The Molecular Mechanism of FABP4 Inhibition Effects of GAS and 4-HBA in Gastrodia elata Blume Was Discussed Based on NMR and Molecular Docking

To isolate gastrodin (GAS), 4-hydroxybenzyl alcohol (4-HBA), and phenolic compounds from Chinese medicine Gastrodia elata Blume, and to explore the binding mode of fatty acid binding protein 4 (FABP4/aP2) that is closely related to macrophage inflammation, we study their anti-inflammatory targets. After the ultrasonic extraction of the main active components with 70% ethanol, three resins and three eluents were selected, and eight phenolic monomers with similar structures, such as gastrodin and 4-hydroxybenzyl alcohol, were isolated from Gastrodia elata by AB-8 macroporous resin and silica gel column chromatography and eluted with the CHCl3-MeOH gradient. Their structures were identified by HPLC and nuclear magnetic resonance (NMR). The FABP4 protein was added to GAS and 4-HBA, and the NMR experiment was performed to observe ligand binding. Finally, according to the spectral information of STD-NMR and molecular docking technology, the interaction between ligands and protein was studied. The fluorescence competition experiment confirmed that both GAS and 4-HBA were in the binding cavity of FABP4. Moreover, 3-phenoxy-2-phenylbenzoic acid (PPA) is a possible inhibitor of FABP4, reducing macrophage-related inflammation and endoplasmic reticulum stress. This work provides a new basis for the anti-inflammatory mechanism of Gastrodia elata, paving the way for the research and development of FABP4 inhibitor drugs.


