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Ganoderma lucidum on the taxonomic belongs to the Aphyllopho rales Ganodermataceae Ganoderma. Ancient and modern pharmacological and clinical studies have proved that Ganoderma lucidum does have the effect of preventing and curing diseases and prolonging life. Ganoderma lucidum polysaccharide has significant pseudo-SOD activity, which can significantly remove free radicals generated by the body, prevent free radical damage to the body, prevent lipid peroxidation, protect cells and delay cell aging. It can improve the body's immunosuppression tumor and enhance the body's ability to resist hypoxia、 It regulates blood sugar and blood lipids, protects of liver, prevents radiation and raises white blood cells, anti-herpes virus, anti-mutation, anti-ulcer and so on.
There are many methods for determining the content of polysaccharides, such as visible UV spectrophotometry and titration. More reports are currently available on the UV spectrophotometer. The method mainly uses the principle that the polysaccharide reacts with some reagents to determine the linear relationship between the absorbance and the content, According to the different color -developing agents, it can be divided into phenol sulfuric acid method, anthrone sulfuric acid method, salicylic acid method, etc. Liu Yanping used 722 spectrophotometer to measure the absorbance at 625 nm by color -developing agents of anthrone sulfuric acid. The polysaccharide content of Ganoderma lucidum in Ganoderma lucidum cultured by different strains was determined, and the pigment was removed by activated carbon adsorption and removed oligosaccharide by 85% ethanol, basically eliminate interference. Mao Weilun et al. used a 751-GW spectrophotometer and a DV-6l0 spectrophotometer to develop color with an anthrone-sulfate solution. Determination of Ganoderma lucidum polysaccharides in Ganoderma lucidum capsules by determination of absorbance at 625 nm and refluxed two times with 80% acetic acid, Remove oligosaccharide and remove disturbance. Shi Juan et al. used UV-260 spectrophotometer and 752 UV-visible spectrophotometer to determine the content of Ganoderma lucidum polysaccharides at 488.7nm by phenol sulfuric acid colorimetry. Zheng Xiukui et al. determined the content of Ganoderma lucidum polysaccharides in Ganoderma lucidum Capsules at 520 nm with UV-2100 spectrophotometric juice and 3,5-dinitrosalicylic acid colorimetry. Zheng Xuezhen et al. measured the polysaccharide content by UV-2l0O UV-visible spectrophotometric juice and 75lG spectrophotometry. It is reported that the anthrone sulfur method exist disadvantages such as large fluctuations in data and poor repeatability. If through determination conditions in the phenol sulfuric acid method can be studied in detail and find the best conditions, the accuracy and repeatability of the method can be improved. To achieve the purpose of rapid and accurate determination of polysaccharide content of Ganoderma lucidum, the author studied the conditions of determination of Ganoderma lucidum polysaccharide by phenol sulfuric acid method, and made the data of Ganoderma lucidum polysaccharide content determined by modified phenol sulfuric acid method more stable and accurate.
1. Materials and methods1.1 Experimental material
The test Ganoderma lucidum was provided by the Edible Fungi Research Institute of Hunan Agricultural University; the Hitachi UV-3310 Ultraviolet Spectrophotometer; the reagents such as phenol, sulfuric acid, and absolute ethanol were all provided by the China National Institute for the Control of Pharmaceutical and Biological Products.
1.2 Reagent preparation
5% phenol solution: Weigh 50 g of phenol (AR), add 100 ml of distilled water to dissolve, and prepare a mother liquor. 50% phenol solution, take 10 ml of 50% phenol solution, add distilled water to 100 ml, and prepare a 5% phenol solution, which is now ready for use.
Preparation of the reference solution: Weigh at 105℃ dried to the constant anhydrous glucose , add water to make a 1 mg / ml solution, then weigh 5 ml and 10 ml part, place in a 100 ml volumetric flask, dilute with water to Scale and shake to obtain 0.05 mg/ml and 0.10 mg/ml glucose solution.
