Abstract

To use C.tubulosa resources in Xinjiang efficiently, several extraction methods of polysaccharide from Hericium erinaceus were compared. The aim of this study is to use a Response Surface Methodology (RSM) to optimize the Acetone reflux (ARE), Acetone microwave reflux (AMRE). Acetone ultrasonic microwave reflux (AUMRE) extraction of phenylethanoid glycosides compounds (PEG) from Xinjiang c.tubulosa. Different extraction methods have different effects on the yield and antioxidant activity of phenyl glycoside. The results showed that the yield of phenylethanoid glycosides ARE > AUMRE > AMRE, Vc as a positive control to obtain antioxidant capacity Vc > ARE > AMRE > AUMRE.

1. Introduction

Cistanchetubulosa (Schenk) Wightis known as red willow broomrape, it belongs to orobanchaceae cistanche, perennial parasitic herbs. The stem has medicine effect for blood, kidney yang, laxative. It was known as the "desert ginseng". In recent years, its chemical composition and pharmacological activity were studied by many domestic and foreign scholars, a hundred kinds of chemical components were isolated, mainly including phenylethanoid glycosides, iridoid glycosides, lignan glycosides, monosaccharides, disaccharides, polysaccharides, amino acids, peptides, proteins, triterpene, sterols, polyol, et al ¹. The main active constituent of Cistanche genus was phenylethanoid glycosides. It has anti-bacterial, anti-inflammatory, anti-virus, anti-tumor, anti-oxidation, immune regulation, hypoglycemic and the effect of enhancing memory ^{2, 3, 4, 5}. Therefore, the study on the extraction of phenylethanoid glycosides compounds has important significance for its efficacy and new product research and development. But most of the researchers on the study of extraction of Cistanchetubulosa confined to the optimization of process conditions for a kind of extraction methods. Due to the different experimental materials, detection environment, equipment and other factors, the yield of phenylethanoid glycosides compounds has a great difference.

Acetone is a non-proton solvent, it has strong polarity and dissolve ability, so it can remove some small molecular sugar and fat-soluble impurities. In industrial production, it can be d because of its lower boiling point. Microwave extraction is easy to cause uneven heating of the solution, but the cavitation effect of ultrasonic vibration causes the sample medium to heat at each point, and it does not destroy the structure of the material itself ⁶. Ultrasonic microwave synergistic extraction, which has both advantages and disadvantages. Thereby it can increase the extraction yield of the active material, reduce the extraction time greatly, reduce energy consumption. It is widely used in the research of extraction ⁷.

In this study, with the same kind of Cistanchetubulosa as the raw material, acetone as solvent, using a variety of extraction methods comparative of Xinjiang Cistanchetubulosa phenylethanoid glycosides compounds (PEG), and the antioxidant activity of extracts obtained by different extraction methods were compared. It provides a theoretical basis for the reasonable development of the rare endangered Chinese medicinal resources of Cistanchedeserticola use. It provides more updates and scientific basis for industrial extraction.

2. Materials and Methods

Cistanche is provided by Xinjiang nuclear Life Tech Co, Ltd. All chemicals (analytical grade) used in the experiment were purchased from Urumqi sand District Albert experimental equipment supply station. CW-2000 ultrasonic-microwave synergistic extraction device (fixed ultrasonic frequency 40kHz, power 50W), Shanghai New Extension Analysis Instrument Co, Ltd.

Cistanchetubulosa was ground in a high speed universal grinder (Beijing Guangming Medical Instrument Co, Ltd. ) over 60 mesh sieve to obtain a fine powder. All tested samples were defatted by blending the ground material with light petroleum(1:3 W/V, 2 h, ×5) Reflux in the round bottom flask at ambient temperature. Defatted Cistanchetubulosa samples were air dried for 24h and stored in vacuum packaged polyethylene pouches at -4°C until used for further analysis.

Weighing 10 mg of Echinacea reference substance by colorimetry accurately and dissolve it in methanol in a 50 mL volumetric flask, then diluted it to the scale with distilled water and shook it well. Absorbed the above solution 0, 1, 2, 3, 4, 5 mL. Setting 25 mL capacity bottle respectively, added the mass fraction of 5% sodium nitrite 1 mL, shook well and placed for 6 min, added the mass fraction of 10% aluminum nitrate solution 1 mL, haked and standed still 6 min. Added the mass fraction of 10% sodium hydroxide solution 10 mL, diluted with distilled water to the scale, shook well and placed for 18 min, took the first tube as a blank, measured the absorbance values at 510 nm, where: absorbance as the vertical coordinate, the concentration of the echinacoside as the horizontal coordinate. Obtain the regression equation of the standard curve is y=0.0086x-0.0227, R2=0.9974. Each taking 2 g c.tubulosa extraction, the extracted solution centrifuged at 3000 r.s-1 centrifuge 15 min, the supernatant was volumed in 100 mL flask. After took 1 mL solution of volumetric flask was diluted 300-fold, measured absorbance value with the same method, calculation of phenylethanoid glycosides in c.tubulosa yield.

