and PVC developed crazes and color changes which were attributed to filler separation from the PVC resin, whereas those plastics made by coprecipitation or co-concentration remained clear and uniform during testing with no crazing or color change. In summary, starch can be incorporated into PVC-DOP plastics in amounts of up to 100 parts of starch per 100 parts of PVC by weight with maintenance of strength properties nearly equivalent to those obtained with PVC containing conventional inorganic fillers. Of the techniques studied, the best for mixing starch into plastics were coprecipitation of starch xanthate-PVC latex or coconcentration of PVC-starch gel. The starch-filled plastics are readily attacked by microorganisms commonly found in soil. This microbial attack should cause the plastics, upon disposal, to lose strength, become brittle, and be easily broken up by natural erosive forces into small particles that can become part of the soil.
Acknowledgments We thank R. A . Buchanan for his assistance with the preparation and coprecipitation of starch xanthate and R. G. Fecht for his help with tensile measurements. Literature Cited Buchanan, R. A , , Weislogel, 0. E . , Russell. C. R.. Rist, C. E.. Ind. Eng. Chern.. Prod. Res. Develop.. 7, 155 (1968). Otey, F. H., Westhoff, R. P., Kwolek, W. F., Mehltretter. C. L.. Rist. C. E., Ind. Eng. Chern.. Prod. Res. Deveiop.. 8, 267 (1969). Otey, F. H., Westhoff, R . P.,Mehltretter,,,C. L.,Staerke. 24, 107 (1972). Sarvetnick. H . A , , "Polyvinyl Chloride, p 107, Van Nostrand-Reinhold, New York, N. Y., 1969.
Received f o r reuieu: November 23,1973 Accepted March 12, 1974
Mention of firm names or commercial products does not constitute an endorsement by the U. S. Department of Agriculture.
Dihydrochalcone Sweeteners: Preparation of Neohesperidin Dihydrochalcone George
H. Robertson,* J. Peter Clark, and Robert Lundin
Western Regional Research Laboratory Agricultural Research Service
U S
Department of Agriculture BerkeIey Calif 94710
A three-step batch preparation sequence for conversion of citrus-byproduct naringin to the intensely sweet neohesperidin dihydrochalcone was used to prepare product for pharmacological testing. The reaction sequence was cleavage of naringin to produce phloracetophenone 4'-neohesperidoside (PN), aldol condensation of PN with isovanillin to produce neohesperidin, and hydrogenation of neohesperidin to the dihydrochalcone. Reactions were successful in producing 220 Ib of product at greater than 98% purity. An overall reaction yield of 10% was obtained. Caustic concentration, rate of addition of isovanillin, and catalyst condition were observed to be the critical variables controlling the reaction yields. Overall yields as high as 26% may be attainable.
Introduction The dihydrochalcone derivatives of several flavanone glycosides obtained from citrus fruit are intensely sweet (Horowitz and Gentili, 1963, 1969) and have potential for use as nonnutritive sweeteners in foods where low sugar intake is desirable or necessary. This potential seems to be greatest for use in citrus flavored beverages, chocolate, and soft drinks. Non-food flavoring use in oral products such as toothpaste, mouthwash, and chewing gum can capitalize on the characteristic persistence and slow initial onset of the sweetness sensation. Utilization of selected dihydrochalcones as food additives requires extensive animal feeding trials and certification by the Food and Drug Administration. Preliminary toxicology (Agricultural Research Service, 1968) indicated that neither naringin dihydrochalcone nor neohesperidin dihydrochalcone (100 and 1500X sweeter than sugar, re-
spectively) caused ill effects when fed to rats at levels up to 5% of the diet for 180 days. More extensive testing seemed appropriate but was prevented by the lack of sufficient quantities of the dihydrochalcones. This paper details preparation data obtained at the Engineering and Development Laboratory of the Western Regional Research Laboratory, USDA, during the manufacture of neohesperidin dihydrochalcone for full-scale rat and dog feeding trials. Emphasis is placed on variables which have a critical effect on the rates of reaction, equilibria, and the obtainable yield. The synthesis used was cleavage of naringin to phloracetophenone 4'-neohesperidoside (PN), followed by condensation of P N with isovanillin to neohesperidin, and concluded with catalytic hydrogenation of neohesperidin to neohesperidin dihydrochalcone (Horowitz and Gentili, 1969; Krbechek, et al., 1968). Ind. Eng. Chem., Prod. Res. Develop., Vol. 13, No. 2,1974
