CN114958362B

CN114958362B - Angiotensin converting enzyme inhibitor

Info

Publication number: CN114958362B
Application number: CN202210652389.2A
Authority: CN
Inventors: 湛志华; 薛茗月; 毛惠会
Original assignee: Lingnan Normal University
Current assignee: Lingnan Normal University
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2024-01-26
Anticipated expiration: 2042-06-08
Also published as: CN114958362A

Abstract

The invention discloses an angiotensin converting enzyme inhibitor, which is a yellow aeolian wood leaf extract, wherein the inhibition rate of 2.5mg of the yellow aeolian wood leaf extract to 0.5U/L ACE is 88.20%; the preparation method of the yellow aeolian wood leaf extract comprises the steps of taking yellow aeolian wood leaves, shearing the yellow aeolian wood leaves, and then placing the cut yellow aeolian wood leaves in a beaker; then adding ethanol for extraction; recovering solvent after extraction, adsorbing the concentrated solution by D101 macroporous resin, eluting with ethanol, recovering solvent by rotary evaporator, and evaporating to dryness to obtain yellow brown crude product; then, the yellow aeolian-wood leaf extract is obtained through absolute ethyl alcohol reflux refining, concentration and freeze drying, and the obtained yellow aeolian-wood leaf extract is stored in a dryer for standby.

Description

Angiotensin converting enzyme inhibitor

Technical Field

The invention belongs to the field of biochemical analysis, and particularly relates to an angiotensin converting enzyme inhibitor.

Background

Hypertension is a major factor in the occurrence of cardiovascular disease worldwide. Angiotensin converting enzyme (Angiotensin converting enzyme, ACE) hydrolyzes angiotensin I (Ang I) to angiotensin II (Ang II), which is an effective vasoconstrictor, when ACE activity is increased, increases hydrolyzed Ang II, resulting in an increase in blood pressure, and thus ACE is a potential target for developing antihypertensive drugs. The normal level of ACE activity in serum as determined spectrophotometrically is 7-25U/L. In addition to hydrolyzing Ang I, ACE is also capable of hydrolyzing many other polypeptides, such as substance P, enkephalin, and bradykinin, affecting a variety of physiological processes including reproduction, renal metabolic function, hematopoiesis, and immune response. Therefore, efficient and rapid detection of ACE activity levels in human serum is of great importance for the prevention of hypertension and the discovery of new angiotensin converting enzyme inhibitors (Angiotensin converting enzyme inhibitor, ACEI).

ACEI screening relies on corresponding enzyme activity detection methods. At present, the detection method of ACE activity mainly comprises a high performance liquid chromatography, an enzyme coupling method, a high performance liquid chromatography mass spectrometry and the like. Most of these methods require sample processing through multiple steps, are cumbersome to operate, and have problems of low sensitivity. In addition, these methods all require expensive precision equipment investment, and the detection requires a large amount of organic solvents, which is costly and environmentally unfriendly.

Prior document 1 (CN 1831529 a) discloses a method for rapid screening of angiotensin converting enzyme inhibitors using high performance liquid chromatography and mass spectrometry. The method comprises the steps of taking the PMIDA or the PMIDA leucine as a substrate, taking purified rabbit pulmonary angiotensin converting enzyme or human blank plasma as a source of the angiotensin converting enzyme, heating in a water bath for reaction, filtering a membrane or centrifuging, separating the substrate, a product and an added internal standard in an angiotensin converting enzyme reaction mixed solution by using high performance liquid chromatography, and detecting the hippuric acid generated by the hydrolysis of the substrate catalyzed by the angiotensin converting enzyme in a negative ion mode of electrospray mass spectrum.

The prior art document 2 (CN 108659099 a) discloses a mass spectrometry probe for detecting the activity of angiotensin converting enzyme and application thereof, wherein a polypeptide with an amino acid sequence of Asp-Ser-Asp-Lys-Pro and a piperazine compound modified on aspartic acid at the N-terminal of the polypeptide are taken as mass spectrometry probes, which are taken as enzyme substrates and specifically identified by angiotensin converting enzyme, and the activity of angiotensin converting enzyme is detected by measuring the amount of probes or cleavage products before and after cleavage reaction by mass spectrometry.

