Papaver Bracteatum Lindl Research Articles

Chromatographic and spectroscopic study of impurities present in thebaine obtained from Papaver bracteatum Lindl.

Chromatographic and spectroscopic study of impurities present in thebaine obtained from Papaver bracteatum Lindl.

Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. The effects of induced polyploidy as well as the effect of methyl Jasmonate (MeJA) elicitation on the root production of thebaine and on the expression of five alkaloid biosynthesis related genes were studied. The in vitro tetraploidy induction caused a significant increased expression of norcoclaurine synthase (NCS) and salutaridinol (SAT), and a significant decreased expression of berberine bridge enzyme (BBE) in the leaves. In the root tissues, the BBE, NCS, and SAT showed an increased expression in tetraploid plants, while codeinone reductase (COR) showed a decreased expression. A similar alteration pattern was found in mixoploid plants when compared to their diploid counterparts. MeJA at concentrations of 0.1 and 0.5 mM caused a remarkable increase in the thebaine content in the roots of treated plants, where the highest thebaine content was identified in plants elicited with 0.5 mM MeJA. Elicitation treatment caused a substantial increase in the expression of NCS and SAT in the leaves, while it had no major effect on BBE, codeine 3-O-demethylase (CODM) and COR. Expression analysis in the roots showed that MeJA caused a significant increase in the expression of only BBE and NCS, while expression of other studied genes remained unchanged. Our results may be exploited for improved thebaine production and the processing of Persian poppy.

Enhanced thebaine and noscapine production and modulated gene expression of tyrosine/dopa decarboxylase and salutaridinol 7-O-acetyltransferase genes in induced autotetraploid seedlings of Papaver bracteatum Lindl

Papaver bracteatum, belonging to Papaveraceae, is considered to be a primary ingredient in the production of codeine and anti-drug drugs (e.g., naloxone and Naltrexone) without the involvement of morphine. For polyploidy induction, papaver seedlings were treated with various colchicine concentrations (0, 0.05, 0.1, 0.2 and 0.5% w/v) in three time durations (24, 48 and 72 h). The effects of polyploidy induction on morphological, physiological, alkaloid production and gene expression levels in the pathway of benzylisoquinoline alkaloids were analyzed. High rates of seedling survival were observed at lower colchicine concentrations (0.05 and 0.1% w/v) versus higher concentrations (0.2 and 0.5% w/v). The best polyploidy treatment to induce P. bracteatum shoot tips was observed in plants treated with 0.2% colchicine for 72 h. P. bracteatum-induced tetraploids showed higher stomata and an increase in leaf area (mm2), chlorophyll (a, b and total) and a decrease in stomata density, leaf length (cm) and petiole length. Flow cytometry and chromosome counting analysis revealed that chromosome number was 2n = 2x = 14 in diploid plants and 2n = 4x = 28 in tetraploid plants. Gene expression analysis through semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that the transcription level of sat and tydc genes of the benzylisoquinoline biosynthesis pathway was enhanced in tetraploid plants. The tetraploid plants of P. bracteatum also showed 5.86 and 30.55 times higher thebaine and noscapine contents than the diploid plants, respectively. Regarding the higher commercial value of thebaine and noscapine, tetraploids can efficiently be used to improve the thebaine and noscapine production of P. bracteatum.

Effects of methyl jasmonate and phloroglucinol on thebaine and sanguinarine production in cell suspension culture of Persian poppy (Papaver bracteatum Lindl.)

The biosynthesis path engineering could be very promising for mass production of alkaloids by applying elicitors in the cell suspension culture of Persian poppy (Papaver bracteatum Lindl.). In this work, the effects of different concentrations of methyl jasmonate (MJ) and phloroglucinol (PG) on thebaine and sanguinarine productions in vitro were investigated. Roots as explant and supplementing 3mg L-1 2,4-Dichlorophenoxyacetic acid with 0.5 mg L-1 Benzyl amino purine to modified MS medium were selected to achieve the most efficient combination for callus induction and production of callus fresh and dry weights. At 48 h after treatment, the addition of PG and MJ individually and in combination together significantly increased both thebaine and sanguinarine contents than the control. The results of high-performance liquid chromatography (HPLC) detection indicated that the highest production rate has been achieved through a synergic effect of two elicitors after 48 h. Results revealed that adding 200 μM of MJ and 100 mg L-1 PG increased thebaine and sanguinarine contents by 56.36 and 107.71-fold than control cells, respectively.

