Thin-layer Identification, Extraction Process and Content Determination of Chlorogenic Acid in Laggera alata (D. Don) Sch. Bip. ex Oliv.

Qiaofeng QIN, Xue JIANG, Jiangcun WEI, Jiabao MA,3*, Jingrong LU, Lifu WEI, Xiumei MA, Xuanyang ZENG

1. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 2. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 3. Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning 530200, China

Abstract [Objectives] This paper aims to establish thin-layer identification and content determination method for Laggera alata (D. Don) Sch. Bip. ex Oliv. with chlorogenic acid as the index component and compare the content of chlorogenic acid in L. alata from different places in Guangxi. [Methods] Silica gel GF254 thin-layer plate was used for identification under an ultraviolet lamp (365 nm), with butyl acetate-formic acid-water (V∶V∶V=7∶2.5∶2.5) as a developing agent. The content of chlorogenic acid was determined under the following chromatographic conditions: column, Inertsil ODS-3 C18 column (4.60 mm × 250 mm, 5 μm); mobile phase, methanol-0.1% phosphoric acid (28∶72); detection wavelength, 329 nm; flow rate, 1.0 mL/min; column temperature, 25 ℃; and injection volume, 10 μL. [Results] Chlorogenic acid can be detected by thin layer chromatography with clear spot and good specificity. Chlorogenic acid showed a good linear relationship in the injection amount range of 0.099-0.99 μg (R2=0.999 9). The content of chlorogenic acid in L. alata varied greatly among the 10 different producing areas in Guangxi. L. alata produced in Dee Township, Longlin, Baise, Guangxi showed the highest chlorogenic acid content, and that produced in Shangsi County and Pingle County showed the lowest chlorogenic acid content. [Conclusions] This method can effectively identify L. alata and accurately determine the content of chlorogenic acid, thereby providing a scientific basis for the development and utilization of L. alata resources.

Key words Laggera alata, Chlorogenic acid, Content determination, Thin-layer identification, Extraction process

1 Introduction

Laggeraalata(D. Don) Sch. Bip. ex Oliv. (Compositae:Laggera) is used as medicine with the entire plant. It is mainly distributed in the east, southeast and southwest of China, as far north as Anhui and Hubei[1]. Fresh branches and leaves ofL.alatagive off a special smell after rubbing, so it is used as an antibacterial, anti-inflammatory, heat-clearing and detoxifying medicine in Yunnan, hailed as "Smelly Panacea"[2].L.alatais commonly used in diseases such as bruises, snake bites, rheumatoid arthritis, skin eczema, abdominal pain and diarrhea. At present, it is mostly used in the treatment of respiratory diseases[3], colds[4]and viral infections[5], but its effective ingredients are not yet clear. Studies[2, 6-7]have found thatL.alatamainly contains compounds such as flavonoids, sesquiterpenes and phenolic acids, and phenolic acids include chlorogenic acid, caffeic acid and ferulic acid[8]. Chlorogenic acid has a wide range of biological activities, and has pharmacological effects such as antibacterial, antiviral[9], anti-inflammatory, protecting liver, increasing choleresis, anti-tumor, lowering blood pressure and scavenging free radicals[10]. In addition, chlorogenic acid has obvious effect on diseases of blood system, digestive system and reproductive system[11].

In this study, thin-layer identification and content determination of chlorogenic acid inL.alatafrom 10 different areas of Guangxi were carried out, so as to provide a reference for the quality evaluation ofL.alataand lay a certain foundation for the development of new methods for quality control ofL.alata.

2 Materials

2.1 Instruments and equipment2695 High-performance liquid chromatograph (Waters, USA); Simplicity ultrapure water system (Millipore China Limited); Practum224-1CN analytical balance[Satorius Scientific Instruments (Beijing) Co., Ltd.]; DHG-9203A electric heating constant-temperature air blast oven (Shanghai Qixin Scientific Instrument Co., Ltd.); TGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory).

