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Abstract

Objective

Although the discrimination of crude drugs by metabolomics analysis using liquid chromatography–mass spectrometry (LC–MS) has been widely reported, there are few reports on natural products analysis using supercritical fluid chromatography (SFC)–MS. This study aimed to compare the results of metabolomic analysis of licorice obtained using SFC–MS and LC–MS.

Methods

We analyzed 14 CHCl₃ extracts of licorice roots (Glycyrrhiza glabra, G. uralensis, and G. inflata) using SFC-time-of-flight (TOF)/MS and LC-TOF/MS in conjunction with principal component analysis (PCA). The three species were assessed for discrimination based on the PCA results. The characteristic constituents of each variety were purified, and their chemical structures were elucidated using various spectroscopic analyses.

Results

The PCA score plots of the results of both SFC and LC classified the licorice samples into three groups. Seven compounds (six known compounds and one new phenolic compound, glyglabone) were isolated from G. glabra, five known compounds were isolated from G. uralensis, and four known compounds were isolated from G. inflata.

Conclusions

The 16 isolated compounds may be characteristic compounds in the three licorice species. The SFC and LC methods showed comparable results in distinguishing the three species. SFC was highly effective for directly analyzing CHCl₃ extracts, reducing analysis time and organic solvent consumption by over 50% compared to the LC analysis. Thus, SFC-TOF/MS in combination with PCA can be used to differentiate between G. glabra, G. uralensis, and G. inflata.

Keywords

licorice SFC-TOF/MS LC-TOF/MS PCA phenolics glyglabone

Introduction

Licorice is one of the most commonly used crude drugs in Japan. In China, the roots and stolons of Glycyrrhiza glabra, G. uralensis, and G. inflata are used as crude drugs.¹ G. glabra and G. uralensis are listed in the Japanese Pharmacopoeia, and G. inflata is used as a food additive (as a sweetener and antioxidant agent) in Japan.^1,2 Glycyrrhizin, an oleanane-type saponin, is used as a sweetener that is approximately 170 times sweeter than sucrose.³ It is also used as a pharmaceutical and cosmetic ingredient for its anti-inflammatory, anti-bacterial, and hepatoprotective activities.^4,5 Additionally, licorice oil extracts containing flavonoids and phenolic compounds are used as antioxidants.² Glabridin, glycicumarin, and licochalcone A are characteristic constituents of G. glabra, G. uralensis, and G. inflata, respectively.^2,6 Several other phenolic compounds with isoprenoid substituents, such as the 3-methyl-2-butenyl (prenyl) group, have been isolated from the licorice species,⁷ which may aid species identification.

In recent years, several studies have utilized nuclear magnetic resonance (NMR), gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–mass spectrometry (LC–MS) for metabolomic analysis of licorice.^8–10 Sample preparation for NMR is relatively simple; however, in crude samples, it may be difficult to identify compounds from a characteristic ¹H-NMR spectrum.¹¹ GC–MS is used for the analysis of compounds with low polarity¹²; however, derivatization is required for non-volatile constituents, which makes comprehensive analysis of plant metabolites difficult. Reversed-phase (RP) LC–MS is often used to examine plant extracts but is unsuitable for analyzing compounds with low polarity.¹³ Furthermore, normal-phase LC is limited by the availability of mobile phase solvents for MS analysis, and its retention time repeatability is often less feasible than that of RP-LC.¹⁴

Supercritical fluid chromatography (SFC) can aid the analysis of compounds with low polarity, as it has a shorter acquisition time than LC.^15–17 Supercritical carbon dioxide (scCO₂), generally used as a mobile phase in SFC systems, is non-toxic, non-reactive, non-flammable, inexpensive, and environment friendly.^13,18 The polarity of scCO₂ is similar to that of hexane. Adding modifiers such as MeOH, MeCN, and acids enables analysis across a wide range of polarities. Therefore, several applications of scCO₂ to plant extracts, one of the multi-constituent substances, have been reported.^16,17,19 However, unlike studies using GC–MS or LC–MS, there are few reports on the use of SFC-TOF/MS for metabolomic assays of natural products. The SFC analysis of an aqueous–ethanol extract of licorice was reported by Li et al.²⁰ Although EtOH can be used to extract flavonoids, highly polar compounds such as sugars are also co-extracted, which leads to concerns regarding the robustness of SFC analysis. As aqueous solvents cannot be used for SFC analysis, water must be evaporated first and then the residue must be redissolved in an organic solvent. In contrast, CHCl₃ can be used to extract phenolic compounds, specifically from licorice and then the extract can be analyzed directly using SFC.

