Optimization of Extraction Process and Antioxidant Activity of Polysaccharide in Embelia parviflora Wall. by RSM

Wenli LI, Xianxian LIU, Xiaolian LIANG, Yong CHEN, Danqing PANG, Zhouyan HUANG, Qian HAN, Wenjun LIU, Liangni CHEN

College of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] To optimize the extraction process of polysaccharide in Embelia parviflora Wall. by response surface methodology, and to study the antioxidant activity in vitro of polysaccharide in E. parviflora, so as to provide a basis for the further development and utilization of E. parviflora. [Methods] The solid-to-liquid ratio, extraction temperature, extraction time and extraction times were used as single factors to investigate the effects. On this basis, the response surface methodology (RSM) was used to optimize the extraction process of polysaccharide in E. parviflora. DPPH and ABTS free radicals were used to investigate the antioxidant capacity of polysaccharide in E. parviflora. [Results] The optimal solid-to-liquid ratio for extraction of polysaccharide from E. parviflora was 32∶1 (mL/g), extraction temperature was 77 ℃, and extraction time was 36 min. The IC50 values of DPPH and ABTS were 0.04 and 0.03 mg/mL, respectively. [Conclusions] The optimized extraction process of polysaccharide in E. parviflora is stable, the extraction rate is high, and has strong antioxidant activity. It is expected to provide a reference for the industrial extraction of polysaccharide from E. parviflora.

Key words Embelia parviflora Wall., Polysaccharide, Response surface methodology (RSM), Antioxidant activity

1 Introduction

EmbeliaparvifloraWall. is also calledEmbeliapulchellaMez,EmbeliamyrtifoliaHemsl. et Mez,Samaraparviflora,EmbeliadistichaH.R.Fletcher,Ribesioidesmyrtifolium(Hemsl. & Mez) Kuntze. It can be collected in the whole year, and is used as medicines after cutting into sections and drying.E.parviflorahas functions of relaxing sinews and activating collaterals, dispelling dampness and relieving pain, and can be used for treatment of gynecological diseases such as irregular menstruation and amenorrhea.E.parvifloramay contain sugars, glycosides, saponins, phenols, tannins, flavonoids, organic acids,etc.[1]. Polysaccharides have anti-fatigue, anti-oxidation, anti-inflammatory, anti-coagulation, and tumor cell growth inhibition effects[2-7]. At present, there are few studies on the polysaccharides ofE.parviflora. Based on the single factor experiment, we optimized the extraction process of polysaccharide inE.parvifloraby the response surface methodology, in order to provide a reference for the industrial extraction of polysaccharide fromE.parviflora[8]. It is hoped that traditional Chinese medicine can go to the world, improve pharmaceutical innovation capabilities, form an integrated development model of industry, academia, research and marketing, go to the world and the future, and realize resource sharing and win-win cooperation[9].

2 Instruments and materials

Tecan Infinite M200 PRO microplate reader; HWS-26 electroheating thermostatic water bath (Shanghai Keelrein Instruments Co., Ltd.); KQ5200B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); UV-1780 ultraviolet-visible spectrophotometer (GL Sciences (Suzhou) Co., Ltd.); Elmasonic P 180H ultrasonic cleaner (Germany); YK4-400B high-speed pulverizer (Shandong Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd.); CE2117-Z induction cooker (Airmate Electrical (Shenzhen) Co. Ltd.,); SQP electronic balance (Sartorius Sartorius Scientific Instruments (Beijing) Co., Ltd.) D-anhydrous glucose (production batch No.HA062207198, Baoji Chenguang Biotechnology Co., Ltd.); other reagents were of analytical grade. Medicinal herbE.parviflorawas collected from Guangxi Jinxiu Yao Autonomous County, and identified by associate professor Guo Min from Guangxi University of Chinese Medicine as dry ground part ofEmbeliaparvifloraWall.

3 Methods

3.1 Determination polysaccharide content

3.1.1Plotting of standard curve. Precisely weighed 5.0 mg of glucose standard substance and fixed the volume in a 50-mL volumetric flask to obtain a glucose standard solution with a concentration of 0.1 mg/mL. Pipetted 50, 100, 150, 200, 250, 300, 350, and 400 μL of glucose standard solution in a test tube, added distilled water to 1 mL, and shook up to obtain concentrations of 5, 10, 15, 20, 25, 30, 35, 40 μg/mL standard solution. Added 0.6 mL of 3% phenol solution, slowly added 5 mL of 90% concentrated sulfuric acid, shook up, heated in water bath for 25 min, and cooled to room temperature in an ice water bath. Subtracted the blank with distilled water and measured the absorbance at the maximum absorption wavelength. Plotted the standard curve and obtained the regression equation:Y=0.017 1X+0.309,R2=0.999 6.

