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Article Traditional Medicine Research 2018 May; 3(3): 115-130 Modernization of Traditional Medicine

Screening for 2 inhibitors from natural compounds of Radix Glycyrrhizae using computer simulation

Ming Yang1, 2, Yi Jin1, 2, Li-Ping Yang1*

1Department of Pharmacy of Beijing Hospital, National Center of Gerontology, Assessment of Clinical Drugs Risk and Individual Application Key Laboratory; National Clinical Research Center of Respiratory Diseases, Beijing, China. 2School of Life Sciences and Biopharmaceuticals, Shenyang Pharmaceutical University, Shenyang, China.

*Correspondence to: Li-Ping Yang, Department of Pharmacy, Beijing Hospital, Assessment of Clinical Drugs Risk and Individual Application Key Laboratory, National Clinical Research Center of Respiratory Diseases, Beijing, China. E-mail: [email protected].

Highlights Editor’s Summary Besides the well-known components which possess Chinese herb Gancao (Radix Glycyrrhizae) the glucocorticoids-like anti-inflammatory effect of possesses both the steroid-like and non-steroid anti- Chinese herb Gancao (Radix Glycyrrhizae), there inflammatory effects, which reflects the multi- are still other components which possess the targets characteristics of the role of Chinese herb in NSAIDs-like anti-inflammatory effect and their the treatment of disease. underling mechanism is related to the inhibition on COX-2.

Citation: Yang M, Jin Y, Yang LP. Screening for cyclooxygenase 2 inhibitors from natural compounds of Radix Glycyrrhizae using computer simulation. Traditional Medicine Research 2018, 3(3): 115-130. DOI: 10.12032/TMR201811070 Submitted: 13 April 2017, Accepted: 23 November 2017, Online: 1 May 2018.

Submit a manuscript: http://www.tmrjournals.com TMR | March 2018 | vol. 3 | no. 3 |115 Article Traditional Medicine Research 2018 May; 3(3): 115-130

Abstract Objective: To explore the molecular basis of the effects of Gancao (Radix Glycyrrhizae, GC) on inflammation through the inhibition of cyclooxygenase 2 (COX-2). Methods: The Discovery Studio 4.5 System was used to predict the physicochemical properties of GC molecular compounds. The Ligand Profiler was used to screen for natural GC components that could combine with the COX-2 pharmacophores. The AutoDock Vina 1.1.2 software was used for the molecular docking of the natural GC components with the COX-2 protein. Results: The aromatics were the closest to the non-steroidal anti-inflammatory drugs in terms of the three properties, namely molecular weight, molecular surface area, and molecular solubility, followed by the flavonoids; whereas the terpenoids/saponins differed most from the non-steroidal anti-inflammatory drugs in terms of the three properties; and the aliphatics were inconsistent. One hundred and eighteen small molecules were obtained through the pharmacophore screening using GC. The molecular binding energy (MBE) results demonstrated that the MBE value of the flavonoids/aromatics, obtained from their binding with the COX-2 protein, was lower than that obtained from their binding with the substrate, metabolism of , whereas the MBE value of the aliphatics/terpenoids, obtained from their binding with the COX-2 protein, was higher than that obtained from their binding with the substrate, arachidonic acid. Finally, further filtration, based on the physicochemical properties and the molecular binding energies of the small molecules, was carried out. Forty-two natural GC components, including 35 flavonoid and 7 aromatic constituents, with low binding energies and potential inhibitory effects on COX-2, were screened. Conclusion: Using the three-step program, pharmacophore screening, molecular docking, and physicochemical properties analysis, we screened out 35 flavonoid molecules and 7 aromatic molecules, which may be potential COX-2 inhibitors, from GC. Two of the 35 flavonoid molecules (licochalcone A and glabridin) have been confirmed in the laboratory to have inhibitory effects on COX-2. Our findings provide a material basis for the development of non-steroidal GC drugs. Keywords: Radix Glycyrrhizae, COX-2, Pharmacophores, Molecular docking, Physicochemical properties

摘要

目的：探索甘草通过抑制环氧酶-2（COX-2）发挥抗炎作用的分子基础。 方法：运用Discovery Studio 4.5系统预测化合物的分子性质，运用Ligand Profiler筛选与COX-2药效团作用 的甘草天然成分；运用AutoDock Vina 1.1.2软件进行甘草天然成分与COX-2蛋白的分子对接。 结果：甘草中的芳香族在分子量、分子表面积和分子溶解度这三方面与非甾体类抗炎药物（NSAIDs）最 为接近，其次是黄酮，萜类/皂苷类则相差最大，而脂肪族类波动较大；将513个甘草小分子与59个COX-2 药效团进行模拟结合，筛选出了118个小分子（FitValue > 0.5）；用这118个甘草成分（A组）与40个NSAIDs （B组）与COX-2蛋白（1CVU）对接后发现，COX-2蛋白与甘草中的黄酮类和芳香族的结合能较低，而 与脂肪族和萜类的结合能较高；结合分子量、分子表面积、分子溶解度的预测值，最终筛选出结合能低 的、对COX-2酶具有潜在抑制作用的42个甘草天然成分，包括35个甘草黄酮成分和7个芳香族成分，其中 有2个黄酮成分已被实验证实对COX-2酶具有选择性抑制作用。 结论: 采用药效团初筛、分子对接再筛、结合小分子的理化性质精筛的三步曲方案，可以从中药天然成 分中筛选出已知靶标（如COX-2酶）的潜在抑制剂。 关键词：甘草；COX-2；药效团；分子对接；理化性质

Abbreviations: GC, Gancao (Radix Glycyrrhizae); AA, Arachidonic acid; COX-2, Cyclooxygenase 2; NSAIDs, Non-steroidal anti-inflammatory drugs; DS 4.5, Discovery Studio 4.5; MW, Molecular - Weight; MSA, Molecular - Surface - Area; MS, Molecular - Solubility; RMSD, Root mean square deviation; MBE, Molecular binding energy; MV, Median value; PGE2, E2. Funding: This work was supported by Building Project of National Clinical Priority Specialty for Clinical Pharmacy. Competing interests: The authors declare that there is no conflict of interests regarding the publication of this paper. Copyright: © 2018 TMR Publishing Group Limited. This is an open access article distributed under the terms of the Creative Commons Attribution Non Commercial License. Executive Editor: Cui-Hong Zhu.

