Optimization of Ultrasonic-enzymatic Extraction of Total Flavonoids from Cardamine tangutorum O. E. Schulz by Response Surface Methodology and Content Determination

Xiaoxia CAI, Bochao LI, Xuexue LI, Hongna SU, Wenbing LI, Yuan LIU

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

Abstract [Objectives] This study aimed to optimize the extraction process of total flavonoids in Cardamine tangutorum O. E. Schulz. [Methods] On the basis of single-factor experiments, the ultrasonic-enzymatic extraction process of total flavonoids in C. tangutorum O. E. Schulz was optimized by Box-Behnken design from aspects of ethanol volume fraction, extraction time, extraction temperature and enzyme addition, with yield of total flavonoids as the evaluation index. [Results] The optimal extraction process of total flavonoids in C. tangutorum O. E. Schulz was as follows: ethanol volume fraction of 70%, ultrasonic extraction time of 43 min, ultrasonic extraction temperature of 37 ℃ and cellulose addition of 0.07 mg. Under the optimal conditions, the yield of total flavonoids from C. tangutorum O. E. Schulz reached 75.90 mg/g. [Conclusions] The optimized extraction process was reasonable and feasible, and it could provide a reference for the extraction of total flavonoids in C. tangutorum O. E. Schulz. This study provided a foundation for the quality control of food/medicinal materials and the development and research of big health products of C. tangutorum O. E. Schulz.

Key words Cardamine tangutorum O. E. Schulz, Total flavonoid, Response surface methodology, Ultrasonic-enzymatic hydrolysis, Extraction process

1 Introduction

Dried whole plant ofCardaminetangutorumO. E. Schulz (Cruciferae), also known as Shigecai and Shijiecai, could be used to treat indigestion and diarrhea, and its external application could treat broken tendons, bruises, carbuncle swelling[1-3].C.tangutorumO. E. Schulz had a long history of dual use for medicine and food. It was commonly used as raw material for sauerkraut in southwestern China.C.tangutorumO. E. Schulz was the most commonCardamineplant on the southeastern edge of Qinghai-Tibet (about 3 600-4 200 m above sea level) for medicine and food. Modern researches[4-7]showed thatCardamineplants contained flavonoids, volatile oil, nitrogen-containing compounds and other ingredients. Flavonoids were one of the effective ingredients in many traditional medicine and food[8-9]. They had neuroprotective, anti-myocardial ischemia, blood pressure-lowering, learning memory-improving, anti-gastric ulcer, reproductive tissues-protecting, anti-inflammatory, anti-tumor, blood sugar-lowering and other pharmacological effects. Therefore, in order to further develop and utilize theC.tangutorumO. E. Schulz resource unique to Qinghai-Tibet Plateau, taking the yield of total flavonoids as an indicator, the extraction process of total flavonoids inC.tangutorumO. E. Schulz was optimized by four-factor and three-level central composite design-response surface methodology in this study, on the basis of single-factor experiments, and the contents of flavonoids in different batches ofC.tangutorumO. E. Schulz samples were determined, with a view to laying the foundation for the quality control of food/medicinal materials and the development and research of big health products ofC.tangutorumO. E. Schulz.

2 Materials

Dual-beam UV-Vis spectrophotometer (TU-1950, Beijing Puxi General Instrument Co., Ltd.), electronic analytical balance [AE240S, Mettler-Toledo Instruments (Shanghai) Co., Ltd.], ultrasonic cleaner (KQ-250B, Kunshan Ultrasonic Instrument Co., Ltd.), digital display constant temperature water bath (HH-2, Jintan Honghua Instrument Factory) and high-speed Chinese medicine grinder (HX-200, Zhejiang Yongkang Xi’an Medicinal Hardware Products Factory) were the main instruments and equipment used.

Rutin standard (batch No.100080-200707) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. The other important drug used was cellulase (batch No.4131022). The water used was pure water. All the reagents were of analytical grade.C.tangutorumO. E. Schulz samples were collected from Aba Tibetan and Qiang Autonomous Prefecture and Ganzi Tibetan Autonomous Prefecture in Sichuan Province from 2016 to 2017 (Table 1). They were identified by Professor Liu Yuan of Southwest Minzu University. TheC.tangutorumO. E. Schulz samples were pulverized and passed through No.3 sieve.

Table 1 Sources of Cardamine tangutorum O. E. Schulz samples

3 Methods and results

3.1 Determination of total flavonoids

3.1.1Preparation of reference solution. An accurate amount (10.20 mg) of rutin standard was dissolved in 70% ethanol to 50 mL to obtain the rutin reference solution (0.204 mg/mL).

