Crown gall tissue of transgenic ginseng polysaccharide fraction extracted Optimization of inspection_618

By Nancy Lane,2014-10-30 14:33
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Crown gall tissue of transgenic ginseng polysaccharide fraction extracted Optimization of inspection_618

    Crown gall tissue of transgenic ginseng polysaccharide fraction extracted Optimization of inspection

     Abstract Objective To optimize the study of genetically modified American ginseng polysaccharides crown gall tissue extraction conditions. Orthogonal design optimization method using crown gall tissue of Panax optimal extraction process of polysaccharide, select the extraction temperature, add water, extraction time and extraction times four study factors. The results of orthogonal experiment results showed that: Ginseng crown gall tissue in optimal extraction process of polysaccharide: extraction temperature 70 ?, 40 times the

    solvent, extracting 3 times, 3 h / times. Conclusion The optimized process is simple, good stability.

     Key words Panax quinquefolium; crown gall tissue; polysaccharide; extraction; Optimization

     Optimization of Extraction Methods of Polysaccharides Constituents from the Crown Gall Cultures of Panax quinquefolium

     Abstract: ObjectiveTo investigate the extraction methods

    of polysaccharides constituents from the crown gall cultures of Panax quinquefoliums. MethodsThe orthogonal test was employed to optimize and establish the extraction technology of water-soluble polysaccharides from the crown gall of P.

    quinquefolium.ResultsWhen employed 70 ? of extracting

    temperature, 40 times of water, refluxing and extracting for 3 times, 3 hours for every turn, the optimal extraction efficacy was obtained. ConclusionExtraction method optimized will be used for the isolation and purification of polysaccharides from the transgenic crown gall cultures of P. quinquefolium.

     Key words: Panax quinquefolium; Crown gall;

    Polysaccharides; Extraction; Optimization

     American ginseng Panax quinquefolium L. also known as

    ginseng, American ginseng, Western ginseng, for Araliaceae Panax herbs, native to the eastern United States and Canada, perennial perennial, is a rare medicinal herbs, with qi Health blood repose of the effectiveness of spermatogenic . Modern

    chemistry and pharmacology studies have shown that American ginseng to improve immunity with a strong heart, stomach, blood fat, sedation, blood and inhibit tumor growth and other effects, and some of its pharmacological effects are ginseng can not be replaced. Its main active ingredient is saponins, polysaccharides and amino acid composition. For clinical anti-

    fatigue, anti-aging, anti-cancer nourishing Jiapin [1,2].

    Slow-growing wild or cultivated ginseng, 5 ~ 6 years for medicine, herbal medicine ginseng so tight market supply and

    the price is more expensive.

     The 20th century, 60 years later, people gradually discovered that polysaccharide in anti-tumor, anti-virus, the

    treatment of cardiovascular diseases, anti-aging, etc. has a

    unique biological activity, and its role mostly through the immune system to achieve. There is now isolated from natural products to extract more than 300 kinds of polysaccharide compounds, which extracted from the medicinal plants the most important water-soluble polysaccharides have been found to

    have more than 100 kinds of Chinese medicine in more carbohydrates role in promoting immune . Type of polysaccharide is not cytotoxic and the quality of medicines easier to control by chemical means, has become the main directions of development of new drugs.

     American Ginseng polysaccharide (Polysaccharide from Panax quinquefolium, PPQ), American ginseng saponins (Panax quinquefolium saponin, PQS) and American ginseng compound mixture of immune pharmacological research is the focus of

    scientists. If PPQ cyclophosphamide (Cyclophosphamide, CY)-

    induced reduction in peripheral white blood cells significantly protective effect, and can antagonize CY under the action of the thymus, spleen weight reduction, enhance the

    normal and immunocompromised mice reticuloendothelial system phagocytic function [3,4]; increase in non-specific immunity

    in mice and cellular immune function, the immune enhancing effect increased with the dose increased, with a certain

    amount of effect relationship [4]. It was also reported in the American ginseng polysaccharide extract from the fruit of the obese mice are hypoglycemic effect, one can treat diabetes, but does not change their body weight [5].

