Abstract
A simple and effective method for the simultaneous quantitative analysis of six caffeoylquinic acids (CAs) in Matricaria chamomilla L. (M. chamomilla) using high-performance liquid chromatography (HPLC) with diode-array detection (DAD) was established. The chromatographic separation was performed on a Waters XBridge Shield RP C18 column (4.6 mm × 250 mm, 5 μm) with the mobile phase of acetonitrile (0.5% phosphoric acid) and water (0.5% phosphoric acid) using a gradient elution at a flow rate of 1.0 mL/min and UV detection at 327 nm. The correlation coefficients of all analytes were 0.999, and the results showed excellent linearity. The lower limits of detection (LLOD) and quantification (LLOQ) of all analytes fall within the range of 0.014∼0.017 μg/mL and 0.068∼0.086 μg/mL, respectively. The extraction recoveries of all analytes fall within the range of 100.74%∼101.55%, with relative standard deviation not exceeding 2.83%. The intraday and interday precisions fall within the range of 0.03%∼0.65% and 0.02%∼0.09%, respectively. This validated method was successfully applied to the investigation of 34 samples of M. chamomilla collected from different geographical areas. The results showed that the established method is appropriate for the analysis of the six CAs in M. chamomilla and helpful for quality assessment of capitula of M. chamomilla (CMC), whole herb of M. chamomilla (WHMC), and related herbal formulas.
1. Introduction
Matricaria chamomilla L. (M. chamomilla) is a kind of famous herbaceous plant indigenous to Europe. It has been naturalized to many countries and regions of the world for thousands of years as one of the most popular medicinal plants in folk and traditional medicine [1, 2]. The capitula of M. chamomilla (CMC), named as “German Chamomile” in Europe, is included in the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP) to treat a series of diseases, such as digestive ailments, restlessness, mild insomnia due to nervous disorders, inflammation, and irritations of the skin and mucosa [3]. The dried whole herb of M. chamomilla (WHMC) named as “Yangganju” is recorded in Medicine and Pharmacy of Traditional Uyghur Medicine in Xinjiang China to treat stomach upset, dysuria, skin itching, blurred vision, cystitis, and stomatitis [4]. Moreover, essential oil of M. chamomilla is used extensively in cosmetics and aromatherapy in Europe [5]. In sight of its significant therapeutic applications, a large group of active constituents have been identified from M. chamomilla by researchers [6–9]. Our group has been devoted to the study on potential bioactive natural products in traditional medicinal plants for several decades [10–13]. In our previous chemical constituent study of M. chamomilla, except chlorogenic acid, five other caffeoylquinic acids (neochlorogenic acid, cryptochlorogenic acid, and isochlorogenic acid A, B, and C) were identified from M. chamomilla for the first time (Figure 1). The CAs represent a class of interesting natural products with wide pharmacological activities including antioxidant [14], anti-inflammatory [15, 16], antimicrobial [17], enzyme inhibition [18], hepatocyte protection [19], platelet aggregation inhibition [20], antihepatic fibrosis [21], and anti-SARS [22]. According to these previous reports, the pharmacological activities of CAs were consistent with the efficacy of CMC and WHMC, and thus, the CAs should be the active ingredients. However, the method for the determination of CAs in M. chamomilla has not been reported. Herein, to develop improved solution for the quality evaluation of CMC and WHMC, a simple and effective HPLC method was developed for simultaneous quantitative analyses of the six CAs.
2. Experimental
2.1. Chemicals, Reagents, and Materials
Acetonitrile (HPLC grade) was purchased from Fisher (Canada). Phosphoric acid (HPLC grade) was purchased from CNW Technology (Germany). Purified water was purchased from Wahaha Company (China). Syringe filter (0.45 μm) was purchased from ANPEL (China). Standard compounds of neochlorogenic acid (NCA), cryptochlorogenic acid (CCA), and isochlorogenic acid A, B, and C (ICA, ICB, and ICC) were purchased from Chengdu Herbpurify CO., LTD. Standard compound of chlorogenic acid (CA) was purchased from National Institutes for Food and Drug Control. The purity of the six reference compounds was determined to be more than 98% by normalization of the peak areas detected by HPLC-DAD.
