Optimization of the Extraction Process of Rutin from Flos Sophorae by Orthogonal Experiments

Shan LI Meiyuan LU Lin YAO Xinying MO Guilin YANG Haisheng ZENG

Abstract [Objectives] This study was conducted to optimize the extraction process of rutin in Flos Sophorae.

[Methods]With the percentage content of rutin extracted from Flos Sophorae as the evaluation index and rutin as the reference substance， the content of rutin in Flos Sophorae extracted ultrasonically was determined by high performance liquid chromatography. L9（34） orthogonal experiments were carried out to optimize the three factors （solvent concentration， material-to-liquid ratio， extraction time） that affect the effect of ultrasonic extraction of rutin from Flos Sophorae.

[Results] The extraction effect of the crude medicinal powder was best with the methanol concentration of 80%， the ratio of material to liquid at 0.1∶30， and the extraction time of 40 min.

[Conclusions]This study provides a reference for optimizing the extraction process of rutin in Flos Sophorae.

Key words Flos Sophorae; Rutin; Extraction process; High performance liquid chromatography

Received： April 2 2021  Accepted： June 17， 2021

Supported by 2020 Guangxi University Middle-aged and Young Teachers Basic Research Ability Improvement Project （No.2020KY13034）; Scientific Research Task of Administration of Traditional Chinese Medicine of the Guangxi Zhuang Autonomous Region （No. GZZC2019147）; the First Batch of High-level Talent Research Project in the Affiliated Hospital of Youjiang Medical University for Nationalities in 2019 （No.Y20196311）; School-level Scientific Research Project of Youjiang Medical University for Nationalities （No.yy2018ky018）.

Shan LI （1985-）， male， P. R. China， pharmacist in charge， devoted to research about pharmaceutical analysis.

*Corresponding author. E-mail： 334043616@qq.com.

Flos Sophorae refers to the dried flowers and flower buds of the legume Sophora japonica L. The medicine is slightly cold in nature， bitter in taste， and has the effects of cooling blood for hemostasis， and removing heat from the liver. Flos Sophorae contains rutin， quercetin and mucilage[1]. The main component of Flos Sophorae is rutin， which has anti-inflammatory， antibacterial， antiviral， antitumor， and antiarrhythmic effects[2]. The methods for determination of rutin in traditional Chinese medicine reported in current literatures mainly include high performance liquid chromatography， ultraviolet spectrophotometry， capillary electrophoresis， colorimetry， thin-layer scanning photometry and fluorospectrophotometry， etc.[3-4]. Therefore， rutin has been widely used in medicine， health food and cosmetics， and has high development value. In this study， with the content of rutin in Flos Sophorae as an indicator， orthogonal experiments were carried out to optimize the extraction process for three influencing factors： the ratio of material to liquid， the concentration of the extract， and the extraction time， aiming to provide a certain reference basis for further research on the quality standards of Flos Sophorae medicinal materials. At present， in this study， in order to ensure the quality and clinical efficacy of its medicinal materials， we established a method for determining the content of rutin flavone in Flos Sophorae， which provides a scientific basis for the later development and utilization of the medicinal resources of Flos Sophorae.

Materials and Methods

Instruments and reagents

Instruments

Agilent high performance liquid chromatograph （1260 type G1311X quaternary pump， G1329B automatic temperature control autosampler， ultraviolet detector， etc.）; KQ-5200 ultrasonic cleaner; PRACTUM224-KN SOP electronic balance （Sartorius Scientific Instruments （Beijing）Co.， Ltd.）.

Reagents

Rutin reference substance （batch number： 195126）， purity>98%， was purchased from Shanghai Winherb Medical Technology Co.， Ltd.; methanol was chromatographically pure; glacial acetic acid was analytically pure; experimental water was ultrapure water; and the rest of the reagents used in the experiment were analytically pure. Flos Sophorae medicinal materials were purchased from LBX pharmacy， and were identified as the dried flowers and buds of legume Sophora japonica L.

Experimental methods

Chromatographic conditions

Elite Hypersil ODS2 column （4.6 mm×250 mm， 5 μm）; filler： octadecylsilane bonded silica; mobile phase： methanol-0.3% glacial acetic acid solution （volume ratio 42∶58）; detection wavelength： 257nm; column temperature： 30 ℃; injection volume： 10 μl; theoretical plate number： not less than 2 000 based on the rutin peak. According to the above conditions， the separation of each component was good， as shown in Fig. 2.

Preparation of reference solution

An appropriate amount of the rutin reference substance was accurately weighed， added with methanol and prepared to a solution with a concentration of 0.120 mg/ml.

Preparation of test solution

A certain amount of the crude powder of Flos Sophorae medicinal material （about 0.2 g） was accurately weighed， and added into an Erlenmeyer flask with a stopper. Then， the corresponding concentration and volume of the methanol solution was accurately added， and the weight was measured. The mixture was ultrasonically treated for corresponding time， followed by cooling and then weighing. The lost weight was complemented with methanol， and the system was shaken well， and filtered. Finally， 2 ml of the filtrate was accurately measured， added into a 10 ml measuring flask， and added with methanol to constant volume， giving the test solution after shaking well.

Results and Analysis

Methodological investigation

Preparation of standard curve

First， 4， 6， 8， 1 14， 16 μl of the above-mentioned rutin reference solution were precisely drawn and injected into the liquid chromatograph， respectively， for the measurement of the peak area. With the injection amount of the rutin reference substance （μg） as the abscissa （X）， and the peak area integral value as the ordinate （Y）， a standard curve was drawn， and regression analysis was performed on X with Y， obtaining the regression equation Y=1 652.798 6X+14.038 768， r=0.999 4. The linear range was 0.408-1.428 μg， within which rutin had a good linear relationship.

