, which is a new compound of the genus A~otytis, was determined as [,3dlacetoxytetradeca-6,12-dien-B,[O-diin (3) by spectroscopic
and chemical methods.
CH~
CH1
0
_1
2
Pharmaceutisch Weekblad Scientific Edition
ANTIHYPERGLYCEMIC AND INSULIN RELEASE EFFECTS OF TORMENTIC
ACID
M. Payg*, M.D. Ivorra* and A. Villar O
In a previous study (I) tormentic acid, a hypoglycemic
principle from PotePiwnanes
Desf. (Rosaceae), was
i s o l a t e d and e l u c i d a t e d . I t s h y p o g l y c e m i c a c t i v i t y was
established in normoglycemlc Wistar rats.
In the present study female Wistar rats were used to determine the antihyperglycemic and insulin release effects o f
tormentic acid compared with those of glibenelamide.
Tormentic a c i d (30 mg/kg) and glibenclamide ([0 mg/kg)
were administered orally with an overdose of glucose
(2 g/kg). The control group received only the overdose of
glucose (2 g/kg). The blood samples for the glucose and
insulin determination were collected from the jugular v e i n
at intervals of 30 min during 2 h. The glycemia level was
determined with an enzymatic glucose-oxidase method and the
insulinemla level by RIA.
The experiments showed that tormentic acid increased the
circulating insulin levels with a corresponding decrease
in the glyeemie levels. However this effect was less
potent than that of glibenclamlde.
I. A. Villar, M. PayS, M.D. HortlgEela and D. Cortes,
Planta Med., (1986) 43-45
CH3-- CH=CH-(C=C)z- CH= CH-CH3-GHI--CtH-CHI-CH~
_3
o
?
~=o
!
c=0
CH}
CH"1
I. Hikino et al., Chem. Pharm. Bull., 12 ([964) 755-60
2. Endo et al., Bull. Chem. Soc. Jpn., 52 (1979) 2439-40
3. Bohlmann et al., Phytochemistry, 22 (1983) 2858-59
* Pharmazeutisches Instltut der Universit~t Bonn, Kreuzbergweg 26, 5300 Bonn (F.R.G.)
O Department of Pharmacy, Kang-Weon National University,
Chuncheon 200 (Korea)
ANTIFUNGAL EFFECT OF ISO-EUGENOL AND TERPENOID ALCOHOLS
ON PeniciZ~ium vlmldicatum
A. Pauli*O and K. Knobloch*
A combination of essential oils may lead to a synergistic
activity as observed in a few living systems ([,2). In
order to evaluate antlfungal effects caused by mixtures
of varying concentrations of Iso-eugenol and structurally
related monoterpenold (L-cltronellol, linalool, uerol,
geraniol) and sesqulterpenoid ~-blsabolol, farnesol)
was grown and handled
alcohols, PenicilZium virids
as described before (3). Depending on the ratio isoeugenol/terpenold alcohols, a specific inhibitory influence
on the fungal growth was observed. When iso-eugenol was
applied at a 6.5 nM concentration and 32.5 nM of nerol oe
geraniol were added, an enhanced antlfungal activity could
be noticed. 32.5 nM L-cltronellol caused an even more
pronounced effect. The strongest antlfungal effect was
obtained for a mixture of 19.5 nM geraniol and 32.5 nM
iso-eugenol. Linalool, e-blsabolol and farnesol were not
found to exhibit synergistic activities in combination
with iso-eegennl.
|. S.R. Charon, N.F. Shah and S.T. Nfkam, Bull. Half. Inst.,
I[ (1984) 18-21
2. N.K. Saksena and S. S a k s e n a , I n d . P e r f u m . , 28 ([984)
42-45
3. A. Paull and K. Knobloch, Inhibitory Effects of
Essential Oil Components on Growth of Food-Contamlnatlng
Fungi. Z. Lebensm. Unters. Forsch., in press
* Institut fHr Botanik und Pharmazeutische Biologie der
Universlt~t Erlangen-Nfirnberg, Staudtstr. 5, 8520
Erlangen (F.R.G.)
O Instltut ffir Umwelthygiene und Pr~ventivmediztn der
Unlverslt~t Erlangen-NOrnberg, Wasserturmstr. 5, 8520
Erlangen (F.R.G.)
* Laboratorlo de Farmacognosia y Farmacodinamia, Departamento de Farmacologfa y Farmacoteenia,
Facultad de Farm a c i a , U n i v e r s i t a t de V a l ~ n c i a , Av. B l a s c o Iba~ez 13,
4 6 0 1 0 V a l e n c i a (Spain)
O L a b o r a t o r i o de F a r m a c o g n o s i a y F a r m a e o d l n a m l a , D e p a r t a mento de F a r m a c o l o g f a , F a c u l t a d de F a r m a c i a , U n l v e r s l d a d
Complutense de M a d r i d , Ciudad U n l v e r s i t a r i a ,
28040
Madrid ( S p a i n )
DETECTION OF LUCIDIN, A GENOTOXIC PRINCIPLE IN ROOT
EXTRACTS OF Rubio tinetoi,'~n AND THE CONVERSION TO ITS
ETHYL ETHER DURING ETHANOL EXTRACTION
B. Poglnsky*,O~ J. Westendorf*, LJ. Kraus O and H. Marquardt
Root extracts of madder (Rubia tinotol'um L.) are widely and
chronically used in therapy and prophylaxis of urollthiasls.
