Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound
Abstract
:1. Introduction
2. Results
2.1. Optimization of HPLC Separation
2.2. Regression Equation, Linearity, and Limits of Detection (LOD) and Quantification (LOQ)
2.3. Determination of the Seven Marker Compounds of the UGS Extract
2.4. Precision, Accuracy, and Recovery
2.5. Antioxidant Activity of the Marker Compounds of UGS
2.6. Biological Activitis of the UGS Extract in Neuronal Cell Lines
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Chemicals and Reagents
4.3. Apparatus and Chromatographic Conditions
4.4. Preparation of Standard Solutions
4.5. Preparation of the UGS 70% Ethanol Extract and Sample Solutions
4.6. Calibration Curve, LOD, and LOQ
4.7. Precision, Accuracy, and Recovery
4.8. ABTS-Scavenging Activity
4.9. DPPH-Scavenging Activity
4.10. Cell Lines and Culture
4.11. Cell Counting Kit (CCK) Assay
4.12. LDH Release Assay
4.13. Enzyme-Linked Immunosorbent Assays (ELISAs) for Cytokine Production
4.14. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I are commercially available. |
Latin Name | Scientific Name | Amount (g) | Origin |
---|---|---|---|
Uncariae Ramulus et Uncus | Uncaria sinensis | 6 | China |
Atractylodis Rhizoma Alba | Atractylodes japonica | 8 | China |
Poria Sclerotium | Poria cocos | 8 | China |
Bupleuri Radix | Bupleurum falcatum | 4 | China |
Angelicae Gigantis Radix | Angelica gigas | 6 | Bonghwa, Korea |
Cnidii Rhizoma | Cnidium officinale | 6 | China |
Glycyrrhizae Radix et Rhizoma | Glycyrrhiza uralensis | 3 | China |
Total amount | 41 |
Compound. | Linear Range (μg/mL) | Regression Equation (y = ax + b) a) | r2 | LOD b) (μg/mL) | LOQ c) (μg/mL) | |
---|---|---|---|---|---|---|
Slope (a) | Intercept (b) | |||||
Liquiritin apioside | 3.125–50 | 15290 | 4536.9 | 1.0000 | 0.177 | 0.537 |
Liquiritin | 1.5625–25 | 18759 | 2614.9 | 0.9999 | 0.052 | 0.157 |
Ferulic acid | 0.78125–25 | 56995 | 1865.6 | 1.0000 | 0.039 | 0.118 |
Glycyrrhizin | 6.25–200 | 4882.5 | 1533 | 1.0000 | 0.619 | 1.876 |
Decursin | 12.5–400 | 30409 | 77457 | 0.9998 | 0.925 | 2.804 |
Decursinol angelate | 6.25–200 | 35125 | 38848 | 0.9998 | 0.232 | 0.705 |
Atractylenolide I | 0.78125–12.5 | 62615 | 1322.9 | 1.0000 | 0.015 | 0.046 |
Compound | Content (mg/g) |
---|---|
Liquiritin apioside | 1.671 ± 0.004 |
Liquiritin | 2.014 ± 0.004 |
Ferulic acid | 0.605 ± 0.002 |
Glycyrrhizin | 10.267 ± 0.05 |
Decursin | 16.431 ± 0.04 |
Decursinol angelate | 7.606 ± 0.002 |
Atractylenolide I | 0.190 ± 0.001 |
Compound | Fortified Conc. (μg/mL) | Intra-Day (n = 5) | Inter-Day (n = 5) | ||||
---|---|---|---|---|---|---|---|
Observed Conc. (μg/mL) | Precision a) (%) | Accuracy b) (%) | Observed Conc. (μg/mL) | Precision (%) | Accuracy (%) | ||
Liquiritin apioside | 5 | 4.84 | 2.11 | 96.88 | 5.01 | 1.25 | 100.18 |
10 | 10.14 | 1.49 | 101.36 | 10.19 | 0.96 | 101.87 | |
20 | 20.10 | 0.96 | 100.50 | 20.15 | 0.61 | 100.76 | |
