US2727891A

US2727891A - Vegetable

Info

Publication number: US2727891A
Authority: US
Publication date: 1955-12-20
Anticipated expiration: 1972-12-20

Description

United States Patent PROCESS OF ISOLATING HYPOTENSIVE AGENT FROM VERATRUM PLANTS Gerhard W. Kusserow, Santa Monica, Murle W. Klohs, Glendale, and Philip K. Bates and Kenneth J. Gross, Santa Monica, Calif., assignors to Riker Laboratories,

Inc., Los Angeles, Calif., a corporation of Delaware Application April 6, 1950, Serial No. 154,324

13 Claims. (Cl. 26l )236) This invention relates to hypotensive compositions and to the preparation of hypotensive compositions; more particularly it relates to the extractives and to the extraction of such hypotensive alkaloidal compositions from the drug Veratrzmz viria'e, also known as American hellebore and green hellebore, and from others of the Veratrum genus, such as Veratrum album, Verajrum californi cu m, Veratrum fimbriqtum, and Veratrum eschscholtzz'i, and from similar plant drugs containing hypotensive alkaloids.

This application is a continuation-in-part of our application Serial No. 92,256, filed May 9, 1949, now abandoned.

While many compositions have been developed and used to lower blood pressure, and the search for more satisfactory remedies for circulatory ailments, including hypertension in particular, has been active, those compositions so far placed at the disposal of the medical profession have been characterized by many disagreeable side reactions, or have been so difilcult and hence ex: pensive to prepare that their usefulness in the hands of the physician has been greatly impaired.

We have discovered that hypotensive alkaloidal compositions of outstanding therapeutic value may be obtained from various species of the genusVeratrum and kindred drugs, more particularly Veralrum viride, Verarur n escholrzii, and Veratrum fimbriazum, and we have also discovered novel and efiective processes for readily segregating such hypotensive compositions.

The compositions derived by our process from Vera- 2mm virz'de and similar species are alkaloidal and have definite activity in lowering blood pressure when administered orally, and have definite physico-chemical characteristics when subjected to the standard tests for purity of organic compounds. Because of their definite therapeutic activity, the physician is enabled to determine readily and speedily the proper dosage for the individual patient. Further, our compositions are nonhabit forming and are especially valuable in that optimum dosages may be administered orally over a considerable period of time without untoward results, building up of tolerances or disagreeable side reactions.

7 One object of the invention, as indicated in the foregoing, is to provide the medical profession with an improved hypotensive agent derived from Veratrum species such as vixide.

Another object of the invention is to provide a process for the extraction of hypotensive alkaloids from the dried rhizome and roots of Veratrum viride and other species.

Another object of the invention is to isolate in a substantially pure form the desirable alkaloidal components from the extractives of the dried rhizome and roots of Veratrum viride and other Veratrum and similar alkaloidal drug plants.

Another object of the invention is to isolate in the form of an alkaloidal composition those alkaloids derivable from Veratrum species and similar alkaloidbearing plants which are useful and desirable as bloodpressure-lowering agents, or hypotensive agents, while eliminating almost completely all of those accompanying alkaloids which produce therapeutically objectionable side reactions. We have found that many of the better known .Veratrum alkaloids are not importantly valuable as hypotensive agents and in addition produce objectionable side reactions. They include jervine, rubijervine, isorubijervine, pseudojervine, veratramine, veratrosine, and the like, and these we seek to eliminate.

Other objects will become apparent from the following specification, in which Veratrum viride is taken as typical of the various Veratrum species and similar drug plants containing alkaloidal hypotensive agents.

We have found that a menstruum comprising a solvent for the alkaloidal materials, such as chloroform to which aqueous ammonia (ammonium hydroxide) has been added, extracts the alkaloidal compounds substantially completely from dried ground rhizome and roots of Vera: trum viride. C hloroform substitutes may be employed in some instances as hereinafter indicated, and, especially, instead of chloroform with aqueous ammonia, anhydrous chloroform or equivalent saturated with ammonia gas may be substituted. The resultant extracted substance, which is a resin-like material, contains not only the de.

- sired alkaloids but also impurities and other alkaloidal constituents which produce objectionable side reactions when used therapeutically. Without at present committing ourselves to any particular theory, while it is possible that the ammonia (aqueous ammonium hydroxide) merely increases the solvent efiect of the chloroform, it now seems more probable that the desired alkaloidal constituents are naturally present inthe drug plant as acidic salts and that these are converted by ammonia to free, alkaloid bases which are soluble in chloroform (or similar solvent). 7

When fresh or undried rhizome and roots are to be treated, owing to their Wet condition they are initially extracted with aqueous acetic acid, precipitated with ammonia, and then extracted with ammoniacal chloroform or the like as more fully developed later. This acetic acid extract from the fresh or undried materials is similar in alkaloidal content to the resin-like material obtained by starting with chloroform and ammonia. It is probable that the acetic acid (or equivalent acid herein disclosed) converts the natural alkaloid salts in the drug plant to water-soluble salts which are readily soluble in the water of the acid solution.

