Abstract
Ligularia fischeri is a perennial plant in the Asteraceae family, native to Japan, China, Eastern Siberia, and Korea. It is generally known to be beneficial for anti-aging, bronchial diseases, anti-cancer, and constipation. In this study, Ligularia fischeri grown in five regions of Korea (Hahyang, Hongseong, Jeongseon, Nonsan, and Yangsan) were collected, and the absence of interfering substances was confirmed through HPLC analysis and chromatograms compared with standard substances. Potential compounds were identified through MS/MS analysis, and their identities were confirmed using three DCQA (Di-caffeoylquinic acid) standard samples. The linearity, precision, limit of quantification (LOQ) and limit of detection (LOD), and recovery were then measured by quantitative analysis to confirm the content of the three DCQAs. The results of the analysis of three types of DCQA content in Ligularia fischeri obtained from five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) using three different solvent concentrations (100% DW, 30% EtOH, and 50% EtOH) are as follows (5 g of raw material/50 mL of extraction solvent). In 100% distilled water, 3,4-DCQA was highest in Nonsan (9.29 mg/g), 3,5-DCQA was highest in Hoengseong (5.32 mg/g), and 4,5-DCQA was highest in Nonsan (3.38 mg/g). In 30% ethanol, 3,4-DCQA was highest in Nonsan (19.15 mg/g), 3,5-DCQA was highest in Hoengseong (9.98 mg/g), and 4,5-DCQA was highest in Nonsan (11.79 mg/g). In 50% ethanol, 3,4-DCQA was highest in Nonsan (21.52 mg/g), 3,5-DCQA was highest in Hoengseong (17.06 mg/g), and 4,5-DCQA was highest in Nonsan (11.25 mg/g).
Keywords: Ligularia fischeri; 3,4-DCQA; 3,5-DCQA; 4,5-DCQA
Subject terms: Biochemistry, Developmental biology, Drug discovery, Biomarkers
Introduction
Ligularia fischeri is a plant of the Asteraceae family native to the high mountain regions of Northeast Asia, particularly Korea, China, and Japan. Its roots and leaves have been widely used traditionally for medicinal and edible purposes1. Recent studies have revealed that Ligularia fischeri contains various bioactive compounds and is attracting attention for its physiological effects, such as antioxidant, anti-inflammatory, and anti-cancer properties2.
The main bioactive compounds of Ligularia fischeri include polyphenolic compounds, flavonoids, terpenoids, and sesquiterpenoids, which have been also reported to contain various bioactive effects such as antioxidant, anti-inflammatory, and antibacterial activities3–5. In particular, polyphenols, flavonoids, and chlorogenic acid are known to have positive effects on the prevention of chronic diseases6. Additionally, some studies suggest that Ligularia fischeri extract may exhibit physiological functions such as inhibiting cancer cell proliferation, lowering blood sugar levels, protecting the liver, and treating rheumatoid arthritis7–10. However, scientific research on Ligularia fischeri is still in its early stages, and standardized methods are required to ensure reliable and reproducible experimental results, particularly in analytical procedures.
Generally, DCQAs (dicaffeoylquinic acids) are characterized by the esterification of quinic acid and caffeic acid and exist as isomers such as 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, depending on the position of caffeic acid11. A search on PubMed using the keywords “Ligularia fischeri” and “DCQA” found a total of two papers. Among these, only one paper actually addressed the separation and identification of the compounds. Specifically, Shang et al. (2010) analyzed Ligularia fischeri harvested from Daegwallyeong, Gangwon-do, South Korea, and identified three types of DCQAs (dicaffeoylquinic acids), were 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, with 4,5-DCQA having the highest content12. Additionally, according to a study conducted in Korea, leaf extracts of Ligularia fischeri collected in summer (June) had higher polyphenol and flavonoid content and stronger antioxidant and antibacterial activity compared to samples collected in winter (December)13. Thus, to date, there has been limited research on regional differences in the content of DCQA in Korea.
Therefore, in this study, five regions (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) where Ligularia fischeri is cultivated in Korea and analyzed the compounds using HPLC-MS/MS and chromatograms, confirming the absence of interfering compounds. As a result, seven main peaks were identified, including three DCQAs (dicaffeoylquinic acid): 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. After that, the content differences of three types of DCQAs (3,4-DCQA, 3,5-DCQA, and 4,5-DCQA) were confirmed using standard compounds, and linearity, precision, limit of quantification (LOD), limit of detection (LOQ), and recovery rate were measured for quantitative analysis. This study aims to scientifically confirm the potential value of Ligularia fischeri as a functional food ingredient, as well as regional and solvent-specific differences in content and validation methods, thereby providing foundational data for future industrial applications.