In this study, 14 CHCl₃ extracts of the roots of G. glabra, G. uralensis, and G. inflata were assessed using SFC-TOF/MS and LC-TOF/MS to validate the advantages of SFC. In addition, principal component analysis (PCA) based on MS data was utilized to differentiate between the three licorice species and to investigate the characteristic compounds in each variety. To the best of our knowledge, this is the first study on metabolomic analysis of licorice extract using SFC-TOF/MS.

Results and Discussion

The root extracts of G. glabra (five batches), G. uralensis (five batches), and G. inflata (four batches) were prepared using CHCl₃. Each extract was diluted with CHCl₃ and filtered through a 0.2-µm membrane before injecting it directly into the SFC-TOF/MS. The analysis was conducted in negative ion mode, and the analytical results are illustrated in the base peak ion (BPI) chromatogram (Figure 1). The BPI chromatogram plots the maximum intensity peak against the time axis for all the acquired mass spectra. The SFC-TOF/MS was completed within 7 min, during which several major peaks, including peaks for glabridin, glycycoumarin, and licochalcone A, were detected.

Figure 1.

Base peak ion (BPI) chromatogram obtained for SFC-TOF/MS analysis of extracts from Glycyrrhiza glabra (A), G. uralensis (B), and G. inflata (C).

PCA was used to evaluate the SFC-TOF/MS data. The PCA score plot enables visual discrimination based on the component profiles detected using MS analysis. The ellipse was set to represent the 95% confidence interval of the data. The PCA score plots depicted that the samples were divided into three groups (G. glabra, G. uralensis, and G. inflata), based on the first and second principal components, which accounted for 67.0% of the total variance. The retention time and m/z of the detected peaks were further examined using PCA loading plots. The PCA loading plot facilitated the profiling of markedly components that contributed to the distinction on the score plots, revealing that several compounds, including glabridin, glycycoumarin, and licochalcone A, are key variables for discrimination. In addition, the compounds (1–16) that aid the classification of G. glabra, G. uralensis, and G. inflata were isolated and analyzed using NMR (Figure 2).

Figure 2.

Principal component analysis (PCA) score plot (A) and loading plot (B).

