J. W. ROTHROCK, P. A. HAMMES, and W. J. McALEER Merck Sharp & Dohme Research laboratories, Merck & Co., Inc. Rahway, N. J.
Isolation of Diosgenin by Acid Hydrolysis of Saponin
Elimination of organic solvents in the initial extraction of the plant material, and utilization of hydrocarbon solvents with high selectivity for sapogenin, give a process that is simpler, less expensive, and more adaptable to large scale operations than classical processes
EARLY
in the commercial production of cortisone it was clearly evident that an alternative starting material was desirable to supplement the available supplies of bile acids. Therefore, a n intensive investigation was undertaken to find other steroids, especially of plant origin, that could be easily converted to cortisone. Among the genera of plants investigated were Strophanthus, Agave, Dioscorea, and Yucca, from which were derived sarmentogenin, hecogenin, and diosgenin, and yeasts from which ergosterol was obtained. This discussion deals with the process developed in these laboratories for the isolation of diosgenin (Ab-22a-spirosten-3/3-01) from Dioscorea tubers. Dioscorea barbasco amarillo, native to Guatemala, was selected as best of 65 investigated species of plants belonging to the family Dioscoreaceae from the standpoint of ready availability and yield of diosgenin. The classical methods of Tsukamoto and Ueno (77), Marker and others (6-9)) Wagner (72-75), and others (3, 5 ) available a t that time for the isolation of sapogenins were not commercially practical. In the isolation procedure
186
employed most often by these investigators, the steroidal saponins were extracted by ethyl alcohol from ground plant material, then acid hydrolyzed to liberate the sapogenin from the glycoside. The crude sapogenins were obtained by the ether extraction of the hydrolyzate and were purified by being defatted with ligroin, treated with charcoal, and recrystallized several times. Often recrystallization through the acetate was required before pure sapogenin was obtained. A procedure for isolating diosgenin, somewhat similar to the one described here, is presented in a recent patent by Inoue and others (4). The process described involves direct acid hydrolysis of pulverized tuber, followed by preferential extraction of diosgenin from the dried hydrolyzate by hydrocarbon solvents, such as petroleum ether and heptane. The sapogenin can then be crystallized directly from the hydrocarbon solvent in a highly purified form. This simplified procedure is particularly suited to a species of tuber such as Dioscorea barbasco amarillo, containing only one sapogenin, because fractional crystallizations, chro-
INDUSTRIAL AND ENGINEERING CHEMISTRY
matography, and other purification steps are eliminated in preparing a satisfactory product for further chemical work. An alternatite process by which saponin may be isolated, if desired, involves hot water extraction of sliced tuber. The aqueous solution thus obtained can be concentrated and extracted to yield saponin, or hydrolyzed and extracted to produce diosgenin.
Isolation of Diosgenin From Tubers The process for the isolation of diosgenin from tubers consists of three major operations: preparation of the tubers, hydrolysis of the saponin, and extraction of the diosgenin. ,4 typical example f0llOU~S. A finely pulverized, uniform sample of tuber was prepared by passing 4 kg. of fresh Dioscorea barbasco amarillo through a micropulverizer (Bantam) equipped with a 0.013-inch (slot width) herringbone screen ; this was followed by thorough hand mixing. Moisture: loss on drying determinations revealed 24.4 and 24.6% of nonvolatile material when dried to constant weight in an oven a t 80" C. Duplicate 200-gram
Table 1.
Influence of State of Subdivision of Dioscorea Tuber during Drying on Diosgenin Recovery
Dry Wt.
Sample No.
of Fresh Tuber,
%
37.0 26.7 24.5 22.8 19.4 22.8 24.0 24.0 a
Micropulverized Fresh Tuber Assayed Fresh Dried and Assayed Yield", M.P., Yield", Y.P., % O c. % C.
..
4.56 4.93 4.65 4.80 4.18 4.32 5.16 5.84 5.66 5.726.67
*..
