ANALYSIS OF THE FRUIT OF RHAMNUS FRANGULA BY J. LOWE HALL B. S. University of Illinois, 1919 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN CHEMISTRY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS 1921 - ■ CONTENTS . Page Acknowledgment Introductory and Historical - - - - The Material ----------- Method of Procedure -------- A. The Water-Insoluble Fraction 3. The Water-Soluble Fraction - C. The Seeds --------- Discussion of Results Summary -------------- Bibliography ----------- i 1 8 9 10 14 16 oo Cj O 24 25 ACKNO WL E DG M EN r The writer takes sincere pleasure in expressing thanks and appreciation to Dr. G . D. Beal, under whose able help and direction this work has "been carried out. Digitized by the Internet Archive in 2016 https://archive.org/details/analysisoffruitoOOhall 1 . ANALYSIS OP THE FRUIT OP RHAMNUS FRANGULA. Introductory and Historical . While this work has to do only with the fruit of Rhamnus Frangula, no consideration of the subject could he complete with- out some mention of the other parts of the plant as well as some of the more closely related snecies. For the use of the various parts of these plants in medicine has a very ancient origin, and has been of prime importance in its chemical history and econom- ic application. The writer is indebted to an article written by E. N. Gathercoal (1) in the Journal of the American Pharmaceuti- cal Association from which the following brief historical con- sideration is taken. For centuries the bark of a wild shrub, known in England as Alder Buckthorn or Berry Alder, has been used in Eurone as a purgative. This bark is now recognized in most of the leading pharmacopoeias of the world, under the name of Frangula, or Frangulae Cortex. Rhamnus Frangula, the plant yielding the drug, ranges along roadsides and in thickets over all of Europe, except in the very northernmost parts, and east over northern Asia . Associated with Rhamnus Frangula is Rhamnus Catharticus, a thorny shrub, named in England, Buckthorn or Way thorn. This plant is also found in northern Africa, India, and eastern 2 United States. The fruit, especially, has been employed for many centuries in Europe as a cathartic. It is now official in a few of the Euronean pharmocopoeias . As a medicine the fresh, rice berries are made into a decoction or the abundant juice is ex- pressed and made into a syrup. The bark, also, possesses purga- tive properties, which, in the fresh bark, are said to be more drastic than Frangula. Another group of Rhamni furnishin^nedicinal barks, is found along the western coast of North America. -With the settlement of California by the Spaniards, the new-comers noted that the native Indians used the bark of a certain kind of a shrubby tree as a cathartic. The Spaniards named this plant and its bark Cascara Sagrada (Sacred Bark). This drug is now official in nearly all the pharmacopoeias of the world. It is obtained from the Plant Rhamnus Purshiana, which ranges over the west slopes of the Cascade Mountains, from central California well up into British Columbia, and forms extensive low forests on the valley and mountain sides. The term "ramnos" used by the early Greek physicians and naturalists, is thought to be derived from the Celtic "ram", signifying a tuft of thorns or branches. The name was applied to certain thorny plants by these writers, but from their meager or inaccurate descriptions it is impossible to establish that the plants mentioned by them were any of the Rhamni as we know them today . The early Anglo-Saxons were acquainted with purgative properties of, at least, the Rhamnus Catharticus, for we find 3 . the plant mentioned in their medical writings before the Norman Conquest. The juice of Way thorn berries is described as an aperient by Welsh physicians at the beginning of the thirteenth century . Crescentius (1305) mentions Rhamnus Catharticus under the name Spina Gervinae and describes Rhamnus Frangula under the name Avornus, mentioning the use of the middle bark as an evacuant. It is not until Matthiolus (2) (1543) in his commentary on the materia medica of Dioscorides, that a good description of Rhamnus Frangula, with mention of the purgative property of bark and berries, is found in literature. He first uses the name "frangula” in connection with the plant; (frango, frangere, meaning "to break", an allusion to the