[ 25O ] THE SEPARATION AND IDENTIFICATION OF SOLANACEOUS ALKALOIDS FROM NORMAL AND GRAFTED PLANTS BY G. M. JAMES AND B. H. THEWLIS Oxford Medicinal Plants Scheme, Department of Botany, University of Oxford [Received 5 December 1951) For many years experimental grafting of alkaloid-forming plants on non-alkaloid-bearing stocks and vice versa has been carried out in several laboratories (Hieke, 1942; Dawson, 1942, 1944; James, 1944, 1945; Cromwell, 1943), and considerable evidence has accumulated to suggest that the main seat of alkaloid synthesis in the plants normally forming mydriatic alkaloids is in their root systems (Dawson, 1948). In every case this work involved the grafting of a member to whom mydriatic alkaloids are foreign, and the results showed simply presence or absence of alkaloids. Recently, methods have been described by which the alkaloids (including atropine) and hyoscine can be estimated separately in mixtures and so the ratio of the alkaloids in the plant can be determined. Trautner & Roberts (1948) described a method for the estimation of these alkaloids, a separation of the mixed bases being first achieved by adsorption chromatography, using activated silica, followed by titration. The method fails if the hyoscine proportion is less than 10% of the total; hence, while applicable to extracts from Duboisia species and many species of Datura (such as D. innoxia)*, it is unsuitable for extracts from Atropa belladonna. Evans & Partridge (1948) have more recently described a method using partition chromatography which can be used for extracts containing lower proportions of hyoscine. In the present investigation their work has been developed so that the method can be used to estimate these alkaloids in actual plant extracts; this made it possible to determine the ratio in the plants, and it became clear that in Datura innoxia and Atropa belladonna, characteristic and very different ratios were normally developed. This suggested that reciprocal grafts of these two species in which both alkaloids are normally formed, but in very different proportions, might produce further and rather more elegant experimental evidence than was obtainable in the earlier work involving non-alkaloidal plants. A series of reciprocal grafts was made, therefore, and stock and scion dried and analysed separately as described below. METHODS The grafts were all made as 'slip grafts'; a slip with terminal bud and two or three leaves was taken from a young, rapidly growing plant, the stem was trimmed to the shape of a narrow wedge and inserted in a slit made in a young stock plant. The slit was made below a leaf in the soft part of a growing stem. The whole was then bound tightly with raffia, and kept in a moist atmosphere. After 10 days or a fortnight the shoot of the stock The name Datura innoxia is used in this paper for the plant commonly known as D. metel, as it appears from the work of Timmerman (1927) that the former name is correct.
Separation and identification of solanaceous alkaloids from plants 251 was cut back to one or two leaves and lateral buds, the union was retied as required, and, when the scion began to grow, the shoot of the stock was cut right off just above the graft union. When the scion was growing rapidly the grafts were potted on in John Innes sterile compost number 2 and at intervals were watered with 'Liquinure' made up at medium strength with tap water. They were grown on for 3-4 months, and were then shaken out of the pots, the soil removed as carefully and completely as possible and the plants divided up as indicated in the tables. All plant material was first dried at 55-60 C. and ground to a no. 40 powder, i g. was then weighed into a 50 ml. beaker and stirred with I ml. 95 % alcohol and o-i ml. 10% aqueous ammonia until evenly wetted. 5-0 ml. of chloroform was added and the mixture heated to boiling; it was poured hot into a percolator containing a 2-O-2-5 cm. layer of activated alumina, to remove frothing agents, and the percolate was collected in a 100 ml. measuring cylinder. The beaker was rinsed with successive portions of chloroform and the rinsings put through the percolator until a total of 30 ml. was collected. 30 ml. of i-2n-hydrochloric acid was added and the whole was shaken vigorously for i min. It was then allowed to settle, the acid layer was separated and filtered through a dry filter-paper to remove drops of chloroform. The filtrate was made alkaline with 5 % aqueous ammonia and shaken up with 30 ml. of ether. The ethereal layer was separated, evaporated almost to dryness to remove ammonia and made up to 50 ml. At this stage two portions of i ml. each were removed and estimated by the Vitali- Morin method described by Roberts & James (1947); this gave the total tropane alkaloid content, which could be compared with the value obtained by titration which was known sometimes to include non-tropane bases. The remaining 48 ml. was allowed to run through a column similar to that described hy Evans & Partridge (1948). This contained 10 g. of celite 545 impregnated with 3-3 ml. M/2-phosphate buffer, ph 6*8. All the tropane alkaloids were adsorbed on the column and, when the liquid had run through, the column was developed with pure ether saturated with the same buffer solution, portions of 20 ml. eluate being collected separately. Each was evaporated just to dryness in a titration tube, the residual oil dissolved in I ml. alcohol and treated with i ml. N/50-sulphuric acid, added from a narrowbore burette; the liquid was made up to 15 ml. with COj-free distilled water and treated with 6 drops of methyl red. The residual acid was back-titrated with N/ioo-sodium hydroxide as described by Roberts & James (1947). When successive titrations showed that no more alkaloid was coming through, the sum of the titres was obtained; this, multiplied by 50/48 gave the hyoscine content of the original material. Elution was then continued using chloroform saturated with buffer as eluting solution. The eluate was treated as before, and the resulting figure was the hyoscine content of the original. The ratio was determined for each part of the plant analysed. RESULTS The results obtained from a series of analyses of normal plants of A. belladonna and Datura innoxia are summarized in Tables i and 2: It is evident from the figures in the last column of Table i that all parts of a normal plant of Atropa belladonna show a ratio approximating to o-i, whatever the variation in the total alkaloid content.
