Application of an active ingredient based on radish seed extract as an angiotensin-converting enzyme inhibitor

Abstract

This invention belongs to the field of biomedicine, specifically relating to the application of an active ingredient based on radish seed extract as an angiotensin-converting enzyme inhibitor. The active ingredient is either sinapine or glucosaturtiin. This invention employs affinity ultrafiltration combined with UPLC-Orbitrap-MS to rapidly screen and identify potential inhibitors from radish seeds online. Through ACE activity inhibition experiments, combined with molecular docking studies, the affinity of the active compound for ACE was investigated. Finally, by combining affinity ultrafiltration RBA values, IC50, and molecular docking binding forces, two potential ACE inhibitors, sinapine and glucosaturtiin, were identified. Furthermore, further research after screening significantly narrows the research scope, saves time in the new drug discovery and development process, and reduces screening costs and risks.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of an active ingredient based on radish seed extract as an angiotensin-converting enzyme inhibitor. Background Technology

[0002] Hypertension is one of the major diseases threatening human health and the most dangerous factor in cardiovascular diseases. Angiotensin-converting enzyme (ACE) is a key enzyme in the production of angiotensin II. Inhibiting ACE activity can reduce the production of angiotensin II, thereby effectively reducing vasoconstriction and blood pressure, achieving the goal of antihypertensive treatment. Traditional Chinese medicine (TCM) has promising application prospects as a source for screening ACE inhibitors. However, the components of TCM herbs are complex. Traditional methods require extraction and separation of TCM herbs, followed by further performance studies such as activity assays. This process is time-consuming, and the therapeutic mechanisms and material basis are difficult to elucidate, thus hindering the development of TCM in this field. Furthermore, how to rapidly screen angiotensin-converting enzyme inhibitors from natural products or TCM has always been a challenge in this field.

[0003] Affinity ultrafiltration-liquid chromatography-mass spectrometry (LC-MS) is a screening technique based on protein-ligand interactions in solution. It separates bound and unbound molecules through ultrafiltration and combines this with mass spectrometry analysis. Its advantages include the ability to screen for target molecules under near-physiological conditions, making it suitable for complex samples; and the use of mass spectrometry as a label-free detection method avoids label interference with protein function, improving the accuracy and reliability of screening.

[0004] Radish seeds exhibit a variety of pharmacological activities, including enhancing gastrointestinal motility, lowering blood lipids, anticancer effects, antitussive properties, expectorant effects, and lowering blood pressure. The renin-angiotensin system (RAS) is a hormone system that regulates blood pressure, and ACE is one of the key targets for regulating these RASSes. However, to date, the corresponding inhibitory active components of radish seeds related to ACE remain to be explored. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides an application of the active ingredient based on radish seed extract as an angiotensin-converting enzyme inhibitor.

[0006] Another object of the present invention is to provide a method for screening active ingredients based on radish seed extract as angiotensin-converting enzyme inhibitors.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] This invention provides the application of an active ingredient based on radish seed extract as an angiotensin-converting enzyme inhibitor, wherein the active ingredient is sinapine or glucosaturtiin.

[0009] This invention also provides a method for screening active ingredients based on radish seed extract, comprising the following steps:

[0010] (1) Take the crushed radish seeds and defatt them. Add the defatted radish seeds to the solvent, extract them by ultrasonication, combine the extracts, concentrate them, and evaporate them to dryness in a water bath to obtain radish seed extract. Dissolve the radish seed extract in boric acid buffer solution to obtain the test solution for later use.

[0011] (2) Mix the test solution and ACE and incubate; after incubation, centrifuge with an ultrafiltration centrifuge tube, mix the complex formed by the monomeric compound bound to ACE with 70% acetonitrile (volume fraction), dissociate and centrifuge, and collect the filtrate;

[0012] (3) The active ingredients were analyzed and identified by ultra-high performance liquid chromatography-electrostatic field orbital trap high-resolution mass spectrometry system. Then, sinigrin and gentianin were screened by in vitro ACE enzyme inhibition activity experiment and molecular docking.

[0013] Preferably, in step (1), the mass ratio of the defatted radish seeds to the solvent is 1:10; the solvent is 70% methanol (volume fraction).