soft and fragile nature of its wood). By 1700 the botanical characters of most of the European Rhamni were well established. Linnaeus (3) (1753) places them in the Petandria Monogynia. He includes both Rhamnus Frangula and Catharticus as natives of Sweden in his Flora Svecica (1745), and mentions as pharmaceutical products derived from them: Spina Gervinae Baccae, Syrupus Domesticus, Frangulae Cortex. The bark of Rhamnus Frangula has been recognized in the pharmacopoeias of central Europe since the middle of the last century, including the Banish (1368), Norwegian (1870), Swedish (1871), German (1870), Prussian (1862), Hanoverian (1861), and Dutch (1871)*, Austrian (1389), French Codex (1908), U. S. (1880), and Eritish (1885, though it was omitted from the last edition). The chemistry of rhamnus barks presents much of interest 4 . because from the first analysis "by Gerber in 1828, it has been observed that the active urinciples are resinous in nature, difficult to separate from one another and to determine their true consti tution . Even at the present day these analyses are far from being in a satisfactory condition. Gerber, (4) obtained, among numerous other vegetable con- stituents, 2.7$ of yellow resinous coloring matter and 4.6$ of bitter acrid extractive, which he considered contained the active constituents. He noted the yellow coloring matter became dark- red with alkalies. Hubert (5) (18.30) analyzed the juice from the fruit of Rhamnus Catharticus. He found a bitter substance, apparently the active constituent, and closely resembling the cathartin of senna leaves, a green coloring matter, which in the ripe fruit is purple red, due to the action of acids in the ripening fruit, and a brown material insoluble in alcohol but easily sol- uble in water. Pleury (6) (1842) obtained from the unripe berries of Rhamnus Catharticus, rhamnine in pale yellow crystals. Winckler (7) (1849) obtained rhamnine from the unripe berries of Rhamnus Catharticus and cathartin from the ripe fruit. He considered that rhamnine by the ripening process is converted into cathartin and glucose. (This is the first pub- lished evidence of the glucosidic nature of these resinous con- stituents of the Rhamni ) . 5 . Binswanger (8) (1849) found in frangula bark the crystalliz- able yellow coloring principle which was named (by L. A. Buchner) rhamnoxanthin , an ether-soluble amorphous resin, one or more alcohol soluble resins, a bitter substance of resinous nature in which the ourgative nrooerties of the bark seem to lie, sugar, gum, tannin, plant acids, extractive, etc. He compared the bark of Rhamnus Catharticus with Prangula bark and found that the constituents were similar, but included also a bitter, water soluble, crystallizable substance to which he attributed the greater hydragogue properties of the Rhamnus Catharticus bark. This principle was differentiated from the cathartin of senna leaves and named rhamno-cathartin . He found rhamno- xanthin also in the seeds of Rhamnus Catharticus and Rhamnus Prangula. The juice of the rioe berries contained a violet coloring matter turning red with acids and green with alkalies, a bitter extractive, etc. The unripe berries contained only the rhamnin of Pleury. Buchner (9) (1853), who worked with Binswanger at Munich in 1849, obtained from the root bark of Rhamnus Prangula, rhamnoxanthin in sublimable, golden-yellow needles, very slightly soluble in water, but easily so in alcohol or ether (especially hot), readily in solutions of ammonia and the fixed alkalies with a fine -nurole-red color, and in concentrated sulfuric acid with a red color. By neutralization of the al- kaline solution, the rhamnoxanthin was thrown out as a yellow powder, and by dilution of the concentrated sulfuric acid so- lution with water it was likewise separated out. 6 . Casselmann (10) (1857) obtained the resinous constituent of Prangula in crystalline form, designated it frangulin, and decomposed it with the formation of glucose and an acid product he named frangulinic or ni tro-frangulic acid. Phipson (11) (1858) found rhamnoxanthin in the branches of Rhamnus Frangula and of Rhamnus Catharticus and corroborated Buchner's description of it. Kubly (12) (1866) separated from frangula bark the glucos- ide, which he named avornin, an amornhous resin, and a principle similar to cathartic acid, which he had a