Rev. Biol. Trop. 50(3/4): 963-967, 2002 www.ucr.ac.cr www.ots.ac.cr www.ots.duke.edu

Volatile constituents of the leaves of Siparuna thecaphora () from Turrialba, Costa Rica

José F. Cicció1* and Jorge Gómez-Laurito2 1 Centro de Investigaciones en Productos Naturales (CIPRONA) and Escuela de Química, Universidad de Costa Rica, 2060 San José, Costa Rica. Fax: (506) 225 9866; [email protected] 2 Centro de Investigaciones en Productos Naturales (CIPRONA) and Escuela de Biología, Universidad de Costa Rica, 2060 San José, Costa Rica. * Corresponding author.

Received 6-VIII-2002. Corrected 18-XI-2002. Accepted 26-XI-2002.

Abstract: The composition of the essential oil from leaves of Siparuna thecaphora (Poepp. et Endl.) A. DC. collected in Turrialba, Costa Rica, was determined by capillary GC/MS. Seventy-six compounds were identified corresponding to ca. 95% of the oil. The major components were germacrene D (32.7%), a -pinene (16.3%), b- pinene (13.8%) and b-caryophyllene (4.1%). Thirty-one minor compounds were identified for the first time in this genus of .

Key Words: Siparuna thecaphora, Siparunaceae, essential oil composition, germacrene D, a -pinene, b-pinene, b-caryophyllene.

