Optimization of Extraction Process of Quercetin from Flos Sophorae Immaturus Using Orthogonal Design

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

Xiamen Key Laboratory of Marine Medicinal Natural Products and Cell Engineering/Xiamen Medical College, Xiamen 361023, China

Abstract [Objectives] This study aimed to optimize the ultrasonic-assisted extraction process of quercetin from Flos Sophorae Immaturus. [Methods] The natural product quercetin in Flos Sophorae Immaturus was extracted by ultrasonic-assisted method, and the content of quercetin in the extract was determined by ultraviolet spectrophotometry. The effect of ethanol concentration, solid/liquid ratio, temperature, time and ultrasonic power on the yield of quercetin in Flos Sophorae Immaturus was investigated by using single-factor experiments. Based on the results of single-factor experiments, an orthogonal experiment was designed to optimize the extraction process. The experimental data obtained were subjected to range analysis, analysis of variance and SSR test using SPSS 20.0 software to obtain the optimal extraction process. The results concluded were verified. [Results] The optimal ultrasonic-assisted extraction process for quercetin in Flos Sophorae Immaturus was as follows: temperature of 85 ℃, time of 30 min, solid/liquid ratio of 1∶20 g/mL, ethanol concentration of 50%. Under the optimal extraction conditions, the yield of quercetin in Flos Sophorae Immaturus was the highest. [Conclusions] This study provides a theoretical basis for the application of quercetin in Flos Sophorae Immaturus.

Key words Flos Sophorae Immaturus, Quercetin, Ultrasonic-assisted extraction, Orthogonal design

1 Introduction

Flos Sophorae Immaturus (dried flower buds of the leguminous plantSophorajaponica) is ovoid or oblong and shaped like rice grains, with the effect of cooling blood, stopping bleeding, clearing liver heat and purging fire[1]. Research[2-5]has shown that the effective active ingredients of natural products extracted from Flos Sophorae Immaturus are mainly flavonoids, among which quercetin is the most abundant. Quercetin is a polyhydroxy flavonoid, which is widely found in the flowers, leaves and fruits of plants and has a variety of biomedical activities in anti-inflammatory, anti-viral, anti-oxidant, anti-bacterial, anti-cancer and other aspects[6-12]. Flos Sophorae Immaturus has an extremely rich source in China, with good quality and low price. Quercetin, a natural product extracted from Flos Sophorae Immaturus, has considerable application prospects in the fields of medicine, food and health when used as an effective active ingredient. In this paper, quercetin in Flos Sophorae Immaturus was extracted with the ultrasonic-assisted extraction method[13-14], and the extraction process was optimized[15-16], in order to provide a theoretical basis for the application of quercetin in Flos Sophorae Immaturus.

2 Materials and methods

2.1 MaterialsThe instruments and equipment used in this study included UV-Vis spectrophotometer[UV-2100, Unico (Shanghai) Instrument Co., Ltd.], centrifuge (3K15, SIGMA), electronic balance[ALC-2100, Sartorius Scientific Instruments (Beijing) Co., Ltd.], digital ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.) and electric heating constant-temperature water bath (HWS-24, Shanghai Yiheng Scientific Instrument Co., Ltd.).

Anhydrous ethanol, anhydrous methanol,etc. of analytical grade were all purchased from Sinopharm Group Co., Ltd. Powder of Flos Sophorae Immaturus was purchased from Jiayu Biological Technology Co., Ltd. in Qiaocheng District, Bozhou City. Quercetin standard was produced by the Chengdu Herbpurify Biotechnology Co., Ltd.

2.2 Methods

2.2.1Extraction process of quercetin. The extraction process was as follows: Flos Sophorae Immaturus powder → sieving → ultrasonic extraction → centrifuging and collecting supernatant → repeating the step once → merging the supernatant → determining the content.

The powder of Flos Sophorae Immaturus was sieved through 45-mesh pharmacopoeia sieve, sealed and stored in a refrigerator at 4 ℃. An accurate amount of the Flos Sophorae Immaturus powder that had been dried to a constant weight was dissolved in ethanol of certain concentration according to certain solid/liquid ratio and subjected to extraction under the set ultrasonic power, time and temperature. The fluid was centrifuged at 10 000 r/min for 10 min, and the supernatant was collected. The precipitate obtained was extracted one more time. The filtrate of the two times was merged, and the absorbance was measured at the wavelength of maximum absorption. The concentration of quercetin in the extract was calculated according to the regression equation concluded, and the content of quercetin in Flos Sophorae Immaturus (mg/g) was then calculated. The experiment was repeated three times.

2.2.2Determination of the maximum absorption wavelength. A certain volume of the extract was diluted appropriately and scanned using an ultraviolet spectrophotometer within the wavelength range of 200-600 nm, and the wavelength of maximum absorption was determined to be 374 nm.

