Detection of Morphine in Papaver setigerum DC

Sections

INTRODUCTION
BOTANICAL CLASSIFICATION OF Papaver setigerum
Chemical method of analysis
Results and discussion
CONCLUSIONS
ACKNOWLEDGEMENTS

Details

Author: C. G. Farmilo, , H. L. J. Rhodes, , H. R. L. Hart and, H. Taylor
Pages: 26 to 31
Creation Date: 1953/01/01

Detection of Morphine in Papaver setigerum DC

C. G. Farmilo,1
H. L. J. Rhodes,2
H. R. L. Hart3and
H. Taylor4

1. Chemical analyses by C .G. Farmilo and H. R. L. Hart

2. Botanical classification of papaver setigerum by H. L. J. Rhodes

3. Photography by H. Taylor

INTRODUCTION

Morphine was first isolated from the dried latex obtained from capsules of Papaver somniferum soon after 1800. It was the first alkaloid discovered. Recent reviewers ([1] ),5 ([2] ), ([3] ), ([4] ) do not entirely agree as to the man who first discovered morphine. Although Derosne ([5] ) described the isolation of a "salt of opium" in 1803, apparently the chief credit for the discovery of morphine should be shared by Séguin ([6] ), whose report to the Académie des Sciences was made in 1804 but not published until 1814, and Sertürner ([7] ) who was the first to publish a clear account, in 1805, and who gave the name "morphine" in his later work in 1817. Sertürner characterized morphine as a "vegetable alkali".

There have been some reports of finding morphine in plants other than Papaver somniferum but they were later found to be incorrect ([8] ). Fulton ([9] ) concluded in 1944 that. "At most, two or three very close relatives of P. somniferum (in the section Mecones of genus Papaver) possibly also produce morphine, but as they have not been analysed even this extension is in doubt and it is really probable only for Papaver setigerum" and further, "of some twenty-eight species of Papaver that have been examined for chromosome numbers, only P somniferum and P. setigerumhave the number 11 or a multiple [of this number] ([10] ) Perhaps these are the only opium poppies. It seems fairly certain that Papaver setigerum is a true opium poppy; yet there do not seem to be any reports of actual chemical analyses"([11] ). Fulton's statements summarize the facts which led to the present investigation.

1 Chemist in Charge, Organic Chemistry and Narcotic Section, Food and Drug Laboratories, Department of National Health and Welfare, Ottawa, Canada

2 Assistant Botanist, Division of Botany and Plant Pathology, Department of Agriculture, Ottawa, Canada

3 Supervisory Inspector, Inspection Services (formerly Narcotic Analyst), Food and Drug Laboratories, Ottawa, Canada

4 Photographer, National Film Board, Ottawa, Canada

To definitely determine the presence of morphine in Papaver setigerum and to find out whether it is a "true" opium poppy, a cytogenetic, botanical, and chemical investigation has been carried out. Hart, in 1945 in co-operation with Senn and Zinck had made a preliminary survey for the presence of morphine in all poppy species that could be obtained. The results of his survey are reported here for the first time. The object of the present paper is to present botanical and cytogenetic evidence for the botanical identification of the plant P setigerum and a preliminary report of the presence of morphine in P setigerum which has been detected and identified for the first time. The chemical identification of morphine was carried out by means of characteristic colour and crystal reactions. The first portion of the paper presents botanical results with illustrations of the plants used and the second portion of the paper deals with chemical extraction and identification methods. Botanical descriptions of the poppies, other than P. setigerum, are not included.

BOTANICAL CLASSIFICATION OF Papaver setigerum

Papaver setigerum DC is a poppy which grows wild in the Mediterranean region, especially in southwestern Europe. It has been cultivated as a flowering annual, but apparently is not grown in North America at the present time.

The plant is closely related to opium poppy P. somniferum L. and some authors have treated it as a variety or sub-species of that species. Rouy and Foucand ([13] ) and Bonnier [(12)] listed it as a sub-species in floras dealing with southwestern Europe.

