Essential oils of Daucus carota ssp. maximus

YEIARhlACEUTI XTAHELVETIAE ELSEVIER

Pharmaceutics

Acta Helvetiae

70 (1995) 79-84

Essential oils of Daucus carota H-E.A.

Saad *, S.H. El-Sharkawy,

ssp.

maximus

A.F. Halim

Vniuersity of Mansoura, Faculty of Pharmacy, Department of Pharmacognosy, El-Mansoura-35516, (Received

4 May 1994; revised 24 August

1994; accepted

21 October

Egypt

1994)

Abstract The fruit, leaf and stem essential oils of Daucus carota ssp. maximus have been studied by GC-MS. The fruit oil consists chiefly of phenylpropanoids and sesquiterpene hydrocarbons. It is characterized by the presence of trans-methylisoeugenol, methyleugenol, p-asarone, shyobunone and preisocalamendiol. Phenylpropanoids are completely absent from both the leaf and stem oils and the oxygenated sesquiterpenes constitute about 52 and 80%, respectively of the oils composition, with shyobunones and preisocalamendiol as major component. These monocyclic ketones as well as a-bourbonene, aristol-9-en-3-01 and aristolenol have never before been reported as components of any Daucus oil. Unlike most of the studied varieties, carotol, daucol and geranyl acetate are completely absent. These results reflect the unique composition of the essential oils of this Lebanese variety. Keywords:

Daucus carota ssp. maximus (Apiaceae);

Essential

oils; GC-MS

1. Introduction Daucus L. (Tackholm, 1972) (Apiaceae) includes about 60 species mostly distributed in Europe, Africa and West Asia and few ones in North America and Australia (Zohary, 1972; Jafri, 1985). D. carota L. ssp. muximus (Desf.) Ball CD. maximus Desf., D. mauritanicus L.) is a tall robust annual spiny-fruited herb growing wild in the Mediterranean and West Irano-Turanian Territories (Zohary, 1972; Tutin et al., 1981; Jafri, 1985). D. curotu L. (Zohary, 1972; Tutin et al., 1981) was separated, on the basis of intraspecific morphological variations, into twelve interrelated and interhybridized subspecies (Tutin et al., 1981). The domestic and popular varieties, D. carom ssp. boissieri (Muschler, 1912) and D. carotu ssp. sativus (Tutin et al., 1981) are usually cultivated for their fleshy edible roots (Bailey, 1960). Carrot (D. carotu L.) fruits and roots are well known,

* Corresponding

author.

0031-6865/95/$09.50 0 1995 Elsevier SSDI 0031.6865(94)00054-9

Science

B.V. All rights reserved

since old time, by their use as stimulant, carminative, diuretic, vermifuge and in the treatment of digestive disorders (Volak et al., 1984). The fruit essential oil was proved to be hypotensive, cardiac and CNS depressant and has a moderate protective anticonvulsant effect in strychnine and metrazol poisoning. Carrot fruit oil is widely used as a flavouring agent in food products, grape wine and nonalcoholic beverages (Bodrug, 1982) and in perfumery since it blends very well in all kinds of perfumes (Guanther, 19.50). Moreover, fruit oils of some varieties proved to have antibilharzial (Halim et al., 19881, fungicidal (Guerin and Reveillere, 1985; Dwivedi et al., 1991) and antibacterial (Syed et al., 1986) activities. The genus Daucus was intensively studied for its flavonoid (Crowden et al., 1969; Harborne, 1971; Harborne and Williams, 1972; El-Moghazi et al., 1980; Gupta and Niranjan, 1982; Saleh et al., 1983; Ram and Devi, 1983; Feeny et al., 1988; Roessingh et al., 1991; Shaaban et al., 1994) and essential oil contents (Guanther, 1950; Pigulevskii and Kovaleva, 1955a,b, 1959, 1961; Pigulevskii et al., 1960, 1962; Pigulevskii and Motskus, 1962; Harborne, 1971; Williams and Har-

