Evaluation of Non-polar Composition in Plumbago Zeylanica Leaves by Gas Chromatography and Mass Spectrometry

Background: Plumbago zeylanica plant belongs to Plumbaginaceae. The plant is reported for many pharmacological activities. Aim: The objective of the study was to identify fatty acids and non-polar chemical compounds in Plumbago zeylanica leaves. Materials and methods: Petroleum ether extract was prepared using soxhlet apparatus. Saponifiable and unsaponifiable matter was separated with saponification process. To identify fatty acids in saponifiable matter further esterification was performed. Gas chromatography and mass spectrometry analysis was performed of both saponifiable and unsaponifiable fractions. All the fatty acid methyl esters and non-polar chemical compounds were identified using NIST library data. Results: A total of 14 compounds were identified with comparison of NIST data. From that, 8 fatty acid methyl esters and 6 non-polar chemical compounds were identified. Here we have analyzed fatty acids and non-polar chemical compounds by the same GC-MS method. Conclusions: The present analysis showed that Plumbago Zeylanica leaves contain 8 fatty acids and 6 non-polar chemical compounds. Principal determination of the research was development of efficient method to identify non-polar compound from plant by single injection using chromatographic technique.


INTRODUCTION
Plumbago zeylanica is an important medicinal plant. It is found in tropical Africa and Asia, especially in the west zones. 1,2 The plant have antimicrobial and antiulcer activity, anti-obesity, anti-inflammatory, and hypocholesterolemic activity, hepatoprotective, wound healing activity, and cytotoxicity. 3 Plumbago zeylanica plant is mainly known for its bioactive compound -plumbagin. It is reported to have anti-carcinogenic activity, antioxidant, cardio-pro-tective, antimalarial, antifungal, anti-atherosclerotic, central nervous system stimulatory, anti-hyperglycemic, and anti-inflammatory activities. [3][4][5][6][7] The Plumbago zeylanica plant contains many chemical compounds. 8 Phytochemical screening of the plant shows presence of fatty acids, saponins, carbohydrate, steroid, tannins, flavonoids, and alkaloids. [9][10] Chemical compounds can be categorized into polar and non-polar compounds. Non-polar profile of plant leaves was still not reported. Here we are reporting for the first time non-polar compounds presence in plant leaves of Plumbago zeylanica.

MATERIALS AND METHODS
Petroleum ether, methanol, KOH, n-hexane, ethyl acetate, diethyl ether, and chloroform of Merck Ltd. were used. Plumbago zeylanica plant (leaves) was obtained from RK University garden in Rajkot and it was identified by Dr. Anjisha Maharshi, School of Science, RK University.

Preparation of extracts 11
Plumbago zeylanica plant was dried for one to two weeks at room temperature. The dried material was finely powdered. 126.50 gm of powder was extracted with petroleum ether for at 60-80°C using soxhlet apparatus and the obtained extract was 3.40 gm.

Saponification 12-14
The petroleum ether extract obtained by soxhlet extraction was then suspended in 20% alcoholic KOH in round bottom flask. This solution was then refluxed for 8 hours with constant stirring. After it was refluxed, the solution was collected in a separating funnel and 30 ml distilled water was added. 100 ml of diethyl ether was added to the separating funnel stirred, shaken and then left to stand for separation of layers. Two layers were formed, an aqueous layer containing saponifiable fraction (A) and an organic layer containing unsaponifiable fraction (B). Multiple times extracted with diethyl ether and separated saponifiable fraction (A) and unsaponifiable fraction (B).

Saponifiable fraction (A) 15,16
Saponifiable fraction (A) was acidified with 5N H 2 SO 4 and the aqueous layer was extracted 3-4 times with diethyl ether. Ethereal layer was collected and washed with distilled water, dried over anhydrous Na 2 SO 4 and evaporated to obtain fatty acid portion (100 mg). An accurately (90 mg) weighed fatty acid portion was taken with 10 ml methanol in presence of catalytic amount conc. H 2 SO 4 and refluxed on water bath for 10 hrs. Cooled the same and diluted with (40 ml) water and extracted using hexane. The extract was dried over Na2SO4 and volume was made up to 10 ml with hexane and gas chromatography, and mass spectrometry analysis was performed.

Unsaponifiable fraction (B)
The unsaponifiable fraction (B) was washed with water till it became neutral and then dried with sodium sulphate. 17 It was filtered and dried. The obtained dried unsaponifiable matter was 0.812 gm. The unsaponifiable extract was further dissolved in di-ethyl ether and gas chromatography and mass spectrometry analysis was performed.

GC-MS Protocol
The GC-MS analysis was carried out using Agilent Technologies Model 5977B MS occupied to 7820A GC equipped with HP-5 capillary column (30 m×0.32 mm i.d.; 0.25 µm film thickness). The column temperature was initially held at 60°C for 5 min, then increased to 100°C with a rate of 10°C/ min, held for 1 min further, raise the temperature to 150°C at a rate of 10°C/min, held for 10 min after that to increase temperature up to 200°C at a rate of 10°C/min, hold time 10 min, continue to 250°C with rate of 10°C/min, hold time 10 min. Raise the temperature to 300°C at a rate of 10°C/min and hold time 5 min. Helium flow rate was 1.3 ml/min. The ion source of the MS was operated at 230°C and the transfer line at 260°C. Electron impact (EI) ionization was carried out at 1195.2 EMV, and quantitative determination was based on the total ion current corrected for the detector response of each compound. For analysis, compounds were dissolved in diethyl ether (2 ml) and 1 µl aliquots by auto-injector. The mass range from 50-850 Amu was scanned at a rate of 1562 [N= 2] unit/second. Compounds were identified by direct comparison of mass and their patterns were matched with data from the NIST 14 Library having more than 2,50,000 mass patterns of reported compounds.

Saponifiable fraction (A)
Plant contains non-polar compounds as lipid and other compounds. Here we have converted lipid into their esters by esterification and then identified using GC-MS. Direct injection of lipid was not possible into GC-MS. So, it was necessary to convert lipids into their esters. The result showed that by the esterification procedure eight fatty acids were identified by GC-MS. Single injection of esters mixture in GC-MS gives accurate results for identification of fatty acids with use of NIST library. Here we have identified eight fatty acids methyl esters from leaves of Plumbago zeylanica (Fig. 1, Table 1).

Unsaponifiable fraction (B)
Unsaponifiable fraction was injected into GC-MS and six non-polar compounds were identified confirmed by GC-MS. The comparison of the mass spectrums with the NIST database gave more as confirmatory compound structure match (Fig. 2, Table 2).
Here we used petroleum ether solvent to identify particularly non-polar compounds with respect to fatty acids and other non-polar chemical entity. On the other hand, our study used the leaves of Plumbago zeylanica. Here we are reporting for the first time the non-polar compound profile of Plumbago zeylanica leaves. So, the objective of study was completely achieved with the use of chromatographic techniques i.e. TLC and GC-MS.

CONCLUSION
An efficient and rapid method was developed for efficient separation of fatty acids and nonpolar chemical entity using GC-MS. The fatty acids and non-polar chemical compounds identified in this experiment may be responsible for the biological activity of the plant. The GC-MS method can be used in pharmaceutical and natural product industry. Here we have done a non-polar study of Plumbago zeylanica plant leaves. Researchers can take initiative to study the polar compound in leaves of this plant for further studies.