The cytological evidence of a close affinity between P setigerum and P somniferum is discussed by Fulton ([14] ). P. somniferum is diploid ( n=11) and P. setigerum is tetraploid ( n=22) with exactly twice the number of chromosomes. No other species of Papaver is known to have the number n=11 or a multiple of this number. This information indicates that P setigerum is not the wild ancestral species of the cultivated P somniferum, as was formerly believed since a diploid would not be derived from a tetraploid plant.

Figures in parentheses indicate items within the list of references to be found at the end of this article.

The seed for the strain tested was received by Dr. C. Heimburger from the Rouen Botanical Garden, France, in 1933. Dr. Heimburger sent seed of the strain under the name Papaver setigerum to the Division of Botany and Plant Pathology, Ottawa, in 1945. Seed from the plants grown in 1945 was sown in 1951 to produce the plants used in the chemical analysis.

Dr. Heimburger determined the chromosome number to be 2n=44. In 1951, Dr. R. Moore of the Division of Botany and Plant Pathology made a count of n=22 on flower bud material from the culture, thus confirming the earlier count. Figure 1 is a photograph of the meiotic division on which the 1951 count was based.

Originally there were both plants with violet flowers and plants with mauve flowers in the strain, but Dr. Heimburger had attempted to purify the strain by eliminating the plants with mauve flowers. He found that the violet form differed from the mauve form by one gene, with the heterozygotes intermediate.

Figure 1. Meiotic division in pollen mother cell of Papaver setigerum DC.

Full size image: 68 kB, Figure 1. Meiotic division in pollen mother cell of Papaver setigerum DC.

The plants show the characters described for P. setigerum by A. P. de Candolle ([15] ) in the original description. In general they are less coarse than plants of P somniferum as usually cultivated, the flowers being smaller and the leaves thinner in texture and not glaucous (see figures 2 and 3). The teeth of the leaves are long and jagged, and each tooth is tipped with a characteristic bristle not found in P somniferum (see figure 2). The buds and young peduncles are beset with setose hairs. These hairs are found more sparingly on the mature peduncles (figure 3) and along the lower midrib of the leaves. The plants closely resemble the illustration of P setigerum in Bonnier ([12] ) (figure 4) except that the hairs on the peduncles are more sparse than in Bonnier's figure.

The plants of the strain used for chemical analysis are thus identified as Papaver setigerum DC.

Chemical method of analysis

The following procedure was used for the extraction of morphine from Papaver setigerum, and only slightly modified ([14] ) for other poppies: 50-100 grammes of dried, broken-up plant material were placed in a Waring blender with 100 ml of 95 per cent ethanol. After 10 minutes of blending the solution reached a temperature of 50°C. The blending was continued for 20 minutes and the extraction repeated twice with 100 per cent ethanol. The combined extracts were filtered using Whatman No. 40 filter paper, and the filtrate was refiltered through sintered glass. The solution of alcohol was then concentrated in an evaporating dish. The residue was treated with ether and filtered to remove the chlorophyll and plant resins. The ether-insoluble residue was treated with water (5-15 ml.) slightly acidified with acetic acid and filtered into a separatory funnel. The solution was then made alkaline to litmus with ammonia and extracted with chloroform-isopropyl alcohol (2 + 1) solution (8, 5, 2 ml. portions) and filtered into another separatory funnel. The water layer was then extracted twice with chloroform-isopropyl alcohol (3 + 1) using 10 and 5 ml. consecutive portions. The aqueous solution was then discarded. The combined chloroform-isopropanol solution was extracted with three portions (7, 5, 3 ml.) of 2 per cent aqueous sodium hydroxide solution. The first two portions of alkali solution were combined and placed in a separatory funnel, marked A. The second funnel marked B contains a 3 ml. portion of alkaline extract. The chloroform-isopropanol solution was set aside for examination of other alkaloid present. The solution in separatory funnel A was shaken twice with chloroform. Each 3 ml. portion of solvent was washed by shaking with the alkaline wash solution in separatory funnel B. The chloroform was then discarded. The alkaline solutions were made acid using a litmus paper indicator.

