United States Patent [191 [11] Patent Number: 4,818,533 Boulware et al. [45] Date of Patent: * Apr. 4, 1989

[54] PRODUCTION OF HIGH PURITY [56] References Cited ALKALOIDS us. PATENT DOCUMENTS [75] Inventors; Richard T. Boulware, High point, 3,843,788 10/1974 lwasa et a1...... 424/195.1 N_C_. George Schlowsky’ 3,849,561 11/1974 Iwasa BI a1. 514/284 - ’ 4,145,412 3/1977 Landanyi ...... 424/58 Rldgewood’ N'J' 4,335,110 6/1982 Collins 424/145 . ' . . 4,376,115 3/1983 McCrorey . 424/145 [73] Asslgnee~ VIP?“ Pharmaceumal, Inc-2 Fm 4,406,881 9/1983 Ladanyi ...... 424/49 Collms, Colo. FOREIGN PATENT DOCUMENTS [ * ] Notice: The portion of the term of this patent 2755577 6/1979 Fed Re of German sabsequent 1° Aug- 30’ 2005 has been 2856577 6/1980 Fed. Reg. of Germanz . dlsclalmed- 495311 4/1976 U.S.S.R. . 2078109 1/1982 United Kingdom ...... 514/280 21 A l.N .: 96262 [ 1 pp 0 ’ Primary Examiner-—John W. Rollins [22] Filed: Sep. 4, 1987 [57] ABSTRACT A method for extracting valuable alkaloid materials Related U.S. Application Data from containing the same comprising slurrying [63] continuation of Ser. No. 753,264’ JuL 9, 1935, abam the comminuted material in amixture of water and cloned, which is a continuation-in-part of Ser. No, ? cosolvem at a PH of 8-51 addmg a n9nP0la1',.WaieT' 596,539, Apr‘ 4, 1984, abandoned, insoluble, solvent to d1ssolve the alkalold, washing the nonpolar phase with acidulated water to convert the [51] Int. GL4 ...... A61K 35/78; A61K 31/44 alkaloid to the imminium ion or acid salt form, precipi [52] U.S. C1...... 424/195.1; 514/279; rating the alkaloids, and recovering and drying the alka 514/280; 514/282 loid. [58] Field of Search ...... 424/1951; 514/282, 5 14/ 279, 280; 566/61 10 Claims, 1 Drawing Sheet US. Patent ' Apr.4, 1989 4,818,533

LeafRoot or MILL COLLECTOR

Me OH VAPOR

NcIOH MeOH cHzcl-z AGITATED H20 DISTILLATION CONDENSER MeoH TANK Organic COLUMN TANK Waste

STEAM _ H2O TO MeOH Organic SEWER Recycle Waste

CH2CL2 AIkuIoids CHZCLZ VAPOR I—_——_I “2° DISTILLATION CONDENSER Citric Acid COLUMN TANK