The inventors have found that the above problems can be solved by carbon quantum dots (Carbon quantum dots, CQDs). CQDs have unique optical properties, but also exhibit low cytotoxicity and good biocompatibility due to the main carbon element content, thereby promoting the application of carbon dots in biological fields such as biological cell labeling, biological imaging, biological detection, disease diagnosis, and drug release applications. Therefore, the development of novel low-toxicity fluorescent nano materials with good biocompatibility based on CQD, and the construction of fluorescent nano probes with good stability and high sensitivity for detecting ACE activity are a new idea.

On the basis, an ACE is utilized to catalyze a product Hippuric Acid (HA) generated by a specific substrate, namely, hippocampal-histidyl-leucine (Hip-His-Leu, HIL), and the activity of ACE can be detected through an indirect reaction by detecting the change of fluorescence intensity after a BL-CQDs fluorescent probe is combined with the HA.

Disclosure of Invention

The invention aims at comprising the following steps:

1. providing a BL-CQDs fluorescent probe and a preparation method thereof, wherein BL is the acronym of English words of ficus microcarpa leaves in the abbreviation of the BL-CQDs fluorescent probe;

2. based on BL-CQDs fluorescent probes, ACE activity detection is realized by using a fluorescence analysis method;

3. ACEI screening is realized based on an ACE activity detection method, and an angiotensin converting enzyme inhibitor is obtained.

Aiming at the technical problems existing in the prior art, the principle and the method related by the invention comprise the following steps: the fresh ficus microcarpa leaves are used as a carbon source, and finally the single-excitation double-emission fluorescent carbon nanomaterial with excellent fluorescent performance is prepared; the carbon quantum dot is used as a fluorescent probe, so that the detection limit and the detection range of the activity detection of the angiotensin converting enzyme are enhanced, and meanwhile, the environment-friendly and safe raw materials, the simplicity and convenience in operation, the stable performance and the low cost are realized.

In order to achieve the above purpose, the specific technical scheme of the invention is as follows:

the BL-CQDs fluorescent probe takes ficus microcarpa leaves as raw materials, carbon quantum dots prepared through extraction and carbonization reactions, namely BL-CQDs, and the lattice spacing of the obtained BL-CQDs is 0.209nm and has a graphene-like structure; the microcosmic appearance is uniformly and spherically distributed in a single dispersion way, and the average size of BL-CQDs particles is 1.6-1.8nm; the surface of the modified polyurethane contains hydrophilic functional groups of hydroxyl, carbonyl and aldehyde groups; meanwhile, BL-CQDs have the characteristic of single excitation and double emission fluorescence, namely BL-CQDs fluorescent probes;

the BL-CQDs fluorescent probe has optical characteristics and anti-interference performance, wherein the anti-interference performance comprises photo-bleaching resistance, pH stability and salt stability;

the optical characteristic is that the BL-CQDs fluorescence quantum yield is 4.5%, and the fluorescent light has single excitation and double emission, specifically, the best emission peak under the best excitation wavelength of 410nm is positioned at 480nm and 673nm, and BL-CQDs emit bright red fluorescence under an ultraviolet lamp;

the photobleaching resistance is shown by that the fluorescence intensity and the initial intensity of BL-CQDs are not obviously changed after the BL-CQDs are irradiated under a xenon lamp and an ultraviolet lamp;

the pH stability is expressed by that the best buffer solution environment for BL-CQDs detection is a boric acid-sodium borate buffer system with the pH value of 8.3;

the salt resistance stability is expressed by specific selectivity and stability to ACE enzymatic product HA;

the anti-interference performance, namely the specific selectivity to HA, is that common metal ions, anions and biological small molecules do not basically influence the fluorescence signals of BL-CQDs, wherein the common metal ions, anions and biological small molecules are specifically shown as follows, and the metal ions comprise Na ⁺ 、K ⁺ 、Ca ²⁺ 、Fe ³⁺ 、Mg ²⁺ 、Al ³⁺ 、Mn ²⁺ 、Ba ²⁺ 、NH ₄ ⁺ 、Zn ²⁺ 、Cd ²⁺ 、Pb ²⁺ 、Bi ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the Anions include SO ₄ ^2- 、SO ₃ ^2- 、NO ₃ ^- 、NO ₂ ^- 、HPO ₄ ^2- 、H ₂ PO ₄ ^- 、CO ₃ ^2- And HCO ₃ ^- The method comprises the steps of carrying out a first treatment on the surface of the Biological small molecules include Ala, val, leu, pro, trp, cys, AA, biotin, glucose, DA, HA.