Materials and methods
Chemicals and reagents
The chemical standards of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-MS/MS grade solvents (acetonitrile and triple deionized water) and methanol were obtained from Duksan Pure Chemical Co. Ltd. (Dongdaemun-gu, Seoul, Korea).
Preparation of plant materials
Ligularia fischeri was collected from Hamyang (Code No. 00951 A) and Yangsan (Code No. 00952 A), Gyeongsangnam-do; Hoengseong (Code No. 00953 A) and Jeongseon (Code No. 00954 A), Gangwon-do; and Nonsan (Code No. 00955 A), Chungcheongnam-do harvested May to July was authenticated by Dr. Gwang Il Kim, who belongs to the R&D Technology Commercialization Division, Kick the Hurdle, Changwon, South Korea. After obtaining permission from each local farm to collect and provided by Kick the Hurdle Inc. The samples were stored in the Animal Bio Resources Bank, Korea, a nationally designated research materials bank, and each source was assigned a code number (Fig. 1). The leaves and stems of the plants provided were washed with water, cut into pieces, and freeze-dried. They were then placed in sealed polyethylene bags containing silica gel and stored at −20 °C until use.
Fig. 1.
Leaf shapes of Ligularia fischeri in each cultivation area. (A) Yangsan, (B) Nonsan, (C) Jeongseon, (D) Hoengseong and (E) Hamyang.
Preparation of sample and standard solutions
The preparation of plants and standards solution for LC-MS was performed using a modified technique14. The Ligularia fischeri leaves and stems powder (5 g) was extracted for 72 h at 60 °C in a water bath using three solvents (v/v): 100% DW, 30% EtOH, and 50% EtOH, each in 50 mL. The extract was centrifuged at 3000 rpm for 10 min. After centrifugation, the supernatant was filtered through filter paper (Whatman, Qualitative, Circles, 110 mm Dia, Cat No. 1001 − 110). A rotary evaporator (N-1110, Eyela, Tokyo, Japan) was rotated at 100 rpm and used under reduced pressure at 45 °C to completely remove any residual ethanol. After extraction, Ligularia fischeri was freeze-dried repeatedly and obtained a powder. Prepared Ligularia fischeri sample powder and standard of 3,4-DCQA, 3,5-DCQA, 4,5-DCQA were prepared at a concentration of 1 mg/mL in methanol. All samples were filtered through a 0.45 μm PVDF syringe filter prior to analysis and prepared in the same method as above prior to injection for HPLC-MS/MS analysis.
HPLC-MS/MS instrumentation and analysis
HPLC and LC-MS/MS was performed on a Shimadzu Nexera Lite LC-40D HPLC system (Shimadzu Corp., Kyoto, Japan) and X500R Ultra Quadrupole Time of flight LC/MS/MS System (Sciex Corp., MA, USA) operated in positive ion mode (spray voltage set at − 4.5 kV). The solvent used was DW and Acetonitrile containing 0.1% formic acid, a gradient system was used at a flow rate of 0.5 mL/min for analysis, and a Prontosil C18 column (length, 250 mm; inner diameter, 4.6 mm; particle size, 5 μm; Phenomenex Co., Ltd., California, USA, Biochoff Chromatography) was used. Mobile phase B gradient elution was programmed in 10-minute intervals as follows: 10–15% B; 15–20% B; 20–25% B; 25–40% B; 40–70% B; 70–95% B. The final 10 min were conducted isocratically at 95% B. The analysis was performed with combined UV and photodiode array (DAD) detectors at a wavelength of 284 nm, and temperature of 35 °C.
The mass spectrometry conditions for qualitative analysis of peaks identified by HPLC were performed in positive mode using electrospray ionization (ESI) and multiscan between m/z 100–2000, with a desolvation temperature set at 500 °C, spray voltage at 5500 V, ion source gas at 50 psi, curtain gas at 30 psi, and declustering potential (DP) at 80 V; MS/MS spectra were set with a collision energy of 35 ± 15 V. The obtained ion chromatogram data were generated using SCIEX OS software (3.0.0).