125
Experimental Section Chemicals. Naringin was obtained from Sunkist Growers of California and isovanillin from Fluka AG, Chemische Fabrik. Both materials were used directly as supplied. Potassium hydroxide (technical grade), hydrochloric acid (reagent grade), and Raney nickel active catalyst (from W. R. Grace) were also used. Equipment. Condensations were performed in 200-gal stainless steel vessels. Mixers agitated the contents of the vessels, and a double-pipe heat exchanger heated or cooled the liquors in a piped loop external to the reactor. Hydrogenations were performed in a sealed 75-gal stainless steel kettle. Hydrogen entered the liquors through a stainless steel sparging ring. The vessel was sealed with Teflon or flexible plastic gaskets a t stationary fittings and with a carbon-carbon mechanical seal a t the propeller shaft. Crystalline solids were collected on a plate-and-frame filter. A forced-air tunnel dryer was used to dry intermediate products and a vacuum freeze-dryer was used for drying the final product. Procedures. The procedures used during the final series of production are described below. The procedures are for the three-step sequence: naringin cleavage to produce P N followed by P N collection, P N condensation with isovanillin to produce neohesperidin followed by neohesperidin collection, and neohesperidin hydrogenation to neohesperidin dihydrochalcone followed by neohesperidin dihydrochalcone collection. An alternative two-step sequence is possible and was used in preliminary work. In this sequence the hydrogenation was performed directly on diluted and cooled condensation liquors without recovery of neohesperidin. Procedures for Phloracetophenone 4'-Neohesperidoside Isolation. A 15-207' solution of potassium hydroxide in water was prepared in a 200-gal reactor. Naringin was added with stirring and dissolved; then the liquor was heated to 100°C. This temperature was maintained for the 1.0-7.0 hr duration of the reaction. The reaction was quenched by external cooling and direct addition of an amount of cold water equal to that originally in the reactor. The vigorously stirred reaction liquors were neutralized to p H 6.3 f 0.5 by direct addition of concentrated hydrochloric acid. The crystals which formed were redissolved by heating to 70"C, and then were slowly recrystallized to increase crystal size and improve filtration. Finally, the crystals were filtered, washed, and dried. Procedures for Neohesperidin. A solution of potassium hydroxide in water was prepared in a 200-gal reactor. P N was added with violent agitation and the liquors were heated to maintain a 100°C temperature. Isovanillin was added slowly with violent agitation. Reaction temperature was maintained for 10-20 min after the isovanillin addition was completed. The reaction liquors were quenched by external cooling and direct addition of a volume of cold water equal to twice the original amount of water. Vigorously stirred liquors were then neutralized to pH 6.0 f 0.5 with 12 N hydrochloric acid. When neohesperidin crystallized, the slurry was heated to 75°C to dissolve unreacted PN and then filtered a t that temperature to recover undissolved neohesperidin. The filtrate was cooled and refiltered to recover PN. Recovered P N was added to the feed stock. Procedures for Neohesperidin Dihydrochalcone. A solution of potassium hydroxide (10-20%) in water was prepared in the 75-gal hydrogenation reactor containing Raney nickel catalyst. Neohesperidin was added with violent agitation. The reactor was sealed, evacuated, purged 126
Ind. Eng. Chem., Prod. Res. Develop., Vol. 13, No. 2, 1974
