LAGO, R. C. A.

PINTO, A. C.

SIMONI, M. L. P. S. C.

English

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O N T H E O C C U R R E N C E O F C Y A N O L I P I D S I N Paullinia carpopodea C A M B E S S AND P. cupana K U N T H 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 1 Ή-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 1 Ή-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. 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 INTRODUCTION Paul Unia carpopodea is known in Brazil as "timbó" and grows wild in the states of Minas Gerais, São Paulo and Paraná. The plant is used for its ictiotoxic properties (Reitz, 1980). P. cupana is native to Amazonas and Pará states and is commonly known as "guaraná". Its seeds are used for preparing a powder used in re­freshments, recommended as an en­ergy reconstituem and for alleviating stomach disorders. "Guaraná" also has a reputation as a cardiotonic and arte­riosclerosis prevention agent (Reitz, 1980). Angelucci et al. (1978) men­tioned ca. 4% of caffeine in its seeds. Cyanolipids are a unique class of plant lipids that seem to occur almost exclusively in the seed oils of the Sapindaceae (Mikolajczak et al. 1970; Gowrikumar et al. 1976; Nishizawa et al. 1983), although cyanolipids were identified for the first t ime in Boraginaceae, Cordia verbenacea (Mikolajczak, 1969). Seigler (1976) showed that, in fact, the plant studied belonged to the Sapindaceae family. According to Mikolajczak (1977), not all species of Sapindaceae contain cyanolipids. The four known cyanol ipid structures (types) present the same branched five-carbon nitrile skeleton. Types I and II are long chain fatty di-esters of l-cyano-2-hydroxymethyl prop-2-en-l-ol (I) and of l-cyano-2-hydroxymethyl prop-l-en-3-ol (II). Types III and IV are mono-esters of 1-cyano-2-hydroxymethy 1 prop-1 -en (III) and of l-cyano-2-methylprop-2-en-l-ol (IV). Cyanogenesis is defined as the capacity shown by certain plants of yielding HCN through the hydrolysis of chemical compounds produced by them. For a long time this process was associated with the presence of cyano-genic glycosides in plant tissues. The first and conclusive evidence that cyanogenici ty in kusum seed oil (Schleichera trijuga) could not be at­tributed to a glycoside but rather to a lipid-based material was provided by Kundu & Bandyopadhyay (1969). Cyanolipids I and IV are cyanogenic. Different seeds may yield con­flicting results, as in the case of P. cupana; for some authors, its seeds con­tain type I cyanolipids while for others the material is devoid of such com­pounds (Seigler & Kawahara, 1976). This study was undertaken in order to study P. carpopodea lipids and to con­firm whether P. cupana contains or not cyanogenetic compounds. EXPERIMENTAL P. carpopodea fruits were col­lected in Ouro Preto, Minas Gerais (voucher is deposited at the herbarium of the Pharmacy and Biochemistry School, Federal University of Ouro Preto, under the register number 1031 (flowers) and 1949 (fruits)). P. cupana fruits were from Pará state. The seeds were separated from the fruits, ground in a lab mill and their oil extracted in a Butt type apparatus, with light petro­leum, b.p. 40-60°C. The alkaline qualitative picrate test (Feigl, 1954) was conducted to detect the presence of the -CN group. Thin-Layer Chromatography (TLC) was carried out using silica gel G plates and benzene as eluent (no other solvent gave better or equivalent separation). Triacylglycerols and cyanolipids bands were scrapped out from preparative plates (0.5mm), ex­tracted with CH 2C1 2 and weighed, af­ter evaporation of the solvent. Purifi­cation was carried out through HPLC, in a Waters 403 system, equipped with Refractive Index Detector, using two Altex ODS columns (5xl50mm;5mm) in series, and acetone: acetonitrile: dichloromethane 3:2:1 as eluent, at flow rate of lml/min. The infrared spectrum of P. carpopodea cyanolipids showed car-bonyl (1740cm"1) and =CH 2 absorp­tions (1157cm-1 and 920-965 cm 1 ) . Ή-NMR spectra were recorded at 200MHz in CDCl, with TMS as in­ternal standard. I 3 C-NMR spectrum was taken at 100MHz in CDC1 3. P. carpopodea cyanolipids. Ή -NMR: δ 2.33 (m, Η and H b); 4.70 (s, H , H d), 5.52 (s, H g), 5.67 (s, H f), and 5.96 ( s , H ) . , 3C-NMR: δ 120.8 (-CN), 130.0 (=CH 2), 135.3 (C=), 171.5 (CO) and 173.0 (CO). P. cupana cyanolipids. Ή-NMR: δ 2.34 ( m , H , H b ) , 4.63 (s, H , H d), 5.50 ( s , Η ) , 5.66 (s, H f) and 5.94 (s, H ) . RESULTS AND DISCUSSION P. carpopodea and P. cupana seeds yielded 40.5% and 1.2% oil, re­spectively. The seed oil from P. carpopodea was clearly positive to the picrate test which, nevertheless, failed 102 Lago et al. o H d H e o Figure 1. Protons assignment in Ή-NMR spectrum of P. Carpopodea cyanolipids. to prove the cyanogenicity of the P. cupana oil or the presence of cyano-genic cyanolipids. A band which ap­peared, for both oi ls , above the triacylglycerols on the TLC plate could only be related to ei ther cyanolipid I or IV, since cyanolipids II and III are more polar than the triacylglycerols. The UV spectrum exhibited a very weak nitrile absorption band at 208nm. The positive Feigl test and the absence of a -CN absorpt ion at 2230cm' 1 in the IR region (Bellamy, 1956) indicated the presence of a non-conjugated cyano group, correspond­ing either to type I or IV cyanolipids. Through preparat ive TLC, cyanolipids in the oils were quantified. While P. carpopodea oil contained 70.7% of cyanolipids and 21.3% of other lipids, mostly triacylglycerols (Lago et al, 1995), P. cupana oil ex­hibited only 12%) of cyanolipids (or 0.2% in the seeds). Lago et al. (1995) observed that eicosenoic acids are the main constitu­ents (52.8%) in P. carpopodea seed oil. They are the major components in the cyanolipid fraction (64.95%) but the third in the triacylglycerol fraction (16%). In P. elegans, Spitzer (1995) identified c/s-13-eicosenoic acid as the main component of total l ipids (44.4%), which the author named Paullinic acid, and small amounts of cis-11 -eicosenoic acid (gadoleic acid, 3.7%) and c/s-15-eicosenoic acid (0.7%). The Ή - N M R spectrum of the cyanolipid fraction showed a multip-let (probably a double triplet) at δ 2.33 which was assigned to protons of me­thylene group a to carbonyl group (H a, H b) shown in Figure 1. The -CH 2- pro­tons (H c, H d) gave an apparent singlet at δ 4.70. The remaining protons of the dihydroxynitrile moiety of cyanolipids produced apparent singlets at δ 5.52 (Η ), 5.67 (H f) and 5.96 (H ). Equiva-lent data have been presented by Seigler et al. (1970) and Mikolacjzak (1977) for type I cyanolipids. Ή-NMR spectrum of P. cupana seed oil showed also equivalent data with a multiplet at δ 2.34 (H a, H b), a singlet at δ 4.63 (H c, H d) and singlets centered at δ 5.10, δ 5.66 and δ 5.94 (terminal -CH 2- protons, H e, H p and proton bonded to α-carbon to the ni­trile group, H c, respectively). In the l 3C-NMR spectrum of the cyanolipid fraction assignment for some carbon atoms are as follows: -On the occurrence of cyanolipids in Paullinia 103 CN group- 120.8ppm; =CH 