Sample preparation
Chemicals and reagents
Raw licorice was purchased from Sichuan Xinhehua Decoction Pieces Co. Ltd. (Sichuan, China) and DNA-Authenticated as Glycyrrhiza uralensis Fisch root [19]. Voucher specimens (No. L20190706) were deposited in the herbarium of the Institute of Chinese Materia Medica of the China Academy of Chinese Medical Sciences. Honey was purchased from Beijing Tongrentang Bee Products Co. Ltd. (Zhejiang, China). Food-grade glucose, fructose, and sucrose were acquired from Sigma-Aldrich Corp. (St. Louis, MO, USA). Reference compounds including liquiritin (2), liquiritigenin (3), isoliquiritin (5), isoliquiritigenin (6), and glycyrrhizic acid (7) were obtained from Shanghai Yuanye Biotech Co. Ltd. (Shanghai, China). Isoliquiritin apioside (4), 2,4-dihydroxyacetophenone (9), D-fructose (10), and D-glucose (11) were obtained from Chengdu Chroma-Biotechnology Co. Ltd. (Chengdu, China). Liquiritin apioside (1), glycyrrhetinic acid (8), 5-hydroxymethylfurfural (12), and rutin, oleanolic acid (internal standard, IS) were procured from Beijing Century Aokang Biotech Co. Ltd. (Beijing, China). Their purity was determined by high-performance liquid chromatography (HPLC) to be > 98%. Chromatography-grade acetonitrile and methanol were acquired from Fisher Scientific Worldwide Co. Ltd. (Shanghai, China). All other reagents were of analytical grade.
NADES preparation
The honey analog known as pure NADES was prepared by mixing a 35:35:1 molar ratio of glucose, fructose, and sucrose with 29 mL water [15]. The NADES product and honey were characterized by Bruker Ascend-600M 1H Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker Corp., karlsruhe, Germany), and by differential scanning calorimetry (DSC) [18].
Preparation of different processed licorice and their decoctions
Licorice processing: Processed licorice was prepared with dried licorice root, honey, and NADES. The licorice samples comprised raw licorice (R) (unprocessed), fried licorice (F) (stir-fried without additives), honey-fried licorice (H) (mixed with honey and fried), and NADES-fried licorice (N) (mixed with NADES and fried). The processing methods are described in the 2020 version of the “People's Republic of China Pharmacopoeia” (Provision No. 0213).
Licorice decoctions: Each of the foregoing products (100 g based on sliced licorice weight) was kept in 700 mL distilled water at 25 °C for 30 min and refluxed at 100 °C for 30 min. The decoctions were filtered with 2 layers of gauze, and the residues were refluxed with 600 mL distilled water at 100 °C for 20 min. The filtrates were combined, condensed to < 200 mL in 80 ℃ water bath, and adjusted to 200 mL with water. The decoctions were stored at -80 °C before use. The decoctions were resuspended by shaking before use in the cell-based bioactivity tests.
Reference solutions: Stock solutions of the reference standards (liquiritin apioside (1), liquiritin (2), liquiritigenin (3), isoliquiritin apioside (4), isoliquiritin (5), isoliquiritigenin (6), glycyrrhizic acid (7), glycyrrhetinic acid (8), 2,4-dihydroxyacetophenone (9), D-fructose (10), D-glucose (11), and 5-hydroxymethylfurfural (12)) were prepared by dissolving 5 mg of each standard in 5 mL methanol. The stock solutions were stored at -20 °C before use.
Immunological experiment
Animals and experimental design
Male ICR mice (18–22 g) were purchased from SPF Biotechnology Co. Ltd. (Beijing, China). They were housed in a specific pathogen-free grade laboratory at room temperature (20–25 °C) and constant relative humidity (RH) under a 12-h light/12-h dark cycle. They had ad libitum access to food and distilled water. They were allowed to acclimate to their new environment for 3 d prior to the experiments. They were weighed and randomly divided into 10 groups (n = 6 per group), namely, normal, model, high-dose raw licorice (7.5 g/kg), low-dose raw licorice (5.0 g/kg), high-dose fried licorice (7.5 g/kg), low-dose fried licorice (5.0 g/kg), high-dose honey-processed licorice (7.5 g/kg), low-dose honey-processed licorice (5.0 g/kg) groups, high-dose NADES-processed licorice (7.5 g/kg), and low-dose NADES-processed licorice (5.0 g/kg) groups. Mice in group 2-10 were intragastrically (i.g.) administrated with rhubarb extract (15 g/kg, once a day) for 7 d to induce Pi-deficiency model. Then, the mice in group 1 (normal) and group 2 (model) were administered (i.g.) sterile physiological saline once daily for 7 d. Mice in the raw, fried, honey-processed, and NADES-processed licorice groups were orally administered decoctions at 7.5 mL/kg body weight (BW) once daily for 7 d. All procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China (2006) and approved by the Animal Welfare Ethics Committee of the Institute of Chinese Materia Medica (China Academy of Chinese Medical Sciences, Beijing, China).
