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

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.

Opium

R/ values

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

 

No.

Poppy

Rf relative to 100 and Colouration

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, Japan
 
 
 
 
 
 
 
 
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
Tokyo, Japan
12
orientale "Princess Victoria Louise"
L
19BG
 
27V
 
 
96V
Tokyo, Japan
 
 
 
16BG
 
23V
 
 
96V
 
 
 
 
17BG
 
24V
 
 
96V
 
 
 
 
17BG
 
25V
 
 
92V
 
13
orientale "Rembrandt"
L
19BG 20DB 28V
 
 
91V
Tokyo, Japan
14
orientale "Pink Beauty" .
L
18BG
 
27V
 
 
91V
Tokyo, Japan
15
orientale "Mrs. Perry"
L
19DB
 
29V
 
 
93V
Tokyo, Japan
16
orientale "Columbia (Olympia fl. pl.)"
L
19DB
 
 
 
 
94V
Tokyo, Japan
17
orientale "Perry's White"
L
18BG 26DB 28V
 
 
93V
Tokyo, Japan
 
 
 
18BG
 
26V
 
 
89V
 
18
somniferum
L
 
 
 
77DB
 
94V
Tokyo, Japan
19
somniferum .
L
 
 
 
78DB
 
96V
Tokyo, Japan
20
somniferum
L
 
 
 
72DB
 
94V
Tokyo, Japan
 
 
 
 
 
 
70DB
 
92V
 
21
somniferum
L
 
 
 
71DB
88V 93V
Tokyo, Japan
22
setigerum
L
 
 
 
74DB
89V 93V
Saitama, Japan
 
 
 
 
 
 
73DB
87V 92V
 
 
 
 
 
 
 
67DB
88V 94V
 
23
somniferum
L
 
 
 
72DB
 
94V
Tokyo, Japan
24
somniferum
L
 
 
 
76DB
 
95V
Tokyo, Japan
25
somniferum
L
 
 
 
75DB
91V 96V
Tokyo, Japan
26
somniferum
L
 
 
 
77DB
 
96V
Tokyo, Japan
27
somniferum .
L
 
 
 
75DB
91V 95V
Tokyo, Japan
28
somniferum .
L
 
 
 
70DB
 
92V
Tokyo, Japan
29
somniferum .
S
 
 
 
73DB
 
 
Tokyo, Japan
30
somniferum
S
 
 
 
70DB
84V
 
Japan, perhaps originally from North·China
 
 
C
 
 
 
73DB
88V
 
 
31
rhoeas
C
none
 
 
 
 
 
Tokyo, Japan
32
orientale
L
20BG 28DB
 
 
92V
 
Kassel, Germany
 
 
 
22BG 30DB
 
 
95V
 
 
 
 
 
20BG 29 DB
 
 
94V
 
 
33
argemone
L
none
 
 
 
 
 
Louvain Belgium
34
rhoeas
L
 
 
 
 
 
93V
Rome, Italy
35
pyrenaicum
L
 
 
 
 
 
90V
Hamburg, Germany
36
polare
L
17V 21DB
 
75DV
 
95V
Hamburg, Germany
37
atlanticum
L
16V 21DB
 
74DV
 
94V
Vienna, Austria
38
rubriaurantiacum
L
16V 20DB
 
72DV
 
92V
Helsinki, Finland

TABLE II (continued)

Detection of morphine in poppies by paper chromatography

Rf value and colouration of the alkaloidal spots

No.

Poppy

Rf relative to 100 and Colouration

Origin

 
Papaver
 
 
 
 
 
 
 
 
39
rupifragum var. atlanticum
L
 
18V 21DB 41DB 72DV 96V
Helsinki, Finland
 
 
 
 
17V 23DB
 
69DV 91V
 
40
rhoeas
L
 
 
 
 
 
92V
Helsinki, Finland
41
alpinum
L
 
none
 
 
 
 
Uppsala, Sweden
42
apulum
L
 
none
 
 
 
 
Uppsala, Sweden
43
glaucum
L
 
 
 
 
 
86V
Uppsala, Sweden
44
lecoqii
L
 
 
 
 
 
95V
Uppsala, Sweden
45
pilosum
L
 
17V 22DB
 
70DV 93V
Uppsala, Sweden
46
rhoeas "Shirley Wilks"
L
 
 
 
 
 
89V
Wageningen, Netherlands
47
lateritium
L
 
18V 23DB 40V
 
89V
Berlin-Dahlem, Germany
48
rubro-aurantiacum
L
 
18V 24DB 41V
 
91V
Basle, Switzerland
49
pavoninum
L
 
none
 
 
 
