2231-3354 GC-MS analysis of phytocomponents in the ethanolic extract of Nelumbo nucifera seeds from Russia

Article history: Received on: 16/01/2015 Revised on: 12/02/2015 Accepted on: 04/03/2015 Available online: 27/04/2015 The present study was designed to determine the phytocomponents of the ethanol extract of N. nucifera seed using GC-MS. The phytochemical screening of the ethanolic extract of N. nucifera seed was performed using Perkin-Elmer Gas Chromatography Mass Spectroscopy. GC-MS chromatogram of the ethanolic extract of N. nucifera seed showed thirty-eight peaks which indicating the presence of thirty-eight phytochemical constituents. The major phytochemical constituents are lupeol (22.95%), octadecatrienoic acid-2-[(trimethylsilyl)oxy]-1[[(trimethylsilyl)oxy] methyl]ethyl ester (Z,Z,Z) (7.86%), 2,3-dihydroxypropyl--cis-13-docosenoate (5.35%), oleanolic acid(5.19%), tristrimethylsilyl ether derivative of 1,25-dihydroxyvitamin D2 (3.36%) ,trimethylsilyl derivative of 2-monoolein (3.26%), lucenin 2 (3.02%), lupanol (2.95%), tetraneurin-a-diol (2.78%), carotene1,1',2,2'-tetrahydro-1,1'-dimethoxy (2.32%), 1-oxo-forskolin (1.99%), oleic acid-3-hydroxypropyl ester (1.78%), oleic acid trimethylsilyl ester (1.69%), betulin (1.61%), à-amyrin-trimethylsilyl ether (1.49%), stigmast-5-en-3ol-(3á,24S)(1.43%), flavone-4'-OH,5-OH,7-di-O-glucoside (1.11%). Among thirty-eight compounds identified, only twelve were reported to have biological activities. Our results indicate that N. nucifera seed contain various bioactive components.


INTRODUCTION
Nelumbo nucifera (N.nucifera), a widely used plant in folk medicine, belongs to the family Nymphaeaceae.It is heavily cultivated on the banks of Volga river delta in the south of Russia especially in Astrakhan region.All parts of this plant are eaten such as leaves, seeds, flowers and rhizomes and used as traditional Chinese herbs (Sridhar and Bhat, 2007).N. nucifera has many pharmacological activities including antidiabetic, antipyretic, anti-inflammatory, anticancerous, antimicrobial, antiviral and anti-obesity properties (Kashiwada et al., 2005).Several bioactive compounds have been derived from N. nucifera parts belonging to different chemical groups, including alkaloids, flavonoids, glycosides, triterpenoid, vitamins etc., which all have their own therapeutic impact (Mukherjee et al., 2009).There are variations in antioxidant activity and concentration of phytocomponents in the ethanol extracts of lotus seeds and based on the growing region and dryness (Zhao et al., 2014).Linoleic acid, palmitic acid, and oleic acid are the main fatty acid components in N. nucifera seeds and most of them are in ester form, while phytosterols existed mainly in the free form rather than in steryl-fatty acid ester form (Zhao et al., 2013).
The ethanol extract of N. nucifera seed reduces adipose tissue weights, ameliorates blood lipid profile, and modulates serum leptin level in rats fed a high-fat diet (You et al., 2014).Embryo of N. nucifera seed has a neuropreventive and memory improving effects (Kim et al., 2014).A petroleum ether extract of seed has been reported to possess anti-fertility activity in female albino mice at the dose of 3 mg/kg (Mazumder et al., 1992).Methanol extract of N. nucifera seed contain neferine which has various therapeutic effects such as induction of sedation, hypothermia, antifever effects, and anxiolytic effects comparable with those of diazepam but with a different mechanism (Sugimoto et al., 2008).
Sivasankari et al. evaluated the hypoglycemic activity of inorganic constituents in N. nucifera seeds on streptozotocininduced diabetes in rats (Sivasankari et al., 2010).
Several alkaloids have been isolated from the seed embryo of N. nucifera as liensinine and isoliensinine (Guo and Chen, 1984).Isoliensinine isolated from the seeds reduced bleomycin-induced pulmonary fibrosis in mice (Xiao et al., 2005).
Hydroalcoholic extract of N. nucifera seed changed total and differential white blood cell counts, improved phagocytosis, and potentiated immune inflammatory reactions (Mukherjee et al., 2010).However, there have been not much reports on the phytochemical screening of the ethanolic extract of N. nucifera seeds.

Collection and processing of plant material
Seeds of Nelumbo nucifera were collected from lotus flowers which cultivated on the banks of Volga river in the region of Astrakhan in the south of Russia.Seeds were washed under tap water to remove dust particles, then they were dried and seed coat was removed and discarded.Finally, seeds without coats were pulverized to powder by using of a mechanical grinder.

Preparation of the extract
20 g of the powder of seeds were soaked in 95% ethanol for 12 h.The extracts were then filtered through Whatman filter paper No. 41 along with 2 gm sodium sulfate to remove the sediments and traces of water in the filtrate.The filtrate was then concentrated by rotator evaporator. 2 μl of filtrate was used for GC-MS analysis.

GC-MS analysis
GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i auto sampler and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column TG 5MS composed of 5% phenyl methyl polysiloxane, operating in electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 1 µl was employed (split ratio of 10:1) injector temperature 200°C; ionsource temperature 200 °C.The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10 °C/min, to 200 °C, then 5 °C/min to 280 °C, ending with a 9 min isothermal at 280 °C.Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 650 Da.MS transfer line temperature 280 °C.

Identification of phytocomponents
Identification of compounds was conducted using the database of Wiley9 library combined with the National Institute of Standards and Technology (NIST) library.The name, molecular weight, molecular formula, and area under peak of the components of the test materials were ascertained.

RESULTS AND DISCUSSION
GC-MS chromatogram analysis of the ethanolic extract of N. nucifera seed [Figure 1] showed thirty-eight peaks which indicating the presence of thirty-eight phytochemical constituents.On comparison of the mass spectra of the constituents with Wiley9 and NIST library, the thirty-eight phytocompounds were characterized and identified and their retention time (RT), molecular weight (MW), molecular formula and concentration (peak area %) are presented in table 1.
Among the compounds, twelve compounds were reported to have biological activities.The various phytochemicals which contribute to the medicinal activities of N. nucifera seed were shown in table 2.
The chemical constituents identified in the ethanolic extract of N. nucifera seed belongs to different chemical groups.The most prevailing component in the extract, lupeol (22.95%), is a triterpene was reported to possess several pharmacological activities including anticancer, antiprotozoal, anti-inflammatory, antimicrobial and chemopreventive properties (Gallo and Sarachine, 2009).

Table 2 :
Bioactivity of phytocomponents identified in the ethanolic extract of N. nucifera seed by GC-MS.