Method for screening hyaluronidase inhibitor by combining fluorescence sensing with affinity chromatography technology

Abstract

The invention provides a method for screening a hyaluronidase (HAase) inhibitor, and belongs to the technical field of medicines. The method comprises the following steps: firstly, rapidly identifying a natural product with HAase inhibition activity by adopting a fluorescence sensing technology according to the principle that carbon quantum dots (CDs) are used as a fluorescence probe, hyaluronic acid (HA) modified gold nanoparticles are used as a quencher, and the fluorescence of the CDs can be quenched by the quencher; when HAase exists, HA can be enzymatically decomposed to release CDs, and fluorescence recovery is caused; when a screened sample has HAase inhibition activity, CDs fluorescence recovery can be prevented. And 2, capturing components in a sample capable of inhibiting HAase activity by virtue of an affinity chromatography technology, immobilizing HAase on a carrier material by virtue of a method, eluting by virtue of a solvent to obtain components capable of being combined with HAase, and identifying the components by virtue of a liquid chromatography-mass spectrometry technology. According to the method, the HAase inhibitor can be rapidly and accurately screened from natural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and particularly relates to a method for screening hyaluronidase inhibitors based on fluorescence sensing combined with affinity chromatography technology. Background Art

[0002] Hyaluronic acid (HA) is a negatively charged linear non-sulfated glycosaminoglycan composed of repeating disaccharide units of (β-1,4-D-glucuronic acid and (β-1,3)-N-acetyl-D-glucosamine. As a key component of the extracellular matrix (ECM), the synthesis and degradation of HA are closely related to various biological processes, including fertilization, embryonic development, immune regulation, cell migration and differentiation, wound healing, inflammation, and tumor growth. The biological functions of HA largely depend on its molecular weight, and different-sized HA molecules play different roles: high-molecular-weight HA (HMW-HA) has anti-inflammatory properties, while low-molecular-weight HA (LMW-HA) promotes inflammatory responses.

[0003] Hyaluronidase (HAase), as an endoglycosidase that degrades HA, is widely distributed in the animal kingdom and plays an important role in the progression of various diseases. For example, the overexpression of HAase in tumor cells is closely related to cancer progression, and an increase in the level of HAase in urine can be used as a potential biomarker for bladder cancer. HAase also participates in the degradation of dermal protein matrix as an enzyme related to skin aging. In addition, HAase destroys the structural integrity of HA by cleaving anti-inflammatory HMW-HA into pro-inflammatory LMW-HA, thereby stimulating the production of pro-inflammatory cytokines and chemokines. Therefore, it is crucial to develop a simple and efficient method for screening HAase inhibitors.

[0004] Currently, the detection methods for HAase include viscosity method, Raman spectroscopy, electrochemical technology, fluorescence biosensing method, etc. Among them, the fluorescence biosensing method has attracted much attention due to its rapid, sensitive, and convenient characteristics. Among fluorescence probes, carbon dots (CDs) are considered a promising class of nanomaterials due to their small size, low toxicity, and excellent biocompatibility. Fluorescence biosensing methods can achieve rapid screening of enzyme inhibitors in complex systems, but they face major challenges in separating individual active components. In recent years, immobilized enzyme affinity chromatography technology has received increasing attention due to its advantages such as fast separation speed, low cost, good thermal stability, and reusability. This technology performs dynamic screening by immobilizing target molecules on various carrier materials such as silica, magnetic nanoparticles, and multi-walled carbon nanotubes.

[0005] Therefore, the present invention combines the advantages of biosensing and affinity chromatography to achieve high-throughput screening of hyaluronidase inhibitors in traditional Chinese medicine, as well as the capture and identification of active components. It has important scientific significance and application value for new drug development and contributes to safeguarding public health. SUMMARY OF THE INVENTION