short time previously isolated from senna leaves. The avornin he split into avornic acid and glucose. Paust (13) (1869) stated that the frangulin of Casselmann, and the avornin of Kubly are identical, and assigned them the formula C^H^O^. He named the acid resin from the decompo- sition of this glucoside, frangulic acid. Liebermann and Waldstein (14) (1876) identified emodin ( trioxymethylanthraquinone ) from frangula bark and stated that frangulic acid is probably emodin. Prescott (15) (1879) was the first to analyze cascara sa- grada bark. He found a brown resin of strongly bitter taste, colored a vivid purple-red by potassium hydrate solution, sparingly soluble in water or ether, but freely so in alcohol, chloroform, benzol, carbon disulphide, and solutions of caustic alkalies, though precipitated from the latter by acids. He found also some other resins, tannin, oxalic and malic acids. etc . 7 . Schwab e (16) (1888) found frangula to yield frangulin 0.04$, and eraodin 0.1$. He corroborated, the physical characters of frangulin as stated by Casselman and Faust, and amplified on them. His -proximate analysis indicated the formula Frangulin by hydrolysis, yields emodin. He round in case <■. r a bark emodin but no frangulin. Thorpe and Miller (17) (1892) corroborated Schwabe's form- ula for frangulin and determined that the sugar from the deco — position of frangulin was a true rhamnose. Cabannes (18) (1895) renorted that in sections of frangula bark treated with alcoholic potassa solution, the parenchyma of the cortex, medullary rays, and bast, all acquire a strong red color, but that in cascara bark sections only one or two layers of the cortical parenchyma, the medullary rays, and the five or six inner rows of bast parenchyma take the color. Ammonia and soda solutions react the same as potassa. Oesterle (19) (1399) found that frangula-emodin differs from aloe-emodin. Perrot (20) (1900) stated that powdered frangula bark with alkalies produces a deen red color, but that powdered cascara bark gives a yellow color, and that the powders could be dis- tinguished in this manner. Tschirch and Polacco (21) (1900) analyzed Rhamnus Catharti- cus fruit and determined the presence of emodin, several color- ing matters, a sugar, etc. The purgative action was ascribed to the emodin. 3 . Tschirch and Pool (22) (1908) found that the emodins from frangula and cascara "barks were identical, that neither of the barks yielded rhein, but that chrysophanic acid was present in frangula bark. Schmidt (23) (1912) describes frangulin (rhamnoxanthin ) , (C H 0 ), as occuring in lemon-yellow, glistening fine needle- Cj X oU »/ crystals, odorless and tasteless, melting at 228° to 230° C. It is almost insoluble in water and in cold ether, but soluble in 180 parts of 80^ hot alcohol. Concentrated sulfuric acid dis- solves it with a dark-red color and with caustic alkalies it forms solutions of a ourple red color. By boiling with an al- coholic solution of hydrochloric acid it becomes converted into rhamnose and frangula-emodin , (C-^H^O^ ) , which forms bright red glistening needles melting at 255°C. It is insoluble in water, slightly soluble in alcohol, and easily soluble in chloroform and benzol . The Material. The berries of Rhamnus Frangula were gathered as they became ripe and immediately immersed in 95 per cent alcohol, thus preventing any enzymatic action. About a half liter of the berries covered with alcohol was available for this analysis Consequently the work was handicanned considerably in attempting to purify and identify small amounts of material. . ’ A O * r i .-tv , - ■ — r i ,, . 9 . Method of Pr oc edure . The alcoholic extract was filtered from the berries and the berries washed with about 200 c.c. of alcohol (till the filtrate was colorless). The extract was browni sh -black by- reflected light, and when quite shallow transmitted a dull, dark-red light. The berries were then dried in a vacuum oven at 70 c '~ 80°C, and mulled in a mortar in order to brea v the hardened skins from the seeds. The skins were winnowed from the seeds and exhausted with boiling alcohol, which was then combined with the first extract . This combined alcoholic extract was evaoorated to a thick syrup consistency, and to it was added 300-400 c.c. of water. This mixture was placed in a refrigerator for preservation while the water-soluble