Recycle

Nu CL

Aikoloids DIGEST ‘ TANK (S)- FILTER DRY PRODUCT

H2O NuCL TARS TO SEWER 4,818,533 1 2 mixed alkaloids of the plant Sanguinaria: namely, san PRODUCTION OF HIGH PURITY ALKALOIDS guinarine, chelerytherine, and protopine. While few references can be found in the literature regarding the This application is a continuation, of application Ser. usage of any of the pure benzophenanthridine alkaloids, No. 753,264, ?led July 9, 1985, now abandoned, which plants containing such compounds have been used for a is a continuation-in-part of Ser. No. 596,589, ?led Apr. wide variety of medical ailments. 4, 1984, now abandoned. Several patents have disclosed the use of extracts of Sanguinaria for such purposes, notably U.S. Pat. No. BACKGROUND OF THE INVENTION 4,145,412; U.K. Pat. No. 2,042,336; ‘U.S. Pat. No. This invention relates to a method for recovering 4,376,115; U.S. 4,406,881; German Pat. No. 2,907,406; active alkaloids which form a free base soluble in non Belgain Pat. No. 888,843. These patents describe the use polar solvent and a water-soluble acid salt, from plants Sanguinaria extracts as antimicrobial agents as well as containing these compounds. mouth treating agents. Many plants contain valuable alkaloids, which can be The prior art cited above describes a method of ex used in treating a variety of conditions. Among the uses tracting active ingredients from Sanguinaria canadensis for plant-derived alkaloids are the chelidonene group, by extracting cut or ground bloodroot with methanol which have been found to be useful against malignant for at least 24 hours at an elevated temperature, ?ltering tumors, papillomatosis of the larnyx, and early forms of the liquid extract contained, evaporating the extract to lupus erythematosus. dryness, dissolving the dried residue in chloroform, Alkaloids of the chelidonene group have so far been 20 adjusting the chloroform solution to an acid pH by the found in the ?mbriated poppy (Glaucium ?mbrilligerum addition of hydrocloric acid, ?ltering the acidi?ed ex Boiss.) and in Corydalis ledebouranl'a Kar. et Kir. and tract, evaporating it to dryness, and dissolving the dried Corydalis severtzovii Regal. These alkaloids have also residue in glycerine for mixing with a carrier. been found in the , such as the V.A. Chelombit’ko et al., in Khimiko~Farmatsevtiches () microcarpa (Maxim) Fedde. 25 kii Zhurnal, No. 2, 49-52, Feb., 1968, disclose a method Sanguinaria canadensis, Linn (family Papvaraceae) is of extracting chelerythrine and sanguinarine from commonly known as bloodroot, redroot, puccoon, plants by soaking the plants in 10% ammonia solution teterwort, etc., and is a perennial herb native to North covered with dichloroethane and then in dichloroeth America. The plant and its juices have been used for ane. The solution was alkali?ed and the alkaloid bases various purposes in pre-historical and historical times. 30 precipitated. The plant has been used, in particular, as a folk remedy. U.S.S.R. Pat. No. 495,311, discloses a method of The plant has generally been used whole, either undried obtaining greater celandine alkaloids by extracting the (fresh) or dried. The usual procedure is to powder the leaves of plants containing the alkaloids with aqueous dried plant and mix it with a carrier. This folk remedy acetone acidi?ed with acetic acid, removing the ace has been tried for such conditions as asthma, bronchitis, tone by distillation, and raising the pH to 10-11 with dysentery, ringworm, and a substantial list of other ammonium hydroxide to precipitate berberine. The ailments. precipitate is extracted with trichloromethane to obtain The rhizome of the plant has not been used much in the soluble berberine group alkaloid bases, which can be modern times. The present principal use of Sanguinaria worked up into berberine, chelidonine hydrochloride, is as stimulate expectorant in cough syrups and in home 40 and a mixture of sanguinarine, chelerytherine, and opathic medicine. chelilutin. As early patent, U.S. Pat. No. 209,331, disclosers the German Pat. No. 2,856,577 discloses a method of use of bloodroot, zinc chloride, and Kerosene oil in preparing benzophenanthridine alkaloids from plant equal proportions for treating open sores. U.S. Pat. No. materials by moistening chopped plant materials with 433,257, describes a salve of pulverized bloodroot, ar 45 an ammonia solution and subjecting the mixture to a menian bole, powdered rosin, lard, and Stockholm tar trichloromethane extraction to enrich the alkaloids. for use in the treatment of piles. US. Pat. No. 2,344,830, Sulfuric acid is added and the solvent is simulataneously discloses the use of a mixture of zinc chloride, stibnite, distilled off. The residue is basi?ed with ammonia to and bloodroot to ?x and outline diseased tissue for exci precipitate the alkaloid free bases which are then ?l sion by surgery. tered off. More recently, it has been discovered that extracts of sanguinaria and other plants of the families Papavera SUMMARY OF THE INVENTION ceae. Fumariaceae, and Berberidaceae such as Macleaya The present invention is directed to a more re?ned cordata, , Corydalis sevctcozii, C. method of obtaining alkaloids form plants of the families ledebouni, Argemone mexicanus, and Chelidonium majus Papaveracease, Fumariaceae, and Berberidaceae such contain benzo-c-phenanthridine alkaloids which are as Sanguinaria canadensis, , Macleaya, believed to have valuable properties in conditioning microcarpa Corydalis sevctvozzii, C. ledebouni, Argemone oral tissue, as well as in preventing and treating gingivi mexicanus, Chelidonium majus,. Bocconiafrutescens, and tis, periodontitis, and mouth odors. mixtures thereof, and other plants which contain alka The pure chemical sanguinarine, chelerythrine, pro