Submit a manuscript: http://www.tmrjournals.com TMR | March 2018 | vol. 3 | no. 3 | 116 Article Traditional Medicine Research 2018 May; 3(3): 115-130

Studio 4.5 (DS 4.5, San Diego, CA, USA) System, and Background AutoDock Vina software (Scripps Research Institute). Unless specified, the calculation process was carried out Chinese herb Gancao (Radix Glycyrrhizae, GC) is the dry with default values. root and the rhizome of Glycyrrhiza species. It is used by Chinese medicine doctors to invigorate and regenerate the The natural ingredients of GC and NSAIDs spleen, clear heat and relieve toxicity, relieve cough and Six hundred and fifty-three natural GC components were sputum production, and to control pain [1]. The history of obtained in our previous studies; however, only 513 of the application of GC in traditional Chinese medicine can those components, including flavonoids (166), terpenoids be traced back to the ancient book named and saponins (114), aliphatic compounds (172), and Shennongbencaojing of the Han Dynasty of China (the aromatic compounds (61), were included in this study. third century A.D.). Besides, among the 113 prescriptions The inflammatory pathways of the 40 NSAIDs were of Shanghanlun, which were written by Zhang Zhongjing, obtained from the KEGG website (http://www.kegg.jp/). more than half of the prescriptions contained GC. The SDF three-dimensional formats of the 513 GC Current researches have reported that GC has several components and those of the 40 NSAIDs were either pharmacological effects, including anti-inflammatory downloaded from the PubMed database or generated with effects, antiviral effects, liver protective effects, the DS 4.5 software. Finally, the Minimize Ligands antitussive effects, and detoxification effects [2-7]. GC module of the DS 4.5 software was used to optimize the protects against both acute and chronic inflammations [8]. energy of the small molecular ligands, which were used The most widely used GC preparations in clinical practice for the screening and docking of the pharmacophores. include compound licorice tablets, compound licorice mixtures, glycyrrhizin tablets, glycyrrhizin injections, and Pharmacophore screening monoammonium glycyrrhizinate [9]. They are mainly Using the Ligand Profiler module, under the Prepare used as antitussives and hepatoprotective drugs. The Ligands program in DS 4.5, the small molecular ligands mechanism of action of these licorice preparations is not were docked with the pharmacophores corresponding to yet clear, but their actions are attributed to their the COX-2 protein in the PharmaDB database, and the anti-inflammatory effects. BEST parameter was chosen to generate the lowest Inflammation is involved in the mass production of energy conformations with an energy threshold of 20 -1 leukotriene and , related to the metabolism kcal∙mol . When the Fit Value was high, the compounds of arachidonic acid (AA). A variety of inflammatory combined better with the pharmacophore, so we chose the factors induced lipoxidase and cyclooxygenase 2 (COX-2) small molecules with Fit Values of more than 0.5 to carry to converts AA into leukotriene and prostaglandins, on with the molecular docking. resulting in a series of clinical symptoms of inflammation [10]. The commonly used anti-inflammatory drugs in Preparation of target protein (COX-2) receptor clinic main contain steroidal anti-inflammatory drugs and We selected the 1CVU, which is the crystal structure of non-steroidal anti-inflammatory drugs (NSAIDs). the AA substrate bound to the COX-2 protein, as the Previous studies on the anti-inflammatory effects of GC docking receptor for the AutoDock Vina. The components focused on the terpenes and saponins of GC, three-dimensional crystal structure (PDB ID: 1CVU) of including glycyrrhizic acid (also named glycyrrhizin), COX-2 was downloaded from the Protein Database (PDB) glycyrrhetinic acid and oleanolic acid, etc. Their chemical and processed with the AutoDock Vina software. The structures were similar to that of steroid and had structure of COX-2 was optimized by removing excess glucocorticoids-like anti-inflammatory effect by protein conformations, deleting ligands, removing water inhibiting lipoxidase and COX-2 products [11-14]. molecules, adding charges, and hydrogenating atoms. It Except for terpenes and saponins, GC also contains many was then stored in a PDBQT format. other components such as flavonols. Though their chemical structures differed totally from that of steroid, Molecular docking by AutoDock Vina software they also possessed the powerful anti-inflammatory The GC small molecules, obtained from the effects [15, 16]. To explore whether the other components pharmacophore screening, were combined with the of GC possess the NSAIDs-like anti-inflammatory effect COX-2 protein (1CVU), together with the NSAIDs, using and their underling mechanism is related to the inhibition the AutoDock Vina version 1.1.2 software on COX-2, the present study designed a screening route (http://vina.scripps.edu/). According to the literature, the and method to select the bioactive components of GC and center of the COX-2 active site was set to center_x = 27.7, further explore their anti-inflammatory mechanism using center_y = 24.6, and center_z = 46.9, and the size of the the three-step program, pharmacophore screening, active site was set to size_x = 24, size_y = 24, and size_z molecular docking, and physicochemical properties = 24. The parameters were set to num_modes = 10 and analysis. exhaustiveness = 50. The other parameters were set to default values. The virtual binding energy was calculated Methods with the Lamarckian genetic algorithm and Autodock Vina software. When the docking binding energy was low, All the researches were carried out using the Discovery the affinity of the compound for the target was high,

