A CHEMICAL STUDY OF BOCCONIA LATISEPALA WATS.

XORGE ALEJANDRO DOMINGUEZ, JEST~S G;IRCIA DELGADO, ALBERTO R!IONROY C., LUIS G. ARMENDARIZ, ABRAHAM ALCALA, JOSEFINA QUEVBDO, AND P-~ULINO ROJAS

Departan7ento de Qz~inzica, Escz~ela de Ciencias, Institz~to Tecnolo'gico de il[o?zterrey, ilfowterrey, N.L. Mexico

Received October 26, 1064

ABSTRACT

The Bocconia latisepala Wats. , has been chenlically studied. Each part was extracted separately with petroleum ether and with ethanol. T h e extracts were crystallized and purified b y chrolnatographic methods. Cerylic alcohol was isolated from petrole~lm ether extracts o f the bark, stems, and leaves. T h e bark and stems, leaves, roots, and seeds afforded the alkaloids chelerythrine, sanguinarine, protopine, a-allocryptopine, and oxysanguinarine, identified b y their physical and chemical properties.

The Bocconia genera of the alkaloid-rich Papaveraceae family contains nine species most of which are native to RlIexico. They were used by the Indians as dyes and, of inore interest, in a variety of medicinal preparations (1, 2).'

Armendariz (3) and later Manske (4, 5 ) in chemical studies on B. arborea reported the presence of chelerythrine (3-6), sanguinarine (3, 6), protopine (3,4, 6), and allocryptopine (4) as well as other unidentified substances. Studies of B. cordata Wild (formerly Macleya cordata) (7, 8, 9), B. frutescens L. (lo), and B. parcei Hecht (11) have also been reported. No cheinical studies of B. lat*isepala Wats., a common medicinal plant of northeastern Mexico, have been made so it was selected for study. Pulverized samples of the roots, bark and stems, leaves, and seeds mere each extracted with petroleum ether and then ethanol. The course of the extraction and later fractionations were followed by thin-layer chromatography (t.1.c.) (12).

The petroleum ether extracts of the bark and stems and the leaves afforded a precipitate shown to be cerylic alcohol. The filtrate from the extract of the bark and stems on alumina chromatography afforded more cerylic alcohol, chelerythrine (yellow, m.p. 205" decoinp.), and sanguinarine (orange, m.p. 255-258' decomp.) identified by their infrared spectra, pseudocyanides, and t.1.c. Xi values. A white compound, m.p. 360-363O, was not further investigated since i t gave negative results in the usual alkaloid tests. In addition to these two alltaloids, the alcoholic extract of the leaves and the roots afforded oxysanguinarine, protopine, and a-allocryptopine.

The petroleum ether extract of the seeds afforded a reddish, fluorescent oil (33.5%) shown upon saponification to contain principally stearic and palmitic acids and smaller a~nounts of oleic and lauric acids. From the non-saponifiable fraction was isolated a small amount of chelerythrine and sanguinarine. Thin-layer chromatography indicated that a-allocryptopine and oxysanguinarine were also present.

E X P E R I M E N T A L

Melting points (uncorr.) were determined on a ICoRer hot stage. Ultraviolet spectra wcre deterlnined on a Beclrman DU spectrophoto~neter and infrared spectra on a Beckman IR-5 spectrophotometer or a Perkin-Elmer Infracord. Micronalyses were b y Dr. A. Bernhardt, Miilheim/Ruhr, Germany. Identification methods and reactions were those o f Cheronis (13), Feigl (14), and Cromwell (15).

'These plants known locally as "llora sangre" (weeping blood), "tlacoxil~z~itl" "palo del diablo" (devil's stick), and "cocoxihiritl" were used by early Indians as healing agents (1). Herndndez (2 ) mentioned that the stenzs were used to dissipate filnzs and ulcers of the eyes. T h e 'Ijzrices", fruits, and crzrsked leaves were applied as cataplas?ns lo heal old wounds and dissolve warls.

Canadian Journal of Chemistry. Volume 43 (19G5)

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680 CASADIAN JOURS.-\L O F CHEMISTRY. VOL. 43. 1965

Pla+zt i%faterials Samples of Bocconia lutisepala Wats. (16) were collected in Chipinque, Nuevo Le6n, Mkxico a t an altitudc

of 1 500 ~neters in July 1960, November 1961, and August 1963.

Thin-Layer Chroinutography Glass plates (20 X 20 X 0.3 cm) were used to support films of silica gel G (Merck) or aluminium oxide

G coated with a DESAGA applicator. Plates were developed with ?L-butanol -acetic acid - water (75:15:10 v/v).