200.53 203 - 5 201 - 3 201 - 3 201 - 3 201 - 3 200 - i 194 - 6 199 -201.5 200 -202 200 - 2
Cubed Fresh Tuber, Dried, Micropulverized, Assayed Yielda, Y.P., % C
5.56 4.96
203 201
-5 -3
3.22 2.93
4.92
202
-4
..
...
4.68
201.5-3.5
..
...
..
5.05
194
*.
5.70
201
...
202 203
-4.5 -5
-6
...
3.01 3.74 3.55
200 194 203
-3 -6 -5
-3
2.07
198-200
Based on dry weight of tuber.
samples of the tuber mash, each equivalent to 49 grams of dry material, were placed in three-necked, 2-liter roundbottomed flasks equipped with Trubore stirrers. The mixture was made 2N by adding 31 ml. of concentrated hydrochloric acid and 750 ml. of 2N hydrochloric acid solution, which provided a slurry that could be conveniently stirred. The mixtures were stirred a t reflux temperature for 2 hours; the flasks and contents were then cooled under a stream of water. The hydrolyzates were filtered on Buchner funnels and the precipitates, after being washed free of acid with about 500 ml. of cold tap water, were allowed to dry a t room temperature overnight. This material can be dried more rapidly in an oven a t 80" C. These dry, brown residues, weighing about 30 grams each and consisting of crude diosgenin, soil particles, and undigested tuber cellulose, were placed together with the filter paper in 43 X 123 mm. thimbles and extracted overnight in a Soxhlet apparatus with 300 ml. of petroleum benzin [ (Merck) boiling point, 30-60' C.]. As the extraction proceeded, the diosgenin began to crystallize in the boiling flask and a t completion most of the sapogenin had crystallized out. Maximum recovery was attained by reducing the volume of the solvent to about 50 ml. by evaporation in a hooded steam bath and by chilling the flasks in a refrigerator for several hours. White crystalline diosgenin was recovered on a tared sintered-glass funnel and washed with two small portions of cold solvent. Pigments and oily materials passed into the filtrate. From one sample was obtained 2.35 grams (4.80% yield on a dry weight basis) of crystalline diosgenin, melting a t 201203' C. and from the other sample 2.28 grams (4.65% yield), melting a t 201203 " C. All melting points are corrected capillary readings.
Preparation of Tubers for Processing Either fresh or dry tubers may be utilized in this process. Dried tuber, prepared in a n oven a t 80' C. or in the sun on a patio, properly stored, and handled under dry conditions loses no diosgenin over a long period of time. Fresh tubers can be utilized only if there is a relatively short delay from harvesting to processing, because of a rapid decrease in diosgenin content as rotting proceeds. Whether fresh or dry tuber is used, it should be in a finely divided state to ensure rapid hydrolysis and extraction. Thus, dried tubers should be milled to 100 to 200-mesh. Fresh tuber can be comminuted satisfactorily by a micropulverizer equipped with a 0.013-inch herringbone screen. Small quantities of fresh tuber can be prepared in a Waring Blendor. If dry tuber is used, the yield of diosgenin will depend on the state of subdivision of the tuber during the drying operation. Thus, tubers micropulverized in the fresh state and assayed either fresh or dry yielded almost twice as much diosgenin as the identical tubers that were diced (1 cm.) and dried before micropulverizing (see Table I), The various assays run on each sample were obtained on 200- to 250-gram aliquots taken from a uniform mixture. I n all cases final particle size was about the same and all samples were dried under the same conditions. The only difference in the two dry sample assays was particle size of tuber during the drying operation: in one case 1-cm. cubes and in the other case a finely micropulverized mash. Whatever change takes place must occur in the organized tissue during drying. Certainly no saponin is synthesized in the disintegrated tuber during drying, because the results of micropulverized, dried samples are in good agreement with micropulverized fresh samples that
were immediately subjected to acid hydrolysis.