[0014] Preferably, in step (1), the mass concentration of the test solution is 20 mg·mL. -1 The pH of the boric acid buffer solution is 7.4.

[0015] Preferably, in step (2), the volume ratio of the test solution to ACE is 1:1 to 1:4; the enzyme activity of ACE is 0.01-0.5 U / mL; the incubation is at 37 °C for 0.5 to 2.5 h; and the molecular weight cutoff of the ultrafiltration centrifuge tube is 10 kDa.

[0016] Preferably, in step (2), the dissociation time is 5 to 20 minutes.

[0017] Preferably, in step (3), the ultra-high performance liquid chromatography (UHPLC) conditions are as follows: an ACQUITY UPLC® HSS T3 column with dimensions of 2.1 × 100 mm and 1.8 µm is used; the mobile phase is a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (B); the column temperature is 40 ℃; and the flow rate is 0.3 mL·min. -1 The injection volume was 3 μL.

[0018] Preferably, in step (3), the specific process of gradient elution is as follows: 0-3 min, 0% B; 3-8 min, 0-5% B; 8-10 min, 5% B; 10-15 min, 5-15% B; 15-17 min, 15% B; 17-24 min, 15-30% B; 24-28 min, 30-70% B; 28-35 min, 70-100% B.

[0019] Preferably, in step (3), the mass spectrometry conditions are as follows: ESI ion source, positive and negative ion modes; capillary voltage 3500 V; sheath gas: 35.0 si; auxiliary gas: 10.0 L·min -1 Capillary temperature: 350 °C; Collision energy: 20.0, 40.0, 60.0 eV; Mass scan range: 80~1200 Da.

[0020] This invention employs affinity ultrafiltration, which can efficiently screen for bioactive compounds by selectively separating and enriching conjugates of target proteins or enzymes. Using affinity ultrafiltration technology, specific components that can bind to ACE and inhibit its activity can be rapidly screened from complex plant extracts. After characterizing the chemical composition of radish seeds using UPLC-MS / MS, the inhibitory activity of its main components on ACE was evaluated, leading to the discovery of natural ACE inhibitors.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) This invention uses affinity ultrafiltration combined with UPLC-Orbitrap-MS to screen and identify eight potential inhibitors from radish seeds online and rapidly. ACE activity inhibition experiments were then used to further verify the inhibitory effects of sulforaphene, glucoraphenin, gluconapin, glucoiberverin, glucosamine, sinapine, glucosaturtiin, and Siberian polygalactosyl glycoside A5. (A5) The inhibitory activity of 8 potential compounds on ACE was further studied by molecular docking to investigate the affinity between active compounds and ACE. Finally, based on the affinity ultrafiltration RBA value, IC50, and binding force of molecular docking, two potential ACE inhibitors, sinigrin and gentianin, were identified. Furthermore, further research was conducted after screening, which greatly narrowed the research scope, saved time in the discovery and development of new drugs, and reduced screening costs and risks. (2) The method provided by this invention is simple, fast, efficient, and sensitive, and is suitable for screening and identifying active small molecules from complex systems of traditional Chinese medicine. The research results show that radish seeds may be an important source of potential natural ACE inhibitors. Attached Figure Description

[0023] Figure 1 This is the mass spectrum of sinigrin;

[0024] Figure 2 This is the mass spectrum of watercress glycosides;

[0025] Figure 3 Total ion chromatogram of affinity-ultrafiltration LC-MS for radish seed and ACE;

[0026] Figure 4 The ACE inhibitory activity of 3-butenyl thioglucoside, a component of radish seeds;

[0027] Figure 5 The ACE inhibitory activity of 3-methylthiopropylthioglycoside, a component of radish seeds;

[0028] Figure 6 The ACE inhibitory activity of radish seed component daidzein;

[0029] Figure 7 The ACE inhibitory activity of sinigrin, a component of radish seeds;

[0030] Figure 8 The ACE inhibitory activity of radish seed component radish extract;

[0031] Figure 9The ACE inhibitory activity of sulforaphane, a component of radish seeds;

[0032] Figure 10 The ACE inhibitory activity of Siberian polygalactosyl glycoside A5, a component of radish seeds;