with hydrogen, reevacuated, and pressurized to 10-14 psig with hydrogen gas. The reaction was maintained a t room temperature for 22-70 hr until judged complete by chromatography of samples of the liquors. At completion of the reaction the liquors were transferred to a neutralization vessel. The liquors were neutralized with agitation to p H 6.0 f 0.5 with concentrated hydrochloric acid. These agitated liquors were set aside for one to two days to crystallize. The crystals were then filtered, washed, and dried. All liquors and wash waters were retained and refiltered. Purification of the material was accomplished by recrystallizing from water: the water was heated to 80°C to dissolve the dihydrochalcone, filtered to remove suspended insolubles, then cooled to recover dihydrochalcone crystals. The crystals were filtered and freeze-dried. All crystals from the entire production were blended and pulverized. Analysis. Qualitative analysis of reaction mixtures was routinely made using a thin-layer chromatographic system. Commercial cellulose precoated plastic foils (Brinkman M N polygram 300 cel F-254) were spotted and suspended in a sealed chamber containing 10% acetic acid solution. The developed spots were detected under ultraviolet or visible light after spraying with (a) a mixture of equal volumes of 0.5% FeC13 and 0.5% K3Fe(CN)G or (b) a mixture of equal volumes of 10% NaNOz and 3.3'-dimethoxybenzidine (1.3 g + 3 ml of 12 N HCl + 200 ml of HzO). This method differentiated all substances except naringin dihydrochalcone and neohesperidin dihydrochalcone. All substances could be distinguished with a second thin-layer system using polyamide, precoated, plastic foil chromatograms eluted with a nitromethane-methanol solution. Samples were dissolved in 2-methoxyethanol, which is inert to the chromatogram backing. These were then spotted with drawn-out capillary tubes. Only Eastman plates K541-V gave satisfactory results. A diazotized benzidine spray was used to identify spots (Gentili and Horowitz, 1971). Quantitative 'H nuclear magnetic resonance (nmr) analyses of freeze-dried samples were performed on an internally locked Varian HR-100 spectrometer. Pyridine-& a t 60°C was found to be the most useful solvent. However, exact peak positions in this solvent are significantly dependent on the concentration of tetramethylsilane (TMS) which is used as the internal reference for defining peak positions. Some positions also depend on the sample temperature and the amount of water present. Using a sample temperature of 60°, we found it difficult to control the TMS concentration because of its high volatility (bp 29"). Use of sealed tubes or a less volatile reference, such as hexamethylcyclosilane (bp 208"), would be required for reference quality spectra. Relative compositions of all the encountered species were obtained using pure component spectra as standards. Instrument sensitivity was increased by multiple-scan averaging to ensure detection of as little as 0.4% of known impurities. All samples were dried in vacuo for 2 hr a t 105°C to remove the remaining water of hydration so that the baseline in the aromatic region could be examined. Some components slowly reprecipitated from the hot solution if the concentration exceeded 3%. The presence of uncleaved naringin in phloroacetophenone preparations can be determined quantitatively from the ratio of the intensity of the rhamnosyl methyl doublet resonance of naringin which is centered a t approximately 1.65 ppm, to that of the corresponding doublet from phloroacetophenone occurring 0.08 ppm upfield. Partial over-
T a b l e I. Summary of Phloracetophenone 4'-Neohesperidoside Preparation Runs Feed KOH,c Feed N,(& Time, Temp, Run wt % 1b-moles hr "C
Yield
Yield N ,
%
%
IA il,2,8-10,17)
14.1
0.425
1.2
115
59.1
5.3
16.8
0.717
1.5
118
55.5
8.5
17.0
0.269
4.7
89
44.4
7.3
24.0
1.52
5.7
94
43.9
15.8
22.0
0.379
17.0
95
16.6
9.8
IIB (3-6, 11-16)
IIC (20.21)
IID (22,23,25,28 31,321
IIE 129,30)
N = naringin. PN = phloracetophenone 4'-neohesperidoside. Naringin fed a t same weight concentration. Time of reaction: reactions 1-24 premixed for 0.5 t o 1.3 hr before liquors heated; reactions 25-32 premixed for 0.3 hr or less.
