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Alkaloids content in the ripening seeds of Nigella damascena,
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315
ALKALOIDS CONTENT IN THE RIPENING SEEDS OF NIGELLA
LIPASES IN BIOORGANIC CHEMISTRY: STRUCTURAL MODIFICATION OF POLYHYDROXYLATED NATURAL COMPOUNDS A. Dudda’, S. Gebhardt’, P. SupperI’, J. Rein&, B. Daniel?. S. Rivad and M. Schubert-Zsilavecz’ ‘Institute of Pharmaceutical Chemistry, J. W. GoetheUniversity Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany. b Department of Organic Chemistry I, University of Bayreuth, D-95440 Bayreuth, Germany. ‘Dipartimento di Chimica Organica e Industriale, Universita Degli Studi di Milano, Via Venezian 21, l-201 33 Milano, Italy. %NR, lnstituto di Chimica degli Ormonl. I-20131 Milano. Italy.
DAMASCENA G. and F. Tome
Department of Biology, University of Milan. via Celoria, 26, 20133 Italy Ni@/e damescane L. (Ranunculaceae) is characterized by the presence of volatile alkaloids (damascenine and related compounds) mainly localized in the seeds. Little information is available about the pharmacological action of this plant: some authors report only an analgesic and diuretic effect of the seeds. This work was undertaken to determine the time course of alkaloid accumulation during flowering and fruiting. The plant material was extracted with light petroleum in a Soxhlet apparatus and subsequently with methanol, at mom temperature. The alkaloids were recovered with chlomfonn fmm the acqueous acidified layers and evaluated by HPLC, using an RP-C 18 column with H20: CH3CN (37) (pH 8 with diethylamin) as eluent in isocratic condiions. The alkaloids were monitored at 230 nm and their quantification has been made using naphthalene as internal standard. The identity of alkaloids was conhrmed by rpectmscopic methods. In the vegetative phase of growth, small quantities nf Nkaldids a-m accumulatedin~the planfs (6.4 m#tgb individuals). The transition from vegetative to reproductive phase is characterized by a significant increase of the secondary metabolites. Perianth and ripening pericarp remain devoid of alkaloid, while the damascenine wntent in the ripening seeds increases mom than five times in about 5-10 days, reaching a value of 8 mg1100seeds. In conclusion there is no appreciable production of alkaloid during the life cycle of the plant, except during the fruiting phase, when the content in the ripening seeds greatly increase in a very short while. Therefore it appears that ripening seeds of N&~/la damascena may be a suitable material for studies on the mechanisms leading to alkaloid n~P~~mulation.
In recent years, lipases have been used for the transformation of a great number of organic compounds. One of the main fields of application of lipase-catalysed regioselective acylation is sugar chemist@. Non-derivatised monosaccharides and alkyl-glycosides are preferably acylated at the primary hydroxy group. If there are no free primary hydroxy groups in the molecule, it is also possible to distinguish between various free secondary hydroxy groups. By the example of selected monosaccharides and glycosidic natural compounds (such as Digitoxose, various cardiac glycosides and alpha-Hederin) we are investigating the transesterification activity and regioselectivity of various commercially available lipases. Operating in f-amylalcohol with vinylacetate or vinylbutyrate as acyl donor, we obtained mono-, di- and triacyl-derivatives of the used substrates. Their structures were characterised by modern NMR and MS techniques. [IIN. B. Bashir. S. J. Phytian, A. J. Reason, S. M. Roberts, J. Chem. Sot. Perkin Trans. I 1995, 2203 This work was supportedby Boehringer Mannheim and DAAD
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316
ALKALOIDS IN NIGELLA DAMASCENA SEEDS: AN HYPOTHESISON THEIR BIOLOGICAL ROLE G. and F. Tome Department of Biology, University of Milan, via Celoria 26, 20133 Italy N&e//a damasc-ana L. (Ranunculaceae)is a common herbaceous plant growing in grasslands in tempemte zones in Europe. It is chemicalty chamcterized by the presence of alkaloids, mainly damesceninr and damascinine. Previous investigations had pointed out that the seeds are the pmferenttal site of alkaloid accumulation #andthat a great quantity of damascenine and damascinine is located in the seed coats. The aim of this work was to elucidate whether this peculiar distribution of alkaloids has an ecological role. Fimtly it has been noticed that the seeds release in the medium a large amount of compounds during the first hours of imbibton. To evaluate the relationship of the above fact with seeds germination and plantlet growth, seedo of N&alla damescane, Pisum sativum, Zaa mays, Rephanus satiwm and Lactuca sativa wem allowed to germinate in controlled wndtttons in presence of imbibitton water obtained fmm different wncentrations of Nips//e seeds. The results show that the seeds mact to the treatment in diffemnt manner: the germination of Pisum and Lactuca seeds appear to be simply delayed at low concentration and inhibited of about 10% at higher wncentratton; the germination of Z. meys and Rephanus is stmngly inhibited (mom than 80%) even at lower concentrations; the germination of Nigdle se& appeam to be delayed and slightly inhibtd at low wnwntratton and inhibtd of about 40% at the higher wnwntrattons. Surprisingly enough, in Lactuca, the plantlets from seeds escaped from the inhibition, grow more vigomusty and show a fresh and dry weight higher in comparison v&h the contml. From the above msults it wpeam confirmed the hypothesis that the compounds, released from the seed coats of Ni@a, play a role allelopathic species.
