Abstracts of short lectures

Vol. 9 " I987 Pharmaceutisch Weekblad Scientific Edi~on

P O S S I B I L I T I E S AND LIMITATIONS 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.

ON THE BIOCHEMISTRY AND BIOLOGY OF STRESS REACTION: ADAPTOGENS AS MEDICINES FOR HEALTHY PEOPLE R. H~nsel

215

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 unspecifl 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, Khnlgln-Lulse Strasse 2 + 4, I000 Berlin 33 (F.R.G.)

Institute of Pharmacognosy and Phytochemistry, Free University Berlin, K6nigln-Luise Strasse 2 + 4, [000 Berlin 33 (F.R.G.)

CULTIVATION AND BREEDING OF MEDICINAL PLANTS 1987

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. The 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 geno- types for specific environmental and 6griculturel conditions will be shown. Some effects of pre- and post-harvest treatments on phyto- chemicals yield will he presented.

Abstracts of short lectures NEW HYDROXYALOINS: THE 'PERIODATE-POSITIVE SUBSTANCE' FROM CAPE ALOES ANn CINNAMOYL ESTERS FROM CURACAO ALOES H.W. Rauwald

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-glucosyl- anthrone 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

Department of Medicinal, Aromatic a n d Spice Crops, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50 250 (Israel)

Institute of Pharmaceutical Biology, University of Frank- furt, 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 e x u d a t e s o f s e v e r a l E a s t A f r i c a n shrubby AZoe s p e c i e s and o b s e r v e d the e x u d a t e of Aloe rabas Rendle to be s i m i l a r to our s amples of 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

* 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)

V o l . 9 - I 987 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 preas- says 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, consi- dering 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 he 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 has 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 (1986) 51

2. Sz. Nyiredy, S.Y. M6szgros, g. Nyiredy-Miklta, g. Dallenbach-T~ike and O. Stlcher, HRC & CC., 9 (19861 605

Pharmazeutlsches Institut, Eidgen~ssische Technlsche Hoch- schule Z ~ r l c h , 8092 Zfirlch (Switzerland)

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

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 N- oxide is the primary product of blosynChesls, iC accumu- lates as an end product. Comparative studies indicate that presumably in most PA plants the PA are accumulated as N- oxides. 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 they 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

SEPARATION OF PHOKBOL ESTERS BY TWO-DIMENSIONAL TLC AND RAPID PURIFICATION USING LPLC AND SUBSEQUENT REVERSED- PHASE OPLC C.A.J. Erdelmeier*, P.A.S. van Leeuwen 0 and A.D. Kinghorn*

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) employ- ing 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. Chroma-

togr., 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.)

* Program f o r C o l l a b o r a t i v e R e s e a r c h in the P h a r m a c e u t i c a l S c i e n c e s , and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illl- nois st Chicago, Chicago, IL 60612 (U.S.A.)

0 Clinical Pharmacoklnetics Laboratory, College of Pharma- cy, 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

In recent years several new analytical and on-line preparative chromatographic methods were developed and applied in phytochemlstry. In planar chromatography the forced- flow 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, Prepa- rative Chromatography Techniques, 1986, Springer, Berlin

Pharmazeutlsches Institut, EidgenSssische Technische Hoch- schule ZQrich, 8092 ZHrieh (Switzerland)

HYDROXYCINNAMIC ACID ESTERS IN TISSUE CULTURES OF Leontopodium a~pinwr#, "Edelweiss" Deirdre Hennessy, Ingrld Hook and Helen Sheridan

217

Little information is available on the phytochemistry of Leontopodiw~ agplnw, 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. alps were initiated from sterile micro- propagated 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.

Department of Pharmacognosy, School of Pharmacy, Trinity College Dublin, 18, Shrewsbury" Rd., Dublin 4 (Ireland)

A RADIOIPR4UNO ASSAY FOR QUINOLIZIDINE ALKALOIDS FOR STUDIES WITH PLANT CELL CULTURES M. Wink

ARIA 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.

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 l- airlift-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 mini- column chromatography on silica gel and NPLC on reversed phase material.

Universitgt MQnchen, Institut fur Pharmazeutische Biologie, Karlstr. 29, 8000 MOnchen 2 (F.R.G.)

* Pharmaceutical Institute, University of TQbingen (F.R.G.)

O Hoechst AG, Frankfurt a. M. (F.R.G.)

2 1 8

REGULATORY ENZYMES IN SHIKONIN BIOSYNTHESIS L. Heide* and M. Tabata O

Cell c u l t u r e s of Lithospelmllan erythrorhizon are capab le 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 . Th i s p rocess has become the 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 re- pressing shlkonln production have been investigated (I), but enzymatic studies of shlkonln formation have not been possible so far, due to 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.

V o l . 9 - 1987 Pharmaceutisch Weekblad Scientific Edition

CHROMATOGRAPHIC ANALYSIS OF POLYPHENOLS OF SOME IBERIAN ~y~8 species T. Adzet~ R. Vila and S. Ca~gueral

The taxonomical complexity of the genus Thymus as well as the pharmaceutical applications of its species, make Its phytochemlcal study interesting. Moreover, the West Medl- terranean 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 he 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 caf- feic 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

* Inscitut for Pharmazeucische Biologie, Universit~t Bonn, Nussallee 6, 5300 Bonn I (F.R.G.)