Sixteen compounds were purified using open-column chromatography and preparative HPLC based on PCA loading plots, which served as indicators of the characteristic compounds. Additionally, the retention time and molecular weight of these isolated compounds were compared with those isolated using the SFC-TOF/MS analysis of the extract. The structures of 1–16 were evaluated using NMR analysis, which revealed a new compound 1, and 15 other known compounds (Figure 3). Compound 1 was detected at m/z 325.1075 ([M-H]⁻) using high-resolution (HR)-electrospray ionization (ESI)-TOF/MS analysis, and its molecular formula was revealed to be C₁₉H₁₈O₅. Infrared (IR) spectra revealed the presence of functional groups such as conjugated carbonyl (1630 cm⁻¹) and hydroxy (3350 cm⁻¹). One carbonyl carbon signal (δ_C 204.3 ppm) and one methylene signal (δ_H 4.04 ppm and δ_C 40.1 ppm) in the ¹H- and ¹³C-NMR spectral data indicated that compound 1 had a 1,2-diphenylethanone moiety.^21,22 The 1D NMR spectrum, ¹H–¹H correlation spectroscopy (COSY), and heteronuclear multiple bond correlation (HMBC) experiments exhibited a set of ortho-coupled signals (δ_H 6.33 and 6.86 ppm; δ_C 109.0 and 130.1 ppm), cis-coupled olefinic signals (δ_H 5.57 and 6.72 ppm; δ_C 129.3 and 117.0 ppm), gem-dimethyl signal (δ_H 1.38 ppm; δ_C 27.8 ppm), a hydroxy proton (δ_H 8.17 ppm), and three quaternary carbon bonded hydroxyl or oxygen group signals (δ_C 76.0, 151.7, and 153.5 ppm) on a 5-hydroxy-2,2-dimethyl-2H-chromen substituent. Furthermore, the 1D NMR spectrum displayed a hydroxy signal (δ_H 6.04 ppm), intramolecular hydrogen-bonded hydroxyl signal (δ_H 12.24 ppm), two hydroxyl group-connected aromatic carbon signals (δ_C 164.0 and 166.6 ppm), and three methine signals, a meta-coupled doublet signal (δ_H 6.35 ppm; δ_C 103.9 ppm), an ortho- and meta-coupled double doublet signal (δ_H 6.43 ppm; δ_C 108.6 ppm), and an ortho-coupled doublet signal (δ_H 7.85 ppm; δ_C 133.5 ppm), suggesting a 2,4-dihydroxyphenyl moiety. Altogether, these spectral data were similar to those of erybacin B (C₁₉H₁₈O₅).²² Erybacin B has been reported to be a specific marker for G. glabra.²³ However, the ¹³C-NMR spectrum of four aromatic carbon atoms in association with hydroxyl or oxygen groups was discordant between compound 1 and erybacin B. Compared to those in erybacin B,²² the spectral data of the chromene group in compound 1 revealed an upfield shift (−7.4 and −9.0 ppm, respectively), whereas the dihydroxyphenyl group demonstrated a downfield shift (+7.6 and +10.1 ppm, respectively); the chemical shift of protons in these groups indicated a slight shift. The structure was validated using ¹H–¹H COSY, HMBC, and nuclear overhauser effect spectroscopy (NOESY) correlations (Figure 4), and the chemical shifts are displayed in Tables 1 and 2. Thus, we established the structure of the newly identified compound 1 as 1-(2,4-dihydroxyphenyl)-2-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)ethenone and named it glyglabone (Suppplemental Figures S1–S7). The other known 15 compounds were identified as glabrol (2),²⁴ 3,4-didehydroglabridin (3),²⁵ 4′-O-methylglabridin (4),²⁶ kanzonol Y (5),²⁷ hispaglabridin A (6),²⁶ 3-hydroxyglabrol (7),²⁶ 6-prenyllicoisoflavanone (8),²⁸ licoisoflavanone (9),²⁹ licoricidin (10),³⁰ 3-(γ,γ-dimethylallyl)-kievitone (11),³¹ glisoflavanone (12),³² licoisoflavone B (13),³³ glabrone (14),²⁶ licochalcone E (15),³⁴ and echinatin (16),³⁵ after comparison with spectroscopic data available in literature (Supplemental 1H- and 13C-NMR data for known compounds). These 16 compounds were presumed to be constituent markers of G. glabra, G. uralensis, and G. inflata (Table 3).

Figure 3.

Chemical structures of compounds 1–16.

Figure 4.

¹H–¹H COSY, HMBC, and NOESY correlations of compound 1.

Table 1.

¹H-NMR Data of Glyglabone (1) and Erybacin B in CDCl₃.

	Glyglabone (1)	Erybacin B²²
2-H	4.04 (2H, s)	4.07 (2H, s)
3′-H	6.35 (1H, d, J = 2.6 Hz)	–
5′-H	6.43 (1H, dd, J = 2.6, 8.2 Hz)	6.39 (1H, d, J = 8.8 Hz)
6′-H	7.85 (1H, d, J = 8.2 Hz)	7.75 (1H, d, J = 8.8 Hz)
3″-H	–	6.45 (1H, d, J = 2.2 Hz)
5″-H	6.33 (1H, d, J = 8.2 Hz)	6.37 (1H, dd, J = 2.2, 8.1 Hz)
6″-H	6.86 (1H, d, J = 8.2 Hz)	6.98 (1H, d, J = 8.1 Hz)
1‴-H	6.72 (1H, d, J = 10.0 Hz)	6.68 (1H, d, J = 9.5 Hz)
2‴-H	5.57 (1H, d, J = 10.0 Hz)	5.58 (1H, d, J = 9.5 Hz)
tert-Dimethyl	1.38 (6H, s)	1.45 (6H, s)
2′-OH	12.24 (s)	12.62 (s)
4′-OH	6.04 (br s)	–
2″-OH	8.17 (s)	7.80 (br s)
4″-OH	–	5.41 (br s)

