Optimization of Extraction of Total Flavonoids from HERBA BLUMEAE RIPARIAE Using Response Surface Methodology

Hongna SU, Yining ZHANG, Xuexue LI, Xuelian ZHAO, Yisong LI, Wenbing LI, Yuan LIU

1. College of Pharmacy, Southwest Minzu University, Chengdu 610041, China; 2. Institute of Qinghai-Tibetan Plateau Research, Southwest Minzu University, Chengdu 610225, China; 3. Sichuan Provincial Qiang-Yi Medicinal Resources Protection and Utilization Technology Engineering Laboratory, Chengdu 610225, China; 4. Ethnic Medicine Institute, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] This study aimed to optimize the ultrasonic-assisted extraction process of total flavonoids from a Zhuang medicine HERBA BLUMEAE RIPARIAE using response surface methodology. [Methods] Using rutin as a reference substance, a standard curve was drawn at a wavelength of 292 nm. Content determination was performed through absorbance under different conditions. The effects of ultrasonic time, ethanol volume fraction and liquid-to-solid ratio on the yield of total flavonoids from HERBA BLUMEAE RIPARIAE were investigated by single-factor tests. By using Box-Behnken test, the ultrasonic-assisted extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE was optimized. [Results] The optimal extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE was as follows: ultrasonic time of 30 min, ethanol volume fraction of 50% and liquid-to-solid ratio of 40∶1 (mL/g). Under the optimal extraction conditions, the average yield of total flavonoids from HERBA BLUMEAE RIPARIAE was 85.47 mg/g. [Conclusions] The optimized extraction process was convenient and feasible. This study will provide a reference for the extraction of total flavonoids from HERBA BLUMEAE RIPARIAE, and provide certain guidance and basis for further development and utilization of the plan resource of Blumea riparia (Bl.) DC.

Key words Zhuang medicine, HERBA BLUMEAE RIPARIAE, Total flavonoids, Response surface optimization, Extraction process

1 Introduction

HERBA BLUMEAE RIPARIAE was dried whole grass ofBlumeariparia(Bl.) DC. (Compositae), also known as Baihua Jiuliming and Guangya[1]. It was a key medicinal material in Guangxi[2], with effects of activating menstrual blood, expelling wind, drying dampness, stopping bleeding and promoting urination. In clinics, it was used to treat symptoms such as abnormal menstrual period, postpartum hemorrhage, infertility, ulcers, erosion of vulva, rheumatism and bone pain[3]. HERBA BLUMEAE RIPARIAE had a long history of use in Guangxi, especially in Baise[4]. The active ingredients in HERBA BLUMEAE RIPARIAE include catecholamines, total flavonoids, sterols, polysaccharides and water-soluble compounds. Among them, total flavonoids had the effects of improving sleep[5], protecting heart and cerebral vessels[6], scavenging free radicals[6], and fighting oxidation[7]. In this study, taking yield of total flavonoids as the index of investigation, and selecting ethanol volume fraction, liquid-to-solid ratio and ultrasonic time as the factors investigated, the extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE was optimized using response surface methodology, in order to provide some experimental guidance and theoretical basis for further development and utilization ofB.riparia(Bl.) DC.

2 Materials

TU-1810 UV-Vis spectrophotometer (Beijing Purkinje General Instruments Co., Ltd.); KQ-259DE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); NewClassic MF analytical balance (Mettler, Switzerland).

HERBA BLUMEAE RIPARIAE was purchased from Longlin County, Baise City, Guangxi Province. It was identified by Professor Liu Yuan of Southwest Minzu University as dried whole grass ofB.riparia(Bl.) DC. (Compositae). The HERBA BLUMEAE RIPARIAE sample was pulverized and passed through a No.3 sieve for future use. Lutin (batch No. 100080-200707, content ≥ 98%) was purchased from National Institutes for Food and Drug Control. Ethanol, of analytical grade, was purchased from Chengdu Kelong Chemical Co., Ltd.

3 Methods and results

3.1 Determination of total flavonoids content

3.1.1Preparation of reference solution. An accurate amount of rutin reference was dissolved in 40% ethanol to 10 mL. Thus, rutin mother liquor of 1 mg/mL was prepared.

3.1.2Investigation of linear relationship. Accurate volumes (0, 0.1, 0.2, 0.5, 0.8 and 1.0 mL) of rutin mother liquor were diluted with 40% ethanol to 10 mL, respectively, thus reference solutions with concentrations of 0.01, 0.02, 0.05, 0.08 and 0.1 mg/mL were prepared. Taking 40% ethanol as a blank control, the absorbances of the standard solutions were measured at 292 nm. With the concentration (C) as the abscissa and the absorbance (A) as the ordinate, the standard curve was plotted, and the linear regression equation obtained was as follows:

A=12.184C-0.017 6 (r=0.999 8).