0 Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto (Japan)

Laboratory of Pharmacology and Pharmacognosy, Faculty of Pharmacy, University of Barcelona, Zona Unlversltaria de Pedralbes, 08028 Barcelona (Spain)

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 to 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 fo l l owed by f u r t h e r s e p a r a t i o n on columns w i th Con A-Sepharose 4B and B io -Ge 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 turn- over 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 Charac-

terization and its Determination by HPLC. Planta Med., submitted

2. M. Maze~is and L. Crews, Biochem. J., 108 ([968) 725- 730

3. L.P. Nock and M. Mazells, Arch. Biochem. Biophys., 249 (1986) 27-33

AROMATIC ACID DERIVATIVES FROM Pomietomia officir~Zi8 L. (URTICACEAE) J. Budzlanowski

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

Instltur for Botanik und Pharmazeutlsche Biologle der UniversltAt Erlangen-NOrnberg, Staudtstr. 5, 8520 Erlangen (F.R.G.) Department of Pharmaceutical Botany, K. Marcinkowski Medi-

cal Academy, ul. Wlenlawsklego I, 61-712 Pozna6 (Poland)

Vol. 9- I987 Pharmaceutisch Weekblad Scientific Edition 219

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 Ri- plasmid, 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.)

ANALYSIS OF TWO FUNCTIONALLY DIFFERENT IM~UNOMODOLATORS FROM Aloe ve~ 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 of A~oe vera g e l ( L . ; Burm. f . ) f o r t r e a t - ment of infections has 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 ve~ 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 stir- ring. 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 pnly- saecharides 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

(1986) I17 Department of Pharmacognosy, Faculty of Pharmacy, State University of Utrecht, Catharij~eslngel 60, 3511GH Utrecht (The Netherlands)

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 intra- caecally 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.,

s u b m i t t e d

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 of a Tabernaemontana ds (Apocynaceae) suspension culture with biotic elicitors like cellulase, AspergiZ~us niger or Candida aZblean8 r e s u l t e d in 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 the 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.

Laboratorium voor Farmaceutische giologie sn Fytofarmaco- logic, K.U. Leuven, Van Evenstraat 4, 3000 Leuven (Belgium)

* 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 Univer- sity, Nonnensteeg 3, 2311VJ Leiden (The Netherlands)

2 2 0 V O I . 9 - 1987 Pharmaceutisch Weekblad Scientific Edition

PRODUCTION OF ARTEMISINI~ (QINGHAOSU) IN Artemlsiu annua L. E.M. Croom, Jr.

The sesqulterpene peroxide artsmlsinin (Qinghaosu) is the parent 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. ann~ will be presented.

THE CHEMICAL DISCRIMINATION OF Eohinuoea angustlfolia AND E. pallida R. Bauer*, V. Wray O and H. Wagner*

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 polyacety- lanes 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-dleue- 2-one. Another compound, g-hydroxy-pentadeca-9,13-diene-ll- yne-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. ~gustifoZ~ and the presence of echinacoslde in both species. E. angustifoZia contained also cynarin which was not found in E. paIZida. Chromato- graphic 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

Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, MS 38677 (U.S.A.)

* 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.)

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 sssen- tlal 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

NOVEL FUNGICIDAL POLYPNENOLICS FROM CoPdia goetzei A. Marston and K. Hostettmanu

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 ouou~,~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~cke~idgea a~eba~ca (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.

Institut for Pflanzenbau und ZQchtung der JL-Unlversit~t Giessen,Versuchsstation Rauischholzhausen, 3557 Ebsdorfergrund 4 (F.R.G.)

Instltut de Pharmacognosie et Phytochlmie, Unlverslt~ de Lausanne, Lausanne (Swltzerland)

Vol . 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 Gram- negative bacteria E. eoZi. However, the Gram-posltlve bacteria S. auPeu8 was affected by the polyhydroxylated steroidal glycosides, polyhydroxylated sterols and di- sulfated 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, Unlver- sita 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)

AN ANTITUMOR ACTIVE 8-1,3/I,6-GLUCAN FROM Phytophthora paraaitica J. Kraus, W. Blaschek and G. Franz

221

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 shownto 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 demon- strafed that the glucans had no direct cytotoxic effects. In mice, treated simultaneously with the glocan and trypan blue, a macrophage inhibiting agent, the 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 host- mediated 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, GC- MS, 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 . van den Berg and R .P . L a b a d i e , P l a n t a Med. , 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, 3511GH Utrecht

(The Netherlands)

222

ANTIVIRALLY ACTIVE CONSTITUENTS OF Spondias mombin L. J. Corthout*, L. PietersP, M. Claeys*, D. Vanden Eerghe O and A.J. Vlietinck*

The results of a broad screening program for various chemo- therapeutical 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 under reduced pressure and the residue acid- ified 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 hese substances i s currently determined and will also be discussed.

V o l . 9 - I987 Pharmaceutisch Weekblad Scientific Edition

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

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. Inorder 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 of Pharmaceutical Sciences, University of Antwerp (UIA), 2610 Wllrijk (Belgium)

O Department of Medicine, University of Antwerp (UIA), 2610 Wilrijk (Belgium)

* Department o f Pharmacognosy, Faculty o f Pharmacy, State Unlversity of Utrecht, Catharljnesingel 60, 3511GR Utrecht (The Netherlands)

O Laboratorlum Biohorma, P.O. Box 33, 8080 AA Elburg (The Netherlands)

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 pyrroli- zldine 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 N- oxide 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

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 food- plants, 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 Pharmacochemistry-Molecular Toxicology, Free University, Amsterdam (The Netherlands)

0 Department of Pharmacognosy, Faculty of Pharmacy, State University of Utrecht, CatharlJneslngel 60, 3511GH Utrecht (The Netherlands)

V Laboratorium Biohorma, Elburg (The Netherlands) Department of Population Biology, University of Leiden, Sehelpenkade 14a, 2313 ZT Leiden (The Netherlands)

Vol . 9 - I987 Pharrnaceutisch Weekblad Scientific Edition

SIMULTANEOUS FLAME-IONISATION AND NITROGEN-SPECIFIC DETECTION AND RETENTION INDICES IN GAS CHROMATOGRAPHY OF STEROIDAL ALKALOIDS W.M.J. van Gelder

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 were 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 two- channel 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)

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

2 2 3

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 chromo- phores. 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)

Division of Pharmacognosy, Center for Blo-Pharmaceutlcal Sciences, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden (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 the 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 to 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 gluco- mannans. 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

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), fol- l~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.

