A CHEMICAL STUDY OF THE BITTER PRINCIPLE OF PI! (Tacca Leontopetaloides (L.) O. Ktze) A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHIWSOPHY JUNE, 1959 by" Carl.E. S1fanholm 1iBwn • .ACl H3 nO o 19 cop,,2 59- 8884 Thesis Committee: Dr. Paul Seheuer, Chairman Mr. Irwin Lane Dr. Hiromu Matsumoto Dr. John Naughton Dr. John P~e To Sally, Carlene and Kimi TABLE OF CONTENTS PAGE LIST OF FIGURES . • t • • • . .. iii LIST OF TABLES · · · v I. INTRODUCTION ·· · 1 II. EXPERIMENTS AND RESULTS . · ·· 7 A. Extraction Procedure o 0 0 " \) I) (t · · · 7 B. Isolation and Purification of the Bitter Principle 8 (Taccalin) ill 0 0 " ·.. .. C. Criteria for Homogeneity 11 D. Characterization Studies . • e ••• 14 1. Physical and Chemical Properties ·.. · ..... 14 Studie~ 2. Structural "0000000"0 · . 18 a. Acetylation Experiments 0 • " 0 0 •••• 18 b. Opening of the Anhydride 000'0' · . .. 19 c. Degradation Experiments •• · .. 25 (1) beta-·Diketone (IV) 0 0 . " . " . 25 (2) Ketone (VI) o 0 " •• •,• • • 0 • • 36 0) Alcohol (VII) · .. .. .. 36 (4) Lactone (VIII) •• 39 (5) Hydroxy Ester (IX) .. e ••• 41 III. DISCUSSION OF RESULTS . · . 43 A. Determination of the Molecular Formula . 43 B. Nature of the Oxygen Functions .. " . " . 46 C. Establishment of the Number of Rings •••0•••••••• 49 D. Structural Studies ••• e •••••• o •••••• o.oo 50 11 PAGE 1. Basic Hydrolyses • 0 0 0 0 0 •• . 50 2. Degradation of Taccalin ••• 0 0 • • ·. 55 3. Partial Structures of the Degradation Products ••• · . 60 Eo Conclusions (I (I I) Cl . .. 62 SUMMARY • 0 0 •• o • • 64 APPENDIX: COMPANION SUBSTANCES OF TACCALIN 0 o (l (I • 67 L Introduction 0 0 • 0 •• 0 .. (;I (I (;I • • 0 67 II. Experilrents and Results (I " 0 0 67 Ao Ester (I) •. 0 0 0 0 000;)0"00 67 B. Alcohols (X) and (XI) OOOOOQOl.) 000000 73 III. Discussion of Results o 0 0 0 0<1000000 75 A. Ester (I) •• 0 ·. 75 B. Alcohols (X) and (XI) • 0 00.000 77 V. BIBLIOGRAPHY •• 0 eo.o.oo 78 0 VI. ACKNOWLEDGMENTS • •• 0 83 iii LIST OF FIGURES PAGE FIG. 1 THE PIA PLANT, ~ Leontopeta1oides (L.) O. Ktze. (a) TUBERS, (b) FLOWERING PLANT, (c) INFLORESCENCE, (d) CROSS-SECTION OF TUBER, (e) GROWING PLANT • 6 FIG. 2 UV ABSORPTION SPECTRUM OF ESTER (I) IN ETHYL ETHER 1 (c = 5.0 X 10- g./l.) . 0 0 ••••••••• • 0 • • 0 12 FIG. 3 UV ABSORPTION SPECTRUM OF TACCALIN IN METHANOL 2 0 • 0 • 0 •• ( c =4.42 X 10- g./l.) .•.• " " • <) •"• 13 FIG. 4 IR SPECTRUM OF TACCALIN • O.'CI'O~OOOO"O'O.' •• 16 FIG. 5 IR SPECTRUM OF PRODUCT FROM "ACETYLATION" OF TACCALIN - ACID (II); FILM FROM CHLOROFORM 0 •• , • 0 • 0 ••• o • • • " • 20 FIG. 6 IR SPECTRUM OF ACID (III); FILM FROM CHLOROFORM • o • • 0 23 FIG. 7 GLPC OF: 1. MESITYL OXIDE/ACETONE (99/1); II. VOLATILE FRAGMENTS FROM THE BASIC HYDROLYSIS OF TACCALIN ••••• •• 24 FIG. 8 IR SPECTRA OF MESITYL OXIDE (CURVE A) AND VOLATILE FRAGMENT FROM BASIC HYDROLYSIS OF TACCALIN (CURVE B); LIQUID FILMS.. 26 FIG. 9 UV ABSORPTION SPECTRUM OF BETA· DlKETONE (IV), _ IN 2-PROPANOL (c =2.5 X 10-2 g./l.), •.•.• IN 2-PROPANOL (c =12.4 X 10-2 g./l.), - ---- IN 0.1 N NaOH (c =6.8 X 10-2 g./l.) •••..••• .•.• . .