The results showed that the rutin reference solution had a good linear relationship with the absorbance in the range of 10-100 μg/mL.

3.1.3Determination of total flavonoids. An accurate amount (around 0.5 g) of the powder of HERBA BLUMEAE RIPARIAE was added a certain amount and a certain volume fraction of ethanol under the set experimental conditions, ultrasonicated for a certain time (100 W, 40 kHz) and filtered. An accurate volume (0.1 mL) of the filtrate was diluted with 40% ethanol to 10 mL, shaken well, and measured according to the description in Section3.1.2. The yield of total flavonoids (mg/g) was calculated.

E=C×V×n/m

whereErepresents the yield of total flavonoids, mg/g;Crepresents the concentration of flavonoids in the extract, g/mL;Vrepresents the volume, mL;nis dilution times; andmis the weight of the sample used, g.

3.2 Single-factor tests

3.2.1Effect of ethanol volume fraction on yield of total flavonoids. Five portions of the powder of HERBA BLUMEAE RIPARIAE, 0.5 g for each, were weighed. They were added with 5 mL of ethanol with different volume fractions, respectively. The ultrasonic temperature and ultrasonic time were set to 25 ℃ and 1 h, respectively. After filtration, the residues were washed with a small amount of extraction solution, and then the filtrates were diluted to 10 mL, respectively. The yield of total flavonoids was measured according to the description in Section3.1.3. The results are shown in Fig.1.

Fig.1 Effect of ethanol volume fraction on yield of total flavonoids from HERBA BLUMEAE RIPARIAE

The results show that the yields of total flavonoids extracted with different volume fractions of ethanol were 3.62, 6.28, 5.26, 4.80 and 3.65 mg/g, respectively. It showed that the yield of total flavonoids first increased significantly and then decreased gradually with the increase of ethanol volume fraction. When the volume fraction of ethanol was 40%, the yield of total flavonoids was the highest. Then, with the increase of ethanol volume fraction, the yield of total flavonoids decreased instead. This was because that with the gradual increase in ethanol volume fraction, the dissolution amount of fat-soluble impurities from HERBA BLUMEAE RIPARIAE gradually increased, and these impurities might be combined with the ethanol-water molecular system and compete for dissolution with flavonoids, reducing the dissolution of flavonoids from HERBA BLUMEAE RIPARIAE, leading to a reduction in the extraction rate of total flavonoids[8]. In short, when the volume fraction of ethanol was 40%, the yield of total flavonoids was the highest (31.38 mg/mL).

3.2.2Effect of ultrasonic time on yield of total flavonoids. Five portions of the powder of HERBA BLUMEAE RIPARIAE, 0.5 g for each, were added with 5 mL of 40% ethanol and subjected to ultrasonic extraction in a 25 ℃ water bath for 10, 20, 30, 60 and 90 min, respectively. After filtration, the residues were washed with a small amount of extract, and the filtrates were diluted to 10 mL, respectively. The yield of total flavonoids was measured according to the description in Section3.1.3. The results are shown in Fig.2.

Fig.2 Effect of ultrasonic time on yield of total flavonoids from HERBA BLUMEAE RIPARIAE

The results show that under different ultrasonic times, the yields of total flavonoids were 4.28, 6.05, 5.34, 5.22 and 4.50 mg/g, respectively. When the ultrasonic time was 20 min, the yield of total flavonoids was the highest. With the extension of the ultrasonic time, the yield of total flavonoids tended to increase slightly. With the further extension of the ultrasonic time, the yield of total flavonoids was gradually decreasing. Excessive ultrasonic time might cause a large amount of non-flavonoids to dissolve, leading to a reduction in the extraction rate of total flavonoids from HERBA BLUMEAE RIPARIAE[9].

3.2.3Effect of liquid-to-solid ratio on yield of total flavonoids. Five portions of the powder of HERBA BLUMEAE RIPARIAE, 0.5 g for each, were added with different volumes of 40% ethanol, sonicated at 25 ℃ for 1 h, and filtered, respectively. The residues were washed with a small amount of extract, and the filtrates were diluted to 10 mL, respectively. The yield of total flavonoids was measured according to the description in Section3.1.3. The results are shown in Fig.3.

Under different liquid-to-solid ratios, the yields of total flavonoids were 48.11, 62.90, 68.73, 73.65 and 72.75 mg/g, respectively. When the liquid-to-solid ratio was 40∶1 (mL/g), the yield of total flavonoids was the highest. When the liquid-to-solid ratio was smaller than 40∶1 (mL/g), the yield of total flavonoids increased gradually; and when the liquid-to-solid ratio exceeded 40∶1 (mL/g), the yield of total flavonoids declined slightly. Taking both consumption of materials and yield of total flavonoids into account, the liquid-to-solid ratio was finally determined as 40∶1 (mL/g), under which the yield of total flavonoids was 73.65 mg/g.

