TLC Identification of Laggerae Alatae Herba and Extraction Process of Chlorogenic Acid

Huimin ZHOU, Xiumei MA, Ting PENG, Chunmei LIU, Xue JIANG, Jiangcun WEI*, Wen ZHONG*, Chunli TANG

1. Guilin Municipal Hospital of Traditional Chinese Medicine, Guilin 541002, China; 2. Guangxi International Zhuang Medical Hospital, Nanning 530201, China; 3. The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530023, China

Abstract [Objectives] Taking chlorogenic acid as index component, TLC and content determination method of Laggerae Alatae Herba were established. [Methods] TLC identification used silica gel G thin-layer plate, and butyl acetate∶formic acid∶water (7∶2.5∶2.5) was taken as developing agent, and it was inspected under ultraviolet lamp (365 nm). The content was determined by chromatographic 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 speed: 1.0 mL/min; column temperature: 25 ℃; injection volume: 10 μL. [Results] Chlorogenic acid can be detected by TLC, with clear spots and good specificity. When injection volume of chlorogenic acid was between 0.099 and 0.990 μg (R2=0.999 9), there was good linear relationship. In low, medium and high sample adding groups of Laggerae Alatae Herba, average recovery rates of chlorogenic acids were 98.80% (RSD=2.09%), 98.24% (RSD=1.96%) and 99.65% (RSD=2.15%). [Conclusions] The method could effectively identify medicinal material Laggerae Alatae Herba, and accurately measure the content of chlorogenic acid in Laggerae Alatae Herba, thereby providing a scientific basis for developing and using medicinal resources of Laggerae Alatae Herba.

Key words Laggera alata, Chlorogenic acid, Content determination, TLC identification, Extraction process

1 Introduction

Laggeraalata(D. Don) Sch. Bip. Ex Oliv belongs toLaggeraof Asteraceae. The whole herb is used as medicine, and it is bitter, pungent and mild in nature. Medicinal material Laggerae Alatae Herba has the effects of dispelling wind, dehumidification and detoxification[1-3], and often is used to cure many diseases, such as traumatic injury, poisonous snake bite, rheumatoid arthritis, skin eczema, abdominal pain and diarrhea. Experiment shows that Laggerae Alatae Herba mainly contains flavonoids[4], phenolic acids[5]and other chemical compounds[6]. In the experiment, TLC identification and content determination method of chlorogenic acid in Laggerae Alatae Herba were established. The research could provide reference for quality evaluation of Laggerae Alatae Herba, and lay a certain foundation for the study of new methods for quality control of Laggerae Alatae Herba.

2 Instruments and reagents

2.1 Instruments2695 type of high performance liquid chromatography (USA Waters Company); Simplicity ultra pure water system (Millipore China Co., Ltd); Practum224-1CN analytical balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.].

2.2 ReagentsReference substance of chlorogenic acid (China Institute for Food and Drug Control, lot number: 1110885-200102, purity: >98.0%); both methanol and acetonitrile were chromatographically pure (Fisher, 4 L); ethanol, phosphoric acid, acetic acid and other reagents were analytically pure, and experimental water was ultra pure water. Laggerae Alatae Herba was collected in Shangsi County of Guangxi, and it was identified as whole grass ofL.alataby the teacher from Teaching and Research Office of Traditional Chinese Medicine Identification, Guangxi University of Traditional Chinese Medicine.

3 Methods and results

3.1 TLC identification3 g of Laggerae Alatae Herba powder was weighed, and 60 mL of 40% ethanol was added for backflow for 1 h. After filtration, the filtrate was evaporated to about 2 mL as the test solution of Laggerae Alatae Herba. An appropriate amount of chlorogenic acid reference substance was taken, and methanol was added to dissolve it, and then 1.1 mg/mL of control solution was prepared. 5 μL of test solution and 5 μL of control solution were respectively pointed on the same silica gel GF254 thin-layer plate, and toluene∶ethyl acetate∶formic acid (5∶3∶1) was taken as developing agent for expansion. After taken out, it was dried in air and then inspected under ultraviolet lamp (365 nm). In chromatograph of Laggerae Alatae Herba test sample, fluorescent spots of the same color were displayed at the position corresponding to the chromatogram of the reference substance. The result was shown in Fig.1.