     With the development of molecular biology, genetic engineering will be used in tissue culture to produce metabolites rather concern. One advantage of the Ri plasmid of Agrobacterium rhizogenes and Agrobacterium tumefaciens Ti plasmid was transformed into the form of genetically modified organs (such as the hairy root and crown gall tumor) research more, so as the production of plant metabolites has opened up new avenues . Transgenic ginseng crown gall tissue culture and its production of saponins from the study group a more

    systematic study [6 ~ 10], but American ginseng crown gall tissue culture studies of polysaccharide has not been reported. In this paper, on the basis of preliminary work, reported that American ginseng polysaccharides extracted crown

    gall tissue Optimization study.

     An apparatus and materials

     1.1 Materials of transgenic ginseng crown gall tissue transduction by the research group income. By the nopaline-

    type Agrobacterium tumefaciens strain C58 induced by American ginseng stem infections, access to crown gall tissue, and after many years of domestication selection to form a stable, excellent culture system [7].

     1.2 Instruments and equipment TDL80-2B centrifuge; Tokyo

    physical and chemical N-1000 Rotary Evaporator; Denmark Heto

    MAXI-DRYPLUS freeze dryer; Japan SHIMADZU company's UV-2450

    UV-Vis spectrophotometer; Germany Sartorius ACCULAB ALC-110.4

    electronic balance.

     2 Methods

     2.1 ginseng polysaccharides extracted crown gall tissue conditions of single-factor analysis on the ginseng

    polysaccharides extracted from crown gall tissue factors, selection and extraction in this experiment directly related to the extraction temperature, add water, extraction time, extraction times these four factors were analysis at different

    levels in order to determine the best factor level orthogonal experiment.

     2.1.1 Extracting the temperature you choose to sample by sample volume of 50 doubly water, extraction time 3 h, at different temperatures (40,50,60,70,80,90,100 ?) to extract.

     2.1.2 Canadian water samples in the extraction temperature of 70 ?, extraction time 3 h under the conditions of use of different water content (10,20,30,40,50,60 times of water) to extract.

     2.1.3 Sample extraction time taken by 50 doubly water,

    extraction temperature 70 ?, according to the length of

    extraction time (1,2,3,4,5 h) to extract.

     2.1.4 Extracting the number of samples in the extraction temperature of 70 ?, 50 doubly water, extraction time 3 h under the conditions of extraction 1,2,3,4,5 times.

     2.2 Orthogonal experimental method preferred Panax crown gall polysaccharide extraction

     2.2.1 Orthogonal experimental design selected water consumption, extraction time and extraction times for evaluation of factors, each prepared three levels (Table 1), choose L9 (33) orthogonal design table.

     Table 1 orthogonal factor level (abbreviated)

     2.2.2 Determination of polysaccharide Weigh redistilled phenol 12.5 g, placed in 250 ml flask, add distilled water to

    dissolve fixed volume scale, shake, dark cold, or get a 5% phenol solution.

     Preparation of standard curve [11]: the preparation of 100 μg / ml of glucose solution: Precision Weigh dried to constant weight standard glucose 25 mg, dissolved in water,

    placed in 250 ml flask, the constant volume. Were taken 0.2,0.4,0.6,0.8,1.0 ml volume of glucose solution in 10 ml flask, the supply water to 2 ml (in 2 ml water as a blank control), each with 1 ml 5% phenol solution, and quickly join

    the 5 ml concentrated sulfuric acid, placed in 50 ? water

    bath and heat 15 min, remove the cooled to room temperature. Constant volume by adding water to the scale, shake. Was measured at 490 nm absorbance. Absorption of glucose concentration on the degree of mapping, was standard curve.

     Y = 57.809X 0.048 24 (r = 0.998 6)

     Sample test [12]: analyte sample preparation: The crown gall tissue harvested washed three times with distilled water to remove the surface of agar, placed in drying oven (

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