[0006] In view of this, the present invention has developed a method for screening hyaluronidase inhibitors in natural products including traditional Chinese medicine based on a fluorescence sensor combined with affinity chromatography technology. This method can quickly, efficiently and accurately screen for hyaluronidase inhibitors.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows: 1. A method for screening hyaluronidase inhibitors by fluorescence sensing combined with affinity chromatography technology, comprising the following steps: (1) Screening of samples with hyaluronidase inhibitory activity: Using p-aminophenol and ethylenediamine as raw materials, quantum dot fluorescent probes (CDs) are synthesized in one step under mild conditions and further purified by column chromatography. Hyaluronic acid (HA) and gold nanoparticles (AuNPs) are self-assembled to synthesize HA-AuNPs, which are purified with a 10 kDa ultrafiltration centrifugal tube and used as a quencher. Hyaluronidase (HAase) and a traditional Chinese medicine extract solution are added to a centrifuge tube and incubated at 37 °C for 30 minutes, then diluted CDs, HA-AuNPs and PBS are added and incubated for another 2 hours, and the fluorescence is measured. Traditional Chinese medicines with HAase inhibitory activity are screened according to whether the fluorescence is quenched. (2) Preparation of a hyaluronidase affinity chromatography support material: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are respectively dissolved in morpholineethanesulfonic acid, then multi-walled carbon nanotubes (MWCNTs) are added and reacted with shaking at room temperature for a certain time to add aldehyde groups to the MWCNTs. After centrifugation, washing and filtration, HAase is added and reacted for a certain time to covalently bind the aldehyde groups of the MWCNTs to the carboxyl groups of HAase, obtaining the HAase@MWCNTs affinity chromatography support material. (3) Separation and identification of components with hyaluronidase inhibitory activity: Affinity chromatography is used to separate the components that can bind to HAase. The traditional Chinese medicine extract screened in step (1) with HAase inhibitory activity is added to the HAase@MWCNTs affinity chromatography support material obtained in (2), and after sufficient shaking reaction for a certain time, the unadsorbed supernatant is centrifuged and taken out. An appropriate solvent is used for elution to obtain the solution bound to HAase@MWCNTs, which is the adsorption solution. The initial solution of the traditional Chinese medicine extract, the unadsorbed solution and the adsorption solution are respectively injected into a high performance liquid chromatograph for detection to screen out the components that bind to HAase@MWCNTs; liquid chromatography-mass spectrometry technology is used to identify the adsorbed components; by measuring the half inhibitory concentration of the screened binding components against HAase, HAase inhibitors are finally obtained.

[0008] Preferably, in the screening method of the hyaluronidase inhibitor in step (1), the solution concentration of the quantum dots is 1 mg / mL.

[0009] Preferably, for the screening of the sample with hyaluronidase inhibitory activity in step (1), it is characterized in that the ratio of the addition amounts of the quantum dots, quencher, hyaluronidase and traditional Chinese medicine extract is 0.67:1:1:1.

[0010] Preferably, for the screening of the sample with hyaluronidase inhibitory activity in step (1), it is characterized in that the concentration of the traditional Chinese medicine extract solution is 1 mg / mL.

[0011] Preferably, for the screening of the sample with hyaluronidase inhibitory activity in step (1), it is characterized in that the concentration of the hyaluronidase solution is 7.8125 μg / mL to 500 μg / mL.

[0012] Preferably, for the separation and identification of the components with hyaluronidase inhibitory activity in step (3), it is characterized in that the affinity chromatography method is specifically as follows: the chromatographic column is C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength is 270 nm, mobile phase A is 0.1% phosphoric acid, mobile phase B is acetonitrile, and the gradient elution program is 0 min - 98% A; 10 min - 89% A; 13 min - 87% A; 38 min - 72% A. The column temperature is 35°C and the flow rate is 1.0 mL / min.

[0013] Preferably, for the separation and identification of the components with hyaluronidase inhibitory activity in step (3), it is characterized in that the liquid chromatography - mass spectrometry method is specifically as follows: the chromatographic column is C18 (2.1 mm × 50 mm; 3.5 μm), mobile phase A is 0.1% formic acid, mobile phase B is 0.1% formic acid acetonitrile, and the gradient elution program is 0 min - 98% A; 10 min - 89% A; 13 min - 87% A; 38 min - 72% A. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320°C, the sheath gas temperature is 350°C, the gas flow rate is 11 L / min, and the mass range selection is 50 - 3000 (m / z).

[0014] The anti - inflammatory effect of the HAase inhibitor is verified by measuring the half - inhibitory concentration of hyaluronidase and in vitro cell experiments in the present invention.