Among the active principles are hydroxyanthraqulnones and
their glycosides which have also been used as dyes.
We prepared an ethyl acetate extract of madder, and divided
it into 40 fractions by HPLC. Mutagenicity testing of the
fractions by the Salmonella/mlcrosome assay showed one
fraction to be highly mutagenlc. The corresponding compound
was identified as lucidin (I,3-dlhydroxy-2-hydroxymethyl9,10-anthraquinone) by mass and NMR spectroscopy. The compound was then tested in a battery of genotoxiclty assays
(mutageniclty in bacterial and mammalian cells, Induct'Ion
of DNA-repair in primary rat hepatocytes and in vitro transformation of C3H/M2-~ouse flbroblasts) and was found to be
positive in all assays.
During extraction of madder with ethanol, lucidln was partly cofiverted to the corresponding 0J-ethyl ether. This
reaction l~ highly temperature-dependant. The ~0-ethyl ether
was also mutagenlc. However, In contrast to lucidln, this
effect r e q u i r e d metabolic activation, probably by o x i d a t i v e
conversion to lucldin.
The experiments demonstrated the genotoxic potential of
lucldin and'suggested a possible risk to human health, when
madder or other lucidin-containing plant extracts are
t h e r a p e u t i c a l l y used.
* Abt. f~r Allgemelne Toxlkologle, Unlversit~tskrankenhaus
Eppendorf, Grindelallee 117, 2000 Hamburg 13
(F.R.G.)
0 Lehrstuhl ffir Pharmakognosle der Onlverslt~t Hamburg,
Bundesstrasse 43, 2000 Hamburg 13 (F.R.G.)
This work is based on a doctorate study by B. Poglnsky in
the Faculty of Biology, University of Hamburg
Vol. 9 - I987
Pharmaceutisch Weekblad Scientific Edition
TWO NEW ANTIFUNGAL NAPHTHOXIRENE DERIVATIVES AND THEIR
GLUCOSIDES FROM Sesumum ungo~ense
O. Potterat*, H. Stoeokli-EvansO, J.D. Msonthl V and
K. Hostettmann*
In our search for biologically active constituents from
African medicinal plants, we have investigated Sesamum
angoZense Welw. (Pedallaceae). The llpophillc extract from
the root bark of S. angoZenae exhibited antlfungal activity
against CZadaspoPiwn cuoumer~num as well as cytofoxicity
towards Co-I15 colon tumour ceils.
A combination of flash chromatography and low-pressure LC
on RP-8, applied to the dichloromethane extract, led to the
isolation of three antlfungal compounds, naphthoxirenes
and 2 and glucoside 2"
Naph~hoxirene ~ was shown to be responsible for the antiproliferative activity of the dlchloromethane extract.
DCCC separation of the methanol extract, followed by lowpressure LC on RP-8, provided glucnslde 4 which showed
neither fungicidal nor cytotoxic properties.
The structure o f ~ was established by X-ray d i f f r a c t i o n
a n a l y s i s . The i d e n t i t i e s o f the other naphthoxlrenes were
deduced by c h e m i c a l and s p e c t r o s c o p i c methods.
OR
O
OR
243
PROANTHOCYANIDINS FROM Pote~um
G. Reher
sps
(ROSACEAE)
Some Pote~um species are used in folk medicine in the
Mediterranean region for their hypoglycemic effect.
The effect may be attributed to tormentlc acid and other
pentacyclic trlterpenes (1,2).
The r o o t b a r k o f Poterff.um sps
was i n v e s t i g a t e d f o r
i t s p h e n o l i c compounds. The most i m p o r t a n t p r o a n t h o c y a n i d l n s , a n t h o c y a n i d i n s and c a t e e h o l s were s e p a r a t e d ,
i s o l a t e d and i d e n t i f i e d . DCCC and g r a d i e n t HPLC were used
f o r t h e s e p a r a t i o n o f t h e p r o a n t h o e y a n i d i n s . The compounds
were i d e n t s
by c h r o m a t o g r a p h i c and s p e c t r o s c o p i c
methods.
l . G. C a r r a z , A. B o u c h e r l e and A. Dardas, C.R. Acad. S e l . ,
P a r i s , Ser." D, 266 (19687 293-4
2. A. Villar, M. PayS, M.D. HortigOela and D. Cortes,
Planta Med., (1986) 43-45
O
OH O
H
I
R - II
2
R = II
3
R - R-D-glucopyranosyl
&_
R - B-O-glucopyranosyl
* Institut de Pharmacognosie et Phytochimle, Unlversitfi
de Lausanne, Lausanne (Switzerland)
0 Instltut de Chimie, Unlverslt6 de Neuch~tel, NeuehRtel
(Switzerland)
V Department of Chemistry, University of Malawl,
Chancellor College, Zomba (Malawl)
GC-MS ANALYSIS OF LIPOPHILIC FLAVONOID AGLYCONES IN PLANT
EXTRACTS
M. Randrlamlnahy, P. Proksch, L. Wltte and T. Hartmann
As part of a study on flavonolds from HeZichPIjsum species
from Madagascar we developed a GC-MS procedure that allows
a rapid and unambiguous identification of underlvatlzed
flavonoid aglycones in plant extracts. The method combines
the high resolution of capillary GC and the mass spectral
information on the compounds present, and adds a new and
useful tool for flavonold analysis.