80

H-E.A. Saad et al. / Pharmaceutics Acta Heluetiae 70 (1995) 79-84

borne, 1972; Hogg et al., 1974; Hilal et al., 1975; Ashraf et al., 1977, 1979; Benecke et al., 1987; Halim et al., 1988; Lawrence and Reynolds, 1988; Cu et al., 1989; Dhillon et al., 1989). It was observed as the richest genus of Apiaceae in essential oil and the fruit oil pattern was found very useful for separating and characterizing the genus within the family (Harborne, 1971; Williams and Harborne, 1972). However, it was shown that carrot fruit oil varies qualitatively and quantitatively within the different varieties (Harborne, 1972; Williams and Harborne, 1972; Ashraf et al., 1977, 1979; Halim et al., 1988) and even within the same variety (Pigulevskii and Kovaleva, 1955a, 1959, 1961; Pigulevskii et al., 1960, 1962; Pigulevskii and Motskus, 1962). It was suggested that this variation could be due to geographical factors. Nevertheless, the major oil constituents as the monoterpene ester geranyl acetate and the sesquiterpene alcohols carotol and daucol as well as the monoterpene hydrocarbons (Y- and ppinenes and limonene are rather common in the different varieties (Harborne, 1971; Williams and Harborne, 1972). To the best of our knowledge, the fruit essential oil and sometimes those of the leaf and root of seven subspecies of D. carota of different localities were investigated, but nothing could be traced in the current literature about the ssp. maximus. Accordingly, the composition of the fruit, leaf and stem essential oils of this Lebanese plant present the subject of this study.

2. Experimental 2.1. Plant material Ripe and mature fruits of Daucus carota L. ssp. maximus (Desf.) Ball were collected in August 1991 from fruiting plants growing wild in Akar region, North Lebanon, and cultivated in October 1991 in the Experimental Station of Medicinal Plants, Pharmacognosy Department, Faculty of Pharmacy, University of Mansoura. Ripe and mature fruits as well as the leaves and stems, required for the present study, were collected in early July 1992, from the cultivated flowering and fruiting plants. The plant identity was kindly verified by Dr. I. Mashaly, Department of Botany, Faculty of Science, University of Mansoura. 2.2. Oils preparation Fresh and crushed fruits (300 g, each) were separately

(100 g>, leaves and stems subjected to hydrodistilla-

tion for 8 h adopting (1984) method.

the

Egyptian

Pharmacopoeia

2.3. Determination of physical constants Specific rotation, [ LY]~, was performed on the methanolic solution of the oil (1%) and measured in 1 dM tubes at the Sodium D Line using Perkin-Elmer 141 polarimeter. Specific gravity was determined using a micro specific gravity bottle. 2.4. Gas chromatographic analyses (GC) A Varian 3300 gas chromatograph equipped with FID and a fused silica capillary column (28 m X 0.25 mm ID> coated -with bonded phase DB-1 of 0.25-m film thickness. Injection of hexane solution (1 pl2.8 kg/p11 was applied and the chromatogram was produced by holding the oven temperature at 50” for 1 min, then programmed from 50-270” at 4”/min. Helium was used as a carrier gas at a linear flow rate of 30 ml/s, measured at 150”, with splitting ratio of 1:30 and the septum sweep was held constant at 10 ml/min. Quantitation and retention time determination were carried out with a Spectra Physics SP 4290 integrator. 2.5. Calculation of Kovats retention indices (I,) The oils were separately spiked with a standard mixture of a homologous n-alkane series (C&s) and then analyzed by GC under the above mentioned conditions. Retention indices were directly obtained by application of Kovats procedure (Kovats, 1965; Jennings and Shibamoto, 1980). 2.4. Gas chromatographic-mass (GC-MS)

spectrometric

analyses

A Girdel series 32 gas chromatograph (Delsi, France) coupled with Nermag RlO-10C mass spectrometer was used and spectra were produced under the same conditions mentioned above to obtain comparable results. Mass spectral analyses were run by EI technique at 70 eV. 2.7. Components identification The oils constituents were identified by matching their mass spectral and retention indices data with those reported in the literature (Stenhagen et al., 1974; Masada, 1976; Adams, 1989).

