Abstract
Objective
To establish a high-performance liquid chromatographic method (HPLC) for the simultaneous determination of 16 compounds from Artemisia ordosica.
Methods
HPLC was used to analyze 16 quality indicators of A. ordosica. The HPLC conditions were as follows: Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile (A)-water (B) as mobile phase, gradient elution: 0–10 min, 75%−65% B; 10–30 min, 65%−35% B; and finally 30–40 min, 35%−15% B. The flow rate was 1.0 mL/min, the column temperature was 40 °C, the injection volume was 10 μL, and monitored by absorbance at 285 nm for compounds 1–10, 12 and 225 nm for compounds 11, 13–16.
Results
Under the selected experimental chromatographic conditions, compounds 1–16 showed good linearity (r > 0.9993) in a wide concentration range. Their average recoveries were 99.50%, 95.38%, 97.75%, 96.00%, 98.20%, 97.50%, 95.50%, 99.33%, 96.75%, 96.50%, 98.50%, 97.83%, 99.20%, 95.33%, 97.33% and 96.30%, respectively, and the RSD were 1.99%, 1.81%, 1.63%, 1.98%, 1.67%, 1.92%, 1.74%, 1.67%, 1.90%, 1.72%, 1.88%, 1.83%, 1.79%, 1.76%, 1.81% and 1.96%, respectively.
Conclusion
Based on the results of the HPLC analysis, it was concluded that p-hydroxycinnamic acid (1), O-hydroxycinnamic acid (2), coniferyl alcohol (5), 5,4′-dihydroxy-7,3′-dimethoxyflavanone (8), 5,4′-dihydroxy-7-methoxyflavanone (9), 5-hydroxy-7,4′-dimethoxyflavanone (12), dehydrofalcarindiol (13), arteordoyn A (14), dehydrofalcarinol (15) and capillarin (16) are best suited for the role of quality indicators of A. ordosica grown in different ecological environments.
Keywords: Artemisia ordosica Krasch, high performance liquid chromatography, quality indicators, simultaneous determination
1. Introduction
The genus Artemisia, belonging to the family Compositae, is an annual, biennial or perennial herb, which consists of 50 species and 15 varieties in Inner Mongolia, and account for 30% of Artemisia plants in China. Artemisia plants have many economic uses, such as some species are used as medicines, some are often used as excellent forage in grasslands and some play an important role in the protection of ecological environment in sandy areas. Artemisia ordosica Krasch is one of the main arido-active shrubs which grows in the arid and semi-arid areas of the north China, including Inner Mongolia, Xinjiang, Ningxia, Gansu and Shanxi (Abad et al., 2012, Zhang et al., 2013), which is utilized as a folk medicine for rheumatism, swelling treatment and clearing heat (Zhang et al., 2017).
Phytochemical studies have shown that this plant collected from Xinjiang contained volatile oils (Zhang et al., 2017), coumarins (Zhao et al., 2005a), flavonoids (Zhong et al., 2016, Zhao et al., 2005, Zhang et al., 2006), terpenoids (Tan, Zheng, & Tang, 1998) and acetylenes (Zhang et al., 2017). The genus Artemisia is rich in coumarins, flavonoids and acetylenes, which have been shown to be the main bioactive constituents (Maierdan et al., 2006, Wang et al., 2007). However, prior to each analysis of the compounds in Artemisia plants, reference substances have to be purchased, which are costly and quite difficult to obtain and causes potential measurement errors by the inconsistency between analyte and reference substance.
At present, there are many quantitative analysis of one or two active components in an herb using corresponding reference substances as quality indicators (Wu et al., 2018, Lin et al., 2018). Then, compound with the same retention time on the same column is not necessarily the same component. To avoid the potential measurement errors and obtain the most useful chemical information, A. ordosica was extracted by 95% ethanol and the extractive was isolated by silica gel column chromatography combined with TLC and semi-preparative HPLC, and the structures of the isolated compounds were determined by spectroscopic methods, including extensive 1D and 2D NMR techniques reported in our previous research (Wu et al., 2018, Lin et al., 2018).