Molecular diversity and phytochemical variability in the Iranian poppy (Papaver bracteatum Lindl.): A baseline for conservation and utilization in future breeding programmes

In the present investigation, 72 accessions of the Iranian poppy (Papaver bracteatum Lindl.) were analyzed for genetic diversity and population structure using start codon targeted polymorphism (SCoT) and inter simple sequence repeat (ISSR) markers along with four important phytochemical traits to provide baseline knowledge for the Iranian poppy’s breeding and conservation plans. Twelve ISSR and thirteen SCoT primers generated a total of 98 and 186 fragments with a mean of 8.17 and 14.31 fragments per primer, respectively. Polymorphic information content for ISSR and SCoT primers ranged from 0.39 to 0.45 and 0.28 to 0.34, with the resolving power ranging from 21.61 to 3.97 and 13.08 to 28.02, respectively. Neighbour-joining (NJ) based clustering grouped 72 accessions into three main groups based on two markers studied (ISSR and SCoT) and the combined data (ISSR + SCoT), which associated with their eco-geographical regions. Population structure based analysis divided 72 accessions into 3 subpopulations using ISSR markers, when SCoT was used eight subpopulations were observed. However, when the combined data was used only three subpopulations were found, which corresponded to the grouping observed with the NJ method and these results were supported by principal coordinate analyses (PCoA). Phytochemical analysis revealed that plant capsule has higher total amounts of the alkaloids; thebaine, morphine and oripavine than stem tissues. Interestingly, for the geographical parameters, latitude showed a significant and positive correlation with thebaine extracted from both stem and capsules and the regression results confirmed these associations. Taken together, our results indicated that three populations Ploor, Eil-Teymoor and Anjomane due to their high contents of alkaloids like thebaine as well as the Taham population due to its high content of morphine and oripavine have a strong enough potency to be used in the pharmacy industry.

The effect of intra‐peritoneal administration of Papaver bracteatum Lindl. extract on development of NMRI mice oocytes treated with Doxorubicin

The effect of water-alcohol Papaver bracteatum Lindl. extract on development of mice oocytes treated with Doxorubicin (dox) was examined in this study. The mice were classified into four groups. Control group, mice injected intraperitoneally (IP) with saline. Extract group alone, mice treated with 200 mg/kg of body weight (bw), IP, twelve consecutive days. Dox group alone, mice were given dox, IP, 10 mg/kg bw. Experimental group treated with extract and dox together. Effect of the extract on the development of mice oocytes treated with dox were evaluated through assisted reproductive technology techniques (ARTs). Developmental rate and blastocyst formation was improved by using the extract. A significant increase in in vitro developmental competence in comparison with dox group (P < 0.05) was observed. The results of this study indicated that P. bracteatum Lindl. extract could prevent dox toxicity of dox affecting both follicle or oocytes, and therefore it can result in improved embryo development which was observed in mice treated with dox plus P. bracteatum Lindl. compared to mice treated with dox alone.

In vitro regeneration of Persian poppy (Papaver bracteatum)

AbstractPersian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.

Influence of Agrobacterium rhizogenes on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy (Papaver bracteatum Lindl.): preliminary report

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l−1 paromomycin to select for pBI121 transformants and 200 mg l−1 cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l−1 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l−1 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.