 
London, United Kingdom
50
umbrosum
L
 
 
 
 
 
94V
Klagenfurt, Austria
51
aurantiacum
L
 
16V 19DB 37DB
 
90V
Munich, Germany
52
mairei
L
 
 
21DB
 
 
88V
Munich, Germany
53
monanthum
L
 
 
 
 
 
93V
Munich, Germany
54
persicum
L
 
17V 20DB
 
 
94V
Munich, Germany
55
oreophilum
L
 
 
 
 
 
92V
Munich, Germany
56
argemone
L
 
 
 
 
 
93V
Cologne, Germany
57
bracteatum
L
 
 
22DB 30DB
 
94V
Cologne, Germany
58
nudicaule
L
 
none
 
 
 
 
Cologne, Germany
59
californicum
L
 
 
 
 
 
92V
Dublin, Eire
60
hybridum
L
 
none
 
 
 
 
Dublin, Eire
61
pyrenaicum
L
 
 
 
 
 
92V
Dublin, Eire
62
triniaefolium
L
 
 
 
 
 
91V
Dublin, Eire
63
lateritium
L
 
16V 19DB
 
 
90V
Dublin, Eire
64
nudicaule
L
 
12V
 
 
 
86V
Heidelberg, Germany
65
monanthum
L
 
12DV 14DB 3lB 67V 86V
Sofia, Bulgaria
66
dubium
L
3V
 
 
 
 
 
Toulouse, France
67
collinum
L
2V
 
 
 
 
 
Nantes, France
68
hybridum L
L
 
none
 
 
 
 
Uppsala, Sweden
69
rhoeas var. latifolium
L
 
11DB
 
 
 
91V
London, United Kingdom
70
schinzianum
L
 
14DB
 
 
 
91V
Braumchweig, Germany
71
rupifragum
L
 
13DB
 
 
 
88V
Warsaw, Poland
72
orientale
L
3V 12BG 18B
 
 
88V
Graz, Austria

TABLE III

Chromosome numbers of Papaver species (determinations of T. Kawatani and T. Ohno)

Species
Chromosome number 2n
Previous determinations a b 2n
species
Chromosome number 2n
Previous determinations a b 2n
Papaver
 
 
Papaver
 
 
alpinum
14
14 (Ljungdahl, 1922) (Sugiura, 1936,
hybridum
14
14 (Ljungdahl, 1922)
   
1940) (Faberge, 1944)
   
(Sugiura, 1937, 1940)
apulum
14
12 (Sugiura, 1936)
lateritium
14
14 (Ljungdahl, 1922) Yasui, 1941)
argemone
42
12 (Beale, 1939);
lecoqii
14  
   
42 (Sugiura, 1936, 1940)
mairei
14  
atlanticum
14
14 (Ljungdahl, 1922)
monanthum
14  
   
12, 14 (Sugiura, 1940)
nudicaule
14
14 (Ljungdahl, (1922)(Fabere, 1944)
aurantiacum
14      
28 (Horn, 1938)
bracteatum
42
14 (Yasui, 1936)
oreophilum
14
14 (Sugiura, 1936)
californicum
14  
orientale
42
42 (Yasui, 1921) (Ljungdahl, 1922)
collinum
28      
28 (Snoad, 1952) 42 (Yamazzaki, 1936)
glaucum
14
14 (Sugiura, 1931, 1944)
pavoninum
12
12 (Sugiura, 1931, 1936)
   
(Kuzmina, 1935)
persicum
14
14 (Ljungdahl, 1922)

TABLE III (continued)

Chromosome numbers of Papaver species (determinations of T. Kawatani and T. Ohno)

Species
Chromosome number 2n
Previous determinations a b 2n
Species
Chromosome number 2n
Previous determinations a b 2n
Papaver
 
 
Papaver
 
 
pilosum
14
28 (Ljungdahl, 1922)
rupifragum var.
   
polare
14  
atlanticum
14  
pyrenaicum
14  
schinzianum
14  
rhoeas
14
14 (Tahara, 1915) (Vilcins & Abele,
setigerum
44
22 (Sugiura, 1940); 44 (Kuzmina, 1935)
   
1927) (Yamagaki, 1936) (Sugiura,
   
44 (Ljungdahl, 1922)
   
1940) (Lawrence, 1930) (Felfoldy:
   
44 (Heimburger, Moore, 1953) c
    1947)
somniferum
22
22 (Furusato, 1940) (Tahara, 1915)
rhoeas var.
       