Here, we report a finding that the potential quality indicators were selected according to the results of HPLC analysis of simultaneously determining 16 compounds, namely p-hydroxycinnamic acid (1), O-hydroxycinnamic acid (2), acacetin (3), 3,5-dihydroxy-7,4′-dimethoxyflavone (4), coniferyl alcohol (5), arteordosin A (6), arteordosin B (7), 5,4′-dihydroxy-7,3′-dimethoxyflavanone (8), 5,4′-dihydroxy-7-methoxyflavanone (9), dihydroconiferyl alcohol (10), O-hydroxycapillene (11), 5-hydroxy-7,4′-dimethoxyflavanone (12), dehydrofalcarindiol (13), arteordoyn A (14), dehydrofalcarinol (15) and capillarin (16) in A. ordosica samples from five different provinces in China. To date, the concentrations of these compounds were not monitored in real time. The analysis of the contents of secondary metabolites from A. ordosica grown in different ecological environments can be an important source of information for quality indicators.
2. Materials and methods
2.1. Plant materials
The aerial parts of A. ordosica were collected from five different provinces in China in June 2019, and identified by Prof. Buhebateer (Inner Mongolia University for Nationalities). We collected five batches of A. ordosica: Inner Mongolia (Inner Mongolia, China; Sample IM), Xinjiang (Xinjiang, China; Sample XJ), Ningxia (Ningxia, China; Sample NX), Gansu (Gansu, China; Sample GS) and Shanxi (Shanxi, China; Sample SX). Voucher specimens were deposited in a warehouse (The storage temperature was 10–30 °C, and the relative humidity (RH) was 30%−50%, and 24 h was dark) in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities.
2.2. General experimental procedures
HPLC (LC-20AT, Shimadzu, Kyoto, Japan) was performed on Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 µm). Acetonitrile (HPLC grade) was purchased from Guangfu Fine Chemical Institute (Tianjin, China). Ultrapure water was prepared by Milli-Q instrument (Millipore, Massachusetts, USA). All other solvents were of analytical grade and obtained from Tianjin Damao Chemical Reagents Factory (Tianjin, China). The purity of the compounds 1–16 (Fig. 1) was determined by HPLC with DAD, in which the purity of p-hydroxycinnamic acid (1), O-hydroxycinnamic acid (2), acacetin (3), 3,5-dihydroxy-7,4′-dimethoxyflavone (4), coniferyl alcohol (5), arteordosin A (6), arteordosin B (7), 5,4′-dihydroxy-7,3′-dimethoxyflavanone (8), 5,4′-dihydroxy-7-methoxyflavanone (9), dihydroconiferyl alcohol (10), O-hydroxycapillene (11), 5-hydroxy-7,4′-dimethoxyflavanone (12), dehydrofalcarindiol (13), arteordoyn A (14), dehydrofalcarinol (15) and capillarin (16) was more than 98%.
Fig. 1.
Structures of compounds 1–16 from A. Ordosica.
2.3. Preparation of standard solutions
Standard stock solutions (2.0 mg/mL) of compounds 1–16 were prepared by dissolving the compounds in acetonitrile, which were stored at 4 °C. A series of working standard solutions containing each of the compounds were prepared by dilution with acetonitrile to the proper concentrations from the stock solution before using.
2.4. Sample preparation
Dried powder of five batches samples (60 mesh, 1.0 g) was extracted under ultrasonication with 95% ethanol (25 mL) for 30 min. Subsequently, each extracted solution was filtered, evaporated to dryness, and redissolved in acetonitrile (25 mL). The acetonitrile solutions were stored at 4 °C and filtered through a 0.45 µm membrane filter before LC analysis.
