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Structural Identification and Submerged Culture of an Antidiabetic Bioactive Substance from Coprinus Comatus

Author: DingZhongYang
Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Coprinus comatus diabetic mellitus 4,5-Dihydroxy-2-Methoxy-Benzaldehyde (4, 5-DHMB) non-enzymatic glycation liquid fermentation
CLC: R151
Type: PhD thesis
Year: 2007
Downloads: 88
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Abstract


Coprinus comatus, a precious macro-fungus, has been designated as natural, nutritious and healthy food by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). It has recently acquired interests due to its attractive potential application in pharmaceutical industries. It was reported that the immunomodulation activites, such as antihyperg-lycemia, liver injury recovering, immunomodulating, antitumor and hypolipidemic, have been observed in C. comatus. In this dissertation, with inhibition of non-enzymatic glycation reaction as the main index, the separation, purification, structure analysis and high efficiency production process of an antidiabetic substance from C. comatus fermentation broth were investigated in detail. The main results were described as follows:1. In this study, a substance which gained from C. comatus fermentation broth with macropopous resin, has been proved that could inhibit the non-enzymatic glycation (NEG) reaction. And this substance was identified to be neither polysaccharide not protein. Comatin, which has high inhibition percentage on the NEG reaction, was obtained from the fermentation broth, which absorbed by macropopous resin and then separated with normal pressure C18 reversed-phase chromatography. It was observed that 95.86% and 60.53% inhibition percentage of comatin at the dose of 2mg/mL, and 0.5mg/mL, respectively. Comatin was further purified through medium pressure C18 RP chromatography to gain chromatographic grade sample.2. Based on the spectral data of NMR, MS, infrared and ultraviolet, comatin was identified as 4,5-Dihydroxy-2-Methoxy-Benzaldehyde (4,5-DHMB). The quantum chemistry calculation showed that 4, 5-DHMB has the stable structure. As we best knowledge, this is first report that 4,5-Dihydroxy-2-Methoxy-Benzaldehyde obtained from C. comatus.3. A high performance liquid chromatographic procedure was developed to determine the content of 4, 5-DHMB in C. comatus fermentation broth, and has proved to be validated. Chromatographic separation is performed on a C18 column (150mm×2.1mm i.d., 5μm particle).The mobile phase is composed of methanol-water (30:70, v/v) to 100% methanol in 20 min for gradient elution at a flow rate of 0.3mL/min. The method has good liner in the range of 1.23~123μg·mL-1 with R2=0.999. Intra- and inter-day precision ranged from 1.5 to 5.6% and 2.5 to 4.6%, respectively. The recovery was within 93.8%-96.6%. The assay procedure is simple, quickly, precise, and assure the continuous research for Coprinus comatus submerged culture and 4, 5-DHMB.4. In vitro experiment showed that the IC50 of 4,5-DHMB on inhibition the non-enzymatic glycation reaction was 0.39mg/mL, and also found that 4,5-DHMB could combine with Amadori products to change their structure and finally attenuate their fluorescence intensity. Furthermore, the quantum chemistry calculation showed that the possible mechanism of 4, 5-DHMB inhibition non-enzymatic glycation lie in its influence on the reaction after Amadori reaction or the process of shift base rearrangement. The IC50 of 4, 5-DHMB on inhibiting the a-glycosidase was 2.96, and it has been proved to be a non-competitive inhibitor. The values of Km and Ki were 3.5x 104mol/L and 1.35 mg/mL, respectively.5. The blood glucose levels were significantly elevated above initial blood glucose levels in response to the glucose challenge. There was a significant improvement in glucose tolerance of about 21 and 10.3% at 1 h peak value in 4, 5-DHMB mice. The maximum fall in fasting blood glucose (FBG) was after 1 h and maintained up to 2 h during fasting. It therefore appeared that 4, 5-DHMB was effective on FBG and healthy mice.6. In vitro experiment demonstrated that: 1) 4, 5-DHMB could protect diabetic mice which induced by alloxan; 2) 4, 5-DHMB could remarkedly decreased the level of fasting blood glucose, plasma fructosamine, triglyceride, cholesterol in 1 type diabetic mice and attenuated the injured degree of 13 cell in pancreatic islet.7. The results of Plackett-Burrnan suggested that the conditions for 4, 5-DHMB accumulation and mycelia growth were different. In order to achieve a high concentration, high yield and high productivity of 4,5-DHMB, a response surface methodology (RSM) was developed to optimize the nutritional and environmental conditions of 4,5-DHMB production. Firstly, the medium composition were optimized utilizing CCD design and the optimum medium composition were corn powder 15.40 g/L, wheat bran 13.01 g/L, KH2PO4 4.02 g/L, MgSO4·7H2O 1.87 g/L, glucose 5 g/L. With the optimum medium, 674.49 μg/mL 4,5-DHMB was achieved. The optimum environmental conditions for 4,5-DHMB production were described as follows: Temperature 26.84"C, fermentation time 99.57h, initial pH 6.30, volume of medium 173.93 mL/500 mL. 894.01μg/mL 4, 5-DHMB was production under the optimized conditions.8. Fermentations were carried out in a 7 i fermentor (71, Bioflo 101, NBS, USA) with 4 1 fermentation medium. It was found that the broth state play key role on the 4,5-DHMB production. A low agitation speed was facilitated the secretion of 4,5-DHMB from C. comatus, This may be due to the fact that shearing force severely change the state of mycelia in fermentation broth. Litter influence was observed aeration and dissolved oxygen on the mycelia growth and the biomass accumulation, but aeration and dissolved oxygen play important role on 4, 5-DHMB synthesis. On another hand, a little organic acid was detected in the C. comatus fermentation broth.9. The optimized fermentation conditions for the 7 1 fermentor were determined: 4 1 fermentation medium, temperature 27℃, initiate pH 6.30, inoculum concentration 7.5%, agitation speed 75rpm, initiate aeration 0.6wm and lowered to 0.4wm after 88~6h, fermentation time112~120h. A highest 4,5-DHMB concentration (830.41μg/mL) was achieved by combinational consideration of the optimum nutritional and environmental conditions in 7 1 fermentor.

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