Volatile Organic Compounds of Leaves and Flowers of Montanoa Tomentosa

FLAVOUR AND FRAGRANCE JOURNAL VOLATILE OILS OF MONTANOA TOMENTOSA Flavour Fragr. J. (In press) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1560 Volatile organic compounds of leaves and ﬂowers of Montanoa tomentosa

Ramón Enrique Robles-Zepeda,1 Jorge Molina-Torres,2 Edmundo Lozoya-Gloria2 and Mercedes G. López2*

1 Universidad de Sonora, Encinas y Rosales, 83000, Hermosillo, Sonora, México 2 Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, 36500 Irapuato, Guanajuato, México Received 15 January 2004; Revised 7 September 2004; Accepted 4 October 2004

ABSTRACT: Montanoa tomentosa is a valued plant due to its medicinal properties, mainly as a menstruation and childbirth inducer. The volatile constituents of the aerial parts (leaves and flowers) of this medicinal plant were analysed by SPME–GC–MS. The major constituents of the volatile fraction were monoterpenes, such sabinene, α-pinene and α- thujene, which accounted for 65% of the total fraction. Other identified compounds were sesquiterpenes, like α-gurjunene, caryophyllene and germacrene D, but in minor amount. The total proportion of identified compounds was 86.7% (17 volatiles) and 82.1% (14 volatiles) for leaves and flowers, respectively. Some of the most abundant terpenes found in this plant are well known for their relevant roles in the environment and in many industrial applications. The characterization of volatile from M. tomentosa is established in this study for the first time, therefore their utility in different aspects, opens an interesting area to carry out further investigation of the plant. Copyright © 2005 John Wiley & Sons, Ltd.

KEY WORDS: Montanoa tomentosa; medicinal plant; aerial parts; volatile constituents; sabinene; α-thujene; SPME–GC–MS

Introduction pounds in different products. A large number of different methods are known for the extraction of VOCs from The genus Montanoa belongs to the tribe Heliantheae of plants. However, in the last decade solid-phase micro- the family Asteraceae and contains 25 species,1 localized extraction (SPME) has been an excellent choice among mainly in Latin America. Montanoa tomentosa Cerv., all extracting methods.5,6 SPME is a simple, rapid, a perennial shrub, is widely distributed in Mexico. solvent-free, inexpensive and effective method for the Originally the plant was known by its Nahuatl name, selective extraction of volatile and semivolatile com- cihuapatli, which means ‘woman medicine’ (cíhuatl, ponents from matrices containing non-volatile high- woman; patli, medicine). However, nowadays it is known molecular weight components. as zoapatle.2 Although zoapatle has been used on the M. tomentosa is a highly aromatic plant, therefore it is treatment of many diseases for centuries, it was only last possible that it accumulates diverse volatile compounds century, that Mexican researchers showed interest on in its aerial parts. In the present work we examined the the study of this plant. M. tomentosa has been used in volatile composition of leaves and flowers of M. traditional medicine in Mexico to treat diverse female tomentosa by SPME–GC–MS. health problems, being particularly helpful on childbirth induction.3 Some authors attribute some of the pharmaco- logical properties of M. tomentosa principally to the Experimental diterpenic acids, kaurenoic acid (KA; kaur-16-en-18-oic acid) and grandiflorenic acid [GA; kaura-9 (11), 16-dien- Plant Material 18-oic acid].4 However, other compounds may also play relevant roles. Fresh M. tomentosa leaves and flowers were col- The study of volatile organic compounds (VOCs) lected from plants obtained in vitro and transferred in plants is of high interest, basically due to their large to the experimental field of CINVESTAV-IPN, Unidad industrial applications as flavouring and aromatic com- Irapuato, Mexico. Species authentication was performed by Dr Jerzy Rzedowski from the Instituto de Ecologia, Centro Regional del Bajio in Patzcuaro, Michoacan, Mexico, where a voucher specimen (IEB-57689) was deposited. * Correspondence to: M. G. López, Departamento de Biotecnología y Leaves were collected in a size range of 7–7.5 cm and Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, 36500 Irapuato, Guanajuato, México. flowers in a size range of 1–1.5 cm. This fresh material E-mail: [email protected] was analysed immediately.