3.1.2Preparation and determination of sample solution. Precisely weighed 1 g coarse powder ofE.parviflora, extracted with ultrasonic waves in distilled water, filtered, and dried in a 70 ℃ water bath. Added 3 times the volume of 70% ethanol for precipitation, and let it stand at 4 ℃ overnight, evaporated the precipitate to dryness, to obtain the crude polysaccharide. Added the distilled water to dissolve the crude polysaccharide, fixed the volume to 100 mL, precisely pipetted 0.08 mL of extract, added distilled water to make up to 1 mL, and placed into a conical flask, measured the absorbance at 486 nm wavelength using the ultraviolet-visible spectrophotometer in accordance with the method of Section3.1.1. Calculated the polysaccharide content inE.parviflorausing formula (1).

W(mg/g)=CV/2 000m

(1)

whereWdenotes the polysaccharide content ofE.parviflora(mg/g),Cis the mass concentration of polysaccharide in the sample solution (μg/mL),Vis the constant volume of the sample (mL) andmis the mass of the raw material (g).

3.1.3Determination of maximum absorption wavelength. Scanned the 400-800 nm wavelength of the ultraviolet-visible spectrophotometer, the reference solution and the test solution showed maximum absorption at 486.5 nm, so 486 nm was determined as the measurement wavelength. The UV full-wavelength scan of the reference substance and sample solution was shown in Fig.1.

Fig.1 UV full-wavelength scan of the reference substance (A) and sample solution (B)

3.1.4Determination of measurement methods. Selected phenol mass fraction (3%, 4%, 5%, 6%, 7%), phenol volume (0.6, 0.8, 1, 1.2, 1.4 mL), sulfuric acid mass fraction (80%, 85%, 90%, 95%, 100%), and the time of color development (20, 25, 30, 35, 40 min) on the polysaccharide content ofE.parviflora, determined the optimal method is the same as that in Section3.1.1.

3.1.5Single factor experiment. Selected the factors of solid-to-liquid ratio (10∶1, 20∶1, 30∶1, 40∶1, 50∶1), temperature (40, 50, 60, 70, 80 ℃), extraction time (10, 20, 30, 40, 50 min) to investigate their effects on the polysaccharide content ofE.parviflora.

3.1.6Response surface experiment. On the basis of single factor experiment, used the Design-Expert 8. 0.6 software to conduct Box-Behnken experimental design. Taking the solid-to-liquid ratio, extraction temperature, and extraction time as independent variables, and absorbance A as the dependent variable, 3 factors and 3 levels were used to optimize the extraction conditions of polysaccharide inE.parviflora. Experimental factors and level design were listed in Table 1.

Table 1 Response surface analysis factors and levels

3.2 Determination of DPPH free radical scavenging ability of polysaccharide inE.parvifloraThe sample solution was prepared using the method in Section3.1.2. Firstly, extracted the crude polysaccharide, precisely weighed the 0.05 g of crude polysaccharide, added a small amount of distilled water to dissolve ultrasonically, placed it in a 50-mL volumetric flask, and diluted with water to the scale graduation line to obtain 1 mg/mL sample solution. The sample solution prepared during the actual measurement was then diluted with distilled water to a suitable group of concentrations (0.7, 0.5, 0.3, 0.1, 0.08, 0.06, 0.04, 0.02 mg/mL). Precisely weighed 50 mg of ascorbic acid, placed it in a 50-mL volumetric flask, diluted to the scale graduation line with ultrapure water, prepared a 1 mg/mL solution, and stored at 4 ℃.

3.2.1Determination of DPPH free radical scavenging capacity. (i) Preparation of 0.1 mmol/L DPPH solution. Precisely weighed 4 mg of DPPH powder, placed it in a 100-mL volumetric flask, diluted with anhydrous ethanol to the scale graduation line, to obtain 0.1 mmol/L DPPH solution, and stored at 4 ℃. (ii) Measurement. Took 40 μL of sample solution of different concentration and 160 μL of DPPH solution in 96-well plate, mixed thoroughly. The reference solution was 40 μL of distilled water and 160 μL of DPPH solution mixed evenly. Pipetted different concentrations of the test solution in the 96-well plate to remove the outermost circle. Each column had 6 replicate wells, of which 4 replicate wells were added with 40 μL of the same sample, and the other 2 replicate wells were added with 40 μL of distilled water to remove the effects of the drug background on the experiment. Each replicate well was added with 160 μL of DPPH solution (A+B=0.2 μL). An equal volume of distilled water was used to take the place of the sample solution as a blank group, and an equal volume of anhydrous ethanol was used to take the place of DPPH as a control group. After completion of the addition of each solution, kept out of the light for the reaction at room temperature for 30 min, and measured the absorbance at the wavelength of 517 nm, expressed asA1,A2, andA0. Ascorbic acid was used as a positive control to evaluate the DPPH free radical scavenging rate of the active substance. Calculated the DPPH scavenging rate using the formula (2):