22 (Yasui, 1921) (Ljungdahl, 1922)
latifolium
14      
22 (Kuzmina, 1935) 20 (Ghimpu, 1933)
rubriaurantiacum
14  
triniaefolium
14
28 (Sugiura, 1940)
rubro-aurantiacum
14  
umbrosum
14
14 (Sugiura, 1931, 1936)

a C. D. Darlington and A. P. Wylie. Chromosome Atlas of Flowering Plants, 2nd. ed. 33-34, London: George Allen & Unwin Ltd. 1955.

Georg Tischeer, "Pflanzliche Chromosomen-Zahlen" in Tabulae Biologicae

4, 24 (1927); 12, 65 (1936); 16, 172-3 (1958); also Die Chromosomen-Zahlen der Gefassp flanzen Mitteleuropas ('s-Gravenhage: Dr. W. Junk, 1950).

c See footnote I, p. 20.

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

For 98 varieties or strains of Papaver somniferum, using in most cases seeds from foreign botanical gardens to obtain the plants, the morphine content of the opium was determined and compared with that of opium poppy plants of ordinary Japanese origin.

When sufficient opium could be collected from plants of one strain, the method of assay of the Japanese Pharmacopoeia VI was used. When the amount of opium was smaller, this method was modified to use only 1 gramme of opium. When less than 1 gramme was the most that could be collected, our method of paper chromatography and spectrophotometry was used).[9]

The results are given in Table IV.

The poppies were sown in October 1954 and 1955 and grown under uniform cultural conditions at the Kasukabe Experiment Station (numbers 1 to 26, 58 to 98) and the Wakayama Experiment Station (numbers 27 to 57). The method of incision was the usual practice described in section 1.

It is remarkable that the ten highest results for morphine percentage are on opium of poppies of which the seeds came from certain quite northern botanical gardens, and they are above 20%; but the quantity of opium was low.

Variety No.
Morphine percentage
Seed origin
25
30.0
Copenhagen, Denmark
64
27.5
Tabor, Czechoslovakia
11
24.9
Cologne, Germany
22
24.0
Montreal, Canada
19
23.6
Cologne, Germany
20
23.0
Munich, Germany
67
22.1
Nantes, France
90
21.2
Li?, Belgium
91
21.0
Seattle, United States of America
63
20.7
Hamburg, Germany

In these cases the determinations were all made by our paper chromatographic method. This method possibly yields somewhat higher results than the precipitation method of the Japanese Pharmacopoeia, on the same opium.

However, it is to be noted that the highest results by the modified J.P. VI method were the following:

Variety No.
Morphine percentage
Seed origin
36
20.04
Copenhagen, Denmark
47
19.82
Cologne, Germany

It is very interesting to observe that two samples of variety monstrosum, No. 25 and 67, gave very high results; 30% and 22.1%.

Contrary to the high morphine percentage in some opiums of foreign seeds, the ordinary Japanese opium "Ikkanshu" gave medium results.

However, opium weight per capsule was also calculated. The poppies of Japanese origin, "Ikkanshu", produced a large quantity of opium. As can be seen in Table IV, the poppies which gave more opium than 150 mg per capsule were all of the "Ikkanshu" strain. Therefore the Japanese strain "Ikkanshu" is superior in productivity of opium to all other poppies that we have grown in Japan from foreign seeds; but in these trials so far we have used seeds from botanical gardens and not seeds from poppies used for commercial opium production in other countries.

The poppies producing opium of exceptionally high morphine percentage, such as Danish and Czech poppies, produced a very small quantity of opium per capsule; but such poppies have great interest for trials as parent stock for commercial production, and our paper chromatographic method is a very convenient means for selecting plants of high productivity.

TABLE IV

Examination of poppies for opium of highest morphine yield

Method used:
Period of incision:
A. Japanese Pharmacopoeia VI method.
 
B. Slightly modified J.P.VI method using 1 g of opium.
Nos. 1-26, 27-57, 92-98 in 1955.
C. Our paper chromatographic method.
Nos. 58-91 in 1956.

