Method for screening xanthine oxidase inhibitor from traditional Chinese medicine / natural products

Abstract

The invention relates to the field of medicine and health, and discloses a method for screening an XOD inhibitor from a traditional Chinese medicine / natural product and application of the XOD inhibitor in a stigma-removed flower flavone extract of crocus sativus. Specifically, a two-dimensional liquid chromatography method combining affinity chromatography (active XOD column + inactivated XOD column + reference column) and reversed-phase chromatography is established, the XOD inhibitor is screened from the traditional Chinese medicine / natural products, three chromatographic columns, namely an active XOD chromatographic column, an inactivated XOD chromatographic column and a reference chromatographic column, are adopted in a one-dimensional affinity chromatography system, and the active XOD chromatographic column, the inactivated XOD chromatographic column and the reference chromatographic column are used for detecting the XOD inhibitor in the traditional Chinese medicine / natural products. The phenomena of'false positive 'and'sieve leakage' of the XOD inhibitor are effectively solved. By adopting the technology provided by the invention, five flavonoid glycoside compounds are screened from the stigma-removed flower flavone extract of the crocus sativus, and in-vivo and in-vitro experimental results show that the flavonoid glycoside compounds have XOD inhibitory activity, so that the flavonoid glycoside compounds can be used for screening XOD inhibitors in traditional Chinese medicines and natural product extracts; the screened chemical components can be applied to development of products for treating diseases such as gout.

Description

Technical Field

[0001] The invention relates to the establishment and application of a method for screening xanthine oxidase (XOD) inhibitors from traditional Chinese medicines / natural products, and belongs to the field of medicine and health. Background Art

[0002] XOD is a key enzyme in the process of uric acid production. Currently, most of the uric acid-lowering drugs in the clinical frontline are XOD inhibitors, such as allopurinol, febuxostat, etc. Febuxostat and allopurinol can cause adverse reactions in patients with hyperuricemia during use, and it is particularly important to develop a new generation of highly effective and low-toxic XOD inhibitors. Currently, affinity ultrafiltration is often used to screen XOD inhibitors from traditional Chinese medicine / natural products, but there are "false positives" and "missing screening" of weak affinity compounds. The present invention adopts two-dimensional liquid chromatography technology to establish a method for screening XOD inhibitors from traditional Chinese medicine / natural product extracts, which effectively avoids the occurrence of "false positives" and "missing screening" phenomena. Summary of the invention

[0003] The present invention establishes a two-dimensional liquid chromatography system consisting of affinity chromatography and reversed-phase chromatography, takes the flavonoid extract of the flower part of saffron without stigma as the research object, and screens and characterizes the compounds bound to XOD in the flavonoid extract. The one-dimensional affinity chromatography consists of a XOD affinity column, an inactivated XOD affinity column and a reference chromatography column, which effectively reduces the "false positive" and "missed screening" phenomena in the screening process; the two-dimensional reversed-phase chromatography separates and qualitatively analyzes the compounds bound to XOD in the flavonoid extract. At the same time, surface plasmon resonance (SPR) technology, isothermal titration calorimetry (ITC) technology and an acute hyperuricemia mouse model are used to preliminarily characterize the in vivo and in vitro XOD inhibitory activity of the target compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 This is a connection flow diagram of the two-dimensional liquid chromatography screening system in Example 2 of the present invention.

[0005] Figure 2 This is a chromatogram of the screening results of XOD inhibitors in the flavonoid extract of the stigma-free flower part of Crocus sativus in Example 3 of the present invention. A, B and C: represent the peaks of the extract sample solution after passing through the active XOD column, the deactivated XOD column and the reference column. 18 Separation chromatogram on the column 1: Kaempferol-3-O-sophorobiose-7-O-glucoside; 2: Quercetin-3-O-sophoroside; 3: Kaempferol-3-O-sophoroside; 4: isorhamnetin-3-O-(2-O-rhamnosyl)-glucoside; 5: Kaempferol-3-O-rutinoside; 6: Kaempferol-3-O-glucoside DETAILED DESCRIPTION

[0006] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are limited to illustrating the present invention rather than limiting the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are deemed to be included within the scope of the present invention.

[0007] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0008] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0009] Example 1

[0010] Preparation and activity evaluation of affinity chromatography columns

[0011] (1) Preparation of XOD affinity chromatography column

[0012] Using a diol-based chromatographic column, XOD is coupled to the chromatographic column using the "Schiff" reaction principle. The process mainly includes the steps of activating the chromatographic column; oxidizing the diol group of the chromatographic column to an aldehyde group; coupling XOD to the chromatographic column; and blocking the aldehyde active sites that are not bound to XOD.

[0013] (2) Preparation of deactivated XOD column and reference column

[0014] The XOD affinity chromatography column was rinsed with pure methanol to obtain a deactivated XOD column.