An important aspect of this invention is the isolation from the above-described extract of the desirable alkaloid constituents which lower blood pressure without produc ing therapeutically undesirable side reactions when administered to the patient, the objectionable, more commonly known alkaloids of the various Veratrum species and various impurities being substantially or entirely eliminated. Thus, a plurality of desired alkaloids is recovered as the new alkaloidal composition. This composition is made up of amorphous bases not at present positively identified and perhaps one or more of the less known alkaloids of the group commonly derived from the various Veratrum species.

In the accompanying drawing, the figure is a flow diagram indicative of the general steps of the process of isolation and purification, and also indicative of one acceptable procedure, although variations thereof are disclosed in the following specification.

As a first step in one method of purification, the solvent is removed from the resin-like extract as by evaporation under partial vacuum at a slightly elevated temperature, for example, 30 C. to 50 C., which insures against decomposition of the alkaloids. The solvent-free extract is then dissolved in one of the lower alcohols, such as methanol, previously slightly heated.

results are obtained. A few per cent, such as 4% or 5%, constitute a practical limit. As a matter of fact, if the percentage is carried too high, there may be a tendency to reduce the proportion of alkaloids extracted from the plants. If a solvent such as methanol is employed, where it is possible to absorb as much as 20% of ammonia gas, such an ammonia gas concentration is to be avoided both because of the tendency to lower extraction capacity, and to avoid saponification which may occur if the plant drug contains any significant proportion of moisture, as when the plant drug is damp or wet.

Purification of crude extract The resinous chloroform extract is dissolved in about 440 ml. of warm methanol (about 50 C.) (2 ml. per gram of extract). The methanol solution is then poured into about 4.4 liters of 5% aqueous acetic acid, with stirring. This is about one volume of methanol to about volumes of acid solution. After standing for one hour the solution is filtered through ordinary towel cloth, then through a polishing filter using an asbestos filter disk. The clear orange brown filtrate is adjusted to a pH of about 9.2 with concentrated ammonium hydroxide diluted with an equal volume of water to yield about 14% ammonia by weight. A cream-colored precipitate forms and is recovered by filtration. After being thoroughly washed with water the precipitate is dried in a vacuum oven at about 35 C. for about 15 hours with some flow of air through the oven. When dry, the material is a cream-colored powder which will be referred to as the water-insoluble fraction containing the desired alkaloids. The yield is about 0.35% to 0.40% (Product A).

The intermediate cream-colored product (Product A) may be used as such without further purification because it possesses valuable blood-pressure-lowering properties without significantly objectionable factors. its physicochemical characteristics are as follows:

Descripti0n.An off-white, cream-colored powder which is very sternutative;

Solubility.Easily soluble in many organic solvents such as methyl alcohol, ethyl alcohol, chloroform, acetone, propylene glycol, ethylene glycol monomethyl ether (methyl Cellosolve); partially soluble in benzene and ether (ethyl ether); substantially insoluble in water;

Optical rotation range.[al =73 to ll0 at a concentration of 0.5 gram per 100 ml. in 95% ethyl alcohol;

Ultraviolet absorption-Strong absorption peak at 250 millimicrons at which the calculated specific extinction coeflicient is approximately 19,000 to 23,000 based on a concentration of 0.000016 gram per ml. of 95% ethyl alcohol;

Nitrogen c0ntent.--Approximately 2.9%;

Sulfuric acid color test.When dissolved in concentrated sulfuric acid on a spot plate, gives a yellow color which changes to brown on standing.

As previously indicated, this product, in addition to the stated solubilities above, is also soluble in dioxane, in acetic acid and other described acids, and in ammoniacal chloroform, but is insoluble in ammonium hydroxide solutions and similar aqueous basic solutions, at least at a pH above about 8.

Separation of the desirable alkaloids from the waterinsoluble fraction The water-insoluble fraction obtained above (approximately 52 gms.) is dissolved with stirring in about 1000 ml. of 5% aqueous acetic acid (about ml. per gram of the precipitate constituting the above Product A). To this solution is added about 100 to 105 gms. of reagent grade ammonium sulfate dissolved in 250 ml. of water (about solution). After standing for one hour the resultant precipitate is removed by filtration and washed with water on the filter. The filtrate and washings are combined and adjusted to a pH of 9.4 to 9.5 with concentrated ammonium hydroxide and the precipitate formed is recovered by filtration. After most of the Water has been removed from the gelatinous precipitate on the filter funnel, the precipitate is dissolved in an excess of chloroform by shaking in a separatory funnel. The chloroform layer is removed and dried with anhydrous sodium sulfate. The dried chloroform layer is evaporated to dryness under vacuum and the residue dissolved as much as possible by shaking with 250 ml. of benzene (benzol). The benzene solution is filtered and the filtrate evaporated to dryness under vacuum, yielding a pale yellow product that contains the alkaloids most desirable for blood pressure lowering. The yield is about 0.06% to 0.10% (Product B).