A crude methanollc extract was separated by column chromatography on Sephadex LH-20 into a non-phenollc and a
phenolic fraction. The latter was directly submitted to
GC-MS. The usefulness and limitations of the procedure will
be discussed.
Instltut fQr Pharmazeutische Biologie der T.U.,
Mendelssohnstr. I, 3300 Braunschwelg (F.R.G.)
Division of Pharmacognosy, University of Hamburg,
Bundesstr. 43, 2000 Hamburg 13" (F.R.G.)
STUDY ON THE ANTIOXIDANT PROPERTIES OF POLLEN EXTRACTS
L. Samochowlec, J. W6jclckl, D. Kadlubowaka and A. Kownaeka
Pollen extracts (Cernitins) contain mainly water-soluble
substances (Cernitln T60) as well as fat-soluble components
(Cernltln GBX). It has been demonstrated that Cernltlns
have a remarkable lipid lowering effect, both in animals
and in humans. In addition, it has been established that
they have a beneficial effect against the development of
atherosclerosls.
The study on the antloxldant and hypollpldemlc effect of
pollen extracts was performed using male mongrel rabbits
and Wistar rats. The animals were fed upon a hlgh-fat diet
composed of cholesterol, coconut oli and chollc acid, and
received pollen extracts (Cernltlns) orally, the rabbits
over a period of 12 weeks and the rats over a period of
two weeks. The levels of malondialdehyde (MDA) as an
indication of the degree of peroxidatlon, and of llplds
(cholesterol, trlglycerldes, separation of llpoprotelns
into fractions) were measured.
The study showed a reduction of MDA concentrations under
the influence of Cernltlns suggesting antloxldant properties of them. The total cholesterol and trlglycerlde
content was also decreased.
Institute of Pharmacology and Toxlcologle,
Wlkp. 72, 70-111Szczecln (Poland)
Powstafic6w
244
COMPOSITION OF THE ESSENTIAL OIL OF
E. ~ a r e r
VOI. 9 " 1987
Salvia pisidiea
The genus Salvia (Labiatae) is represented in Turkey by
more than 80 species and some of them are endemic (I).
Salvia pisidiea Boiss. et Reldr. is an endemic plant of
southern Anatolia, the essential oil of which hasn't been
investigated up to now. For the present study, flowering
parts of $. ~isidioa were collected near Elmall (Antalya,
Turkey) in June 1985. The plant material was subjected to
hydrodistillation in a Clevenger apparatus in order to
obtain the essential oil. The chemical composition of the
ell was studied by GLC after fractionation by column
chromatography under conditions reported before (2).
The content of essential oil was 0.7% (v/w). The majority
of the oil (85%) was formed of oxygen-containing component&
whereas only a small quantity (15%) of hydrocarbons was
present. The main constituents of the oil were sabinyl
acetate (28.8%), e - and ~-thujooe ([8.3%), 1,8-cineole
(II.1%) and camphor (9.2%).
Further analytical data and chromatograms of the sample
studied will be presented.
1. P.H. Davis, Flora of Turkey and the East Aegean Islands,
Vol. 7, 1982, University Press, Edinburgh
2. E. ~ a r e r , DSga C I I , I (1987) 97-103
Pharmaceutisch Weekblad Scientific Edition
THE ESSENTIAL ROOT OIL OF Peuoedanum paZuatre
G. Schmaus*, W. Schultze*, K.-R. Kubeczka* and R. Hlltunen O
Peueedanum palustre
(L.) Moencb (Apiaceae) is a perennial
plant growing at wet places in most of Europe, extending
eastwards to central Asia. The root has a pungent, bitter
taste, and an odour somewhat resembling to incense.
Formerly it was offlclnal under the name Radix Oisnitli
and used in folk medicine particularly as a remedy against
epilepsy. According to literature the root contains resinous substances, coumarins and an essential oil of rather
unknown composition.
We analysed the essential root oil of P. paZustre. Recently
we reported on the occurrence of sesqailavandulol as the
main constituent of this oil (I). Besides this uncommon
sesqulterpene alcohol further compounds were identified
by GC-MS, IR and NMR spectroscopy.
The essential oil consisted of 30% monoterpene hydrocarbons, 5% sesquiterpene hydrocarbons and 65% oxygenated
compounds. The structures of several hitherto unknown
compounds were elucidated.
I. K.-H. Kubeczka, G. Schmaus and V. Formacek, Structural
elucidation of an irrigular seaquiterpene alcohol from
Pe~eedanwn palustre (L.) Moench. In: Progress in Essential Oil Research, E.-J. Brunke, ed,, 1986, Walter de
Gruyter & Co., Berlln/New York, pp. 271-278
Department of Pharmacognosy, Faculty of Pharmacy, Ankara
University, Tando~an-Ankara (Turkey)
* Department of Pharmaceutical Blology, University of
WOrzbdrg, Mittlerer Dallenbergweg 64, 8700 WOrzburg
(F.R.G.)
O Division of Pharmacognosy, School of Pharmacy, University
of Helsinkl, Fabianinkatu 35, 00170 Helslaki (Finland)
FLAVONOID CONTENTS OF TURKISH WINES FROM THE TRAKYA REGION
E. Sayron-AtasO and V. Cihangir
H~ytophthora
Wines are still officinal in some pharmacopoeias. In our
study, wines from grapes of Trakya (Turkey) were screened
for their flavonold content. White wine was found to
contain trace amounts, whereas red wine contained a number
of flavonol aglycones which were determined by (two dimensional) TLC. An assay was developed making use of TLC and
UV spectroscopy. In this way, the total amount of flavonols,
and the content of the major flavonol were determined.
