The GC–MS analysis of the diethylether and ethylacetate fraction of the peel of Solanum incanum and the study of their antibacterial activity

The diethyl ether and ethyl acetate fractions of the peel of the fruit of Solanum incanum. (S. incanum) were analyzed using gas chromatography-mass spectrometry (GC–MS). 105 compounds were identified in the diethyl ether fraction and 75 compounds were identified in ethyl acetate fraction. Among them, 5 compounds were analyzed by fragmentation pattern, discussed, and compared with NIST database. The antibacterial screening was also conducted for bothdiethyl ether and ethyl acetate fractions of the fruit peel of S.incanum using four pathogens, two Gram-positive bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) and two Gram negative bacteria Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumonia), at various concentrations (250, 500, 750 and 1000 μg/ml). The diethyl ether and ethyl acetate fractions of the peel of S. incanum exhibited activity against E. coli and K. pneumonia at 1000 μg/ml concentration.


Introduction
Natural Products are of considerable significance to the health of human beings and animals. The medicinal importance of plants lies in the secondary metabolites, which produce a definite physiological action on the human or animal body. Investigation of the phytochemical constituents and relating them to pharmacological activities furnish a plethora of information. In this line, our group has also reported the phytochemicals and their uses (Tesfaye and Ravichandran 2018;Tamrat and Ravichandran 2018;Sulochana 2016a, 2016b). Many of the endemic medicinal plants are traditionally used as spices and food plants (Sakha et al. 2018).
Solanum L. (Solanaceae) is a large and diverse genus of flowering plants. The species of Solanum are known for their medicinal importance (Sheeba 2010). S. incanum has various traditional applications in many Ethiopian communities. The species is endemic and well distributed in Ethiopia (Abebe et al. 2014). S. incanum is commonly seen around local neighborhoods, green-lands, and roadsides (Yrjonen 2004).
The fruit of S. incanum is the source of many medicinally important secondary metabolites (Asaolu 2003). Further, solanine and solasonine have already been isolated (Alghamdi 2013). The leaves of S. incanum L. were found to contain minerals such as K (Auta and Ali 2011) and Ca (Abdalla 2015).
In recent times, GC-MS analysis is progressively engaged in the analysis of medicinal plants (Asha et al 2017). The GC-MS analysis of the leaf extract (Sundar and Pillai 2015) and the fruits extract of S.incanum from India has been reported (Buvaneshwari et al. 2017). We have earlier reported the GC-MS analysis of the essential oil derived from the peel of the fruit from S. Incanum (Yetayih and Ravichandran 2020) but there is no report on the GC-MS analysis of the diethyl ether and ethyl acetate fraction of the peel of the fruit of the S.incanum. Hence, we report the 1 3 GC-MS analysis of the diethyl ether extract and the ethyl acetate fraction of the peel of the S.incanum.

Experimental
The survey, collection, and preparation of plant material The Fruits of S incanum (5 kg) were collected on May 22, 2018, identified by Dr. Tena Regasa(botanist), Department of Biology. The collected ripe fruits were cleaned and washed with water to remove all the impurities, and ripe fruits were cut with the sterilized blade and the internal content was removed. The peel of the fruit was cut into small pieces and dried till constant weight is achieved. The dried peel was powdered using a laboratory mill and sieved and stored in air-tight containers till it is used.

Preparation of the extract
A sample of 1.5 kg S. incanum peel powder was soaked in 7-L of 70% methanol solution (Hassim et al 2014;Khalil et al 2017). The solution was kept for 7 days with random shaking and the extract was filtered by cotton plugged filtration followed by Whatman No 1 filter paper. The same procedure was repeated twice and the extracts were combined and evaporated to dryness using Rota-evaporator at 60 °C. The obtained methanol extract was used for further portioned into different fractions based on polarity.

Liquid _ liquid extraction
The methanol extract was partitioned into various solvents based on their polarity. The methanol extract was transferred to a separatory funnel and successively partitioned with various solvents including hexane, diethyl ether, and ethyl acetate. The extracts in each solvent were done successively till the added solvent is colorless. The different fractions obtained were dried over anhydrous sodium sulfate (Khalil et al. 2017). Each of the fractions was combined, and concentrated by a rota-evaporator. Among them, diethyl ether and ethyl acetate fractions were considered for further work.