This pale yellow product is preferably further treated to remove the remaining less desirable rubijervine and isorubijervine constituents by shaking with about 250 ml. of acetone to dissolve the constituents soluble therein, filtering and recovering the solution, and evaporating to dryness under vacuum to yield a final pale product which may be designated as straw-colored. The yield is about .04% to 07% (Product C).

Products B and C, as apparent from the foregoing descriptive matter, are water-insoluble, soluble in solutions of nonoxidizing and nonreducing acids of the character indicated, soluble in the lower alcohols, soluble in dioxane, and soluble in chloroform and ammoniacal chloroform, but insoluble in aqueous alkaline solutions such as ammonium and alkali metal hydroxide solutions, at least above a pH of about 8. They are also soluble in benzol, and acetone, and moreover are soluble in indicated aqueous acid solutions containing ammonium sulfate, whereas some of the objectionable alkaloids which are soluble in the acid solutions themselves are precipitated as sulfates in the presence of ammonium sulfate. Similarly, while the desirable alkaloids are precipitated from such an acid solution, containing ammonium sulfate, upon addition of ammonium hydroxide to render the solution alkaline, certain undesirable alkaloids remain in the solution and are discarded upon filtration.

The processes herein described are desirably carried on in glass or similar apparatus, or in stainless steel apparatus, to insure against possible contamination by materials of which the apparatus might otherwise be constructed.

Except as otherwise indicated as important and limiting, operations are ordinarily conducted under room temperatures and pressures, and proportions may be varied considerably as will be understood by the chemist. In connection with drying temperatures, where reference has been made to drying at about 30 C. to 40 C., it will be appreciated that both higher and lower temperatures may be employed, according to the particular drying method used. if higher temperatures are employed, they should not exceed about C. because decompositlon starts at about C. and becomes serious at about C. Similarly, considerably lower temperatures may be employed where low temperature drying processes are acceptable, and possibly drying may then occur at temperatures as low as 70 C. in connection with employment of warm solvents, the upper temperatures ordinarily will be readily controlled by reason of the comparatively low boiling point of the solvent. Otherwise, it is obvious that the temperatures will be regulated to avoid decomposition.

Obviously, many modifications of the process as above described, may be made without departing from the spirit of the generic invention presented. Thus, acetone, benzene, ether, or other volatile organic alkaloid solvent capable of absorbing ammonia and inert or nonreactive toward the alkaloids, such as carbon tetrachloride and the like, may be substituted for chloroform in the initial extraction of the ground Veratrum viria'e or other Veratrum species. Other lower aliphatic alcohols up to about eight carbons per molecule, such as ethyl, propyl, isociablyihigher thanpH 7.

propyl, amyl, and hexyl alcohols and mixtures thereof, or oth er Water-.mis cibIesQIVents or :mixtures thereof, maybe Substituted for methanol in the process. Such water miscible solvents are particularly the water-miscible others, suchas m-dioxane and p-dioxane,-and the alkyl ethers of diethylene glycol (commonly known as the fCarbitols) suchas the monomethyl,nronobutyhmonoethyl, and diethyl'diethylene glycols. Other examples of such water+miscible solvents are .glycerine, propylene glycol and the methyl, ethyl, butyl, and benzyl glycol ethers known as the Cellosolvesl Instead of the acetic acid particularly mentioned, other dilute acids; maybe used, forexample, citric acid, tartaric acid, sulfuric acid, vhydrochloric acids and mixtures thereof. In practice these acids are commonly used in relatively=di1ute water solutions,'su'ch as the 5% acetic acid solution previously mentioned. in each instance Where acid generically is mentioned, it is understood that, 'for'tliis purpose, the acid shall be a nonoxidizing, nonreducing, dilute, aqueous acid solution, the acid being a freely water-soluble organic or inorganic acid capable of combining with the alkaloidal constituents present to form the water-solubleacid addition salts of the desirable alkaloidsas well understood in this art. By this definition it is intended to exclude such acids as nitric acid, sulfurous acid, chromic acid, perchloric acid, and thellike, 'at least under conditions in which oxidization or'reduction might takeplace. This, of course, will be obvious to the organic chemist.

