Sijunzi decoction (SJZT), a traditional Chinese formula (TCMF) consisting of four herbs, has been widely used for the treatment of various gastrointestinal symptoms. However, its modernization process is hindered by the lack of a powerful quality control method that covers the major active components in the formula. The aim of this study was to establish a UPLC method for the quantitative determination of ten active components in Sijunzi decoction including ginsenoside Rg1, Re, Rb1, liquiritin, liquiritigenin, glycyrrhizic acid, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid. Separation was achieved using an ACQUITY UPLC BEHC18 column (2.1 mm × 100 mm, 1.7
Traditional Chinese herbal formulation (TCMF) has been widely used in the clinic for its well-proven efficacy with few side effects. Sijunzi decoction (SJZT) is one of the most famous TCMFs consisting of four herbs:
Supporting these well-confirmed pharmacological efficacies, recent years have seen an increasing knowledge of the chemical components from SJZT. HPLC-MS was exploited to analyze the major components of SJZT, and eight ginsenosides (ginsenosides Rg1, Re, Rf, Ro, Rb1, Rc, Rb2, and Rd) and glycyrrhizic acid were identified through structural elucidation [
The aim of this research was to develop a convenient, reliable, and sensitive analytical method to determine the quantity of major compounds in SJZT by using ultraperformance liquid chromatography (UPLC). Specifically, ginsenoside Rg1, Re, Rb1, liquiritin, liquiritigenin, glycyrrhizic acid, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid were selected as the marker constituents for the relatively high contents in the individual herbs and their validated pharmacological effects, such as anti-inflammation, brain protection effects, antioxidation effect, and hypoglycemic effect [
The four crude herbs,
The chemical structure of ginsenoside Rg1, Re, Rb1, liquiritin, liquiritigenin, glycyrrhizic acid, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid.
The analytes were separated on an ACQUITY UPLC H-class system with a PDA detector using 2.1 mm × 100 mm × 1.7
According to the original composition of SJZT, the four constituting herbs including
Ethanol was added to an aliquot of 5 mL SJZT overnight in order to remove the polysaccharides. The supernatant was transferred into a test tube and evaporated to dryness with vacuum at room temperature. Finally, the residue was reconstituted in 5 mL methanol by vortex mixing for 5 min and centrifuged at 16,000 rpm for 10 minutes. 5
The standard stock solutions of ginsenoside Rg1 (300.00
The negative control samples of SJZT were prepared by deriving one herb from the prescriptions. The herbs were accurately weighed according to the prescription of SJZT and prepared with the same procedure as for the sample preparation.
Specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision (repeatability and intra- and interassay), and accuracy (recovery) of this UPLC method were evaluated in accordance with International Conference on Harmonization (ICH) [
The standard calibration curve for the linearity assay was prepared with seven different concentrations of diluted standard solutions (ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, liquiritin, glycyrrhizic acid, liquiritigenin, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid). The lower limit of quantification (LLOQ) was determined as the lowest concentration point of the standard curve and the signal-to-noise ratio was higher than 10. The lower limit of detection (LLOD) was defined as the amount that could be detected with a signal-to-noise ratio of 3.
The precision of the analytical method was evaluated by intrabatch and interbatch variability. Three different concentrations of standards (low, medium, and high) were prepared. The quantity of each component was determined by the respective calibration curve. RSD was used to measure precision. The interbatch reproducibility test was carried out on three different batches.
Recovery studies were carried out by spiking three concentrations of mixed standards at low (50% of the known amounts), medium (100% of the known amounts), and high (200% of the known amounts) in the 5 mL of SJZT. Then, the spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above.
Optimization of the separation conditions for HPLC analysis was performed including the mobile phase composition, gradient elution program, and wavelength. To obtain chromatograms with better resolution of adjacent peaks within shorter time, the chromatographic conditions were optimized. Methanol and acetonitrile were compared in the experiment. The result showed that acetonitrile was much better as it could result in a better resolution and shorter time for analysis. In addition, water and 0.1% phosphoric acid/water were investigated and the result showed that 0.1% phosphoric acid/water was better than water. As a result ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7
Moreover, different gradient profiles were also optimized. Actually, we tried to simplify the gradient elution system and shorten the analysis time, but peaks for atractylenolide III and atractylenolide I have not been completely separated except for the current condition to in the gradient program mentioned above.
In this experiment, the specificity of UV absorption was also investigated, using the present chromatographic conditions and comparing a SJZT sample with a standard mixture. The UV absorbance and the best UV detection wavelength of each compound in SJZT were confirmed as follows: ginsenoside Rg1, Re, and Rb1 (203 nm), liquiritin, glycyrrhizic acid, and liquiritigenin (254 nm), atractylenolide I and atractylenolide III (222 nm), atractylenolide II (276 nm), and pachymic acid (242 nm).
