HENRY HYENANCSHIN AND OTHER CONSTITUENTS ETC. 1619 CLXXXII. -Hyenanchin and other Constituents of Hyenanche globosa. By THOMAS ANDERSON HENRY. Hyenanche glo bosa Lamb (Toxicodendrom cnpense Thun) is the sole member of a genus of the natural order Euphorbiacm and in distribution is confined tot South Africa. The name of the plant is said to have originated from the use olf the seeds in South Africa as a poison for wild animals especially hyenas. I n 1858 Henkel obtained by fractionation of a concentrated alcoholic extract of the fruits a syrupy preparation which proved to be highly toxic (Arch. Pkamz,. 1858 144 16). Engelhardt subsequently prepared the toxic constituent which he named hyenanchin in a crystalline condition but beyond stating that it was neither an alkaloid nor a glucoside made no1 attempt to characterise it (Arb.pharm I n s t . Dorp. 1892 8 5). Shcztly afterwards E. Merck (Merck’s Ann. Report 1895 123) pointed out that if the observation made by Henkel and by Engelhardt, that hyenanchin exerts a strychnine-like action on the brain but is without action on the spinal cord is trustworthy the substance might be of some therapeutic value as a substitute for strychnine in cases where cerebral action alone is required. For a supply of the plant the author is indebted to Mr. I. B. Pole Evans Chief of the Division of Botany Department of Agriculture Pretoria. E X P E R I M E N T A L . The material used included steins (1 kilo.) leaves (2 kilos.) and fruits (4 kilm.). Each of these parts of the plant was extracted separately with chloroform in a continuous hot percolatioa apparatus yielding in the case of the stem and leaves a hard, dark green wax and in that of the fruits a neutral dark yellow oil which was not examined in detail.Extraction was continued with hot 95 per cent. alcohol and the liquid concentrated to a thin syrup which was polurd into five volumes of water causing the separation of sparingly soduble tannin containing a small amount of a yellow colouring matter. The filtrate after purifi-cation with lead acetate in the usual manner was concentrated under reduced pressure to a viscid syrup which was then extracted repeatedly with an equal volume of ethyl acetate until it was free from bitterness and no longer toxic. The brown sticky residue left on distilling off the ethyl acetate was dissolved in three time 1620 HENRY HYENANCHIN AND OTHER its weight of water and set aside when after some weeks a bitter, poisonous crystalline material separated.Mere traces of this were obtained from the leaves and stems and only 6 grams from the fruits (0.15 per cent.) but this does not represent all that is present as the mother liquor was still toxic and intensely bitter, and in the case of the fruits a further 1.7 grams (making a total yield of 0.19 per cent.) were obtained by repeating the treatment with ethyl acetate. This crude material could only be recrystallised by adding it to fifty times its weight of boiling water and' filtering rapidly, when on cooling there separated a crop of slender colourless needles.Slight concentration of the filtrate gave a further crop of the same substance and reduction of the mother liquor from this crop to half its original volume led to the separation of a second substance crystallising in short colourless hexagonal prisms of which more was obtained by further concentration. This second substance forms about two-thirds of the original crystalline material and it is proposed to apply to it Engelhardt's name hyenanchin; as the substance crystallising in needles is an isomeride of this i t may appropriately be called isohyenanchin. Hyenanchin. This substance isolated as described above and purified by repeated recrystallisation from boiling water has no melting point, but when heated becomes yellow a t 200° then darkens and finally decomposes sharply with effervescence at 234O.It dissolves in water to the extent of 1-18 per cent. a t 15O is more soluble in boiling water and sparingly so in alcohol butyl alcohol ethyl acetate or acetone. It has [a] + 14.7" in water (Found C=58.2, 58.3 58.05 57-9; H=6*03 6.01 6.4 6.3. Loss at llOo in a vacuum nil. Hyenanchin contains no nitrogen reduces Fehling's solution on boiling and silver nitrate solution on gently warming or on keep-ing in the cold decolorises permanganate immediately and gives an amorphous yellow precipitate with bromine water. Boiling dilute hydrochloric acid converts it into an amorphous brown sub-stance which gives a characteristic orange-coloured amorphous precipitate with phenylhydrazine in the cold. No sugar is pro-duced.On warming with alkalis hyenanchin furnishes a distil-late containing a minute quantity of a substance which gives the iodoform reaction reduces Fehling's solution and furnishes a semi-carbazone crystallising in short colourless needles and decom-posing at ZOOo which may be acetolsemicarbazone C,H,02N3 (decornp. 