Thebaine Content Research Articles

Phylogeography and genetic structure of Papaver bracteatum populations in Iran based on genotyping-by-sequencing (GBS)

Papaver bracteatum, known for its high thebaine content and absence of morphine, has emerged as a promising alternative to opium poppy for codeine production. In this study, our objective was to create a diverse panel representing the natural variation of this species in Iran. To achieve this, we employed genotyping-by-sequencing to obtain genome-wide distributed single-nucleotide polymorphisms (SNPs) for phylogeographic analysis, population structure assessment, and evaluation of genetic diversity within P. bracteatum populations. A total of 244 P. bracteatum individuals from 13 distinct populations formed seven genetic groups, along with one highly admixed population. We observed a clear split between the populations inhabiting the Alborz Mts. in the east and Zagros Mts. in the west. In between these mountain ranges, the population of Kachal Mangan exhibited a high degree of genetic admixture between both genetic groups. At or after the end of the last glacial maximum, when climate conditions rapidly changed, all P. bracteatum populations experienced a strong demographic bottleneck reducing the already small effective population sizes further before they increased to their recent strengths. Our results suggest that the ongoing climate change together with human pressure on the species’ habitats and limited seed-dispersal ability are potential factors contributing today to rising genetic isolation of P. bracteatum populations. Our results provide genetic data that can be used for conservation measures to safeguard the species’ genetic diversity as a resource for future breeding approaches in this medicinally important species.

A cluster of acute thebaine poisonings from non-food grade poppy seeds in the Australian food supply

Introduction Poppy seed tea is used for its opioid effects and contains multiple opium alkaloids, including morphine, codeine, papaverine, and thebaine. Animal studies indicate thebaine has strychnine-like properties, but there is limited literature describing human thebaine poisoning. We describe a cluster of acute thebaine poisoning in people ingesting tea made using poppy seeds with high thebaine content that entered the Australian food supply chain. Methods This is an observational study of patients poisoned after drinking poppy seed tea. Cases were identified by three prospective toxicovigilance systems: the Emerging Drug Network of Australia collaboration, the New South Wales Prescription, Recreational and Illicit Substance Evaluation program, and the Emerging Drugs Network of Australia Victoria study. We report characteristics of clinical toxicity in cases with reported ingestion of poppy seed tea and analytical confirmation of thebaine exposure. Results Forty cases presenting with multi-system toxicity following poppy seed tea ingestion were identified across seven Australian states/territories from November 2022 to January 2023. Blood testing in 23 cases confirmed high thebaine concentrations. All 23 were male (median age 35, range 16–71 years). All patients experienced muscle spasms. Rigidity was described in nine, convulsions in six, while rhabdomyolysis, acute kidney injury, and metabolic acidosis occurred in five patients. There were two cardiac arrests. The thebaine median admission blood concentration was 1.6 mg/L, with a range of 0.1–5.6 mg/L, and was the dominant opium alkaloid in all samples. Convulsions, acute kidney injury, metabolic acidosis, and cardiac arrest were associated with increasing median thebaine concentrations. Four patients were managed in the Intensive Care Unit, with two receiving continuous kidney replacement therapy (one also received intermittent haemodialysis) for kidney injury. There was one death. Conclusions Thebaine toxicity, like strychnine poisoning, resulted in neuromuscular excitation characterized by muscle spasm, rigidity, and convulsions. Severe toxicity, including acute kidney injury, metabolic acidosis, and cardiac arrest, appears dose-dependent.

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.