3.1.2Preparation of sample solution. An accurate amount (around 0.5 g) of the powder ofC.tangutorumO. E. Schulz (Note: The medicinal material used in single-factor and response surface experiments were all the samples S3) was weighed and extracted under the set ethanol volume fraction, ultrasonic extraction time, ultrasonic extraction temperature and enzyme addition. The extract obtained was vacuum-filtered, and the residue was rinsed with a small amount of extract. The liquid was finally diluted to 50 mL and shaken well for later use.

3.1.3Investigation of linear relationship. Different volume (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mL) of rutin reference solution was transferred to a 20 mL colorimetric tube, added with 2.5 mL of 1% aluminum chloride solution to develop color, added with 70% ethanol to 10.0 mL, shaken well, and let standard for 10 min. Blank solution was prepared by replacing rutin reference solution with 70% ethanol. The absorbance at 510 nm was determined. With absorbance (A) as the ordinate and mass fraction of rutin (C) as the abscissa, linear regression was performed to get the regression equation:A=1.323 8C-0.116 1 (R2=0.999 7). The results showed that the rutin reference solution had a good linear relationship at 102.00-714.00 μg/mL.

3.1.4Determination of total flavonoids[10-11]. An appropriate amount of the flavonoids extract ofC.tangutorumO. E. Schulz was placed in 20 mL test tube, added with 70% ethanol to 10 mL and shaken well. With 70% ethanol as a blank control, color was developed according to the method in Section3.1.3. The absorbance at 510 nm was determined. According to the regression equation, the content of total flavonoids was calculated.

3.2 Methodological investigation

3.2.1Precision test. Six portions of the rutin reference solution prepared in Section3.1.1, 2 mL for each, were sampled. Color was developed according to the method under Section3.1.3. The absorbances (A) at 510 nm were determined, and theRSDvalue was calculated to be 0.71%, indicating good precision.

3.2.2Reproducibility test. Six portions of the powder ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed, prepared into solutions according to the method under Section3.1.2, and color-developed according to the method in Section3.1.3. Their absorbances (A) at 510 nm were determined, and theRSDvalue was calculated to be 0.15%, indicating that the method has good reproducibility.

3.2.3Stability test. Six portions of the powder ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed, prepared into solutions according to the description in Section3.1.2, and color-developed according to the method in Section3.1.3. The absorbances were determined once every 15 min (n=6). The result showed that theRSDvalue was 0.96%, indicating that the stability was better within 90 min.

3.2.4Sample recovery test. Nine portions of the powder ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed, added with different amounts of rutin reference solution, prepared into test solutions according to the method in Section3.1.2, and color-developed according to the description in Section3.1.3. The absorbances (A) at 510 nm were determined. The average recovery rate was calculated to be 98.98% (RSD=1.33%), indicating that the method was accurate and reliable.

3.3 Single-factor experiments

3.3.1Effect of ethanol volume fraction on the yield of total flavonoids. Five portions of the power ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed. They were added with 15 mL of ethanol with a volume fraction of 40%-80% and 0.05 mg of cellulase, ultrasonicated in water bath (37 ℃) for 40 min, and vacuum-filtered, respectively. The residue obtained was rinsed with a small amount of the extract. The filtrate was diluted to 50 mL. A certain volume (0.8 mL) of the liquid obtained was transferred to a 10 mL colorimetric tube, color-developed according to the method described in Section3.1.3, and determined at 510 nm. The yield of total flavonoids was calculated. The results (Fig.1) showed that the yield of total flavonoids was highest at the ethanol volume fraction of 70%. As the volume fraction of ethanol increased, the osmotic pressure increased, the degree of dissolution of some pigments and alcohol-soluble impurities had increased, leading to a decrease in the extraction rate of total flavonoids[12].

Fig.1 Effect of ethanol volume fraction on the yield of total flavonoids from Cardamine tangutorum O. E. Schulz

3.3.2Effect of ultrasonic extraction time on the yield of total flavonoids. Five portions of the power ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed. They were added with 15 mL of 70% ethanol and 0.05 mg of cellulase, ultrasonicated in water bath (37 ℃) for 20, 30, 40, 50 and 60 min, and vacuum-filtered, respectively. The residue obtained was rinsed with a small amount of the extract. The filtrate was diluted to 50 mL. A certain volume (0.8 mL) of the liquid obtained was transferred to a 10 mL colorimetric tube, color-developed according to the method described in Section3.1.3, and determined at 510 nm. The yield of total flavonoids was calculated, and the results were shown in Fig.2. Under the extraction time of 40 min, the yield of total flavonoids was the highest. With the extension of the extraction time, the amount of undissolved total flavonoids had been very small, but other impurities might increase, leading to a decrease in the yield of total flavonoids[12-13].