1.3 Extraction of polysaccharides
Precision Weigh Ganoderma lucidum powder 2 g, placed in the Soxhlet extractor, add water 90ml, heated reflux for 6 h; combine extract and transfer to a 100 ml volumetric flask, dilute to the mark with water, shake well, accurately measure 10 ml, add 150 ml of ethanol, shake well, place at 4 ° C for 12 h, remove, centrifuge, the supernatant fluid is decanted, dissolve the precipitate with water and transfer to a 50 ml volumetric flask, shake well to volume, that is, the sample solution is reserved.
2. Determination of test conditions
2.1 Selection of maximum absorption wavelength
The anhydrous glucose reference solution and the Ganoderma lucidum test solution obtained by phenol sulfuric acid were respectively taken and scanned at 400 to 700 nm to determine the maximum absorption wavelength. The results show that both have maximum absorption at 490 nm.
2.2 Selection of the amount of phenol addition
Take 0.05 mg/ml glucose solution and Ganoderma lucidum test sample 1.0 ml each place 6 parts in 15 ml stoppered test tubes, respectively add 5% phenol solution 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ml, Shake well, add 5.0 ml of sulfuric acid, dilute to 15 ml with distilled water, Shake and mix quickly and in boiling water for 30 min. 1.0 ml of water was added with 1.0 ml of a 5 % phenol solution, and add into 5 ml of concentrated sulfuric acid as a blank solution, and the absorbance at 490 nm was measured by spectrophotometry, and distilled water was used as a blank. The test results show that, when the other conditions are constant, the absorbance value does not increase with the increase of the amount of 5% phenol, and the absorbance is the largest when the dosage is 1.0 ml, so the optimum amount of phenol is 1 ml.
2.3 Selection of sulfuric Acid addition amount
Take 0.05 mg/ml glucose solution and Ganoderma lucidum test sample 1.0 ml each for 8 parts, place them in 10 ml stoppered test tubes, add 5% phenol solution 1.0 ml, shake and mix, respectively add 1.0, 2.0, 3.0 , 4.0, 5.0, 6.0, 7.0, 8.0 ml of sulfuric acid, make up to 10 ml with distilled water, Shake and mix quickly, at room temperature for 30 min, measure the absorbance at 490 nm by spectrophotometry, blank according to item 2.2 Method preparation. The test results show that the absorbance increases with the increase of the amount of sulfuric acid solution under other conditions unchanged, and the absorbance is unchanged at 5 ml. Therefore, the optimum amount of sulfuric acid is 5 ml.
2.4 Determination of developing stability.
Take a glucose standard solution of 0.05 mg/ml and 1.0 ml of distilled water in a 10 ml stoppered test tube, add 1.0 ml of a 5% standard phenol solution, mix well, and quickly add 5.0 ml of concentrated sulfuric acid,respectively reacting for 10,30,60,90,120,150 and 180 minutes in a boiling water bath, cooled to room temperature immediately after removal,and then the absorbance was measured at 490 nm. The blank was prepared according to the method of 2.2. The measured absorbance values respectively were 0.374 、0.394、 0.392、0.393、 0.392、0.391and 0.392. Since the prolongation of time is favorable for the dissolution of polysaccharide, the color development is stable within 3 hours, but the absorbance is the largest at 30 min. Therefore, to improve the measurement efficiency of the polysaccharide content, the experimental selection was performed at 30 min.
2.5 Selection of Colour temperature
Take a glucose standard solution of 0.05 mg/ml and 1.0 ml of distilled water in a 10 ml stoppered test tube, add 1.0 ml of 5% standard phenol solution, mix well, and quickly add 5.0 ml of concentrated sulfuric acid, mix and separately place temperature is a 25, 50, 75, 100 ° C water heat for 30 min and Immediately cool to room temperature after removal, then measure the absorbance at 490 nm. Refer to item 2.2 for blank liquid, and the results respectively are 0.434、0464、0.535 and 0.556, From this, it can be seen that the higher the temperature, the more the polysaccharide is dissolved, and the absorbance is the highest at 100 ° C, indicating that the polysaccharide is most dissolved in the boiling water, so the color development temperature is selected to be 100 ° C.
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