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Analasys of Morphine Yield in Various Poppies

Various papaver varieties were tested for the presence or absence of morphine by method of paper chromatography. The chromosome numbers of various species were counted. Further, for varieties or strains of P. somniferum, the morphine content was determined for the purpose of the plant breeding, chiefly by method of paper chromatography and spectrophotometry. The morphine content of commercial opiums produced by the Japanese cultivators in the crop seasons of 1953/54 and 1954/55 are summarized in this paper.

Studies of poppies and opium

Sections

1. Alkaloidal analysis of the opium from Papaver setigerum DC.
2. A natural hybrid of Papaver setigerum DC. and P. somniferum L.
3. Examination of poppies of various species for the presence of morphine
4. Examination of poppy varieties of Papaver somniferum L. for opium of the highest morphine yield
5.The porphyroxine content of Japanese opium and of opium from successive lancings
The morphine content of Japanese opium

Details

Author: Haruyo Asahina,, Toyohiko Kawatani,, Miss Masako Ono,, Sanaenosuke Fujita,
Pages: 20 to 33
Creation Date: 1957/01/01

Studies of poppies and opium

Haruyo Asahina, Chief of the Narcotic Section of the National Hygienic Laboratory;
Toyohiko Kawatani, Director of the Kasukabe Experiment Station of Medicinal Plants, Attached to the National Hygienie Laboratory;
Miss Masako Ono, Narcotic Section, National Hygienie Laboratory;
Sanaenosuke Fujita, Kasukabe Experiment Station of Medicinal Plants, Tokyo, Japan

Introduction

Since the promulgation of the Japanese Opium Act in 1954, which allows the cultivation of the opium poppy under licence, we have grown the opium poppy and other poppies for study and experiments, and the opiums or juices therefrom have been analysed.

Papaver setigerum DC. and its hybrid with P. somniferum L. have been grown. The main alkaloids in opiums from these different poppies were estimated and the percentages compared.

On the opium poppy varieties and other poppies, we tested for the presence or absence of morphine by our method of paper chromatography. The chromosome numbers of various species were counted. Further, for varieties or strains of P. somniferum, the morphine content was determined for the purpose of the plant breeding, chiefly by our method of paper chromatography and spectrophotometry.

The content of "porphyroxine-meconidine ", a red-turning alkaloid when heated in diluted mineral acid, of Japanese opium and of opium from successive lancings of P. somniferum was measured spectrophoto-metrically.

The morphine content of commercial opiums produced by the Japanese cultivators in the crop seasons of 1953/54 and 1954/55 was assayed by us by the method of the Japanese Pharmacopoeia, and this information is summarized in the paper.

Our paper is divided into the following sections:

  1. Alkaloidal analysis of the opium from Papaver setigerum DC.

  2. A natural hybrid of Papaver setigerum DC. and P. Somniferum L.

  3. Examination of poppies of various species for the presence of morphine.

  4. Examination of poppy varieties of Papaver somniferum L. for opium of the highest morphine yield.

  5. The porphyroxine content of Japanese opium and of opium from successive lancings.

  6. The morphine content of Japanese opium.

Acknowledgements

It is a pleasure to acknowledge our indebtedness to Dr. Charles G. Farmilo, Organic Chemistry and Narcotic Section, Food and Drug Directorate, Canada, for the seed of P. setigerurn DC. and to the chemists of the laboratory of the Division of Narcotic Drugs, United Nations, for their invaluable and continuous help and guidance.

We wish to express our thanks to S. Mizumachi and Y. Shiuchi, National Hygienic Laboratory, for their assistance in making many of the analyses during this investigation. Thanks are also due to T. Ohno, Kasukabe Experiment Station, for his aid in cytogenetical studies.

1. Alkaloidal analysis of the opium from Papaver setigerum DC.

The presence of morphine in Papaver setigerum DC., previously suspected, was confirmed by Farmilo et al.[1] in 1953. Morphine was detected in the pod, bud, and leafy parts of this poppy plant.

The Japanese Opium Act (Act No. 71) promulgated in 1954, prohibits the cultivation of the opium poppy, including P. setigerum, except under licence.

For actual production of opium only P. somniferum has been cultivated, and there is no history of the cultivation of P. setigerum even experimentally, in Japan. No estimation of the alkaloids in opium from this poppy plant has been reported prior to the writing of the present paper. The capsules of P. setigerum are small and dehiscent, and this species can scarcely have any commercial use. Besides our desire to clarify the legal point of its inclusion in the Opium Act, it was studied for scientific reasons and as a possible parent in hybridization.