Department o f Pharmacognosy, F a c u l t y o f Pharmacy,
University, Tandogan-Ankara (Turkey)
INFLUENCE OF VARIOUS CULTURE CONDITIONS ON GROWTH OF
SPECIES
M. SchOtz, W. Biaschek, J. Kraus and C. Franz
In recent studies it was shown that cell walls of the
p h y t o p a t h o g e n l c fungus ['hytophthopa parasitica, b e s i d e s
cellulose, contain ~-l,3/l,6-glucans acting as elicitors
for the appropriate host plants (I). The isolated glucans
exhibited a pronounced antitumor activity against Sarcoma180 in mice via stimulation of the specific Immune system
(2). In order to obtain high amounts of these and related
antitumor active glucans, the nutritional and physical
culture conditions for different Phytophthot'a species were
investigated.
The fungi were shown to be thiamine heterotroph. Glucose,
sucrose as well as starch hydrolysates could be used as
carbon source. Growth was stimulated up to seven fold by
20 mg/l of ~ -sitosterol. After some days of adaptation, asparagine, potassium nitrate and ammonium nitrate were accepted as nitrogen source. The preculture conditions, the time
of inoculation and permanent aeration were essential for
good growth. At optimum growth conditions up to 30 g
(dry w e l g h ~ fungal mycelium could be harvested within
five days ~ n 8 1 of culture medium, which represented
about 6 g of cell wall material.
I. M.L. Bouillant, J. Fabre-Bonvin and P. gicci, Tetrahedron Letters, 24 (1983) 51-52
2. M. Brune~au, I. Fabre, J. Perret, O. Michel, P. Rlccl,
J.P. Joseleau, J. Kraus, M, Schneider, W. Blaschek and
G. Franz, Carbohydr. Res., in press
Ankara
Institute of Pharmaceutical Biology, University of
Regensburg, Universit~tsstr. 31, 8400 Regensburg (F.R.O.)
Vol. 9 - I987 Pharmaceutisch Weekblad Scientific Edition
MONOTERPENE GLYCOSIDES OF DIFFERENT LAMIACEAE*
G. Schulz and E. Stahl-Biskup
It has been shown that monoterpene glycosides evidently
are involved in the formation, transport, and accumulation
of essential oils in plants (I). However, so far little is
known about the systematic distribution of monoterpene
glycosides.
The composition of glycoside fractions was investigated
for some L a m i a c e a e : Hys~opus officlnalis, Laoandu~a
angustifoZia, Melisaa officinaZis, Ocimum basiticwn,
Rosmarinun offieinalis, Salvia officinalis, Origanam
vuZgape.
By means of HPLC on RP-18, 5-8 monoterpene glycosides were
detected in all species studied. The terpenoid moieties
were identified, after hydrolysis by B-glucosidase at
pB 6.5, by GC and GC-MS.
The experiments showed that even when the major components
of an essential oil have a hydroxylic group, they do not
always occur as glycosides. This suggests various roles
of the glycosides in question.
I. E. Stahl-Biskup, Monoterpene Glycosides, State-of-theArt, Flay. Fragr. J., in press
* Part of the thesis of G.S.
Department of Pharmacognosy, University of Hamburg,
Bundesstr. 43, 2000 Hamburg 13 (F.R.G.)
BIOCHEMICAL CHARACTERIZATION OF EMBRYOGENIC
lanata SUSPENSION CULTURES
S. Seidel and E. Reinhard
Digitalis
Undifferentiated
c e l l c u l t u r e s of D i g i t a l i s lanata a r e n o t
a b l e to p r o d u c e c a r d e n o l i d e s ( 1 ) , w h e r e a s m o r p h o l o g i c a l
differentiation
l e a d s to c a r d i a c g l y c o s i d e formation ( 2 ) .
Differentiation was initiated by transferring long-term.
suspension cultured cells of an embryogenic strain from a
modified MS-medlum to an appropriate differentiation medium
Embryo formation started after six weeks and led to stable
cultures with leaf-like structures after 15 weeks. Their
cardenolide content was monitored by digitoxin RIA through
all differentiation stages. In addition, several enzyme
activities, i.e. UDP-glucose:digitoxin 16'-O-glucosyltransferase and digilanidase (3), glucose-6-phosphate dehydrogenase, malate dehydrogenase, a-mannosidase, acid phosphatas~
8-glucosidase, and lanatoside acetylesterase were measured.
Digilanidase activity was absent in undifferentiated cell
cultures, but increased strictly parallel to the cardenolide accumulation, thus providing an additional marker for
beginning cardiac glycoside formation.
After fractlonating a methanolic extract of these differentiated embryogenic cultures via preparative HPLC, we
found odorosid H, odorobiosid G, glucodigifucosid, verodoxin and strospesid as major cardenolides (4). Their
identity was verified by comparison with authentic samples
on TLC, HPLC and by MS.
I. E. Relnhard, M. Boy and F. Kaiser, Planta Med. Suppl.,
(1975) 163-168
2. M. L u c k n e r and B. D i e t t r i c h ,
I n : P r i m a r y and S e c o n d a r y
Metabolism of Plant Cell Cultures, K.-H. Neumann, W.