2.5. Chromatographic conditions
The chromatographic separation was carried out using an Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 µm). The mobile phase was comprised of acetonitrile (A) and water (B) with a gradient elution: 0–10 min, 75%−65% B; 10–30 min, 65%−35% B; and finally 30–40 min, 35%−15% B. Compounds 1–10, 12 were monitored at a wavelength of 285 nm and compounds 11, 13–16 at 225 nm, with a flow rate of 1 mL/min at the injection volume 20 µL and column temperature of 30 °C.
2.6. Validation procedure
To evaluate the validity of the method, validation tests were performed (Wang, Ao, & Dai, 2013). The working standard solutions of compounds 1–16 were used to determine the calibration curves (Jin, Du, Shi, & Liu, 2010). Under the present chromatographic conditions, the limit of detection (LOD) and limit of quantitation (LOQ) were estimated at S/N ratio of 3 and 10, respectively (Guo et al., 2017). Intermediate precision was implemented by intra-day and inter-day precision studies (Agrawal & Laddha, 2017). In order to evaluate the repeatability of the developed assay, six different working solutions prepared from the same sample (Sample IM) were analyzed. The relative standard deviation (RSD) was taken as a measure of repeatability (Guo et al., 2017). Accuracy was estimated by recovery studies using a standard addition method (Agrawal & Laddha, 2017).
3. Results and discussion
3.1. Optimization of extraction conditions
Selection of a suitable extraction method for a certain type of sample is always important. The variables involved in the procedure such as solvent, solvent volume and extraction time were optimized (Fig. 2). Different concentrations of aqueous ethanol solutions were selected as extraction solvents. The best solvent was found to be 95% ethanol, which allowed complete extraction of all compounds at higher yields and 75% ethanol and 55% ethanol had worse performance due to poor solubility of all compounds in the solvents. In order to investigate the solvent volume, about 1.0 g of dried sample (Sample IM) was extracted with 10, 20, 30 mL of 95% ethanol, and the results showed that 20 or 30 mL of 95% ethanol had the highest extraction yield with almost equal extraction capacities. Therefore, 25 mL were selected as the solvent volume. The influence of the extraction time on the efficiency of extraction was also examined. The results showed that the highest amounts of all compounds were obtained with extraction for 30 min.
Fig. 2.
Results of optimization of extraction conditions in sample IM. A, Effect of solvent on compounds content; B, Effect of solvent volume on compounds content; C, Effect of extraction time on compounds content. 1, p-hydroxycinnamic acid; 2, O-hydroxycinnamic acid; 3, acacetin; 4, 3,5-dihydroxy-7,4′-dimethoxyflavone; 5, coniferyl alcohol; 6, arteordosin A; 7, arteordosin B; 8, 5,4′-dihydroxy-7,3′-dimethoxyflavanone; 9, 5,4′-dihydroxy-7-methoxyflavanone; 10, dihydroconiferyl alcohol; 11, O-hydroxycapillene; 12, 5-hydroxy-7,4′-dimethoxyflavanone; 13, dehydrofalcarindiol; 14, arteordoyn A; 15, dehydrofalcarinol; 16. capillarin.
3.2. Optimization of HPLC conditions
In order to obtain the compounds which are the best suited for the role of quality indicators of A. ordosica, a novel HPLC method was developed for simultaneously determining 16 compounds, which were isolated from A. ordosica (Sample IM). The operating conditions such as the mobile phase compositions, the gradient mode and the maximum absorption wavelength were optimized. The chromatogram of samples was shown in Fig. 3, 16 compounds were well separated under the described chromatographic conditions.
Fig. 3.
