Study on the Technological Conditions of Ultrasound-assisted Extraction of Polysaccharides from Ajuga ciliata Bunge (Fujian)

Wenwen LU, Ruonan HE, Zhongwei CHEN, Chao YI, Zhuangyu ZHANG, Yanyan ZHENG

Xiamen Key Laboratory of Marine Medical Natural Products Resources, Xiamen Medical College, Xiamen 361000, China

Abstract [Objectives] To determine the optimum technological conditions of ultrasound-assisted extraction of polysaccharides from Ajuga ciliata Bunge. [Methods] In this study, Ajuga ciliata Bunge was used as raw material, and an orthogonal experiment was designed based on the single factor experiment to investigate the effects of ultrasonic time, solid-liquid ratio, ultrasonic temperature and ultrasonic power on the extraction rate of polysaccharides from A. ciliata Bunge (Fujian). Range analysis, variance analysis and SSR test of significant differences between groups were carried out with SPSS 20.0, and the test results were verified. [Results] Under the conditions of ultrasonic temperature of 85 ℃, ultrasonic time of 80 min, solid-liquid ratio of 1∶15 g/mL and ultrasonic power of 500 W, the yield of polysaccharides from A. ciliata Bunge (Fujian) was the best. [Conclusions] This study can provide theoretical basis for the application of A. ciliata Bunge (Fujian).

Key words Ajuga ciliata Bunge, Polysaccharide, Ultrasonic extraction, Orthogonal test

1 Introduction

AjugaciliataBunge is a plant in the Labiatae family, and its whole grass can be collected, dried or used in fresh state in spring, summer and autumn.A.ciliataBunge has the effects of relieving cough, eliminating phlegm, relieving asthma, inhibiting bacteria, resisting inflammation[1], and promoting immunity[2-3]. It can be used to treat respiratory tract diseases, digestive tract diseases, hepatobiliary diseases, sores and pyogenic infections. In addition, it is also used for the treatment of snakebite and mad dog bite[4]. The polysaccharide compound contained inA.ciliataBunge is one of its main active components. There are many extraction methods of polysaccharides, among which ultrasonic extraction is a relatively simple, efficient and reproducible extraction technology that can realize industrial application[5-7]. In this study, withA.ciliataBunge (Fujian) as raw material, ultrasonic-assisted extraction technology was used to optimize the technological conditions through single-factor and orthogonal experiments to determine the optimal technological conditions for the extraction of polysaccharides, so as to provide theoretical basic data for the application ofA.ciliataBunge (Fujian).

2 Materials and methods

2.1Materials

2.1.1Raw materials and reagents.A.ciliataBunge was purchased from Haicang District, Xiamen City, Fujian Province; glucose standard solution was purchased from Chengdu Ruifen Biotechnology Co., Ltd.; phenol, sulfuric acid and other reagents were analytically pure and purchased from Xilong Chemical Co., Ltd.

2.1.2Instruments and equipment. UV-2100 UV-vis spectrophotometer, Unico (Shanghai) Instrument Co., Ltd.; 3K15 centrifuge, SIGMA; ALC-2100 electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; KQ-500DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; HWS-24 electric thermostat water bath, Shanghai Yiheng Scientific Instrument Co., Ltd.

2.2Methods

2.2.1Extraction method of polysaccharides. After crushing and sifting through the 45-mesh screen, the dried powder was stored in a laboratory bottle at 4 ℃. 1.0 g dried powder ofA.ciliataBunge was taken and mixed with purified water according to the solid-liquid ratio, and it was shaken well for ultrasonic extraction under the set ultrasonic power, time and temperature. After ultrasonic treatment, the supernatant was sucked out by centrifugation (10 000 r/min, 4 ℃) for 10 min, and set aside. The precipitation residue was extracted twice again, and the supernatant was combined for single factor and orthogonal experiments, to investigate the effects of solid-liquid ratio, ultrasonic power, ultrasonic temperature and ultrasonic time on the yield of polysaccharides fromA.ciliataBunge. The above experiments were repeated 3 times.

2.2.2Single-factor test method. The effects of ultrasonic extraction temperature on the yield of polysaccharides fromA.ciliataBunge were investigated∶solid-liquid ratio (20∶1 mL/g), ultrasonic power (350 W) and ultrasonic time (30 min). The effects of ultrasonic temperature at 50, 60, 70, 80, 90 ℃ on the yield of polysaccharides fromA.ciliataBunge were investigated, respectively.

The effects of ultrasonic extraction time on the yield of polysaccharides fromA.ciliataBunge were investigated∶solid-liquid ratio (20∶1 mL/g), ultrasonic power (350 W) and ultrasonic temperature (70 ℃). The effects of ultrasonic extraction time at 10, 30, 50, 70, 90 and 110 min on the yield of polysaccharides fromA.ciliataBunge were investigated, respectively.

