Extraction Technology and Content Determination of Total Flavonoids from Traditional Chinese Medicine Yipichou

Qianqian LIU, Wen ZHONG, Zongxi SUN,, Xiumei MA, Xinli LU, Yusheng HUANG, Jingrong LU, Jiangcun WEI,*

1. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. Guangxi University of Chinese Medicine, Nanning 530200, China; 3. Guangxi Jialu Human Resource Consulting Co., Ltd., Nanning 530031, China

Abstract [Objectives] The research aimed to optimize extraction technology of total flavonoids from traditional Chinese medicine Yipichou, and establish methods of extraction and content determination of its medicinal materials. [Methods] Using reflux extraction method, total flavonoids of Yipichou were extracted. Via single-factor test, influence of extraction method, extraction solvent concentration, extraction volume and extraction time on content of total flavonoids from Yipichou was inspected. Using orthogonal test, extraction conditions were optimized, and optimal extraction technology of total flavonoids from Yipichou was obtained. [Results] The optimal extraction process of total flavonoids from Yipichou was as below: 70% of methanol, 1∶40 of solid-liquid ratio, and 1.0 h of reflux extraction time. [Conclusions] In the test, total flavonoids were extracted from Yipichou, with better repeatability, and stable and feasible method, which could provide certain scientific basis for studying quality standard of this medicinal material.

Key words Yipichou, Total flavonoids, Extraction process, Ultraviolet-visible spectrophotometry

1 Introduction

Traditional Chinese medicine Yipichou belongs to Convolvulaceae Juss., and it is stem and leaves ofArgyreiaacetaLour[1-2]. Yipichou has many kinds of curative effects, such as stopping bleeding, relieving cough, resolving phlegm, relieving pain, detoxification, dispelling wind, and removing dampness. Traditional Chinese medicine Yipichou could be used to cure rheumatic pain, chronic tracheitis, breast pain and other symptomatic diseases[1-2]. Yipichou is mainly distributed in Guangxi, Guangdong, Hunan, Yunnan and other regions, and is often used as an antidote in clinic[3-4]. Flavonoids are broad-spectrum bioactive substances and are widely distributed in plants. It has anti-cancer, cancer preventing, antibacterial, antiviral, cholesterol lowering and other pharmacological effects and biological activities, and can protect endocrine system[5]and immune system[6-7].

In this paper, content of total flavonoids from traditional Chinese medicine Yipichou was taken as the index. Using single-factor test, the influence of extraction method, extraction solvent, extraction volume and extraction time on content of total flavonoids from traditional Chinese medicine Yipichou was inspected. Orthogonal test was used to further select the best extraction process, which could provide certain reference basis for further studying quality standard of Yipichou. At present, there is less domestic research on determining content of total flavonoids from Yipichou. To guarantee quality and clinical efficacy of its medicinal materials, content determination method of total flavonoids from Yipichou was established, which could provide scientific basis for the later development and utilization of its medicinal resources.

2 Instruments and reagents

2.1 InstrumentsUV1780 type of ultraviolet-visible spectrophotometer (Shimadzu); one-100 000th balance (XS105D, Mettler-Toledo Weighing System Co., Ltd.); KQ5200B ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.); Youpu ULUPURE laboratory ultrapure water meter (Shanghai Youpu Industrial Co., Ltd.).

2.2 ReagentsRutin (lot No.205125), purity >98%, and it was bought from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. Methanol, ethanol, sodium nitrite, aluminum nitrate, and sodium hydroxide were all AR. The medicinal materials were collected in Yulin of Guangxi, and it was identified as whole herb ofA.acutaLour. by professor from Guangxi University of Chinese Medicine. After 60 ℃ of drying, it was reserved for use.

3 Methods and results

3.1 Preparation of control sample solutionAppropriate amount of rutin control sample which was dried to constant weight at 105 ℃ was weighed precisely, and 70% methanol was added to dissolve and fix the volumn to 25 mL of volumetric flask. After shaken evenly, it was prepared into 0.45 mg/mL of control solution.

3.2 Preparation of test sample solution1.0 g of Yipichou powder was weighed precisely and set in conical flask. 25 mL of 70% methanol was added, and it was weighed. After reflux extraction for 1.0 h, it was weighed again. 70% methanol was used to make up for the lost weight. After shaken evenly and filtered, 2.0 mL of subsequent filtrate was measured precisely and set in 50 mL of graduated flask. According to determination method of total flavonoids, its absorbance was determined, and content of total flavonoids in sample solution was calculated by using linear equation, namely content of total flavonoids in Yipichou.