Note: 1-10 was 10 batches of Laggerae Alatae Herba samples; A was reference substance of chlorogenic acid.Fig.1 TLC of 10 batches of Laggerae Alatae Herba

3.2 Content determination

3.2.1Preparation of Laggerae Alatae Herba sample. Whole grass ofL.alatawas collected. After natural drying, it was crushed to pass through No.2 sieve for content determination.

3.2.2Chromatographic conditions. Chromatographic 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 speed: 1.0 mL/min; column temperature: 25 ℃; injection volume: 10 μL. According to above condition, separation of each component was good (Fig.2).

Note: A. Reference substance; B. Laggerae Alatae Herba; C. 25% of ethanol.Fig.2 HPLC of chlorogenic acid reference substance and Laggerae Alatae Herba sample

3.2.3Preparation of control solution. 11.0 mg of chlorogenic acid was weighed precisely and set in 10 mL of graduated flask. Methanol was added to dissolve and dilute it to the graduate, and 1.1 mg/mL of reference stock solution was obtained. 0.45 mL of chlorogenic acid reference stock solution was precisely weighed and set in 10 mL of graduated flask. Methanol was added to dilute it to the graduate, and control solution of chlorogenic acid was obtained.

3.2.4Preparation of test solution. 1.0 g of Laggerae Alatae Herba powder was weighed precisely, and 25% of ethanol was used for extraction. The extracting solution was centrifugated for 10 min at the speed of 13 000 r/min. After that, supernatant was taken and filtrated by 0.45 μm of millipore filter, and the filtrate was test solution.

3.2.5Preparation of standard curve. 2, 5, 10, 15, and 20 μL of chlorogenic acid reference solution (concentration was 49.5 μg/mL) was injected into liquid chromatograph. Peak area was taken as vertical coordinate, and injection volume (μg) was taken as horizontal coordinate. Standard curve was drawn, and regression equation was calculated:Y=1.0×106X-7 705.6 (R2=0.999 9). The results showed that there was good linear relationship when injection volume of chlorogenic acid was between 0.099 and 0.990 μg.

3.2.6Precision test. An appropriate amount of the reference solution (0.49 μg/mL) was precisely measured, and it was continuously injected for six times under the condition of Section3.2.2.RSDof peak area of chlorogenic acid was 0.37%, which was lower than 3%. It illustrated that instrument had good precision.

3.2.7Stability test. 1.0 g of Laggerae Alatae Herba powder was weighed precisely and prepared into test solution by the method under Section3.2.4. The determination was carried out at 0, 2, 4, 8, 12 and 24 h after the preparation of the test solution according to the conditions in Section3.2.2.RSDof peak area of Laggerae Alatae Herba was 2.78%, which was lower than 3%. It illustrated that test solution had good stability in 24 h.

3.2.8Repeatability test. 1.0 g of Laggerae Alatae Herba powder was weighed precisely, and there were 6 copies in total. Test solution was prepared by the method under Section3.2.4, and it was determined under the condition of Section3.2.2. Average content of chlorogenic acid was 1.576 0 mg/g, and itsRSDwas 1.56%, which was lower than 3%. It showed that the method had good repeatability.

3.2.9Sample recovery test. 0.5 g of Laggerae Alatae Herba powder from the same batch (chlorogenic acid content was 1.576 0 mg/g) was precisely weighed, and there were 9 copies in total. They were divided into three groups: low, medium and high sample adding groups (80%, 100% and 120% of sample adding in 0.5 g of medicinal material), and sample adding amount was 0.630 3, 0.787 6 and 0.946 0 mg, respectively. Average recovery rates of chlorogenic acid in low, medium and high sample adding groups of Laggerae Alatae Herba were 98.80%, 98.24% and 99.65%, andRSDwas 2.09%, 1.96% and 2.15% (n=3). It showed that the method had good accuracy. The result was shown in Table 1.

Table 1 Sampling recovery rate of chlorogenic acid in Laggerae Alatae Herba

4 Extraction process of chlorogenic acid in Laggerae Alatae Herba

4.1 Single factor investigation

4.1.1Determination of the maximum absorption wavelength. Methanol was used to dissolve and dilute chlorogenic acid reference solution. Methanol was taken as reference solution, and ultraviolet-visible spectrophotometer was used for scanning in the wavelength range of 200-400 nm. According to absorption curve of chlorogenic acid, it was determined that the best absorption wavelength was 329 nm.