The effects of the solid-liquid ratio on the yield of polysaccharides fromA.ciliataBunge were investigated∶ultrasonic power (350 W), ultrasonic temperature (70 ℃) and ultrasonic time (30 min). The effects of solid-liquid ratio at 10∶1, 20∶1, 30∶1, 40∶1, 50∶1 mL/g on the yield of polysaccharides fromA.ciliataBunge were investigated, respectively.

The effects of ultrasonic power on the yield of polysaccharides fromA.ciliataBunge were investigated∶solid-liquid ratio (20∶1 mL/g), ultrasonic temperature (70 ℃) and ultrasonic time (30 min). The effects of ultrasonic power at 100, 200, 300, 400, 500 W on the yield of polysaccharides fromA.ciliataBunge were investigated.

2.2.3Orthogonal test method. On the basis of single factor experiment, 4 factors which had obvious effect on the yield of polysaccharides fromA.ciliataBunge were selected, and 3 levels were selected for each factor. L9(34) was used to carry out orthogonal experiment, and the experiment was repeated 3 times in each group. Range analysis, variance analysis and SSR test of significant differences between groups were carried out with SPSS 20.0, the extraction conditions were optimized and the test results were verified.

2.2.4Method for determination of polysaccharides. Phenol-sulfuric acid coloration method: the sample solution was pipetted, and mixed with 1.0 mL of 6% phenol solution, and then 5.0 mL of concentrated sulfuric acid. After shaking well and cooling, the mixture was taken in a boiling water bath for 15 min for color reaction[8-9].

Determination of the maximum absorption wavelength: the newly extracted polysaccharide solution was diluted or concentrated to a proper concentration, and after the color reaction, the maximum absorption wavelength was determined by ultraviolet spectrophotometer at the wavelength between 200 and 600 nm.

The drawing of the standard curve: the glucose standard solution was dried to constant weight at 105 ℃, and 20.000 mg of standard substance was accurately weighed in the beaker. After it was completely dissolved with proper amount of water, the glucose standard mother solution[10-11]was prepared by transferring it to a 500 mL volumetric flask. 0.0, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mL of mother solutions of glucose standard sample were accurately pipetted into 10 mL test tubes, respectively, and it was replenished up to 2.0 mL with distilled water, respectively. The absorbance was determined immediately at the determined maximum absorption wavelength by phenol-sulfuric acid color-developing method. Taking the concentration of glucose standard solution as abscissa and the corresponding absorbance as ordinate, the standard curve was drawn and linear regression equation was established[12].

Determination of polysaccharides content ofA.ciliataBunge: after the color reaction of 1 mL of extract of polysaccharides fromA.ciliataBunge using phenol-sulfuric acid coloration method, the absorbance was measured at the determined maximum absorption wavelength, and the content of polysaccharides in the extract was calculated by standard curve regression equation. It is calculated as follows:

whereCis the concentration of polysaccharides fromA.ciliataBunge calculated by substituting the measured absorbance into the regression equation (mg/mL);Vis the volume of the extract (mL);Kis the dilution factor;mis the weight of dried powder ofA.ciliataBunge (g).

3 Results and analysis

3.1DrawingofglucosestandardcurveThe ultraviolet spectrophotometer was used to scan the spectrum between 200 and 600 nm, and the maximum absorption wavelength was determined to be 485 nm. With the concentration of glucose standard solution as the abscissa and the corresponding absorbance as the ordinate, the standard curve is drawn (Fig.1). The linear regression equationy=0.112 1x-0.008 4 (R2=0.999 5) was obtained. The concentration of glucose standard solution showed a good linear relationship with absorbance in the range of 2.0-9.0 μg/mL.

Fig.1 Glucose standard curve

3.2Singlefactortestresults

3.2.1Effect of ultrasonic extraction temperature on the yield of polysaccharides. As shown in Fig.2, the extraction rate of polysaccharides fromA.ciliataBunge increased with the increase of ultrasonic extraction temperature, but decreased when the temperature exceeded 80 ℃, so the optimum ultrasonic extraction temperature was 80 ℃.

Fig.2 Effect of ultrasonic extraction temperature on the yield of polysaccharides from A. ciliata Bunge

3.2.2Effect of ultrasonic extraction time on the yield of polysaccharides. As shown in Fig.3, the extraction rate of polysaccharides fromA.ciliataBunge increased with the increase of ultrasonic extraction time. When the ultrasonic extraction time was more than 90 min, the extraction rate of polysaccharides fromA.ciliataBunge decreased, so the best ultrasonic extraction time was 90 min.