Introduction
Adipocyte fatty-acid-binding protein (FABP4) is expressed in adipocytes and macrophages and integrates infammatory and metabolic responses [1].Studies in FABP4-defcient mice have shown that this lipid chaperone has a signifcant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis [1].Foam cells on the inner membrane of the aorta in the atherosclerosis mice were signifcantly reduced in the presence of the FABP4 inhibitor (BMS309403, 25 mM).Te expression of these cytokines (MCP-1, interleukin (IL)1β, IL6, and tumor necrosis factor (TNF) in macrophages) was signifcantly reduced in the FABP4-expressing macrophages treated with the FABP4 inhibitor [1].On the other hand, gastrodin attenuates the foam cells on the inner membrane of the inner membrane of aorta in atherosclerosis mice [2,3].Gastrodin was found to markedly attenuate the expression of the interleukin (IL)1β, IL6, and tumor necrosis factor (TNF) both in vivo and in vitro [4].In vitro and in vivo studies show that the use of FABP4 inhibitors can efectively reduce the infammatory response, indicating that FABP4 may be a potential candidate target for the treatment of infammationrelated diseases [5][6][7][8].
Infammation is closely related to many metabolic diseases, such as atherosclerosis, type 2 diabetes (T2DM), and obesity [9,10].Te main immune cell type that causes infammation in the islets of obesity and type 2 diabetes is macrophages [11].In addition, macrophages are a major activator of chronic infammation of the innate immune system and play a central role [12].FABP4, as a fatty acid companion, is greatly increased when infammation is activated [13].In macrophages, the deletion or chemical inhibition of FABP4 will increase the level of intracellular free fatty acids, and the increase of free fatty acids will induce the expression of uncoupling protein 2 (UCP2) through the PPARc-mediated pathway.Te increase in UCP2 expression reduces mitochondrial oxidative stress, endoplasmic reticulum stress, and infammation and changes the mitochondrial function and the intracellular level of free fatty acids inside macrophages [14][15][16][17].Te infammation endoplasmic reticulum and mitochondrial stress in macrophages can be reduced by inhibiting the binding of FABP4 with free fatty acids in macrophages.Terefore, the development of FABP4 inhibitors can inhibit macrophage infammation and improve the endoplasmic reticulum and mitochondrial stress of macrophages.
Based on molecular docking technology, it was found that levofoxacin, a broad-spectrum antibiotic, is also a high-afnity inhibitor of FABP4 [18].NMR can be used as a method to monitor the intermolecular interaction, and it is a powerful means to study the protein-ligand interaction.Among them, the ligand observation of nuclear magnetic resonance experiments is used to identify small ligand molecules bound to biological macromolecules.Commonly used methods include those based on nuclear Overhauser efect (NOE), saturation transfer diference (STD-NMR), water ligand observed via gradient spectroscopy (WaterLOGSY), and transferred-nuclear Overhauser efect spectroscopy (tr-NOESY) experiments, and also the INPHARMA experiment.Te STD-NMR experiment depends on the magnetization exchange from the protein-bound state to the ligand-free state.In this spectrogram, only the signal of the conjugate is observed, but the signal of protein and nonconjugate is subtracted.Te conjugate in the mixture can be determined by STD-NMR, and the binding epitope can also be defned [19,20].Gastrodia elata belongs to Orchidaceae, frst recorded in Shennong Materia Medica.As a traditional Chinese herbal medicine, it is generally considered to have antiinfammatory, antibacterial, and neuroprotective efects.GAS, 4-HBA, etc. are the main components [4,21].GAS can increase the activity of superoxide dismutase (SOD) and decrease the level of reactive oxygen species (ROS) in the liver.Some researchers have suggested that the pathway of GAS's efcacy is through the anti-infammatory efect [22][23][24][25].In this study, GAS, 4-HBA, and phenols with similar structures were isolated from Gastrodia elata by column chromatography.After their structures were identifed by HPLC and NMR, the 1H-NMR experiment of ligand observation was used to study whether they interacted with FABP4, and the molecular docking technique was used to predict the interaction mode between the active ingredients and proteins with FABP4.Te purpose of this study is to fnd out the active ingredients that inhibit FABP4 from Gastrodia elata, which can clarify the molecular mechanism of the anti-infammatory efect of Gastrodia elata, providing the basis for developing the new structure of FABP4 inhibitory drugs (Figure 1).Gastrodia elata is produced in Tongcheng County, Xianning City, Hubei Province.It was identifed as Gastrodia elata by Associate Professor Xiao Ruolei of the Hubei University of Science and Technology.Standard GAS, 4-HBA, and phenols were purchased from Chengdu Aifa Biotechnology Co., Ltd., with purity >97%; chromatographic grade acetonitrile and methanol were purchased from the United States Symantec Company; HPLC water for Wahaha pure wateer Hangzhou Wahaha Group Co., Ltd.(Hangzhou, China) was used to provide purifed water; dichloromethane, trichloromethane, petroleum ether, benzene, ethyl acetate, and ethanol (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd.Dimethyl sulfoxide and Tris-EDTA (pH = 7.5) were purchased from Sigma-Aldrich, USA.FABP4 was purchased from Wuhan Kaisheng Biotechnology Co., Ltd.AB-8, D101, and ADS-7 macroporous resins were purchased from Tianjin Guangfu Fine Chemical Research Institute.Silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Co., Ltd.

Macroporous Resin Pretreatment.
Te newly bought macroporous resin was put in 95% ethanol solution overnight, completely immersed in ethanol, and then washed with distilled water and 95% ethanol repeatedly until the fltrate was colorless.Te extractant was washed with distilled water until there was no alcohol taste.

Cleaning and Regeneration of Macroporous Resins.
Te used macroporous resin was soaked with dilute acid and dilute alkali repeatedly, cleaned until the cleaning liquid was colorless, and fnally rinsed with distilled water until PHneutral.

