"Timbó" and Grows Wild Identified for the First Time in in the States of Minas Gerais, São Boraginaceae, Cordia Verbenacea Paulo and Paraná

ON THE OCCURRENCE OF CYANOLIPIDS IN Paullinia carpopodea CAMBESS AND P. cupana KUNTH SEED OILS

Regina C. A. LAGO1, Maria de Lourdes P. S. C. SIMONI2, Ângelo da C. PINTO3

ABSTRACT — Paullinia carpopodea seed oil contains 70% type I cyanolipids with cyanogenetic properties, as proven by chemical and spectrometric techniques. P. cupana seed oil also contains cyanogenic type I cyanolipids, according to its 1Ή-NMR spectrum. The existing controversy in the literature about the presence and/or type of cyanolipids in P. cupana seed oil is probably due to the low amount of these compounds (0.2%) in the seeds.

Key-words: Sapindaceae, cyanolipids, cyanogenesis, Paullinia carpopodea, Paullinia cupana. Ocorrência de Cianolipidios em Óleos das Sementes de Paullinia carpopodea Cambess e P. cupana Kunth RESUMO — O óleo das sementes de Paullinia carpopodea contem 70% de cianolipidios cianogenéticos, do tipo I, como mostrado através de métodos químicos e espectrométricos. O óleo de P. cupana contém o mesmo tipo de cianolipídios, como evidenciado através de seu espectro de 1Ή-NMR. A controvérsia existente na literatura sobre a presença e/ou tipo de cianolipídios no óleo de P. cupana foi, provavelmente, causada pelo baixo teor com que estes componentes ocorrem nas suas sementes (0,2%). Palavras-chave: Sapindaceae, cianolipídios, cianogênese, Paullinia carpopodea, Paullinia cupana.

INTRODUCTION Sapindaceae (Mikolajczak et al. 1970; Gowrikumar et al. 1976; Nishizawa et Paul Unia carpopodea is known al. 1983), although cyanolipids were in Brazil as "timbó" and grows wild identified for the first time in in the states of Minas Gerais, São Boraginaceae, Cordia verbenacea Paulo and Paraná. The plant is used for (Mikolajczak, 1969). Seigler (1976) its ictiotoxic properties (Reitz, 1980). showed that, in fact, the plant studied P. cupana is native to Amazonas belonged to the Sapindaceae family. and Pará states and is commonly According to Mikolajczak (1977), not known as "guaraná". Its seeds are used all species of Sapindaceae contain for preparing a powder used in re cyanolipids. freshments, recommended as an en The four known cyanolipid ergy reconstituem and for alleviating structures (types) present the same stomach disorders. "Guaraná" also has branched five-carbon nitrile skeleton. a reputation as a cardiotonic and arte Types I and II are long chain fatty di- riosclerosis prevention agent (Reitz, esters of l-cyano-2-hydroxymethyl 1980). Angelucci et al. (1978) men prop-2-en-l-ol (I) and of l-cyano-2- tioned ca. 4% of caffeine in its seeds. hydroxymethyl prop-l-en-3-ol (II). Cyanolipids are a unique class of Types III and IV are mono-esters of 1- plant lipids that seem to occur almost cyano-2-hydroxymethy 1 prop-1 -en exclusively in the seed oils of the (III) and of l-cyano-2-methylprop-2-

1 Embrapa Agroindústria de Alimentos, Αν. das Américas 29.501, Rio de Janeiro 2 Escola Prof. Celso Tozzi, R. Floriano Peixoto 230, CEP 13820-000, Jaguariúna, SP 3 IQ/UFRJ, Centro Tecnológico, Bloco A, Cidade Universitária, Ilha do Fundão, Rio dc Janeiro en-l-ol (IV). detect the presence of the -CN group. Cyanogenesis is defined as the Thin-Layer Chromatography capacity shown by certain plants of (TLC) was carried out using silica gel yielding HCN through the hydrolysis G plates and benzene as eluent (no of chemical compounds produced by other solvent gave better or equivalent them. For a long time this process was separation). Triacylglycerols and associated with the presence of cyano- cyanolipids bands were scrapped out genic glycosides in plant tissues. The from preparative plates (0.5mm), ex first and conclusive evidence that tracted with CH2C12 and weighed, af cyanogenicity in kusum seed oil ter evaporation of the solvent. Purifi (Schleichera trijuga) could not be at cation was carried out through HPLC, tributed to a glycoside but rather to a in a Waters 403 system, equipped with lipid-based material was provided by Refractive Index Detector, using two Kundu & Bandyopadhyay (1969). Altex ODS columns (5xl50mm;5mm) Cyanolipids I and IV are cyanogenic. in series, and acetone: acetonitrile: Different seeds may yield con dichloromethane 3:2:1 as eluent, at flicting results, as in the case of P. flow rate of lml/min. cupana; for some authors, its seeds con The infrared spectrum of P. tain type I cyanolipids while for others carpopodea cyanolipids showed car-