•• •• 29 FIG. 10 IR SPECTRUM OF BETA-DlKETONE (IV) o • • " •••• 0 " " " " 0 30 iv PAGE FIG. 11 IR SPECTRUM OF COPPER (II) CHELATE OF BETA-DlKETONE (IV); MINERAL OIL MULL •. " 0 • ".oooo.ooo •• e •• oO 32 FIG. 12 ABSORPTION SPECTRUM OF IRON (III) CHELATE OF BETA-DlKETONE (IV) IN 2-PROPANOL (c =1005 X 10-1 go/l.) ···· ·· · · · 33 FIG. 13 IR SPECTRUM OF ACID (V); MINERAL OIL MULL · · · · · · 35 FIr1. 14 tN ABSORPTION SPECTRUM OF KETONE (VI) IN ETHANOL 1 , (c = 3.0 X 10- g./l.) . 0 0 . 0 . · 0 · · · ·· · · 37 FIG. 15 IR SPECTRUM OF KETONE (VI); MINERAL OIL MULL 0 · · 0 · ·· 0 38 FIG. 16 IR SPECTR~l OF ALCOHOL (VII); SOLID FILM 0 ·. · · · · · · · 40 FIG. 17 IR SPECTRUM OF HYDROXY ESTER (IX); SOLID FILM · · · · · · 0 42 FIG. 18 IR SPECTRUM OF ESTER (I); SOUD l"ILM • . · · · · ··· · · 0 70 FIG. 19 IR SPECTRUM OF ALCOHOL (XII); SOLID FILM • 0 . · · · · · ·· 71 FIG. 20 IR SPECTRUM OF ACID (XIII); SOLID FILM • · ·· · · · · · 72 FIG. 21 IR SPECTRUM OF ALCOHOL (X); MINERAL OIL MULL • · 0 0 · ·· · . 74 FIG. 22 IR SPECTRUM OF ALCOHOL (XI); MINERAL OIL MULL • ·· · ·· · 76 v LIST OF TABLES PAGE TABLE I. SOME NON-NITROGENOUS BITTER PRINCIPLES ISOLATED FROM PLANTS 3 TABLE II. COMPARISON OF POSSIBLE MOLECULAR FORMULAS OF TACCALIN • •• 44 I. INTRODUCTION Pia belongs to the plant family Taccaceae, which consists of approx­ imately thirty species (1), widely distributed over the warmer regions of the globe (2). Taccaceae contains only two genera, Schizocapsa and Tacca. The former consists of only two species located in the South China-Malaya area, while ~, which is sub-divided into three sections ­ Ataccia, Palmotacca, andEutacca- is more widely distributed (3). Limpricht (4) accepted the name Tacca pinnatifida J. & G. Forst. for pia, but the presently accepted name is Tacca Leontopetaloides (L.) O. Ktze (5)~ Pia is probably native to tropical Asia and of aboriginal intro­ duction to Hawaii (6), although the exact origin of this species is not known (7). Pia is an herb consisting of tuberous underground stelJ\s similar to the potato, with leaves that rise directly from the ground and which resemble papaya leaves. 'The stem, which may reach a height of four feet or more, is topped by a striking inflorescence of small green and purplish flowers (8) (see Fig. 1). The pia plant was cultivated for its starch content and was produced in Hawaii in sizable quantities in the 1850's for export to Europe under the name "arrowroot" (9). The plant is rarely cultivated now, but small * Another common synonym of pia is Tacca hawaiiensis. However, £1. reference (1), p. 30. 2 quantities can still be found growing wild near Hilo in the Puna district. The pure starch from the pia tubers was combined with coconut cream, bananas or ti root juice to yield food staples eaten by most Polynesians. It was well known to the Hawaiians, however, that a bitter material had to be removed from the raw tuber in order to make the starch edible. The raw tuber has been reported to be toxic (10) and only the Easter Islanders are reputed to eat the unwashed tuber, but only after baking (11). The Hawaiians grated the tubers and washed them in running water for prolonged periods. The resultant bitter extract was used in this dilute state as a medicine to combat diarrhea and dysentery, particularly in infants (12). I In 1857, Lepine carried out a superficial chemical analysis of pia and reported 2.2% of a bitter extract (13). This