Macroporous Resin Selection.
Te efects of AB-8, D101, and ADS-7 macroporous resins on GAS and 4-HBA extraction were compared.Te dry rhizome of Gastrodia elata was sliced and crushed, and 60 g of Gastrodia elata powder (3 portions) was taken.400 mL of 70% ethanol was added for ultrasonic extraction for 1 h.After fltration, the 2 Journal of Analytical Methods in Chemistry fltrate was concentrated to 30 mL (3 portions) at 70 °C and extracted with three kinds of macroporous resin columns (inner diameter: 2.5 cm, 70 g).After gradient elution with water, 30% and 60% ethanol were successively followed by freeze-drying.Te separation efect of ab-8 resin with 30% alcohol was the best after HPLC screening.Te elution procedure was performed at 0-10 min (for the 6%-15% fraction), at 10-15 min (for the 15%-25% fraction), at 15-25 min (for the 25%-40% fraction), and 25-30 min (for 40% and above).Te fow rate was 0.8 mL/min.Te column temperature was 30 °C.Te detection wavelength was 270 nm.Te injection volume was 20 µL.

1 H-NMR and 13 C-NMR Identifcation of GAS and 4-HBA.
HPLC can only detect substances in a certain wavelength range.To ensure that fne structure attenuation and chemical shift perturbation can be observed in the 1 H-NMR spectra of GAS and 4-HBA, 1 H-NMR and 13 C-NMR spectra were needed to determine the composition of extracted GAS and 4-HBA, which was convenient for subsequent observation.
To prepare for 1 H-NMR and 13 C-NMR sample solutions, 5 mg of sample powder was dissolved in 500 μL of DMSO-d 6 .Journal of Analytical Methods in Chemistry performed at 298 K on a Bruker 400 MHz spectrometer, with a relaxation delay of 2 s.Te binding phenomenon can be observed by 1 H-NMR spectrum broadening and chemical shift perturbation.

Molecular Docking Experiment.
We generated the structural formula of GAS, 4-HBA, and PPA in Chem Draw 2D 20.0 software (https://revvitysignals.com/products/ research/chemdraw).Te structure of BMS309403 was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and we determined the energy optimization of the GAS, 4-HBA, PPA, and BMS309403 structure by using the MM2 force feld in Chem Draw 3D 20.0 (https:// revvitysignals.com/products/research/chemdraw)[18].Te ligand is saved as a mol2 fle after energy optimization.We import the mol2 format of the ligand into Auto Dock Tools 1.5.7 (https://autodocksuite.scripps.edu/adt/)to set the torsion and output it as a pdbqt format fle.Te crystal structure of FABP4 was downloaded from the protein database RCSB (https://www.rcsb.org)(PDB ID 2NNQ, 1.80 Å).PyMol (https://pymol.org/2/)was then used to remove water molecules and original eutectic ligands from the protein.Proteins were hydrogenated in AutoDock Tools 1.5.7 and output as a fle in the pdbqt format.Te pdbqt fles of the receptor and ligand are imported into Auto Dock Tools 1.5.7.Molecular docking is performed in Auto Dock Tools 1.5.7, and the magnitude of the binding energy refects the possibility of binding between the receptor and ligand.Te lower the binding energy, the higher the afnity between the receptor and the ligand.Te lower the binding energy, the more stable the conformation of the receptor and the ligand.In summary, docking was completed in four steps.Te frst step was protein hydrogenation, charge calculation, and protein type assignment (assign ad4 type).Te second step was small molecule hydrogenation to keep it fexible.Te third was to set the docking range X88, Y92, and Z116, the docking box completely covers the receptor protein, and the ligand is located outside the docking box.Te last step was to use the genetic algorithm for molecular and protein docking using default parameters.After docking for 50 times, visualization was performed in PyMol.4