DPPH scavenging rate (%)=[1-(A1-A2/A0)]×100

(2)

whereA1denotes the absorbance value of the test group;A2denotes the absorbance value of the control group;A0denotes the absorbance value of the blank group.

3.2.2Determination of ABTS free radical scavenging capacity. (i) Preparation of the ABTS solution. Precisely weighed 193.3 mg of ABTS powder, placed it in a 100-mL volumetric flask, and prepared ABTS to a 7 mmol/L stock solution with distilled water. (ii) Preparation of 2.45 mmol /L K2S2O8aqueous solution. Precisely weighed 34.5 mg of potassium persulfate (K2S2O8) powder. Placed it in a 100-mL volumetric flask, diluted ultra-pure anhydrous to the scale graduation line, to obtain 2.45 mmol /L solution. (iii) Measurement. Mixed the above 7 mmol/L ABTS stock solution and 2.45 mmol/L K2S2O8solution in equal volume, and stored at room temperature in the dark for 14 h. Before testing, the ABTS stock solution was diluted 20 times with ultrapure water to adjust it to a wavelength of 734 nm, and the absorbance was in the range of 0.7±0.05. Pipetted 20 μL of different concentrations of the test solution in the 96-well plate. Each column had 9 replicate wells, of which 6 replicate wells were added with 20 μL of the same sample, and the other 3 replicate wells were added with 20 μL of ultrapure water to remove the effects of the drug background on the experiment. Each replicate well was added with 180 μL of ABTS solution. After completion of the addition of each solution, shook up and kept the reaction in the dark at room temperature for 10 min, and then measured the absorbance of the reaction solution at a wavelength of 734 nm with an enzyme-linked immunoassay, recorded asA1andA0, respectively. Took ascorbic acid as a positive control to evaluate the ABTS free radical scavenging rate of the active substance. Calculated the ABTS scavenging rate using the formula (3):

ABTS scavenging rate (%) =[1-(A1/A0)]×100

(3)

where whereA1denotes the absorbance value of the test group;A0denotes the absorbance value of the blank group.

4 Results and analysis

4.1 Single factor experiment

4.1.1Effects of solid-to-liquid ratio on extraction of polysaccharide content. Under the conditions of the extraction temperature of 80 ℃, the extraction time of 30 min, and two extraction times, at the solid-to-liquid ratio of 10∶1, 20∶1, 30∶1, 40∶1, 50∶1, we extracted polysaccharide, measured the absorbance and calculated the polysaccharide content. Plotted the curve taking the solid-to-liquid ratio as the abscissa and the polysaccharide content as the ordinate, as shown in Fig.2. With the solid-to-liquid ratio increases, the polysaccharide content increases. When the solid-to-liquid ratio is 30∶1, the polysaccharide content is the highest, and then it drops, indicating that the solid-to-liquid ratio of 30∶1 is the most suitable for extracting polysaccharide inE.parviflora.

Fig.2 Effects of solid-to-liquid ratio on polysaccharide content in Embelia parviflora Wall.

4.1.2Effects of the extraction temperature on the extraction of polysaccharide content. Under the conditions of solid-to-liquid ratio of 30∶1, extraction time of 30 min, and two extraction times, at the extraction temperature of 60, 70, 80, 90, and 100 ℃, we extracted polysaccharide, measured the absorbance and calculated the polysaccharide content. Plotted the curve taking the extraction temperature as the abscissa and the polysaccharide content as the ordinate, as shown in Fig.3. With the extraction temperature rises, the polysaccharide content increases. When the temperature reaches certain value (80 ℃), the polysaccharide content is the highest, and then it drops, indicating that the extraction temperature of 80 ℃ is the most suitable for extracting polysaccharide inE.parviflora.

Fig.3 Effects of extraction temperature on polysaccharide content in Embelia parviflora Wall.