[0015] As can be seen from the above technical solutions, the present invention provides a method for screening hyaluronidase inhibitors based on fluorescence sensing and affinity chromatography techniques, belonging to the technical field of drug screening. In the first step of this method, fluorescence sensor technology is used to quickly identify samples with hyaluronidase inhibitory activity. The principle is that synthesized carbon quantum dots are used as fluorescence probes, and hyaluronic acid-modified gold nanoparticles (HA-AuNPs) are used as quenchers. Among them, HA-AuNPs can quench the fluorescence of carbon quantum dots through fluorescence resonance energy transfer. In the presence of hyaluronidase, hyaluronic acid is enzymatically cleaved into small fragments, thereby releasing carbon quantum dots and resulting in fluorescence recovery. If there are components with hyaluronidase inhibitory activity in the sample to be screened, the fluorescence recovery of carbon quantum dots can be blocked, thus initially screening samples with hyaluronidase inhibitory activity. In the second step, affinity chromatography technology is used to identify the components in the samples with hyaluronidase inhibitory activity. The method immobilizes hyaluronidase on a carrier material, elutes with a solvent to obtain the components that can bind to hyaluronidase, and then uses liquid chromatography-mass spectrometry technology to identify the components. The screened active components are further verified through in vitro cell experiments. This method can efficiently and accurately screen hyaluronidase inhibitors, with high screening efficiency and low false positive rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0017] Attached Figure 1 is a schematic diagram of the screening method process of the present invention. (Figure A is a schematic diagram of fluorescence sensing screening; Figure B is a schematic diagram of affinity chromatography screening; Figure C is a schematic diagram of in vitro cell experiment of gallic acid) Attached Figure 2 is the characterization of quantum dots and their fluorescence characteristics. (Figure A is the transmission electron microscope image of CDs; Figure B is the atomic force microscope image of CDs; Figure C is the fluorescence excitation and emission spectra of CDs; Figure D is the fluorescence emission spectra of CDs at different excitation wavelengths) Attached Figure 3 is the investigation of the addition ratios of the synthesized quantum dots, hyaluronic acid, and hyaluronidase.

[0018] Attached Figure 4 is the verification of the fluorescence sensing screening method. (Figure A shows the fluorescence spectra of the system measured in the range of HAase concentration from 0 to 500 µg / mL, and the inset shows the calibration curve of HAase; Figure B is the selectivity verification of the fluorescence sensing model; Figure C is the positive drug verification of the fluorescence sensing model; Figure D is the negative drug verification of the fluorescence sensing model) Appendix Figure 5 Characterization of multi-walled carbon nanotubes and multi-walled carbon nanotubes loaded with hyaluronidase. (Figure A is a schematic diagram of the characterization; Figures B and C are transmission electron micrographs of MWCNTs; Figures D and E are transmission electron micrographs of HAase@MWCNTs; Figure F is the X-ray photoelectron spectroscopy of MWCNTs and HAase@MWCNTs; Figure G is the synchronous thermal analysis diagram of MWCNTs and HAase@MWCNTs) Appendix Figure 6 Evaluation of the affinity chromatography screening model. (Figure A is tannic acid affinity chromatography; Figure B is argatroban affinity chromatography; Figure C is avibactam sodium affinity chromatography; Figure D is acetazolamide affinity chromatography; Figure E is the adsorption rate of the positive drug and the negative drug on HAase@MWCNTs) Appendix Figure 7 For the screening results of hyaluronidase inhibitors in 43 traditional Chinese medicines and the determination of the half-inhibitory concentration IC 50 value of one kind of hyaluronidase inhibitor screened. (Figure A is a schematic diagram of the affinity chromatography for screening the active components that can inhibit hyaluronidase activity in traditional Chinese medicines; Figure B is the fluorescence sensing model for screening 43 traditional Chinese medicines; Figure C is the adsorption chromatogram of the extract of Rhodiola rosea added to HAase@MWCNTs; Figure D is the ion chromatogram and mass spectrum of gallic acid extraction; Figure E is the half-inhibitory concentration of gallic acid) Appendix Figure 8 For the results of gallic acid inhibiting allergic inflammation. (Figure A is the determination of cell viability 24 h after gallic acid administration; Figure B is the determination of cell viability 48 h after gallic acid administration; Figures C, D, E, and F are the detections of the concentrations of IL-4, IL-5, IL-6, and TNF-α in the cell supernatant) Specific implementation manners

[0019] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0020] The embodiments of the present invention disclose a method for screening hyaluronidase inhibitors based on fluorescence sensing combined with affinity chromatography technology, including the following steps: Example 1