STD-NMR Screening
Journal of Analytical Methods in Chemistry 2.9.1,8-ANS Fluorescence Competition Experiment.Te fuorescent probe 8-anilinonaphthalene-8-sulfonic acid (1,8-ANS) has been proven to be the ligand of FABP4.When ANS binds to FABP4, its maximum emission light will be blue-shifted.Te excitation and emission wavelengths of free ANS were determined to be 370 nm/540 nm in the early stage of the experiment, respectively.After binding protein, it becomes 360 nm/460 nm, so by observing the change in the fuorescence intensity at 360 nm/460 nm before and after adding the ligand, the ability of the ligand to replace bound 1,8-ANS from the binding cavity can be determined.Competition assay fuorescent experiments: 100 μg FABP4 protein was dissolved in 5 mL Tris-EDTA (pH � 7.4), 10 mg 8-anilino-1-naphthalenesulfonic acid (1,8-ANS) was dissolved in 50 mL distilled water, and 0.5 mL of such 1,8-ANS solution was added to the 4 mL above-mentioned target protein solution; the excitation and emission wavelengths were determined.Te GAS solid was added to make the fnal concentration of GAS (0, 1, 8, 16, 24, 32, 40,4 8, and 56 μM) and mixed in the test tube after adding solid GAS every time; the fuorescence intensity of the solution was measured in a cuvette of Shi Ying using a Hitachi F-7000 fuorescence spectrometer.With the substitution of 1,8-ANS in the FABP4 binding cavity by GAS, the fuorescence intensity gradually decreased, and the fuorescence data of each concentration were recorded.Te determination of 4-HBA is the same as above, except that the fnal concentration of 4-HBA is 0, 10, 20, 30, 40, 50, 60, 70, and 80 μM.
IC 50 determination experiment: 50 µg of the target protein was dissolved in 2.5 ml of Tris-EDTA (pH = 7.4) bufer solution, 30 mg of ANS was dissolved in 1 mL DMSO to obtain a mother liquor of about 100 mM, 1 µL of the mother liquor was diluted with 2999 µL of Tris-EDTA (pH = 7.4) bufer solution, and 60 µL of diluent was added to a 96-well plate to make the fnal concentration of ANS 10 µM.Ten, 80 µL of the target protein solution was added to a 96-well plate, 60 µL of compound solution obtained by gradient dilution was added as the sample well (the compound was dissolved by Tris-EDTA (pH = 7.4) bufer solution), 60 µL of ANS diluent was added to the 96-well plate, and 80 µL of Tris-EDTA (pH = 7.4) bufer solution was added to the 96-well plate as the local well.60 µL ANS diluent was added to the 96-well plate to make its fnal concentration 10 µM; then, 80 µL target protein solution was added to the 96-well plate; compound solution obtained by gradient dilution as blank hole was added; 60 µL ANS diluent was added to the 96-well plate to make its fnal concentration 10 µM; then, 80 µL Tris-EDTA bufer was added to the 96well plate; and compound solution obtained by gradient dilution as the control hole was added.Te infuence of the fuorescence of the compound itself on the results was eliminated.Te solution was shaken and incubated at room temperature for 90 s, and then, the fuorescence intensity of each hole was recorded with a BioTek Synergy 2 Multifunctional Enzyme Labeling Instrument for every 45 s at the excitation and emission wavelengths of 360 nm/460 nm for 10 min, and the inhibition rate by the following formula was calculated: Inhibition rate of the sample = (1 − (Fluorescence value of sample hole-Fluorescence value of local hole-Fluorescence value of control hole-Fluorescence value of local hole)/(Fluorescence value of blank hole-Fluorescence value of local hole)) × 100%.Te IC 50 values were expressed as mean from three independent experiments and determined via the nonlinear regression analysis using GraphPad Prism software 9.5.