Poppies grown at the Kasukabe Experiment Station

No.
Morphine percentage
Determination method used
Opium weight per capsule
Capsules incised
Period of. incision
Plant height
Seed origin
 
 
 
mg
 
 
cm
 
1c
9.26
B
170.2 23
23 - 30. V
125
Wakayama, Japan
2c
15.48
B
232.5 12
23 - 30. V
120
Wakayama, Japan
3c
11.97
B
115.5 27
23 - 30. V
132
Wakayama, Japan
4c
7.90
B
166.1 24
23 - 30. V
122
Wakayama, Japan
5c
12.45
B
159.1 23
23 - 30. V
125
Wakayama, Japan
6
16.71
B
60.5 30
23 - 30. V
120
Aichi, Japan
7
12.98
B
116.0 27
23 - 30. V
125
Aichi, Japan
8
13.43
B
72.2 25
27.V - 1. VI
133
Utrecht (Baarn), Netherlands
9
12.31
B
44.6 53
1 - 6. VI
125
Louvain, Belgium
10
15.6
C
42.3 17
1 - 6. VI
135
Turin, Italy
11
24.9
C
2.9 24
7 - 12. VI
150
Cologne, Germany
12
19.4
C
6.9 25
1 - 6. VI
142
Dublin, Eire
13a
18.4
C
5.3 29
1 - 6. VI
147
Rome, Italy
14
15.2
C
4.3 30
1 - 6. VI
151
Li?, Belgium
15
12.7
C
4.6 34
1 - 6. VI
125
Basle, Switzerland
16
16.1
C
2.3 26
1 - 6. VI
143
Berlin-Dahlem, Germany
17
10.76
A
89.9 120
23 - 30. V
128
Japan, perhaps originally from North China
18
17.8
C
2.9 30
1 - 6. VI
133
Bremen, Germany
19
23.6
C
1.4 33
1 - 6. VI
147
Cologne, Germany
20
23.0
C
1.5 27
1 - 6. VI
140
Munich, Germany
21
14.6
C
4.4 37
1 - 6. VI
134
Delft, Netherlands
22
24.0
C
1.3 22
1 - 6. VI
133
Montreal, Canada
23
15.1
C
0.3 33
1 - 6. VI
100
Edinburgh, United Kingdom
24
15.4
C
0.2 39
1 - 6. VI
135
Copenhagen, Denmark
25b
30.0
C
0.9 44
7 - 12. VI
144
Copenhagen, Denmark
26a
10.4
C
0.2 28
1 - 6. VI
120
Naples, Italy
58
11.0
C
47.4 17
15 - 19. V
65
Formosa, China
59c
11.14
B
157.4 25
28. V- 3. VI
114
Wakayama, Japan
60
15.41
B
83.2 15
28.V - 3. VI
126
Utrecht (Baarn), Netherlands
61
10.3
C
38.1 17
6 - 10. VI
102
Parma, Italy
62a
18.8
C
10.9 23
6 - 10. VI
91
Adelaide, Australia
63
20.7
C
51.5 12
11 - 15. VI
126
Hamburg, Germany
64
27.5
C
22.9 8
11 - 15. VI
141
Tabor, Czechoslovakia
65
16.4
C
34.9 4
11 - 15. VI
137
Delft, Netherlands
66
15.56
B
60.4 19
6 - 12. VI
135
Strasbourg, France
67b
22.1
C
32.0 5
11 - 15. VI
146
Nantes, France
68a
11.7
C
40.5 6
11 - 15. VI
137
Bratislava, Czechoslovakia
69
14.5
C
51.4 18
17 - 22. V
51
Formosa, China
70
17.34
B
102.0 23
3- 9. VI
128
Louvain, Belgium
71c
13.30
B
184.9 12
28.V- 3. VI
121
Wakayama, Japan
72
19.2
C
38.0 18
11 - 15. VI
151
Seattle, United States of America
73
19.8
C
25.5 9
11 - 15. VI
151
Braunschweig, Germany
74
17.1
C
25.6 9
11 - 15. VI
154
Lodz, Poland
75
14.6
C
53.8 15
3 - 9. VI
130
Florence, Italy
76
16.0
C
26.0 12
6 - 12. VI
121
Florence, Italy
77
17.82
B
84.2 15
3 - 9. VI
135
Louvain, Belgium
78c
14.13
B
205.5 12
28.V- 3. VI
116
Wakayama, Japan
79
18.6
C
16.8 9
6- 10. VI
145
Lodz, Poland
80
17.5
C
31.2 12
6 - 10. VI
136
Bonn, Germany
81a
13.9
C
21.4 12
14- 16. VI
127
Nantes, France
82
17.7
C
56.9 16
11 - 15. VI
108
Besancon, France
83
18.2
C
47.2 10
11 - 15. VI
94
Dublin, Eire
84
14.70
B
98.3 12
3 - 9. VI
131
Turin, Italy

TABLE IV (continued)

Examination of poppies for opium of highest morphine yield

a Poppies grown at the Kasukabe Experiment Station (continued)

