Extraction Optimization of Polysaccharides from Rhizoma Polygonati by Response Surface Methodology and Content Determination

Qianru SHEN, Lian LIAN, Hongyan YU, Cheng YANG

School of Biomedicine and Chemical Engineering, Liaoning Institute of Science and Technology, Benxi 117004, China

Abstract [Objectives] This study aimed to investigate the water extraction-alcohol precipitation technology and content determination method of polysaccharides in Rhizoma Polygonati. [Methods] Taking the content of polysaccharides in the extract as the indicator, the extraction process was optimized using single-factor tests and Box-Behnken Design. The content of polysaccharides extracted was determined by the anthrone-sulfuric acid method. Among the three factors in the test design, the solid/liquid ratio and extraction time had a significant effect (P<0.05, P<0.01), while the extraction temperature had no effect on the content of polysaccharides extracted (P>0.05). [Results] The optimal extraction process for polysaccharides in Rhizoma Polygonati was as follows: liquid/solid ratio 20∶1 (mL/g), extraction time 2 h, and extraction temperature 85 ℃. Under the optimal extraction process, the content of polysaccharides in the extract reached 12.50%. [Conclusions] The extraction process optimized by response surface methodology for polysaccharides in Rhizoma Polygonati is reliable and has practical value, and can be used to guide production practices.

Key words Rhizoma Polygonati, Response surface methodology, Anthrone-sulfuric acid method

1 Introduction

Rhizoma Polygonati is the dried rhizome ofPolygonatumkingianumColl. Et Hemsl,PolygonatumsibiricumRed. orPolygonatumcyrtonemaHua, perennial herbaceous plants in the Liliaceae family, also known as Dahuangjing, Jitou Huangjing and Jiangxing Huangjing. It is mainly distributed in southwest, northeast, Inner Mongolia, Anhui and Zhejiang of China[1-3]. Rhizoma Polygonati contains polysaccharides, saponins, flavonoids, alkaloids, lignans and other chemical components. The 2015 edition ofChinesePharmacopoeiahas used polysaccharides as a quality control indicator for Rhizoma Polygonati and its content limit has been specified[1, 4-5]. Rhizoma Polygonati is a tonic traditional Chinese medicine. It is also widely used in folk as a medicinal and food plant, with functions of invigorating qi, nourishing yin, invigorating the spleen, moisturizing the lung and replenishing the kidney. It is mainly used to treat symptoms such as spleen and stomach qi deficiency, physical fatigue, lung deficiency, dryness and cough, and soreness and weakness of waist and knees[1, 6]. Modern pharmaceutical research shows that Rhizoma Polygonati has the effects of delaying aging, enhancing immunity, anti-fatigue, lowering blood sugar, anti-tumor, protecting cardiovascular, improving memory,etc., and it has huge development potential and broad market prospects[7-12]. However, the backward extraction and separation process has plagued the industrialization of Rhizoma Polygonati in China. In this paper, the water extraction-alcohol precipitation process of polysaccharides from Rhizoma Polygonati was optimized by response surface methodology to obtain the optimal parameter combination, and the content of polysaccharides in the extract was determined with anthrone-sulfuric acid method, with a view to improving the scientificity of quality evaluation and quality control of Rhizoma Polygonati and promoting the comprehensive development and utilization of Rhizoma Polygonati products.

2 Materials

2.1 Instruments and equipmentThe instruments and equipment used mainly included T6 UV-Vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd.), CPA225D micro electronic balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.], rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), SHZ-D circulating water vacuum pump (Gongyi Yuhua Instrument Co. Ltd.), HH digital display constant temperature electric heating water bath (Jiangsu Jintan Guosheng Experimental Instrument Factory), FW135 Chinese herbal medicine grinder (Tianjin City Taisite Instrument Co., Ltd.), KQ-300VDB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), FDA-50 vacuum freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.).

2.2 Reagents and drugsThe Rhizoma Polygonati sample (dried, 0.5 kg) was provided by Beijing Tongrentang Pharmacy (Benxi), and identified as the dried rhizome ofPolygonatumsibiricumRed. by Professor Li Feng of Liaoning University of Traditional Chinese Medicine. It was crushed and passed through a 60-mesh sieve for use. The other reagents and drugs used were mainly anhydrous glucose, absolute ethanol, anthrone, and concentrated sulfuric acid (analytically pure, Sinopharm Chemical Reagent Co., Ltd.).

