Optimization of the Ultrasonic-assisted Extraction Process for Total Flavonoids in Primula sikkimensis Hook. by Response Surface Methodology

Qing XIA, Dandan TANG, Zhiqiang ZHANG, Jianhui JIANG

Sichuan College of Traditional Chinese Medicine, Mianyang 621000, China

Abstract [Objectives] To optimize the ultrasonic-assisted extraction process for total flavonoids in Primula sikkimensis Hook. [Methods] The effects of ethanol volume fraction, extraction time, extraction temperature, ultrasonic time and solid-to-liquid ratio on the yield of total flavonoids were investigated by single factor method, and the extraction process for total flavonoids in P. sikkimensis was optimized by Box-Behnken experimental design. [Results] The optimal extraction process for total flavonoids in P. sikkimensis was ethanol volume fraction 45%, extraction time 29 min, extraction temperature 80 ℃, solid-to-liquid 1∶40 (g/mL) and ultrasonic time 10 min. Under such conditions, the average yield of total flavonoids in P. sikkimensis was 3.09%. [Conclusions] The optimized extraction process is simple and feasible, and is expected to provide a reference for the extraction of total flavonoids in P. sikkimensis.

Key words Primula sikkimensis Hook., Total flavonoids, Response surface methodology (RSM), Extraction process

1 Introduction

PrimulasikkimensisHook. (genusPrimulain family Primulaceae) is also called Xijinbaochun (Xiangzhisaibao in Tibetan language). It is a common Tibetan medicinal material with the effects of clearing heat, reducing swelling, and stopping diarrhea. It is used for the treatment of fever, blood disease, pulse disease, pediatric dysentery, edema and diarrhea[1].

This medicinal material contains many kinds of components, such as flavonoids and anthraquinones, among which flavonoids are the main active components[2-3]. Using the orthogonal experiment, Qiu Jianpingetal.[4]optimized the extraction process for total flavonoids inP.sikkimensis, and the extraction process was 60% ethanol reflux extraction for 60 min, and extraction time was three. But his operation was relatively complex, the average yield of total flavonoids inP.sikkimensisflower was 6.32%. However, if only using flowers as medicinal materials, medicinal resources are relatively scarce. Stems and leaves ofP.sikkimensismay also contain corresponding flavonoids. Therefore, the whole plant ofP.sikkimensismay have potential medicinal value.

Due to its high extraction efficiency and simple operation, the ultrasonic extraction technology has been widely applied in the extraction of active components of Chinese medicinal materials[5]. Therefore, in this experiment, we intend to use ultrasound-assisted method to extract the total flavonoids from the whole plant ofP.sikkimensis, and optimize the extraction process by response surface methodology (RSM), in the hope of providing certain experimental guidance and theoretical basis for the further development and utilization of the resources of the Tibetan medicinal plantP.sikkimensis.

2 Materials

TU-1950 Double Beam UV-Vis Spectrophotometer (Beijing Purkinje General Instrument Co., Ltd); KQ-300DB Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); ESJ200-4 Electronic Balance (Shenyang Longteng Electronic Co., Ltd.); HWS-12 Digital Electroheating Thermostatic Water Bath (Changzhou Guohua Electric Appliance Co., Ltd.).

P.sikkimensismedicinal material was collected from the foot of Mengbi Mountain, Xiaojin County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province in August 2019. The specific information of the sample is shown in Table 1. Identified by Ren Jinsong, teacher from Teaching and Research Section for TCM Identification of Sichuan College of Traditional Chinese Medicine, it wasPrimulasikkimensisHook. The whole plant was crushed, passed through No. 3 sieve, and stored for later use; rutin reference substance (batch No.MUST-14031012, Chengdu Must Bio-Technology Co., Ltd.); water was purified water; absolute ethanol was analytical reagent (AR).

Table 1 Sample information of Primula sikkimensis Hook.

3 Methods and results

3.1Determinationofyieldoftotalflavonoids

3.1.1Preparation of reference substance solution. Precisely weighed of 15.56 mg rutin reference substance, diluted with absolute ethanol to 25 mL, shook well, to obtain the reference substance solution with the mass concentration of 0.622 4 μg/mL.