s: singlet, d: doublet, dd: double doublet, br s: broad singlet.

Table 2.

¹³C-NMR Data of Glyglabone (1) and Erybacin B in CDCl₃.

	Glyglabone (1)	Erybacin B²²
C-1	204.3 (C)	204.0 (C)
C-2	40.1 (CH₂)	39.4 (CH₂)
C-1′	112.7 (C)	112.4 (CH)
C-2′	164.0 (C)	160.7 (C)
C-3′	103.9 (CH)	109.4 (C)
C-4′	166.6 (C)	160.9 (C)
C-5′	108.6 (CH)	109.1 (CH)
C-6′	133.5 (CH)	131.9 (CH)
C-1″	112.7 (C)	113.2 (C)
C-2″	151.7 (C)	156.5 (C)
C-3″	111.4 (C)	104.7 (CH)
C-4″	153.5 (C)	156.4 (C)
C-5″	109.0 (CH)	108.0 (CH)
C-6″	130.1 (CH)	131.6 (CH)
C-1‴	117.0 (CH)	115.5 (CH)
C-2‴	129.3 (CH)	128.3 (CH)
C-3‴	76.0 (C)	78.2 (C)
tert-Dimethyl	27.8 (CH₃ × 2)	28.4 (CH₃ × 2)

Table 3.

Phenolic Compounds Isolated from G. glabra, G. uralensis, and G. inflata.

No.	Compound	Molecular formula	Structure type	Plant source
1	Glyglabone	C₁₉H₁₈O₅	Other	G. glabra
2	Glabrol	C₂₅H₂₆O₄	Flavanone	G. glabra
3	3,4-Didehydroglabridin	C₂₀H₁₈O₄	Isoflavan	G. glabra
4	4′-O-Methylglabridin	C₂₁H₂₂O₅	Isoflavan	G. glabra
5	Kanznol Y	C₂₅H₃₀O₅	Chalcone	G. glabra
6	Hispaglabridin A	C₂₅H₂₈O₄	Isoflavan	G. glabra
7	3-Hydroxyglabrol	C₂₅H₂₈O₅	Flavan-3-ol	G. glabra
8	6-Prenyllicoisoflavanone	C₂₅H₂₆O₆	Isoflavanone	G. uralensis
9	Licoisoflavanone	C₂₀H₁₈O₆	Isoflavanone	G. uralensis
10	Licoricidin	C₂₆H₃₂O₅	Isoflavan	G. uralensis
11	3-(γ,γ-Dimethylallyl)-kievitone	C₂₅H₂₈O₆	Isoflavanone	G. uralensis
12	Glisoflavanone	C₂₅H₂₈O₆	Isoflavanone	G. uralensis
13	Licoisoflavanone B	C₂₀H₁₆O₆	Isoflavanone	G. inflata
14	Glabrone	C₂₀H₁₆O₅	Isoflavone	G. inflata
15	Licochalcone E	C₂₁H₂₂O₄	Chalcone	G. inflata
16	Echinatin	C₁₆H₁₄O₄	Chalcone	G. inflata

For comparison with the SFC-TOF/MS results, the CHCl₃ extract was concentrated, redissolved in EtOH, filtered through a 0.2-µm membrane, and analyzed using LC-TOF/MS. In the LC method, longer columns than those used for SFC were employed to achieve better separation. BPI chromatogram data under negative ion mode are shown in Figure 5. The PCA score plots using LC-TOF/MS showed that the samples were classified into three groups, similar to the SFC results. The first and second principal components accounted for 62.1% of the total variance. In addition, compounds (1–16) were also plotted as characteristic compounds in the PCA loading plots (Figure 6). These results were equivalent to those obtained using the SFC method followed by PCA; however, the analysis time and amount of organic solvents consumed in the LC method were more than twice that of the SFC method. Therefore, the SFC method distinguished between G. glabra, G. uralensis, and G. inflata more rapidly and sustainably than the LC method.