Figure 2. Photograph of Papaver setigerum DC plant similar to those collected for chemical analysis and determination of morphine (1950) Figure 3. Photograph of dried poppy herbarium sample of Papaver setigerum DC collected by M. Zinck (1945)

Full size image: 56 kB, Figure 2

The acid solutions were extracted with chloroform to help purify the morphine extract. The solution in B was made ammoniacal, added to the separatory A, and the whole solution made ammoniacal. It was then extracted once with (2 + 1) and twice with (3 + 1) chloroform-isopropanol. The extracts were washed in funnel B with slightly ammoniacal water and then filtered into an evaporating dish. The aqueous layer was discarded and the solvent was evaporated to near dryness on a steam bath. When amounts greater than 10 mg. were obtained, recrystallization from methanol amyl-alcohol, or anisole was found successful. Ammonium chloride was added to crystallize small amounts (less than 10 mg.) of morphine from an alcohol-ether-water system.

The recrystallized residue was identified in the following manner. About 0.1 mg. of the residue was placed on a microscope slide and a drop of iodine reagent was added. The reagent was prepared from a stock solution of iodine in potassium iodide containing 10 gm. iodine and 50 gm. potassium iodide in 100 ml water, by mixing 1 ml of the I 2-KI solution with 3 ml. of concentrated hydrochloric and 3 ml. syrupy phosphoric acid.

The characteristic rosettes of fine needles later changing into, or growing from the solution at the same time were fine plate like structures and some individual needle crystals. These crystals were also obtained from a sample of known morphine. Another portion of the residue was tested with Marine's reagent. The fine needles characteristic of the morphine Marine complex, morphine-CdI 2 were observed. Colour tests using Fröhdes', Marquis', and Meckes' reagents, as well as ferric chloride gave the colours characteristic of morphine. It was concluded that morphine was present in the residue extracted from Papaver setigerum.

Results and discussion

Table I shows the results obtained by Hart in 1945 in the analysis of twenty-six species of poppies. The conclusion made on the basis of crystal and colour reactions on the residue obtained exactly according to Fulton's procedure ([14] ) are shown in the column entitled "morphine". It is seen that only two of the species tested contained morphine; these are Papaver somniferum and Papaver setigerum The presence of morphine in Papaver setigerum was confirmed by Farmilo in 1951.

Although the seeds of the opium poppy do not come under narcotic control laws generally, the question of whether certain poppy seeds are those of the opium poppy has, nevertheless, been raised on several occasions. Previously it seemed that the only way to tell was to plant the seeds and see whether morphine could by found in the resulting plant. Although the poppy seeds do not contain identifiable amounts of opium alkaloids, it now seems on the basis of cytogenetic findings that determination of the chromosome number will tell whether or not the seeds belong to an opium poppy. As far as is known, if the haploid number is 11 or a multiple of 11, the seeds belong to an opium poppy, while if the haploid number is 7 or a multiple of 7 the seeds belong to some other poppy. The chromosome numbers may be determined if some actively growing tissue (cells in the state of subdivision) such as root tip on the sprouting seed, or leaf bud on the seedling can be obtained. This is easily done within a week if the seeds sprout promptly, but sometimes it is difficult to get them to sprout in the laboratory. This process is a routine matter for the chromosome expert, but may not have occurred to the narcotic expert or chemist as a method of identification of the poppy plant, or seed. It will, therefore, be useful to narcotic agents.

