Content Determination of Quercetin and Kaempferol in Flueggea virosa (Roxb. ex Wild.) Baill.

Zhiying WEI, Hailin LU, Hongrui LIANG, Xiaojiao PAN

Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] The purpose was to determine the contents of quercetin and kaempferol in Flueggea virosa (Roxb. ex Wild.) Baill., thereby providing reference data for the research and application of F. virosa. [Methods] Quercetin and kaempferol in F. virosa were extracted with ultrasonic method, using dilute hydrochloric acid-methanol (1∶10) solution as the extraction solvent, and their contents were determined with RP-HPLC. [Results] The contents of quercetin and kaempferol in F. virosa were 0.233 6 and 0.327 2 mg/g, respectively. [Conclusions] The method has high speed, high resolution, high sensitivity, accurate and reliable detection results, and can be used as a method for the determination of quercetin and kaempferol in F. virosa.

Key words Flueggea virosa (Roxb. ex Wild.) Baill., Quercetin, Kaempferol

1 Introduction

Flueggeavirosa(Roxb. ex Wild.) Baill. (Euphorbiaceae:Flueggea) is a perennial evergreen shrub. It is distributed in tropical regions around the world, mainly in the provinces of southern, eastern and southwestern China. It is mostly found in moist areas such as streams and bushes. The fruit is small, round and white, caespitose in leaf axils. Therefore,F.virosais also called Baiyanyu, Quefanshu, Baihuotan, Baipaoguo,etc. It tastes bitter and astringent, and has minor toxicity. Generally, it is boiled for external use[1-3].F.virosahas activities of clearing heat, removing toxicity, relieving pain, relieving itching and stopping bleeding, and it has significant efficacy for impetigo, dermatitis, rheumatism, burns, bruises and traumatic injury[1-5].

Scholars at home and abroad in modern times have conducted extensive experimental research onF.virosa, and a variety of chemical components with significant pharmacological activity, such as sterols, phenols, alkaloids, and flavonoids, are isolated from it[6-15]. Chen Mengjing,etal.[7]isolated virosecurinine, norsecurinine, fluggeaineether, fluggeainol from the rhizome ofF.virosa. Wang Guocaietal.[8]isolated bergenin, quercetin, kaempferol, gallic acid, virosecurinine and other ingredients from leaves and shoots ofF.virosa. Ji Yuejuetal.[9]isolated gentisic acid and protocatechuic acid from the branches and leaves ofF.virosa. Alkaloids, such as virosecurinine, norsecurinine, fluggeaineether and fluggeainol are more enriched in the roots, stems and leaves ofF.virosa. The existence of a large number of alkaloids may be the reason for its small toxicity in clinical application. The pharmacological mechanism of action has yet to be verified by experiments. Experimental pharmacological studies have shown thatF.virosaextract has the functions of protecting the liver, improving the detoxification ability of damaged liver, and fighting against fibrosis, malaria, HIV, and hepatitis C virus (HCV), has antibacterial, anti-inflammatory and analgesic effects and can inhibit adjuvant arthritis, and has anti-acetylcholinesterase activity and can regulate nerve conduction function[14-29].

At present, the research on the composition ofF.virosafocuses on bergenin and gallic acid. Research on other chemical components has yet to be carried out. In this experiment, reverse-phase high-performance liquid chromatography was used to determine the contents of quercetin and kaempferol inF.virosa, in order to provide basic experimental data for the in-depth study and application ofF.virosa.

2 Materials

2.1 Main drugsF.virosa, identified by Associate Professor Guo Min from Guangxi University of Chinese Medicine was collected from Fucheng Town, Wuming County, Nanning City, Guangxi Province. The specimens were kept in the Laboratory of Pharmaceutical Analysis, College of Pharmacy, Guangxi University of Chinese Medicine. Quercetin standard (content ≥99.0%, batch No. 171230) was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. Kaempferol standard (content ≥98.0%, batch No. YN1114SA13) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

2.2 Main instruments and equipmentThe instruments and equipment used mainly included high-performance liquid chromatograph (Agilent, Agilent, USA), electronic analytical balance (SQP, Sartorius, PRC), desktop high-speed centrifuge (H1650-W, Xiangyi, PRC), ultra pure water instrument (Direct-Q5, Merck Millipore, German), circulating water multipurpose vacuum pump (HWS, Greatwall Scientific, PRC), ultrasonic cleaner (KQ-500E, Kun Shan Ultrasonic, PRC), electric-heating constant-temperature water bath tank (HWS-24, Qixin, PRC) and high-speed pulverizer (YK-100A, Yikang, PRC).

3 Methods and results

3.1 Preparation of mixed reference solutionAppropriate amounts of quercetin standard and kaempferol standard were dissolved in methanol (analytical grade) to prepare into mixed reference solution with quercetin concentration of 0.028 5 mg/mL and kaempferol concentration of 0.040 0 mg/mL.

3.2 Preparation ofF.virosatest solutionF.virosacollected was air-dried and pulverized. A certain amount (about 3 g) of the powder was added with 25.0 mL of diluted hydrochloric acid-methanol (1∶10) mixed solution and weighed accurately. Then, the liquid was heated in water bath, kept boiling for 45 min, and cooled to room temperature. After making up the lost weight with methanol, the new liquid was filtered, and the subsequent filtrate was centrifuge at 20 000 r/min for 10 min. The supernatant was collected as the test solution ofF.virosa.

3.3 Chromatographic conditionsThe chromatographic conditions were as follows: column, Yilite C18chromatographic column (4.6 mm × 250 mm, 5 μm); column temperature, 30 ℃; detection wavelength, 360 nm; mobile phase, methanol-0.4% phosphoric acid solution (47∶53); flow rate, 0.8 mL/min; sample size, 10 μL.

