Jiří Havel, Oldřich Kolovrat, Ludmila Kameníková*, Miroslav Bechyně** Oseva PRO s.r.o., Research Institute of Oil Crops Opava, * Oseva PRO s.r.o., Keřkov ** Czech Agricultural University Prague
Development of the screening method and search
for genetic resources of the opium poppy
(Papaver somniferum) with high thebaine content
Opracowanie metody wstępnego testowania
i poszukiwanie źródeł genetycznych maku
(Papaver somniferum) z wysoką zawartością tebainy
Słowa kluczowe: zawartość tebainy, chromatografia cienkowarstwowa, surowce genetyczne Key words: Papaver somniferum, thebaine content, thin-layer chromatography (TLC), genetic
resources
Tebaina jest ważnym surowcem w przemyśle farmaceutycznym używanym do produkcji nieuzależniających analgetyków. Celem projektu jest opracowanie metody wstępnej selekcji na skład alkaloidów oraz poszukiwanie źródeł ge-netycznych maku (Papaver somniferum) z wysoką zawartością tebainy. Wydaje się, że chromato-grafia cienkowarstwowa jest najbardziej przy-datną metodą do wstępnej selekcji na skład alkaloidów. Metoda polega na ekstrakcji alka-loidów mieszaniną chloroform + metanol + amo-niak, rozwinięciu chromatogramu w komorze i wybarwieniu chromatogramu odczynnikiem Dragendorfa. Metodę tą stosowano do testo-wania materiałów hodowlanych i odmian z kolekcji. W materiałach zebranych w 1998 r. znaleziono około 9% roślin z wyższą zawar-tością tebainy, a w materiałach z 1999 r. około 15,5%. Genotypu z wysoką i stabilną zawar-tością tebainy nie udało się dotychczas znaleźć.
Thebaine is an important source material in the pharmaceutical industry for the production of non-addictive analgesics. The aim of the project is to develop a screening method for the determination of alkaloid composition in the poppy and to find genetic resources of the opium poppy (Papaver somniferum) with high thebaine content. The most suitable method for the preliminary determination of alkaloid composition seems to be thin-layer chromatography. The principle of the method is the extraction of alkaloids in the mixture of chloroform + methanol + ammonia, the development of a chromatogram in the developing chamber and the detection of a chromatogram by Dragendorff reagent. This method was applied to test the breeding materials and varieties from the collection of poppy genetic resources. In the materials from the year 1998 there were approximately 9% of plants with higher percentage of thebaine, in the materials from the year 1999 there were about 15.5%. A genotype with high and stable content of thebaine has not been found yet.
The increasing risk of drug abuse makes the drug manufactures produce safer drugs, which cannot be easily abused. An important source material for the
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production of such drugs is the alkaloid thebaine. Thebaine is used in the production of a number of semisynthetic morphinan derivatives — butorphanol (strong and safe analgesic) naloxone (morphine antagonist), nalmefene (morphine antagonist, which reduces the permanent damage to the central nervous system after a stroke) and buprenorphin (strong and safe analgesic). Thebaine, as well as other morphine-type alkaloids (codeine, morphine) cannot be synthetically produced. For the needs of the pharmaceutical industry thebaine is imported. The attempts to grow poppy (Papaver bracteatum) as a domestic source of thebaine failed, because it is a perennial plant with slow initial development and its agronomy makes it unsuitable for commercial growing.
The aim of the study was to develop a screening method for the preliminary determination of thebaine content in poppy capsules. With respect to the fact that thebaine is a precursor of codeine and morphine there might be a genotype in which thebaine conversion into codeine and morphine is inhibited. A screening method was used to test the genetic resources and the breeding lines of the opium poppy (Papaver somniferum) with the aim of finding this genotype.
Material and methods
The initial material for searching the genetic resources of the poppy with high content of thebaine was supplied from three localities. In Opava and Keřkov, the varieties coming from a collection of oil crop genetic resources and the breeding lines at different stages of development were grown. At the Czech Agricultural University in Prague the studies involved the poppy plants derived from the local varieties of the Bohemian Moravian Highlands and the poppy plants from Turkey and India. In all localities a large number of different genotypes were grown with the aim of recording the highest possible genetic variability.5 to 20 plants of each localities were isolated immediately prior to blooming. From straightening buds prior to the bloom calyx and corolla petals were removed and the remaining part of the flower was isolated using a paper bag. The isolated flowers were marked with a color ribbon. After natural self-pollination and stamina mortality (one week later) the bags were removed. After maturation the plants were harvested and all capsules including the non-isolated ones were manually shelled. The isolated seed from each plant was marked and stored separately for each growing. To ensure a sufficiently large size of samples for chemical analysis, the capsules from the whole plant (including the non-isolated ones) were used. Very hard nodes, which cannot be ground in the mills, were removed from the capsules. The remnants were ground and passed through a fine sieve so that the particles might be of uniform size.
On the basis of the literary search it was found that apart from other methods (e.g. spectrophotometry, isotachophoresis) most studies employed chromatographic methods. Of gas, thin-layer, column and paper chromatography the most suitable is thin-layer chromatography.