No.
Morphine percentage
Determination method used
Opium weight per capsule
Capsules incised
Period of. incision
Plant height
Seed origin
 
 
 
mg
 
 
cm
 
85c
15.97
B
184.2 23
28.V - 3.VI
116
Wakayama, Japan
86
14.9
C
63.4 12
6 - 10.VI
120
Besancon, France
87
17.1
C
45.2 6
11 - 15.VI
138
Bremen, Germany
88
15.2
C
101.6 4
3 - 9.VI
125
Taranto, Italy
89a
14.3
C
16.1 52
3 - 7.VI
97
Lisbon, Portugal
90
21.2
C
36.5 4
11 - 13.VI
134
Li?, Belgium
91
21.0
C
12.9 8
11-13.VI
143
Seattle, United States of America
92
9.0
C
36.6 29
7 - 9.VI
162
Rome, Italy
93a
12.9
C
11.4 65
7 - 12.VI
110
Adelaide, Australia
94
17.6
C
39.2 32
7 - 12.VI
132
Braunschweig, Germany
95
15.8
C
8.0 57
7 - 12.VI
125
London, United Kingdom
96
16.0
C
1.9 48
7 - 12. VI
145
Amsterdam, Netherlands
97
14.3
C
18.4 33
7 - 12.V1
140
Amsterdam, Netherlands
98
14.8
C
1 7 64
1 - 6.VI
160
Turin, Italy

a Var. plenum

bVar. monstrosum

c"Ikkanshu"

(2) Poppies grown at the Wakayama Experiment Station

No.
Morphine percentage
Determination method used
Seed origin
No.
Morphine percentage
Determination method used
Seed origin
27
10.25
A
Wakayama, Japan
42 16.0
C
Poznan, Poland
28
9.85
A
Wakayama, Japan
43 14.7
C
Louvain, Belgium
29
10.42
A
Wakayama, Japan
44 17.6
C
Braunschweig, Germany
30
11.57
A
Wakayama, Japan
45 11.4
C
Li?, Belgium
31
11.10
A
Wakayama, Japan
46 16.61
B
Delft, Netherlands
32
9.65
A
Wakayama, Japan
47 19.82
B
Cologne, Germany
33
10.47
A
Wakayama, Japan
48 17.51
B
Berlin-Dahlem, Germany
34
11.58
A
Wakayama, Japan
49 17.83
B
Basle, Switzerland
35
11.24
A
Wakayama, Japan
50 18.3
C
London, United Kingdom
36
20.04
B
Copenhagen, Denmark
51 16.6
C
Bremen, Germany
37
17.6
C
Copenhagen, Denmark
52 17.73
B
Amsterdam, Netherlands
38
9.60
A
Wakayama, Japan
53 13.6
C
Cologne, Germany
39
17.1
C
Frankfurt, Germany
54 12.66
B
Dublin, Eire
40
16.98
B
Seattle, United States of
55 16.91
B
Amsterdam, Netherlands
     
America
56 11.65
A
Wakayama, Japan
41
16.08
B
Seattle, United States of
57 11.43
B
Japan, perhaps originally
     
America
 
 
 
from North China

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

The content of "porphyroxine-meconidine" of opium from successive lancings of the same poppy capsules was determined.

The method used was based on the one described by L. Fuchs and W. Ullrich.10 Some modifications were introduced.

Experimental

About 0.15 g of opium was accurately weighed. In a small mortar, 0.1 g of calcium hydroxide and 2.5 cc of water, precisely measured, were added and the mixture rubbed to a smooth paste. After addition of 12.5 cc of water, precisely measured, the mixture was stirred for 20 minutes.

It was then filtered. 10 cc of the filtrate, precisely measured and brought into the extraction apparatus (fig. 5), was extracted continuously with peroxide-free ether for about 4 hours until a negative test for "porphyroxine-meconidine" was obtained with the return ether.

The ether was then evaporated on the steam-bath. To the residue, 0.5 cc of dilute HC1 (1 part by volume of conc. HC1 to 99 of water) was added, and the solution was heated in a boiling water-bath. After 5 minutes the colour was fully developed. The red solution was rinsed in to make exactly 40 cc or 20 cc in a volumetric flask.

The absorbance of the red-coloured solution was measured by a Beckman 13 spectrophotometer at 511 mµ, using a 1 cm cell.

Full size image: 13 kB

The value for 10 cc of solution obtained from 0.1 g opium was calculated.