[0033] Figure 11 To investigate the ACE inhibitory activity of brassinosteroids, a component of radish seeds;

[0034] Figure 12 To study the interaction between active compounds and ACE in molecular docking. Detailed Implementation

[0035] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0036] Example 1

[0037] 1.1 Experimental Materials

[0038] 1.1.1 Medicinal materials and reagents

[0039] Radish seeds (produced in Shandong Province, purchased from Shandong Provincial Hospital of Traditional Chinese Medicine, batch number No. 230502), angiotensin-converting enzyme (derived from rabbit lung) ≥2.0 U / mg (Shandong Deren Pharmaceutical Technology Co., Ltd.), 10 kDa ultrafiltration centrifuge tubes (purchased from Millipore Ltd.), HEPES buffer solution, borate buffer solution (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), formic acid, acetonitrile (chromatographic grade, Thermo Fisher Scientific), raphanin, sulforaphane, 3-butenyl thioglucosinolate, 3-methylthiopropyl thioglucosinolate, brassinoglucosinolate, sinigrin, stigmasterone, and Siberian polygalactosyl glycoside A5 (all purchased from Chengdu Ruifenside Biotechnology Co., Ltd.), and Watson's water (purchased from Watson's China Ltd.); all other reagents were of analytical grade.

[0040] 1.1.2 Experimental Apparatus

[0041] Ultimate 3000 Ultra-High Resolution Liquid Chromatography System - Q-Exactive Orbitrap-MS Quadrupole-Electrostatic Orbit Trap High Resolution Mass Spectrometer (Thermo Fisher Scientific), SQP 0.0001 ppm Electronic Analytical Balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), KQ-250E Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

[0042] 1.2 Experimental Methods

[0043] 1.2.1 Preparation of Radish Seed Test Solution

[0044] Take 100 g of crushed radish seeds, defatt them three times with four times the amount of petroleum ether (60-90 ℃), take 10 g of the defatted radish seeds, add 10 times the amount of 70% methanol, and extract by ultrasound three times, 30 min each time. Combine the extracts, concentrate, and evaporate to dryness in a water bath to obtain an extract of radish seeds in pure water, 70% methanol, and methanol. Accurately weigh 0.1 g of the extract and dissolve it in a boric acid buffer solution at pH=7.4 to prepare a solution with a mass concentration of 20 mg·mL⁻¹. -1 Test solution, for later use.

[0045] 1.2.2 Affinity Ultrafiltration

[0046] A concentration of 20 mg·mL -1 The test solution and ACE (0.1 U / mL) were mixed at a ratio of 1:2 (v / v) and incubated at 37 °C for 1 h. Another inactivated ACE (100 °C, water bath for 10 min) served as a blank control. After incubation, the mixture was centrifuged three times at 13000 r / min for 30 min each time using a 10 kDa ultrafiltration centrifuge tube to remove small molecules that were not bound to ACE. The complex formed by the monomeric compound bound to ACE was mixed with 2 mL of 70% acetonitrile, dissociated for 10 min, centrifuged at 13000 r / min for 30 min, and the filtrate was collected. This process was repeated three times.

[0047] 1.2.3 In vitro ACE inhibition rate

[0048] The control wells contained 10 μL ACE, 20 μL HEPES buffer, and 40 μL FAPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine). The sample wells contained 10 μL ACE, 20 μL control, and 40 μL FAPGG. The initial absorbance (a1 and b1) of the control and sample wells was measured at 340 nm. After incubating the plate at 37°C for 30 min, the absorbance was measured again (a2 and b2). Each sample was measured in five replicates. The absorbance reduction value A for the control wells was calculated as A = a1 − a2, and the absorbance reduction value B for the sample wells was calculated as B = b1 − b2. The formula for calculating the ACE inhibition rate of the samples is as follows:

[0049] ACE inhibition rate (%) = 1 - B / A.

[0050] 1.2.4 Chromatographic conditions

[0051] An ACQUITY UPLC® HSS T3 (2.1×100 mm, 1.8 µm) column was used. The mobile phase consisted of a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (B) (0–3 min, 0% B; 3–8 min, 0–5% B; 8–10 min, 5% B; 10–15 min, 5–15% B; 15–17 min, 15% B; 17–24 min, 15–30% B; 24–28 min, 30–70% B; 28–35 min, 70–100% B). The column temperature was 40 °C and the flow rate was 0.3 mL / min. -1 The injection volume was 3 μL.