A preparation method of BL-CQDs fluorescent probe comprises the following steps:

step 1, raw material treatment, namely taking fresh ficus microcarpa leaves as raw materials, cleaning the ficus microcarpa leaves, crushing, and extracting under a certain condition to obtain an extract;

the extraction condition in the step 1 is that the volume ratio of the extractant is 2:1, and extracting for 30min; the extract obtained in the step 1 meets the raw material proportion of 6g/30mL;

step 2, preparing carbon quantum dots, namely under certain conditions, performing solvothermal carbonization reaction on the extract liquid obtained in the step 1, filtering after the reaction is finished, and filtering filtrate through an ultrafiltration membrane to obtain a carbon quantum dot solution, wherein the obtained carbon quantum dot solution is simply called BL-CQDs (BL-CQDs fluorescent probes);

the condition of the solvothermal carbonization reaction in the step 2 is that the solvothermal carbonization reaction temperature is 180 ℃ and the reaction time is 10 hours;

the preservation condition of the carbon quantum dot solution is that the preservation temperature is 4 ℃, and the use condition is that the carbon quantum dot solution is diluted by 10 times for use when the subsequent detection is needed.

An application of an ACE activity detection method based on BL-CQDs fluorescent probes, wherein the ACE activity detection method comprises the following steps:

step a, establishing an enzymatic reaction system of a standard Angiotensin Converting Enzyme (ACE) solution, namely mixing a boric acid-sodium borate buffer solution BBS and a maleimido-histidyl-leucine (HIL) to obtain a mixed solution, adding ACE into the mixed solution, and uniformly mixing to obtain the enzymatic reaction system of the standard ACE solution;

in the step a, the BBS concentration is 0.1mol/L, the pH=8.3, the HHS concentration is 5mmol/L, and the ACE concentration is 0.5U/L; the volume ratio of BBS, HIL and ACE is 3:2:1, a step of;

b, establishing an enzyme inactivation reaction system, namely incubating the standard ACE solution obtained in the step a in a water bath under a certain condition, and adding HCl to terminate the reaction after the water bath is completed, so as to obtain the enzyme reaction system;

the water bath incubation condition in the step b is that the water bath temperature is 37 ℃ and the water bath time is 40min; the condition for stopping the reaction in the step b is that the concentration of HCl is 1.0mol/L, and the temperature is kept for 30min after HCl is added;

step c, testing the fluorescence intensity of an enzyme inactivation system, namely adding BL-CQDs (basic CQIDs) into the enzyme inactivation system obtained in the step b according to the condition that BL-CQDs and HHT meet a certain volume ratio, and testing the fluorescence intensity under a certain condition to obtain the concentration data of the product HA of the enzymatic reaction, namely realizing ACE activity detection;

in the step c, the volume ratio of BL-CQDs to HHT is 1:2, wherein the fluorescent intensity test in the step c is carried out under the condition lambda _ex ＝377nm，λ _em =460 nm and λ _em =676 nm, slit 5.0nm.

Ratio I of BL-CDs fluorescence intensities ₆₇₆ /I ₄₆₀ Linear with ACE activity in the range of 0.02-0.8U/L, and the regression equation is DeltaF= 2.5371C _ACE -0.031 1, lod of 0.016U/L;

wherein I is ₆₇₆ /I ₄₆₀ Represents the ratio of the fluorescence intensity at 676nm to the fluorescence intensity at 460nm, F represents I ₆₇₆ /I ₄₆₀ Delta F represents F/F ₀ -1。

The ACEI screening method based on BL-CQDs fluorescent probes is applied, ACEI screening is achieved through an ACE activity inhibition experiment, the ACE activity inhibition experiment is specifically characterized in that in the ACE activity detection method, when an enzyme deactivation reaction system is established, an inhibitor is added into a standard ACE solution enzymatic reaction system, then an enzymatic reaction is carried out, and then the effect of the inhibitor can be obtained through a fluorescence intensity test of a subsequent enzyme deactivation system.

An angiotensin converting enzyme inhibitor, wherein the inhibitor is an extract of yellow aeolian wood leaves, and the inhibition rate of 2.5mg of the extract of yellow aeolian wood leaves to 0.5U/L ACE is 88.20%.