3.3 Selection of detection wavelength3 mL of control sample solution was weighed precisely into 25 mL of volumetric flask, and 1 mL of 5% sodium nitrite solution was added. After shaken evenly and set for 6 min, 1 mL of 10% aluminum nitrate solution was added. After shaken evenly and set for 6 min, 10 mL of 4% sodium hydroxide solution was added, and water was added to graduate. It was shaken evenly and set for 12 min, and corresponding reagent was taken as blank. According to spectrophotometric method, absorption spectrogram between 400-800 nm was determined, and its maximum absorption wavelength was 512 nm (Fig.1).

Fig.1 Absorption spectrogram of rutin control sample

3.4 Formulation of linear equation0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 mL of control sample solutions in Section3.1were respectively set into 25 mL of volumetric flask, and 0.4 mL of 5% sodium nitrite was added. After set for 6 min, 0.4 mL of 10% aluminium nitrate was added. After set for 6 min, 4 mL of 4% sodium hydroxide was added. Water was added to graduate, and it was shaken evenly and set for 10 min. After that, absorbance was determined at 512 nm[8]. Absorbance to concentration was used to make linear equation, and the equation was as below:y=13.045x-0.039 2,R2=0.999 2. Within the range of 0.009-0.072 mg/mL, concentration and absorbance had good linear relationship.

3.5 Methodology investigation

3.5.1Precision test. 1.0 g of Yipichou powder was weighed precisely to prepare test sample. According to the methods of Section3.3, chromogenic assay was conducted. At the wavelength of 512 nm, its absorbance was determined for 6 times. The result showed thatRSDwas 1.21%, less than 3%, showing that the method had good precision.

3.5.2Stability test. 1.0 g of Yipichou powder was weighed precisely to prepare test sample. According to the methods of Section3.3, chromogenic assay was conducted, and absorbance of the same test sample solution under different time was determined. At the wavelength of 512 nm, absorbance was determined at 0, 10, 20, 30, 40, 50 and 60 min. The results showed thatRSDwas 2.75%, less than 3%, and absorbance basically unchanged within 60 min. It was clear that sample was basically stable within 60 min.

3.5.3Repeatability test. 6 copies of Yipichou powder were weighed precisely to prepare test sample, each for 1.0 g. According to the methods of Section3.3, chromogenic assay was conducted, and its absorbance was determined parallelly at the wavelength of 512 nm. The results showed that average content of total flavonoids from Yipichou was 24.85 mg/g, andRSDwas 2.79, less than 3%. It showed that the method had good repeatability.

3.5.4Recovery test of sample addition. 0.5 g of Yipichou powder whose content of total flavonoids was known (24.85 mg/g) from the same lot was weighed precisely, and there were 9 copies in total. They were divided into 3 groups: low, moderate and high sample addition groups (according to 80%, 100% and 120% of total flavonoids content from 0.5 g of medicinal materials, rutin was added precisely, and sample addition amounts were 9.94, 12.43 and 14.91 mg). Test sample was prepared by operation steps of Section3.2, and chromogenic assay was conducted according to the methods of Section3.3. At the wavelength of 512 nm, its absorbance was determined parallelly, and content of total flavonoids was calculated. Seen from experimental results, average recovery rates of total flavonoids in low, moderate and high sample addition groups of Yipichou were 98.90%, 100.07%, and 101.02%, andRSDwas 2.12%, 2.74%, and 2.51% (n=3), showing that the method had good accuracy. The results were shown in Table 1.

Table 1 Experiment on recovery rate of sample addition for extraction of total flavonoids from Yipichou (n=9)

3.6 Results of single-factor inspection test

3.6.1Extraction method. 1.0 g of Yipichou powder was weighed precisely and set in conical flask. Extraction time was 1 h, and 25 mL of 70% methanol was used. Immersion method, ultrasonic extraction and reflux extraction were used to extract total flavonoids from Yipichou. Test sample was prepared by operation steps of Section3.2, and chromogenic assay was conducted according to the methods of Section3.3. The results displayed that extraction rate of reflux extraction method was the highest, so reflux extraction was seclected ( Fig.2).

Fig.2 Influence of extraction method on content determination of total flavonoids from Yipichou

3.6.2Concentration of methanol solvent. 1.0 g of Yipichou powder was weighed precisely and set in conical flask. Extraction time was 1 h, and 25 mL of methanol solvent in different concentrations was used. Reflux extraction method was used, and different concentrations of methanol solvent to yield of total flavonoids was used to do single-factor test, and concentrations of methanol were designed as 30%, 50%, 60%, 70%, 80% and 100%. Test sample was prepared by operation steps of Section3.2, and chromogenic assay was conducted according to the methods of Section3.3. The results displayed that extraction rate of total flavonoids by 70% methanol reflux was the highest (Fig.3).