Submit a manuscript: http://www.tmrjournals.com TMR | March 2018 | vol. 3 | no. 3 | 117 Article Traditional Medicine Research 2018 May; 3(3): 115-130 indicating that the inhibitory effect of small molecules on Prediction of results of physicochemical properties for large molecules is very strong. GC compounds and NSAIDs The MW, MSA, and MS of the GC components (n = 513, Comparison of physical and chemical properties using including aliphatics, flavonoids, aromatics, and DS 4.5 software terpenoids/saponins) and the marketed NSAIDs (n = 40) Calculation of the physicochemical properties of the 513 were predicted by the molecular properties modules of GC components and the 40 NSAIDs was with the DS 4.5 the DS 4.5 software (Table 1). software. The operation steps are as follows: selected The values of the aliphatics (MW = 184.4, MSA = Molecular Properties → Molecular - Solubility (MS) → 239.5) and those of the terpenoids/saponins (MW = 468.7, Molecular - Weight (MW) → Molecular - Surface - Area MSA = 532.8) differed from those of the NSAIDs (MW = (MSA). Parameters were set to default values. 293.3, MSA = 277) in terms of MW and MSA. The Note: MW is the sum of the atomic weights of all median value (MV) of the aliphatics (MV = 184.4) was atoms in a molecule. MSA refers to the total surface area less than that of the NSAIDs (MV = 293.3). The MV of of each molecule. MS refers to the solubility of a the terpenoids/saponins (MV = 468.7) was far greater component. The solubility was greater when the absolute than that of the NSAIDs (MV = 293.3). Otherwise, the MS value was smaller. values of the aromatics (MW = 278.3, MSA = 281.9) were similar to those of the NSAIDs, and the values of Results the flavonoids (MW = 354.4, MSA = 360.2) were a little higher than those of the NSAIDs. The overall value of the aromatics was smaller than that of the flavonoids.

Table 1 Prediction of results of physicochemical properties for GC compounds and NSAIDs

Compound number Overall range Median value

MW

NSAIDs 40 137.1~844.4 293.3

Aliphatics 172 32~507 184.4

Aromatic 61 92.1~422.5 278.3

Flavonoids 166 222.2~726.7 354.4

Terpenoids/Saponins 114 134.2~1028.1 468.7

MSA

NSAIDs 40 139.1~894.8 277

Aliphatics 172 54.2~653.1 239.5

Aromatic 61 113.3~442.7 281.9

Flavonoids 166 213.2~681.4 360.2

Terpenoids/Saponins 114 160.5~1028 532.8

MS

NSAIDs 40 -11.2 ~ -1.1 -4.5

Aliphatics 172 -19 ~ 0.7 -4.9

Aromatic 61 -7 ~ 1.3 -3.7

Flavonoids 166 -9.6 ~ -0.9 -3.7

Terpenoids/Saponins 114 -11.8 ~ -0.7 -7.8 GC, Gancao (Radix Glycyrrhizae); NSAIDs, Non-steroidal anti-inflammatory drugs; MW, Molecular - Weight; MSA, Molecular - Surface - Area; MS, Molecular - Solubility.

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The median MS value of the terpenoids/saponins (MS = 0, Fit Value > 0, and Fit Value > 0.5, were 6259, 1326, = -7.8) was much smaller than that of the NSAIDs (MS = and 680 respectively. The duplicate items of the small -4.5), and the median MS value of the aliphatics (MS = molecules in the 680 results were subtracted and 118 -4.9) was the same as that of the NSAIDs, whereas the small molecules with a Fit Value of > 0.5 were obtained. median MS values of the aromatics and flavonoids (MS = The 118 GC small molecules consisted of 43 aliphatics, -3.7, MS = -3.7) were both slightly higher than that of the 44 flavonoids, 24 aromatics and 7 terpenoids/saponins. NSAIDs. However, the variation in the MS value of the The 118 GC small molecules were used for the following aliphatics was the highest, from +0.7 to -19. molecular docking and physicochemical properties These results indicated that the aromatics were the analyses. closest to the NSAIDs in terms of the three properties Further filtration by molecular docking with COX-2 (MW, MSA, and MS), followed by the flavonoids. The crystal (1CVU). The ligand molecule, AA, was docked terpenoids/saponins differed most, and there were into the binding site of the COX-2 protein using the inconsistencies in the values of the aliphatics. Autodock Vina software (Figure 1). The root mean square deviation (RMSD) values of the ligand molecules and the Pharmacophore screening and molecular docking original ligand molecules were used to determine the Screening potential inhibitors of GC components with rationality of the parameter settings and the suitability of COX-2 pharmacophores. The DS 4.5 PharmaDB the procedure for the protein receptor-ligand complex. database contains 8 different crystals of the COX-2 The RMSD value for the docking of the AA into the protein (3mqe, 3rr3, 3q7d, 1pxx, 3ntg, 3ln0, 3ln1, 4fm5) COX-2 protein was 1.62 Å (Figure 1). It is generally and a total of 59 pharmacophores. Using the Prepare believed that RMSD ≤ 2 Å indicates that the docking Ligands program in the DS 4.5 software, the 513 GC results and the original conformation have overlapped small molecules were combined with the 59 very well, and that the design of the docking parameters pharmacophores and 7585 results were obtained; the was reliable and feasible. corresponding figures for the various Fit values, Fit Value