2.2 Reagents and drugsThe reagents and drugs used included chlorogenic acid reference substance (National Institutes for Food and Drug Control, batch No.1110885-200102, purity>98.0%), methanol (chromatographically pure, Fisher), acetonitrile (chromatographically pure, Fisher), ethanol (analytically pure), phosphoric acid (analytically pure), acetic acid (analytically pure), pure water, thin-layer silica gel GF 254 (Qingdao Ocean Chemical Factory), and so on (analytically pure). A total of 10 batches of entireL.alataplants were sampled (Table 1), and they had all been identified by the professor of Guangxi University of Chinese Medicine. The collectedL.alatasamples were air-dried for use.

Table 1 Collection information of Laggera alata (D. Don) Sch. Bip. ex Oliv. samples

3 Methods and results

3.1 Thin-layer identificationA certain amount (3 g) of powder ofL.alatawas weighed, added with 60 mL of 40% ethanol, refluxed for 1 h and filtered, and the filtrate collected was evaporated to about 2 mL as a test solution. An appropriate amount of the chlorogenic acid standard was dissolved in a certain volume of methanol to obtain a 0.1 mg/mL of reference solution. Certain volumes of the test solution and reference solution prepared above, 5 μL for each, were loaded on the same silica gel GF254 thin-layer plate and developed with toluene-ethyl acetate-formic acid (V∶V∶V=5∶3∶1) as the developing agent. Then, the gel was taken out, air-dried and inspected under an ultraviolet lamp at 365 nm. At the corresponding position of the standard, spots of the same color were observed. The results are shown in Fig.1.

Note: 1-10. L. alata samples; A. chlorogenic acid standard.

3.2 Content determination

3.2.1Sample preparation. The entire plants ofL.alatawere collected. They were dried naturally, crushed and passed through No.2 sieve for chlorogenic acid content determination.

3.2.2Chromatographic conditions. The chromatographic conditions were as follows: column, Inertsil ODS-3 C18(4.60 mm×250 mm, 5 μm); mobile phase, methanol-0.1% phosphoric acid (28∶72); detection wavelength, 329 nm; flow rate, 1.0 mL/min; column temperature, 25 ℃; and injection volume, 10 μL. Under the chromatographic conditions described above, the resolution of each component was good (Fig.2).

Note: A. L. alata; B. 25% ethanol; C. chlorogenic acid standard.

3.2.3Preparation of reference solution. An accurate amount (11.0 mg) of the chlorogenic acid standard was dissolved in methanol to 10 mL to prepare into stock reference solution of 1.1 mg/mL. An accurate volume (0.45 mL) of the prepared stock reference solution was diluted to 10 mL with methanol, and thus, the reference solution of chlorogenic acid with concentration of 49.5 μg/mL was obtained.

3.2.4Preparation of test solution. An accurate amount (1.0 g) ofL.alatapowder was weighed, extracted with 25% ethanol, refluxed and centrifuged at a radius of 0.65 cm, 13 000 r/min for 10 min, and the supernatant was collected and filtered through 0.45 μm microporous membrane. The filtrate obtained was sample solution.

3.2.5Drawing of standard curve. Different volumes (2, 5, 10, 15 and 20 μL) of the chlorogenic acid reference solution were injected to the liquid chromatograph, respectively. With peak area and injection amount as the ordinate and abscissa, the standard curve was drawn, and the regression equation obtained was as follows:Y=1.0×106X-7 705.6 (R2=0.999 9). The result show that the peak area and injection amount showed a good linear relationship in the range of 0.099-0.99 μg.

3.2.6Precision test. The reference solution was detected under the chromatographic conditions described in Section3.2.2six times repeatedly. TheRSDvalue of the peak areas of chlorogenic acid was 0.37%, smaller than 3%, indicating that the instrument has good precision.

3.2.7Stability test. An accurate amount (1.0 g) of powder ofL.alatafrom certain producing area was weighed and prepared into test solution according to the method described in Section3.2.4. The content of chlorogenic acid in the test solution was detected 0, 2, 4, 8, 12 and 24 h after the preparation, respectively under the conditions in Section3.2.2. TheRSDvalue of the peak areas of chlorogenic acid obtained was 2.78%, smaller than 3%, indicating that the stability of the test solution was good within 24 h.