Genetic diversity in Indian poppy (P. somniferum L.) germplasm using multivariate and SCoT marker analyses

The current study was undertaken to estimate the morphological and molecular diversity present among the 51 accessions of Indian opium poppy germplasm using Mahalanobis D2 and SCoT (Start Codon Targeted Polymorphism) marker analyses, respectively. A good range of morphological variations were observed among the accessions. The accessions were clumped into nine clusters and the morphological diversity recorded was 69%. Clusters VII and IX showed the maximum inter-cluster distance (117.97) whereas it was found to be minimum (21.53) in case of clusters II and III. The morphological trait, seed yield per capsule, contributed maximum (17.30%) towards the genetic divergence followed by thebaine content (14.56%) and papaverine content (14.06%). In contrast to the morphological diversity, genetic diversity at molecular level was found to be limited (33%), although successfully detected by utilizing DNA markers targeting coding regions of genome (SCoT markers). The outcome of the study has prospects in identification of lines with desirable traits to be utilized in future breeding programmes.

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.

Endophytic Consortium With Diverse Gene-Regulating Capabilities of Benzylisoquinoline Alkaloids Biosynthetic Pathway Can Enhance Endogenous Morphine Biosynthesis in Papaver somniferum

Secondary metabolite biosynthesis in medicinal plants is multi-step cascade known to be modulated by associated endophytes. While a single endophyte is not able to upregulate all biosynthetic steps, limiting maximum yield achievement. Therefore to compliment the deficient characteristics in an endophyte we tried consortium of endophytes to achieve maximum yield. Here, efforts were made to maximize the in planta morphine yield, using consortium of two endophytes; SM1B (Acinetobacter sp.) upregulating most of the genes of morphine biosynthesis except T6ODM and CODM, and SM3B (Marmoricola sp.) upregulating T6ODM and CODM in alkaloid-less Papaver somniferum cv. Sujata. Consortium-inoculation significantly increased morphine and thebaine content, and also increased the photosynthetic efficiency of poppy plants resulted in increased biomass, capsule weight, and seed yields compared to single-inoculation. The increment in morphine content was due to the modulation of metabolic-flow of key intermediates including reticuline and thebaine, via upregulating pertinent biosynthetic genes and enhanced expression of COR, key gene for morphine biosynthesis. This is the first report demonstrating the endophytic-consortium complimenting the functional deficiency of one endophyte by another for upregulating multiple genes of a metabolic pathway similar to transgenics (overexpressing multiple genes) for obtaining enhanced yield of pharmaceutically important metabolites.

Spatiotemporal oscillations of morphinan alkaloids in opium poppy

Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT, COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part of stem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulous bud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage, while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times. Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, while negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from upper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achieve high amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all the five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling times. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebaine biosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes of T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the producing (source) tissues to the roots (sink), known as a phenomenon of 'source-to-sink transportation'. The results, altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal oscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opium poppy.

Genetics of alkaloids in poppy straw with other morphological traits in opium poppy (Papaver somniferum L.)

Opium poppy belongs to the family − ‘Papaveraceae’ and is well known mainly for its pharmaceutically important alkaloids and seeds. In order to delineate the gene-effects conditioning the expression of seed yield and straw alkaloids, six crosses viz. IS 14×Sampada, I 14×Mtu, Sampada×Sujata, CIM Ajay×Dfr 44, Sapna×Sanchita and SPS 49×TS 1 involving the eleven parents, were sampled for P1, P2 and F1 (=P1×P2) non segregating, and F2 (=F1), BCP1 (=F1×P1), and BCP2 (=F1×P2) segregating generations. The days to flowering (50%) and plant height showed all the three type of interactions (i, j, l) in three crosses, namely C1 − IS 14×Sampada and C2 − I 14×Mtu but the traits peduncle length, codeine and thebaine content showed in other three crosses as C1 − IS 14×Sampada,C2 − I 14×Mtu,C3 − Sampada×Sujata and C4 − CIM Ajay×Dfr 44 in order. The two type of interactions additive×additive and additive×dominance interactions (i, j,) exhibited by the C6 − SPS 49×TS 1 for plant height and thebaine content, C5 − Sapna×Sanchita for peduncle length, codeine and narcotine content. The genetic basis of character expression indicated that the interacting crosses showing non-allelic interactions particularly duplicate epistasis in most of the crosses. While most of those without epistatic interactions exhibited superiority over parental mean. On the basis of study results the two specific cross combinations like Sampada×Sujata and CIMAP Ajay×Dfr 44 are selected to ensure tangible superiority for the most economic traits like high seed yield and poppy straw/capsule husk alkaloids including other contributing traits also like early flowering, capsule index for poppy crop improvement programme.