Opium

  1. ( b) From Papaver somniferum L.-" Ikkanshu" cultivated during 1954/55 at the Wakyama Experiment Station attached to the National Hygienic Laboratory.

  2. By "Kiritori" method.-" Kiritori" is a method of collecting the latex from poppy capsules. Its meaning is cutting (Kiri) and taking from (tori), that is, to incise the unripe capsule and immediately take the latex away from the poppy capsule.

  3. By" Oigaki" method.-" Oigaki" is another method. It means an additional (Oi) collection (gaki), that is, to collect the latex (" Asabuki ") which blows out (buki) early the following morning (Asa) from the same incision made in the "Kiritori" method.

  4. ( d) From P. somniferum cultivated during 1954/55 by a drug company (Nippon Shinyaku) in Kyoto.

  5. White flowered variety, originally from Pakistan.

  6. Violet-flowered variety, originally from Pakistan.

  7. Opium samples distributed by the United Nations. UN No. 180 (first lancing), and UN No. 181 (second lancing); India, Rajasthan, variety Dhaturia.

 

Date of lancing

 
c
d
I
29 May 2 June
II
31 May 4 June
III
2 June
 
IV
4 June
 

Results

Calculated values for the absorbance of the coloured solution of 10 cc, obtained from 0.1 g of opium, measured with Beckman B spectrophotometer at 511 mµ.

Lancing
(a)
(b)
(c)
(d)
(e)
I
1. 524
2.175 2.129 1.157 0.755
II
1.915 3.167 2.267 0.380 1.115
III
2.487 2.407 2.145
 
 
IV
2.228 2.376 2.973
 
 

Volume in which measurement carried out (cc.)

40 40 40 20 20

Later experiments were made on opium front poppies grown from foreign seed obtained from botanical gardens with the following results:

Opium sample
Absorbance lancing
Country where seeds obtained
 
I
II.
 
f
0.728 1.550
Rome, Italy
g
0.123
0. 589
Amsterdam, Netherlands
h
0.594 1.448
Braunschweig, Germany
i
0.702 1.152
Turin, Italy

Calculated by the equivalent worked out by Farmilo and Kennett,11 the "Lovibond values" of the above readings should be the following (multiplication of the "1%" absorbance in a 1-cm cell by 30.2):

Calculated Lovibond values

 

Lancings

 

Opium sample

       

Seed origin

 
I
II
III
IV
 
a
46.0 57.8 75.1 67.3
Japan
b
65.7 95.2 72.7 71.7
Japan
c
64.3 68.5 64.8 89.8
Pakistan
d
34.9 11.5
 
 
Pakistan
e
22.8 33.7
 
 
India
f
22.0 46.8
 
 
Italy
g
3.7 17.8
 
 
Netherlands
h
17.9 43.7
 
 
Germany
i
21.2 34.8
 
 
Italy

In Table V are given the results of comparative colorimetric determinations of "porphyroxine-meconidine" in Japanese opiums, in comparison with opiums of five other countries (China, India, Iran, Korea, Turkey). Twelve Japanese samples of recent production are included, with one sample of the former legal production, and two samples (Nos. 402 and 435) from poppies grown experimentally in Japan. Only in the case of one of the latter samples is the value as low as for the opiums of China, Iran, Korea, or Turkey (in the samples examined).

In our earliest determinations (in 1954) comparisons were made with a photoelectric colorimeter, using filter S.53. These have been converted to calculated" Lovibond values ", for ready, comparison with values given by the UN Secretariat, by the factor obtained by Fuchs & Ullrich,12 equivalent to multiplication of the "1%" absorbance by 39.5. In the second part of the table are determinations made with the Beckman B spectrophotometer. For these the "Lovibond values" have been calculated using the equivalent found by Farmilo and Kennett, already mentioned.

Full size image: 99 kB

Full size image: 19 kBFull size image: 37 kB

MAP II. Villages of Opium Poppy Cultivation

Black spot shows the village in which the opium poppy was actually cultivated under licence during 1954/55.

Nagano
 
Wakayama
 
1.
Ooka, Sarashina
1.
Tasugawa, Arita
2.
Murakami, Sarashina
2.
Yuasa, Arita
3.
Sakagi, Hanishina
3.
Hiro, Arita
4.
Tanaka, Chiisagata
4.
Yura, Hidaka
 
 
5.
Ena, Hidaka
Aichl
 
Hiroshima
 
 
 
1.
Ono, Saeki
1.
Akabane, Atsumi
2.
Itsukaichi, Saeki
1.
Tawara, Atsumi
3.
Kurose, Kamo
 
 
4.
Suigano, Mitsugi
Osaka
 
5.
Fuchu
1.
Toyokawa, Mishima
Okayama
 
2.
Fukui, Mishima
1.
Sakuto, Aida
3.
Takatsuki
 
 
4.
Ibaragi
Hyogo
 
5.
Suita
1.
Anji, Shiso

Conclusions

  1. The content of porphyroxine in Japanese opium is nearly always high, and sometimes extremely high. This is very characteristic.