Y-Similarly, where the use of ammonium hydroxide has been indicated, the alkali metal hydroxides maybe employed to replace ammonium hydroxide except Where the solventemployed must be volatile as in the case of theinitial chloroform extraction step in which ammonium hydroxideis used. Otherwise mixtures of these alkaline materials,.including ammonium, sodium, and potassium hydroxides andmixtures thereof, orother appropriate alkaline.solutions,.may be used interchangeably. Commonly these alkaline materials are used in concentrations -to yield a pH value of about 9.2, and sometimes a;pH value of 9.4 or 9.5.. The usual operative range is between about pH8 and vpH 10, or in other-Words, appre- Ordinarily a pH above 10 will not be employed because a higher pH tends .to saponification or kindred reaction with the desiredalkaloidal: materials undergoing treatment.

In connection with the previouslymentioned use of anhydrous drying agents, in addition to employment of anhydrous sodium sulfate, other anhydrous drying agents,'such as calcium sulfate, calcium chloride, and

copper-sulfate, may be employed, as may .many of the well-knownmeans for removing fluids from precipitates or separating immiscible liquids, such as centrifuging, whichyieldsatisfactory-results when employed in the processv insteadof filtering.

Respecting the employmentofammonium sulfate to precipitate undesired alkaloids, the proportion used is between aboutone-quartergram and about. four grams per, gram of dissolved precipitate in the solution being treated. The optimum is about two grams ofammonium sulfatqper gram of dissolved precipitate. The proportion of about four grams is approximately the .upper practical limitbecause, as the proportion of four grams is exceeded, such greater proportion of ammonium sulfate beginstothrow out the desired alkaloids. It will be noted that theammoniurn sulfate solution indicated in theabove example is a fairly strong solution containing about of; ammonium sulfate. Such strong solution is desirable inasmuch. as it reduces the volume of total solutionnto be handled. Instead of ammonium sulfate it issometirnes feasible to use suchzsulfates as the alkali metal sulfates and the alkaline earth metal sulfates. Since. it isthe small concentrationof the sulfate. radical which important, it will sometimes be possible, though apparently not so desirable, to use a minor proportiou of sulfuric acid for :example around 1% to 1.5% 11-1280. based on thetotal-solution. I g 4 in addition to the pale yellow productobtained as abovedescribed, which contains the most desirable alkaloids, other alkaloidsmay be obtained'from'the ammoniated filtrates indicated .in the purification and separation paragraphs of the above example, appropriate solvents and precipitants, such asthose herein disclosed, being-employed to separate the respective alkaloids :by steps similar to those steps described above.

Having particular reference to the pale yellow product (Product B) of the example, this blood-pressurelowering alkaloidal fraction possesses the following physico-che'rnical characteristics Sdlubility.Easily soluble in most'organic solvents such as methyl alcohol,.ethyl alcohol, chloroform, ben-' zene (benzcl), ether, acetone, ethylene glycol monomethyl ether (methyl Cellosolye), ethyl acetate, methyl ethyl ketone and propylene glycolysubstantially.insoluble in water, the lower boiling petroleum fractions such-as petroleum ether, and cyclic paraflins;

Optical rotation range..-[a] =l5 to 35 ata concentration of 0.5 gramper 100 ml. in 95% ethyl alcohol;

Ultraviolet adsorption-.Absorption,peak at 250 .to 254-millimicrons at-Which thecalculated specific extinctioncoeificient isv approximately 4,000 to 6,000basedon a concentration 01' 000008 .gram permleof 95% ethyl alcohol; there is also a definite leveling oifin the absorption spectrum curve at 280 to 3 00 millimicronsand the calculated specific extinction coefiicient using 290 millimicrons as an average is approximately 820 to .1640 based on a concentration of 0.0004 gramgper ml. of 95% ethyl alcohol; I v g Nitrogen content. Approximately 2.9%;

Sulfuric acid color rest.-When.dissolved ,in concentrated sulfuric acid on aspot plate, gives a bright orange brown colorwhich changes to alight brown upon-stand- Having reference to the straw-colored .finalzproduct (Product C) of the example produced by a. final-extraction withacetone, the characteristics of thiscomposition are in general the same as those above given for the; pale yellow product, except as differently indicated below. These characteristics areasfollows:

.Color, appearance, taste.-Straw-colored ,powder, strongly sternutatory, and having a bitter acrid taste;

Optical 1 r0tazion.['c] ='-25 :7 (Concentration =0.5% in ethanol);

Spectrophotomezric characteristics.-

Log E250 ma 3.75 0.20 Log E290 mu=3.02i0.20 (Unit of concentration 1 gm./cc. in'ethanol);

Equivalent weight.550i50;

Nitrogen based an equivalent weight.--2.33% :10 2.80%; v

Percent v0latilematter.Not greater than 5% when determined at 1 mm. Hg at 70 C.;

Melting p0int.135 C.;

S0lubility.Distilled watch-Insoluble. 99%+ soluble at a concentration of 40 mg. per cc. of solvent at'25C. Benzene.%+ soluble at a concentration of 80 mg.'per cc. of solvent at-25 C.