Fig.3 Effect of ultrasonic extraction time on the yield of polysaccharides from A. ciliata Bunge

3.2.3Effect of solid-liquid ratio on the yield of polysaccharides. As shown in Fig.4, the extraction rate of polysaccharides fromA.ciliataBunge increased with the increase of the solid-liquid ratio. When the solid-liquid ratio exceeded 20∶1 mL/g, the extraction rate of polysaccharides fromA.ciliataBunge decreased, so the best solid-liquid ratio was 20∶1 mL/g.

Fig.4 Effect of solid-liquid ratio on the yield of polysaccharides from A. ciliata Bunge

3.2.4Effect of ultrasonic power on the yield of polysaccharides. As shown in Fig.5, the extraction rate of polysaccharidesfromA.ciliataBunge increased with the increase of ultrasonic power. When the ultrasonic power was more than 450 W, the extraction rate of polysaccharides fromA.ciliataBunge decreased, so the best ultrasonic extraction power was 450 W.

Fig.5 Effect of ultrasonic power on the yield of polysaccharides from A. ciliata Bunge

3.3OrthogonalexperimentaldesignandresultanalysisBased on the results of single factor experiment, L9(34) was used to carry out orthogonal experiment. The factors and levels are shown in Table 1, the design results and range analysis are shown in Table 2, and the analysis of variance is shown in Table 3. The experiment was repeated 3 times in each group. According to theRvalue in Table 2, the influencing factors are in the following order: A ultrasonic temperature>D ultrasonic power>B ultrasonic time>C solid-liquid ratio. Based on the range comparison of various factors at various levels, the optimum extraction conditions were determined as follows: ultrasonic temperature of 85 ℃ (A3), ultrasonic time of 80 min (B1), solid-liquid ratio of 15∶1 g/mL (C1), ultrasonic power of 500 W (D3). The analysis of variance in Table 3 showed that ultrasonic temperature had a significant effect on the extraction process.

Table 1 Factors and levels of orthogonal test

3.4SSRtestforsignificanceofdifferencesamongtreatmentsinorthogonaltestAs shown in Table 4, there was no significant difference between level 1 and level 2 of factor A (ultrasonic temperature), but there was a significant difference between level 3 and level 1 or level 2. There was no significant difference among the levels of factor B (ultrasound time), factor C (solid-liquid ratio) and factor D (ultrasound power). Considering the process cost and the energy factors in the extraction process, the optimum extraction conditions were as follows: ultrasonic temperature of 85 ℃ (A3), ultrasonic time of 80 min (B1), solid-liquid ratio of 15∶1 g/mL (C1), ultrasonic power of 400 W (D1).

3.5VerificationandanalysisoftestresultsTaking the range analysis results of orthogonal test and the adjustment results after SSR test for significant differences between groups as the technological conditions, three groups of parallel experiments were carried out to calculate and verify which group of technological conditions were the best technological conditions. Range analysis showed that the content of polysaccharides (167.15 mg/g) extracted by the technological conditions (A3, B1, C1, D3) was higher than the content of polysaccharides (143.68 mg/g) extracted by the technological conditions adjusted by SSR test (A3, B1, C1, D1). At the same time, comparing the results of orthogonal test in Table 2, the content of polysaccharides extracted by the technological conditions of range analysis (A3, B1, C1, D3) was higher than the content of polysaccharides extracted by the orthogonal test, while the content of polysaccharides extracted by the technological conditions adjusted by SSR test (A3, B1, C1, D1) was lower than the content of polysaccharides extracted by the technological conditions in No. 9 orthogonal test.

Table 2 Results of orthogonal experimental design and range analysis

Table 3 Analysis of variance of orthogonal test results

Table 4 SSR test for significant differences between groups

4 Conclusions

Ultrasound-assisted extraction is a rapid, efficient and environmentally friendly extraction method. In this study, ultrasound-assisted extraction method was used to extract polysaccharides fromA.ciliataBunge (Fujian). The strong vibration effect, cavitation effect and crushing effect generated by ultrasound are fully utilized to improve the extraction efficiency of polysaccharides fromA.ciliataBunge (Fujian). In this study, an orthogonal experiment was designed based on the single factor experiment to investigate the effects of ultrasonic time, solid-liquid ratio, ultrasonic temperature and ultrasonic power on the extraction rate of polysaccharides fromA.ciliataBunge (Fujian). Range analysis, variance analysis and SSR test of significant differences between groups were carried out with SPSS 20.0. The extraction conditions were optimized, and the test results were verified. Finally, the optimum conditions of ultrasound-assisted extraction of polysaccharides fromA.ciliataBunge (Fujian) were determined as follows: ultrasonic temperature of 85 ℃ (A3), ultrasonic time of 80 min (B1), solid-liquid ratio of 15∶1 g/mL (C1), ultrasonic power of 500 W (D3).