Results and Discussion
3.1.Te Extraction and Separation Results of GAS and 4-HBA.Gastrodia elata has a complex chemical composition, so it is difcult to extract and purify.We compared diferent resins and eluents to optimize the separation condition, obtaining high purity in the fnal extraction.Figure 3 is the HPLC characteristic spectrum of the alcohol eluent with the highest content of GAS and 4-HBA among the three resins.Te contents of GAS and 4-HBA separated by AB-8 elution are the highest (Figure 3(b)).AB-8 resin is selected for extraction.
Figure 4 shows the HPLC characteristic spectra of different eluents.Te separation result of benzene-EtOAc has an obvious tailing phenomenon, which may be caused by impurities (Figure 4(e) and (f )).Te 4-HBA separated by petroleum ether-EtOAc contains a small amount of impurities (Figure 4(a) and (b)), and the purity of GAS and 4-HBA separated by CHCl 3 -MeOH as eluent is the highest (Figure 4(c) and (d)), so CHCl 3 -MeOH is selected.
Figure 5 is the HPLC characteristic spectra of the extraction and separation results of GAS and 4-HBA.R4, R5, and R6 are HPLC chromatograms of macroporous resin AB-8 eluted with water, 30% ethanol, and 60% ethanol, respectively.It is found that the chromatographic signals of GAS and 4-HBA in 30% ethanol eluate Figure 5(e) are relatively strong, so 30% components are selected to be separated.Figure 6(a) shows that the retention time of standard GAS is 8.340 min and that of 4-HBA is 13.937 min; R3 is 100 ∶ 12. Te retention time of the CHCl 3 -MeOH extract is 13.928 min (Figure 5

Analysis of Phenolic Extraction Results of Gastrodia elata.
Te structure of phenols was identifed by high-efciency liquid chromatography.A comparison of chromatogram of mixed calibrating standards and phenolic extract (Figure 2(s)).Te reference substance found that Figure 2(a)-(d) in HPLC diagram corresponds to substances i, j, k, and l, respectively; Figure 2(e)-(h) corresponds to substances m, n, u, and v, respectively. 13C-NMR Experimental Results.

1 H-NMR Experimental Results of Ligand Observation.
As a method to monitor the intermolecular interaction, NMR is a powerful means to study the protein-ligand interaction.Among them, the NMR experiments of ligand observation can be used to identify small ligand molecules combined with biological macromolecules [28].NMR observations such as transverse and longitudinal relaxation rates (R1 and R2), nuclear Overhauser efect (NOE), and difusion coefcient are highly dependent on the molecular size and shape.Terefore, the NMR parameters of small  molecules are very sensitive to their interactions with large molecules.If a small molecule binds to a protein, the chemical environment of the hydrogen nucleus at this site will change, which will disturb the chemical shift of this site.Te changes in the chemical shift, line width, relaxation rate, and NOE value can be used to characterize and quantify the binding of small molecules to larger target molecules such as proteins.Macromolecules show smaller difusion coefcients and larger R2 values, and there is a more obvious NOE efect between nuclei, while small molecules are the opposite.When a small molecule binds to a protein, it will temporarily appear as the NMR observation of the protein.
Due to the large longitudinal relaxation rate (R2) of the protein and the more obvious NOE efect between nuclei, small molecules bound to the protein will show wider spectral lines and attenuation of fne structures.Trough these and chemical shift perturbation, the ligand small molecules bound to protein can be selected.100 ∶ 12 and 100 ∶ 40 CHCl 3 -MeOH extracts were identifed as GAS and 4-HBA by HPLC and NMR.GAS and 4-HBA combined with FABP4 protein will temporarily show the NMR observation values of protein molecules, so we can compare the changes in 1 H-NMR spectra of GAS and 4-HBA before and after protein addition.Te attenuation of fne structure, broadening of spectral line, and perturbation of chemical shift, and whether GAS and 4-HBA are bound to protein can be judged from Figure 8(a), which shows the 1 H-NMR spectrum of GAS before and after protein addition.It is observed that the fne structure of all hydrogen nuclei has been attenuated, and the chemical shift perturbation of No. 8, No. 10, No. 19, and No. 20 hydrogen atoms has been observed (their chemical shifts move to the low feld, possibly due to the formation of hydrogen bonds between No. 8, No. 10, No. 19, and No. 20 hydrogen atoms and proteins, leading to the decrease in the electron cloud density of this hydrogen atom, so the chemical shift of hydrogen nuclei moves to the low feld).Te above indicates that there is a binding phenomenon between GAS and protein.Figure 8(b) shows the 1 H-NMR spectrum of 4-HBA before and after protein addition.We observed the attenuation of the fne structure of No. 1, No. 3, No. 4, and No. 6 hydrogen atoms, as well as the perturbation of chemical shifts of hydroxyl hydrogen atoms 8 and 9, and the broadening of the spectral line of hydrogen atom 7. Terefore, GAS and 4-HBA are binding with protein.