A B C

D E F

Figure 1 Active site of COX-2 crystal (1CVU) and the interaction with arachidonic acid A, COX-2 crystal (1CVU) by ban; B, COX-2 crystal (1CVU) by stick; C, Active site of COX-2 crystal (1CVU); D, Arachidonic acid, Yellow: original morphology, Grey: docking morphology; E, Arachidonic acid: interacting with the 1CVU crystal in active site of COX-2. F, Arachidonic acid: after docking with COX-2 crystal (1CVU). COX-2, Cyclooxygenase 2. Table 2 Group by binding energy of GC compounds and NSAIDs with COX-2 Groups GC compounds (n = 118) NSAIDs (n = 40) Group 1: Low binding energy1 A1 (n = 59) B1 (n = 34) Group 2: High binding energy2 A2 (n = 59) B2 (n = 6) 1: Binding energy of compounds is lower than arachidonic acid binding energy; 2: Binding energy of compounds is higher than arachidonic acid binding energy. GC, Gancao (Radix Glycyrrhizae); COX-2, Cyclooxygenase 2; NSAIDs, Non-steroidal anti-inflammatory drugs.

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The 118 GC components, screened with the binding with the COX-2 protein, was lower than that pharmacophores, were classified as group A small obtained from their binding with the AA, which may have molecules and the 40 NSAIDs were classified as group B a good inhibitory effect on the COX-2 protein. However, small molecules, which were docked with the the MBE value of the aliphatics/terpenoids, obtained from macromolecules, COX-2 crystal protein (1CVU) and AA, their binding with the COX-2 protein, was higher than respectively . We obtained 158 molecular binding energy that obtained from their binding with the substrate, AA, (MBE) values. Among them, the MBE value of the AA which may have a less inhibitory effect on the COX-2 and 1CVU was -8, which was used as the reference value. protein. The docking pattern of COX-2 and the small The two groups, A and B, were subdivided into groups A1 molecules is shown in Figure 2. and B1, with a MBE value lower than the reference value, and groups A2 and B2, with a MBE value higher than the Further filtration based on the physicochemical reference value. The results showed that there were 59 properties and the MBE of the small molecules GC components in the A1 group, including 43 flavonoids, The 118 GC components and the 40 NSAIDs were 14 aromatics, 1 aliphatic, and 1 terpenoid; the A2 group grouped into four ranges, including small range, medium also contained 59 GC components, including 42 range, large range, and extra-large range according to the aliphatics, 10 aromatics, 1 flavonoid, and 6 terpenoids. MW, MSA, and MS (small range MW < 200, MSA < 200, The NSAIDs were mainly found in group B1 (34); and MS > -3; medium range MW: 200 to 300, MSA: 200 however, six of them (, , , to 300, MS: -4 to -3; large range MW: 300 to 400, MSA: acetaminophen, , and ) 300 to 400, MS: -6 to -4; and extra-large range MW > were found in group B2 (Table 2). 400, MSA > 400, MS < -6). The analysis and statistics From the results of the MBE determination, the MBE were carried out according to molecular docking results value of flavonoids/aromatics, obtained from their (Table 3). xxxxxx Table 3 GC components and NSAIDs were grouped according to their physicochemical properties Medium range Super large range Groups Properties Small range group Large range group group group MW < 200 200 ~ 300 300 ~ 400 > 400 MSA < 200 200 ~ 300 300 ~ 400 > 400 MS > -3 -4 ~ -3 -6 ~ -4 < -6 GC properties (59) N = 8 N = 26 N = 45 N = 11 Group A1 MW N = 0 N = 13 N = 39 N = 7 with low binding MSA N = 0 N = 13 N = 39 N = 7 energy MS N = 8 N = 21 N = 23 N = 7 Satisfying three properties N = 0 N = 10 N = 22 N = 3 NSAIDs properties (34) N = 0 N = 24 N = 27 N = 5 Group B1 MW N = 0 N = 20 N = 11 N = 3 with low binding MSA N = 0 N = 19 N = 12 N = 3 energy MS N = 0 N = 8 N = 22 N = 4 Satisfying three properties N = 0 N = 5 N = 5 N = 2 GC properties (59) N = 22 N = 39 N = 33 N = 30 Group A2 MW N = 20 N = 25 N = 8 N = 6 with high binding MSA N = 9 N = 21 N = 21 N = 8 energy MS N = 12 N = 7 N = 11 N = 29 Satisfying three properties N = 7 N = 0 N = 1 N = 5 NSAIDs properties (6) N = 4 N = 2 N = 1 N = 0 MW N = 4 N = 1 N = 1 N = 0 Group B2 MSA N = 4 N = 1 N = 1 N = 0 with high binding MS N = 4 N = 2 N = 0 N = 0 energy Satisfying three properties N = 4 N = 1 N = 0 N = 0 GC, Gancao (Radix Glycyrrhizae); NSAIDs, Non-steroidal anti-inflammatory drugs; MW, Molecular - Weight; MSA, Molecular - Surface - Area; MS, Molecular - Solubility.

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A B C

D E F

G H I Figure 2 The amino acid residues binding with GC compounds or NSAIDs in COX-2 active site A, Arachidonic acid; B, Ibuprofen; C, ; D, Glycycoumarin; E, Licochalcone A; F, Glypallichalcone; G, Licochalcone B; H, Glabridin; I, Homopterocarpin. GC, Gancao (Radix Glycyrrhizae); NSAIDs, Non-steroidal anti-inflammatory drugs; COX-2, Cyclooxygenase 2.

