Composition of the Leaf and Peel Oils of Citrus medica L. 'Diamante' from Crete

Journal of Essential Oil Research: JEOR, Nov/Dec 2004 by Vekiari, Stavroula A, Protopapadakis, Eftichios E, Gianovits-Argyriadou, Niki

Abstract

The essential oil isolated from the leaves and peel of Cretan origin Citrus medica, var. 'Diamante,' was analyzed by eombination GC and GC/MS. Twenty-seven and 29 components were identified in the leaf and peel oils, respectively. Limonene was the major constituent in the oil of leaf and peel, while the content of the other constituents varied. The oxygenated monoterpene geranial, neral, geranyl acetate, neryl acetate and the monoterpenic hydrocarbon myrcene followed. Significant differences among oil components from citron leaves and peel were observed. Research was carried out to determine the aroma profile of citron oil constituents as a contribution in the species taxonomy.

Key Word Index

Citrus medica, Rutaceae, citron, essential oil composition, limonene, neral, geranial.

Introduction

The cultivation of citron in Crete has been in progress for many centuries. Improved genetic material has been created by mutation and by the introduction of foreign strains into the island. The citron (Citrus niedica L.) was the first citrus fruit cultivated in the Mediterranean region and especially in Crete according to Protopapadakis (1).

There are not many reports regarding the quantitative analysis of C. medica nor of the variety studied here compared with the other major citrus oils as described by Shaw (2). In the work of Gioffré (3), information about the cultivation of citron plants in different places of Greece was given. The composition of the oils from different citron fruit cultivars growing in Italy and Corsica has been investigated by Huet (4). Fleisher and Fleisher (5) have examined the leaf and peel oils of Citrus medica, var. ethrog by GC/MS. Also, leaf and peel oils of five different citron varieties have been studied by Lota et al. (6) by GC, GC/MS and 13C-NMR.

Several research groups have worked in order to study Citrus taxonomy and the identification problem through the analysis of essential oils as described by Scora and Torrisi (7) and Pieringer et al. (8). Also a significant attempt has been made by Protopapadalds (9) to characterize some sour orange varieties and cultivars (Citrus aurantiwn) by the use of isoeuzyme mid essential oil analysis. Other relevant information to the studied subject report has been found in which the influence of varieties in the oil composition was investigated by Usai et al. (10).

In this report, a detailed quantitative analysis of citron leaf and peel oils of Cretan origin is presented. The present study represents the first approach for the characterization of the chemical composition of citron peel and leaf oils of the most widespread cultivar, Diamante, growing in Crete.

Experimental

The citron samples used in our study were of cultivar 'Diamante' derived from trees planted at orchards of the Institute of Subtropical Plants at Chania. In order to certify this cultivar, an isoenzyme electrophoresis method was developed. Citron cultivars have been previously characterized by Protopapadakis (1) by isoenzymic analysis. Electrophoresis was carried out in a refrigerated box (4°-6°C). Four isoenzymatic systems in correlation with biometric characteristics were applied: malate dehydrogenase, tetrazolium oxidase, glutamate oxoloacetate transaminase and esterases.

The cultivar studied had the following characteristics: smooth fruits, purple flowers and young shoots. Eight trees were selected for sampling from which 20 leaves were collected from each tree and from all aspects of the canopy. Care was taken to get leaves of approximately five to seven months of age for individual tree, representative of the cultivar. The leaves were collected at different time intervals over a one-year period. The fruits were collected three months after anthesis from each tree and from the four compass points, to minimize position variability.

The hydrodistillation method known as the Clevenger-type system described by Huet (11) was used in order to isolate the oils from the leaves and peel seperately. The oil components were identified by GC/MS as is described by Sandra and Bicchi (12) with an HP5890 gas Chromatograph coupled with an HP5970 mass spectrometer system. The GC conditions were as follows: column type, fused silica capillary HP- (25 m × 0.20 mm; 0.33 µm film thickness); carrier gas, helium; flow rate, 1 mL/min; injection mode, split/splitless with split ratio 1/100; MS acquisition parameters; source EI or 70 eV. The injector temperature was 200°lC and the transfer line temperature was 250°C. For the analysis, the following multistep temperature program was used: initial temperature, 60°C, then to 150°C at a rate of 3°C/min, hold for l min, then to 180°C at a rate of 5°C/min and then to a final temperature of 230°C at a rate of 10°C/min.

For quantitative analysis a Perldn Elmer Autosystem model GC with FID detector equipped with an H-P terminal and a glass-lined capillary inlet operated in the split mode with a splitting ratio 1/20 was used. Chromatographie analyses were run six times and the results were calculated on the basis of an internal standard method (IS). By the use of octyl acetate as IS of known high purity standard, the volatile compounds were quantified. The concentration of the individual compounds was expressed relative to IS. An absolute concentration (gr/mL diluted oil with octyl acetate 1/25) of the relevant compound was determined. The injector and detector temperature was maintained at 200°C and 240°C, respectively. The oven temperature was held at 50°C at the beginning for 5 min, then raised to 95°C at a rate of l°C/min and finally increased to 230°C at a rate of 4°C/min and held for 15 min. Helium was used as the carrier gas. A capillary column (30 m × 0.32 mm; 0.25 µm film thickness) was used with the liquid phase: SE-52. oil components were identified by a combination of MS data and RI (retention index) data obtained from measurements with respect to n-alkane standards.