Liquiritin | 5 | 4.99 | 2.52 | 99.78 | 5.09 | 0.74 | 101.84 |
10 | 10.50 | 1.65 | 105.04 | 10.36 | 1.14 | 103.63 | |
20 | 20.49 | 1.13 | 102.46 | 20.44 | 0.73 | 102.18 | |
Ferulic acid | 1.5 | 1.46 | 3.36 | 97.63 | 1.47 | 2.73 | 97.81 |
3 | 3.15 | 1.36 | 105.12 | 2.97 | 2.98 | 98.93 | |
6 | 6.28 | 0.84 | 104.63 | 5.85 | 2.90 | 97.42 | |
Glycyrrhizin | 12.5 | 12.26 | 0.97 | 98.08 | 12.25 | 0.96 | 97.98 |
25 | 24.98 | 2.00 | 99.92 | 24.47 | 0.86 | 97.90 | |
50 | 49.44 | 0.38 | 98.88 | 49.51 | 0.42 | 99.02 | |
Decursin | 20 | 19.74 | 1.42 | 98.71 | 19.68 | 1.48 | 98.42 |
40 | 41.51 | 0.97 | 103.78 | 40.47 | 1.22 | 101.17 | |
80 | 81.37 | 0.72 | 101.71 | 81.34 | 0.53 | 101.67 | |
Decursinol angelate | 10 | 9.58 | 0.93 | 95.76 | 9.51 | 2.61 | 95.12 |
20 | 19.55 | 0.73 | 97.73 | 19.50 | 0.45 | 97.51 | |
40 | 39.13 | 0.43 | 97.83 | 39.11 | 0.46 | 97.78 | |
Atractylenolide I | 1 | 0.98 | 2.53 | 98.40 | 1.00 | 1.14 | 100.07 |
2 | 2.05 | 1.48 | 102.60 | 2.02 | 0.95 | 101.23 | |
4 | 4.02 | 0.98 | 100.50 | 4.01 | 0.69 | 100.37 |
Compound | Retention Time (min) | Peak Area (AU) | ||
---|---|---|---|---|
Mean ± SD | RSD (%) | Mean ± SD | RSD (%) | |
Liquiritin apioside | 12.48 ± 0.05 | 0.43 | 234009.83 ± 1419.74 | 0.61 |
Liquiritin | 13.00 ± 0.06 | 0.49 | 143858.00 ± 903.60 | 0.63 |
Ferulic acid | 14.09 ± 0.08 | 0.55 | 212067.33 ± 1604.31 | 0.76 |
Glycyrrhizin | 33.46 ± 0.18 | 0.52 | 144019.50 ± 726.13 | 0.50 |
Decursin | 42.93 ± 0.07 | 0.16 | 1905525.83 ± 12044.19 | 0.63 |
Decursinol angelate | 43.45 ± 0.07 | 0.16 | 1094712.83 ± 4431.44 | 0.40 |
Atractylenolide I | 50.27 ± 0.12 | 0.24 | 234499.67 ± 1056.72 | 0.45 |
Compound | Original Conc. (μg/mL) | Spiked Conc. (μg/mL) | Found Conc. (μg/mL) | Recovery a) ± SD (%) | RSD (%) |
---|---|---|---|---|---|
Liquiritin apioside | 17.75 | 4 | 21.59 | 95.99 ± 0.75 | 0.78 |
10 | 27.86 | 101.14 ± 1.34 | 1.33 | ||
20 | 37.56 | 99.07 ± 1.28 | 1.29 | ||
Liquiritin | 21.27 | 4 | 25.46 | 104.94 ± 0.84 | 0.80 |
10 | 31.61 | 103.49 ± 1.72 | 1.66 | ||
20 | 41.42 | 100.80 ± 1.41 | 1.40 | ||
Ferulic acid | 6.63 | 1.5 | 8.16 | 101.49 ± 1.55 | 1.53 |
3 | 9.73 | 103.14 ± 0.76 | 0.74 | ||
6 | 12.79 | 102.70 ± 0.61 | 0.60 | ||
Glycyrrhizin | 9.21 | 2.5 | 11.63 | 97.04 ± 1.32 | 1.36 |
5 | 14.08 | 97.45 ± 1.21 | 1.24 | ||
10 | 18.90 | 96.94 ± 1.40 | 1.44 | ||
Decursin | 14.81 | 4 | 18.84 | 100.68 ± 3.23 | 3.21 |
8 | 22.56 | 96.77 ± 1.05 | 1.08 | ||
16 | 30.48 | 97.91 ± 0.75 | 0.77 | ||
Decursinol angelate | 6.90 | 2 | 8.85 | 97.26 ± 1.16 | 1.20 |
4 | 10.76 | 96.53 ± 0.47 | 0.48 | ||
8 | 15.22 | 104.00 ± 0.35 | 0.33 | ||
Atractylenolide I | 2.08 | 1 | 3.11 | 102.93 ± 1.30 | 1.26 |
2 | 4.14 | 102.68 ± 1.49 | 1.46 | ||
4 | 5.97 | 97.24 ± 0.42 | 0.43 |
μM | 1 | 2 | 3 | 4 | 5 | 6 | 7 | nM | L-ascorbicAcid * |
---|---|---|---|---|---|---|---|---|---|
ABTS | |||||||||
0 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0.0 ± 0.9 | 0 | 0.0 ± 0.9 |