loids which form a free base soluble in non-polar sol topine, chelerubine, chelilutine, sanguilatine, macar vents and a water soluble acid salt. pine, sanguirubine, allocryptopine, homochelidonene, The benzo-c-phenanthridine alkaloids which have and berberine, can be isolated form plants other than been identi?ed in Sanguinaria include sanguinarine, Sanguinaria. They are also available, although rarely, chelirubine, sanguirubine, chelilutine, chelerythrine, from some chemical supply houses, semi-puri?ed forms 65 and sanguilutine. of the alkaloids are commercially available, and these In the process of the present invention, alkaloid con are generally referred to as sanguinarine nitrate and taining plant material is ground and slurried with water sanguinarine sulfate. These “salts” are the salts of the or water/solvent mixture. The pH is adjusted to 8.5 4,818,533 3 4 with any available base, such as sodium carbonate, am Plant material, generally roots or leaves, is broken up monium hydroxide, potassium hydroxide, sodium hy with a hammer mill and collected in a bag collector. droxide, and the like. This converts the alkaloidal con The comminuted plant material is optionally recovered stituents to the free base or pseudo base form which in a wet scrubber before being sent to an agitator tank. may then be extracted in non-polar solvents such as the The comminuted plant material is agitator in a tank with nonpolar solvent, chloroform, or dichlorethane. It is water, methanol and methylene chloride, and caustic, thus possible to extract the alkaloidal constituents away wherein the alkaloids are extracted into the methylene from more polar, water soluble substances, which re chloride. The methylene chloride containing the alka main with the aqueous slurry of ground plant matter. loids is directed to an agitated tank where water and A phase transfer agent is added to the slurring of 0 citric acid are added to the methylene chloride solution. ground plant material as a cosolvent for the alkaloids of The two phases are agitated and allowed to reform. The interest. The phase transfer agent is soluble in both alkaloids are now disolved in the water citric acid methylene chloride and water, and can be selected from phase. The methylene chloride phase is taken to a re the group consisting of lower (C1-C4) alcohols, lower covery still and the alkaloids which are dissolved in the (C1-C4) ketones, tetrahydrofuran, and dirnethyl sulfox water and citric acid phase are precipitated with sodium ide. The alkaloids of interest for the present process, chloride, digested, and ?ltered to remove water, excess such as the benzophenanthridines, which are insoluble sodium chloride and soluble tars. The ?lter cake is dried in water in the pseudobase form, are soluble in the phase to obtain the puri?ed alkaloid product. transfer agent, which in turn is soluble in the nonpolar 20 solvent as well as water. DETAILED DESCRIPTION OF THE One method of processing is for an aqueous slurry of INVENTION base converted alkaloids to be emulsi?ed with the im EXAMPLE I misible non-polar phase. This facilitates extraction of the alkaloids into the nonpolar solvent. One hundred grams of root from bloodroot, Sangui The emulsion is broken by ?ltration. This frees the naria canadensis, were ground or pulverized to a 60/40 slurry of ground plant matter and facilitates a phase mesh and slurried with 500 ml. water, 100 ml methanol, separation of the aqueous phase from the immiscible and 2.5 g sodium hydroxide. The pH of the mixture was non-polar phase. The alkaloids remain with the non 8.5. The slurry was transferred to a 2000 ml separatory polar solvent. 30 The nonpolar phase is washed with acidulated water funnel and 600 ml methylene chloride were added. The to convert the alkaloids to the imminium ion or acid salt phases were mixed until the methylene chloride turned form. In this form, the alkaloids are water soluble and a deep purple color. transfer to the aqueous phase. Nonpolar impurities are The purple emulsion was broken by passing the liquid through a coarse ?lter. This ?ltering step broke the retained in the nonpolar phase. 35 At this point, the alkaloids have passed through two emulsion and removed extraneous ground plant mate steps which have left polar impurities behind and left rial. nonpolar impurities behind, and the alkaloids exist in a The methylene chloride phase was transferred to a highly re?ned form. They may at this point be precipi clean 2000 ml separatory funnel and washed with 500 tated as acid salts or once again be made basic and ex ml of water which contained 15.0 g citric acid. The tracted back into a nonpolar solvent for the isolation of alkaloids imparted a deep orange color to the water speci?c alkaloids. phase. The alkaloids may be precipitated as the chloride salt The water phase was transferred to a clean ?ask and with sodium chloride or hydrochloric acid, or as the heated with stirring to 60° C. nitrate or sulfate salt with nitric or sulfuric acid, respec Next, 75.0 g sodium chloride (or 10 g/ 100 ml) is tively. added with stirring at 60° C. and an orange ?occulant It has been found that recovery of benzophenanthri precipitate was observed to form. dine alkaloids from bloodroot and Macleaya cordata by Stirring was discontinued, and the precipitate was the process of the present invention is nearly quantitia allowed to sit for 24 to 48 hours. tive. 50 The precipitate was collected and dried at 40° C. In a preferred embodiment of the invention, the base The precipitate was about 42% sanguinarine chloride - converted alkaloids are slurried in a nonpolar solvent by weight and approximately 80% by weight mixed phase containing suf?cient water and cosolvent (phase benzophenanthridine