This investigation was undertaken to grow and identify botanically P. setigerum, and to determine quantitatively the content of alkaloids in the opium from this poppy plant.

With this in mind, the following two experiments, experiment I and experiment II, were made in successive years. The seeds were sown in October 1954 and 1955. The flowering continued almost throughout May of the following year.

There were plants with violet flowers and also plants with mauve flowers in the strain. In experiment I both kinds were used without separating them, whereas in experiment II only plants with violet flowers were used.

Three or four incisiom, each covering a quarter of the capsule, were made at intervals of one day, till no more latex was yielded. The first incision began ten days after the petals had fallen. In experiment I it began at different dates in the sequence of flowering, irrespective of the strains, whereas in experiment II all sets of incisions made began on the same date. Each incision consisted of four longitudinal slashes.

The capsules were incised in the evening and the latex was gathered just after incision (" Kiritori" collection) and the following morning (" Oigaki" collection). The opiums thus obtained were mixed, as is the usual practice in collecting commercial opium from P.somniferum.

Details of the Collection of Opium from Papaver setigerum DC.

Experiment
Seeds sown
Strains used
Period o/ incision
Plant height (cm)
I
20.X.1954
Violet and mauve-flowered
16-31.V.1955
65
II
25.X.1955
Violet-flowered
18-24.V.1956
67
Capsules incised
Opium per capsule (air.dried) (Mg)
I
90
15.56
II
130
15.88

The presence of alkaloids, at the proper Rf values for morphine, codeine, and other opium alkaloids in the opium from P. setigerum was readily demonstrated by using paper chromatography [2]

Opium was rubbed thoroughly with dilute hydrochloric acid, and the solution containing the alkaloidal salts was centrifuged. The supernatant solution was used for the paper chromatography.

The paper chromatography was carried out as follows:

Solvent: n-butanol 50, 28 % NH4OH 9, distilled water 15 parts by volume. In the preparation of the solvent, the constituents were mixed, the mixture allowed to stand for some time, and the top layer of the mixture used for chromatography.

Paper: No. 51 manufactured by Toyo Filter Paper Co., Ltd., 2.5 x 40 cm.

Movement of solvent: Ascending.

Spot-developing reagent: Potassium iodoplatinate prepared according to Munier: 1 cc of 10% platinic chloride and 25 cc of 4% potassium iodide were mixed and the resulting solution made up to 50 cc with H 20.

The following spots were obtained. The Rf values and colouration of the spot are given.

Chromatography of Opium from Papaver setigerum DC.

R/ values
Opium
Experiment I
0.73
0.87'
0.92
Experiment II
0.72 0.88 0.93
(Opium tincture
0.71 0.87
0.93 for comparison; P. somniferum)
Colouration
Dark blue
Violet
Violet

The characteristic dark blue colouration of the morphine spot on a filter paper, when treated with potassium iodoplatinate solution, and occurring at the proper Rf value for morphine, near 0.71 with this solvent, was easily recognized.

The other alkaloidal spots were coloured violet. The Rf value of the codeine spot was 0.87 and the spot of Rf 0.93 corresponded to other by-alkaloids. Thebaine, papaverine, and narcotine were inseparable from each other by this solvent.

Opium tincture was used for comparison to fix the position of the morphine spot more precisely under the particular conditions.

The morphine content of the opium from P. setigerum was then determined by paper chromatography and spectrophotometry. [3] [4]

The contents of codeine, thebaine, papaverine, and narcotine were also assayed by our unified method.[5]

The results, as determined on the air-dried opium, are given below:

Alkaloidal Analyses of Opium from Papaver setigerum DC.

Opium
Morphine
Codeine
Thebaine
Papaverine
Narcotine
Experiment I
5.1 0.9 2.1 1.9 0.1
Experiment II
7.3 0.8 1.6 2.6 0.1

The values were quite similar for the two crop seasons. Perhaps this is due to a dose control of growing and harvesting methods and use of a particular strain of the species, but it is naturally not known whether this species may vary much in the alkaloids present in the opium or not. We hope later to examine one or more other strains of P. setigerum.

For comparison, the alkaloidal analyses of a few opium samples from the United Nations Opium Distribution Centre assayed by our unified method are given below. These relate, of course, to opium from P. somniferum.

Comparative Alkaloidal Analyses of Opium from Papaver somniferum L.

