Analysis on Quality Control of Argyreia acuta Lour

Ru ZHANG, Jiangcun WEI, Chunli TANG, Bihua NONG, Guangli LU, Yanxiu WANG

1. Guangxi Maternal and Child Health Care Hospital, Nanning 530003, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China

Abstract [Objectives] To establish a method for caffeic acid content determination and thin-layer chromatography identification of the Zhuang medicine Argyreia acuta Lour. [Methods] Silica gel GF254 (thin-layer plate), toluene-formic acid-ethyl acetate (V∶V∶V=5∶1.2∶3) (developing agent) and 365 nm ultraviolet light were used for thin-layer chromatography identification. Under the chromatographic conditions of column of Thermo SCIENTIFIC Hypersil C18 (5 μm, 4.60 mm × 250 mm), mobile phase of methanol-0.1% phosphoric acid (23∶77), detection wavelength of 320 nm, column temperature of 30 ℃, sample size of 10 μL and flow rate of 1.0 mL/min, the content of caffeic acid in A. acuta Lour was determined. [Results] Caffeic acid can be detected in thin layer chromatography, with strong specificity and clear spots. When the sample size of caffeic acid is within the range of 0.015 1-0.453 0 μg (R2=0.999 9), the content of caffeic acid showed a relatively good linear relationship with the peak area. The average recovery rate of A. acuta Lour was 99.94% (RSD=2.68%), 97.56% (RSD=1.57%) and 99.79% (RSD=2.05%), respectively. [Conclusions] This method can effectively identify A. acuta Lour, and can accurately determine the content of caffeic acid in A. acuta Lour. It has characteristics of high accuracy, high precision, good color rendering stability, good reproducibility and fast analysis speed.

Key words Argyreia acuta Lour, Caffeic acid, HPLC, Thin-layer chromatography

1 Introduction

ArgyreiaacutaLour (Convolvulaceae), also known as Yipichou, Baibei Sichou and Baimian Shuiji, has a variety of curative effects such as stopping bleeding, relieving cough, resolving phlegm, relieving pain, detoxification, dispelling wind and eliminating dampness. It can be used to treat rheumatic pain, chronic bronchitis, milk pain and other symptoms and diseases[1-2].A.acutaLour is commonly used as an antidote in clinical practice in Zhuang medicine[3-4]. By reviewing related literature[5-6], it is found thatA.acutaLour contains a certain amount of caffeic acid in different parts, so in this study, caffeic acid was used as an indicator component, and its content was determined by HPLC, and thin-layer chromatography was used for qualitative identification ofA.acutaLour, in order to provide a basis for preparation development and quality standard research ofA.acutaLour. Caffeic acid is a natural resource with extremely medicinal value. China has abundant caffeic acid resources. The content of caffeic acid inA.acutaLour was determined, and the differences in the caffeic acid content ofA.acutaLour in various regions of Guangxi were analyzed, so as to improve the quality control ofA.acutaLour.

2 Instruments and drugs

2.1 InstrumentsHigh-performance liquid chromatograph (2695); ultrapure water system (Simplicity, Millipore China Limited); analytical balance [Practum224-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.]; high-speed desktop centrifuge (TGL-16G, Shanghai Anting Scientific Instruments Factory); electronic constant-temperature water bath (HWS-26, Shanghai Qixin Scientific Instrument Co., Ltd.).

2.2 DrugsThe drugs used included caffeic acid standard (batch No.1910885-800102, China National Institutes for Food and Drug Control, purity > 98.0%); ethanol, phosphoric acid, acetic acid (analytical grade); ultrapure water; methanol, acetonitrile (chromatographic grade); thin-layer silica gel GF 254 (Qingdao Ocean Chemical Plant) . A total of 6 batches ofA.acutaLour (Table 1) were sampled, and they were identified by the teacher of the Department of Chinese Medicine Identification, Guangxi University of Chinese Medicine as the aboveground part ofA.acutaLour. The medicinal part was the whole plant, and the drying was performed in an oven at 56 ℃. The executive standard of all the samples in this experiment wasPharmacopoeiaofPeople’sRepublicofChina(2015 Edition).