4.1.3Effects of the extraction time on the extraction of polysaccharide content. Under the conditions of the solid-to-liquid ratio of 30∶1, the extraction temperature of 70 ℃, two extraction times, and at extraction time of 10, 20, 30, 40, and 50 min, we extracted polysaccharide, measured the absorbance and calculated the polysaccharide content. Plotted the curve taking the extraction time as the abscissa and the polysaccharide content as the ordinate, as shown in Fig.4, indicating that when the extraction time is 40 min, the polysaccharide content is the highest.

Fig.4 Effects of extraction time on polysaccharide content in Embelia parviflora Wall.

4.1.4Effects of the extraction times on the extraction of polysaccharide content. Under the conditions of the solid-to-liquid ratio of 30∶1, the extraction temperature of 70 ℃, the extraction time of 30 min, and at one, two and three extraction times, we extracted polysaccharide, measured the absorbance and calculated the polysaccharide content. Plotted the curve taking the extraction times as the abscissa and the polysaccharide content as the ordinate, as shown in Fig.5, indicating that when the extraction is one times, the polysaccharide content is the highest.

Fig.5 Effects of extraction times on polysaccharide content in Embelia parviflora Wall.

4.2 Optimization of polysaccharide extraction process by response surface methodology

4.2.1Model equation establishment and significance test. Design-Expert8.0.6 software was used for Box-Behnken response surface test design. The test plan design and results are indicated in Table 2. Regression analysis was carried out on the total polysaccharide content ofE.parviflorain Table 2 to the independent variables solid-to-liquid ratio, extraction temperature, and extraction time. Polysaccharide content (mg/g)=37.72+3.72A-4.67B-4.78C-2.47AB+1AC+4.48BC-9.00A2-10.12B2-7.92C2. Analysis of variance was carried out on the model, it can be seen from Table 3 that theFvalue of the model is 27 andP<0.001, indicating that the model is significant. TheFvalue of the lack of fit is 5.41, andP>0.05 is not significant, indicating that the model fits well. From Table 4, it can be seen that the multivariate correlation coefficientR2=0.972, indicating that the correlation of the simulation coefficients is high. Under this regression model,A,B,C, andBChave a significant effect on the total polysaccharide content, andA2,B2, andC2have a very significant effect on the total polysaccharide content. From theFvalue, we can see the degree of effects: Extraction time>extraction temperature>solid-to-liquid ratio.

Table 2 Design and results of response surface optimization test

Table 3 Variance analysis of regression equation

Note:**denotes extremely significant difference (P<0.001), and*denotes significant difference (P<0.05).

Table 4 Variance analysis of regression model

4.2.2Response surface analysis. With the aid of Box-Behnken, we plotted the response surface graph according to the regression model selected by the response surface, as shown in Fig.6, it shows that the extraction time, extraction temperature and solid-to-liquid ratio have a significant effect on polysaccharide content inE.parviflora. In the figure, the extraction time surface is steep, followed by the extraction temperature, and finally the solid-to-liquid ratio, indicating that the extraction time has the greatest effect on the polysaccharide content inE.parviflora.

4.3 Determination of antioxidant activity of polysaccharide inE.parvifloraFrom Fig.7, it can be seen thatIC50=0.04. The ability ofE.parviflorapolysaccharide to scavenge DPPH free radicals increases with the increase of concentration. It tends to be stable at 0.5 mg/mL, and the ability ofE.parviflorapolysaccharide to scavenge DPPH free radicals is weaker than that of ascorbic acid. From Fig.8, it can be seenIC50=0.03. The ability ofE.parviflorapolysaccharide to scavenge ABTS free radicals increases with the increase of concentration. At 0.08 mg/mL, the ability ofE.parviflorapolysaccharide to scavenge ABTS free radicals is equivalent to that of ascorbic acid.

Fig.6 Response surface diagram for the interaction of various factors on the content of polysaccharides

Fig.7 DPPH free radical scavenging rate of polysaccharide in Embelia parviflora Wall.

Fig.8 ABTS free radical scavenging rate of polysaccharide in Embelia parviflora Wall.

5 Conclusions

Through analysis of the regression model, the optimal extraction process is as follows: solid-to-liquid ratio is 32∶1 (mL/g), extraction temperature is 77 ℃, and extraction time is 36 min, and the predicted polysaccharide content is 39.87 mg/g. According to the optimal extraction process of the model, the calculated polysaccharide content inE.parviflorais 39.5 mg/g, which is not much different from the ideal value, indicating that the extraction process obtained by response surface optimization is reliable and has practical value. The polysaccharide inE.parviflorahas good antioxidant capacity. This study is expected to provide a basis for better utilization ofE.parvifloraresources.