UPLC-Q-Orbitrap HRMS Combined With Molecular Docking To Investigate Material Basis Of Bitter Taste And Flavor-effect Relationship in Cistanchia Desertica Ⅱ
Dec 18, 2024
2 Methods and results
2.1 Identification of Cistanche deserticola substances
2.1.1 UPLC-Q-Orbitrap HRMS detection conditions Chromatographic conditions:
Phenomenex Kinetex C18 column (2.1 mm×100 mm, 2.6 μm); mobile phase A (aqueous phase, containing 0.1% acetic acid), B (isopropanol-acetonitrile 1:1), gradient elution (0-1 min, 1% B; 1-8 min, 1%-99% B; 8-9 min, 99% B; 9-9.1 min, 99%-1% B; 9.1-12 min, 1% B); flow rate 0.2 mL·min-1; column temperature 30 ℃; injection volume 2 μL.
Mass spectrometry conditions: Orbitrap Exploris 120 mass spectrometer was used to collect primary and secondary mass spectrometry data under the control of software (Thermo Scientific Xcalibur software, version: 4.4). The spray voltage was 3.8 kV (positive) or -3.4 kV (negative), the ion source temperature was 320 °C, the sheath gas flow rate was 50 arb, the auxiliary gas flow rate was 15 arb, and the ion transfer tube temperature was 320 °C. The scanning mode was full scan/data-dependent secondary scan (Full MS/dd-MS2), the primary resolution was 60 000, the secondary resolution was 15 000, the ion scanning range was m/z100~1 500, and the collision energy gradient was 20, 30, 40 eV.
High-quality Cistanche Materials With Higher echinacoside, verbascoside, tubuloside A, isoverbaceous glycoside
2.1.2 Preparation of reference solution
Accurately weigh appropriate amounts of echinacoside, verbascoside, tubuloside A, isoverbaceous glycoside, gentianoside, and geniposide reference substances, add 50% methanol to make a mixed reference solution with a mass concentration of 500 ng·mL-1, shake well, and obtain.
2.1.3 Preparation of test solution
Weigh 1.0 g of sample powder (M1) collected in spring and 1.0 g of sample powder (M5) collected in autumn, add extract solution (methanol-water 4:1), soak completely, ultrasonicate at 250 W and 35 kHz for 1 h at room temperature, then centrifuge at 4 000 r·min-1 for 10 min, and filter to obtain alcohol extract of Cistanche deserticola.
2.1.4 Identification of substances
By comparing the primary and secondary mass spectrometry information data of each Cistanche deserticola sample, and combining with relevant literature and reference materials, it was identified that different samples of Cistanche deserticola contain 69 chemical components in common, see Table 2, and the total ion flow diagram of Cistanche deserticola is shown in Figure 2.
Table 2 Constituents of Cistanche deserticola extracts identified based on UPLC-Q-Orbitrap HRMS
| No. | Compound | Molecular Formula | Measured Value (m/z) | Error (×10⁻⁶) | Ion Mode | Fragment Ions | Type |
|---|---|---|---|---|---|---|---|
| 0.02 | Isomenthone | C₁₀H₁₈O | 223.06 | -8.7 | [M + H]⁺ | 223.06, 207.02, 137.02 | Monoterpenoids |
| 0.57 | Uracil | C₄H₄N₂O₂ | 157.04 | 1.8 | [M + H]⁺ | 157.03, 59.02, 49.01 | Other |
| 0.67 | Glucosamine | C₆H₁₃NO₅ | 195.05 | 1.1 | [M + H]⁺ | 75.01, 195.05, 59.01 | Carbohydrates |
| 0.68 | Galactosamine | C₆H₁₃NO₅ | 181.07 | 1.1 | [M + H]⁺ | 181.07, 71.01, 89.02 | Carbohydrates |
