GC-MS Assisted Phytoactive Chemical Compounds Identification and Profiling with Mineral Constituents from Biologically Active Extracts of Aerva javanica (Burm. f) Juss. ex Schult

Aerva javanica (Burm. f) Juss. ex Schult. (Family: Amaranthaceae) family is one of the traditional medicinal plant growing in the United Arab Emirates. Apart from studies related to some medicinal properties, phytochemical, GC MS compound characterization and biological activities still to be investigated. An experiment was conducted to determine the possible bioactive components with their chemical structures and elucidation of phytochemicals from the aerial parts of the plant. The macro and micro-mineral constituents and antioxidant activities were also evaluated. Aerial parts of A. javanica were extracted sequentially with hexane, chloroform, ethyl acetate, acetone, methanol by cold percolation method. Free radical scavenging and antioxidant properties of methanolic extract were evaluated by using in vitro antioxidant assays such as hydroxyl radical scavenging activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radical scavenging activity and ABTS radical scavenging activity. Primary phytochemical and micro-macro element was tested using standard protocol. The chemical characterization was done with the help of Gas Chromatography-Mass Spectrometry (GC–MS), and the mass spectra of the total compounds in the extract were matched with the National Institute of Standard and Technology (NIST) library. Mineral constituents were identified and estimated by ICP-OES. Ninety-nine metabolites were obtained by GC-MS anslysis; indole was found to be major components followed by 2-Chlorallyl diethyldithiocarbamate (CDEC), Carbaril, Bis(2ethylhexyl) phthalate, Quinoline, 4H-Cyclopenta[def]phenanthrene,2-[Bis(2-chloroethylamino)]-tetrahydro-2H-1,3,2oxazaphosphorine-2-oxide, Phenobarbital, 1H-Indole, 2-methyl-, 2,3,7,8-Tetrachlorodibenzo-p-dioxin Disulfide, diphenyl. The presence of various bioactive compounds in the extract validates the traditional medicinal uses of this plant.


Introduction
Day by day new drugs are being developed from different plant species.Highly significant anticancer drugs and different antimicrobial agents are the need of the day.The potential of herbal agents should be analyzed for their candidature in treatment of various ailments (Malongane et al., 2017).With the all the benefits of modern synthetic medicine, still people prefer plant based natural medicines due to the side effects of synthetic medicines (de Oliveira et al., 2017).Plants deliver dynamic molecule, develop at different degrees in many nations of the world, and are the premise of nearby traditional herbal medicine and drug in their particular societies (Teschke and Zhang, 2015).The synthetic additives and antioxidants are widely used in the current industries and health systems.In modern times, there are many food antioxidant substances and are Antioxidant activity of extracts ABTS • + radical (Wolfenden and Willson 1982) DPPH • assay (Brand-Williams et al., 1995), Superoxide anion scavenging activity (Nishimiki et al., 1972) and hydroxyl radical scavenging activity (Halliwell, 1982) were determined from the extracts.

Phytochemical screening test for the extracts
The dried extracts were subjected to the preliminary phytochemical screening by using Harbourne (Harborne, 1998) methods to test for alkaloids, flavonoids, tannins, terpenoids, steroids, saponins, protein, cardiac glycosides and anthraquinones.

Microelements and macroelements
Samples were prepared as explained previously (Ameri et al., 2014).The percentages of each element in this sample was determined by plotting the absorbance of each element versus the element concentration by using standard AR grade solutions of the elements, for example K, Mg, Ca, Na, Fe, Mn, Zn, P and S. The elemental analysis was done for aerial parts of the plant species (ICP-OES -Agilent Technologies, 710).