3 Methods and results

3.1 Extraction process of polysaccharides from Rhizoma PolygonatiRhizoma Polygonati powder→extraction with hot water 3 times→suction filtration→filtrate combination and centrifugation→supernatant concentration→alcohol precipitation for 24 h→centrifugation→precipitation and freeze-drying→crude polysaccharides of Rhizoma Polygonati.

Under the conditions (extraction temperature, extraction time, liquid/solid ratio) designed in the single-factor and orthogonal tests, polysaccharides in Rhizoma Polygonati were extracted with hot water, and suction-filtered with Buchner funnel. The filtrate was combined, concentrated to about 20 mL, added with absolute ethanol to about 80% ethanol content, alcohol-precipitated for 24 h and centrifuged at 5 000 r/min for 5 min, and the flocculent precipitate was vacuum freeze-dried to obtain the crude polysaccharides[13-15].

3.2 Drawing of standard curve

3.2.1Preparation of reference solution. An accurate amount (10.0 mg) of anhydrous glucose standard dried to constant weight at 105 ℃was dissolved in deionized water to 100 mL to prepare into reference solution with a concentration of 0.1 mg/mL[16].

3.2.2Preparation of anthrone-sulfuric acid solution. An accurate amount (0.2 g) of anthrone was dissolved in concentrated sulfuric acid to 100 mL to prepare into anthrone-sulfuric acid solution (2.0 mg/mL), which was stored in a brown reagent bottle and kept in dark. The anthrone-sulfuric acid solution was used immediately after its preparation[14, 17-18].

3.2.3Selection of detection wavelength. An accurate volume (200 μL) of glucose reference solution was transferred to a 10 mL colorimetric tube, added with distilled water to 2.0 mL, shaken well, bathed in ice water, added with 6.0 mL of anthrone-sulfuric acid solution, bathed in boiling water for 8 min, cooled quickly to room temperature, and scanned at the wavelength range of 400-800 nm under the T6 UV-Vis spectrophotometer. The results show that there was a maximum absorption at 625 nm, so 625 nm was selected as the detection wavelength[17, 19].

3.2.4Preparation of standard curve. Different volume (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL) of the glucose reference solution was transferred to a 10 mL colorimetric tube, added with distilled water to 2.0 mL, and detected at 625 nm according to the description in Section3.2.3. Distilled water (2.0 mL) was used as the blank control. The regression equation was calculated[20]:Y=0.04X+0.058 5 (R2=0.999 3). It indicates that the glucose solution has a good linear relationship in the concentration range of 2.5-15.0 μg/mL.

3.3 Methodological investigation

3.3.1Precision test. The absorbance of the reference solution was determined six times repeatedly according to the method in Section3.2.4, and the absorbances obtained were 0.152, 0.151, 0.155, 0.152, 0.150 and 0.152, respectively (RSD=1.10%), indicating that the precision of the instrument is good and the measurement result is reliable.

3.3.2Stability test. The Rhizoma Polygonati test solution was determined 0, 1, 2, 4, 6, and 8 h after its preparation in line with the method in Section3.2.4, respectively, and the absorbances determined were 0.473, 0.472, 0.473, 0.471, 0.475 and 0.472, respectively (RSD=0.29%), indicating that the test solution is stable for at least 8 h.

3.3.3Reproducibility test. Six Rhizoma Polygonati test solutions, 0.2 mL for each, were determined according to the method described in Section3.2.4, respectively, and their absorbances were 0.513, 0.512, 0.513, 0.521, 0.515 and 0.520 (RSD=0.75%), indicating that the method has good reproducibility.

3.3.4Recovery test. Nine portions of Rhizoma Polygonati powder with known polysaccharides content were added with different amounts of reference substance, prepared into test solutions, and determined in line with the method in Section3.2.4, respectively. The recovery rate andRSDvalue were calculated (Table 1). The average recovery rate was 99.83%, and theRSDvalue was 1.70% (n=9), indicating that the method is accurate and feasible and can be used for the determination of Rhizoma Polygonati polysaccharides.

