"Isolation of Alkaloids from the Leaves of Mucuna pruriens

Green leaves (2 kg, wet wt.) were macerated in a waring blender with a mixture of chloroform (3 l) and ammonia (15N, 50 mL). The mixture was corked and kept at room temperature, with occasional shaking, for one week. It was filtered and the solvent was removed under reduced pressure. The viscous brown slurry (150 g) was poured into aqueous acetic acid (4%, 100mL) with stirring, and the mixture was kept at room temperature overnight. The clarified solution was extracted with chloroform to remove weaker bases as chloroform soluble acetates.

Chloroform-soluble Acetates

The chloroform-soluble acetates, obtained as a brown amorphous solid (1.2 g), was chromatographed over Brockmann neutral alumina (1.4 X 13 cm). Petroleum (40-60°), benzene, chloroform, methanol andmixtures therof were used as eluents. The course of separation of the alkaloids was followed through paper chromatography.

N,N-Dimethyltryptamine: The chloroform eluates from the column afforded a thi[c]k brown oil (0.08 g), R_F , 0.58; Dragendorff, orange; Ehrlich, purple changing to blue; a -nitroso-b -naphtol-nitrous acid, brown; u.v.: l _max 225, 288, and 294-96 mm ; yellow picrate from alcohol, m.p. and m.m.p. 168-170°.

Anal. Calcd. For C₁₂H₁₆N₂, C₆H₃N₃O₇: N, 16.78. Found: N, 16.88.

N,N-Dimethyltryptamine-N_b-oxide: The chloroform-methanol eluates (99:1) afforded a brown basic gum (0.006 g), R_F, 0.88; Dragendorff, brownish-orange; Ehrlich, cherry red; u.v.: l _max 224-26, 272, and 292 mm ; yellow picrate from alcohol, m.p. and m.m.p. with authentic sample, 178-80°.

Reduction of the compound with zinc dust and acetic acid gave N,N-dimethyltryptamine, isolated as its picrate, m.p. 166-68°.

Chloroform-soluble Strong Bases

The pH of the aqueous solution, after separation of the chloroform-soluble acetates, was brought to 9 with ammonia and the liverated bases were taken in chloroform (three 50 mL portions). The chloroform extract was washed with water, dried (anhydrous Na₂SO₄), and the solvent was removed at reduced pressure to give a brown basic gum (2.7 g).

Chromatography of the basic gum: A portion of the basic gum (1.2 g) was taken in benzene and was chromatographed on Brockmann neutral alumina (1.4 X 18 cm).

5-Methoxy-N,N-dimethyltryptamine: The petroleum-benzene (3:1) eluates, on evaporation, afforded a brown amorphous material (0.05 g) which showed a single spot at R_F, 0.58; Dragendorff, orange; Ehrlich, blue; a -nitroso-b -naphthol-nitrous acid, dull violet; u.v.: l _max 224, 277, 292, and 305 mm ; i.r.: l _max 2.88 (NH), 3.55 (Nme), 6.15 (Ome), 6.18 m (C=C); the picrate crystallized from alcohol as orange needles, m.p. 168-70°. M.m.p. with an authentic sample, m.p. 172°, remained undepressed.

Unidentified b -carboline: The benzene eluates afforded a b -carboline base, R_F, 0.29; Dragendorff, orange; Ehrlich, negative; Frohde, bluish-purple; blue-violet flu[o]rescence under u.v. lamp on papers (characteristic of b -carbolines); u.v.: l _max 239, 245, 270, 292, and 240-42 mm ; i.r.: l _max 2.92 (OH), 6.08 (C=N), 6.15 (C=C), and 13.3 m (4 adjacent aromatic protons); the picrate crystallized from alcohol as yellow flakes, m.p. 256-58°. M.m.p. with harman picrate, m.p. 256-58° showed depression by about 10°.

Anal. (fpr base-picrate):Found: N, 15.87.

The chloroform eluates of the column afforded a second crop (0.048 g) of N,N-dimethyltryptamine.

Bufotenenine: The methanol washings of the column gave a brown basic gum (0.22 g) which showed two spots, R_F, 0.27 and 0.33 on paper chromatograms. They were separated by preparative paper chromatography on Whatman 2 MM paper. The component, R_F, 0.27 showed u.v.: l _max 224, 272, 277, 292, and 305-10 mm ; the base picrate crystallized from alcohol as orange-yellow needles, m.p. 175-77°: M.m.p. with authentic bufotenine pircrate, m.p. 174-77°, remained undepressed.

Unidentified 5-oxyindole-3-alkylamine: The component having R_F, 0.33 was also a 5-oxyindole-3-alkylamine; Dragendorff, orange; Ehrlich, blue; a -nitroso-b -naphthol-nitrous acid, violet; u.v.: l _max 222-226, 277, 288-92, 305-10 mm . Attempted preparation of picrate with this component failed.

Water-soluble Bases

The aqueous mother liquor, after separation of the chloroform soluble tertiary bases, was treated with a saturated aqueous solution of ammonium reineckate. The precipitated reineckate complex was washed with water, dried (2.4 g), m.p. 268-70° (dec.), and its acetone solution was passed through a column of De-Acidite FF (pH 8) (Ghosal and Banerjee, 1969).

Choline: The regenerated hygroscopic base was identified as choline by co-chromatography with authentic sample, RF, 0.29. The base picrate crystallized from alcohol as orange needles, m.p. 240-42. M.m.p. with choline picrate, m.p. 241° remained undepressed.

The mother liquor, after the separation of choine reineckate, was cooled, acidified (pH @ 1) with sulphuric acid, and some more ammonium reineckate solution was added to it.

Unidentified indole-3-alkylamine: The light pink reineckate comlex (0.92 g), m.p. 290-95° (dec.), was decomposed by passing its acetone solution over De-Acidite FF. The regenerated base showed only one spot on papergrams, RF. 0.36; Dragendorff, orange; Erhlich, faint purple; a -nitroso-b -naphthol-nitrous acid, dull violet; u.v.: l _max 220, 280, and 305 (sh) mm . No crystalline derivative of this compound could be prepared.

Isolation of serotonin from the trichomes of pods: The golden yellow trichomes of pods (ca. 50 g) were extracted with alcohol without prior defatting at room temperature for 4 weeks. The alcoholic extract was concentrated under reduced pressure. The alcoholic concentrate showed only one spot, R_F, 0,42 (Whatmann, 3 MM), which corresponded to that of authentic serotonin. Preparative paper chromatography was done on the same paper. A band around R_F, 0.42 was cut out and it was eluted with alcohol. The eluate was concentrated under reduced pressure. The concentrate was divided into two portions. One was subjected to the usual pharmacological screening for serotonin (smooth muscle preparations) and with the other portion the following tests were performed. Colour reaction: Dragendorff, brownish-orange; Erlich, purple changing to blue; a -nitroso-b -naphthol-nitrous acid, violet; Jepson and Stevens (1953) test for N_b-unsubstituted tryptamines, positive; u.v.: l _max 224-26, 288-94, and 305 (sh) mm . The picrate crystallized from aqueous alcohol as orange-yellow needles, m.p. 191-93°. M.m.p. with authentic serotonin picrate, m.p. 190-92°, remained undepressed.

References:

Ghosal, S., and Banerjee, P.K.: Aust. J. Chem., 22, 2029 (1969).

Jepson, J.B., and Stevens, B.J.: Nature (Lond.), 172, 772 (1953).