1.5625 | 2.3 ± 0.6 | 1.9 ± 0.3 | 20.8 ± 0.7 | 0.7 ± 0.9 | 0.1 ± 0.3 | –0.2 ± 0.3 | –1.1 ± 0.6 | 0.78125 | –0.9 ± 0.9 |
3.125 | 3.2 ± 0.6 | 3.5 ± 0.5 | 36.7 ± 0.4 | 0.0 ± 0.8 | 0.3 ± 0.2 | –1.1 ± 0.2 | 2.2 ± 1.3 | 1.5625 | 0.8 ± 0.6 |
6.25 | 7.0 ± 0.6 | 5.5 ± 0.4 | 38.0 ± 0.8 | –0.1 ± 0.7 | 0.7 ± 0.6 | 0.0 ± 0.2 | –0.4 ± 1.1 | 3.125 | -0.2 ± 0.5 |
12.5 | 10.1 ± 0.3 | 10.5 ± 0.1 | 64.4 ± 0.2 | –0.8 ± 0.9 | –1.5 ± 0.7 | –0.3 ± 0.2 | 0.4 ± 0.6 | 6.25 | 2.7 ± 0.8 |
25 | 18.8 ± 0.8 | 19.7 ± 0.8 | 99.7 ± 0.1 | 1.3 ± 0.1 | 0.1 ± 0.4 | –0.4 ± 0.6 | 1.0 ± 1.1 | 12.5 | 7.4 ± 0.5 |
50 | 28.3 ± 0.4 | 29.0 ± 0.6 | 99.8 ± 0.0 | 2.7 ± 1.0 | 0.5 ± 0.9 | 1.5 ± 0.2 | 2.0 ± 0.5 | 25 | 22.4 ± 0.6 |
100 | 43.6 ± 0.0 | 43.1 ± 0.7 | 100.0 ± 0.1 | 3.6 ± 0.2 | 0.0 ± 0.1 | 2.6 ± 0.3 | –0.7 ± 0.3 | 50 | 65.9 ± 1.0 |
DPPH | |||||||||
0 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0.0 ± 2.5 | 0 | 0.0 ± 2.5 |
1.5625 | 0.9 ± 0.2 | 0.9 ± 0.2 | 4.8 ± 0.5 | –0.1 ± 0.7 | 0.9 ± 0.7 | –0.2 ± 1.0 | 2.7 ± 0.4 | 0.78125 | 5.7 ± 1.1 |
3.125 | –0.2 ± 0.3 | –0.2 ± 0.3 | 8.6 ± 0.5 | –0.7 ± 0.7 | 0.1 ± 1.4 | 3.7 ± 1.0 | –0.5 ± 0.5 | 1.5625 | 9.1 ± 3.0 |
6.25 | 2.5 ± 1.0 | 2.5 ± 1.0 | 16.9 ± 0.1 | –1.7 ± 1.2 | 0.3 ± 1.3 | 2.3 ± 0.5 | 0.4 ± 0.8 | 3.125 | 12.4 ± 1.0 |
12.5 | 2.8 ± 0.5 | 2.8 ± 0.5 | 34.6 ± 2.0 | –0.9 ± 0.6 | 0.6 ± 0.8 | 3.6 ± 1.4 | 1.0 ± 0.7 | 6.25 | 21.3 ± 1.0 |
25 | 1.7 ± 0.7 | 1.7 ± 0.7 | 55.1 ± 1.7 | 0.5 ± 1.3 | 0.5 ± 0.3 | 5.5 ± 1.4 | 1.3 ± 1.0 | 12.5 | 41.5 ± 3.6 |
50 | 4.1 ± 1.2 | 4.1 ± 1.2 | 75.5 ± 0.9 | 1.9 ± 0.9 | 0.4 ± 0.2 | 4.1 ± 1.2 | 3.1 ± 0.5 | 25 | 61.8 ± 1.4 |
100 | 2.2 ± 1.3 | 2.2 ± 1.3 | 87.8 ± 0.5 | 4.1 ± 0.4 | 0.2 ± 1.6 | 0.9 ± 1.6 | –1.8 ± 0.6 | 50 | 82.0 ± 1.2 |
Latin Name | Scientific Name | Amount (g) | Origin |
---|---|---|---|
Uncariae Ramulus et Uncus | Uncaria sinensis | 6 | China |
Atractylodis Rhizoma Alba | Atractylodes japonica | 8 | China |
Poria Sclerotium | Poria cocos | 8 | China |
Bupleuri Radix | Bupleurum falcatum | 4 | China |
Angelicae Gigantis Radix | Angelica gigas | 6 | Bonghwa, Korea |
Cnidii Rhizoma | Cnidium officinale | 6 | China |
Glycyrrhizae Radix et Rhizoma | Glycyrrhiza uralensis | 3 | China |
Total amount | 41 |
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Kim, Y.J.; Jeong, S.-J.; Seo, C.-S.; Lim, H.-S.; Sohn, E.; Yun, J.; Kim, B.-Y. Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound. Molecules 2018, 23, 1659. https://doi.org/10.3390/molecules23071659
Kim YJ, Jeong S-J, Seo C-S, Lim H-S, Sohn E, Yun J, Kim B-Y. Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound. Molecules. 2018; 23(7):1659. https://doi.org/10.3390/molecules23071659
Chicago/Turabian StyleKim, Yu Jin, Soo-Jin Jeong, Chang-Seob Seo, Hye-Sun Lim, Eunjin Sohn, Jiyeon Yun, and Bu-Yeo Kim. 2018. "Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound" Molecules 23, no. 7: 1659. https://doi.org/10.3390/molecules23071659