[0015] The preparation of the reference column (uncoupled with XOD) was the same as that of the XOD affinity column, except for the coupling step with XOD.

[0016] (3) Evaluation of affinity chromatography column activity

[0017] The activity of the XOD affinity chromatography column was evaluated using a positive compound (febuxostat). The active XOD coupled to the column was 8.6 nmol. The affinity of the interaction between febuxostat and XOD (dissociation constant, K d ) is 3.87μmol / L.

[0018] Example 2

[0019] 2D-LC system connection flow path Figure 1 As shown. The one-dimensional affinity chromatography is composed of an XOD affinity column, an inactivated XOD affinity column and a reference chromatographic column. The components / sites in the sample to be tested that bind to XOD (i.e., the peak cutting time point) are determined by analyzing the chromatographic behaviors of the sample to be tested on the XOD affinity column, the inactivated XOD affinity column and the reference chromatographic column, and the collected eluent is stored in a loop through a ten-way valve; the compounds in the loop are separated by two-dimensional reverse phase chromatography, and the target compounds are qualitatively identified by reference substance control and mass spectrometry analysis. The relative binding degree (RBD) and specific binding degree (RBD) are used as indicators to evaluate the specific binding ability of the target compound with XOD. The calculation formulas of RBD and SBD are shown in Formula 1) and Formula 2).

[0020]

[0021]

[0022] Among them, A active The target compound flows through the active XOD column and then 18 Peak area on the chromatographic column; A control The target compound flows through the reference column and then 18 Peak area on the chromatographic column; A inactive The target compound flows through the deactivated XO column and then 18 The peak area on the chromatographic column. The larger the RBD value, the stronger the binding ability of the compound to XOD; the larger the SBD value, the stronger the specific binding ability of the compound to XOD. When the compound satisfies RBD>30% and SBD>10% at the same time, the compound has specific binding ability with XOD.

[0023] In order to evaluate the accuracy and precision of the two-dimensional liquid chromatography screening system constructed by the present invention, the performance of the screening method was evaluated using a mixed reference solution of febuxostat and a negative compound (tryptophan). The results showed that the accuracy and precision of the two-dimensional liquid chromatography screening system established by the present invention were good.

[0024] Example 3

[0025] The established two-dimensional liquid chromatography screening system was used to screen the compounds that specifically bind to XOD in the flavonoid extract of the stigma-free flower of Crocus sativus, such as Figure 2As shown. Among them, kaempferol-3-O-sophorobiose-7-O-glucoside and XOD are non-specifically bound, and the compounds that specifically bind to XOD in the flavonoid extract of the stigma-free flower of Crocus sativus include but are not limited to the following compounds: kaempferol-3-O-sophoroside (KS), kaempferol-3-O-rutinoside (KR), kaempferol-3-O-glucoside (KG), quercetin-3-O-sophoroside (QS, isorhamnetin-3-O-(2-O-rhamnosyl)-glucoside. The RBD and SBD parameters show that the above five compounds all have specific binding to XOD.

[0026] Example 4

[0027] Characterization of XOD inhibitory activity of target compounds in vitro and in vivo

[0028] In order to evaluate the in vitro and in vivo XOD inhibitory activity of the target compounds, the SPR technique was used to characterize the interaction between the above flavonoid glycosides and XOD; the ITC "one-step titration method" was used to evaluate the in vitro enzyme inhibitory activity of flavonoid glycosides on XOD; the in vivo XOD inhibitory activity of the representative compound - KS was investigated using the acute hyperuricemia mouse model induced by potassium oxonate. The results showed that the dissociation constant (K d ) values ​​were 39.8μmol / L, 9.5μmol / L, 38.9μmol / L and 11.4μmol / L respectively; the inhibitory constants (K I ) were 4.92mmol / L, 1.11mmol / L and 0.294mmol / L respectively; the catalytic rate constant (K cat ) values ​​are 3.16s -1 、3.15s -1 and 2.45s -1 (K of xanthine cat 3.81s -1 ); Compared with the mice in the acute hyperuricemia model group, KS can significantly reduce the uric acid content in the blood of hyperuricemia mice (P < 0.05) and inhibit the activity of XOD in the blood (P < 0.001) (n = 9). The above experimental data show that the target compound screened from the flower part of saffron without stigma has the effect of inhibiting the activity of XOD, indicating that the present invention can quickly and effectively screen out XOD inhibitors in traditional Chinese medicine / natural products.

Claims

1. A method for screening xanthine oxidase inhibitors in traditional Chinese medicine / natural products based on two-dimensional liquid chromatography technology combined with affinity chromatography (active xanthine oxidase column + inactivated xanthine oxidase column + reference column) and reversed-phase chromatography.

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