Origin of opium
Morphine
Codeine
Thebaine
Papaverine
Narcotine
Turkey export U.N.15
13.5 1.7 0.8 0.8 4.9
India excise U.N.36
11.6 4.2 1.8 0.4 6.3
Iran Fars U.N.47
12.8 4.0 3.5 1.5 7.1

The results show that opium from P. setigerum is comparatively low in morphine and codeine, but high in thebaine and papaverine, and very low in narcotine. While morphine is still the principal alkaloid, narcotine is not the second one, but either thebaine or papaverine, and in general the picture of alkaloidal composition is almost opposite to that of somniferum opium.

The chromosome number was confirmed to be n = 22 in the haploid; 2n = 44 in the diploid (fig. 1 and 2).

Full size image: 7 kB

FIG.1. The Chromosomes in the Pollen Mother Cell of Papaver setigerum DC., showing n = 22 chromosomes, x 1,500

Full size image: 13 kB

FIG. 2. The Chromosomes in the Root Tip Cell of Papaver setigerum DC., showing 2n = 44 chromosomes (x 2,500)

Summary:

  1. Morphine, codeine, and other opium by-alkaloids were detected in the opium from Papaver setigerum DC.

  2. The content of these alkaloids was determined quantitatively.

  3. The chromosome number was confirmed to be n = 22 in the haploid, 2n = 44 in the diploid.

2. A natural hybrid of Papaver setigerum DC. and P. somniferum L.

Vesselovskaya mentioned in 1933 that P. setigerum, although distinct, could be crossed readily with P. somniferum, indicating their close genetic relationship, and that the crosses were perfectly normal fertile hybrids with viable seed.[6] However, we do not know of either a chemical or a detailed botanical investigation of such a hybrid before our own.

It was observed in 1955 that the violet- and mauve-flowered strains of P. setigerum were almost pure phenotypically, that is, the violet- or mauve-flowered strain consisted almost exclusively of plants with violet or mauve flowers respectively.

An F 1 hybrid was found to grow in the violet-flowered strain in 1956. The chromosome number in the root-tip cell was 2n= 33, which was the stun of the gametic chromosome numbers of P. setigerum n = 22, and P. somniferum n = 11 (fig. 3). At the first meiotic metaphase in the pollen mother cell (PMC), 11 bivalent and 11 univalent chromosomes were observed (fig. 4). The chromosome behaviour in meiosis of PMCs of the F 1 hybrid was of the type of Pilosella.

Apparently the female parent is the violet-flowered strain of P. setigerum, whereas the male parent is considered to be P. somniferum, "Ikkanshu" variety, a strain commonly cultivated for actual production of opium in Japan. In 1955, no species with a chromosome number 2n = 22 or its multiple other than P. somniferum and P. setigerum was grown by us, and "Ikkanshu" was cultivated in the adjoining field to the female parent at our experiment station.

The hybrid was 125 cm in height, intermediate between the parents: P. setigerum 67 cm, P. somniferum 135 cm.

External characteristics as well as habits of the hybrid were observed in detail. It was matroclinous. The leaves, stems, flowers and capsules were like those of the female parent but were larger in size.

Seven capsules of the hybrid were incised in the usual way, as described in section 1, and 0.24 g of opium was obtained.

Full size image: 12 kB

FIG. 3. Equatorial Plate in a Root Tip Cell of the F1 Hybrid, showing 33 somatic chromosomes (ca. x 3,700)

Full size image: 9 kB

TABLE I

Morphologieal characteristics of hybrid and its parents ( P. setigerum and P. somniferum )

Female parent (P. setigerum a
Fl hybrid
Male parent (P. somni/erum b)
Flower, colour
violet
violet
white
Pollen grain, length and width µ
42.9 x 26.8
34.3 x 19.6
41.8 x 21.91
length: width
1.6:1
1.75: 1
1.9:1
Stoma, length and width (µ)
69.0 x 45.2
60.7 x 42.6
46.5 x 33.5
Capsule, character
dehiscent
dehiscent
indehiscent
length and diameter (mm)
25 x 13
35 x 19
82 x 44
Disc, diameter (ram)
12 18 27
Ray, range of number
7-10
9-11
10 - 14
average number
8.4 9.9 11.7

a Violet-Flowered strain.

b "Ikkanshu" strain.

The analytical values for alkaloides assayed by our unified method[7] on the opiums from the hybrid and its parents are listed as follows. The parents were grown again in the same year as the hybrid(1956).