Solid–phase Microextraction (SPME) Table 1. Volatile organic compounds extracted and identified in leaves and flowers of M. tomentosa by Volatile organic compounds were extracted by SPME–GC–MS SPME, with a coated fibre of divinylbenzene/carboxen/ Area (%)c polydimethyl siloxane (DVB/CAR/PDMS) from Supelco Co., with a coating thickness of 50/30 µm. The fibre was Rta KIb Compound Leaves Flowers conditioned as recommended by the manufacturer. Fresh leaves and flowers were cut from the field and placed 1.85 799 α-Pinene 15.9 16.5 1.95 878 α-Thujene 10.4 7.8 in glass vials (20 ml), which were sealed with a screw- 2.06 880 Santoline triene 4.6 2.9 capped top containing a Teflon-lined septum and main- 3.45 977 β-Pinene 0.9 0.9 tained at room temperature for 24 h. After this time, the 4.01 983 Sabinene 39.5 40.1 5.51 997 β-Myrcene 0.4 0.4 SPME fibre was exposed to the headspace of the sample 6.13 1080 (+) 2-Carene 0.8 1.2 (leaves or flowers) for 1 h. This was done also at room 6.53 1083 Limonene 3.7 6.0 temperature. The fibre was then retracted and inserted 6.87 1087 β-Thujene 0.4 0.3 8.42 1094 γ-Terpinene 5.1 3.7 immediately into the inlet of the GC–MS for the 9.89 1185 (+) 4-Carene 1.1 0.8 desorption of the volatiles. The desorption process in 17.74 1299 trans-Sabinene hydrate 0.1 0.2 GC–MS was carried out for 1 min. The fibre was left 18.31 1391 α-Cubebene 0.3 nd 19.85 1395 α-Gurjenene 0.8 0.7 in the injector port for 30 min. 22.60 1493 Caryophyllene 1.2 nd 27.12 1594 Germacrene D 0.8 0.6 27.48 1595 β-Selinene 0.7 nd 27.75 1596 Unknown (204)d 0.6 nd GC–MS Analysis Conditions 29.24 1598 Unknown (204)d 2.9 0.4

a The GC–MS analyses were performed in a Hewlett- Rt, Retention time (min). b KI, Kováts index. Packard 5890 Series II gas chromatograph coupled to a c Relative to total detected compounds. mass spectrometer detector (Hewlett-Packard/MSD 5972 d In parenthesis molecular weight of unidentified compounds. Series). Compound separation was carried out using a capillary column HP-FFAP (25 m × 0.32 mm i.d., film thickness 0.52 µm) from Hewlett-Packard. Carrier gas, 82.32% of the total area; in this case the main com- helium at a constant flow rate of 1 ml/min; injector tem- pounds were sabinene (40.1%), α-pinene (16.5%), perature, maintained at 180 °C. The oven temperature α-thujene (7.8%), limonene (6.0%), γ-terpinene (3.7%) programme was: 3 min at 50 °C, followed by a tempera- and santolina triene (2.9%). The major sesquiterpenes in ture increment of 3 °C/min to 120 °C and a second rate the leaves were α-gurjunene, caryophyllene, β-selinene, of 5 °C/min to 200 °C, then held for 13 min. Mass spectra α-cubenene and germacrene D, while in the flowers α- were obtained at 70 eV and the ion source was at 230 °C. gurjunene and germacrene D were detected. Compounds were identified by comparison with the NIST The presence and abundance of some volatile organic 98 mass spectral database, commercial standards and compounds in plants can be used as chemical markers for literature reports. Kováts indices were calculated by com- species identification and/or geographic origin of the parison with a hydrocarbon series from n-pentane to same genus.7 The genus Montanoa comprises 25 species.1 n-tricosene, eluted under the same conditions as the M. tomentosa has been extensively studied for its medici- samples. The results are presented as the area % of nal properties, mainly those attributed to diterpenic acids totally identified compounds. Three replicates were done such as kaurenoic, grandiflorenic and monoginoic acids.4 for each sample. The functional properties of these acids have opened a very interesting research area. Kaurenoic acid is common to all plants, because it is the precursor of gibberellins Results and Discussion biosynthesis.8 On the other hand, grandiflorenic and monoginoic acids are not always present along with Solely volatile organic compounds were mono- and kaurenoic acid, therefore their presence or absence can be sesquiterpenes. The most abundant identified volatiles used as chemical markers of species within this genus. extracted from the leaves and flowers by SPME-GC-MS The volatile compounds produced by plants and other are shown in Table 1. Nineteen compounds were found live species comprise a large number of organic sub- in the leaves, 82.9% monoterpenes and 7.3% sesquiter- stances, including isoprene and isoprenoid compounds, penes. The major constituents in the leaves were sabinene alkanes, alkenes, carbonyl compounds, alcohols and (39.5%), α-pinene (15.9%), α-thujene (10.4%), γ- esters.5 These volatile compounds are responsible for terpinene (5.1%), santoline triene (4.6%) and limonene multiple interactions between plants and other organisms, (3.7%). On the other hand, only 14 compounds were such as pollinating animals, herbivores and the predators totally characterized in the flowers, corresponding to of herbivores.9,10 Furthermore, because some volatile