Barz and E. Reinhard, eds., 1985, Springer-Verlag,
Berlin, Heidelberg, pp. 154-163
3. W. Kreis, U. May and E. Reinhard, poster, this congress
4. S. Seidel and E. Reinhard, Planta Med., in press
Pharmaceutical Institute, University of T~blngen, Auf der
Morgenstelle 8, 7400 TUbingen (F.R.G.)
245
16~-HYDROXYLATION OF DIGITOXIN BY D i g i t a l i s puppupea CELL
CULTURES
J. Sepasgosarian and E. Reinhard
In 1970, the 168-hydroxylation of digitoxin by cell cultures of Digitalis put'puree was described by Furuya (I) for
the first time. We report here on an efficient method to
obtain comparatively high yields of purpureaglycoside B,
the 16~-hydroxylated and glucosylated product. D. pur'purea
cell strain JS 13 was tested in five different media
(listed below) in order to find the best conditions for a
16~-hyd~oxylation of digitoxin.
Culture media: I. G 55: modified MS-medium (with casein/
bidlstilled water); 2. OCLWP: modified MS-medium (lacking
casein / increased cont. of phosphate, glycin and B-vitamins / tap wa6er).
Production media: 3. 8% glucose solution (2); 4. glucosefree OCLWP-medium; 5. tap water.
Cell strain JS 13 was cultivated either in the light or in
the dark, at 25"C, as shake cultures in 300 ml Erlenmeyerflasks containing I00 ml of liquid medium. 120-150 mg/l
digltoxin was added on the second day after transferring
the cells into the culture medium. In the experiments using
production media, the substrate was added immediately after
transfer. The cardenolide content of the freeze-dried cells
and the medium was monitored by HPLC. Highest yields of
16~s-hydroxylated products (5.14 mg/g d.w.) were obtained
from the "tap water-production medium".
i. T. Furuya, M. Hirotani and T. Shinohara, Chem. Pharm.
Bull., 18 (1970) 1080
2. U. Barthlen, Dissertation, 1983, University of T0bingen
Pharmaceutical Institute, University of T~blngen, Auf der
Morgenstelle 8, 7400 TOblngen ('F.R.C.)
COMPARISON OF FATTY ACIDS IN COMMON FINNISH DIETARY FATS
T. Sepp~nen*, T. Laitalalnen O, I. Laakso* and R. Hiltunen*
After a recent legislation, new dietary fat products with
40 or 60% fat instead of earlier 80% have become available
in Finland. Such mixtures contain vegetable oll and butter
or'margarine in various ratios combined with milk protein.
The determination of saturated and tPan8 fatty acids is of
gre@t importance, because these constituents are presumed
to have an inhibitory effect on further desaturation of
essential fatty acids ( 1 ) .
The analyses were performed on a DANI 3865 gas chromatograph
fitted with an SP-2340 fused silica column. Identification
of isomers was based on the retention data from literature
(2) and those of pure compounds (Cl8:ln-7t, Ci8:In-9t,
C 1 8 : 1 n - 1 2 c , C 1 8 : l n - 7 c , C l 8 : l n - 9 c and C l 8 : 2 n - 6 t t ) .
The r e l a t i v e amounts o f monoenoic C18 iron8 f a t t y a c i d s
were s m a l l e s t (1%) and t h o s e o f s a t u r a t e d ones h i g h e s t ( 4 0 45%) i n b u t t e r - v e g e t a b l e
otl mixtures (not hardened).
M a r g a r i n e s u s u a l l y had a trans f a t t y a c i d c o n t e n t o f 10-15%.
However, in one margarine sample a fat content of 80% was
found (linoleic acid 60%; a-linolenlc acid 0.2%); no tpatls
fatty acids were detected.
I. D.F. Horrobin, Essential fatty acids: a review. In:
Clinical Uses of Essential Fatty Acids, D.F. Horrobln,
ed., 1982, Eden Press, Montreal-London, pp. 3-36
2. H.T. Slover and E. Lanza, Quantitative analysis of food
fatty acids by capillary gas chromatography, JAOCS, 56
(1979) 933-943
* Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, Fablaninkatu 35, 00170 Helsinkl (Finland)
O Mineral Laboratory MILA OY, Hitsaajank. 5, 00810 Helsinki
(Finland)
246
V o l . 9 - I987
BIOLOGICAL ACTIVITY OF AN AQUEOUS EXTRACT FROM AiZanthu8
aZtissima CELL
E. Speroni*,
Minghe t t iV
An a q u e o u s
CULTURES
N. Crespi-Perellino O, A. Guleciardi O and A.
extract
from cell cultures
of
in mice, a dose-related
Ailanthus altissima
Swlngle induced,
change in behaviour after intraperitoneal administration. The aqueous
phase, obtained after extraction with toluene and n-butanol,
contained indolic structures. These compounds, precursors of
the indole alkaloids produced (l), were supposed to be 8carbollne or tetrahydro-8-carboline structures, known to
exhibit central effects, often conflicting: {.e. pro/anticonvulsive, hypo/hyper-tbermie. An ethanolic extract of the
cell cultures, depleted of lipophllic substances as previously reported (2), was active on the CNS.
Further chemical and pharmacological investigations on the
aqueous extract will be performed.