The family Siparunaceae comprises two from southern Mexico throughout Central genera, the monotypic West African genus America to Brazil and Bolivia. It is called col- G l o s s o c a l y x and the Neotropical genus loquially “limoncillo” (little lemon) probably S i p a runa (formerly placed in the family because of the strong lemon-scented fruits. ), which contains about 72 In Panama, the infusion of crushed leaves species of , straggling shrubs, and is used to treat colds and rheumatism (Duke (Renner et al. 1997). Up to now, six species of 1972). In Guatemala, the fresh leaves are S i p a ru n a from Costa Rica have been placed on the forehead to relieve headaches described. (Morton 1980). Siparuna thecaphora (Poepp. et Endl.) A. To the best of our knowledge, no reports DC. is an aromatic or small (3-5 m have been made regarding the essential oil tall), with opposite, simple leaves, and unisex- composition from this genus of plants in Costa ual flowers. The fruits are fleshy and reddish Rica. when ripe, with arilate seeds. Due to its highly Early phytochemical studies of the genus variable morphology, a great number of syn- Siparuna revealed the presence of aporphine, onyms are found in the literature (e.g., S. oxoaporphine and morphinandienone alkaloids nicaraguensis, S. andina, S. gilgiana). This (Braz et al. 1976, Chiu et al. 1982, Gerard et is a widespread species in Costa Rica. It al. 1986, Lopez et al. 1988, 1990, 1993); cadi- can be found in both Pacific and Caribbean nane sesquiterpenes (El-Seedi et al. 1994) and sides of the Country, from near sea level up to kaempherol glycosides (Leitão et al. 2000). close to 2 000 m elevation. It is distributed Reports of the leaf essential oils constituents 964 REVISTA DE BIOLOGÍATROPICAL obtained from the Panamanian and Brazilian S. ture: 250ºC; detector temperature: 260ºC; ion- guianensis were varied. They range from tho- ization voltage: 70eV; scanning over 38-400 se containing as main constituents curzerenone amu range; split injection 1:20. Integration of and curzerenone derivatives (Antonio et al. the total ion chromatogram, expressed as area 1976), epi-a -bisabolol, spathulenol, selin-11- percent, has been used to obtain quantitative en-4a -ol, elemol, b-eudesmol, atractylone and compositional data. germacrone (Zoghbi et al. 1998) or (E)-neroli- Identification: Identification of the com- dol (Fernandez-Machado et al. 1998). ponents of the oil was performed using the Some chemical studies of S. thecaphora retention indices on a phenyl methyl silicone have been done previously. The oxoaporphine column, and by comparison of their mass alkaloid liriodenine was isolated from the spectra with those published in the literature twigs (under the synonym S. nicaraguensis) (Stenhagen et al. 1974, Swigar and Silverstein (Gerard et al. 1986). The alkaloids liriodenine 1981, McLafferty 1993, Adams 1995, 2001) or and oxonantenine, reported for the first time in those of our own database. the genus, were isolated from the roots (under the synonym S. gilgiana) (Chiu et al. 1982). The volatile oil of the fruits (under the syn- RESULTS onym S. nicaraguensis) was studied previous- ly (Manjarrez and Mendoza 1967). The main The oil is constituted mainly by hydrocar- constituents of the oil were b-elemene, citral bons (89.0%) whereas the oxygenated com- and b-ionone. pounds accounts only for 6.4% (Table 1). Among the 76 compounds identified, sesquiterpenoids represented 56.7% and MATERIALS AND METHODS monoterpenoids 38.7%. Germacrene D (32.7%), a - p i n e n e Plant Material: Leaves of Siparuna the - (16.3%), b-pinene (13.8%) followed by the caphora from Costa Rica, were collected in widespread sesquiterpene b- c a r y o p h y l l e n e Santa Cruz, Turrialba, province of Cartago, in (4.1%) were identified as the main const i t u e n t s 1998. A voucher specimen was deposited in of the oil. Among the oxygenated compounds the Herbarium of the University of Costa Rica a -cadinol (1.6%), epi-a -muurolol (1.0%) and at the School of Biology (USJ 68612). 1,8-cineole (0.8%) were the major ones. Th e Essential Oil Isolation: Fresh leaves other constituents were 69 compounds found in were subjected to hydrodistillation for 3 hr minor or trace amounts. Of these ones, thirty- using a modified Clevenger-type apparatus. one are newly identified compounds in the The distilled oil was collected as a colorless genus Si p a r una (see Table 1). liquid, dried over anhydrous sodium sulfate, and stored at -10ºC [ 0.2% (v/w) yield]. General Analytical Procedures: The oil was analyzed by gas chromatography/mass TABLE 1 spectrometry (GC/MS). The mass spectra were Percentage composition of the Siparuna thecaphora obtained using a Hewlett-Packard G1800A leaf oil from Costa Rica GCMSD System, employing a fused silica aCo m p o u n d bRI Le a v e s dMe t h o d (30im x 0.25 mm) column coated with a 5% phenyl methyl silicone (film thickness a -t h u j e n e * 93 0 0. 3 1, 2 0.25iµm). Operation conditions were: carrier a -p i n e n e 93 9 16 . 3 1, 2, 3 c gas: He (1.0 mL/min); oven temperature pro- a -f e n c h e n e * 95 3 t 1, 2 ca m p h e n e 95 4 0. 4 1, 2 gram: 75ºC (4 min), 75º-200ºC at 3ºC/min, sa b i n e n e * 97 5 1. 3 1, 2 200ºC (8 min); sample injection port tempera- b-p i n e n e 97 9 13 . 8 1, 2, 3 INTERNATIONALJOURNALOF TROPICALBIOLOGYAND CONSERVATION 965 my r c e n e 99 1 2. 5 1, 2 a -c a l a c o r e n e 15 4 6 t 1, 2 a -p h e l l a n d r e n e 10 0 3 t 1, 2 germacrene B 15 6 1 1. 