The validation study allowed the evaluation of the method for its suitability for routine analysis.
Representative chromatograms of the standard solution, sample solution, and negative control samples at different UV wavelength were shown in Figure
Ultraperformance liquid chromatography (UPLC) chromatograms at different wavelengths of standard mixture (a), SJZT extraction (b), and negative control samples (c). Peaks: (1) liquiritin, (2) liquiritigenin, (3) ginsenoside Rg1, (4) ginsenoside Re, (5) ginsenoside Rb1, (6) glycyrrhizic acid, (7) atractylenolide III, (8) atractylenolide I, (9) atractylenolide II, and (10) pachymic acid.
The linearity of the developed method was assessed using seven different concentrations each of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, liquiritin, glycyrrhizic acid, liquiritigenin, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid and the observed concentrated ranges were as follows: 4.68~300.00, 4.72~302.10, 4.68~300.00, 1.41~90.30, 1.72~110.00, 1.36~87.00, 1.12~71.40, 0.79~50.60, 0.92~59.00, and 3.13~200.00
The linear regression data, LODs, and LOQs of ten compounds.
Components | Regression equations |
|
Linear range |
LODs |
LOQs |
---|---|---|---|---|---|
Ginsenoside Rg1 |
|
1 | 4.68–300.00 | 0.07 | 0.23 |
Ginsenoside Re |
|
0.9998 | 4.72–302.10 | 0.07 | 0.23 |
Ginsenoside Rb1 |
|
0.9998 | 4.68–300.00 | 0.06 | 0.20 |
Liquiritin |
|
0.9998 | 1.41–90.30 | 0.02 | 0.07 |
Liquiritigenin |
|
0.9998 | 1.36–87.00 | 0.02 | 0.07 |
Glycyrrhizic acid |
|
0.9998 | 1.72–110.00 | 0.03 | 0.10 |
Atractylenolide III |
|
0.9999 | 0.92–59.00 | 0.01 | 0.03 |
Atractylenolide I |
|
0.9998 | 1.12–71.40 | 0.02 | 0.07 |
Atractylenolide II |
|
0.9999 | 0.79–50.60 | 0.01 | 0.03 |
Pachymic acid |
|
0.9996 | 3.13–200.00 | 0.05 | 0.16 |
Intraday and interday variations were chosen to determine the precision of the developed assay. The analyzed data showed that relative standard deviation (RSD) of intra- and interday was in the range of 0.21–1.18% and 0.20–1.10% (
Precision, repeatability, and stability of ten compounds in SJZT (
Analytes | Precision | Repeatability RSD (%) | Stability RSD (%) | ||
---|---|---|---|---|---|
Levels (ug/mL) | Intraday RSD (%) | Interday RSD (%) | |||
Ginsenoside Rg1 | 7.03 | 1.05% | 0.29% | 0.98% | 1.01% |
37.50 | 0.30% | 0.20% | |||
240.00 | 0.85% | 1.04% | |||
|
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Ginsenoside Re | 7.08 | 0.60% | 0.22% | 0.87% | 1.04% |
37.76 | 0.36% | 0.42% | |||
241.68 | 0.81% | 0.93% | |||
|
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Ginsenoside Rb1 | 7.03 | 0.28% | 0.22% | 1.20% | 0.92% |
37.50 | 0.17% | 0.16% | |||
240.00 | 1.15% | 0.98% | |||
|
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Liquiritin | 2.12 | 0.33% | 0.70% | 0.48% | 0.57% |
11.29 | 0.54% | 0.35% | |||
72.24 | 1.18% | 1.03% | |||
|
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Liquiritigenin | 2.04 | 0.83% | 0.70% | 0.27% | 0.92% |
10.88 | 0.39% | 0.49% | |||
69.60 | 0.39% | 0.31% | |||
|
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Glycyrrhizic acid | 2.58 | 0.91% | 1.04% | 0.28% | 0.73% |
13.75 | 0.21% | 1.13% | |||
88.00 | 0.71% | 0.72% | |||
|
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Atractylenolide III | 1.38 | 1.08% | 0.46% | 0.63% | 0.84% |
7.38 | 0.96% | 0.50% | |||
47.20 | 0.93% | 0.83% | |||
|
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Atractylenolide I | 1.67 | 1.03% | 0.58% | 0.81% | 0.92% |
8.93 | 1.07% | 0.97% | |||
57.12 | 0.87% | 0.72% | |||
|
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Atractylenolide II | 1.19 | 1.03% | 0.64% | ||
6.33 | 0.63% | 0.62% | |||
40.48 | 0.97% | 0.72% | |||
|
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Pachymic acid | 4.69 | 0.52% | 0.48% | ||
25.00 | 0.97% | 1.10% | |||
160.00 | 0.61% | 0.37% |
RSD: relative standard deviation.