[0021] Synthesis, Characterization and Fluorescent Properties of Quantum Dots. Mix 5 mL of p-aminophenol solution (1 M) and 5 mL of ethylenediamine solution (1 M), dilute to 20 mL with phosphate buffer, and stir at 50 °C for 5 hours. Subsequently, evaporate the solvent using a freeze dryer and purify to obtain a black solid powder product, namely quantum dots (CDs), by column chromatography (using ethyl acetate as the eluent). The properties of the CDs were studied through a series of characterization experiments. The surface morphology of the CDs was characterized using a transmission electron microscope (TEM) (Talos F200S), and the results are as Figure 2 shown in Figure A. The results show that the prepared CDs have a uniform morphology and good monodispersity. The CDs are spherical in shape, with a diameter range of 1 - 5 nm. The morphology of the CDs was observed using a Bruker Dimension EDGE atomic force microscope (AFM), and the results are as Figure 2 shown in Figure B. The results show that the CDs are evenly distributed and spherical. The excitation and emission wavelengths of the CDs were further investigated using a fluorescence spectrophotometer, and the results are as Figure 2 shown in Figure C. The results show that the excitation and emission wavelengths of the CDs are 415 nm and 520 nm, respectively. In addition, an emission spectrum was obtained by screening a series of excitation wavelengths (375, 380, 385, 390, 395, 400, 405, 410, 415, 420 nm) using a fluorescence spectrophotometer, and the results are as Figure 2 shown in Figure D. The results show that as the excitation wavelength increases, the fluorescence intensity first increases and then decreases significantly, and finally reaches a maximum at 415 nm. The above results indicate that the CDs were successfully prepared, with a maximum excitation wavelength of 415 nm and a maximum emission wavelength of 520 nm. Example 2

[0022] Experiment on the Proportion of Quantum Dots, Hyaluronic Acid and Hyaluronidase Added. Take 16 EP tubes, add 20 μL of the prepared CDs (1 mg / ml) solution to each EP tube, and then add 10 μL, 20 μL, 30 μL, 40 μL of HA-AuNPs and 10 μL, 20 μL, 30 μL, 40 μL of HAase (500 μg / mL) respectively. Incubate at 37 °C for 2 hours, and record the maximum fluorescence intensity at 450 - 600 nm under an excitation wavelength of 415 nm. The results are as Figure 3 shown. The results show that when 20 μL of CDs, 30 μL of HA-AuNPs and 30 μL of HAase are added, the fluorescence intensity recovery is the largest. Therefore, the ratio of the addition amounts of CDs, HA-AuNPs and HAase is selected as 0.67:1:1. Example 3

[0023] Hyaluronidase linear range and standard curve experiment. Prepare a series of HAase solutions with concentrations of 1.9 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 15.6 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL. Take 30 μL of each solution and add them to a mixed solution containing 20 μL of CDs, 30 μL of HA-AuNPs, and 120 μL of PBS, and incubate at 37 °C for 2 hours. Record the maximum fluorescence intensity at 450 - 600 nm under an excitation wavelength of 415 nm. The results are shown in Figure 4 Figure A. The results show that in the concentration range of 1.9 μg / mL to 500 μg / mL, as the concentration of the HAase solution gradually increases, the fluorescence value gradually increases. That is, there is a proportional relationship between the concentration of the hyaluronidase solution and the fluorescence intensity value. The linear equation is y = 1.41433 + 0.00302x, and R 2 = 0.9954. Example 4

[0024] Hyaluronidase selectivity verification experiment. Take 30 μL each of hyaluronidase, bovine serum albumin, β-lactamase, tyrosinase, hemoglobin, β-galactosidase, and carbonic anhydrase, with a concentration of 500 μg / mL for each. Add them to 20 μL of CDs, 30 μL of HA-AuNPs, and 120 μL of PBS solution respectively, and incubate at 37 °C for 2 hours. Record the maximum fluorescence intensity at 450 - 600 nm under an excitation wavelength of 415 nm. The results are shown in Figure 4 Figure B. The results show that after adding HAase, the fluorescence intensity of the solution recovers to the greatest extent. When other enzymes are added, the fluorescence intensity of the solution hardly recovers. Therefore, the established fluorescence sensing method can be used for the screening of HAase inhibitors. Example 5