Production of Pharmaceuticals from Papaver Cultivars in Vitro

AbstractIranian (Papaver bracteatum Lindl.) and opium poppy (P. somniferum L.) plantlets obtained from germinated seeds grown on a Murashige and Skoog basal medium (BM) readily manifest alkaloids. Temperature had a profound effect on growth and alkaloid production after 8 weeks in culture. Plantlets of poppy cultivars (cvs.) grew best at 18.5 and 20°C compared to 15 or 25°C. An alkaloid survey study with 24 Iranian and 21 opium poppy cvs. revealed that total morphinan alkaloids ranged from 0 to 6.55 mg/g dw. Prolific axillary branching was achieved from poppy cvs. by maintaining shoots on BM containing 1.0 mg/L N6‐benzyladenine and 0.01 mg/L α‐naphthalene acetic acid for an additional 16 weeks. The influence of vessel size on the growth response of established shoot clumps was determined by subculture in a variety of culture vessels for 8 weeks. The tested culture vessels included culture tubes (55 mm3 capacity (cap.)), babyfood jars (143 mm3 cap.), Magenta GA‐7 containers (365 mm3 cap.), and polycarbonate jars (1890 mm3 cap.) employing an in vitro hydroponics system (i.e. an automated plant culture system (APCS)). Highest growth rates occurred employing the APCS. The culture vessel capacity had a significant positive correlation on shoot length, fresh weight, number of leaves, and number of shoots. Shoot length, fresh weight, leaves, and shoots grown in the APCS exhibited increases of 1‐, 21.5‐, 7.8‐, and 8.3‐fold, respectively, compared to shoots grown in culture tubes. Higher culture growth rates that occurred in the larger‐size vessels were correlated with lower alkaloid production (mg alkaloids/g dw). However, the overall total alkaloids/vessel [(mg alkaloid/g dw)×g culture dw] increased because of greater biomass production per vessel. The alkaloid content was found to remain stable for shoots grown over a 6–month evaluation period.

Cytogenetic studies in “X-7”, a new line of Papaver bracteatum Lindl. with increased thebaine content

Papaver bracteatum Lindl. is of substantial medical and pharmaceutical interest as material for commercial production of codeine. During various experiments, some involvingcolchicine treatment of seeds, a diploid plant (called “X-7”) developed. This plant had a substantial increase in the amounts of thebaine, the main alkaloid produced in the capsules. By cloning this plant, a new series of bracteaturn plants were obtained, and the thebaine content of the new individuals vaned markedly from ca 3 % to about 17 % against normal variation from 1 70 to 3 70. Subsequent cytological analyses revealed the X-7 plants to be diploid (2n=14). Their karyotype may contain minor structural differences from the mother line, but these were difficult to characterize. Marked differences of meiotic behaviour occurred. The “X-7” line appears to have arisen through diploidization and chromosomal rearrangements from polyploid tissue.

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with α-naphthaleneacetic acid (NAA) (1.0 mg 1-1) and benzylamine purine (BA) (0.5 mg 1-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1−1), BA (0.5 mg 1−1) and casein hyrdrolyzate (2.0 mg 1-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.

SalutaridineN-Oxide from the Capsules ofPapaver bracteatum

A novel compound, salutaridine N-oxide ( 1) has been isolated from PAPAVER BRACTEATUM Lindl (Papaveraceae) in which the major alkaloids were salutaridine and thebaine. Semisynthetic 1 was prepared for structure confirmation. Other components of the alkaloidal fraction were isothebaine, oripavine, neopine, and thebaine N-oxide.

Thebaine content and yield in induced tetraploid and triploid plants of Papaver bracteatum Lindl.

The effect of colchicine induced autotetraploidy on the thebaine content and yield of the capsules of Papaver bracteatum was studied. A significant increase in the thebaine content of the capsules was found in tetraploid plants during two consecutive growing seasons, but the thebaine yield per plant was slightly reduced in the autotetraploids. Autotriploid plants showed a very high concentration of thebaine (8.8%) only in the first growing season.

Alkaloids of Cell Suspensions Derived from Four Papaver spp. and the Effect of Temperature Stress

Summary Cell suspension cultures derived from Papaver bracteatum Lindl var. Arya II P. somniferum L. var. Halle and var. Berlin, P. setigerum D. C., and P. nudicaule L. were found to contain protopine, sanguinarine, isothebaine and orientalidine. Growth of P. bracteatum suspensions at 36°C followed by 5°C caused release of thebaine into the media.