Figure 5.

Base peak ion (BPI) chromatogram obtained for LC-TOF/MS analysis of the extracts from G. glabra (A), G. uralensis (B), and G. inflata (C).

Figure 6.

Principal component analysis (PCA) score plot (A), loading plot (B), and boxed portion of the loading plot on a larger scale (C).

Conclusions

This study, to our knowledge, is the first to utilize SFC-TOF/MS coupled with PCA to distinguish between G. glabra, G. uralensis, and G. inflata. All 16 identified compounds, including the new compound, glyglabone (1), are expected to be characteristic constituents of the three licorice species. The relationship between the time and location of plant collection and the constituents of licorice roots is currently under investigation and will be reported in the future. The SFC analysis revealed tailing peaks, which was inappropriate for the quality control (QC) of licorice roots. Consequently, suitable stationary and mobile phases and additives are required to enhance peak tailing. However, compared with the LC method, the SFC method was advantageous for the direct analysis of CHCl₃ extracts and was considered to be more rapid and eco-friendly for distinguishing between G. glabra, G. uralensis, and G. inflata.

Experimental

Plant Materials

The roots of G. glabra (five batches), G. uralensis (five batches), and G. inflata (four batches) were used in this study. Each species was provided and identified by Prof. Ying Wang of the South China Botanical Garden.

General

The following instruments were used to obtain physical data: ultra-violet (UV) spectra, Shimadzu UV-1800 UV-Vis spectrometer; IR spectra, JASCO FT/IR-460 spectrometer; NMR spectra, JEOL ECS-400 (400 MHz) spectrometer; preparative HPLC, YMC LC-Forte/R equipped with a YMC-Actus Triart C18 column (250 × 20 mm i.d., YMC Co. Ltd, Kyoto, Japan); recycling preparative HPLC, Japan Analytical Industry LC-908 equipped with two JAIGEL GS-310 columns (500 × 20 mm i.d., Japan Analytical Industry Co. Ltd, Tokyo, Japan). The following materials were used for chromatography: synthetic absorbent resin column chromatography, DIAION HP-20 (Mitsubishi Chemical Co. Ltd, Chiyoda-ku, Tokyo, Japan); normal-phase silica gel column chromatography, Silica gel 60 (Merck, KGaA., Darmstadt, Germany); and reversed-phase silica gel column chromatography, Chromatorex ODS (Fuji Silysia, Ltd, Kasugai, Japan).

Sample Preparation for PCA

The roots of G. glabra, G. uralensis, and G. inflata were extracted using CHCl₃ for 1 hour at room temperature. Prior to SFC-TOF/MS analysis, the extracts were diluted tenfold with CHCl₃ and filtered through a 0.2-µm membrane. Subsequently, the solvent was removed and redissolved in EtOH (0.5 mg/mL), filtered through a 0.2-µm membrane, and subjected to LC-TOF/MS analysis. The QC sample consisted of equal proportions of each solution, which was used to adjust the retention time of each peak detected in all samples for PCA.