Quantification and validation of the analytical method
The analytical method validation was conducted in accordance with the ICH and U.S. Food and Drug Administration bioanalytical method validation guidelines15,16. The quantification of compounds detected in Ligularia fischeri extract was performed at 284 nm, and the concentrations of the three DCQAs were calculated using the following formula: (Calibration curve results (ug/mL) × Final volume (mL) × dilution factor × standard solution purity)/(sample weight (g) × 1000 (ug/mg)). Specificity, linearity, detection limit (LOD), quantification limit (LOQ), precision, and recovery rate were measured to evaluate the performance of this method.
Specificity
The specificity of the chromatogram and PDA spectrum patterns of the Ligularia fischeri extract was confirmed as follows: This was achieved by comparing them with standard solutions, based on the selective quantification of various compounds in complex mixtures.
Linearity and range
Standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA at concentrations of 100, 250, 500, 750, and 1000 µg/mL (n = 5) were prepared and analyzed to confirm the linearity of the calibration curve. The standard solution analysis was repeated five times for each concentration. The eluted peaks were subjected to linear regression (n = 5) and measured as the ratio of peak area to analyte concentration. The linearity of the association was assessed using the correlation coefficients (R2) that were computed from the calibration curves.
LOD and LOQ
The minimum quantity of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA that may be found in the Ligularia fischeri extract sample is known as the LOD. The LOQ is the minimum quantity that can be accurately and quantitatively suitable for precision. The standard deviations of the y-intercepts and slopes of the calibration curves were used to determine the LOD and LOQ values. The calibration curve through standard deviation (σ) and slope (S) was used to verify linearity (LOD: 3.3σ/S and LOQ: 10σ/S).
Precision and recovery
The precision of the validation method was determined using standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA by intraday. Intraday was five times a day to analyze precisionThe content of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA contained in Ligularia fischeri was validated by conducting an interday test five times a day for three consecutive days. To determine precision, the relative standard deviation (RSD) was calculated. The accuracy of the suggested approach was verified by a recovery analysis. Recovery was assessed after measuring the Ligularia fischeri extract with five concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. Each analysis was repeated five times. Recovery was calculated based on the sample peak area value, standard peak area value, and peak area value.
Results
Separation and characteristic analysis of phenolic compounds in Ligularia fischeri extract
The qualitative and quantitative analysis of compounds contained in Ligularia fischeri by region (Hamyang, Hoengseong, Jeongseon, Nonsan, and Yangsan) and extraction solvent (100% DW, 30% EtOH, and 50% EtOH) was performed using HPLC-MS/MS. A total of seven peaks were identified by HPLC retention time and UV-vis spectra (Fig. 2). The seven compounds obtained at a wavelength of 284 nm were identified as Chlorogenic acid17,18, Caffeic acid19,20, Quercetin hexoside (Hyperoside or (Isoquercitrin)21,22, 3,4-Dicaffeoylquinic acid23,24, 3,5- Dicaffeoylquinic acid, and 4,5- Dicaffeoylquinic acid24,25. Table 1 provides the analysis results of mass spectrometry data based on existing literature. This is followed by the predicted fragmentation of compounds obtained through LC-MS/MS (Fig. 3).
Fig. 2.
Chromatograms of Ligularia fischeri extract based on origin and extraction solvent. Orange represents 100% DW, blue represents 30% EtOH, and green represents 50% EtOH peaks. Each region is labeled as (A) Hamyang, (B) Hoengseong, (C) Jeongseon, (D) Nonsan, and (E) Yangsan. The compounds detected at 284 nm are Chlorogenic acid (1), Caffeic acid (2), Quercetin hexoside (Hyperoside or (Isoquercitrin) (3), 3,4-Dicaffeoyl quinicacid (4), 3,5-Dicaffeoylquinic acid (5), 4,5-Dicaffeoylquinic acid (6).
Table 1.