[0052] 1.2.5 Mass Spectrometry Conditions

[0053] ESI ion source, positive and negative ion modes; capillary voltage 3500 V; sheath gas: 35.0 si; auxiliary gas: 10.0 L·min -1 Capillary temperature: 350 °C; Collision energy: 20.0, 40.0, 60.0 eV; Mass scan range: 80~1200 Da.

[0054] 1.2.6 Molecular docking

[0055] Molecular docking was used to study the interaction between potential inhibitors and ACE. The 3D structures of the compounds were downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the ACE crystal structure (PDB ID: 1O86) was downloaded from the protein database (http: / / www.rcsb). Ligands and water molecules were removed using PyMol, and hydrogen atoms were added using AutoDockTools software. The results were saved as pdbqt files and then semi-flexible docking was performed. Based on the docking results, the optimal docking result between the compound and the enzyme was selected for analysis, and PyMol was used for plotting and visualization.

[0056] 1.3 Experimental Results

[0057] 1.3.1 Screening of ACE-inhibiting active ingredients in radish seeds

[0058] This invention employs UPLC-Exactive Orbictive-MS technology to rapidly screen potential active ACE ligands from a 70% methanol extract of radish seeds. Radish seed extract and ACE are co-incubated, and after affinity ultrafiltration screening, the large molecular complexes formed by the active ingredients and ACE are retained in the ultrafiltration tube. The mixture is washed with PBS buffer and dissociated with 70% acetonitrile solution. A UPLC-Exactive Orbictive-MS method is then established to analyze the bound active ingredients in the dissociated solution.

[0059] First, relevant domestic and international literature was searched on PubMed and CNKI to systematically organize the known chemical components of radish seeds and their related species and import them into the PubChem database. Their structural information was then verified. Precise mass numbers were calculated using Xcalibur 3.0 software based on the types and numbers of atoms in each compound. Using Xcalibur 3.0 software, precise mass numbers were calculated based on the elemental composition of each component. Combined with molecular ion and fragment ion information, and compared with relevant literature reports, the chemical components were identified. First, the quasi-molecular ion peak and its precise mass number were determined using primary mass spectrometry (MS). Then, the chemical formula prediction function in the Xcalibur 3.0 mass spectrometry workstation was used to predict the chemical formula and molecular weight. Based on the predicted value (i.e., theoretical value) with a mass number error within ±5 ppm, the EIC chromatogram of the corresponding compound was extracted from the TIC chromatogram of the primary mass spectrometer. Then, based on MS... 2 By analyzing fragment information, deducing relevant functional groups and the structure of the parent nucleus, summarizing characteristic fragment ions, and finally inferring and identifying the structure by referring to the mass spectrometry fragmentation patterns of the components contained in radish seed extract.

[0060] The specific identification process is as follows:

[0061] In positive ion mode, a quasi-molecular ion peak [M] is displayed in the primary mass spectrum. + , m / z 310.1654, predicted chemical formula is C 16 H 24 NO5 + In the secondary mass spectrum, its characteristic fragment ions were found to be: m / z The possible fragmentation pathways of sinigrin and their mass spectra are shown in Figure 1, with values ​​of 251.0916, 236.0677, 207.0655, 175.0392, and 147.0443, a retention time of 15.52 min, and a mass number error of 1.74 ppm. Based on literature data and verification with online databases, the possible fragmentation pathways of sinigrin are deduced.

[0062] The possible cleavage pattern of sinigrin is as follows:

[0063] .

[0064] The quasi-molecular ion detected by primary mass spectrometry in negative ion mode is m / z 422.0586, and its predicted chemical formula is C. 15 H 21 NO9S2, retention time 2.59 min, mass number error 0.223 ppm. Related literature indicates that characteristic fragment ions of glucosinolates include m / z 275, 259, 241, 195, 96, and 75. Furthermore, fragment ions at m / z 259 and m / z 97 constitute an effective preliminary screening method, commonly used to determine the presence of glucosinolates in plant extracts. Based on the fragment ions m / z 274.9904, 259.0125, 241.0017, 96.9584, and 74.9893 in the secondary mass spectrometry, the possible fragmentation pathway of piracetamin and its mass spectrum are inferred as follows. Figure 2 As shown.

