Phytochemical studies of extracts of the Kirkia wilmsii tuber

Abstract

Ethnobotanical surveys have shown that K. wilmsii is used traditionally for the treatment of hypertension, diabetes, fever, hypertension, malaria, toothache and diabetes mellitus. Despite all this interesting information, there is no known literature on the phytochemistry of the tubers is recorded. This research seeks to gain an in-depth understanding of the structure of the bioactive compounds that can be extracted from K. wilmsii tubers. The major objective of this research was to do an extensive study on K. wilmsii plant by extracting, isolating and identifying phytochemical(s) that are bioactive from the plant. This was achieved through, the determination of suitable phytochemical extraction solvents, development and optimisation of analytical HPLC methods, biological activity studies, investigation of levels of metal contamination and structural elucidation of the bioactive compounds by nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis) and gas chromatograph-mass spectrometry (GC-MS). In this research, tubers were air dried and ground into a fine powder. Extraction was performed on the powder. In the extraction and phytochemical extraction tests, extraction under the ultrasonic method was done by adding 1g of powder to 25ml solvent and extracting for the specified time. For the extraction test studies the tests were performed for 15, 13 and 60mins. Extraction for phytochemical tests were done according to adapted recent methods. Quantitative tests were also carried out according to recent methods with minor modifications. Phytochemical screening confirmed the presence of phenolics, flavonoids, terpenoids, tannins, cardenolide deoxy sugars and reducing sugars. The extraction results showed that from the 12 solvent extracts used, six gave yields higher than 5%, while the other six gave yields less than 1%. The highest extract yield of 52.9%was obtained using 80%methanol while the lowest yield of 7.3% was obtained using ethanol at 60 min. The 80% methanol, methanol/chloroform/water (12:5:3) (MCW) and 60% methanol extracts were significantly higher than those of ethanol, methanol and water (P < 0.05). Therefore ethanol, methanol, methanol/chloroform/water, 80% methanol, 60% methanol and water can be used as suitable phytochemical extraction solvents for K. wilmsii tubers. Total phenolic content and total flavonoid content analysis proved the presence of high levels of phenolic compounds as well as flavonoids. The presence of phenols and flavonoid could be , space Revise responsible for the radical scavenging activities observed. Total phenolic content recorded extracts ranged from (45.32 ± 0.50) to (122.84 ± 0.31) mg gallic acid equivalent per gram. A maximum total flavonoid content of (917.02 ± 0.10) mg quercetin equivalent per gram and a minimum of (206.26 ± 0.10) mg quercetin equivalent per gram were recorded for methanol and water, respectively. The flavonoid content for methanol was significantly higher than all the other extracts (P < 0.05). The scavenging profiles of K. wilmsii extracts were significantly lower (P < 0.05) than that of ascorbic acid and IC50 values ranged from 129.94 mg/mL for methanol to 225.04 mg/mL for water. An IC50 value of 56.52 mg/mL was obtained with ascorbic acid. Antimicrobial activity tests were performed using the disc diffusion (Kirby-Bauer) method using 9 micro-organisms. Sub-culturing of the microbes was performed before using them in the test. The minimum inhibitory concentrations were determined using the micro titre plate method. In the antimicrobial tests the positive control, amoxycillin, inhibited growth of all the tested bacteria except for K. pnuemoniae. All the K. wilmsii extracts also tested negative for inhibition of K. pneumoniae. The most potent extracts were found to be extracts from 80% methanol and 50%acteone followed by MCW and 80% ethanol. As expected, the minimum inhibitory concentration (MIC) values for the positive control were very low (0.031-1mg/ml). Only the 80% acetone extract had one comparable MIC values (0.031 -1 mg/ml) against S. aureus. The 80% methanol extract also had 2 encouraging results with MIC value of 0.031mg/ml against E. coli and S. typhi. The absolute solvent extracts did not yield interesting results as their antimicrobial activities were not pronounced. The water extracts also tested negative for inhibition of all pathogens used in the current work. These water extract results are in contrast to a lot of other work done on natural products. Proximate analysis tests were done are moisture content and ash content, which done according to recent methods. Elemental analysis was performed on the XRF. Proximate analysis studies revealed that the average moisture content of the dried K. wilmsii tuber was found to be 3.44±0.11% which is less than the recorded moisture content for most natural plants. Our moisture result is well below the maximum 10% suggested for herbal remedies (EDQM, 2007). The average total ash, acid insoluble ash and water soluble content of the K.wilmsii roots was found to be 12.43±0.41, 1.22±0.3 ad 8.18 ±0.39% respectively. The higher the total ash value, the xiv higher the chances of contamination, especially by minerals, less than the allowed 14% maximum. The result is close to the upper limit, possibly due to inorganic matter getting embedded in the tubers as they grow in the soil. The acid insoluble ash was found to 1.22% lower than 2% maximum. The XRF results for Cr, Pb, As, Cd and Hg levels for the K. wilmsii tuber were 1.48, 3.55, 0.14, ND and ND ppm respectively (mg/kg) are lower than the maximum allowed concentration of 25, 0.5, 1.5, 0.5, and 3ppm (EDQM, 2007; ICH., 2014) respectively for the metals except for Pb whose limits are above specifications. In our samples, Cd and Hg were not even detected. These results agree with our sub-chronic toxicity studies that showed that all the monkeys that were fed with a K. wilmsii meal did not die and did not show signs of poisoning. Toxicological tests were carried out on vervet monkeys at the South African Medical Research Council, Primate Unit and Delft Animal Centre in Cape Town. One set of monkeys was fed with K. wilmsii while the other set of monkeys was fed with the normal food. The biochemical, haematological and physical and physiological tests were done at the centre. In the toxicology studies there were no signs or symptoms as that were indicative of unwell-being or distress during the 120day study period. The LD50 value was not determined as no mortalities were recorded. In the tested group of monkeys, no significant difference (p>0.05) in body weight gain, food and water intake were recorded. Similarly, the haematological and biochemical parameters and organ weights did not record any significant alterations (p>0.05) in the test (K. wilmsii) group when compared to the control results. The biochemical and haematological tests did not indicate any damage of the kidneys and liver and consequently any cell damage. Although not significant, the results indicate possible anti-hypertension properties of K. wilmsii. HPLC methods were developed from scratch using the researcher prior knowledge. Column purification methods were performed and adapted from recent methods cited in publications. The purification of extracts yielded several extracts with different proportions and only 3 were selected for the final structural elucidation tests. The 3 extracts had UV-VIS spectra that exhibited maxima at 247nm (KTC3), 290, 295 and 358nm (KTC4) and 287nm (KTC5). KTC3, KTC4 and KTC5 chromatograms were recorded and the resolution was generally satisfactory and a few components need to be separated. The HPLC profiles were meant to provide a tool for the purification of the extracts. xv FTIR, GC-MS and NMR were performed according either according to normal laboratory methods or were adapted from recent research. The GC produced a chromatogram with a peak at 5.77minutes for KTC3, whose MS produced the base peak with a mass to charge ratio of 77 and the parent peak gave a mass to charge ratio of 110 implying that KTC3 compound is likely to have a molar mass of 110g/mol. KTC4’s main peak was at 5.75 minutes exhibiting a mass spectrum with a base peak of 97 and parent peak of 110. The GC chromatograms for KTC5 had a retention time of 6.75minutes, a base peak of 100 and the parent molecule of molar mass 113.