The HPLC-MS/MS data of phenolic compounds from Ligularia fischeri extract with different origins.
| Peak No. |
Rt(min) | Formula | Compound | UV max | [M + H]+ | MS/MS | Reference |
|---|---|---|---|---|---|---|---|
| 1 | 16.95 | C16H18O9 | Chlorogenic acid | 325, 250 | 355 |
181 (C9H8O4) [M + H-C7H10O5]+ 163 (C9H6O3) [M + H-C7H10O5-H2O]+ 135 (C8H6O2) [M + H-C7H12O6-CO]+ |
17,18 |
| 2 | 21.16 | C9H8O4 | Caffeic acid | 330 | 181 |
163 (C9H6O3) [M + H-H2O]+ 145 (C9H5O2−) [M + H-H2O- H2O] 135 (C8H6O2) [M + H-COOH]+ |
19,20 |
| 3 | 31.62 | C21H20O12 |
Quercetin hexoside (Hyperoside or (Isoquercitrin) |
355, 255 | 465 |
303 (C15H10O7) [M + H-C6H10O5]+ 153 (C7H3O4−) [M + H-C6H10O5-RDA] 109 (C7H8O) [M + H- C6H10O5-RDA-CO2] 271 (C15H10O5) [M + H-C6H10O5-H2O2]+ 255 (C15H10O4) [M + H-C6H10O5-H2O2-O]+ |
21,22 |
| 4 | 33.18 | C25H24O12 | 3,4-Dicaffeoylquinic acid | 325, 290 | 517 |
499 (C25H22O11) [M + H-H2O]+ 337 (C16H16O8) [M + H-H2O-C9H6O3]+ 193 (C7H12O6) [M + H-C18H12O6]+ 175 (C7H10O5) [M + H-C9H8O4-C9H6O3]+ |
23,24 |
| 5 | 35.01 | C25H24O12 | 3,5- Dicaffeoylquinic acid | 330, 290 | 517 |
355 (C16H18O9) [M + H-C9H6O3]+ 193 (C7H12O6) [M + H-C9H6O3-C9H6O3]+ 181 (C9H8O4) [M + H-C9H6O3-C7H10O5]+ 137 (C8H8O2) [M + H-C16H16O8-O]+ |
24,25 |
| 6 | 38.31 | C25H24O12 | 4,5- Dicaffeoylquinic acid | 330, 290 | 517 |
355 (C16H18O9) [M + H-C9H6O3] 193 (C7H12O6) [M + H- C9H6O3-C9H6O3] 175 (C7H10O5) [M + H-C18H18O6-H2O] |
24,25 |
Fig. 3.
Fragmentation scheme of identified compounds in Ligularia fischeri extract.
Optimization of HPLC–MS/MS condition
Standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA (100 µg/mL) were used to optimize the HPLC-MS/MS conditions. A Pronto SIL column (250 × 4.6 mm, 5 μm, 120-5-C18 SH, Bischoff Chromatography, Leonberg, Germany) was used to identify detectable phenolic compounds, and the system was set up using a gradient method with a column temperature of 35 °C and a mobile phase consisting of water and acetonitrile, each containing 0.01% formic acid. Based on a literature review, the maximum absorbance of chlorogenic acid was found to be around 290 nm, so the UV wavelength range of 200–400 nm was set to detect its derivatives, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA26.
For the identification of compounds in the sample, positive ESI conditions were applied because ion fragments showed higher safety than negative mode. Subsequently, when the sample was prepared using methanol, it exhibited sufficiently excellent peak shapes.
Method of validation
Specificity
Through the chromatograms of the standard solution and sample solution, we confirmed that the peaks in the Ligularia fischeri extract were clearly separated and that there was no interference from other compounds. Specificity was confirmed by verifying that the retention time (RT) patterns between the standard solution and sample solution matched and that when the standard solutions were mixed, each standard solution was detected at different times (Fig. 4).
Fig. 4.
Chromatograms of Ligularia fischeri extracts and three DCQAs as potential functional compounds. (A) The black peak is from Ligularia fischeri extract, and the blue peak is from a mixture of three standard DCQAs. (B) The mixture of three standard DCQAs is shown as a pink peak, and 3,4-Dicaffeoyl quinic acid is shown as a black peak. (C) The pink peak represents the mixture of the three standard DCQAs, and the black peak represents 3,5-dicaffeoyl quinic acid. (D) The pink peak represents the mixture of the three standard DCQAs, and the black peak represents 4,5-dicaffeoyl quinic acid.