Estraction of Bark and Stems General Proced~ire The powdered, dry bark and stems of B. latisepala (800 g) were extracted first with petroleum ether

(b.p. 30-60') and then with ethanol. The marc was discarded. After t.1.c. examination, each extract was concentrated a t 40-50' under reduced pressure to 250 ml, a quarter of its original volume. After cooling, any precipitate present was collected and the filtrate evaporated to dryness and worked up separately.

Petrolezr?,~ Ether Extract On concentration of the petroleu~n ether extract 2.5 g (0.31y0) of a greenish precipitate I T P (m.p. 72-76")

was obtained. Crystallization from petroleum ether raised the m.p. to 79-80'. Evaporation of the filtrate (five fluorescent spots on t.1.c.) afforded 0.99 g (0.12%) of a greenish, resinous material. This was dissolved in 20 n ~ l of petroleum ether, placed on chromatographic colun~n of 80 g of neutral alumina (Mcrck), and eluted with solvents of increasing polarity. Fractions with similar t.1.c. characteristics were combined for attempts to separate their components. From fractions 1-12 an orange precipitate, principally of chel- erythrine and sanguinarine (t.l.c.), was obtained. From fractions 13-20, there was obtained 140 nlg of ;L

white precipitate ( ITC), 1n.p. 79-80", later identified as cerylic alcohol. The second crop, a brown precipitate (32 mg) on fractional crystallization from cyclohesane gave 8 mg of yellowish needles (dTC, 1n.p. 205-207", decomp.) and a second crop of 20 mg of orange crystals (STC, m.p. 255-258', decornp.). On treatment with acids BTC acquired a golden-yellow color and STC an orange-red color. They were subsequently identified as chelerythrine and sanguinarine. From fractions 21-25, the same yellow and orange precipitates (58 mg) were obtained. A white precipitate (10 mg, 4TC m.p. 330-333") was isolated from fractions 26-31. I t was subsequently identified as ox),sanguinarine by its color reactions and R f value on t.1.c.

I T P , on crystallization from chloroform or methanol (Norite), alforded white crystals, m.p. 79-60". The infrared spectrum exhibited bands a t 2.83 p (OH), 3.45 p (C-H), and 6.85 p (CH?). I T P was optical inactive (chloroform or metha~lol), contained one C-methyl group (Kuhn-Roth), no double bands (tetra- nitromethanc), and one active hydrogen (Zerewitinoff). The reduction test with chro~nic acid was positive and the ~llolecular weight (Rast, in camphor) was 346 (calcd. 382). I T P was identified as cerylic alcohol.

Anal. Calcd. for C?GIH;JO: C, 81.60; H, 14.2; 0 , 4.18. Found: C, 81.19; H , 13.91; 0 , 4.01. ITIJ, when treated with acetyl chloride in pyridine and worlred up in the usual way, alforded an acetyl

derivative, 1n.p. 64-65" (lit. (17) 65"), infrared 5.77 and 8.05 p. Anal. Calcd. for C:&IsGOl: C, 79.18; H , 13.29; 0 , 7.53. Found: C, 79.12; H, 13.36; 0 , 7.65. Similarly I T P afforded a benzoate when treated with benzoyl chloride in the presence of petroleunl

ether and sodiunl hydroxide. Crystallization from petroleum ether alfoided white crystals, 1n.p. 47-49'. 'The infrared spectrum indicated absence of hydroxyl groups and the presence of an ester f u n c t i o ~ ~ (5.80 p and 7.92 p).

Anal. Calcd. for C3,HssO?: C, 71.42; H , 12.01. Found: C, 81.36; N, 11.95. Compound ZTC, 1n.p. 205-207°, gave a yellow color with acids or with potassium persulfate. The ultra-

violet spectrum of ZTC in ethanol (violet solution) shows absorption with A,, (log E) = 255 mp (4.6), 298 (4.68). I ts infrared s p e c t r ~ ~ m (ICBr) shows A ,,,, = 3.30, 3.35, 6.25, 6.40, 6.60, 6.90, 7.10, 7.50, 7.90, 8.30, 9.40, 9.65, and 10.60p.

Anal. Calcd. for C21H19NO~: C, 69.03; H, 5.24; X, 3.83. Found: C, 69.12; H , 5.28; N, 3.71. A mixed melting point with an authentic sanlple of chelerythrine was undepressed. The Rr of 2TC and

chelerythrine on t.1.c. were similar as were their fluorescence under ultraviolet light and their behavior with ~nodified Dragendorlf's reagent.

Compound STC, n1.p. 255-258O, exhibits ultraviolet absorption in ethanol a t A,,,, (log E) = 259 (4.34), 312 (4.61), and 338 (4.18). I ts infrared spectrum (ICBr) shows bands a t A,,, = 3.32, 6.15, 6.20, 6.44, 6.75, 8.45, 9.30, and 9.42 p.