Sample preparation for GCMS
Different solvents (methanol, acetone, ethyl acetate, chloroform and Hexane) separately received and stored at -20 °C for further use.For GCMS, each extract was prepared separately by dissolving 1 mg of each extract dissolved in 1ml of fresh HPLC grade solvent and, vortexes for 2 min and the extract was filtered by 0.2 µl filter.The 25 ng/µl concentration each extract made with HPLC grade solvents was added 1 ml of extract in respective GC tube for analysis.Methanol, acetone and chloroform samples were completely dried using Nitrogen gas.Sample was derivatised using 30 µl pyridine and 60µl of BSTFA: TMCS (99:1) and incubated at 60 °C for 60 min.Derivatised samples were subjected 1 µLin to GC-MS (Lisec et al., 2006).

GC-MS analysis
GC-MS measurements were performed with a Shimadzu instrument equipped with GC: Aligent 7890 A, MS: MS detector 5975C, Ionization for MS: Electron Impact Ionization, Mass Analyzer: Single Quadrupole, Software: Data Analysis, AMDS, Library: NIST 2011, column: DP 5 ms, Dimensions: 30 m L X 0.25 mm ID × 0.25 µm film thickness, initial temperature is 240 °C 2 min hold time, ram temperature is 50 °C to 280 °C 5 min is the hold time, total time is 40 min, carrier gas is helium, flow (13 ml/min) is 1.0, split flow: 1.5 ml/min, injection volume: 1µl, Scan mass range: 30 m/z-600 m/z and polarity + ve.GC-MS performed based on the database having more than many patterns.The spectrum of the unknown compound was compared with the spectrum of the known compounds in library.

Data analysis
Raw GC-MS data were analysed as explained by Jin et al. (2016) and following the method of Jumtee et al. (2009).harmless if used in minor quantities but in large quantities they have risks to human health like skin rashes and itching, and many other ailments (Lidon and Silva, 2016).
In the search for new compounds, and also for quality control, the suitable methods of screening are very important (Keskes et al., 2017).The selection of accurate medicinal plants, the extract screening within vitro methods for estimation of its biological activities will lead to the discovery of potentially useful compounds (Mathekaga and Meyer, 1998).In traditional medical systems, almost all the plants used may contain potential substances which are responsible for their biological activities and can be utilized for various diseases (Duraipandiyan et al., 2006).History reveals that plants are sources of successful drugs, and will continuously be important for screening of new lead compounds (Atanasov et al., 2015).
Aerva javanica (Burm.f) Juss.ex Schult. is one among the important plants in Amaranthaceae family.This plant is widely seen many countries (Judd et al., 2008).Many phytochemical compounds have been isolated from this plant (Emam, 1999).A. javanica has been widely used for many diseases in traditional and modern medical systems (Deshmukh et al., 2008).There are various reports stating the medicinal properties of A. javanica like antioxidant, antiviral, antiplasmodial, antibacterial (Baltina et al., 2003;Al-Fatimi et al., 2007;El-Hadi et al., 2010;Singh et al., 2010;Srinivas and Reddy, 2012).There are traditional uses for this plant (Samejo et al., 2012).The leaves of plant are used for fodder to goats and whole plant is used as a fuel.The whole A. javanica plant is used for the purpose of chest pain, ascaris and diarrhea with blood (Teklehaymanot and Giday, 2010).
In this study, there was analyzed the metabolic profile of A. javanica using gas chromatography-mass spectrometry (GC-MS) with their chemical structures and elucidation of phytochemicals from the aerial parts of the plant.The macro and micro-mineral constituents and antioxidant activities were also evaluated.

Plant material
Samples of Aerva javanica (Burm.f) Juss.ex Schult.were collected from the Emirate of Abu Dhabi (UAE).The plant material collects in all collection site was authorized by the Department of Biology (College of Science, UAE University).The field studies did not involve endangered or protected species.

Preparation of plant extracts
The coarsely powdered the aerial parts of A. javanica were extracted sequentially with hexane, chloroform, ethyl acetate, acetone, methanol by cold percolation method.All the organic extract extracts were filtered using rotary evaporator.The residues were dissolved in Dimethyl Sulfoxide (DMSO) and these crude extracts were subjected to in-vitro assays.Different organic extract of samples for metabolomics analysis.