HPLC of solutions of sample and mixed chemical standards. (A, Solution of sample, 285 nm; B, Solution of mixed chemical standards, 285 nm; C, Solution of sample, 225 nm; D, Solution of mixed chemical standards, 225 nm. 1, p-hydroxycinnamic acid; 2, O-hydroxycinnamic acid; 3, acacetin; 4, 3,5-dihydroxy-7,4′-dimethoxyflavone; 5, coniferyl alcohol; 6, arteordosin A; 7, arteordosin B; 8, 5,4′-dihydroxy-7,3′-dimethoxyflavanone; 9, 5,4′-dihydroxy-7-methoxyflavanone; 10, dihydroconiferyl alcohol; 11, O-hydroxycapillene; 12, 5-hydroxy-7,4′-dimethoxyflavanone; 13, dehydrofalcarindiol; 14. arteordoyn A; 15, dehydrofalcarinol; 16, capillarin).
3.3. Method validation
The linearity, LOD, LOQ, precision, repeatability, stability, and accuracy of the developed HPLC method were validated. As shown in Table 1, all calibration curves exhibited excellent linearity (r: 0.9993–0.9997) in a relatively wide concentration range. The overall LODs and LOQs of 16 compounds were 2.0–6.0 µg/mL and 5.0–20 µg/mL, respectively. The results of the tests of precision, repeatability and stability of 16 compounds were showed in Table 2. The values of precision (RSD), repeatability (RSD) and stability (RSD) were all < 2.36%. Table 3 demonstrated that the overall recoveries lay between 95.33% and 99.50% for the 16 compounds with RSD values < 2.00%. Therefore, the HPLC method was precise, accurate and sensitive enough for simultaneously quantitative evaluation of sixteen compounds in A. ordosica from five different provinces in China.
Table 1.
Calibration curves, LOD and LOQ data of sixteen compounds investigated by high performance liquid chromatography (n = 3).
| Compounds | Calibration curves a | r | Linear range (μg/mL) | LOD (μg/mL) | LOQ (μg/mL) |
|---|---|---|---|---|---|
| p-Hydroxycinnamic acid | y = 3.45 × 103 x − 536 | 0.9994 | 20.0–100.0 | 6.00 | 20.0 |
| O-Hydroxycinnamic acid | y = 3.23 × 103 x − 475 | 0.9996 | 20.0–100.0 | 6.00 | 20.0 |
| Acacetin | y = 1.14 × 103 x − 168 | 0.9995 | 10.0–50.0 | 3.00 | 10.0 |
| 3,5-Dihydroxy-7,4′-dimethoxyflavone | y = 4.11 × 102 x + 127 | 0.9993 | 5.00–15.0 | 2.00 | 5.00 |
| Coniferyl alcohol | y = 2.75 × 103 x − 186 | 0.9998 | 20.0–100.0 | 6.00 | 20.0 |
| Arteordosin A | y = 1.44 × 103 x − 161 | 0.9995 | 10.0–50.0 | 3.00 | 10.0 |
| Arteordosin B | y = 7.42 × 102 x − 159 | 0.9997 | 5.00–15.0 | 2.00 | 5.00 |
| 5,4′-Dihydroxy-7,3′-dimethoxyflavanone | y = 2.88 × 103 x − 131 | 0.9995 | 20.0–100.0 | 6.00 | 20.0 |
| 5,4′-Dihydroxy-7-methoxyflavanone | y = 1.0 × 103 x − 327 | 0.9993 | 10.0–50.0 | 3.00 | 10.0 |
| Dihydroconiferyl alcohol | y = 7.22 × 102 x − 954 | 0.9996 | 5.00–15.0 | 2.00 | 5.00 |
| O-Hydroxycapillene | y = 1.19 × 103 x − 150 | 0.9995 | 10.0–50.0 | 3.00 | 10.0 |
| 5-Hydroxy-7,4′-dimethoxyflavanone | y = 2.85 × 103 x − 115 | 0.9995 | 20.0–100.0 | 6.00 | 20.0 |
| Dehydrofalcarindiol | y = 1.20 × 103 x − 118 | 0.9993 | 10.0–50.0 | 3.00 | 10.0 |
| Arteordoyn A | y = 1.15 × 103 x − 132 | 0.9994 | 10.0–50.0 | 3.00 | 10.0 |
| Dehydrofalcarinol | y = 1.78 × 103 x − 254 | 0.9995 | 20.0–100.0 | 3.00 | 10.0 |
| Capillarin | y = 3.93 × 103 x − 102 | 0.9993 | 20.0–100.0 | 6.00 | 20.0 |
Note: ay = peak area and x = concentration (μg/mL).