195-200O) (Found N=31.12. Calc. N=32*06 per C,,HI8O7 requires C = 58.06 ; €€= 5.8 per cent.) CONSTITUENTS O F HYENANCHE GLOBOSA. 1621 cent.). A specimen of acetdsemicarbazone prepared under similar conditioas and without recrystallisation gave N = 30.8 per cent., and a mixture of the two decomposed a t 198O. No derivatives off hyenanchin with hydrosylamine phenylhydrazine o r semicarb-azide could be obtained. Actiom of Baryta.-Although hyenanchin is not acid to indicators it neutralises alkalis but its titratioa presents difi-culty olwing to the ease with which it becomes coilourad and decom-poses in presence of alkalis.If a solutioln of hyenanchin to which excess of baryta has been added is warmed or kept for several days it becomes brown and turbid the latter due to the gradual separation of barium carbonate. By dissolving it in A7/5-baryta and a t once titrating with N/5-sulphuric acid in the presence of phenolphthalein a fairly satisfactory end-point is obtained (Foand Ba(OH) required for neutralisation 55.2 55.05. Calo. for two C0,H groups 55.3 per cent.). On evaporating in a vacuum the filtrate from the barium sulphate foirmed barium carbonate is gradually deposited and the filtrate from this yields on complete evaporation in a vacuum, a nearly coloarless varnish which appears tor consist chiefly of a barium salt of a monocarboxylic acid since it yields on addition of excess of N/5-sulphuric acid 29.8 per cent.of its weight of barium sulphate (C,,H,,O,,Ba requires BaSO = 31.5 per cent.). The corresponding acid obtained by adding the calculated quantity of N/5-sulphuric acid to an aqueous solution of this salt and evaporating the filtrate to dryness in a vacuum is a colourless varnish which is readily soluble in water and reduces Fehling’s sollution on boiling and ammoniacal silver nitrate in the cold. The acid does not’ regenerate hyenanchin on drying even atl 150° in a These results indicate that hyenanchin is probably a dilactolnne, convertible by the carefully regulated action of weak alkali into the corresponding dihydroxydicarboxylic acid C,,H,,O, which is unstable and readily loses one carboxyl group.Action of Acetic Anhydride.-When hyenanchin is heatedl with acetic anhydride at looo most of it crystallism out unchanged on coolling but if a drop of pyridine be added and the heating con-tinued it is converted into a soft sticky resin from which with great difficulty theire cam be separated by repeated crystallisation from dilute alcolhol a small yield of crystalline material conslisting of a t least three substances (a) cream-coloared needles softening a t 136O and finally melting and decomposing a t 169O; ( b ) colour-less nsedles m. p. 1 2 6 O ; (c) colourless short needles m. p. 104O.Only a few centigrams of each of these substances were obtained, vwuum 1622 HENRY HYENANCHIN AND OTHER so that they could not be satisfactorily purified for examination, but it is probable that only the second is a true acetyl derivative od hyenanchin (Found C = 56.7 57.05 ; H == 5.7. Cl,H1707Ac and C,,H1607Ac require C=57-9; 13=5.6 per cent.). isoHye?tanchin crystallises from boiling water in long slender needles with a silky lustre has no melting point butl becomes brown a t 245O and decomposes sharply with effervescence at 299O. It. dissolves in water to the extent of 0.26 per cent. a t 15O is ' some-what more soluble in boiling water and less so in alcohol or ethyl acetate. [a] -61'3O in water (Found C=58*08 58-25; H=5*97, 6.16. It reduces Fehling's solution on boiling and ammoniacal silver nitrate in the cold.A ction of Baryta .