A total of 34 samples were collected from different geographical areas and identified as Matricaria chamomilla (L.) by Professor Chunsheng Liu and his group at Beijing University of Chinese Medicine, as shown in Table 1. The samples were air-dried (indoor) at the origin of collection. Among them, 3 representative geographical batches (D1, D10, and D12) were categorized into different parts of M. chamomilla (roots, stems, and leaves) in the laboratory. These medicinal materials were deposited in Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.
2.2. Apparatus
All medicinal materials were ground by high-speed disintegrator (Tianjin Taisite FW100). All sample extraction processes were carried out in a water bath (Tianjin Taisite DK-98-1). All medicinal materials and standard compounds were weighed by electronic analytical balances (Mettler Toledo AL204-IC and XS105 Dual Range). The HPLC system was SHIMADZU Prominence LC-20A (Shimadzu Corporation, Tokyo, Japan) equipped with CBM-20Alite system controller, LC-20AT pump, CTO-20A column oven, SPD-M20A UV-Vis detector, SIL-20A autoinjector, DGU-M20A5 degasser, and Shimadzu LC-solution work station.
2.3. Chromatographic Conditions
The separation of CAs was carried out on a Waters XBridge Shield RP C18 column (4.6 mm × 250 mm, 5 μm) at 35°C with a flow rate of 1.0 mL/min. The detection wavelength was 327 nm with an injection volume of 10 μL. The optimized mobile phase system was A (acetonitrile/phosphoric acid, 99.5/0.5, ) and B (water/phosphoric acid, 99.5/0.5, ) with a gradient elution program: A/B = 12/82 (0–13 min), A/B = 12/82–25/75 (13–15 min), and A/B = 25/75 (15–30 min). All data were acquired and processed by Shimadzu LC solution software.
2.4. Preparation of Standard Solution
Standards were weighed accurately and dissolved in 10 mL of 70% aqueous methanol to prepare the standard mix stock solution of NCA (270 μg/mL), CCA (212 μg/mL), CA (240 μg/mL), ICA (260 μg/mL), ICB (218 μg/mL), and ICC (246 μg/mL). The standard mix stock solution was stored at 4°C and filtered through a 0.45 μm syringe filter before HPLC analysis.
2.5. Preparation of Sample Solution
A conical flask was charged with 0.3 g of sample and 15 mL of 70% aqueous methanol. Then, the sample was refluxed for 45 min. After cooling to room temperature, replenish the loss of the solvent with 70% aqueous methanol. Finally, the sample solution was filtered through a 0.45 μm syringe filter prior to HPLC analysis.
2.6. Method Validation
The proposed method was validated according to CFDA guidelines for the validation of analytical methods for pharmaceutical quality standard, with respect to linearity, lower limit of detection (LLOD) and quantification (LLOQ), precision, repeatability, stability, and accuracy.
The standard mix stock solutions at 12 different concentrations were injected for two replicates. The calibration curve was constructed by least square fit of the data with the peak area (y-axis) versus the injection amounts (x-axis) for each compound. The standard mix stock solution was further diluted to explore the LLOD and LLOQ. The LLOD and LLOQ were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively.
The precision was evaluated with standard solution under the selected optimal conditions in six replicates continuously. To further evaluate the repeatability of the developed assay, the sample was analyzed in six replicates. Stability was tested with the sample at room temperature (25°C) and analyzed at 0, 4, 12, 24, 48, and 72 h, respectively. Recovery tests were performed to evaluate the accuracy of the developed method. The accurate amounts of six CAs were weighed and spiked to certain amounts of the sample powder and were then extracted and analyzed in accordance with the method described above. The spiked amount of each standard was adjusted to provide a similar concentration present in the sample. The recovery rate (%) was measured for six replicates.
3. Results and Discussion
3.1. Optimization of the Extraction Procedure
During sample preparation, the extraction parameters, e.g., extraction method, solvent, extraction time, and solvent volume, were optimized. The efficiency of the extraction procedure was evaluated using different extraction methods, i.e., reflux and ultrasonic-assisted method. The results demonstrated that the reflux method provided the higher value in the content of the target compounds than the ultrasonic-assisted method. Then, the other factors were investigated using monofactor analysis, i.e., extraction solvent (30%, 50%, and 70% aqueous methanol () and pure methanol), extraction time (30 min, 45 min, and 60 min), and solvent volume (5 mL, 10 mL, 15 mL, and 20 mL). As a result, the optimized extraction procedure was confirmed to be refluxed with 15 mL of 70% aqueous methanol solution () for 45 min.