Developmental accumulation of thebaine and some gene transcripts in different organs of Papaver bracteatum

Noted as a rich source of thebaine and a potential alternative to Papaver somniferum for production of codeine and some semi-synthetic antagonist drugs (namely naloxone and naltrexone), Papaver bracteatum is a perennial species belongs to the section oxytona. Benzylisoquinoline alkaloids (BIAs) as a diverse class of alkaloids comprises about 2500 various structures contain medically important pharmaceuticals. Developmental and inducible factors control the alkaloid biosynthesis in opium poppy. To make better understanding on comparative developmental accumulation of alkaloid content and tyrosine/dopa decarboxilase (TYDC), berberine bridge enzyme (BBE), salutardinol acetyl transferase (SAT) and codeinone reductase (COR) gene transcripts in different organs of the species, current study was conducted. The accumulation of thebaine was higher in the capsule at pendulous bud (10.66mg/gr dry weight) following in the root at bud initiation (10.25mg/gr dry weight). The minimum amount of thebaine was observed in the petal of flowering stage (4.57mg/gr dry weight). Although in some cases the fluctuation of thebaine content could be coordinated to the up or down-regulation of gene transcripts, however, the presence of considerable exceptions complicate making of a comprehensive conclusion.

Comparative study for stability and adaptability through different models in developed high thebaine lines of opium poppy (Papaver somniferum L.)

Successful development of stable and adaptable crop varieties solely depend on the positive results obtained from the interaction between genotype and environment that consequently has significant impact on breeding strategies. GEI is a finest breeding approach that helps in the development of varieties for specific or wide adaptation, depends on the expression of stability. The present study was aimed to find out the stable thebaine rich line out of the seven advanced breeding high thebaine lines which have been developed for the first time through rigrous selection from advance generations of interspecific population derived from cross between Papaver somniferum L. and Pa paver setigerum DC. based on the estimate of genotype×environment interaction (GEI). The results of ANOVA showed wide variability in the genotypes under testing which suggested that the genotypes interacted differentially with respect to yield performance, so further stability analysis across environments should be followed before being released for commercial cultivation. The overall stability analysis following different stability models (including regression and graphical models) concluded that the lines NBIHT-1, NBIHT-3 and NBIHT-4 were highly stable and adaptable for seed, opium and thebaine content in different agro-climatic conditions Lines. NBIHT-5 and NBIHT-6 can also perform drastically well in specific climatic conditions to obtain maximum gain. The study concluded that the GGE Biplot model proposed by Yan and Hunt is best suited for stability analysis than other different stability models due to ease in visualization of stable genotypes from the graphical representation. Commercialization of such developed high thebaine lines having more than 10% thebaine in comparison to the existing genotypes having 1–2% thebaine could revolutionize pharmaceutical industries worldwide.

Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition

The in vitro shoot culture of a T-DNA insertional mutant of Papaver somniferum L. established by the infection of Agrobacterium rhizogenes MAFF03-01724 accumulated thebaine instead of morphine as a major opium alkaloid. To develop a non-narcotic opium poppy and to gain insight into its genetic background, we have transplanted this mutant to soil, and analyzed its alkaloid content along with the manner of inheritance of T-DNA insertion loci among its selfed progenies. In the transplanted T0 primary mutant, the opium (latex) was found to be rich in thebaine (16.3% of dried opium) by HPLC analysis. The analyses on T-DNA insertion loci by inverse PCR, adaptor-ligation PCR, and quantitative real-time PCR revealed that as many as 18 copies of T-DNAs were integrated into a poppy genome in a highly complicated manner. The number of copies of T-DNAs was decreased to seven in the selected T3 progenies, in which the average thebaine content was 2.4-fold that of the wild type plant. This may indicate that the high thebaine phenotype was increasingly stabilized as the number of T-DNA copies was decreased. In addition, by reverse transcription PCR analysis on selected morphine biosynthetic genes, the expression of codeine 6-O-demethylase was clearly shown to be diminished in the T0 in vitro shoot culture, which can be considered as one of the key factors of altered alkaloid composition.

