Rhizoma Imperatae Optimization of ultrasonic extraction of polysaccharide
【Abstract】 Objective To determine the optimum ultrasonic extraction conditions of Rhizoma Imperatae polysaccharide extract Rhizoma Imperatae polysaccharide, for Rhizoma
Imperatae research, development and utilization provide a theoretical basis. Methods Rhizoma Imperatae polysaccharide content of the indexes, using phenol - sulfuric acid assay
method using the orthogonal experimental method to determine the best ultrasound Rhizoma Imperatae polysaccharide extraction conditions. The results of extraction times and the solid-liquid ratio on the extraction rate of polysaccharides Rhizoma Imperatae play a major role. The optimum ultrasonic extraction as follows: extraction temperature 80 ?, with 20
times the amount of water, ultrasonic extraction 3 h, extracted three times, Rhizoma Imperatae polysaccharides yield of 6.33%. Conclusion determined Rhizoma Imperatae optimal ultrasonic extraction process of polysaccharide.
【Key Words】 Rhizoma Imperatae Rhizoma Imperatae
polysaccharides extracted orthogonal ultrasonic phenol -
sulfuric acid method
Rhizoma Imperatae only contained in the "Shen Nong's Herbal Classic", formerly known as Chine, for grasses Imperata
Imperata cylindrical (L.) P. Beauv var.major CEHubb.'s Roots , Sweet, cold, with the cooling blood to stop bleeding , heat-diuretic achievements. For the treatment of hematemesis, gonorrhea, negative urine, edema, and jaundice. Studies have shown that the active ingredient triterpenoids and sugar accounted for 18.8% of the total chemical composition . Eglinton and Natory team has done a great deal of research to its constituents, has isolated elements of the Arundo donax,
Imperata cylindrical element, fern-ol, and other
triterpenoids. Rhizoma Imperatae aqueous crude extract by gel filtration and anion-exchange chromatography for separation of a series of high molecular weight polysaccharide material, and its molecular weight is greater than 15 × 104Da. Using
chemical analysis to determine the existence of these polysaccharides in the six kinds of single-glucoside,
respectively, glucoside, galactoside, gums aldehyde glucoside, mannosidase, wood glucoside, rhamnoside. To give full play to medical effectiveness to Rhizoma Imperatae polysaccharide content in this study for the detection of targets, with the phenol - sulfuric acid assay method to study solid-liquid
ratio (g / ml), extraction temperature (?), ultrasonic time
(h ), extraction times and other factors discussed under the
conditions of ultrasonic extraction to determine the optimum conditions for extraction of polysaccharide.
An instrument and reagent
Spectrum54 UV - VIS spectrophotometer (Shanghai Lingguang Technology Co., Ltd.); KQ-250DB NC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); glucose standard (Sinopharm, Shanghai Chemical Reagent Co., Ltd.) , Rhizoma Imperatae (Jining bonres Chinese Herbal Pieces Co., Ltd.) was identified as grasses Imperata Imperata cylindrical (L.) P. Beauv
var.major CEHubb.'s roots.
2 Methods and Results
2.1 Optimization of Extraction of polysaccharide Rhizoma Imperatae
2.1.1 Preparation of standard glucose solution, said precision drying to constant weight to take the glucose
standard 25.2 mg, add appropriate amount of water dissolved and transferred to 250 ml flask, add water to scale, dubbed the concentration of 100.8 μg / ml standard glucose solution
2.1.2 5% phenol reagent preparation to take phenol 100 g,
plus 0.1 g of aluminum and sodium bicarbonate 0.05 g, distilled. Collection of 182 ? steam copies, said to take 7.5
g, dissolved in 150 ml water, brown bottle Fang Bingxiang back home.
2.1.3 Standard Curve drawing precision to take the amount
of glucose standard solution 0.1,0.2,0.3,0.4,0.5,0.6,0.7 ml test tube placed in dry, respectively, add water to make into a 1.0 ml. And then 5% phenol solution were added to 1.6 ml, shake, and then add concentrated sulfuric acid 7.0 ml, fully shaken, at room temperature 25 min, was measured at 490 nm maximum absorption at the same time make a blank. To absorb
the degree of (A) for the vertical axis, the solution concentration (μg / ml) as the abscissa, was the regression equation: A = 0.007 7C-0.045 2, the correlation coefficient r = 0.999 75, shows the concentration of glucose in the 10.08 ~
70.56 (μg / ml) within the solution absorbance (A) and concentration (μg / ml) showed good linear relationship.
2.1.4 Orthogonal experimental design Rhizoma Imperatae main active ingredient of a series of high molecular weight polysaccharide material soluble in water, hardly soluble in organic solvents. Therefore, the reference to ultrasonic extraction of polysaccharides related to literature, Rhizoma Imperatae polysaccharide extraction process can be used repeatedly in different water extraction, selection mainly
affects extraction efficiency of solid-liquid ratio (g / ml)
(A), extraction temperature (?) ( B), ultrasonic time (h)
(C), extraction times (D) for evaluation of the factors, factor levels of the arrangements in Table 1. The use of L9
(34) orthogonal experimental design, shown in Table 2. Table 1 factor level (abbreviated)
2.1.5 Rhizoma Imperatae polysaccharide sample preparation
Rhizoma Imperatae sample processing: taking Rhizoma Imperatae said sub-paragraph block of 300 g (0.2 ~ 0.5 cm), by
petroleum ether (60 ~ 90 ?) 500 ml skimmed back twice, 1 h / time. 80% ethanol was then extracted twice, 2 h / times to remove the simple sugars and oligosaccharides, the dregs (a) drying stand.
Weighing: Weighing has been handled well in Rhizoma
Imperatae 27 copies, each of 5.00 g.
Ultrasonic extraction: According to Table 3 to set the extraction temperature, ultrasonic time, extraction times and solid-liquid ratio, add distilled water to operate, according
to conditions of ultrasonic extraction, each experiment done three times in parallel experiments numbers. Reposted elsewhere in the paper for free download http://
Concentration: The combined extract for each experiment, vacuum concentrated to 150 ml, plus a small amount of
activated carbon bleaching, filtration.
Alcohol Shen: filtrate plus 5 times the amount of 95% ethanol, placed filtration, centrifugation, remove supernatant, centrifugation following floc, at 4 000 r / min, the centrifuge 15 min.
Washing: precipitation, dissolved in 100 ml of water ethanol precipitation repeated a times, precipitated with ethanol, acetone, washed several times.
Drying: In the 4 000 r / min, the centrifuge 15 min later, taking deposits on the heating temperature within a
drying oven dried at 60 ?, that was Rhizoma Imperatae
2.1.6 Preparation of sample solution will be Rhizoma Imperatae and determination of polysaccharides in the mortar, after the inquiry into the powder were placed in 500 ml of the
flask, and then draw from Precision 10.0 ml, placed in 100 ml of volumetric flask as a polysaccharide reserve liquid. Exact amount of liquid to take polysaccharide reserves 1.0 ml, according to the standard curve measured the same way as measured absorbance and the following substituted into the regression equation, the results in Table 2. Table 2 Orthogonal experimental results L9 (abbreviated)
From Table 3 orthogonal experiment results show that affect the extraction rate of polysaccharides Rhizoma
Imperatae primary and secondary factors in the order: extract the number of (D)