| 0.81 | Caprylic Acid | C₈H₁₆O₂ | 144.11 | 1.0 | [M + H]⁺ | 104.11, 103.08, 87.03 | Other |
| 0.83 | L - Aspartic Acid | C₄H₇NO₄ | 132.03 | 0.7 | [M + H]⁺ | 132.03, 115.00, 71.01 | Amino Acids |
| 0.97 | Ethyl Acetate | C₄H₈O₂ | 198.19 | 1.4 | [M + H]⁺ | 197.02, 54.02, 153.02 | Other |
| 0.98 | Malic Acid | C₄H₆O₅ | 155.00 | 0.0 | [M + H]⁺ | 111.00, 49.00, 68.99 | Organic Acids |
| 0.99 | D - Galactose | C₆H₁₂O₆ | 180.16 | 1.1 | [M + H]⁺ | 179.06, 89.02, 59.01 | Carbohydrates |
| 1.00 | L - Glutamic Acid | C₅H₉NO₄ | 188.09 | -0.4 | [M + H]⁺ | 118.09, 119.09, 72.08 | Amino Acids |
| 1.01 | L - Isoleucine | C₆H₁₃NO₂ | 132.10 | -0.9 | [M + H]⁺ | 132.10, 86.03, 115.07 | Amino Acids |
| 1.05 | Glycine | C₂H₅NO₂ | 182.08 | -0.9 | [M + H]⁺ | 182.08, 95.04, 29.03 | Amino Acids |
| 1.48 | Uridine | C₉H₁₂N₂O₆ | 243.06 | 0.3 | [M + H]⁺ | 243.06, 103.01, 102.10 | Nucleosides |
| 1.93 | Methyl D - Iodide | C₂H₅I | 131.03 | 0.0 | [M + H]⁺ | 131.03, 59.00, 41.01 | Other |
| 2.29 | Naringin | C₂₇H₃₂O₁₄ | 345.12 | 3.2 | [M + H]⁺ | 345.12, 327.11, 147.05 | Flavonoids |
| 3.29 | Protocatechuic Acid | C₇H₆O₄ | 153.02 | 1.1 | [M + H]⁺ | 153.02, 109.02, 41.00 | Phenolic Acids |
| 3.46 | Triacontanol | C₃₀H₆₂O | 445.10 | 2.7 | [M + H]⁺ | 445.10, 271.08, 101.00 | Alkanols |
| 3.47 | Kaempferol - 3 - O - glucoside | C₂₁H₂₀O₁₁ | 373.11 | 2.3 | [M + H]⁺ | 123.01, 149.06, 167.07 | Flavonoids |
| 3.48 | Quercetin | C₁₅H₁₀O₇ | 109.03 | 0.6 | [M + H]⁺ | 109.03, 79.05, 77.03 | Flavonoids |
| 3.66 | Chlorogenic Acid | C₁₆H₁₈O₉ | 375.09 | -0.7 | [M + H]⁺ | 375.11, 359.07, 309.03 | Phenolic Acids |
| 3.70 | Cinnamic Acid | C₉H₈O₂ | 347.13 | 4.1 | [M + H]⁺ | 347.13, 181.09, 113.07 | Phenolic Acids |
| 3.74 | 8 - Epigallocatechin - 3 - gallate | C₂₂H₁₈O₁₁ | 315.37 | 13.6 | [M + H]⁺ | 315.37, 136.05, 103.04 | Flavonoids |
| 3.76 | Limonene | C₁₀H₁₆ | 163.04 | 0.2 | [M + H]⁺ | 163.04, 93.07, 62.03 | Monoterpenoids |
| 3.77 | Hesperidin | C₂₈H₃₄O₁₅ | 299.11 | -1.9 | [M + H]⁺ | 299.11, 181.08, 101.04 | Flavonoids |
| 3.91 | Vanillin | C₈H₈O₃ | 307.81 | 3.8 | [M + H]⁺ | 307.83, 173.03, 149.02 | Phenolic Acids |
| 4.11 | Isoquercitrin | C₂₁H₂₀O₁₂ | 419.13 | 4.1 | [M + H]⁺ | 419.13, 149.02, 101.00 | Flavonoids |
| 4.21 | Isopropyl Methylphenidate | C₁₄H₂₁NO₂ | 221.08 | -0.8 | [M + H]⁺ | 221.08, 205.09, 104.00 | Other |
| 4.22 | Syringic Acid | C₉H₁₀O₅ | 785.25 | 1.4 | [M + H]⁺ | 785.25, 663.21, 661.19 | Phenolic Acids |
| 4.30 | Eugenol | C₁₀H₁₂O₂ | 179.04 | 0.2 | [M + H]⁺ | 179.04, 153.03, 113.03 | Phenolic Acids |
| 4.40 | Caffeine | C₈H₁₀N₄O₂ | 769.26 | 1.4 | [M + H]⁺ | 769.26, 629.24, 622.14 | Alkaloids |
| 4.43 | Chrysin | C₁₅H₁₀O₄ | 442.05 | 1.1 | [M + H]⁺ | 442.05, 173.03, 113.03 | Flavonoids |
2.2 Molecular docking screening of bitter substances in Cistanche deserticola
2.2.1 Construction of ligand database
According to the chemical components of Cistanche deserticola identified in 2.1.4, their SDF structures were downloaded from the PubChem database to establish a ligand small molecule compound library. All ligand small molecules were structurally optimized using the molecular mechanics program Minimize.
