GC-MS ANALYSIS AND ANTIMICROBIAL ACTIVITY OF SUDANESE CYPERUS ESCULENTUS

Abdel Karim M 1,* and Fath El-Rahman A 2 . 1. Sudan University of Science and Technology, Faculty of Science, Dept. of Chemistry. 2. Omdurman Islamic University , Faculty of Education, Dept. of Chemistry. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1713 and gastrointestinal disorders (Anderson et.al. 1994). Tiger nut has also been reported to be used in the treatment of flatulence, indigestion, diarrhea, dysentery, and excessive thirst ( Chevallier,1996). They are also used in Brazil for the treatment fever (De Abreu Matos ,2008) and in loosing weight (Borges et.al. ,2008).

Materials and Methods:-
Plant material:-Tubers of Cyperus esculentus were purchased from the local market -Omdurman, Sudan. The plant was kindly authenticated by Institute of Aromatic and Medicinal Plants-Khartoum ,Sudan.
Test organisms:-Cyperus esculentus oil was screened for antibacterial and antifungal activities using the standard microorganisms shown in Table(1).

Esterification of oil:-
A Methanolic solution of sodium hydroxide was prepared by dissolving (2g) of sodium hydroxide in 100ml methanol. A stock solution of methanolic sulphuric acid was prepared by mixing (1ml)of concentrated sulphuric acid with (99ml) methanol.
The oil(2ml) was placed in a test tube and (7ml) of alcoholic sodium hydroxide were added followed by (7ml) of alcoholic sulphuric acid. The tube was stoppered and shaked vigorously for five minutes and then left overnight.(2ml) of supersaturated sodium chloride were added, then (2ml) of n-hexane were added and the tube was vigorously shaked for five minutes .The hexane layer was then separated.(5μl) of the hexane extract were mixed with 5ml diethyl ether . The solution was filtered and the filtrate(1μl) was injected in the GC-MS vial.

GC-MS analysis:-
Cyperus esculentus fixed oil was analyzed by gas chromatographymass spectrometry. A Shimadzo GC-MS-QP2010 Ultra instrument with a RTX-5MS column (30m,length ; 0.25mm diameter ; 0.25 μm, thickness)was used. Helium (purity; 99.99 %) was used as carrier gas. Oven temperature program is given in Table 2, while other chromatographic conditions are depicted in Table 3.  Antimicrobial assay:-Preparation of bacterial suspensions:-One ml aliquots of 24 hours broth culture of the test organisms were aseptically distributed onto nutrient agar slopes and incubated at 37°C for 24 hours. The bacterial growth was harvested and washed off with sterile normal saline, and finally suspended in (100 ml) of normal saline to produce a suspension containing about 108-109 colony forming units per ml. The suspension was stored in the refrigerator at 4°C until used. The average number of viable organism per ml of the stock suspension was determined by means of the surface viable counting technique.
Serial dilutions of the stock suspension were made in sterile normal saline in tubes and one drop volumes (0.02 ml) of the appropriate dilutions were transferred by adjustable volume micropipette onto the surface of dried nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the drop to dry, and then incubated at 37°C for 24 hours.

Preparation of fungal suspensions:-
Fungal cultures were maintained on dextrose agar incubated at 25°C for four days. The fungal growth was harvested and washed with sterile normal saline, and the suspension was stored in the refrigerator until used.

Testing for antibacterial activity:-
The cup-plate agar diffusion method was adopted , with some minor modifications, to assess the antibacterial activity. (2ml) of the standardized bacterial stock suspension were mixed with (200 ml) of sterile molten nutrient agar which was maintained at 45°C in a water bath. (20 ml) Aliquots of the incubated nutrient agar were distributed into sterile Petri dishes. The agar was left to settle and in each of these plates which were divided into two halves, two cups in each half (10 mm in diameter) were cut using sterile cork borer (No 4), each one of the halves was designed for one of the test solutions. Separate Petri dishes were designed for standard antibacterial chemotherapeutics (ampicillin and gentamycin).
The agar discs were removed, alternate cups were filled with( 0.1 ml) samples of each test solution using adjustable volume microtiter pipette and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37°C for 24 hours.
The above procedure was repeated for different concentrations of the test solutions and the standard chemotherapeutics. After incubation, the diameters of the resultant growth inhibition zones were measured in triplicates and averaged.

Constituents of oil:-
The GC-MS spectrum of the studied oil revealed the presence of 21 components( Table 4).The typical total ion chromatograms (TIC) is displayed in Fig.1.
1715  Oleic acid (9-octadecenoic acid) acid is very common in human diet. The hypotensive potential of olive oil is probably due to this acid (Terese et.al.,2008). This acid finds some applications in soap industry and it is used in small amounts in some pharmaceutical products. It is also used as soldening flux in stained glass work. Oleic acid is employed as emollient (Currasco,2002). It is claimed that the consumption of oleate in olive oil has been associated with decreased risk of breast cancer (Martin et.al.,1994) 9,12-Octadecadienoic acid methyl ester (13.62%):- Fig. 3 shows the EI mass spectrum of 9,12-octadecadienoic acid methyl ester. The peak at m/z294, which appeared at R.T. 17.518 in total ion chromatogram, corresponds to M + [C 19 H 34 O 2 ] + .The peak at m/z263 corresponds to loss of a methoxyl function. 9,12-Octadecadienoic acid is an essential fatty acid that can not be synthesized by humans and is available through diet (Burr et.al.,1930).It belongs to one of the two families of essential fatty acids. It occurs in lipids of cell membrane and is used in the biosynthesis of arachidonic acid . It is converted enzymatically into mono-hydroxy 1717 products which are subsequently oxidized by some enzymes to keto metabolites..Deficiency of linolate caused hair loss and poor wound healing in model animals (Cunnane and Anderson,1997;Ruthig and Mecklung-Gill,1999).

Hexadecanoic acid methyl ester(19.27%):-
Mass spectrum of hexadecanoic acid methyl ester is depicted in Fig. 4.The peak at m/z 270, which appeared at R.T. 15.868 corresponds to M + [C 17 H 34 O 2 ] + while the peak at m/z239 is attributed to loss of a methoxyl group. Hexadecanoic acid (palmitic acid) is a saturated fatty acid which is very common in plants. It is produced first during the synthesis of fatty acids (Gunstone et.al.,2007) and is considered as precursor of long-chain fatty acids. Palmitic acid is a major lipid component of human breast milk (Kingsbury et.al.,1961;Jensen et.al.,1978). The acid finds applications in soaps and cosmetics industries. It is also used in food industry .
Methyl stearate(10.88%):-Mass spectrum of methyl stearate is shown in Fig. 5  Antibacterial activity:-In cup plate agar diffusion assay ,the oil was screened for antimicrobial activity against six standard human pathogens. The average of the diameters of the growth of inhibition zones are depicted in Table ( 5) .The results were interpreted in commonly used terms (>9mm: inative;9-12mm:partially active; 13-18mm: active; < 18mm:very active) .Tables (6) and (7) represent the antimicrobial activity of standard antibacterial and antifungal chemotherapeutic agents against standard bacteria and fungi respectively.   The oil was partially active against Staphylococcus aureus , but it exhibited significant activity against the fungus: Candida albicans.