-isoHyenanchin dissolves immediately in excess of N/5-baryta solution forming a solution which only beconies slightly yellolw after remaining several days a t Oo and under these conditions combines with sufficient baryta to neutralise one carboxyl group (Found on immediate titration 29.8; after three days a t Oo 29.2. Calc. for one CO,H group 27.7 per cent.). On again adding excess od A7/5-baryta solutioa and boiling gently for it few minutes and titrating back baryta equivalent to a second carboxyl grotup is folund to have been absorbed (Found: 25.9. Calc. 27.7 per cent. Total for two CO,H groups=55'4. Calc. 55-3 per cent.). A second estimation made by heating isohyenanchin wifh excess of iVI5-baryta solution for one hour a t looo and tit<rating back gave 56.2 per cent.The filtrate from these estimations on concentration behaved like the similar p r s paration from hyenanchin (p. 1621) and gave as a final residue an amorphous barium salt yielding 29.1 per cent. 09 barium sulphate on precipitation with N / 5-sulphuric acid. C,,H1807 requires C = 58.06 ; H = 5-80 per cent.). Physiological A c t i o n of Hyenanchin and isoHyenanchin. The author is greatly indebted to1 Dr. J. Trevan of the Well-come Physiological Research Laboratories who kindly undertook the examination of a series of preparations of Hyenanche and finally of the pure substances isolated. Dr. Trevan reports that hyenanchin has a physiological action almost identical in kind with that o,f picrotoxin but is much weaker.GoI-Iyenanchin on the contrary is not toxic in such doses as can be injected intra-venously C0"ST~TOEN'l'S OF HYENANCHE GLOBOSA. 1623 Relationship of Hyenanchin and isoiyenanchin t o other #on-nitrogenous Convulsant Poisons. The reactions described above and the physiological action of hyenanchin indicate that the latter belongs to the group of con-vulsant non-nitrogenous poisons of which picrotoxinin Cl5Hl6O6 (m. p. 206'5O [a] +4040r in alcohol) coryamirtin C,,H,,O, (m. p. 225O dextrorotatory) and tutin C,&,O7 (m. p. 208-209°, [a] +9*25O in alcohol) are the only well-defined members knolwn. Associated with picrotoxinin in the molecular compound picro-toxin C,H,,O,, is the substance picrotin C,,HI8O7 (m.p. 245-246O [aJD -55.2O in water) which is not toxic. Picrottoxinin and picrotin are both dilactones and are now believed to contain, respectively one and two hydroxyl groups the function of the sixth and seventh oxygen atoms respectively being still unknown (Horrmann Annalen 1916 411 273). Both these substances yield small quantities of acetone on distillation with alkali. Picrotin is isolmeric with hyenanchin and isohyenanchin and in many ways is very similar t o the latter. The1 two have therefore been carefully compared and found not tot be identical the chief differences being that picrotin has a definite melting point and is completely converted into the corresponding dicarboxylic acid by baryta in the cold whilst isohyenanchin has no definite; melt-ing point and is converted into the corresponding dicarboxylic acid by baryta in two well-defined stages.A mixture of both substances begins to sinter a t 2 2 5 O which is well below the melb ing and decomposing points of t,he two components. A number of substances of the formula C1,HJ807 have also been prepared from picrotoxinin and picrotin (compare Horrmann Zoc. cit .), namely picrotin-lactone (33B0 decomp.) picrotoxic acid (m. p. 230-231° [a] + 81.7O crystallises with 2H,O) a-picrotoxink acid (m. p. 209O [a] -48O) and /3-picrotoxinic acid (m. p. 235O, [a] -48O). None of these closely resembles either hyenanchin or isoh yenanchin. Sub s id i ar y C o n s ti t u e n t s of H ye nan c h e g 1 o b o s a. Examination of the Wax. Isolatiom of a New Phtytosterol and a New Wax AZcohol.The dark green wax from the leaves and stems was mixed with an equal weight of animal charcoal and extracted with boiling ethyl acetate yielding a greenish-yellow solution. This on cool-ing deposited a. mixture od two substances which were separated by heating the solution to boiling and adding enough ethyl acetat 1624 HENRY HYENANCHIN AND OTHER to keep both substances dissolved a t 35O. On keieping the liquid then depmited gelatinous granules which filtered with difficulty, and on drying in the air formed a pale brownish-green hoirny mass. This was purified by distillation in a vacuum and recrystal-lisation frmn ethyl acetate when it formed colourless masses od minute needles melting atl 82-83O (corr.). A number of solid alcohols melting near this1 temperature are known but all oif them differ slightly in compolsition and most of them in crystalline form from hyenanche alcohol and of those tried ceryl myricyl and wheat* alcojhols all depressed the melting point.It is readily