Antioxidant assays of methanolic extract
The extracts showed significant antioxidant potential when compared to standard gallic acid (Table 1).In ABTS+ scavenging activity the values are varied significantly (P < 0.05) and ranged from 10 to 160 µg GAE/g extract.The hydrogen-donating ability is revealed by this assay.The antioxidant activity was almost comparable directly to the standard gallic acid (IC50 value 7.94 µg/mL) (Table 1).Gallic acid was used as reference standard in DPPH method.In comparison with gallic acid, the methanolic extracts of A. javanica showed high scavenging activity in vitro (Table 1).
javanica was 39.1-76.4% and that of the standard gallica was 37.2-58.3%.The methanolic extract of A. javanica exhibited concentration-dependent radical scavenging activity, that is, percentage inhibition increased with sample concentration (Table 1).

Phytochemical screening of methanolic extract
Phytochemical screening for the methanolic extracts of A. javanica reveals the active phytochemical constituents of the plant understudy in which flavonoids, tannins, alkaloids, steriods, protein, terpenoid, phenols, cardiac glycosidel were present.However, the extract tested showed the absence of saponin and anthraquinones (Table 2).Both A. javanica and GAE showed significant inhibitory activity in a concentration-dependent manner to inhibit hydroxyl-radical-mediated deoxyribose damage was assessed at a concentration of 10-160 μg/mL.In the present investigation, the hydroxyl radical scavenging activity observed was in the range of 27.3-62.1% at the concentration of 160 μg/ml.While scavenging hydroxyl radical, the ability of methanol extract (62.1%) was found to be higher than reference standard gallic acid (54.0%) (Table 1).Superoxide radical reduced NBT to a blue-colored formation that was measured at 560 nm.At 10-160 μg, the superoxide scavenging activity of methanolic extract of A.

Mineral compositions
Nutritional composition of the aerial plant parts of A. javanica was presented in Table 3. Ca, Cr, Cu K, Mg, Mn, Ni, P, S and Zn were presented in appreciable quantities.Low concentrations of Cd, Co, Fe, N, and Pb were observed in the methanolic extract of A. javanica.