Note: The yield of phenylethanoid glycosides (%) = the phenylethanoid glycosides content of extraction (g) /weight of powder(g).

On the basis of the single-factor experiment, three factors that had a great influence on each extraction method was selected as the response variables of each method from the five factors of liquid ratio, the volume fraction of acetone, extraction time, extraction temperature and microwave power, and the yield of phenylethanoid glycosides as the response value. Then three groups of RSM experiments (Table 1) with three-factor and three-level were designed according to Box-Benhnken Central combination design principle. Finally, the extraction rate of the three methods were compared.

Reducing powers of phenylethanoid glycosides were determined according to the method ⁸, with some modifications. Phenylethanoid glycosides 1 mL were mixed with potassium ferricyanide (K3Fe(CN)6, 2.5 mL, 1%) in sodium phosphate buffer (NaH2PO4-Na2HPO4, 2.5 mL, 0.2 mol/L, pH 6.6), and the mixture was incubated at 50°C for 20 min. At the end of the incubation period, TCA (2.5 mL, 10 g/100 mL)was added to the mixture and centrifuged at 3000 g for 10 min at room temperature. The resulting supernatant was taken and mixed with 2.5 mL of H2O and 0.5 mL of ferric chloride (0.1 g/100 mL), then, incubated at 37°C for 10 min. The absorbance of the final mixture was measured at 700 nm. The increase of the absorbance of the reaction mixture indicated that the reduction ability of phenylethanoid increased.

Analysis of variance, the linear regression coefficient, and the quadratic regression equation of three different methods by the response surface Box-Benhnken were obtained. Moreover, response surface interaction and contour plots were obtained. The data were fitted using the Design-Expert 8.0.6.

3. Results and Discussion

Based on the results of Box-Behnken Central composite design, the actual set of experiments performed are lists in Table 2, a modified design from ⁹. The experimental design consists of twelve factorial points and five replicates of the central point. Experimental runs were randomised, to minimise the effects of unexpected variability in the observed responses.

Using Design-Expert 8.0.6 software analyze the data of Table 2, obtain the Table 3 of regression model analysis of variance, the results of analysis of variance (ANOVA) showed that the contribution of the quadratic model was significant. The F-value and p-value in Table 3 were used to determine the significance of each coefficient. The corresponding variables would be more significant if the absolute F-value becomes greater and the p-value becomes smaller. Lack of fit was also given in Table 3, in order to check the quality of the fitted models. The fitted quadratic models equation for ARE, AMRE, AUMRE as follows:

(1)

(2)

(3)

Table 3 shows the significance of the linear relationship between the dependent variable and the independent variables. The model could account for 97.77% of the variance in the response value, which indicates a good fit of the model. The non-significance of the lack of fit (0.0794) showed that the quadratic regression equation could forecast the response value well. The significance terms of A, B, C, AB, BC, A2, B2 and C2 demonstrated that they could have a big influence on the response value. The results also suggest that the most important independent indices were the change of volume fraction (A), extraction time (B) and liquid feed ratio (C), in that order. These values would give a relative good fit to the mathematic model in Eq. (1).

Table 3 shows the significance of the linear relationship between the dependent variable and the independent variables. The model could account for 99.43% of the variance in the response value, which indicates a good fit of the model. The non-significance of the lack of fit(0.2013)indicated that the quadratic regression equation could forecast the response value well. The significance terms of A, B, C, AB, BC, A2, B2 and C2 demonstrated that they could have a big influence on the response value. The results also suggest that the most important independent indices were the change of volume fraction (A), extraction time (B) and microwave power (C) , in that order. These values would give a relative good fit to the mathematic model in Eq. (2).

Table 3 shows the significance of the linear relationship between the dependent variable and the independent variables, the non-significance of the lack of fit (0.1160) showed that the quadratic regression equation could forecast the response value well. The model could account for 99.46% of the variance in the response value, which indicates a good fit of the model. The significance terms of A, B, C, AB, AC, BC, A2, B2 and C2 demonstrated that they could have a big influence on the response value. The results also suggest that the most important independent indices were the change of volume fraction (A), microwave power (B) and liquid feed ratio (C), in that order. These values would give a relative good fit to the mathematic model in Eq. (3).