lap does not prevent an accurate determination of the intensities from the lowest and highest field lines. Detection of unreacted phloroacetophenone in neohesperidin can be carried out in the same way since the doublet from neohesperidin occurs 0.08 ppm to lower field. Traces of phloroacetophenone in chalcone or dihydrochalcone samples are most conveniently identified by a sharp singlet which occurs a t 2.81 pprn and which arises from its acetyl methyl hydrogens. In such mixtures no other resonances occur in this region. Incomplete hydrogenation or cleavage of a chalcone can be recognized by the presence a t low field, in the vicinity of 8.15 and 8.6 ppm, of a typical AB quartet ( J = 16 Hz) which arises from the isolated hydrogens on the conjugated "chalcone" double bond. This multiplet is readily distinguishable from the aromatic peaks of isovanillic acid, the only other signals expected in this region. The lowest-field resonance of these occurs as a doublet ( J = 2 Hz) a t 8.17 ppm. The corresponding resonance from isovanillin comes a t 7.70 ppm in a normally unobstructed spectral region although its aldehydic resonance a t 10.00 ppm is a more sensitive indicator of its presence. A sample temperature of 60" for nmr analyses provides improved solubility and resolution for some compounds encountered in this project. However the 31" spectrum of neohesperidin dihydrochalcone has fewer overlapping peaks and the broad, low-field resonance which arises both from sample hydroxyl groups and any residual water is broader and mainly falls below the aromatic proton region of interest in this study (7.30-6.50 ppm). The flattest possible baseline is required if trace signals in this region are to be usefully enhanced by computer averaging of multiple scans. In the enhanced spectrum of the final neohesperidin dihydrochalcone, weak peaks at 7.00 and 7.09 ppm were assigned to approximately 2 f 1% (w/w) naringin dihydrochalcone by extrapolation of the signals from samples to which known amounts of this compound had been added. A 0.3 f 0.1% residual concentration of phloroacetophenone was determined by the same technique except that the acetyl methyl peak at 2.81 pprn was measured. The minimum amounts which could be detected by this technique are estimated to be 0.1% phloroacetophenone and 0.6% naringin dihydrochalcone. N o other impurities in the final product could be detected by nmr. Results and Discussion Phloracetophenone 4'-Neohesperiodoside. Thirty-two batch retrograde condensations consuming 2150 lb of naringin and producing 1101 lb of P N were performed. Table I summarizes the conditions used and the results of the reactions. The production was conducted in two series testing dif-
IO
8 0 .e
e E-
6
i a 4
2
-
20
0
IO
25
30
I5 Log Caustic Concentration, wt%
Figure 1. Influence of caustic concentration on conversion of naringin ( N ) to phloracetophenone4'-neohesperidoside( P N )
ferent equipment and reaction conditions. Series I (runs 1-17) used a 75-gal sealed, agitated, stainless steel vessel. The reactions of this series consumed 26 to 52 lb of naringin each and were performed a t llS"C, in 17% or less caustic solutions, and for a reaction time of 1 hr. Series I1 (runs 20-32), initiated by a need for greater production, used a 200-gal open, agitated, stainless steel tank, and the reactions, each of which consumed 110 lb of naringin, were performed a t 93"C, in 17% or more caustic solutions, and for reaction times of 5-24 hr. The combined product of series I contained a 57% yield of P N and a 7% yield of unreacted naringin. A total of 36% of the feed naringin was lost to soluble decomposition products. The combined product of series I1 contained 39% yield of P N and a 13% yield of naringin; a total of 47% of the feed naringin was lost. A notable result of these runs was the observation of a strong effect of caustic concentration on the equilibrium distribution ratio of P N to naringin ( N ) . This effect is illustrated in Figure l. As the caustic concentration increased in the range 14-23% the P N / N ratio decreased from 11 to 2.5. The data point for conditions IIE, Table I and Figure 1, appears out of place; however, since the final OH- concentration was as much as two times greater than the nominal or makeup value due to evaporative losses during this long run, the point should appear farther to the right and may indeed correlate with the remaining data. Although the reaction time does not appear to affect the P N / N ratio for the range of conditions employed, it is imInd. Eng. Chern., Prod. Res. Develop., Vol. 13, No. 2 , 1974
127
Table 11. Summary of Neohesperidin Preparation Runs. ISO, stoic equiv
Feed compn, lb-moles Run
PN
N
IVA
Neo
1-4 5 6-7 8 9 10-11
0.170 0.214 0.142 0.160 0.121 0.161
0,093 0.084 0,032 0.052 0.035 0,049
...
...
... . .
0.018
fc
IS0
=
isovanillin; IVA
50
...
, . .
0,016 0,002
0,040
=
isovanillic acid; Neo
=
Product compn, lb-moles
time,
1.8 1.5 2.3 2.1 2.7 2.2
...
0.08
IS0 addition
Yield of Neo,
min
pHb
Neo
IVA
PN
%
10.5 5.0 11.5 10.0 14.0 12.5
6.2 6.3 6.0 4.4 4.3 6.2
0.047 0.039 0.053 0.058 0,058 0.049
...
0.011
...
... ...
38.3 22.9 30.1 38.7 34.7 46.2
neohesperidin; stoic
=
... 0.019 0.038
...
...
... ...
stoichiometric. pH of neutralization.