3.4 Methodological investigation

3.4.1Linear relationship. The mixed reference solution (quercetin concentration of 0.028 5 mg/mL and kaempferol concentration of 0.040 0 mg/mL) was detected at different sample sizes, 4, 8, 10, 12, 16 and 18 μL. The peak areas were recorded. The linear regression equation for the quantitative relationship between peak area and concentration was drawn. The results show that under the experimental conditions (the sample size of quercetin and kaempferol was within the range of 0.114-0.456 and 0.160-0.640 μg, respectively), there was a good linear relationship between the sample size and the peak area. The linear equations werey=11 296x-14.043 (r=0.999 8) andy=5 680.2x-56.818 (r=1.000 0), respectively.

3.4.2Precision. The mixed reference solution was detected under the chromatographic conditions described in Section3.3six times repeatedly. The peak areas of quercetin and kaempferol were recorded, and theirRSDvalues were calculated to be 1.8% and 0.3%, respectively. It indicates that the precision of the method meets the requirements of content determination.

3.4.3Stability. An accurate amount (3 g) of the powder ofF.virosawas sampled and prepared into test solution in line with the method in Section3.2.Within 24 h, the test solution was detected six times at an equal interval under the experimental chromatographic conditions. The contents were calculated, and theRSDvalues of the duplicates were calculated to be 1.2% and 2.1%, respectively. The results show that the test solution ofF.virosawas stable within 24 h.

3.4.4Reproducibility. Six portions ofF.virosapowder, 3 g for each, were weighed, and prepared into test solutions according to the same method in Section3.2. Under the experimental chromatographic conditions, separation was performed, followed by analysis. Based on the experimental data, theRSDvalues were calculated, 2.2% and 0.6%. This result shows that the reproducibility of this experimental method meets the content determination requirements.

3.4.5Recovery test. Six portions ofF.virosapowder, around 1.5 g for each, were weighed accurately. Each of them was added with approximately the same amounts of quercetin standard and kaempferol standard as they contained (theoretical contents of quercetin and kaempferol were 0.350 4 and 0.490 8 mg), added with dilute hydrochloric acid-methanol (1∶10) to 25.0 mL, weighed accurately, heated in water bath to boiling, extracted ultrasonically while keeping boiling for 45 min, cooled to room temperature, and weighed accurately again. The lost weight was made up with extraction solvent. The new liquid was filtered, and the subsequent filtrate was centrifuged at 20 000 r/min for 10 min, and the supernatant was collected as mixed test solution. The mixed test solution was separated and analyzed according to the experimental chromatographic conditions of Section3.3.The calculation results show that the average recovery rates were 96.78% and 100.6%, respectively; and theRSDvalues were 2.41% and 2.04%, respectively, indicating that the accuracy of the experimental method meets the content determination requirements (Table 1-2).

Table 1 Results of recovery test of quercetin (b) in Flueggea virosa (Roxb. ex Wild.) Baill.

Table 2 Results of recovery test of kaempferol (c) in Flueggea virosa (Roxb. ex Wild.) Baill.

3.5 Determination of quercetin and kaempferol inF.virosa

Six portions ofF.virosapowder (three for quercetin determination and three for kaempferol determination), around 3 g for each, were weighed accurately and prepared into test solutions, respectively. Under the experimental conditions, the contents of quercetin and kaempferol in the test solutions were determined. The results show that the average contents of quercetin (Table 3) and kaempferol (Table 4) inF.virosawere 0.233 6 and 0.327 2 mg/g, respectively (n=3); and theRSDvalues were 0.33% and 0.56%, respectively (n=3). The HPLC chromatograms of the mixed reference solution andF.virosatest solution prepared are shown in Fig.1-2.

Fig.1 HPLC chromatogram of quercetin (b) and kaempferol (c) mixed reference solution

Note: b, quercetin; c, kaempferol.

Table 3 Content of quercetin in samples of Flueggea virosa (Roxb. ex Wild.) Baill. (n=3)

Table 4 Content of kaempferol in samples of Flueggea virosa (Roxb. ex Wild.) Baill. (n=3)

4 Conclusions and discussion

The effects of different extraction solvents [ethanol, methanol, dilute hydrochloric acid-methanol (1∶10)], different extraction methods (reflex traction method and ultrasonic extraction method), and different solid to liquid ratios on the dissolution of the components to be tested were investigated. The results show that when method was used as the solvent and acid was added for catalytic hydrolysis, the dissolution of quercetin and kaempferol were significantly improved. It is speculated that the quercetin and kaempferol inF.virosaexist in the forms of aglycone and glycoside. The experimental results obtained from boiling by water bath combined with ultrasonic extraction were more repeatable than those obtained from water bath reflex extraction.

In this experiment, the chromatographic conditions were optimized. It was found that when the methanol-0.4% phosphoric acid solution with a ratio of 47∶53 was selected as the mobile phase for isocratic elution, and the flow rate was 0.8 mL/min, the separation and analysis results were better, the resolution was greater than 1.5, other components had no interference with the components to be tested, and the number of theoretical plates was not less than 5 000.

Quercetin and kaempferol inF.virosawere separated and analyzed according to the above-mentioned experimental method. The results show that the average contents of quercetin and kaempferol inF.virosawere 0.233 6 and 0.327 2 mg/g, respectively.

The HPLC method is easy to operate, and has a wide range of applications, high speed, high resolution, high sensitivity, and accurate and reliable detection results. It can be used as a method for the determination of quercetin and kaempferol inF.virosa.