3.2.8Reproducibility test. A total of six portions of powder ofL.alatafrom certain producing area, 1.0 g for each were weighed and prepared into test solutions, respectively according to the method described in Section3.2.4. The content of chlorogenic acid in the test solutions was detected under the chromatographic conditions in Section3.2.2. The result show that the average content of chlorogenic acid was 1.567 0 mg/g, and theRSDvalue was 1.56%, smaller than 3%, indicating that the method has good reproducibility.

3.2.9Recovery test. A total of 9 portions of powder ofL.alatafrom certain producing area, in which the content of chlorogenic acid was known, were weighed, 0.5 g for each. The 9 samples were randomly and evenly divided into 3 groups, namely low, medium and high-sample loading groups (the addition amount of chlorogenic acid was 80%, 100% and 120%, respectively of the amount of chlorogenic acid contained in the 0.5 g ofL.alatasample,i.e., 0.630 3, 0.787 6 and 0.946 0 mg). In the low, medium and high-sample loading groups, the average recovery rate was 98.80%, 98.24% and 99.65%, respectively, and theRSDvalue was 2.09%, 1.96% and 2.15%, respectively (n=3). It indicates that the accuracy of this method is good. The results are shown in Table 2.

Table 2 Sampling recovery of chlorogenic acid in Laggera alata (D. Don) Sch. Bip. ex Oliv.

4 Extraction process of chlorogenic acid from L. alata (D. Don) Sch. Bip. ex Oliv.

4.1 Single-factor tests

4.1.1Determination of the maximum absorption wavelength. The chlorogenic acid reference solution was diluted with methanol and scanned with an ultraviolet-visible spectrophotometer in the 200-400 nm wavelength range, with methanol as the reference solution. The result was analyzed, and the maximum absorption wavelength of chlorogenic acid was determined. The result is shown in Fig.3.

As shown in Fig.3, chlorogenic acid has maximum absorption at the peak positions 1, 2 and 3. According to the absorption curve of chlorogenic acid and the three detection wavelengths (329, 243 and 218 nm), the optimal absorption wavelength was determined to be 329 nm.

Fig.3 Absorption curve of L. alata (D. Don) Sch. Bip. ex Oliv.

4.1.2Investigation of extraction methods. Under the conditions of extraction time of 1 h and extraction solvent of ethanol (25%, 20 mL), chlorogenic acid inL.alatawas extracted by the reflux extraction method, ultrasonic extraction method and solvent impregnation method, respectively. The experimental results show that the reflux extraction method had a high extraction rate for chlorogenic acid (Fig.4). Therefore, the reflux extraction method was chosen in this experiment.

Fig.4 Effect of different extraction method on extraction rate of chlorogenic acid from Laggera alata (D. Don) Sch. Bip. ex Oliv.

4.1.3Investigation of solvent concentration. The extraction time, solvent volume and extraction method were fixed as 1 h, 20 mL and reflux extraction method, respectively. The chlorogenic acid inL.alatawas extracted with ethanol and methanol, respectively. The concentrations of ethanol and methanol were designed as 0%, 25%, 45%, 65% and 85%. The results show that when 25% ethanol was used as extraction solvent, the extraction rate of chlorogenic acid was the highest (Fig.5).

4.1.4Investigation of extraction volume. Under the fixed conditions of extraction time of 1 h, extraction solvent of 25% ethanol and extraction method of reflux extraction method, chlorogenic acid inL.alatawas extracted with 15, 25, 35 and 45 mL of ethanol, respectively. The results show that the extraction rate of chlorogenic acid was the highest when the solvent volume of 25 mL (Fig.6).

Fig.5 Effect of different extraction solvent on extraction rate of chlorogenic acid from Laggera alata (D. Don) Sch. Bip. ex Oliv.

Fig.6 Effect of different solvent volume on extraction rate of chlorogenic acid from Laggera alata (D. Don) Sch. Bip. ex Oliv.