  2. The changes in the porphyroxime content with repeated lancings do not seem to be entirely regular, but in eight cases out of nine the content in opium of the second lancing was greater than in opium of the first lancing.

TABLE V

Absorbance determinations of "Porphyroxine-meconidine" in opium samples

Lab. No.
Absorbance (1%)
Calculated Lovibond value
Origin

A. Determinations in photoelectric colorimeter using filter S. 53

1
0.209 8.3
Turkey, Aydin, UN 1 D
15
0.331 13.1
Turkey, export type, UN 15
18
0.192 7.6
Iran, Fars, UN 47
19
0.154 6.1
Iran, Khorassan, UN 51
24
0.835 33.0
India, Malwa, UN 35
35
0.220 8.7
China (Manchuria)
36
0.240 9.5
China (Inner Mongolia)
Lab. No.
Absorbance (1%)
Calculated Lovibond value
Origin
37
0.324 12.8
Korea (produced before World War II)
38
0.128 5.1
China (Northern)
39
1.917 75.7
Japan (former legal production)

B. Determinations with Beckman B spectrophotometer

37
0.365 11.0
Korea, produced in Korea before World War II
39
2.500 75.5
Japan, former legal production
117
1.220 36.9
India, United Nations, Malwa (Excise)
401
1.464 44.3
Japan, Aichi
402
0.338 10.2
Japan, Kyoto, Takeda Herbal Garden
403
2.008 60.7
Japan, Osaka
404
1.376 41.5
Japan, Osaka
405
2.276 68.8
Japan, Osaka
408
2.778 84.0
Japan, Wakayama
409
2.704 81.7
Japan, Wakayama
435
1.000 30.2
Japan, Wakayama, Experiment Station of National Hygienic Laboratory
436
3.344 101.0
Japan, Wakayama
437
0.328 9.9
Iran, from Iranian Commissioner
438
0.411 12.4
Turkey, import
501
3.377 102.0
Japan, Wakayama
502
2.674 80.8
Japan, Osaka
503
0.971 29.3
Japan, Aichi
504
1.583 47.8
Japan, Nagano
505
0.820 24.8
Japan, Hiroshima

The morphine content of Japanese opium

TABLE VI

Morphine percentages in Japanese opium, crop season 1953/54

Method: All except 402, 435 by our K/15 method 13 402 and 435 by a modification of the Eder & Wäckerlin method 14

Lab. No.
Morphine %
Origin (in Japan)
401
13.99
Kambe, Atsumi, Aichi
402
10.75
Ichijoji, Sakyo, Kyoto
403
10.75
Fukui, Mishima, Osaka
404
14.24
Fukui, Mishima, Osaka
405
10.29
Ibaragi, Osaka
406
11.97
Ibaragi, Osaka
407
11.53
Takatsuki, Osaka
408
15.61
Minamihiro, Arita, Wakayama
409
15.37
Minamihiro, Arita, Wakayama
Lab. No.
Morphine %
Origin (in Japan)
410
15.64
Minamihiro, Arita, Wakayama
411
12.07
Tsugi, Arita, Wakayama
412
15.73
Minamihiro, Arita, Wakayama
413
11.80
Tsugi, Arita, Wakayama
414
13.87
Minamihiro, Arita, Wakayama
415
15.91
Minamihiro, Arita, Wakayama
416
17.60
Minamihiro, Arita, Wakayama
417
15.49
Tsugi, Arita, Wakayama
418
15.95
Hiro, Arita, Wakayama
419
12.55
Tsugi, Arita, Wakayama
420
11.64
Tsugi, Arita, Wakayama
421
15.23
Minamihiro, Arita, Wakayama
422
13.66
Minamihiro, Arita, Wakayama
423
14.08
Hiro, Arita, Wakayama
424
14.60
Hiro, Arita, Wakayama
425
13.30
Hiro, Arita, Wakayama
426
13.51
Hiro, Arita, Wakayama
427
14.25
Hiro, Arita, Wakayama
428
14.55
Hiro, Arita, Wakayama
429
15.18
Hiro, Arita, Wakayama
430
15.29
Minamihiro, Arita, Wakayama
431
15.08
Minamihiro, Arita, Wakayama
432
13.94
Minamihiro, Arita, Wakayama
433
10.62
Tsugi, Arita, Wakayama
434
11.96
Tsugi, Arita, Wakayama
435
14.12
Yada, Hidaka, Wakayama

In Table VI the laboratory analyses are given for Japanese opiums of the crop season of 1953/54, and in Table VII analyses are Summarized for the crop season of 1954/55.