Fig.2 Effect of ultrasonic extraction time on the yield of total flavonoids from Cardamine tangutorum O. E. Schulz

3.3.3Effect of the amount of enzyme added on the yield of total flavonoids[14]. Three portions of the power ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed. They were added with 15 mL of 70% ethanol, bathed in water (42 ℃), added with 0, 0.05 and 0.10 mg of cellulase, ultrasonicated for 40 min, and vacuum-filtered, respectively. The residue obtained was rinsed with a small amount of the extract. The filtrate was diluted to 50 mL. A certain volume (0.8 mL) of the liquid obtained was transferred to a 10 mL colorimetric tube, and color-developed according to the method described in Section3.1.3. The yield of total flavonoids was calculated, and the results were shown in Fig.3. The yield of total flavonoids under the addition of cellulase was higher than that without the addition of cellulase, but there was no significant difference in the yield of total flavonoids between different addition amounts of cellulase. Considering the cost and use amount of actual experiment, the addition amount of cellulase was determined to be 0.05 mg in the ultrasonic-enzymatic extraction of total flavonoids inC.tangutorumO. E. Schulz.

Fig.3 Effect of amount of enzyme added on the yield of total flavonoids from Cardamine tangutorum O. E. Schulz

3.3.4Effect of ultrasonic extraction temperature on the yield of total flavonoids. Five portions of the power ofC.tangutorumO. E. Schulz, 0.5 g for each, were weighed. They were added with 15 mL of 70% ethanol and 0.05 mg of cellulase, ultrasonicated in water bath (27, 37, 42, 57 and 72 ℃) for 40 min, and vacuum-filtered, respectively. The residue obtained was rinsed with a small amount of the extract. The filtrate was diluted to 50 mL. A certain volume (0.8 mL) of the liquid obtained was transferred to a 10 mL colorimetric tube and color-developed according to the method described in Section3.1.3. The yield of total flavonoids was calculated, and the results were shown in Fig.4. At the extraction temperature of 42 ℃, the yield of total flavonoids was highest.

Under the addition of cellulase, in the range of 27-72 ℃, with the increase of ultrasonic extraction temperature, the yield of flavonoids showed a trend of increasing first and then decreasing, and reached the highest at 42 ℃. As the extraction temperature increased, cellulase activity was destroyed and inactivated by high temperature, and the structure of flavonoids had also changed, leading to a decrease in the yield of total flavonoids[15-16].

Fig.4 Effect of ultrasonic extraction temperature on the yield of total flavonoids from Cardamine tangutorum O. E. Schulz

3.4 Optimization of the extraction process by response surface methodologyBased on the results of single-factor experiments, a four-factor (ethanol volume fraction, ultrasonic extraction time, ultrasonic extraction temperature and cellulase addition) three-level Box-Behnken response surface experiment was designed to optimize the extraction process of total flavonoids inC.tangutorumO. E. Schulz. The factors and levels of the response surface design were shown in Table 2, and the design and results were shown in Table 3.

Table 2 Factors and levels in the response surface design

Table 3 Results of response surface experiment for optimizing extraction process of total flavonoids in Cardamine tangutorum O. E. Schulz

3.5 Response surface analysisBased on the data in Table 2, taking the yield of total flavonoids inC.tangutorumO. E. Schulz as response value (Y), stepwise regression fitting was performed on ethanol volume fraction (A), ultrasonic extraction time (B), ultrasonic extraction temperature (C) and enzyme addition (D) using Design-Expert V8.0.5b software. The regression equation obtained was as follows:

Y=2.555 11+0.070 067A+0.018 667×B+0.067 693×C+26.06×D-5×10-5×AB+10-4×AC-0.055×AD-1.666 67×10-5×BC-0.035×BD-0.1 ×CD-4.9×10-4×A2-1.381 25×10-4×B2-9.011 11×10-4×C2-114.6×D2.