Table 111. Summary of Neohesperidin Dihydrochalcone Preparation Runs
I ~
Run 3 4 5 6b 7b 8
Reaction Total DHCa Concn Charge hydrogen reaction molar of KOH, of Neo, pressure, time, yield, wt % lb Psig hr % 14.5 14.5 7.7 7.5 7.5 7.5
50 54 55 68 60 70
12.0 12.0 14.5 13.5 12.5 14.0
65 66 22 53 36 37
74 94 65.5 36.8 36.8 48.5
DHC = neohesperidin dihydrochalcone. b Yield for runs 6 and 7 is calculated from combined product. 0
of:
'
0 2
1 3
1
4
5
1
,
I
6
7
8
Reciprocal rate of addition,min/equiv.
Figure 2. Influence of rate of addition of isovanillin on yield of neohesperidin (Neo) portant to the overall yield of P N plus unreacted naringin because these natural products are not particularly stable a t the severe reaction conditions. Comparison in Table I of IIC and IID with IIE for a threefold increase in reaction time to 17 hr shows a decrease in overall yield from 56 to 26%. Additionally, comparison of IIB with IIC for a threefold increase in reaction time to 4.7 hr shows a decrease in overall yield from 64 to 52%. The lower yields of series I1 may also be influenced by the presence of a fresh supply of air increasing the opportunity for oxidative losses. Series I was performed in a closed reactor. Neohesperidin. The second production step was the aldol condensation of isovanillin with P N to yield neohesperidin. The eleven batch condensations are summarized in Table 11. Feed stock for the condensations was contaminated with uncleaved naringin when the P N was supplied from product of the cleavage step and with isovanillic acid and neohesperidin when the P N was supplied from unreacted condensation reagents. Yields ranged from 22.9 to 46.2%. Temperature and caustic concentration were held constant throughout these experiments; however, composition and reaction times varied over wide limits. The yield of neohesperidin from the condensation was found to depend primarily on the rate of addition of isovanillin. This dependence is illustrated in Figure 2 where an increase in reciprocal rate of addition from 4.0 to 6.0 min/ equiv results in a proportionate increase in yield from 25 to 44%. This effect appears to be the result of competition between the desired condensation and the homomolecular self-condensation of isovanillin (the Cannizzaro reaction). Rapid addition would discourage dispersion and encourage homomolecular interaction, whereas slow addition would 128
Ind. Eng. Chem., Prod. Res. Develop., Vol. 13, No. 2, 1974
encourage dispersion and the desired heteromolecular interaction. Yields higher than 50% would appear reasonable if the reciprocal rate of addition was greater than 6.5 min/equiv. We expect, however, that the yield could not be indefinitely improved in this way since the ultimate amount of neohesperidin produced will be limited by the condensation equilibrium (as affected by the caustic concentration) and by decomposition reactions. Neither limit was attained in this production series. Product was recovered from the neutralized (pH 6.0) reaction liquors in two steps: filtration a t 75°C to remove undissolved neohesperidin and refiltration of this filtrate a t 20°C to recover unreacted P N and isovanillin. This filtration gave pure neohesperidin for subsequent hydrogenation and PN containing about 10% neohesperidin for recycle to the P N feed stock. The alternative two-step reaction sequence eliminated this collection-purification step and yielded a sweet final product contaminated, however, with bitter PN. The rate of crystallization of neohesperidin was governed by the pH of the neutralized reaction mixture. At pH 6.0, crystallization of pure neohesperidin required 18 hr. At p H 4.2 the product which crystallized instantly contained 50% isovanillic acid, characterized by its bright yellow color. Neohesperidin Dihydrochalcone. The final step in the three-step sequence was the catalytic hydrogenation of neohesperidin chalcone. Five separate hydrogenations consuming a total of 357 lb of neohesperidin are reported in Table 111. A yield of 65.5% was obtained from all the hydrogenations including 29 lb of product obtained by rehydrogenation of the combined reaction liquors. The finishing step, consisting of filtration a t 75°C to remove entrained catalyst followed by recrystallization, conserved 94% of this dihydrochalcone. Nmr analyses of the product indicated dihydrochalcone purity of 98%. The hydrogenation yield appeared to be affected by the catalyst condition. An 84% yield of dihydrochalcone was