Table 1 Results of recovery test

3.4 Extraction and purification of polysaccharides in Rhizoma PolygonatiA certain amount (10.0 g) of Rhizoma Polygonati powder was weighed, added with 250 mL of 80% ethanol and refluxed in water bath at 90 ℃ for 1 h. The extraction was repeated twice, followed by suction-filtration. After the filter residue was dried, it was added with 200 mL of distilled water, bathed in boiling water for 1 h and suction-filtered. The extraction was repeated twice. The filtrate was combined, centrifuged at 5 000 r/min for 5 min, concentrate under reduced pressure to 10 mL, protein-removed with chloroform-n-butanol (V∶V= 4∶1), dialyzed with deionized water for 24 h, added with absolute ethanol to about 80% ethanol concentration, let standard overnight at 4 ℃, and centrifuged. The precipitate was washed with ether, acetone and absolute ethanol successively, and freeze-dried in vacuum to obtain refined polysaccharides.

3.5 Determination of conversion factorAn accurate amount (10 mg) of the refined Rhizoma Polygonati polysaccharides was dissolved in distilled water to 100 mL to prepare Rhizoma Polygonati polysaccharide stock solution, of which the absorbance was determined according to the method in Section3.2.4. According to the regression equation, the glucose concentration in the refined Rhizoma Polygonati polysaccharide stock solution was calculated. The conversion factor was calculated according to the following formula:

Conversion factor (f)=W/(C×D)

(1)

In the formula,Wis the weight refined Rhizoma Polygonati polysaccharides (mg);Cis the concentration of glucose in the refined polysaccharide stock solution (mg/mL); andDis the dilution factor of refined polysaccharides (mL).

The concentration of glucose in the stock solution was calculated based on the absorbance value of the refined Rhizoma Polygonati polysaccharides. According to formula (1), the conversion factor (f) was calculated to be 2.25.

3.6 Content determination of polysaccharides in samples

3.6.1Preparation of sample solution. An accurate amount (1.0 g) of Rhizoma Polygonati powder was extracted under different conditions, suction-filtered and centrifuged at 5 000 r/min for 5 min, and the supernatant was diluted with distilled water to 100 mL to prepare into Rhizoma Polygonati polysaccharides test solution[21].

3.6.2Content determination of polysaccharides in samples. The absorbance of the test solution prepared in Section3.6.1was determined according to the method in Section3.2.4. According to the regression equation, the concentration of glucose (C) in the tests solution was determined, and the content of polysaccharides was calculated according to the following formula:

Polysaccharides content (%)=(C×D×f/m)×100

(2)

In formula (2),Cis the concentration of glucose in the test solution (mg/mL);Dis the dilution factor of Rhizoma Polygonati polysaccharide solution (mL);fis the conversion factor; andmis the mass of Rhizoma Polygonati (mg).

3.7 Single-factor tests on extraction of polysaccharides from Rhizoma PolygonatiPolysaccharides in Rhizoma Polygonati were extracted according to the flow in Section3.1. The effect of extraction temperature, extraction time and liquid/solid ratio on the content of polysaccharides extracted was investigated by single-factor tests[14, 22].

3.7.1Effect of solid/liquid ratio on content of polysaccharides. Under the fixed extraction temperature (80 ℃) and extraction time (1.5 h), the effect of different liquid/solid ratio on the content of polysaccharides extracted was investigated. As shown in Fig.1, when the liquid/solid ratio increased from 10∶1 to 15∶1, polysaccharides in Rhizoma Polygonati rapidly penetrated into the solution due to the expansion of the concentration difference, leading to increased content. Within the liquid/solid ratio range of 20-30 (mL/g), it had little effect on the content of polysaccharides extracted. Therefore, the liquid/solid ratio should be controlled within 10-20 (mL/g).

Fig.1 Effect of liquid/solid ratio on content of polysaccharides extracted from Rhizoma Polygonati

3.7.2Effect of extraction time on content of polysaccharides. The extraction temperature and liquid/solid ratio were fixed at 80 ℃ and 15∶1 mL/g, and the effect of different extraction time on the content of polysaccharides extracted was investigated. The results are shown in Fig.2. The content of polysaccharides increased when the extraction time extended from 1 to 2 h, reached the maximum under the extraction time of 2.5 h, and decreased when the extraction time further extended. With the extension of extraction time, polysaccharides in Rhizoma Polygonati were continuously extracted. When the concentration difference inside and outside the cell reached equilibrium, the polysaccharides no longer penetrated into the solution. After 2.5 h, the content of polysaccharides decreased. On the one hand, the polysaccharides might be hydrolyzed and the structure changed; and on the other hand, the other types of components extracted affected the content of polysaccharides. Therefore, the extraction time should be controlled within 1.5-2.5 h.