Hydrolysis of Saponin Diosgenin exists in the plant tissues in a combined, glycosidal form (saponin) which can be cleaved by mineral acid into its component parts, the sugar moieties, and diosgenin. Earlier workers, Marker and others (8, 9 ) , Wagner (72-75), and others (3, 4, 7 7 ) , hydrolyzed under conditions varying from 2 N hydrochloric acid refluxed for 2 hours to 5% alcoholic sulfuric acid heated for 20 hours. I n none of these cases was it shown that optimal hydrolysis conditions were used. Rothman and coworkers (70), in studying the conditions for the hydrolysis of a number of different purified saponins, found that dioscin was completely hydrolyzed by 4 N hydrochloric acid solution refluxed for 4 hours; 2N acid refluxed for 2 hours released only 35y0 of the total sapogenin. The use of 1 N acid was ineffective; 6 N acid destroyed sapogenin with excess heating time. However, the authors have found that the highest yields of diosgenin were obtained from micropulverized tuber when the slurry was refluxed for 2 hours in 2 N hydrochloric acid solution. Hydrolysis conditions more severe than these (see Table 11), with respect to either reflux time or acid concentration, resulted in a somewhat lower yield or a more highly colored, lower melting product; less vigorous conditions led to incomplete hydrolysis. T h e discrepancy between Rothman's observations and the authors' is difficult to explain, unless the presence of some substance in the tuber mash facilitates hydrolysis. Other acids were investigated, but only strong, nonoxidizing mineral acids, such as hydrochloric, hydrobromic, and sulfuric were found satisfactory. Nitric acid and phosphoric acid were totally VOL. 49,
NO. 2
FEBRUARY 1957
187
Table II. Reflux
a
Effect of Acid Concentration and Reflux Time on Hydrolysis of Saponin"
Time, Hours
Isolated Diosgenin
1
Yield, % M.p., O C.
2 4
Concentration of Hydrochloric Acid in Slurry 1N
2N
4N
1.46
199-201
3.90 201-204
2.42 199-201
Yield, 70 M.p., O C.
2.72 198-200
4.10 199-202
1.96 195-198
Yield,
7*
M.p.,
C.
3.10 201-203
3.90 199-203
165-175
1.44
50-grain samples of a mill house blend of dry, whole micropulverized tubers were hydrolyzed in 1 liter of hydrochloric acid solution.
ineffective. Lower acid concentrations and shorter periods of hydrolysis are realized when the reaction is carried out under pressure. Thus, the same yield of diosgenin is obtained from either a 0.2N hydrochloric acid slurry of dry micropulverized tuber maintained at 150" C. (55 pounds per square inch gage) for 1 hour or 2 N solution refluxed for 2 hours at atmospheric pressure. The diosgenin content of crude dried hydrolyzate is not decreased by long storage under dry conditions. If this material is insufficiently washed before drying, especially when sulfuric acid is used for hydrolysis, the steroid content decreases rapidly.
Extraction and Purification of Diosgenin Hydrocarbon solvents selectively extract diosgenin from crude, dry hydrolyzates and retain in solution all of the oily materials and pigments but essentially no steroid and thereby facilitate the recovery and purification of the compound. Of the many solvents investigated, petroleum ether, Skellysolve B, Skellysolve C, and Esso heptane proved most useful. In a Soxhlet apparatus using petroleum ether (boiling point 30'-60" C.), the solvent of choice for laboratory work, the extraction of sapogenin is completed in approximately 6 hours. However, extractions may be conveniently set up late in the afternoon and permitted to run overnight so as to ensure complete recovery of the steroid. Esso heptane (boiling point, 98" C.) was well suited to production operations involving batchwise extractions of hydrolyzate milled to 100 mesh or finer (7) * The yield of diosgenin for assay and development work was based on a white crystalline product melting a t 200' C. or higher. This material, of about 95 to 100% purity, was found satisfactory in a use test for the preparation of A7-dehydrodiosgenin acetate (2). If diosgenin of still higher purity is desired, it can be prepared by crystallization from methyl ethyl ketone, ethyl alcoholacetic acid (1 to 1)) or chloroformmethanol (1 to 1).
