Mitsue Haraguchi et al- Alkaloidal Components in the Poisonous Plant, Ipomoea carnea (Convolvulaceae)

 

J. Agric. Food Chem.  2003,   51,  4995−5000   4995

Alkaloidal Components in the Poisonous Plant, Ipomoea carnea  (Convolvulaceae) MITSUE   HARAGUCHI,† SILVANA  L. GORNIAK,‡ KYOKO   IKEDA,§ YASUHIRO   MINAMI,§ ATSUSHI   KATO,⊥ ALISON  A. WATSON,| ROBERT   J. NASH,# RUSSELL   J. MOLYNEUX,X AND   NAOKI   ASANO*,§ Animal Health Center, Biological Institute of Sa˜o ˜o Paulo, Sa˜o Paulo, Brazil; Research Center for Veterinary Toxicology (CEPTOX), School of Veterinary Veterin ary Medicine, University of Sa˜o ˜o Paulo, Sa˜o ˜o Paulo, Brazil; Faculty of Pharmaceutical Sciences, Hokuriku University, Universit y, Ho-3 Kanagawa-machi, Kanazawa Kanaza wa 920-1181 920-1181,, Japan; Department of Hospita Hospitall Pharm Pharmacy, acy, Toyama Medica Medicall and Pharm Pharmaceuti aceutical cal University, Univers ity, Toyama 930-0194, Japan; Molec MolecularNa ularNature ture Limited Limited,, Gogerd Gogerddan, dan, Aberys Aberystwyth, twyth, Cardiganshire SY23 3EB, United Kingdom; Institute of Grassland and Environmental Research, Gogerddan, Gogerdd an, Aberystwyth, Cardiganshire Cardiganshire SY23 3EB, United Kingdom; and Wester Western n Region Regional al Resear Research ch Center,, Agricul Center Agricultural tural Research Service, U.S. Depart Department ment of Agricul Agriculture, ture, 800 Buchan Buchanan an Stree Street, t, Albany, California 94710

Natural intoxication of livestock by the ingestion of  Ipomoea carnea   (Convolvulaceae) sometimes occurs in tropical regions of the world. Polyhydroxylated alkaloids were isolated from the leaves, flowers, and seeds of the poisonous plant and characterized. Chromatographic separation of the leaf extract resulted in the isolation of swainsonine ( 1), 2-epi -lentiginosine -lentiginosine (2 (2), calystegines B1  (  (3 3), B2  (  (4 4), B3  (  (5 5), and C1  (  (6 6), and  N -methyl-methyl-trans -4-hydroxy-4-hydroxy-L-proline (7 (7). The contents of 1 of  1  in the fresh leaves and flowers were 0.0029 and 0.0028%, respectively, whereas the contents of 1 of  1,,  3  3,, and 4 and  4  in the seeds were ∼10 times higher than those in the leaves and flowers. Alkaloids  3  3,,  4  4,, and 6 and  6 showed  showed a potent inhibitory activity toward rat lysosomal   -glucosidase, with IC50   values of 2.1, 0.75, and 0.84 µ 0.84  µM, M, respectively, and alkaloid 5 alkaloid  5  was a moderate inhibitor of R - and  and  -mannosidases. -mannosidases. Although alkaloid 1 alkaloid  1 is  is known as a powerful inhibitor of lysosomal R -mannosidase -mannosidase (IC50 ) 0.02 0.02 µ  µM), M), alkaloid 2 alkaloid  2,, which has been thought to be an intermediate in the biosynthesis of  1  1,, was also a potent inhibitor of -mannosidase with an IC50  value of 4.6 µ 4.6  µM. M. R -mannosidase KEYWORDS:   Ipomoea carnea  carnea ; poisonous plant; intoxication in livestock; polyhydroxylated alkaloids; glycosidase inhibition

INTRODUCTION Certain poisonous plants often cause serious livestock losses. The Austr Australian alian legume speci species, es,   Swainsona, ar aree kn know own n as “poison “poiso n peas”, and sheep eating them develop a syndrome called “pea struck” (1,  2). There is also the livestock poisoning by the closely related   Astragalus   and   Oxytropis   species, species, whic which h are found fou nd thr throug oughou houtt mos mostt of the wor world, ld, and int intoxi oxicat cation ion of  livestock by certain of those species known as locoweeds in the wes wester tern n Uni United ted Sta States tes is cal called led “lo “locoi coism” sm” (2,   3). Th Thee common clinical symptoms in livestock on ingestion of these poisonous legumes are depression, tremors, nervousness, ema* Author to whom correspondence should be addressed (fax   +81 76 229 2781; e-mail [email protected]). † Biological Institute of Sa˜o ˜o Paulo. ‡ Research Center for Veterinary Toxicology (CEPTOX). § Hokuriku University. ⊥ Toyama Medical and Pharmaceutical University. | MolecularNature Limited. # Institute of Grassland and Environmental Research. X Agricultural Research Service.

ciation, gastrointestinal malfunction, and reproductive alterations (4,   5), and the poi poison soning ing is cha charac racter terize ized d by cyt cytopl oplasm asmic ic vacuolation of neuronal cells due to accumulation of mannoserich oligosaccharides in lysosomes ( 6 ). ). The trihydroxyindolizidine alkaloid swainsonine (1) (Figure 1) occurs in these legumes and has been identified as a causative agent in locoism ( 3,  7 ). ). Swainsonine is a potent inhibitor of lysosomal ( 8 ) and Golgi R -mannosida -mannosidase se II ( 9). Lysoso Lysosomal mal   R -mannosida -mannosidase se and Golg Golgii R -mannosidase -mannosidase II belong to class II R -mannosidases -mannosidases and cleave R 1,2-, 1,2-,   R 1,3-, 1,3-, and   R 1,6-l 1,6-link inked ed man mannos nosee res residu idues es (10-12). Prolonged ingestion of swainsonine by animals leads to a phenocopy cop y of the gen geneti etical cally ly ind induce uced d lys lysoso osomal mal sto storag ragee dis diseas easee mannosidosis (6 ). ). The concentration of swainsonine detected in all plants implicated in poisoning is not high. The yield of    Astragalus lentiginosus swainsonine from the vegetative parts of  Astragalus was found to be 0.007% (dry weight) weight) (3). A threshold of toxicity is diff difficult icult to estab establish lish for swain swainsonin sonine, e, but a conser conservativ vativee approach suggests that levels in excess of 0.001% should be of  concern (13). Swainsonine is a lysosomotropic compound and

10.1021/jf0341 10.1021 /jf0341722 722 C CCC: CC: $25.00 © 2003 Americ American an Chemica Chemicall Society Society Published on Web 07/11/2003

 

4996   J. Agric. Agric. Food Food Chem., Chem.,  Vol. 51, No. 17, 2003

Haraguchi et al.