Table 2.
Precision, repeatability and stability of developed method.
| Compounds | Content (mg/g) | Intra-day (n = 3) |
Inter-day (n = 3) |
Repeatability (n = 5) |
Stability (n = 5) |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Detecteda (mg/g) | Accuracy (%) | RSD (%) | Detecteda (mg/g) | Accuracy (%) | RSD (%) | Contenta (mg/g) | RSD (%) | Contenta (mg/g) | RSD (%) | ||
| p-Hydroxycinnamic acid | 1.636 | 1.626 ± 0.026 | 99.38 ± 1.75 | 1.76 | 1.623 ± 0.025 | 99.20 ± 1.81 | 1.82 | 1.624 ± 0.028 | 1.72 | 1.632 ± 0.030 | 1.84 |
| O-Hydroxycinnamic acid | 1.571 | 1.556 ± 0.022 | 99.04 ± 1.41 | 1.42 | 1.549 ± 0.020 | 98.60 ± 1.64 | 1.66 | 1.563 ± 0.025 | 1.60 | 1.560 ± 0.028 | 1.79 |
| Acacetin | 0.672 | 0.658 ± 0.011 | 97.92 ± 1.82 | 1.86 | 0.655 ± 0.012 | 97.47 ± 1.95 | 2.00 | 0.683 ± 0.013 | 1.90 | 0.666 ± 0.014 | 2.10 |
| 3,5-Dihydroxy-7,4′-dimethoxyflavone | 0.209 | 0.206 ± 0.004 | 98.56 ± 1.69 | 1.71 | 0.203 ± 0.03 | 97.13 ± 1.89 | 1.95 | 0.212 ± 0.005 | 2.36 | 0.207 ± 0.004 | 1.93 |
| Coniferyl alcohol | 1.062 | 1.058 ± 0.015 | 99.62 ± 1.38 | 1.39 | 1.055 ± 0.014 | 99.34 ± 1.50 | 1.51 | 1.053 ± 0.020 | 1.90 | 1.058 ± 0.022 | 2.08 |
| Arteordosin A | 0.778 | 0.770 ± 0.012 | 98.97 ± 1.53 | 1.55 | 0.767 ± 0.011 | 98.59 ± 1.49 | 1.51 | 0.783 ± 0.016 | 2.04 | 0.788 ± 0.015 | 1.90 |
| Arteordosin B | 0.310 | 0.297 ± 0.005 | 95.81 ± 1.60 | 1.67 | 0.299 ± 0.006 | 96.45 ± 1.92 | 1.99 | 0.306 ± 0.007 | 2.29 | 0.307 ± 0.006 | 1.95 |
| 5,4′-Dihydroxy-7,3′-dimethoxyflavanone | 1.225 | 1.217 ± 0.016 | 99.38 ± 1.31 | 1.32 | 1.214 ± 0.018 | 99.10 ± 1.48 | 1.49 | 1.199 ± 0.020 | 1.67 | 1.195 ± 0.019 | 1.59 |
| 5,4′-Dihydroxy-7-methoxyflavanone | 0.710 | 0.699 ± 0.010 | 98.45 ± 1.40 | 1.42 | 0.695 ± 0.012 | 97.89 ± 1.73 | 1.77 | 0.700 ± 0.013 | 1.86 | 0.694 ± 0.012 | 1.73 |
| Dihydroconiferyl alcohol | 0.314 | 0.311 ± 0.005 | 99.04 ± 1.62 | 1.64 | 0.309 ± 0.005 | 98.41 ± 1.63 | 1.67 | 0.312 ± 0.006 | 1.92 | 0.308 ± 0.006 | 1.95 |
| O-Hydroxycapillene | 0.698 | 0.691 ± 0.010 | 99.00 ± 1.39 | 1.40 | 0.693 ± 0.012 | 99.28 ± 1.73 | 1.74 | 0.699 ± 0.013 | 1.86 | 0.701 ± 0.012 | 1.71 |
| 5-Hydroxy-7,4′-dimethoxyflavanone | 1.230 | 1.219 ± 0.017 | 99.11 ± 1.40 | 1.41 | 1.223 ± 0.019 | 99.43 ± 1.55 | 1.56 | 1.222 ± 0.020 | 1.64 | 1.218 ± 0.021 | 1.72 |