Department of Pharmacognosy, Faculty of Pharmacy, State University of Utrecht, Catharljneslngel 60, 3511GH Utrecht (The Netherlands)

* 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 el- ement in the first step of the 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 to f u r t h e r i n v e s t i g a t i o n s . 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, 3511GB 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 skin- healing 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, 3511GH 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, �9 R. Verpoorte* and A. Baerheim Svendsen*

In previous e x p e r i m e n t s , L - t r y p t o p h a n proved to 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.

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

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 are 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, Center 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)

V o l . 9 - I 9 8 7 Pharmaceutisch Weekblad Scientific Edition 2 2 5

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 he reaction was stopped by addition of methanol. A known amount of 5- methyl-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 -15~ the half llfe time is about 7 d a y s . P a r t i a l l y 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 DEAE- Sephacel 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.

INVESTIGATION OF THE FLAVONOIDS OF BPukenthalia apicuZifoZia AND THEIR MOLLUSCICIDAL ACTIVITY M.M. 8aleh*, R.Z. Abbas* and J.B. Zwavlng 0

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 tech- nology.for the snail control. Two flavonoids were isolated from the methanolic extract of the 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

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 tumor- promoting phorbol-12,13,20-trlesters were isolated from croton oil, the seed oll of CPoton tig~iwn L. (Euphor- blaceae), 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 substl- tuent, 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

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

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.

Depar tmen t of P h a r m a c e u t i c a l S c i e n c e s , U n i v e r s i t y of Antwerp (UIA), 2610 WilriJk (Belgium)

* 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 . Schr ipsema*, A.H. H o l i e r * , C.R. Langezaa l* , P.A.A. Harkes O, R. Verpoor te* and A. Baerheim Svendsen*

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 photo- synthesis, a carbon source must be provided, which is con- verted 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.

* 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, 2311VJ Leiden (The Netherlands)

3-METHOXYFLAVONES, ANTIVIRALLY ACTIVE SUBSTANCES FROM HIGHER PLANTS L. Van Hoof*, D.A. Vanden Berghe* and A.J. Vlietinck O

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

* Deparfment of Microbiology, University of Antwerp (UIA), 2610 WilriJk (Belgium)

O Department of Pharmacognosy, University of Antwerp (UIA), 2610 Wilrljk (Belgium)

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 sub- terranean 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 both assays. The present study was aimed at the characterization of the immunomodulatory components. Therefore an aqueous and a chloroform extract were tested for anti- complementary activity and for their effect on zymosan- induced 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 zymosan- induced 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)

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. pos- 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, eucanna- b 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 ldehyde , enhanced c y t o t o x i c l t y , as d id a c e t a l l s a t i o n o f bo th hydroxy l groups w i th a c e t o n e . I n t r o d u c t i o n of more bulky grodps v i a a c e t a l l s a t l o n r e v e r s e d the enhancement . F s u b s t i t u t i o n of acetophenone as bu lky 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

V o l . 9 - I 9 8 7 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~ cannabi- n~ 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

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

227

* Department of Pharmacognosy, State University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen (The Netherlands)

O Department of Radiopathology

* 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 Outre- Met (ORSTOM),Paris (France)

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

PHYTOCHEMICAL INVESTIGATION OF Sideritis hyssopifo~ia VAR. pyrenaica T. Adzet*, S. Ca~igueral*, C. Ib~ez O, I. Monasterio*O and R. Vila*

The polyphenols and the essential oil composition of aerial pas t s of siderites h~ssopifoZia L. va r . pyrenaica Briquet (Lab ia tae ) 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 lavone d i g l y c o a i d e s , 7 - O - ( a c e t y l - a l l o s y l - g l u c o s y l ) - l a o s c u t e l l a r e i n and the cor responding unacetylated glycoside, and a caffeoylquinic acid (chlorogenic acid) were isolated from methanol and methanol- water (l:l) extracts by column chromatography, and identified by UV-Vis, H-NMR, 13C-NMR, FAB-MS, and hydrolysis. The essential oil (0.01% v/w), obtained by hydrodlstil- lation, 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.

*Escuela P o l i t e c n i c a del Chimborazo, Riobamba Ecuador ODipart imento d i Chimica Organica , U n i v e r s i t ~ d e g l i S tud i ,

Via Ta rame l l i [0, 27100 Pavia ( I t a l y ) VLaborator io 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 tud i , Via Ta rame l l i 10, 27100 Pavia ( I t a l y )

* 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 Pedra lbes , 08028-Barcelona (Spain)

0 LUCTA S.A., Apartado 1112, 08080-Barcelona (Spain)

2 2 8 Vol. 9 " I 987 Pharmaceutisch Weekblad Scientific Edition

EFFECT OF DRYING ON YIELD OF LENONGRASS OIL AND ITS ALDEHYDE CONTENT S.M. Husse in Ayoub

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). In a p r e v i o u s paper we r e p o r t e d our r e s u l t s on the compo- s 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 IMPORTANCE OF YIELD COMPONENTS FOR THE SELECTION OF PupuveP bPaotea~um ON HIGH THEBAINE YIELD PER UNIT AREA C. Bartsch

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 stig- matic 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 correla- tions 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 as 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 the 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 the number and w e i g h t o f the cap - s u l e s had the largest i n f l u e n c e on the y i e l d ; the 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 tendency , bu t they gave on ly 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 Biologic, Nussallee 6, 5300 Bonn (F.R.G.)