The characterization and testing results of the invention are as follows:

1. BL-CQDs are tested by TEM, XRD, FTIR, XPS, and the BL-CQDs are uniformly and spherically distributed in a single dispersion way; BL-CQDs have very small nano-particle sizes with average sizes of 1.6-1.8nm; the lattice spacing of BL-CQDs is 0.209nm; BL-CQDs carbonize the graphene-like structure in the synthesis process; the BL-CQDs are rich in hydrophilic groups such as hydroxyl, carboxyl, aldehyde groups and the like on the surface, and have good water solubility.

2. BL-CQDs fluorescence property test shows that the fluorescent dye composition has the advantages of good optical property, photobleaching resistance, pH stability, salt resistance stability and interference resistance,

the optical characteristic is that the fluorescence quantum yield Q of BL-CQDs is 4.5%, the fluorescence performance of single excitation and double emission is achieved, specifically, the best emission peak under the best excitation wavelength of 410nm is located at 480nm and 673nm, and BL-CQDs emit bright red fluorescence under an ultraviolet lamp;

the anti-interference performance, namely the specific selectivity to HA, is that common metal ions, anions and biological small molecules do not basically influence the fluorescence signals of BL-CQDs, wherein the common metal ions, anions and biological small molecules are specifically shown as follows, and the metal ions comprise Na ⁺ 、K ⁺ 、Ca ²⁺ 、Fe ³⁺ 、Mg ²⁺ 、Al ³⁺ 、Mn ²⁺ 、Ba ²⁺ 、NH ₄ ⁺ 、Zn ²⁺ 、Cd ²⁺ 、Pb ²⁺ 、Bi ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the Anions include SO ₄ ^2- 、SO ₃ ^2- 、NO ₃ ^- 、NO ₂ ^- 、HPO ₄ ^2- 、H ₂ PO ₄ ^- 、CO ₃ ^2- And HCO ₃ ^- The method comprises the steps of carrying out a first treatment on the surface of the The biological small molecules include alanine Ala, valine Val, leucine Leu, proline Pro, tryptophan Trp, cysteine Cys, ascorbic acid AA, biotin Biotin, glucose, dopamine DA and hippuric acid HA.

3. When BL-CQDs fluorescent probe is used as ACE activity detection method, the fluorescence intensity ratio I of BL-CODs ₆₇₆ /I ₄₆₀ Linear with ACE activity in the range of 0.02-0.8U/L, and the regression equation is DeltaF=2.5371C _ACE -0.031 1, LOD of 0.016U/L.

4. When BL-CQDs fluorescent probe is used as ACEI screening method, an angiotensin converting enzyme inhibitor is found, and the effect is that the inhibition ratio of 2.5mg yellow aeolian wood leaf extract to 0.5U/L ACE is 88.20.

From the above characterization and testing, the present invention has the following advantages over the prior art: the BL-CQDs fluorescent probe provided by the invention has extremely high fluorescence response, greatly improves the detection sensitivity, has extremely good stability, selectivity and specificity on enzymatic hydrolysis product hippuric acid, and is very suitable for detecting the activity of angiotensin converting enzyme from a complex system or screening compounds with the activity of inhibiting the angiotensin converting enzyme from complex systems such as traditional Chinese medicines.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of BL-CQDs and detection of HA;

FIG. 2 is (a) TEM of BL-CQDs; (b) particle size distribution; (c) HR-TEM image;

FIG. 3 is an XRD pattern of BL-CQDs;

FIG. 4 is a FTIR spectrum of BL-CQDs;

FIG. 5 is a high resolution spectrum of (a) XPS full spectrum (b) C1s, (C) N1s and (d) O1s of BL-CQDs;

FIG. 6 is a graph of the ultraviolet absorption spectrum and the optimal fluorescence excitation emission spectrum of BL-CQDs (inset: photographs of BL-CQDs under daylight (left) and 365nm ultraviolet lamp (right);

FIG. 7 is a graph of the emission spectra of BL-CQDs at excitation wavelengths of 360nm to 450 nm;

FIG. 8 is the effect of BBS at different pH on BL-CQDs fluorescence intensity: (a) Lambda (lambda) _em =470 nm and λ _em Graph of fluorescence intensity versus pH at=677 nm; (b) I ₄₇₀ /I ₆₇₇ A plot of ratio versus pH;

FIG. 9 is the effect of saline solutions of different concentrations on BL-CQDs fluorescence intensity: (a) NaCl; (b) KCl;

FIG. 10 is a graph showing the effect of different ions on BL-CQDs fluorescence intensity: (a) metal ions; (b) an anion; (c) amino acids and biological small molecule substances; (d) The effect of different substances on BL-CQDs fluorescence intensity in the presence of HA;

FIG. 11 shows the ratio of BL-CQDs to fluorescence intensity of various substances at different concentrations: (a) ACE; (b) HHL;

FIG. 12 is a graph showing the ratio of the fluorescence intensity (a) of products produced by catalyzing HHT with different concentrations to BL-CQDs; (b) a linear relationship graph.