Figure 1-7 show the interaction response surface between each pair of response values in three methods. As can be seen from Table 3, when ARM extraction was used, the model interaction term AC had no significant affect on the response value. Therefore, the 3D plots shows at only Figure 1-7.

3D response surfaces and contour plot were shown in Figure 1-2 to demonstrate the changes in the yield of phenylethanoid glycosides as a function of model interaction terms extraction time and volume fraction extraction time and liquid feed ratio of ARE. Figure 1 showed that the region of extraction time (30 min~72 min) and volume fraction (55%~60%) would give a higher yield (51.9 mg/100 g). As the volume fraction increased, the yield decreased to 39.2%. Figure 2 showed that the region of liquid feed ratio (10:1~16:1) and extraction time (30 min~72 min) would give a higher yield (51.9 mg/100 g). As the liquid feed ratio and volume fraction increased, the yield had no significant change.

3D response surfaces and contour plot shown in Figure 3-4 demonstrated the changes in the yield of phenylethanoid glycosides as a function of model interaction terms extraction time and volume fraction, microwave power and extraction time of AMRE. Figure 3 showed that the region of extraction time (10 min~25 min) and volume fraction (55%~61%) would give a higher yield (47.48 mg/100 g). With a further increase of extraction time and volume fraction, the yield showed a slight decrease. Figure 4 showed that the region of microwave power (350 W~425 W) and extraction time (10 min~25 min) would give a higher yield (47.48 mg/100 g). As microwave power and extraction time increased, the yield decreased from 47.48 mg/100 g to 44.15 mg/100 g.

3D response surfaces and contour plot shown in Figure 5-7 demonstrated the changes in the yield of phenylethanoid glycosides as a function of model interaction terms microwave power and volume fraction, volume fraction and liquid feed ratio, microwave power and liquid feed ratio of AUMRE. Figure 5 showed that the region of microwave power (90 W~105 W) and volume fraction (45%~51%) would give a higher yield (49. 8 mg/100 g). With a further increase of microwave power, the yield showed a sharp decrease. Figure 6 show that the region of volume fraction (45%~51%) and liquid feed ratio (10:1~16:1) would give a higher yield (49.8 mg/100 g). As the liquid feed ratio increased, the yield decreased. Figure 7 show that the region of microwave power (90 W~105 W) and liquid feed ratio (10:1~16:1) would give a higher yield (49.8 mg/100 g). With a further increase of microwave power, the yield showed a sharp decrease.

As it can be seen from Figure 8, in the concentration range of 0 mg/mL~ 0.5 mg/mL, the antioxidant activity of different methods for extracting PEG and Vc. The reduction force of PEG and Vc increased linearly with the increase of concentration. Reduction force the size of the order is as follows: Vc> ARE> AMRE> AUMRE. This is due to the ultrasonic microwave process, the part of the active substance degeneration, reduced antioxidant capacity, reduced power did not change significantly when the concentration exceeds 0.5 mg/mL, and reducing power Vc the strongest, ARE slightly lower than Vc, indicating that PEG reducing power is relatively large.

To obtain the optimal process for each group, for three parallel experiments and get the phenylethanoid glycosides yield prediction values and theoretical values are close, has a very good fitting. Experimental results demonstrate the correctness of the built model (Table 4).

4. Conclusions

The results show that the optimum extraction conditions by quadratic regression model for the reflux of acetone are as follows: volume fraction 59.59%, extration time 66.19 min, liquid feed ratio 15.72:1(mL.g-1). For the Acetone microwave reflux is as follows: volume fraction 60.88%, extration time 22.15 min, microwave power 418.24 W, the ultrasonic frequency 40 kHz, the ultrasonic power is 50 W, the extraction time is 15 min. For the ultrasonic microwave reflux of acetone is as follows: extraction concentration 50%, extraction microwave power 100 W, ratio of liquid-solid14:1 (mL.g-1). Reflux extraction method takes the longest, but PEG yield is relatively high. Microwave and ultrasonic microwave method is shorter, but due to the microwave and ultrasonic vibration, will destroy the structure of some extractions, the yield of the resultant lower than reflux extraction, it provided a theoretic basis for future industrial productions required, better service in practical operation.

Acknowledgements

The authors thank Professor Liang Wang for his suggestions and revising the manuscript. And thank the college of life science and technology of Xinjiang University for its financial support.

References