SFC-TOF/MS and LC-TOF/MS Conditions

SFC analysis was performed using the ACQUITY UPC² system (Waters Corp.), equipped with a binary solvent pump, sample manager, column manager, convergence manager, and a backpressure regulator, connected to a TOF/MS (Xevo G2 Tof, Waters Corp., Milford, MA, USA). SFC analysis was conducted on an ACQUITY Torus DIOL column (100 × 2.1 mm i.d., 1.7 μm; Waters Corp.) at a flow rate of 1.0 mL/min at 40 °C using a mobile phase of scCO₂ (purity ≥99.99%, A) and MeOH (B) (containing 0.1%, v/v formic acid). The gradients of A and B were as follows: 0–20% B at 0–6 min, 20% B at 6–8 min, 20–0% B at 8–8.1 min, and 0% at 8.1–10 min. The makeup liquid consisted of MeOH containing 0.1% formic acid. The flow rate and the back pressure were maintained at 0.2 mL/min and 2000 psi, respectively. The injection volume for each sample was 2.0 μL.

The ACQUITY UPLC system (Waters Corp.) comprised a binary solvent manager, a sample manager, and a column manager connected to a TOF mass spectrometer. Qualitative analysis was performed on an ACQUITY BEH C18 column (150 × 2.1 mm i.d., 1.7 μm; Waters Corp.) at a flow rate of 0.4 mL/min at 40 °C using a mobile phase of pure water (A) (containing 0.1% formic acid) and MeCN (B) (containing 0.1% formic acid). The gradients of A and B were as follows: 30–90% B at 0–16 min, 90% B at 16–21 min, 90–100% B at 21–22 min, 100% B at 22–27 min, 100–30% B at 27–28 min, and 30% B at 28–30 min. The injection volume for each sample was 2.0 μL. To perform PCA, data were processed using Masslynx 4.1 and Progenesis QI software (Waters Corp.) and exported to the SIMCA software, version 17 (Sartorius Stedim Biotech).

Extraction and Isolation

The dried roots of G. glabra (1.0 kg) were extracted with CHCl₃ (2.5 L) under reflux (1 h), and the solvent was removed in vacuo to obtain the CHCl₃ extract (14 g). This extract was subjected to DIAION HP-20 column chromatography and eluted with MeOH–H₂O (8:2 to 10:0, v/v) and Me₂CO. The MeOH eluate (6.0 g) was chromatographed on a silica gel column and eluted with n-hexane–EtOAc (2:1 to 1:1, v/v) to yield fractions G1 to G6. Fraction G2 (1.0 g) was separated using an octadecyl-silica (ODS) column with MeOH–H₂O (6:4 to 10:0, v/v) and further purified using recycling preparative HPLC (MeOH) to obtain compound 1 (5.9 mg). Fraction G3 (1.6 g) was fractionated using ODS column chromatography and eluted with MeOH–H₂O (6:4 to 10:0, v/v) to yield fractions G3-1 to 6. Fraction G3-1 (167 mg) was successively purified using preparative HPLC [MeCN–H₂O (60:40, v/v)] and recycled preparative HPLC (MeOH) to obtain compound 1 (3.0 mg). Fraction G3-3 (227 mg) was subjected to preparative HPLC [MeOH–H₂O (70:30, v/v)] to yield compounds 2 (37 mg, 0.0037%) and 3 (16 mg, 0.0016%). Fraction G3-4 (76 mg) was purified using preparative HPLC [MeCN–H₂O (60:40, v/v)] and recycling preparative HPLC (MeOH) to obtain compounds 4 (14 mg, 0.014%), 5 (11 mg, 0.011%), and 6 (5 mg, 0.005%). Compound 7 (66 mg, 0.066%) was obtained by separating fraction G5 (1.0 g) on an ODS column with MeOH–H₂O (6:4 to 10:0, v/v) and purifying it by recycling preparative HPLC (MeOH).