[0025] Verification experiment of hyaluronidase positive inhibitor. A series of tannic acid (TA, hyaluronidase positive inhibitor) solutions with concentrations of 0 μg / mL, 1.9 μg / mL, 3.9 μg / mL, 7.8 μg / mL, 31.25 μg / mL, and 62.5 μg / mL were prepared. 30 μL of each TA solution and 30 μL of HAase solution (500 μg / mL) were pre-incubated for 30 min, and then 20 μL of CDs, 30 μL of HA-AuNPs, and 90 μL of PBS solution were added respectively, and incubated at 37 °C for 2 hours. The maximum fluorescence intensity at 450 - 600 nm under the excitation wavelength of 415 nm was recorded. The results are shown in Figure 4 Figure C. The results show that as the concentration of TA increases, the maximum fluorescence value gradually decreases. The above results indicate that TA inhibits the activity of HAase, resulting in a decrease in the fluorescence intensity of the solution. That is, the established fluorescence sensing method can accurately screen out HAase inhibitors. Example 6

[0026] Verification experiment of hyaluronidase negative inhibitor. 30 μL of tannic acid (hyaluronidase positive inhibitor), and 30 μL of acetazolamide, avibactam sodium, and argatroban, which have no inhibitory activity on hyaluronidase, were taken. The above four drugs were pre-incubated with 30 μL of HAase solution (500 μg / mL) for 30 min respectively, and then 20 μL of CDs, 30 μL of HA-AuNPs, and 90 μL of PBS solution were added respectively, and incubated at 37 °C for 2 hours. The maximum fluorescence intensity at 450 - 600 nm under the excitation wavelength of 415 nm was recorded. The results are shown in Figure 4 Figure D. The maximum fluorescence intensity in the mixed solutions containing acetazolamide, avibactam sodium, and argatroban is higher than that in the tannic acid solution, that is, acetazolamide, avibactam sodium, and argatroban cannot inhibit the activity of hyaluronidase and cause the fluorescence intensity of the solution to decrease. The above shows that the established fluorescence sensing method is not affected by other non-HAase inhibitors. Example 7

[0027] Characterization of the affinity chromatography support material - immobilized enzyme HAase@MWCNTs. The properties of HAase, MWCNTs, and HAase@MWCNTs were investigated through a series of characterization experiments. The surface morphology of HAase@MWCNTs was characterized using a transmission electron microscope (TEM) (Talos F200S); the elemental compositions of MWCNTs and HAase@MWCNTs were analyzed using a Thermo Fisher K-ALPHA X-ray photoelectron spectrometer (XPS); the surface structures and properties of MWCNTs and HAase@MWCNTs were studied using a NETZSCH STA 449 F3 / METTLER1100LF synchronous thermal analyzer (TG-DSC). The TEM results showed that HAase@MWCNTs ( Figure 5 B and 5C) still maintained the basic morphology consistent with the original MWCNTs ( Figure 5 D and 5E), making it difficult to confirm whether HAase was successfully immobilized on MWCNTs solely through morphological analysis. To further clarify the surface chemical composition, elemental distribution, and compositional changes, XPS analysis was conducted. As Figure 5 shown in F, the change in nitrogen content confirmed the successful immobilization of HAase on the surface of MWCNTs. In addition, the TG-DSC was used to study the thermal behavior of MWCNTs and HAase@MWCNTs during heating. As Figure 5 shown in G, HAase@MWCNTs exhibited two characteristic mass loss stages: an initial mass reduction of 2.07% in the range of 33 - 201 °C, followed by a significant reduction of 8.25% in the range of 201 - 634 °C. These results indicated that the immobilization of HAase enzyme on MWCNTs led to an obvious exothermic trend. The above results showed that HAase was successfully immobilized on the surface of MWCNTs, and the affinity chromatography support material of immobilized enzyme HAase@MWCNTs was prepared. Example 8

[0028] Positive and negative adsorption verification experiments for affinity chromatography. To 4 EP tubes containing HAase@MWCNTs, 200 μL of tannic acid, argatroban, avibactam sodium, and acetazolamide were added respectively. After mixing, they were placed in an oscillator and shaken for 30 minutes, then left to stand, and the supernatant was obtained by centrifugation, which was the non-adsorbed solution. Subsequently, 200 μL of a mixed solution of acetonitrile - 10% acetic acid (volume ratio of the two was 1:1) was added to the remaining solid. After mixing, it was placed in a room temperature oscillator and shaken for 30 minutes, and the supernatant was obtained by centrifugation, which was the adsorbed solution. The initial solutions, non-adsorbed solutions, and adsorbed solutions of the above drugs were analyzed by high performance liquid chromatography. The results are shown inFigure 6 As shown. The results indicate that the HAase positive inhibitor tannic acid has good adsorption with HAase@MWCNTs, while the three HAase negative inhibitors argatroban, avibactam sodium, and acetazolamide have poor adsorption with HAase@MWCNTs. The above shows that HAase@MWCNTs can strongly adsorb HAase inhibitors, so it can be used for the capture of HAase inhibitors in complex systems including traditional Chinese medicine extracts. Example 9

