Ipomoea, Rivea and Tissue Cultures: Influence of Various Chemical Factors on Production and Growth

R. H. DOBBERSTEIN1 AND E. JOHN STABA2 (University of Nebraska. Lincoln. Nebraska 68508)

Seeds and of certain members of the contain . The principal -alkaloid containing plants are Ipomoea violaeea L. (morning-glory, Badoh Negro), Rioea eorymbosa (L.)Hall. f. (Ololiuqui, wild morning-glory) and Argyreia nervosa Boj. (baby Hawaiian wood rose). Extracts of these plants may (6, 11, 17) or may not (9, 12) elicit a diethylamide- like response. Stab a and Laursen (19) reported the presence of indole alkaloids in static tissue cultures of Ipomoea violaeea var. pearly gates and flying saucer and Rioea eorymbosa. The principal objective of this study was to examine suspension cultures of Ipomoea violaeea var, pearly gates, Rivea eorymbosa and Argyreia. nervosa and static cultures of Rivea corymbosa in media containing various chemical factors that might stimulate indole alkaloid production.

MATERIAL A D METHODS tissue origin and media.-Seeds of l. violacea var. pearly gates and R. corymbosti were sterilized and grown in static culture as previously described (19). A. neruosa seeds! were scraped with a razor blade to remove the hair. notched with a scalpel and soaked in tap water for 24 hr before sterilization. They were sterilized in a .5.25%sodium hypochlorite solution! diluted 1:3 with sterile distilled water, for 15 min in vacuo. These seeds were placed in Petri plates con- taining 20 ml sterile tap water.After germination, root, stem and leaf callus cultures were ob- tained by placing sterile roots, stems and leaves in one-oz dry squares containing 18 ml revised tobacco medium (RT)(13) with 0.1 ppm 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.1% agar. The Argyreia callus tissue used for this study was of leaf origin; the Ipomoea and Rivea callus tissues were of seed origin. All callus tissues had been maintained on solid medium for at least one year before they were grown as suspension cultures. The basal medium used for Ipomoea was RT medium with 0.1 ppm 2,4-D and 10 ml/liter of the following stock solution in g/liter: L-glutamine, 20.0; L-glutamic acid, 5.0; L-argenine HCI, 2.5; L-asparagine, 20.0; glycine, .5.0;L-methionine, 0..5; L-cystine, 0..5; and D-alanine, 5.0. The basal medium used for Rivea was RT medium with 0.1 ppm 2,4-D; that used for Argyreia was RT medium with 0.1 ppm 2,4-D and 0.2 ppm kinetin. All media were adjusted to pH 6.5 with 10% KOH or 10% NaOH and autoclaved. Basal media modifications.- The following modifications were made to each of the three basal media: (i) Low potassium medium (K-1).-The RT medium contains 783.8 mg/liter of potassium. The inorganic stock solution was made as previously reported (13), but the 38.0 g/2 liters KNO, was replaced with 32.0 g/2 liters NaN03 and the pH was adjusted with 10% NaOH instead of 10% KOH.Thus prepared, the low potassium medium contained 49.0 mg Zliter of potassium. This medium was used because it has been shown that in intact higher plants, a low potassium level (1,564 mg/lOOg dry weight of soil) favors free accumulation or biosynthesis, a low indoleacetic acid (IAA) content and an increased amount of total alkaloids in Datura tatuZa and Rauwolfia serpentine (30). (ii) High phosphate medium (P-2).-The RT medium contained 118.6 mg/liter of phosphate and the high phosphate medium prepared, contained 593.2 mg /liter of phosphate.To obtain this additional phosphate without altering the potassium concentration, 1.3 g a2HPO. were added to each liter of medium. It is known that in some species of Cltunceps pur-puree (Fr.) Tul. (ergot), a high phosphate concentration (0.1-3.0 g/liter) causes increased alkaloid formation (18, 22).