H-E.A. Saad et al. / Pharmaceutics Table 1 Composition Peak No.

of the fruit essential

oil of D. carota

Component

IR

(DB1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

a-Pinene Sabinene Monoterpene hydrocarbon P-Pinene p-Cymene Limonene Methyleugenol P-Caryophyllene a-Bergamotene Sesquiterpene hydrocarbon cY-Humulene trans-p-Farnesene trans-Methylisoeugenol Shyobunone P-Bisabolene P-Sesquiphellandrene P-Asarone a-Bisabolene Preisocalamendiol

ssp. maximus

930 967 968 971 1010 1020 1375 1420 1434 1437 1447 1458 1466 1491 1498 1510 1520 1532 1.587

1)

Relative percentage traces traces traces traces traces traces 1.97 1.48 0.28 0.34 0.31 0.43 37.22 0.65 34.70 0.25 17.65 3.00 0.39

a a a a a a

a Less than 0.2%.

3. Results and discussion Fruits, leaves and stems of D. carota ssp. maximus afforded on hydrodistillation pale yellow oils (2.63, 0.17 and 0.1% v/w>, lighter than water and having characteristic fruity odour. [a]g: - 91.14”, - 29.32” and -34.58” (c = 1, CH,OH), specific gravity: 0.964, 0.906 and 0.922, respectively at 25°C. 3.1. The fruit oil GC of the freshly distilled oil revealed the presence of at least 19 components, 17 of which, accounting for about 98.33% of the oil composition, were identified (Table 1). The fruit essential oil consists chiefly of phenylpropanoids (56.84%) and sesquiterpene hydrocarbons (40.79%), accompanied by relatively much smaller amounts of monoterpenes (1.33%) and oxygenated sesquiterpenes (1.04%). Its composition appeared to be of peculiar nature and is distinctly different from those of the other studied varieties (Pigulevskii et al., 1960; Harborne, 1971; Williams and Harborne, 1972; Ashraf et al., 1979). Phenylpropanoids are present in a surprisingly significant amount (56.84%) with trans-methylisoeugenol as the major oil constituent (37.22%). It is accompanied by a noticeable amount of P-asarone (17.65%)

Acta Helueriae 70 (I 9953 79-84

81

and a much smaller amount of methyleugenol(1.97%). The phenolic ethers trans-methylisoeugenol and pasarone were reported only from the fruit essential oil of D. carota var. boissieri but in a relatively poor yield (1.46% and 0.71%, respectively) (Halim et al., 19881, while methyleugenol, though present as a minor constituent (1.97%), was detected in carrot root oil (Harborne, 1971) but has never before been reported as a component of any Daucus fruit essential oil. The sesquiterpene hydrocarbons are distinctly dominated by p-bisabolene (34.70%) while P-caryophyllene and a-bisabolene are detected in much lower amounts (1.48% and 3.00%, respectively). The oxygenated sesquiterpene fraction is only represented by shyobunone (0.65%) and preisocalamendiol (0.39%) of elemane and germacrane types, respectively. These monocyclic ketones are reported here for the first time as new components of Daucus fruit oil. They were previously isolated and identified from the rhizome essential oil of Aconts calamus (Iguchi et al., 1968, 1970). The sesquiterpene alcohols carotol and daucol, main constituents of D. carota, are totally absent. The monoterpene hydrocarbons are present in small amounts and constitute about 1.33% of the oil composition. The oil is also characterized by the absence of oxygenated monoterpenes and in particular the monoterpene ester geranyl acetate, a main constituent of several varieties of D. carota fruit oils (Pigulevskii and Kovaleva, 1955a,b, 1959; Pigulevskii et al., 1960; Harborne, 1971; Williams and Harborne, 1972; Ashraf et al., 1979). Nevertheless, geranyl acetate and other oxygenated monoterpenes were not detected in the fruit essential oils of D. carota var. boissieri cultivated in Egypt (Halim et al., 1988) and the wild red, black and yellow varieties of D. carota growing in Pakistan (Ashraf et al., 1977). 3.2. The leaf and stem oils GC of both oils revealed the presence of at least 40 (leaves) and 21 (stems) components, 30 and 17 of which, constituting about 84% and 89% respectively of the oils composition, were identified (Table 2). The leaf essential oil consists chiefly of oxygenated sesquiterpenes and sesquiterpene hydrocarbons accounting for about 51.20% and 25.25% of the oil composition as well as relatively smaller amounts of monoterpene hydrocarbons (6.31%), n-alkanes (4.55%), monoterpene alcohols (4.19%) and a straight chain aldehyde (3.22%). The stem oil is qualitatively and quantitatively different in its composition. It is strongly

H-E.A. Saad et al. / Pharmaceutics Acta Helvetiae 70 (1995) 79-84

82 Table 2 Composition maximus

of the leaf and

Peak No.