material was going into solution. The gelatinous precipitate was filtered out, washed with water, and again taken un in alcohol, although an insoluble residue re- mained, as was often the case in similar subsequent operations; itj^ay have been the result of resinif ication, hydrolysis, or similar reaction. Sufficient alcohol was added to the aqueous solution for preservation . The dry seeds (to be analyzed later) were put in a dry stoppered flask 10 . A. The Water-Insoluble F raction . The accompanying diagram illustrates the procedure followed in manipulating the alcoholic extract, the Roman numerals to he used later in referring to the individual oortions in their sub- sequent separations. The water-insoluble portion of the original extract having been taken up in alcohol as previously mentioned, was then mixed with a sufficient quantity of purified sawdust to absorb it effectively, allowed to dry until the odor of alcohol was no longer apparent, and then placed in a large Soxhlet extraction apparatus. The material was then extracted with ether until it came through colorless (II, see diagram). Extraction was then completed with alcohol (V, see diagram). The ether extract (II) gave a strong color test (red) with ammonia, and was therefore shaken out successively with 5 per cent solutions of ammonium carbonate, sodium carbonate, and sodium hydroxide; these fractions were acidified and shaken out with ether. Vol . of base Color Comnarat i ve Fraction required precipi tation Ammonium carbonate 75 c . c . Faint pink Hone Sodium carbonate 230 c.c. Very deep red O c» Sodium hydroxide 200 c.c. Pale red 1 The precipitates from the second and third fractions , ( the first was discarded) , were soluble in glacial acetic acid. but did not crystallize out on concentration, a small fraction merely separating in a semi -gelatinous state. This material - . : ' - ; ( * ’ • ■ 11 . DIAGRAM SHOWING MANIPULATION OP COMBINED ALCOHOLIC EXTRACT. Alcoholic Extract ■ r Precipitate with w ater 1 Insolubl e in water Take up in 9 5 ^ alcohol 1 Soluble in water ( V III ) Shake out with ether and combine extract with extraction (II) . Sol, in alcohol (I) Mix with purified sawdust and extract with ether in soxhlet ( II ) . Complete extraction with alcohol (V). Hydrolyze with 1^ HCL. Concentrate to syrup and pour into water. Insol, in alcohol Resin; discarded. J n r — Precipi tate Decolorize aqueous solution with lead subacetate, filter. ~i i Recover acids by passing H ? S into aqueous suspension. Filter off PbS. Aqueous solution (III). Filtrate (IV). Determine sugars 1 Solu ble in water (VI ) Insol uble in wat e'r Shake out with ether Dry and extract with and combine washings ether in soxhlet (VII). with (VII). Examine for carbohydrates. Test small amount of each ether extract for_ anthraquinone derivatives with a little ammonia (deep red or purple). 12 . in each case was filtered off on a hard filter and dried, but amounted -practically to nothing. The acetic acid solution was a very deep, rich red color, nearly opaque. The addition of water to each of these acetic acid solutions caused the pre- cipitation of a bright yellow substance in a flocculent state, which was filtered out, redissolved in 5^ sodium hydroxide, acidified, and shaken out with ether. On allowing the spon- taneous evaporation of the ether solution of the sodium carbon- ate fraction on a large watch glass, it was noticed that the material aopeared to be deposited in two forms, an outer ring of a deep red leaf-like material, and an inner circle of small yellow particles, - mostly the former. The material in each case was again taken up in glacial acetic acid and concentrated on the steam bath. During this purification, the sodium hydroxide fraction decreased markedly in mass, and the acetic acid solution, even when concentrated to about 1 c.c., yielded only a few particles of apparently flocculent, non-crystalline material. However the sodium carbonate fraction under similar treat- ment yielded a small cron of very finely divided material, melting at 250°- 2^5° C. An attempt to nrenare an acetyl derivative yielded barely enough material of questionable nature for a melting point determination: it did not melt under 320°0 . From these data it is evidently impossible to arrive at any definite conclusions concerning the identity of the sub- stances