Fig.3 Influence of methanol concentratin on content determination of total flavonoids from Yipichou

3.6.3Extraction time. 1.0 g of Yipichou powder was weighed precisely and set in conical flask. 25 mL of 70% methanol solvent was taken as extraction solvent, and reflux extraction method was used. Extraction time to yield of total flavonoids was used to do single-factor test, and extraction time was set as 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Test sample was prepared by operation steps of Section3.2, and chromogenic assay was conducted according to the methods of Section3.3. The results displayed that extraction rate was the highest when extraction time was 1.5 h (Fig.4).

Fig.4 Influence of extraction time on content determination of total flavonoids from Yipichou

3.6.4Extraction volume. 1.0 g of Yipichou powder was weighed precisely and set in conical flask. Extraction time was 1 h, and 25 mL of 70% methanol solvent was taken as extraction solvent, and reflux extraction method was used. Different volumes of 70% methanol to yield of total flavonoids was used to do single-factor test, and extraction volume of 70% methanol was designed as 20, 30, 40, 50, 60, and 70 mL. Test sample was prepared by operation steps of Section3.2, and chromogenic assay was conducted according to the methods of Section3.3. The results dispalyed that extraction rate was the highest when extraction volume was 40 mL (Fig.5).

Fig.5 Influence of extraction volume on content determination of total flavonoids from Yipichou

3.7 Orthogonal experimental designMain extraction method of total flavonoids from Yipichou was organic solvent extraction. According to related literature, different concentrations of methanol was taken as extraction solvent finally. According to single-factor investigation results, methanol concentration (60% methanol, 70% methanol, and 80% methanol), solid-liquid ratio (1∶30, 1∶40, 1∶50), and extraction time (1.0, 1.5, and 2.0 h)were set[9-12]. According to assistant software of orthogonal design, L9(34) orthogonal experimental design was selected (Table 2).

Table 2 Factor and level of L9 (34)orthogonal test for extraction of total flavonoids from Yipichou

3.8 Orthogonal experiment and result analysisAccording to results of orthogonal analysis, methanol concentration and soild-liquid ratio had significant influence on extraction of total flavonoids from Yipichou, while extraction time had no significant influence on extraction of total flavonoids from Yipichou. Experimental variance analysis showed that both methanol concentration and solid-liquid ratio had significant difference, while extraction time had no significant difference, and sequence of each influecne factor was solid-liquid ratio (B)>methanol concentration (A)> extraction time (C). So, methanol concentration and solid-liquid ratio had statistical significance for extraction of total flavonoids from Yipichou, while extraction time had no significant significance. Considering from efficiency, time and cost of experiment, extraction time was selected as 1.0 h. Therefore, the most ideal extraction case was A2B2C1, namely 70% of methanol concentration, 1∶40 of solid-liquid ratio, and 1.0 h of reflux extraction time. Under the extraction condition, the extraction effect was the best (Table 3-4).

Table 3 L9 (34) orthogonal test result for extraction of total flavonoids from Yipichou

Table 4 Variance analysis of orthogonal experiment for extraction of total flavonoids from Yipichou

3.9 Determination of sample content3 copies of Yipichou powder were weighed precisely and set in conical flask, each for 1.0 g. Test sample was prepared by operation steps of Section3.2, and coloration was conducted by the methods of Section3.3. Content of total flavonoids was determined, and the results were shown in Table 5.

Table 5 Content determination results of total flavonoids from Yipichou

4 Conclusions and discussion

Yipichou was taken as research object, and three influecne factors were inspected: methanol concentration, extraction volume and extraction time. Total flavonoids were extracted from Yipichou by reflux extraction, and orthogonal experiment was used to select the optimal extraction technology. According to related literatures and prior research basis, main influence factors for extraction of total flavonoids from Yipichou were solvent concentration, extraction volume and extraction time. According to the selection of influence factors, L9(34) orthogonal experiment was used for multiple experiments. The results showed that the optimal extraction process of total flavonoids from Yipichou was as below: reflux extraction, 70% of methanol concentration, 1∶40 of solid-liquid ratio, and 1.0 h of extraction time.

On the basis of single-factor test, extraction process optimization of total flavonoids from Yipichou was established via orthogonal test. The analysis of variance was significant, and the equation had better effect on the experiment, and sequence of each factor on extraction rate of total flavonoids was solid-liquid ratio B (g/mL)> methanol concentration A (%)> extraction time C (min).