Antibacterial and radical scavenging activity of fatty acids from Paullinia pinnata L.
Annan Kofi1, Gbedema Stephen2, Adu Francis2
1 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
2 Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
|Date of Submission||27-Apr-2009|
|Date of Decision||20-May-2009|
|Date of Acceptance||01-Jun-2009|
|Date of Web Publication||16-Feb-2010|
Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Twelve fatty acids identified from the methanol extract of Paullinia pinnata L. root extract by the use of Gas Chromatography coupled with mass spectrophotometer were screened for antibacterial activity against both Gram positive and Gram negative bacteria as well as three resistant strains of Staphylococcus aureus SA1199B, XU212 and RN4220 resistant to norfloxacin, tetracycline and erythromycin respectively. Using the 96-well microtitre plate method, azelaic acid (a dicarboxylic acid), showed the highest activity against all organisms tested with minimum inhibitory concentration range of 32-256 μg/ml while palmitic, oleic, eicosanoic and stearic acids also showed weak to moderate activity against Staph. aureus, B. subtilis, E. coli and P. aeruginosa. However, docosanoic, tetradecanoic and eicosenoic acids had no activity against all organisms tested. The results also indicated that while the crude methanol extract possess potent antioxidant activity by bleaching DPPH radical (IC 50 of 3.8μg/ml), the fatty acids did not show any significant radical scavenging action.
Keywords: Antibacterial, DPPH, Fatty acids, Free radical, Paullinia pinnata
|How to cite this article:|
Kofi A, Stephen G, Francis A. Antibacterial and radical scavenging activity of fatty acids from Paullinia pinnata L. Phcog Mag 2009;5, Suppl S2:119-23
|How to cite this URL:|
Kofi A, Stephen G, Francis A. Antibacterial and radical scavenging activity of fatty acids from Paullinia pinnata L. Phcog Mag [serial online] 2009 [cited 2021 May 13];5, Suppl S2:119-23. Available from: http://www.phcog.com/text.asp?2009/5/19/119/59795
| Introduction|| |
Infectious diseases caused by bacteria, fungi, parasites and viruses are still a major threat to public health, despite the huge progress in human medicine. Their impact is particularly large in developing countries due to unavailability of medicines and the emergence of widespread drug resistance  . Natural products especially, those used in ethnomedicine provide a major source of innovative therapeutic agents for various conditions including infectious diseases.
P. pinnata L. (Sapindaceae) is used in phytotherapy for the management of various diseases in different parts of the world  . The whole plant is used in Ghana to treat dysentery. The roots, mashed with seeds of Piper guineense, are applied as a styptic to cut veins and to treat leprosy  . The roots are also chewed for coughs and pulmonary diseases, gonorrhoea, fractures or abscesses or used on open sores. It is also used as aphrodisiac  .The roots are also reported to be used in ethnomedicine to treat bacterial infections and healing of chronic wounds  . However, the various compounds previously isolated from P. pinnata have not been linked directly to the purported traditional usage. These include triterpene saponins, catechol tannins; Flavone glycosides; paullinoside A, paullinomide A, β-sitosterol and β-amyrin , . In an effort to understand the medicinal role of the fatty acids present in P. pinnata, this report focused on their identification, in vitro antibacterial and free radical scavenging properties.
| Material and Methods|| |
Collection of plant
Plant material was collected in October 2005 and authenticated at the Centre for Scientific Research into Plant Medicine (CSRPM), Akwapim-Mampong, Ghana, where voucher specimen number 10/03/005 have been deposited.
Isolation and identification of fatty acids
Twelve fatty acids were isolated and identified from the methanol extract of the root by the Gas chromatography-mass spectroscopy (GC-MS) method  . The sample was derivatised into the methyl ester by refluxing with tetramethylsilane (TMS) at 100°C for 30 min. Methyl esters of fatty acids were analysed with a Fisons MD 800 mass (quadrupole)-GC 8000 series instrument equipped with silica column (30 m Χ 0.25 mm I.D.) coated with OV1. Samples were injected by hot split method, with the injector and detector maintained at 270°C and 250°C respectively. The temperature program was 40°C for 4 min. after injection, then increased to 280°C (8°C/min) with a hold at 280°C for 20 min. Helium (20 psi) was used as a carrier gas with the flow rate of 0.5 ml/min. The gas chromatograph was directly coupled to a mass spectrometer working in electron impact ionization mode at 70 eV, and scanned masses in the range of 40-650 Da.
Data were acquired in full scan mode and processed with the Hewlett Packard Chemstation Sotfware. Compounds were identified by their mass spectra, interpretation of MS fragmentation patterns and comparing retention times of their peaks to those of standard compounds analyzed previously. Quantification of methyl esters of fatty acids was performed by integration of appropriate peak areas in Total Ion Chromatogram (TIC).
The bacteria used for the tests were obtained from the National Culture Type Collection (NCTC), UK and included both Gram positive and Gram negative bacteria. The Gram positive bacteria used were Bacillus subtilis (NCTC 10073), Staphylococcus aureus (NCTC 4163) Streptococcus faecalis (NCTC 775), Micrococcus flavus (NCTC 7743), as well as resistant strains of Staph. aureus SA1199B, RN4220 and XU212. Gram negative bacteria used were Escherichia coli (NCTC 9002) and Pseudomonas aeruginosa (NCTC 10662).