Fig.3 Effect of liquid-to-solid ratio on yield of total flavonoids from HERBA BLUMEAE RIPARIAE

3.3 Response surface optimization testAccording to the results of the single-factor tests, by choosing three factors of ethanol volume fraction, ultrasonic time and liquid-to-solid ratio, the extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE was optimized using the response surface design of Design-Expert.V8.0.6.1. The factors and their levels for the response surface design are shown in Table 1. The test design and results are shown in Table 2.

Table 1 Factors and levels of response surface test for optimizing extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE

LevelUltrasonic time(A)∥minLiquid-to-solid ratio(B)∥mL/gEthanol volume fraction(C)∥%-11030∶13002040∶14013050∶150

The Design-Expert.V8.0.6.1 was used to design the test. The test data shown in Table 2 were analyzed using the software to obtain the regression equation of total flavonoids (Y) against ultrasonic time (A), liquid-to-solid ratio (B) and ethanol volume fraction (C):

Y=13.500-0.830A+ 0.966B+1.447C+0.013AB-0.011AC+0.024BC+0.032A2-0.029B2-0.019C2

Table 2 Results of response surface test for optimizing extraction process of total flavonoids from HERBA BLUMEAE RIPARIAE

TestcodeUltrasonictimeLiquid-to-solid ratioEthanol volumefractionYield of totalflavonoids∥mg/g1-10-160.562-10177.52301-155.59411081.48501174.4861-1078.15700073.09800072.359-1-1065.881010-171.341100072.371200069.731300072.131410184.00150-1-164.79160-1173.9417-11063.82

Note: a. Ethanol volume fraction and liquid-to-solid ratio; b. Ethanol volume fraction and ultrasonic time; c. Liquid-to-solid ratio and ultrasonic time.

Fig.4 Response surface plots of the effects of pairwise interactions of various factors on the yield of total flavonoids from HERBA BLUMEAE RIPARIAE

3.4 Verification testUsing the Design-Expert.V8.0.6.1 software, the optimal ultrasonic-assisted extraction process for total flavonoids from HERBA BLUMEAE RIPARIAE was obtained: ultrasonic time of 29.97 min, liquid-to-solid ratio of 38.24∶1 (mL/g) and ethanol volume fraction of 49.04%. Under the optimal extraction conditions, the yield of total flavonoids was 84.55 mg/g.

In order to verify the reliability of the optimization results using response surface methodology, considering the feasibility of actual operation, the extraction conditions were modified: ultrasonic time of 30 min, liquid-to-solid ratio of 40∶1 (mL/g) and ethanol volume fraction of 50%. Three verification tests were performed using the above optimized conditions. The experimental results were shown in Table 4. The measured yield of total flavonoids from HERBA BLUMEAE RIPARIAE was 84.86 mg/g, with a relative deviation from the theoretical prediction of 0.26%, basically consistent with the theoretical prediction.

Table 3 Results of variance analysis

SourceSum ofsquaresFreedomMeansquareFvaluePvalueSignifi-canceModel827.02991.8916.350.000 7∗∗∗A278.571278.5749.560.000 2∗∗∗B6.8316.831.210.306 9C415.711415.7173.96< 0.000 1∗∗∗AB7.2611.291.290.293 0AC4.6310.820.820.394 4BC23.6914.224.220.079 2A245.22145.228.040.025 2∗B234.84134.846.200.041 6∗C214.50114.502.580.152 3Residual39.3575.62Lack of fit32.74310.916.610.049 7Error6.6041.65Sum866.3616Correction coefficientR2=0.383 4; R2adj=0.896 2

Note:***P<0.001,**P<0.01,*P<0.05.

Table 4 Results of verification test

TestcodeATotalflavonoids∥mg/gAverageyieldSTDEVvalueRSDvalue10.2484.8684.862.953.48%20.2381.9130.2587.82

4 Discussion

In this paper, ultrasonic-assisted extraction was used to extract total flavonoids from HERBA BLUMEAE RIPARIAE. Three factors of ethanol volume fraction, liquid-to-solid ratio and ultrasonic time were selected. Based on the results of single-factor tests, using response surface methodology, the extraction conditions for total flavonoids from HERBA BLUMEAE RIPARIAE were optimized. The optimal extraction process was as follows: ultrasonic time of 30 min, liquid-to-solid ratio of 40∶1 (mL/g) and ethanol volume fraction of 50%. The extraction process optimized with this method is easy to operate and has low pollution. The optimized extraction conditions of total flavonoids using response surface methodology have theoretical guiding significance for the comprehensive utilization of HERBA BLUMEAE RIPARIAE and the industrialization of flavonoids.