Figure 1.  Structures of alkaloids isolated from  I. carnea .

accumulates rapidly in lysosomes of normal human fibroblasts in cul cultur turee to pro produc ducee inh inhibi ibitio tion n of int intrac racell ellula ularr lys lysoso osomal mal R -mannosidase -mannosidase (14). Swainsonine is also found in certain species of the plant family fam ily Con Convol volvul vulace aceae. ae. Wei Weirr vin vinee [ Ipomoea   sp. sp. Q6 (a (aff ff.. calobra)] grows in a sma small ll area of sou southe thern rn Queenslan Queensland d in Australia and is reported to produce neurological disorders in livestock. The clinical symptoms are similar to those caused by swainsonine-containing legumes. Molyneux et al. detected swainsonin swain soninee in the seeds and estim estimated ated the level as 0.058 0.058% % by gas chromatography-mass spectrometry (GC-MS) (15). There are serious livestock poisonings reported reported for other   Ipomoea species.  Ipomoea carnea  is a plant of tropical American origin but is now widely distributed in the tropical regions of the world. Thi This s pla plant ntas isshrubs erect, ere ct,toden densel sely leafed fed,,(16  and almost alm ost unb unbran ranche ched, d, growing 23 ymlea high ). Natural ). intoxication occurs in livestock that chronically ingest the plant, and the early poisoning reports were from Sudan and India ( 17 ,   18 ). ). The toxicity has been confirmed in feeding experiments with goats and sheep (19,  20). The nortropane alkaloids calystegines B2   (4) and C1   (6), together with swainsonine ( 1) (Figure 1), have been detected in the leaves collected in Mozambique where goats were intoxicated (20). Calystegines B2  and C1  are potent inhibitor inhi bitorss of glyco glycosidase sidasess (21). Hence, whether the poisoning by this plant is due to a sin single gle effect effect of swa swains insoni onine ne or a combination of toxic effects by swainsonine and calystegines is a very important problem (15,  20 ,  22 ). Poisonous plants are a serious problem for livestock breeding in Brazil, and ∼75 plants have had their toxicity confirmed by experimen exper iments ts with the anima animall specie speciess affe affected cted under natur natural al conditions (23).   I. carnea   is foundthroughout in most regions Brazil and is an evergreen plant blooming the yearof(Figure 2). The anima animals ls eat this plant especially especially in drought periods beca be caus usee it is on onee of th thee fe few w pl plan ants ts th that at st stay ay gr gree een. n. In experimental experi mental studies in which the plant was given to adult goats, all animals showed disorders of behaviors and consciousness as well as abnormalities of gait, ability to stand, and posture (Fig Figure ure 3), and on onee go goat at died died (23). The histo histopatho pathologic logical al changes were cytoplasmic vacuolation of the neurons in the central and peripheral nervous system, suggesting a lysosomal storage disorder by lysosomal glycosidase inhibition. In this study we describe the isolation and characterization of alkaloids from the leaves, flowers, and seeds of   I. carnea   in Brazil as well as the alkaloid level in each part, and inhibitory activities of alkaloids toward rat lysosomal glycosidases were investigated.

Figure 2.   I. carnea  growing  growing in a culture at CEPTOX, School of Veterinary

Medicine, University of Sa˜ o Paulo, Pau lo, Sa˜ o Paulo, Brazil. B razil.

Figure 3.   Contracture defects in the front legs of a goat produced by

consumption consum ption of  I. carnea . NMR NM R (1 (125 25 MH MHz) z) sp spec ectr traa we were re re reco cord rded ed on a JEO JEOL L EC ECPP-500 500

MATERIALS AND METHODS

General Methods. The Methods.  The purity of samples was checked by HPTLC on silica gel 60F254  (E. Merck) using the solvent system PrOH/AcOH/  H2O (4:1:1), and a chlorine-o-tolidine reagent or iodine vapor was used for detect detection. ion. Optical rotations were measured with a Jasco DIP370 digital polarim polarimeter eter (Tokyo, Japan).   1H NMR (500 MHz) and   13C

spectrometer (Tokyo, Japan). Chemical shifts are expressed in parts per million downfield from sodium 3-(trimethylsilyl)propionate (TSP) in D2O as an internal standard. FABMS were measured using glycerol as a matrix on a JEOL JMS-SX 102A spectrometer. GC-MS Analysis.   Samples were dried and trimethylsilylated by treatment with N -methyl-methyl- N -(trimethylsilyl)trifluoroacetamide -(trimethylsilyl)trifluoroacetamide in pyridine

 