GC-MS Analysis of different solvents extract
From five batches of approximately 1 kg of air dried powdered leaves, mean percentage yields of 4.78% of nhexane extract, 5.12% of chloroform ethyl acetate extract, 3.52% of Ethyl acetate, 2.74 % of acetone extract and 8.47% of methanol extract were obtained.Methanol extract gave the highest percentage yield.Most of the constituents were polar in nature.
There are many unknown compounds in the polar extracts from the plant material of A. javanica.Comprehensive coverage showed similar discrimination as when using the 155 known and putative metabolites and solely the 99 identified features.
It is for the first time that the composition of the five extracts of the aerial parts of A. javanica has been investigated through GC / MS analysis.The mass spectra were compared with those of the known compounds stored in the National Institute of Standards and Technology (NIST) research library.The bioactive compounds present in n-hexane, chloroform, acetate, acetone and methanol extracts obtained from A. javanica aerial parts are shown in Tables 1-5.The prevailing compounds were 2-Chlorallyl diethyldithiocarbamate (CDEC), Carbaril, As whole Hexane extract of the aerial parts of A. javanica was found to be rich in benzene metabolites.On comparison of the mass spectra of the constituents with the NIST library, the ten phytocompounds were characterized and identified (Table 4).The results revealed that Piperidine was found as the one major component is the hexane extract and the nine minor components are Tridecane, o-Xylene, 2-Hexanone and other benzene derivatives were also present.
The chloroform extract of A. javanica pertaining to the GC-MS analysis were given in Table 5. Thirty-eight compounds were detected in the chloroform extract of the 520 aerial parts of A. javanica and Piperidine, Carbaril, Bis(2ethylhexyl) phthalate, Quinoline were found to be the major compounds followed by Dimethyl phthalate, Propanedinitrile, 5-Fluorouracil, 1,3-Dioxolane, Eicosane and various benzene and propenoic acid were also identified.
The ethyl acetate extracts showed twenty chemical compounds in the GC MS analysis (Table 6) which were detected according to their retention time on fused silica capillary column.These compounds mainly comprised of o-Xylene, Dimethyl sulfon, Undecane, Pentadecane was identified as a major chemical constituent followed by Propanoic acid, Ethylbenzene Tridecane, Dibutyl phthalate etc.
The acetone ether crude extract of the aerial parts of A. javanica were analysed by GC-MS which leads to the identification of 17 organic compounds.The chemical compounds were identified according to their retention time on a fused silica capillary column.The list of the compounds with their retention time, molecular formula, molecular weight, area and area (%) were presented in Table 8.The identified major compounds are 4H-Cyclopenta[def]phenanthrene, CDEC, 2-[Bis(2chloroethylamino)]-tetrahydro-2H-1,3,2oxazaphosphorine-2-oxide, Phenobarbital along with other minor constituents were also present.
The antioxidant properties of this plant extracts can be attributed to the presence of different phytochemicals.In this context, the presence of phytochemicals such as alkaloids, flavonoids, tannins, steroids, terpenoids, reducing sugar, saponins and glycosides are to be mentioned, as they have a prominent role in plant antioxidant responses (Robert and Lin, 1994).The presence of flavonoid itself function acts as scavenger of free radicals by rapid donation of a hydrogen atom affording a phenoxy radical intermediate that is relatively stable (Bors et al., 1990).Similar antioxidant actions and phytochemicals were reported previously from A. lanata (Yamunadevi et al., 2001;Kumar et al., 2013).Crude extracts of different parts of A. javanica made with hexane, chloroform and methanol showed the presence of phytochemicals (Srinivas and Reddy, 2012).
Different extracts were obtained from the aerial parts of A. javanica through selective sequential extraction with solvents of increasing polarity, namely, n-hexane, chloroform, ethyl acetate, acetone and methanol.GC-MS analysis of the n-hexane, ethyl acetate and methanol extracts revealed the presence of various bioactive compounds.
The major compound of Piperidine is present both in Hexane and chloroform extracts but in different quantities.In addition, Phenobarbital is present both acetone and the polar methanol extract.Piperidine as they are drug intermediates (Lin et al., 2010;de Sa et al., 2009;Meth-Cohn, 2000).Several tryptophan and tryptamine derivatives of prenylated indole compounds, and their scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) showing good activity (Estevão et al., 2010).In present study showing that methanolic extract and chloroform extract showed indole compounds and its derivative.

Conclusions
In conclusion, the methods revealed several new constituents, while one of the A. javanica extracts displayed comparative antioxidant activity to that of the bioactive molecule of Indole, 1H-Indole, 2-methyl-, 2,3,7,8-Tetrachlorodibenzo-p-dioxin Disulfide, diphenyl.Finally, statistical analysis demonstrated certain correlations among the variables selected.The studies on phytochemical constituents of A. javanica are scanty.Identification of these compounds in the plant serves as the basis in determining the possible health benefits of the plant leading to further biologic and pharmacologic studies.

Table 1 .
In vitro free radical scavenging activities of methanolic extract from Aerva javanica

Table 4 .
Biologically active chemical compounds of hexane extract of the aerial parts of A. javanica

Table 5 .
Biologically active chemical compounds of chloroform extract of the aerial parts of A. javanica

Table 6 .
Biologically active chemical compounds of ethyl acetate extract of the aerial parts of A. javanica

Table 7 .
Biologically active chemical compounds of methanol extract of the aerial parts of A. javanica

Table 8 .
Biologically active chemical compounds of acetone extract of the aerial parts of A. javanica