252 G. M. JAMES AND B. H. THEWLIS The values for the ratio in Datura innoxia are more variable than in Atropa belladonna; but in every case the hyoscine is the bulk of the alkaloid and the ratio is high, and clearly different from that of Atropa belladonna. It seemed worth investigating the ratios of grafted plants, and accordingly Table i. Alkaloid analysis of normal plants of Atropa belladonna 4. xi. 49 15. vii. 50 I. viii. 50 Leaves Fibrous roots Young and mature leaves Old leaves Fibrous roots Taproot Immature leaves Mature leaves Stems Fibrous roots Taproot 2-9 4-26 2-8 1-8 2-5 2-9 1-9 3-2 4-2 6-9 4-2 2-85 4-20 2-91 1-94 2-63 2-95 1-95 3-20 4-45 7-OI 4-15 0-30 0-5C/ 0-36 0-18 0-26 0-31 0-25 0-31 0-48 0-31 0-38 2-54 3-95 2-56 1-75 2-37 2-64 1-71 2-89 3-97 2-89 3-73 0-12 0-14 o-io 0-12 0-15 O-IO Table 2. Alkaloid analysis of normal plants of Datura innoxia 16. ix. 49 17. X. 49 7. xi. 49 8. viii. 50 22. ix. 50 Young leaves Young stems and petioles Fibrous root Young leaves Mature leaves Stems Fibrous root Flowers 1-75 0-925 3-25 0-82 2-00 3-07 4-35 2-05 1-17 2-30 6-45 1-75 0-96 4-61 0-86 3-31 4-51 2-24 1-17 2-42 6-50 1-65 0-78 2-52 0-81 1-69 3-015 4-07 I-09 2-16 5-88 0-049 0-17 0-54 0-047 0-34 0*30 0-46 0-27 0-08 0-25 0-62 16-93 4-61 4-62 17-36 4-96 9-83 9-13 7-59 13-81 8-51 9-46 samples of grafts Dattira innoxia on Atropa belladonna and of A. belladonna on Datura innoxia were analysed, together with corresponding samples of normal plants dried during the same season, as a comparison. These results are summarized in Tables 3 and 4. It is clear from these figures that the shoot of the scion in grafted plants contains the alkaloids hyoscine and in the proportions characteristic of the stock species, rather than in those characteristic of normal plants of its own species. It was thought that the ratios might possibly change during ageing or starvation, and that some part of the difference might be due to effects of this kind in the scion. Accordingly, experiments were set up using leaves from normal plants of Atropa belladonna to test the effects of age and of starvation conditions on the ratio in the leaves. A sample of four whole plants, selected at random, was taken and the material was divided into the following fractions: green leaves, both juvenile and mature; yel-
Separation and identification of solanaceous alkaloids from plants 253 lowing leaves, about one-fifth yellow; tap root, fibrous root. Each fraction was dried separately, sampled and analysed as described above. The results are summarized in Table 5. Table 3. Alkaloid analysis of {a) graft plant of Datura innoxia on Atropa belladonna; {b) shoot of normal plant of Datura innoxia; (c) root of normal plant of Atropa belladonna 22. ix. 50 15. vii. 50 (a) Large leaves of scion shoot, D. innoxia (b) Large leaves of normal plant, D. innoxia (a) Stems of scion shoot, D. innoxia (6) Stems of normal plant, D. innoxia (a) 'Tops' (young leaves, young stems, petioles) of scion shoot, D. innoxia (b) ' Tops' of normal plant, D. innoxia (a) Roots (tap and fibrous) of stock A. belladonna (c) Roots (tap and fibrous) of normal plant, A. belladonna) I-20 I 00 I 90 1-90 I-20 4-85 2-70 1-22 I-OI 204 1-29 4-97 213 2-79 O-I2 0-90 019 1-83 4-47 0-23 0-28 I'lO 1-85 O-20 I-I7 0-49 1-89 2-50 7-8s o-io 9 20 010 9-09 013 o-ii 1 Table 4. Alkaloid analysis of {a) graft plant 0/Atropa belladonna on Datura innoxia and {b) shoot of normal plant of Atropa belladonna 16. ix. 49 (a) Leaves and tops of scion shoot of A. beltadonna (b) Leaves and tops of j normal plant of.-j. 6e/- ladonna Hyoscme. t j Hyoscy-., ' ; hn>oscy hvoscvamine i- 75 2-90 i-oi 2-85 0-91 0-30 O-IO 2-54 9'49 O-I2 An experiment was also carried out in wbich samples of ten isolated mature leaves were kept in a dark incubator at 21 C. with their petioles dipping in water for the periods shown in Table 6. At the end of the period the leaves were dried, powdered and analysed as before. DISCUSSION It is clear from the figures quoted above that normal plants of A. belladonna contain the alkaloids hyoscine and in the ratio of approximately O-I2, and that this ratio remains surprisingly constant, being unaffected by the total
