Gas Chromatography aided Mass Spectral Fingerprinting of Volatile Phytoconstituents from Torilis leptophylla Roots

Abstract. Torilis leptophylla commonly known as bristle fruit hedge parsley is widely acknowledged for its potential to treat liver and gastrointestinal disorders. Till today no scientific evidence has been reported to support this claim. So the present investigation was carried out to get an inclusive picture of volatile phytoconstituents of Torilis leptophylla. Hyphenated chromatographic technique revealed the presence of 11 VOC’s from n -Hexane extract and 5 from chloroform extract. Identified compounds belonged to different classes including terpenes, esters, phenols, hydrocarbons and alcohols . Among alcohols, falcarinol was revealed to be a de-novo antifungal agent not previously reported in the genus Torilis. These analytical/structural chemical investigations highlighted a first metabolic pattern associated with the use of this plant as a folk medicine for the cure of various infectious diseases. Moreover, they ratified that the metabolic pool of phytochemicals can play a noteworthy role in drug discovery and development.


Introduction
Torilis leptophylla is commonly known as 'bristle fruit hedge parsley' due to its spine bearing fruits and wide spread along hedges. Torilis leptophylla is endemic annual herb to the Indo-Pak subcontinent and shows diversified habitats including man-made places, disturbed habitats, rocks, pastures and fields [1]. It is used since ancient times to relieve hepatic, gastric and intestinal disorders.
The plant also serves as a disinfectant, so it is highly efficacious against some pathogens [2].
Furthermore, leaves are taken as vegetable while stem and branches serve as fodder [3].
Phytochemical profile of Torilis leptophylla roots has not been documented earlier.
Understanding full medicinal prospects of Torilis leptophylla roots requires a complete/wide picture of its phytoconstituents. The present study aimed at exploration of volatile phytoconstituents through gas chromatography mass spectrometry (GC-MS) to provide authentic basis for medicinal usage of plant.

Materials and Methods
Roots extracts of Torilis leptophylla were prepared employing infusion technique by soaking 100g of roots in 300 mL n-hexane and chloroform respectively. Contents were irradiated at a frequency Clear and concentrated Torilis leptophylla n-Hexane (TLH) and Torilis leptophylla chloroform (TLC) extracts were submitted to GC-EI-MS analysis.

Results and Discussion
GC-MS analysis revealed the presence of 11 and 5 VOCs from TLH and TLC, respectively.
They belonged to different classes of compounds including terpenes, esters, phenols and hydrocarbons.
Identity of the compounds was established on the basis of literature cited [5]. The molecular formula of the compounds was deduced from their corresponding molecular masses by applying rule of thirteen.
Evidences obtained from the mass spectra for the presence of heteroatom(s) were used to modify the base formulae. The degree of unsaturation was identified by using Hydrogen deficiency index (HDI).
Total ion current chromatograms of the n-Hexane and chloroform extracts are shown in 1 and 2 respectively. Molecular ion peak was observed at m/z 204 and was quite abundant. This abundance was correlated with the structure of the compound. The abundance of molecular ion peak showed that there was only a little congestion within the molecule. Furthermore, the molecular ion was even suggesting that either nitrogen was absent or even number of nitrogen atoms were there in the compound TLH-1.
Base peak was observed at m/z 161 formed by the loss of 43 mass units corresponding to the loss of propyl radical. The peak at m/z 55 was quite abundant and absence of ion corresponding to 149 mass units provided ample information for the presence of cyclohexane ring system in the compound.
To assist already manifested evidences, explanation of diagnostic signal is given below.

Allylic cleavage Double bond
Deductions from the lost fragments towards the sanction of actual structure are presented below

Rule of ThirteenC15H24
Molecular formula of the compound was determined to be C15H24by applying rule of thirteen.
C15H24 is a multiple of C5H8 that depicted 2-methyl-1,3-butadiene also called isoprene unit in the compound. The presence of isoprene unit provided vital clue that the compound belonged to the terpenes class of secondary metabolites more precisely to the sesquiterpenes [6].

Modified formulaC15H24
As there was no indication for the presence of heteroatom, modified formula was the same as the base formula.

Hydrogen deficiency index4
HDI was calculated to be 4 which could be justified by any of the given ways.

Proposed structure
By joining all the above mentioned patches structure of the compound was proposed as αcubebene.

Fragmentation Scheme
For the justification of major fragments formed in EI-MS, fragmentation scheme of the compound in detail is given in scheme 1.

Scheme 1Fragmentation scheme of α-cubebene
The low intensity signal at m/z 189 suggested the loss of methyl radical from molecular ion at m/z 204. As methyl group is not readily lost from acyclic system so it suggested that it was present as branch at cyclic or acyclic system or at bridge head position. The stability of this peak was associated with the formation of secondary carbocation that rearranges to tertiary carbocation by a hydride transfer.
The intensity of peak at m/z 119 is a little less than that of base peak; the fragment that portrayed m/z 119 not only contained an internal alkene but also had a secondary carbocation that was in resonance which favored the increased stability of this fragment. Interpretation of the lost fragments is given below. Interpretation of diagnostic signals for the endorsement of actual structure is given below.

139
Higher homologue of allylic cleavage

Base FormulaC17H40
Base formula was determined by applying rule of thirteen to be C17H40.

Modified formulaC17H24O
Base formula was modified for the presence of one oxygen atom. The possibilities that included the ring(s) were excluded because no evidence for ring has been detected. On the basis of diagnostic signals, the first three possibilities of HDI were ruled out so left with just one possibility (2 triple bonds + 2 double bonds).

Proposed structure
On the basis of above-mentioned evidences, structure of compound TLH-8 was proposed as Falcarinol.

Scheme 2bCarbocation rearrangement in Falcarinol
Allylic cleavage led to the formation of fragment corresponding to m/z 159. Loss of 18 mass units corresponding to loss of water molecule from the fragment of m/z 159 resulted in formation of fragment at m/z 141.
Fragment at m/z 119 was formed from fragment corresponding to m/z 159 as a result of Propargylic cleavage and loss of water molecule from this fragment leading to a peak at m/z 101. On the basis of above mentioned evidences the structure of the compound was proposed as falcarinol [7].

List of identified compounds based upon GC-MS analyses
Identified compounds from GC-MS analysis of TLH and TLC extract are tabulated below.

Conclusions
Identification of these important biologically active compounds revealed that T. leptophylla is a promising source of noteworthy bioactive compounds that could be utilized in future for the development of new and safer drugs.