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The results showed that the GC components were energies were between 300 and 400, and the molecular mainly distributed in the large-range group, followed by solubility was between -6 and -4. the medium range group, small range group, and The 34 NSAIDs in group B1 were in the medium, large extra-large range group. Flavonoids mainly fell to group range, and extra-large range groups, and the 6 NSAIDs in A1, whereas aliphatics mainly fell to group A2. The group B2 were in the small range group (Figure 3, Table aromatics mainly fell to the large range and extra-large 4). Most of them were in the following ranges; MW < group, A1, and the terpenoids were predominantly found 400, MSA < 400, and MS > -6. In the 59 GC components in the extra-large range group, A2. If the binding energy with low COX-2 binding energy, the MW and MSA of values had been used to predict the inhibitory effect, the the 35 flavonoids were distributed in the range of 200 ~ inhibitory effects of aliphatics and terpenoids on COX-2 400 and the MS was in the range of -6 ~ -3. The MW and would have been weaker, and the inhibitory effect of MSA of the 7 aromatic components were distributed in flavonoids and some aromatics on COX-2 would have the range of 300-400 and the MS was in the range of -6 ~ been stronger. The MW and MSA of the flavonoids with -4. Thus, we predicted that the 35 flavonoids and 7 low binding energies were between 200 and 400, and the aromatics may have a strong inhibitory effect on COX-2 molecular solubility was between -5 and -3. The MW and (Figure 4, Table 5). MSA of the aromatic molecules with low binding xxxxxxx

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Figure 3 The structures of 40 non-steroidal anti-inflammatory drugs

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Figure 4 The structures of 42 Gancao (Radix Glycyrrhizae) compounds

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Table 4 Prediction of physicochemical properties and binding energy with COX-2 of NSAIDs

Name MBE value MS MW MSA Indomethacin farnesil -11 -11.19 562.139 618.89 Celecoxib -10.8 -6.441 381.372 352.62 -9.8 -4.343 314.356 297.7 -9.8 -5.254 339.342 345.67 -9.8 -5.768 357.788 351.28 -9.7 -5.288 314.359 301.28 -9.7 -6.571 358.842 347.55 -9.7 -6.003 415.824 407.07 -9.5 -5.86 356.411 352.46 -9.3 -4.944 298.356 285.94 -9.2 -5.316 293.317 295.41 -9.2 -4.788 281.23 262.21 -9.1 -3.768 255.269 249.14 axetil -9 -5.745 330.35 345.67 -9 -5.372 296.149 276.15 -8.9 -4.315 241.285 256.14 -8.9 -4.579 261.704 255.45 Ibuprofen piconol -8.9 -5.686 297.391 334.17 -8.8 -4.394 254.281 262.36 -8.7 -3.886 371.819 327.02 -8.6 -3.578 250.198 234.03 -8.5 -3.302 331.346 309.42 -8.5 -3.607 255.269 259.57 -8.5 -3.978 257.284 276.97 -8.5 -5.192 296.149 272.61 -8.4 -4.507 447.462 437.01 Flurbiprofen -8.4 -4.63 244.261 248.68 -8.3 -4.659 228.286 251 -8.3 -4.039 242.27 249.02 -8.3 -4.291 260.308 259.22 -8.3 -4.515 287.354 301.34 -8.2 -4.061 230.259 242.53 -8.1 -3.14 337.374 306.77 -8.1 -3.933 246.302 256.33 Ibuprofen -7.8 -3.876 206.281 240.17 Meloxicam -7.7 -3.631 351.401 327.71 Aspirin -6.5 -1.889 180.157 185.08 Acetaminophen -6.4 -1.385 151.163 163.09 Salicylamide -6.2 -1.107 137.136 144.75 Sodium salicylate -6 -1.072 138.121 139.07 MW, Molecular - Weight; MSA, Molecular - Surface - Area; MS, Molecular - Solubility; MBE, Molecular binding energy; NSAIDs, Non-steroidal anti-inflammatory drugs; COX-2, Cyclooxygenase 2.

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Table 5 The prediction of physicochemical properties and binding energy with COX-2 of GC molecules including 35 flavone and 7 aromatic molecules with potential inhibitory effect on COX-2 NO. Name Category MBE MS MW MSA Arachidonic acid ( Reference) COX-2 substrate -8 -8.075 303.459 377.58 35 Flavonoids 1. Medicarpene Pterocarpans -10.3 -4.179 354.396 351.5 2. Licobenzofuran Flavonols -10.2 -5.39 366.407 385.78 3. Uralenol-3-methylether Flavones -10.2 -3.661 386.395 387.35 4. Phaseollinisoflavan Isoflavans -10.1 -4.641 324.37 319.73 5. Glabranin Flavanones -10 -4.213 324.37 330.57 6. Gancaonol C Isoflavans -10 -4.68 354.396 355.09 7. Licoflavone C Flavones -9.8 -3.916 338.354 337.68 8. 4'-O-Methylglabridin Isoflavans -9.7 -5.352 338.397 343.8 9. Glepidotin B Flavanones -9.6 -3.912 340.37 341.38 10. Licorisoflavan B Isoflavans -9.6 -3.546 340.37 344.25 11. 1-Methoxyphaseollidin Pterocarpans -9.6 -4.171 354.396 351.5 12. Neouralenol Flavones -9.5 -3.06 370.353 360.24 13. 2-[3,4-dihydroxy-2-(3-methylbut-2-enyl)pheny Flavonols -9.5 -3.06 370.353 360.24 l]-3,6,7-trihydroxychromen-4-one 14. Licochalcone D Chalcone -9.5 -4.91 354.396 380.61 15. 3'-Methoxyglabridin Isoflavans -9.4 -4.694 354.396 355.06 16. Glabridin Isoflavans -9.3 -4.736 324.37 319.73 17. 7-Hydroxy-2-methyl-3-phenyl-chromen-4-one Isoflavones -9.2 -3.433 252.265 245.27 18. Licoisoflavanone Isoflavanones -9.2 -3.452 354.353 338.1 19. Uralene Flavones -9.2 -3.452 384.379 384.31 20. Medicarpin Pterocarpans -9.1 -3.029 270.28 249.06 21. Homopterocarpin Pterocarpans -9.1 -3.641 284.307 273.13 22. Licochalcone A Chalcone -9 -5.839 338.397 374.39 23. 7-Methoxy-4-hydroxyflavonol Flavonols -8.9 -3.085 284.263 270.56 24. Licocoumarone Other flavones -8.9 -4.8 340.37 353.62 25. Dihydrolicoiso Isoflavanones -8.8 -3.087 356.369 353.83 26. Dehydroglyasperin C Isoflavenes -8.8 -4.272 354.396 368.32 27. Afrormosin Isoflavones -8.7 -3.469 298.29 295.27 28. 2H-1-Benzopyran-7-ol, Isoflavenes -8.4 -3.141 270.28 265.87 3-(2-hydroxy-4-methoxyphenyl)- 29. Licoricone Isoflavones -8.4 -4.572 382.406 397.71 30. Isovestitol Isoflavans -8.3 -3.283 272.296 268.37 31. Vestitol Isoflavans -8.3 -3.22 272.296 268.37 32. Glypallichalcone Chalcone -8.2 -3.782 270.28 278.17 33. Isomucronulatol Isoflavans -8.2 -3.204 302.322 303.7 34. Neoisoliquiritin Chalcone -8.2 -3.178 418.394 398.23 35. Licochalcone B Chalcone -8 -3.205 286.279 289.43