Inocula of the microorganisms were prepared from the 24 h Mueller-Hinton broth (Sigma) cultures and suspensions were adjusted to 105CFU/ml. Fatty acid samples were solubilized in 2% DMSO in water. Minimal inhibition concentration (MIC) values of the extracts were determined based on a micro-well dilution method , .
The 96-well sterile plates were prepared by dispensing 180 μl of the inoculated broth plus a 20 μl aliquot of the fatty acid made up in broth or 20 μl broth in the case of negative control in each well. Tetracycline (Sigma) was included as positive control. Plates were incubated for 24 h at 37°C. Bacterial growth was determined after addition of 50 μl p-iodonitrotetrezolium violet (0.2 mg/ml, Sigma).
Antioxidant activity (DPPH radical scavenging activity)
The DPPH scavenging activity of the extract and the identified fatty acids was measured from the bleaching of a purple-coloured methanol solution of 2,2'-diphenylpicrylhydrazyl (DPPH) which was used as a reagent in a spectrophotometric assay ,. Fifty microlitres of various concentrations of the extract and fatty acids in methanol were added to 5ml of a 0.004% methanol solution of DPPH. This was incubated at room temperature for 30 minutes after which absorbance was read against a blank at 517nm on a Thermo Spectronic UV spectrophotometer. L-ascorbic acid was used as positive control in these experiments.
Inhibition of free radical DPPH, in percentage was calculated as
Scavenging activity (%) = A0 - A1 Χ 100 A0
A0 is the initial absorbance of methanolic solution of DPPH at 517nm.
The IC50 value was obtained through extrapolation from linear analysis, using the Prism Software, and denoted the concentration of sample required to scavenge 50% of DPPH radicals.
| Results and Discussions|| |
Twelve fatty acids were identified from the methanolic extract of the root, using the GC-MS. The relative percentage and the retention times of the methyl ester of each fatty acid are reported in [Table 1] and were obtained from the peak area in the Total Ion Chromatogram [Figure 1]. The two most abundant fatty acids were 9-octadecenoic (oleic) acid [30.8%] and hexadecanoic (palmitic) acid [30.0%]. Two of the identified fatty acids, 9-octadecenoic (oleic) acid and 13-eicosenoic (paullinic) acid are mono unsaturated fatty acids. Nonanedioic (Azelaic) acid [4.4%] is a dicarboxylic acid. Fatty acids, including eicosanoic and eicosenoic acids, and cyanolipids have been reported in the genus Paullinia, including P. elegans, P. meliaefolia and P. cupana , . They are usually found in the seed oils of these plant species. However, this is the first report on the fatty acid composition of P. pinnata.
Azelaic acid significantly inhibited the growth of all organisms tested. Palmitic, oleic, eicosanoic and stearic acids also showed weak to moderate activity against Staph. Aureus, B.subtilis, E.coli and P. aeruginosa [Table 2]. However, docosanoic acid, tetradecanoic acid and eicosenoic acid had no activity against all organisms tested. With the exception of azelaic acid, none of the fatty acids had activity against the multi-drug resistant strains of Staph aureus, SA1199B, XU212 and RN4220. These results are in agreement with the previous studied activity of fatty acids where they were found to inhibit the growth of both Gram positive and Gram negative bacteria  . Fatty acids of various chain lengths are known for their antimicrobial action, particularly against Gram positive bacteria. The observed inhibition is explained as a consequence of the uptake of undissociated fatty acids which dissipate the transmembrane proton gradient and thereby affect ATPase activity  . The undissociated form of fatty acids is highly soluble in membrane phospholipids and has been shown to enter the cell by passive diffusion  .
In the DPPH assay, the methanol extract exhibited a significant radical scavenging action [Table 3] with an IC50 value of 3.8 μg/ml. P. pinnata was much more potent than L- ascorbic acid with IC50 of 21.1μg/ml. This compared favorably with the literature value of 21.04μg/ml for L-ascorbic acid  . The observed strong antioxidant action of P. pinnata may be attributed to the presence of phenolic compounds and flavonoids in the plant but these have not been tested for antioxidant activity . Such compounds have been found by different workers to be highly antioxidant  .
| Conclusions|| |
Our investigations have established the presence of twelve fatty acids in the crude methanol extract of P. pinnata, some of which contribute to the overall antibacterial action of the plant. Although the total methanol extract exhibit a highly significant antioxidant action, this activity could not be linked with the fatty acids present. We have also justified the ethnomedicinal use of P. pinnata in the treatment of bacterial infections.
| Acknowledgement|| |
We are grateful to the Ghana Educational Trust Fund (GETFUND) for financially supporting this work and the staff of Chemistry Department, King's College London, for the technical assistance.
| Abbreviations|| |
ATP Adenosine triphosphate
CFU Colony forming units
IC50 Concentration that gives 50% inhibition
MIC Minimum inhibitory concentration
TMS Tetramethyl silane
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[Table 1], [Table 2], [Table 3]
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