on N@e//a itself and,
at variuos
extent.
on other
PERSPECIIVES OF DEVELOPMENT OF NOVEL PREPARATIONS OUT OF MEDICINAL LEECHES Yu.N.Khomvakov, T.V.Phyodorov, I.E.Zykova and S.E.Severin Moscow Research Institute of Medical Ecology, Simpheropolsky blvd, 8, Moscow, 113149, Russia It is well known, that medicinal leech Hirudo Medicinalis is a source of different types of biological active substances such as inhibitors of proteinases: hirudin, hirustasin, bdellins, eglins etc. Due to an important role of the proteinases in the maturation of tissues and in an angiogenesis, we decided to investigate the influence of preparations out of medicinal leeches on the development of the experimental tumours (cancer of mouse lung-67 and sarcoma of hamster). We studied also the possibility of usage these preparations like an immunomodulating agent at vaccine process (S.dublin 6). We compared the properties of well known preparation “Piyavit” and the preparation out of leeches with specific activity 240740 AT NIH U/mg protein. It was found , that intraperitoneal inoculation of extract out of head parts of leeches inhibits the development of tumours in doses from 30 to 300 &kg. Maximal effect was obtained after inoculation of the preparation at the beginning of an experiment. There was no effect after inoculation of “Piyavit”. As an immunomodulating agent the extract increases the resistance against the experimental salmonellosis at the same doses. So, the p.mparation out of medicinal leeches can be used for a basis for a creation of antitumour and immunomodulating medicines. Acknowledgements: We express our sincere gratitude to Dr. N.V.Borisov and V.B.Rodin for kindly providing the preparations and Dr. V.V.Gusev for technical assistance.
313
315
ALKALOIDS CONTENT IN THE RIPENING SEEDS OF NIGELLA
LIPASES IN BIOORGANIC CHEMISTRY: STRUCTURAL MODIFICATION OF POLYHYDROXYLATED NATURAL COMPOUNDS A. Dudda’, S. Gebhardt’, P. SupperI’, J. Rein&, B. Daniel?. S. Rivad and M. Schubert-Zsilavecz’ ‘Institute of Pharmaceutical Chemistry, J. W. GoetheUniversity Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany. b Department of Organic Chemistry I, University of Bayreuth, D-95440 Bayreuth, Germany. ‘Dipartimento di Chimica Organica e Industriale, Universita Degli Studi di Milano, Via Venezian 21, l-201 33 Milano, Italy. %NR, lnstituto di Chimica degli Ormonl. I-20131 Milano. Italy.
DAMASCENA G. and F. Tome
Department of Biology, University of Milan. via Celoria, 26, 20133 Italy Ni@/e damescane L. (Ranunculaceae) is characterized by the presence of volatile alkaloids (damascenine and related compounds) mainly localized in the seeds. Little information is available about the pharmacological action of this plant: some authors report only an analgesic and diuretic effect of the seeds. This work was undertaken to determine the time course of alkaloid accumulation during flowering and fruiting. The plant material was extracted with light petroleum in a Soxhlet apparatus and subsequently with methanol, at mom temperature. The alkaloids were recovered with chlomfonn fmm the acqueous acidified layers and evaluated by HPLC, using an RP-C 18 column with H20: CH3CN (37) (pH 8 with diethylamin) as eluent in isocratic condiions. The alkaloids were monitored at 230 nm and their quantification has been made using naphthalene as internal standard. The identity of alkaloids was conhrmed by rpectmscopic methods. In the vegetative phase of growth, small quantities nf Nkaldids a-m accumulatedin~the planfs (6.4 m#tgb individuals). The transition from vegetative to reproductive phase is characterized by a significant increase of the secondary metabolites. Perianth and ripening pericarp remain devoid of alkaloid, while the damascenine wntent in the ripening seeds increases mom than five times in about 5-10 days, reaching a value of 8 mg1100seeds. In conclusion there is no appreciable production of alkaloid during the life cycle of the plant, except during the fruiting phase, when the content in the ripening seeds greatly increase in a very short while. Therefore it appears that ripening seeds of N&~/la damascena may be a suitable material for studies on the mechanisms leading to alkaloid n~P~~mulation.