Anal. Calcd. for C2aEllsNOs: C, 68.76; H , 4.33; N, 4.01. Found: C, 68.75; H , 4.25; N, 4.15. A inixed melting point with sanguinarine was itndepressed. The R f of STC and sanguinarine were identical

as was their behavior ~ ~ n d e r ultraviolet light and with modified Dragendorff's reagent. Compound 4TC, was identified by a mixed melting point with oxysanguinarine and by comparison of

their Rr's on t.1.c. The concentrated petroleum ether extract of 759 g of dried leaves afforded 5 g (0.66%) of impure cerylic

alcohol. The greenish hltrate (negative tests for allraloids) was discarded. The Inarc from the leaves yielded an ethanol extract which gave a positive all;aloid test. On concentration to a quarter of its volume, a pre- cipitatc (11-HP-I, 10.0 g, 1.33%) was obtained which gave a strong positive test for allcaloids. Thc filtrate

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DOMINGUEZ ET AL.: CIIEMICAL STUDY OF BOCCONIA LATISEPALA WATS. 681

(positive test for all;aloids) was evaporated to dryness and the residue (11-HR, 8.32 g, 1.1%) was dissolved in chloroforni and chro~natographed on 400 g of alumina. The fractions containing the two components with thc lowest Rr values on t.1.c. (2HC and SHC), were combined for separation by the pseudocyanide procedure (4, 18, 19, 20). Fractional crystallization from acetone gave 40 nlg of white crystals (A), n1.p. 256-357" (reported (4, 20) for the pseudocyanide of chelerythrine, m.p. 25S0), which was not changed after three recrystallizatio~is from acetone. Concentration of the filtrate gave 70 mg of white crystals (B) m.p. 234-236" (reported (4, 18) for the pseudocyanide of sanguinarine, m.p. 237-238"). On hydrolysis of A with diluted hydrochloric acid, the hydrocl~loride of chelerythrine, m.p. 256-258" (reported (6) 251-256", yellow needles), was obtained. Thin-layer chromatography showed only one compone:lt which had the sanie Rr value as a simultaneously run sample of the authentic allcaloid. (B) was recrystallized twice from acetone, to give a product, m.p. 225-22G0, identical with that reported (20) for sanguinarine pseudocyanide. On hydrolysis with dilute hydrochloric acid, sanguinarine hydrochloride was obtained as darlc-red crystals, 111.p. 204-206" (reported (17) 203-305"). Thin-layer chromatography showed only one orange spot having the same Rr value as an authentic specimen of sanguinarine. A methanol solution of 100 mg of a mixture of 2HC and SHC was chromatographed on 60 g of silica gcl. On develop~nent with chlorofor~n there was ob- served an upper red band and a lower yellow band which after elution with chloroform was shown to be chelerythrine. Ethyl acetate eluted the red band (sanguinarine). Thin-layer chro~natography of each fraction showed a single spot and co~nparison with authentic samples of thc alkaloids established their identity.

The precipitate (11-HP-I) showed four fluorescent spots on t.1.c. I t was dissolved in chloroform and chro~natographed on 300 g of al~iimina. Fractions 6-12 afforded two din'erent allcaloids, 65 mg of white crystals (11-HP-2), m.p. 160°, subsequently identified as or-allocryptopi~ie and 459 mg of a tan precipitate (11-HP-S), m.p. 330°, later identified as oxysanguinarine. From fractions 13-34, after fractional crystallization, com- pounds 11-P-4, m.p. 75-78" (identilied as itiipure cerylic alcohol), 11-HP-5, m.p. 155-15g0, and 11-HP-6, m.p. 171-l74", weighing 184 tng, coinpound 11-HP-7, m.p. 206-20T, were obtained. Compound 11-HP-7 was ider~tified as protopine, on the basis of t.1.c. and mised nielting point determillation. Compounds 11-HP-5 and 11-HP-6 did not give positive allialoid testsand so it was not further examined.

Compound 11-HP-2 (or-allocryptopine), m.p. lGOo, showed a single violet spot on t.1.c. when observed under ~~l t raviole t light. The color of the spot slowly changed to yellow on standing. 11-HP-2 in ethanol exhibit A,,,, m p (log E) 236 (4.2) and 288 (3.6). The infrared spectrum (ICBr) showed bands max. = 3.0, 3.5, 5.53, 6.25, 6.8, 7.9, and 9.7 p.

