VOLATILE OIL OF PILOCARPUS PENNATIFOLIUS 325

Copyright © 2004 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 325–326

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2004; 19: 325–326Published online 29 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1306

Analysis of the volatile oil from Pilocarpus pennatifoliusLemmaire (Rutaceae) leaves by GC–MS

Ana Paula Santos,1 Melânia Cornélio Lopes,1 Renata P. Limberger,2 Miriam Anders Apel,2

Amélia T. Henriques2 and Paulo R. H. Moreno1*

1 Instituto de Química, Universidade de São Paulo, São Paulo, Brazil2 Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Received 10 September 2002; Revised 29 April 2003; Accepted 2 May 2003

ABSTRACT: The oil obtained by steam distillation of dried and powdered Pilocarpus pennatifolius Lemmaire leaves was

analysed by GC and GC–MS. The major components were tridecane (56.7%) and pentadecane (25.5%). Additionally, γγγγγ-

muurolene (6.6%), spathulenol (2.6%), βββββ-elemene (2.1%), βββββ-caryophyllene (1.2%) and eugenol (0.3%) were also found

in the crude oil. Copyright © 2004 John Wiley & Sons, Ltd.

KEY WORDS: Pilocarpus pennatifolius Lemmaire; Rutaceae; jaborandi; volatile oil constituents; GC; GC–MS; tridecane;

pentadecane

* Correspondence to: P. R. H. Moreno, Instituto de Química, Universidade

de São Paulo, Caixa Postal 26007, CEP: 05505-900, São Paulo, SP, Brazil.

E-mail: prmoreno@iq.usp.br

Contract/grant sponsor: Fundação de Amparo à Pesquisa do Estado de São

Paulo, Brazil.

Contract/grant sponsor: Conselho Nacional de Desenvolvimento Científico

e Tecnológico, Brazil.

compound was made by comparison of their mass spec-

tra with available data in system library and their calcu-

lated Kováts retention index.13,14

Experimental

P. pennatifolius Lemmaire leaves were collected in

Parque Ecológico of the Universidade de Campinas,

Campinas-SP, Brazil, in October 2000. A voucher speci-

men (No. 30683) is deposited in the Herbarium of the

Universidade Estadual de Campinas (UEC), São Paulo,

Brazil. The oil was obtained from dried (room tempera-

ture) and powdered leaves (390 g) by steam distillation

in a Clevenger-type apparatus for 3 h.15 The oil was ex-

tracted from the condensed water layer with n-pentane,

dried over anhydrous sodium sulphate and the solvent

was subsequently eliminated under vacuum at room

temperature. A pale yellow oil was obtained in a yield of

0.31%. The qualitative and the quantitative analysis were

evaluated by capillary GC and GC–MS.

Gas Chromatography

The GC analysis was performed in a Hewlett-Packard

5890 Series II chromatograph, equipped with a FID

detector, an automatic injector (HP 7673), an electronic

integrator (HP 3396A) and a capillary HP-5 column

(cross-linked 5% phenyl in 95% methyl silicone; 30 mg

× 0.32 mm i.d., film thickness 0.25 µm). Helium was used

as carrier gas. The oven temperature was programmed

from 50 °C (isothermal for 2 min), rising at 2 °C/min to

290 °C (isothermal for 10 min). FID detector and injector

temperatures were 350 °C and 100 °C, respectively.

Introduction

Pilocarpus pennatifolius Lemmaire (Rutaceae)1 is one of

the various Pilocarpus spp. called ‘jaborandi’ by native

populations from South America (especially Brazilians).

This plant was used by those people with the aim to in-

duce salivation and sweating against snake poisoning and

fevers.2 Nowadays, the great importance of Pilocarpus

spp. (specially P. microphyllus) is due to the extraction

of the alkaloid pilocarpine from the leaves. Since its

discovery, this substance is commonly used in glaucoma

treatment and, most recently, against xerostomy, the

reduction of saliva production due to chemo- or radio-

therapy.3,4 Coumarins, flavonoids and amides have been

also isolated as secondary metabolites from several

Pilocarpus spp.5–7

The volatile constituents present in the leaves of other

Pilocarpus spp. P. trachyllophus Holmes, P. spicatus

St. Hill, P. mycrophyllus Stapf, P. jaborandi Holmes and

P. pauciflorus St. Hill, were also investigated.8–12 The

crude oil of P. pauciflorus exhibited a potential activity

against Staphylococcus aureus, while that obtained from

P. spicatus leaves showed strong antibacterial activity

against Pseudomonas aeruginosa.9

In this paper, we describe the analysis of essential oil

composition of P. pennatifolius Lemmaire by capillary

GC and GC–MS techniques. The identification of each

326 A. P. SANTOS ET AL.

Copyright © 2004 John Wiley & Sons, Ltd. Flavour Fragr. J. 2004; 19: 325–326

(56.8%) and pentadecane (25.5%), the major components,

were previously not found in volatile oils from other

Pilocarpus spp. Hydrocarbons were previously isolated

from epicuticular waxes from several Pilocarpus spp.

and followed a C14–C33 pattern,16,17 but only 1-phenyl-5-

vinyl-5,9-demethyl-decane was considered a taxonomic

marker. The analogous aliphatic ketones (2-undecanone

and 2-tridecanone), described as major constituents for

others species8–12 (all collected in north-eastern Brazil),

were not detected in this particular species. The GC

analysis indicated that 15% of the oil was consisted of

sesquiterpenoids (12.3% of sesquiterpene hydrocarbons

and 2.6% oxygenated sesquiterpenes). Two isomeric

oxygenated monoterpenes, cis-β-ocimene and trans-β-

ocimene (both 0.2%), were also found. Eugenol, a pro-

penylphenol, was present in P. pennatifolius oil as 0.3%

of the total composition and, to our knowledge, this is

the first report of this compound as a constituent of

Pilocarpus volatile oil.

