1、1Optimization on the Extraction of Polysaccharides from Fructus Corni Using Uniform DesignAbstract： Uniform Design was used to optimize the effects of processing parameters for extraction of polysaccharides from Fructus Corni. Four independent variables including extraction temperature （） ， extracti
2、on time （min） ， liquid ratio （times） and pH value were studied. The optimal conditions were determined and tri-dimensional response surfaces were plotted according to the try grid method. The results indicated that liquid ratio （times） and pH value variables markedly affect the polysaccharides value
3、. Relevant parameters of the extract of the highest concentration of polysaccharide are as follows that the liquid ratio is 1： 33 with the pH value of 8.5. Under optimized conditions the experimental yield was in agreement with the predicted value. Key words： Fructus Corni； Polysaccharides； Extracti
4、on； Uniform Design Uniform design is a multi-factor optimization experimental design method， which was created by famous Chinese 2mathematicians Fang Kaitai and Wang Yuan 1.Through this design method， the test points can be fully “dispersed uniformly“ within the experimental range and each point is
5、of better representativeness. Hence， the number of test points can be reduced greatly and also the factor level can be adjusted properly， preventing high-grade level and low-grade level from meeting. Thus， it is a powerful tool to select optimization conditions when seeking the best experimental con
6、ditions， the best proportion， etc 2. In recent years， it has been applied widely in the optimization of extraction technologies. Fructus Corni is mainly distributed in Zhejiang， Henan， Anhui， etc. Japanese scholars used to research Fructus Corni 3-5. Organic chemicals in Fresh fructus corni mainly i
7、nclude reducing sugar， polysaccharide， organic acids， phenols， glycoside， iridoids， saponins， tannin， etc. Also， its pulp and core contain over 10 amino acids and rich vitamin B， vitamin C and over 20 mineral substances. Polysaccharides are indispensable substances for life metabolism， and can adjus
8、t the immune function， improve the organism immunity， and have the effects of anti-aging， anti-tumor and anti-AIDS， etc. Also， there have been some reports on the extraction of fructus corni polysaccharides， but the 3extraction rate was not ideal. To extract polysaccharides more effectively from fru
9、ctus corni， the authors analyze effects of extraction temperature， time， solid-liquid ratio and pH value on the polysaccharides yield and make a uniform design for the fructus corni polysaccharides extraction 6 to select the best extraction process conditions at a lower cost. 1. Materials and method
10、s 1.1 Materials and instruments Dried fructus corni is made in Anhui； D （+）-glucose and phenol （Fluka split packaging） are imported by Shanghai HUASHUO Fine Chemicals Co.， LTD； and others are the domestic analytical reagents. TU-190 ultraviolet and visible spectrophotometer is from Beijing PERSEE； T
11、DL80-2B centrifuge， thermostat water bath and precision acidity meter are from Shanghai TIANDA Instrument Co.， LTD； and others are electronic scale， miniature turbine mixer and all-powerful pulverizer， etc. 1.2 Extraction of fructus corni polysaccharide 1.2.1 Technological process The technological
12、process is shown as below： Weigh dried Fructus Corni sifted with a 100-mesh sievewater extract adjust solid-liquid ratioadjust pHset 4temperatureset timeextractcentrifuge add supernatant 7 1.2.2 Extracting method by using uniform design to optimize fructus corni polysaccharides Grind dried Fructus C
13、orni and sift with a 100-mesh sieve to gain the powder. Add eightfold-volume 95% ethanol to reflux for 8 hours to defat. Cool and filter it， and take residues and dry them. Set aside. Weigh a certain amount of pre-processed Fructus Corni powder， and then select the extraction temperature， time， soli
14、d-liquid ratio and pH value. Conduct the four-factor and ten-level uniform design experiment by using the contents of Fructus Corni as investigation indexes to optimize the extraction conditions of the Fructus Corni polysaccharides. 1.2.3 Analytical method （1） Content determination of fructus corni
15、polysaccharides： Use phenol-sulfuric acid method 9 and take glucose as standard substance to absorb a certain volume of polysaccharide extract， and determine the light-sucking value based on the standard curve method， and calculate the relative content of polysaccharide based on standard curve. （2）
16、Determination of the Maximum Absorption Wavelength： 5Prepare different-concentration glucose standard solution and polysaccharide extract and then operate based on the making methods of standard curve， and scan 400 nm-500nm wavelengths to absorb spectrums. The result is that standard substances and
17、sample substances are similar in terms of absorbing spectrums and have the maximum absorption at 489nm； therefore， 489nm is selected as measurement wavelength. （3） Establishment of standard curve： Accurately absorb 0.1ml， 0.2ml， 0.3ml， 0.4ml， 0.5ml， 0.6ml， 0.7ml， 0.8ml and 0.9ml glucose standard flu
18、ids （concentration： 0.114 mg/ml） into test tube slowly and add distilled water until it is 1ml， add 1ml 5% phenol （saved in refrigerator to avoid light） ， add 5ml 98% concentrated sulfuric acid and stir uniformly， and set aside at room temperature quietly for 30 min. Make a blank control with distil
19、led water， and use TU-190 ultraviolet and visible spectrophotometer to measure the absorbance at 489nm. The result is shown in Table 1. Make a regression processing on the above data and gain the regression equation C=0.0975A+0.0011， and related coefficient r is 0.9995. 1.3.4 Determination of polysa