2.2.3Determination of quercetin content. An accurate amount (10.000 0 mg) of the quercetin standard that had been dried to constant weight was placed in a beaker, added with an appropriate amount of 60% methanol, heated in water bath to make the quercetin standard completely dissolve, transferred to a 50-mL volumetric flask and diluted to 50 mL in success to obtain the stock solution of quercetin. Accurate volumes (0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mL) of the stock solution were diluted to 10 mL, respectively to prepare into standard solutions of 0, 0.002, 0.004, 0.006, 0.008, 0.010 and 0.012 mg/mL. The absorbance of the standard solutions was measured at 374 nm, respectively. Taking the concentration of the standard solutions as the abscissa and the corresponding absorbance as the ordinate, the standard curve was drawn, and the linear regression equation was obtained:y=72x+0.003 7 (R2=0.999 5). It shows that the concentration of quercetin has a good linear relationship with absorbance in the range of 0.002-0.012 mg/mL. For determining the content of quercetin in the extract, 1 mL of the fresh Flos Sophorae Immaturus extract was taken and measured at 374 nm after diluted appropriately. The absorbance obtained was substituted into the regression equation, and the content of quercetin in the extract was calculated according to the following formula:

Content of quercetin (mg/g) =C×V×K/m

In the formula,Cis the concentration of quercetin calculated by substituting the measured absorbance into the regression equation (mg/mL);Vis the volume of the extract (mL);Kis the dilution factor; andmis the mass of Flos Sophorae Immaturus powder (g).

3 Results and analysis

3.1 Effect of single factors on the yield of quercetin inS.japonica

3.1.1Ultrasonic-assisted extraction temperature. The solid/liquid ratio, ethanol concentration, ultrasonic power and ultrasonic time were fixed at 1∶20 g/mL, 70%, 350 W and 30 min, respectively, and the effect of different ultrasonic temperatures (40, 50, 70, 80 and 90 ℃) on the yield of quercetin was investigated. As shown in Fig.1, as the ultrasonic temperature rose, the content of quercetin increased; and when the ultrasonic temperature exceeded 80 ℃, the content of quercetin decreased instead as the ultrasonic temperature rose. Therefore, the optimal ultrasonic temperature was 80 ℃.

3.1.2Ultrasonic-assisted extraction time. After fixing the solid/liquid ratio, ethanol concentration, ultrasonic power and ultrasonic temperature at 1∶20 g/mL, 70%, 350 W and 70 ℃, the effect of different ultrasonic times (10, 20, 30, 40 and 50 min) on the yield of quercetin was investigated. As shown in Fig.2, with the extension of ultrasonic time, the content of quercetin increased; and when the ultrasonic time exceeded 30 min, the content of quercetin declined instead as the ultrasonic time extended. Therefore, the optimal ultrasonic time was 30 min.

3.1.3Solid/liquid ratio. The ethanol concentration, ultrasonic power, ultrasonic temperature and ultrasonic time were fixed at 70%, 350 W, 70 ℃ and 30 min, respectively, and the effect of different solid/liquid ratios (1∶10, 1∶20, 1∶30, 1∶40 and 1∶50 g/mL) on the yield of quercetin in Flos Sophorae Immaturus was investigated. With the decrease of solid/liquid ratio, the content of quercetin increased; and when the solid/liquid ratio was smaller than 1∶20 g/mL, the content of quercetin decreased instead as the solid liquid ratio decreased (Fig.3). Therefore, the optimal solid/liquid ratio was 1∶20 g/mL.

Fig.1 Effect of ultrasonic extraction temperature on yield of quer-cetinFig.2 Effect of ultrasonic extraction time on yield of quercetinFig.3 Effect of solid/liquid ratio on yield of quercetin

3.1.4Ethanol concentration. After fixing the solid/liquid ratio, ultrasonic power, ultrasonic temperature and ultrasonic time at 1∶20 g/mL, 350 W, 70 ℃ and 30 min, the effect of different ethanol concentrations (10%, 30%, 50%, 70% and 90%) on the yield of quercetin was investigated. Fig.4 shows that with the increase of ethanol concentration, the content of quercetin increased; and when the ethanol concentration was higher than 50%, the content of quercetin declined instead as the ethanol concentration increased further. Therefore, the optimal ethanol concentration was 50%.

3.1.5Ultrasonic power. The solid/liquid ratio, ethanol concentration, ultrasonic temperature and ultrasonic time were fixed at 1∶20 g/mL, 70%, 70 ℃ and 30 min, respectively, and the effect of different ultrasonic powers (100, 200, 300, 400 and 500 W) on the yield of quercetin was investigated. As shown in Fig.5, at the ultrasonic powers of 200 and 400 W, the contents of quercetin were lower; and when the ultrasonic power was 300 W, the content of quercetin reached the maximum. Therefore, the optimal ultrasonic power was 300 W.