1 the material is devoid of such com bonyl (1740cm" ) and =CH2 absorp pounds (Seigler & Kawahara, 1976). tions (1157cm-1 and 920-965 cm1). This study was undertaken in order to Ή-NMR spectra were recorded study P. carpopodea lipids and to con at 200MHz in CDCl, with TMS as in firm whether P. cupana contains or not ternal standard. I3C-NMR spectrum cyanogenetic compounds. was taken at 100MHz in CDC13. P. carpopodea cyanolipids. Ή- EXPERIMENTAL NMR: δ 2.33 (m, Η and Hb); 4.70 (s,

P. carpopodea fruits were col H, Hd), 5.52 (s, Hg), 5.67 (s, Hf), and lected in Ouro Preto, Minas Gerais 5.96 (s,H). ,3C-NMR: δ 120.8 (-CN),

(voucher is deposited at the herbarium 130.0 (=CH2), 135.3 (C=), 171.5 (CO) of the Pharmacy and Biochemistry and 173.0 (CO). School, Federal University of Ouro P. cupana cyanolipids. Ή-NMR: δ

Preto, under the register number 1031 2.34 (m,H,Hb), 4.63 (s, H, Hd), 5.50

(flowers) and 1949 (fruits)). P. cupana (s,Η), 5.66 (s, Hf) and 5.94 (s, H). fruits were from Pará state. The seeds RESULTS AND DISCUSSION were separated from the fruits, ground in a lab mill and their oil extracted in P. carpopodea and P. cupana a Butt type apparatus, with light petro seeds yielded 40.5% and 1.2% oil, re leum, b.p. 40-60°C. spectively. The seed oil from P. The alkaline qualitative picrate carpopodea was clearly positive to the test (Feigl, 1954) was conducted to picrate test which, nevertheless, failed

102 Lago et al. o Hd He o Figure 1. Protons assignment in Ή-NMR spectrum of P. Carpopodea cyanolipids. to prove the cyanogenicity of the P. identified c/s-13-eicosenoic acid as the cupana oil or the presence of cyano- main component of total lipids genic cyanolipids. A band which ap (44.4%), which the author named peared, for both oils, above the Paullinic acid, and small amounts of triacylglycerols on the TLC plate cis-11 -eicosenoic acid (gadoleic acid, could only be related to either 3.7%) and c/s-15-eicosenoic acid cyanolipid I or IV, since cyanolipids II (0.7%). and III are more polar than the The Ή-NMR spectrum of the triacylglycerols. cyanolipid fraction showed a multip- The UV spectrum exhibited a let (probably a double triplet) at δ 2.33 very weak nitrile absorption band at which was assigned to protons of me

208nm. The positive Feigl test and the thylene group a to carbonyl group (Ha, absence of a -CN absorption at Hb) shown in Figure 1. The -CH2- pro

1 2230cm' in the IR region (Bellamy, tons (Hc, Hd) gave an apparent singlet 1956) indicated the presence of a non- at δ 4.70. The remaining protons of the conjugated cyano group, correspond dihydroxynitrile moiety of cyanolipids ing either to type I or IV cyanolipids. produced apparent singlets at δ 5.52

Through preparative TLC, (Η ), 5.67 (Hf) and 5.96 (H ). Equiva- cyanolipids in the oils were quantified. lent data have been presented by While P. carpopodea oil contained Seigler et al. (1970) and Mikolacjzak 70.7% of cyanolipids and 21.3% of (1977) for type I cyanolipids. other lipids, mostly triacylglycerols Ή-NMR spectrum of P. cupana (Lago et al, 1995), P. cupana oil ex seed oil showed also equivalent data hibited only 12%) of cyanolipids (or with a multiplet at δ 2.34 (Ha, Hb), a

0.2% in the seeds). singlet at δ 4.63 (Hc, Hd) and singlets Lago et al. (1995) observed that centered at δ 5.10, δ 5.66 and δ 5.94 eicosenoic acids are the main constitu (terminal -CH2- protons, He, Hp and ents (52.8%) in P. carpopodea seed proton bonded to α-carbon to the ni

oil. They are the major components in trile group, Hc, respectively). the cyanolipid fraction (64.95%) but In the l3C-NMR spectrum of the the third in the triacylglycerol fraction cyanolipid fraction assignment for (16%). In P. elegans, Spitzer (1995) some carbon atoms are as follows: -