present, but it seems probable that the sodium carbonate 13 . fraction, at least, contained e’nodin (red; melts at 255°C), and the sodium hydroxide fraction possibly chrysophanic acid (yellow, though melting much lower, 198°C, and, according to Beal and Okey (24), insoluble in cold solutions of alkali car- bonates, but soluble in sodium hydroxide). The alcoholic extract (V, see diagram) was hydrolyzed 3-l/2 hours on the steam bath with 100 c.c. 1 % hydrochloric acid. It was then evaporated to a syrup, diluted with considerable water, and the precipitate filtered off and dried. The water solution was pale yellow. The dry precipitate wa s extracted with ether in a^Soxhlet apparatus. The aqueous solution (VI ) was shaken out with ether, the extract being combined with the Soxhlet extract (VII ). The aqueous solution did not reduce Fehling's solution. The Molisch •i-naohthol test for carbohydrate (Sherman (25) p. 57) was negative in comparison to a blank on distilled water. Therefore, as. the subsequent examination of the ether extract (VII ) shov/ed considerable anthraquinone de- rivative, it must have -been linked in some form insoluble in ether, but not with a carbohydrate. The ether extract (VII ) gave a very pronounced color test with dilute alkali. Accordingly It was extracted with the usual sequence of dilute bases. Fraction Vol . of base Color Comparative requ ired Precipit ation Ammonium carbonate 150 c.c. Pale yellow Trace Sodium carbonate 450 c.c. Very deep red Very heavy Sodium hydroxide 100 c.c. Pale red Trace 14 . The sodium carbonate fraction contained perhaps 0.2 gram of precipitated material, which was crystallized from hot gla- cial acetic acid, yielding a small crop of minute crystals, which appeared under the microscope as broad, ragged nlates, o o and which melted at 252 - 255 G. They were deem red in color, hut reflected a yellow fluorescent color similar to "fool’s gold". The substance was evidently emodin. The other two fractions yielded negligible quantities of precipitate. B. The Water-Soluble Frac t .ion . The aqueous solution (VIII) was shaken out with ether, but the extract gave no color test with alkali. The ether, was evaporated on a large watch glass, leaving a thin film of light-yel lowi sh brown material possessing a deasant odor re- sembling peaches, and a bitter taste. It had the appearance of a gum and did not reduce Fehling’s solution before or after hydrolysis, was insoluble in water, hot ten per cent sodium hydroxide, and acetic anhydride. The aqueous solution (VIII ) was treated with lead sub- acetate to remove its deep color, the precipitate filtered off, and hydrogen sulfide passed into the filtrate to remove the excess lead. After the lead sulfide was filtered off, the filtrate was colored only slightly yellow. This solution (IV) was warmed gently on the steam bath and air bubbled through it to remove the hydrogen sulfide. 15 . The Molisch oc-naphthol test gave decided indication, of carbohydrate in the solution even to the extent that consider- able precipitate was formed. The solution exerted a very marked reduction on Fehling's solution, and formed an osazone in 8-9 minutes which under the microscope appeared as the sheaves of long yellow needles characteristic of glucosazone; it melted at 198-202° C. Consequently the sugar might be either glucose or levulose. However, Seliwanoff’s test for levulose (Hawk (26) p. 55) was negative; therefore there must have been glucose present in appreciable quantity. A very brilliant red anilin acetate test for furfural was obtained, indicating a pentose (Sherman (25) p. 57). No positive test for rhamnose was obtained by the alcohol - sulfuric acid method (Browne (27) o. 577). The lead subacetate precipitate was pulverized, suspended in water, and hydrogen sulfide passed in until the lead was converted to the sulfide, thereby liberating the organic acids carried down by the subacetate. Tannic, malic, and a trace of succinic acids were found by a system of analysis according to Barfoed (28). Briefly Barfoed's method is as follows: Neutralize the solution with ammonia, concentrate to small volume, neutralizing again if necessary. Mix with 7-3 volumes of alcohol and allow to stand 12-24 hours; filter. The pre- cipitate contains oxalic, tartaric, and citric acids; malic acid will appear in the filtrate and may be precipitated with