Alkaloids in   Ipomoea carnea at 60  ° C for 1 h. Analyse Analysess were performed on a Hewlett Hewlett-Packa -Packard rd 5890 series ser ies II inst instrum rument ent equi equippe pped d with a 5971 mas mass-se s-selec lective tive det detect ector or operating at 70 eV, an on-column injector, and a 60 m × 0.32 mm i.d. SE-30 fused silica column. The column was temperature programmed from 120 to 300   °C at 10   °C/min. Plant Materials. Identification Materials.  Identification of  I.   I. carnea  Jacq. subsp.   fistulosa (Mart. ex. Choisy) D. Austin was confirm (Mart. confirmed ed by taxonom taxonomist ist Rosangela Sima˜o ˜o Bianchini of the Instituto Insti tuto de d e Botanica de Sa˜o Paulo, Brazil. The leaves and flowers of  I.   I. carnea  were collected in May 2001 and April 2002, 200 2, res respec pective tively, ly, fro from m the cult culture ure in the Res Resear earch ch Cen Center ter for Veterinary Veterin ary Toxicology (CEPTO (CEPTOX), X), School of Veterina Veterinary ry Medicin Medicine, e, University of Sa˜o Paulo, Brazil. The seeds were collected in July 2002 from the fields of Votupora Votuporanga nga City, Sa˜o ˜o Paulo, Brazil. A voucher specimen was deposited in the Herbarium of the Instituto de Botanica de Sa˜o Paulo, Brazil (SP-360911). (SP-36091 1).  The fresh leaves (2 kg) of  I. carnea  were Extraction and Isolatio Isolation. n. The homogenized in 50% aqueous EtOH (6 L) and allowed to stand for a week. The filtrate was evaporated to give a brown syrup (50 g). This syrup was dissolved in water (500 mL) and applied to a column of  Amberlite IR-120B (150 mL, H + form). The 0.5 M NH 4OH eluate was conc co ncen entr trat ated ed to gi give ve a br brown own sy syru rup p (4 (4.5 .5 g) g),, wh which ich wa wass fu furth rther er chromatographed over a Dowex 1-X2 (150 mL, OH- form) column with H2O (1.2 L) as eluant to give a colorless syrup (1.13 g). The syrup was applied to an 87 cm   ×  1.8 cm Amberlite CG-50 column (NH 4+ form) with H2O as eluant (fraction size   )  9.5 mL). The H2O eluate was divided divided into four pools: I (fraction (fractionss 12-21); II (fractions 2838); III (fractions 39-54); and IV (fractions 64 -115). The column was eluted with 0.5 M NH 4OH, and fractions 4-21 were collected and designated pool V. Total yields of pools I, II, III, IV, and V were 320, 50, 16, 65, and 46 mg, respectively. The successive chromatography of pool I with Dowex 1-X2 (OH - form) and Amberl Amberlite ite CG-50 (NH4+ form) gave  N -methyl-methyl-trans-4-hydroxy-L-proline (4 mg, 7 mg,  7)) and calystegine C1 (25 mg, 6 mg,  6). ). Other pools were similarly chromatographed with Dowex 1-X2 (OH- form) and Amberlite CG-50 (NH4+ form) to give calystegine B2  (41 mg, 4 mg,  4)) from pool II, calystegine B1  (13.7 mg, mg,  5)) and swainsonine (55 mg, 3) from pool III, calystegine B 3  (7.8 mg, 5 -lentiginosine inosine (5.7 mg, mg, 2 1) from pool IV, and 2- epi-lentig  2)) from pool V. In the isolation procedures described above, it was found that most of   7  in the 50% aqueous EtOH extract was adsorbed on the anionof   exchang exc hangee res resin in Dowe Dowex x 1-X 1-X2 2 (H- for form). m). Henc Hence, e, we imp improv roved ed the isolation isolati on method of  7 to  7  to determine the content in the fresh leaves. The 50% aqueous EtOH extract (1 g) obtained from 40 g of the fresh leaves was applied to a column of Dowex 50W-X2 (50 mL, H + form) and eluted with 0.5 M NH4OH. The eluate was concentrated to give a brown syrup (280 mg). This syrup was chromatographed over a Dowex 50WX8 (50 mL, pyridine form) with 0.1 M pyridinium formate buffer (pH 3.1) as eluant. Fractions 8-15 (fraction size ) 5 mL) were concentrated and further chromatographed on an Amberlite CG-50 column (1 -65 cm, NH4+ form) with H2O as eluant to give 24.8 mg (0.062% of fresh weight) of  7.  7 . The fresh flowers (590 g) and seeds (130 g) were similarly extracted with 50% aqu with aqueou eouss EtOH EtOH,, and the ext extrac racts ts wer weree chr chroma omatogr tograph aphed ed according to the former method to give  1  (16.6 mg), 3 mg),  3  (2.1 mg), and 4  (4.3 mg) from the flowers and  1  (43.1 mg), 3 mg),  3  (6.8 mg), 4 mg),  4  (27.7 mg), 5  (0.8 mg), and  6  (5.2 mg) from the seeds. Swainsonine (1):  13 C NMR (D2O) δ  25.6 (C6), 34.9 (C7), 54.1 (C5), 63.0 (C3), 68.8 (C8), 71.5 (C2), 72.1 (C1), 75.2 (C8a). The trimethylsilyl (TMSi) derivative of  1,  1 , with a GC retention time of 14.21 min, gave the molecular ion at  m /  z  389 and the base peak at  m /  z  185. These data were identical to those of the tri-TM tri-TMSi Si derivative of swainsonine. 2-epi-Lentiginosine ( 2): [ R ]D -31.7°  ( c  0.25, H2O);  1 H NMR (D2O) δ  1.19 (1H, m, H-6 ax), 1.31 (1H, m, H-7ax), 1.44 (1H, m, H-8ax), 1.67 (1H, m, H-8eq), 1.83 (1H, m, H-7eq), 1.97 (1H, m, H-6eq), 2.06 (1H, ddd,  J  )  2.7, 9.1, 11.1 Hz, H-8a), 2.14 (1H, ddd,  J  )  2.7, 11.4,

11.4 Hz, H-5ax), 2.19 (1H, dd,  J   )  5.0, 10.5 Hz, H-3), 2.98 (1H, m, H-5eq), 3.42 (1H, dd,  J  ) 6.9, 10.5 Hz, H-3′), 3.60 (1H, dd,  J  ) 6.9, 9.1 Hz, H-1), 4.18 (1H, ddd,   J   )   5.0, 6.9, 6.9 Hz, H-2);   13C NMR (D2O)   δ   25.9 (C-7), 27.1 (C-8), 30.6 (C-6), 55.2 (C-5), 63.0 (C-3), 69.1 (C-8a), 69.7 (C-2), 77.4 (C-1). The TMSi derivative of  2,  2 , with a GC retention time of 11.21 min, gave the molecular ion at   m /  z   301