2.2.2 Selection of Bitter Receptors
The bitter taste receptor (TAS2R) is a member of the GPCRs family and mediates the bitter taste perception process of taste bud cells on the tongue. There are 25 receptor subtypes. Among them, TAS2R1, TAS2R7, TAS2R8, and TAS2R14 can be activated by a variety of bitter substances and belong to the broad-spectrum bitter perception receptors [14-15]. Therefore, this study selected TAS2R1, TAS2R7, TAS2R8, and TAS2R14 bitter taste receptor proteins for docking with the chemical components of Cistanche deserticola, and the bitter protein crystal structure was obtained from the Alphafold database.
2.2.3 Molecular docking
The chemical component of Cistanche deserticola identified by UPLC-Q-Orbitrap HRMS was used as a ligand, and the bitter taste receptor subtypes TAS2R1, TAS2R7, TAS2R8, and TAS2R14 were used as protein receptors. The molecular docking was performed using the AutoDock Vina software. The smaller the docking score, the better the binding energy. The receptor protein was pre-processed using Auto Dock Tools (ADT), including dehydration and hydrogenation. The ligand was also processed using ADT, including hydrogenation, charge calculation, detection, and selection of rotatable bonds. The active site of the receptor was determined using ADT, and the docking box was set to ensure that the box size was sufficient to contain the active site. After the docking was completed, the visualization software PyMOL was used to view and analyze the docking results. In order to reduce the false positive results of molecular docking, this study used the docking score of bitter positive ligand quinine as the screening criterion and selected chemical components that bind well to at least two bitter receptors as the bitter substances of Cistanche deserticola. As a result, a total of 20 chemical components were potential bitter substances, including 6 phenylethanoid glycosides, 5 flavonoids, 3 phenolic acids, 2 cyclopentadiene ether terpenes, 2 alkaloids and 2 other components, indicating that phenylethanoid glycosides and flavonoids are the main bitter substances in Cistanche deserticola. The docking scores are shown in Table 3.
Table 3 Molecular docking scores of bitter presenting substances of Cistanche deserticola extracts with bitter taste receptors kcal·mol-1
| No. | Bitter Substance | TAS2R1 | TAS2R7 | TAS2R8 | TAS2R14 |
|---|---|---|---|---|---|
| 1 | Prunasin | -7.6 | -6.6 | -7.8 | -7.1 |
| 2 | Trifolirhizin | -8.8 | -7 | -8.7 | -8.5 |
| 3 | Benzoic Acid | -7.9 | -6.9 | -8.2 | -7.1 |
| 4 | Vitexin | -7.6 | -6.8 | -8.5 | -7.1 |
| 5 | Trifolin | -8.4 | -7.5 | -8.2 | -7.7 |
| 6 | Isoquercitrin | -9.2 | -7.5 | -8.1 | -7.8 |
| 7 | Isomenthone | -7.3 | -6.7 | -7.4 | -6.9 |
| 8 | Curcumin | -9.2 | -8.1 | -8.3 | -7.4 |
| 9 | Dehydroacetic Acid | -6.6 | -6 | -7.8 | -7.9 |
| 10 | Ginsenoside | -7.3 | -7.9 | -8.8 | -8.3 |
| 11 | Piperine | -8.8 | -7.9 | -7.9 | -7.9 |
| 12 | Epicatechin | -9.8 | -6.9 | -9.1 | -8.7 |
| 13 | Isoquercitrin (again) | -8.1 | -8 | -8.2 | -8 |
| 14 | Rhein | -8.7 | -7 | -8.9 | -8.5 |
| 15 | Isosalicylic Acid | -8.7 | -6.5 | -8.3 | -9.3 |
| 16 | Pinocembrin | -9.1 | -7.6 | -8.1 | -8.6 |
| 17 | Eriocitrin | -7.9 | -7.6 | -8.2 | -8.4 |
| 18 | Genistin | -8.1 | -8.2 | -9.1 | -8.2 |
| 19 | Kaempferol | -8.4 | -8.1 | -9.1 | -8.4 |
| 20 | Isoquercitrin (third mention) | -8.2 | -8.4 | -7.9 | -8.7 |
2.3 Electronic tongue evaluation of the bitterness of Cistanche deserticola
2.3.1 Signal acquisition
Refer to the literature method and optimize it [16]. The parameters are: collection temperature 25 ℃, data collection time 120 s, collection period 1 s, stirring speed 1 r·s-1, using ultrapure water as the cleaning fluid, before each sample measurement Clean the sensor for 10 seconds.
2.3.2 Sample determination
Weigh 1.0 g of Cistanche deserticola sample powder respectively, weigh it accurately, add 80 mL of water, reflux and extract for 1 h, let cool, centrifuge, filter, take 40 mL of filtrate, add distilled water to make the volume to 100 mL. Place the prepared sample into a 100 mL standard beaker of the instrument and perform the measurement. Repeat the measurement three times for each sample.