I. N. Crespi-Perellino et aZ., J. Nat. Prod., 49 (1986) 814
2. N. Crespi-Perelllno et aZ., J. Nat. Prod., in press
* Istituto Farmacologia, Via Irnerio 48, 40126 Bologna
(Italy)
0 Sviluppo Biotecnologlco Farmltalia C. Erba, Via del
Gracchi 35, 20146 Milano (Italy)
V Dipartimento Sclenze Farmaeeutiehe, Via Belmeloro 6,
40126 Bologna (Italy)
IRIDOIDS OF THE GENUS Verbaaeum
L. ~wiaZek*, B. G~abias* and P. Junlor O
Pharmaceutisch Weekblad Scientific Edition
GLC ANALYSIS OF THE EPA AND DHA CONTENTS OF SOME FISH OILS
COMMON IN TURKEY
M. Tanker, S. Kurucu and N. Tanker
Essential fatty acids are important dietary factors as
precursors of the human prostaglandins and leukotrienes.
Fish oils are a good source for a series of essential fatty
acids, llke eicosapentaenolc acid (C20:5;EPA) and docosahexaenole acid (C22:6;DHA). They have been shown to reduce
plasma triglyeerides and may reduce plasma cholesterol as
well. These fatty acids also inhibit platelet function and
prevent the formation of thrombosis.
The fish oils used in this study were either supplied as
commercial products or obtained by extraction by chloroform-methanol (2:1) from fresh fish. Methyl esters of the
fatty acids were prepared by BF3-methanol and analyzed by
GLC on a OV-17 column.
The fatty acids composition and the relative amounts of EPA
and DHA in fish oils from European anchovy, mullet, horse
mackerel, Atlantic mackerel, bluefish, common pandora et~.
will b e presented.
Department
of Ankara,
of Pharmacognosy, Faculty of Pharmacy,
Tando~an-Ankara (Turkey)
PHENYLPROPANOIDS
University
IN PLANTS AND ROOT ORGAN CULTURES OF
Coreopsis SPECIES
U. Thron, J. Relchllng and g. Martin
Iridoid fractions isolated from the 31 species belonging to
the sectio B o t h r o ~ p e ~ of the genus Verbascum (Scrophulariaceae) were analysed. The presence of 14 irldoids was
detected by chromatographic methods (PC, TLC, RPTLC).
Aucuhin, aJugol and harpagide occur c o B o n l y in Verbasoum.
The presence of these three glucosides both in this taxon
and in the previously investigated genus Sc~ophuZaPia (I)
indicates a phylogenetlc relationship between these genera
and supports the view that they belong to one tribe,
Verbaseeae (2).
In the leaves of ten species a glycoside was detected, which
was later isolated and identified by IH- and ]3C-N~, and,
MS as geniposld acid.
It is the first irldoid glycoside with a 10-C aglycone
structure found in the genus VePbasc~.
I. S. Luczak and L. ~wiaXek, Herba Pol., 24 (1978) 27
2. J.W. Thieret, Sida Contr. Bot., 3 (1967) 87
* Institute of Environmental Research and Bioanalysis,
Medical Academy in L6df (Poland)
0 Institut f~r Pharmazeutische Biologie der Unlversit~t,
Ueutschhausstrasse 17 I/2, 3550 Marburg (F.R.G.)
Our special interest concerns comparative studies on the
biosynthesis of secondary products in plants and corresponding cell cultures. We established in vitro cultures
of Coreopsis grandiflora, C. LanceoZa~a and C. tinc~oria
and compared root organ cultures with roots of these plants
wlth respect to the accumulation of phenylpropanolds.
In all species we identified three phenylpropanoids, already found in other CoPeopsis species: l'-isobutyryloxyeugenol isobutyrate; l'-acetoxy-eugenol isobutyrate;
els-|',2'-epoxy-conlferylalcohol
dllsobutyrate (1).
Additionally we found three new compounds: l'-(2-methylbutyryloxy)-eugenol isobutyrate; l'-Isovaleroyloxyeugenol Isobutyrate; cis-l',2'-epoxy-eonlferylalcohol3'-(2-methylbutyryl)-4-isobutyrate.
These compounds were the main constituents of the roots,
whereas the shoots 6(mtalned only small quantities. They
could not be detected in the inflorescences. In the root
organ cultures the dllsobutyrylester predominated, whereas
the other eugenol derivatives only occurred in traces.
The isolated compounds were identified by MS, and IHand 13C-NMR.
I. F. Bohlmann and Ch. Zdero, Chem. Ber., [01 (1968) 3243
Institut f~r Pharmazeutlsche Biologie der Universlt~t
Heidelberg, Im Neuenheimer Feld 364, 6900 Heidelberg
(F.R.G.)
Vol. 9 "
I987 Pharmaceutisch Weekblad Scientific Edition
NORDITERPENES AND NORDITERPENE GLYCOSIDES FROM
Dr,ymar~a
arenapioides (CARYOPHYLLACEAE)
D. Vargas*, X.A. Dominguez O and K. Hostettmann*
Known as "alfombrilla" in the semidesert areas of northern
Mexico and southern United States, DpymaPia aPenaPicides
is said to be responsible for the death of livestock that
accidentally feed on it. It has been reported that the
saponin-containing extract was toxic to sheep and chicken
(I). We found terpenoid glycosides in the extract'whlch
were ent-kaurene norditerpenoids of the atractyllgenln _I
type (2,3).
The ethanol extract of the plant was partitioned between
n-butanol and water. The organic phase was concentrated
and subjected to flash column chromatography on silica gel.