[0029] Experiment on screening hyaluronidase inhibitors in traditional Chinese medicine by fluorescence sensing. Take 44 EP tubes and first add tannic acid, Rhodiola rosea Rhodiola rosea L., Paeonia lactiflora Paeonia veitchii Lynch, Cyperus rotundus Cyperus rotundus L., Paeonia veitchii Cynanchum otophyllum Schneid., Pheretima aspergillum Lonicera japonica Thunb, Polygonum multiflorum Fallopia multiflora (Thunb.) Haraldson, Morinda officinalis Morinda officinalis How, Achyranthes bidentata Achyranthes bidentata Blume, Gastrodia elata Bl., Myrrh Commiphora myrrha (Nees) Engl., Cuscuta chinensis Cuscuta chinensis L., Ephedra sinica Ephedra sinica Stapf, Plantago asiatica Plantago asiatica L., Sophora alopecuroides Sophora alopecuroides L., Glycyrrhiza uralensis Glycyrrhiza uralensis Fisch. ex DC., Saposhnikovia divaricata Saposhnikovia divaricata (Trucz.) Schischk., Juglans mandshurica Juglans regia L., Salvia miltiorrhiza Salvia miltiorrhiza , Ginkgo biloba Ginkgo biloba L., Xanthium sibiricum Xanthium strumarium L., Cinnamomum cassia Ramulus Cinnamomi , Magnolia liliflora Magnolia biondii Pamp., Panax notoginseng Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow, Foeniculum vulgare Foeniculum vuLgare Mill., Atractylodes macrocephala Koidz., Wolfberry leaf ( Atractylodes macrocephala Koidz.), Lycium barbarumL.))、Epimedium ( Epimedium brevicornu Maxim.), Codonopsis pilosula ( Codonopsis pilosula (Franch.) Nannf.), Phellodendron chinense ( Phellodendron chinense Schneid.), Polygala tenuifolia ( Polygala tenuifolia Willd.)、Sophora flavescens( Sophora flavescens Aiton、Mitsuko ( Fructus Leonuri )、Silver Bupleurum( Stellaria dichotoma var. lanceolata Bunge), break ( Dipsacus asper Wall.exHenry), Nutmeg ( Myristica fragrans Houtt.), Astragalus ( Astragalus membranaceus (Fisch.) Bunge), costus root ( Aucklandia lappa Decne.), Coptis chinensis ( Coptis chinensis Franch.), wolfberry ( Lycium barbarum L.), Magnolia officinalis ( Houpoea officinalis (Rehder & EH Wilson) NH Xia & CY Wu), Psoralea corylifolia ( Cullen corylifolium (Linnaeus) Medikus), Stephania tetrandra ( Stephania tetrandra S.Moore )、Qinjiao( Gentiana macrophylla Then, 30 μL of HAase solution (500 μg / mL) was added to each sample and incubated for 30 min. Then, 20 μL of CDs, 30 μL of HA-AuNPs, and 90 μL of PBS solution were added and incubated at 37°C for 2 h. The maximum fluorescence intensity at 450–600 nm under an excitation wavelength of 415 nm was recorded. The results are shown in Table 1. Figure 7 As shown in B. The results showed that the maximum fluorescence value of the system solution after adding Rhodiola rosea was significantly weakened, indicating that the activity of HAase decreased after reacting with the Chinese medicine Rhodiola rosea extract, that is, the Rhodiola rosea extract has the effect of inhibiting HAase activity, and there are components in the Rhodiola rosea extract that can inhibit HAase activity. Example 10