Fig.4 Effect of ethanol concentration on yield of quercetin

Fig.5 Effect of ultrasonic power on yield of quercetin

3.2 Optimization of the extraction process of quercetin inS.japonicaby orthogonal experimentOn the basis of single-factor experiment, A L9(34) orthogonal experiment was designed to optimize the extraction process. The ultrasonic extraction temperature, time, solid/liquid ratio and ethanol concentration, which had a significant effect on the yield of quercetin, were selected as the experimental factors, and 3 levels were designed for each factor. Taking the quercetin yield as an indicator, an orthogonal experiment was conducted. Each experiment was repeated 3 times. The experimental data obtained were subjected to range and variance analysis to determine the optimal process parameters for extracting quercetin from Flos Sophorae Immaturus by ultrasonic-assisted extraction. The results are shown in Table 1-3.

Table 1 Factors and levels of L9(34) orthogonal experiment for optimizing ultrasonic-assisted extraction process of quercetin in Flos Sophorae Immaturus

Table 2 Results and range analysis of L9(34) orthogonal experiment for optimizing ultrasonic-assisted extraction process of quercetin in Flos Sophorae Immaturus mg/g

From the perspective ofRvalue, the intensity of influence of various factors affecting the extraction of quercetin from Flos Sophorae Immaturus by ultrasonic-assisted extraction method was in the following order: C>D>A>B, that is, solid/liquid ratio>ethanol concentration>ultrasonic temperature>ultrasonic time. The range values of the levels of each factor were compared, and the results are shown in Fig.6. The optimal process was determined to be A3, B2, C2 and D2, that is, ultrasonic temperature of 85 ℃, ultrasonic time of 30 min, solid/liquid ratio of 1∶20 g/mL and ethanol concentration of 50%.

The results of analysis of variance show that ultrasonic temperature, ultrasonic time, solid/liquid ratio and ethanol concentration had a significant effect on the extraction of quercetin from Flos Sophorae Immaturus by ultrasonic-assisted extraction.

Table 3 Analysis of variance of results of orthogonal experiment

Fig.6 Comparison of ranges of various levels of each factor

3.3 Multiple comparisons between the treatments of the orthogonal experimentThe results of the orthogonal experiment were subjected to SSR test, and the results are shown in Table 4. As shown in Table 4, there was no significant difference among different levels of ultrasonic temperature (A) (P>0.05), and there was a significant difference between level 2 and level 3 (P<0.05); there was no significant difference among different levels of ultrasonic time (B) (P>0.05); in terms of solid/liquid ratio (C), there was a significant difference between level 1 and level 2 (P<0.05), and there was no significant difference between level 1 and level 3, or level 2 and level 3 (P>0.05); in terms of ethanol concentration (D), level 1 was different from level 2 and level 3 insignificantly (P>0.05), and there was a significant difference between level 2 and level 3 (P<0.01). Combined with the results of the range analysis, fully considering factors such as extraction process cost and energy saving, the optimal extraction process was modified as A3, B1, C2 and D1, that is, ultrasonic temperature of 85 ℃, ultrasonic time of 25 min, solid/liquid ratio of 1∶20 g/mL and ethanol concentration of 40%.

Table 4 SSR test of significance of differences between groups

3.4 Verification resultsSix portions of Flos Sophorae Immaturus powder were weighed, and extracted under the optimal extraction process concluded by range analysis and the modified optimal extraction process, respectively, three portions for each. The average yield of each process was calculated in order to verify whether the yield was the highest. The results are shown in Table 5. The verification results show that the content of quercetin extracted by the modified optimal extraction process was higher than the content of quercetin extracted by the optimal extraction process concluded by range analysis. By comparing with the results of Table 2, it could be found that the content of quercetin extracted by the modified optimal extraction process was higher than that of each experiment in the orthogonal design.

Table 5 Verification of experimental results

4 Conclusions

The ultrasonic-assisted extraction method was used to process Flos Sophorae Immaturus to extract quercetin from Flos Sophorae Immaturus. The effect of ultrasonic extraction time, ultrasonic extraction temperature, ultrasonic power, solid/liquid ratio and ethanol concentration on the yield of quercetin was investigated. Through orthogonal experiment, the optimal process parameters for extracting quercetin from Flos Sophorae Immaturus were concluded as A3, B2, C2 and D2, that is, ultrasonic temperature of 85 ℃, ultrasonic time of 30 min, solid/liquid ratio of 1∶20 g/mL and ethanol concentration of 50%. The results of analysis of variance show that ultrasonic temperature, ultrasonic time, solid/liquid ratio and ethanol concentration had a significant effect on the extraction of quercetin from Flos Sophorae Immaturus by ultrasonic-assisted extraction. Through multiple comparisons between various factors, the optimal extraction process was modified to be A3, B1, C2 and D1,i.e., ultrasonic temperature of 85 ℃, ultrasonic time of 25 min, solid/liquid ratio of 1∶20 g/mL and ethanol concentration of 40%. The results of verification experiment show that the yield of quercetin extracted by the optimal extraction process A3, B2, C2 and D2 concluded by range analysis was lower than that extracted by the modified optimal extraction process A3, B1, C2 and D1, but the yields of quercetin were all higher than the yields of quercetin under other extraction conditions in the orthogonal design table. This process can be used to extract quercetin from Flos Sophorae Immaturus.