The fractions obtained were further resolved using different preparative techniques that included centrifugal
partition chromatography, centrifugal TLC and low-pressure
LC, which yielded the norditerpenes _l and 6_, and the
glycosides 2 and _3. Their identities were established by
spectroscopic and chemical methods.
! R: H
-2
RO\ ~ l i
247
IDENTIFICATION OF THE MAIN C O U ~ R I N S IN THE pHARMACOLOGICALLY ACTIVE EXTRACTS OF Peucedanum ~zZustPe
H. Vuorela*, H. Pernil~*, N. Nystrand*, R. Hlnkk~nen O and
R. Hiltunen*
Some components of Paueec~zn~npa~ustre (L.) MSnch have been
shown to have calcium antagonistic activity according to
in vitPo tests (I). However three components, the activity
of which was comparable to verapamil, a commercially
available calcium antagonist, were so far not identified.
In the present study these components were identified.
Their isolation from dlethyl ether and n-pentane root
extracts was carried out using low pressure silica column
c h r o m a t o g r a p h y and r e v e r s e d p h a s e HPLC. The t h r e e main
compounds were i d e n t i f i e d
by MS, IR, UV, IH- and 13C-NMR
s p e c t r o s c o p y a s c o l u m b i a n a d i n , ( + ) - o x y - p e u c e d a n i n and
ostruthol.
I. H. Vuorela, S. Alaranta, K. TSrnqvlst end R. Hlltunen,
Planta Med., (1986) 549
.,.]"")~
R : glc
--3 R : g l c l - - 2 g l c
4
RO: ~o
OH
COOH
I. M.C. Williams, J. Range Manage., 31 (1978) 182
2. L. Camarda, L. Ceraulo, M. Ferrugia, C. Pascual and
V. Sprlo, Planta Med., (1986) 363 and references therein
3. J.R. Hanson, M. Siverns, F. Piozzi and G. Savona,
J.C.S. Perkin I (1976) 114
* Instltut de Pharmacognosie et Phytochimie, Unlversit~
de Lausanne, Lausanne (Switzerland)
0 Instituto Tecnologice y de Estudios Superiores de
Monterrey (Mexico)
* Division of Pharmacognosy, School of Pharmacy, University
of Helsinki, Fabianlnkatu 35, 00170 Helslnkl (Finland)
O Finnish Sugar Co. Ltd., Technical Center, Kantvik
(Finland)
STEAM-VOLATILE COMPOUNDS FROM Geum urbanum ROOTS
C. Vollmann, W. Schultze and K.-H. Kubeczka
ANTIBIOTIC COMPOUNDS FROM NEW ZEALAND PLANTS
J.R.L. Walker and A.L.J. Cole
Ggwn urbc~urn L., Rosaceae, is a perennial plant, which
grows mainly at the edges of forests, in light woods and
lowland forests from Europe extending to Asia. The roots of
this plant are appreciated in folk medicine as an antidiarrholc drug because of their tanning agents; in the
beverage industry they are used for flavouring liqueurs.
The latter use is based on the content of geln, an eugenol
glycoside. This glycoside yields eugenol after steam distillation. Besides this phenylpropanold, we found a series of
further constituents, mainly monoterpenoids. By GC-MS
.
several compounds with a pinane skeleton were identified.
Comparison of the volatiles of the fresh roots with those
of a number of commercial drugs showed no marked differences except for one sample in which neither eugenol nor
eugenol glycoside could be detected. On the basis of this
observation the applicability of the root volatiles for
detecting drug falsifications will be discussed.
New Zealand has a unique indigenous flora and we are
currently screenlng its plants for antibacterial and
antifungal acitivity. To date we isolated and characterized
four novel antibiotics.
I. Falcarindiol, a polyacetylene, was isolated from leaves
of ScheffZera digitata, but it was absent in other, relaird Araliaceae. Low concentrations of falcarlndiol
inhibited the spore germination and mycelial growth of
many common dermatophytes.
II. Polygodlal, a bicyclic sesqulterpene dialdehyde, was
isolated from leaves of Pseudowintera eolorata and exhibited strong antibiotic activity against the yeast
Cand~da aZbicans. Other species of Winteraceae were also
examined.
Ill. A novel cyclopentenyl compound active against pathogenic Pseudomonads.
IV. A compound showing antidermatophyte activity from a
Department of Pharmaceutical Biology, University of
W~rzburg, Mittlerer Dallenbergweg 64, 8700 W~rzburg
(F.R.G.)
StereocauZo~
species
(a l i c h e n ) .
Department of Plant and Microbial Sciences, University
of Canterbury, Christchurch (New Zealand)
248
Vol.
THE FLAVONOID PATTERN IN DIFFERENT PARTS OF PARSLEY
D. Warncke and K.-H. Kubeczka
Parsley, PetposeZinum eriapum (Miller) A.W. Hill (syn. P.
8ativurn Hoffm.) is a biennial plant of the Apiaceae, which
is cultivated throughout the world. The leaves are commonly
used as a spice, whereas the fruits and the roots are used
as diuretics. The physiological activity is attributed to
the essential oil as well as to the flavonolds, the contents of which vary in different parts of the plant.
Besides calculation of the total flavonoid content in
fruits, herbs and roots, the qualitative and quantitative
flavonold composition was analysed by HPLC and photo-diodearray detection.
