肖怀秋，李玉珍

湖南化工职业技术学院应用化学系，株洲412004

Introduction

In recent years，some bioactive polysaccharide(PS)isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology.They exhibit various biological activities［1］，such as strong antioxidant properties，and can be explored as a novel potential antioxidant［2］.

Fern is a sporous plant with fascicular，and now exists approximately 12，000 species around the globe.In China，there is about 61 families，223 genus，2600 species，there exist around 300 species may be selected for the purpose of medicine usage［3］.Lygodium japonicum (Thumb.)SW，one of the most valued traditional Chinese medicines(TCMs)，can be found in hillside，forest，bosk，and lawn in the Yangtze River drainage region and several southern provinces in China.The whole plant and mature spore of this plant have been used for the treatment of pneumonia，acute gastroenteritis，dysentery，urinary tract infection，skin eczema，nephritis dropsy，urine calculus，and so on［4］.

Currently，many works mainly centralized in its curative effects［5］，but less on the extraction parameters optimization using response surface methodology(RSM)and the function mechanism for supporting its versatile bioactivity，such as antioxidant activity.In this study，a RSM based on a three-factor-three-level Box-Bohnken Design(BBD)was employed to identify and optimize the critical，and significant extraction conditions that will maximize the production of PS.And meanwhile，antioxidant activities in vitro against Oand·OH of purified PS were also investigated.

Materials and Methods

Plant materials and chemicals

Aerial part of L.japonicum was collected from Jiulang Mountain located in Zhuzhou city，Hunan Province and identified by LI Lan who graduated from Hunan University of Chinese medicine.The collection was naturally dried，and ground into powder.Vitamin C was purchased from Nanjing Jianchen Ltd.，Co..And all other chemicals and reagents were of analytical grade.

Extraction and preparation of crude PS

L.japonicum was weighed(approx.5.0 g)and dipped in proper amount of double distilled water，and extracted at a certain temperature for a period，and the resulting suspension was centrifuged(4000 g for 30 min)，the resulting supernatant was then membrane-filtered (0.45 μm，Millipore)，the filtered aqueous solutions were cooled，and then，vacuum freeze-drying at-14℃，giving a final volume of about 50 mL.Three volumes of anhydrous alcohol were added to this concentrate，the resulting mixture was placed in a refrigerator at-4℃overnight.The resulting precipitate was separated by centrifugation，washed exhaustively using 96%alcohol，dissolved in deionized water，and prepared for further purification.

Purification of crude PS

Trichloroacetic acid(TCA)was added to the crude PS solution to denature and precipitate protein，and dialyzed using dialysis bag at-4℃ overnight.Trace pigment was absorbed using activated charcoal.One hundred milligrams of crude PS was dissolved in distilled water and the resulting solution was membrane-filtered (0.45 μm)and applied to a DEAE-52 cellulose column(1.6×40 cm).The column was eluted firstly with deionized water，and then successively with 0.5 mol/L KCl at a flow rate of 30 mL/h(data omitted).PS concentration in each fraction was monitored by the phenol-sulfuric acid method［6］，and then，purified PS was gained and collected for further investigations.

Extraction parameters optimization of PS using

RSM

The experimental design employed in the present study was a Box-Bohnken Design with three affected factors，i.e.，extraction time(ETm)，the ratio of sample to water(S/W)and extraction temperature(ETp).The experimental plan consisted of 17 trails and the value of the independent response was the mean of the triplicate.The second-order polynomial coefficients were calculated and analyzed using the‘Design expert’(Version 7.0.0，Stat-Ease Inc.，Minneapolis，USA) statistical package.Statistical analysis of the fitted model was employed to evaluate the analysis of variance (ANOVA).

Determination of OFR scavenging activities in vitro Sample solutions with different concentrations(0～300 μg/mL)were prepared for antioxidant appraisal.

The ability of the purified PS to scavenge Owas determined according to the method represented by Nishikimi［7］.The scavenging effect was calculated using the following equation.

Scavenging effect(%)=［(Acontrol-ASample)/Acontrol］× 100%

Determination of·OH

Measurement of·OH scavenging activity of purified PS was investigated based on the approach described by Chung［8］with some modifications.The reaction mixture consisted of 0.1 mL of 10 mM FeSO4，0.1 mL of 10 mM EDTA，0.5 mL of 10 mM α-deoxyribose，0.9 mL of sodium phosphate buffer(pH7.4)and 1.0mL of various concentrations of PS sample and standard were thoroughly mixed in a tube separately.Hydrogen peroxide(0.2 mL，10 mM)was then added and the reaction mixture was incubated at 37℃ for 1 h.One milliliter of 2.8%TCA and 1.0mL of 1.0%thiobarbituric acid(TBA)were added to the test tubes and boiled for 15 min.After cooling the mixture，the absorbance was measured at 532 nm.Sodium phosphate buffer(pH 7.4)instead of sample was used as blank.The scavenging activity was evaluated as the inhibition rate of α-deoxyribose oxidation by·OH.The scavenging effect was determined using the formula described by Ren［9］.

Statistics

All the data were expressed as mean±standard deviation(SD)of the triplicate.The values were regarded to be significantly different when P＜0.05.