Detailed Description

Embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. The following examples are only to be construed as limiting the scope of the invention.

For ease of description, the present invention employs the following abbreviations:

BL-CQDs are banyan leaf carbon quantum dots;

HIL is a hippocampal-histidyl-leucine, an abbreviation for Hip-His-Leu;

HA is Hippuric acid, i.e., an abbreviation for Hip, hippuric acid;

example 1

A preparation method of BL-CQDs fluorescent probe and its application in ACE activity detection and ACEI screening are shown in figure 1.

step 1, raw material treatment, namely taking fresh banyan leaves as raw materials, crushing 6.0g of banyan leaves She Qingjing, adding the crushed banyan leaves into a mixed solution of 20mL of absolute ethyl alcohol and 10mL of acetone, standing and extracting for 30min, and filtering after the extraction is finished, so as to obtain an extract with a raw material ratio of 6g/30mL;

and 2, preparing the carbon quantum dots, namely performing solvothermal carbonization reaction on the extract liquid obtained in the step 1 by using a boric acid-sodium borate buffer solution BBS under the condition that the reaction temperature is 180 ℃ and the reaction time is 10 hours, filtering after the reaction is finished, and filtering the filtrate by using an ultrafiltration membrane to obtain a carbon quantum dot solution, wherein the obtained carbon quantum dot solution is abbreviated as BL-CQDs (BL-CQDs) fluorescent probes.

The preservation condition of the obtained carbon quantum dot solution is that the carbon quantum dot solution is preserved at the temperature of 4 ℃, and when the subsequent detection is needed, the carbon quantum dot solution is diluted by 10 times for use.

To demonstrate that BL-CQDs of the present invention are water soluble, the dispersibility of BL-CQDs was tested by Transmission Electron Microscopy (TEM) while the morphology and structure of BL-CQDs could be characterized, and the test results are shown in FIG. 2.

As can be seen from FIG. 2 (a), BL-CQDs are uniformly spherically distributed in a single dispersion; by statistical software analysis, as shown in FIG. 2 (b), the BL-CQDs particles had an average size of 1.7nm, i.e., BL-CQDs had very small nano-particle size;

as can be seen from FIG. 2 (c), the lattice spacing of BL-CQDs is 0.209nm, which indicates that the BL-CQDs carbonize a graphene-like structure during the synthesis process.

To further demonstrate that BL-CQDs have a grapheme-like carbon structure, XRD characterization was performed on solid BL-CQDs, setting a scan range of 10-60. The test results are shown in fig. 3, showing a broad diffraction peak at 2θ=20.16°, indicating that the carbon atoms of the synthesized BL-CQDs show a highly disordered state; the peak was found to correspond to the (002) face of graphene by comparison with standard cards, consistent with the HR-TEM results of BL-CQDs, confirming the grapheme-like carbon structure of BL-CQDs.

In order to prove that the surface of BL-CQDs is rich in hydrophilic groups such as hydroxyl groups, carboxyl groups, aldehyde groups and the like, the BL-CQDs have good water solubility, and the functional group structure on the surface of BL-CQDs is tested through Fourier transform infrared spectroscopy (FTIR). The test result is shown in FIG. 4, 3.29 cm in infrared spectrum ^-1 The peak at this point was assigned to-OH/-NH, 2.928.29 cm ^-1 And 2.854.08 cm ^-1 The peak at the position is-CH in alkane compound ₂ -a stretching vibration peak of 1.34 cm ^-1 The peak at this point indicates the presence of carboxylic acid c=o; 1627.16cm ^-1 、1 458.39cm ^-1 And 720.25cm ^-1 The absorption peak at the position belongs to the stretching vibration peak of C=C double bond and the out-of-plane bending vibration peak of C-H, 1.374 cm ^-1 The peak at this point may be attributed to flexural vibration in the-OH bond plane, 1.074.56 cm ^-1 The band at the site is attributed to an ether C-O group.