Copyright © 2005 John Wiley & Sons, Ltd. Flavour Fragr. J. (In press) VOLATILE OILS OF MONTANOA TOMENTOSA compounds have antipathogenic properties, they are References produced and emitted by plants as a defence mechanism against attack by herbivores.11 1. Funk VA. In The Systematics of Montanoa (Asteraceae, Heliantheae). Memoirs of the New York Botanical Garden, vol. Frequently, the biological activity of essential oils is 36. New York Botanical Garden, Bronx, New York, 1982. due to the presence of a mixture of compounds and not 2. Lozoya X. In Xiuhapatli, Herba officinalis. Secretaría de Salud- to a single one. However, characterization of the volatile UNAM: México, DF, 1999; 33. 3. Gallegos A. Contraception 1983; 27: 211. components of M. tomentosa has also provided signifi- 4. Enríquez RG, Mirya EG, Ortíz B et al. Planta Med. 1996; 62: cant insight into its practical medicinal uses, e.g. myrcene 569. has been shown to possess potent analgesic activity 5. Alcaraz C, Augusto F, Christensen E et al. Anal. Chem. 2001; 73: 12 4729. in rats. Another monoterpene found in this plant, 6. Cornu A, Carnat AP, Martin B et al. J. Agric. Food Chem. 2001; limonene, has previously been shown to inhibit rat mam- 49: 203. mary carcinogenesis,13 and it has also been shown to 7. Roussis V, Tsoukatou M, Petrakis PV et al. Biochem. System. Ecol. 2000; 28: 163. induce the activity of a detoxifying enzyme, indicating 8. Helliwell CA, Poole A, Peacock JW, Dennis ES. Plant Physiol. its potential as an anticarcinogenic agent.14 Another 1999; 119: 507. terpenoid compound of commercial interest is sabinene, 9. Mookherjee BD, Wilson RA, Schrankel KR, Katz I, Butler JF. In Bioactive Volatile Compounds from Plants. ACS Symposium which was the most abundant identified volatile in the Series No. 525. American Chemical Society: Washington, DC, leaves and flowers of M. tomentosa and is widely used in 1992; 35. the synthesis of perfume components, with cosmetic and 10. McCaskill D, Croteau R. TIBTECH. 1998; 16: 349. 15 11. Gershenzon J, Croteau R. In Herbivores: Their Interaction with possible medicinal applications. On the other hand, Secondary Plant Metabolites, vol. 1, Rosenthal G, Berenbaum M sabinene hydrate is used as an effective insecticide for (eds). Academic Press: San Diego, CA, 1991; 165. cloth.16 Caryophyllene is applied as a pheromone in the 12. Lorenzetti BB, Souza GEP, Sarti SJ, Filho DS, Ferreira SH. 17 J. Ethnopharmacol. 1991; 34: 43. management of beneficial insects. Finally, germacrene 13. Russin WA, Hoesley JD, Elson CE, Tanner MA, Gould MN. D is used as a constituent of eye-disease pills and as a Carcinogenesis 1989; 10: 2161. masking substance in cerambycid beetle attractants.18,19 14. Zheng G, Kenney PM, Lam LKT. J. Agric. Food Chem. 1993; 41: 153. The presence of glandular and non-glandular trichomes 15. Retamar JA, Elder HV, Molli JS, Gasparri MG. Essent. Deriv. is common to species of the Asteraceae. In M. tomentosa Agrum. 1994; 64: 61. we have observed the presence of these structures in the 16. Sato H, Watanabe Y, Itoi H, Higo M, Kato T. Sublimate of sabinene hydrate (Lion Dentifrice Co. Ltd, Japan). Jpn. Kokai leaves and flowers. Glandular trichomes are structures Tokkyo Koho, JP 54148183 791120; application: JP 78-57374 that accumulate a large diversity of volatile organic 780515. 1979; 3. compounds associated with several biological functions. 17. Pickett JA, Dawson GW, Free JB et al. In Pheromones in the Management of Beneficial Insects. Proceedings of the British It is probable that the zoapatle volatiles found in this Crop Protection Conference—Pests and Diseases, 1984; 1: study might accumulate in this type of trichomes. 247. To our knowledge, this is the first report on volatile 18. Yamasaki T, Sato M, Sakoguchi H. Appl. Entomol. Zool. 1997; 32: 423. compounds of M. tomentosa and the beginning of a new 19. Martens-Lobenhoffer J, Behrens-Baumann W, Loesche D, Meyer and exciting research area. FP. Pharmazie 1998; 53: 136.