[0065] The possible cleavage pattern of watercress glycosides is as follows:

[0066] .

[0067] The changes in peak area of ​​compounds obtained from liquid chromatography-mass spectrometry data of radish seed extract before and after ACE activation and inactivation were used to identify potential bioactive ligands with various binding affinities to ACE.

[0068] Active group: The experimental group with added ACE, where the enzyme is in a normal and active state.

[0069] Inactivated group: ACE that has been treated at 100℃ is added to make ACE lose its activity (protein denaturation), and therefore no longer has enzymatic function.

[0070] Blank group: Control group, which does not contain ACE, used to exclude interference from background factors or non-specific effects.

[0071] Eight potential compounds were initially screened, with RBA = A1 / A2. A1 and A2 represent the chromatographic peak areas of the radish seed extract containing active and inactive ACEs, respectively. Figure 3 As shown in Table 1, Sinapine and Gluconasturtiin exhibited high affinity binding activity, with RBA values ​​of 5.49 and 4.84, respectively.

[0072] 1.3.2 In vitro ACE inhibition assay using radish seed ligands

[0073] To verify the reliability of affinity ultrafiltration and UPLC-Exactive Orbictive-MS methods, the ACE inhibitory activity of the screened active compounds was evaluated through in vitro enzyme activity inhibition experiments. Eight compounds—Sulforaphene, Glucoraphenin, Gluconapin, Glucoiberverin, Glucobrassicin, Sinapine, Gluconasturtiin, and Sibiricose A5—were selected to evaluate their ACE inhibitory activity.

[0074] like Figures 4-11 As shown, the ACE inhibitory activity levels are as follows: Sinapine > Gluconasturtiin > Sulforaphene > Glucoraphenin > Gluconapin (3-butenyl glucosinolate) > Glucoiberverin (3-methylthiopropyl thioglycoside) > Glucobrassicin > Sibiricose A5, among which Sinapine (IC5) 50 = 30.86±3.17 μM) showed the best inhibitory effect on ACE, followed by glutasturtiin (IC50). 50 =39.60±2.79 μM).

[0075] 1.3.3 Molecular docking

[0076] Molecular docking is an important technique for elucidating the affinity of acceptor-ligand complexes. It allows for the simulation of interactions between potential inhibitors and ACE. (See Table 1 and...) Figure 12 As shown, the main amino acid residues bound to sinigrin, 3-butenyl glucosinolate, brassinosteroid, and stigmosiderin are Ala149, Val350, Tyr, Asn85, Arg124, Ser516, Phe512, Arg124, Ala126, Glu123, and Lys118, with affinity binding energies of -9.78, -7.00, -6.88, and -6.92 kcal / mol, respectively, all less than -6.0 kcal / mol, indicating that they can bind well to ACE and thus inhibit ACE activity.

[0077] Table 1 IC50 of active compounds 50 and affinity

[0078]

[0079] 1.4 Conclusion

[0080] This invention employs affinity ultrafiltration combined with UPLC-Orbitrap-MS to rapidly screen and identify eight potential inhibitors from radish seeds online. Further ACE inhibition experiments were conducted to verify the inhibitory activity of these eight potential compounds—rhamnoside, glucosinolate, 3-butenylthioglucosinolate, 3-methylthiopropylthioglucosinolate, brassinoglucosinolate, sinigrin, stigmosiderin, and Siberian polygalactosyl A5—on ACE. Molecular docking was then used to study the affinity between the active compounds and ACE. Finally, the RBA value and IC50 values ​​of the affinity ultrafiltration were considered. 50 The binding forces of molecular docking identified two potential ACE inhibitors: sinigrin and gentianin. This method is simple, rapid, efficient, and sensitive, making it suitable for screening and identifying active small molecules from complex systems of traditional Chinese medicine. Furthermore, the results suggest that radish seeds may be an important source of potential natural ACE inhibitors.

Claims

1. Use of a glucoraphanin as the sole active ingredient in the manufacture of a medicament for the treatment of hypertension.

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