MOLLUSCICIDAL PROPERTIES OF Hyd~ora o~yssinica S.M. Nussein Ayoub, L.K. Yankov and R.O. Ovtcharov

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 (Hydnoraceae) . 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, the 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

LAVANDULIFOLIOSIDE, A NEW PHENYLPROPANOID GLYCOSIDE FROM S tachy s lavandu ~ i f o Z la V AR . Zavar~u Z i f o l ia A.A. Ba~aran*, I. Calls*, 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 as m t ea i n the v i c i n - i t i e s of An ta lya (Turkey) . T h e r e f o r e , i t was i n v e s t i g a t e d fo r i t s e s s e n t i a l o i l ( 1 ) . I n v e s t i g a t i o n s of 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 the 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 the 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 of the 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 - d i h y d r o x y - 8 - p h e n y l - e thoxy-O- a - L - a r a b i n o p y r a n o s y l - ( l § 2 ) - a - L - r h a m n o p y r a n o s y l - ( l ~ 3 ) - 4 - O - c a f f e o y l - 8 - D - g l u c o p y r s n o s i d e ) 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 water- soluble 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 chromatography. The chemica 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 proposed s t r u c t u r e . UV, IR and MS da t a s u p p o r t e d the proposed s t r u c t u r e o f l a v a n d u l i f o l i o s i d e , C34H44019. I. A.A. Basaran, Pharmacognostical investigations on

Stachya ZavanduZifoZia Vahl v a t . lavandullfoZia, Ph.D. Thesis, 1984, Hacettepe University, Ankara

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 of 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)

* D i v i s i o n of Pharmacognosy, F a c u l t y o f Pharmacy, Hace t tepe 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 Pha rmazeu t i s ches I n s t i t u t , ETH-Zentrum, 8092 ZOrlch ( S w i t z e r l a n d )

V o l . 9 - I987 Pharmaceutisch Weekblad Scientific EdiHon

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

The pregnane ester glycoside fraction from Condurango cortex, dried bark of Marsden~ co~go Rchb. f. (Asclepiadaceae), was reinvestigated. Four as yet unknown g l y c o s i d e s o f the new ag lycone condurangogen in E were i s o l a t e d by column chromatography and subsequen 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, E O, E 2 and E 3. ~he 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~gradat ion of 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

* Fach be re i ch Chemie, Phillipps-Universit~t, 3550 Marburg (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.)

229

ISOLATION AND DENSITOMETRIC DETERMINATION OF HYFOLAETIN-g- O-GLUCURONIDE FROM LEAVES OF MaZva 8iZvestPi8 M. Billeter, B. Meier and O. Stiohsr

Malva si~vestr~a leaves are used in folk medicine for treatment of cough and external inflammatory diseases due to t h e i r h i g h c o n t e n t o f m u c i l a g i n o u s s u b s t a n c e s . The re fo re most s t u d i e s on M. s i l v e s t r i e concern 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 flavo- hold 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/poly- ethyleneglycol-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

Echinaceu angustifolia and Baptisla tinctoria contain, llke 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 electro- phoresls 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

DETERMINATION OF THE FRUCTAN CONTENT IN SYMPHYTI RADIX BY A GASCHROMATOGRAPRIC AND AN ENZYMATIC METHOD

W. Blaschsk, M. Dhll and G. Franz

The roots of 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 freeze- dried, 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 phospho- glucose-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.

Schaper and BrQmmer, Bahnhofstr. 35, 3320 Salzgltter 61 Institute of Pharmaceutical Biology, University of Regens- (F.R.G.) burg, 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 and 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 degra- dations 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


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,

39 (1984) 869 3. J. Budzianowski, L. Skrzypc~ak and M. Wesolowska,

Acta Biol. Crac. Ser. But., in press

Institute of Pharmaceutical Biology, University of Regensburg, Unlverslt~tsstr. 31, 8400 Regensburg (F.R.G.)

Department of Pharmaceutical Botany, K. Marcinkowski Medical Academy, ul. Wienlawskiego I, 61-712 Pozna~ ( P o l a n d )

ECOLOGICAL FACTORS INFLUENCING GROWTH AND HYPERICIN PIGMENTS OF H~pePieum PROVENIENCES H. Braunewell and A. VSmel

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 Raulsch- holzhausen Experimental Station on loamy soil; plants from five sites were planted at Welhenstephan on humic soil. Analysis of the hypericin pigments was performed photo- metrically (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

Institut for Pflanzenbau und ZOchtung der JL-Universit~t Giessen, Versuchsstation Raulschholzhausen, 3557 Ebsdorfer- grund 4 (F.R.G.)

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

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-methoxy- trans- 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

* 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)


STDDIES ON PLANT CELL CULTURES OF CheZidoniura mo~u8 V M.L. Colombo*, F. Tome~ N. Crespi-Perellino V, A. Guicciardi and A. Minghetti ~

Chelidonium magus L. drew the a t t e n t i o n 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 alka- lold from C. ~u8 roots, is the basis for chemical 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.