2.3.3 Bitterness evaluation results using electronic tongue
After methodological inspection, the relative standard deviation (RSD) of the precision, repeatability, and 36-h stability experiments were 1.7%, 3.6%, and 1.4% respectively, all less than 5.0%, indicating that this method is feasible. The quantitative bitterness values of different Cistanche deserticola samples ranged from 8.856 to 27.32, as shown in Table 4, with significant differences.
3 Discussion
In this study, 69 chemical components were identified in Cistanche deserticola using UPLC-Q-Orbitrap HRMS technology, and 20 bitter substances that stimulate bitter taste receptors in Cistanche deserticola were screened out by combining molecular docking technology, including 6 phenylethanol glycosides, 5 flavonoids, 3 phenolic acids, 2 iridoids, 2 alkaloids and 2 other components, including echinacoside, verbascoside, tubuloside A, isoverboscoside, geniposide, etc.; 9 batches of fresh Cistanche deserticola samples from the same origin in different months were collected and divided into different months and different parts. The electronic tongue method was used to determine the quantitative value of the bitterness of the samples, and HPLC was used to determine the 6 potential bitter pharmacological components (echinacoside, verbascoside, tubuloside) in the samples. A, iso-verbascoside, jinshicanoside, geniposide) content, and UV-visible spectrophotometry was used to determine the total phenylethanoid glycosides, total polysaccharides, total alkaloids, total flavonoids, and total phenolic acid content in the samples. On this basis, combined with chemometric analysis, Pearson correlation analysis, grey correlation analysis, OPLS-DA, etc. were used to find the bitter components in Cistanche deserticola. The correlation analysis results showed that the content of total phenylethanoid glycosides and total flavonoids in Cistanche deserticola was positively correlated with the bitter response value, and the total polysaccharides of Cistanche deserticola were negatively correlated with the bitter response value. Among them, the content of phenylethanoid glycoside components echinacoside, verbascoside, and jinshicanoside was significantly positively correlated with the bitter response value, which was consistent with the molecular docking results, verifying the accuracy of the molecular docking results. The two methods supported each other and provided a reference for the subsequent debittering and flavor correction of Cistanche deserticola.
Through network pharmacology prediction, 245 bitter substance targets were obtained, and the key targets were EGFR, PIK3CB, PTK2, PIK3CD, PIK3R1, PTPN11, IK3CA, SRC, JAK2, PDGFRB, etc.; the KEGG pathway and GO function enrichment results of the bitter substance targets showed that insulin resistance, the role of PI3K-AKT signaling pathway in diabetic complications, and AGR-RAGE signaling pathway in diabetic complications were the key pathways of the bitter substance targets of Cistanche deserticola. A total of 267 GO functions were obtained for the bitter substance targets. Including 123 biological processes, 75 molecular functions and 69 cell components; by performing Wayne analysis on the bitter substance targets and the disease targets of the bitter effect of Cistanche deserticola, the results showed that 54.69%, 65.30%, 48.16%, 40.40%, 20.40% and 19.59% of the bitter substance targets may have bitter effects of anti-diabetic nephropathy, neuroprotection, immunomodulation, anti-oxidation, anti-aging and anti-osteoporosis. This shows that the bitter substances in Cistanche deserticola have a certain material basis for bitter effects.
In this study, the extract of Cistanche deserticola was used as the research object, and the bitter substances in Cistanche deserticola were screened out by UPLC-Q-Orbitrap RMS and molecular docking; HPLC was used to determine the content of the main bitter medicinal components, and UV-visible spectrophotometry was used to determine the content of each major category of bitter substances. The electronic tongue was used to evaluate the degree of bitterness, and the bitter components were verified by chemometrics. Modern technical research methods were fully utilized to explore the taste information of Cistanche deserticola and reveal its bitter material basis, which will provide a reference for the subsequent debittering and flavor correction of Cistanche deserticola. Through the study of network pharmacology, the mechanism of action of bitter substances in Cistanche deserticola and its relationship with the target of bitter efficacy were preliminarily explored, which will provide ideas and references for the future study of the "taste-effect" relationship of the five flavors of Chinese medicine. However, there are still some shortcomings: this experiment only selected plants with good quality in the traditional harvest season to measure the bitter substance content and bitterness of different parts. There is a certain degree of insufficient sample size, and the results of the differences in bitterness of different parts lack certain reliability. It is planned to use large-scale samples for verification and conduct more systematic research in the future; it is generally believed that alkaloid components are bitter, but the correlation analysis of this experiment showed that alkaloids and bitterness are negatively correlated, which may be because the alkaloid component in Cistanche deserticola is mainly betaine, and molecular docking shows that it binds poorly to bitter receptors. In addition, the bitter results screened by molecular docking lack specific bitterness quantitative evaluation experimental verification data, and the screening results may have false positives, and further research and exploration of the experiment is needed.