Induction of variability through mutagenesis in opium poppy (Papaver somniferum L.)

Opium poppy (Papaver somniferum L.), an important plant of the family Papaveraceae, is valued for its extensive medicinal properties due to the presence of more than 80 different alkaloids, which are products of the polygenic interaction of different genes involved in plant metabolism. Broadening the genetic base through induced mutation is a supplementary tool that can lead to the development of genetic variability. The present study was undertaken to generate a broad genetic variability through mutation breeding using physical doses (gamma radiation of kR10 to kR50 at an interval of kR10), chemical doses (EMS of 0.2, 0.4, 0.6, and 0.8% (w/v)), and combined doses (gamma and EMS) of mutagen, and to evaluate the plants’ advance generations for different traits as well as for specific alkaloids, especially thebaine and codeine. The kR30 dose, which caused the highest results for all 3 genetic parameters (GCV, h2, and GA%) for 7 traits, was the most effective in NBRI-1. Similarly, the kR10 + 0.4% EMS dose proved to be the best for NBRI-5, affecting 10 different characters. The kR10 + 0.4% EMS dose created positive mutations for high thebaine and codeine content and low morphine content, while the kR40 + 0.6% EMS dose did the same for narcotine. The study also confirmed that the pathway of morphinan alkaloids and narcotine formation was bifurcated at the lower combined dose (kR30 and kR10 + 0.4% EMS), which was effective in causing micromutation in morphinan alkaloid pathways. The higher combined dose (kR40 + 0.4% EMS) affected narcotine production.

Papaver bracteatum Lindl.--a new plant source of opiates.

Abstract Certain strains of P. bracteatum Lindl. are reported to contain in the roots significant quantities of thebaine, which can be used for the production of codeine and other opiates. Results from plants raised from Iranian seeds and grown near London for 4 to 5 years are presented. The botanical and chemical characters correspond closely to those of the active strain Halle III described by Böhm (1970) and enable this plant to be clearly distinguished from the closely related P. orientale L. The thebaine content of the roots was 0.25 % which is much lower than the value of 0.7 to 1.3% reported in Halle III roots. The fruiting tops, however, were also investigated and found to contain significant amounts of thebaine in field conditions. If tops were harvested in the summer and roots in the autumn, about 15 kg thebaine per hectare should be produced from our strain grown in the conditions described. This compares favourably with about 1 kg alkaloids per hectare from Turkish opium production or even the 3 kg per hectare from the high yielding Indian farms.

Papaver bracteatum Lindley: thebaine content in relation to plant development.

Four thebaine-rich varieties of P. bracteatum have been grown in the open over two seasons and the thebaine distribution in aerial parts examined to determine the most suitable source material for commercial production. The leaves contained only 0-1 to 0-15%; the capsules 0-5 to 3-0% and the bled latex 28 to 53%. The maximum for the latter occurred about 3-4 weeks after petal opening and during the day, at about 15,00 h. A product 'bractium' prepared exactly as opium from P. somniferum contained up to 55% thebaine and calculations from the 1974 results gave theoretical yields up to 58 k of thebaine per hectare. However this is a very labour intensive method; furthermore bled latex only represents about 46% of the total thebaine of the capsule. In addition the pedicels contain significant amounts of thebaine, so that fruiting tops may be recommended as source material. In the capsule the thebaine content reaches a peak 3 to 4 weeks after petal opening and again two weeks later. At this fully ripe stage there is a theoretical yield of 50 kg per hectare. Two further advantages accrue from collection at this time: the ripe seeds can probably be used for similar purposes as poppy seed; and the pericarps at this stage contain no 'bound thebaine' (i;e., thebaine insoluble in MeOH; NH4OH but soluble in acetic acid--in unripe capsules bound thebaine represents 18 to 36% of the total thebaine). There is some evidence that, as this perennial plant increases in age, the capacity for thebaine production seems to continue increasing. Storage of raw material, even in ideal conditions, led to a loss of thebaine of 12 to 20% in one year.

