Determination of Kaempferol Content in 10 Batches of Different Production Areas of Yao Medicine Lysimachia foenum-graecum Hance

Yangning OU, Chenghao ZHANG, Xiumei MA, Jiangcun WEI*, Wen ZHONG, Lihua YU, Yusheng HUANG, Lizhen CAO

1. Hezhou Hospital of Traditional Chinese Medicine, Hezhou 542899, China; 2. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China

Abstract [Objectives] To determine the kaempferol content in Yao medicine Lysimachia foenum-graecum Hance. [Methods] The high performance liquid chromatography (HPLC) method was used. The chromatographic column: Inertsil ODS-3 C18 column (4.60 mm×250 mm, 5 μm); mobile phase: acetonitrile-0.1% glacial acetic acid; flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 360 nm; sample injection volume: 10 μL. [Results] The kaempferol content in Yao medicine L. foenum-graecum was determined, Y=20 652X-151 098 (R=0.999 7) shows a good linear relationship within the linear range of 9.80-156.8 μg/mL. [Conclusions] The high performance liquid chromatography (HPLC) method was established for determining the kaempferol content in Yao medicine L. foenum-graecum. This method is expected to provide a scientific basis for the formulation of quality control methods for L. foenum-graecum.

Key words Lysimachia foenum-graecum Hance., Kaempferol, High performance liquid chromatography (HPLC)

1 Introduction

Guangxi Yao medicineLysimachiafoenum-graecumHance., also called Linglingxiang, Xunxiang, Xiangcao, Peilan, and Paicao, is a plant ofLysimachiaL. genus in Primulaceae family, and mainly produced in Guangxi and Yunnan of China[1].L.foenum-graecumis a precious Chinese herbal medicine. Its stems and leaves can be used as medicine. It has the effects of invigorating the stomach, sweating, and relieving pain. It has significant effects on the treatment of colds, abdominal pain, eczema, cold and damp beriberi, traumatic abscess,etc., thus it has high medicinal value[2-3]. Besides,L.foenum-graecumis a precious raw material in the spice industry. It is widely used in the fragrance of high-end tobacco and wine[4]. In addition, it is one of the three major spices (the other two areCinnamomumcassiaPresl andIlliciumverumHook.f.) in Guangxi. It is pungent in flavor and sweet in taste and warm in nature, so it has high medicinal value. The extracts and essential oils obtained fromL.foenum-graecumas the raw material have long-lasting and stable aroma and can be used to make tobacco and balsam[3,5].L.foenum-graecumis rich in a variety of active substances, such as flavonoids, triterpenoids, polyphenols and polysaccharides[6-7].

Guangxi Yao medicineL.foenum-graecumis a commonly used Chinese herbal medicine in the folk areas. At present, there is little research on its medicinal resources, and there is no relatively complete quality control standard method. In order to develop Guangxi Zhuang medicineL.foenum-graecummedicinal resources and establish a scientific method for its quality control, we performed an experimental research on the properties of Zhuang medicineL.foenum-graecummedicinal materials, and detected the effective component kaempferol content inL.foenum-graecum.

2 Materials

2.1 Medicinal materialsThe medicinal materials were identified by teacher from the Department of Pharmacy in the First Affiliated Hospital of Guangxi University of Chinese Medicine asLysimachiafoenum-graecumHance, a plant of theLysimachiaL. genus in Primulaceae family. The specific source is shown in Table 1. The collected part is whole herb, and the dry place is dried at 60 ℃. Kaempferol reference solution (bought from China National Institute for the Control of Pharmaceutical and Biological Products, batch No.110861-201911) was used for content determination.

Table 1 Information of samples of Lysimachia foenum-graecum Hance

2.2 InstrumentsWaters 2695-2996 high performance liquid chromatograph (Waters Corporation, USA); UPC-11-10T laboratory ultra water purifier (Sichuan ULUPURE Technology Co., Ltd.); SQP electronic balance (Sartorius Sartorius Scientific Instruments (Beijing) Co., Ltd.); KQ500DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); SHB-III circulating water type vacuum pump (Zhengzhou Great Wall Scientific Industrial & Trade Co., Ltd.).

2.3 ReagentsEthanol (Chengdu Kelong Chemical Co., Ltd., batch No.2019060701, AR); glacial acetic acid (Tianjin Guangfu Fine Chemical Research Institute, chromatographically pure); acetonitrile (Thermo Fisher Scientific Inc., USA, chromatographically pure); methanol (Thermo Fisher Scientific Inc., USA, chromatographically pure).

3 Methods and results

3.1 Preparation of test sample solutionPrecisely weighed 2.0 g of coarse powder ofL.foenum-graecum, placed in a proper conical flask with stopper, precisely added 50 mL of methanol, sealed, weighed, ultrasonic treated (250 W, 40 kHz) for 1.0 h, cooled down, weighed again, made up the lost weight with proper volume of methanol, shook up and filtered, centrifuged at 13 000 r/min for 10 min, passed through a 0.22 μm microporous membrane, took the filtrate, and obtained the test sample solution.

3.2 Preparation of reference solutionPrecisely weighed 12.25 mg of kaempferol reference substance and placed into a 25-mL volumetric flask, diluted to the mark with methanol, passed through a 0.22 μm microporous membrane, and took the filtrate as the stock solution of kaempferol reference substance. (Kaempferol: 490 μg/mL).

3.3 Chromatographic conditionsThe chromatographic column: Inertsil ODS-3 C18column (4.60 mm×250 mm, 5 μm); mobile phase acetonitrile-0.1% glacial acetic acid; flow rate: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 360 nm; sample injection volume: 10 μL. The gradient elution was shown in Table 2.