J. Agric. Food Chem.,  Vol. 51, No. 17, 2003   4997 and the base peak at  m /  z  97. These data were identical to those of the di-TMSi derivative of 2- epi-lentiginosine. Calystegine B1  ( 3):   13C NMR (D2O)  δ  38.9 (C-4), 43.6 (C-7), 62.9 (C-5), (C5), 72.6 (C(C-3), 3), 75.9 (C(C-6), 6), 81.3 (C(C-2), 2), 93.8 (C(C-1). 1). The TMS TMSii derivative of   3, with a GC retention time of 14.93 min, gave [M   CH3]+ at  m /  z  448, [M   -  HOTMSi] at  m /  z  373, and the base peak at m /  z 244. These data were identical to those of the tetra-TMSi derivative of calystegine B1. Calystegine B2  ( 4):   13C NMR (D2O)  δ  24.5 (C-6) (C-6),, 31.5 (C-7) (C-7),, 58.6 (C-5), (C5), 77.6 (C(C-4), 4), 77.7 (C(C-3), 3), 80.4 (C(C-2), 2), 93.2 (C(C-1). 1). The TMS TMSii derivative deriva tive of  of    4, with a GC retention time of 15.97 min, gave the molecular ion at   m /  z  463 and the base peak at   m /  z   217. These data were identical to those  of the tetra-TMSi derivative of calystegine B 2. Calystegine B3  ( 5): 13C NMR (D2O)  δ  22.9 (C-6) (C-6),, 34.1 (C-7) (C-7),, 58.2 (C-5), (C5), 73.0 (C(C-3), 3), 75.2 (C(C-4), 4), 77.2 (C(C-2), 2), 92.9 (C(C-1). 1). The TMS TMSii derivative deriva tive of  of    5, with a GC retention time of 14.85 min, gave the molecular ion at   m /  z  463 and the base peak at   m /  z   217. These data were identical to those of the tetra-TMSi derivative of calystegine B 3. Calystegine C 1  ( 6 ): ):   13C NMR (D2O)  δ  43.6 (C-7), 67.4 (C-5), 71.7 (C-6), (C6), 75.5 (C(C-4), 4), 77.7 (C(C-3), 3), 79.4 (C(C-2), 2), 93.6 (C(C-1). 1). The TMS TMSii derivative of   6, with a GC retention time of 17.21 min, gave [M   CH3]+ at   m /  z   536 and the base peak at   m /  z   217. These data were identical to those of the penta-TMSi derivative of calystegine C 1.  N-Methyl-trans-4-hydroxy N-Methyl-trans4-hydroxy- L-proline (7 ): ):   [R ]D -67.9° ( c 0.34, H2O); 1 H NMR (D2O)  δ  2.26 (1H, ddd,  J  ) 4.6, 11.0, 14.2 Hz, H-3R ), ), 2.49 (1H, dddd,   J   )  1.8, 2.3, 7.3, 14.2 Hz, H-3 ), 3.05 (3H, s,   N -CH -CH3), 3.22 (1H, ddd,   J   )  1.8, 2.3, 12.8 Hz, H-5 ), 3.97 (1H, dd,   J   )  4.6, 12.8 Hz, H-5R ), ), 4.20 (1H, dd,   J   )  7.3, 11.0 Hz, H-2), 4.64 (1H, m, H-4);   13C NMR (D2O)  δ  41.1 (C-3), 46.0 ( N -CH -CH3), 65.5 (C-5), 72.3 (C-4),, 73.0 (C-2), 175.8 (C-1); HRFAB (C-4) HRFABMS, MS, m /  z  146.0821 [M  +  H] + (C6H12NO3   requires 146.0817). Glycosidasee Inhibitory Activities.   The rat epididymal fluid was Glycosidas purified from epididymis according to the literature (11) and used as the source of lysosomal glycosidases. The reaction mixture consisted of 50  µ L of 0.2 M acetate buffer (pH 5.0), 50  µ L of 2% Triton X-100 (Sigma Chemical Co.), 30  µ L of the enzyme solution, and 20  µ L of an inhibitor solution or H2O. The reaction mixture was preincubated at 0   °C for 10 min and started by the addition of 50   µL of 6 m M 4-methylumbelliferyl glycoside (Sigma Chemical Co.) (1 mM in the case of   -galactoside), followed by incubation at 37   °C for 1-2 h. The reaction was stopped by the addition of 2 mL of 0.1 M glycine buffer (pH 10.6). Liberated 4-methylumbellife 4-methylumbelliferone rone was measur measured ed (excitation, 362 nm; emission, 450 nm) with a Hitachi fluorescence spectrophotometer F-4500 (Tokyo, Japan).

RESULTS AND DISCUSSION   I. carn carnea ea

GC-MS GCMS Analyse Ana lysess of of Extrac Ext racts ts of  flowers, and .   After Aft er 50% aqueous EtOH extracts the leaves, seeds of   I. carnea  were preliminarily purified with Amberlite IR-120B (H+ form) and Dowex 1-X2 (OH- form), they were trimethylsilylated with   N -methyl-methyl- N -(trimethylsilyl)trifluoroacetamide -(trimethylsilyl)trifluoroacetamide (25). Ana Analys lysis is of the extracts extracts by cap capil illar lary y GCGC-MS MS of the their ir trimethyl trim ethyl (TMSi) deri derivativ vatives es demon demonstrat strated ed the prese presence nce of  swainsonine (1) and calyst calystegines egines B1  ( 3), B 2  ( 4), B 3  ( 5), and C1 (6) from all parts of  I.   I. carnea. GC analysis of the seed extract is shown in   Figure 4. The major component in all extracts, with a GC retention time of 14.21 min, gave a molecular ion at m /  z  389 and a base peak at  m /  z  185. These data were identical to th thos osee of th thee tr trii-TM TMSi Si de deri riva vati tive ve of sw swai ains nson onin ine. e. Th Thee secondary components (15.97 min) in all extracts had a mass spectr spe ctrum um ide identi ntical cal in all res respec pects ts to tha thatt of the tet tetrara-TMS TMSii deriva der ivativ tivee of a sta standa ndard rd sam sample ple of cal calyst ystegi egine ne B2, wit ith h a   m /  z   463   m /  z   217 mol molecu ecular lar ionof at 463 having and an d athree base ba seadjacent peak pe ak at characteristic compounds trimethylsilylated secondary hydroxyl groups. The other two peaks with retention times of 14.93 and 17.21 min had the same fragmentation tio n pat patter terns ns as tho those se of aut authen hentic tic cal calyst ystegi egines nes B1   and C1, respectively. The tetra-TMSi calystegine B1   and penta-TMSi