Morphological, chemical, and molecular analyses of Turkish Papaver accessions (Sect. Oxytona)

The species of the section Oxytona Bernh., classified under the genus Papaver L., are of great importance due to their medical alkaloid contents such as morphine, codeine, and thebaine. A clearly established classification system is not available for Oxytona species. The aim of this study was to find relationships among 53 accessions belonging to 3 important Oxytona species, i.e. Papaver bracteatum Lindl., P. orientale L., and P. pseudo-orientale (Fedde) Medw., using 19 different morphological characters, 1 chemical character, and 15 random amplified polymorphic DNA (RAPD) markers. Nineteen characters, including bract and sepal leaves, petal marks, and capsule shape and size were analysed, and distinct morphological differences among accessions were identified. The thebaine content of the accessions was determined via HPLC analysis. Of the 53 accessions, 21 contained thebaine alkaloid ranging from trace amounts to 2.5% and the rest contained no thebaine. RAPD-PCR markers were used for analysis of accessions at molecular level. A total of 81 of bands generated by 15 successful RAPD-PCR reactions were detected as polymorphic. The polymorphism rate was approximately 85%. The combination of morphological, chemical, and molecular marker analyses resulted in a better classification for the section Oxytona.

Selection for high thebaine/low morphine content in Papaver somniferum L.

High contents of thebaine in the ripe capsules were observed in the L4 and I5 generations from a spontaneous mutant, similar to the observations in earlier generations. Outcrossings of plants with normal morphine contents still take place. Some correlation was found for ‘high thebaine content in mother capsule/high frequency of plants with thebaine as major alkaloid in progeny’ but no correlation was obtained for thebaine content and capsule size. Thebaine in these plants accumulates in a way similar to that of morphine in normal plants. Varying light intensities had no influence on the the-baine/morphine relationship in the plant. A retarded development in the pre-flowering period was observed in breeding lines with high frequencies of thebaine plants.

Selection for high thebaine/low morphine content (cpv. Morph: The) in Papaver somniferum L.

Selection for high contents of the industrially valuable alkaloid thebaine has been performed in Papaver somniferum. The breeding lines studied are probably the result of a spontaneous mutant, which has been cross pollinated by wind or insects, as segregations have been observed in the subsequent generations. Low frequencies of thebaine plants were found in the I1 and I2 generations. In the I3 generation one line resulted in the production of 92% plants with high thebaine content. Offsprings from this line produced only 17.5% thebaine plants when cultivated in green house conditions during winter. The thebaine content in the dry capsules averages 0.65% as compared to 0.37% morphine in normal alkaloid plants when cultivated under identical conditions.

Some varieties of Papaver somniferum L. with changed morphinane alkaloid content.

Plants with a genetically determined low morphine content have been studied. During two years one spontaneous mutant has produced a morphine content in opium of 2 per cent and 0.03 per cent in dry, ripe capsules. Other alkaloids are only found in trace amounts. At least 9 per cent cross pollination occurred in Papaver somniferum as a two year average. Another spontaneous mutant has been isolated with a low production of morphine and a high thebaine content. The fresh latex of this mutant shows a reddish colour.

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.

Narcotic Alkaloids of Four Papaver Species from Iran

Four native Papaver species of Iran, i. e. P. glaucum, P. tenuifolium, P. dubium and P. fugax, were collected from their natural habitat and subjected to HPLC analysis for determination of their morphine, codeine and thebaine content. P. dubium and R. glaucum contained all of the three mentioned narcotic alkaloids, while morphine was not found in P. fugax, and P. tenuifolium was free from codeine.