Studies On The Burdock Active Components And Their Antioxidant, Antibacterial Activities

Burdock (Arctium lappa L.) is a popular vegetable in China and Japan, exhibiting various biochemical activities. The extraction, separation, activities of the active components in burdock leaves and the inulin in burdock root were studied. The present study could provide help for the deep development of burdock and its application in food industry.1. The extraction and separation of active components from burdock leaves and burdock rootsThe simultaneous ultrasonic and microwave assisted extraction (UMAE) technique was employed to obtain phenolics. The effects of UMAE variables on the yield of phenolics were investigated. The optimized conditions were as follows: solvent to solid ratio was 20:1 (mL/g), extraction time was 30s, microwave power was 500 W and two times of extraction. Moreover, the phenolic yield of UMAE (10.28mg/g) was higher than that by maceration extraction (ME) and ultrasonic extraction, indicating a significant reduction of extraction time and an improvement of efficiency. The phenomenon is related to the strong disruption of leaf tissue structure by microwave induced expansion and ultrasonic shaking, which had been observed with the scanning electron microscopy. The results suggest that UMAE is a good alternative for the extraction of phenolics, with a great potential for industrial application. Also, UMAE provides a new sample preparation technique for characterization of the phenolic compounds from plants. The inulin from burdock root was extracted by simultaneous ultrasonic/ microwave assisted extraction (UMAE). It was found that UMAE required a much shorter extraction time than conventional stirring extraction. The suitable condition for UMAE of inulin was under a simultaneous ultrasonic power of 50w and a microwave power of 400w, at a ratio of 1g of solid material to 15mL of water for an extraction time of 60s. A comparison of scanning electron microscopy images of raw and simultaneous ultrasonic/microwave treated burdock roots indicated microfractures and disruption of cell walls in burdock root flakes.After ultrafiltration, the purified inulin product was obtained, and the purity of inulin product was 91.3%. The burdock leaves extract was fractioned with petroleum ether, ethyl acetate, n-butanol and water, named as PF, EF, BF and WF. After activity-guide fractionation, separation by column chromatogram and high-speed counter-current chromatography, two purified active compounds (chlorogenic acid, p-coumaric acid) were obtained from EF. The compounds were then identified by UV,ESI-MS,1HNMR and 13CNMR.2. Identification of the compounds and development of a method for the determination of five compounds in burdock leavesThe compositions of burdock leaves were then identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The composition was identified based on retention time, UV and MS spectra compared with those of authentic compounds or literature data, and 11 compounds were characterized, providing a more complete identification of phenolic compounds in burdock leaves than previously reported. The compounds were quercetin, cynarin, o-hydrobenzoic acid, benzoic acid, quercitrin, caffeic acid, luteolin, chlorogenic acid, p-coumaric acid, arctiin and rutin. The occurrence of benzoic acid, o-hydrobenzoic acid and p-coumaric acid is reported for the first time. By optimizing the extraction, separation and analytical conditions, a sensitive and accurate high performance liquid chromatographic method has been developed for the simultaneous determination of five active compounds (benzoic acid, caffeic acid, chlorogenic acid, p-coumaric acid and rutin) in burdock leaves. The analysis was performed on a Waters symmetry C18 column at 30℃using 20mmol/L aqueous formic acid solution/methanol gradient system at a flow rate of 1.0mL·min-1 and photodiode array detection (PDA) at wavelength of 280 nm. The method showed good linearity and satisfactory accuracy and recoveries.3. The antimicrobial and antioxidant activities of active components from burdock leavesThe burdock leaf extract exhibited high lipid peroxidation inhibition activity, DPPH radical, hydroxyl and superoxide anion radicals scavenging ability. The antioxidant activities of each burdock leaves fraction were investigated alone and in combination with tertiary butylhydroquinone (TBHQ). The EF exhibited the highest antioxidant activity. Although TBHQ exhibited higher lipid peroxidation inhibitory activity than EF, the reducing power, superoxide anion scavenging capability, DPPH and hydroxyl radicals scavenging ability of EF were higher than those of synthetic antioxidant (TBHQ). Moreover, a synergistic antioxidant effect between EF and TBHQ was first demonstrated by isobolographic analysis, indicating that EF dramatically enhances the antioxidant efficiency of TBHQ. The results indicate that the EF could be used as sources of nature antioxidant in food industry, and allows a decrease of about 4 folds in the amounts of the synthetic compounds used. The extract of burdock leaves was tested for its antimicrobial potential against three Gram-positive (streptococcus pneumoniae, bacillus subtilis, Staphylococcus aureus), three Gram-negative bacteria (Shigella dysenteriae, Escherichia coli, salmonella typhimurium) and three kinds of fungi (Aspergillus niger, saccharomyces cerevisiae, penicillium). The extract could not significantly inhibit the growth of fungi. But, the extract was effective against both Gram-positive and Gram-negative bacteria. The MIC values ranged from 1.5 to 2.5 mg/mL. The antibacterial activities of burdock leaves fractions against six food-related bacteria were first investigated. The data from minimum inhibitory concentration (MIC) values showed that EF and other fractions effectively inhibited the growth of all test bacterial pathogens, the antibacterial activity of EF being much greater than BF and WF. The time-kill assay indicated that EF exhibited significant bactericidal activity against all the six pathogens. At 12 h, EF was bactericidal for 4/6, 6/6 and 6/6 strains at 1×MIC, 3×MIC and 5×MIC, respectively. The ethyl acetate fraction based on its lower MIC values, concentration and time-dependent antibacterial ?and bactericidal activities could be useful in control of bacterial pathogens. The antibacterial activities of chlorogenic acid, p-coumaric acid, rutin and benzoic acid against six food-related bacteria were investigated. The antibacterial activity of p-coumaric acid was higher than other conpounds. The MIC values of p-coumaric acid against streptococcus pneumoniae, Staphylococcus aureus, bacillus subtilis, Shigella dysenteriae, Escherichia coli and salmonella typhimurium were 20, 10, 20, 20, 20 and 80μg/mL, respectively. The oral acute toxicity test indicated that the LD50 of EF was beyond 10000mg/kg, and EF was actually non-toxic. For all the fractions, the antioxidant and capacity had a significant correlation with total phenolic content. The antibacterial ability also had a significant correlation with total phenolic content, suggesting that the activities were probably due to the combined action of phenolic compounds. The phenolic compounds of the fractions were then identified. Among them the contents of chlorogenic acid, o-hydrobenzoic acid, p-coumaric acid and rutin were high.4. The antibacterial mechanism of chlorogenic acid and p-coumaric acidChlorogenic acid and p-coumaric acid could increase the membrane permeabilization of G- bacteria, causing the ion leakage. The treatment of Shigella dysenteriae cells with phenolic compounds induced a major influx of PI, demonstrating that the phenolic compounds executed their antibacterial effects via binding to the surface of the cells, followed by the formation of membrane pores. Also, it was supported by the observations made under transmission electron microscopy. Taken together, the antibacterial activity of chlorogenic acid and p-coumaric acid was concluded to result from the disintegration of the cell membrane.The nucleic acid was the action target of two phenolic compounds after penetrating the Shigella dysenteriae cells. They didn't breakdown the genomic DNA, but strongly binding to DNA. The results of fluorescence quenching experiments and circular dichroism indicated that chlorogenic acid and p-coumaric acid could bind to bacterial genomic DNA, change its conformation, and loosen the double helix of DNA. Both phenolic compounds binding to DNA influenced the function of gene. The results demonstrated that chlorogenic acid and p-coumaric acid have dual bactericidal mechanisms: disrupting bacterial cell membranes and binding to bacterial genomic DNA to inhibit cellular functions, ultimately leading to cell death.