Sample collection and measurement
Twenty-four hours after the final administration, the mice were weighted and then injected in the cauda with India ink (0.01 mL/g BW). Twenty microliters blood were taken from the eyepit vein plexus at 2 min and 20 min after the India ink injection. The blood was placed in a centrifuge tube containing 2 mL of 1% (w/v) sodium bicarbonate for 1 h. The absorbances (optical density [OD]) of the mice sera were measured at 650 nm by Thermo Scientific Multiskan GO Multiskan Spectrum (Thermo Fisher Scientific, Waltham, MA, USA). Thirty minutes after the India ink injections, Mice were euthanized, then the spleen and thymus were excised and weighed,
Data analysis
The charcoal particle expurgation index (K) of the macrophage count was calculated as follows:
where, t1 and t2 are the time points (2 min and 20 min, respectively) at which blood was drawn from the mouse eyes after the India ink injection.
The spleen index was calculated as follows [2]:
Data were expressed as means ± SD. Differences between groups were evaluated with SPSS v. 25.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
Cell-based bioactivity tests
Cell culture
HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (FBS) and 100 μg/mL kanamycin in an incubator at 37 °C under a humidified CO2 (5%) atmosphere. Before seeding, pER-α-Luc HEK-293 cells were washed with phosphate-buffered saline (PBS) and cultured in DMEM containing 5% cdFBS for 2 d starvation. The pER-α-Luc HEK-293 cells were seeded in a 96-well plate. Each well contained 40,000 cells and 0.1 mL estrogen-free medium. Other cell lines including pNrf2-Luc HEK-293 and pTLR-4-Luc HEK-293 were inoculated in 96-well plates. Each well contained 20,000 cells and 0.1 mL DMEM with 10% (v/v) FBS.
Biological activity testing
The estrogen receptor (ER)-α, nuclear factor erythroid 2-related factor (Nrf2), and toll-like receptor 4 (TLR4) promoter assays were conducted on the HEK293 cells. An appropriate licorice extract concentration was added to the pER-α-Luc HEK-293 cells, and the suspension in a humidified CO2 (5%) incubator at 37 °C was incubated for 24 h. Then, 17-β-estradiol (Sigma-Aldrich Corp.) was dissolved in dimethyl sulfoxide (DMSO) and used as a positive control. The medium was aspirated before adding 0.5 mL lysis buffer (pH 7.8) to each well for 10 min. Luciferase assay buffer (0.1 mL/well) was added, and the luciferase activity was immediately determined. Estrogenic activity was calculated as follows [20]:
For the TLR4 promoter assay, an appropriate licorice extract concentration was added to the pTLR4-Luc HEK-293 cells, and they were incubated for 4 h at 37 °C. Lipopolysaccharide (LPS) (Sigma-Aldrich Corp.) dissolved in DMSO was the positive control. For the Nrf2 promoter assay, an appropriate licorice extract concentration was added to the pNrf2-Luc HEK 293 cells, and they were incubated for 16 h at 37 °C. Andrographolide (20 μM) dissolved in DMSO was the positive control. The other steps followed were the same as those used for the ER-α assay.
Total polysaccharide and flavonoid content measurement
The levels of total polysaccharides and flavonoids in the decoctions were determined by UV-Vis spectrophotometry [21]. The total polysaccharides were detected at 590 nm by the anthrone-sulfuric acid method using anhydrous glucose as a reference [22]. Liquiritin was the reference, as it is the main dihydroflavone in licorice. All major flavonoids in licorice have maximum absorptions at ~336 nm.