3.6 Verification experimentAfter analysis using Design-Expert V8.0.5b software, the optimal ultrasonic-enzyme extraction process for flavonoids inC.tangutorumO. E. Schulz was obtained: ethanol volume fraction of 68.84%, ultrasonic time of 43.45 min, ultrasonic temperature of 36.85 ℃ and enzyme addition of 0.07 mg. Under the optimal conditions, the yield of total flavonoids reached 7.590 5%.

In order to verify the reliability of the response surface methodology, while considering the feasibility of actual operation, the process was revised as follows: ethanol volume fraction of 70%, ultrasonic extraction time of 43 min, ultrasonic extraction temperature of 37 ℃, and enzyme amount of 0.07 mg. Under the revised conditions, three verification experiments were conducted. The average yield of total flavonoids measured was 7.590 5%, and the relative deviation from the theoretical prediction was 0.29%, basically consistent with the theoretical prediction. It proves that the extraction conditions of flavonoids inC.tangutorumO. E. Schulz optimized by response surface methodology were reliable.

3.7 Mass fraction of total flavonoidsThree portions of the powder ofC.tangutorumO. E. Schulz were weighed accurately. They were prepared into test solutions according to the optimal process, and determined accordingly. The total flavonoid content was calculated according to the standard curve. The content of total flavonoids inC.tangutorumO. E. Schulz collected from different producing areas had little difference, ranging between 72.06 and 75.58 mg/g (Table 5).

Table 4 Analysis of variance of regression equation

Fig.5 Response surface plots showing the interactive effects of various factors on the yield of flavonoids in Cardamine tangutorum O. E. Schulz

Table 5 Contents of total flavonoids in Cardamine tangutorum O. E. Schulz samples of different habitats

4 Discussion

(i)C.tangutorumO. E. Schulz was the most common edible and medicinal species on the southeastern edge of Qinghai-Tibet Plateau (about 3 600-42 00 m above sea level). In previous survey and investigation in Aba Tibetan and Qiang Autonomous Prefecture and Ganzi Tibetan Autonomous Prefecture of Sichuan Province, it has been found that local Tibetan and Qiang people have a long history of eatingC.tangutorumO. E. Schulz, and believe that it could regulate blood pressure, promote gastrointestinal peristalsis, detoxify, clear away heat, reduce fire,etc. Modern literature[17]showed that the main active ingredient ofC.tangutorumO. E. Schulz was flavonoids. To this end, in this study, based on single-factor experiments, the extraction process of flavonoids inC.tangutorumO. E. Schulz was optimized by using central composite design-response surface method, with a view to providing raw material for the subsequent development of big health products.

(ii) At present, ultrasonic-assisted extraction, enzyme-assisted extraction, Soxhlet extraction,etc. were mostly used for the extraction of total flavonoids in drugs[18-19], all of which have disadvantages such as high extraction cost and low efficiency. In this experiment, the ultrasonic-enzymatic hydrolysis method was used to effectively remove the fetters of the cell wall to flavonoids through enzymes, realizing the rapid release of flavonoids[20], and by making use of the cavitation produced by ultrasound, cells were ruptured quickly, making flavonoids more soluble under the thermal effect of ultrasound[21]. This method has the advantages of short extraction time, mild extraction conditions, less solvent consumption, and high extraction rate. There have been studies on the extraction of total flavonoids by combined enzyme and ultrasonic methods, but there were few reports on the use of ultrasonic-enzymatic hydrolysis to extract total flavonoids fromC.tangutorumO. E. Schulz. In this study, the central composite design-response surface method was used to optimize the ultrasonic-enzymatic extraction of flavonoids fromC.tangutorumO. E. Schulz, and the optimal extraction process obtained was as follows: ethanol volume fraction of 70%, ultrasonic extraction time of 43 min, ultrasonic extraction temperature of 37 ℃ and cellulase addition of 0.07 mg. Under the optimal extraction process, the yield of flavonoids reached 75.90 mg/g. This study provides a demonstration for the extraction of total flavonoids from other varieties.

(iii) The results of determination of total flavonoids in each sample showed that there were little differences in the content of total flavonoids among theC.tangutorumO. E. Schulz samples collected from different producing areas, ranging between 72.06 and 75.58 mg/g. There were also little differences in the content of total flavonoids among theC.tangutorumO. E. Schulz samples harvested at different times. However, the content of flavonoids inC.tangutorumO. E. Schulz harvested in July was slightly higher than that harvested in June. In short, the content of total flavonoids inC.tangutorumO. E. Schulz is high, and the determined extraction method is stable and feasible. This study will provide a scientific basis for the comprehensive development and utilization of the plant.