soluble in chloroform boiling ethyl acetate or alcohol and spar-ingly so in ether does n o t combine with bromine and appears t~ be a new saturated alcohol of the formula C,H,,*OH (Found in substance dried a t GOo in a vacuum C=81*02 81.27 81.19 -f ; H = 13.5 13.55 13.89.1- C,,H,,O requirm C = 81.3 ; H = 14.1 per centl. ) . When boiled f o r several hours with acetic anhydride in presence of pyridine it yields an acetyl derivative which is readily hydro-lysed on recrystallisation from most solvents but separates from acetic anhydride in s o f t masses of colourless needles melting a t 75" (corr.) (Found C = 78.8 ; I3 = 13.0.C,,H,,O requires C=78.7; H=13*1 per cent.). Carnaubyl alcohol which also has this formula crystallises in leaflets (m. p. 68-69O) and is quite distinct from the alcohol od 31. gkobosa. The other known alcoholls of this formula are either liquid o r of much lower melting point than Ilyenanche alcohol. The filtrate from the alcohol1 oln concentration deposited a second substance which after repeated crystallisation from boiling ethyl acetate forms long lustrous needles melting at 265O (corr.) is readily soluble in chlolroform olr boiling ethyl acetate and spar-ingly so in alcohol even on boiling (Found C=83*66 83.95; R=11*35 11-66.CBH,,O requires C=84-4; H=11-55 per cent,). [a]g -22.4O in chloroform. It gives a typical phytosterol reaction with sulphuric acid in the presence of acetic anhydride and furnishes a monoacetyl deriv-ative crystallising from hob ethyl acetate in small spheroidal massw of colourless needles melting a t 244O (corr.) (Foand: G=81.12; H=10*96. C,,H,,O requires C=81*8; H=10*9 per centl.). This substance appears t o be a new phytosterol belonging to the series represented by the general folrmula CnH2n-100 of which a t least eight are now knolwn beginning with alcornol C,H,,O * For a specimen of this alcohol the author is indebted to Mrs. M. T. Ellis (Biochem. J . 1918 12 160). t Regenerated from acetyl derivative CONSTITUENTS O F HYENANCHE GLOBOSA. 1625 (Hartwich and Dunnelnberger A rch.Pharm. 1900 238 348), and terminating with amyrin C,H~,O (Windaus and Welsch, ibid. 1908 246 506). The new phytosterol is exceptional in this series in being lzvorotatory and in giving an acetyl derivative melting at a lower temperature than the parent substance but in all other respects it resembles other members of the series. Isolation of a New Yellow Colouring Matter. The colouring matter was obtained in small amount (total 2.3 grams crude 1.0 gram pure from all three sources) by extracting the sparingly soluble tannin (p. 1619) with boiling dry ether and was recrystallised from alcohol from which it separated on slotw evaporation in microscopic yellow needles which became brown a t 200° and finally melted and decomposed at 270-280O.It is moderately soluble1 in alcohol sparingly so in ether and insoluble in chloroform. The solution in alcohol gives a brownish-black pre-cipitate with ferric chloride (Found C = 62-43 62*1,* 62.1 % ; H=4*4 4*4,* 4-25.* C,,H,,O, requires C = 62.5 ; H = 4-16. C,,H120,,2H20 requires loss 11.1 per cent.). The collouring matter furnished an acetyl derivative crystal-lising from holt alcohol in masses of cream-cololured needles melt-ing a t 234-236O (dwomp.; corr.) (Found C=63*4; H=4.0. Loss a t 60° in a vacuum nil). On regeneration with hydrochloric acid in the presence of hot acetic acid the acetyl derivative yielded 71.3 per cent. of colouring matter identical with the cilriginal sub-stance. Sulphuric acid could not be used for this purpose as i t appeared to convert the colouring matter into a soluble sulphmic acid (compare A. G. Perkin T. 1899 75 448). These results indicate that the acetyl derivative should be represented by the formula C15H,0,Ac3 (requires C = 63.6 ; H = 4.04 ; colouring matter, 72.7 per cent.) which is a triacetyl derivative of the parent sub-stance less one molecule of water. The reactions of the colour-ing matter indicate that it? belongs tot the flavone group in which the loss of a molecule of water o n acetylation does not appear to have been recorded previously except doubtfully in the case of morin (A. G. Perkin loc. cit.). Unfortunately the small amount oS material available precluded further investigation of this and other points. Loss in a vacuum at looo 10.08. WELLCOME CHEMICAL RESEARCH LABORATORIES. [Received November 26th 1920.1 * Regenerated from acetyl derivative