GC-MS STUDIES AND ANTIMICROBIAL ACTIVITY OF SUDANESE CAPSICUM ANNUM FIXED OIL

Abdel Karim, M. 1,* , Nafesa, A.G. 2 , Weam M. K 3 and Khadiga A 4 . 1. Sudan University of Science and Technology, Faculty of Science, Dept. of Chemistry. 2. Bisha University , Faculty of Science. 3. Omdurman Islamic University , Faculty of Pharmacy. 4. UFA Pharmaceutical Industries, Khartoum ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The present study was designed to investigate the chemical constituents of Capsicum annum seed oil and to evaluate its potential antimicrobial activity. 27 components were detected by GC-MS analysis. Major constituents are: 9,12-octadecadienoic acid(56.57%) , , hexadecanoic acid(17.60%), 9-octadecenoic acid (5.62%) , methyl stearate(5.50%) and stigmasterol(2.37%) . Butylated hydroxytoluene, a potent antioxidant, was detected as a minor constituent(0.22%). The antibacterial activity of the oil was evaluated via cup plate agar diffusion assay against six standard human pathogens(Gram positive: Staphylococcus aureus and Bacillus subtilis; Gram negative : Escherichia coli and Pseudomonasa aeruginosa and the fungi Candida albicans and Aspergillus niger) . The oil showed different antimicrobial responses against test organisms. It was partially active against the fungus Candida albicans but significant activity against Staphylococcus aureus was observed.
Capsicum annuum is a species native to southern North America and northern South America. This species is the most common and extensively cultivated of the five domesticated capsicums. The species encompasses a wide variety of shapes and sizes of peppers, both mild and hot, ranging from bell peppers to chili peppers (McLeod et.al.,1982;Minguez et.al.1994 ;Hayman and Kam ,2008). Cultivars are descended from the wild American bird pepper still found in warmer regions of the Americas (Francis,2013 ;Zhi-Yun,2005) . In the past some woody forms of this species have been called C. frutescens, but the features that were used to distinguish those forms appear in 901 many populations of C. annuum and there is no consistently recognizable C. frutescens species . Although the species name annuum means "annual" (from the Latin annus "year"), the plant is not an annual and in the absence of winter frosts can survive several seasons and grow into a large perennial shrub(Alice , 2004). The parts used are berry fruits. Removal of seeds and veins results in a less pungent and more brightly coloured product. The pungent constituents found in Capsicum annum are the capsaicinoids, present only in the fruit of the plant in small amounts, as low as 0.001 to 0.005% in "mild" and 0.1% in "hot" cultivars (Andrews, 1984 ;McLeod et.al.,1982).
Hot peppers, used as relishes, pickled or ground into a fine powder for use as spices, derive their pungency from the compound : capsaicin (8-methyl-N-vanillyl-6-enamide), a substance characterized by acrid and burning taste, that is located in the internal partitions of the fruit. Capsaicin stimulates gastric secretions and, if used in excess, causes inflammation. Most of the capsaicin in a pepper is found in the interior ribs that divide the chambers of the fruit, and to which the seeds are attached(Alice , 2004).
Hot peppers are used in medicine as well as food in Africa and other places around the world. C. annuum is used traditionally for gout, dyspepsia accompanied by flatulence, paralysis etc. Its most valuable application appears however to be in cynanche maligna (acute diphtheria) and scarlatina maligna (malignant scarlet fever) used either as a gargle or administered internally.
Alicia et.al. (2004) characterized and quantified some constituents of C. annum at four maturity stages(Immature green,green ,immature red and red). Individual hydroxycinnamic acids ,flavonoids,vitamin C and individual; carotenoids were characterized and quantified. Twenty three flavonoids and 5 hydroxycinnamic derivatives were identified from pericarp by HPLC-diode array detection-electrospray ionization-mass spectrometry. Four new cyclic diterpene glycosides together with 12 known compounds were isolated from Capsicum annum fruits. Structures of the new isolates were deduced on the basis of their spectral data. The knowncapsidolshowed significant in vitro bacteriostatic activity comparable to that of standard drug. Some of the isolated compounds were evaluated for antioxidant potential (Simona et.al.,2006).

Materials and Methods:-
Preparation of plant extract for phytochemical screening:-(150 g) of powdered shade-dried fruits of Capsicum annum were macerated with n-hexane until exhaustion. This prepared extract(PE) was used for phytochemical screening according to the method described by Harborne( 2001 ) Extraction of oil from Capsicum annum seeds:-Powdered seeds of Capsicum annum (200g) were exhaustively extracted with n-hexane at room temperature. The solvent was removed under reduced pressure and the oil was kept in the fridge at 4 o C for further manipulation. 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.

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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 shaken vigorously for five minutes and then left overnight.(2ml) of supersaturated sodium chloride were added, then (2ml) of normal hexane were added and the tube was vigorously shaken 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:-
Capsicum annum seed 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 1, while other chromatographic conditions are depicted in Table 2.  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 10 8 -10 9 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 antimicrobial activity of the oil. (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 903 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 a sample. Separate Petri dishes were designed for standard antimicrobial chemotherapeutic agents. (ampicillin , gentamycin and clotrimazole ).
The agar discs were removed, alternate cups were filled with (0.1) ml samples 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. After incubation, the diameters of the resultant growth inhibition zones were measured in duplicates and averaged.

GC-MS analysis of Capsicum annum fixed oil:-
Constituents of Capsicum annum seed oil were identified and quantified by comparison with the MS library (NIST) .The fragmentation pattern resulting from GC-MS analysis was also studied. Comparison of the mass spectra with the database on MS library revealed about 90-95% match.

Constituents of oil:-
GC-MS analysis of the studied oil revealed the presence of 27 components( Table 4).The typical total ion chromatogram(TIC) of hexane extract is shown in Fig.1. 904 Table 4:-Constituents of Capsicum annum fixed oil.
The following compounds were detected in the chromatogram as major constituents: 9,12-Octadecadienoic acid methyl ester(56.57%) Fig. 2:-Mass spectrum of 9,12-octadecadienoic acid methyl ester The peak at m/z 294, which appeared at R.T. 17.534 in total ion chromatogram (Fig.2) Fig. 3:-Mass spectrum of hexadecanoic methyl ester. The EI mass spectrum of hexadecanoic acid methyl ester is displayed in Fig. 3. In total ion chromatogram, the peak at m/z 270 (R.T. 15.852) corresponds to M + [C 17 H 34 O 2 ] + .The peak at m/z239 corresponds to loss of a methoxyl function. Fig. 4:-Mass spectrum of 9-octadecenoic acid methyl ester The EI mass spectrum of 9-octadecanoic acid methyl ester is depicted in Fig. 4.The peak at m/z 296, which appeared at R.T. 17.558 in total ion chromatogram, corresponds to M + [C 19 H 36 O 2 ] + .The peak at m/z265 is due to loss of a methoxyl function. Antimicrobial activity:-Capsicum annum fixed oil was evaluated for antimicrobial activity against standard organisms. The average of the diameters of the growth 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 showed significant activity against Staphylococcus aureus. It was also active against Pseudomonas aeruginosa and Bacillus subtilis. Though active against the fungal strain Aspergillus niger, it was patially active against the fungus Candida albicans. However, no activity was observed against Escherichia coli.