Fruits and roots showed similar flavonoid patterns, the
herb a different one. The average total amounts of flavonolds - calculated as aplln - were 2.5 - 7.1% in the herb,
and 0.7 - 2.2% in the fruits. Analyses of numerous root
samples indicated the existence of two different groups:
one without flavonoids, the other one containing 0.3 - 2.2%
flavonoids.
Department of Pharmaceutical Biology, University of
W~rzburg, Mittlerer Dallenbergweg 64, 8700 W~rzburg (F.R.G.)
9 -
I987 Pharmaceutisch Weekblad Scientific Edition
DETERMINATION OF ALLIIN IN GAR.LIC BY RP-HPLC/ELECTROCHEMISTRY AND PRE-COLUMN DERIVATIZATION
S.J. Ziegler, B. Meier and O. Stlcher
Sulfur amino acids, e.g. S-alk(en)yl-cystelne-sulfoxides,
are known as precursors of the volatile constituents of
garlic. While a number of methods (GC, NPLC) have been
developed for the analysis of the lipophilic compounds,
determination of the genuine amino acids has largely been
neglected. Efficient methods are of increasing interest in
quality control, characterization, and standardization of
garllc preparations used in phytotherapy.
As a further result of our investigations on the use of
HPLC with selective and sensitive detection techniques in
the analysis of medicinal plants 41,2) we present a method
for quantitative determinatlon of the major compound of
the sulfur amino acids, alllin ((+)-S-allyl-L-cystelne
sulfoxlde). Substances needed for the optimization of the
chromatographic and detection conditions were synthesized,
and pure (+)-L-alliin was obtained by fractional crystallization.
Garlic samples were extracted with 50% methanol following
enzyme inactivation with heat. After sample clean-up and
pre-column derivatlzatlon, the alliln-derlvative could be
separated from the other amino acids using RP-HPLC. The
mobile phase was optimized by means of the "Prisms-model"
(37. Sensitive amperometrlc detection of the electroactive
S-substltuted isoindoles was achieved with a glassy-carbon
electrode at a potential of 750 mV perau$ Ag/AgCI.
Results of the various analyses will be presented.
i. S.J. Ziegler, B. Meier and O. Sticher, J. Chromatogr.,
391 (1987) 419
2. S.J. Ziegler, B. Meier and O. Stlcher, Plants Med.,
(1986) 383
3. Sz. Nylredy, B. Meier, C.A.J. Erdelmeler and O. Stlcher,
HRC & CC., 8 (1985) 186
Pharmazeutisches Institut, Eidgen~ssische Technlsche
Hochschule Z~rich, 8092 ZSrich (Switzerland)
APPLICATION OF POLLEN EXTRACTS IN CHRONIC EXPERIMENTAL
ALCOHOLISM
J. W6Jcickl, L. S~mochowiec and D. Kadlubowska
OPTIMIZATION OF ~[PLC CONDITIONS FOR SEPARATION OF CLOSELY
RELATED FUROCOUMARINS
G. Zogg, Sz. Nylredy and O. Stlcher
Pollen extracts
(Cernltin T60 and Cernitln GBX) were made
from six plant species. Numerous chemical substances have
been identified and isolated from pollen: amlnoacids,
coenzymes, enzymes, all known vitamins, sterols, minerals
and trace elements.
To evaluate the effectiveness of pollen extracts, serum and
liver homogenate lipid contents were measured in rats receiving for three months ethanol and pollen in separate
groups, as compared with controls. Serum enzymes (aminotransferases, alkaline phophatase) activity and bllirubin
level were also determined. In serum of rats given ethanol,
the total lipid level increased 32%, and triglycerides
47%; a marked increase of serum enzymes activity and
bilirubin level was also noted. The hepatic total lipid
and triglyceride content was distinctly higher. In rats
exposed to an intake of ethanol and to the administration
of pollen extracts over a two month period, a marked
improvement was observed, manifested by the return to,
or a tendency to return to normal biochemical parameters
in the blood serum and liver homogenate.
During the last few years, different methods of preparative
pressure LC ([) have been developed for separation of closely related compounds. Different parameters, e.g. solvent
strenght, velocity of the mobile phase, loading capacity,
column dimensions, were optimized for the MPLC isolation
of the closely related furocoumarlns from HePaoZeuln
Institute of Pharmacology and Toxicology,
Wlkp. 72, 70-III Szczeclo (Poland)
Powsta~c6w
sphondyZi~.
For the separation of the investigated compounds (isobergapten, pimpinellln, bergapten, iso-pimpinellln and
sphondin) different analytical and preparative methods have
been evaluated in previous studies (2,3). In the present
study the optimization of the mobile phase was carried out
on TLC plates in unsaturated chambers using the "Prlsm~model". It was then applied to analytical overpressured
layer chromatography.(OPLC) and subsequently to MPLC.
The MPLC separatlorrs were not only carried out with
equilibrated columns, but also with dry filled columns,
uslng.dlfferent operating parameters. The chromatograms
obtained for an extract from h'. sphondyZi~ will be presented.
I. K. Rostettmann, M. Hostettmann and A. Marston, Preparative Chromatography Techniques, [9B6, Sprlnger Verlag,
Berlin, pp. 27-79
2. C.A.J. Erdelmeier, P h . D . thesis no. 7924, 1983, ETH
ZUrlch
3. G. Zogg, Sz. Nylredy and O. Stlcher, J. Liq. Chromatogr.,
submitted
Pharmazeutlsches Instltut, EidgenSssische Technische Hochschule ZUrich, 8092 Z~rich (Switzerland)