obtained from runs 3 and 4 in which a 14% caustic concentration and old Raney nickel catalyst were used. Lower yields of 37-65% were obtained from runs 5-8 in which a 7-870 caustic concentration and 24 lb of fresh catalyst were used. Furthermore, the longer the exposure to the fresh catalyst, the lower the yield (Table 111). Additional evidence of the degradation by nickel catalyst has been obtained by others (Simms, 1970). In our experience, the degradation which occurred was to unidentified and unrecovered species. The reaction liquors at this step and indeed a t every step in the sequence were dark brown and had an intense phenol-like odor. The overall reaction sequence yielded 220 lb of a uniform white powder. The overall yield of product based on 2200 lb of naringin starting material was 10%. Conclusions Based on the data presented above, we reach the following conclusions. 1. Neohesperidin dihydrochalcone with less than 2% impurities can be manufactured successfully by a batch, three-step production sequence. 2. The yield of product was 10%. Accumulated data indicate that this may be raised to 20-3070 by the following: ( a ) cleavage of naringin: by the use of caustic a t less than 14% for more favorable equilibrium or the use of a sealed, pressurized reactor to increase reaction temperature and
decrease time of reaction, thereby reducing degradation of reactant and product; (b) condensation of phloracetophenone 4'-neohesperidoside by the use of as long a time of addition of isovanillin as is practical; and (c) hydrogenation of neohesperidin by the use of palladium catalyst and minimum exposure time. Acknowledgment Lynn Williams, Nancy Bennett, Leonard Jurd, Joseph Corse, Jack Simms (through the courtesy of Mead Johnson Research Laboratory), Bill Rockwell, Dante Guadagni, and Ben Stark assisted in various aspects of the project. Literature Cited Agricultural Research Service, U. S. Department of Agriculture, "Dihydrochalcone Sweeteners," CA 74-18, June 1968. Gentili, E.,Horowitz, R. M., J. Chromatogr., 63, 467 (1971). Horowitz. R . M., Gentili, 8..U. S.Patent 3,087,621 (1963). J. Agr. FoodChem.. 17, 696 (1969). Horowitz, R. M., Gentili, 6.. Krbechek, L., Inglett. G., Holik. M . , Dowling, B., Wagner, R., Ritter. R . , J . Agr. FoodChem.. 16, 108 (1968). Simms, J., private communication from Mead Johnson Research Laboratory, Evansville, lnd., 1970.
Receiued for reuieu October 30, 1972 Accepted J a n u a r y 18,1974 Reference t o a c o m p a n y or p r o d u c t n a m e does n o t imply approval or recommendation of t h e p r o d u c t by t h e U. S. D e p a r t m e n t of A g r i c u l t u r e t o t h e exclusion of others t h a t m a y be suitable.
Isolation of Antitumor Alkaloids from Cephalotaxus harringtonia Richard G. Powell,* S. Peter Rogovin, and Cecil R. Smith, Jr. Northern Regional Research Laboratory. Peoria. 111. 61604
Purified antitumor alkaloids (harringtonine, isoharringtonine, and homoharringtonine) from Cephalotaxus harringtonia have been isolated in the pilot plant. Major processing steps included extraction of the plant material with ethanol, isolation of a crude alkaloid fraction by chloroform extraction, preliminary separation of the crude alkaloids by 10-tube countercurrent distribution, concentration of the active alkaloids by column chromatography, and final separation of the active alkaloids by 200-tube countercurrent distribution. From 455 kg of plant material, about 330 g of crude alkaloid was obtained which yielded 36 g of three purified active cephalotaxine esters.
Several alkaloids isolated from the evergreen tree, Cephulotaxus harringtonia, have shown significant activity against L-1210 or P388 leukemia in mice (Mikolajczak, et al., 1972; Powell, et al., 1972). The active alkaloids, which are esters of cephalotaxine (I), include harringtonine (II), isoharringtonine (111), homoharringtonine (IV), and deoxyharringtonine (V). These and a number of other Cephalotaxus alkaloids are found throughout the tree but are most concentrated in the seed and are found in lesser amounts in roots, stems, and leaves (Perdue, e t al., 1970). Large quantities of seed have been unavailable for processing. Leaves also contain high concentrations of lipids and other ethanol-soluble materials which form tenacious emulsions that are difficult to process. Alkaloids I1 and IV have both been cleared for preclinical pharmacological evaluation by the National Cancer Institute on the basis of their initial activity. Alkaloid IV is currently favored over alkaloid I1 as it shows activity a t Ind. Eng. Chem., Prod. Res.
Develop.,Vol. 13, No. 2, 1974 129