| Dehydrofalcarindiol | 0.920 | 0.909 ± 0.015 | 98.80 ± 1.67 | 1.69 | 0.906 ± 0.016 | 98.48 ± 1.77 | 1.80 | 0.908 ± 0.018 | 1.98 | 0.910 ± 0.016 | 1.76 |
| Arteordoyn A | 0.569 | 0.561 ± 0.008 | 98.59 ± 1.43 | 1.45 | 0.558 ± 0.009 | 98.07 ± 1.81 | 1.85 | 0.565 ± 0.011 | 1.95 | 0.563 ± 0.010 | 1.78 |
| Dehydrofalcarinol | 1.158 | 1.149 ± 0.019 | 99.22 ± 1.65 | 1.66 | 1.146 ± 0.022 | 98.96 ± 1.92 | 1.94 | 1.147 ± 0.023 | 2.01 | 1.150 ± 0.018 | 1.57 |
| Capillarin | 2.362 | 2.319 ± 0.035 | 98.18 ± 1.51 | 1.54 | 2.315 ± 0.040 | 98.01 ± 1.73 | 1.77 | 2.348 ± 0.043 | 1.83 | 2.355 ± 0.039 | 1.66 |
Note: RSD = relative standard deviation; a Data are presented as mean ± standard deviation.
Table 3.
Recovery data of developed method (n = 6).
| Compounds | Concentrations of sixteen compounds |
Recovery b (%) | RSD (%) | ||
|---|---|---|---|---|---|
| Original (mg) | Added (mg) | Found a (mg) | |||
| p-Hydroxycinnamic acid | 0.812 ± 0.014 | 0.80 | 1.608 ± 0.032 | 99.50 | 1.99 |
| O-Hydroxycinnamic acid | 0.784 ± 0.013 | 0.80 | 1.547 ± 0.028 | 95.38 | 1.81 |
| Acacetin | 0.343 ± 0.005 | 0.40 | 0.734 ± 0.012 | 97.75 | 1.63 |
| 3,5-Dihydroxy-7,4′-dimethoxyflavone | 0.106 ± 0.001 | 0.10 | 0.202 ± 0.004 | 96.00 | 1.98 |
| Coniferyl alcohol | 0.525 ± 0.009 | 0.50 | 1.016 ± 0.017 | 98.20 | 1.67 |
| Arteordosin A | 0.391 ± 0.006 | 0.40 | 0.781 ± 0.015 | 97.50 | 1.92 |
| Arteordosin B | 0.153 ± 0.002 | 0.20 | 0.344 ± 0.006 | 95.50 | 1.74 |
| 5,4′-Dihydroxy-7,3′-dimethoxyflavanone | 0.601 ± 0.011 | 0.60 | 1.197 ± 0.020 | 99.33 | 1.67 |
| 5,4′-Dihydroxy-7-methoxyflavanone | 0.351 ± 0.005 | 0.40 | 0.738 ± 0.014 | 96.75 | 1.90 |
| Dihydroconiferyl alcohol | 0.155 ± 0.002 | 0.20 | 0.348 ± 0.006 | 96.50 | 1.72 |
| O-Hydroxycapillene | 0.352 ± 0.006 | 0.40 | 0.746 ± 0.014 | 98.50 | 1.88 |
| 5-Hydroxy-7,4′-dimethoxyflavanone | 0.611 ± 0.010 | 0.60 | 1.198 ± 0.022 | 97.83 | 1.83 |
| Dehydrofalcarindiol | 0.453 ± 0.008 | 0.50 | 0.949 ± 0.017 | 99.20 | 1.79 |
| Arteordoyn A | 0.283 ± 0.004 | 0.30 | 0.569 ± 0.010 | 95.33 | 1.76 |
| Dehydrofalcarinol | 0.575 ± 0.011 | 0.60 | 1.159 ± 0.021 | 97.33 | 1.81 |
| Capillarin | 1.175 ± 0.010 | 1.00 | 2.138 ± 0.042 | 96.30 | 1.96 |
Note: RSD = relative standard deviation; a Data are presented as mean ± standard deviation; b Recovery = found value - original value/added value × 100%.