Table 1 Information on Argyreia acuta Lour samples

3 Methods and results

3.1 Identification by thin-layer chromatographyA certain amount (1.0 g) of the powder ofA.acutaLour collected from Fuchuan County, Guangxi was sampled, added with 20 mL of 30% ethanol, reflux-extracted for 1 h and filtered with filter paper, and the filtrate obtained was evaporated to dryness. The evaporated product was dissolved in methanol to about 1 mL as a sample solution. A certain amount of caffeic acid standard was dissolved in an appropriate volume of methanol to prepare into caffeic acid standard solution with a concentration of 1.15 mg/mL. A certain volume (5 μL) of each of the sample solutions was loaded to the same silica gel GF254 thin-layer plate and developed with toluene-formic acid-ethyl acetate (V∶V∶V=5∶1.2∶3) as the developing agent. After developing to the front line, the thin-layer plate was taken it out, and the developing agent was evaporated. Then, the plate was observed under 365 nm ultraviolet light environment. In the chromatogram ofA.acutaLour, above the position corresponding to the standard, there were fluorescent spots of the same color displayed. The result is shown in Fig.1.

Note: 1-6, A. acuta Lour samples; A, caffeic acid standard.

3.2 Content determination

3.2.1Preparation ofA.acutaLour sample. Whole plants ofA.acutaLour were collected, dried at 56 ℃ to constant weight, pulverized, passed through No.2 sieve, and stored under sealed condition.

3.2.2Chromatographic conditions. Column: Thermo SCIENTIFIC Hypersil C18(5 μm, 4.60 mm × 250 mm); column temperature: 30 ℃; mobile phase: methanol-0.1% phosphoric acid (23∶77); sample size: 10 μL; detection wavelength: 320 nm; flow rate: 1.0 mL/min. Under the chromatographic conditions above, the resolution is good (Fig.2).

Note: A, caffeic acid standard; B, A. acuta Lour sample.

3.2.3Preparation of standard solution. An accurate amount (7.55 mg) of caffeic acid standard was dissolved in methanol to 5 mL to prepare into standard solution of 1.51 mg/mL. An accurate volume (0.2 mL) of the standard solution above was diluted to 10 mL with methanol, and thus, the caffeic acid standard solution of 30.2 μg/mL was obtained.

3.2.4Preparation of sample solution. An accurate amount (1.0 g) of the powder ofA.acutaLour collected from Fuchuan County, Guangxi was reflux-extracted with 30% ethanol and centrifuged at 13 000 r/min for 10 min, and the supernatant was collected and filtered through 0.45 μm microporous membrane. The filtrate obtained was the sample solution.

3.2.5Drawing of standard curve. A certain volume (0.5, 1.0, 3.0, 5.0, 8.0, 12.0 and 15.0 μL) of the caffeic acid standard solution (30.2 μg/mL) was injected the high-performance liquid chromatograph, respectively. Taking the sample amount (μg) as the abscissa and the peak area as the ordinate, the standard curve was drawn, and the regression equation was calculated. The regression equation obtained was as follows:Y=6.0×106X-30 036 (R2=0.999 7). When the sample amount of caffeic acid was within the range of 0.015 1-0.453 0 μg (R2=0.999 9), it showed a good linear relationship with the peak area.

3.2.6Precision test. Under the conditions described in Section3.2.2, the sample solution (30.2 μg/mL) was detected six times repeatedly. TheRSDvalue of the peak areas of caffeic acid was calculated to be 1.28%, smaller than 3%, indicating good precision of the instrument.

3.2.7Stability test. A certain amount (1.0 g) of the powder ofA.acutaLour collected from Fuchuan County, Guangxi was prepared into sample solution according to the method in Section3.2.4. At 0, 2, 4, 8, 12 and 24 h after the sample solution was prepared, it was detected under the conditions in Section3.2.2, respectively. TheRSDvalue of the peak areas of caffeic acid was calculated to be 2.07%, smaller than 3%, indicating that the sample solution was stable within 24 h.