4.1.2Investigation on extraction method. Extraction time of chlorogenic acid was determined as 1 h, and 20 mL of 25% ethanol was used for extraction. Reflux extraction, ultrasonic extraction and solvent impregnation were respectively used to extract chlorogenic acid from Laggerae Alatae Herba. Experimental results showed that reflux extraction method had higher extraction rate of chlorogenic acid. So, reflux extraction method was selected in the experiment.

4.1.3Investigation on solvent concentration. Extraction time of chlorogenic acid was determined as 1 h, and 20 mL of solvent and reflux extraction method were used. Single factor experiment on the yield of chlorogenic acid in Laggerae Alatae Herba with the concentration of ethanol and methanol was conducted, and concentration of ethanol and methanol was designed as 0%, 25%, 45%, 65% and 85%. The results displayed that the reflux by 25% of ethanol had higher extraction rate.

4.1.4Investigation on extraction volume. Extraction time of chlorogenic acid was determined as 1 h, and 25% of ethanol and reflux extraction method were used. Single factor experiment on the yield of chlorogenic acid in Laggerae Alatae Herba with solvent extraction volume was conducted, and extraction volume was 15, 25, 35 and 45 mL. The results displayed that the extraction rate was the highest when volume was 25 mL.

4.1.5Investigation on extraction time. 25 mL of 25% ethanol and reflux extraction method were used. Single factor experiment on the yield of chlorogenic acid in Laggerae Alatae Herba with the extraction time was conducted, and the extraction time was set as 0.5, 1.0, 1.5, 2.0 and 2.5 h. The results displayed that extraction rate of chlorogenic acid was higher when extraction time was 1.0 h.

4.2 Orthogonal experimental designThe main methods of extracting chlorogenic acid from Laggerae Alatae Herba are mainly reflux extraction, ultrasonic extraction and impregnation. By referring to the relevant literature and theChinesePharmacopoeiaof 2020, ethanol with different concentrations was used as solvent for extraction in this experiment, and solvent concentration (10%, 25%, and 40% of ethanol), solvent volume (20, 30 and 40 mL), and extraction time (1.0, 1.5 and 2.0 h) were inspected. According to orthogonal design assistant software, L9(34) orthogonal experimental design was selected (Table 2).

Table 2 Factor and level of orthogonal test for extracting chlorogenic acid from Laggerae Alatae Herba

1.0 g of Laggerae Alatae Herba powder was precisely weighed, and chlorogenic acid was extracted from Laggerae Alatae Herba according to above method. Then, it was operated according to preparation method of test solution. Test was conducted according to L9(34) orthogonal design scheme, and content of chlorogenic acid in Laggerae Alatae Herba was determined by HPLC. The results were shown in Table 3.

Table 3 Orthogonal experimental design and results for extracting chlorogenic acid from Laggerae Alatae Herba

4.3 Orthogonal experiment and result analysisANOVA results (Table 4) displayed that the concentration of ethanol and the volume of extraction solvent had significant effects on the extraction rate of chlorogenic acid from Laggerae Alatae Herba, followed by extraction time. ANOVA results showed that there was significant difference in ethanol concentration and extraction solvent volume, but no significant difference in extraction time, and strength of each influencing factor was A>B>C. So, the factors that had statistical significance for the extraction effect of chlorogenic acid from Laggerae Alatae Herba were the concentration of ethanol and the volume of extraction solvent. Therefore, the most ideal extraction scheme in this experiment was A3B1C1, namely 40% of ethanol concentration, 20 mL of extraction solvent volume, 1 h of reflux extraction time. At this time, the extraction effect was the best.

Table 4 ANOVA results

5 Validation experiment

According to the best technology determined by orthogonal experiment, 3 batches of validation tests were conducted. The results displayed that content of chlorogenic acid from Laggerae Alatae Herba was 1.34 mg/g, andRSDwas 1.60%. The results showed that the process had good reproducibility, and it can be used as the extraction process of chlorogenic acid from Laggerae Alatae Herba.

6 Conclusions

Chlorogenic acid can be detected by TLC, with clear spots and good specificity. When injection volume of chlorogenic acid was between 0.099 and 0.990 μg (R2=0.999 9), there was good linear relationship. In low, medium and high sampling groups of Laggerae Alatae Herba, average recovery rates of chlorogenic acids were 98.80% (RSD=2.09%), 98.24% (RSD=1.96%) and 99.65% (RSD=2.15%). The method could effectively identify medicinal material Laggerae Alatae Herba, accurately measure the content of chlorogenic acid in Laggerae Alatae Herba, and provide a scientific basis for developing and using medicinal resources of Laggerae Alatae Herba.