To further determine the surface functional groups of BL-CQDs, X-ray photoelectron spectroscopy (XPS). The peaks of C1s, N1s and O1s (FIG. 5 (a)) at 285.37eV, 399.72eV and 531.34eV, respectively, can be seen by XPS characterization, demonstrating that BL-CQDs consist essentially of element C, N, O. The 3 peaks at 284.77eV, 286.16eV and 288.34eV of fig. 5 (b) represent the presence of C-C/c=c bonds, C-O-C bonds and c=o bonds, respectively. The 2 deconvolution peaks at 399.57eV and 400.2eV of fig. 5 (C) correspond to c= N, N-H bonds, respectively, and the 2 peaks at 531.54eV and 532.79eV of fig. 5 (d) correspond to C-O and c=o bonds, respectively. It is well demonstrated that BL-CQDs are surface-rich with hydrophilic functional groups that enhance the water solubility and stability of BL-CQDs, consistent with the characterization results of FTIR.

To demonstrate that BL-CQDs are characterized for use as fluorescent probes, the following optical characterization experiments, photobleaching resistance experiments, pH stability experiments, salt stability experiments, and interference immunity experiments were performed on the optical properties, stability, and specific selectivity of BL-CQDs.

Optical characterization experiments optical property testing was performed on BL-CQDs using an ultraviolet-visible absorption spectrometer and a fluorescence spectrophotometer. The test results are shown in FIG. 6, in which the ultraviolet absorption spectrum of BL-CQDs has a strong absorption peak at 263nm, which can be attributed to the n-pi transition of the C=C double bond; weak uv absorption peaks at 418nm and 673nm are caused by pi-pi transitions of the c=o/c=n double bond. The fluorescence spectrum shows that BL-CQDs have single-excitation double-emission fluorescence performance, and the optimal emission peak under the optimal excitation wavelength of 410nm is positioned at 480nm and 673 nm; BL-CQDs can fluoresce bright red under an ultraviolet lamp.

Photo bleaching resistance test BL-CQDs were tested for photo bleaching resistance under the conditions of fluorescence intensity after continuous irradiation for 60min under a xenon lamp 7 and a 365nm ultraviolet lamp, respectively. BL-CQDs show that their fluorescence intensity does not change much compared with the initial intensity after long-term irradiation under a xenon lamp and an ultraviolet lamp, indicating that BL-CQDs have excellent light stability.

pH stability experiments the change in fluorescence intensity of BL-CQDs was tested in BBS environments with pH values of 6.8-9.3. The test results are shown in FIG. 8 when pH<8.3 lambda _em The fluorescence intensity at 470nm is almost unchanged, but λ _em Fluorescence intensity at=677 nm gradually decreases; while when pH is>8.3 lambda _em =470 nm and λ _em Fluorescence intensity at=677 nm was increased correspondingly, but λ _em Fluorescence intensity at 470nm was higher with pHIs obvious; at pH 8.3, I ₄₇₀ /I ₆₇₇ The ratio was maximized and BBS at pH 8.3 was chosen as the optimal buffer for BL-CQDs detection.

Salt stability resistance experiments, the salt stability of BL-CQDs was tested under conditions of NaCl concentration varying from 0-2.5mol/L and KCl concentration varying from 0-1.5mol/L, respectively. As shown in FIG. 9, the fluorescence intensity of BL-CQDs was not much different from the initial intensity, i.e., it was shown that BL-CQDs had good salt stability.

Interference resistance experiments the effect of the following ions or molecules on BL-CQDs fluorescence intensity was determined. Metal ion Na with concentration of 10mmol/L ⁺ 、K ⁺ 、Ca ²⁺ 、Fe ³⁺ 、Mg ²⁺ 、Al ³⁺ 、Mn ²⁺ 、Ba ²⁺ 、NH ₄ ⁺ 、Zn ²⁺ 、Cd ²⁺ 、Pb ²⁺ 、Bi ²⁺ The test results of (a) are shown in fig. 10 (a); SO (SO) ₄ ^2- 、SO ₃ ^2- 、NO ₃ ^- 、NO ₂ ^- 、HPO ₄ ^2- 、H ₂ PO ₄ ^- 、CO ₃ ^2- And HCO ₃ ^- The test results of common anions are shown in fig. 10 (b); the test results of the amino acids and biological small molecules such as Ala, val, leu, pro, trp, cys, AA, biotin, glucose, DA, HA are shown in fig. 10 (c); the test results of the mixed ions are shown in fig. 10 (d), and the fluorescence intensity Δf=f/F is analyzed ₀ (wherein F is a different metal ion at lambda _em =673 nm and λ _em Ratio of fluorescence intensities at 470nm (I ₆₇₃ /I ₄₇₀ )；F ₀ For blank set at lambda _em =673 nm and λ _em Ratio of fluorescence intensities at 470nm (I ₆₇₃ /I ₄₇₀ ) A) is provided; the overall results are shown in FIG. 10, and the addition of common ions and biological small molecules, except HA, HAs substantially no effect on the fluorescence signal of BL-CQDs, i.e., it is demonstrated that BL-CQDs fluorescent probes are specifically selective for HA.