Precision test

The 0.102 0 mg/ml rutin reference solution was taken and repeatedly injected 6 times （10 μl each） according to the chromatographic conditions under "Chromatographic conditions". The peak areas were measured and the RSD value was calculated. The RSD value of the peak area was 0.38%， indicating that the precision of the instrument was good.

Stability test

A certain amount of the crude powder of Flos Sophorae medicinal material （about 0.2 g） was accurately weighed， and prepared by the same method into a test solution， which was injected at 0， 3， 6， 9， 12 and 24 h， respectively. The areas of the rutin peak were recorded， and the RSD value of the rutin peak area was calculated. The RSD value was 1.05%， less than 3%， indicating that the Flos Sophorae sample had good stability.

Repeatability test

A certain amount of the crude powder of Flos Sophorae medicinal material （about 0.2 g） was accurately weighed， and prepared by the same method into a test solution. With the rutin reference solution as a control， the content of rutin was determined， and the RSD value was calculated. The average content of rutin was 152.6 mg/g， that is， every 1 g of Flos Sophorae contained 152.6 mg， and the RSD value of the content of rutin was 2.61%， less than 3%， which meant that the repeatability was good.

Recovery test

Six parts of Flos Sophorae medicinal material （0.1 g each） were accurately weighed. Each part of the medicinal material was added with an appropriate amount of rutin reference solution and prepared into a sample solution according to the sample solution preparation method under "Preparation of test solution". The sample solutions were injected separately， and the content was measured and the recovery rate was calculated. The results are shown in Table 5. The RSD of the recovery was less than 3.0%， that is， the recovery test was good. The results are shown in Table 1.

Investigation of the extraction process by orthogonal experiments

Design of the orthogonal experiments[5-6]

According to relevant literatures and reference to the 2020 edition of the Chinese Pharmacopoeia， we finally adopted the extraction method with different methanol concentrations as solvents， and investigated such three conditions as the solvent concentration （60% methanol， 80% methanol， 100% methanol）， the ratio of material to liquid （0.1∶20， 0.1∶30， 0.1∶40）， and the extraction time （30， 40， 50 min）. According to the orthogonal design assistant software， L9（34） orthogonal experiment design was selected， as shown in Table 2.

A certain amount of the crude powder of Flos Sophorae medicinal material （about 0.2 g） was accurately weighed， extracted according to the above extraction methods of rutin in Flos Sophorae， and then prepared according to the preparation method of test solution. The experiments were carried out according to the L9（34） orthogonal design scheme. The rutin in the extracts of Flos Sophorae was determined by high performance liquid chromatography. The results are shown in Table 3.

Analysis of experimental results

According to the software analysis， the results are shown in Table 4. With the content of rutin in Flos Sophorae as the experimental index， various experimental factors had different effects on the extraction of Flos Sophorae. The intuitive analysis results showed that methanol concentration had a significant impact on the extraction process of rutin， followed by the material-to-liquid ratio， and then the extraction time. The results of the analysis of variance showed that methanol concentration caused significant differences， while the liquid-to-material ratio and extraction time showed no significant differences， and the strength of various influencing factors ranked as A>B>C， so the factor that had statistical significance for the extraction effect of Flos Sophorae was methanol concentration. Considering the cost and efficiency， the most ideal extraction scheme in this study was A2B2C that is， when the methanol concentration was 80%， the material-to-liquid ratio was 0.1∶30， and the extraction time was 40 minutes， the extraction effect was the best.

Sample determination

Three parts of Flos Sophorae medicinal material （0.2 g each） were accurately weighed， added into conical flasks， and prepared into test solutions according to the operation steps under "Preparation of test solution" and the optimal extraction process （i.e.， 80% methanol， material-to-liquid ratio 0.1∶30， extraction time 40 min）. According to the method under "chromatographic conditions"， the content of rutin in Flos Sophorae medicinal material was determined by color development. The three parts of Flos Sophorae medicinal material were measured in parallel， and the average value of rutin was taken. The results are shown in Table 5.

Conclusions and Discussion

In this study， methanol-0.1% formic acid solution， methanol-0.2% formic acid solution， methanol-0.1% glacial acetic acid solution， methanol-0.2% glacial acetic acid solution， methanol-0.3% glacial acetic acid solution， methanol-0.1% phosphoric acid solution， and methanol-0.2% phosphoric acid solution were used as the mobile phase and compared. It was concluded that using methanol-0.3% glacial acetic acid solution as the mobile phase for gradient elution， the rutin separation effect was better. The chromatographic peak symmetry was good， and the baseline was relatively stable and baseline separation from the impurity peaks was observed. Therefore， this method is considered to be good and can be used for the determination of rutin in Flos Sophorae.

In this study， with the traditional Chinese medicine Flos Sophorae as the research object， the three influencing factors of methanol concentration， material-to-liquid ratio and extraction time were investigated. The ultrasonic extraction method was used to extract the rutin component in Flos Sophorae， and rutin was used as the experimental reference substance. The content of rutin in

Flos Sophorae was determined by liquid chromatography， and the extraction process of Flos Sophorae was optimized by the method of orthogonal experiments. According to relevant literatures， it was determined that the main factors affecting the extraction process of Flos Sophorae were solvent concentration， material-to-liquid ratio and extraction time. According to the needs of the experiment， the L9（34） orthogonal experiment table was adopted to conduct multiple experiments. Finally， the experimental results showed that the best process for ultrasonic extraction of Flos Sophorae was the methanol concentration of 80%， the material-to-liquid ratio at 0.1∶30， and the extraction time of 40 min， with which the best extraction effect was achieved. This study provides a reference basis for optimizing the ultrasonic extraction process of rutin from Flos Sophorae.

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