report is the sole published record of a chemical nature concerning pia. Except for a similar paper dealing with the bitterness and toxicity of Tacca umbrarum* (14), no chemical study has been made of any species of this plant family. Since no chemical compounds have been isolated from Taccaceae, a prediction of the general nature of the bitter principle cannot be made. It was hoped that a search of the literature for bitter principles from other plants might indicate some relationship from which the bitter principle of pia could be categorized as to type. Some representative bitter principles of current interest, some * Tacca umbrarum is syno~mous with 1. madagascariensis (15), which is endemic to Madagascar where pia is found in abundance. Pia is closely related to !. madagascariensis taxonomically, which indicates that their bitterness might arise from the same (or a similar) chemical substance. TABLE I SOME 'NON-NI'FRQGE~SBITTEllPlUNCIPLES-ISOLATED FROM PLANTS Chemical TypeoT Pharmacological Name Source Formula Functional Groups Action Reference Pikrosa1vin Salvia officinalis C Z.OH Z604 Diphenolic lactone Bacteriostat 16 Limonin Citrus aurantium °Z6H3008 II Fur ana dilactoneII Not reported 17 val'. Natsudaidai Lactucin Lactuca virosa C 1SH 160S Dihydroxy-a1pha, beta­ Not reported 18 unsaturated ketone­ guaianolide Tenulin He1enium G 16H Z00S Lactone-hemiaceta1­ Not reported 19 tenuifolium alpha, beta-unsaturated ketone - guaiane skeleton Geigerin Geigeria aspera ClSHZ004° HZ ° IIMono1actaneI' "Pharmacologi­ ZO cally active" Absinthin Artemisia C30H4006 Dihydroxy-dimeric Inconclusive ZI absinthium guaianolide Darutoside Siegesbeckia GZ6H 4403 Hydrolysis yields Not reported ZZ o,rielrtalis~ glucose, a glycol and a trihydroxy diterpenoid compound Alantopicrin Inu1a he1enium C 17H Z40 4 Lactone? Fish poison Z3 \N - T ABLE I (conti d) picrolichenic Pertusaria amara C25H3007 Depsidone Anti= malarial 24 acid (crustose lichens). Marrubiin Marrubium vulgare C H 0 Lactone- carboxylic, ? 25 17 22 6 carbonic acid anhydride containing spiro linkage Nimbin Melia indica C28H4008 Steroidal lactone Ca.rdiac 26 Kondurangin Marsdenia ? Glycoside. Aglycon is Carcinogen 27 condurango dodeeahydrofluorenone derivative Elaterin Ecballium C32H4408 Diosphenol grouping Anti-tum.or 28 elaterium- activity Acetylandromedol Rhododendron C22H3607 Acetate of hexahydroxy Hypotensive 29 maximtIm compound agent til- 5 of which are still incompletely characterized, are listed in Table I. Only those containing carbon, hydrogen and oxygen are recorded, since they are more pertinent to this investigation, as will be seen later. It is apparent from Table I that non-nitrogenous bitter principles occur in widely diverse plant types - flowering plants and even lichens. Chemically, several skeletal types can be distinguished: steroid, terpenoid, aromatic and depsidone, any of which may be glycosidically bound to a sugar moiety. Although most of the compounds are highly oxygenated, the nature of the oxygen functions vary widely and any re­ lationship which might exist is not immediately apparent. Due to this great diversity of chemical types found in plants, little ~ priori in­ formation can be surmised about the bitter principle of pia.