Figure 4. Illustration of some poppy plants from Gaston Bonnier (1911) showing comparison of P. setigerum and P. somniferum and others

Full size image: 165 kB, Figure 4. Illustration of some poppy plants from Gaston Bonnier (1911) showing comparison of P. setigerum and P. somniferum and others

CONCLUSIONS

  1. Papaverum setigerum has been grown and identified botanically.

  2. A chromosome count revealed the haploid number to be n=22. for Papaver setigerum.

  3. Morphine has been detected in the pod, bud, and leafy parts of Papaver setigerum DC for the first time.

  4. Twenty-four other species of poppies were examined and no morphine was found in any of the

  5. A chromosome count may be used as a means of identifying an opium poppy

Table I

Results of survey of poppies for presence of morphine

      Chemical observations

Poppy botanical name

No. of samples

Stage of growth

Marquis

HNO 3

FeCl 3

Marmes

Bouchardats

Morphine

Argemone alba
1
Bud
Brown
Brown
Green
Amorphous
Amorphous
None
 
1
Pod
Brown
Brown
Green
Amorphous
Amorphous
None
Argemone barclayana
1
Bud
Brown
Brown
None
Amorphous
Amorphous
None
Argemone grandiflora
2
Bud
Brown
Red brown
None
Amorphous
Amorphous
None
 
2
Pod
Brown
Brown
Green
Amorphous
Amorphous
None
Argemone mexicana
2
Bud
Brown
Red brown
Green
Amorphous
Amorphous
None
 
2
Pod
Brown
Red brown
Green
Amorphous
Amorphous
None
Argemone ochroleuca
1
Pod
Brown
Brown
Green
Amorphous
Amorphous
None
Argemone platyceras rosea
1
Bud
None
(No residue obtained)
       
 
1
Pod
Purple
Brown
Green
Amorphous
Amorphous
None
Chelidonium majus
1
Bud
Brown
Brown
None
None
None
None
 
2
Pod
Brown
Brown
None
Amorphous
None
None
Dicranostigma franchetiana
1
Bud
Red brown
Red brown
None
Amorphous
Amorphous
None
Eschscholzia californica
5
Bud
Crimson
Brown
Olive green
Amorphous
Amorphous
None
 
5
Pod
Red
Brown
Green
Amorphous
Amorphous
None
Eschscholzia pulchella
2
Bud
Purple
Brown
None
Amorphous
Amorphous
None
 
2
Pod
Red
Brown
Greenish
Amorphous
Amorphous
None
Glaucium sp
1
Pod
Purple
Orange to brown
Green
Amorphous
Amorphous
None
Papaver argemone
1
Bud
Brown red
Red brown
Green
Amorphous
Amorphous
None
 
2
Pod
Purple
Brown
Green
Amorphous
Amorphous
None
Papaver commitatum
2
Bud
Brown
No colour
On evapora-tion, green
Amorphous
Amorphous
None
 
2
Pod
Brown
   
Amorphous
Amorphous
None
Papaver dubium
4
Bud
Brown
Red brown
None
Amorphous
Amorphous
None
 
3
Pod
Brown
Brown
Finally green
Amorphous
Amorphous
None
Papaver glaucum
2
Bud
Brown
Yellow
Orange
Amorphous
Amorphous
None
 
2
Pod
Brown
Brown
None
Amorphous
Amorphous
None
Papaver lateritium
1
Bud
Brown
Brown red
       
 
     
Brown orange
Blue green
Amorphous
Amorphous
None
 
1
Pod
Brown
Brown orange
None
Amorphous
Amorphous
None
Papaver Monothum
1
Pod
Red to brown
Red brown
On standing green,
Amorphous
Amorphous
None
  1
Bud
Brown
Yellow
On standing green,
Amorphous
Amorphous
None
Papaver nudicaule
5
Bud
Crimson
Brown
No colour to green streaks
Amorphous
Amorphous
None
Papaver orientale
4
Pod
Red brown
Red, yellow, orange,
Green
Amorphous
Amorphous
None
Papaver rhoeas
5
Bud
Red brown
Brown
None immediately finally green
No ppt.
Amorphous
None
  5
Pod
Red brown
Brown
None immediately, finally green
Amorphous
Amorphous
None
Papaver schinzianum
1
Bud
Red brown
Brown
Green
Amorphous
Amorphous
None
  1
Pod
Red brown
Brown
Finally green
Amorphous
Amorphous
None
Papaver setigerum
2
Bud
Purple
Red
Blue green
Needles
Feathered plates
Present
  2
Pod
Purple
Red. orange, yellow, crimson orange
Blue
Needles
Feathered plates
Present
Papaver somniferum
11
Bud
Red purple
Yellow
Blue green
Needles
Crystalline
Present
  9
Pod
Red purple
Yellow red, orange yellow
Blue green
Needles
Crystalline
Present
Papaver umbrosum
1
Bud
Brown
Brown
Green
Amorphous
Amorphous
None
  1
Pod
Brown
Brown
None
Amorphous
Amorphous
None
Roemaria refracta
1
Bud
Brown
Orange
Green
Amorphous
Amorphous
None
  1
Pod
Brown
Brown yellow
Green
Amorphous
Amorphous
None