lead subacetate. Tannic acid is mostly precipitated immediately 16 . in slightly ammoniacal solution by calcium chloride. Lead sub- succinate is soluble in hot water, the submalate is not. Or treat the alkali salts of succinic and malic acid with lead sub- acetate till precipitation is complete, add ammonium acetate until precipitate dissolves, and add two volumes of alcohol; the lead malate precipitates, and the succinate remains in solution. Calcium malate is insoluble in a 50-70 oer cent solution of alcohol (by volume) in water. Neutralized benzoic, acetic, and formic acids do not precipitate on addition of calcium chloride and 1-2 volumes of alcohol. C. The S eeds . _ The seeds are dicotyledonous , the cotyledons being flat and round, and lying parallel to the flat side of the seed; ordinarily two seeds, sometimes three, occur in each berry and are flattened against each other. A thin cross-section of a cotyledon was mounted on a slide and treated with 5 % sodium hydroxide. Under the microscope a row of red snots appeared along the axis of the mount, which followed the location of the vacuoles, thus demonstrating the distribution of the anthra- quinone derivatives in the seed. The dry seeds were ground up in a coffee mill but with considerable difficulty caused by clogging; their oil content was such as to allow the ground seed to cohere tightly together. Thirty-two grams of the ground seed were obtained; it possessed a oleasant/nut-like odor, and was of a dark chocolate brown color. 17 . This material was extracted in a Soxhlet apparatus with ether. The extract gave a deep red coloration with dilute alka- li, and was shaken out with dilute sodium hydroxide, in order to get an approximate idea of the total anthraquinone-deri vative content. The alkali extract was acidified with dilute hydro- chloric acid, the yellow nrecipitate filtered off, carefully dried and weighed; it amounted to 0.6 gram, or about two per cent of the weight of the dry seeds. This material would not again go entirely into solution in ether, chloroform, or mix- tures of "both, or in benzene. This conduct was noted at other times when the material was allowed to dry and warmed below 100° G. Apparently some constituent of it oxidizes readily. However the material seemed most soluble in benzene, and in such solution was shaken out with the usual succession of dilute bases . Fraction Vol . of base required Comparative Color of Color of nre- Ex trac t Benzene _ cini tation Ammonium carbonate 150 c.c. Sodium carbonate 200 c.c. Sodium hydroxide 225 c.c. Pink Red trace Rich red Yellow 1 Dark red Colorless 2 The first fraction was discarded. The second fraction failed to crystallize from hot benzene or alcohol, merely se- parating as a small amount of bright yellow amorphous material. The third fraction likewise would not crystallize from benzene or alcohol. During the evaooration of the alcohol from this fraction on the steam bath, the material, on being exnosed to the air, even before all the alcohol was gone, turned black . 18 . and acquired a sticky consistency. It would not then completely redissolve in alcohol, a 'black sediment remaining. The solution was decanted from the sediment and allowed to evaporate snontan- eously; the denosit was dark purplish brown. This experience serves to show how carefully the material must be handled to prevent oxidation. The remaining ether extract of 'the seeds was evaporated on the steam bath, leaving a clear mobile, pale-yellow oil possess- ing the pleasant odor characterizing the ground seeds. This oil was dried in vacuo over concentrated sulfuric acid, and the following constants determined: The index of refraction was taken by an Abbe ref ractome ter at 19° C. and corrected to 15.5° G. Reading at 19° C -------- 1.4726 Reading corrected to 15.5° G - - 1.4749 The specific gravity was determined with a Westphal balance by the alcohol-water method. A few drops of the oil were nut in a cold mixture of alcohol and water, and the mixture adjusted by adding alcohol or water until the droplets of oil remained stationary in suspension at 15.5° G. At this point the snecific gravity of the mixture was read on the Westphal balance. Snecific gravity of oil at 15.5° G - - - 0.9197 The iodine number was determined according to the method of Wijs. The iodine monochloride solution in glacial acetic acid