Analysis of Docking Results of GAS and 4-HBA Molecules.
FABP4 shows a typical β-barrel structure, and the cavity in the middle is the binding site of ligands such as fatty acids.According to the ligand entrance hypothesis of FABP4, the fatty acid entrance consists of the angle between β3-β4 and β5-β6 and the amino acid residues of α helix Val32, Phe57, Lys58, and Ala75 (Figure 9).Te entrance position change allows the ligand to enter mainly based on the conformational change mechanism of Phe57.Te carboxyl group of FAs is oriented inward and coordinates with one tyrosine and two arginine residues through electrostatic interaction (the bonded fatty acid carboxyl group forms a salt bridge with Arg106, Arg126, and Tyr128, and the mutation of each of these amino acids will cause the protein to be unable to bond with fatty acids) [29].
Te molecular docking results are shown in (Figure 10).Te binding site of BMS309403 and FABP4 is at the fatty acid inlet (Figure 10(a), Table S1).Te carboxyl group of BMS309403 forms hydrogen bonds with Arg126 and Tyr128, and the total binding energy is −10.95 kcal/mol.Te aromatic group at position 3 of the central nucleus of BMS309403 pyrazole is in the hydrophobic region 1 formed by Phe16, Tyr19, Met20, Val25, Phe57, and Asp76 (hydrophobic region 1 composed of yellow and purple Phe57 amino acid residues in Figure 10(a)).Te aromatic ring at position 4 is located in hydrophobic region 2 formed by Ala33, Gly34, Ala36, Pro38, Ser55, Phe57, and Arg126 (hydrophobic region 2 composed of green and blue-violet Phe57 amino acid residues in Figure 10(a)).Te eutectic structures of BMS309403 and FABP4 (PDB ID: 2NNQ (Figure 11) are consistent with molecular docking results [30,31].
Te docking results of FABP4 and GAS (Figure 10(b), Table S2) show that hydrophilic glucose forms hydrogen bonds with Arg106, Ala75, Gln95, Glu72, and Tr60, and the total binding energy is −6.45 kcal/mol.Te benzene ring of GAS is located in hydrophobic region 2, which is consistent with the position of the 4-position benzene ring of BMS309403 in protein.Although the number of hydrogen bonds formed between GAS and protein is more than BMS309403, the total bond energy is lower than the latter, which may be due to the hydrophobic interaction between BMS309403 and hydrophobic regions.It can be seen that the hydrophobic interaction formed by the two hydrophobic regions is one of the key acting forces for the combination of BMS309403 and FABP4.
Te docking result of FABP4 and 4-HBA is shown in Figure 10(d) and Table S3; two hydroxyl groups of 4-HBA form hydrogen bonds with protein amino acid residues Trp97, VAL23, and Asp76 to obtain a total binding energy of −4.2 kcal/mol.Te binding position is at the entrance of protein and fatty acid binding.Te docking results of FABP4 and PPA (Figure 10(c), Table S4) show that carboxyl groups of PPA form hydrogen bonds with Arg126 and Try128, and the total binding energy is −7.81 kcal/mol, which is higher than −4.2 kcal/mol.However, the number of hydrogen bonds is less than that of GAS, and the two benzene rings are in hydrophobic regions 1 and 2, which shows that the binding mode is similar to BMS309403.Te binding sites of BMS309403, GAS, 4-HBA, and PPA are all at the entrance where fatty acids bind to FABP4 protein.
Te docking results indicate that GAS, 4-HBA, and PPA may exert similar inhibitory efects as BMS309403 and, thus, exert similar pharmacological activities.GAS, 4-HBA, and phenols with similar structures are all active ingredients in Gastrodia elata.Terefore, after taking Gastrodia elata, they may play a synergistic role in inhibiting FABP4, and their binding energy is lower than that of BMS309403.Terefore, compared with BMS309403, the efect of the Gastrodia elata active ingredient is mild.As the protein and ligand are constantly bound and dissociated, the small molecule is constantly saturated, but the molecule that is not bound to the protein will not change.To attenuate that resonance signal of the small molecule bound to the saturated protein, and through protein resonance unsaturation (the protein and the ligand are not resonantly saturated so that the nuclear magnetic spectrum shows the signals of all proteins and ligand) and resonance saturation (the protein is resonantly saturated under a certain radio frequency signal so that the ligand signal bound to the protein is also saturated), STD-NMR can be used to determine the conjugate in the mixture, and the binding epitope (the part that binds to the protein most closely) can also be defned.
Because STD-NMR only shows the proton signal of the ligand bound to protein, the bound ligand can be visually observed from the STD-NMR spectrogram, and the 1.8-ANS fuorescence competition experiment is used to further verify the small molecule with strong signal.
Te results of STD-NMR showed that the hydrogen atoms of GAS and 4-HBA interacted with FABP4, and the STD-NMR signal of GAS was strong, while that of 4-HBA was weak, which indicated that the interaction between gas and FABP4 was stronger than that of 4-HBA, which is consistent with the molecular docking results (Figures 12(a   those of other hydrogen atoms, indicating that these two hydrogen atoms have a stronger interaction with FABP4, which is consistent with the docking result that these two hydrogen atoms form hydrogen bonds with FABP4 (Figure 12(b)).In the STD-NMR spectrum of GAS, it is found that hydroxyl groups mainly interact with FABP4, and the proportion of hydrogen atoms in the benzene ring is also high, indicating that there may be π-π interaction between GAS and amino acid residues of FABP4(Figure 12(a)).All STD efects were quantifed using the equation A STD � (I 0 − I sat )/I 0 � I STD /I 0 .Terefore, signal intensities of the STD-NMR spectrum (I STD ) were compared with the corresponding signal intensities of the reference spectrum (I 0 ).Te strongest STD signal in the spectrum was assigned a value of 100% and used as the reference to calculate the relative STD efects accordingly.