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Continue 7 Aromatics 1. Kanzonol U Other aromatic -10.5 -5.156 308.328 302.53 2. Glyinflanis B Other aromatic -10.5 -4.445 338.354 341.39 3. Glyurallin A Other aromatic -9.8 -5.083 352.38 354.62 4. Glycyrin 3-arylcoumarins -9.4 -4.881 382.406 397.11 5. Glycyrrhizol B Other Coumarins -9.1 -5.559 350.365 338.89 6. Glycycoumarin 3-arylcoumarins -9.1 -4.205 368.38 373.05 7. Gancaonol A 3-arylcoumarins -9 -5.147 380.391 381.39 GC, Gancao (Radix Glycyrrhizae); COX-2, Cyclooxygenase 2; MW, Molecular - Weight; MSA, Molecular - Surface - Area; MS, Molecular - Solubility; MBE, Molecular binding energy. Table 6 Amino acid residues interacted with NSAIDs and GC compounds in COX-2 active site NSAIDs Licorice Name Abbreviation Sequence Accumulative frequency Name Abbreviation Sequence Accumulative number with small molecule number frequency with small interaction molecule interaction Valine VAL 349 39 Valine VAL 349 42 Alanine ALA 527 35 Alanine ALA 527 35 Valine VAL 523 29 Valine VAL 523 33 Leucine LEU 352 22 Leucine LEU 352 23 Arginine ARG 120 18 Argnine ARG 120 21 TRP 387 16 Tryptophan TRP 387 18 Serine SER 530 13 Leucine LEU 531 17 Methionine MET 522 11 Isoleucine ILE 345 14 Leucine LEU 359 10 Leucine LEU 359 13 Isoleucine ILE 345 9 Valine VAL 116 11 Leucine LEU 531 9 Leucine LEU 117 11 Serine SER 353 8 Serine SER 353 10 Glycine GLY 526 8 Tyrosine TYR 385 9 Phenylalanine PHE 518 7 Phenylalanine PHE 518 8 Tyrosine TYR 385 7 Phenylalanine PHE 381 8 Valine VAL 116 6 Serine SER 530 8 Leucine LEU 534 5 Methionine MET 522 7 Phenylalanine PHE 381 5 Tyrosine TYR 355 7 Tyrosine TYR 355 4 Leucine LEU 384 6 Phenylalanine PHE 209 3 Histidine HIS 90 5 Histidine HIS 90 3 Tyrosine TYR 348 5 Phenylalanine PHE 205 3 Glycine GLY 526 5 Tyrosine TYR 348 3 Leucine LEU 534 5 Arginine ARG 513 3 Glutamine GLN 192 4 Leucine LEU 117 2 Phenylalanine PHE 209 4 Glutarnine GLN 192 2 Phenylalanine PHE 205 3 Methionine MET 535 2 Valine VAL 344 3 Isoleucine ILE 377 1 Alanine ALA 516 1 Leucine LEU 384 1 Isoleucine ILE 517 1 Methionine MET 113 1 Asparagine ASN 375 1 Valine VAL 344 1 Argnine ARG 513 1 Total 286 Total 339 GC, Gancao (Radix Glycyrrhizae); NSAIDs, Non-steroidal anti-inflammatory drugs; COX-2, Cyclooxygenase 2.