Acknowledgements—This work was financially supported by Fundaçãode Amparo à Pesquisa do Estado de São Paulo (FAPESP) and ConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq).

References

1. Skorupa LA. Revisão taxonômica de Pilocarpus Vahl (Rutaceae).PhD Thesis, São Paulo University, São Paulo, 1996; 426.

2. Holmsted B, Wassen SH. J. Ethnopharmacol. 1979; 1: 3–21.3. Miller LJ. Cancer Bull. (Houston) 1993; 45(6): 549–550.4. Valdez IH, Wolff A, Atkingson JC, Macynsky AA, Fox PC.

Cancer (Philadelphia) 1993; 71(5): 1848–1851.5. Amaro Luis JM, Massanet GM, Pando E, Rodriguez Luiz F.

Planta Med. 1990; 56: 304–306.6. Andrade Neto M, Mendes PH, Silveira ER. Phytochemistry 1996;

42(3): 885–887.7. Bertrand C, Fabre N, Moulis C. Fitoterapia 2001; 72: 844–

847.8. Andrade Neto M, Cunha UA, Mafezoli J, Silveira ER. J. Essent.

Oil Res. 2002; 14: 319–324.9. Craveiro AA, Andrade CHS, Matos FJA, Alencar JW. J. Nat.

Prod. 1979; 42(6): 669–671.10. Andrade Neto M, Mafezoli J, Cunha UA, da Silveira SC, Silveira

ER. J. Essent. Oil Res. 1995; 7: 671–674.11. Andrade Neto M, Cunha UA, Mafezoli J, Silveira ER. J. Essent.

Oil Res. 2000; 12: 769–774.12. Santos FA, Cunha GMA, Viane GSB et al. Phytother. Res. 1997;

11: 67–69.13. Adams RP. In Identification of Essential Oil by Ion Trap Mass

Spectrometry. Academic Press: San Diego, CA, 1989.14. Jennings W, Shibamoto T. In Qualitative Analysis of Flavor and

Fragrance Volatiles by Glass Capillary Gas Chromatography.Academic Press: New York, 1980.

15. Farmacopéia Brasileira, Parte I. Atheneu: São Paulo, 1988.16. Negri G, Salatino MLF, Salatino A, Skorupa LA. Phytochemistry,

1998; 49(1): 127–129.17. Skorupa LA, Salatino MLF, Salatino A. Biochem. Syst. and Ecol.

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Gas Chromatography–Mass Spectrometry

The GC–MS analysis was carried out in a Shimadzu

QP5000 gas chromatograph connected to a quadrupole

detector operating in electronic impact mode at 70 eV,

with helium as the carrier gas at a rate of 1 ml/min. Two

capillary columns (DB-5 and CARBOWAX 20M, 30 m

× 0.25 mm i.d., film thickness 0.25 µm) were used and

the oven temperature was programmed from 60 °C to

300 °C at 5 °C/min for the DB-5 column and 60 °C to

250 °C at 3 °C/min for the Carbowax 20 M column.

Injector and detector temperatures were 180 °C and

260 °C, respectively. The Kováts retention index were

calculated by co-injection with a n-alkanes series and

concentrations were determined according to GC peak

areas.

Results and Discussion

The composition of P. pennatifolius Lemmaire oil

was determined by means of capillary GC and GC–MS

analysis. The combination of techniques allowed the

identification of 99.3% of the total composition of the

crude volatile oil (Table 1). The hydrocarbons tridecane

Table 1. Composition of Pilocarpus pennatifoliusLemmaire leaf essential oil, Kovats index for eachcompound and relative percentage provided by GC–MS

Constituent RI DB5 RI CW 20M %

Hexanol 864 1281 0.1

cis-β-Ocimene 1031 1195 0.2

trans-β-Ocimene 1041 1209 0.2

Tridecane 1299 1561 56.8

Undecanol 1301 1661 0.6

Eugenol 1348 2078 0.3

α-Copaene 1367 1432 0.7

β-Bourbonene 1375 1460 0.2

β-Cubebene 1381 1484 0.1

β-Elemene 1384 1570 2.1

β-Caryophyllene 1409 1580 1.2

α-Humulene 1442 1598 0.1

γ-Muurolene 1473 1637 6.6

β-Selinene 1476 1678 0.2

Bicyclogermacrene 1486 1734 0.6

Pentadecane 1496 1759 25.5

δ-Cadinene 1515 1778 0.2

Germacrene B 1546 1815 0.3

Spathulenol 1569 2039 2.6

Methyl hexadecanoate 1921 2171 0.5

Nonadecanal 2107 2540 0.2

KI DB-5 and KI CW are the Kováts index in DB-5 and Carbowax 20 M

columns, respectively.