Phytochemical insvestigation of the lipoidal fraction of Passiflora caerulea L . grown in Egypt

Phytochmemical investigation of the petroleum ether extract of the leaves of Passiflora caerulea L. grown in Egypt revealed the presence of fatty acids, esters of fatty acids, sterols and hydrocarbons. GLC analysis of lipid fraction led to the identification of phytosterols (19.97%), hydrocarbons (75.39%), saturated (54.42%) and unsaturated (36.62%) fatty acids. Moreover, three esters of fatty acids were isolated by column chromatographic fractionation, and their structures were established based on spectroscopic data including EI-MS, H NMR and C NMR; they were identified as myristic acid ethyl ester (T1), ethyl linoleate (T2) and ethyl oleate (T3).


Introduction
Passiflora caerulea L. is a member of family Passifloraceae.This family contains about 580 species in 12 genera, many of which are tendril climbers and shrubs or trees that occur naturally in tropical and warm temperate regions (Mabberley, 1997).Passiflora caerulea L. (blue Passion flower), which synonyms includes: Abu sab'at alwan, Zahrat es saah is native to South America (Brazil, Argentina and Paraguay) and introduced into Britain in 17 th century, it is the most vigorous and tender species having traditional use of its fruit as a sedative and anxiolytic (Rendle, 1959;Hickey and King, 1988).
The authors previously studied the biological activities and chemical composition of the polar fractions of the leaves (El-Askary et al., 2017).From the reported data, Passiflora species are rich in the fatty acid either saturated or unsaturated; it was reported that the passion fruit (Passiflora edulis) seed oils established the presence of saturated and unsaturated fatty acids as linoleic, oleic, palmitic, stearic and myristic acids (Steven et al., 2004).
Reviewing the current literature about the non-polar fraction of Passiflora caerulea L. growing in Brazil, few data was found revealing that oleic and linoleic and palmatic were the major constituents (Nolasco et al.,1999) but nothing was traced concerning the nonpolar fraction of the plant cultivated in Egypt.
Therefore, this study was carried out dealing with the isolation and identification of the major compounds in the non-polar fraction of Passiflora caerulea L. grown in Egypt, where environmental conditions are comparatively different from those of Brazil.

Study of lipoidal content
Air-dried leaves of Passiflora caerulea L. were extracted by light petroleum till exhaustion, the solvent was then evaporated under vacuum.Two grams of the residue were used for preparation of unsaponifiable matter and the separated fatty acids were methylated prior to GLC analysis (Finar, 1973).
Unsaponifiable matter (USM) and fatty acids methyl esters (FAME) were separately subjected to GLC analyses according to the conditions previously described (El-Sakhawy et al., 2016).Peak identification was performed by comparing the retention time (Rt) of each compound with those of standard material.

Column chromatography of petroleum ether extract
Six grams of petroleum ether extract were purified with charcoal giving 4.8 g of purified fraction that was fractionated on silica gel column (50 cm L × 3 cm D).Gradient elution was performed using nhexane-CHCl3 mixtures.Fractions were collected and monitored by TLC.Fractions with similar chromatographic pattern were pooled, evaporated under reduced pressure.Three oily compounds were obtained (T1-T3) by elution with 7, 10 and 12% CHCl3 in hexane, respectively.

Results and discussion
The yield of light petroleum extract constituted 0.7% While, the results of GLC analysis of FAME (Table 2) revealed the presence of at least 14 components, representing (91.042%).The percentage of identified saturated fatty acids (54.42%) was found to be higher than that of unsaturated (36.62%).Palmitic and stearic were the major identified saturated fatty acids (42.02% and 4.72%, respectively).Linoleic (21.85%) and oleic (7.72%) acids were the major identified unsaturated fatty acids.These findings were in accordance with a previous report revealing that oleic, linoleic and palmitic were the major in the Passiflora species (Nolasco et al., 1999;Sant'Anna et al., 2001).
Column chromatographic fractionation of the non-  polar extract led to the isolation of three compounds (T1-T3).Their structures (Figure 1) were elucidated by means of spectral data, viz EIMS, 1 H and 13 C NMR. Compound T1 (19 mg, yellow oil) soluble in hexane and chloroform, its mass spectrum showed a molecular ion peak of 256 corresponding to a molecular formula C16H32O2.The 1 H-NMR showed a triplet at 0.8 (J= 7Hz) that was assigned to CH3 -14 representing vicinal coupling with an adjacent methylene group.The crowded multiplets at δH 1.11-1.20 were suggestive of a long carbon chain of methylene groups.A triplet at 2.28 (J =7 Hz) that is assigned to CH2-2 indicated the attachment of this methylene group to a carbonyl function while the quartet at δH 4.11 (J =7 Hz) that was assigned to CH2-1', was for a direct attachment to an oxygen atom.
The 1 H-NMR is typical for a saturated acid ethyl ester and identified as myristic acid ethyl ester.The 13 C-NMR revealed the presence of 16 carbon including one methyl assigned at δc 14.3 ppm, methylene at δc 22.8-34.5 and one oxygenated carbon at δc 60.2 in addition to an ester carbonyl carbon at δc 174.T1 was then identified as myristic acid ethyl ester (Richard et al., 2001).
Compound T2 (18 mg, yellow oil) soluble in hexane and chloroform, its mass spectrum showed a molecular ion peak of 308, corresponding to a molecular formula C20H36O2.and reported data (Choundhury et al., 1994;Siddiqui et al., 2004) the compound T3 was identified as ethyl oleate.

Conclusion
Three fatty acid esters were isolated from the nonpolar extract of the leaves of Passiflora caerulea L.
Their corresponding fatty acids were previously reported to be present in the Passiflora species (Nolasco et al., 1999;Steven et al., 2005).

Conflict of interest
The authors report no declaration of conflict of interest.
Passiflora caerulea L. leaves used in the study were collected during 2008-2010 from El-Orman Botanical Garden, Giza, Egypt and the identification of the plant material was kindly verified by Eng.Therese Labib, consultant of plant taxonomy at Ministry of Agriculture and the former director of El-Orman Botanical Garden, Giza, Egypt.A herbarium (No.21.09.13) is deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University.