ABSTRACT
Polysaccharides and free monosaccharides are important active components in Cistanches Herba, which have functions of anti-aging and immunological activity regulation. The study of monosaccharide composition in polysaccharide and free monosaccharide can lay a foundation for the study of primary structure, spatial structure of Cistanche polysaccharide and biological activity of Cistanches Herba. In this study, a method of water extraction and alcohol precipitation was used to extract Cistanche polysaccharide. Trifluoroacetic acid was selected as the hydrolytic acid for polysaccharide hydrolysis. An orthogonal experimental method is established. Three levels of acid concentration, hydrolysis temperature and hydrolysis time were selected to investigate the optimal hydrolysis condition. The optimal hydrolysis condition was 0.08 mol·L-1 trifluoroacetic acid hydrolysis at 100 ℃ for 3 h. The free monosaccharides of Cistanches Herba were extracted by water extraction. The established ion chromatogram integrated pulsed amperometry method can efficiently separate 11 monosaccharides in a short time. The method has good repeatability and high sensitivity, methodological experiment results meet the requirements of quantitative determination. It can accurately determine the monosaccharide composition of Cistanche polysaccharide and free monosaccharide content. Ion chromatography does not require derivatization operation and the pre-treatment steps are simple. This method can measure fructose, but PMP derivation-HPLC method can't. The monosaccharide composition of Cistanche polysaccharide include fucose, arabinose, rhamnose-galactose, glucose, xylose, mannose, fructose, ribose and glucuronic acid, among which the contents of glucose and fructose are relatively high. The free monosaccharides in the water extract of Cistanches Herba include glucose, fructose and mannose.
ABSTRACT
Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 μM and 0.047 μM min−1ml−1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37°C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109±3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrode–electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 μAμM−1 with a sensor affinity [Km(app)] of 50 μM and 95% reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6–1000 μM, whereas the biosensor generated linear response in the 1.5– 1000 μM range with a response time of 24 s and limit of detection of 0.56 μM. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.