 

4998   J. Ag Agric. ric. F Food ood Ch Chem., em.,  Vol. 51, No. 17, 2003

Haraguchi et al.

Figure 4.   GC-MS analysis of the trimethylsilylated resin-treated extract of the seeds of I. carnea . Peaks: 1, swainso swainsonine; nine; 3, calyste calystegine gine B1; 4, calystegine

B2; 5, calystegine B3; 6, calystegine C1.  z  448 calystegine C,1 373  showed characteristic ionspeak at  m /   m /  z [M   -   CH3]+ [M    HOTMSi]+fragment with a base at + + 244,  m /  z  536 [M   -  CH 3] , and 461 [M   -  HOTMSi] with a base peak at  m /  z  217, respectively. The minor component with a retention time of 14.85 min had a mass spectrum ( m /  z   463 [M]+ and a base peak at   m /  z   217) consistent consistent with those of  calystegine B3. Isolation Isolatio n and Chara Characteriza cterization tion of Alkalo Alkaloids. ids.   The fresh leaves (2 kg) of   I. carnea  were extracted with 50% aqueous EtOH.. The chromatographi EtOH chromatographicc separ separation ation of the extract using various cation and anion ion-exchange resins afforded seven alkaloids:   1  (55 mg),   2   (5.7 mg),   3  (13.7 mg),   4  (41 mg),   5 (7.8 mg),  6  (25 mg), and  7  (4 mg). The GC-MS and  13 C NMR spectra of alkaloids  1 ,  3 ,  4 ,  5 , and  6  were in accord with those of swainsonine and calyste calystegines gines B 1, B 2, B3, and C1, respectively.  1   13  2   were consistent with those The H and C NMR spectra of  2

(H+ form) column and eluted with 0.5 M NH 4OH to give a total alkaloid fraction. This fraction was applied to a Dowex

of 2-epi-lentiginosine isolated thelegume fungus  Rhizoctonia leguminicola   (26 from ) andfermentations the leaves ofofthe  Astragalus lentiginosus   (27 ). ) . GCGC-MS MS ana analys lysis is of   2   gave a retention time (11.21 min) and mass spectrum identical to those of an authentic sample of 2- epi-lentiginosine. Comparison of  the optical rotations of the natural product [[R ]D -31.7° (c 0.25, H2O) from   I. carnea; [R ]D   -32.5°   (c   0.13, CHCl3) from   A. lentiginosus] an and d sy synt nthe heti ticc sa samp mple le [[R ]D   -39.4°   (c   0.58, CHCl3)] ( 29) established the structure of  2  2  as (1S ,2 ,2 R,8aS )-1,2)-1,2dihydroxyindolizidine. dihydroxyindolizidin e. The  1 H and  13 C NMR spectra of  7  7  were superimposable with those of a novel imino acid,   N -methyl-methyltrans-4-hydroxy-L-proline, isolated from the leaves of  Copaifera   Copaifera multijuga   (Caesalpi (Caesalpinioid nioideae), eae), and the optic optical al rotat rotation ion [[R ]D -67.9° ( c 0.34, H2O)] of  7  7  was also similar to that [[ R ]D -74.0° (c  0.01, MeOH)] in the literature ( 28 ). ). The contents of alkaloids in the leaves, flowers, and seeds

50 50WW-X8 X8 (p (pyr yrid idin inee fo form rm)) co colu lumn mn and elut el uted edtowi with th 0. 0.1 M pyridinium formate buffer (pH 3.1)an asd eluant give a 1pure sample of  7  7 . The content in the fresh leaves was estimated as 0.062%. The contents of swainsonine ( 1) in the fresh leaves and fre fresh sh fl flowe owers rs wer weree 0.0 0.0029 029 and 0.0 0.0028 028%, %, res respec pectiv tively ely,,  1  and calystegines B1  ( 3) and B2  ( 4) in whereas the contents of  1 the seeds were   ∼10 times higher than those in the leaves and  1  in  I. carnea  described above are in flowers. The contents of  1 fresh weight and exceed the level of 0.001% in dry weight ( 13) estimated to produce neurological damage in livestock, which is derived from lysosomal storage by the chronic inhibition of  R -mannosidase. -mannosidase. 2-epi-Lentiginosine is probably an intermediate in the bioleguminico inicola la   because synthe syn thesis sis of swa swains insoni onine ne in   R. legum because the experimental refeeding of [3H]-2-epi-lentiginosine resulted in a very high level (45%) of incorporation of radioactivity into

 I.  7  I. carnea of   are summarized  Tableas1.aAlthough a novel imino acid  was isolated from theinleaves minor component, the isolation method using an anion-exchange resin Dowex 1-X2 (OH- form) was found to cause a significant loss of yield. Hence, we developed a concise isolation method for this imino acid. The 50% EtOH extract extract was appli applied ed to a Dowex 50W-X2 50W-X2

swainsonine (26 ). ). Very interestingly, Blanco and Sardina haveenantiospecifically synthesized 8-epi-swainsonine from  trans 4-hydroxy-L-proline (30). Biological Activities.  The 50% EtOH extracts of the leaves, flowers, and seeds were preliminarily treated with Amberlite IR-120 IR120B B (H+ for form) m) and tes tested ted for inh inhibi ibitor tory y act activi ivity ty of 

Table 1.  Alkaloid Content in Fresh Plant Materials content (%) in fresh wta  alkaloid

leaves

swainsonine, 1   2-epi -lentiginosine, -lentiginosine, 2   calystegine B1, 3   calystegine B2, 4   calystegine B3, 5   calystegine C1, 6   N -methyl-methyl-trans -4-hydroxy-4-hydroxy-L-proline, 7

0.0029 0.0003 0.0007 0.0021 0.0004 0.0013 0.0620 0.