Comparative Alkaloidal Analyses of opium from the F 1 Hybrid and its Parents

(Percentage)

Opium
Morphine
Codeine
Thebaine
Papaverine
Narcotine
P. setigerum (?)
7.3 0.8 1.6 2.6 0.1
F 1 Hybrid
13.2 3.6 1.5 2.6 0.2
P. somniferum (?)
"Ikkanshu"
16.0 3.7 1.0 0.9 1.1

By comparison with the result for opium from the female parent, P.setigerum, the opium of the hybrid showed a fairly considerable increase in morphine content (almost doubled) and a large increase in codeine content (four times as great). Thebaine and papaverine, already high, were not changed.

FIG. 4. First Meiotic Metaphase of a Pollen Mother Cell of the F 1 Hybrid, showing 11 bivalent (in black) and 11 univalent (in black) chromosomes(ca. x 2,860)

The narcotine content increased from 01% to 0.2% by our determination, but thus remained remarkably low.

As compared with the opium from the male parent. P. somniferum, the opium of the hybrid was almost equal in morphine and codenine, very low in narcotine, but quite high in thebaine and papaverine.

It is very interesting to observe that in the production of some alkaloids the hybrid became almost equal to the female parent, but in others it remained like like the female parent, whether the production was high, as with thebaine and papaverine, or low, as with narcotine.

Summary :

  1. A natural hybrid of P. setigerum DC (?) and P. somniferum L. (?) was found. Connecting the external characteristics, it was matroclinuos.

  2. The chromosome number was 2n=33. At the first meiotic metaphase in PMCs ,11 bivalent and 11 univalent chromosomes were observed.

  3. The chromosome behaviour in meiosis of PMCs was of the type of Plosella.

  4. Morphine was detected in the opium from the hybrid. The content was 13.2%.

  5. The opium of the hybrid was high in thebaine and papaverine, but very low in narcotine. In morphine and codeine production, the hybrid was superior to the female parent and was almost equal to the male parent; but in production of other alkaloids, thebaine, papaverine, and narcotine, it corresponded to the female parent.

3. Examination of poppies of various species for the presence of morphine

During 1955 and 1956 a considerable number of varieties and strains of Papaver somniferum, P. orientale, and P. rhoeas, together with 29 other species grown at the Kasukabe Experiment Station from seeds sent by foreign botanical gardens, were examined for the presence of morphine by our method of paper chromatography. The method has been given in a preceding section of this paper and was previously published in a United Nations document.[8]

The results are set out in Table II. In nearly all cases the latex was used for the test. In no poppy but P. somniferum and P. setigerum was any clear evidence of morphine found. In five other cases (Nos. 36 to 39, and No. 45) there was a spot at about the right Rf value, but the colouration was different from that of a morphine spot.

Examination was made by the ordinary paraffin method to determine or confirm the chromosome numbers of 31 of these species of Papaver. The results are set out in Table III -This table also shows in which cases previous determinations are known to us. It will be noted that only P.somniferum and P. setigerum were found to have chromosome numbers which are multiples of 11.

TABLE II

Detection of morphine in poppies by paper chromatography Rf value and colouration of the alkaloidal spots

L: Latex
B: Blue
C: Capsule
DB: Dark blue
S: Straw
BG :Blue green
V: Violet
DV: Dark violet
Italics: Morphine spot
Poppy
Rf relative to 100 and Colouration
No. Origin
Papaver
1
somniferum .
L
71DB
88V 94V
Japan, perhaps originally
69DB
86V
from North China
69DB
87V 92V
71DB
86V 91V
2
Somniferum
L
72DB
95V
Saitama, Japan
73DB
95V
70DB
92V
3
Rhoeas .
L
none
Tokyo, Japan
4
Rhoeas
L
91V
Tokyo, Japan1
93V
5
orientale "Mahoney"
L
15DB
96V
Tokyo, Japan
6
orientale bracteatum
L
18DB
36V
94V
Tokyo, Japan
7
orientale "Queen Alexandra"
L
18DB
95V
Tokyo, Japan
8
orientale "Feltham"
L
17BG
25V
94V
Tokyo, Japan
9
orientale "Beauty of Livermere"
L
18BG
25V
96V
Tokyo, Japan
10
orientale "Salmon Queen"
L
95V
Tokyo, Japan
11
orientale "Apricot Queen" .
L
18DB
26V
92V

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