3 1, 2 a -t e r p i n e n e * 10 1 7 0. 1 1, 2 E-n e r o l i d o l 15 6 4 0. 3 1, 2 p-c y m e n e 10 2 5 t 1, 2 b-c a l a c o r e n e * 15 6 6 t 1, 2 li m o n e n e 10 2 9 0. 4 1, 2, 3 germacrene D-4-ol* 15 7 6 0. 1 1, 2 b-p h e l l a n d r e n e 10 3 0 0. 3 1, 2 sp a t h u l e n o l 15 7 8 0. 1 1, 2 1, 8-cineole 10 3 1 0. 8 1, 2, 3 caryophyllene oxide 15 8 3 0. 2 1, 2 (Z)- b-o c i m e n e 10 3 7 1. 0 1, 2 ep i - a - m u u r o l o l * 16 4 0 1. 0 1, 2 (E)- b-o c i m e n e * 10 5 0 0. 2 1, 2 cu b e n o l 16 4 7 0. 3 1, 2 g-t e r p i n e n e 10 6 0 0. 2 1, 2 b-e u d e s m o l 16 5 1 0. 2 1, 2 ci s -sabinene hydrate* 10 7 0 t 1, 2 a -c a d i n o l * 16 5 4 1. 6 1, 2 p-m e n t h a - 3 , 8 - d i e n e * 10 7 3 t 1, 2 mint sulfide* 17 4 1 t 1, 2 te r p i n o l e n e 10 8 9 0. 1 1, 2 Monoterpene hydrocarbons 36 . 9 li n a l o o l 10 9 7 0. 2 1, 2, 3 Oxygenated monoterpenes 1. 8 n-n o n a n a l * 110 1 t 1, 2 Sesquiterpene hydrocarbons 52 . 1 en d o - f e n c h o l * 1117 t 1, 2 Oxygenated sesquiterpenes 4.6 ci s -p-m e n t h - 2 - e n - 1 - o l * 112 2 t 1, 2 al l o - o c i m e n e * 113 2 t 1, 2 a Compounds are listed by elution order in a 5% tr a n s -p i n o c a r v e o l * 113 9 t 1, 2 phenyl methyl silicone column; b RI = Retention tr a n s - p -m e n t h - 2 - e n - 1 - o l * 114 1 0. 2 1, 2 indices (Adams 2001); c t = trace (<0.05%); d pi n o c a r v o n e * 116 5 t 1, 2 Method: 1 = Retention Indices on 5% methyl phenyl te r p i n e n - 4 - o l * 117 7 0. 4 1, 2 silicone column; 2 = MS spectra; 3 = standard. a -t e r p i n e o l 118 9 0. 1 1, 2 * Newly identified constituents in the genus Siparuna. my r t e n a l * 119 6 t 1, 2 tr a n s -p i p e r i t o l * 12 0 8 t 1, 2 pu l e g o n e * 12 3 7 0. 1 1, 2 ne r a l 12 3 8 t 1, 2 ge r a n i o l 12 5 3 t 1, 2 DISCUSSION ge r a n i a l 12 6 7 t 1, 2 bornyl acetate* 12 8 9 t 1, 2 These results differ markedly from those d-e l e m e n e 13 3 8 2. 3 1, 2 a -c u b e b e n e 13 5 1 0. 2 1, 2 obtained with oils isolated from S. guianensis. cy c l o s a t i v e n e 13 7 1 0. 1 1, 2 Antonio et al. (1976) reported that the leaf oil lo n g i c y c l e n e * 13 7 4 0. 1 1, 2 of plants collected in Panama, contains as a -y l a n g e n e 13 7 5 0. 3 1, 2 major constituents curzerenone and curz- a -c o p a e n e 13 7 7 1. 8 1, 2 erenone type compounds or products decom- b-b o u r b o n e n e 13 8 5 0. 2 1, 2 b-c u b e b e n e 13 8 8 0. 3 1, 2 posing to curzerenone. Zoghbi et al. (1998) b-e l e m e n e 13 9 1 0. 8 1, 2 reported the composition of three types of oils b-c a r y o p h y l l e n e 14 1 9 4. 1 1, 2, 3 from S. guianensis collected at different loca- b-c o p a e n e * 14 3 2 0. 5 1, 2 tions of the Amazon, Brazil: type A, with epi- g-e l e m e n e 14 3 7 0. 8 1, 2 a -bisabolol and spathulenol as main con- a -h u m u l e n e 14 5 5 0. 6 1, 2 al l o a r o m a d e n d r e n e 14 6 0 0. 1 1, 2 stituents; type B, with spathulenol, selin-11- ci s -m u u r o l a - 4 ( 1 4 ) , 5 - d i e n e * 14 6 7 0. 1 1, 2 en-4a -ol, b-eudesmol and elemol; and type C, germacrene D 14 8 5 32 . 7 1, 2 with germacrone, germacrene D, bicycloger- b-s e l i n e n e 14 9 0 0. 6 1, 2 macrene, germacrene B and atractylone as tr a n s -m u u r o l a - 4 ( 1 4 ) , 5 - d i e n e * 14 9 4 0. 4 1, 2 major constituents. Fernandez-Machado et al. ep i - c u b e b o l 14 9 4 0. 5 1, 2 bi c y c l o g e r m a c r e n e 15 0 0 1. 2 1, 2 (1998) reported that (E)-nerolidol was the a -m u u r o l e n e 15 0 0 0. 5 1, 2 main component of the leaf oil of S. guianen - germacrene A 15 0 9 0. 1 1, 2 sis, during flowering. They observed a large d-a m o r p h e n e * 15 1 2 0. 5 1, 2 seasonal variation of nerolidol. g-c a d i n e n e 15 1 4 0. 3 1, 2 Our results also differ from those obtained cu b e b o l 15 1 5 0. 3 1, 2 d-c a d i n e n e 15 2 3 2. 0 1, 2 by Manjarrez and Mendoza (1967) from the tr a n s -c a d i n a - 1 ( 2 ) , 4 - d i e n e * 15 3 5 0. 1 1, 2 fruits of S. thecaphora from Mexico (under the a -c a d i n e n e * 15 3 9 0. 1 1, 2 synonym S. nicaraguensis). The oil was 966 REVISTA DE BIOLOGÍATROPICAL characterized by the presence of b-elemene, Adams, R.P. 2001. Identification of Essential Oil citral and b-ionone. Components by Gas Chromatography/ Quadrupole In the current study of S. thecaphora, Mass Spectroscopy. Allured, Carol Stream IL. 456 p. Germacrene D (32.7%), a -pinene (16.3%) and Antonio, T.M., G.R. Waller & C.J. Mussinan. 1976. b-pinene (13.8%) were identified as the main Composition of Essential Oil from the Leaves of constituents of the oil. Preliminary studies (still Siparuna guianensis (Monimiaceae). Chemistry and in progress) concerning the composition of the Industry 16: 1187-1188. oil of S. thecaphora from three locations in Braz, R., S.J. Gabriel, C.M.R. Gomes, O.R. Gottlieb, Costa Rica also revealed chemical diffe r e n c e s M.D.G.A. Bichara & J.G.S. Maia. 1976. (Chavarría 2002). This chemical diffe r e n c e s Oxoaporphine Alkaloids from Fusea longifolia and could be attributed to the effect of geographical S i p a runa guianensis. Phytochemistry 15: 11 8 7 - location and microenvironment or to the effe c t 1188. of interspecific and infraspecific differences or Chavarría, A. 2002. Variación morfológica, química y to the phenological stage of the plant. molecular de Siparuna thecaphora (Siparunaceae) en tres zonas de Costa Rica. Tesis de licenciatura, ACKNOWLEDGMENTS Universidad de los Andes, Bogotá, 170 p.