ACKNOWLEDGEMENTS

The authors wish to acknowledge the co-operation of Dr. H. A. Senn, Dr. W. G. Dore and Dr. C Frankton of the Division of Botany and Plant Pathology of the Central Ex- perimental Farm for advice, loan of equipment for drying plants and technical help during the growing, identification and preparation of the plant materials The help of Dr R. J. Moore of the Cytogenetics Section in making the chromosome count is appreciated. We wish to acknowledge the continued help and advice of Dr. L. I. Pugsley, Dr C. A. Morrell, and Dr. J. H. Craigie for permission to publish these results.

The help of Mr. Charles Fulton from United Nations Secretariat, Narcotics Division, in giving freely expert advice and helpful discussion during the course of experiments is gratefully acknowledged, and his suggestion that a chromosome count would probably be useful for identification of an opium poppy is acknowledged.

REFERENCES

001

Holmes, H L, The Alkaloids, Chemistry and Physiology , Academic Press Inc., Publishers, New York, 1952, p 2

002

Henry, T. A., The Plant AIkaloids, Churchill, J. A, Ltd., 104 Gloucester Place, London, 1949, p. 213.

003

Small, L F. and Lutz, R. E, "Chemistry of the Opium Alkaloids", U.S Govt Printing Office, Washington, 1932, Supplement No 103 to the Public Health Reports, Part II, p. 138

004

Weill, P B, "The Structure of Morphine", United Nations Bulletin on Narcotics , vol I[, no 2, April 1950, p. 8.

005

Derosne, Ann Chun , 45, 257 (1803).

006

Séguin, M. A, Ann Chum , Académie des Sciences, France 1804, 92, 225 (1814)

007

Sertürner, F. W, Trommsdorffs Journal der Pharmazie, 13 (I) 234 (1805) 14, (I), 47 (1806); 20, (I) 99 (1811), Ann Chun et phys., (=) 5, 275 (1817); ( Gilbert's) Ann Physik , 55, 56 (1817); 57, 192 (1817); 59, 50 (1818).

008

Fulton, C. C, The Opium Poppy and other Poppies , U S. Treasur3 Department, Bureau of Narcotics, U S Government Printing Office, Washington, U.S A, 1944

009

Fulton, C. C., Ibid, page 55, second paragraph beginning, "At most... (ending) ... sentigerum".

010

Tichler, George, Pflanzlicher Chromosomenzahlen Tabulae Biologicae, 4, 24, (1927); 12, 65 (1936); 16, 172-173 (1938).

011

Fulton, C. C., Same as ref. 8, p. 38, 4th paragraph beginning "Of some... (ending) ... analyses".

012

Bonnier, G, Flore complete illustree en couleurs de France, Suisse et Belgoique , Neuchatel, Paris, Bruxelles, nouvelle edition, revue et corrigee, 1922, fasc. 4, p 47, t. 24 fig. 104 b.

013

Rouy, G, and Foucand, Flore de France , Asnieres, 1893, 1 '1952-153.

014

Fulton, Charles C., The Opium Poppy and other Poppies , U.S. Treasury Department, Bureau of Narcotics, U S Government Printing Office, Washington, U S.A., 1944, pp 37-38

015

Candolle, A. P, de, Flore Francaise , Paris, 1815, 5 (suppl): 585.