Gas Chromatography-Mass Spectrometry (GC-MS) analyses
Gas chromatography-mass spectrometry (GC-MS) is an important analytical technique to analyze the chemical composition of the plant extract. The GC-MS analysis was done using an Agilent 6890 GC with Agilent 5973 mass selective detector [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 45-400 amu, and scan rate = 3.99 scans/sec], and an Agilent ChemStation data system by the method reported in the literature (Yetayih and Ravichandran 2020;Essien et al. 2016). The GC column was an HP-5 ms fused silica capillary with a (5% phenyl) polymethyl siloxane stationary phase, the film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 ml/min. The inlet temperature was 200 °C and the interface temperature was 280 °C. The GC oven temperature program was used as follows: 40 °C initial temperature, hold for 10 min; increased at 3 °C/min to 200 °C; increased 2 °C/min to 220 °C. A 1% w/v solution of the sample was prepared and 1 μL was injected using a splitless injection technique. The compounds were identified based on their retention time (RT), and by the comparison of their fragmentation patterns with those of the reported values and NIST Library (G1036A, revision D.01.00)/Chem Station data system (G1701CA, version C.00.01.08)] (Yetayih and Ravichandran 2020;Okhale et al. 2018).

Antibacterial activity
Four strains of pathogenic bacteria were used in this study including Gram-positive, S. aureus (ATCC 31,488), and S. pyogenes (ATCC 27,853), and Gram-negative bacteria, E. coli (ATCC 25,922), and K. pneumonia (ATCC 27,853). The disk diffusion method was employed for the evaluation of antibacterial activity (Dar et al. 2017). The bacterial strains were inoculated on Mueller Hinton broth, homogenized on a sterilized Petri dish to yield a uniform depth. The different fractions of the plant extracts were dissolved in 10% of dimethyl sulfoxide (DMSO). 250, 500, 750, and 1000 μg/ml (extract soaked by each disc) concentration of the fractions were tested for antibacterial activity respectively. The zone of inhibition was measured. The experiments were done in triplicate. DMSO (10%) and Gentamicin (10 μg/disc) were taken as negative and positive control respectively. Statistical analysis was performed using MS Excel. The graphs were also plotted with MS Excel. The structures are drawn with ChemDraw Ultra 8.0.

The diethyl ether fraction
The GC-MS analysis of diethyl ether fraction was confirmed with the presence of 105 components (Table 1). The components were identified by representing 100% of the total sample composition.
The principal compounds identified in the diethyl ether fraction were 8-nonenoic acid (19.59%),

Benzyl alcohol(MW108)
The empirical formula C 7 H 8 O was derived using the nitrogen rule and rule of thirteen. The absence of the M + 2 peak indicated the absence of halogen, silicon, and sulfur. The base peak at 79 indicated the presence of phenyl groups. The other fragments benzyl cation(91), phenyl cation (77), cyclopentadienyl cation (65), and cyclobutadienyl cation (51) confirmed the presence of Benzyl alcohol ( Fig. 1 and Scheme 1).

Antibacterial assay
The diethyl ether and ethyl acetate fractions of the peel of S. incanum exhibited antibacterial activity against E. coli, K. pneumonia, S. pyogenes, and S. aureus. The results were shown in Table 4. The results indicated that the antibacterial activity was specific in action against the growth of bacterial species.
The antibacterial activity of diethyl ether fraction and ethyl acetate fraction was analyzed by in vitro disk diffusion method using paper discs (Indhumathi and Mohandass 2014;Elisha et al. 2017). Figures 6 7 and 8 summarize the results. The diethyl ether fraction exhibited higher activity against E. coli at 1000 μg/ml whereas the ethyl acetate fraction showed higher activity against K. pneumonia at 1000 μg/ml. Further, the extracts were found to show greater activity against E. coli, K. pneumonia.