3.1.2Linear relationship test. The content of total flavonoids was determined with reference to the method inChinesePharmacopoeia(2015)[6]. Precisely weighed 0.25, 0.5, 1, 2, 3 and 4 mL of the reference solution, separately placed into a 25-mL volumetric flask, added water to 4 mL, added 5% sodium of nitrite to mix fully, let it stand for 6 min, added 0.5 mL of 10% aluminum nitrate solution, shook up, placed for 6 min, then added 5 mL of 4% sodium hydroxide solution, added water to the scale, shook well, placed for 15 min, prepared the blank control, and measured the absorbance at the wavelength of 500 nm by the spectrophotometry. Taking the mass concentration of rutin (X) as the abscissa and absorbance (Y) as the ordinate, we plotted the standard curve, and obtained the linear regression equation:Y=0.118X-0.017 2,R2=0.999 9, indicating that in the range of 6.23-74.69 μg/mL of the rutin reference solution, there is a good linear relationship with the absorbance.

3.1.3Extraction of total flavonoids and determination of the yield. Precisely weighed 0.5 g ofP.sikkimensispowder, added the ethanol with a certain volume fraction, and under the set test conditions, according to certain extraction time, extraction temperature, solid-to-liquid ratio and ultrasonic time, we performed the ultrasonic assisted extraction. Filtered, washed with a small amount of extract solution, diluted to a 25-mL volumetric flask, shook well, and obtained the total flavonoids extract solution ofP.sikkimensis. Precisely weighed 1 mL of the total flavonoids extract and placed it in a 25-mL volumetric flask. In accordance with the method in Section3.1.2, starting from "adding water to 4 mL", measured the absorbance using the method and calculated the yield of total flavonoids.

3.2Methodologytest

3.2.1Precision test. Precisely weighed 6 pieces of 1 mL reference solution in the Section3.1.1, and measured the absorbance at 500 nm in accordance with the method in Section3.1.2, the calculatedRSDwas 1.5%, indicating the instrument has high precision.

3.2.2Repeatability test. Precisely weighed 6 pieces of 0.5 g ofP.sikkimensispowder, prepared the test solution in accordance with the method in Section3.1.3, measured the absorbance at 500 nm in accordance with the method in Section3.1.2, the calculatedRSDwas 2.7%, indicating this method has high repeatability.

3.2.3Stability test. Took the same test solution, measured the absorbance at 500 nm in accordance with the method in Section3.1.2, measured once at 10, 20, 30 and 60 min (n=6), and the calculatedRSDwas 2.5%, indicating that the stability is good within 60 min.

3.2.4Recovery rate test. Precisely weighed 6 pieces of 100 mg ofP.sikkimensispowder, added proper amount of rutin reference substance, prepared the test solution in accordance with the method in Section3.1.3, measured the absorbance at 500 nm in accordance with the method in Section3.1.2, the average recovery rate was 95.2%, and theRSDwas 3.6%, indicating this method is accurate and reliable.

3.3SinglefactortestWe performed the single factor tests with five factors of ethanol volume fraction, ultrasonic time, extraction time, extraction temperature and solid-to-liquid ratio to determine the appropriate single factor conditions influencing the yield of total flavonoids inP.sikkimensis.

3.3.1Effects of ethanol volume fraction on the yield of total flavonoids. Precisely weighed 5 pieces of 0.5 g ofP.sikkimensispowder, added with 20 mL ethanol with volume fraction of 40%, 50%, 60%, 70%, and 80% separately, and heated in water bath at 60 ℃ for 30 min and performed ultrasonic extraction for 20 min. Then, filtered and washed with little extract solution, diluted to a 25-mL volumetric flask, shook well, measured the absorbance in accordance with the method in Section3.1.3, calculated the yield of total flavonoids, the results are shown in Fig.1.

Fig.1 Effects of ethanol volume fraction on the yield of total flavonoids in Primula sikkimensis Hook.

As shown in Fig.1, the yield of total flavonoids inP.sikkimensispowder first increases and then decreases with the increase of the ethanol volume fraction. When the ethanol volume fraction is 50%, the yield of total flavonoids reaches the maximum. However, as the ethanol volume fraction continues to increase, the yield of total flavonoids decreases. The reason may be that as the ethanol volume fraction increases, the dissolution of fat-soluble substances is increased, and the polarity of the solvent decreases, leading to a decrease in the solubility of flavonoids[7]. Therefore, we determined that the appropriate ethanol volume fraction is 50%.