Linearity, range and LOD, and LOQ
Linearity verification was performed five times at each concentration, and the linearity of 3,4-DCQA (R2 ≥ 0.9998), 3,5-DCQA (R² ≥ 0.9998), and 4,5-DCQA (R² ≥ 0.9997) were confirmed. Subsequently, we obtained the linear expressions for 3,4-DCQA (y = 4390.5x − 34206), 3,5-DCQA (y = 5093.5x − 106812), and 4,5-DCQA (y = 5549.9x − 143191). The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were determined, with the LOD and LOQ for 3,4-DCQA being 0.795 mg/L and 2.386 mg/L, respectively. The LOD and LOQ for 3,5-DCQA were 0.637 mg/L and 1.910 mg/L, respectively. The LOD and LOQ for 4,5-DCQA were 0.589 mg/L and 1.766 mg/L, respectively (Table 2).
Table 2.
Calibration curve data for the quantification of 3 type of dcqas.
| Compound | Slopes of calibration | Correlation coefficient (R2) | LOD (mg/L) |
LOQ (mg/L) |
|---|---|---|---|---|
| 3,4-DCQA | 4390.5 | 0.9998 | 0.795 | 2.386 |
| 3,5-DCQA | 5093.5 | 0.9998 | 0.637 | 1.910 |
| 4,5-DCQA | 5549.9 | 0.9997 | 0.589 | 1.766 |
LOD: Limit of Detection; LOQ: Limit of Quantitation, (n = 5).
Precision
Table 3 shows the intraday precision (repeatability) values of the standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which were measured under the same conditions within a single day. The tests were conducted five times within a concentration range of 100–1000 µg/mL, and the relative standard deviation (RSD) values for each standard solution concentration were within 0.25%. Table 4 shows the interday precision (intermediate precision) values of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract obtained from five regions, using the same equipment under the same conditions but on different days. This confirmed the reliability and reproducibility of the HPLC-PDA analysis method.
Table 3.
Intraday precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA.
| Compound | Nominal concentration (ug/mL) |
Intraday | |
|---|---|---|---|
| Observed concentration | Precision RSD (%) | ||
| 3,4-DCQA | 100 | 98.04 ± 0.24 | 0.2458 |
| 250 | 255.24 ± 0.66 | 0.2579 | |
| 500 | 498.58 ± 0.86 | 0.1729 | |
| 750 | 744.18 ± 0.86 | 0.1151 | |
| 1000 | 1003.97 ± 1.03 | 0.1024 | |
| 3,5-DCQA | 100 | 99.52 ± 0.19 | 0.1936 |
| 250 | 255.29 ± 0.45 | 0.1780 | |
| 500 | 494.97 ± 0.62 | 0.1260 | |
| 750 | 745.89 ± 0.77 | 0.1027 | |
| 1000 | 1003.09 ± 0.91 | 0.0905 | |
| 4,5-DCQA | 100 | 103.17 ± 0.18 | 0.1727 |
| 250 | 254.27 ± 0.33 | 0.1280 | |
| 500 | 489.48 ± 0.59 | 0.1208 | |
| 750 | 748.14 ± 0.70 | 0.0938 | |
| 1000 | 1004.39 ± 0.81 | 0.0808 | |
Data were expressed as the mean SD (n = 5).
Table 4.
Interday precision for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract.