'Based on portions of a thesis submitted by R. H. Dobberstein to the Gradua te College, university of Nebraska in partial fulfillment of the requirements for the M.S. degree. 'Present address: Department of Pharmacognosy, University of Minnesota, Minneapolis, Minn. 55455. 30btained from Dr. Ara Der Marderosian, Philadelphia College of Pharmacy and Science, Philadelphia, Penn. +Purcx Corp., Ltd., South Gate,Calif. 141 142 LLOYDIA [VOL. 32, xo 2

(iii) Special carbohydrate medium (C-3).-In this medium, the concentration of sucrose was reduced from 3% to 0.5%.In addition, 1.0% sorbitol and 0.5% fumaric acid were added. In order to dissolve all of the fumaric acid,NaOH was added, one pellet at a time, until the furmaric acid dissolved. This medium was used because a reduced carbon source should be depleted early, forcing the tissue into nitrogen shunt (21). At the same time, car- bohydrate alcohols (1, 5, 21, 23, 24, 27) and Krebs-cycle acids (3) have been reported to in- crease alkaloid production in ergot submerged cultures. (iv) Precursor medium 0II-4).-In this experiment, 100 mg /liter i.-tryptophan and 1.0 mg /liter mevalonic acid lactone were added to the medium. Mevalonic lactone (3.5 mg) was added to 10 ml double distilled water and the pH was adjusted to approximately 11. The solu- tion was allowed to stand overnight at room temperature, and then the pH was adjusted to approximately 6.5.This treatment hydrolyzes mevalonic lactone to mevalonic acid.The L-tryptophan (350 mg) was added to the solution, and diluted to 46.6 ml. After cold steriliza- tion, the solution was aseptically transferred to a specially constructed flask connected to a multiple dose inoculation syringe (14), and 0..5 ml of this solution was aseptically added to each flask. Mevalonic acid and L-tryptophan are ergoline alkaloid precursors (8, 16, 25, 26, 2S). (v) Tween-chick pea meal medium (T-5).-This medium consisted of 0.1% Tween-SO' and 1.0% chick pea meal added to basal RT medium. The untreated chick peas! were ground to a ::lO-mesh powder in a Wiley mill.The chick pea meal did not completely dissolve, so the medium was well shaken before being dispersed into each flask or dry square. Since Tween-80 is a sur- face active agent, it might favorably change the cell membrane permeability. Chick pea meal has been shown to increase alkaloids in certain submerged ergot strains (4).

TABLE 1. Growth index and age of A rgyreia, Ipomoea and Rivea suspension tissues and Rioea callus tissue.

Argyreia Ipomoea Rivea Rivea Callus

Media- , G.I.b Age G.I.h Age c.r.» Age G.I.b Age (wk) (wk) (wk) (wk) ---- Control I . .. . 2.9 3 1.7 4 1.1 3 3.6 10 Control II. ... 5.8 5 2.1 8 8.0 5 - - C-3 ...... 1.1 5 0.9 8 1.5 5 0.8 10 M-4 .... 3.7 5 2.0 8 2.3 5 2.5 10 T-5. 2.1 5 1.8 S 2.0 5 3.7 10 Control III ...... 2.1 6 1.6 7 1.8 S 2.5 15 K-l...... - . l.5 6 0.9 7 l.7 8 2.3 15 P-2 ...... 1.1 6 0.7 7 2.1 S 2.7 15 1.1 1.4 :\-6...... ' ...... 0.4 6 0.6 7 S 15

"Tissues grown in media Control III, K-l, P-2 and N-6 were grown for two generations. Argyreia was grown for three weeks, the old media decanted and fresh media added. Ipomoea was grown for four weeks, the old media decanted and fresh media added. Rivea was grown for three weeks, the old media decanted and fresh media added. Rivea callus was grown for ten weeks, transferred to fresh media. "Grow th Index (G.I.)=final wet weight of tissue/wet weight of inoculum.

(vi) Auxin medium (N-6).-To the basal RT medium, 2.0 mg /liter naphthalene acetic acid (NAA) and 0.2 mg /liter kinetin were added in place of 2,4-D, or kinetin and 2,4-D. It has been shown that lAA, NAA, 2,4-D, indolepropionic acid and indolebutyric acid can cause increased alkaloid production in certain submerged ergot strains (29). Growth Conditions.-For each test medium modification and its control, five loopfuls of tissue were transferred with a sterile, sieved, stainless steel transfer cup of one-ml capacity, to twenty 250 ml Erlenmeyer flasks containing 75 ml of the test or control media. After inoculation, the flasks were placed on a reciprocating shaker (SO strokes/min; stroke length 5 ern) at room temperature (25-30°). The light conditions were not controlled, thus the cell suspensions received partial light exposure during the day and none at night.The Growth Index (G.!.) and the age of the tissue are given in table 1. The Rivea static cultures were grown at room temperature (25-30°) in one-oz dry squares containing 18 ml of media.For each test media modification and its control, 75 dry squares were inouc1ated with tissue by using a sterile scalpel.