Having reference to the .previouslyindicated acetic acid extraction of fresh or undriedVeratr imyiridegland similar plants containing hypotensive a gcnts,.fth e extraction is made with dilute acetic acid. (or dilute. equivalent acid asheretofore disclosed). Uponcompletionof the, acid extractiomthe solution is removed. madetarni moniacal [(e. -g. pH 9.2) with ammoniumfhydroxide, thereby producing aprecipitate .which. is yrecovereld z by, filtration. Theiiltrate. maybe treated for recovery ofita product as hereinafter disclosed. The precipitate is taken Acetone up in ammoniacal chloroform produced as heretofore described, or equivalent solvent, the undissolved residue being discarded and the filtrate being evaporated to dryness under vacuum to yield a solid product. (This procedure may also be used on the dried plant drugs, but is not economically desirable for that purpose.) This product corresponds with the cream-colored intermediate product above described (Product A) which is precipitated from an acetic acid-alcohol solution or from an acetic acid-dioxane solution by rendering alkaline with ammonium hydroxide, and then recovered as a precipitate. This acid-extracted residual product obtained by the evaporation of the ammoniacal chloroform may be used as such, or mixed with the described intermediate cream-colored product. Also, it may be further purified with, or in the same manner as, such intermecfiate product to yield final products corresponding with those pre viously described (Products B and C). Like the creamcolored intermediate product, this residual product recovered by the evaporation of the ammonia-chloroform solvent is substantially alkali-insoluble, water-insoluble, soluble in the described acids, soluble in chloroform, and soluble in the described alcohols or others such as methanol or dioxane.

EXAMPLE As an example of a specific procedure of acid extraction of a freshly harvested (undried) plant drug, the following is given:

Extraction of plant Approximately 8 pounds of ground, undried, green hellebore containing about 85% moisture is placed in a glass percolator and covered with 10% aqueous acetic acid (about 8 pounds). After standing for 16 hours the extract is drained.

Nora-Taking into consideration the moisture content of the drug in this example the acetic acid in the total water present is about 5.5% and the ratio of total menstruurn to dry material (solids) is in the order of 12 to 1 by weight. These figures are not critical. The acid strength can vary from 5% to 6% and the ratio of menstruum to solids from, say, 10 to 1 up to 14 to 1. However, the concentration and quantity of menstruum added should preferably be adjusted within these limits based on the approximate moisture content of the drug.

Purification of crude extract The aqueous acid extract is adjusted to a pH of 9.5 with concentrated aqueous ammonium hydroxide (28% NHa). The precipitate formed is recovered by filtration, and after being thoroughly washed with water is dried in a vacuum oven at about C. with some flow of air through the oven. The yield is 0.3% to 0.5% (dry basis for starting drug).

The dried material is then treated with about 300 ml. of chloroform to which 10 ml. of 28% aqueous ammonia has been added (or preferably with a chloroform solution of gaseous NHa) to dissolve as much as possible of the material. The chloroform-insoluble residue (which is chiefly inert impurities) is removed by filtration and the chloroform extract is evaporated to dryness under vacuum. The material, when dry, is a cream-colored powder corresponding with that heretofore referred to (Product A) as the water-insoluble fraction containing the desired alkaloids and also designated as the intermediate product. The yield is 0.15% to 0.25% (dry basis).

As previously indicated, other useful alkaloids may be obtained from the ammonia filtrates obtained upon precipitation with ammonium hydroxide from acetic acidalcohol solutions (or other acid-alcohol solutions of disclosed acids and alcohols), or from acetic acid-dioxane solutions (or other acid and water-miscible ether or similar solutions disclosed). Similar desirable alkaloids are similarly obtainable from the above described ammonia filtrate obtained upon neutralization with ammonium hydroxide of the acetic acid solution produced in extracting fresh or undried starting materials. Both types of ammonia filtrates mentioned are to be treated in the same manner, and may be combined for such treatment. The treatment consists in extracting desired constituents from the ammonia filtrates with chloroform. Chloroform is admixed in any preferred manner with the aqueous ammoniacal filtrate, as by agitation therewith in sufiicient quantity to extract from the aqueous ammonia filtrate all of the chloroform-soluble constituents. The chloroform solution is then allowed to stratify and separate from the aqueous layer.

The chloroform solution is then evaporated to dryness under vacuum to yield a chloroform-soluble residue which constitutes a therapeutically useful intermediate product. This may be designated as Product D.