3.4. Sample analysis
This developed HPLC method was subsequently applied to the simultaneous determination of sixteen compounds in A. ordosica from five different provinces in China. Their contents were listed in Table 4. It was noteworthy that at 225 and 285 nm, compounds 4, 6 and 14 were not monitored in samples XJ, NX and GS, and compound 3 in sample SX, compound 7 in samples XJ, NX and SX, compound 10 in samples XJ and SX, compound 11 in samples XJ and GS were also not monitored. Then, compounds 1, 2, 5, 8, 9, 12, 13, 15 and 16 were detected in all samples. Compounds 1, 2 and 5 belonged to phenylpropanoids, compounds 8, 9 and 12 were somewhere to flavonoids, while compounds 13, 15 and 16 belonged to alkynes. Flavonoids (Albacha, Grayerb, & Jensen, 2003; Lai et al., 2007) possessed anti-inflammatory, analgesic and antioxidant, and the diverse biological activities including antibacterial, anti allergic, anti-inflammatory, analgesic, anti-tumor and inhibition of superoxide were reported for phenylpropanoids (Wang et al., 2021, Zhao et al., 2005) and alkynes (Nakamura et al., 1998; Wei, Li, Liu, & Liang, 2009), which are suitable as indicators of quality for A. ordosica grown in different ecological environments, are widely used in Mongolian traditional medicine for treatment of diverse diseases, especially for arthritis and rheumatoid. The content of these compounds in all samples varied from (1.099 ± 0.015) to (1.623 ± 0.025) mg/g of 1, (1.102 ± 0.011) to (1.568 ± 0.018) mg/g of 2, (0.998 ± 0.013) to (1.263 ± 0.018) mg/g of 5, (1.201 ± 0.018) to (1.457 ± 0.023) of 8, (0.693 ± 0.013) to (0.881 ± 0.015) mg/g of 9, (1.165 ± 0.018) to (1.306 ± 0.022) mg/g of 12, (0.836 ± 0.015) to (1.007 ± 0.018) mg/g of 13, (1.150 ± 0.018) to (1.222 ± 0.023) mg/g of 15, (1.768 ± 0.033) to (2.350 ± 0.041) mg/g of 16. It can be surmised that variations of the marker compounds may be associated with the quality and potency of A. ordosica from different ecological environments which leads to a conclusion that they should be considered as quality indicators for A. ordosica.
Table 4.