NEW COL91ARINS FROM Meaua ferpea L. M. De Bernardi*, C. Vidari*~ P. Vita-Finzi*, G. Mellerio O B. Gabetta V and A. Scilingo V

2 3 1

Mesua ferrea L., Guttiferae, is an indigenous tree very 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~ R

II R- C.~OR)Et o ~ Ho~

* 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)

* 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 Ripa- monti 99, 20100 Milano (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 i 9 the desert of 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 sus- pensions. A typical fermentation curve of E. caZyptPata in liquid medium will be discussed. i. P. Eiacalone, Thesis, 1982, Unlversit~ di Firenze

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 of Sesbania sesban M e r r i l (Leguminosae) ware 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 spermi- cldal 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 (1977) 131

* Farmitalia C. Erba, Sviluppo Riotecnologico, Milano (Italy)

0 Facolt~ dl Farmacia, Universit~ di Bologna (Italy) V Facolt~ dl Farmacia, Universlt~ dl Firenze (Italy)

Institut de Pharmacognosie et Phytochimie, Universit~ de Lausanne, Lausanne (Switzerland)

232 V o l . 9 - ~987 Pharrnaceutisch Weekblad Scienttfic Edition

SESQUITERPENE LACTONES FROM Arnica nevadensis and Arnica ~i8~osa M. Ebert and G. Willuhn

In p rev ious papers we repor ted on the ae squ i t e rpene l ac tones (S1) of Arnica s p e c i e s be longing to the subgenera Chcunissons 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), 2-0- acetylivalin and ll~,13-dihydroivalin (I) were isolated and identified by IR, MS and IR-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-hydroxy- 6-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).

"~ o ido c I o I 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.)

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY. ON-LINE AND OFF- LINE 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

* 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

Bystropogon plumosus (L.fil.)L'Rer. (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-~-D- glucopyranoside, 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

STRUCTURE AND CYTOTOXIC ACTIVITY OF TRITERPENES FROM Alisma pZantago-aquatica L. SSP. p~antago-aquatica E. Eich*, G. Kohaupt*, K. Stopp*, A. Maidhof O, W:E.G. Mflller O, P. Schade V and K. Mfillen V

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 for 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.)

*Institut fQr Pharmazie der Universit~t Mainz, Saaratr. 21, D-6500 Mainz (F.R.G.)

OInstitut fQr Physiologiache Chemie der Universit~t Mainz

V Institut for Organischa Chemie der UniversitKt Mainz

Vol . 9 " I987 Pharmaceutisch Weekblad Scientific Edition

ELICITOR INDUCTION OF S-ADENOSYLMETHIONIN:ANTHRANILIC ACID N-METHYLTRANSFERASE ACTIVITY IN SUSPENSION CULTURES OF 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 bio- synthetic 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 dark-grown 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 of the enzyme activity. Maximum activity (10-24 ~kat/kg in dark- grown, 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), fo rming d i f f e r e n t a l k a - l 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

Institut fflr Pharmazeutische Biologic der T.U., Mendelssohnstr. l, 3300 Braunschwelg (F.R.G.)

IRIDOID AND SECOIRIDOIDIIGLUCOSIDES FROM Genti~ olivieri T. Ers6z*, A.J. Chulia O, J. Garcia 0 and I. Calzs*

2 3 3

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 of Turkey. The crude drug "Afa r " ( a e r i a l p a r t s of 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 tomach ic or 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 seen- iridoids 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, gentiopi- croside, 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

* Division of Pharmacognosy, Faculty of Pharmacy, Uni- versity 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*

Capp~ia spinosa L. v a r . aeg~ptia (Cappar ldaceae ) which 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 liquid- liquid 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

DIASTEREOMERIC 3,11 - OXYGENATED CRINANE ALKALOIDS FROM CPi~um a s i a t i c u m L. EX THAILAND AND OTHER SPECIES A.W. Frahm and D. Bsutner

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

* L a b o r a t o i r e de Pharmacognos ie , Fa c u l t~ de Pharmacie , Oniversit~ Louis Pasteur, B.P. I0, 67048 Strasbourg C~dex (France)

O Laboratoirs de Culture Cellulaire. Institut de Physlo- logic, 21 Rue Descartes, 67084 Strasbourg C6dex (France)

V Department of Pharmacognosle, Faculty of Pharmacy, Cairo University, Cai ro (Egypt)

Institut f~r Pharmazeutische Chemle der Universit~t Bonn, Kreuzbergweg 26, 5300 Bonn I (F.R.G.)

234

STUDY OF THE VARIATION OF THE ESSENTIAL OIL 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*

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 (~ydrodistil- lation) 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.


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

Plants were grown in a field plot in Boncourt (Switzerland) from seeds of Eehinaoea p~.rea 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 (Carbo- wax 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. an~sti- 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)

* Institute of Plant Biology, University of ZHrich, 8008 ZOrich (Switzerland)

0 Spagyros SA, 2926 Boncourt (Switzerland)

THE CHEMICAL COMPOSITION OF THE VOLATILE OIL OF Haronga madagascariensis B. Gehrmann and LJ. Kraus

The composition of the volatile oil of Haronga madagaseariensis 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

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-disul- fate 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 phloro- gluclnols, 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 desaliniza- tion 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

Division of Pharmacognosy, University of Hamburg, Bundesstr. 43, 2000 Hamburg 13 (P.R.G.)

* 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.)

V o l . 9 - *987 Pharmaceutisch Weekblad Scientific Edition

METABOLISM OF QUINOLIZIDINE ALKALOIDS IN THE GENUS Laburnum (LEGUMINOSAE)

R. Grelnwald, W. Schultze and F.-C. Czygan

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~ic- tro~des (Berberidaeeae), which was described by Flom et aZ. (1). We isolated baptifollne from both species. The abso- lute configuration of these two alkaloids should be determined by spectroscopic methods (IR, MS, and |H- and |3C- NMR).

I. M.S. Flom, R.W. Doskotch and J.L. geal, J. Pharm. Sci., 56 (1967) 1515-1517

2 3 5

INVESTIGATION ON THE VARIABILITY OF APIGENIN AND SELECTION OF APIGENIN TYPES OF TETRAPLOID CHAMOMILE

J. Hblzl and S. Hess

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 A- content 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

Embelia schimpepi, Myrsine africana and Maesa lanceolata (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.

APPLICATION OF HEADSPACE GC IN ESSENTIAL OIL ANALYSIS. Xl. ANALYSIS OF SILYLATED SAMPLES

Y. Holm, E. Aho and R. Hiltunen

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.