The dried roots of G. uralensis (840 g) were extracted using CHCl₃ (2 L), and the solvent was removed. The CHCl₃ extract (32 g) was applied to a DIAION HP-20 column and eluted with MeOH–H₂O (5:5 to 10:0, v/v) and Me₂CO. The MeOH eluate (6.0 g) was chromatographed on a silica gel column and eluted with n-hexane–EtOAc (2:1 to 1:1, v/v) to obtain fractions U1 to U8. Fraction U1 (3.9 g) was separated on an ODS column with MeOH–H₂O (6:4 to 10:0, v/v) and further purified using preparative HPLC [MeCN–H₂O (65:35, v/v)] and recycling preparative HPLC (MeOH) to obtain compound 8 (6.0 mg, 0.0007%). Fraction U2 (2.4 g) was fractionated using ODS column chromatography and eluted with MeOH–H₂O (6:4 to 10:0, v/v) to yield fractions U2-1 to 7. Fraction U2-2 (336 mg) was purified using preparative HPLC [MeOH–H₂O (65:35, v/v)] and recycling preparative HPLC (MeOH) to obtain compound 9 (78 mg, 0.0092%). Fraction U2-4 (150 mg) was purified using preparative HPLC [MeOH–H₂O (70:30, v/v)] and recycling preparative HPLC (MeOH) to yield compound 10 (44 mg, 0.0052%). Fraction U2-5 (168 mg) was purified using preparative HPLC [MeOH–H₂O (75:25, v/v)] and recycling preparative HPLC to obtain compounds 11 (49 mg, 0.0058%) and 12 (30 mg, 0.0036%).

The dried roots of G. inflata (400 g) were extracted with CHCl₃ (2 L), and the solvent was removed. The CHCl₃ extract (11 g) was fractionated using silica gel column chromatography and eluted with n-hexane–EtOAc (1:1, v/v) to obtain fractions I1 to I5. Fraction I2 (1.6 g) was separated on an ODS column with MeOH–H₂O (7:3 to 10:0, v/v) and further purified using preparative HPLC [MeCN–H₂O (68:32, v/v)] and recycling preparative HPLC (MeOH) to obtain compound 13 (35 mg, 0.0088%). Fraction I3 (4.5 g) was chromatographed on a silica gel column and eluted with n-hexane–EtOAc (2:1 to 1:1, v/v) to obtain fractions I3-1 to 4. Fraction I3-2 (283 mg) was purified using preparative HPLC [MeCN–0.1%aq.TFA (55:45, v/v)] and recycling preparative HPLC (MeOH) to obtain compound 14 (23 mg, 0.0058%). Fraction I3-3 (1.0 g) was separated using an ODS column with MeOH–H₂O (5:5 to 10:0, v/v) and successively purified using preparative HPLC [MeCN–H₂O with 0.05% trifluoroacetic acid (60:40, v/v)] and recycling preparative HPLC (MeOH) to yield compound 15 (10 mg, 0.0025%). Fraction I4 (200 mg) was purified using preparative HPLC [MeCN–0.05% aqueous trifluoroacetic acid (60:40, v/v)] and recycling preparative HPLC (MeOH) to obtain compound 16 (10 mg, 0.0025%).

Glyglabone (1): amorphous powder; HR-ESI-TOF/MS: calculated for: C₁₉H₁₈O₅: m/z 325.1076 [M-H]⁻, found: 325.1075; UV (MeOH) λ_max nm (log ε): 317 (3.25), 278 (3.28), 226 (3.50); IR (KBr): ν_max 3350, 1630, 1510, 1480, 1360, 1230, 1130 cm⁻¹; ¹H- and ¹³C-NMR (CDCl₃): shown in Tables 1 and 2.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231205727 - Supplemental material for Differentiating Between Three Licorice Species Using SFC-TOF/MS Analysis With Principal Component Analysis

Supplemental material, sj-docx-1-npx-10.1177_1934578X231205727 for Differentiating Between Three Licorice Species Using SFC-TOF/MS Analysis With Principal Component Analysis by Hideki Saito, Shozo Miyoshi, Toshiyuki Murakami and Ryuichiro Suzuki in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to thank Prof. Ying Wang for assisting with the identification of licorice and Editage () for English language editing.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: H.S., S.M., and T.M. are employees of Maruzen Pharmaceuticals Co., Ltd., the sponsor of this study. H.S. has a pending patent that belongs to the employer.

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies conducted on human patients or animal models.

Statement of Informed Consent

There are no human patients involved in this article and informed consent is not applicable.

ORCID iDs

Hideki Saito

Ryuichiro Suzuki

Supplemental Material

Supplemental material for this article is available online.

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