3.3.2Effects of ultrasonic time on the yield of total flavonoids. Precisely weighed 5 pieces of 0.5 g ofP.sikkimensispowder, added with 20 mL ethanol with volume fraction of 50%, heated in water bath at 60 ℃ for 30 min and performed ultrasonic extraction for 10, 20, 30, 40, and 50 min, filtered and washed with little extract solution, diluted to a 25-mL volumetric flask, shook well, measured the absorbance in accordance with the method in Section3.1.3, calculated the yield of total flavonoids, the results are shown in Fig.2.

From Fig.2, it can be seen that as the ultrasonic time increases, the yield of total flavonoids decreases. When the ultrasonic time is 10 min, the yield of total flavonoids is the maximum, possibly because the ultrasonic wave has a mechanical shearing effect, and the long-term effect destroys the structure of some flavonoids[5]. Therefore, we determined that the appropriate ultrasonic time is 10 min.

Fig.2 Effects of ultrasonic time on the yield of total flavonoids in Primula sikkimensis Hook.

3.3.3Effects of extraction time on the yield of total flavonoids. Precisely weighed 5 pieces of 0.5 g ofP.sikkimensispowder, added with 20 mL ethanol with volume fraction of 50%, heated in water bath at 60 ℃ for 15, 30, 45, 60, and 75 min, performed ultrasonic extraction for 10 min, filtered and washed with little extract solution, diluted to a 25-mL volumetric flask, shook well, measured the absorbance in accordance with the method in Section3.1.3, calculated the yield of total flavonoids, Results are shown in Fig.3.

Fig.3 Effects of extraction time on the yield of total flavonoids in Primula sikkimensis Hook.

From Fig.3, it is known that when the extraction time is 30 min, the yield of total flavonoids is the highest. With the increase of temperature, the yield of total flavonoids decreases, possibly because the excessive time leads to decomposition of some flavonoids[7]. Therefore, we determined that the appropriate ultrasonic time is 30 min.

3.3.4Effects of extraction temperature on the yield of total flavonoids. Precisely weighed 5 pieces of 0.5 g ofP.sikkimensispowder, added with 20 mL ethanol with volume fraction of 50%, heated in water bath at 50, 60, 70, 80, and 90 ℃ for 30 min, performed ultrasonic extraction for 10 min, filtered and washed with little extract solution, filtrate diluted to a 25-mL volumetric flask, shook well, measured the absorbance in accordance with the method in Section3.1.3, the yield of total flavonoids, the results are shown in Fig.4.

Fig.4 Effects of extraction temperature on the yield of total flavonoids in Primula sikkimensis Hook.

From Fig.4, it is known that when the extraction temperature is 80 ℃, the yield of total flavonoids is the highest. With the increase of temperature, the yield of total flavonoids decreases, possibly because the too high temperature leads to decomposition of some flavonoids, or because after the temperature reaches the boiling point of solvent, it leads to loss of solvent, and accordingly leading to decline of the yield[7]. Therefore, we determined that the appropriate extraction temperature is 80 ℃.

3.3.5Effects of the solid-to-liquid ratio on the yield of total flavonoids. Precisely weighed 5 pieces of 0.5 g ofP.sikkimensispowder, added with 20 mL ethanol with different volume fractions, and heated in water bath at 80 ℃ for 30 min and performed ultrasonic extraction for 10 min. Then, filtered and washed with little extract solution, diluted to a 25-mL volumetric flask, shook well, measured the absorbance in accordance with the method in Section3.1.3, calculated the yield of total flavonoids, the results are shown in Fig.5.

Fig.5 Effects of solid-to-liquid ratio on the yield of total flavonoids in Primula sikkimensis Hook.