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, Pharma-

ceuticals 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

V o l . 9 - I987 Pharmaceutisch Weekblad Scienttfic Edition

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 Helsinkl, Fabianinkatu 35, 00170 Relsinki (Finland)

* Division of Pharmacognosy, School of Pharmacy, University of Helsinki, Fabianinkatu 35, 00170 Helsinki (Finland)

0 Orion Pharmacautlca, Research Centre, P.O. Box 65, 02101Espoo (Finland)

A NEW I~/NOASSAY 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 mono- 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 bis- indollc 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.

* 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, Techni- cal University of Helslnkl, 02150 Espoo (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 AM{ca species ontil now (4,5).

ORI

OH 0 OH 0

I : RI=H ; R2= H 4-9_: RI=H or CH 3 ~: RI= CH3;" R2= H R2= H o r CH 3 _3: R l= H ; R 2- OCH 3 R3= H or CH 3 i. 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. Yatskie-

vych, 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.)

V o l . 9 - I 9 8 7 Pharmaceutisch Weekblad Scientific Edition

QUANTITATIVE DETERMINATION OF BORJATRIOL IN Sideriti8 mugronensi8 HERB A. Jim~nez*, S. Ma'bez* and A. Villar O

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 (Lamiaceae ) which i s c o n s i d e r e d to be one of t he 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 spectrophoto- metric 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

EFFECTS OF ONION AND GARLIC EXTRACTS ON SPONTANEOUSLY HYPERTONIC RATS

J. Kiviranta*, T. Sepp~nen*, H. Karppanen O, H. Huovinen* and R. Hiltunen*

237

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

* Laboratorio de Farmacognosia y Farmacodinamia, Departamen- to de Farmacologla y Farmacotecnia, Facultad de Farmacia, Universitat de Val~ncla. Av. Blasco Ib~ez 13, 46010 Valencia (Spain)

O Laboratorio de Farmacognosla y Farmacodinamia, Departamen- to de Farmacologla, Facultad de Farmacia, Unlversidad Complutense de Madrid. Ciudad Universitarla, 28040 Madrid (Spain)

* Division of Pharmacognosy, School of Pharmacy, University of Helsinki, 00170 Helsinkl (Finland)

0 School of Pharmacology, University of Helsinkl, 00170 Helslnki (Finland)

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 inveS-

tigated. 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

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 anthra- ce,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, School of Pharmacy, University of Helsinki, Fabianinkatu 35, 00170 Helsinkl (Finland) Division of Pharmacognosy, University of Hamburg,

Bundesstrasse 43, 2000 Hamburg 13 (F.R.G.)

238 Vo l . 9 - I987 Pharmaceutisch Weekblad Scienufic Edition

CARDIAC GLYCOSIDE GLUCOSYLTRANSFERASE AND GLUCOHYDROLASE ACTIVITIES IN PLANTS ANU CELL CULTURES OF THREE Olgi~Zis SPECIES W. Kreis, U. May and E. Reinhard

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. ~oodil) 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~oodii, 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 prop- ertles 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.)

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 germa- crene-D. Caryophyllene epoxldeand 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

* 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

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 were Bacillus subtiZs Candida aZbicans, ShigeZ~a sones 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

HERBAL PRODUCTS - A PHARMACOGNOSTICAL INVESTIGATION genre Lorentzen and Anne Marie R~rdam

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 pharma- cognosticgl 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)

Vol . 9 - I987 Pharmaceutisch Weekblad Scientific Edition

ALTHAEAE RADIX MUCILAGE POLYSACCHARIDES, ISOLATION, CHARACTERIZATION AND STABILITY

A. Madaus, W. Blaschek and G. Franz

The r o o t s o f Althaea o f f l c ina~ 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 measu remen 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 appar- ent 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.

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

2 3 9

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 frag- ments, �9 but 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 3-0- monoglucosldes and 3-O-rhamnoglucosldes. Capillary GC-MS revealed the presence of minor anthocyanins, up to now un- described. 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 (1975) 393-399 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 (1977) I l l - 1 2 0

Institute Of Pharmaceutical Biology, University of Regens- burg, Universlt~tsstr. 31, 8400 Regensburg (F.R.G.)

* Invernl della Beffa, Research and Development Labora- tories, Via Ripamonti 99, 20141 Milan (Italy)

O Laboratolres Fournier, 42 Rue de Longvic, 2[300 Chengve (France)

A GERMACRANOLIDE, GUAIANOLIDES AND XANTHANOLIDES FROM FLOWERS OF Arnica moZlls Chr. Marcinek and G. Willuhn

Up to now pseudoguaianolides, xanthanolides, and eudes- manolides 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~, "13- dlhydroxanthinosin from the flowers of Arnica mollis Book. (subgenus C~ssonis), which were identified by spectroscopic methods (1H-NMR, L3C-NMR). I i s the 2 - 0 - a c e t y l d e r i v a t i v e o f b a l l e y i n ; the b a i l e y i n c o n f i g u r a t i o n a t C-I and C-10 has 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 ) . A c c o r d i n g to our i n v e s t i g a t i o n I i s a ge rma- c r o l i d e ( l , 1 0 - t P a n s ) , which was e s t a b l i s h e d by X- ray s t r u c - t u r e a n a l y s i s . 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 bio- synthetic pathway for xanthanolides (5).

RO H ~/OH

0 ~ , 0 0 " I ii R : He IV

II| R ; 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, Bot . R e v . , 48 (1982) 121-595 Institut for Pharmazeutische Biologle d e r Unlverslt~t DUsseldorf, 4000 D~sseldorf I (F.R.G.)