It has been demonstrated through the above experiments that BL-CQDs of the present invention can be used as fluorescent probes, i.e., BL-CQDs fluorescent probes. Next, an ACE activity detection method constructed based on the BL-CQDs fluorescent probe of the present invention is provided.

An ACE activity detection method based on BL-CQDs fluorescent probe comprises the following steps:

step a, establishing an enzymatic reaction system of a standard Angiotensin Converting Enzyme (ACE) solution, firstly mixing 60 mu L of BBS with the concentration of 0.1mol/LpH =8.3 and 40 mu L of HHT with the concentration of 5mmol/L to obtain a mixed solution, then adding 20 mu L of ACE with the concentration of 0.5U/L into the mixed solution, and uniformly mixing to obtain the enzymatic reaction system of the standard ACE solution;

b, establishing an enzyme inactivation reaction system, namely placing the standard ACE solution obtained in the step a into a 250 mu L centrifuge tube, performing water bath incubation under the condition that the water bath temperature is 37 ℃ and the water bath time is 40min, adding 60 mu L of 1.0mol/L HCl after the water bath is finished, and preserving heat for 30min to terminate the reaction to obtain the enzyme inactivation reaction system;

step c, fluorescence intensity test of enzyme inactivation reaction System 20 μLBL-CQDs was added to the enzyme inactivation reaction System obtained in step b, at λ _ex ＝377nm，λ _em =460 nm and λ _em And (3) carrying out fluorescence intensity test under the condition that the slit is 5.0nm and the 676nm, so as to obtain concentration data of the product HA of the enzymatic reaction, namely, realizing ACE activity detection.

In order to prove that the ACE and HIL residues in the enzyme reaction system have no influence on ACE activity detection, the ACE activity detection method is respectively carried out on different ACE concentrations and different HIL concentrations, and the test results are shown in FIG. 11. Wherein, the concentration range of ACE is 0-20U/L; HHT concentration ranges from 0 to 5mmol/L. The influence of the concentration of ACE and HIL on BL-CQDs fluorescence intensity in a reaction system in the ACE activity detection process is eliminated,

the test results showed that the ratio of fluorescence intensity of ACE to HIL at different concentrations (I ₆₇₆ /I ₄₆₀ ) All remained between 0.09-0.10, i.e. had no effect on the detection of ACE activity by the BL-CQDs probes.

To further demonstrate the sensitivity and minimum detection concentration of the ACE activity detection method of the present invention, HA production corresponding to different ACE concentrations, as well as fluorescence intensity ratios, were determined in the enzymatic reaction of step b above. As shown in fig. 12, as ACE concentration increases, the amount of HA produced in the reaction also increases, and the fluorescence intensity ratio gradually increases; at an ACE concentration of 0.8U/L, the substrate HIL reacted completely, and the ratio of fluorescence intensity was not substantially changed by adding a further high concentration of ACE. As can be seen from an analysis of the results of the experiment,

1. BL-CQDs fluorescence intensity ratio (DeltaF=F/F) ₀ -1，F＝I ₆₇₇ /I ₄₆₀ ) Maintains good linear relation with ACE concentration in the range of 0.02-0.8U/L, and the linear equation is DeltaF=2.5371C _ACE -0.031 1，(R ² ＝0.993 6)；

2. The minimum limit of detection of ACE activity by BL-CQDs is 0.016U/L.

Experimental results show that the concentration of ACE and HHT has no influence on the fluorescence intensity of BL-CQDs in the reaction system, namely, BL-CQDs have good sensitivity and stability on the detection of ACE activity.

In order to prove the practical application effect of the ACE activity detection method based on BL-CQDs fluorescent probe construction, an ACE activity detection experiment using human serum as a detection object is adopted in the embodiment.