On the occurrence of cyanolipids in Paullinia 103 CN group- 120.8ppm; =CH2, Fcigl, F. 1954. Spot Tests, v.l, 4. cd., Elsevier, BO.Oppm; =C, 135.3ppm and CO New York, p.263, 269. groups, 171.5 and 173.0ppm. The pos Gowrikumar, G.; Mani, V.V.S.; Rayana, G.L. sibility of having cyanolipids II and/ 1976. Cyanolipids in Sapindus emarginatus seed oil. Phytochemistry, or III had been discarded previously. 15:1566. Differences in chemical shifts between Kundu, M.K.; Bandyopadhyay, C. 1969. Stud carbon atoms of types I and IV ies on some chemical aspects of kusum cyanolipids (Mikolajczak & oil. J. Am. Oil Chem. Soc, 46(1):23. Weisleder, 1978), such as the presence Lago, R.C.A.; Simoni, M. de L.P.S.; Pinto, of two signals corresponding to dis A.C. 1995. Lipids in Paullinia carpopodea tinct CO carbon atoms in the spectrum Cambess seed oil. In: Latin-American of cyanolipids I instead of only one Congress and Exhibition on Fats and Oils Processing, 6., AOCS/SBOG, Campinas, signal, as would be the case for type 1995. Proceedings... p. 195. IV cyanolipids, are sufficient for dis Mikolajczak, K.L.; Scigler, D.S.; Smith Jr., tinguishing one from the other. C.R.; Wolf, I. Α.; Bates, R.B. 1969. A cyanogenetic lipid from Cordia vcrbenacea CONCLUSIONS DC seed oil. Lipids, 4(6): 617. Mikolajczak, K.L.; Smith Jr., C.R.; Tjarks, The oil content and its composi L.W. 1970. Cyanolipids of tion make P. carpopodea seeds a Cardiospermum halicacabum and other source of type I cyanolipids, while due sapindaceous seed oils. Lipids, 5(10): 812. to its low content in P. cupana seed oil Mikolacjzak, K.L. 1977. Cyanolipids. Prog. its presence is only detectable after Chem. Fats and other Lipids, 15: 97. concentration. The existing contro Mikolacjzak, K.L.; Weisleder, D. 1978. 13C Nuclear Magnetic Resonance spectros versy in the literature is attributed to copy of cyanolipids and cyanolipid-con- the low content (0.2%) of these com taining seed oils. Lipids, 13:514. pounds in the seeds. Nishizawa, M.; Adachi, K.; Sastrapradja, S.; Hayashi, Y. 1983. Isolation of individual ACKNOWLEDGMENT type II cyanolipids from Nephelium lappaceum. Phytochemistry, 22(12): 2853. The second author acknowledges Reitz, R. 1980. Sapindáceas. Itajaí: Herbário CNPq for the award of an M.Sc. Barbosa Rodrigues, 1980. (Flora scholarship. Ilustrada Catarinense), p.1-25. Scigler, D.S. 1976. Cyanolipids in Cordia Literature cited verbenaceac. A correction. Biochem. Syst. Ecoi, 4(4):235. Angclucci, E.; Tocchini, R.P.; Lazarine, V.B.; Scigler, D.S.; Kawahara, W. 1976. New reports Prado, M.A.F. 1978. Caracterização of cyanolipids from sapindaceous plants. química dc sementes de guaraná (P. Biochem. Syst. Ecol, 4(4):263. cupana var. sorbilis Duckc). Boi. ITAL Scigler, D.S.; Mikolajczak, K.L.; Smith Jr.; (56):1183. C.R.; Wolf, 1. A. 1970. Structure and re Bellamy, L. J. 1956. The Infrared Spectra of actions of a cyanogenetic lipid from Cor• Complex Molecules. John Wiley & Sons, dia verbenacea seed oil. Chem. Phys. Lip• New York, p.225. ids, 4: 147.

104 Lago ef al. Simoni, M.L.P.S.C. 1993. Componentes lipidicos em Paullinia carpopodea and Paullinia cupana. Dissertação de Mestrado, Instituto de Química/UFRJ. Rio de Janeiro. 114p. Spitzer, V. 1995. GLC-MS Analysis of the fatty acid of the seed oil, triglycerides, and cyanolipids of Paullinia elegans (Sapindaceae) - a rich source of cis-13- cicosenoic acid (Paullinic Acid). J. High Resol. Chrom., 18(7): 413.

Aceito para publicação cm 23/02/2000

On the occurrence of cyanolipids in Paullinia ...