ASCARIDOLE AND RELATED PEROXIDES FROM THE GENUS CHENOPODIUM

Aim: Detection of monoterpenoid ascaridole and other terpenoids in the genus Chenopodium from the East Mediterranean. Method: Distribution of ascaridole in leaves of 13 species medicinal plant belonging to the genus Chenopodium was examined with the help of the GC-MS method. Results: cis-Ascaridole was found as a major peroxy monoterpenoid (up to 46.9 %) in the oil. Three minor isomers: cis-isoascaridole (1.1-6.4 %), trans-ascaridole (1-2 %), and trans-isoascaridole (1-2 %) were also detected. The biological activities and biosynthesis of ascaridole are further discussed. Conclusions: The results on Ascaridol and the major volatiles and semi-volatiles of wild species belonging to the genus Chenopodium provide important information on biologically active monoterpenoid compounds and volatile metabolites biosynthesized in wild medicinal plants growing in the East Mediterranean.


INTRODUCTION
In the past several decades, natural peroxides have been isolated from a wide variety of plants, and marine organisms 1,2 .Extensive pharmacological screening performed on microorganisms and other species resulted in discovery of novel peroxides with antitumor, antibacterial, antimalarial, and antiviral agents 1,3 .Many natural and synthetic peroxides were used in the past as therapeutic agents 1,3 .
Species of the family Chenopodiaceae are widely distributed in the East Mediterranean area, where they are often used commercially as spices or drugs because of the presence of useful secondary metabolites.The most characteristic constituents are fl avonols, essential oils and terpenes [4][5][6][7] .The genus Chenopodium includes varieties of weedy herbs (more than 200 species) native to much of Europe, Asia, India, China and both North and South America 8 .Goosefoot is common name for the genus Chenopodium, as well as for the goosefoot family Chenopodiaceae.Various plant parts of diff erent species of Chenopodium have been traditionally used in the treatment of several disorders 9 .C. ambrosioides (also known as American wormseed oil, chenopodium oil, or Baltimore oil) is rich of monoterpenes 10 .The seed and fruit contain a large amount of essential oil which has a main active compound in it called ascaridole.Herb C. ambrosioides is a plant widely known in popular medicine as antihelminthic, vermifuge, emmenagogue and abortifacient 11,12 .It is used for the treatment of digestive, respiratory, uro-genital, vascular and nervous disorders, for metabolic disturbances such as diabetes and hypercholesterolemia, and as sedative, antipyretic and antirrheumatic 13 .Kishore et al. 14 described the fungitoxicity of chenopodium oil against dermatophytes such as Aspergillus fumigatus and Cladosporium trichoides.
Ascaridole was fi rst isolated in 1895 by a German pharmacist living in Brazil and it has been attributed with most of the vermifuge (worm-expelling) actions of the plant.In the early 1900's it was one of the major antihelmintics used to treat ascarids and hookworms in humans, cats, dogs, horses, and pigs [15][16][17][18] .Essential oil from the fresh Chenopodium ambrosioides contains the ascaridol (40-70 %), α-perpinene, p-cymeme, glycol, and isoascaridol 19 .Ascaridole (also known as ascarisin; 1,4-epidioxy-p-menth-2-ene) is a bicyclic monoterpene that has an unusual bridging peroxide functional group.Ascaridole has been documented with sedative and pain-relieving properties as well as antifungal eff ects 20 .Ascaridole was found to be a potent inhibitor in vitro development of Plasmodium falciparum 21 , Trypanosoma cruzi 22 , and Leishmania amazonensis 23 .Ascaridole also showed activity against diff erent tumor cell lines in vitro (CCRF-CEM, HL60, MDA-MB-231).The fi ndings are the fi rst hint that ascaridole may be an interesting novel candidate drug for cancer treatment 24 .A few review articles devoted to pharmaceutical application of ascaridole have been published 25,26 .
In this paper, we report the detection of monoterpenoid ascaridole and other terpenoids in the genus Chenopodium from the East Mediterranean.

Plant material
All species of the genus Chenopodium were collected from January to May during 2003-2006.Two species C.

Preparation of the essential oil
Extraction of volatiles and semi volatiles was performed.Air-dried leaves of each species (6-12 g) were mixed with 250 ml of double distilled water (DDW) and 0.03 g 4-isopropyl phenol as internal standard and subjected to steam distillation for 3 hrs at atmospheric pressure.The water distillate containing oil was distrib-uted two times with hexane (50 ml each) with the help of a separatory funnel.A 50-ml of 10 % sodium hydroxide solution was added to the hexane layer and stirred at room temperature for 5 minutes and the two phases were separated.The aqueous fraction was washed with 50 ml of hexane, and the hexane fractions were combined, dried with anhydrous sodium sulfate and were labeled as "non-phenolic" fraction.To the basic water layer, 1 N HCl was added with stirring until pH = 3.The solution was then extracted three times with hexane (50 ml each).The hexane fractions were combined and dried over anhydrous sodium sulfate and labeled as "phenolic" fraction.The oil was dried over anhydrous sodium sulphate and studied by GC-MS as described previously 27 .