 

flflowers  

se s eeds

0.0028

0.0332

−b 

−

0.0004 0.0007

0.0052 0.0213 0.0006 0.0040 nd

 

−

 

−

ndc 

   

a  [Isolated c  nd,

alkaloid (g)/fresh wt (g) of plant material]  ×  100.   b  −, not detected. not determined.

 

Alkaloids in   Ipomoea carnea

J. Agric. Food Chem.,  Vol. 51, No. 17, 2003   4999

Table 2.  Concentration of the Resin-Treated Extracts of  I. carnea 

Givingg 50% Inhibition of Rat Epididymis Glycosidases Givin Glycosidases IC50 ( µg/mL) enzymea 

leaves

R -glucosidase -glucosidase  -glucosidase R -galactosidase  -galactosidase R -mannosidase  -mannosidase R -fucosidase -fucosidase

 

 

flowers

seeds

−b 

−

−

1.7 290 570 0.51 730

4.6 760

0.6 230

−

−

   

−

0.034

0.016

− −

− −

a 

Activities of rat epididymis glycosidases were determined using 4-methylumbelliferyl glycoside as substrate at pH 5.0.   b  −, no inhibition (<50% inhibition at 1000  µ g/mL).

Table 3.  Concentration of Alkaloids Isolated from  I. carnea  Giving   Giving

50% Inhibition of Rat Epididymis Glycosidases Glycosidases IC50 ( µM) enzymea 

1

R -glucosidase -glucosidase

 

 -glucosidase R -galactosidase -galactosidase  -galactosidase R -mannosidase  -mannosidase R -fucosidase -fucosidase

         

2

−b 

−

−

−

− −

− −

0.02

4.6

− −

3

4

−

 

−

− −

2.1  

0.75 120

 

710

−

− −

−

−

 

5 −

 

−

0.84

− −

150 90 −

not yet been established, but their potent glycosidase inhibitory properties, in particular toward  -glucosidase, suggest that they may have a significant effect. Furthermore, whether  N -methyl-methyltrans-4-hydroxy- L-proline (7), which is a nonprotein imino acid and contained at a high level (0.062% of fresh weight) in the leaves, is toxic or nontoxic to livestock is undetermined at this time.. Exper time Experiment imentss of glycos glycosidase idase inhibition inhibition and toxi toxicity city by isolated alkaloids using human cells in culture are ongoing. ACKNOWLEDGMENT

−

20  

− −

6

nine and calystegines is very important. The levels of swainsonine measured in all parts of the plant, but especially the seeds, far exceed the minimum levels, estimated at 0.001%, required to produce symptoms of locoism by locoweeds (13). However, although many of the symptoms described for  I. carnea toxicosis of goats in Brazil are typical of locoweed poisoning, others are not characteristic and may be due to lysosomal storage defects induced by calystegines. It is noteworthy that the total levels of calystegines detected in   I. carnea   leaves and seeds in this investigation approach or exceed those of swainsonine in the same plant. The toxicity levels of calystegines in animals have

− −    

− −

We are grateful to Marcos B. Ferreira for the photographs of   Ipomoea carnea  and a poisoned goat. LITERATURE LITERATU RE CITED

−

a  Activities

of rat epididymis glycosidases were determined using 4-methylumbelliferyl glycoside as substrate at pH 5.0.   b  −, no inhibition (<50% inhibition at 1000  µ M).

lysos lysosomal omal glyco glycosidase sidasess prepar prepared ed from rat epidi epididymis dymis.. The inhibitory activities were determined fluorometrically by using the appropriate fluorogenic substrates. As shown in   Table 2 all resin resin-tre -treated ated extr extracts acts showed poten potentt inhib inhibitor itory y activ activitie itiess toward lysosomal -glucosi -glucosidase dase and R -mannosidase -mannosidase and a weak  inhibitory activity toward R -galactosidase. -galactosidase. In particular, the seed extract powerfully inhibited R -mannosidase -mannosidase and   -glucosidase with IC50  values of 0.016 and 0.6  µ g/mL, respectively. The IC50  values of alkaloids isolated from  I. carnea  toward rat lysosomal glycosidases are shown in  Table 3. In our present study, swainsonine (1) showed an IC50 value of 0.02  µM toward rat epididymis R -mannosidase, -mannosidase, whereas Dorling et al. have also

(1) James, L. F.; Van Kampen, K. R.; Hartley, W. J. Comparative Comparative pathology of   Astragalus   (locoweed) and   Swainsona   poisoning in sheep.  Pathol. Vet.  1970  1970,,  7 , 116-125. (2)) Hartle (2 Hartley, y, W. J. J.;; Ba Bake ker, r, D. C. C.;; Ja Jame mes, s, L. F. Co Comp mpar arat ative ive pathological aspects of locoweed and   Swainsona  poisoning in livestock. In  Swainsonine and Related Glycosidase Inhibitors ; James, L. F., Elbein, A. D., Molyneux, R. J., Warren, C. D., Eds.; Iowa State University University Press: Press: Ames, IA, 1989; pp 50-56. (3) Molyneu Molyneux, x, R. J.; James, L. F. Loco intoxication: intoxication: Indoliz Indolizidine idine alkaloids of spotted locoweed ( Astragalus lentiginosus l entiginosus). Science 1982,,  216 , 190-191. 1982 (4) Stegelm Stegelmeier, eier, B. L.; James, L. F.; Panter, K. E.; Molyneux, Molyneux, R. J. Serum swainsonine concentration and   R -mannosidase -mannosidase activity in cattle and sheep ingesting   Oxytropis sericeae  and  Astragalus lentiginosus  (locoweeds).  Am. J. Vet. Res.  1995  1995,,  56 , 149-154. (5) Panter Panter,, K. E.; Gardne Gardner, r, D. R.; James, L. F.; Stegelm Stegelmeier, eier, B. L.; Molyneux, Molyneu x, R. J. Natural toxins from poisonous plants affecting reproductive reprodu ctive function in livestoc livestock. k. In   Natural Natural and Select Selected  ed 