5Atlas Power Co., Wilmington,Delaware. 6Garbanzas, EI Molino Mills, Alhambra, Calif. JUNE 1969] DOBBERSTEIN AND STABA: INDOLEALKALOIDS 143

Analytical Methods.-(i) Growth.-After the specified growth periods, the contents of each flask were filtered and the wet weight of the tissue from each flask determined. The Rivea callus tissues from each dry square were combined and the total wet weight determined. All tissues C and media were frozen (-30 ) until extracted. (ii)Extraction.'-The suspension tissues and Rivea callus were extracted twice with 2'lc tartaric acid by homogenizing at low speed in a Waring Blender (Model PB-5) for ten min. The tartaric acid tissue extract and the suspension medium were made basic (pH 8.5) with am- monium hydroxide and extracted twice with n-butanol.The butanol extracts were combined with petroleum ether (approximate ratio 3:;5) and extracted first with 10% tartaric acid, then with 2% tartaric acid. The combined aqueous tartrate phase was used for spectre-analysis. For thin-layer chro- matography, the combined aqueous tartrate phase was made basic with ammonium hydroxide (pH 8.5) and concentrated to a syrup-like residue under vacuum, in the dark at 30°. This resi- due was extracted three times with small volumes of ethyl acetate and the final volume of extract diluted to 10 m!. This ethyl acetate extract was spotted on the plates. Before extraction, the agar media were thawed and filtered. This caused some of the medium to be released from the agar. The freezing-thawing-filtering procedure was repeated four times until only a small amount of solid agar remained. The solid agar was extracted twice with basic n-butanol. The media which had been filtered from the agar was made basic with ammonium hydroxide (pH 8.5) and extracted twice with n-butanol. The butanol extracts from the solid agar and from the medium were combined and then extracted as above with petroleum ether and tartaric acid solutions.

FIG. l. Rivea suspension culture: thin layer plates from Agurell's chromatographic procedure. 1-Control III extract, 2-IAA and indole, 3-K-l extract, 4-agroc1avine, 5·-an- thranilic acid, 6-P-2 extract, 7-elymoc1avine and , 8-;-.J-6 extract, .9- and . (left to right).

(iii) Spectrophotometric assay.-The assay procedure of Michelon and Kelleher (1,5) was used for the quantitative determination of total ergoline alkaloids, calculated as ergonovine maleate. The absorbance was measured at ;j90 rnu with a Beckman-DB Spectrophotometer. (iv) Thin-layer chromatography.-Two chromatographic procedures were used. For Hof- mann's procedure (20) 40 1-'1of the ethyl acetate extracts were spotted on Aluminum Oxide G plates" and developed in a chamber with chloroform: methanol (19:1). For Agurell's procedure (2), 40 1-'1of the ethyl acetate extracts were spotted on Silica Gel G plates' and developed in a chamber with chloroform: methanol (4:1). The standards applied to each plate were ergonovine maleate, ergotamine, elymoc1avine, , , tryptophan, indole, IAA and anthranilic acid. Each extract was chromatographed in each system. After development, the plates were sprayed with l.0% PDAB (p-dimethylaminobenzaldehyde) dissolved in ethanol which was acidified with concentrated H'C], After 10 min, the plate was sprayed with 0.1% sodium nitrite in 50% ethanol until the plate was damp. When the plate was dry, it was heated at 100° for two min, resprayed with PDAB reagent and photographed (fig. 1). RESULTS ANDDISCUSSION In these experiments, Ipomoea grew as light yellow tissue aggregates. Rivea suspension tissues grew as creamy-colored finely divided cell suspensions; Rivea 'Procedure furnished by W. J. Kelleher, 1966. BE. Merck, Distributed by Brinkman Instruments, Inc., Long Island, ;-.J. Y. 144 LLOYDIA [VOL. 32, NO.2 callus tissues grew as creamy-colored to white, friable tissues. Argyreia tissues grew as light gray to white, finely divided cell suspensions. Growth.-It is somewhat difficult to draw conclusions about the effects of the nutrients on tissue wet weight growth due to their large standard deviations. It appeared that all the media variations depressed growth of Argyreia and Ipomoea (fig. 2 and 3). The greatest growth depression was caused by C-3 medium. All of the test media appeared to have little effect on Rivea suspension growth, except for media P-2, which may have slightly enhanced growth. For Rivea callus cultures, medium T-5 may have enhanced growth slightly; medium P-2 signifi- cantly increased growth. All other medium variations appeared to have depressed growth of the tissues studied.