| Compound | Origin | Solvent ratio (%) | Interday | |
|---|---|---|---|---|
| Observed concentration | Precision RSD (%) | |||
| 3,4-DCQA | Hamyang | 100 | 400.93 ± 0.74 | 0.184 |
| 70 | 694.71 ± 1.05 | 0.151 | ||
| 50 | 628.71 ± 0.69 | 0.111 | ||
| Hoengseong | 100 | 799.18 ± 0.49 | 0.061 | |
| 70 | 1675.84 ± 1.26 | 0.075 | ||
| 50 | 1876.16 ± 1.18 | 0.063 | ||
| Jeongseon | 100 | 782.42 ± 0.58 | 0.582 | |
| 70 | 894.37 ± 0.67 | 0.075 | ||
| 50 | 952.01 ± 1.08 | 0.113 | ||
| Nonsan | 100 | 929.38 ± 0.99 | 0.108 | |
| 70 | 1914.76 ± 1.23 | 0.064 | ||
| 50 | 2152.28 ± 2.05 | 0.095 | ||
| Yangsan | 100 | 194.00 ± 0.72 | 0.723 | |
| 70 | 300.77 ± 0.57 | 0.190 | ||
| 50 | 381.56 ± 0.41 | 0.109 | ||
| 3,5-DCQA | Hamyang | 100 | 347.12 ± 0.22 | 0.065 |
| 70 | 637.33 ± 1.12 | 0.175 | ||
| 50 | 1147.29 ± 0.99 | 0.087 | ||
| Hoengseong | 100 | 532.29 ± 0.90 | 0.169 | |
| 70 | 995.43 ± 0.52 | 0.052 | ||
| 50 | 1706.04 ± 1.12 | 0.066 | ||
| Jeongseon | 100 | 320.79 ± 0.41 | 0.129 | |
| 70 | 550.61 ± 0.48 | 0.086 | ||
| 50 | 1070.25 ± 0.50 | 0.047 | ||
| Nonsan | 100 | 495.30 ± 0.55 | 0.112 | |
| 70 | 987.99 ± 0.70 | 0.516 | ||
| 50 | 1470.61 ± 1.06 | 0.072 | ||
| Yangsan | 100 | 171.60 ± 0.49 | 0.291 | |
| 70 | 373.13 ± 0.52 | 0.138 | ||
| 50 | 843.39 ± 0.78 | 0.093 | ||
| 4,5-DCQA | Hamyang | 100 | 180.36 ± 0.63 | 0.352 |
| 70 | 749.18 ± 0.42 | 0.056 | ||
| 50 | 1044.93 ± 0.96 | 0.092 | ||
| Hoengseong | 100 | 304.43 ± 0.48 | 0.158 | |
| 70 | 950.79 ± 0.38 | 0.039 | ||
| 50 | 988.96 ± 0.90 | 0.091 | ||
| Jeongseon | 100 | 213.68 ± 0.40 | 0.188 | |
| 70 | 705.77 ± 0.75 | 0.106 | ||
| 50 | 927.41 ± 0.73 | 0.079 | ||
| Nonsan | 100 | 338.26 ± 0.47 | 0.138 | |
| 70 | 1179.49 ± 0.85 | 0.072 | ||
| 50 | 1125.37 ± 0.69 | 0.062 | ||
| Yangsan | 100 | 86.76 ± 0.44 | 0.508 | |
| 70 | 435.07 ± 0.35 | 0.081 | ||
| 50 | 466.12 ± 0.67 | 0.145 | ||
Solvent; DW (with Ethanol), (n = 5).
Recovery
The recovery study was conducted using HPLC-PDA analysis with a mixture of Ligularia fischeri extract and standard solutions of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. The overall recovery rates for 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA were 96.9675–99.1368%, with RSD values all below 1% (Table 5).
Table 5.
Recovery study of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract.
| Compound | Concentration | Recovery (%) | SD | RSD (%) |
||
|---|---|---|---|---|---|---|
| Extract | Standard | Extract with standard | ||||
| 3,4-DCQA | 1079.54 ± 1.27 | 508.75 ± 4.19 | 1540.11 ± 6.45 | 96.97 | 0.61 | 0.631 |
| 3,5-DCQA | 745.79 ± 3.03 | 514.37 ± 2.49 | 1228.55 ± 9.41 | 99.14 | 0.71 | 0.721 |
| 4,5-DCQA | 576.41 ± 5.88 | 516.39 ± 6.36 | 1126.87 ± 11.71 | 97.44 | 0.89 | 0.913 |
Quantitative analysis of the 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract by solvent ratio and origin
Analysis of the content of three types of DCQA by the concentration of extraction solvent in Ligularia fischeri. from five regions showed that in 100% distilled water, 3,4-DCQA was highest in Nonsan (929.38 ± 0.99 ug/mL), Hoengseong (799.18 ± 0.49 ug/mL), and Jeongseon (782.42 ± 0.58 ug/mL); 3,5-DCQA was higher in the order of Hoengseong (532.29 ± 0.90 ug/mL) Nonsan (495.30 ± 0.55 ug/mL) and Hamyang (347.12 ± 0.22 ug/mL); and 4,5-DCQA was higher in the order of Nonsan (338.26 ± 0.47 ug/mL), Hoengseong (304.43 ± 0.48 ug/mL), and Jeongseon (213.68 ± 0.40 ug/mL). In 30% ethanol, 3,4-DCQA was Nonsan (1914.76 ± 1.23 ug/mL), Hoengseong (1675.84 ± 1.26 ug/mL), and Jeongseon (894.37 ± 0.67 ug/mL); 3,5-DCQA was Hoengseong (995.43 ± 0.52 ug/mL), Nonsan (987.99 ± 0.70 ug/mL), and Hamyang (637.33 ± 1.12 ug/mL); and 4,5-DCQA was Nonsan (1179.49 ± 0.85 ug/mL), Hoengseong (950.7943 ± 0.3793 ug/mL), and Hamyang (749.1779 ± 0.4227 ug/mL). In 50% ethanol, 3,4-DCQA was Nonsan (2152.28 ± 2.05 ug/mL), Hoengseong (1876.16 ± 1.18 ug/mL), and Jeongseon (952.01 ± 1.08 ug/mL); 3,5-DCQA was Hoengseong (1706.04 ± 1.12 ug/mL), Nonsan (1470 ± 61 ± 1.06 ug/mL), and Hamyang(1147.29 ± 0.99 ug/mL); and 4,5-DCQA was Nonsan (1125.37 ± 0.69 ug/mL), Hamyang(1044.93 ± 0.96 ug/mL), and Hoengseong (988.96 ± 0.90 ug/mL) (Table 6).