R  50  value of  1 calculated  1  toward jackbean as 0.02  µ Mthe (8 IC ). Swainsonine ). is known to inhibit-mannosidase human liver lysosomal   R -mannosidase -mannosidase in a competitive manner with a   K i valu va luee of 0. 0.07 07   µM (31). In 19 1984 84,, th thee ra race cema mate te of 2-epilentiginosine lentigino sine (2) was synthesized from 3-pyrroline and reported to be a very weak inhibitor of acid R -mannosidase -mannosidase with an IC50 value of 7.5 mM ( 32). Pas Pastuz tuzak ak et al. reported reported the nat natura urall  2  in the leaves of  A.   A. lentiginosus  and the lack of  occurrence of  2 glycosidase inhibitory activity (27 ). ). However, in the present work 2-epi-lentiginosine proved to be a potent inhibitor of rat lysosomal   R -manno - mannosid sidase ase wit with h an IC50   valu valuee of 4. 4.6 6   µM. Polyhydroxylated nortropane alkaloids calystegines B1, B 2, and C1  are known to be potent inhibitors of almond and bacterial  -gluc -glucosida osidases ses (21) bu butt we were re al also so po pote tent nt in inhi hibi bito tors rs of ra ratt lysosomal   -glucosidases, with IC50   values of 2.1, 0.75, and 0.84  µ M, respectively. Calystegine B3 was a moderate inhibitor

  R 

of lysosomal lysos omal - an and d   by -mannosida -mann osidase. se. Poten Potent inhibitio inhib n of    -glucosidase lysosomal calystegines B1, Bt2, and Cition 1  poses a problem that it would produce a phenocopy of the human genetic lysosomal storage disorder Gaucher disease.   I. carnea  to livestock is purely due Whether the toxicity of  I. to swainsonine or due to a combination of effects by swainso-

(6)

(7)

(8)

(9)

(10)

Synthetic Toxins; Tu, A. T., Gaffield, W., Eds.; ACS Symposium Series 745; American American Chemical Society: Washington, DC, 1999; pp 154-172. Dorling, P. R.; Huxtable, Huxtable, C. R.; Vogel, P. Lysosomal Lysosomal storage in Swainsona   spp. spp. toxicosis: An induced mannosidosis. mannosidosis.   Neuro pathol. Appl. Neurobiol.  1978  1978,,  4 , 285-295. Colegate, S. M.; Dorling, P. R.; Huxtable, C. R. A spectroscopic investigation investig ation of swainsonine: swainsonine: An   R -mannosidase -mannosidase inhibitor of  isolated from  Swainsona canescens.   Aust. J. Chem.   1979, 1979,   32, 2257-2264. Dorling P. R.; Huxtable, C. R.; Colegate, Colegate, S. M. Inhibition of  lysosomal   R -mannos - mannosida idase se by swai swainson nsonine ine,, an indo indolizi lizidine dine alkaloid isolated from   Swainsona canescens.  Biochem. J.  1980  1980,, 191, 649-651. Fuhrmann, Fuhrma nn, U.; Bause, E.; Legler, G.; Ploegh, H. Novel mannosidas nosi dasee inhi inhibito bitorr bloc blocking king con conver version sion of high man mannose nose to complex oligosaccharides.   Nature  1984 ,  3 07  , 755 758.  1984, Neves, V. L.; Schmidt, M. C. Human lysosomal alpha-mannosialpha-mannosi-

dase: Isolation and nucleotide nucleotide sequence of the full-length cDNA. cDNA.  Biochem. Biophys. Res. Commun.  1994  1994,,  200 , 239-245. (11)) Liao (11 Liao,, Y. F.; Lal, A.; Mremen, Mremen, K. W. Clon Cloning, ing, expressio expression, n, purification, and characterization of the human broad specificity lysosomall acid   R -mannosidase. lysosoma -mannosidase.   J. Biol Biol.. Che Chem. m.   1996, 1996,   271, 28348-28358.

 

5000   J. Agric. Agric. Food Food Chem., Chem.,  Vol. 51, No. 17, 2003 characterization, and (12) Moremen, K. W.; Robbins, P. W. Isolation, characterization, expression of cDNA encoding murine R -mannosidase -mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex  N -glycans. -glycans.  J. Cell Biol.  1991  1991,,  1 15, 1521-1534. (13)) Mol (13 Molyneu yneux, x, R. J.; James, James, L. F.; Ral Ralphs, phs, M. H.; Pfi Pfister ster,, J. A.; Panter, K. P.; Nash, R. J. Polyhydroxy alkaloids glycosidase inhibitors inhibito rs from poisonous plants of global distribution: distribution: analys analysis is and identification. In Plant-Associated Toxins-Agricultural, Phytochemical and Ecological Aspects; Colegate, S. M., Dorling, P. R., Eds.; CAB International: International: Walling Wallingford, ford, U.K., 1994; pp 107-112. (14) Chotai, K.; Jennings, C.; Winchester, B.; Dorling, P. The uptake   R 

(15)

(16) (17)