40.0

35.0 .. ~ 30.0 c z '", ~ 2~O LOg t;; 0.9 ~::.: ~ 20.0 0.800 ,. e ... 0.7 :g ~ 15.0 O.6~ 0.5 ~ 10.0 0.4 ~

• O.3~

5.0 0.2

0.0l:•..L,-'::JRc.LRs?--...t.;.A.t.,J,..L;:-RSL...... J~;.'::-L:!-l-.....l.,-L;-.L,;-L?----,O,.!.7..o '~:-4>

CONTROL I CONTROL II

FIG. 2. Argyreia,Ipomoea and Rivea tissue cultures: growth-alkaloid relationship. Control 1- A=Argyreia (3 wks old), I=Ipomoea (4 wks old), Rc=Rivea callus (10 wks old), Rs=Rivea suspension (3 wks old). Control II, C-3, M-4 and T-5-A = Argyreia (5 wks old), I=Ipomoea (8 wks old), Rc=Rivea callus (10 wks old), Rs=Rivea sus- pension (5 wks old). Media Designation: C-3 = Special carbohydrate medium (sor- bitol, fumaric acid), M-4=Precursor medium (t-t.ryptophan, mevalonic acid), T-5= Tween-chick pea meal medium.

4C.0 -

35,0

30.0 o z " e 25.0 1.0 ~ e ... 0.9, i- l: ~ 20.0 O.S E 0.7 ~a I;'"; 15 3; 15.0 0.6 ~ C.!:! ~ 10.0 4 ~ 0.3 g 0.0

0.0L1.:-L:-~-----'L1.:-L:-.L,;-L---~:-L:-.L,;-L---.Ll-:-L~ Al~Rs AI~Rs AJ~Rs CONTROL m K-[ . P-2 fIG. 3. Argyreia, Ipomoea and Rivea tissue cultures: growth-alkaloid relationship. Control III,K-l, P-2 and N-6-A = Argyreia (6 wks old), I=Ipomoea (i wks old), Rc= Rivea callus (15 wks old),Rs=Rivea suspension (8 wks old). Media Designation: K-l=Low potassium medium, P-2=High phosphate medium, -6=Auxin medium (naph thaleneacetic acid, kinetin). JU\E 1969] DOBBERSTEIN AND STABA: INDOLE ALKALOIDS 145