Component

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

cY-Pinene 1,2,3-Trimethyl benzene Sabinene P-Phinene Myrcene Limonene cis-Ocimene Vans-Ocimene Linalool Undecane trans-2-Nonenal 4-Terpineol u-Terpineol Dodecane Unknown Tridecane u-Copaene P-Bourbonene Sesquiterpene hydrocarbon P-Caryophyllene y-Decalactone cY-Bergamotene a-Humulene trans-P-Farnesene P-Cubebene Shyobunone isomer (Z, E)-a-Fernesene Shyobunone Sesquiterpene hydrocarbon Sesquiterpene hydrocarbon Caryophyllene epoxide Unknown Preisocalamendiol Oxygenated sesquiterpene Oxygenated sesquiterpene Oxygenated sesquiterpene Unknown Unknown Unknown Aristol-9-en-3-01 Aristolenol isomer Heptadecane

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

stem

essential

oils of D. carota ssp.

IR

Relative

(DB - 1)

Leaf

930 955 967 971 983 1020 1026 1038 1085 1100 1136 1160 1171 1200 1209 1300 1376 1383 1389

traces a 0.56 0.85 0.53 1.84 2.30 0.31 0.48 3.34 0.22 3.22 0.27 0.58 0.48 0.51 3.41 0.80 0.99 0.56

4.37 1.79 1.95 0.60 1.06 2.09 _ _ _ _ _ _ _

1420 1428 1434 1447 1458 1474 1475 1483 1491 1493

2.15 1.74 0.51 0.70 2.49 12.72 3.77 0.56 16.84 traces

0.63 _ _

1496

3.77

_

1537

2.98

3.85

1539 1587 1597

2.83 17.95 0.90

32.69

1616

3.51

6.86

1619

2.69

2.64

1626 1627 1639 1651 1660 1700

0.70 0.70 _

_

0.82 _

percentage Stem

_ _ 0.73 _ 0.38

0.32 1.30 3.55 7.62 _ 24.33 _

_

1.19 1.09 0.97

0.44

a Less than 0.2%.

dominated by oxygenated sesquiterpenes (80.05%) accompanied by relatively small amounts of monoterpenes (10.07%) and sesquiterpene hydrocarbons