GC-MS analysis
Essential oils were analyzed using Shimadzu GC-17A connected to MS-QP5050A.The GC-MS was operated   1, and explanation in the text) in the electron impact ionization mode (EI) at 70 eV.Sampling the hexane extract was carried out by taking out 1 μl sample from 2 ml vials using an AOC-20i autosampler while sampling of the headspace of the dry leaves was performed by taking 0.2 ml from 27 ml HS vials using a Shimadzu HSS-4A autosampler.The HS vials were sealed with silicon rubber septa and aluminum caps after introduction of the sample while the AOC vials were sealed with 8mm double-faced rubber septa and screw cap with 12 mm hole.The GC was equipped with a fused silica capillary column; DB-5, 30m x 0.25 mm i.d., coating thickness is 0.25 μm, Supelco (Sigma-Aldrich Inc., USA).The GC-MS operating conditions were as follows: the carrier gas fl ow rate was 1.6 ml He/min.Injector and detector temperatures were 230 o C and 250 o C respectively.Split ratio was 1:30.The column temperature was held at 60 °C for 2 minutes, then raised from 60 °C to 100 °C at 3 °C/min and from 100 to 280 °C at 30 °C/min and held there for 2 min.Solvent cut time was 4 minutes and the starting time of the chromatogram was 5 minutes.Mass range was from 30 to 350 Daltons, and scan interval was 0.5 seconds.Detector voltage was set to 1.50 kV.For HS samples, vial temperature was set to 100 o C and the sy-ringe temperature was set to 110 o C. The identifi cation of the compounds was based mainly on their retention times in comparison with those from authentic standards.The standards were injected separately in addition to adding them to the leaves extracts (spiking) to enhance the relevant peaks of interest.Identifi cation of some peaks was based on matching of their MS spectra with NIST/EPA/ NIH Mass Spectral Library (NIST 2005).
The oil composition of the genus Chenopodium varied quantitively and qualitively within and between natural populations and showed no correlation to the geographical distribution.Distribution of ascaridole in the genus Chenopodium which was collected in diff erent regions around the World is shown in Table 2.It is from these results clear that a more accurate quantitative determination of dihydroascaridole (18), ascaridole (19), and isoascaridole (20) in essential oils can be obtained by combination of GC-MS.Thus, the total content of the three compounds is available from GC-MS analysis and was found in the samples employed in the present studies using a non-polar column.
The biosynthesis of ascaridole from the conjugated symmetric diene α-terpinene (a major component of the oil from C. rubrum and C. fi cifolium) was shown to be catalyzed by a soluble iodide peroxidase isolated from homogenates of C. ambrosioides fruit and leaves 40 .The enzymatic synthesis of ascaridole was confirmed by GC-MS of the product, which was also shown to be racemic.Optimal enzymatic activity occurred at pH 4.0 in the presence of 2.5 mM H 2 O 2 and 1 mM NaI. Peroxidase activity was susceptible to proteolytic destruction only after periodate treatment, suggesting an association of the enzyme(s) with polysaccharide material.Ascaridole biosynthesis from α-terpinene was inhibited by cyanide, catalase, and reducing agents, but not by compounds that trap superoxide or quench singlet oxygen.A peroxide transfer reaction initiated by peroxidase-generated I + is proposed for the conversion of α-terpinene to ascaridole.Photolysis of ascaridole in hydrocarbon solvents at 185 nm gave isoascaridole as the major product and p-mentha-1,3-diene as the minor product, whereas at 354 nm additional 41 .iso-Ascaridole and p-MeC 6 H 4 SO 3 H in Et 2 O mixed with ice cooling gave an oil from which on standing was deposited 1,4:2,3-diepoxy-p-menthane (30) (ref. 42).Based on our and other authors' experimental data, we have proposed biosynthetic pathway of ascaridole and related products in plants (see Fig. 1).

CONCLUSIONS
In this study, we have described the separation and identifi cation of ascaridol and the major volatiles and semi-volatiles of wild species belonging to the genus Chenopodium.Comparable results of the percentages of semi-volatile phenols were obtained using conventional SD-GCMS.This report provides important information on biologically active monoterpenoid compounds and volatile metabolites biosynthesized by medicinal plant growing wild in the East Mediterranean.

31 Fig. 1 .
Fig. 1.The proposed mechanism involves the formation of ascaridole and other monoterpenoid constituents in the genus Chenopodium (number under structure see inTable 1, and explanation in the text)

Table 1 .
Ascaridole and other terpenoids in essential oils of the genus Chenopodium from the East Mediterranean