Each experiment consisted of twenty flasks, of which no more than three were contaminated at one time. Contaminated flasks were not used for either growth calculations or for alkaloid analysis. Alkaloid production.--(i) Quantitative.-In the following discussion, total alkaloids (mg) extracted from 100 g wet weight of tissue and 100 ml of media is reported as alkaloid percent.Total alkaloids refers to the mg of total alkaloids found in both tissue and media, independent of the weight of tissue or volume of media from which the alkaloids were extracted. In control media, Rivea callus produced the greatest total alkaloids (1.63 mg, control II); Argyreia produced the least (0.16 mg, control I). In addition, Rivea suspensions produced the greatest alkaloids percent in control media (0.97 mg %, control I) and Argyreia the least (0.03 mg %, control II) (fig. 2 and 3). On a alkaloid percent basis, the following tissues were more efficient than their respective controls: Rivea suspension tissue in C-3 medium (1.02 mg/lOO g); Ipomoea tissue in M-4 (0.71 mg/lOO g), N-6 (0.63 mg/lOO g) and P-2 (0.46 mg/lUO g) media; Rivea callus tissue in N-6 medium (0.27 mg/lOO g); and Argyreia tissue in N-6 (0.14 mg/IOO g) and K-1 (0.12 mg/lOO g) media. The following medium variations caused a greater total alkaloid production than in the control: K-l medium (1.07 mg) for Argyreia tissue; M-4 (2.29 mg), N-6 (1.73 mg) and P-2 (1.33 mg) media for Ipomoea tissue and N-6 (1.84 mg) and P-2 (1.63 mg) media for Rivea suspension tissue. All of the variations depressed total alkaloid production in Rivea callus tissue. In this study, the tissues generally contained a greater amount of alkaloids than the media, although the ratio of tissue alkaloids to medium alkaloids varied from one tissue and one medium to another. The second generation media usually contained a greater amount of alkaloids than the first generation media. (ii) Qualitative.- The various test media did not appear to have a regular or predictable effect on the variety of alkaloids contained in either tissue or media. The greatest variety of alkaloids are apparently produced by Rivea suspension cultures in K-l medium. The media variations did appear to cause differences in the alkaloids produced. In most cases, control II, grown for a longer time period than control I, appeared to contain a few more alkaloids than control I. Both tissue and media extracts produced spots which had Rp values similar to standards applied on thin layer plates. Because of similarities in chromatographic R» values, agroc1avine may be present in either the tissue or the media of Rivea suspension cultures for all six media variations and the control. Agroc1avine appeared to be the most commonly occurring alkaloid in all of the other tissues as well. Co-chromatography of the extracts with agroc1avine was not done.A sample chromatogram may be seen in fig. 1. (iii) Comparative.-The seeds of Ipomoea tnolacea var. pearly gates were reported to contain from 0.015 to 0.024 % total alkaloids (7), Rivea corymbosa from (U)21 to 0.060% total alkaloids, (7) and Argyreia neruosa 0.30% total alka- loids (10). Although the percentages of alkaloids reported in this study are low as compared to the above seeds, the amounts produced by Rivea suspension cultures do compare favorably with the amounts produced by Ipomoea and Rivea callus cultures in an earlier study; var. pearly gates callus and media were found to contain 0.92 mg/lOO g wet weight total alkaloids and Rivea callus and media were found to contain 0.38mg/IOO g wet weight total alkaloids (19) . SUMMARY Suspension cultures of Ipomoea violacea var. pearly gates, Rivea corymbosa and A rgyreia neruosa produce indole-type alkaloids.Six different chemicals added to the medium were found to have an irregular, unpredictable quantitative and qualitative effect 011 alkaloid production. The greatest total alkaloid production 146 LLOYDIA [VOL. 32, NO.2