4.1.5Investigation of extraction time. The extraction solvent and extraction method were fixed at 25 mL 25% ethanol and reflux extraction, respectively, and the effect of different extraction time (0.5, 1.0, 1.5, 2.0 and 2.5 h) on the extraction rate of chlorogenic acid was investigated. The results show that under the extraction time of 1.0 h, the extraction rate of chlorogenic acid was the highest (Fig.7).

Fig.7 Effect of different extraction time on extraction rate of chlorogenic acid from Laggera alata (D. Don) Sch. Bip. ex Oliv.

4.2 Design of orthogonal testThe methods of extracting chlorogenic acid fromL.alataare mainly reflux extraction method, ultrasonic extraction method and solvent impregnation method. According to the relevant literature and with reference to the 2015 edition of Chinese Pharmacopoeia, in this experiment, ethanol was selected as the extraction solvent, and the effects of different solvent concentration (10%, 25% and 40%), solvent volume (20, 30 and 40 mL) and extraction time (1, 1.5 and 2 h) on the extraction rate of chlorogenic acid were investigated. To optimize the extraction process, a L9(34) orthogonal test was designed (Table 3).

An accurate amount (1.0 g) ofL.alatapowder was weighed, and chlorogenic acid was extracted with the extraction process described in Table 3, respectively. The content of chlorogenic acid was determined by high-performance liquid chromatography (Table 4).

Table 3 Factors and levels of orthogonal test

Table 4 Design and results of orthogonal test

4.3 Orthogonal test and results analysisThe results of variance analysis (Table 5) show that the intensity of effect of different factors on the extraction ofL.alatawas different, with chlorogenic acid content as the experimental index. The intuitive analysis results show that ethanol concentration and extraction solvent volume had a significant effect on the extraction rate of chlorogenic acid inL.alata, followed by extraction time. The results of variance analysis show that the influence of ethanol concentration and extraction solvent volume was significant, and that of extraction time was insignificant. The influencing intensity of various factors ranked as A>B>C. The factors that had a statistically significant effect on the extraction of chlorogenic acid inL.alatawere etha-nol concentration and extraction solvent volume. The optimal extraction process determined in this experiment was A3B1C1, that is, ethanol concentration of 40%, extraction solvent volume of 20 mL and reflux extraction time of 1 h.

Table 5 Results of analysis of variance

5 Determination of chlorogenic acid content in samples

An accurate amount of powder (passed through No.2 sieve) ofL.alatafrom each producing area was weighed, produced into test solution according to the method described in Section3.2.4, determined by high-performance liquid chromatography under the conditions in Section3.2.2, respectively. The content of chlorogenic acid was calculated using the one-point external standard method. The results are shown in Table 6.

Table 6 Content of chlorogenic acid in Laggera alata (D. Don) Sch. Bip. ex Oliv. from different producing areas in Guangxi

6 Conclusions and discussion

The results of this study show that when butyl acetate-formic acid-water (V∶V∶V=7∶2.5∶2.5) was used as the developing agent, the spots were clear and the resolution was good. When methanol-0.1% phosphoric acid solution was used as the mobile phase, the resolution was relatively good, the baseline was relatively stable and the peak appearance time was appropriate, so the mobile phase was determined to be methanol-0.1% phosphoric acid. Among the detection wavelengths of 329, 243 and 218 nm, the baseline drifted, and more miscellaneous peaks appeared at the detection wavelengths of 243 and 218 nm, and at the detection wavelength of 329 nm, the baseline was relatively stable, with fewer miscellaneous peaks, smaller solvent peak, and larger chlorogenic acid response value. Therefore, 329 nm was finally used as the detection wavelength in this experiment.

L.alatafrom different places in Guangxi all contained chlorogenic acid, and the content of chlorogenic acid varied greatly. TheL.alataproduced in De’e Township, Longlin, Baise had the highest chlorogenic acid content, followed by that produced in Yining District, Nanning City, and theL.alataproduced in Shangsi County and Pingle County had the lowest chlorogenic acid content. There was no significant difference in the content of chlorogenic acid inL.alataamong Wuming, Yongning District and Lipu, or between Shangsi County and Pingle County.