1.8-ANS Fluorescence Competition Experiment Results.
1.8-ANS is proved to be the ligand of FABP4, which can lead to an increase in the fuorescence intensity and the blue shift of the maximum emission light after binding with protein.
Before the experiment, the excitation wavelength and emission wavelength of free 1.8-ANS were determined by spectral scanning, and after binding to the protein, the excitation wavelength and emission wavelength of the increased fuorescence intensity were determined.Te addition of inhibitors can compete with 1.8-ANS for binding sites, resulting in 1.8-ANS in its free form, leading to a decrease in the fuorescence intensity.Increasing the concentration of inhibitors would increase free-form 1.8-ANS, leading to a decrease in the fuorescence intensity.Te fuorescence intensity will not decrease if it is not bound to protein, so it can be used for the simple screening of small molecule inhibitors bound to protein [32][33][34][35].
Te results of fuorescence competition experiments show that the fuorescence intensity decreases with the addition of diferent concentrations of GAS and 4-HBA compared with that without GAS and 4-HB, indicating that GAS and 4-HBA compete with 1,8-ANS for binding sites, which leads to a decrease in the concentration of ANS-FABP4 in the system and a decrease in the fuorescence intensity (Figure 13(a) GAS, Figure 13(b) 4-HBA).Te IC 50 of GAS and 4-HBA were calculated to be 29.84 μM and 50.48 μM (Figure 13(c) d), respectively, which is consistent with the results of molecular docking and STD-NMR, and both of them competed with ANS for binding sites, indicating that their binding sites were in the binding cavity of FAPB4 and ligand, and ANS bound to FAPB4 could be replaced.