The authors are grateful to Vicerrectoría Chiu, S.Y.C., R.H. Dobberstein, H.H.S. Fong & N.R. Farnsworth. 1982. Oxoaporphine Alkaloids from de Investigación UCR (Project 809-99-264) Siparuna gilgiana. J. Nat. Prod. 45: 229-230. for financial support. To M. Segnini and Centro de Investigación en Contaminación Duke, J.A. 1972. Isthmian Ethnobotanical Dictionary. Ambiental (International Atomic Energ y Fulton, Maryland. pp. 96. A g e n c y, project. 802-96-550) for GC/MS El-Seedi, H., F. Ghia & K.B.G. Torssell. 1994. Cadinane facilities. To N. R. Farnsworth (College of Sesquiterpenes from S i p a runa macro t e p a l a. Pharmacy, University of Illinois at Chicago, Phytochemistry 35: 1495-1497. USA) for his help to access the NAPRALERT database. Fernandes-Machado, S.M., V. A . F. A d a u t o - R i b e i r o , J.S.L.T. Militão, S. Maia de Morais & M.I. Lacerda- Machado. 1998. Seasonal Variation of (E)-Nerolidol in S i p a runa guianensis Aublet and 1 3C - N M R RESUMEN Spectral Assignments of (E)- and (Z)-Nerolidol. J. Essent. Oil Res. 10: 708-710. La composición química del aceite esencial de las hojas de Siparuna thecaphora (Poepp. et Endl.) A. DC., Gerard, R.V., D.B. MacLean & T.M. Antonio. 1986. recolectada en Turrialba, Costa Rica, se estudió mediante Examination of three Siparuna Species for Alkaloid la técnica de cromatografía capilar gaseoso-líquida, aco- Content. Phytochemistry 25: 2155-2156. plada a un espectrómetro de masas (GC/MS). Se identifi- caron 76 compuestos que correspondieron a cerca del 95% Leitão, G.G., S.S.V. Soares, T.d.B.M. Brito & F. Delle del aceite. Los componentes mayoritarios fueron germa- Monache. 2000. Kaempferol Glycosides from creno D (32.7%), a -pineno (16.3%), b-pineno (13.8%) y Siparuna apiosyce. Phytochemistry 55: 679-682. b-cariofileno (4.1%). Dentro de los compuestos minorita- rios, treinta y uno se describen por primera vez como cons- Lopez, J.A., Y. Aly & P.L. Schiff Jr. 1988. Alkaloids of tituyentes de este género de plantas. Siparuna pauciflora. Planta Med. 54: 552-553.

