ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 3, с. 329-337
UDC 543.42,543.544:581.573.4
GAS CHROMATOGRAPHY-MASS SPECTROMETRY CHARACTERISATION OF THE ANTI-Listeria COMPONENTS OF Garcinia kola SEEDS © 2014 D. Penduka*, K. A. Basson**, B. Mayekiso*, L. Buwa* and I. A. Okoh*
*Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology,
University of Fort Hare, P. Bag X1314 Alice, 5700, South Africa **Department of Biochemistry and Microbiology, University of Zululand, P/Bag X1001, KwaDlangezwa, 3886,
KwaZulu-Natal Province, South Africa e-mail: aokoh@ufh.ac.za Received September 9, 2013
Adsorption chromatography was used to separate the bioactive constituents of the crude n-hexane extract of Garcinia kola seeds. The silica gel 60 column fractions were eluted using the solvent combination of benzene : ethanol : ammonium hydroxide (BEA) in the ratio combination of 36 : 4 : 0.4 v/v. The fractions were tested for anti-Listeria activities by determining their MIC50, MIC90 or MIC against 4 Listeria isolates. The fractions were labelled BEA1 to BEA5 and 3 out of the 5 fractions eluted were active against the test Listeria species with MIC's ranging from MIC 0.157 mg/mL to MIC50 0.625 mg/mL. The most active fractions, BEA2 and BEA3, were subjected to gas chromatography coupled to mass spectrometry (GC-MS) to identify their composition. Fraction BEA2 constituted of 18 compounds mostly sterols and the BEA3 fraction contained 27 compounds with the most abundant compounds being fatty acids derivatives. The BEA2 fraction's interactions with antibiotics proved to be 100% synergistic with ciprofloxacin and ampicillin whilst it exhibited 50% additivity and 50% synergism with penicillin G. However, all the interactions of the BEA2 fraction with each of the conventional antibiotics used were synergistic against the human listeriosis causative bacteria Listeria monocytogenes.
DOI: 10.7868/S0555109914030271
Isolation and identification of compounds responsible for antimicrobial activities in crude plants extracts is an important factor and it requires the use of different multi-step chromatographic techniques. Chromatography is a physical method of separating compounds in a mixture due to their different affinity to stationary and mobile phases [1]. Chromatography is a versatile technique that can separate gases and volatile substances by gas chromatography (GC), nonvolatile compounds and compounds of extremely high molecular weight (including biopolymers) by liquid chromatography (LC) [1]. In GC the mobile phase is a gas whilst in LC the mobile phase is a liquid.
There are many different variations of LC which are mainly dependent on the stationary phase chemistry. The simplest forms of LC are paper chromatogra-phy and thin-layer chromatography (TLC). Paper chromatography is based upon the separation of a sample's components on paper with a solvent whilst TLC is based on the separation of the plant's components on a thin two dimensional sheet of stationary phase (usually silica) coated onto a slide/solid material which is placed in a closed tank with the solvent system [2].
TLC is an effective and inexpensive procedure that gives the researcher an idea of how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf value of a
compound is compared with the Rf value of a known compound [3]. Bioautography is a useful technique to determine antimicrobial compounds within a plant extract. TLC combined with bioautographic methods combine chromatographic separation and in situ activity determination facilitating the localization and target-directed isolation of active constituents in a mixture [3].
Column chromatography-based LC is a much more powerful technique than the paper chromatog-raphy and TLC methods, and has a greater sample capacity [2]. Column chromatography may employ either isocratic elution which uses a mobile phase of a constant single composition or employ gradient elu-tion whereby the mobile phase composition is altered during the chromatographic separation process. The flow of the mobile phase can either be due to gravity or vacuum where columns are not designed to withstand high pressures [2]. The mobile phase used in plants extracts separation is usually the one that would have shown good separation of the extract's components during TLC and also showed compounds with antimicrobial activity during bioautography. The usual stationary phases used in column chromatography are adsorbents such as silica, alumina, calcium carbonate, calcium phosphate, magnesia, sugar, carbon, magnesium silicate, magnesium carbonate or starch.
High performance liquid chromatography (HPLC) is distinguished from ordinary liquid chromatography because the pressure of HPLC is relatively high, while ordinary liquid chromatography typically relies on the force of gravity to provide pressure. HPLC is usually coupled to mass spectrometry (MS). MS provides abundant information for structural elucidation of the compounds, whilst HPLC is efficient in separating chemical compounds in a mixture. Therefore, the combination of HPLC and MS (HPLC-MS) facilitates rapid and accurate identification of chemical compounds in medicinal herbs, especially when a pure standard is unavailable [4].