A further purified product may be obtained from the chloroform-soluble fraction by ether extraction, any undissolved residue being discarded and the ether solution evaporated to dryness under vacuum to yield the desired further purified product. This may be designated as Product E.

More specifically, the clear, aqueous ammoniacal filtrate used as the starting material for this phase of the invention, which commonly has a pH of about 9.2, is extracted in a continuous extractor with chloroform for about sixteen hours or more until all of the chloroformsoluble constituents have been taken up in the chloroform. This chloroform extract is then evaporated to dryness under vacuum. It is partially water-soluble. It contains desirable hypotensively active alkaloids. The yield is about 0.09% to 0.14%. The physicochemical characteristics of this chloroform-soluble extract (Product D) are as follows:

Solubility-Readily soluble in methyl alcohol, ethyl alcohol, chi reform, propylene glycol and methylCellosolve; partially soluble in dioxane, acetone, ether, benzol and water;

Optical rotation range.-lul =l0 to 25 at a concentration of 0.5 gram per 100 ml. ethyl alcohol;

Ultraviolet abs0rpzion.I-las an absorption peak at 250 to 254 millimicrons at which the calculated specific extinction coefiicient is approximately 5,000 to 7,000 based on a concentration of .00008 gram per ml. of 95% ethyl alcohol;

Nitrogen. c0ntent.Approximately 2.4%

Sulfuric acid color test.When dissolved in concentrated sulfuric acid on a spot plate gives a reddish-brown color which changes to brown upon standing.

When it is desired to eliminate some of the less desirable agents from the last described chloroform-soluble, partially water-soluble extract, the residual product obtained by the evaporation of the chloroform to dryness is taken up in anhydrous ether and the ether extract evaporated to dryness under vacuum.

More particularly, this residual chloroform-soluble product is extracted five times with successive portions of anhydrous ether (ethyl ether), using 200 ml. of ether per 10 grams of material in each portion. The ether extract is then evaporated to dryness under vacuum. The residue is a light tan-colored resinous material containing desired hypotensively active alkaloids free from some of the less desirable alkaloids contained in the chloroform-soluble starting residue. The yield is about 0.04% to 0.06%.

The light tan-colored product just described (Product E) is a desirable therapeutic, blood-pressure-lowering alkaloidal fraction possessing the following physico-chemical characteristics:

Solubility.Readily soluble in chloroform, dioxane, ether, benzene, ethyl alcohol, methyl alcohol, propylene glycol, acetone and methyl Cellosolve; partially soluble in water;

Optical rotation range.[a] =-6 to -20 at a 11 concentration of 0.5 gram per 100 95% lethyhaleohol; I

Ultraviolet -absorption.-Has an absorption peak at approximately ,250 millimicrons at which the calculated specific-extinction coetficient is 4,500'to 7,500 based on a concentration of 0.00008 gram -per -ml. of 95% ethyl alcohol;

Nitrogen content-Approximately 2.3%;

. Sulfuric acid color test.When dissolved in concentrated sulfuric acid on a spot plate gives a reddish-brown color which-changes to brown upon standing.

If desired, anhydrous benzol maybe substituted for the'anhydrous ether for extracting the described :chlo'roform-soluble, partially water-soluble fraction, and .the product thereby obtained has physico-chemical characteristics which aresubstantially the same asthose of the anhydrouscether extract.

Theabove described processes-may be used also for the recovery audvisolation of kindred alkaloids from similar alkaloid bearing plants.

,Havingparticular reference to ProductC of this invention, the constituents are amorphous or noncrystalline forms 'of alkaloids which are almost wholly water-insoluble. Apparently they are principally unidentified amorphous bases possibly containing one or more of the known amorphous alkaloids from Veratrum species.

However, .this composition is substantially free from cevadine (which is water-soluble) and veratridine, these two together being sometimes referred to as veratrine. Thisproduet is also substantially free from the commonly known Veratrum alkaloids particularly -jervine, rubijervine, isorubijervine, pseudojervine, veratramine, veratrosine, and the like. Thus, our process results in eliminating from the Veratrum alkaloids those alkaloids which are commonly crystalline and at the same time produce therapeutically undesirable side reactions. In other words, we have been able to isolate an alkaloidal composition whcih contains only the desirable hypotensively active alkaloids, only traces of the usual crystalline, objectionable alkaloids being found.

In-Product B, some ofthe crystalline, better known Veratrum alkaloids are found, more particularly rubijervine and isorubijervine, but the proportion of these is small enough and the objectionable characteristics are so minor, at least in the presence of the other constituents, that Product B is highly desirable asa hypotensive agentcxceptpossibly with highly sensitive patients.

