Isolation and characterization of total volatile components from leaves of citrus limon linn.

The isolation of the essential oil of whole fresh leaves of Citrus lemon by steam distillation is described. The chemical composition of the oil was investigated by means of Gas-Liquid Chromatography (GLC), Column Chromatography (CC) and coupled Gas Chromatography-Mass Spectrometry (GC-MS). The 27 most important volatile components were identified. The volatile components were identified by comparing their retention times of GC chromatograph with those of literature. Further identification was done by GC- MS. The components of the oil, percentage of each constituent, their RI values and their Eight Peak Index were also summarized and reviewed with standard available literature.

Introduction

Citrus limon Linn. is a member of family Rutaceae. It is a small evergreen tree with shining leaves. Oil glands are present in the leaves and other aerial parts like fruits and fruit peels etc. It is distributed all over India, particularly in home gardens and small- sized orchards in Uttar Pradesh, Bombay, Madras and Mysore. Found wild in the north- west regions of India ascending to 1300 m. (1). A straggling, bushy, small tree (3-4 m high) with thorny branches. Leaves ovate; petiole margined or winged. Flowers are small, white or pinkish, sweet- scented. Fruits are oblong or ovoid, bright yellow, rind thick; pulp acid, pale yellow. The Cultivation time of flowers and fruits in April- May and May- June (2). The essential oil of the leaves contains neral, geranial and limonene as major components with riboflavin and thiamine (3). Eriocitrin (7-rioictyol- β-rutinoside), apigenin, luteolin, chrysoseriol, qurcetin, isorhamnetin, limocitrin, limocittrol, sinapic acid, p-coumaric acid, scopoletin, umbelliferone, Citrusins A, B, C and D isolated from peels. Limonin, nomilin, decaacetylnomilin, obacunone, decaacetylnomilinic acid and their 17- β-D- glucopyranosides and ichangin are isolated from seeds (4). Juice of ripe fruits is used as antidiarrhoeal, antidysentric, antiscorbutic, and astringent. Also used in the treatment of rheumatism, scurvy gout and leprosy. Its rind is used as carminative and stomachic (5). Lemon juice contains antipneumonia factor (6). Juice of Citrus is a source of citric acid. Decitrated lemon juice is used for the manufacture of vitamin C. Peel is used as an agreeable flavoring agent (7), in cosmetics as hair- rinse and as mouth-freshner. It possesses insect repellent property (8). With a view to explore the scientific basis for identification of various active volatile components present, it was considered to isolate and characterized various volatile components of leaves.

Materials and Methods

Plant Material

Fresh leaves of Citrus limon Linn. were taken from locality of Noida, Uttar Pradesh. A voucher specimen was preserved in the herbarium of the Department of Pharmacognosy and Phytochemistry, Ram Eesh Institute of Vocational and Technical Education, Greater Noida.

G.C. Analysis

Analytical GC was carried out on a Varian 3300 gas chromatograph fitted with a silicon DB-1 capillary column (30m × 0.25m). Film thickness 0.25 micrometer and carrier gas nitrogen was used. Flow rate 1.5 ml/ min. Split mode, temperature programmed 80°C-250°C at 4°C/ min. Injector temperature and detector (FID) temperatures were 250°C and 300°C respectively.

GC-MS Analysis

Analytical GC-MS was carried out on a QP- 2000 instrument at 70 eV and 250°C. GC column Ulbon HR-1 equivalent to ov-1 fused capillary 0.25 mm × 50m with film thickness 0.25 micron. The initial temperature was 100°C for 6 minutes and heated at a rate of 10°C per minute to 250°C. Carrier gas helium, flow rate 2ml/min and FID detector were used (9).

Isolation of oil

The plant material was hydro-distilled according to the method recommended in the British Pharmacopoeia, 2003. The oil was dried over anhydrous sodium sulphate and stored at 4°C in the dark (1011).

Results

Identification of volatile components

The volatile components were identified by comparing their retention times of GC chromatograph with those of literature (Figure 1). Further identification was done by GC-MS (Figure 2). The fragmentation patterns of mass spectrum were compared with those of the spectrometer database using the NBS 54 AL and Wiley L- built libraries and also with those reported in literature. Constituents were identified by comparing their retention indices with those of authentic standards available in author's laboratory. The components of the oil, percentage of each constituent and their RI values are summarized in Table 1–2 and their Eight Peak Index in Table 3.

Figure 1

GLC Spectra of volatile oil of Citrus limon Linn.

Figure 2

GC-MS Spectra of volatile oil of Citrus limon Linn.

Table 1

Percentage of various components of Citrus limon Linn.

Table 2

Chemical composition of volatile oil from the leaves of Citrus limon Linn.

Table 3

Eight Peak Index of Volatile Constituents of Leaves of Citrus limon Linn.

Discussion

Twenty seven (27) components comprising 100% of total volatile components were identified by GC-MS analysis of the oil. The oil was characterized by large amount of monoterpenes (82.10%). There were 15 monoterpenes (82.10%), including 6 hydrocarbons (48.1%), 2 alcohols (6.0%), 2 aldehydes (6.6%), 2 acids (8.3%) and 3 esters (13.1%). Among twenty seven components, there were 8 sesquiterpene (8.7%) including 3 hydrocarbons (5.8%), 3 alcohol (2.4%), 1 oxide (0.4%), 1 epoxide (0.1%). Four components present in 9.2% remain unidentified (Table 1). Highest percentage area of Limonene (13.5), Geranial (6.4), Citronellic acid (7.0), Neryl acetate (5.4) and Geranyl acetate (6.2), shows these as major constituents, whereas Humulene epoxide (0.1) was the minor constituents present in oil (Table 2). Structures of different component from there mass fragmentation was analyzed and supported its presence in Citrus fruit from various extensive standard literature citations (Table 3).

Conclusion

Many of the volatile chemicals are detected, which were naturally derived from leaves of Citrus plants. Although twenty seven components comprising 100% of total volatile components were identified by GC-MS analysis of the oil. Thus if one or more of these chemicals are on various fragrant plants products, it could be possible to carry out a testing program on the set of 15-20 chemicals reported here as appearing repeatedly in these products. It would also be possible to use the methods described here to identify chemicals of interest in other products associated by calculating relative corrected retention volumes and comparing these with values for known compounds.