As may be seen approximately (or calculated) from the latter table, the average content is only between 11% and 12 %, although opium of a much higher morphine percentage can be obtained by collecting the first lancings separately.

These determinations were made by the method of the Japanese Pharmacopoeia VI.

TABLE VII

Classification of Japanese opium samples according to morphine percentages, crop season 1954/55

Morphine percentage

Province and number of samples

 
Nagano
Aichi
Osaka
Wakayama
Hirochima
Okayama
Hyogo
Total
Below 4
 
1
 
 
 
 
 
1
4-5
 
 
 
1
 
 
 
1
5-6
 
2
 
2
 
 
 
4
6-7
 
 
 
1 3
 
1 5
7-8
 
 
2 3 6 2 5 18
8-9
3 13 12 16 9 3 4 60
9-10
6 27 50 36 6 5 8 138
10-11
10 59 76 161 7 8 10 331
11-12
11 64 59 243 10 8 3 398
12-13
17 48 34 184 6 1 1 291
13-14
5 23 7 127 3 1 2 168
14-15
4 19 4 41
 
1
 
69
15-16
 
9
 
11
 
 
 
20
16-17
1 6 1 3 1
 
 
12
17-18
 
4
 
 
1 1
 
6
Above 18
 
1
 
 
 
 
 
1
TOTAL....
57 276 245 829 52 30 34 1,523
1

C. G. Farmilo, H. L. J. Rhodes, H. R.. L. Hart & H. Taylor. "Detection of morphine in Papaver setigerum DC. ", Bulletin on Narcotics, Vol. V, No. 1, 26, 1953.

2

Haruyo Asahina & Masako Ono, "Detection of morphine in poppy plants by paper chromatography ", United Nations document ST/SOA/ SER.K/39, 30 June 1955.

3

Haruyo Asahina and Masako Ono, " Determination of morphine in opium tincture by paper chromatography ", United Nations document · ST/SOA/SER.K/40, 25 August 1955.

4

Haruyo Asahina and Masako Ono, "Quantitative determination of morphine in opium by paper chromatography and spectrophotometry ", Bulletin on Narcotics, Vol. VIII, No. 4, 1956.

5

An outline of this procedure is to be published by the United Nations Narcotics Division in the series ST/SOA/SER.K/...

6

M. A. Vesselovskaya. "The Poppy - Its classification and its importance as an oleiferous crop " - Supplement 56 to the Bulletin of Applied Botany, of Genetics and Plant Breeding. The Lenin Academy of Agricultural Sciences in the USSR, Leningrad; 1933 (in Russian and English).

7

An outline of this procedure is to be published by the United Nations Narcotics Division in the series ST/SOA/SER.K/...

8

See footnote 2, p. 21.

9

See footnote 4, p. 21.

10

L. Fuchs & W. Ullrich, "The Colorimetric Determination of' Porphyroxine-Meconidine', 2nd communication. Experiments on the Determination of ‘ Porphyroxine-Meconidine' in Small Quantities of Opium". United Nations document ST/SOA/SER.K/19, 1 September 1953.

11

Charles G. Farmilo & Patricia M. L. Kennett, "The Colorimetric Determination of ' Porphyroxine-Meconidine ', The Relation between Tintometer and Spectro-photometric Measurements obtained in the Determination of 'Porphyroxine-Meconidine' in Opiums ", United Nations document ST/SOA/SER.K/14, 26 January 1953.

12

L. Fuchs & W. Ullrich, "The Colorimetric Determination of 'Porphyroxine-Meconidine'" United Nations document ST/SOA/ SER.K/10, 25 September 1952.

13

Haruyo Asahina, "An assay procedure for opium based on that of the Japanese Pharmacopoeia, and morphine percentages obtained on samples from various countries ", United Nations document ST/SOA/SER. K/15, 11 March 1953. This procedure was adopted as official for the Japanese Pharmacopoeia as from 15 March 1955.

14

R. Eder and E. Wackerlin, "The determination of morphine in opium ", Quarterly Journal of Pharmacy and Pharmacology, 10, 680 - 730, 1937.