Anal. Calcd. for C21H?:,05N: C, 68.24; H, 6.28; 0, 21.66; N, 3.79. Found: C, 68.48; H, 5.95; 0, 21.90; N, 3.77.

Compound 11-HP-S (oxysanguinarine), 111.p. 330°, gaveasalinon or brownish spot on t.1.c. when observed u~?der ultra\.iolet light. I ts ultraviolet spectrum in ethanol exhibited A,,:,, mp (log e) = 256 (4.33) 286 (3.82), 310 (4.0), and 330 (3.8). I ts iilfrared spec t r~ i~n exhibited bands A,,sl~nrat 2.06, 3.45, 6.05, 6.75, 7.6, and 7.9 p.

Anal. Calcd. for CnoMjj05N: C, 69.16; 1-1, 3.77; 0 , 23.03; N, 4.03. Found: C, 70.57; H, 3.54; 0 , 23.23; N , 3.66.

The dried, powdered roots (500 g) were cxtracted and worked up by the general isolation procedure. The reddish pctroleurn cther solution left, on evaporation, 5.5 g (1.1%) of a reddish residue (IRP). A 5 g sample of IRB was chromatographed on aluinina; from the eluates 150 nig of chelerythrine was obtained, besides two other non-allcaloidal fractions, IRB (brownish-red precipitate), m.p. 140-145', and IRC (light- brown precipitate) m.p. 165-175". They were not further investigated.

From thc ethanolic extracts of the roots 40 g (8%) of a dark resin (11-RR) was obtained. This residue gave positive all;aloid tcsts. A chlorofor~n solution of the residue was chro~natographed on alumina and from the eluate; or-allocryptopine, chelerythrine, sal~guinarine, protopine, and oxysanguinarine were isolated and identified.

I'etrolezi71i Etl~er Extract o f the Seeds From 906 g of ground, dried seeds a reddish ethcreal extract was obtained. I t gave weakly positive

all;aloid tests. Evaporation to a quarter of its volume gave 0.85 g of a reddish, greasy precipitate (0.94%). On evaporation of the filtrate there remained 305 g (33.570) of a reddish fluorescent oil, specific gravity, 0.935 a t 31"; n1,"' 1.4326; iodine index 69.02; saponification indes 176. A t.1.c. of a sample of the oil showed t11e presence of chelerythrine and sanguinarine. The oil was saponified and by chromatography of the liberated fatty acids, principally stearic and palmitic acids were isolated with smaller amounts of oleic and lauric acids.

The marc \vas extracted, first with acetone and then with ethanol. Both extracts were chromatographed on alumina yielding sanguinarine, chelerythrine, and oxysanguinarine.

ACICNOMTLEDGMENTS

This research was made possible by a grant (GR1l 11332-01) froill the U.S. Public I-Iealth Service, National Iilstitutes of Health. We thanlc Dr. R. I-I. R'Ianske, Dominion Rubber Company Ltd. (Canada) and Dr. J. Slavick, Prague, Czecl~oslovakia, who furnished several reference alkaloids.

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682 C:lNADI.-IS J0UR':rIL OF CHEMISTRY. VOL. 13, 1965

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p. 610. 3. E. ARAIENDARIZ. Datos para la materia medica Mexicana.Vo1. 1. Sria. de Fomento, Mexico. 1895. p. 15. 4. R. H. F. MANSKE. Can. J. Research B, 21, 140 (1943). 5. R. H. F. MANSICE. Alkaloids, 4, 147, 255 (1954). 6. T. A. HENRY. The plant alkaloids. 4th ed. Churchill, London. 1949. 7. J. F. EIJICIIANN. Rec. Trav. Chim. 3, 12 (1884). 8. P. MURRILL and J. 0. SCHLOTTERBECK. Ber. 33, 2802 (1900). 9. I i . HOGFGARTHER. NIonatsh. 19, 179 (1898).

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1957. 14. F. FEIGL. Spot tests in organic chemistry. 6th ed. Elsevier, N.Y. 1960. 15. R. T. CROMWELL. I n Moderne Methoden der Pflanzenanalyse. Vol. 4. Edited by Ii . Pech and M. V.

Tracey. Springer-Verlag, Berlin. 1055. p. 967. 16. P. STANLEY. Trees and shrubs of Mexico. Vol. 1. Smithsonian Institute, Washington. 1923. p. 290. 17. J. HEILBRON and H. M. BUNBURY. Dictionary of organic compounds. Oxford Press, N.Y. 1953. 18. J. SLAVIK and L. SLAVIKOVA. Collection Czech. Chem. Commun. 20, 17 (1955). 19. P. ILIRRER. Ber. 50, 212 (1917). 20. E . SPATH and F. I~UFFNER. Ber. 64, 1123 (1931).

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