3.2.8Reproducibility test. Six portions of the powder ofA.acutaLour collected from Fuchuan County, Guangxi, 1.0 g for each, were weighed, prepared into solutions according to the method of Section3.2.4, and detected under the conditions of Section3.2.2, respectively. The average content of caffeic acid was calculated to be 124.52 μg/g, and theRSDvalue was 1.21%, smaller than 3%, indicating that the method has good reproducibility.

3.2.9Recovery test. Nine portions of the powder ofA.acutaLour (caffeic acid content of 124.52 μg/g) collected from Fuchuan County, Guangxi, 0.5 g for each, were weighed. They were evenly divided into three groups: low addition group (50% of that contained in 0.5 g of sample, addition amount of 33.184 μg), middle addition group (100% of that contained in 0.5 g of sample, addition amount of 66.640 μg) and high addition amount (150% of that contained by 0.5 g of sample, addition amount of 100.096 μg). In the low addition group, the average recovery rate was 99.94%, and theRSDvalue was 2.68% (n=3); in the middle addition group, the average recovery rate was 97.56%, and theRSDvalue was 1.57% (n=3); and in the high addition group, the average recovery rate was 99.97%, and theRSDvalue was 2.05% (n=3) (Table 2). TheRSDvalues were all smaller than 3%, indicating that the accuracy of the method is good.

Table 2 Recovery test of caffeic acid in Argyreia acuta Lour

4 Extraction process for caffeic acid in A. acuta Lour

4.1 Investigation on single factors

4.1.1Extraction method. Under the fixed conditions of extraction volume of 20 mL, extraction time of 1 h and extraction solvent of 30% ethanol, the effect of different extraction methods on the extraction rate of caffeic acid inA.acutaLour from Fuchuan County, Guangxi was investigated. The results show that among the three methods, the reflux extraction method was more preferable (Fig.3).

Fig.3 Effect of different extraction methods on extraction rate of caffeic acid in Argyreia acuta Lour

4.1.2Extraction solvent. The extraction volume, extraction time and extraction method were fixed at 20 mL, 1 h and reflux extraction, and the effect of different extraction solvents on the extraction rate of caffeic acid inA.acutaLour from Fuchuan, Guangxi was investigated. The results show that between the two extraction solvents, 50% ethanol was more preferable (Fig.4).

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4.1.3Solvent concentration. The extraction volume, extraction time, extraction solvent and extraction method were fixed at 20 mL, 1 h, ethanol and reflux extraction, and the effect of different solvent concentrations on the extraction rate of caffeic acid inA.acutaLour from Fuchuan County, Guangxi was investigated. The results show that the extraction rate of caffeic acid was the highest under the extraction concentration of 10%, and so in this study, 10% ethanol was more preferable (Fig.5).

Fig.4 Effect of different extraction solvents on extraction rate of caffeic acid in Argyreia acuta Lour

Fig.5 Effect of different concentrations of solvent on extraction rate of caffeic acid in Argyreia acuta Lour

4.1.4Extraction volume. The extraction time, extraction solvent and extraction method were fixed at 1 h, 10% ethanol and reflex extraction, and the effect of different extraction volumes on the extraction rate of caffeic acid inA.acutaLour collected from Fuchuan County, Guangxi was investigated. The results show that the extraction rates of caffeic acid at the extraction volumes of 20 and 30 mL were higher than those at the other extraction volumes (Fig.6).

Fig.6 Effect of different extraction volumes on extraction rate of caffeic acid in Argyreia acuta Lour

4.1.5Extraction time. After fixing the extraction volume, extraction solvent and extraction method at 20 mL, 10% ethanol and reflux extraction, the effect of different extraction times on the extraction rate of caffeic acid inA.acutaLour collected from Fuchuan County, Guangxi was investigated. The results show that among the extraction times of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h, the extraction rate of caffeic acid at the extraction time of 1.5 h was the highest (Fig.7).