[0030] Experiment on screening components in Rhodiola rosea extract that can bind to hyaluronidase by affinity chromatography. Using the Rhodiola rosea extract with HAase inhibitory activity screened in Example 9 as the research object, add 200 μL of Rhodiola rosea extract (1 mg / ml) to an EP tube containing HAase@MWCNTs. After mixing, place it on an oscillator and shake for 30 minutes. Centrifuge to obtain the supernatant (unadsorbed solution). Subsequently, add 200 μL of a mixed solution of acetonitrile - 10% acetic acid (volume ratio of the two is 1:1) to the remaining solid. After mixing, place it on a room temperature oscillator and shake for 30 minutes. Centrifuge to obtain the supernatant (adsorbed solution). Analyze the initial solution, unadsorbed solution, and adsorbed solution of the Rhodiola rosea extract by high performance liquid chromatography. In addition, prepare a gallic acid reference solution (100 μg / mL) and analyze it using the same high performance liquid chromatography method. The results are shown in Figure 7 Figure C (a: initial solution of Rhodiola rosea extract; b: unadsorbed solution of Rhodiola rosea extract; c: adsorbed solution of Rhodiola rosea extract; d: gallic acid reference solution). The results show that in the high performance liquid chromatography chart of the adsorbed solution of Rhodiola rosea extract, 1 chromatographic peak can be clearly seen. Through verification with the reference substance, it is gallic acid, that is, HAase@MWCNTs successfully captured gallic acid, the component in Rhodiola rosea that can bind to HAase. Example 11

[0031] Experiment on further determining the components in Rhodiola rosea that can bind to hyaluronidase by liquid chromatography - mass spectrometry. Add 200 μL of Rhodiola rosea extract to an EP tube containing HAase@MWCNTs. After mixing, place it on a room temperature oscillator and shake for 30 minutes. Centrifuge to obtain the supernatant (unadsorbed solution). Subsequently, add 200 μL of a mixed solution of acetonitrile - 10% acetic acid (volume ratio of the two is 1:1) to the remaining solid. After mixing, place it on an oscillator and shake for 30 minutes. Centrifuge to obtain the supernatant (adsorbed solution). Analyze the adsorbed solution of the Rhodiola rosea extract by liquid chromatography - mass spectrometry. The results are shown in Figure 7 Figure D. The results show that gallic acid was detected in the adsorbed solution of Rhodiola rosea extract. Therefore, it was determined that the component in traditional Chinese medicine Rhodiola rosea that can bind to HAase is gallic acid. Example 12

[0032] Half - inhibitory concentration IC of gallic acid, a hyaluronidase inhibitor 50Determination experiment. A series of gallic acid reference solutions with concentrations of 1.95 μg / mL, 3.90 μg / mL, 7.8125 μg / mL, 15.625 μg / mL, 25 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 100 μg / mL, 125 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL were prepared. 30 μL of each solution was incubated with 30 μL of HAase solution (500 μg / mL) in advance for 30 min, and then 20 μL of CDs, 30 μL of HA-AuNPs, and 90 μL of PBS solution were added respectively, and incubated at 37 °C for 2 hours. The maximum fluorescence intensity at 450 - 600 nm under the excitation wavelength of 415 nm was recorded. The results are shown in Figure 7 shown in 50 Figure E. The results showed that the IC Example 13

[0033] Determination experiment on the anti-inflammatory activity of gallic acid as a hyaluronidase inhibitor. HNEpC cells were seeded in 96-well plates at a density of 1×10 5 cells / well and cultured overnight at 37 °C and 5% CO2 in DMED medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Pen / Strep). Subsequently, a series of gallic acid reference solutions with concentrations of 7.8125 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL were added to the medium with a total volume of 100 μL, and incubated at 37 °C for 24 hours and 48 hours. The cell viability was detected using the cell counting kit-8 (CCK-8): after adding the CCK-8 reagent to each well, it was incubated at 37 °C for 1 - 2 hours, and the absorbance was measured at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 8 Figures A and 8B. The results showed that when the concentration of gallic acid ≤ 31.25 μg / mL, it had no effect on cell activity. That is, gallic acid with a concentration of 31.25 μg / mL was selected for the anti-inflammatory experiment, and the administration time was 24 hours. The inflammatory marker analysis experiment was divided into three groups: (1) control group, (2) lipopolysaccharide (LPS) model group, and (3) LPS + gallic acid administration group. The cell culture supernatants of each group were collected, and the corresponding enzyme-linked immunosorbent assay (ELISA) kits were used to quantitatively detect the concentrations of interleukin (IL)-4, IL-5, IL-6, and tumor necrosis factor-α (TNF-α) in the cell supernatants. The results are shown inFigure 8 as shown in C, 8D, 8E, and 8F. The results showed that compared with the control group, the levels of IL-4, IL-5, IL-6, and TNF-α in HNEpC cells were significantly increased after LPS stimulation ( p <0.001). After intervention with gallic acid, the secretion levels of IL-4, IL-5, IL-6, and TNF-α induced by LPS were significantly recalled to normal levels ( p <0.001). The above results indicate that gallic acid can inhibit the inflammatory response induced by LPS.