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 the subgenus Longiflor~, A. grandlfLora A l e x a n d e r , and from the subgenus hrticuLaria, A. breus (Gray) and A. tomentosa T o r t . and T ram. , 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, re- garding 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 than A. taber'naemontana. Based on t h i s s tudy i t became pos@ible to c h a r a c t e r i z e not on ly the s p e c i e s of the subgenus Euamsonia growing in Hungary , but a l s o to 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

FORMATION OF 5-METHYLDIGINATIN VIA BIOTRANSFORMATION BY Digitalis Zanata CELL cuLTURES

P. Meiss and E. Relnhard

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 B- methyldlginatin 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 hydroxy- ls.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


A NEW ACYLATED AND SOME OTHER FLAVONOID GLYCOSIDES OF ARNICAE FLOS

I. Merfort

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. incanaa 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"-0- acetyl)-6-D-glucopyranoside, a new natural compound. To- gether 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, Jaceo- sidin, ~, 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, Auf der Morgenstelle g, 7400 TObingen (F.R.C.)

Institut for Pharmazeutische Biologie der Universitgt DUsseldorf, 4000 D0sseldorf I (F.R.G.)

A DITERYENOID WITH A NEW CARBON SKELETON FROM P~gmaeopremna he~baoea 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-hydroxyanthran- 2,5,6-trl-one, a diterpenoid with a new carbon skeleton.'

SEPARATION OF NON-POLAR COMPONENTS OF ANISI FRUCTUS BY DCCC H. Miething, V. Seger and R. Hgnsel

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 (fim- pi~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 2- methylbutyrate and some other compounds. Additionally mixtures of other non-polar natural products were separated using these solvent systems.

* 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

Institute of Pharmacognosy and Phytochemistry, Free University of Berlin, K~nigin-Luise-Str. 2-4, I000 Berlin 33 (F.R.G.)

V o l . 9 " I987 Pharmaceutisch Weekblad Scientific EdiSon

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

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. However, some phytopathogen bacteria 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

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

2 4 I

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 wellas 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

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

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 reversed- phase HPLC; estimation of valeranone was performed quali- tatlvely 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 respective-

ly 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

FURANODITERPENE GLYCOSIDE AS A BITTER PRINCIPLE OF YinospoPa cpispa MIERS P. Pachaly and A.Z. Adnan

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, dlar- rhoea 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 spectro- scoplc methods.

H ] C ~ O

,

I. N.G. Bisset and J. Nwaiwu, Planta Med., 48 (1983) 275

Institut f~r Pflanzenbau und ZUchtung der JL-Universit~t Giessen, Versuchsstation Rauischholzhausen, 3557 Ebsdorfer-

grund 4 (F.R.G.)

Pharmazeutisches Instltut der Universit~t Bonn, Kreuzberg- wen 26, 5300 Bonn (F.R.G.)

242 V o l . 9 - z987 Pharmaceutisch Weekblad Scientific Edition

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 of 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 [,3- dlacetoxytetradeca-6,12-dien-B,[O-diin (3) by spectroscopic and chemical methods.

CH~ 0 CH1

_1 2

C H3 -- C H =CH-(C=C)z- CH= CH -CH3-GHI--Ct H-CHI-CH ~

o ? _3 ~=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, Kreuz- bergweg 26, 5300 Bonn (F.R.G.)

O Department of Pharmacy, Kang-Weon National University, Chuncheon 200 (Korea)

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 hypoglycemic 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 of 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

* Laboratorlo de Farmacognosia y Farmacodinamia, Departa- mento de Farmacologfa y Farmacoteenia, Facultad de Far- macia , U n i v e r s i t a t de Va l~nc i a , Av. Blasco 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 Farmacognosia y Farmaeodlnamla , Depa r t a - mento de Fa rmaco logfa , F a c u l t a d de Farmacia , U n l v e r s l d a d Complutense de Madrid, Ciudad U n l v e r s i t a r i a , 28040 Madrid (Spa in )

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) alcohols, PenicilZium virids was grown and handled as described before (3). Depending on the ratio iso- eugenol/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. Saksena, Ind . Per fum. , 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

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-hydroxymethyl- 9,10-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.

* 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.)

* 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 anti- proliferative activity of the dlchloromethane extract. DCCC separation of the methanol extract, followed by low- pressure LC on RP-8, provided glucnslde 4 which showed neither fungicidal nor cytotoxic properties. The structure of ~ 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 of the other naphthoxlrenes were deduced by chemica l and s p e c t r o s c o p i c methods.

OR O OR O

OH O H

I R - I I 2 R = II

3 R - R -D-g lucopy ranosy l &_ R - B -O-g lucopy ranosy l

* 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)

PROANTHOCYANIDINS FROM Pote~um sps (ROSACEAE) G. Reher

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 bark of 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 fo r t h e s e p a r a t i o n of the 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 ch romatograph ic and s p e c t r o s c o p i c methods. l . G. Ca r raz , A. Boucher le 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

Division of Pharmacognosy, University of Hamburg, Bundesstr. 43, 2000 Hamburg 13" (F.R.G.)

243

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.

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.

Instltut fQr Pharmazeutische Biologie der T.U., Institute of Pharmacology and Toxlcologle, Powstafic6w Mendelssohnstr. I, 3300 Braunschwelg (F.R.G.) Wlkp. 72, 70-111Szczecln (Poland)

244 VOI. 9 " 1987 Pharmaceutisch Weekblad Scientific Edition

COMPOSITION OF THE ESSENTIAL OIL OF Salvia pisidiea E. ~are r

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

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 resin- ous 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 Essen- tial 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

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.

INFLUENCE OF VARIOUS CULTURE CONDITIONS ON GROWTH OF H~ytophthora 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 Sarcoma- 180 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, aspa- ragine, potassium nitrate and ammonium nitrate were accept- ed 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 welgh~ 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, Tetra-

hedron 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

Department of Pharmacognosy, Fa c u l t y of Pharmacy, Ankara University, Tandogan-Ankara (Turkey) 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-the-

Art, Flay. Fragr. J., in press * Part of the thesis of G.S.

