Response surface optimization of ultrasonic assisted extraction of flavonoids from Brucea javanica

This text was mainly studied the response surface optimization of ultrasonic assisted extraction of flavonoids from Brucea javanica. The optimum extraction process conditions were obtained by color reaction to prove the presence of flavonoids and by response surface simulation and quantification, the optimum condition was: ethanol concentration was 90%, the ratio of liquid to solid was 10:1, and the extraction time was 2h, the total extraction rate was the largest, up to about 1.43%.


INTRODUCTION
Brucea javanica is the mature seed of Brucea javanica (Simaroubaceae), also known as Hazelnut, Sophora flavescens and so on. It is mainly in China's Guangdong and Guangxi provinces, and is rich in resources. The main chemical constituents from Brucea javanica are bitter lignans, flavonoids, triterpenoids and alkaloids. These ingredients are great of using in the field of medicine and human health. Many researchers have studied the chemical constituents of Brucea Javanica [1][2][3][4] . With the continuous development of plant extraction technology, various chemical components in Brucea Javanica are extracted by people for scientific research [5][6][7] . Especially, the anti-cancer effect of Brucea javanica oil has been paid more attention [8][9][10][11][12][13] , and the anti-cancer effect of Brucea javanica oil has been extensively studied. It is reported that flavonoids have anti-inflammatory and analgesic effects. In this study, flavonoids were extracted from Brucea javanica, hoping to provide references for further processing and utilization of traditional Chinese medicine [14] .

Materials
Brucea javanica was bought from the local supermarket. Anhydrous ethanol, sodium nitrite, aluminum sulfate, sodium hydroxide were bought from Beijing North Chemical Fine Chemicals Co., Ltd. Rutin, saffron red, o-phenol, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium dihydrogen phosphate were bought from National drug group chemical reagents Co., Ltd. All the reagents were analytically pure.

Extraction procedure of flavonoids
Brucea javanica was smashed with mill grinding, the powder weight was 1.00 g, a certain volume fraction of ethanol solution was added, and adjusting the ratio of liquid to solid, put triangular flask which contained the extraction sloution into ultrasonic cleaner, set temperature as 50 ℃, set power as 400 W. After extraction, the extraction liquid was filtered by filter paper.

Calculation of flavonoids content
The flavonoids content C% was calculated according to the formula (1).

Color analysis identification
The above extracted liquid of Brucea javanica was taken as sample, and color analysis was carried out by UV lamp irradiation, ammonia reaction, and sodium hydroxide reaction. As shown in Figure 2, with the increase of ratio of liquid to solid, flavonoids extraction rate in Brucea javanica was first increased and then showed a gentle trend. When the ratio of liquid to solid was too low, the sample could not be completely immersed in the solvent, the extraction was incomplete, and the extraction rate was low. When the ratio was higher than 20:1, the curve showed a gentle trend, indicating that this volume of solvent had reached the extraction effect, which led to waste of solvent. So when the extraction temperature was 50 ℃ , the ultrasonic power was 400 W, the ethanol concentration was 70%, extraction time was 2 h, flavonoids extraction rate was better. As shown in Figure 3, with the increase of ultrasonic time, the extraction rate of flavonoids became higher. After 2h, the extraction rate of flavonoids decreased slightly, the reason was that the ultrasonic time was too long, caused the instrument temperature more than 50 ℃, depressed flavonoids material degeneration. After 2h, the flavonoids in the sample had been fully extracted, and the further time would only increase the energy consumption. So when the temperature was 50 ℃ , ethanol concentration was 70%, ultrasonic time was 2 h, flavonoids extraction rate was better. With the increase of ethanol concentration, the extraction rate of flavonoids increased first and then decreased (see Figure 4). It might be that if the ethanolconcentration was different and the polarity was different. After the ethanol concentration was over 70%, the extraction rate began to decline greatly, and the dissolution of flavonoids was reduced, while some impurities were increased, leading to the reduction of the extraction rate. So when extraction temperature was 50 ℃, ultrasonic time was 2 h, the ethanol concentration was 70%, flavonoids extraction rate was better.

Experimental results of response surface
The Box Behnken model by software Design Expert 8.0.6 was used to design the response surface experiment. The design parameter contained the ratio of liquid to solid, ultrasonic time and ethanol concentration. By the Design Expert 8.0.6 software analysis, the optimum condition for flavonoids was: the extraction temperature was 50 ℃, ultrasonic power was 400 W, ratio of liquid to solid was 10:1, ethanol concentration was 90%, ultrasonic time was 2 h, under the condition, the flavonoid extraction rate could reach to 1.43%.      It could be seen from Figure 5, 6 and 7 that three factors ethanol concentration, ultrasonic time, ratio of liquid to solid had the influence on the extraction rate. The best extraction rate could be achieved by seeking the optimal experimental conditions. A few drops of sample solution were put on the filter paper and irradiated by ultraviolet lamp. yellow-green fluorescence could be seen, it appeared that the extraction solution contained flavonoids. A few drops of sample solution were taken and dripped on the filter paper for air drying. Ammonia solution was poured into the small beaker. The filter paper was smoked on the small beaker for 1min, yellowgreen fluorescence appeared.

Conclusions
This experiment mainly studied the effects of various factors on the extraction rate of flavonoids from Brucea javanica, providing some theoretical basis for its extraction in the food and drug industry. The results showed that for Brucea javanica extraction, when extraction temperature was 50 ℃, the ultrasonic power was 400 W, the ratio of liquid to solid was 10:1, ethanol concentration was 90%, ultrasonic time was 2 h, the extraction rate could reach to 1.43%. By color reaction it could be known that the flavonoids extracted from the Brucea javanica were mainly flavonols.