(6.91%). Nevertheless, both oils are characterized by the complete absence of phenylpropanoids, the major components of the fruit oil, and the dominance of oxygenated sesquiterpenes (leaves, 51.20%, stems, 80.05%). The oxygenated sesquiterpene fraction, in both oils, is unexpectedly dominated by the monocyclic ketones, shyobunones and preisocalamendiol (leaves, 20.61 and 17.95%; stems, 31.95 and 32.69%, respectively). These ketones were detected in the fruit oil but in relatively trace amounts (0.65% and 0.39%). The sesquiterpene alcohols, carotol and daucol, like the fruit oil, are totally absent. However, the tricyclic sesquiterpene alcohols, aristol-9-en-3-01 (leaves, 0.82%; stems, 1.09%) and aristolenol isomer (stem, 0.97%), though present in minor quantities, have never before been reported as components in any Daucus essential oil. P-Cubebene constitutes about 50% of the total sesquiterpene hydrocarbon fraction of both oils. It is accompanied by noticeable amounts of trans-p-farnesene (leaves, 2.49%; stem, 1.30%), P-caryophyllene (2.15%, 0.63%) and unidentified sesquiterpene hydrocarbon (leaves, 3.77%; t,, 26.506 min; I,, 1496; m/z, 204, 134, 119, 107, 93, 77, 55, 41). p-Bisabolene, the major sesquiterpene hydrocarbon of fruit oil (34.70%), is totally absent. The tricyclic sesquiterpene hydrocarbon a-bourbonene, detected in a minor amount in the leaf oil (0.99%), has never before been reported as a component in any Daucus essential oil. The monoterpene hydrocarbons of both oils varied qualitatively and quantitatively in their composition. cY-Pinene represents the major component of the stem oil (4.37%). It is associated with significant amounts of limonene (2.09%), sabinene (1.95%), myrcene (1.06%) and small amount of P-pinene (0.60%). On the other hand, limonene is the major component of the monoterpene hydrocarbon of the leaf oil (2.30%) and accompanied by myrcene (1.84%) and small amounts of sabinene (0.85%), /3-pinene (0.53%) and traces of a-pinene. cis- and trans-Ocimenes are only detected in the leaf oil. The oxygenated monoterpenes are totally absent from the stem oil but they are represented by the monohydric alcohols linalool, as a major alcohol (3.34%), as well as (Y- and 4-terpineol (0.58% and 0.27%) in the leaf oil. The monoterpene ester geranyl acetate could not be traced in both oils. The aldehyde, trans-Znonenal (3.22%) and n-alkanes (4.55%) are only confined to the leaf oil. truns2-Nonenal is reported here as a new component of Daucus leaf oil. However, it was previously detected in the root essential oil of D. carota and its vapour displayed a powerful insecticidal activity against the

H-E.A. Saad et al. / Pharmaceutics Acta Helvetiae 70 (1995) 79-84

carrot rust fly, Psilu rosae with an LC,, of 2.17 mg/24 h (Guerin and Ryan, 1980). The aforementioned data pointed out the peculiar composition unique to the fruit, leaf and stem essential oils of the cultivated D. carotu ssp. muximus of Lebanese origin. The occurrence of the closely related truns-methylisoeugenol, methylphenylpropanoids, eugenol and P-asarone, in the fruit oil, in such appreciable amounts is of great significance in insect-plant interactions as they are known as oviposition stimulants in carrot leaves for the carrot rust fly, Psilu rosae F. (Guerin and Stadler, 1984). The fruits of D. carota ssp. maximus are, therefore, considered as inexpensive and available rich source of essential oil and of these highly efficient attractants and oviposition stimulants phenylpropanoids for use in traps designed for practical application on carrot rust fly management (Whitehead and Bowers, 1983; Hedin, 1991). This important value together with the chemosterilant effect of P-asarone (Saxena et al., 1977) of the fruit oil and the insecticidal properties of truns-2-nonenal (Guerin and Ryan, 19801, limonene, myrcene, a-terpineol and linalool (Hedin, 1991) of the leaf oil prompted us to propose the use of D. carota ssp. maximus as a useful trap crop to reduce pest population by mass trapping of carrot rust fly leading to the protection of domestic carrot varieties against the insect damage and the reduction in the use of pesticide which increase the production costs and environmental contamination. On the other hand, the remarkable occurrence of shyobunones and preisocalamendiol as well as the complete absence of phenylpropanoids from the leaf and stem oils could be considered as characteristic chemotaxonomic markers of the studied variety, maximus. References Adams, R.P. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press, London. Ashraf, M., Aziz, J., Karim, A. and Bhatty, M.K. (1977) Studies on the essential oils of the Pakistani species of the family Umbelliferae Part 9. Daucus carota, carrot, gajor seed oil. Pak. J. Sci. Ind. Res. 20, 103-105. Ashraf, M., Zaidi, S.A., Mahmood, S. and Bhatty, M.K. (1979) The essential oils of the Pakistani species of the family Umbelliferae 31. Wild Daucus carota, carrot seed oil. Pak. J. Sci. Ind. Res. 22, 258-259. Bailey, L.H. (1960) Manual of Cultivated Plants Growing in the United States and Canada. MacMillan, New York, pp. 747-751. Benecke, R., Reichold, K., Kessel, M. and Schmidt, W. (1987) Comparative investigation on the yield and composition of carrot seed oil of various cultivars of Daucus carota L. ssp. sativus (Hoffm.) Arcang. Pharmazie 42, 256-259.

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