Contents of compounds 1–16 in A. ordosica collected from five different provinces in China.
| Compounds | Contents of compounds 1–16 (mg/g, n = 3) |
||||
|---|---|---|---|---|---|
| Sample IM a | Sample XJ a | Sample NX a | Sample GS a | Sample SX a | |
| p-Hydroxycinnamic acid | 1.623 ± 0.025 | 1.356 ± 0.019 | 1.506 ± 0.020 | 1.099 ± 0.015 | 1.331 ± 0.018 |
| O-Hydroxycinnamic acid | 1.568 ± 0.018 | 1.302 ± 0.013 | 1.102 ± 0.011 | 1.448 ± 0.017 | 1.245 ± 0.013 |
| Acacetin | 0.687 ± 0.012 | 0.748 ± 0.013 | 0.582 ± 0.010 | 0.665 ± 0.013 | ND |
| 3,5-Dihydroxy-7,4′-dimethoxyflavone | 0.214 ± 0.004 | ND | ND | ND | 0.209 ± 0.003 |
| Coniferyl alcohol | 1.054 ± 0.016 | 0.998 ± 0.013 | 1.205 ± 0.016 | 1.263 ± 0.018 | 1.086 ± 0.016 |
| Arteordosin A | 0.784 ± 0.014 | ND | ND | ND | 0.662 ± 0.013 |
| Arteordosin B | 0.307 ± 0.005 | ND | ND | 0.219 ± 0.004 | ND |
| 5,4′-Dihydroxy-7,3′-dimethoxyflavanone | 1.201 ± 0.018 | 1.457 ± 0.023 | 1.405 ± 0.022 | 1.367 ± 0.021 | 1.237 ± 0.019 |
| 5,4′-Dihydroxy-7-methoxyflavanone | 0.703 ± 0.011 | 0.881 ± 0.015 | 0.736 ± 0.012 | 0.781 ± 0.014 | 0.693 ± 0.013 |
| Dihydroconiferyl alcohol | 0.311 ± 0.005 | ND | 0.308 ± 0.006 | 0.268 ± 0.004 | ND |
| O-Hydroxycapillene | 0.702 ± 0.012 | ND | 0.633 ± 0.010 | ND | 0.674 ± 0.011 |
| 5-Hydroxy-7,4′-dimethoxyflavanone | 1.223 ± 0.019 | 1.188 ± 0.017 | 1.207 ± 0.020 | 1.165 ± 0.018 | 1.306 ± 0.022 |
| Dehydrofalcarindiol | 0.907 ± 0.017 | 0.849 ± 0.015 | 0.868 ± 0.016 | 0.836 ± 0.015 | 1.007 ± 0.018 |
| Arteordoyn A | 0.566 ± 0.010 | ND | ND | ND | 0.534 ± 0.009 |
| Dehydrofalcarinol | 1.150 ± 0.018 | 1.218 ± 0.022 | 1.222 ± 0.023 | 1.209 ± 0.020 | 1.184 ± 0.019 |
| Capillarin | 2.350 ± 0.041 | 1.778 ± 0.034 | 1.802 ± 0.035 | 1.768 ± 0.033 | 2.145 ± 0.039 |
Note: ND = not determined; a Data are presented as mean ± standard deviation.
4. Conclusion
A new HPLC method was developed and validated for the quantitative estimation of compounds 1–16 in A. ordosica from different ecological environments. This study indicates that the proposed HPLC approach gives us reliable, precise and accurate results for the simultaneous quantification of the analyzed compounds in different samples with adequate runtime and low solvent consumption. According to the results of the HPLC analysis, it was concluded that p-hydroxycinnamic acid, O-hydroxycinnamic acid, coniferyl alcohol, 5,4′-dihydroxy-7,3′-dimethoxyflavanone, 5,4′-dihydroxy-7-methoxyflavanone, 5-hydroxy-7,4′-dimethoxyflavanone, dehydrofalcarindiol, arteordoyn A, dehydrofalcarinol and capillarin are best suited for the role of quality indicators of A. ordosica grown in different ecological environments. In the future, the described approach will be used for the quantification of quality indicators of A. ordosica collected from different ecological environments.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Key R&D Program of China (2019YFC1712300).
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