The accuracy of the method was assessed by a recovery assay. The spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above. The measured data showed that the recovery of the investigated components ranged from 95.07% to 102.67%, and their RSD values were all less than 3.0% (Table
Recovery of eight components in Sijunzi (
Components | Contents (ug/mL) | Quantity added |
Theoretical amount |
Recorded amount (ug/mL) | Recovery (%) | RSD (%) |
---|---|---|---|---|---|---|
Ginsenoside Rg1 | 64.83 | 37.5 | 102.33 | 102.42 | 100.25 | 0.65 |
64.83 | 75 | 139.83 | 139.44 | 99.48 | 0.78 | |
64.83 | 150 | 214.83 | 210.05 | 96.81 | 0.54 | |
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Ginsenoside Rb1 | 48.17 | 37.5 | 85.67 | 85.24 | 98.85 | 0.67 |
48.17 | 75 | 123.17 | 119.96 | 95.72 | 0.82 | |
48.17 | 150 | 198.17 | 191.81 | 95.76 | 0.87 | |
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Ginsenoside Re | 61.63 | 18.88 | 80.51 | 80.95 | 102.33 | 0.53 |
61.63 | 37.76 | 99.39 | 99.73 | 100.9 | 0.20 | |
61.63 | 75.53 | 137.16 | 137.13 | 99.96 | 0.26 | |
|
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Liquiritin | 57.39 | 22.58 | 79.97 | 78.98 | 95.62 | 0.76 |
57.39 | 45.15 | 102.54 | 100.78 | 96.1 | 0.87 | |
57.39 | 90.3 | 147.69 | 143.72 | 95.6 | 0.84 | |
|
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Liquiritigenin | 47.48 | 21.75 | 69.23 | 69.27 | 100.18 | 0.25 |
47.48 | 43.5 | 90.98 | 91.67 | 101.59 | 0.79 | |
47.48 | 87 | 134.48 | 136.8 | 102.67 | 0.89 | |
|
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Glycyrrhizic acid | 111.18 | 55.00 | 166.18 | 164.12 | 96.25 | 0.62 |
111.18 | 110.00 | 221.18 | 217.71 | 96.84 | 0.63 | |
111.18 | 220.00 | 331.18 | 330.52 | 99.7 | 0.36 | |
|
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Atractylenolide III | 42.08 | 29.50 | 71.58 | 72.45 | 102.95 | 0.83 |
42.08 | 59.00 | 101.08 | 100.81 | 99.54 | 0.30 | |
42.08 | 118.00 | 160.08 | 160.29 | 100.18 | 0.24 | |
|
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Atractylenolide I | 10.50 | 8.93 | 19.43 | 19.37 | 99.33 | 0.21 |
10.50 | 17.85 | 28.35 | 28.47 | 100.67 | 0.20 | |
10.50 | 37.50 | 48.00 | 46.15 | 95.07 | 0.62 |
Recovery (%) = (recorded amount − original amount)/spiked amount * 100%.
The established analytical method was subsequently applied for the simultaneous determination of the ten markers in 3 batches of SJZT. The results are presented in Table
Contents of eight components in three batches of SJZT.
Sample | Source | Contents (mg/g) | |||||||
---|---|---|---|---|---|---|---|---|---|
Ginsenoside Rg1 | Ginsenoside Re | Ginsenoside Rb1 | Liquiritin | Liquiritigenin | Glycyrrhizic acid | Atractylenolide |
Atractylenolide I | ||
1 | Batch 1 | 0.254 | 0.115 | 0.184 | 0.659 | 0.078 | 0.258 | 0.020 | 0.013 |
2 | Batch 2 | 0.445 | 0.185 | 0.261 | 0.858 | 0.100 | 0.345 | 0.043 | 0.017 |
3 | Batch 3 | 0.209 | 0.094 | 0.116 | 0.600 | 0.067 | 0.572 | 0.039 | 0.011 |
In this study, a UPLC method for the simultaneous determination of ten active ingredients in SJZT has been developed and the results showed that it could be used for the quality control of the SJZT. Thus, this validated that UPLC method could be expected to provide a new basis for the quality control of SJZT.
The authors declare that there is no conflict of interests regarding publication of this paper.
Kang An and Guo Jin-rui contributed equally to this work.
This study was financially supported by the Youth Natural Science Fund of Nanjing University of Chinese Medicine (no. 11XZR09), National Natural Science Fund of China (no. 81202983), Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (no. SKLNMKF201209), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).