was added to the sample (0.2 - 0.4 gram) dissolved in chloro- form, and contained in a glass stoppered flask. After stand- ing thirty minutes a solution of potassium iodide was added. ■* 19 and the free iodine titrated with standard sodium thiosulphate solution. A blank de termination was made at the same time. Thiosulfate solution, 1 c.c. = 0.01376 gram iodine. Weight of samnle I II 0.2382 0.2932 Thiosulfate titration Blank 47.00 c.c. 47.00 c.c. Samnle 26.78 22.13 Net 20.22 24.82 Iodine absorbed 0.2780 g. 0.3416 g. Iodine number 116.7 117.1 The saponifi cation number was determined by refluxing the sample with alcoholic potash on a steam bath for thirty minutes, cooling, adding phenolphthalein, and titrating with standard acid. A blank determination was also made. Normality factor of acid 0.1003. Weight of 'Sample I II 2.1132 g. 2.0137 g. Acid titration Blank 97.31 c.c. 9J . 81 c.c. Samnle 25.00 28.50 Net 72.31 69.31 Saponification number 193.9 193.7 The ner cent soluble and insoluble acids were determined from the combined solutions from the sanonif ication value de termination Two cubic centimeters more of standard acid ' 20 . than required to liberate the fatty acids from the soap were added to the solution. The mixture was slightly warmed to facillitate cohesion of the insoluble acids, and transferred to a separatory funnel whereby the floating insoluble acids were senarated and washed. The acids were transferred by the aid of a little ether to a small weighing bottle, nlaced in a vacuum desiccator over concentrated sulfuric acid, and dried to constant weight . The nercent soluble acids was determined by titrating the aqueous solution obtained above with standard alkali, allowing for the two cubic centimeters excess of standard acid used. Weight of combined samples Weight of insoluble acids 4.1269 g. 3.3712 g. Per cent of insoluble acids 93.3 Normality factor of alkali Weight of combined samnles 4.1269 g. 0.0993. Titration with alkali 4.90 c . c . Excess standard acid 2.00 Titration of soluble acids 2.90 Calculated as butyric 0.02543 g. Per cent soluble acids 0.62 The index of refraction of the insoluble acids was taxen by an Abbe ref ractometer , and corrected to 15.5° C, as the acids solidify below 19° C. Reading ----- Corrected to 15.5° C 1.4653 1.4660 1.4635 1.4635 21 . The melting ooint of the insoluble acids was taken by drawing the melted acids into melting point tubes about one millimeter in diameter, leaving in a refrigerator 24 hours, and melting in the usual way in an onen beaker of water. The melting point was taken as the point at which the acids became transparent . Melting point of insoluble acids - - - 26.5° C. The solidifying point of the acids, or titer test, was taken by surrounding the beaker with ice-water and noting the point at which turbidity apoeared. o Solidifying point of insoluble acids - - 19.5 0. The iodine number of the insoluble acids was determined in the same way as for the oil. Thiosulfate solution, 1 c.c. = 0.01376 grams iodine. I II Weight of samole Thiosulfate titration Blank Sample Net Iodine number Because of the long oeriod of 0.2176 g. 0.2246 g-. 46.90 c.c. 46.90 c.c. 50.65 29.90 16.25 17.00 101.9 101.6 time required to dry the took place, since the higher than that of the efficient aspirator, carbon dioxide insoluble acids, oxidation evidently iodine number of the acids should be oil. The desiccator was evacuated with an and the nrecaution of drying in an atmosnhere of was not thought necessary. 22 . The neutral equivalent of the insoluble acids was determin- ed by dissolving the samples in neutral alcohol, adding phenol- phthalein, and titrating with standard alkali. Normality factor of alkali 0.0998. I II Sample 0.5365 g. 0.4286 g. Titration 13.36 c.c. 14.65 c.c. Neutral equivalent of insoluble acids 292.3 293.1 The oil is a characteristic seed-oil, resembling in these constants sunflower-seed oil, and cottonseed oil, but having a slightly lower specific gravity and less viscous behaviour. Discussion of R es ults . It is a notable fact that the sodium carbonate fraction of the alkali extract was distinctly the largest fraction in the case of the alcoholic extract and the hydrolyzed alcoholic extract, but in the case of the seeds the sodium hydroxide fraction was the largest. However, the solvent in the first two cases was ether, whereas benzene was used in the latter, which might explain the difference. But it seems likely that at least two tyres of alkali extractive materials were present, that probability being further strengthened by the fact that the sodium carbonate removed the red color from the benzene solution, leaving it a strong yellow, even though the sodium carbonate fraction was much smaller than the sodium hydroxide fraction . 