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We found that only 6 of the 42 small molecules acute phases and paracmasis of the entire inflammation (licochalcone A, glabridin, glycycoumarin, process, and AA is converted to different prostaglandins glypallichalcone, homopterocarpin, licochalcone B) were at different stages of the inflammation process, from reported for the determination of their contents. The PGE2 in acute stage to in paracmasis. content of licochalcone A was in the range of 0.01-14.47 The COX are -generated mg/g, glabridin was 0.2-2.3 mg/g, glycycoumarin was rate-limiting enzymes, having a direct impact on the role 0.01-1.74 mg/g, glypallichalcone was 0.06-0.8 mg/g, of prostaglandins and in inflammation [22]. homopterocarpin was 0.0006-0.09 mg/g, and licochalcone Selective COX-2 inhibitors, such as rofecoxib, celecoxib, B was 0.08-2 mg/g. These data indicated that the role of , valdecoxib, and etherside, predominantly GC was probably derived from the contents of these inhibit the production of COX-2, but have little effects on ingredients. COX-1, resulting in less side effects, such as gastrointestinal bleeding. In this study, we used software Frequency of interaction of small molecules with simulation systems (DS 4.5 and AutoDock Vina) to target amino acid residues at the activity center of COX-2 COX-2, using NSAIDs as references and the crystal The selected 42 GC components and the 40 NSAIDs structure of COX-2 and AA (PDB ID: 1CVU) as a interacted with 31 amino acid residues in the active site of template, to screen all the known active ingredients of GC; COX-2, and these amino acid residues were sorted the active ingredients of GC obtained from our previous according to the number of times of action (Table 6). The study. results showed that the names and sequences of the top 6 First, we calculated the MW, MSA, and MS of the 40 amino acid residues were the same between the 42 NSAIDs and those of the 513 GC small molecules at the licorice components and the 40 NSAIDs; they were in the same time, and compared the physicochemical properties order of valine acid 349, alanine 527, valine 523, leucine of the two groups. The results showed that aromatics 352, arginine 120, and tryptophan 387. Starting from the were close to NSAIDs in terms of the three properties, beginning of the seventh amino acid residue, the order of followed by flavonoids, whereas the aliphatics and interaction between the small molecules and the amino terpenoids/saponins differed from the NSAIDs in terms acid residues used in both Chinese and Western medicine of these three properties. We then screened 118 GC small was slightly different, but the roles of the amino acid molecules that could bind to COX-2. We found that the residues were basically the same. These results implied binding energy values of half of the GC small molecules, that the 42 GC components had a potent inhibitory effect obtained from their binding with the COX-2 protein, were on COX-2. lower than those obtained from their binding with the substrate, AA. Moreover, for the flavonoids and Discussion aromatics, the binding energy values obtained from their binding with the COX-2 protein were lower than those GC is one of the most commonly used Chinese herbal obtained from their binding with the AA. For the medicines in clinical practice, and our previous studies aliphatics and terpenoids, the binding energy values have shown that its main constituents are flavonoids, obtained from their binding with the COX-2 protein were terpenoids/saponins (glycyrrhizic acid - glycyrrhizin, higher than those obtained from their binding with the glycyrrhetinic acid), aliphatics, and aromatic compounds AA. [17]. Clinically, GC is widely used in the treatment of To screen accurately for the possible COX-2 inhibitor liver disease, gastrointestinal disease, enteritis, exogenous GC components, we compared the differences between fever, cough, sore throat, chronic skin diseases, and food the 118 GC small molecules and the 40 NSAIDs in terms poisoning [18]. of binding energy and physicochemical properties. The The COX enzyme has two isoenzymes, COX-1 and results showed that the three physicochemical properties COX-2. COX-1 is a structural enzyme, which exists in of the 35 flavonoids and the 7 aromatics were close to many normal tissues and maintains the integrity of those of the NSAIDs, and the binding energy value normal gastrointestinal mucosa. When it is blocked, it obtained from their binding with COX-2 was also lower leads to serious adverse effects in the gastrointestinal tract. than that obtained from their binding with the substrate, COX-2 is an inducible enzyme in most organs, and its AA; the binding energy values were close to those of level is significantly increased in inflammatory tissues most NSAIDs, particularly those of the selective COX-2 [19, 20]. The expression of COX-2 increased when it was inhibitors. Therefore, we predicted that the 35 flavonoids stimulated with both intracellular and extracellular stimuli, and the 7 aromatics in GC may have a better inhibitory such as lipopolysaccharide, TNF-α, and interleukin-1β. effect on COX-2. COX-2 can promote the transformation of AA to To verify the accuracy of our predictions, we checked . prostaglandin G2 is further converted the existing literature for research on GC ingredients and to prostaglandin H2, followed by the formation of a series found a number of reports about licoflavone, licochalcone of prostaglandins including (PGE2), A, and epoxidizing enzymes. The study by Cui Y et al. prostaglandin D2, prostaglandin F2α, and thromboxane, showed that licochalcone A significantly reduced paw through the actions of synthetase and reductase, and edema induced by carrageenan; licochalcone A could through the processes of dehydration and isomerization significantly inhibit both COX-2 activity and expression [21]. COX-2 is sustained and abundantly expressed in the induced by lipopolysaccharide in murine macrophages