While Product A contains larger proportions of the commonly known crystalline type of alkaloids, nevertheless, the high content of amorphous constituents renders the composition much more desirable than Veratrum products heretofore available, in that objectionable side reactions are minor.

Products D and E are partially water-soluble,ainor phous alkaloid compositions, with scarcely a trace of crystalline alkaloids, and have been "found to havevery good hypotensive characteristics, although apparently not the equivalent of those 'ofProduct C.

Wherethe term crystalline or crystallineetype is applied to various alkaloids, it signifies those which are commonly'known as such and are recoverable in crystalline form, and the term amorphous applies vto those alkaloids which are now characteristically separable in amorphous 'form. V

Inasmuch as many variations of the generic, invention herein "disclosed 'willbecome apparent to 'those skilled in this art, it 'is intended to protect all modifications falling withinthe scope of the patent claims.

We claim as our invention:

1. A-processfor isolating hypotensive alkaloids substantially-free-from those having objectionable side reactions,--said process including the steps of: obtaining-fa solution of alkaloids from plants of the Veratrum family in arnmoniacal organic'solvent for said alkaloids; dissolving in an aqueous solution of a non-oxidizing, nonreducing a i t os a ka o u ble therein which are found in ;said ammoniacal organic solvent; :addingammonium sulfate to such acid solution toprecipitatealkaloids having-therapeutically objectionable side reactions; rejecting ;the insolubles formed in such acid solution; precipitating alkaloidfractions from the resultant acid solution, following removal of said insolubles, by alkalizing with an agent from the class consisting of ammonia and alkali metal hydroxides; dissolving the last-mentioned precipitate in chloroform; evaporating to dryness; taking up from the residue in benzene those fractions soluble therein; and recovering the benzene soluble fractionsas a hypotensive composition of alkaloids.

'2. A process for isolating a mixture of hy'potensive alkaloids from Veratrum plants containing the same, which process comprises the steps of; obtaining a solution of the Veratrum alkaloids in an aqueoussolution of a non-oxidizing, non-reducing acid; adding to said solution sulfate ions'to precipitate sulfates of undesired alkaloids; rejecting the insolubles formed thereby; rendering the-resulting solution alkaline to produce a precipitate of alkaloids; dissolving the last-mentionedprecipitate in chloroform; evaporating to dryness; taking up fromthe residue in benzene, those alkaloids soluble therein; and recovering the benzene-soluble alkaloids as a hypotensive composition.

3. A process as defined'in claim 2, in which the solution of Veratrum alkaloids in said aqueous solution of said acid is obtainedby a series of steps including: extracting Veratrum plants withan organic alkaloid solvent made alkaline with ammonia; replacing such solvent and ammonia with a water-miscible organic alkaloid solvent; mixing the resulting solutionwith an aqueous solution 'Of a non-oxidizing, non-reducing acid; and rejecting materials insoluble in said last-mentioned aqueous solution.

4. ,A rprocess as defined in claim 3, in which the firstmentioned organic alkaloid solvent is chloroform containing ammonia gas dissolved therein.

5. A process as defined in claim 3, in which the firstmentioned organic alkaloid solvent is chloroform containing ammonia gas dissolved therein; and said water-miscible organic alkaloid solvent is dioxane.

'6. A, process;as defined in claim 2, in which the solution of Veratrum alkaloids in said aqueous solution of said acid is obtained by a series of steps including: extracting Veratrum plants with chloroform containing ammonia gas "dissolved therein; replacing said chloroform and ammonia with dioxane; mixing the resulting solution with an aqueous solution of acetic acid;:and rejecting materials insoluble in said aqueous solution of acetic acid.

.7. The process as defined in claim .6, in which the alkaloids soluble in said aqueous solution of acetic acid are precipitated by rendering said aqueous solution alkaline; and-the precipitated alkaloids are separated and again dissolved inanaqueous solution ofacetic acid.

58. A processas defined inclaim 2, in which the solu tionof the vVeratrum alkaloids in-said aqueous-solution of said acidis obtained by a series of steps including: extracting Veratrum plants with an aqueoussolution of acetic acid; rendering the extract alkaline to-precipitate Veratrum alkaloids; recovering in solution in chloroform the chloroform-soluble alkaloids, the insolubles being rejec-ted; evaporating the resulting solution -ofalkaloids to dryness; 'anddissolving the residue in an aqueous solution of a non-oxidizing, non-reducing acid.

9. A process as defined in claim 2, in which said nonoxidizing,-non-reducing acid is acetic acid.

10. *Aprocess as defined in claim 2, in which the sulfate ions-are added in the form of ammonium sulfate.

--1l. A process as defined in claim '2, in which the lastrncntioned composition is dissolved in acetone and any insoluble material and crystalline precipitate forming therein is discarded; and the soluble material recovered as a further purified hypotensive composition.