Lopez, J.A., J.G. Laurito, F.T. Lin, F.K. Duah, M. Sharaf, Y.Aly, L.K. Wong & P.L. Schiff Jr. 1990. Alkaloids REFERENCES of Siparuna tonduziana. Planta Med. 56: 492.

Adams, R.P. 1995. Identification of Essential Oil Lopez, J.A., J.G. Laurito, F.T. Lin, M. Sharaf, L.K. Wong Components by Gas Chromatography/ Mass & P.L. Schiff Jr. 1993. Alkaloids of Siparuna griseo- Spectroscopy. Allured, Carol Stream, IL. 468 p. flavescens. Planta Med. 59: 100. INTERNATIONALJOURNALOF TROPICALBIOLOGYAND CONSERVATION 967

Manjarrez, A. & V. Mendoza. 1967. The volatile oil of the Siparuna (Siparunaceae: ). Int. J. Plant. Sci. fruits of S i p a runa nicaraguensis Heml. Perfum. 158 (6 Suppl.): S89-S98. Essent. Oil Rec. 58: 23-24. Stenhagen, E., S. Abrahamsson & F. W. MacLaff e r t y McLafferty, F.W. 1993. Registry of Mass Spectral Data. (Eds.). 1974. Registry of Mass Spectral Data. Wiley, Wiley, New York. New York. 3358 p.

Morton, J. 1980. Atlas of Medicinal Plants of Middle Swigar, A.A. & R.M. Silverstein. 1981. Monoterpenes. America. Bahamas to Yu c a t a n. C.C. T h o m a s , Aldrich, Milwaukee, Wisconsin. 130 p. Springfield, IL. 1420 p. Zoghbi, M.d.G.B., E.H.A. Andrade, A.S. Santos, M.H.L. Renner, S.S., A.E. Schwarzbach & L. Lohmann, 1997. Silva & J.G.S. Maia. 1998. Essential Oil of Siparuna Phylogenetic position and floral function of guianensis Aubl. J. Essent. Oil Res. 10: 543-546.