188
Extraction of Crude Saponin Since the saponin of Dioscorea is water-soluble. a crude extract can easily be obtained. Dilute solutions can be prepared by heating fresh or dry sliced tuber for several hours at reflux or steam bath temperature and decanting the supernatant liquid. At higher tempera tures-i. e,, higher pressures-extraction is more rapid and more complete. Chips heated for a few minutes a t 150' C. in a steel bomb or for 1 hour at 112' C. in a pressure cooker can be extracted satisfactorily with a smaller volume of water. Additional saponin is recovered from the spent chips by expressing the juice with a press. A still more concentrated saponin solution can be obtained by continuous, hot extraction. An apparatus built for this purpose consisted of a wide mouth (55/50) 1 liter round bottomed flask, a column made by sealing 55/50 male and female joints end to end and a watercooled condenser. The sliced tuber, either fresh or dry, was supported in the extraction column by a 20-mesh stainless steel wire screen. Steam generated in the bottom flask passed up through the sliced tubers to the condenser; the condensate dripped down through the tubers into the boiling flask. Complete extraction of the saponin was realized overnight. A small drop of DowCorning Antifoam A added to the flask depressed foaming. Diosgenin can be obtained easily from these saponin extracts by adjusting the solution with hydrochloric acid to 2 N and proceeding by the method outlined above. Under some circumstances this method of isolation may be preferred. I t is obvious, however, that hydrolysis of the mashed tuber affords a more direct and more economical process.
Summary A simplified process for the isolation of diosgenin in a highly purified form from Dioscorea tubers involves direct acid hydrolysis of the pulverized tubers and preferential extraction of the sapogenin with hydrocarbon solvents such as petroleum benzin or Esso heptane.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Either fresh or dry tuber can be used; both should be in a fine state of subdivision for processing. Fresh pulverized tuber yielded considerably more sapogenin than pulverized dry chips. Dioscorea barbasco amarillo is especially well suited to this process. I n an alternative method saponin can be obtained by hot water extraction of fresh or dry sliced Dioscorea tuber. Diosgenin is obtained by acid hydrolysis and selective solvent extraction.
Acknowledgment The authors wish to thank R. G. Denkewalter and E. E. Howe for encouragement and advice during this investigation.
Literature Cited (1) Burtsfield, D. R., Merck Sharp &
Dohme Research Labs., Merck & Go., Inc., Rahway, N. J., private communication. ( 2 ) Chamberlin, E. M., Ruyle, W. V., Erickson, A. E., Chemerda, J. M., Aliminosa, L. M., Erickson, K. L., Sita. G. E.. Tishler. M.. J . Am. Chem. Sac. 73, 2396 (i951): (3) Fujii, K., Matsukawa, T., J . Pharm. SOC.Japan 56, 59 (1936). (4) Inoue, M., others (to Takeda Pharmaceutical Industries Co.), Japan Patent 2977 (1954). ( 5 ) Jacobs, W. A., Simpson, 9. C . , J . Bid. Chem. 105, 501 (1934). ( 6 ) Marker, R. E., Tsukamoto, T., Turner, D. L., J . Am, Chcm. Sac. 62. 2525 11940). ( 7 ) Marlkr, R.' E., Turner, D. L., U1shafer, P. R., Ibid., 62, 2542 (1940). ( 8 ) Marker, R. E., Wagner, R. B., U1shafer, P. R., Ibid., 64, 1283 (1942). ( 9 ) Marker, R. E,, Wagner, R. B., U1shafer, P. R., Wyttbecker, E. L., Goldsmith, D. P., Ruof, C. H., Ibid., 69, 2167 (1947). (10) Rothman, E. S., Wall, M. E., Walens. H. R.. Ibid.. 74. 5791 (1952). (11) Tsukamoto, T., Ueno; Y . , J: Pharm. Soc. Japan 56, 135 (1936). (12) Wagner, R. B. (to Parke Davis & Co.), U. S. Patent 2,408,827 (Oct. 8, 1946). (13) Zbid., 2,408,834 (Oct. 8, 1946). (14) Zbid., 2,408,835 (Oct. 8, 1946). (15) Zbid., Re 23,008 (June 15, 1948).
RECEIVED for review April 5, 1956 ACCEPTED August 4, 1956