12. A process for isolating a mixture of hypotensive alkaloids from Veratrum plants containing the same, which process comprises the steps of: initially extracting the plants with chloroform containing ammonia gas absorbed therein to obtain a solution of alkaloids; replacing the chloroform and ammonia in said solution with dioxane; thereafter replacing said dioxane with an aqueous solution of acetic acid while rejecting materials insoluble in said acid solution; adding ammonium sulfate to said acid solution to precipitate sulfates of undesired alkaloids; rejecting the insolubles formed thereby; rendering the resulting solution alkaline with ammonia to produce a precipitate of alkaloids; dissolving the last-mentioned precipitate in chloroform; evaporating the resulting chloroform solution of alkaloids to dryness; taking up the residue in benzene; discarding insoluble material; evaporating the remaining benzene solution of alkaloids to dryness; taking up the resulting residue in acetone; discarding any insoluble material and any crystalline precipitate which forms in said acetone; and recovering the remaining acetone-soluble alkaloids as a hypotensive composition.

13. A process for isolating a mixture of hypotensive alkaloids from Veratrum plants containing the same, which process comprises the steps of: initially extracting the plants with chloroform containing ammonia gas absorbed therein to obtain a solution of alkaloids; replacing the chloroform and ammonia in said solution with dioxane; thereafter replacing said dioxane with an aqueous solution of acetic acid while rejecting materials insoluble in said acid solution; rendering said acid solution alkaline with ammonia; filtering out the resultant precipitate; dissolving such precipitate in acetic acid; adding ammonium sulfate to said acid solution to precipitate sulfates of undesired alkaloids; rejecting the insolubles formed thereby; rendering the resulting solution alkaline with ammonia to produce a precipitate of alkaloids; dissolving the lastmentioned precipitate in chloroform; evaporating the resulting chloroform solution of alkaloids to dryness; taking up the residue in benzene; discarding insoluble material; evaporating the remaining benzene solution of alkaloids to dryness; taking up the resulting residue in acetone; discarding any insoluble material and any crystalline precipitate which forms in said acetone; and recovering the remaining acetone-soluble alkaloids as a hypotensive composition.

References Cited in the file of this patent UNITED STATES PATENTS 684,650 Spiegel Oct. 15, 1901 2,206,407 Kondo et a1. July 2, 1940 2,226,528 Folkers Dec. 24, 1940 OTHER REFERENCES Chem. Abst., vol. 8, p. 674 (1914) abstract of article by Keller et al., in Arch. Phanm, vol. 251, pp. 207-16 (1914).

Chem. Abst., vol. 17, p. 183 (1923) abstract of article in J. Am. Pharm. Assoc., vol. 11, pp. 166-174 (1922).

Fried et al.: I. Am. Chem. Soc., vol. 71, pp. 3260-61 (1949).

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Manceau et al.: Ann. pharm. franc., vol. 3, pp. 11-14 (1945).

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Claims

1. A PROCESS FOR ISOLATING HYPOTENSIVE ALKALOIDS SUBSTANTIALLY FREE FROM THOSE HAVING OBJECTIONABLE SIDE REACTIONS, SAID PROCESS INCLUDING THE STEPS OF: OBTAINING A SOLUTION OF ALKALOIDS FROM PLANTS OF THE VERATRUM FAMILY IN AN AMMONIACAL ORGANIC SOLVENT FOR SAID ALKALOIDS; DISSOLVING IN AN AQUEOUS SOLUTION OF A NON-OXIDIZING, NONREDUCING ACID THOSE ALKALOIDS SOLUBLE THEREIN WHICH ARE FOUND IN SAID AMMONIACAL ORGANIC SOLVENT; ADDING AMMONIUM SULFATE TO SUCH ACID SOLUTION TO PRECIPITATE ALKALOIDS HAVING THERAPEUTICALLY OBJECTIONABLE SIDE REACTIONS; REJECTING THE INSOLUBLES FORMED IN SUCH ACID SOLUTION; PRECIPITATING ALKALOID FRACTIONS FROM THE RESULTANT ACID SOLUTION, FOLLOWING REMOVAL OF SAID INSOLUBLES, BY ALKALIZING WITH AN AGENT FROM THE CLASS CONSISTING OF AMMONIA AND ALKALI METAL HYDROXIDES; DISSOLVING THE LAST-MENTIONED PRECIPITATE IN CHLOROFORM; EVAPORATING TO DRYNESS; TAKING UP FROM THE RESIDUE IN BENZENE THOSE FRACTIONS SOLUBLE THEREIN; AND RECOVERING THE BENZENE SOLUBLE FRACTIONS AS A HYPOTENSIVE COMPOSITION OF ALKALOIDS.