Fig.2 Effect of extraction time on content of polysaccharides extracted from Rhizoma Polygonati

3.7.3Effect of extraction temperature on content of polysaccharides. Under the fixed extraction time (2 h) and liquid/solid ratio (15∶1, mL∶g), the effect of different temperature on the extraction of polysaccharides from Rhizoma Polygonati was investigated. When the temperature rose from 75 to 85 ℃, the content of polysaccharides continuously increased, indicating that within a certain temperature range, the dissolving ability and permeation rate of polysaccharides increased with the increase of temperature. At the temperature of 85 ℃, the content of polysaccharides reached the maximum. When the temperature continued to increase, the molecular structure of polysaccharides might be destroyed, resulting in a decrease in the content (Fig.3). The extraction temperature should be controlled at 80-90 ℃.

Fig.3 Effect of extraction temperature on content of polysaccharides extracted from Rhizoma Polygonati

3.8 Optimization of extraction process of polysaccharides in Rhizoma Polygonati

3.8.1Test design. On the basis of single-factor tests, the extraction process was optimized using response surface design and analysis module of Design-Expert 8.0.5 software, taking polysaccharide content as the response value[23]. According to the central design principle of Box-Behnken, the factors and levels of response surface analysis were determined (Table 2). Three dependent variables of extraction temperature (A), extraction time (B) and liquid/solid ratio (C) were selected, and content of polysaccharides was taken as response value. Design-Expert 8.0.5 software was used for response surface design and analysis. The extraction process of polysaccharides in Rhizoma Polygonati was optimized, and the results are shown in Table 3.

Table 2 Factors and levels of response surface analysis for optimizing polysaccharides extraction from Rhizoma Polygonati

Table 3 Design and results of response surface test for optimizing polysaccharides extraction from Rhizoma Polygonati

Table 4 Results of analysis of variance

The results of the analysis of variance show that thePvalue of the regression equation was less than 0.01, indicating that the model has statistical significance within the study scope of this experiment. ThePvalue of the lack-of-fit item was greater than 0.05, indicating that the model fits well, and it can reflect the relationship between the content of Rhizoma Polygonati polysaccharides and the liquid/solid ratio, extraction time and extraction temperature. The multivariate correlation coefficientR2=0.961 2, indicating that the model has good predictability, and response surface methodology can be used to optimize the extraction processof polysaccharides in Rhizoma Polygonati. Under this regression model, liquid/solid ratio had a significant effect and extraction temperature had an insignificant effect on the content of polysaccharides. The influencing intensity of the factors on the extraction of polysaccharides in Rhizoma Polygonati ranked as liquid/solid ratio>extraction time>extraction temperature.

Through the fitting analysis of the model, the best extraction process was obtained: liquid/solid ratio 19.8 ∶1 (mL/g), extraction time 1.78 h and extraction temperature 83.35 ℃. The predicted value of Rhizoma Polygonati polysaccharides content was 12.26%. According to the actual operation, the extraction process was adjusted to the follows: liquid/solid ratio 20∶1 (mL/g), extraction time 2 h, and extraction temperature 85 ℃.The extraction method was verified under the modified conditions. The extraction was repeated three times, and the average content of polysaccharides was 12.50%. The relative standard deviation from the theoretical prediction value (12.26%) of the model was 1.37%. Therefore, the extraction process optimized by response surface methodology for polysaccharides in Rhizoma Polygonati is reliable and has practical value.

4 Conclusions and discussion

Rhizoma Polygonati polysaccharides are a medicinal ingredient with great development potential. Because the water extraction and alcohol precipitation method is easy to operate, low in cost, and suitable for large-scale industrial production, it has become the main method for extracting polysaccharides in Rhizoma Polygonati.

In this study, based on single-factor tests, the extraction process of polysaccharides in Rhizoma Polygonati was optimized using Box-Behnken response surface methodology, and a method for content determination was established. The best extraction conditions obtained were as follows: liquid/solid ratio 20∶1 (mL/g), extraction time 2 h and extraction temperature 85 ℃. Under these conditions, the average content of polysaccharides extracted was 12.50%, and there was no significant difference between the measured value and the model predicted value, indicating that this method can be used to optimize the extraction process of polysaccharides in Rhizoma Polygonati, with a view to providing a scientific basis for the selection of high-quality Rhizoma Polygonati resources and the stable development of the health industry.