Results and Discussion

Optimization of extraction parameters

In RSM，a prior knowledge to understand performance of the process and process variables under investigation is necessary for achieving a more realistic model［10］.Preliminary trials［(Plackett–Burman design，PBD) and(steepest ascent design，SAD)］indicated that the range of ETm(90～150 min)，S/W(1∶10～1∶30，w/ v)and ETp(45℃ ～55℃)is beneficial for PS extraction.The range and levels of the three independent variables are presented in Table 1.

Table 1 Level and code of variables chosen for Box-Bohnken Design

The design matrix of the variables in coded format is presented in Table 2.Each run was repeated in triplicate，and thus the values list in Table 2 were the average value of the triplicate，and meanwhile，the predicted values of the response were obtained from quadratic model fitting techniques and also be displayed in Table 2.

Table 2 Box-Bohnken Design matrix along with the experimental and predicted values

9 0 0 0 12.413 12.85 10 0 -1 -1 6.611 6.90 11 -1 -1 0 8.611 8.63 12 0 0 0 12.317 12.85 13 0 0 0 12.216 12.85 14 0 0 0 12.312 12.85 15 0 1 -1 9.743 9.73 16 -1 0 1 8.627 8.59 17 0 0 0 12.825 12.85

It can be seen from Table 2，there is a considerable variation for PS extraction rate depending on the extraction conditions.The replication at the central point conditions resulted in higher the PS extraction rate than at other levels.The predicted value(y)can be fitted and described as:

Where y is the predicted value，and x1，x2and x3are the coded value of the tested variables.

The statistical significance of the fitted Eq.(1)was checked by F-test，and the ANOVA for quadratic model is summarized in Table 3.

Table 3 ANOVA for the fitted quadratic polynomial model

The value of Adj.R2(0.9669)suggests that 96.69% of total variation is attributed to the independent variables and only 3.31%of the total variation can not be explained effectively by this model.The value of R (0.9943)indicates good agreement between the predicted and experimental value.The value of lack-of-fit for quadratic regression Eq.(1)is not significant(p= 0.1305)，which indicates that the model equation was adequate for predicting the response under any combination of values of the variables.The coefficient estimates of Eq.(1)，along with the corresponding p-value are presented in Table 4.

Table 4 Results of regression analysis of a full second-order polynomial model

The p-values can be obtained from Table 4 that x1is significant(P＜0.05)，x21，x22，x23are extremely significant(P＜0.01)，nevertheless，x2，x3，x1x2，x1x3and x2x3are not significant(P＞0.05).The 3D surface plot of the graphical discription for the regression equation is presented in Fig.1-A，1-B and 1-C.

Fig.1 Response surface plot of effects of ETp and ETm，S/W and ETm，and ETp and S/W on PS extraction rate

The main task of response surface is to hunt efficiently for the optimum values of the variables for maximizing the response.Elliptical contours are obtained when there is a perfect interaction between the independent variables［11］.The optimal predicted value was obtained by fitted model as approximately 12.446%.By solving the inverse matrix(from Eq.(1))，the optimum value of the tested variables in coded format are x1=0.11，x2=0.09 and x3=-0.02，in uncoded(natural)units are ETm of 123.3 min，S/W of 1∶20.9(w/v)and ETp of 49.9℃，respectively.Under these conditions，the crude PS extraction rate of verification experiment is 12.85%±0.18%(n=3)，slightly greater than that obtained from the plot analysis.

Fig.2 Oand·OH scavenging capacity of the purified PS and Vitamin C

Scavenging activity of the purified PS to Oand ·OH

Fig.2-A illustrates that the purified PS was capable of scavenging Oin an concentration-dependent manner(linear regression equation is y =5.9838x+ 0.081，R2=0.9449)，which is similar with the results reported by Li X［3］.The 50%of Oinhibition concentration(IC50%-O)of purified PS and Vitamin C are 83.42 μg/mL and 62.06 μg/mL，respectively.The inhibition precentage of Ogeneration by 300 μg/mL doses of the purified PS and Vitamin C was found as 96.26%and 96.14%，respectively.The results demonstrated that the purified PS exhibits strong Oscavenging activity，and could bear comparison with that of Vitamin C.The·OH scavenging activity of purified PS and Vitamin C are presented in Fig.2-B，the inhibition percentage of different purified PS concentrations on·OH scavenging can be formulated as a linear regression equation y=5.5912x-1.2655(R2=0.9207).Results illustrated that the purified PS possesses a strong·OH scavenging capacity.50%of·OH inhibition concentration(IC50%-·OH)for PS and Vitamin C are 91.69 μg/mL and 66.16 μg/mL，respectively.The precentage inhibition of·OH generation by 300 μg/mL doses of the purified PS and Vitamin C was found as 75.63%and 74.82%，respectively.

Conclusion

In this study，a RSM based on a Box-Bohnken Design was applied for optimization of crude PS extraction.The optimum conditions were determined as ETm of 123.3 min，S/W of 1∶20.9(w/v)and ETp of 49.9℃，the predicted value and verification experimental value of PS extraction rate are 12.446%and 12.850%，respectively.The antioxidant evaluation of purified PS in vitro revealed that PS possesses strong Oand·OH scavenging activity，which may be comparable to vitamine C.Through our investigations，it may be rational to assure that PS from L.japonicum can be used as an effective OFRs scavenger and play its curative role in traditional medicine due to the mechanism of antioxidant ability in it.

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