Conclusion
Diferent macroporous resins and silica gel column chromatography eluents were compared.AB-8 macroporous resin and silica gel (200-300 mesh) were selected as the stationary phase, and CHCl 3 -MeOH was used as the eluent of the silica gel column.Eight phenolic monomers, such as GAS and 4-HBA (the main active components extracted from Gastrodia elata), are adopted by column chromatography, and the content and purity are high (95%).Teir structures were identifed by HPLC and NMR, and it was found that their structures were similar to those of GAS and 4-HBA.By observing the attenuation of the peak intensity and the perturbation of chemical shift in the 1 H-NMR of the ligand with or without protein, it was found that the binding phenomenon occurred among GAS, 4-HBA, and FABP4.Te molecular docking technique was used to predict the space position of their binding with FABP4.By using the 1,8-ANS fuorescence competition experiment, it was found that the binding site of GAS and 4-HBA was in the binding cavity of FABP4 and ligand.Te results showed that they were similar to the classical FABP4 inhibitor BMS309403, but their binding energy was low.Te IC 50 concentrations of GAS and 4-HBA were 29.84 μM and 50.48 μM.However, a variety of phenols with similar structures to GAS and 4-HBA were found in Gastrodia elata, which may be the common inhibition of FABP4 by a variety of components with similar structures.Meanwhile, it also provides a new strategy to inhibit FABP4, i.e., a variety of substances with similar structures but mild efects play an inhibitory role together, to play a role in resisting macrophage infammation.At the same time, it is also helpful to the research of other metabolic diseases.At last, PPA was found to be a possible structure of the FABP4 inhibitor by molecular docking technology, providing new strategies and useful data for the drug research of the FABP4 inhibitor, creating a new basis for the molecular mechanism of anti-infammation of Gastrodia elata [36][37][38].

Figure 6 : 1 H
Figure 6: 1 H-NMR of GAS and 4-HBA.Te numbers in the 1 H-NMR spectrum represent the hydrogen atoms at this position in the molecular formula.(a) GAS 1 H-NMR and (b) 4-HBA 1 H-NMR.

Figure 7 :
Figure 7: 13 C-NMR of GAS and 4-HBA.Te number in the 13 C-NMR spectrum indicates the carbon atom at this position in the molecular formula.(a) GAS 13 C-NMR and (b) 4-HBA 13 C-NMR.

8
Journal of Analytical Methods in Chemistry 3.6.Experimental Results of STD-NMR.STD-NMR experiment depends on the magnetization exchange from the protein-bound state to the ligand-free state.Te method includes selectively saturating protein NMR signals by using certain radiofrequency radiation without interfering with the resonance signal of small molecules.After saturation, it passes through NOE and spin difusion.Terefore, all protein resonances are saturated.When the molecule binds to the protein surface, the saturation of the protein proton is transferred to the molecule through the spin difusion of intermolecular NOE, which leads to the proton saturation of the small molecule bound to the protein.
) and 12(b)).In the STD-NMR spectrum of 4-HBA, the signals of No. 8 and No. 9 hydrogen atoms are stronger than

Figure 8 :Figure 9 :Figure 10 :BMSFigure 11 :
Figure 8: 13 H-NMR experiment for ligand observation.Te numbers in the 1 H-NMR spectrum represent the hydrogen atoms at this position in the molecular formula.(a) 1 H-NMR experiment for ligand observation and (b) 4-HBA 1 H-NMR experiment for ligand observation.