Table 6.
Changes in content of the 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA according to extraction by solvent ratio and region.
| Compound Name | Solvent ratio (%) | Concentration (ug/mL) | ||||
|---|---|---|---|---|---|---|
| Hamyang | Hoengseong | Jeongseon | Nonsan | Yangsan | ||
| 3,4-DCQA | 100 |
400.93 ± 0.74 E |
799.18 ± 0.49 A |
782.42 ± 0.58 C |
929.38 ± 0.99 B |
194.01 ± 0.72 D |
| 70 |
694.71 ± 1.05E |
1675.84 ± 1.26 A |
894.37 ± 0.67 C |
1914.76 ± 1.23 B |
300.77 ± 0.57 D |
|
| 50 |
628.71 ± 0.69 E |
1876.16 ± 1.18 A |
952.01 ± 1.08 C |
2152.28 ± 2.05 B |
381.56 ± 0.41 D |
|
| 3,5-DCQA | 100 | 347.1160 ± 0.2238 E | 532.2904 ± 0.9027 C | 320.7918 ± 0.4125 A |
495.30 ± 0.55 D |
171.64 ± 0.49 B |
| 70 | 637.3278 ± 1.1169 E | 995.4297 ± 0.5177 C | 550.6090 ± 0.4755 A |
987.99 ± 0.70 D |
373.13 ± 0.52 B |
|
| 50 | 1147.2962 ± 0.9989 E | 1706.0361 ± 1.1197 C | 1070.2532 ± 0.5016 A |
1470.61 ± 1.07 D |
843.39 ± 0.78 B |
|
| 4,5-DCQA | 100 | 180.36 ± 0.6340 E |
304.43 ± 0.4813 A |
213.68 ± 0.40 C |
338.26 ± 0.46B |
86.76 ± 0.44 D |
| 70 | 749.18 ± 0.4227 E |
950.79 ± 0.38 C |
705.77 ± 0.75 A |
1179.49 ± 0.85 B |
435.07 ± 0.35 D |
|
| 50 |
1044.93 ± 0.96 E |
988.96 ± 0.90 C |
927.41 ± 0.73 B |
1125.37 ± 0.69 A |
466.12 ± 0.67 D |
|
Solvent; DW (with Ethanol), (n = 5), All values are mean ± SD (n = 3). A-E Means with different superscripts in the same row were significantly different at p < 0.05 using Duncan’s multiple range test. Also tested based on superscript A in the same row.
Discussion
Ligularia fischeri contains dicaffeoylquinic acids (DCQAs), one of the main bioactive compounds, which are promising substances that can contribute to health promotion through their antioxidant and anti-inflammatory effects27,28. Dicaffeoylquinic acids (DCQAs) contains two caffeic acid molecules and one quinic acid molecule, and its structure varies depending on its position, such as 3,4-, 3,5-, and 4,5-DCQA. Recent studies have shown that 3,5-DCQA inhibits NO and suppresses the expression of inflammatory mediators such as INOS, COX2, and TNF-α. Additionally, 4,5-DCQA has been found to regulate an anti-inflammatory pathway mediated by the TRPV1 receptor and inhibit COX2 expression27,29. Especially, in the case of Ligularia fischeri, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA are evenly contained and exist at a certain concentration30. Therefore, there is a lot of research being conducted on the development of various functional foods and pharmaceuticals using DCQAs.