Screening method – procedure of analysis:
Sample preparation: 0.5 g of finely ground capsules were added into 25 ml of extraction mixture of chloroform + methanol + ammonia 26 % in the ratio 15:9:1 and kept in a wide-mouth bottle for drugs. The mixture closed in the bottle was allowed to stand overnight, on the second day it was shaken for 8 hours, then it was allowed to stand overnight again and on the next day it was shaken for another 2 hours. The extract was filtered and the filtrate was thickened from the volume of 5 ml to the volume of 1 ml by blowing nitrogen to the surface at an increased temperature.
20 µl of extract sample was spread on the plate for thin-layer chromatography Silicagel 60 F254 and allowed in the chamber to develop by using a mixture of diethyl ether + chloroform + methanol + ammonia 26% at the ratio 30 : 5 : 3 : 1. After the termination of development the chromatogram was detected by Dragendorff reagent and visually assessed. The method is suitable for the preliminary determination of the presence or the absence of thebaine. The promising materials must be further tested by an accurate method. After the first cycle of analyses it was found that this method covers the whole spectrum of alkaloids. One part of the analysis included also the determination of codeine and morphine.
Dragendorff reagent
8 g of basic bismuth nitrate dissolved in 20 ml of concentrated HCl and 27 g of potassium iodide dissolved in 30 ml of distilled water were mixed and adjusted to the volume of 100 ml. For detection the buffer solution was diluted 10x with hydrochloric acid (1 mol/l).
The screening method was developed in such a way that the prepared extract of alkaloids could be analyzed by the accurate method using high-pressure liquid chromatography. This procedure will make the analysis of samples selected by the screening method possible without any further cost covering the special preparation of extracts for HPLC. The size of a poppy capsule sample is limited and in some cases it is not enough for repeated extraction.
Results
Table 1 gives the results of screening of the breeding materials from Opava and Keřkov from the year 1998. In these localities were found approximately 9% of genotypes with higher content of thebaine.Jiří Havel ...
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Table 1 Determination of thebaine in materials from the year 1998
Ocena zawartości tebainy w materiałach pochodzących z 1998 roku
Varieties Odmiany Number of varieties Ilość odmian Number of analysed plants Ilość analizowanych roślin
Number of plants with high thebaine content Ilość roślin z wysoką zawartością tebainy Opava
varieties of the world collection odmiany z kolekcji światowej
32 234 25
Opava
breeding lines — linie hodowlane
52 318 34
Keřkov
breeding lines — linie hodowlane
19 154 8
In 1999 in Opava and Keřkov localities, a survey of varieties from the collection of poppy genetic resources and breeding materials under development were continued. The lines from the year 1998 in which high thebaine content was detected were grown in these localities. The genotypes from crossing of local varieties from the Bohemian Moravian Highlands and poppy plants from Turkey and India were grown at the Czech Agricultural University in Prague. In Opava the poppy stands were severely damaged by adverse weather conditions. Due to the torrential rain and wind during the storm before the bloom the stand was completely lodged. For this reason, only a small part of the necessary number of plants was isolated and yield assessment was not possible. The isolated plants were harvested from the breeding materials and lines with high content of thebaine of the year 1998. The varieties from genetic resources were completely damaged.
Table 2 gives the results from the screening test of genotypes from the year 1999. A more precise screening method was used to analyze the material from the Czech Agricultural University — a total of 225 plants from 34 lines. 37 plants (16.4%) were found to have increased thebaine content. 92% plants had high morphine content, 6 plants showed higher codeine content. It was found that majority of plants with higher thebaine content had also high morphine content, only 2 plants had increased thebaine content and low content of other alkaloids. In the breeding material from Opava there were 13 plants with high thebaine content but they all had also increased morphine content. Similar results were obtained in Keřkov. A total of 193 plants from 38 blue-seeded and white-seeded varieties of the poppy in the world collection and 207 plants from 18 white-seeded breeding lines were analyzed. All materials from Keřkov have high morphine content, 76 plants have high thebaine content and 38 plants have high codeine content. The plants with high thebaine or codeine content and low content of other alkaloids were not found in the material.
Ilość roślin z wysoką zawartością alkaloidów Localities Miejscowości Number of varieties/lines Ilość odmian/linii Number of analysed plants Ilość analizowanych
roślin thebainetebaina codeine kodeina morphinemorfina thebaine+ morphine tebaina + morfina Opava
breeding lines under development wyprowadzone linie hodowlane
44 101 13 0 101 13
Opava
progeny from lines with high thebaine content — potomstwo z linii z wysoką zawartością tebainy
24 61 10 3 60 10
ČZU Praha
individual plants — pojedynki
34 225 37 6 207 35
Keřkov
varieties of the world collection odmiany z kolekcji światowej
38 193 46 13 193 46
Keřkov
breeding lines — linie hodowlane
8 107 29 15 107 29
Keřkov
progeny from lines with high thebaine content — potomstwo linii z wysoką zawartością tebainy 7 120 12 16 120 12 Keřkov comparative experiment doświadczenia porównawcze 10 100 18 9 100 18
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The analysis of the progeny of plants from the year 1998 for high thebaine content showed that the stability of this trait is rather poor. Of 61 plants from Opava subjected to analysis, only 10 (16.4%) had high thebaine content. All the plants had also high morphine content. In the material from Keřkov it was found that out of 120 plants of 7 lines 12 plants had high thebaine content. The plants had also high morphine content. No progenies from both localities showed high and stable thebaine content.