alkaloids. The remaining 20% transfer agent) to effect the extraction of the alkaloids comprised excess sodium chloride and moisture. into the nonpolar phase. The nonpolar solvent contain ing the alkaloids is separated from the ground plant EXAMPLE II material by ?ltration. One hundred grams of Macleaya Cordata leaves were After ?ltration, water is added to the nonpolar sol ground to a 60/40 mesh and were slurried with 500 ml vent containing the alkaloids to remove the polar impu water, 100 ml methanol, and 2.5 g sodium hydroxide. rities and most of the cosolvent (phase transfer agent) The pH of the slurry was 8.5. from the non-polar containing alkaloid phase. The slurry was transferred to a 2000 ml separatory Processing then proceeds as above beginning with funnel and 600 ml methylene chloride was added. The washing the nonpolar phase with acidulated water. phases were mixed until the methylene chloride became a deep purple. DETAILED DESCRIPTION OF THE 65 The slurry, now in the form of a emulsion, was ?l DRAWINGS tered through a coarse ?lter. This ?ltering step broke The drawing is a block diagram of the process on a the emulsion and removed extraneous ground plant commercial scale for extracting alkaloids form plants. material. 4,818, 533 5 6 The methylene chloride phase was transferred to a EXAMPLE IV clean 2000 ml separatory funnel and washed with 500 ml of water which contained 15.0 g citric acid. The The procedure of Example III was repeated using 100 grams of ground Macleaya cordata leaves instead of alkaloids imparted a deep orange color to the water 5 100 grams of ground bloodroot leaves. phase. The precipitate collected was approximately 37% The water phase was transferred to a clean ?ask and sanguinarine chloride by weight. The total mixed ben heated with stirring to 60° C. zophenanthridine alkaloids was about 80% by weight. Next, 75.0 i sodium chloride was added with stir What is claimed is: 10 1. A method for extracting an alkaloid from a plant ring at 60° C., an an orange precipitate formed. containing the alkaloid, which alkaloid forms a free base Stirring was discontinued, and the precipitate was soluble in non-polar solvents and a water soluble acid allowed to sit for 24 to 48 hours. The precipitate was salt comprising grinding the plant material, slurrying collected and dried at 40° C. the plant material in a mixture of water and a cosolvent, The precipitate was about 37% sanguinarine chloride adjusting the pH of the mixture to a pH of about 8.5, extracting the alkaloid in a nonpolar solvent which is by weight and approximately 80% by weight mixed insoluble in water, dissolving the alkaloid into water by bentophenanthridine alkaloids. The remaing 20% of the lowering the pH of the solution with acid, adding the precipitate was excess sodium chloride and moisture. salt of a mineral acid or a mineral acid to precipitate the 20 alkaloid, and collecting the precipitated alkaloid. EXAMPLE III 2. The method of claim 1 wherein the nonpolar sol On hundred grams of ground bloodroot was pre vent is selected form the group consisting of methylene mixed with 10.0 grams of sodium carbonate and slurried chloride, chloroform, and dichloroethane. 3. The method of claim 1 wherein the cosolvent is for one hour with a solution of 660 grams methylene 25 selected from the group consisting of lower (C1-C4) chloride, 72 grams isopropyl alcohol, and 28 grams alcohols, lower (C1-C4) ketones, tetrahydrofuran, and water. dimethylsulfoxide. The slurry was then ?ltered through a coarse ?lter to 4. The method of claim 1 wherein the pH of the wa remove the plant material and the deep purple extract ter-cosolvent mixture is raised with a compound se 30 lected from the group consisting of sodium carbonate, .was transferred to a 2000 ml separatory funnel. The ammonium hydroxide, potassium hydroxide, and so extract was washed twice with 500 ml. neutral water, a dium hydroxide. total of 1000 ml, to remove water soluble impurities as 5. The method of claim 1 wherein the mineral acid is well as residual isopropyl alcohol. The isopropyl al selected form the group consisting of hydrochloric cohol-water phase was retained, and the isopropyl alco acid, sulfuric acid, and nitric acid. hol was recovered by distillation. 6. The method of claim 1 wherein the mineral acid salt is sodium chloride. The washed methylene chloride extract was then 7. The method of claim 1 wherein the alkaloid is a extracted with 300 ml water containing 9.0 grams citric benzophenanthridine alkaloid. acid, and then with 200 ml water containing 6.0 grams 8. The method of claim 7 wherein the plant is selected citric acid. The alkaloids were retained in the acidulated from the plant families consisting of Papaveraceae, water phase and the methylene chloride phase was Fumariaceae, and Berberidaceae. retained for distillation and recycling. 9. The method of claim 7 wherein the plant is selected from the group consisting of Sanguinaria canadensis, The water phase was heated with stirring to 65° C., 45 Macleaya cordata or , Corydalis and 50 grams sodium chloride was added as a precipitat sevctvozzii, C. ledebouni, A rgem z'ne mexicanus, ing agent. The solution and precipitate were allowed to Chelia'onz'um majus, Bocconia frutescens and mixtures cool and digest for 24 hous. The precipitate was then thereof. collected and dried for 16-24 hours at 40° C. 10. The method of claim 1 wherein the amount of water used in the mixture of water, and cosolvent, is the The precipitate collected was approimately 42% minimum amount required to effect extraction of the sanguinarine chloride by weight and about 80% by alkaloids into the nonpolar solvent. weight mixed benzophenanthridine alkaloids. * ll‘ 1‘ * *

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