20、ccharide content of sample solution 6Accurately absorb 0.1ml extract after dilution and add distilled water until it is 1ml based on standard curve method （the same operation method）. 2. Result and analysis 2.1 Single-factor experiment （1） Effects of extraction temperature on extraction process： Sep
21、arately weigh N samples of the processed dried Fructus Corni powder. Each Sample weighs 10g. Add 200ml distilled water to each sample and stir them respectively for 90 min at different temperatures. Centrifuge the mixture at 4000r/min for 20min， and measure the sugar concentration. The result is sho
22、wn in Figure 1. From figure 1， it is known that the change of polysaccharides concentration is not significant under the different temperature extractions. （2） Effects of extraction time on extraction process： Separately weigh processed dried Fructus Corni powder （10g） and add 200ml distilled water，
23、 and stir them for 150 min and extract the mixture at 80 at different times. Centrifuge the mixture at 3500r/min for 20min， and measure the sugar concentration. The result is as shown in Figure 2. From figure 2， it is known that the change of 7polysaccharides concentration is not significant under t
24、he extraction of different times. （3） Effects of different water content on extraction process： Separately weigh processed dried Fructus Corni powder （10g） and add different amounts of distilled water， stir at 80 for 150min and extract the mixture. Centrifuge the mixture at 3500r/min for 20min， and
25、measure the sugar concentration. The result is shown in Figure 3. From figure 3， it is known that the polysaccharide concentration in extraction increases when different water content is extracted， but it changes little when water content reaches a certain extent. （4） Effects of pH Value on Extracti
26、on Process： Separately weigh processed dried Fructus Corni powder （10g） and add 200ml distilled water， and stir them for 150 min and extract the mixture at 80 at different times. Centrifuge the mixture at 3500r/min for 20min， and measure the sugar concentration. The result is shown in Figure 4. From
27、 figure 4， it is known that different pH values have effects on polysaccharide concentration of extract， and the polysaccharide of fructus corni is easier to be extracted under the condition of neutral to alkali. 2.2 Uniform 8experimental design （1） Uniform design experimental plan： In the process o
28、f extracting fructus corni polysaccharides， the investigated factors mainly include extraction temperature， time， water amount and pH value. There are temperature X1 （55-100） ， time X2 （ 30min-200min） ， water amount X3 （6-33 times） and pH value （4-8.5）. According to the values of all factors and the
29、 experimental accuracy requirements， all factors are designed in a level table （see Table 2）. Then， based on the U10*（108） in the uniform design table， the experimental plan can be designed （see Table 3）. （2） Analysis on results of uniform design experiment： Wang Yufangs uniform design software 3.0
30、can be used for multivariate gradual regression analysis based on the data in Table 3， and ultimately the regression equation and corresponding parameters can be gained. Regression equation： y=b（0）+b（1）*X（3）+b（2）*X（4） Regression coefficient： b （i）： b（0）=4.858； b（1）=0.1257； b（2）=0.3591 Standard regre
31、ssion coefficient： B （i）： B（1）=0.8144； B（2）=0.3879； multiple correlation coefficient：R=0.9696； determination coefficient：R2=0.9402； correction 9determination coefficient：R2a=0.9327 Regression equation significant inspection： sample capacity N=10； significance level=0.05 ； check value Ft = 55.01； cri
32、tical valueF （0.05， 2，7）=4.737， FtF（0.05， 2，7） ； Regression equation is significant. Residual standard deviation： s=0.3887. The contribution of elements of all equations to regression （per partial regression square and descending order）： U （1） =11.26， U （1）/U=67.73%； U （2） =2.554， U （2）/U=15.37%. Fr
33、om the standard regression coefficients of all factors， the factor exerting the biggest effect on polysaccharide concentration is solid-liquid ratio within the above levels and follows pHs， but the extraction temperature and time have insignificant effects on the concentration. Thus， when the dried
34、fructus corni power is under the100-mesh sieve condition， the extraction temperature and time have insignificant effects on extraction rate of polysaccharides. To increase the extract concentration and maximize the extraction rate， it is better to only select the proper solid-liquid ratio and pH， bu
35、t not to raise temperature and time. The experimental optimization method is the grid trial method. The 10best extraction condition is X3 = 33 and X4 = 8.5； the pre-test polysaccharide concentration Y is 12.06 （+ /0.9190 mg/ml）. （3） Three-dimensional graphic surface figure can be drawn in the experi
36、mental range based on the solid-liquid ratio （Figure 5）. From figure 5， it can be seen that the factor influencing the polysaccharide concentration most is the solid-liquid radio （X3） in the curve， and follows pH （X4）. Figure-5 shows that the results of the 3-D graphic surfaces for polysaccharide ex
37、traction are consistent with the above analysis. （4） Verifying the experiment： To verify the reliability of optimization results， X1=80， X2=60min， X3=33 and X4=8.5 are used to verify the experiment （operate it in parallel for four times）. The average polysaccharide concentration is 12.68mg/ml and is
38、 close to the predicted value. Thus， this uniform design is of significance. 3. Conclusion Through the above uniform design， the optimization condition to extract polysaccharose from fructus corni is that the solid-liquid radio is 1：33 and pH is 8.5； and extraction temperature and time have insignificant influences on