3.2. Optimization of the Chromatographic Conditions
In order to separate the six CAs, a gradient method was developed to determine all the constituents in one analysis. Various mixtures of mobile phases were tested, such as methanol and water, methanol (0.1% formic acid) and water (0.1% formic acid), and methanol (0.5% phosphoric acid) and water (0.5% phosphoric acid), but the separation was unsatisfactory. However, by replacing methanol with acetonitrile, the special mobile phase system (acetonitrile (0.5% phosphoric acid) and water (0.5% phosphoric acid)) significantly improved the separation. We also tried to simplify the mobile phase system as acetonitrile and water (0.5% phosphoric acid), but the separation was unsatisfactory. Due to the similar structure, the UV absorption spectrograms of the six CAs were almost identical. The detection wavelength was selected at the maximum absorption of 327 nm.
3.3. Method Validation
The analytical method was validated with respect to the linearity, LLOD, LLOQ, precision, repeatability, stability, and accuracy. The linear ranges, regression equations, and correlation coefficients obtained from typical calibration curves and LLOD (S/N = 3) and LLOQ (S/N = 10) are shown in Table 2. All calibration curves showed excellent linearity, and the correlation coefficients were higher than 0.999.
As shown in Table 3, the precision of the method was evaluated with peak areas obtained for each analyte and expressed as relative standard deviation (RSD). The RSD of intraday and interday was 0.49% and 0.09% for NCA, 0.65% and 0.03% for CCA, 0.06% and 0.03% for CA, 0.10% and 0.02% for ICA, 0.03% and 0.04% for ICB, and 0.12% and 0.05% for ICC, respectively. The method is repeatable, with the RSD was in the range of 1.0%∼2.3%. The CAs were proved to be stable in sample solution within 72 h at room temperature with the RSD below 1.1%. As shown in Table 4, the extraction recoveries were performed to evaluate the accuracy of the developed method. The mean recoveries were in the range of 100.7%∼101.5% with the RSD less than 3.0% for all the six CAs. In general, the developed method is precise, repeatable, and accurate for the simultaneous quantitative determination of the six CAs in M. chamomilla.
3.4. Sample Analysis
The established method has been successfully applied for the simultaneous determination of M. chamomilla samples collected from different geographical areas, as shown in Table 5 and Figure 2. The results showed that the contents of six CAs in CMC and WHMC collected from different geographical areas were different, and the contents of six CAs in different parts of specific M. chamomilla were also different.
(a)
(b)
Although the position of the substituent caffeoyl group in NCA, CCA, and CA is different, their mother nucleus structures are the same. In order to simplify the results, NCA, CCA, and CA were defined as total chlorogenic acids (TCAs). Similarly, ICA, ICB, and ICC were defined as total isochlorogenic acids (TICAs). The contents of six CAs, TCAs, and TICAs in CMC were generally higher than WHMC. The TCA contents in WHMC series and CMC series were in the range of 0.17∼1.98 mg/g and 0.84∼3.98 mg/g, respectively. The TICA contents in these series were in the range of 0.47∼5.84 mg/g and 1.38∼9.78 mg/g, respectively.
Compared with the literature report, the results showed that the contents of CAs in CMC were comparable to that of apigenin-7-O-glucoside (about 0.2∼6.2 mg/g), higher than that of most flavonoids (such as luteolin, apigenin, and 7-methoxycoumarin; far less than 1.0 mg/g) [8, 9, 23]. Because the pharmacological activities of CAs were consistent with the efficacy of CMC and WHMC, the CAs together with coumarins and flavonoids could all be considered as the main bioactive ingredients.
4. Conclusion
In this work, an HPLC method was established for the simultaneous determination of six CAs with pharmacological activities in M. chamomilla for the first time. The established method was validated by linearity, reproducibility, recovery, and precision; all parameters found satisfactory. This newly established HPLC method will be helpful in the quality assessment of CMC, WHMC, and related herbal formulas in future.
Data Availability
The chromatographic data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was financially supported by the Key Project at Central Government Level: The Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources (2060302). The authors are grateful to Dr. Rizwan Elahi for providing language assistance and grammar check.