A new rearrangement product of thebaine, isolated from Papaver bracteatum Lindl. Structural assignment of thebaine N-oxides

From Papaver bracteatum an unusual alkaloid is isolated: 6,7,8,9,10,14-hexadehydro-4,5-epoxy-3,6-di-methoxy-17-methylthebinan (1). This alkaloid is also obtained from the mixture of rearrangement products of one of the thebaine N-oxides. The structure of another constituent of the latter mixture is deduced to be 6,7,8,9,10,14-hexadehydro-3,6-dimethoxythebinan-4-ol (15). The presence of both thebaine N-oxides in Papaver bracteatum is confirmed, and their structures are revised on chemical and spectroscopic grounds.

Germination, phenology and thebain accumulation of Papaver bracteatum, in Cluj-Napoca, Romania

Based on genetic resources found in house-gardens of Cluj-Napoca, the cultivation experiment was started with Papaver bracteatum Lindl. in 1980. Best germination of seeds was found in the second year after harvest at 24 degree. The phenology of plant growth and development was registered and the thebain accumulation analysed. The highest thebain yield (6.62 kg/ha), was measured in roots or in dried capsules lacking seeds (4.32 kg/ha).

Cytological analysis of hybrids between Papaver somniferum L. and Papaver bracteatum Lindl. ; phylogenetic relationships between the two species

Cytological analysis of hybrids between Papaver somniferum L. and Papaver bracteatum Lindl. ; phylogenetic relationships between the two species

On papaver bracteatum—XIV : Ready access to benzylidene phthalimidine compounds from rhoeadine alkaloid-derived nitriles

The B D trans lactole alpinigenine ( 1a) isolated from Papaver bracteatum Lindl. was shown to form the oxime 2a which in turn was transformed to the nitrile 3a by dehydration, the methiodide 4a, and — by treatment with alkali under mild conditions — to the benzylidene phthalimidine derivative 5a, whose structure was elucidated by spectral analysis as well as oxidative fragmentation into 6 and 7. The unique reaction leading to 5a apparently is bound with certain structural prerequisites inherent to 3a, including stereochemistry. When cis-alpinigenine ( 1b) was subjected to the same sequence, little of 5a accordingly could be obtained, and even none of any phthalimidine was formed from the non-acetoxylated nitrile 11.

Alpinigenine from Papaver bracteatum Lindl. Restricted rotation in an unusual oxidation product

Alpinigenine (1) was isolated in relatively large amount from a type of Papaver bracteatum Lindl. from Iran. Oxidation of this alkaloid with Jones reagent produced a lactone function and, unexpectedly, converted the NMe into an NCHO, present as a mixture of rotamers. Crystallization afforded one pure component, which equilibrated in solution within 2 h. 13C N.m.r. spectroscopy was used to confirm these results and enabled an insight into the conformation in solution of the seven-membered rings in alpinigenine and its oxidation product. X-Ray analysis determined unambiguously the absolute configuration of alpinigenine.

High Pressure Liquid Chromatographic Determination of the Five Major Alkaloids in Papaver somniferum L. and Thebaine in Papaver bracteatum Lindl. Capsular Tissue

A high pressure liquid chromatographic isocratic procedure is described for determining and quantitating the 5 major alkaloids narcotine, papaverine, thebaine, codeine, and morphine in Papaver somniferum L. and thebaine in Papaver bracteatum Lindl. Other papaveraceous alkaloids, including salutaridine, oripavine, laudanosine, isothebaine, cryptopine, alpinigenine, narceine, protopine, and gnoscopine, were also quantitated. The values for morphine, codeine, and thebaine in P. somniferum were in agreement within 5--9% with values obtained by the United Nations Narcotics Laboratory by other methods. In contrast to previously reported procedures, the advantage of this method is that no precolumn or other purification other than solvent extraction of the capsular tissue is necessary. Isocratic chromatography alone on a single column resolved the 5 major alkaloids.