Table 2 Chromatographic elution conditions

3.4 System suitability test (SST)

3.4.1Blank test. Pipetted methanol solvent and perform determination under chromatographic conditions in Section3.3. The results are shown in Fig.1.

Fig.1 Chromatogram for blank solvent

3.4.2Reference substance test. Pipetted the solution prepared in Section3.2and prepared the mixed reference solution and performed determination under chromatographic conditions in Section3.3. The results are shown in Fig.2.

Note: 1. Kaempferol.

3.4.3Sample test. Pipetted the test solution prepared in Section3.1and performed determination under chromatographic conditions in Section3.3. The results are shown in Fig.3.

Fig.3 Chromatogram for Lysimachia foenum-graecum Hance sample

Result analysis: under the above mentioned chromatographic condition, the chromatographic separation and peak shape of the kaempferol components in the reference substance andL.foenum-graecumare good, and are not interfered by other impurity peaks.

3.5 Methodology test[8-9]

3.5.1Plotting of standard curve. Precisely weighed the stock solution of kaempferol reference substance and placed into a 2-mL volumetric flask, diluted to the mark with methanol, prepared 5 pieces of reference solution, passed through a 0.22 μm microporous membrane, and took the filtrate, performed determination under chromatographic conditions in Section3.3. The linear range was 9.80-156.8 μg/mL, the correlation coefficient was 0.999 7, and the linear equation wasY=20 652X-151 098. Taking the peak area (Y) as the ordinate and the reference substance concentration (X) as the abscissa, we plotted the standard curve. Through analysis, it is found that the kaempferol reference substance has a good linear relationship with the peak area at the concentration of 9.80-156.8 μg/mL.

3.5.2Precision test. Took an appropriate amount of the reference solution in Section3.2and repeated the sample injection and determination under the chromatographic conditions in Section3.3for 6 times, each time 10 μL, made a record of the peak area of kaempferol inL.foenum-graecum, and calculated theRSDvalue. The results indicate that theRSDof the kaempferol peak area was 0.42%, indicating that the precision of the instrument is good.

3.5.3Reproducibility test. Precisely weighed 6 pieces ofL.foenum-graecumLXC1 (Jingxi City, Guangxi) medicinal powder in parallel, each about 2.0 g, the preparation of the test product was using the method in Section3.1, and the content was determined in accordance with the chromatographic conditions in Section3.3, made a record of the peak area, and calculated the kaempferol content inL.foenum-graecumsample according to the regression equation. Through calculation, the average content of kaempferol was 425 μg/mL, and theRSDwas 1.92% (n=6), indicating that the method has good repeatability.

3.5.4Stability test. Took an appropriate amount of the test solution in Section3.1and placed it at room temperature for 0, 2, 4, 8, 10, 12, and 24 h, and then measured the sample under the chromatographic conditions in Section3.3and made a record of the peak area. TheRSDof the kaempferol peak area was 1.52%, indicating that the test solution was basically stable within 24 h at room temperature.

3.5.5Sample recovery test. Took 6 pieces of powder ofL.foenum-graecumsamples with known content, each sample 1 g, precisely weighed, added low, medium, and high (80%, 100%, 120% for kaempferol reference substance) of different amounts of kaempferol reference substance, and prepared the test solution using the method in Section3.1. According to the chromatographic conditions in Section3.3, injected and measured the samples, made a record of the peak area, and calculated the sample recovery rate. The results are shown in Table 3. The recovery rate of the components in the different polar parts ofL.foenum-graecumroots was in the range of 95%-105%, indicating that the method is accurate and reliable in determining the kaempferol content.

Table 3 Results of recovery rate test for kaempferol (n=6)

4 Determination of samples

Separately weighed 10 batches of powder ofL.foenum-graecumfrom different production areas, each sample 2.0 g, prepared the test solution using the method in Section3.1, and measured according to the chromatographic conditions in Section3.3, operated in parallel for 3 times, made a record of the peak area and calculated the content ofL.foenum-graecumsample using one point external standard method. The content of kaempferol was 425.28, 420.72, 422.41, 426.05, 423.91, 422.08, 422.87, 424.06, 423.83, and 424.15 μg/g, respectively.

5 Results and discussion

In this experiment, we investigated effects of methanol-0.05% phosphoric acid, methanol-0.1% phosphoric acid, methanol-0.2% phosphoric acid, acetonitrile-0.05% glacial acetic acid, acetonitrile-0.1% glacial acetic acid, acetonitrile-0.2% glacial acetic acid, acetonitrile-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, methanol-water, acetonitrile-water and other systems on the separation effect of sample components and their peak time, resolution and baseline. The results indicate that using the acetonitrile-0.1% glacial acetic acid system has better resolution of the chromatographic peaks and a relatively stable baseline. Therefore, we used the acetonitrile-0.1% glacial acetic acid system to conduct the gradient elution.

Besides, in this experiment, we investigated the effects of 25 ℃, 30 ℃, and 35 ℃ on resolution, peak time, and baseline stability. The experimental results show that the column temperature has little effect, so the column temperature of this experiment was set at 30 ℃. In addition, we investigated the effects of three different flow rates (1.2, 1.0, and 0.8 mL/min) on the resolution, and the results indicate that the separation was better at 1.0 mL/min and the baseline was also balanced. Therefore, we used 1.0 mL/min as the flow rate.

The results of this experiment show that the content of the same components in the Yao medicineL.foenum-graecumis significantly different, and the content of the same components in the medicinal materials of different production areas is also very different, which may be related to the factors such as extraction and processing methods, the storage conditions of the medicinal materials, picking time and geographical environment. In summary, the method established in this experiment is accurate and reliable, and is expected to provide an experimental basis for the further development and utilization ofL.foenum-graecum.