The ACE activity detection method using human serum as a detection object has the same steps as the ACE activity detection method constructed based on BL-CQDs fluorescent probes, and is different in that: the step a is not performed and, in the step b, human serum is substituted for standard ACE solution.

The fluorescence intensity test results of human serum are shown in Table 1, and the recovery rate is 92.65% -110.3%. Test results show that the ACE activity detection method provided by the invention meets the practical application conditions when taking human serum as a detection object.

Table 1 detection of ACE activity in human serum (n=3)

The BL-CQDs fluorescent probe can be used as an ACE activity detection method and ACEI screening.

An ACEI screening method based on BL-CQDs fluorescent probe can obtain the effect of the inhibitor through ACE activity inhibition experiments, namely ACEI screening is realized.

In the specific method of the ACE activity inhibition experiment, in the ACE activity detection method, when the step b is performed, after an inhibitor is added into a standard ACE solution, an enzymatic reaction is performed, and then the effect of the inhibitor can be obtained through a fluorescence intensity test in the subsequent step c.

To demonstrate the practical effect of BL-CQDs fluorescent probes as an application of ACEI screening, ACEI screening experiments with captopril tablet extract and yellow aeolian tree leaf extract as detection subjects were provided in this example, respectively.

ACEI screening method using captopril tablet extract as subject uses captopril extract as inhibitor to perform ACE activity inhibition experiment.

The preparation method of the captopril tablet extract comprises the steps of taking 3 captopril tablets with the concentration of 25 mg/tablet, and grinding to obtain captopril tablet powder; then accurately weighing a piece of captopril tablet powder, dissolving the captopril tablet powder in ethanol, and carrying out ultrasonic treatment for 10min; extracting supernatant after standing, filtering twice with 0.22 μm ultrafiltration membrane, metering with ultrapure water to 10mL volumetric flask, and preserving at 4deg.C to obtain captopril tablet extract;

the ACE activity inhibition experiment result shows that the inhibition rate of the ACE activity is calculated by a formula (1), the inhibition rate of the 2.5mg captopril tablet to 0.5U/L ACE is 73.88%, wherein U is as follows _ACEa ACE Activity measured for blank without ACEI, U _ACEb ACE activity was measured for the addition of ACEI.

ACEI screening method using yellow aeolian tree leaf extract as object uses yellow aeolian tree leaf extract as inhibitor to make ACE activity inhibition experiment.

The preparation method of the yellow aeolian belltree leaf extract comprises the steps of taking 40g of yellow aeolian belltree leaves, shearing the leaves, and placing the cut leaves in a 500mL beaker; then, 400mL of 60% ethanol is added for extraction; recovering solvent after extraction, adsorbing the concentrated solution by D101 macroporous resin, eluting with 50% ethanol, recovering solvent by rotary evaporator, and evaporating to dryness to obtain yellow brown crude product; then, the extract of the yellow aeolian wood leaves with the weight of 0.920 g is obtained by reflux refining with absolute ethyl alcohol, concentrating and freeze drying, and the obtained extract of the yellow aeolian wood leaves is stored in a dryer for standby.

The ACE activity inhibition experiment result shows that the inhibition rate of 2.5mg of the yellow sycamore leaf extract to the ACE with the same concentration of 0.5U/L is 88.20%.

Screening experiments with captopril tablets and yellow aeolian tree leaf extracts as detection objects show that the yellow aeolian tree leaf extracts have better ACE activity inhibition effect; meanwhile, the BL-CQDs fluorescent probe and the ACE activity detection method provided by the invention can realize ACEI screening efficiently and simply.

Claims

1. An angiotensin converting enzyme inhibitor characterized in that: the inhibitor is a yellow aeolian wood leaf extract, and the inhibition rate of 2.5mg of the yellow aeolian wood leaf extract to 0.5U/L ACE is 88.20%;

the preparation method of the yellow aeolian wood leaf extract comprises the steps of taking yellow aeolian wood leaves, shearing the yellow aeolian wood leaves, and then placing the cut yellow aeolian wood leaves in a beaker; then adding ethanol for extraction; recovering solvent after extraction, adsorbing the concentrated solution by D101 macroporous resin, eluting with ethanol, recovering solvent by rotary evaporator, and evaporating to dryness to obtain yellow brown crude product; then, the yellow aeolian-wood leaf extract is obtained through absolute ethyl alcohol reflux refining, concentration and freeze drying, and the obtained yellow aeolian-wood leaf extract is stored in a dryer for standby.