16~-HYDROXYLATION OF DIGITOXIN BY Digi ta l i s puppupea CELL CULTURES

J. Sepasgosarian and E. Reinhard

245

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. glucose- free 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 Erlenmeyer- flasks 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

Department of Pharmacognosy, University of Hamburg, Bundesstr. 43, 2000 Hamburg 13 (F.R.G.)

Pharmaceutical Institute, University of T~blngen, Auf der Morgenstelle 8, 7400 TOblngen ('F.R.C.)

BIOCHEMICAL CHARACTERIZATION OF EMBRYOGENIC Digitalis lanata SUSPENSION CULTURES S. Seidel and E. Reinhard

U n d i f f e r e n t i a t e d c e l l c u l t u r e s of Dig i ta l i s lanata a r e not a b l e to p roduce c a r d e n o l i d e s ( 1 ) , whereas m o r p h o l o g i c a l d i f f e r e n t i a t i o n l e a d s to c a r d i a c g l y c o s i d e f o r m a t i o n ( 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 differ- entiated embryogenic cultures via preparative HPLC, we found odorosid H, odorobiosid G, glucodigifucosid, vero- doxin 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. Luckner and B. D i e t t r i c h , In : P r ima ry and Secondary

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.)

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 of monoenoic C18 iron8 f a t t y a c i d s were s m a l l e s t (1%) and those of s a t u r a t e d ones h i g h e s t ( 4 0 - 45%) in b u t t e r - v e g e t a b l e o t l m i x t u r e s (no t h a r d e n e d ) . 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 of 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 BIOLOGICAL ACTIVITY OF AN AQUEOUS EXTRACT FROM AiZanthu8 aZtissima CELL CULTURES

E. Speroni*, N. Crespi-Perellino O, A. Guleciardi O and A. Minghe t t i V

An a q u e o u s e x t r a c t f r o m c e l l c u l t u r e s o f Ailanthus altissima Swlngle induced, in mice, a dose-related 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 8- carbollne or tetrahydro-8-carboline structures, known to exhibit central effects, often conflicting: {.e. pro/anti- convulsive, 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


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 docosa- hexaenole 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 be presented.

* 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)

Department of Pharmacognosy, Faculty of Pharmacy, University of Ankara, Tando~an-Ankara (Turkey)

IRIDOIDS OF THE GENUS Verbaaeum L. ~wiaZek*, B. G~abias* and P. Junlor O

Iridoid fractions isolated from the 31 species belonging to the sectio Bothro~pe~ of the genus Verbascum (Scrophular- iaceae) were analysed. The presence of 14 irldoids was detected by chromatographic methods (PC, TLC, RPTLC). Aucuhin, aJugol and harpagide occur coBonly 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

PHENYLPROPANOIDS IN PLANTS AND ROOT ORGAN CULTURES OF Coreopsis SPECIES U. Thron, J. Relchllng and g. Martin

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'-isobutyryloxy- eugenol isobutyrate; l'-acetoxy-eugenol isobutyrate; els-|',2'-epoxy-conlferylalcohol dllsobutyrate (1). Additionally we found three new compounds: l'-(2-methyl- butyryloxy)-eugenol isobutyrate; l'-Isovaleroyloxy- eugenol Isobutyrate; cis-l',2'-epoxy-eonlferylalcohol- - 3'-(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 IH- and 13C-NMR. I. F. Bohlmann and Ch. Zdero, Chem. Ber., [01 (1968) 3243

* 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.)

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 R O \ ~ l i . , . ] " " )~ -2 R : g l c

--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)

IDENTIFICATION OF THE MAIN COU~RINS IN THE pHARMACOLOGI- CALLY ACTIVE EXTRACTS OF Peucedanum ~zZustPe H. Vuorela*, H. Pernil~*, N. Nystrand*, R. Hlnkk~nen O and R. Hiltunen*

247

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 phase 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

* 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

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 anti- diarrholc 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.

ANTIBIOTIC COMPOUNDS FROM NEW ZEALAND PLANTS J.R.L. Walker and A.L.J. Cole

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, re- laird 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 patho- genic Pseudomonads. IV. A compound showing antidermatophyte activity from a StereocauZo~ s p e c i e s (a l i c h e n ) .

Department of Pharmaceutical Biology, University of W~rzburg, Mittlerer Dallenbergweg 64, 8700 W~rzburg

(F.R.G.)

Department of Plant and Microbial Sciences, University of Canterbury, Christchurch (New Zealand)

248

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-diode- array 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.)


DETERMINATION OF ALLIIN IN GAR.LIC BY RP-HPLC/ELECTRO- CHEMISTRY 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 crystal- lization. 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

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 (amino- transferases, 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.

OPTIMIZATION OF ~[PLC CONDITIONS FOR SEPARATION OF CLOSELY RELATED FUROCOUMARINS G. Zogg, Sz. Nylredy and O. Stlcher

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 sphondyZi~. For the separation of the investigated compounds (iso- bergapten, 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, Prepa- rative Chromatography Techniques, [9B6, Sprlnger Verlag, Berlin, pp. 27-79

2. C.A.J. Erdelmeier, Ph.D. thesis no. 7924, 1983, ETH ZUrlch

3. G. Zogg, Sz. Nylredy and O. Stlcher, J. Liq. Chromatogr., submitted

Institute of Pharmacology and Toxicology, Powsta~c6w Pharmazeutlsches Instltut, EidgenSssische Technische Hoch- Wlkp. 72, 70-III Szczeclo (Poland) schule ZUrich, 8092 Z~rich (Switzerland)

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