[0034] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0035] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening hyaluronidase inhibitors by fluorescence sensing combined with affinity chromatography, comprising the following steps: (1)Screening of samples with hyaluronidase inhibitory activity: Using p-aminophenol and ethylenediamine as raw materials, quantum dot fluorescent probes (CDs) were synthesized in one step under mild conditions and further purified by column chromatography. Hyaluronic acid (HA) and gold nanoparticles (AuNPs) were self-assembled to synthesize HA-AuNPs, which were used as quenchers after purification with a 10 kDa ultrafiltration centrifugal tube. After incubating hyaluronidase (HAase) and traditional Chinese medicine extract solution in a centrifuge tube at 37 °C for 30 minutes, diluted CDs, HA-AuNPs and PBS were added and incubated for another 2 hours, and then the fluorescence was measured. Traditional Chinese medicines with HAase inhibitory activity were screened according to whether the fluorescence was quenched. (2) Preparation of hyaluronidase affinity chromatography support material: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were respectively dissolved in morpholineethanesulfonic acid, and then multi-walled carbon nanotubes (MWCNTs) were added and reacted with shaking at room temperature for a certain time to add aldehyde groups to MWCNTs. After centrifugation, washing and filtration, HAase was added and reacted for a certain time to covalently bind the aldehyde groups of MWCNTs to the carboxyl groups of HAase, and the HAase@MWCNTs affinity chromatography support material was obtained. (3) Separation and identification of components with hyaluronidase inhibitory activity: Affinity chromatography was used to separate the components that could bind to HAase. The traditional Chinese medicine extract screened in step (1) with HAase inhibitory activity was added to the HAase@MWCNTs affinity chromatography support material obtained in (2). After sufficient shaking reaction for a certain time, the supernatant of the unadsorbed solution was centrifuged and taken out. The solution bound to HAase@MWCNTs was eluted with a suitable solvent to obtain the adsorbed solution. The initial solution of the traditional Chinese medicine extract, the unadsorbed solution and the adsorbed solution were respectively injected into a high performance liquid chromatograph for detection to screen out the components that bound to HAase@MWCNTs; liquid chromatography-mass spectrometry technology was used to identify the adsorbed components; by measuring the half inhibitory concentration of the screened binding components on HAase, the HAase inhibitor was finally obtained.

2. The screening method of the hyaluronidase inhibitor according to claim 1, wherein: The solution concentration of the quantum dots is 1 mg / mL.

3. The screening method of the hyaluronidase inhibitor according to claim 1, wherein: The ratio of the addition amounts of the quantum dots, quencher, hyaluronidase and traditional Chinese medicine extract is 0.67:1:1:

1.

4. The screening method of the hyaluronidase inhibitor according to claim 1, characterized in that: The solution concentration of the traditional Chinese medicine extract is 1 mg / mL.

5. The screening method of the hyaluronidase inhibitor according to claim 1, wherein The solution concentration of the hyaluronidase is 7.8125 μg / mL - 500 μg / mL.

6. The screening method of the hyaluronidase inhibitor according to claim 1, characterized in that The specific affinity chromatography method is as follows: The chromatographic column is C18 (4.6 × 150 mm; 3.5 μm), the detection wavelength is 270 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is acetonitrile, and the gradient elution program is 0 min - 98% A; 10 min - 89% A; 13 min - 87% A; 38 min - 72% A. The column temperature is 35 °C and the flow rate is 1.0 mL / min.

7. The screening method of the hyaluronidase inhibitor according to claim 1, wherein The specific liquid chromatography-mass spectrometry method is as follows: The chromatographic column is C18 (2.1 mm × 50 mm; 3.5 μm), mobile phase A is 0.1% formic acid, mobile phase B is 0.1% formic acid-acetonitrile, and the gradient elution program is 98% A at 0 min; 89% A at 10 min; 87% A at 13 min; 72% A at 38 min. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320 °C, the sheath gas temperature is 350 °C, the gas flow rate is 11 L / min, and the mass range selection is 50 - 3000 (m / z).

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