Metabolomics analysis
Non-target metabolomics analysis with UHPLC-Q-Orbitrap MS
The licorice decoctions were thawed, diluted 20,000-fold with methanol, and filtered through a 0.22 μm membrane before analysis on an Ultimate 3000 ultrahigh-performance liquid chromatography (UHPLC) instrument coupled with a Thermo Q-ExActiveTM Plus OrbitrapTM high-resolution mass spectrometer (Thermo Fisher Scientific) fitted with a heat electrospray ionization (HESI) interface. Samples were separated on an Acquity UPLCHSS T3 C18 column (2.1 mm × 100 mm; 1.8 μm; Waters Corp., Milford, CT, USA). The column temperature was maintained at 40 °C. The flow rate was 0.30 mL/min. Mobile phase A was 0.1% (v/v) formic acid-acetonitrile, and mobile phase B was 0.1% (v/v) formic acid. The gradient program was as follows: 0–1 min, 98% B; 1–2.5 min, 98–95% B; 2.5–4 min, 95–70% B; 4–8 min, 70–65% B; 8–15 min, 65–50% B; 15–18 min, 50–0% B; 18–23 min, 0% B; 23–23.5 min, 0–98% B; 23.5–26 min, 98% B. The sample temperature was maintained at 20 °C, and the injection volume was 5 μL.
The operating conditions of the mass spectrometry (MS) were as follows: HESI source in positive and negative mode; capillary temperature, 320 °C; sheath gas flow, 35 arb; auxiliary gas flow, 10 arb; scan modes, full MS and dd-MS2; full MS, 70,000 resolution; full MS residence time, 150 ms; dd-MS2, 17,500 resolution; dd-MS2 TopN, 5; mass spectra recorded in m/z range, 120–1,000; step collision energy, 20 V/45 V/70 V; positive mode, 3.5 kV spray voltage; negative mode, 3.2 kV spray voltage. Data were analyzed with Compound Discoverer v. 3.0 (Thermo Fisher Scientific).
The raw mass data were pre-processed with Progenesis QI v. 1.0 (Waters Corp., Milford, CT, USA) for peak alignment and selection as well as deconvolution. Individual ion fragment intensities were normalized for all compounds. The data MS matrices were imported into SIMCA-P v. 13 (Umetrics, Umeå, Sweden) for principal component analysis (PCA) by Pareto scaling to identify group distributions, perform orthogonal partial least square discriminant analysis (OPLS-DA), and identify the potential quality difference markers among groups by variable importance in projection (VIP). One-way ANOVA was conducted on the ion response strength data of the potential mass difference markers using GraphPad Prism v. 7.0 (GraphPad Software, La Jolla, CA, USA) to identify any significant differences among potential mass difference markers. The screened quality difference markers were identified by chromatographic comparison against reference substances, laboratory databases, and literature reports [6,23,24].
Quantitative analysis with UHPLC-QqQ-MS
Licorice decoctions were thawed, diluted 20,000-fold with methanol, and filtered through a 0.22 μm membrane before quantitative analysis. The assay was performed on a 6500 Plus Triple Quad LC–MS/MS system fitted with an ExionLC UHPLC unit (AB SCIEX Corp., Framingham, MA, USA). Sample components were separated on an Acquity UPLCHSS T3 C18 column (2.1 mm × 100 mm; 1.8 μm; Waters Corp.). The column temperature was maintained at 40 °C, and the flow rate was 0.30 mL/min. Mobile phase A was 0.1% (v/v) formic acid-acetonitrile. Mobile phase B was 0.1% (v/v) formic acid. The gradient program was as follows: 0–3 min, 98–95% B; 3–4 min, 95–81% B; 4–7 min, 81–70% B; 7–9 min, 70% B; 9–10 min, 70–50% B; 10–12 min, 50–45% B; 12–17 min, 45–5% B; 17–22 min, 5% B; 22–23 min, 5–98% B; 23–27 min, 98% B. The sample temperature was maintained at 20 °C. The injection volume was 5 μL.
MS data were recorded using electrospray ionization (ESI), negative ion detection, and multiple reaction monitoring (MRM) scanning. The ion spray voltage and temperature were set at 4,500 V and 500 °C, respectively. The curtain, gas 1 (nebulizer), and gas 2 (heater) gas pressures were set to 35 psi, 50 psi, and 50 psi, respectively. The collision gas pressure was set to 9 psi. The compound dwell time was set to 10 ms. The entrance potential and collision cell exit potential were set to 10 V and 18 V, respectively. Data were acquired with Analyst v. 1.6.3 (AB SCIEX Corp., Framingham, MA, USA). The MS/MS parameters for the 18 compounds are listed in Table S1. The method was validated (Supplementary Material).