Gas chromatography is a well established analytical technique commonly used for the characterization and identication of volatile organic compounds [5], as well as a large range of semi-volatile organic compounds through chemical derivatisation which can be achieved through the addition of volatile trimethylsyl (TMS) groups, through the use of compounds such as N-methyl-N-triflouoroacetamide (MSTFA) which causes the formation of volatile trimethylsilyl esters which can be easily characterized and identified by gas chromatography [6]. Gas chromatography coupled to mass spectrometry (GC-MS) allows for separation, identification and quantification of volatile and semi-volatile organic compounds with good resolution in a plant extract, such that it has emerged as the best technique for characterization of low-polarity compounds, including triterpenes, sterols, glycerols, waxes, and derivatized fatty acids [7].
Purification of the plant extract through column chromatography allows for more specific identification of antimicrobial compounds by GC-MS or HPLC-MS. Thorough biological evaluation of plant extracts is vital to ensure their efficacy and safety. These factors are of importance if plant extracts are to be accepted as valid medicinal agents [8]. This paper focuses on the GC-MS characterisation of Garcinia kola seeds' n-hexane extract column chromatography fractions eluted by a solvent combination of benzene : ethanol : ammonium hydroxide (BEA) at the ratio of 36 : 4 : 0.4 v/v. As reported in the previous work [9], this extract exhibited bactericidal properties against Listeria isolates. The aim of this study was to identify the compounds responsible for the anti-Listeria activities through the use of combined chromatographic techniques.
MATERIALS AND METHODS
Plant material. The seeds of Garcinia kola were sourced from the south western part of Nigeria and the ground seed powder was kept in the plant material collection of the AEMREG laboratory, University of Fort Hare, Alice, South Africa.
Bacterial strains. Four Listeria isolates previously isolated from wastewater effluents [10] were used in
the study and included Listeria grayi (LAL 15), Listeria ivanovii (LEL 18), Listeria monocytogenes (LAL 8) and Listeria ivanovii (LEL 30). These were previously found to be susceptible to the n-hexane extract of G. kola seeds [9].
Preparation of the n-hexane extract. The extract preparation followed the method of Basri and Fan [11]. In brief, the method involved steeping 100 g of the seed powder into 500 mL of n-hexane solvent for 48 h with shaking in an orbital shaker (Stuart Scientific Orbital Shaker, UK), after which the mixture was cen-trifuged at 1700 x g for 5 min at 4°C, and the supernatant was filtrated through Whatman No. 1 filter paper (USA). The residue from the extraction was steeped in 300 mL of the solvent and subjected to another 48 h period of extraction with shaking after which it underwent the same filtration process as mentioned above. The extracts were combined and concentrated using a rotary evaporator (Steroglass S.R.L, Italy) at 50°C and then dried to a constant weight in a fume cupboard.
Column chromatography. Seven grams ofthe n-hexane extract was mixed with 14 grams of silica gel 60 (Merck, Gremany; particle size 0.063 to 0.2 mm/70 to 230 mesh) and benzene to form a homogenous thick paste. The mixture was left to dry in the fume cupboard overnight. A suspension of silica gel 60 and benzene was poured into a glass column (40 x 2.5 cm) up to a height of 30 cm being careful to prevent formation of gaps and bubbles and equilibrated with 100% benzene. The dried silica gel 60 with extract was loaded onto the column and eluted with 100% benzene and then with the solvent combination of benzene : ethanol : ammonium hydroxide (BEA) in the ratio of 36 : 4 : 0.4 v/v. The eluted fractions were collected and separated according to differences in colour and also according to the solvent combination used to elute the fraction and then dried in a rotary evaporator (Steroglass S.R.L, Italy) at 50°C, after which they were dried to a constant weight in a fume cupboard. This was done following the column chromatography method as described by Selowa et al. [12] with some modifications in the quantity of extract in which we used 7 g of the extract (instead of 4 g) mixing with 14 g of the silica gel 60 (instead of 8 g) and we used different solvent combinations of starting elution with 100% benzene followed by the BEA solvent combination in the ratio of 36 : 4 : 0.4 v/v instead of elution with chloroform as the mobile phase with the polarities varying
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