254 G. M. JAMES AND B. H. THEWLIS amount of alkaloid in the plant, or by the part of the plant, or by ageing or starvation. Conversely, the alkaloids in normal plants oi Datura innoxia show a characteristically high ratio; these plants show more variation, but the ratio is always very distinct from that of Atropa belladonna. In our plants the average value was 9-71; tap roots always showed particularly high ratios, i3-8i-i7-36; the other parts of the plant showed ratios varying from 4-6 to 9-8. Table 5. Alkaloid analysis of normal Atropa belladonna plants in various fractions [dried 29 September 1950) Green, juvenile and mature leaves Yellowing leaves approx. ^th yellow Fibrous root 4-0 1-5 5-75 7-7 4-16 1-68 6-03 8-11 0-41 0-15 0-51 0-73 3-75 I 53 5-53 7-38 o-i^ 0-092 0-10 Table 6. Alkaloid analysis of leaves of Atropa belladonna starved for varying periods. Samples of ten leaves gathered 14 October 1950 Period of starvation (days) 4 6 3-9 2-45 3-9 4-22 2-65 4-13 0-40 0-27 O-43 3-8i 2-38 3.70 O*IO In grafted plants, the scion always contains the alkaloids in the proportions characteristic of the plant used as stock, not in the proportions characteristic of normal plants of the scion species. This fact supports the view that, under normal conditions, the main synthesis of alkaloids is in the roots, particularly in the young growing root tips; and that the alkaloids formed there are transported unchanged, probably mainly in the transpiration stream, to other parts of the plant. In these experiments no foreign alkaloid has heen introduced into the plant system, both plants normally producing both hyoscine and ; the only difference is the proportions in which the alkaloids are present, and these are pretty constant and very distinct. The very high ratios found in the tap roots of Datura innoxia are always due to the presence of only very small quantities of, suggesting a destruction of in the tap root; but our results are too few for us to put forward any definite hypothesis. SUMMARY 1. A method is described in which the analytical methods of Evans & Partridge are adapted for use with plant materials. 2. Using this method, ratios of Atropa belladonna and Datura innoxia are shown to be reasonably constant and very distinct.
Separation and identification of solanaceous alkaloids from plants 255 3. The ratios in grafted plants are shown to be those characteristic of the stock used, not those characteristic of the normal plants of the scion species. 4. It is shown that starvation and ageing do not cause variations in the ratios characteristic of the species. 5. The bearing of these facts on the formation and distribution of alkaloids in these plants is discussed. REFERENCES CROMWELL, B. T. (1943). Studies in the synthesis of in Atropa belladonna and Datura stramonium. Biochem. Jf. 37, 717. DAWSON, R. F. (1942). Accumulation of nicotine in reciprocal grafts of tomato on tobacco. Amer. jf. Bot. 29, 66. DAWSON, R. F. (1944). Accumulation of anabasine in reciprocal grafts of Nicotiana glauca and tomato. Amer.J. Bot. 31, 351. DAWSON, R. F. (1948). Alkaloid biogenesis. Advanc. Enzymol. 8, 203. EVANS, W. C. & PARTRIDGE, M. W. (1948). The partition chromatography of alkaloids. I. Solanaceous alkaloids. Quart, jf. Pharm. zi, 126. HiEKE, K. (1942). Pflanzenphysiologische Untersuchen iiber die Alkaloide. II. Zur Alkaloidfuhrung der Pfropfpartner bei heteroplastichen Solanaceenpfropfungen. Planta, 33, 185. JAMES, W. O. (1944, 1945). Oxford Medicinal Plants Schema, Annual Reports. ROBERTS, M. & JAMES, W. O. (1947). A method for the estimation of total alkaloids in Belladonna and Stramonium. Quart, jf. Pharm. 20, 1. TiMMERMAN, H. A. (1927). Datura: the nomenclature of the species used in medicine. Pharm. J. 64, 571. TRAUTNER, E. M. & ROBERTS, M. (1948). The separation of hyoscine and and the alkaloidal assay of Duboisia spp. Analyst, 73, 144.