Fig.7 Effect of different extraction times on extraction rate of caffeic acid in Argyreia acuta Lour

4.2 Design of orthogonal testThe main methods of extracting caffeic acid fromA.acutaLour in Fuchuan County, Guangxi are reflux extraction, ultrasonic extraction and impregnation. According to relevant literature andPharmacopoeiaofPeople’sRepublicofChina(2015 Edition), this experiment finally investigated the effect of different solvent volumes, different solvent concentrations and different extraction times on the extraction rate of caffeic acid. An L9(34) orthogonal experiment was designed (Table 3).

According to the design of the L9(34) orthogonal experiment, nine portions of the powder ofA.acutaLour from Fuchuan County, Guangxi, 1.0 g for each, were weighed and prepared into solutions, respectively, and their caffeic acid contents were determined (Table 4).

Table 3 Factors and levels of orthogonal test for optimizing caffeic acid extraction from Argyreia acuta Lour

Table 4 Design and results of orthogonal test for optimizing caffeic acid extraction from Argyreia acuta Lour

4.3 Orthogonal test and result analysisThe analysis results of variance (Table 5) show that the concentration of ethanol had the most significant effect on the extraction rate of caffeic acid inA.acutaLour, followed by solvent volume and extraction time. The effect of solvent volume and extraction time was insignificant, and that of ethanol concentration was significant. The intensity of effect of different factors was in the order as A > B > C. The optimal combination of various influencing factors was A1B2C2, that is, extraction volume of 30 mL, ethanol concentration of 10%, reflux extraction time of 1 h.

Table 5 Results of analysis of variance

5 Determination of sample caffeic acid content

6 Discussions

6.1 Selection of thin-layer chromatographic conditionsThe effect of different development systems including toluene-glacial acetic acid-water-ethyl acetate-formic acid, toluene-ethyl acetate-formic acid, ethyl acetate-formic acid-water, butyl acetate-water-formic acid and n-butanol-glacial acetic acid-water was investigated. It was found that in the development system of toluene-formic acid-ethyl acetate (V∶V∶V=5∶1.2∶3), the separation effect was better and the spots were clearer.

6.2 Selection of mobile phaseThe effect of different mobile phases was investigated. Among different acetonitrile-acid systems (0.1% phosphoric acid, 0.05% phosphoric acid, 0.2% phosphoric acid, 0.1% glacial acetic acid, 0.2% glacial acetic acid and pure water) and methanol-acid systems, the resolution of the methanol-0.1% phosphoric acid was the best, with relatively stable baseline and appropriate peak appearance time, so methanol-0.1%

phosphoric acid aqueous solution was selected as the mobile phase.

6.3 Selection of detection wavelengthThe standard solution and sample solution were subjected to full-wavelength scanning. In the range of 200-400 nm, caffeic acid showed two maximum absorption peaks, at 323 and 320 nm. At the detection wavelength of 323 nm, the peak of the solvent was large, and the baseline drifted. At the detection wavelength of 320 nm, the peak of the solvent was small, the baseline was stable, and the response value of caffeic acid was large. Therefore, 320 nm was selected as the detection wavelength.

7 Conclusions

A.acutaLour from different origins of Guangxi all contains caffeic acid, and the content of caffeic acid differs among different origins.A.acutaLour produced in the Hejiang River basin in Hezhou, Guangxi has the highest caffeic acid content, followed by that in Yining District, Nanning City, Guangxi, andA.acutaLour produced in Fuchuan County, Guangxi shows the lowest caffeic acid content. There is no significant difference in caffeic acid content ofA.acutaLour from Shanglin County, Zhongshan County and Zhaoping County. There is little difference in total flavonoid content ofA.acutaLour from different producing areas in Guangxi. The content of total flavonoids inA.acutaLour from Hejiang River basin of Hezhou, Guangxi was the highest, followed by that from Fuchuan County, Guangxi, and the content of total flavonoids inA.acutaLour from Shanglin County, Nanning City, Guangxi was the lowest.