23 . It was found "by experiment that a very small amount of emodin is sufficient to give a distinct red color to benzene. According to Beal and Obey (24) emodin is soluble in dilute sodium carbonate; chrysophanic acid is insoluble in sodium carbonate, but soluble in dilute sodium hydroxide. Hence it is probable that the emodin was removed by the sodium carbonate, and that the remaining material contained chrysouhanic acid, which is yellow. Unfortunately the latter fraction was over- heated or oxidized in evaporating the solvent, and identifica- tion was made impossible. While the resinous material which was hydrolyzed probably contained frangulin, the aqueous solution could not oe shown to contain rhamnose, or for that matter any reducing sugar, or even a carbohydrate. The solution contained sufficient alcohol for preservative and was kept in a stoppered flasx until exam- ined. Nevertheless this was the only case in which emodin was obtained in sufficiently pure state to permit crystallization. It is possible, during the concentration of the hydrolyzed solution for precipi tation in water, that the small amount of hydrochloric a,cid present was sufficient to convert the mannose to methyl furfural, which would be lost by volatilization. The oil obtained from the seeds has promising possibilities. As shown by its constants it resembles sunflower, maize, and (Sherman) (25). cottonseed oils. . J r, 24 . Rh pi .Prang . eed Oil Sunflower Seed Oil Maize Oil Cottonseed Oil Saponif i cation No. - - - - - 193.8 188 - 196 188 - 194 190 - 197 Iodine No. - - 118.9 104 - 135 111 - 124 104 - 116 Spec. Gravity 15.5° C. 0.9183 0.920-0.927 0.921-0.926 0 . 920—0 . 925 Index of refr. 15.5° G. 1.4749 1.474-1.473 1.475-1.47 7 1.473-1.476 It seems reasonable to exoect that the oil expressed or extracted simply from the ground dry seeds, and containing its normal content of the emodin, etc., would make an excellent lubricant and cathartic. Summary . The analysis of the fruit of Ehamnus Prangula indicates emodin and possibly chrysoohanic acid, glucose, a pentose, a small amount of light brown gum, tannic acid, malic acid, succinic acid, a resin (which was probably frangulin, though no rhamnose was found), a deep purplish-red coloring matter soluble in alcohol, and a fatty oil constituting approximately eighteen Per cent of the weight of the dry seeds. 25 Bibliography . (1) . Gathercoal, E. N., 1915: The Pharmacognosy of the Medicinal Rhamnus Barbs. Jour. Am. Pharm. Assn. V. 4 nt. 1, p.65. (2) . Matthioli , Petri Andreae, 1548: Gommentarii in VI libros Pedacii Dioscorides (Caspar Bauhin edition, 1593), lib. 1, cap. 102, p. 143, and lib. IV, cap. 168, p. 875. (3) . Linnaeus, Carl, 1753: Species Plantarum, Thomae edition (3d, 1764), pp. 279, 280, and 1671. (4) . Gerber, G. ?., 1823: Analysis of the Cortex of Rhamnus Frangula. Brande's Archiv. der Pharmacie, vol. 26, p.l. (5) . 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Neues Repert. fur Pharm., 1866, p. 295. (13) . Faust, August, 1869: Frangulin and its decomposi tion Products. Pharm. Centralhalle , vol. 10, p. 193. (14) . Liebermann, C., and Waldstein, M., 1376: Emodin from Rhamnus Frangula Bark. Ber. der Deutsch Chem. Ges., vol. IX, p. 1775. 26 . (15) . (16) . (17) . (18) . (19) . ( 20 ) . ( 21 ). ( 22 ) . (23) . (24) . ( 25 ) . (26) . (27) . (28) . Prescott, A. B., 1879: Chemical and Microscopical Analysis of the Bark of Rhamnus Purshiana. New Preparations , 1879, p. 27: Am. Jour, of Pharm. , vol. 51, p. 165. Schwahe, Paul, 1888: The Chemical Constituents of the Barks of Rhamnus Prangula and Rhamnus Purshiana. Archiv. der Pharm., vol. 226, p. 569. Thorne, T. E., and Miller, A. K., 1892: Jour, of Chem. Soc. (London), vol. 61, p. 1. Cahannes, E., 1895: The Localization of the Active Principles in Rhamnus Purshiana. Repert. de Pharm., series 3, vol. 7, p. 97. Oesterle , 0. A., 1899: Aloe-Emodin and Frangula-Emodin. 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