In recent years, lipases have been used for the transformation of a great number of organic compounds. One of the main fields of application of lipase-catalysed regioselective acylation is sugar chemist@. Non-derivatised monosaccharides and alkyl-glycosides are preferably acylated at the primary hydroxy group. If there are no free primary hydroxy groups in the molecule, it is also possible to distinguish between various free secondary hydroxy groups. By the example of selected monosaccharides and glycosidic natural compounds (such as Digitoxose, various cardiac glycosides and alpha-Hederin) we are investigating the transesterification activity and regioselectivity of various commercially available lipases. Operating in f-amylalcohol with vinylacetate or vinylbutyrate as acyl donor, we obtained mono-, di- and triacyl-derivatives of the used substrates. Their structures were characterised by modern NMR and MS techniques. [IIN. B. Bashir. S. J. Phytian, A. J. Reason, S. M. Roberts, J. Chem. Sot. Perkin Trans. I 1995, 2203 This work was supportedby Boehringer Mannheim and DAAD
314
316
ALKALOIDS IN NIGELLA DAMASCENA SEEDS: AN HYPOTHESISON THEIR BIOLOGICAL ROLE G. and F. Tome Department of Biology, University of Milan, via Celoria 26, 20133 Italy N&e//a damasc-ana L. (Ranunculaceae)is a common herbaceous plant growing in grasslands in tempemte zones in Europe. It is chemicalty chamcterized by the presence of alkaloids, mainly damesceninr and damascinine. Previous investigations had pointed out that the seeds are the pmferenttal site of alkaloid accumulation #andthat a great quantity of damascenine and damascinine is located in the seed coats. The aim of this work was to elucidate whether this peculiar distribution of alkaloids has an ecological role. Fimtly it has been noticed that the seeds release in the medium a large amount of compounds during the first hours of imbibton. To evaluate the relationship of the above fact with seeds germination and plantlet growth, seedo of N&alla damescane, Pisum sativum, Zaa mays, Rephanus satiwm and Lactuca sativa wem allowed to germinate in controlled wndtttons in presence of imbibitton water obtained fmm different wncentrations of Nips//e seeds. The results show that the seeds mact to the treatment in diffemnt manner: the germination of Pisum and Lactuca seeds appear to be simply delayed at low concentration and inhibited of about 10% at higher wncentratton; the germination of Z. meys and Rephanus is stmngly inhibited (mom than 80%) even at lower concentrations; the germination of Nigdle se& appeam to be delayed and slightly inhibtd at low wnwntratton and inhibtd of about 40% at the higher wnwntrattons. Surprisingly enough, in Lactuca, the plantlets from seeds escaped from the inhibition, grow more vigomusty and show a fresh and dry weight higher in comparison v&h the contml. From the above msults it wpeam confirmed the hypothesis that the compounds, released from the seed coats of Ni@a, play a role allelopathic species.
on N@e//a itself and,
at variuos
extent.
on other
PERSPECIIVES OF DEVELOPMENT OF NOVEL PREPARATIONS OUT OF MEDICINAL LEECHES Yu.N.Khomvakov, T.V.Phyodorov, I.E.Zykova and S.E.Severin Moscow Research Institute of Medical Ecology, Simpheropolsky blvd, 8, Moscow, 113149, Russia It is well known, that medicinal leech Hirudo Medicinalis is a source of different types of biological active substances such as inhibitors of proteinases: hirudin, hirustasin, bdellins, eglins etc. Due to an important role of the proteinases in the maturation of tissues and in an angiogenesis, we decided to investigate the influence of preparations out of medicinal leeches on the development of the experimental tumours (cancer of mouse lung-67 and sarcoma of hamster). We studied also the possibility of usage these preparations like an immunomodulating agent at vaccine process (S.dublin 6). We compared the properties of well known preparation “Piyavit” and the preparation out of leeches with specific activity 240740 AT NIH U/mg protein. It was found , that intraperitoneal inoculation of extract out of head parts of leeches inhibits the development of tumours in doses from 30 to 300 &kg. Maximal effect was obtained after inoculation of the preparation at the beginning of an experiment. There was no effect after inoculation of “Piyavit”. As an immunomodulating agent the extract increases the resistance against the experimental salmonellosis at the same doses. So, the p.mparation out of medicinal leeches can be used for a basis for a creation of antitumour and immunomodulating medicines. Acknowledgements: We express our sincere gratitude to Dr. N.V.Borisov and V.B.Rodin for kindly providing the preparations and Dr. V.V.Gusev for technical assistance.