(18)) (18

(19)

of swainsonine, a specificininhibitor of Cell -DBiochem. -mannosidase, -manno sidase, normal human fibroblasts culture.  J.  21 ,  1983,,into  1983 107-117. Molyneux, Molyneu x, R. J.; McKenz McKenzie, ie, R. A.; O’Sullivan, B. M.; Elbein, A. D. Identification of the glycosidase inhibitors swainsonine and calystegine B2 in Weir vine ( Ipomoea  sp. Q6 [aff. calobra]) and correlation with toxicity toxicity..  J. Nat. Prod.  1995  1995,,  58 , 878-886. Austin, D. F.   Ipomoea carnea  Jacq. v.   Ipomoea fistulosa  Mart. ex Choisy.  Taxon  1977  1977,,  26 , 235-238. Idris, O. F.; Tartour, Tartour, G.; Adam, S. E. I.; Obeid, H. M. Toxicity Toxicity to goats of  Ipomoea   Ipomoea carnea.  Trop. Anim. Health Prod.  1973  1973,,  5 , 119-123. Tirkey, Tirk ey, K.; Yad Yadava ava,, K. P.; Man Mandal, dal, T. K. Effect Effect of aqu aqueou eouss extract of  Ipomoea   Ipomoea carnea  on the haematological and biochemical parameters in goats.  Indian J. Anim. Sci.  1987  1987,,   57 , 10191023. Damir, H. A.; Adam, S. E.; Tartour, Tartour, G. The effects of  Ipomoea   Ipomoea carnea  on goats and sheep.  Vet. Hum. Toxicol.  1987  1987,,  29 , 316-

319. (20)) de Balogh, (20 Balogh, K. K. I. M.; Dimande, Dimande, A. P.; van der Lugt, Lugt, J. J.; Molyneux, Molyneu x, R. J.; Naude´, ´, T. W.; Welma Welman, n, W. G. A lysosom lysosomal al storage disease induced by  Ipomoea carnea  in goats in Mozambique.  J. Vet. Diagn. InV est. est.  1999  1999,,  11 , 266-273. (21) Asano, N.; Kato, A.; Oseki, K.; K.; Kizu, H.; Matsui, K. Calystegins Calystegins of   Physalis alkeke (Solanaceae aceae). ). Structu Structure re alkekengi ngi   var.   francheti   (Solan determination and their glycosidase inhibitory activities.  Eur. J.  Biochem.  1995  1995,,  229 , 369-376. (22)) Wat (22 Watson, son, A. A.; Fle Fleet, et, G. W. J.; Asa Asano, no, N.; Molyneux, Molyneux, R. J.; Nash, R. J. Polyhydroxylated alkaloidssnatural occurrence and therapeutic applications.   Phytochemistry  2001  2001,,  56 , 265-295.

Haraguchi et al. (23) Tokarnia, Tokarnia, C. H.; Dobere Dobereiner, iner, J.; Peixoto, P. V. Poisonous plants affecting livestock in Brazil.  Toxicon  2002  2002,, 40, 1635-1660, and references cited therein. (24) Skudlar Skudlarek, ek, M. D.; Tulsian Tulsiani, i, D. R. P.; Orgebin-Crist, Orgebin-Crist, M.-C. Rat epididymal luminal fluid acid   -D-galac -galactosidase tosidase optimally hydrolases glycoprotein substrate at neutral pH.  Biochem. J.  1992  1992,, 286 , 907-914. (25) Molyneu Molyneux, x, R. J.; Gardner, D. R.; James, L. F.; Colegate, Colegate, S. M. Polyhydroxy Polyhydr oxy alkaloids: Chrom Chromatograp atographic hic analysis.   J. Chro Chro-matogr. A  2002  2002,,  967 , 75-84. (26)) Har (26 Harris, ris, T. M.; Har Harris, ris, C. M.; Hill Hill,, J. E.; Unge Ungemac mach, h, F. S.; Broquist, H. P.; Wickwire, B. M. (1 S ,2 ,2 R,8aS )-1,2-Dihydroxy)-1,2-Dihydroxy-

(27)) (27

(28)

(29)

(30)

indolizidine formation by   Rhizoctonia leguminicola, the fungus that produces slaframine and swainsonine.  J. Org. Chem.  1987  1987,, 52, 3094-3098. Pastusz Pas tuszak, ak, I.; Moly Molyneu neux, x, R. J.; James, James, L. F.; Elbe Elbein, in, A. D. Lentiginosine, Lentigin osine, a dihydro dihydroxyindoli xyindolizidine zidine alkaloid that inhibits amyloglucosidase.   Biochemistry  1990  1990,,  29 , 1886-1891. Figliuolo, Figliuol o, R.; Naylor, S.; Wang, J.; Langenheim, Langenheim, J. H. Unusual nonprote nonp rotein in imi imino no aci acid d and its rel relati ationsh onship ip to phe phenoli nolicc and nitrogenous compounds in Copaifera. Phytochemistry  1987  1987,,  26 , 3255-3259. Heitz, M.-P.; M.-P.; Overm Overman, an, L. E. Complementary Complementary use of iminium ion and N -acyliminium -acyliminium ion cyclization initiators for asymmetric synthesiss of both enantiomers synthesi enantiomers of hydroxy hydroxylated lated indolizidines. indolizidines.  J. Org. Chem.  1989  1989,,  54 , 2591-2596. Blanco, M.-J.; Sardina, F. J. Enantiospecific and stereoselective synthesis of polyhydrox polyhydroxylated ylated pyrrolidines and indolizid indolizidines ines from trans-4-hydroxy-L-proline. J. Org. Chem.  1996  1996,, 61, 4748-4755.

(31) Cenci di Bello, I.; Fleet, G. W. J.; Namgoong, S. K.; Tadano, Tadano, K.; Winchester, B. Structure-activity relationship of swainsonine.  Biochem. J.  1989  1989,,  25 9, 855-861. (32) Colega Colegate, te, S. M.; Dorling, P. R.; Huxtable, C. R. The synthesis and biological activity of ( ()-(1R ,2 ,2R ,8a ,8aR )-indolizidine-1,2-diol. )-indolizidine-1,2-diol.  Aust. J. Chem.  1984  1984,,  37 , 1503-1509. Received for review February 20, 2003. Revised manuscript received April 14, 2003. Accepted April 23, 2003. This work was supported in part by the Special Research Fund of Hokuriku University (N.A.).

JF0341722