The study of analyzing and verifying three types of DCQAs contained in Ligularia fischeri extract as marker compounds is already conducted31. In addition, some studies have identified six types of DCQA isoforms by comparing and analyzing their chemical structures24. Some studies on the antioxidant and physiological activities of Ligularia fischeri have been conducted based on its extraction method, but these studies are limited to simple extracts32. These studies provide an abundance of data for the identification, analysis, and effectiveness of phenolic compounds in Ligularia fischeri extract. However, in this study, we compared the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA, which act as marker compounds of Ligularia fischeri extract. Additionally, we compared and analyzed the concentrations of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA in Ligularia fischeri extract using three different solvents and performed methodological analytical validity verification. First, we verified specificity. This specificity is the ability to selectively measure only the target substance to be analyzed even in a mixed state, which is the most fundamental part of the verification procedure. Subsequently, we verified linearity and range. In the case of linearity, we confirmed that the analysis procedure was proportional to the concentration of the substance in the sample, and through the range, we confirmed the interval between the upper and lower levels. We verified the minimum detectable concentration and the lowest concentration that can be quantitatively determined through detection and quantification limits. For precision, we conducted five analyses to measure intraday and interday precision and presented metrics indicating the degree of dispersion and reproducibility. Finally, we conducted verification to confirm the accuracy of the proposed method through recovery rate33,34.
The results of the content analysis in Table 6 show that the concentration of 3,4-DCQA was highest at 2152.2849 ± 2.0497 ug/mL in the 50% ethanol extract from the Nonsan region, while the concentration of 3,5-DCQA was highest at 1706.0361 ± 1.1197 ug/mL in the 50% ethanol extract from the Hoengseong region, while the highest concentration of 4,5-DCQA was found in the 30% ethanol extract from the Nonsan region at 1179.4910 ± 0.8460 ug/mL. There are various factors contributing to regional differences in DCQAs. For example, Ligularia fischeri tends to grow in relatively cool and humid regions. Additionally, soil conditions such as high organic matter content can increase phenolic compound levels, while climatic factors like high altitude, large diurnal temperature fluctuations, and abundant sunlight have been reported to enhance phenolic compound formation due to stress35,36.
This study examined the importance of validation procedures for ensuring data validity and standardization processes for enhancing data comparability, as well as practical application methods for these procedures. It was confirmed that this approach not only improves data quality but also enhances the reproducibility and generalizability of research results. However, peak no.7 remained unidentified; as this study focused on DCQAs, further structural analysis will be conducted to analyze the unknown peak. Based on such analytical studies, it is necessary to select relevant extracts and compounds for evaluating antioxidant, anti-inflammatory, and bone-forming characteristics in biological research.
Conclusions
We screened six phenolic compounds contained in Ligularia fischeri and applied an HPLC-MS/MS system to perform quantitative analysis of 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. A validation system was established to evaluate specificity, linearity, detection limits, quantification limits, precision, and recovery rates, and differences in content levels were confirmed based on extraction solvents and regional variations. This study contributed to the standardization of three DCQA compounds used as marker components of Ligularia fischeri extract and provided useful insights for analyzing these compounds in related plants. Additionally, this study identified phenolic compounds, including DCQA, contained in Ligularia fischeri, which exhibit various biological activities such as antioxidant, anti-inflammatory, blood sugar regulation, liver protection, and bone health improvement. Further studies are needed to evaluate the potential activities of these compounds.
Acknowledgements
This study was supported by the National Research Foundation of Korea, funded by the Ministry of Science and ICT (grant no. RS-2023-00243376, and RS-2024-00411709) and the Kick the Hurdle Co., Ltd.
Author contributions
Conceptualization, H.H.K. and S.H.J.; methodology, H.H.K.; writing—original draft preparation, S.H.J., M.J.A., J.K.S., and H.H.K.; writing—review and editing, P.B.B., Y.G.M., and K.H.H,; investigation, S.H.J.; validation H.H.K. and S.H.J.; project administration, T.Y.K., J.W.P. and G.I.K.; supervision, G.S.K. All authors have read and agreed to the published version of the manuscript.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.