2 , BO.Oppm; =C, 135.3ppm and CO groups, 171.5 and 173.0ppm. The pos­sibility of having cyanolipids II and/ or III had been discarded previously. Differences in chemical shifts between carbon atoms of types I and IV cyanol ipids (Mikolajczak & Weisleder, 1978), such as the presence of two signals corresponding to dis­tinct CO carbon atoms in the spectrum of cyanolipids I instead of only one signal, as would be the case for type IV cyanolipids, are sufficient for dis­tinguishing one from the other. C O N C L U S I O N S The oil content and its composi­tion make P. carpopodea seeds a source of type I cyanolipids, while due to its low content in P. cupana seed oil its presence is only detectable after concentration. The existing contro­versy in the literature is attributed to the low content (0.2%) of these com­pounds in the seeds. A C K N O W L E D G M E N T The second author acknowledges CNPq for the award of an M.Sc. scholarship. Literature cited Angclucci, E.; Tocchini, R.P.; Lazarine, V.B.; Prado, M.A.F. 1978. Caracter ização química dc sementes de guaraná (P. cupana var. sorbilis Duckc). Boi. ITAL (56):1183. Bellamy, L. J. 1956. The Infrared Spectra of Complex Molecules. John Wiley & Sons, New York, p.225. Fcigl, F. 1954. Spot Tests, v.l, 4. cd., Elsevier, New York, p.263, 269. Gowrikumar, G.; Mani, V.V.S.; Rayana, G.L. 1976. Cyano l ip ids in Sap indus emarginatus seed oil. Phytochemistry, 15:1566. Kundu, M.K.; Bandyopadhyay, C. 1969. Stud­ies on some chemical aspects of kusum oil. J. Am. Oil Chem. Soc, 46(1):23. Lago, R.C.A.; Simoni, M. de L.P.S.; Pinto, A.C. 1995. Lipids in Paullinia carpopodea Cambess seed oil. In: Latin-American Congress and Exhibition on Fats and Oils Processing, 6., AOCS/SBOG, Campinas, 1995. Proceedings... p. 195. Mikolajczak, K.L.; Scigler, D.S.; Smith Jr., C.R.; Wolf, I. Α.; Bates, R.B. 1969. A cy-anogenetic lipid from Cordia vcrbenacea DC seed oil. Lipids, 4(6): 617. Mikolajczak, K.L.; Smith Jr., C.R.; Tjarks, L.W. 1970. Cyano l ip ids of Cardiospermum halicacabum and other sapindaceous seed oils. Lipids, 5(10): 812. Mikolacjzak, K.L. 1977. Cyanolipids. Prog. Chem. Fats and other Lipids, 15: 97. Mikolacjzak, K.L.; Weisleder, D. 1978. 13C Nuclear Magnetic Resonance spectros­copy of cyanolipids and cyanolipid-con-taining seed oils. Lipids, 13:514. Nishizawa, M.; Adachi, K.; Sastrapradja, S.; Hayashi, Y. 1983. Isolation of individual type II cyanol ipids from Nephelium lappaceum. Phytochemistry, 22(12): 2853. Reitz, R. 1980. Sapindáceas. Itajaí: Herbário Barbosa Rodr igues , 1980. (Flora Ilustrada Catarinense), p.1-25. Scigler, D.S. 1976. Cyanolipids in Cordia verbenaceac. A correction. Biochem. Syst. Ecoi, 4(4):235. Scigler, D.S.; Kawahara, W. 1976. New reports of cyanolipids from sapindaceous plants. Biochem. Syst. Ecol, 4(4):263. Scigler, D.S.; Mikolajczak, K.L.; Smith Jr.; C.R.; Wolf, 1. A. 1970. Structure and re­actions of a cyanogenetic lipid from Cor-dia verbenacea seed oil. Chem. Phys. Lip-ids, 4: 147. 104 Lago ef al. Simoni , M.L.P.S.C. 1993. Componentes lipidicos em Paullinia carpopodea and Paullinia cupana. Disser tação 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 cyano l ip ids 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 ... 

On the occurrence of cyanolipids in Paullinia carpopodea Cambess and P. cupana Kunth seed oils.

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On the occurrence of cyanolipids in Paullinia carpopodea Cambess and P. cupana Kunth seed oils.

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