(2.29 mg from 264.8 g wet weight tissue and 1.425 liter media) occurred in Ipomoea violacea var. pearly gates grown in precursor medium (lVI-4) with mevalonic acid and L-tryptophan. ACKNOWLEDGMETS This investigation was supported by U.S. Public Health Services Grant o F1-MH-29,261 (PE) and GM 13440-02. Received 7 August 1968. LITERATURE CITED 1. Abe, M. and S. Yamatodani. 1964. Production of alkaloids by saprophytic culture of ergot fungi. Prog. Ind. Microbiol. 6: 203-229. 2. Agurell, S. 1965. Thin-layer chromatographic and thin-layer electrophoretic analysis of ergot alkaloids. Relations between structure, R" value and electrophoretic mobility in the clavine series. Acta Pharm. Suecica 2: 357-374. 3. Amici, A. M., A. Minghetti, T. Scotti, C. Spalla and R. Tognoli. 1966. Production of ergotamine by a strain of Clcuiceps purpurea (Fr.) Tul. Experientia 22: 41.5-416. 4. Arcamone, F., F. R. S. Chain, A. Ferretti, A. Minghetti, P. Pennella, A. Tonolo and L. Vera. 1961. Production of a new lysergic acid derivative in submerged culture by a strain of Claoiceps paspali. Stevens and Hall. Proc. Royal Soc (London) 166:B 26-.54. :5. Brady, L. R. and V. E. Tyler, Jr. 1960. Alkaloid accumulation in two c1avine-producing strains of Claviceps. Lloydia 23: 8-20. 6. Cohen, S. 1964. Suicide following morning-glory seed ingestion. Am. J. Psych. 120: 1024-1025. 7. Der Marderosian, A. and H. W. Youngken, Jr. 1966. The distribution of indole alkaloids among certain species and varieties of Ipomoea, Rivea and . Lloydia 29: 35-42. 8. Floss, H., U. Mothes and H. Gunther. 1964. Zur biosynthese der mutterkornalkaloide. Uber den mechanismus der reaktion am a-C-atom. Naturjorsch: 19: 784-788. Y. Guthery, K. and S. E. Perling. 1966. Some effects of hallucinogenic morning-glory seeds on the behavior of rats. Psychol, Rep. 19: 949-950. lO. Hylin, J. W. and D. P. Watson. 1965. Ergoline alkaloids in tropical wood roses. Science 148: 499-500. 11. Ingram, A. L., Jr. 1964. Morning-glory seed reaction. J. Am. Med. Assoc. 190: 1133- 1134. 12. Isbell, H. and C. W. Gorodetzky. 1966. Effect of alkaloids of Ololiuqui in man. Psy- chopharmacol. 8: 331-339. 13. Kaul, B. and E. J. Staba. 1968. Dioscorea tissue cultures: 1. Biosynthesis and isola- tion of diosgenin from Dioscorea deltoidea Wall. callus and suspension cells. Lloydia 31: 171-179. 14. Lamba, S. S. and E. J. Staba. 1963. Effect of various growth factors in solid media of Digitalis lanata Ehrh. and Mentha spicata L. cell suspensions. Phyton 20: 175-181. 15. Michelon, L. E. and W. J. Kelleher. 1963. The spectrophotometric determination of ergot alkaloids. A modified procedure employing p-dimethylaminobenzaldehyde. Lloydia 26: 192-20l. 16. Paul, A. G., W. J. Kelleher and A. E. Schwarting. 1954. The culture of Clauiceps pur- purea. II. Preliminary metabolism studies. J. Am. Pharm, Assoc. 43: 205-207. 17. Rice, W. B. and K. Genest. 1965. Acute toxicity of extracts of morning-glory (Ipomoea violacea, Rivea corymbosa) seeds in mice. Nature 207: 302-303. 18. Rosazza, J. P., W. J. Kelleher and A. E. Schwarting. 1967. Production of lysergic acid derivatives in submerged culture. IV. Inorganic nutrition studies with Claoiceps pas pali. Appl. Microbiol. 16: 1270-1283. lY. Staba, E. J. and P. Laursen. 1966. Morning-glory tissue culture: growth and examina- tion for indole alkaloids. J. Pharm. Sci. 66: 1099-110l. 20. Stahl, E. 1962. Thin-layer chromatography. Academic Press, New York, N. Y. pp. 288-290. 2l. Taber, W. A. 1963. Ergot alkaloid production and physiology of Claviceps pur-puree (Fr.) Tul. Develop.Ind. Microbiol. 4: 295-305. 22. Taber, W. A. 1967. Fermentative production of hallucinogenic indole compounds. Lloydia 30: 39-66. 23. Taber, W. A. and L. C. Vining. 1957. In vitro production of ergot alkaloids by cultures of Clauiceps pur-puree (Fr.) Tul. Can. J. Microbiol. 3: 55-60. 24. Taber, W. A. and L. C. Vining. 1958. The influence of certain factors on the in vitro production of ergot alkaloids by .Can. J. Microbiol. 4: 611-626. 25. Taylor, E. H. and E. Ramstad. 1960. Biogenesis of the c1avine-type ergot alkaloids. J. Pharm. Sci. 60: 681-683. JONE 1969] DOBBERSTEIN AND STABA: INDOLE ALKALOIDS 147

26. Taylor, E. H. and E. Ramstad. 1960. Biogenesis of lysergic acid in ergot. Nature 188: 494-495. 27. Tyler, V. E., Jr. 1958. Some factors influencing the saprophytic production of c1avine alkaloids by Claviceps pur-purea. J. Am. Pharm. Assoc. 47: 787-792. 28. Tyler, V. E. and A. E. Schwarting. 1954. The culture of Claviceps purpurea. Ill. Tryptophan metabolism. J. Am. Pharm. Assoc. 43: 207-21l. 29. Teuscher, E. 1965. Influence of auxins on the production of ergot alkaloids in sapro- phytic cultures. Phytochem. 4: 341-343. 30. Wakhloo, J. L. 1965. Evidence for IAA and tryptophan in the shoots of Solanum nigruni and the effect of potassium nutrition on their levels. Plcnta 66: 301-314.