Pharmacokinetic experiments
Drug administration and sample preparation
Male Sprague-Dawley (SD) rats (220–250 g) were obtained from HFK Bioscience Co. Ltd. (Beijing, China). The rats were bred at 25 ℃, 60 ± 5% RH, and 12-h dark-light cycle for 3 d. They had ad libitum access to water and normal chow. All animals were fasted overnight before the experiments. The rats were randomly divided into four groups (n = 6 per group). The raw licorice decoction (R; 1.0 g/mL raw licorice equivalent), the fried licorice decoction (F; 1.0 g/mL raw licorice equivalent), the honey-fried licorice decoction (H; 1.0 g/mL raw licorice equivalent), and the NADES-processed licorice decoction (N; 1.0 g/mL raw licorice equivalent) were orally administered to the rats at 8 g/kg. Blood samples 0.3 mL in volume were collected from the angular vein into heparinized tubes at 0.08 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after oral decoction administration. They were immediately centrifuged at 12,000 rpm at 4 ℃ for 3 min, and the plasma was stored at -80 °C until use.
Calibration standard (CS) and quality control (QC) samples were prepared (Supplementary Materials). One hundred microliters of each plasma, CS, or QC sample was spiked with 300 μL acetonitrile (including rutin and oleanolic acid; 100 ng/mL). The mixture was vortexed for 3 min and centrifuged at 12,000 rpm and 4 ℃ for 10 min. The supernatant was collected and evaporated under a nitrogen stream in a 37 ℃ water bath. The residue was redissolved in 100 μL acetonitrile, vortexed for 3 min, and centrifuged at 12,000 rpm at 4 ℃ for 10 min.
UHPLC-QqQ-MS analysis
The assay was performed on a 6500 Plus Triple Quad LC–MS/MS system fitted with an ExionLC UHPLC unit (AB SCIEX Corp., Framingham, MA, USA). Sample components were separated on an Acquity UPLCHSS T3 C18 column (2.1 mm × 100 mm; 1.8 μm; Waters Corp.). The column temperature was maintained at 40 °C, and the flow rate was 0.30 mL/min. Mobile phase A was 0.1% (v/v) formic acid-acetonitrile. Mobile phase B was 0.1% (v/v) formic acid. The gradient program was as follows: 0–3 min, 98–95% B; 3–4 min, 95–81% B; 4–7 min, 81–70% B; 7–9 min, 70% B; 9–10 min, 70–50% B; 10–12 min, 50–45% B; 12–17 min, 45–5% B; 17–22 min, 5% B; 22–23 min, 5–98% B; 23–27 min, 98% B. The sample temperature was maintained at 20 °C. The injection volume was 5 μL. The electrospray ionization (ESI) source was set in negative ion detection. Quantitation was performed in multiple reaction monitoring (MRM) mode. The transition ions were m/z 549.1→255.2 for liquiritin apioside (1), m/z 417.1→134.9 for liquiritin (2), m/z 255.1→119.1 for liquiritigenin (3), m/z 469.3→425.4 for glycyrrhetinic acid (8), and m/z 609.2→300.2 for rutin (IS), m/z 455.4→407.5 for oleanolic acid (IS). The collision energy (CE) for each compound and IS were -44 V, -43 V, -32 V, -53 V, and -52 V, -57 V, respectively. The declustering potential (DP) were set at -80 V. The ion spray voltage and temperature were set at 4,500 V and 500 °C, respectively. The curtain, gas 1 (nebulizer), and gas 2 (heater) gas pressures were set to 35 psi, 50 psi, and 50 psi, respectively. The collision gas pressure was set to 9 psi. The compound dwell time was set to 10 ms. The entrance potential and collision cell exit potential were set to 10 V and 18 V, respectively. Data were acquired with Analyst v. 1.6.3 (AB SCIEX Corp., Framingham, MA, USA). The method was validated (Supplementary Material).
Statistical analysis
The pharmacokinetic parameters were calculated with Drug Analysis System v. 3.2.6 (Mathematical Pharmacology Professional Committee of China). Noncompartmental analysis was used to determine the maximum time (Tmax), maximum plasma concentration (Cmax), and area under the curve (AUC0-12/24h). Data were means ± SD. Statistical analyses of all data were performed with one-way ANOVA (SPSS v. 25.0; SPSS Corp., Chicago, IL, USA).
Molecular interaction determination of honey and its mixture with licorice compounds
Sample preparation
Honey and corresponding NADES were obtained as described in Subsection of sample preparation. A fructose solution was obtained by dissolving 2 g fructose in 0.5 mL water to ensure that the final water content was the same as that in NADES. Glucose hydrate was prepared in the same manner as the fructose solution. The mixture of liquiritin and NADES was obtained by dissolving 0.12 g liquiritin in 1 mL NADES and stirring thoroughly.
Apparatus and analysis
Fourier transform-infrared (FT-IR) spectra (range 4,000–600 cm-1) were recorded at 25 ℃ in a Frontier FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). The spectra were baseline-corrected with Specture v. 10 (PerkinElmer).