From Fig.5, it can be seen that when the solid-to-liquid ratio is 1∶40 (g/mL), the yield of total flavonoids is the highest. When it exceeds 1∶40 (g/mL), the yield decreases, possibly because the solid-to-liquid ratio is too large, the resistance to ultrasonic breaking of cells is increased, so that the degree of cell fragmentation is reduced, and the yield of effective components is reduced[8]. Therefore, we determined that the appropriate solid-to-liquid ratio is 1∶40 (g/mL).

3.4OptimizationoftheextractionprocessbytheresponsesurfacemethodologyAccording to the results of single factor test, we selected three factors of ethanol volume fraction, extraction temperature and extraction time, and adopted the Box-Behnken response surface test design with 3 levels and 3 factors, to optimize the extraction process of total flavonoids. The response surface analysis factors and level design are shown in Table 2, and the experimental design and results are shown in Table 3.

Table 2 Response surface optimization factors and levels of ultrasonic extraction of total flavonoids in Primula sikkimensis Hook.

Table 3 Response surface experiment design and results for ultrasonic extraction of total flavonoids in Primula sikkimensis Hook.

3.5ResponsesurfaceanalysisAccording to the experimental data in Table 2, with the aid of design-Expert V 8.0.5 b software, we analyzed the data, and the regression equation of total flavonoid yield to the ethanol volume fraction (A), extraction time (B) and extraction temperature (C) isR=-3.439 00+0.257 75A+0.035 400B-0.0206 50C+0.000 083 33AB-0.000 825 000AC-0.000 166 666BC-0.001 827 5A2-0.000 523 333B2+0.004 225 00C2. The regression analysis and analysis results are shown in Table 4. From Table 4, it can be seen that the selected modelP<0.01, which is statistically significant. The lack of fit is not statistically significant (P=0.621 6>0.05). The effect of interaction on the yield of total flavonoids is shown in Fig.6.

Fig.6 Response surface of the interaction effects of various factors on the yield of total flavonoids in Primula sikkimensis Hook.

Table 4 Regression model variance analysis

3.6VerificationtestWe analyzed the data with the aid of Design-Expert V 8.0.5 b software, and obtained the optimal ultrasonic-assisted extraction process of total flavonoids inP.sikkimensis: the ethanol volume fraction 44.58%, extraction time 28.99 min, and extraction temperature 80 ℃. Under such conditions, the yield of total flavonoids was 3.06%. In order to verify the reliability of the response surface method, and considering the feasibility of actual operation at the same time, we modified the process conditions to the ethanol volume fraction 45%, extraction time 29 min, extraction temperature 80 ℃, solid-to-liquid 1∶40 (g/mL) and ultrasonic time 10 min. Under the above optimized conditions, we performed 3 times of verification tests, the measured value of the total flavonoids inP.sikkimensiswas 3.09%, and the relative deviation from the theoretical prediction was 0.98%, which was basically consistent with the theoretical prediction.

3.7DeterminationofyieldoftotalflavonoidsinP.sikkimensisTook three samples ofP.sikkimensisfrom 3 production areas (n=6), and obtained the test solution in accordance with the optimal process conditions, and determined according to the specified method. The results of the total flavonoids content of the samples are shown in Table 5, and the content of total flavonoids in production 3 was the highest.

Table 5 Determination results of yield of total flavonoids in Primula sikkimensis Hook. SD, n=6)

4 Discussion

In this experiment, we adopted the hot-extraction-ultrasonic-assisted extraction method to extract the total flavonoids inP.sikkimensis. Through a single-factor experimental design, and on this basis, we used 3-level 3-factor response surface method to optimize the extraction process of total flavonoids. The results indicate that the effect of extraction time and temperature on the yield of total flavonoids is statistically significant. Thus, we determined that the optimal extraction process for total flavonoids inP.sikkimensisis ethanol volume fraction 45%, extraction time 29 min, extraction temperature 80 ℃, solid-to-liquid 1∶40 (g/mL), and ultrasonic time 10 min. Under such conditions, the mass fraction ofP.sikkimensisis up to 3.09%. In this study, when optimizing the extraction process of total flavonoids inP.sikkimensis, the object of optimization was the whole plant. The optimized method is simpler and faster than the extraction method of Qiu Jianping[4]. We shortened the extraction time, but we did not use this method to determine the yield of total flavonoids inP.sikkimensis, so the extraction efficiency remains to be further studied.