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[23]. The study by Kwon HS et al. showed that 2015. licochalcone A could inhibit inflammatory reactions in 2. Zhang MF, Shen YQ. Advances in studies on macrophages and protect mice from endotoxin shock by Glycyrrhizae Radix et Rhizoma and its active suppressing the generation of nitric oxide and components in anti-inflammation and mechanism. prostaglandin E2. In addition, it could inhibit the Drugs Clinic 2011, 26: 261-267. expression of inducible nitric oxide synthase and 3. Pu JY, He L, Wu SY, et al. Anti-Virus Research of cyclooxygenase [24]. The study by Furuhashi I et al. Triterpenoids in Licorice. Chin J Virol 2013, 29: showed that licochalcone A induces an anti-inflammatory 673-678. effect through the inhibition of COX-2-dependent PGE2 4. Chen YH, Huang MJ, Wang WQ, et al. Comparative production, but it had no effect on COX-1-dependent Study on Major Ingredient Contents and Effect of PGE2 production [25]. Other reports showed the Depressing Transaminase of Licorice from Different relationship between glabridin and COX-2. The study by Sources. Chin J Exp Tradit Med Formulae 2013, 19: Chandrasekaran CV et al. revealed that glabridin 113-116. significantly inhibited PGE2, lipoxygenase, and COX of 5. Yu TF, Tian XD, Li R, et al. Antitussive and human neutrophils (HL-60), whereas isoliquiritigenin expectorant effects of licorice flavonoids, Extractum exerted inhibitory effect against only COX expression, Glycyrrhizae and glycyrrhetinic acid. Chin patent but failed to suppress lipoxygenase expression [26]. drug 1993, 15: 32-33. These reports verified our results, which were predicted 6. He D, Liu FQ, Li HD. Research Progress on with the computer simulation software obtained from detoxification of Glycyrrhiza uralensis. Central South third parties, indicating that the above predictions are Pharm 2009, 7: 927-931. credible. Based on our prediction, licochalcone A and 7. Gao XY, Wang WQ, Wei SL, et al. Review of glabridin were both among the 35 flavonoids which could pharmacological effects of Glycyrrhiza Radixand inhibit COX-2. itsbioactive compounds. Zhongguo Zhong Yao Za The MBE, MW, MSA, and MS of licochalcone A and Zhi 2009, 34: 2696-2700. glabridin were very close to those of the following 8. Dong FY, Wang JJ. Anti-inflammatory mechanism of NSAIDs: zaltoprofen, oxaprozin, florfenic acid, and Glycyrrhetinic acid and its derivatives. J Dalian Med flurbiprofen. GC ingredients, licorice coumarin and Univ 2014, 36: 195-197. homopterocarpin, were close to licochalcone A and 9. Guo HY. Clinical application of Radix glycyrrhizin. glabridin in terms of the four properties indicated above. Clin Med 2010, 30: 109-111. In addition, glypallichalcone and licochalcone B were 10. Wang CY, Kao TC, Lo WH, et al. Glycyrrhizic acid close to the following NSAIDs, naproxen and ibuprofen, and 18beta-glycyrrhetinic acid modulate in terms of the four properties indicated above. The lipopolysaccharide-induced inflammatory response contents of the six GC ingredients mentioned above have by suppression of NF-kappaB through PI3K been reported. The anti-inflammatory effects of most GC p110delta and p110gamma inhibitions. J Agric Food components are derived from these ingredients. Previous Chem 2011, 59: 7726-7733. studies on the anti-inflammatory effects of GC 11. Yu JY, Ha JY, Kim KM, et al. Anti-Inflammatory components focused on steroids, such as glycyrrhizic acid Activities of Licorice Extract and Its Active (also named glycyrrhizin) and glycyrrhetinic acid [11-13]. Compounds, Glycyrrhizic Acid, Liquiritin and This study provides a material basis for the development Liquiritigenin, in BV2 Cells and Mice Liver. of non-steroidal GC drugs. Molecules 2015, 20: 13041-13054. 12. Gujral ML, Sareen K, Phukan DP, et al. Antiarthritic Conclusion activity of glycyrrhizin in adrenalectomised rats. Indian J Med Sci 1961, 15: 624-629. From 513 natural components of GC, we identified 35 13. Huang QC, Wang MJ, Chen XM, et al. Can active flavonoid molecules and 7 aromatic molecules that could components of licorice, glycyrrhizin and inhibit COX-2 activity. Among these, licochalcone A and glycyrrhetinic acid, lick rheumatoid arthritis? glabridin have inhibitory effects on COX-2, and Oncotarget 2016, 7: 1193-1202. glycyrrhizic acid (a terpenoid) has no inhibitory effect on 14. Tsao SM, Yin MC. Antioxidative and COX-2. This conclusion has been verified by other antiinflammatory activities of asiatic acid, studies. The three-step program, pharmacophore glycyrrhizic acid, and oleanolic acid in human screening, molecular docking, and physicochemical bronchial epithelial cells. J Agric Food Chem 2015, properties analysis, is feasible. 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Immunopharmacol 2015, 28: 917-924. 17. Yang M, Jin Y, Yang LP. A systematic summary of natural compounds in Radix Glycyrrhizae. Tradit Med Res 2018, 3: 82-94. 18. Wang Y, Qu CY, Peng XJ. Research progress in pharmacological studies of Glycyrrhiza uralensis Fisch and its derivatives. Gansu Med J 2011, 30: 398-401. 19. Li SJ, Fu ST, Wei W. Application of COX-1 and COX-2,5-LOX inhibitors in prevention and treatment of thrombotic diseases and vascular inflammation. Central South Pharmacy 2005, 3: 111-112. 20. Chen KL, Chen G. Distribution and Action of Natural Cycloxygenase and Lypoxygenase Inhibitor in Traditional Chinese Materia Medica. J South Central Univ Nationalities 2009, 28: 42-46. 21. Wu CY, Chi PL, Hsieh HL, et al. TLR4-dependent induction of vascular adhesion molecule-1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A(2)α/cyclooxygenase-2. J Cellular Physiol 2010, 223: 480-491 22. Yin HY, Zhou YH, Zhu MJ, et al. Role of mitochondria in programmed cell death mediated by arachidonic acid-derived . Mitochondrion, 2013, 13: 209-224. 23. Cui YM, Ao MZ, Li W, et al. Anti-inflammatory activity oflicochalcone A isolated from Glycyrrhiza inflate. Z Naturforsch C 2008, 63: 361-365. 24. Kwon HS, Park JH, Kim DH, et al. Licochalcone Aisolated licorice suppresses lipopolysaccharide- stimulated inflammatory reactions in RAW264.7 cell and endotoxinshock in mice. Mol Med 2008, 86: 1287-1295. 25. Furuhashi I, Iwata S, Shibata S, et al. Inhibition by licochalcone A, a novel flavonoid isolated from liquorice root, of IL-1β-induced PGE2 production in human skin fibroblasts. Pharm Pharmacol 2005, 57: 1661-1666. 26. Chandrasekaran CV, Deepak HB, Thiyagarajan P, et al. Dual inhibitory effect of Glycyrrhiza glabra (GutGardTM) on COX and LOX products. Phytomedicine 2011, 18: 278-284.

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