A study of the chemical components of extracts from kirkia wilmsii and an investigation into their properties

Abstract

Aqueous extraction was performed on the plant Kirkia wilmsii roots at room temperature. The extracts were filtered and stored at 4 °C. An analytical high performance liquid chromatographic (HPLC) method was developed for determination of the components of Kirkia wilmsii roots. A gradient elution programme was developed that employed two mobile phases. Mobile phase A water while the pH was adjusted with trifluoroacetic acid (TFA). Mobile phase B was organic, made up of acetonitrile, where the pH was also adjusted with TFA. A C 18 column and a photo diode array (PDA) detector were used. Flow rate was changed continuously during the run, to ensure the best resolution of the peaks. The resolved peaks were then collected into different containers using the fraction collector on the HPLC. The peaks that were chosen for this research were those at 16.7, 20.3, 28.2, 31.5, 40.4, 50.1, 56.5 and 64.2 mins. Collected components were then freeze dried as opposed to using a rotary evaporator, to avoid heating samples whose chemical properties were not yet known. The net dry weight of each component was determined. The purity of the components was checked by gas chromatography (GC), where each component was dissolved in acetone and then run on the GC. Electrochemical properties of the components were investigated using cyclic voltammetry and anti-oxidant properties were exhibited by four components. All four components showed that they could undergo reversible reactions. The lpc, Epc, lpa, Epa, E, 􀊙 Ep, Ipc/ Ipa and the number of electrons involved in the process for each component were calculated. Possible structures of bioactive compounds were identified using Gas Chromatography - Mass Spectrometry (GC-MS). These results indicated the possible structure of the isolated compounds. Compound 1 contained triphenylphosphine oxide, (carbethoxymethyl)triphenylphosphonium bromide or formadehyde, triphenylphosphoranylidene) hydrazone. Compound 5 was made up of diisooctyl phthalate, phthalic acid, di(2-propylpentyl) ester orphthalic acid octyl 2-propylpentyl ester. Compound 7 contained 1,2,3-benzenetriol, 1,2,4- benzenetriol or 3-methyl-pyrazole-5-carboxylic acid, while compound 8 contained catechol, resorcinol or hydroquinone. All the components analysed by GC-MS showed possible structures that have functional groups that can undergo reversible reactions. This was confirmed by the cyclic voltammetric results. Raw fresh extract was shown to have antimicrobial activity against Escherichia coli, Staphylococcus aureus, Candida albicans and Enterobacter aerogenes. Separated and dried components were each dis·solved in 2 ml of water and antimicrobial activity tests were repeated. Component 1 exhibited antimicrobial activity against Shigella dysenteriae, Aeromonas hydrophilia, Salmonella thyphii, Proteus mirabilis, Escherichia coli and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was calculated to be 3.445 ng/μL for this extract against all these microorganisms. Component 5 was only active against Proteus mirabilis and the MIC was calculated to be 0.08 ng/μl. Component 7 was active against Shigella, Escherichia coli 10, 11 and Staphylococcus aureus. The MIC for component 7 against Shigella was 0.365 ng/μl, 0.046 ng/μI against Escherichia coli 10, and the 0.091 ng/μI against Escherichia coli 11. Component 8 was active against Shigella, Aeromonas hydrophilia, Sal􀑐onella, Proteus mirabilis, Escherichia coli 10 and 11. The MIC against all these microorganisms was found to be 0.155 ng/μI. It was concluded that some the components of Kirkia wilmsii possess antimicrobial and antioxidant properties. Chemical properties and possible structures of the biologically active components were investigated. Further tests still need to be done on the plan, especially the stuctures, which have to be identified.